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Silk fibroin for skin injury repair: Where do things stand?
Journal Pre-proof Silk fibroin for skin injury repair: Where do things stand?
Mazaher Gholipourmalekabadi, Sunaina Sapru, Ali Samadikuchaksaraei, Rui L. Reis, David L. Kaplan, Subhas C. Kundu PII:
S0169-409X(19)30164-4
DOI:
https://doi.org/10.1016/j.addr.2019.09.003
Reference:
ADR 13504
To appear in:
Advanced Drug Delivery Reviews
Received date:
10 May 2019
Revised date:
12 September 2019
Accepted date:
26 September 2019
Please cite this article as: M. Gholipourmalekabadi, S. Sapru, A. Samadikuchaksaraei, et al., Silk fibroin for skin injury repair: Where do things stand?, Advanced Drug Delivery Reviews (2019), https://doi.org/10.1016/j.addr.2019.09.003
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof Silk fibroin for skin injury repair: where do things stand? Mazaher Gholipourmalekabadi* 1, 2, Sunaina Sapru 3, Ali Samadikuchaksaraei1,2, Rui L. Reis4, David L. Kaplan5, Subhas C. Kundu3,4* 1
Cellular and Molecular Research Centre, 2 Department of Tissue Engineering & Regenerative
Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran. Department of Biotechnology, Indian Institute of Technology Kharagpur, India
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3Bs Research Group, I3Bs - Research Institute on Biomaterials, Biodegradable and Biomimetics.
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Department of Biomedical Engineering, Tufts University, Medford, MA, USA
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Medicine, University of Minho, Guimaraes, Portugal.
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Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative
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*Corresponding authors:
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Dr. Mazaher Gholipourmalekabadi Tel: (+98 21) 8862 2755; Fax: (+98 21) 8862 2533 E-mail: [email protected]; [email protected]
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Professor Subhas C Kundu E-mail: [email protected]; [email protected] Tel: +351 253 510 932, Orcid ID: 0000-0002-7170-2291 E-mail of all authors: [email protected]; [email protected] [email protected] [email protected]; [email protected] [email protected] [email protected] [email protected]; [email protected] Number of tables: 5
Number of figures: 6
Running title: Silk fibroin in skin wound healing
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Table of Contents Abstract ......................................................................................................................................3 Statement of significance ............................................................................................................4 Abbreviations used in this article .................................................................................................5 1. Introduction.............................................................................................................................7 2. Silk: types, sources and chemistry ......................................................................................... 12 3. Main characteristics of silk protein fibroin (SF) ..................................................................... 14
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(i) Mechanical properties (tensile and compressive) ............................................................... 14 (ii) Biocompatibility and biodegradation ................................................................................ 15
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(iii) Cell-silk fibroin interaction behavior ............................................................................... 17 4. Silk fibroin for skin tissue engineering .................................................................................. 18
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4.1. Skin structure and healing process .................................................................................. 18
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4.2. SF and SF blends in skin wound healing applications...................................................... 21 4.2.1. SF alone ................................................................................................................... 22
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4.2.2. SF blends ................................................................................................................. 29 5. Biofunctionalization .............................................................................................................. 38
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6. Antibacterial SF derived scaffolds ......................................................................................... 41 6.1. Antibacterial SF fibrous scaffolds ................................................................................... 42
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6.2. Antibacterial SF sponge .................................................................................................. 45 6.3. Antibacterial SF films ..................................................................................................... 47
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6.4. Antibacterial SF Gels ...................................................................................................... 48 7. Other biomedical applications of silk fibroins ........................................................................ 48 8. Benefits and concerns of using silks for biomedical applications ........................................... 49 9. Conclusions and future prospects .......................................................................................... 50 Acknowledgements ................................................................................................................... 52 Authors’ contributions............................................................................................................... 52 Conflict of interests ................................................................................................................... 52 References ................................................................................................................................ 53
Journal Pre-proof Abstract Several synthetic and natural materials are used in soft tissue engineering and regenerative medicine with varying degrees of success. Among them, silkworm silk protein fibroin, a naturally occurring protein-based biomaterial, exhibits many promising characteristics such as biocompatibility, controllable biodegradability, tunable mechanical properties, aqueous
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preparation, minimal inflammation in host tissue, low cost and ease of use. Silk fibroin is often used alone or in combination with other materials in various formats and is also a promising
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delivery system for bioactive compounds as part of such repair scenarios. These properties make
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silk fibroin an excellent biomaterial for skin tissue engineering and repair applications. This review
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focuses on the promising characteristics and recent advances in the use of silk fibroin for skin
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wound healing and/or soft-tissue repair applications. The benefits and limitations of silk fibroin as a scaffolding biomaterial in this context are also discussed.
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regenerative medicine
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Keywords: Silk fibroin; biomaterials; wound healing; skin substitutes; tissue engineering;
Journal Pre-proof Statement of significance Silk protein fibroin is a natural biomaterial with important biological and mechanical properties for soft tissue engineering applications. Silk fibroin is obtained from silkworms and can be purified using alkali or enzyme based degumming (removal of glue protein sericin) procedures. Fibroin is used alone or in combination with other materials in different scaffold forms, such as nanofibrous
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mats, hydrogels, sponges or films tailored for specific applications. The investigations carried out using silk fibroin or its blends in skin tissue engineering have increased dramatically in recent
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years due to the advantages of this unique biomaterial. This review focuses on the promising
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characteristics of silk fibroin for skin wound healing and/or soft-tissue repair applications.
Journal Pre-proof Abbreviations used in this article AaSF: Antheraea assama silk fibroin ADA: alginate dialdehyde
ADSCs: adipose tissuederived mesenchymal stem cells AM: amniotic membrane
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AMP: antimicrobial peptide
HTCC: N-(2-hydroxy)propyl3-trimethyl ammonium chitosan chloride hUCMSCs: human umbilical cord mesenchymal stem cells
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BC: bacterial cellulose
BMSCs: bone marrow mesenchymal stromal cells Bm: Bombyx mori BmSF: B. mori silk fibroin CECTS: N-carboxyethyl chitosan CFFs: child foreskin fibroblasts
pHA: polarized hydroxyapatite
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HFFF2: human caucasian foetal foreskin fibroblast hPL: human platelet lysate
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AgNPs: silver nanoparticles
Bm: Bombyx mori
PEO: polyethylene oxide
HDF: human dermal fibroblast HEKs: human epidermal keratinocytes HFFs: human foreskin fibroblasts
B. cereus: Bacillus cereus
PEI: polyethylenimine
PGS: poly(glycerol sebacate)
Ag2O-SF: silver oxide nanoparticles
bFGF: basic fibroblast growth factor
HaCaT: immortalized human keratinocyte HAMSCs: human amniotic and membrane-derived stem cells
PHBV: poly (3-hydroxybutyrate-co-3-hydroxyvalerate) PLA: poly-L-lactic acid PLGA: poly(lactide-coglycolic acid) PPP: platelet-poor plasma
PRP: platelet-rich plasma PrSF: P. ricini silk fibroin
HA: hyaluronic acid KGF: keratinocyte growth factor
PVA: poly(vinyl alcohol) SA: sodium alginate
LP: L-proline LPS: lipopolysaccharides
SL: salt-leaching
M. albicans: Monilia albicans SLE: salt-leaching electrospinning M. luteus: Micrococcus luteus Sr: strontium MoSe2: molybdenum diselenide
SSF: spunlaced SF
Journal Pre-proof CMC: carboxymethyl cellulose COL: collagen CS: chondroitin sulfate
mSF: SF microfibers
TOCN: oxidized cellulose
M. tuberculosis: Mycobacterium tuberculosis NHDF: normal human dermal fibroblasts
RGD: arginine, glycine and aspartate S. aureus: Staphylococcus aureus S. epidermidis: Staphylococcus epidermidis
CTS: chitosan NHEF: normal human epidermal fibroblasts
SIS: styrene-isoprene-styrene NHEK: normal human epidermal keratinocytes NT: neurotensin
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ECM: extra cellular matrix
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CTZ: ceftazidime
rBMSCs: rat bone marrow mesenchymal stem cells
EGF: epidermal growth factor ESF: electrospun silk fibroin
P. aeruginosa: Pseudomonas aeruginosa PBS: phosphate-buffered saline PDGF: platelet-derived growth factor
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pECM: human placentaderived extracellular matrix
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GA: glutaraldehyde
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FD: freeze-drying
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EDC: 1-ethyl-3-(3dimethylaminopropyl) carbodiimide
GS: gentamicin sulfate
PEG: polyethylene glycol
Sr: strontium TCH: tetracycline hydrochloride
TE: Tissue engineering TERM: tissue engineering and regenerative medicine VEGFα: vascular endothelial growth factor alpha ZWP: homogeneous polysaccharide extracted from the rhizomes of Curcuma zedoaria
Journal Pre-proof 1. Introduction Over the past decade, the limitations with organ transplantation due to the shortage of transplantable organs and the rejection of these organs has been a major issue. The tissue engineering and regenerative medicine (TERM) field has emerged to overcome such limitations for replacing/repairing damaged tissues and organs (Fig. 1). Tissue engineering (TE) consists of
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gathering cells of a patient or the use of acceptable donor cells, expanding the cells in vitro and then seeding these cells into 3-D scaffolds/matrices. The cells are then induced in the presence of
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appropriate medium and bioactive factors to proliferate and differentiate into a desired tissue for
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implantation. There are three main elements for successful tissue regeneration: (i) cells harvested
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from tissue, (ii) scaffolds that supply morphology, structural support, and chemical and mechanical
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appropriate tissue growth [1, 2].
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signals to the cells, and (iii) interactions between the cells and matrix (scaffold) to support
Figure. 1. Illustration of the basic routes of tissue engineering and regenerative medicine. The cells are harvested from patients by biopsy and are grown in 2D culture under aseptic conditions. The cell culture
Journal Pre-proof medium is supplemented with growth factors and other specific features as required by the individual cell type. These cells are sub-cultured for the expansion of cells and are seeded onto 3D matrices for the construction of specific 3D tissues. These matrices/polymeric scaffolds may be natural, synthetic or blends and are fabricated in various material format such as hydrogels, porous sponges, nanofibrous mats, micro/nano particles, micro-/nano patterned surfaces, among others. These matrices may contain bioactive molecules to augment the proliferation of cells. For long-term culture, the matrices are grown in bioreactors
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to support and maintain the cultures under aseptic conditions. The regenerated tissue in vitro may be
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transplanted to restore the damaged tissue in vivo.
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The scaffolds/matrices have a key role in tissue engineering and regenerative medicine. There are many essential characteristics that are rational starting points for designing scaffolds for TE [3, 4].
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First, the biocompatibility of materials is key to support appropriate cellular activities, improve
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tissue regeneration without local or systemic inflammation or immune responses in the host [3, 4]. Second, suitable structure and high surface area is required to allow maximum cell interactions
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while supporting oxygen/nutrient transport. Furthermore, this type of structure is desirable to
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provide space for tissue in-growth. In addition, biodegradability or absorption of the scaffold material is relevant, where the degradation rate of the scaffold should match the rate of tissue regeneration to improve the transfer of functions from the artificial matrices to the native extracellular matrix (ECM) on a time-dependent basis during tissue regeneration [1-6]. Thus, designing scaffolds, which mimic the structure and biological functions of native ECM and provides a suitable environment for regulating cell activities, is the main goal. To obtain such scaffolds, synthetic or natural polymer materials have been pursued to meet the above needs, each with their own benefits and limitations [4, 7].
Journal Pre-proof Although there is no universal biomaterial that meets scaffolding requirements for all types of tissues, proteins as constituent parts of natural tissues are reasonable candidates for applications in TE. Natural proteins such as collagens, elastin, keratins, elastin-like peptides, albumin, fibrin and silks have been used as tissue scaffolds [4, 8, 9]. The biocompatibility, controllable biodegradability, aqueous preparations, and minimal inflammatory response of silk proteins have attracted the attention of researchers toward this need [10-13]. SF is a high molecular weight (>300
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kDa depending on source) amphiphilic protein with repetitive, modular hydrophobic domains,
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which are surrounded by small hydrophilic regions. The natural form of silkworm silks consist of
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a core fiber, silk protein fibroin (SF), and glue-like coating composed of a family of sericin
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proteins. SF also has various secondary structures, including α-helix, β-sheet, and crossed β-sheet. SF fibers can be obtained from the cocoon of the silkworm, Bombyx mori (Bm), which has a
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continuous strand of two fibers, to form the filament providing the unique physical and chemical
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properties [10, 12, 14-16].
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SF-based biomaterials demonstrate excellent biocompatibility [10, 12, 13, 17, 18]. Moreover, the unique structural assembly of this protein provides remarkable mechanical properties when
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compared with other commonly used biopolymer-based biomaterials. The degradation rate in vivo can be controlled from weeks to years after implantation in vivo, depending on crystallization, scaffold form, cross-linking agent, blending and other factors [19-24]. Hence, the silk has been utilized in various applications in TE, such as bone, cartilage, nerve, skin and others (Fig. 2).
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Figure 2. Different applications of silk protein fibroin in tissue engineering and regenerative
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medicine. Silk provides flexibility to fabricate different kinds of matrices and thereby, finds use in multiple biomedical applications including tissue engineering targets like the regeneration and repair of damaged
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tissues. Apart from reconstructing injured areas, silk matrices also effectively deliver bioactive molecules
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including drugs and growth factors to the target site, as well as genes and cells, further enhancing utility in regenerative medicine.
Skin plays a vital role in protecting the human body against the environment, dehydration, and infectious agents. Burns, wounds, or disease may cause the loss of skin integrity and tissueengineered skin substitutes provide an opportunity to restore skin loss [25, 26]. Many natural and synthetic skin substitutes are used for skin wound healing but remain limited in clinical effectiveness in many cases [27, 28]. Natural-based skin substitutes such as autografts, acellular
Journal Pre-proof dermal matrix allograft (alloderm®) and Xenografts (EZ Derm®), natural macromolecules(collagen, silk, cellulose, hyaluronic acid and others) derived scaffolds have several excellent characteristics including biocompatibility and biodegradability with positive impact on wound healing. The limited availability, risk of cross-contamination and cost are disadvantages associated with natural based skin substitutes, which limit clinical applications i [29-32]. Synthetic-based skin substitutes such as synthetic polymer sheets (Tegaderm®, Opsite®), polymer foams or sprays and
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others are available and inexpensive with no risk of infection transmission. However, slow
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degradation, controversial biocompatibility in some cases, and lower impact on wound healing
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compared with natural materials limit applications [29, 33]. Nevertheless, many natural and
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synthetic skin substitutes are already used as skin substitutes with various ranges of success [29,
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31, 34].
Recently Farokhi et al. [13] described the status of SF based wound dressing materials for skin
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regeneration and our goal here is to extend the understanding of silk in this context. The review
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focuses on the promising characteristics of SF for skin wound healing and/or soft-tissue repair and recent SF blend skin substitutes. Based on recent advances and developments with SF-based
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scaffolds, this review addresses the state-of-the-art of SF-derived skin substitutes with additional background, structure of the materials, fabrication procedures/processing, merits and limitations, functional stimuli-response features, functionalized (e.g., antibacterial) bioactive matrices for skin tissue regeneration and would healing. In vitro (Table 1a), in vivo (Table 1b) and clinical trials (Table 1c) carried out between 2000 and 2019 with different types of SF or SF blends, and the major findings and appropriate references, are summarized.
Journal Pre-proof 2. Silk: types, sources and chemistry Silk is secreted by various animals such as silkworms, spiders, scorpions, mites and bees best known as (arthropods) [10, 12, 35] (Fig. 3). The properties of silkworm silk mainly depend on the source of the material. For example, the silk extracted from the cocoons of Bm, which is also known as mulberry silk, is the most extensively characterized and utilized for biomedical
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applications. The silkworms from the family of Saturniidae are known as non-mulberry silks and there are several species in this category namely Antheraea mylitta (tasar), A. assama (A.
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assamensis, muga), A. pernyi, Samia ricini (Philosamia ricini, eri) and others. There are over
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30,000 species of spiders, for example Nephila clavipes and Araneus diadematus, which produce
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silks, with outstanding mechanical properties and lack of sericin protein. However, these silks are
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heterogeneous in nature and to obtain sufficient quantities is a major hurdle and expensive in comparison to silkworm silk. Thus, silk-based biomaterials are usually prepared from silkworm
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silk. The isolation and purification of silk fibers is relatively simple by using alkali or enzyme
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based degumming procedures, with subsequent utility in TE [10, 12, 13, 15, 18, 21, 36, 37].
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Silk is a lightweight (1.3 g/cm3), high strength (up to 4.8 GPa as the strongest fiber known in nature), protein with repetitive modular hydrophobic domains and small hydrophilic groups [35, 38]. The N- and C- termini of SF are highly conserved. The silkworm silk from Bm has two main parts, the fiber core protein, SF and the surrounding sericin proteins. The repetitive hydrophobic sequences are composed of short side-chain amino acids such as glycine and alanine. The hydrophilic blocks are comprised of larger side-chain amino acids such as charged amino acids. The structure of SF depends on the species/genera. For example, the SF of B mori contains a heavy (H) and light (L) chain that connect with a disulfide bond. The H chain has hydrophobic domains such as Gly-X (X being Ala, Ser, Thr, Val) repeats and can create anti-parallel β-sheets.
Journal Pre-proof Additionally, a glycoprotein called P25 is linked to the light and heavy fibroin chains with noncovalent bonds. In contrast, the P25 chain does not exist in non-mulberry silks. Instead, the heavy (H) chain homo-dimer has a main role in the formation of non-mulberry silks [16]. There are differences between mulberry and non-mulberry silks in terms of mechanical properties,
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bioactivity and the degradation behavior [10, 12, 16, 35, 37, 39-43].
Figure. 3. Arrays of silk protein fibroin matrices for tissue engineering and regenerative medicine. Silk as a biomaterial is often obtained from both mulberry and non-mulberry (shown above are some of the main cultivated silkworm species) silkworm silks. Depending on the silkworm species and availability, silk proteins may be isolated from the cocoons after degumming (removal of silk protein sericin) or from the mature 5th instar larvae (mainly from non-mulberry). Silk-based scaffolds are developed in various forms, which range from thin membranes/coatings to 3-D bio printed matrices to support organ specific requirements.
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3. Main characteristics of silk protein fibroin (i) Mechanical properties (tensile and compressive) The biomechanical properties of TE scaffolds have direct implications on their function during tissue regeneration [4, 44]. The biomechanical behavior of biomaterials has a critical impact on
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regulating cell growth, morphology, differentiation, migration and function [45-47]. Cells can
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sense matrix mechanics via receptors and subsequently transform mechanical stimulation to chemical signals [48-50]. For example, mesenchymal stem cell lineage differentiation can be
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affected by matrix stiffness (0.1–1 kPa, 8–17 kPa and 25–40 kPa) in vitro [51]. The mechanical
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behavior of extracellular matrices can also stimulate/repress the secretion of some factors and
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define the stem cell differentiation. The mechanical properties of tissues/organs can undergo changes during normal and pathologic situations [52, 53]. A promising TE scaffold should
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simulate the mechanical behavior of a normal tissue to accelerate tissue regeneration and lead to
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new functional tissue. SF offers a balance of breaking strength, modulus and elongation, which
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make it attractive for tissue engineering applications [10, 12, 17, 54]. The toughness of SF is several times higher than Kevlar [55], and the strength-to-density ratio of native silk is higher than steel (up to ten times) [56]. Silk fibers from different sources have different mechanical properties, an advantage for the development of scaffolds for different tissue needs [10, 54]. Silk fibers isolated from different sources generally have tensile strength, tensile modulus, and breaking strain ranging from 0.9±0.2 to 5±1.2 (g/den), 29–31 to 84–121 (g/den) and 4.1±2.7% to 28.0–34.0%, respectively [10].
Journal Pre-proof The secondary and hierarchical structures of SF directly affect the mechanical properties. The βsheet structure of SF has a critical role in stiffness. In comparison to native silk fibers, scaffolds fabricated from regenerated SF aqueous solution have weaker mechanical properties [54, 57-61]. For example, native SF fibers have a tensile strength and elongation at break about 0.5-0.6 GPa and 10-40%, respectively. The characteristics for dry silk films made from regenerated SF aqueous solution are about 0.02 GPa and 2% [57, 58]. Embedding regenerated silk products in β-sheet
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structures, induced by methanol or ethanol is an option to improve the strength of SF-based
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scaffolds [62-64]. Water-soluble silk films immersed in methanol for 10 min and 60 min had an
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elastic modulus of about 40 MPa and 80 MPa, respectively [65], demonstrating the role of β-sheets
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in the mechanical behavior of SF-based scaffolds.
(ii) Biocompatibility and biodegradation
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Silk sutures have been used since the end of the 19th century in medicine as a safe and
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biocompatible material [41]. Biocompatibility of silk-based scaffold depends on the extraction and
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purification procedures used, and degummed SF is immunologically inert and evokes a minimal inflammatory responses in vitro or in vivo. Sericin shows minimal inflammatory responses as well, and is often considered a biocompatible biomaterial [10, 12, 17, 36, 59, 66-71]. However, the combination of the two proteins, fibroin and sericin results in inflammation. The biocompatibility of SF-based scaffolds has been studied both in vitro and in vivo. The biocompatibility of SF nanofibrous scaffolds when subcutaneously implanted in BALB/c mice for 1 and 4 weeks was demonstrated [59]. An increase in macrophage infiltration into the implanted site of 3D SF-nanohydroxyapatite nanocomposite scaffolds at day 7 and 28 post-implantation in
Journal Pre-proof rat was also reported, while no significant increase of lymphocytes was found when compared to controls (no implants) [72]. SF films have evoked a host immune response at a similar level as collagen-based scaffolds [11]. SF has FDA approval for biomedical applications and some of the silk-based products are already used in the market. In addition, silk-based surgical meshes passed biocompatibility and safety testing for ISO 10993, indicating that this product meets the criteria required for use in medicine [23]. These results suggest good biocompatibility of fully degummed
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SF for TE uses. Subcutaneous implantation of 3D SF scaffolds in rat showed a mild inflammatory
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response after one year post-implantation [72].
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A TE scaffold should degrade at the same speed as new tissue is regenerated/repaired and have
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non-toxic and safe degradation byproducts [73]. Antigenicity of the scaffold degradation
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byproducts is another important concern for the use of SF [10, 12, 17]. Further examination is warranted to understand the low to no cytotoxicity and the reason for the good biocompatibility of
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the SF and its degraded products in long-term period. This insight should be considered in the
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design of biomaterials in general.
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SF is degraded in vitro and in vivo, with the rate depend on the material structure and environment [20, 21, 74-79] [21, 22, 72]. The extraction method, SF source, secondary structure and SF-based scaffold form can affect the biodegradation rate [20, 21, 78, 79]. SF with higher β-sheet content shows slower degradation [80, 81]. Spider silk degrade faster than SF extracted from Bm at least in one study [82]. Many factors, such as β-sheet content, scaffold type, type of silk (virgin or processed), and solvent used for extraction influenced these outcomes [83-85]. Scaffolds made from a regenerated aqueous solution of SF degraded faster than the native degummed SF fibers [77]. In vivo higher concentrations of SF and smaller pore sizes resulted in decreased in vivo degradation [21, 83]. Additionally, 3D SF scaffolds prepared from aqueous SF solution showed
Journal Pre-proof complete in vivo degradation between 2-6 months, while those prepared from organic solvent remained in the implanted site for more than 1 year [19]. The porosity of the 3D SF scaffolds affects degradation rate, higher porosity showed a higher degradation rate [72].
(iii) Cell-silk fibroin interaction behavior
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Cell-ECM interactions control cell proliferation, migration, differentiation and function [86].
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Cells without the ability to attach to surfaces undergo apoptosis [87]. Cells can sense their environment and anchor to matrix components through integrins. Collagen (mediates tensile
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strength of ECM), fibronectin (mediates cell-matrix adhesion) and laminin (organization of basal
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lamina) are the most common components in tissue ECMs. Indeed, there are some sequence
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domains in integrins that bind to specific motifs in collagen, laminin and fibronectin [7, 88]. Cell adhesion properties of TE scaffolds as well as the structure of scaffolds, including porosity, pore
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architecture and size are critical, as these features affect migration, proliferation and differentiation
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of cells. The cells cultured in SF scaffolds with 100-300 µm pores showed enhanced cell growth,
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differentiation and extracellular matrix production than other pore sizes studied. In this regard, Zhang et al. [89], fabricated SF sponges with different ranges of pore sizes (50 to 300 µm) by freeze drying. The BMSCs transfected with BMP7 preferred SF sponges with pore sizes ranging from 100-300 µm with better proliferation, ECM production, osteogenic differentiation and in vivo bone healing. SF provided good attachment sites for a variety of cell types such as fibroblasts and keratinocytes [90], human adipose tissue-derived mesenchymal stem cells (ADSCs) [91], rat bone marrow mesenchymal stem cells (rBMSCs) [59, 72], human osteoblast cell line G 292 [92], human bone marrow mesenchymal stromal cells (BMSCs) [89] and many others. In addition, the presence of arginine, glycine and aspartate (RGD) motifs as cell-binding sites in non-mulberry SF provided
Journal Pre-proof improved cell adhesion when compared to mulberry SF where there is an absence of these sites [16, 39, 43, 93].
4. Silk fibroin for skin tissue engineering 4.1. Skin structure and healing process
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Skin has a vital role in fluid hemostasis, sensation, thermoregulation and other physiological
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functions [25, 26]. Skin is composed of the epidermis and dermis. The epidermis, the outermost
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layer of skin is water resistant and responsible for body liquid evaporation and protection against
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pathogens. The epidermis consists of a thin layer of keratinocytes and is separated from the dermis by a basement membrane. The dermis is the thick layer of skin, which contains connective tissue
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with fibroblasts, hair follicles, sweat glands, blood vessels and extracellular matrix contents such
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as collagen, elastin, and glucoseaminoglycans (GAGs). The dermis layer endows tensile strength
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and elasticity to the skin [25, 26, 94].
Skin injuries may arise from mechanical trauma, burns, diseases and surgical procedures. Skin
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wound healing is a complex process of cellular and molecular events divided into four overlapping phases of hemostasis, inflammation, proliferation and remodeling. The cytokines and growth factors secreted from the cells in the wound bed orchestrate the wound healing process [26, 95]. Upon injury, the damaged vessels are closed by blood clots (hemostasis phase), phagocytes such as neutrophils and macrophages are recruited into the wound site to clear out the dead cells/microorganisms and also secrete growth factors to mediate the proliferation phase (inflammatory phase).
Journal Pre-proof The cells (i.e., fibroblasts, mesenchymal stem cells, endothelial progenitors, and keratinocytes) proliferate, differentiate and migrate to close the wound bed (re-epithelialization), restore vascular networks and form granulation tissue. The loss of contact inhibition and physical tension in keratinocytes triggers signaling pathways mediated by desmosomes and hemi-desmosomes, and subsequently cytoskeleton reorganization, migration and proliferation of keratinocytes, especially from basal layers of the wound edges. Beside keratinocytes, epithelia stem cells from sweat glands
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or nearby hair follicles contribute to the re-epithelialization. Keratinocytes also secrete a variety
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of growth factors and cytokines such as EGF, KGF, VEGF, TGFβ and others, which mediate the
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wound healing process. Fibroblasts grow and secrete collagen (mostly collagen type III) at the
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early phases of proliferation to produce new extracellular matrix at the wound site. Collagen type
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III is synthesized and deposited quickly in the wound bed to form new ECM [26, 96]. In the remodeling phase, the cells that are no longer needed are eliminated from the wound bed
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and the extracellular matrix is remodeled to form the natural structure of the skin. During this
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phase, matrix-remodeling enzymes such as matrix metalloproteinases replace collagen type III with collagen type I. Collagen type I has high tensile strength compared to collagen III. This is
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synthesized and is deposited slowly. A decrease in the number of new vessels is observed in remodeling phases. [26, 95, 97]. The events occur in different phases of skin wound healing processes during the first 30 days post-injury (Figure 4). Acute skin wounds including burns, chemical and surgical injuries heal within 8-12 weeks. Skin wounds are classified into superficial, partial thickness, full-thickness and complex. In superficial skin wounds, only the epidermis is affected. In partial-thickness, all or a portion of the dermis remains intact. In full-thickness wounds, the entire dermis, including sweat glands and hair follicles are lost. In complex wounds, the epidermis, dermis and subcutaneous tissue are damaged.
Journal Pre-proof Spontaneous healing of acute wounds depends on which layer of the skin is affected [5, 95, 98]. In contrast to acute wounds, chronic wounds such as diabetic wounds, infected wounds and bedsores fail to heal fast enough (need more than 12 weeks) and often reoccur. In chronic wounds, the delayed healing and recurrence affect the underlying layers, such as muscles, cartilage and bone and may lead to tissue loss [98, 99]. In general, any phenomenon which postpones the “transition from inflammation to proliferation phase”, such as diabetes, systemic inflammatory
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disease, infections and others can lead to chronic wounds [96, 100]. Immune cells (mostly
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neutrophils and macrophages) secrete a large amounts of pro-inflammatory cytokines (e.g., IL-1,
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IL-6, TNF-α) which induce the expression of matrix metalloproteinases and reduce tissue
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inhibitors of metalloproteinases, leading to formation of a highly proteolytic microenvironment in the wound bed [101, 102]. The cell membranes and structural proteins of the newly formed ECM
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are also damaged by ROS produced by neutrophils [103]. Chronic and full-thickness skin injuries
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require skin grafting to support complete healing [104]. Although several skin substitutes are already commercialized, restoration of non-healing skin wounds remains a major challenge [26,
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28, 105]. As described earlier, SF has many promising mechanical and biological properties for
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skin wound management [13]. The number of research publications in vitro, in vivo and clinical trials using SF and its blends are increasing and a summary of recent research in skin tissue engineering since 2000 are presented in Tables 1 a-c.
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Figure 4. The events of four overlapping phases of normal wound healing process during the first 30 days of post-wounding.
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4.2. SF and SF blends in skin wound healing applications
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SF accelerates skin wound healing through the NF-kB signaling pathway [106]. NF-ĸB regulates various cell behavior such as adhesion, proliferation, inflammation, and elimination of reactive oxygen species via complex signaling pathways. Therefore, NF-ĸB signaling is considered critical in healing processes of various wounds such as scratch injuries [99], corneal epithelial wounds [107], and dermal injuries [106, 108]. Park and his coworkers showed the up-regulation of NF-kB in NIH3T3 cells after exposure to SF [106]. This resulted in enhanced cell proliferation and growth as compared to controls. Their microarray data indicated the increased expression of Toll-like receptors (TLRs) and tumor necrosis factor receptor (TNFR), as two important mediators of NFkB in the SF-induced cells. They also determined the changes of expression of a variety of proteins,
Journal Pre-proof which mediate normal wound healing process and affected by NF-kB such as TGF, EGF, IL-1b, IL-10, vimentin, cyclin D1, fibronectin, VEGF and others s. For example, they showed increased expression of vimentin, cyclin D1, fibronectin, VEGF in cells induced with SF compared to controls. In addition, inhibition of NF-kB signaling in NIH3T3 cells by Bay 11-7082, a pharmacological inhibitor of κB kinase, significantly reduced the expression of vimentin, cyclin D1, fibronectin and VEGF; however, it did not affect the expression of EGF and TGF. Taken
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together, Park et al. concluded that SF accelerated wound healing by modulating the expression of
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proteins responsible for proliferation and remodeling phases of the process by activating the NF-
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kB signaling pathway [106].
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SF materials are used alone or in combination with other materials for skin wound healing
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applications. SF is also bio-functionalized to improve its characteristics for wound healing applications. SF alone is used in the forms of film [109-111] and nanofibrous scaffolds [112, 113]
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and other materials for blends with SF include chitosan (CTS), alginate, carboxymethyl cellulose
4.2.1. Pure SF
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(CMC), gelatin and antibacterial agents like heavy metals and antibiotics [109, 114-119].
4.2.1.1. SF film SF films showed to have superior healing potential compared with conventional hydrocolloids (Duro Active), and similar or better healing compared to lyophilize porcine acellular dermis (Alloaks D) for full-thickness incisional wounds in mice. The full-thickness skin wounds dressed with SF films showed less neutrophil-lymphocyte infiltration (inflammation) and higher collagen regeneration, re-epithelialization and faster healing when compared with Duro Active [120].
Journal Pre-proof Zhang et al. [121] developed a transparent SF film and compared its translational potential to two commercially available wound dressings: Suprathel (PolyMedics Innovations GmbH, Germany) made from polylactide, trimethylene carbonate, and ε-caprolactone; and Sidaiyi (Suzhou Soho Biomaterial Science and Technology Co., Ltd) as a two layer product (silk sponge and silicon layers). A schematic of the fabrication process is shown in Fig. 5a. Roughness and transparency of SF films compared to the two control materials were Sidaiyi>Suprathel>SF film and SF
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film>Suprathel> Sidaiyi, respectively, as SF showed the most transparency and smoothness [121].
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All three materials were waterproof, biocompatible, and effective barriers against bacterial
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entrance. SF films revealed superior gas permeability and fluid handling capacity than Suprathel
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and Sidaiyi. SF films with a thickness of 64.9 µm were much thinner than Suprathel (266 µm) and Sidaiyi (1,789 µm) (Fig. 5b). The wound healing potential of SF films versus Suprathel and Sidaiyi
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was investigated in full-thickness skin wounds in small (rabbit) and large (porcine) (Fig. 5c)
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animals, as well as a randomized single blind clinical trial on 71 patients. Both animal and clinical trial studies showed better and faster wound healing in the SF group compared to the two
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commercial materials [121].
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Figure. 5 (a-e). Silk fibroin film: from fabrication to animal and randomized clinical trial studies. (a) Schematic of the fabrication of transparent silk fibroin film. (b) Silk fibroin film with a thickness of 64.9 µm was much thinner than Suprathel (266 µm) and Sidaiyi (1789 µm). (c) Silk fibroin film exhibited better and faster wound healing compared to Suprathel and Sidaiyi during 90 days in porcine model [121]. (d) Improved wound closure in the wounds treated with SF film compared to Mapitel and the wounds with no treatment after 5, 10, and 15 days [122], (e) Hand burn wounds after 1 month treatment with Biobrane and Dressilk [123].
Journal Pre-proof SF films surface modified with NaOH to form nanofeatured SF membrane for wound dressing applications exhibited enhanced nanotopography, wettability and cellular functions compared to untreated SF scaffolds [111]. Different concentrations of pectin and glycerol were added to SF films to fabricate feeder layers for ADSCs. Pectin and glycerol were used to induce conformational transitions and flexibility to
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SF films, respectively. A formulation of ~94% w/w SF with 6%w/w pectin and ~88% w/w SF 12%w/w glycerol showed superior conformational stability, mechanical properties, respectively,
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and delivery systems for ADSCs [110]. Further investigations were performed to improve the
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flexibility and mechanical behavior of SF films [124, 125]. Various concentrations of dextrose (0-
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15%w/w) as plasticizer were incorporated into SF films to create flexible SF films, with higher
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elongation at break, water holding capacity, surface roughness, water absorbance capacity, hydrophilicity, and better cell proliferation compared to SF films alone [124, 125].
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In another study, silk cocoon sol-gel films were fabricated by immersing silk cocoons in CaCl2-
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ethanol-H2O solutions for different times. Immersion for 90 min supported biocompatibility,
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antibacterial activity, transparency and good swelling with significantly accelerated wound healing rates compared to standard Mepitel® dressings when the films were applied on full-thickness wounds in New Zealand white rabbits (Fig. 5d) [122]. Schiefer et al. [123] compared the effectiveness of Dressilk (PREVOR, France), a commercially available dressing made from silkworm silk to Biobrane (Smith and Nephew ltd, Hull, UK), a silicon based wound dressing coated with nylon mesh and porcine dermal collagen-derived peptides, for burn wound healing patients. Both Dressilk and Biobrane provided safe and effective microenvironments for burn wound healing, with Dressilk being the most cost effective (Fig. 5e) [123].
Journal Pre-proof SF scaffold with amorphous structure had promising characteristics for soft tissue engineering. To provide a tunable amorphous structure and mechanical properties, SF aqueous solutions were freeze dried at -20C to -5C. The freeze dried SF scaffolds at temperature >-9C were water insoluble with crystalline structure, while SF solution freeze dried at -20C were water soluble with minimal crystallinity. This technique is suggested for tuning mechanical and biological
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properties of SF scaffolds to meet criteria required for soft tissue engineering [126].
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4.2.1.2. SF nanofibrous scaffolds
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SF nanofibrous scaffolds are an interesting form of SF derived skin substitutes. Electrospinning
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provides a pseudo 3D structure for cell growth and attachment [59, 68, 112, 113, 127-130]. The
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concentration of SF in the spinning solution has a direct influence on the mechanical and biological characteristics of the electrospun silk fibroin (ESF) mat. ESF scaffolds fabricated from higher
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w/v% SF concentrations exhibited higher nanofiber diameters, improved mechanical properties
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and lower porosity; affecting the biological behavior of cells [59, 62, 128-131]. ESF with smaller diameter nanofibers had better influence on dermal cell proliferation and extracellular matrix
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formation in vitro and in an ex vivo wound model [132]. It is critical to optimize the fabrication process to achieve ESF scaffolds with biomechanical characteristics close to natural skin. Small pore sizes and sheet-like surfaces of ESF mats limit cell infiltration into the nanofibers. Therefore, salt leaching and cold plate electrospinning were introduced to fabricate ESF mats with higher pore sizes than the ESF scaffolds fabricated by traditional electrospinning. Sheikh et al. [133] showed that ESF scaffolds fabricated by cold-plate electrospinning had higher porosity and pore sizes with controllable mat thickness when compared to salt-leaching (SLE) and traditional electrospinning (Fig. 6a). The same research team also compared the characteristics of SF
Journal Pre-proof scaffolds fabricated by freeze-drying (FD), salt-leaching (SL), and SLE [112]. The SLE showed the most homogenous pore distribution, highest porosity (70%), water uptake and swelling ratio compared to FD and SL. Compressive strength in SL was significantly higher than the other methods due to the lower porosity. FD and SLE showed similar compressive strength. All three methods supported NIH 3T3 fibroblast growth and proliferation. The authors concluded that the ESF fabricated by SLE methods had more favorable biomechanical and biological characteristics
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to meet requirements for use in skin tissue engineering [112].
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The effectiveness of SLE SF nanomatrix on the healing of a burn model in rats was evaluated and
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compared to traditional electrospun nanosheets and Medifoam®, a commercially available
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polyurethane hydrocellular dressing foam, Genewel, Korea). SLE SF nanomatrix showed
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accelerated re-epithelialization and wound closure, organized collagen formation and deposition, and reduced inflammatory cytokine and healing time compared to electrospun nanosheets and
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Medifoam® [113]. The superior potential of SLE SF nanomatrix than Matriderm® in healing full
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thickness skin defects was shown by Lee et al. [127]. 3D SF scaffolds with micro-nano fibrous structures were also fabricated by a facile two-step freeze-drying technology to mimic the
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extracellular microenvironment of natural skin. The authors claimed that combinations of micronano fibrous structure in 3D SF scaffolds positively affect dermal cell viability, proliferation, migration and in vivo healing of dorsal full-thickness wounds in rats [134]. Yu et al. [135], found that both electrospun CTS/collagen and CTS/SF showed promising biological and mechanical properties for fibroblasts and keratinocytes in vitro, and full thickness skin wound healing in rats, when compared to a gauze dressing. Some investigations were performed for the fabrication of skin wound dressings using silkworm cocoons [122, 136]. Yu et al. [136] developed an artificial skin with permeation and mechanical
Journal Pre-proof properties close to natural skin, which was demonstrated by treating the silkworm cocoons with different degumming process times (0-100 min) (Fig. 6b). The artificial skins formed from the cocoons still need further in vitro and in vivo experimentation to determine their potential
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Figure 6 (a-d). Silk fibroin fibrous scaffolds for skin wound healing (a) Electrospun silk fibroin scaffolds fabricated by cold-plate electrospinning methods (CPE) had higher porosity and pore sizes with
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controllable mat thickness and easy shaping compared to salt-leaching (SLE) and traditional
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electrospinning techniques (TE) [133]. (b) Silk fibrous mats were fabricated by treating the silkworm cocoons with various degumming process times (0-100 min). SEM micrographs showed the effects of degumming time on morphology and porosity [136]. (c) Electrospun silk fibroin (ESF) on amniotic membranes (AM), as well as cross sectional views of AM/ESF bilayer membranes, indicated successful the coating of AM with ESF [59]. (d) ESF/AM alone or seeded with adipose tissue-derived mesenchymal stem cells (ADSCs) exhibited better wound healing compared to AM and AM/MSCs in a mouse burn model [69].
Autologous platelet-rich plasma (PRP) or platelet-poor plasma (PPP)-immersed SF cocoon mats also have good biocompatibility, support cell growth, and accelerated wound healing when
Journal Pre-proof compared to PPP-SF and control (mats); the PRP-SF mats exhibited higher hydrophilicity compared to the cocoon and PPP-SF mats [137]. Muga fibroin electrospun mats supported L929 fibroblast growth and the delivery of gentamycin sulfate when compared to muga cast films [138]. Darshan et al. [139] fabricated Antheraea mylitta silk mats by degumming sericin from the cocoons and used repeated washing with sterile phosphate-buffered saline (PBS) for wound healing applications. The homogenous morphology of fibroin fibers, favorable mechanical
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properties and biocompatibility for epidermal cell primary culture in vitro was reported.
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Uniform nanofibrous SF mats were fabricated with average nanofiber diameters of 90 ± 0.021 nm
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and demonstrated biocompatibility for bone marrow derived-stromal cells [59], as were 3D bilayer
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skin substitutes made from electrospun SF layer (ESF) coated on decellularized amniotic
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membranes (AM) (Fig. 6c). The fabricated bilayer membranes improved mechanical properties and degradation rates compared to ESF and AM alone. ESF/AM artificial skin increased the
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secretion of pro-angiogenic factors (VEGFα and bFGF) from ADSCs in vitro. The biomechanical
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and biological results suggested ESF/AM artificial skin as a suitable skin substitute in various skin wounds, especially burns [68]. The potential of the ESF/AM bilayer membranes for treatment of
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scarless healing in a preclinical 3rd degree burn model in mice was demonstrated, with the best results in burn wounds implanted with bilayer membranes alone or in combination with ADSCs (Fig. 6d) [69]. The anti-hypertrophic scar property of this bilayer membrane was also proved in a rabbit ear model excisional wounds [140]. 4.2.2. SF blends SF has been used alone in the fabrication of skin substitutes, but it is preferred to blend SF with other materials to improve biological properties. SF blends have superior wound healing potential compared to SF alone [114, 116, 141]. The composition of SF with CTS in various form has a
Journal Pre-proof significant positive impact on biomechanical and biological properties of CTS/SF scaffolds. SF blends for wound healing applications were fabricated in fiber, film, sponge and other formats [116, 141-144]. 4.2.2.1. SF blend film/sponge SF blends in the form of films and sponges are useful for wound healing. Excisional wounds implanted with poly vinyl alcohol/CTS/silk fibroin (PVA/CTS/SF) healed faster than those
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implanted with CTS/SF, SF, PVA/CTS and CTS; SF sponges alone exhibited superior healing
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potential compared to CTS sponges [145]. Spunlaced SF (SSF) scaffolds were coated with a thin
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layer of CTS by hydrogen-bonding assembly to fabricate CTS/SSF 3D blend scaffolds with a mean
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pore size of 50–200 μm. This process improved the mechanical behavior, broad spectrum
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antibacterial activity, and in vivo healing compared to SSF and CTS alone [116]. Nonwoven CTS/SF films also exhibited haemocompatibility and cyto-compatibility as a promising skin
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substitutes [143]. In a multifunctional integrated strategy by Li et al. [27], bioactive glass (BG)
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was incorporated into CTS/SF scaffolds to fabricate composites of BG/CTS/SF with biocompatibility and useful mechanical properties, which promoted vascularization and mature
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blood vessel formation to accelerate wound healing in vivo. Bilayered SF/Gelatin (ratio 20:80) films were mixed with 1% sericin for a randomized clinical trial on 25 patients with split-thickness skin graft donor sites. The healing rate in wounds treated with SF/gelatin/sericin blended films was significantly faster than Bactigras, a commercially available wound dressing with less pain [115]. Accelerated neovascularization, reepithelialization, collagen deposition, wound contraction and healing were also observed in 2% isabgol/2% SF (Isabgol/SF 75/25) composites with a fibrous foam-like architecture. Isabgol (Isab, Psyllium husk) is a natural carbohydrate polymer in India with several health benefits [146].
Journal Pre-proof Sodium alginate (SA) was reported as a promising material for the fabrication of SF blends for wound dressings [114, 147, 148]. SA is cytobiocompatible and effects skin wound healing through wound exudate absorption [149, 150]. Wang et al. [114] fabricated a homogenous, cytocompatible and interconnected 3D 2% SF/2% SA porous composite by freeze drying (-20 to 80C) for wound healing applications. They demonstrated that freezing temperatures affected the pore diameters (54-532 μm) and porosity (66-94 %) of the final scaffold, as pore diameter and
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porosity decreased and increased with decrease of freezing temperature, respectively. Base on their
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findings, crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) provided a
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suitable degradation rate until new ECM and tissue formation progressed.
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Strontium (Sr) is a metal with antibacterial activity and wound healing potential. The incorporation
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of Sr in SF/SA met many characteristics for wound healing, such as water absorption, water vapor transmission rate, and mechanical properties for wound healing. It was also revealed that Sr (5
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mg/ml)/SF/SA had the best antibacterial activity, cyto-compatibility, promoted angiogenesis and
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wound healing potential in vitro [148]. A novel recombinant SF/elastin protein with silk and elastin blocks was developed and its wound healing potential in two forms (aqueous and sponge) was
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compared in vivo. Recombinant SF/elastin self-assembles from liquid to gel, and both forms improved granulation tissue formation. SF/elastin sponges absorbed wound exudate and formed a gel after application on the wound. The authors concluded that recombinant SF/elastin sponges showed better potential as wound dressings than the aqueous form [151]. Rameshbabu and colleagues showed wound dressing sponges made from SF ornamented with human placentaderived extracellular matrix (pECM). The placenta was decellularized and then solubilized by urea buffer. Different ratios of SF/pECM were prepared and freeze dried to fabricate porous sponges. A wide range of cytokines, growth factors and ECM proteins present in the placenta were retained
Journal Pre-proof after the decellularization process and during scaffold fabrication. SF/pECM improved adhesion, proliferation and migration of human foreskin fibroblasts (HFFs), human epidermal keratinocytes (HEKs) and human amniotic and membrane-derived stem cells (HAMSCs) with biocompatibility in vitro and in vivo. Implantation of SF/pECM into a full-thickness wound in rat showed a significant increase in re-epithelialization, wound closure, neovascularization, wound healing compared to SF sponges [152]. In another study, SF films were loaded with an immunomodulator
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peptide (neurotensin, NT) and stimulated by iontophoresis to modulate inflammatory responses
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and accelerate wound healing [153]. CTS/SF films were a useful system for delivery of ADSCs to
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the diabetic wound site in a rat model. The combination of ADSCs and CTS/F was suggested as
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an effective strategy in the management of diabetic wounds [154]. Panico et al. [155] revealed that the addition of glucose in SF film induced crystallization of SF. This increased the absorption
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capacity and flexibility of the SF films. Citrus pectin/SF sponge fabricated by freeze-drying
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method showed a promising wound dressing material due to high water uptake ability (800% in 24 h), low weight loss (21.3% in 40 days), suitable mechanical property and cyto-compatibility
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for fibroblast cells in vitro [156]. To mimic the natural skin extracellular matrix and structure,
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Yang et al. [157] recently fabricated a SF/HA/SA spongy composite by freeze drying method with around 92% in porosity and average pore size of 93 μm. SF/HA/SA composite indicated good physical stability, soft and elastic characteristics with cyto-biocompatibility for NIH-3T3 fibroblast cells in vitro. Implantation of SF/HA/SA composite to full-thickness burn wounds in rat model and histological observations suggest the potential of this composite as a wound dressing. Liu et al. [158] prepared a SF sponge by freeze drying method for controlled release of neurotensin-loaded gelatin microspheres for management of diabetic foot ulcers. Neurotensin, as a neuropeptide, act as an inflammatory modulator and leads to the release of cytokines and
Journal Pre-proof chemotaxis involved in immunomodulation responses, which accelerate neovascularization and wound healing [159, 160]. Neurotensin-loaded gelatin microspheres releasing SF scaffold showed average pore size and porosity of 40–80 μm and ∼85%, respectively. During 28 days posttreatment follow up, neurotensin-releasing scaffold exhibited good drug delivery system and wound dressing material. This accelerated the wound healing in diabetic foot ulcers through the increase of wound closure, fibroblast accumulation and tissue granulation at the wound site with
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minimal scar formation [158].
4.2.2.2. SF blend fibrous scaffolds
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The morphology, surface hydrophilicity, water-uptake capability, fibroblast proliferation and
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attachment of poly (3-hydroxybuty-rate-co-3-hydroxyvalerate) (PHBV) nanofibrous scaffold improved by the addition of ESF (50/50 ratio) and fabrication of PHBV/ESF nanofibrous scaffolds
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by co-electrospinning technique [144]. Shanmugam and Sundaramoorthy developed and
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characterized electrospun mats from Eri (nonmulberry silk) SF and poly-L-lactic acid (PLA), for
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skin wound healing applications. Pure ESF and ESF/PLA mats had an average fiber diameter of 320 nm and 502 nm, respectively. Water uptake and vapor transmission rates in the ESF/PLA were lower than that in the pure ESF mats. ESF showed higher hydrophilicity compared to ESF/PLA, with water contact angles of 23° and 79°, respectively. Both ESF and ESF/PLA were successfully loaded with tetracycline hydrochloride (TCH) and had effective antibacterial activity against Gram positive (S. aureus and S. epidermidis) and Gram negative bacteria (E. coli and P. aeruginosa). Both ESF and ESF/PLA accelerated wound healing compared to control wounds treated with gauze [161]. The addition of SF microfibers (mSF) to pure CTS membranes increased biomechanical properties in vitro and healing efficacy in vivo for the CTS/mSF compared to pure
Journal Pre-proof CTS membranes in full-thickness skin wounds in rats [141]. Zhang et al. [162]successfully fabricated porous mSF/poly(glycerol sebacate) (PGS) and CTS/PGS composite scaffolds with biocompatibility, cell adhesion and favorable degradation for skin tissue engineering applications [162]. Incorporating gold nanoparticles into ESF 3D mats improved degradation profiles, biomechanical
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behavior and elongation at break, similar to normal skin, which accelerates neovascularization and granulation tissue formation in full-thickness wound healing in rats [163]. 3D electrospun PCL
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containing various concentrations of SF particles, fabricated by cold-plate electrospinning and
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automatic magnetic agitation, also have potential as artificial skins. Using this custom-designed
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electrospinning technique, 3D porous mats with thicknesses up to 6 mm with interconnected
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macro-pores ranging from tens to hundreds of microns were successfully fabricated with a homogenous distribution of SF nanoparticles within the PCL mats. While both PCL and SF
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particle-incorporated PCL mats were biocompatible, faster wound healing and collagen deposition
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were shown with the PCL/SF mats with the higher content of SF particles [164]. The addition of SF to oxidized cellulose nanofibers (TOCN) significantly improved its mechanical and wound
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healing effectiveness. TOCN/SF 2% supported improved wound healing in critical sized rat skin excisional models compared to the TOCN nanofibers [165]. SF/chemically active PEGs (PEG-maleimide and PEG-thiol) were introduced as a cytobiocompatible and effective sealants with rapid cross-linking ability through chemical reactions [166]. SF as an anti-adhesion patch was suggested by Wang et al. [167], where various SF nanofibrous blends as ESF/ PVA, ESF/PEG, and ESF/polyethylene oxide (PEO) were prepared by single-spinneret electrospinning. The anti-adhesion properties of the blends were: PVA/ESF> pure ESF> ESF/PEG ~ESF/PEO. The pure ESF and PVA/ESF showed better collagen
Journal Pre-proof regeneration and wound healing compared to the other blends [167]. Selvaraj et al. [168] fabricated an antioxidant scaffold by incorporating Fenugreek, a natural antioxidant, into ESF. Fenugreek-incorporated ESF had better thermal and biomechanical properties, supported higher cell proliferation in vitro and increased re-epithelialization, collagen deposition and wound healing compared to ESF.
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Anti-inflammatory, anti-bacterial, and the wound healing potential of honey make it an interesting material for skin tissue engineering [169]. To improve the wound healing potential of SF,
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honey/ESF wound substitutes were successfully developed by electrospinning [128, 130]. Manuka
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honey (Apis mellifera) incorporated ESF mats exhibited improved water uptake and supported cell
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growth in comparison with ESF, and suggested as a dermal regeneration template [128].
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ESF loaded with various concentrations of Manuka honey by a green electrospinning technique showed smooth surface morphologies with high antibacterial activity [130]. This antibacterial
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activity depended on Manuka honey concentration in the materials. The honey was also
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biocompatible. The average fiber diameter increased with increase of Manuka honey concentration
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in the constructs and the systems showed wound healing potential in vivo. Pignatelli and coworkers introduced human platelet lysate-loaded ESF as a wound dressing for wound healing management. Human platelet lysate is a rich source of growth factors and cytokines involved in wound healing. ESF preserved and released this lysate to the wound site [170]. A separate study also showed that loading of PRP exosomes and a polysaccharide extracted from the rhizomes of Curcuma zedoaria in CST/SF hydrogel sponges resulted in wound dressings for diabetic wound management in rats [171]. SF/PVA/collagen I peptide composite was shown to be a good drug delivery system for localized delivery of S-Nitrosoglutathione, a drug involved in
Journal Pre-proof regulation of the microvascular blood supply through its vasodilator property and activation of VEGF [172], and for the management of ischemic chronic non-healing ulcers in vitro [173]. Inducing anisotropic features to the ESF scaffold can promote its biological behaviors such as cell migration, proliferation and adhesion in vitro, and tissue ingrowth, neovascularization, wound closure and in general wound healing in vivo [174]. In another study, the ESF successfully loaded with amniotic fluid and SA hydrogel to deliver amniotic fluid, a highly enriched fluid with multiple
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therapeutic agents, to the wound bed, promote cell proliferation, migration and wound healing.
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This bioactive dressing showed interesting biomechanical and biological results in vitro. This
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needs further animal study to validate its potential in wound healing in vivo [175].
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Recently, Miguel et al. [176] prepared a two-layered membrane using electrospinning method to
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mimic both epidermis and dermis layers of skin for skin tissue engineering applications. This asymmetric membrane was made from ESF/PCL, as top layer to mimic dense nature. The
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waterproof properties of the natural epidermis, and ESF/HA loaded with thymol (an herbal drug)
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to mimic the natural dermis structure. The electrospun asymmetric membrane showed high wettability, porosity, and the mechanical properties appropriate for the skin wound dressing. The
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release of thymol endowed the ESF/HA side of asymmetric membrane an antibacterial activity against S. aureus and P. aeruginosa with cyto-compatibility for human fibroblasts in vitro.
4.2.2.3. Other blend forms Hydrocolloids, hydrophilic polymers dispersed in water, are suitable for wound healing, especially for burns [177-179]. SF nanoparticles were mixed with CMC and styrene-isoprene-styrene (SIS) to fabricate SF-CMC hydrocolloid wound dressings. SF-CMC hydrocolloid exhibited structural
Journal Pre-proof stability and cyto-compatibility with suitable mechanical properties for wound healing. SF-CMC hydrocolloids also revealed improved burn wound closure and healing when compared to gauze and Neoderm®, a commercially available wound dressing [109]. Subcutaneous administration of microcarriers prepared from SF alone and in combination with gelatin for deep skin wounds in mice also had healing potential with infiltration of immune cells such as mononuclear cells,
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macrophages, and neutrophils without fibrosis [180]. Thangavel et al. [181] developed a hydrogel for wound healing, prepared from silk protein, L-
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proline (LP), a critical amino acid that is used for collagen synthesis by the human body, and CTS.
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SF/LP/CTS hydrogels displayed thermal stability, suitable surface morphology, in vitro cyto-
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compatibility for NIH 3T3s and antioxidant activity. The hydrogels provided sufficient moisture
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for the wound bed. In vitro degradation demonstrated suitable biodegradation rates of the hydrogels. The presence of LP in the fabricated hydrogels had a significant effect on in vitro cell
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viability, migration, proliferation and wound healing, when compared to CTS and SF/CTS
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hydrogels. Kawabata and colleagues [182] showed that the hydrogels fabricated from SF-elastin conjugates (containing 4 SF–like blocks, 7 elastin-like blocks, and 1 modified elastin block) had
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favorable mechanical properties for full-thickness skin wounds in diabetic mice. Incorporation of curcumin in SF hydrogels, fabricated under weak electric fields, was useful for wound dressings with sustained curcumin release, cyto-compatibility with immortalized human keratinocytes (HaCaT). Dhasmana et al. [183] indicated the synergetic effects of SF and acellular dermal matrix in vitro and in vivo. They decellularized goat skin and then dip-coated at different concentrations of SF aqueous solutions. They concluded that the acellular dermal matrix modified with SF showed better wound healing in vivo compared to un-modified matrix, and suggested this scaffold as a biocompatible and cost-effective wound dressing.
Journal Pre-proof There is an inherent self-assembly property between two different silk types at 37 ⁰C. Chouhan et al. [184] fabricated injectable SF hydrogel from two species of silks (BmSF and AaSF, ratio 1:1) and showed that their injectable hydrogel successfully self-assembled at 37 ⁰C. FTIR and X-ray diffraction revealed that the irreversible gelation was due to β-sheet structure formation. The fabricated injectable SF hydrogel supported the proliferation of primary HDF and improved the migration of keratinocytes and re-epithelialization in vitro. In vivo evaluations of injectable SF
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hydrogel on full-thickness third-degree burn wounds indicated the accelerated inflammation,
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proliferation and remodeling phases of wound healing processes when compared to collagen gel,
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as control
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5. Biofunctionalization
Biofunctionalization of SF with growth factors, cytokines and drugs is an effective method for the
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improvement of SF based wound dressings [185, 186]. Two different drug incorporation
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techniques (drug loading or coating) with EGF and silver sulfadiazine were used to incorporate
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the protein into various SF scaffolds, including silk films, lamellar porous silk films and ESF [186]. The functionalized SF scaffolds revealed improved re-epithelialization, wound closure, dermal proliferation and wound healing in a full-thickness excisional wound (8 mm) in mice compared to two commercially available wound dressings (air-permeable Tegaderm tape (3M) (-control) and Tegaderm Hydrocolloid dressing (3M) (+ control)). SF scaffolds (all forms) functionalized with both EGF/silver sulfadiazine (loading and coating methods) showed improved wound healing compared to the other groups [186].
Journal Pre-proof Tripeptide Arg‐Gly‐Asp (RGD), is the most common peptide motif responsible for cell adhesion. Proteins containing higher contents of RGD show improved cell adhesion and attachment. The addition of RGD nucleic acid sequences to the SF gene and the subequent secretion of RGDcontaining SF proteins by transgenic silkworm may be a promising strategy for the fabrication of functionalized materials with high cell adhesion a for human dermal fibroblasts. Transgenic RGDSF can accelerate re-epithelialization, wound closure, neovascularization and wound healing
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compared to SF [187].
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Although growth factors can be incorporated or loaded into SF scaffolds, burst release and rapid
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loss can occur depending on the specific factor and its associated chemistry, which reduces the
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sustained delivery to wound bed. To overcome this limitation, growth factor gene sequences can
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be added to the SF gene and the resulting growth factor-SF proteins can be secreted by transgenic silkworms. The gene sequence of EGF was added to the light chain of the SF gene to develop
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transgenic silkworms carrying EGF-SF conjugated gene sequences. The EGF/SF proteins secreted
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by the transgenic silkworms allow for the sustained presence of EGF in the scaffolds and improved
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fibroblasts proliferation (2.5-fold), to accelerate wound closure in vitro [188]. SF/gelatin electrospun mats were functionalized with astragaloside IV, a bioavailable ingredient from the root of Astragalus membranaceus with anti-inflammatory, antioxidant and anti-aging properties. The compound has been utilized for scar-free healing of deep partial-thickness burn wounds in rats. In vitro evaluations demonstrated promising cell adhesion and biocompatibility for immortalized human fibroblasts. In vivo experiments exhibit accelerated wound closure, neovascularization,
organized
collagen
formation
and
reduced
scar
formation.
SF/gelatin/astragaloside IV electrospun mats were useful topical delivery systems for burn wound healing [185].
Journal Pre-proof As described earlier, non-mulberry SF possesses additional cell-binding motifs (RGDs) compared to mulberry SF [16, 39, 43, 93]. Chouhan et al. [189] functionalized PVA/SF electrospun mats with human recombinant EGF, human recombinant bFGF and LL-37 antimicrobial peptides. SF proteins were extracted from mulberry SF (extracted from Bm) and non-mulberry SF (extracted from A. assama (AaSF) and P. ricini (PrSF)). All the electrospun mats had in vitro cytocompatibility with human dermal fibroblasts (HDF) and HaCaTs. In vitro cell migration scratch
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assays showed significantly increased migration when the cells were treated with a combination
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of EGF, bFGF and LL-37. The wound healing potential of mats in non-diabetic and diabetic full-
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thickness excisional wounds (12 mm) in a rabbit model demonstrated that the functionalized non-
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mulberry SF/PVA mats (PVA/Aa and PVA/Pr) supported faster granulation, wound closure, organized ECM formation, re-epithelialization and angiogenesis compared to controls,
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functionalized PVA and functionalized PVA/Mulberry SF (PVA/Bm) [189].
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Bienert et al. [190] genetically modified Bm larvae to produce functionalized silk with FGF, EGF,
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KGF, PDGF or VEGF growth factors. All of the functionalized silk membranes, as well as native Bm silk, had cyto-compatibility in vitro. EGF-, FGF- and VEGF-functionalized silk membranes
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showed better in vitro and in vivo (full-thickness excisional wounds in mice) wound healing compared to native, PDGF- and KGF-functionalized silk membranes. KGF- and VEGFfunctionalized silk membranes provided improved cell growth and migration support for keratinocyte, while the silk membranes functionalized with EGF and VEGF increased the adhesion of macrophages compared to the other scaffolds. Although the growth factor-functionalized silk membranes supported wound healing, VEGF-functionalized silk membranes showed slightly better wound healing potential.
Journal Pre-proof Liposomes were used to maintain bFGF content in SF hydrogels for sustained release. The bFGFencapsulated liposomes in SF hydrogels showed stable and sustained release of bFGF in mice in deep second-degree scald wound fluids and as a result, accelerated wound healing compared to SF hydrogels and bFGF-encapsulated liposomes [191]. Topical bioactive insulin was encapsulated in SF microparticles by coaxial electrospray for chronic wound healing. Insulin-functionalized SF microparticles were loaded into SF sponges to develop a skin wound dressing. The functionalized
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scaffolds showed sustained release of insulin up to 28 days. Insulin release from the SF scaffolds
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increased the migration of human keratinocytes and endothelial cells in vitro. In vivo examination
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of insulin-SF scaffolds on dorsal full-thickness diabetic wounds in rats demonstrated accelerated
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wound closure, neo-vascularization and healing compared to other experimental groups [192]. A 3D gelatin/sulfonated SF/FGF wound dressing fulfilled the biological needs for wound dressings.
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In this case, 3D gelatin grids were fabricated by 3D printing. The grid was embedded in sulfonated
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SF. FGF was incorporated into the gelatin/sulfonated SF composite through sulfonic acid groups. In vitro assays demonstrated favorable biomechanical and biological properties of the scaffolds,
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and in vivo applications of the gelatin/sulfonated SF/FGF scaffolds in full-thickness excisional
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wounds in rats showed accelerated granulation and healing. The presence of FGF stimulated the neo-vascularization in the wound bed [193].
6. Antibacterial SF derived scaffolds Post-wound infection leads to delayed wound healing, scar formation and cell and tissue death especially in burns [105]. Thus, antibacterial activity is a key need in wound dressings. Some materials, such as CTS, sericin and others, have intrinsic antibacterial activity [37, 194]. SF does
Journal Pre-proof not exhibit inherent antibacterial activity [59], thus many approaches have been developed to endow antibacterial activity to SF-derived scaffolds [147, 195-197]. Table 2 (a-b) summarizes in vitro (Table 2a) and in vivo (Table 2b) studies on antibacterial SF blends for infected skin wound healing applications. Several materials or drugs, such as heavy metals, antibiotics and antibacterial biomaterials were incorporated into SF to fabricate SF composites with antibacterial activity for various TERM applications [117, 118, 147, 196, 198]. SF biomaterials in various forms (films,
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microspheres, hydrogels, coatings) had utility as local antibiotic delivery systems for wound
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healing [148, 198]. Heavy metals, such as selenium, strontium and silver are common antibacterial
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agents [195, 199-201] used in the fabrication of antibacterial SF composites [148] in various
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formats, including films [202], gels [203], electrospun mats [204] and sponges [205].
6.1. Antibacterial SF fibrous scaffolds
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Most SF wound dressings with antibacterial activity are fabricated in fibrous forms. Several studies
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use Ag as a broad-spectrum and strong antibacterial agent to endow antibacterial activity to SF
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mats [206]. The antibacterial activity and toxicity against human cells is dependent on Ag concentration [199, 207, 208]. The adhesion and spreading of human epidermal keratinocytes and human epidermal fibroblasts on silver sulfadiazine–containing SF nanofibrous mat decreased with increase of silver sulfadiazine content in the mats. Optimal concentrations of Ag in wound dressings are critical, to balance high antibacterial activity and no toxicity for human cells [207]. Silk fibers were coated with silver nanocolloids for prevention of infections after surgery [198]. The coated silver nanoparticles (AgNPs) were prepared by two methods (in situ and ex situ). For in situ, the silk fibers were embedded in SF aqueous solutions containing silver nitrate, while for
Journal Pre-proof ex situ, the silk fibers were treated with colloidal AgNPs. Impregnation with AgNPs by both in situ and ex situ methods enhanced the thermal and mechanical properties of the silk fiber-AgNPs mats. The presence of the crystalline AgNPs coated on silk fibers was shown by X-ray diffraction. Both methods showed cyto-compatibility for 3T3 fibroblasts. Although all the mats exhibited strong antibacterial activity against P. aeruginosa and S. aureus, the silk fiber-AgNPs antibacterial mats fabricated by the in situ method displayed better antibacterial activity compared to the
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samples prepared ex situ. The higher antibacterial activity of silk fiber-AgNPs fabricated by in
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situ methods and containing a greater content of silver, compared with those scaffolds prepared ex
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situ, was attributed to the processing method and thus content of silver in the material [198].
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Babu et al. [209] demonstrated antibacterial activity of silver oxide (Ag2O) nanoparticles-
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embedded SF spun (Ag2O-SF spun) against the pathogens (S. aureus and Mycobacterium tuberculosis) and non-pathogenic (Escherichia coli) bacteria. They also showed cyto-
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compatibility of the Ag2O-SF spun mats for 3T3 fibroblasts and in vitro wound healing potential
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when tested by a scratch assay. Çalamak et al. [210] indicated that the incorporation of different concentrations of antibacterial polyethylenimine (PEI) in ESF generated antibacterial wound
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dressings with cyto-compatibility for L929 cancer cells[210]. The incorporation of antibiotics into SF fibers was also useful for the fabrication of antibacterial SF mat wound dressings. Ceftazidime (CTZ), a third generation cephalosporin antibiotic, was loaded into SF/gelatin and electrospun to fabricate antibacterial nanofibrous wound dressings. The CTZ/SF/gelatin material had antibacterial activity against P. aeruginosa and good cyto-compatibility in vitro [211]. The overuse of antibiotics can cause bacterial resistance, as the resistance to CTZ was reported in several studies [212]. Localized release from the above materials may be one option to address this problem.
In other approaches, antimicrobial peptides (AMP), strong and broad-spectrm
Journal Pre-proof antibacterial agents, were used to fabricate antibacterial SF mats [189, 196]. AMPs are natural antibacterial systems in eukaryotes and kill microorganisms (bacteria, virus, protozoa, and fungi) by disrupting the structure or function of microbial cell membranes. These peptides also inhibit ATP-dependent enzymes in cells [213]. AMP can negatively affect cell viability and even cause human cell death when used in high concentrations.
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Song et al. [196] immobilized KR12 (KRIVKRIKKWLR), an antimicrobial peptide, on ESF membranes, which were then tested for antibacterial and biological properties in vitro for wound
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healing. The ESF membranes were treated with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide
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hydrochloride (EDC) to activate carboxylic acid groups in the SF proteins. The activated
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carboxylic acid groups were bound to N-(2-aminoethyl) maleimide (AEM) linkers and then
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reacted with various concentrations of KR12 (ranging 50-500 µg/ml). The ESF/KR12 had antibacterial activity against S. aureus, S. epidermidis, E. coli and P. aeruginosa, while exhibited
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cyto-compatibility for HDF and keratinocytes in vitro, and suppressed expression of TNF-α from
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LPS-induced murine monocytes. ESF/PVA nanofibers were fabricated by electrospinning and were functionalized with the LL-37 antimicrobial peptide. ESF/LL37 showed sustained delivery
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of LL-37 with antibacterial activity against tS. epidermidis MTCC 435 and P. aeruginosa MTCC 1688, and biocompatibility and wound healing potential in vitro and in vivo [189]. Incorporation of ESF with EGF and ciprofloxacin showed controlled release of with antibacterial activity, accelerated re-epithelialization, neovascularization and wound healing in full-thickness excisional wounds in rabbits [214]. Lu et al. [215] fabricates a bi-layered SF/CTS mat by spraying CTS on SF fabric. They show that CTS form a thin layer with connective pores over the SF fabric. The bilayered SF/CTS mat exhibits strong antibacterial activity and no cytotoxicity with high potential
Journal Pre-proof wound healing. This is compared to commercially available foam adhesive dressing (TegadermTM) in full-thickness wounds in a rat model. Transgenic SF with antimicrobial peptides (Cecropin B or Moricin antimicrobial peptides) were used for experimentation as biomaterials [216, 217]. The transgene SF fibers exhibited high antibacterial activity against some bacteria such as E. coli, with excellent biocompatibility. This
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new antibacterial biomaterials for biomedical applications.
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strategy may be useful to endow intrinsic antibacterial activity to the spun SF fibers and provide
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Srivastava et al. [218] coated tasar ESF by AgNPs to fabricate an antibacterial wound dressing mat against P. aeruginosa, S. epidermidis, S. aureus and E. coli and cyto-compatibility for L929
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skin fibroblast. Recently, a promising antibacterial bilayered SF-based scaffold, a SF sponge layer
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covered by ESF loaded with silver sulfadiazine, was developed against S. aureus [219]. Most recently, Zheng et al. [220] incorporated different concentrations of nano-Cu2O (0.5, 1.0, and 5.0
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mg/mL) into poly(ethylene oxide) (PEO)–SF composite and electrospun to fabricate an
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antibacterial wound dressing mat. The decreased uniformity and increased hydrophilicity of mat
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morphology were observed with the increase of nano-Cu2O concentrations. As expected, antibacterial activity of the composite electrospun mat against E. coli and S. aureus bacteria increased with the increase of nano-Cu2O concentrations with no detectable cytotoxicity against pig iliac endothelial cells.
6.2. Antibacterial SF sponge Some antibacterial SF composites were fabricated in the sponge form for infected wound healing management. For example, incorporation of AgNPs into SF sponge showed to have favorable
Journal Pre-proof moisture permeability, satisfying tensile strength , water absorption capacity and stable Ag release with high antibacterial activity and wound healing potential in comparison with porcine acellular dermal matrix [205]. The use of two bactericidal agents of colloidal Ag and ciprofloxacin in SF/SA sponges was a promising strategy to fabricate a highly effective antibacterial wound dressing with favorable wound healing potential [147]. Liu et al. [142] developed a wettable CTS-SF/AgNPs sponge for infected skin wounds. They incorporated different concentrations of AgNPs into CTS-
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SF aqueous solutions, and pour into a container and freeze dried after freezing the casts at -20 C
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and -80 C to fabricate an antibacterial sponge. The sponge had high porosity with good wettability
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for providing moisture in the wound bed and appropriate mechanical property. The sponges also
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showed strong antibacterial activity against various bacteria such as S. aureus, E. coli, P. aeruginosa and Monilia albicans and cyto-compatibility for L929 cells, human fibroblasts and
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human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro. The antibacterial activity and
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wound healing potential of the CTS-SF/AgNPs sponges for the full-thickness wound in mice
[142].
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model infected with P. aeruginosa showed high antibacterial activity with no post wound infection
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Gentamicin sulfate (GS)-loaded SF/gelatin sponge fabricated by freeze drying method was introduced as another candidate for infected wound healing in vitro. The GS/SF/gelatin sponge indicated a decreased compressive modulus property with the increased content of SF and GS. It also exhibited enhanced water swelling capacity with the addition of GS. The sponges with higher content of SF demonstrated to have accumulative release of GS. The sponge had excellent antibacterial activity against S. aureus, S. epidermidis, M. luteus, B. cereus and P. aeruginosa. Only the GS/SF showed cytotoxicity effects for NHDF [221]. Incorporation of AgNPs in collagen/SF sponge fabricated by freeze drying technique also considered as a promising candidate
Journal Pre-proof for the treatment of infected wounds [222]. In the other two studies, chitin/SF 3D scaffolds were loaded with AgNPs and TiO2. These were suggested as effective wound dressings with cytocompatibility. They had also promising swelling, water uptake properties and antibacterial activities against E. coli, S. aureus, and Candida albicans. [223, 224].
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6.3. Antibacterial SF films
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Some antibacterial SF-derived wound dressing materials were fabricated in the form of films. For example, Alam et al. [202] developed a promising sustained drug release system by modifying the
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surface of SF/gelatin film with polyethylene glycol (PEG). The modified film was loaded with
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ciprofloxacin, a drug belongs to a group of antibiotic called fluoroquinolones, exhibited lower
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burst drug release compared to unmodified film. The modified SF/gelatin loaded with antibiotic showed an excellent drug release with high antibacterial activity and better wound healing
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potential in comparison of the unmodified antibiotic-loaded SF/gelatin film [202].
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Strontium is another additive choice with both antibacterial activity and wound healing potential.
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Strontium-loaded SF/SA blend film (Sr/SF/SA) had high antibacterial activity, cyto-compatibility and excellent wound healing potential in vitro. Among Sr/SF/SA films with various contents of Sr, the samples containing 5 mg/ml Sr exhibited the best result to meet the requirement for infected wound healing. Sr/SF/SA film also increased the secretion of important pro-angiogenic factors, VEGF and bFGF in vitro. This accelerated in vivo neovascularization [148]. Molybdenum diselenide (MoSe2) is an inorganic compound of molybdenum and selenium and has high antibacterial activity through its peroxidase-like activity. Activation of carboxylic acid groups of SF and functionalization with MoSe2 is an interesting strategy for endowing antibacterial activity
Journal Pre-proof to SF-derived wound dressing [119]. The addition of selenium nanoparticles in to ESF improved human dermal fibroblast metabolic activity and antibacterial behavior in vitro [201].
6.4. Antibacterial SF Gels Fabrication of antibacterial SF composites in gel form may be a promising strategy to achieve a
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cost-effective antibacterial ointment and excellent wound healing materials. It was reported that
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AgNPs-loaded SF gel had strong antibacterial activity against S. aureus and excellent wound healing potential in full-thickness wounds in the rat when compared to a commercially available
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wound healing gel (soframycin gel) [203]. It was also demonstrated that SF/AgNPs solution can
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be considered as a novel agent for treatment of biofilm-associated diseases, such as sinusitis. The
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sinusitis model was created in New Zealand White rabbits by inoculation with S. aureus. The solution with 384 mg/L AgNPs showed completely elimination of biofilms and repair of defected
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epithelium [225]. An SF hydrogel with improved mechanical properties was fabricated by using
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2-(N,N-dimethylamino) ethyl methacrylate as an excellent delivery system for controlled release
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of cefixime, belongs to a group of antibiotics called cephalosporin [226]. Their results indicated a high degree of water uptake and improved mechanical properties (tensile strength and elongation at break) compared to SF hydrogel without modification and high antibacterial activity.
7. Other biomedical applications of silk fibroins Universally known as textile material, silk threads have embarked on their journey towards other beneficial uses decades ago. Silk also remains a prominent component for several cosmetic products and medical sutures. SF, in particular, finds its niche in the world of biomedical science
Journal Pre-proof for its beneficial properties as a tunable yet biocompatible biomaterial. Besides wound healing and skin repair, fibroin has multiple biomedical applications in various types of TE including bone [227-229], neural [129, 230], cardiac [231, 232], ligament [233, 234], vascular [67, 235], muscle [236, 237], cornea [238, 239], dental [240, 241] , hepatic [242, 243], cartilage [244, 245], osteochondral [246, 247] and others (Fig. 2). Bioprinting techniques create multi-level 3D scaffolds, which can be modified or tailored as per the prerequisites for different TE needs [248].
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Combined with the delivery and sustained release of bioactive molecules (growth factors, drugs
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and antibiotics) in different formulations like microcapsules, nanoparticles, hydrogels, porous
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scaffolds, nanofibrous matrices and others [249-252] further expands the utility for silks. Other
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applications of silk include as food preservatives [253], optical devices [254] and electronics [255],
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among others.
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8. Benefits and concerns of using silks for biomedical applications
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Silk protein fibroin offers an impressive array of biomaterials, which can be applied in diverse
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areas of biomedical science. There are numerous desirable properties exhibited by this protein including ease of materials fabrication, mechanical strength, biocompatibility, flexibility, biodegradability, ease of chemical modification, non-cytotoxicity, no immune response, low production cost and available supplies of material. Although, silk is a natural biomaterial with no major adverse effects, there are still shortcomings associated with biomedical applications. The standardization of the production of cocoons starting from sericulture practices to biomedical grade cocoon materials needs special attention. Even handling of the raw materials after procurement, including storage, transportation and use in the laboratory by trained personnel are
Journal Pre-proof important factors to consider. The degumming process involved in the extraction of fibroin and regeneration from aqueous silk leads to a reduction in the mechanical strength of silk, which limits certain aspects involved in the fabrication process for matrices. Over the years, multiple and diversified animal studies have been reported for various applications of silks, and these validate the safe and effective use of silk [12, 13]. This reflects the promising characteristics of this product,
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but further clinical investigations are needed for specific biomedical products based on silks.
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9. Conclusions and future prospects
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Skin is the first line of defense as well as the most frequently damaged organ of the human body. Wound dressing materials and new techniques/ways to restore the lost or wounded structure of the
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skin have been pursued for many years. Most common clinically used allografts or autografts
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include certain limitations (immune response, restricted host tissue regeneration, lack of degradation), which lead to the need for artificial human skin by TERM. This review described
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the relevance of natural SF in this domain and the numerous categories of scaffolds fabricated and
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investigated. They aid in creating a skin substitute or dressing material. Silks (mainly silkworm silks) have exceptional physical, mechanical, chemical and biological properties, which permit the formation of different biomaterials. Silk protein fibroin is used in skin regeneration, and TERM applications. Films and fibrous matrices are most commonly used for these applications. Other kinds of matrices like sponges, hydrogels, hydrocolloids, microfibers, microcapsules, nanoparticles, bio-printed gel and others are also utilized effectively in partial or full thickness wounds. In order to augment their performance, added functional features like antibacterial activity, drugs and growth factors are included, as well as micro carriers or using genetically
Journal Pre-proof modified SF by creating /transgenic silkworms. Commonly practiced techniques are the inclusion of different inorganic or heavy metals (strontium, selenium, silver), antibiotics, antimicrobial peptides or establishing a transgenic SF having anti-microbial peptides to prevent infection during would healing. The fibroin matrices support the acceleration of cellular adhesion, wound contraction, re-epithelialization and angiogenesis, collagen formation with a reduced immune response. The prospects of using fibroin alone and its blends are quite exciting in wound healing
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due to the positive results achieved in vitro and in vivo. Genetically modified SF combined with
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emerging innovative methodologies may produce cost effective silk-based artificial skin with
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additional functional features. Successful wound healing with minimal scar formation and the
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present clinical trials using fibroin scaffolds further emphasize the utility of silk for the field of skin tissue engineering. The flexibility and ease of modification in silk scaffolds make it possible
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to create improved bi-layered skin-like constructs for clinical use. SF is an FDA approved material
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for some biomedical applications and some silk-based products for cosmetic (silk mask and gel) and medical applications (silk suture and Fibroheal
TM
Ag wound dressing) are already on the
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market. In addition, silk-based surgical meshes passed biocompatibility and safety testing for ISO
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10993. As stated earlier, some SF-based scaffolds such as SF and SF/gelatin/sericin films have successfully passed through preclinical and the randomized clinical trials for full-thickness skin wounds. It is expected that the electrospun SF approaches loaded with or without antibacterial agents may be the most effective SF based skin substitutes and subject to the future clinical trials. In summary, natural silk protein, fibroin has great prospects of expanding its niche in the field of skin tissue regeneration and repairing damaged skin.
Journal Pre-proof Acknowledgements SCK presently holds an European Research Area Chair and Full Professor position at 3B´s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Portugal, supported by the European Union Framework Programme for
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Research and Innovation HORIZON 2020 under grant agreement nº 668983 — FoReCaST.
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Authors’ contributions
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MG and SCK design the concept; MG wrote the paper along with SS; DLK and SCK reviewed
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during the preparation of the manuscript; DLK, AS, SCK and RLR revised the manuscript. All the
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Conflict of interests
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authors discussed, commented, revised and approved the final manuscript.
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Authors declare no conflict of interest.
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+ sign indicates the important references for further consultation for an appropriate area (s) of work.
Journal Pre-proof Legends for figures (1 to 6) and write up for graphical abstract. Figure. 1. Illustration of the basic routes of tissue engineering and regenerative medicine. The cells are harvested from patients by biopsy and are grown in 2D culture under aseptic conditions. The cell culture medium is supplemented with growth factors and other specific features as required by the individual cell type. These cells are sub-cultured for the expansion of cells and are seeded onto 3D matrices for the construction of specific 3D tissues. These matrices/polymeric scaffolds may be natural, synthetic or blends
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and are fabricated in various material format such as hydrogels, porous sponges, nanofibrous mats,
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micro/nano particles, micro-/nano patterned surfaces, among others. These matrices may contain bioactive molecules to augment the proliferation of cells. For long-term culture, the matrices are grown in bioreactors
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transplanted to restore the damaged tissue in vivo.
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to support and maintain the cultures under aseptic conditions. The regenerated tissue in vitro may be
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Figure 2. Different applications of silk protein fibroin in tissue engineering and regenerative medicine. Silk provides flexibility to fabricate different kinds of matrices and thereby, finds use in multiple
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biomedical applications including tissue engineering targets like the regeneration and repair of damaged
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tissues. Apart from reconstructing injured areas, silk matrices also effectively deliver bioactive molecules including drugs and growth factors to the target site, as well as genes and cells, further enhancing utility in regenerative medicine.
Figure. 3. Arrays of silk protein fibroin matrices for tissue engineering and regenerative medicine. Silk as a biomaterial is often obtained from both mulberry and non-mulberry (shown above are some of the main cultivated silkworm species) silkworm silks. Depending on the silkworm species and availability, silk proteins may be isolated from the cocoons after degumming (removal of silk protein sericin) or from the mature 5th instar larvae (mainly from non-mulberry). Silk-based scaffolds are developed in various forms,
Journal Pre-proof which range from thin membranes/coatings to 3-D bio printed matrices to support organ specific requirements. Figure 4. The events of four overlapping phases of normal wound healing process during the first 30 days of post-wounding.
Figure. 5 (a-e). Silk fibroin film: from fabrication to animal and randomized clinical trial studies. (a)
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Schematic of the fabrication of transparent silk fibroin film. (b) Silk fibroin film with a thickness of 64.9 µm was much thinner than Suprathel (266 µm) and Sidaiyi (1789 µm). (c) Silk fibroin film exhibited better
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and faster wound healing compared to Suprathel and Sidaiyi during 90 days in porcine model [121]. (d)
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Improved wound closure in the wounds treated with SF film compared to Mapitel and the wounds with no
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treatment after 5, 10, and 15 days [122], (e) Hand burn wounds after 1 month treatment with Biobrane and
lP
Dressilk [123].
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Figure 6 (a-d). Silk fibroin fibrous scaffolds for skin wound healing (a) Electrospun silk fibroin scaffolds fabricated by cold-plate electrospinning methods (CPE) had higher porosity and pore sizes with
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controllable mat thickness and easy shaping compared to salt-leaching (SLE) and traditional
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electrospinning techniques (TE) [133]. (b) Silk fibrous mats were fabricated by treating the silkworm cocoons with various degumming process times (0-100 min). SEM micrographs showed the effects of degumming time on morphology and porosity [136]. (c) Electrospun silk fibroin (ESF) on amniotic membranes (AM), as well as cross sectional views of AM/ESF bilayer membranes, indicated successful the coating of AM with ESF [59]. (d) ESF/AM alone or seeded with adipose tissue-derived mesenchymal stem cells (ADSCs) exhibited better wound healing compared to AM and AM/MSCs in a mouse burn model [69].
Journal Pre-proof
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Graphical abstract
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Silkworm silk fibroin is a well-established natural protein in the realm of biomaterials with an array of matrices in its repository. These range from primary bio coating to state of the art,
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bioprinting en route for creating an advanced graft for diversified biomedical applications.
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Augmenting these matrices by incorporating functional traits like delivery of bioactive molecules/compounds (growth factor, drug, antibiotic, gene or cell) or conductivity make them smart matrices for skin tissue regeneration and skin repair.
Journal Pre-proof Table 1 (a-c). In vitro (a), in vivo (b) and clinical trials (c) carried out between 2000 and 2019 on different types of 2D and 3D SF or SF blends scaffolds/matrices for skin tissue regeneration, tissue engineering and regenerative medicine applications.
Table 1a: In vitro experimentations on SF or SF blends for skin tissue engineering applications. Design of
Major findings
experiment (s)
References
of
Material (s)
Bm fibroin peptides derived from digestion by
peptides
two important peptides present in N-terminal region
In vitro
[256]
-p
SF-derived
ro
chymotrypsin, VITTDSDGNE and NINDFDED, as
of SF protein promote human fibroblast growth,
lP
SF.
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critical biological properties of N-terminal region of
Chitin/SF Nanofibrous mat made from various
In vitro
[257]
electrospinning of chitin and SF solution (chitin/SF blend); good cyto-compatibility for NHEK cells in 25
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nanofibrous mat
concentration of SF and chitin, and also simultaneous
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Chitin/SF
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% chitin/75 % SF and chitin/SF blends. Conjugation using cyanuric chloride, higher adhesion, growth and viability of subcutaneous fibroblastic cells than pure SF, promoted adhesion of
Lactoseconjugated SF 2D films and 3D scaffolds
fibroblasts compared to myofibroblasts in lactoseIn vitro
conjugated SF film, loss of contractile phenotype of pre-differentiated myofibroblasts in lactoseconjugated SF substrates, suppressed myofibroblast differentiation cultured on 3D lactose-conjugated SF scaffolds and decreased host fibrotic reactions in lactose-conjugated SF scaffolds.
[258]
Journal Pre-proof Plasma treatment of ESF in presence of oxygen and Plasma-treated ESF mat
methane, decreased and increased hydrophilicity In vitro
after methane and oxygen plasma treatment,
[259]
respectively, good cellular activities in oxygen plasma-treated ESF for NHEK and NHEF cells. Successful incorporation of EGF into ESF
EGF-loaded In vitro
[260]
compatibility, accelerated wound closure and wound
nanofibrous mat
of
ESF
nanofibers, controlled release of EGF, good cyto-
healing in human skin-equivalents wound model in
ro
vitro.
transmission rate, biodegradation rate, nanofiber
In vitro
average diameter and morphology, suitable for full-
[131]
re
ESF mat
-p
SF concentration-dependent water vapor
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thickness wound dressing Increased mechanical properties with addition of SF,
nanofibrous mat
In vitro
antibacterial activity depends on type of bacteria,
[261]
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CTS/SF
better biocompatibility with promotion of growth and
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attachment of murine fibroblasts.
SF/PEGs sealant
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Cyto-biocompatible, strong and effective sealant, fast
In vitro
crosslinking (within seconds), better adhesive
[166]
property than the current leading PEG-based sealant, decreased swelling and longer degradation compared to PEG-based sealant. Nanofibers with average diameters of 385 ± 63 nm,
TiO2-loaded ESF mat
higher water content compared to commercially In vitro
available hydrocolloid wound dressing Tegasorb, excellent hemocompatibility and cyto-compatibility for L929 fibroblasts, strong antibacterial activity.
[262]
Journal Pre-proof Chemical interaction between chitosan and fibroin, increased flexibility, swelling ratio and CTS/SF film
In vitro
[263]
biodegradation along with the increase of CTS concentration, favorable cell adhesion and growth support for fibroblasts. Control release of vitamin C, good cyto-
loaded ESF
biocompatibility such as cell adhesion and growth for In vitro
[264]
mouse fibroblast L929 cells, increased expression of
of
Vitamin C-
collagen type I alpha 1, glutathione peroxidase 1 and
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catalase and high anti-oxidative effects.
F nanofibrous
Intermolecular hydrogen bonding among CECTS, In vitro
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CECTS/PVA/S
PVA and SF and excellent cyto-compatibility for L929 cells.
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mat
[265]
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Changed morphology of nanofibers with different concentrations of vitamin E, ribbon-like and round
In vitro
[266]
of vitamin E, excellent water-resistant property, controllable and sustained release of vitamin E,
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loaded ESF mat
shape for lower (2%) and higher (8%) loading dose
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Vitamin E-
CTS/SF/ADA membrane
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promised cell adhesion, proliferation for mouse skin fibroblasts (L929) and anti-oxidant property.
In vitro
permeability, and excellent cell adhesion and
erol
[267]
proliferation for L929 fibroblasts. Good physicochemical and mechanical properties in
SF film/pectin/glyc
Promising stability, water absorption and vapor
In vitro
SF film containing 6% w/w pectin 12% w/w glycerol, promising cell adhesion and growth, and suitable for MSCs delivery.
[110]
Journal Pre-proof Surface modification with NaOH alkaline to form Nanofeatured SF membrane
nano-featured SF membrane, enhanced nano-
In vitro
topography, wettability and cellular function
[111]
compared to untreated SF scaffold. Increased plasticity and flexibility, improved elongation at break, increased water holding SF/dextrose film
In vitro
capacity, hydrophilicity and roughness, enhanced cell
[124]
of
attachment and proliferation and promising system
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for delivery of gentamicin.
Fabrication by cold-plate electrospinning method,
nanomatrix mat
higher porosity and pore size with controllable mat
In vitro
[133]
-p
ESF porous
thickness, easy shaping compared to SLE and
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traditional electrospinning techniques.
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Incorporation of different contents of SF and fabrication of SF/BC with final ratios of 25/75 %,
scaffold
In vitro
50/50 % and 75%, higher water solubility and lower
na
SF/BC porous
uptake with the increase of SF content, non-cytotoxic
[268]
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and genotoxic for L929 fibroblasts, and improved
PHBV/ESF nanofibrous
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cell adhesion with the increase of SF content to 50%.
In vitro
scaffold
Good surface hydrophilicity and water-uptake
[144]
capability, fibroblast cells proliferation and attachment in 50/50 ratio of PHBV/ESF. Average fiber diameter of 160 nm, better growth
Muga SF nanofibrous mat
In vitro
support for L929 fibroblast cells compared to muga SF film, better gentamycin sulphate release compared to muga SF film.
[138]
Journal Pre-proof Fabrication by freeze drying, homogenous, cytobiocompatible and interconnected 3D porous composite scaffold, freezing temperature-dependent SF/SA scaffold
pore diameter (54-532 μm) and porosity (66-94%),
In vitro
decreased pore diameter in lower freezing
[114]
temperature, Increased porosity in lower freezing temperature, suitable degradation rate and
of
crosslinking with EDC. Homogenous pore distribution, highest porosity (70%), water uptake and swelling ration in SLE
nanomatrix vs
compared to FD and SL, higher compressive strength
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fabricated by
of SL, similar compressive strength in FD and SLE,
In vitro
good biocompatibility of all three methods for NIH
[112]
re
3D SF scaffolds
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3D ESF porous
3T3 fibroblast cells, and favorable biomechanical and
methods
biological characteristics in ESF fabricated by SLE
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FD and SL
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method for skin tissue engineering. Treatment with various rates of degumming
mats
In vitro
processes, favorable mechanical property, strong antibacterial activity, and good cyto-compatibility in
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Silk cocoon
[136]
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vitro
SF/CTS/LP hydrogel
Promising thermal stability, favorable surface morphology, in vitro cyto-compatibility for NIH 3T3, antioxidant activity, suitable biodegradation rate of in
In vitro
vitro, capability to keep wounds moisture, better cell viability, migration, proliferation and wound healing of SF/CTS/LP in vitro compared to CTS and SF/CTS hydrogels.
[181]
Journal Pre-proof Improved mechanical and dynamic mechanical CTS/SF film
properties compared to nonwoven SF, good
In vitro
hemocompatibility, cyto-compatibility and
[143]
biodegradability. Promising water absorption, water vapor transmission rate and mechanical properties, antibacterial activity, cyto-compatibility for
In vitro
fibroblasts, better wound healing results in Sr/SF/SA
[148]
of
Sr/SF/SA film
film containing 5 mg/ml Sr, increased the secretion
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of VEGF and bFGF in vitro.
In vitro
and hydrophilicity, enhanced cell attachment and
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film
elongation at break, increased water holding capacity [125]
proliferation and promising system for delivery of
lP
SF/Dextrose
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Favorable plasticity and flexibility, improved
gentamicin.
Antheraea mylitta silk mat
skin
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and proliferation, and good biodegradation rate.
[162]
Homogenous morphology, favorable mechanical
In vitro
AM/ESF bilayered artificial
Good biocompatibility, promising cell attachment
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CTS/PGS
In vitro
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SF/PGS and
In vitro
property, biocompatibility for epidermal cell primary culture, and thermal stability.
[139]
Electrospinning of SF on decellularized amniotic membrane, good cyto-biocompatibility and cell adhesion, mechanical property close to native skin, and angiogenic property.
[269]
Journal Pre-proof Non-covalent interactions between SF and NT, homogeneous NT distribution in SF film surface, sustained release of NT for up to 72h, decreased
Neurotensinloaded SF film stimulated by
production of interleukins from E. coli In vitro
[153]
liposaccharide-stimulated macrophages after exposure to NT-loaded SF film, immunomodulatory
iontophoresis
property, good biocompatibility for fibroblast, and bacteriostatic activity against gram-positive
of
microorganisms.
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Tunable amorphous structure and mechanical property, freeze drying at various temperatures of -20
scaffold
crystalline structure at freeze dry temperature > -9
In vitro
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Amorphous SF
-p
⁰C to -5 ⁰C, water in-soluble behavior with ⁰C, water soluble with minimal crystalline region at
[270]
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freeze dry temperature > -20 ⁰C, potential method for tuning mechanical and biological properties of SF
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scaffolds.
EGF/SF fusion
In vitro wound model
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protein
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Development of EGF- light chain of SF conjugated
Curcuminloaded SF hydrogel
In vitro
gene sequences in transgenic silkworm, prevention of draining of EGF 2.5 fold increase in proliferation of
[188]
fibroblasts, and accelerated wound healing. Fabrication of electro-responsive SF hydrogel loaded with curcumin, 3D scaffold with 80% porosity and 100% swelling ratio, antibacterial activity, and good biocompatibility for HaCaT.
[271]
Journal Pre-proof Fabricated by freeze-drying, high porosity (83%) and Citrus pectin/SF sponge
interconnectivity with pore size around 120 µm, high In vitro
water uptake ability (800% in 24 h) and low weight
[156]
loss (21.3% in 40 days) and cyto-biocompatibility for fibroblast cells.
honey/ESF mat
biocompatibility with improved viability of primary human dermal fibroblasts. Better water uptake and cell growth compared to
In vitro
ESF, and enhanced human dermal fibroblast
Fabrication of ESF by electrospinning, dropping
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amniotic fluid-SA hydrogel onto ESF, sustained and
In vitro
Fabricated by electrospinning, top layer (SF-PCL)
structure
mimicked the dense nature and waterproof properties
ur
In vitro
of the natural epidermis, down layer mimic natural
[176]
structure of dermis, antibacterial activity with cyto-
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thymol)
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proliferation, migration and spreading in vitro.
Two-layered
PCL/SF-HA-
[175]
lP
localized delivery of amniotic fluid, improved cell
hydrogel/ESF
membrane (SF-
[128]
adhesion, growth and infiltration.
Amniotic fluidSA
[170]
of
Manuka
In vitro
ro
mat
Controllable and sustained release of hPL, and good
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hPL-loaded ESF
biocompatibility for human fibroblasts. Induced crystallization of SF, and increased absorption capacity and flexibility and of the SF
Glucose/SF film
In vitro
films with the addition of glucose in SF film, cytobiocompatibility with positive effect in promoting the wound closure.
[155]
Journal Pre-proof SF/PVA/collage
Fabricated by electrospinning, sustained and localized
n I peptide
delivery of S-Nitrosoglutathione, randomly oriented In vitro
loaded with S-
fibers with high porosity, increased cell proliferation with the presence of collagen I peptide, cyto-
ne
biocompatibility with high cell adhesion property.
lP
re
-p
ro
of
Nitrosoglutathio
[173]
Major findings
ur
Design of experiment (s)
Jo
Material (s)
na
Table 1b: In vivo investigations with SF or SF blends for skin tissue engineering.
References
In vivo fullPVA/CTS/S
thickness
Good biocompatibility, flexibility and softness,
F sponge
excisional
increased vascular ingrowth, accelerated wound healing.
wound in rat
[272]
Journal Pre-proof Transparent, easily available and sterilizable, cost effective and transparent, faster healing time, greater
In vivo fullSF film
collagen regeneration and higher biocompatibility than
thickness wound
[273]
conventional hydrocolloid dressing, similar or slightly
in mice
better healing effects compared to lyophilized porcine dermis. Excellent cyto-compatibility for human ADSCs,
scaffold
of
[274]
compared to CTS/SF and control, promoted
ro
freeze-dried
epithelialization in ADSCs-seeded CTS/SF (75:25 ratio)
excisional fullthickness
differentiation of ADSCs into fibro-vascular,
wounds in mice
endothelial, and epithelial cells after culture on CTS/SF
-p
CTS/SF
accelerated wound closure, angiogenesis, re-
In vivo
re
scaffold.
freeze-dried
excisional fullthickness wounds in mice
epithelialization in ADSCs-seeded CTS/SF (75:25 ratio)
[274]
compared to CTS/SF and control, promoted differentiation of ADSCs into fibro-vascular, endothelial, and epithelial cells after culture on CTS/SF
ur
scaffold
accelerated wound closure, angiogenesis, re-
In vivo
na
CTS/SF
lP
Excellent cyto-compatibility for human ADSCs,
Jo
scaffold.
In vivo fullPorous SF
thickness
film
excisional wounds in rat
In vivo fullpHA/SF gel
thickness porcine wound
SF film with porous structure, increased re-
[275]
epithelialization, connective tissue thickness and neovascularization.
3D structure with furry texture and high porosity compared to SF and HA/SF gels, better reepithelialization, matrix formation and wound healing compared to SF and HA/SF gels.
[276]
Journal Pre-proof Recombina
In vivo deep
nt spider
second-degree
silk porous
burn wounds in
sheet
Recombinant silk proteins containing RGD (Arg-Gly-
[277]
Asp) motifs, increased bFGF expression, better wound healing and collagen synthesis compared to control.
rat
Cross-linking with genipin, decreased pore size,
porous scaffold
swelling ratios, degradation and release rates after crossIn vivo burn
linking, excellent cyto-biocompatibility for fibroblasts,
wounds
[278]
accelerated wound closure, re-epithelialization and
of
SF/elastin
wound healing compared to commercially available
ro
collagen base wound dressing Suprasorb C.
-p
Cross-linking with various concentration of GA, improved mechanical properties compared to SF
bi-layered wound dressing
re
controllable biodegradation rate depending cross-linking
thickness
lP
ericin/wax
without wax, homogeneous porous structure,
In vivo full-
degree, good biocompatibility for L929 mouse
excisional wounds in rat
[279]
fibroblasts, better wound closure, re-epithelialization,
na
SF/gelatin/s
collagen synthesis, and wound healing compared to commercially available wound dressing 3MTM
Jo
ur
Tegaderm.
SF/CS/hyal
In vivo full-
uronic acid
thickness
porous
excisional
scaffold
wounds in rat
A ternary scaffold with pore diameter and porosity of 95–248 µm and 88–93%, respectively, promoted cell adhesion, proliferation and survival, good neovascularization, and wound healing in animals implanted with SF/CS/HA(80/5/15 ratio).
[280]
Journal Pre-proof Functionalized with EGF and silver sulfadiazine by drug SF scaffolds (film, silk film, lamellar porous silk
loading or coating techniques, better in vivo reepithelialization, wound closure, dermal proliferation
In vivo full-
and wound healing rate for all functionalized SF
thickness
scaffolds and two incorporated techniques compared to
excisional
two commercially available wound dressings, better
wound in mice
wound healing for SF scaffolds (all forms)
ESF)
functionalized with both EGF/silver sulfadiazine compared to other groups.
of
film and
ro
SF fibers with different diameters (250-1200 nm),
Ex vivo full-
enhanced cell proliferation and migration in samples
thickness wound
with smaller fiber diameter, increased extracellular
healing model
re
matrix proteins in samples with smaller fiber diameter. Increased adhesion and proliferation of mouse
lP
In vivo fullthickness
fibroblasts (L929) compared to PLGA nanofibrous mat,
nanofibrous
excisional
speed up wound healing compared to commercially
mat
wound in
nanomatrix mat
Fabrication by salt-leaching electrospinning methods,
thickness
controllable pore size and morphology, well infiltration
excisional
of cells into ESF nanomatrix, superior healing potential
wound in rat In vivo
ESF mat
available wound dressing Comfeel.
ur
In vivo Full-
Jo
ESF
[281]
na
SF/PLGA
diabetic rat
[132]
-p
ESF mat
[186]
[127]
compared to Matriderm®. Homogenous morphology, excellent support of cell
subcutaneous
adhesion and proliferation of rBMCs and good in vivo
implant in rat
biocompatibility.
[59]
Journal Pre-proof Mean pore sizes of 50–200 μm, strong intermolecular In vivo fullCTS/SSF
hydrogen bonding, good biocompatibility, broad
thickness skin
spectrum antibacterial activity, improved mechanical
wound in rat
behavior, accelerated skin wound regeneration of rats
[116]
compared to SSF and CTS alone.
electrospun mat
properties for immortalized human fibroblast cells in
partial-thickness vitro, accelerated wound closure and neovascularization burn wound in
in vivo, organized collagen formation, reduced scar
rat
formation and excellent topical delivery system for burn wound healing.
Increased biomechanical property compared to CTS
In vivo full-
membrane, favorable biocompatibility, accelerated re-
re
CTS/mSF
[185]
of
e IV
In vivo deep
ro
astragalosid
Promising cell adhesion and good biocompatibility
-p
SF/Gelatin/
thickness skin
epithelialization, wound closure and wound healing
lP
wound in rat
[141]
compared to pure CTS membrane.
na
Average fiber diameter of 320 and 502 nm for pure ESF and ESF/PLA mats, respectively, lower water uptake and vapor transmission rate in ESF/PLA compared to
ur
mat
thickness in rat
Jo
ESF/PLA
In vivo full-
model
pure ESF mat, higher hydrophilicity of ESF compared
[161]
to ESF/PLA, high antibacterial activity of tetracycline hydrochloride-loaded ESF and ESF/PLA, accelerated wound healing in ESF and ESF/PLA groups compared to control wounds treated with gauze. Fibrous foam-like architecture, sufficient porosity and in
In vivo fullIsabgol/SF
vitro biodegradation, support NIH 3T3 fibroblast cells
thickness wound growth and adhesion, accelerated neovascularization, rein rat model
epithelialization, collagen deposition, wound contraction and healing in 2 % isabgol/2 % SF (Isabgol/SF 75/25).
[146]
Journal Pre-proof Excellent healing potential of both SF and SF/Gel SF and SF/Gelatin microcarrier s
microcarriers compared to control, increased infiltration
In vivo deep
of immune cells to wound area, minimal fibrosis
skin wound in
formation, increased expression of pro-inflammatory
mouse
[180]
cytokines TNF, IL-6 and IL-1β, and increased expression of chemokines CXCL1 and CXCL2.
d
In vivo burn
mechanical property for wound healing applications,
wound in rat
better burn wound closure and healing compared to gauze and Neoderm®.
BG/CTS/SF
Excellent biocompatibility, favorable mechanical
-p
In vivo full-
thickness wound property, vascularization and new blood vessel
Wet gold nanoparticle -loaded ESF mat
lP
Fabrication by a facile two-step freeze-drying
full-thickness
technology, increased dermal cell viability, proliferation
wound in rat
In vivo full-
thickness wound in rat
[134]
na
mat
In vivo dorsal
and migration, and accelerated in vivo wound healing.
ur
fibrous SF
formation, and accelerated wound healing.
Jo
/nano-
[27]
re
in rat Micro-
[109]
of
Hydrocolloi
Structural stability, good cyto-compatibility, suitable
ro
SF/CMC
Increased degradation profiles, improved biomechanical property, neovascularization and granulation tissue formation, and elongation at break values similar to normal skin.
[163]
Journal Pre-proof Fabrication by cold-plate electrospinning and automatic SF particleincorporate d PCL electrospun mats
magnet agitation system, 3D porous mats with thickness In vivo full-
up to 6 mm, interconnected macro-pores ranging from
thickness
tens to hundreds μm, homogenous distribution of SF
wounds in rat
nanoparticles within PCL mat, good biocompatibility,
model
higher wound healing rate and collagen deposition in
[164]
PCL/SF mat with higher content of SF particles, great
of
potential as artificial skin. Fabrication by SLE method, higher porosity compared
ro
wound closure, organized collagen matrix deposition,
model in rat
and decreased expression of pro-inflammatory
re
cytokines.
In vivo
retention capacity (440%), water vapor transmission rate (~2330 g m-2 day-1), sustained release of EGF for 3
excisional full-
na
ESF loaded
Good cyto-compatibility for HaCaT cells, high water
lP
Nonmulberry
with EGF
thickness wound days, high elasticity (~2.6 MPa), controllable and
and
in New Zealand white rabbit
[214]
sustained drug release, strong antibacterial activity, and accelerated re-epithelialization, neovascularization and
ur
ciprofloxaci
[113]
-p
ESF mat
to ESF nanosheets, accelerated re-epithelialization and
In vivo burn
wound healing.
Jo
n
In vivo fullSilkworm
thickness wound Good biocompatibility, antibacterial activity,
cocoon sol-
in wounds New
transparent, accelerated wound healing rate compared to
gel film
Zealand white
standard Mepitel® dressing.
rabbit
[122]
Journal Pre-proof Synthesis of growth factor-SF gene construct in transgenic silkworm by genetic engineering technique, secretion of growth factor-SF proteins from transgenic FGF-, EGF-
PDGF- and VEGFfunctionaliz ed silk
and in vivo wound healing potential of EGF-, FGF- or
In vivo full-
VEGF-functionalized silk membranes compared to
thickness
native, PDGF- and KGF-functionalized silk membranes,
excisional
[190]
accelerated keratinocyte cell growth and migration of
wounds in mice
KGF- and VEGF-functionalized silk membranes,
of
, KGF-,
silkworm, good biocompatibility in vitro, better in vitro
membrane
ro
increased adhesion of macrophage for EGF- and VEGFsilk membranes, and slightly better wound healing
-p
potential of VEGF-functionalized silk membrane
Fabrication by 3D bioprinting, favorable biomechanical
In vivo fullthickness
onated
excisional
SF/FGF
wound in rat
property, biocompatibility and cell adhesion for CFFs,
lP
Gelatin/sulf
increased cell migration in presence of FGF, accelerated
[193]
granulation, healing, and neo-vascularization in wound
na
3D
re
compared to other groups.
bed in presence of FGF.
ur
Comparison of two commercially burn wound
Dressilk vs Biobrane
Jo
dressings, Dressilk (PREVOR, France), made from
In vivo
superficial burns in 30 patients
silkworm silk, Biobrane (Smith and Nephew ltd, Hull, UK), a silicon based wound dressing coated with nylon mesh and porcine dermal collagen-derived peptides, both Dressilk and Biobrane provide a safe and effective microenvironment for burn wound healing, and also Dressilk is cost effective
[123]
Journal Pre-proof ESF/COL and CTS/COL nanofibrous
In vivo full-
Support proliferation and adhesion of fibroblasts and
thickness wound keratinocytes, good biocompatibility, and promote in rat
[135]
wound healing compared to gauze dressing.
mat Synthesis by green electrospinning technique, smooth
mat
excellent biocompatibility, increased average fiber
thickness wound
of
honey/ESF
surface morphology, high antibacterial activity,
In vivo full-
diameter with the increase of Manuka honey
in rat
[130]
concentration in construct, and accelerated wound
ro
Manuka
-p
healing.
Excellent biocompatibility and cell growth in PRP/SF
PRP/SF and PPP/SF compared to cocoon mat, enhanced
thickness wound
wound healing in PRP/SF and PPP/SF cocoon mat, and
in rabbit
[137]
better wound healing results in PRP/SF compared to
na
mats
re
PPP/SF
and PPP/SF cocoon mats, increased hydrophilicity in
In vivo full-
lP
PRP/SF and
PPP-SF and control.
ur
Fabrication by freeze drying, amorphous and β-sheet structures, pore radius and porosity ranging from 32.22
sponge
In vivo full-
Jo
SF/HA
μm to 290.76 μm and 74.1 % to 91.15 %, respectively,
thickness
good cyto-compatibility, improved fibroblast cell
wounds in rat
adhesion and proliferation compared to only SF,
[282]
accelerated vessel-like formation and wound healing compared to SF.
TOCN/SF nanofibrous mat
In vivo fullthickness excisional wound in rat
Improved mechanical and wound healing effectiveness compared to TOCN, good wound healing rate in TOCN/SF 2% compared to TOCN nanofibers.
[165]
Journal Pre-proof In vivo full
Potential antioxidant scaffold, better thermal and
Fenugreek-
thickness
biomechanical properties and cell proliferation in vitro
incorporate
excisional
compared to ESF, increased re-epithelialization,
d ESF mat
wounds in rat
collagen deposition and wound healing rate compared to
model
ESF. Encapsulation of insulin in SF microparticles by coaxial
InsulinIn vivo dorsal
ed SF
full-thickness
up to 28 days, increased migration of human keratinocyte and endothelial cells in vitro in insulin-
diabetic wound
[192]
functionalized SF scaffolds, accelerated wound closure,
ro
es-loaded
electrospraying technique, sustained release of insulin
of
functionaliz
microparticl
[168]
in rat
neo-vascularization and healing in insulin-SF scaffold
SF sponge
-p
compared to other experimental groups.
SF/Elastin protein
granulation tissue formation in both sponge and aqueous
lP
nt
In vivo fullthickness skin
forms, ability of wound exudate absorption and gel
wound in guinea formation after application on wound compared to
[151]
na
Recombina
re
Ability of self-assembly from liquid to gel, improved
pig
aqueous form, and better potential as wound dressing
encapsulate d liposome/S F hydrogel
SF solution
99.8 ± 0.5 nm diameter and −9.41 ± 0.10 mV zeta
In vivo deep
potential of bFGF-encapsulated liposome, sustained and
second-degree
controlled release of bFGF, stable and sustained release
scald wound in
of bFGF in wound fluid, accelerated wound healing
Jo
bFGF-
ur
compared to its aqueous form.
mouse
[191]
compared to SF hydrogel and bFGF-encapsulated liposome.
in vivo partial-
Increased viability and migration of NIH3T3 cells,
thickness
accelerated in vitro wound healing though NF-kB
excisional
signaling pathway and in vivo wound healing via NF-kB
wound in rat
regulated proteins.
[283]
Journal Pre-proof
Arg‐Gly‐ Asp motif-
thickness
containing
excisional
SF
Secretion by transgenic silkworm, accelerated migration
In vivo full-
and proliferation of NHDF compared to wild-type silkworm, good re-epithelialization, wound closure,
[187]
neovascularization, and wound healing compared to SF
wound in mouse
secreted by wild-type silkworm.
CTS/SF hydrogel
ZWP and
In vivo diabetic
collagen synthesis and wound healing in PRP-
of
loaded with
Good cyto-compatibility, higher neovascularization,
wound in rat
Exos/ZWP-loaded CTS/SF compared to PRP exosome or ZWP-loaded CTS/SF hydrogel sponge.
PRP
CTS/SF
ADSC
wound in rat
re
Promising delivery system for ADSCs, accelerated collagen synthesis, neovascularization and wound healing.
ur
seeded with
In vivo diabetic
[182]
wound healing rate compared to CMC gel.
Jo
scaffold
biocompatibility, and higher re-epithelialization and
wound in mouse
lP
hydrogel
Promising mechanical property, excellent
In vivo diabetic
na
conjugate
-p
exosomes SF-elastin
[171]
ro
sponge
[154]
Journal Pre-proof Decellularization and solubilization of placenta using SDS and urea buffer, respectively, preparation of different ratios of SF/pECM sponge, retained various In vivo full
cytokines, growth factors and ECM proteins after
SF/pECM
thickness
decellularization process and scaffold fabrication,
sponge
excisional
improved adhesion, proliferation and migration of HFFs,
wound in rat
HEKs and HAMSCs with favorable biocompatibility in
[152]
vitro and in vivo, and significant increase in re-
of
epithelialization, wound closure, neovascularization,
ro
wound healing compared to SF sponge.
Used SF from two different sources (mulberry and nonFunctionaliz
-p
mulberry), excellent in vitro biocompatibility for HDF In vivo diabetic
and HaCaT cells, increased cell migration after
EGF, bFGF
and not-diabetic
treatment with combination of EGF, bFGF and LL-37,
and LL-37
full-thickness
electrospun
excisional
lP
wound in rabbit
faster granulation, wound closure, organized ECM
[284]
formation, improved re-epithelialization and angiogenesis in functionalized non-mulberry SF/PVA
na
mat
re
ed PVA/SF/
mats compared to control, functionalized PVA and
layered artificial skin
In vivo 3rd burn
Jo
AM/ESF bi-
ur
functionalized PVA/mulberry SF.
wound on mouse
Decreased post-burn scar formation, accelerated neovascularization, wound closure and wound healing.
[69]
Journal Pre-proof ESF,
Fabrication by single-spinneret electrospinning
ESF/PVA,
technique, anti-adhesion property of the blends as
ESF/PEG,
In vivo trauma
following order: PVA/ESF> pure ESF> ESF/PEG
ESF/PEO
mouse model
~ESF/PEO, the highest collagen regeneration and
[167]
nonwoven
wound healing rate in ESF and PVA/ESF compared to
mat
others.
CTS/SF
ZWP and
In vivo diabetic
collagen synthesis and wound healing in PRP-
wound in rat
Exos/ZWP-loaded CTS/SF compared to PRP exosome or
ro
loaded with
Good cyto-compatibility, higher neovascularization, [171]
ZWP-loaded CTS/SF hydrogel sponge.
-p
sponge
of
hydrogel
PRP
re
exosomes
lP
Fabricated from two species of silks (AsSF and BmSF, ratio 1:1), irreversible gelation at 37 ⁰C due to β-sheet
degree burn wound in rat
structures formation, cyto-biocompatibility for primary HDF and improved migration of keratinocytes and re-
[184]
epithelialization in vitro, accelerated inflammation,
ur
SF hydrogel
In vivo third-
na
Injectable
Jo
proliferation and remodeling phases of wound healing
In vivo fullAnisotropic
thickness
ESF
excisional wound in rat
process in vivo. Fabrication by electrospinning, inducing anisotropic features by aligning the fibers in an electric field, improved
cyto-compatibility,
cell
migration
and
vascularization in vitro, accelerated wound closure and healing during 28 days follow up.
[174]
Journal Pre-proof Fabrication by freeze drying method, 92% porosity, 93 SF/HA/SA sponge
μm average pore diameter size, soft and elastic
In vivo full thickness burn
characteristics,
wound in rat
biocompatibility
good
physical
stability,
cyto-
for
NIH-3T3
fibroblast
cells,
[157]
accelerated burn wound healing during 21 follow up. Fabricated by freeze drying method, average pore size
Neurotensin
and porosity of 40–80 μm and ∼85%, respectively,
gelatin
excisional
microsphere
diabetic foot
s releasing
wound in rat
accelerated wound healing in diabetic foot ulcers through
of
In vivo
increased wound closure, fibroblast accumulation and tissue granulation at the wound site with minimal scar formation, suggest as drug delivery system and wound
SF sponge
-p
dressing.
Modification of acellular dermis by dip-coating at
goat
thickness
different concentrations of SF aqueous solutions,
acellular
excisional
synergetic effects of SF and acellular dermal matrix on
dermal
wound in albino
wound healing, improved wound healing in vivo
matrix
mice In vivo
lP
compared to un-modified matrix.
excisional
artificial
wound in rabbit
Jo
layered
skin
ear
[183]
na
AM/ESF bi-
re
In vivo Full-
ur
SF modified
[158]
ro
-loaded
Increased re-epithelialization, wound healing, decreased hypertrophic scar formation in the wounds treated with bilayer membrane compared to AM implanted wound.
[140]
Journal Pre-proof Table 1c: Clinical trials being carried out with SF or SF blends for skin tissue engineering.
Material (s)
Design of
Major findings
experiment (s)
References
Randomized Bilayered
trial on split-
Faster wound healing rate and less pain than Bactigras,
SF/Gelatin
thickness skin
a standard wound dressing.
[115]
of
graft donor site Roughness and transparency of SF film compared to Suprathel and Sidaiyi, two commercially available
thickness
wound dressing, as follows: Sidaiyi>Suprathel>SF
wound in
film and SF film>Suprathel> Sidaiyi, respectively,
-p
ro
In vivo full-
rabbit, porcine
re
waterproof, biocompatible, and effective barrier
and
against bacterial entrance, superior gas permeability
randomized
and fluid handling capacity than Suprathel and Sidaiyi,
single blind
SF film with thickness of 64.9 µm, thinner than
[121]
lP
SF film
na
clinical trial on Suprathel (266 µm) and Sidaiyi (1789 µm), better and 71 patients faster wound healing in both animal and clinical trial
Jo
ur
studies compared to Suprathel and Sidaiyi
Abbreviations used in the Table 1(a-c): A. assama silk fibroin (AaSF) ; Alginate dialdehyde (ADA); adipose tissue-derived mesenchymal stem cells (ADSCs); AM (amniotic membrane); Bacterial cellulose (BC); Bombyx mori (Bm); B. Mori silk fibroin (BMSF); N-carboxyethyl chitosan (CECTS); child foreskin fibroblasts (CFFs); carboxymethyl cellulose (CMC); Chondroitin sulfate (CS); Chitosan (CTS); 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC); epidermal growth factor (EGF); freeze-drying (FD); glutaraldehyde (GA); Immortalized human keratinocyte (HaCaT); human amniotic and membrane-derived stem cells (HAMSCs); Human dermal fibroblast (HDF); human epidermal keratinocytes (HEKs); human foreskin fibroblasts (HFFs); Human platelet lysate (hPL); N-(2-hydroxy)propyl-3-trimethyl ammonium chitosan chloride (HTCC); hyaluronic acid (HA); L-proline (LP); normal human epidermal fibroblasts (NHEF); normal human epidermal keratinocytes (NHEK); Neurotensin (NT); Sodium alginate (SA); salt-leaching (SL), spunlaced SF (SSF), Salt-leaching electrospinning (SLE),
Journal Pre-proof
Jo
ur
na
lP
re
-p
ro
of
strontium (Sr); oxidized cellulose (TOCN); human placenta-derived extracellular matrix (pECM); polyethylene glycol (PEG); polyethylene oxide (PEO); Polarized Hydroxyapatite (pHA); poly-Llactic acid (PLA); poly(lactide-co-glycolic acid) (PLGA); platelet-poor plasma (PPP); platelet-rich plasma (PRP); P. ricini silk fibroin (PRSF); poly(vinyl alcohol) (PVA); Homogeneous polysaccharide extracted from the rhizomes of Curcuma zedoaria (ZWP)
Journal Pre-proof
Tables 2 (a-b). In vitro and in vivo experimentations on antibacterial effects of SF or SF blend scaffolds prepared alone and/or adding with other bioactive ingredients.
Table 2a. In vitro experiments are being carried out on antibacterial effects of SF or SF blend scaffolds prepared alone and/or adding with other bioactive ingredients.
SF loaded with antibiotics
ro
ESF mat
[195]
Good cyto-compatibility and strong antibacterial activity against E. coli
[262]
-p
TiO2-loaded
High antibacterial activity against S. aureus and P. aeruginosa growth.
Controllable and sustained antibiotic release, good cyto-compatibility and strong antibacterial activity.
re
mat
References
[197]
lP
ESF/AgNPs
Major findings
of
Material (s)
na
Bionanotextile ESF incorporated with various concentrations of PEI PEI-loaded
(10, 20 and 30 % (w/w)), Further functionalization with sulphate
ESF mat
group, good cyto-compatibility for L929 fibroblasts cancer cell line,
[210]
ur
strong antibacterial activity against S. aureus and P. aeruginosa.
Jo
Fabrication of transgenic silkworm carrying SF-AMP conjugated acid Transgenic
nucleic sequences, secretion of SF-AMP proteins by transgenic
SF-AMP mat
silkworm, high antibacterial activity, low attachment property for bacteria, excellent biocompatibility, and intrinsic antibacterial activity.
[217]
Journal Pre-proof Coating of AgNPs on silk fibers by two in situ and ex situ methods, enhanced thermal and mechanical properties of silk fibers-AgNPs mat AgNPs-
in both fabrication methods, presence of the crystalline AgNPs on silk
loaded Silk
fibers, excellent biocompatibility of the mats fabricated by both
fibrous mat
methods for 3T3 fibroblasts, strong antibacterial activity against P.
[198]
aeruginosa and S. aureus, better antibacterial activity of the mats fabricated by in situ method compared to ex situ.
Vera mat
of
antibacterial agent, with silk fabric using 1, 2, 3, 4-
butanetetracarboxylic acid as cross-linker, good antibacterial behavior
[285]
ro
fabric/Aloe-
Chemical binding of various concentrations of Aloe-Vera, as an
in 15% Aloe-Vera/SF fabric.
-p
Silk
Immobilization of KR12 on ESF membrane, strong antibacterial activity against S. aureus, S. epidermidis, E. coli and P. aeruginosa,
nanofibrous
good biocompatibility for human dermal fibroblasts and keratinocytes
mat
in vitro, suppressed expression of TNF-α from LPS-induced murine
na
monocytes. ESF/Seleniu
Antibacterial activity against S. aureus and S. epidermidis, in vitro
nanoparticles
biocompatibility for human dermal fibroblasts.
Nanoparticles /ESF mat CTZ/SF/Gela tin nanofibrous mat
[204]
Jo
Selenium
ur
m
mat
[196]
lP
re
ESF/KR12
Excellent antibacterial activity against S. aureus, and cyto-compatible for human dermal fibroblast.
[201]
Increased average diameter of fibers with the increase of CTZ content, controllable and sustained release of CTZ, strong antibacterial activity against P. aeruginosa, and good biocompatibility.
[211]
Journal Pre-proof Colloidal Ag/Ciproflox
Gradual release of antibiotic and high antibacterial activity against S.
acin/ SF/SA
aureus and E. coli.
[147]
sponge Sr-loaded
High antibacterial activity, and excellent cyto-compatibility for
SF/SA film
fibroblasts.
hydrogel
methacrylate, excellent delivery system for controlled release of
of
loaded SF
Improved mechanical properties using 2-(N,N-dimethylamino) ethyl [226]
cefixime, high degree of water uptake, improved tensile strength and
ro
Cefixime-
[148]
elongation at break, high antibacterial activity.
-p
Decreased compressive modulus property with the increase of SF and
re
GS concentrations, increased water swelling and weight loss with addition of GS, accumulative release of GS in higher content of SF,
n sponge
excellent antibacterial activity against S. aureus, S. epidermidis, M.
[221]
lP
GS/SF/Gelati
except GS/SF.
na
luteus, B. cereus and P. aeruginosa, no cytotoxicity effects for NHDF
ur
Fabrication by freeze-drying method, high cyto-compatibility for HFFF2 cells, promising swelling and water uptake, good blood
NPs 3D
clotting, biodegradability and mechanical properties with high porosity,
sponge
and strong antimicrobial activity against E. coli, S. aureus, and
[223]
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chitin/SF/Ag
Candida albicans.
Fabrication by freeze-drying method, good cyto-compatibility for chitin/SF/TiO 2
3D sponge
HFFF2 cells, promising swelling and water uptake (93%), good blood clotting, biodegradability and mechanical properties with high porosity (>90%), and strong antimicrobial activity against E. coli, S. aureus, and Candida albicans.
[286]
Journal Pre-proof Fabrication of transgenic silkworm carrying SF-AMP conjugated acid Transgenic
nucleic sequences, secretion of SF-AMP proteins by transgenic
SF/AMP mat
silkworm, good intrinsic antibacterial activity against E. coli, excellent
[216]
biocompatibility. Excellent antibacterial activity against pathogen bacteria such as S. Ag2O-loaded
aureus and M. tuberculosis, high antibacterial activity against non-
SF spun mat
pathogen (E. coli) bacteria, good biocompatibility of the Ag2O-SF spun
[209]
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for 3T3 fibroblasts and excellent in vitro wound healing potential. Fabricated by electrospinning, antibacterial activity against P.
coated by
aeruginosa, S. epidermidis, S. aureus and E. coli, cyto-compatibility for
AgNPs
L929 skin fibroblast
scaffold
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A SF spongy layer covered by ESF loaded with silver sulfadiazine,
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based
[218]
antibacterial activity against S. aureus.
[219]
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Bilayered SF-
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Tasar ESF
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Fabricated by electrospinning, different concentrations of nano-Cu2O (0.5, 1.0, and 5.0 mg/mL), bead formation in higher concentration of nano-Cu2O, decreased uniformity. Showed increase hydrophilicity of
PEO–SF
mat morphology with the increase of nano-Cu2O concentrations,
electrospun
increased antibacterial activity of composite electrospun mat against E.
mat
coli and S. aureus bacteria with the increase of nano-Cu2O
[220]
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nano-Cu2O-
concentrations, no detectable cytotoxicity against pig iliac endothelial cells.
Table 2b. In vivo studies are being carried out on antibacterial effects of SF or SF blend scaffolds prepared alone and/or adding with other bioactive ingredients. Material (s)
Major findings
References
Journal Pre-proof
Sulfadiazin e silverloaded ESF mat
Final ESF mat containing various silver sulfadiazine contents (0.1, 0.5 and 1.0 wt %), decreased adhesion and spreading of human epidermal keratinocytes and epidermal fibroblasts with the increase of silver sulfadiazine concentration in mat and minimum cell adhesion and better wound healing in 1.0 wt % mat compared to 0.1 wt % and 0.5 wt%. Strong antibacterial activity against S. aureus, accelerated epidermal cell
AgNPs/SF
growth, excellent wound healing potential compared to a commercially
gel
available wound healing gel (soframycin gel).
ciprofloxaci
stable drug release with high antibacterial activity compared to unmodified film, and accelerated wound healing.
ciprofloxaci n mat
cells, strong antibacterial activity against E. coli, S. aureus, P, aeruginosa
ur
and
60% ciprofloxacin release within first 6 h, cyto-compatibility for HaCaT
and S. epidermidis and accelerated re-epithelialization, neovascularization
[214]
and wound healing.
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with EGF
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Non-
ESF loaded
[202]
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n sponge
mulberry
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Lower burst drug release compared to unmodified film, excellent and more
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loaded with
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PEG
SF/Gelatin
[203]
of
Gelatin
modified-
[207]
High porosity with good wettability, appropriate mechanical property, Wetable
strong antibacterial activity against S. aureus, E. coli, P. aeruginosa and M.
CTS/SF/Ag
albicans and good cyto-compatibility for L929 cells, human fibroblasts and
NPs sponge
hUCMSCs in vitro and high antibacterial activity in vivo and no post wound infection.
[142]
Journal Pre-proof Col/SF/Ag
Increased neovascularization in vivo, accelerated re-epithelialization and
NPs sponge
wound healing and no post-injury infections in wound bed or edge.
MoSe2/SF nanosheet film
Two dimentional nanosheet film, high antibacterial activity, and great potential as wound disinfectant and wound healing.
Thin film formation of CTS on SF fabric by spraying, connective pores of
SF/CTS
CTS thin film over the SF fabric, strong antibacterial activity, no
mat
cytotoxicity and high potential wound healing compared to TegadermTM.
[215]
LL37
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epithelium in SF/AgNPs solution containing 384 mg/L AgNPs.
Sustained delivery of LL-37, excellent antibacterial activity against S. epidermidis and P. aeruginosa, biocompatibility and wound healing
[284]
potential in vitro and in vivo.
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nanofibrous
[225]
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ESF/PVA/
activity in vitro, complete elimination of biofilms and repair of defected
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solution
Good for treatment of biofilm-associated diseases, strong antibacterial
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SF/AgNPs
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Abbreviations used for Tables 2 (a-b): Silver oxide nanoparticles (Ag2O-SF); silver nanoparticles (AgNPs); antimicrobial peptide (AMP); Bacillus cereus (B. cereus); Collagen (COL); chitosan (CTS); ceftazidime (CTZ); electrospun silk fibroin (ESF); gentamicin sulfate
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mat
[119]
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Bi-layered
[222]
(GS); human caucasian foetal foreskin fibroblast (HFFF2); human umbilical cord mesenchymal stem cells (hUCMSCs); Monilia albicans (M. albicans); Micrococcus luteus (M. luteus); Molybdenum diselenide (MoSe2); Mycobacterium tuberculosis (M. tuberculosis); normal human dermal fibroblasts (NHDF); Pseudomonas aeruginosa (P. aeruginosa); polyethylenimine (PEI); poly(ethylene oxide) (PEO); poly(vinyl alcohol) (PVA); Sodium alginate (SA); Staphylococcus aureus (S. aureus); Staphylococcus epidermidis (S. epidermidis,); Strontium (Sr).
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Mazaher Gholipourmalekabadi, Sunaina Sapru, Ali Samadikuchaksaraei, Rui L. Reis, David L. Kaplan, Subhas C. Kundu PII:
S0169-409X(19)30164-4
DOI:
https://doi.org/10.1016/j.addr.2019.09.003
Reference:
ADR 13504
To appear in:
Advanced Drug Delivery Reviews
Received date:
10 May 2019
Revised date:
12 September 2019
Accepted date:
26 September 2019
Please cite this article as: M. Gholipourmalekabadi, S. Sapru, A. Samadikuchaksaraei, et al., Silk fibroin for skin injury repair: Where do things stand?, Advanced Drug Delivery Reviews (2019), https://doi.org/10.1016/j.addr.2019.09.003
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof Silk fibroin for skin injury repair: where do things stand? Mazaher Gholipourmalekabadi* 1, 2, Sunaina Sapru 3, Ali Samadikuchaksaraei1,2, Rui L. Reis4, David L. Kaplan5, Subhas C. Kundu3,4* 1
Cellular and Molecular Research Centre, 2 Department of Tissue Engineering & Regenerative
Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran. Department of Biotechnology, Indian Institute of Technology Kharagpur, India
4
3Bs Research Group, I3Bs - Research Institute on Biomaterials, Biodegradable and Biomimetics.
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3
Department of Biomedical Engineering, Tufts University, Medford, MA, USA
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5
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Medicine, University of Minho, Guimaraes, Portugal.
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Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative
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*Corresponding authors:
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Dr. Mazaher Gholipourmalekabadi Tel: (+98 21) 8862 2755; Fax: (+98 21) 8862 2533 E-mail: [email protected]; [email protected]
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Professor Subhas C Kundu E-mail: [email protected]; [email protected] Tel: +351 253 510 932, Orcid ID: 0000-0002-7170-2291 E-mail of all authors: [email protected]; [email protected] [email protected] [email protected]; [email protected] [email protected] [email protected] [email protected]; [email protected] Number of tables: 5
Number of figures: 6
Running title: Silk fibroin in skin wound healing
Journal Pre-proof
Table of Contents Abstract ......................................................................................................................................3 Statement of significance ............................................................................................................4 Abbreviations used in this article .................................................................................................5 1. Introduction.............................................................................................................................7 2. Silk: types, sources and chemistry ......................................................................................... 12 3. Main characteristics of silk protein fibroin (SF) ..................................................................... 14
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(i) Mechanical properties (tensile and compressive) ............................................................... 14 (ii) Biocompatibility and biodegradation ................................................................................ 15
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(iii) Cell-silk fibroin interaction behavior ............................................................................... 17 4. Silk fibroin for skin tissue engineering .................................................................................. 18
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4.1. Skin structure and healing process .................................................................................. 18
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4.2. SF and SF blends in skin wound healing applications...................................................... 21 4.2.1. SF alone ................................................................................................................... 22
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4.2.2. SF blends ................................................................................................................. 29 5. Biofunctionalization .............................................................................................................. 38
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6. Antibacterial SF derived scaffolds ......................................................................................... 41 6.1. Antibacterial SF fibrous scaffolds ................................................................................... 42
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6.2. Antibacterial SF sponge .................................................................................................. 45 6.3. Antibacterial SF films ..................................................................................................... 47
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6.4. Antibacterial SF Gels ...................................................................................................... 48 7. Other biomedical applications of silk fibroins ........................................................................ 48 8. Benefits and concerns of using silks for biomedical applications ........................................... 49 9. Conclusions and future prospects .......................................................................................... 50 Acknowledgements ................................................................................................................... 52 Authors’ contributions............................................................................................................... 52 Conflict of interests ................................................................................................................... 52 References ................................................................................................................................ 53
Journal Pre-proof Abstract Several synthetic and natural materials are used in soft tissue engineering and regenerative medicine with varying degrees of success. Among them, silkworm silk protein fibroin, a naturally occurring protein-based biomaterial, exhibits many promising characteristics such as biocompatibility, controllable biodegradability, tunable mechanical properties, aqueous
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preparation, minimal inflammation in host tissue, low cost and ease of use. Silk fibroin is often used alone or in combination with other materials in various formats and is also a promising
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delivery system for bioactive compounds as part of such repair scenarios. These properties make
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silk fibroin an excellent biomaterial for skin tissue engineering and repair applications. This review
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focuses on the promising characteristics and recent advances in the use of silk fibroin for skin
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wound healing and/or soft-tissue repair applications. The benefits and limitations of silk fibroin as a scaffolding biomaterial in this context are also discussed.
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regenerative medicine
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Keywords: Silk fibroin; biomaterials; wound healing; skin substitutes; tissue engineering;
Journal Pre-proof Statement of significance Silk protein fibroin is a natural biomaterial with important biological and mechanical properties for soft tissue engineering applications. Silk fibroin is obtained from silkworms and can be purified using alkali or enzyme based degumming (removal of glue protein sericin) procedures. Fibroin is used alone or in combination with other materials in different scaffold forms, such as nanofibrous
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mats, hydrogels, sponges or films tailored for specific applications. The investigations carried out using silk fibroin or its blends in skin tissue engineering have increased dramatically in recent
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years due to the advantages of this unique biomaterial. This review focuses on the promising
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characteristics of silk fibroin for skin wound healing and/or soft-tissue repair applications.
Journal Pre-proof Abbreviations used in this article AaSF: Antheraea assama silk fibroin ADA: alginate dialdehyde
ADSCs: adipose tissuederived mesenchymal stem cells AM: amniotic membrane
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AMP: antimicrobial peptide
HTCC: N-(2-hydroxy)propyl3-trimethyl ammonium chitosan chloride hUCMSCs: human umbilical cord mesenchymal stem cells
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BC: bacterial cellulose
BMSCs: bone marrow mesenchymal stromal cells Bm: Bombyx mori BmSF: B. mori silk fibroin CECTS: N-carboxyethyl chitosan CFFs: child foreskin fibroblasts
pHA: polarized hydroxyapatite
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HFFF2: human caucasian foetal foreskin fibroblast hPL: human platelet lysate
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AgNPs: silver nanoparticles
Bm: Bombyx mori
PEO: polyethylene oxide
HDF: human dermal fibroblast HEKs: human epidermal keratinocytes HFFs: human foreskin fibroblasts
B. cereus: Bacillus cereus
PEI: polyethylenimine
PGS: poly(glycerol sebacate)
Ag2O-SF: silver oxide nanoparticles
bFGF: basic fibroblast growth factor
HaCaT: immortalized human keratinocyte HAMSCs: human amniotic and membrane-derived stem cells
PHBV: poly (3-hydroxybutyrate-co-3-hydroxyvalerate) PLA: poly-L-lactic acid PLGA: poly(lactide-coglycolic acid) PPP: platelet-poor plasma
PRP: platelet-rich plasma PrSF: P. ricini silk fibroin
HA: hyaluronic acid KGF: keratinocyte growth factor
PVA: poly(vinyl alcohol) SA: sodium alginate
LP: L-proline LPS: lipopolysaccharides
SL: salt-leaching
M. albicans: Monilia albicans SLE: salt-leaching electrospinning M. luteus: Micrococcus luteus Sr: strontium MoSe2: molybdenum diselenide
SSF: spunlaced SF
Journal Pre-proof CMC: carboxymethyl cellulose COL: collagen CS: chondroitin sulfate
mSF: SF microfibers
TOCN: oxidized cellulose
M. tuberculosis: Mycobacterium tuberculosis NHDF: normal human dermal fibroblasts
RGD: arginine, glycine and aspartate S. aureus: Staphylococcus aureus S. epidermidis: Staphylococcus epidermidis
CTS: chitosan NHEF: normal human epidermal fibroblasts
SIS: styrene-isoprene-styrene NHEK: normal human epidermal keratinocytes NT: neurotensin
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ECM: extra cellular matrix
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CTZ: ceftazidime
rBMSCs: rat bone marrow mesenchymal stem cells
EGF: epidermal growth factor ESF: electrospun silk fibroin
P. aeruginosa: Pseudomonas aeruginosa PBS: phosphate-buffered saline PDGF: platelet-derived growth factor
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pECM: human placentaderived extracellular matrix
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GA: glutaraldehyde
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FD: freeze-drying
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EDC: 1-ethyl-3-(3dimethylaminopropyl) carbodiimide
GS: gentamicin sulfate
PEG: polyethylene glycol
Sr: strontium TCH: tetracycline hydrochloride
TE: Tissue engineering TERM: tissue engineering and regenerative medicine VEGFα: vascular endothelial growth factor alpha ZWP: homogeneous polysaccharide extracted from the rhizomes of Curcuma zedoaria
Journal Pre-proof 1. Introduction Over the past decade, the limitations with organ transplantation due to the shortage of transplantable organs and the rejection of these organs has been a major issue. The tissue engineering and regenerative medicine (TERM) field has emerged to overcome such limitations for replacing/repairing damaged tissues and organs (Fig. 1). Tissue engineering (TE) consists of
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gathering cells of a patient or the use of acceptable donor cells, expanding the cells in vitro and then seeding these cells into 3-D scaffolds/matrices. The cells are then induced in the presence of
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appropriate medium and bioactive factors to proliferate and differentiate into a desired tissue for
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implantation. There are three main elements for successful tissue regeneration: (i) cells harvested
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from tissue, (ii) scaffolds that supply morphology, structural support, and chemical and mechanical
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appropriate tissue growth [1, 2].
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signals to the cells, and (iii) interactions between the cells and matrix (scaffold) to support
Figure. 1. Illustration of the basic routes of tissue engineering and regenerative medicine. The cells are harvested from patients by biopsy and are grown in 2D culture under aseptic conditions. The cell culture
Journal Pre-proof medium is supplemented with growth factors and other specific features as required by the individual cell type. These cells are sub-cultured for the expansion of cells and are seeded onto 3D matrices for the construction of specific 3D tissues. These matrices/polymeric scaffolds may be natural, synthetic or blends and are fabricated in various material format such as hydrogels, porous sponges, nanofibrous mats, micro/nano particles, micro-/nano patterned surfaces, among others. These matrices may contain bioactive molecules to augment the proliferation of cells. For long-term culture, the matrices are grown in bioreactors
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to support and maintain the cultures under aseptic conditions. The regenerated tissue in vitro may be
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transplanted to restore the damaged tissue in vivo.
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The scaffolds/matrices have a key role in tissue engineering and regenerative medicine. There are many essential characteristics that are rational starting points for designing scaffolds for TE [3, 4].
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First, the biocompatibility of materials is key to support appropriate cellular activities, improve
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tissue regeneration without local or systemic inflammation or immune responses in the host [3, 4]. Second, suitable structure and high surface area is required to allow maximum cell interactions
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while supporting oxygen/nutrient transport. Furthermore, this type of structure is desirable to
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provide space for tissue in-growth. In addition, biodegradability or absorption of the scaffold material is relevant, where the degradation rate of the scaffold should match the rate of tissue regeneration to improve the transfer of functions from the artificial matrices to the native extracellular matrix (ECM) on a time-dependent basis during tissue regeneration [1-6]. Thus, designing scaffolds, which mimic the structure and biological functions of native ECM and provides a suitable environment for regulating cell activities, is the main goal. To obtain such scaffolds, synthetic or natural polymer materials have been pursued to meet the above needs, each with their own benefits and limitations [4, 7].
Journal Pre-proof Although there is no universal biomaterial that meets scaffolding requirements for all types of tissues, proteins as constituent parts of natural tissues are reasonable candidates for applications in TE. Natural proteins such as collagens, elastin, keratins, elastin-like peptides, albumin, fibrin and silks have been used as tissue scaffolds [4, 8, 9]. The biocompatibility, controllable biodegradability, aqueous preparations, and minimal inflammatory response of silk proteins have attracted the attention of researchers toward this need [10-13]. SF is a high molecular weight (>300
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kDa depending on source) amphiphilic protein with repetitive, modular hydrophobic domains,
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which are surrounded by small hydrophilic regions. The natural form of silkworm silks consist of
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a core fiber, silk protein fibroin (SF), and glue-like coating composed of a family of sericin
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proteins. SF also has various secondary structures, including α-helix, β-sheet, and crossed β-sheet. SF fibers can be obtained from the cocoon of the silkworm, Bombyx mori (Bm), which has a
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continuous strand of two fibers, to form the filament providing the unique physical and chemical
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properties [10, 12, 14-16].
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SF-based biomaterials demonstrate excellent biocompatibility [10, 12, 13, 17, 18]. Moreover, the unique structural assembly of this protein provides remarkable mechanical properties when
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compared with other commonly used biopolymer-based biomaterials. The degradation rate in vivo can be controlled from weeks to years after implantation in vivo, depending on crystallization, scaffold form, cross-linking agent, blending and other factors [19-24]. Hence, the silk has been utilized in various applications in TE, such as bone, cartilage, nerve, skin and others (Fig. 2).
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Figure 2. Different applications of silk protein fibroin in tissue engineering and regenerative
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medicine. Silk provides flexibility to fabricate different kinds of matrices and thereby, finds use in multiple biomedical applications including tissue engineering targets like the regeneration and repair of damaged
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tissues. Apart from reconstructing injured areas, silk matrices also effectively deliver bioactive molecules
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including drugs and growth factors to the target site, as well as genes and cells, further enhancing utility in regenerative medicine.
Skin plays a vital role in protecting the human body against the environment, dehydration, and infectious agents. Burns, wounds, or disease may cause the loss of skin integrity and tissueengineered skin substitutes provide an opportunity to restore skin loss [25, 26]. Many natural and synthetic skin substitutes are used for skin wound healing but remain limited in clinical effectiveness in many cases [27, 28]. Natural-based skin substitutes such as autografts, acellular
Journal Pre-proof dermal matrix allograft (alloderm®) and Xenografts (EZ Derm®), natural macromolecules(collagen, silk, cellulose, hyaluronic acid and others) derived scaffolds have several excellent characteristics including biocompatibility and biodegradability with positive impact on wound healing. The limited availability, risk of cross-contamination and cost are disadvantages associated with natural based skin substitutes, which limit clinical applications i [29-32]. Synthetic-based skin substitutes such as synthetic polymer sheets (Tegaderm®, Opsite®), polymer foams or sprays and
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others are available and inexpensive with no risk of infection transmission. However, slow
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degradation, controversial biocompatibility in some cases, and lower impact on wound healing
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compared with natural materials limit applications [29, 33]. Nevertheless, many natural and
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synthetic skin substitutes are already used as skin substitutes with various ranges of success [29,
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31, 34].
Recently Farokhi et al. [13] described the status of SF based wound dressing materials for skin
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regeneration and our goal here is to extend the understanding of silk in this context. The review
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focuses on the promising characteristics of SF for skin wound healing and/or soft-tissue repair and recent SF blend skin substitutes. Based on recent advances and developments with SF-based
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scaffolds, this review addresses the state-of-the-art of SF-derived skin substitutes with additional background, structure of the materials, fabrication procedures/processing, merits and limitations, functional stimuli-response features, functionalized (e.g., antibacterial) bioactive matrices for skin tissue regeneration and would healing. In vitro (Table 1a), in vivo (Table 1b) and clinical trials (Table 1c) carried out between 2000 and 2019 with different types of SF or SF blends, and the major findings and appropriate references, are summarized.
Journal Pre-proof 2. Silk: types, sources and chemistry Silk is secreted by various animals such as silkworms, spiders, scorpions, mites and bees best known as (arthropods) [10, 12, 35] (Fig. 3). The properties of silkworm silk mainly depend on the source of the material. For example, the silk extracted from the cocoons of Bm, which is also known as mulberry silk, is the most extensively characterized and utilized for biomedical
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applications. The silkworms from the family of Saturniidae are known as non-mulberry silks and there are several species in this category namely Antheraea mylitta (tasar), A. assama (A.
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assamensis, muga), A. pernyi, Samia ricini (Philosamia ricini, eri) and others. There are over
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30,000 species of spiders, for example Nephila clavipes and Araneus diadematus, which produce
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silks, with outstanding mechanical properties and lack of sericin protein. However, these silks are
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heterogeneous in nature and to obtain sufficient quantities is a major hurdle and expensive in comparison to silkworm silk. Thus, silk-based biomaterials are usually prepared from silkworm
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silk. The isolation and purification of silk fibers is relatively simple by using alkali or enzyme
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based degumming procedures, with subsequent utility in TE [10, 12, 13, 15, 18, 21, 36, 37].
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Silk is a lightweight (1.3 g/cm3), high strength (up to 4.8 GPa as the strongest fiber known in nature), protein with repetitive modular hydrophobic domains and small hydrophilic groups [35, 38]. The N- and C- termini of SF are highly conserved. The silkworm silk from Bm has two main parts, the fiber core protein, SF and the surrounding sericin proteins. The repetitive hydrophobic sequences are composed of short side-chain amino acids such as glycine and alanine. The hydrophilic blocks are comprised of larger side-chain amino acids such as charged amino acids. The structure of SF depends on the species/genera. For example, the SF of B mori contains a heavy (H) and light (L) chain that connect with a disulfide bond. The H chain has hydrophobic domains such as Gly-X (X being Ala, Ser, Thr, Val) repeats and can create anti-parallel β-sheets.
Journal Pre-proof Additionally, a glycoprotein called P25 is linked to the light and heavy fibroin chains with noncovalent bonds. In contrast, the P25 chain does not exist in non-mulberry silks. Instead, the heavy (H) chain homo-dimer has a main role in the formation of non-mulberry silks [16]. There are differences between mulberry and non-mulberry silks in terms of mechanical properties,
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bioactivity and the degradation behavior [10, 12, 16, 35, 37, 39-43].
Figure. 3. Arrays of silk protein fibroin matrices for tissue engineering and regenerative medicine. Silk as a biomaterial is often obtained from both mulberry and non-mulberry (shown above are some of the main cultivated silkworm species) silkworm silks. Depending on the silkworm species and availability, silk proteins may be isolated from the cocoons after degumming (removal of silk protein sericin) or from the mature 5th instar larvae (mainly from non-mulberry). Silk-based scaffolds are developed in various forms, which range from thin membranes/coatings to 3-D bio printed matrices to support organ specific requirements.
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3. Main characteristics of silk protein fibroin (i) Mechanical properties (tensile and compressive) The biomechanical properties of TE scaffolds have direct implications on their function during tissue regeneration [4, 44]. The biomechanical behavior of biomaterials has a critical impact on
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regulating cell growth, morphology, differentiation, migration and function [45-47]. Cells can
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sense matrix mechanics via receptors and subsequently transform mechanical stimulation to chemical signals [48-50]. For example, mesenchymal stem cell lineage differentiation can be
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affected by matrix stiffness (0.1–1 kPa, 8–17 kPa and 25–40 kPa) in vitro [51]. The mechanical
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behavior of extracellular matrices can also stimulate/repress the secretion of some factors and
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define the stem cell differentiation. The mechanical properties of tissues/organs can undergo changes during normal and pathologic situations [52, 53]. A promising TE scaffold should
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simulate the mechanical behavior of a normal tissue to accelerate tissue regeneration and lead to
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new functional tissue. SF offers a balance of breaking strength, modulus and elongation, which
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make it attractive for tissue engineering applications [10, 12, 17, 54]. The toughness of SF is several times higher than Kevlar [55], and the strength-to-density ratio of native silk is higher than steel (up to ten times) [56]. Silk fibers from different sources have different mechanical properties, an advantage for the development of scaffolds for different tissue needs [10, 54]. Silk fibers isolated from different sources generally have tensile strength, tensile modulus, and breaking strain ranging from 0.9±0.2 to 5±1.2 (g/den), 29–31 to 84–121 (g/den) and 4.1±2.7% to 28.0–34.0%, respectively [10].
Journal Pre-proof The secondary and hierarchical structures of SF directly affect the mechanical properties. The βsheet structure of SF has a critical role in stiffness. In comparison to native silk fibers, scaffolds fabricated from regenerated SF aqueous solution have weaker mechanical properties [54, 57-61]. For example, native SF fibers have a tensile strength and elongation at break about 0.5-0.6 GPa and 10-40%, respectively. The characteristics for dry silk films made from regenerated SF aqueous solution are about 0.02 GPa and 2% [57, 58]. Embedding regenerated silk products in β-sheet
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structures, induced by methanol or ethanol is an option to improve the strength of SF-based
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scaffolds [62-64]. Water-soluble silk films immersed in methanol for 10 min and 60 min had an
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elastic modulus of about 40 MPa and 80 MPa, respectively [65], demonstrating the role of β-sheets
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in the mechanical behavior of SF-based scaffolds.
(ii) Biocompatibility and biodegradation
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Silk sutures have been used since the end of the 19th century in medicine as a safe and
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biocompatible material [41]. Biocompatibility of silk-based scaffold depends on the extraction and
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purification procedures used, and degummed SF is immunologically inert and evokes a minimal inflammatory responses in vitro or in vivo. Sericin shows minimal inflammatory responses as well, and is often considered a biocompatible biomaterial [10, 12, 17, 36, 59, 66-71]. However, the combination of the two proteins, fibroin and sericin results in inflammation. The biocompatibility of SF-based scaffolds has been studied both in vitro and in vivo. The biocompatibility of SF nanofibrous scaffolds when subcutaneously implanted in BALB/c mice for 1 and 4 weeks was demonstrated [59]. An increase in macrophage infiltration into the implanted site of 3D SF-nanohydroxyapatite nanocomposite scaffolds at day 7 and 28 post-implantation in
Journal Pre-proof rat was also reported, while no significant increase of lymphocytes was found when compared to controls (no implants) [72]. SF films have evoked a host immune response at a similar level as collagen-based scaffolds [11]. SF has FDA approval for biomedical applications and some of the silk-based products are already used in the market. In addition, silk-based surgical meshes passed biocompatibility and safety testing for ISO 10993, indicating that this product meets the criteria required for use in medicine [23]. These results suggest good biocompatibility of fully degummed
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SF for TE uses. Subcutaneous implantation of 3D SF scaffolds in rat showed a mild inflammatory
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response after one year post-implantation [72].
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A TE scaffold should degrade at the same speed as new tissue is regenerated/repaired and have
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non-toxic and safe degradation byproducts [73]. Antigenicity of the scaffold degradation
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byproducts is another important concern for the use of SF [10, 12, 17]. Further examination is warranted to understand the low to no cytotoxicity and the reason for the good biocompatibility of
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the SF and its degraded products in long-term period. This insight should be considered in the
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design of biomaterials in general.
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SF is degraded in vitro and in vivo, with the rate depend on the material structure and environment [20, 21, 74-79] [21, 22, 72]. The extraction method, SF source, secondary structure and SF-based scaffold form can affect the biodegradation rate [20, 21, 78, 79]. SF with higher β-sheet content shows slower degradation [80, 81]. Spider silk degrade faster than SF extracted from Bm at least in one study [82]. Many factors, such as β-sheet content, scaffold type, type of silk (virgin or processed), and solvent used for extraction influenced these outcomes [83-85]. Scaffolds made from a regenerated aqueous solution of SF degraded faster than the native degummed SF fibers [77]. In vivo higher concentrations of SF and smaller pore sizes resulted in decreased in vivo degradation [21, 83]. Additionally, 3D SF scaffolds prepared from aqueous SF solution showed
Journal Pre-proof complete in vivo degradation between 2-6 months, while those prepared from organic solvent remained in the implanted site for more than 1 year [19]. The porosity of the 3D SF scaffolds affects degradation rate, higher porosity showed a higher degradation rate [72].
(iii) Cell-silk fibroin interaction behavior
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Cell-ECM interactions control cell proliferation, migration, differentiation and function [86].
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Cells without the ability to attach to surfaces undergo apoptosis [87]. Cells can sense their environment and anchor to matrix components through integrins. Collagen (mediates tensile
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strength of ECM), fibronectin (mediates cell-matrix adhesion) and laminin (organization of basal
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lamina) are the most common components in tissue ECMs. Indeed, there are some sequence
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domains in integrins that bind to specific motifs in collagen, laminin and fibronectin [7, 88]. Cell adhesion properties of TE scaffolds as well as the structure of scaffolds, including porosity, pore
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architecture and size are critical, as these features affect migration, proliferation and differentiation
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of cells. The cells cultured in SF scaffolds with 100-300 µm pores showed enhanced cell growth,
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differentiation and extracellular matrix production than other pore sizes studied. In this regard, Zhang et al. [89], fabricated SF sponges with different ranges of pore sizes (50 to 300 µm) by freeze drying. The BMSCs transfected with BMP7 preferred SF sponges with pore sizes ranging from 100-300 µm with better proliferation, ECM production, osteogenic differentiation and in vivo bone healing. SF provided good attachment sites for a variety of cell types such as fibroblasts and keratinocytes [90], human adipose tissue-derived mesenchymal stem cells (ADSCs) [91], rat bone marrow mesenchymal stem cells (rBMSCs) [59, 72], human osteoblast cell line G 292 [92], human bone marrow mesenchymal stromal cells (BMSCs) [89] and many others. In addition, the presence of arginine, glycine and aspartate (RGD) motifs as cell-binding sites in non-mulberry SF provided
Journal Pre-proof improved cell adhesion when compared to mulberry SF where there is an absence of these sites [16, 39, 43, 93].
4. Silk fibroin for skin tissue engineering 4.1. Skin structure and healing process
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Skin has a vital role in fluid hemostasis, sensation, thermoregulation and other physiological
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functions [25, 26]. Skin is composed of the epidermis and dermis. The epidermis, the outermost
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layer of skin is water resistant and responsible for body liquid evaporation and protection against
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pathogens. The epidermis consists of a thin layer of keratinocytes and is separated from the dermis by a basement membrane. The dermis is the thick layer of skin, which contains connective tissue
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with fibroblasts, hair follicles, sweat glands, blood vessels and extracellular matrix contents such
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as collagen, elastin, and glucoseaminoglycans (GAGs). The dermis layer endows tensile strength
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and elasticity to the skin [25, 26, 94].
Skin injuries may arise from mechanical trauma, burns, diseases and surgical procedures. Skin
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wound healing is a complex process of cellular and molecular events divided into four overlapping phases of hemostasis, inflammation, proliferation and remodeling. The cytokines and growth factors secreted from the cells in the wound bed orchestrate the wound healing process [26, 95]. Upon injury, the damaged vessels are closed by blood clots (hemostasis phase), phagocytes such as neutrophils and macrophages are recruited into the wound site to clear out the dead cells/microorganisms and also secrete growth factors to mediate the proliferation phase (inflammatory phase).
Journal Pre-proof The cells (i.e., fibroblasts, mesenchymal stem cells, endothelial progenitors, and keratinocytes) proliferate, differentiate and migrate to close the wound bed (re-epithelialization), restore vascular networks and form granulation tissue. The loss of contact inhibition and physical tension in keratinocytes triggers signaling pathways mediated by desmosomes and hemi-desmosomes, and subsequently cytoskeleton reorganization, migration and proliferation of keratinocytes, especially from basal layers of the wound edges. Beside keratinocytes, epithelia stem cells from sweat glands
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or nearby hair follicles contribute to the re-epithelialization. Keratinocytes also secrete a variety
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of growth factors and cytokines such as EGF, KGF, VEGF, TGFβ and others, which mediate the
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wound healing process. Fibroblasts grow and secrete collagen (mostly collagen type III) at the
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early phases of proliferation to produce new extracellular matrix at the wound site. Collagen type
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III is synthesized and deposited quickly in the wound bed to form new ECM [26, 96]. In the remodeling phase, the cells that are no longer needed are eliminated from the wound bed
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and the extracellular matrix is remodeled to form the natural structure of the skin. During this
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phase, matrix-remodeling enzymes such as matrix metalloproteinases replace collagen type III with collagen type I. Collagen type I has high tensile strength compared to collagen III. This is
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synthesized and is deposited slowly. A decrease in the number of new vessels is observed in remodeling phases. [26, 95, 97]. The events occur in different phases of skin wound healing processes during the first 30 days post-injury (Figure 4). Acute skin wounds including burns, chemical and surgical injuries heal within 8-12 weeks. Skin wounds are classified into superficial, partial thickness, full-thickness and complex. In superficial skin wounds, only the epidermis is affected. In partial-thickness, all or a portion of the dermis remains intact. In full-thickness wounds, the entire dermis, including sweat glands and hair follicles are lost. In complex wounds, the epidermis, dermis and subcutaneous tissue are damaged.
Journal Pre-proof Spontaneous healing of acute wounds depends on which layer of the skin is affected [5, 95, 98]. In contrast to acute wounds, chronic wounds such as diabetic wounds, infected wounds and bedsores fail to heal fast enough (need more than 12 weeks) and often reoccur. In chronic wounds, the delayed healing and recurrence affect the underlying layers, such as muscles, cartilage and bone and may lead to tissue loss [98, 99]. In general, any phenomenon which postpones the “transition from inflammation to proliferation phase”, such as diabetes, systemic inflammatory
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disease, infections and others can lead to chronic wounds [96, 100]. Immune cells (mostly
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neutrophils and macrophages) secrete a large amounts of pro-inflammatory cytokines (e.g., IL-1,
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IL-6, TNF-α) which induce the expression of matrix metalloproteinases and reduce tissue
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inhibitors of metalloproteinases, leading to formation of a highly proteolytic microenvironment in the wound bed [101, 102]. The cell membranes and structural proteins of the newly formed ECM
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are also damaged by ROS produced by neutrophils [103]. Chronic and full-thickness skin injuries
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require skin grafting to support complete healing [104]. Although several skin substitutes are already commercialized, restoration of non-healing skin wounds remains a major challenge [26,
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28, 105]. As described earlier, SF has many promising mechanical and biological properties for
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skin wound management [13]. The number of research publications in vitro, in vivo and clinical trials using SF and its blends are increasing and a summary of recent research in skin tissue engineering since 2000 are presented in Tables 1 a-c.
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Figure 4. The events of four overlapping phases of normal wound healing process during the first 30 days of post-wounding.
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4.2. SF and SF blends in skin wound healing applications
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SF accelerates skin wound healing through the NF-kB signaling pathway [106]. NF-ĸB regulates various cell behavior such as adhesion, proliferation, inflammation, and elimination of reactive oxygen species via complex signaling pathways. Therefore, NF-ĸB signaling is considered critical in healing processes of various wounds such as scratch injuries [99], corneal epithelial wounds [107], and dermal injuries [106, 108]. Park and his coworkers showed the up-regulation of NF-kB in NIH3T3 cells after exposure to SF [106]. This resulted in enhanced cell proliferation and growth as compared to controls. Their microarray data indicated the increased expression of Toll-like receptors (TLRs) and tumor necrosis factor receptor (TNFR), as two important mediators of NFkB in the SF-induced cells. They also determined the changes of expression of a variety of proteins,
Journal Pre-proof which mediate normal wound healing process and affected by NF-kB such as TGF, EGF, IL-1b, IL-10, vimentin, cyclin D1, fibronectin, VEGF and others s. For example, they showed increased expression of vimentin, cyclin D1, fibronectin, VEGF in cells induced with SF compared to controls. In addition, inhibition of NF-kB signaling in NIH3T3 cells by Bay 11-7082, a pharmacological inhibitor of κB kinase, significantly reduced the expression of vimentin, cyclin D1, fibronectin and VEGF; however, it did not affect the expression of EGF and TGF. Taken
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together, Park et al. concluded that SF accelerated wound healing by modulating the expression of
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proteins responsible for proliferation and remodeling phases of the process by activating the NF-
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kB signaling pathway [106].
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SF materials are used alone or in combination with other materials for skin wound healing
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applications. SF is also bio-functionalized to improve its characteristics for wound healing applications. SF alone is used in the forms of film [109-111] and nanofibrous scaffolds [112, 113]
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and other materials for blends with SF include chitosan (CTS), alginate, carboxymethyl cellulose
4.2.1. Pure SF
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(CMC), gelatin and antibacterial agents like heavy metals and antibiotics [109, 114-119].
4.2.1.1. SF film SF films showed to have superior healing potential compared with conventional hydrocolloids (Duro Active), and similar or better healing compared to lyophilize porcine acellular dermis (Alloaks D) for full-thickness incisional wounds in mice. The full-thickness skin wounds dressed with SF films showed less neutrophil-lymphocyte infiltration (inflammation) and higher collagen regeneration, re-epithelialization and faster healing when compared with Duro Active [120].
Journal Pre-proof Zhang et al. [121] developed a transparent SF film and compared its translational potential to two commercially available wound dressings: Suprathel (PolyMedics Innovations GmbH, Germany) made from polylactide, trimethylene carbonate, and ε-caprolactone; and Sidaiyi (Suzhou Soho Biomaterial Science and Technology Co., Ltd) as a two layer product (silk sponge and silicon layers). A schematic of the fabrication process is shown in Fig. 5a. Roughness and transparency of SF films compared to the two control materials were Sidaiyi>Suprathel>SF film and SF
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film>Suprathel> Sidaiyi, respectively, as SF showed the most transparency and smoothness [121].
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All three materials were waterproof, biocompatible, and effective barriers against bacterial
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entrance. SF films revealed superior gas permeability and fluid handling capacity than Suprathel
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and Sidaiyi. SF films with a thickness of 64.9 µm were much thinner than Suprathel (266 µm) and Sidaiyi (1,789 µm) (Fig. 5b). The wound healing potential of SF films versus Suprathel and Sidaiyi
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was investigated in full-thickness skin wounds in small (rabbit) and large (porcine) (Fig. 5c)
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animals, as well as a randomized single blind clinical trial on 71 patients. Both animal and clinical trial studies showed better and faster wound healing in the SF group compared to the two
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commercial materials [121].
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Figure. 5 (a-e). Silk fibroin film: from fabrication to animal and randomized clinical trial studies. (a) Schematic of the fabrication of transparent silk fibroin film. (b) Silk fibroin film with a thickness of 64.9 µm was much thinner than Suprathel (266 µm) and Sidaiyi (1789 µm). (c) Silk fibroin film exhibited better and faster wound healing compared to Suprathel and Sidaiyi during 90 days in porcine model [121]. (d) Improved wound closure in the wounds treated with SF film compared to Mapitel and the wounds with no treatment after 5, 10, and 15 days [122], (e) Hand burn wounds after 1 month treatment with Biobrane and Dressilk [123].
Journal Pre-proof SF films surface modified with NaOH to form nanofeatured SF membrane for wound dressing applications exhibited enhanced nanotopography, wettability and cellular functions compared to untreated SF scaffolds [111]. Different concentrations of pectin and glycerol were added to SF films to fabricate feeder layers for ADSCs. Pectin and glycerol were used to induce conformational transitions and flexibility to
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SF films, respectively. A formulation of ~94% w/w SF with 6%w/w pectin and ~88% w/w SF 12%w/w glycerol showed superior conformational stability, mechanical properties, respectively,
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and delivery systems for ADSCs [110]. Further investigations were performed to improve the
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flexibility and mechanical behavior of SF films [124, 125]. Various concentrations of dextrose (0-
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15%w/w) as plasticizer were incorporated into SF films to create flexible SF films, with higher
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elongation at break, water holding capacity, surface roughness, water absorbance capacity, hydrophilicity, and better cell proliferation compared to SF films alone [124, 125].
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In another study, silk cocoon sol-gel films were fabricated by immersing silk cocoons in CaCl2-
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ethanol-H2O solutions for different times. Immersion for 90 min supported biocompatibility,
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antibacterial activity, transparency and good swelling with significantly accelerated wound healing rates compared to standard Mepitel® dressings when the films were applied on full-thickness wounds in New Zealand white rabbits (Fig. 5d) [122]. Schiefer et al. [123] compared the effectiveness of Dressilk (PREVOR, France), a commercially available dressing made from silkworm silk to Biobrane (Smith and Nephew ltd, Hull, UK), a silicon based wound dressing coated with nylon mesh and porcine dermal collagen-derived peptides, for burn wound healing patients. Both Dressilk and Biobrane provided safe and effective microenvironments for burn wound healing, with Dressilk being the most cost effective (Fig. 5e) [123].
Journal Pre-proof SF scaffold with amorphous structure had promising characteristics for soft tissue engineering. To provide a tunable amorphous structure and mechanical properties, SF aqueous solutions were freeze dried at -20C to -5C. The freeze dried SF scaffolds at temperature >-9C were water insoluble with crystalline structure, while SF solution freeze dried at -20C were water soluble with minimal crystallinity. This technique is suggested for tuning mechanical and biological
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properties of SF scaffolds to meet criteria required for soft tissue engineering [126].
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4.2.1.2. SF nanofibrous scaffolds
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SF nanofibrous scaffolds are an interesting form of SF derived skin substitutes. Electrospinning
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provides a pseudo 3D structure for cell growth and attachment [59, 68, 112, 113, 127-130]. The
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concentration of SF in the spinning solution has a direct influence on the mechanical and biological characteristics of the electrospun silk fibroin (ESF) mat. ESF scaffolds fabricated from higher
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w/v% SF concentrations exhibited higher nanofiber diameters, improved mechanical properties
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and lower porosity; affecting the biological behavior of cells [59, 62, 128-131]. ESF with smaller diameter nanofibers had better influence on dermal cell proliferation and extracellular matrix
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formation in vitro and in an ex vivo wound model [132]. It is critical to optimize the fabrication process to achieve ESF scaffolds with biomechanical characteristics close to natural skin. Small pore sizes and sheet-like surfaces of ESF mats limit cell infiltration into the nanofibers. Therefore, salt leaching and cold plate electrospinning were introduced to fabricate ESF mats with higher pore sizes than the ESF scaffolds fabricated by traditional electrospinning. Sheikh et al. [133] showed that ESF scaffolds fabricated by cold-plate electrospinning had higher porosity and pore sizes with controllable mat thickness when compared to salt-leaching (SLE) and traditional electrospinning (Fig. 6a). The same research team also compared the characteristics of SF
Journal Pre-proof scaffolds fabricated by freeze-drying (FD), salt-leaching (SL), and SLE [112]. The SLE showed the most homogenous pore distribution, highest porosity (70%), water uptake and swelling ratio compared to FD and SL. Compressive strength in SL was significantly higher than the other methods due to the lower porosity. FD and SLE showed similar compressive strength. All three methods supported NIH 3T3 fibroblast growth and proliferation. The authors concluded that the ESF fabricated by SLE methods had more favorable biomechanical and biological characteristics
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to meet requirements for use in skin tissue engineering [112].
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The effectiveness of SLE SF nanomatrix on the healing of a burn model in rats was evaluated and
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compared to traditional electrospun nanosheets and Medifoam®, a commercially available
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polyurethane hydrocellular dressing foam, Genewel, Korea). SLE SF nanomatrix showed
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accelerated re-epithelialization and wound closure, organized collagen formation and deposition, and reduced inflammatory cytokine and healing time compared to electrospun nanosheets and
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Medifoam® [113]. The superior potential of SLE SF nanomatrix than Matriderm® in healing full
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thickness skin defects was shown by Lee et al. [127]. 3D SF scaffolds with micro-nano fibrous structures were also fabricated by a facile two-step freeze-drying technology to mimic the
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extracellular microenvironment of natural skin. The authors claimed that combinations of micronano fibrous structure in 3D SF scaffolds positively affect dermal cell viability, proliferation, migration and in vivo healing of dorsal full-thickness wounds in rats [134]. Yu et al. [135], found that both electrospun CTS/collagen and CTS/SF showed promising biological and mechanical properties for fibroblasts and keratinocytes in vitro, and full thickness skin wound healing in rats, when compared to a gauze dressing. Some investigations were performed for the fabrication of skin wound dressings using silkworm cocoons [122, 136]. Yu et al. [136] developed an artificial skin with permeation and mechanical
Journal Pre-proof properties close to natural skin, which was demonstrated by treating the silkworm cocoons with different degumming process times (0-100 min) (Fig. 6b). The artificial skins formed from the cocoons still need further in vitro and in vivo experimentation to determine their potential
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applications in wound management.
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Figure 6 (a-d). Silk fibroin fibrous scaffolds for skin wound healing (a) Electrospun silk fibroin scaffolds fabricated by cold-plate electrospinning methods (CPE) had higher porosity and pore sizes with
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controllable mat thickness and easy shaping compared to salt-leaching (SLE) and traditional
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electrospinning techniques (TE) [133]. (b) Silk fibrous mats were fabricated by treating the silkworm cocoons with various degumming process times (0-100 min). SEM micrographs showed the effects of degumming time on morphology and porosity [136]. (c) Electrospun silk fibroin (ESF) on amniotic membranes (AM), as well as cross sectional views of AM/ESF bilayer membranes, indicated successful the coating of AM with ESF [59]. (d) ESF/AM alone or seeded with adipose tissue-derived mesenchymal stem cells (ADSCs) exhibited better wound healing compared to AM and AM/MSCs in a mouse burn model [69].
Autologous platelet-rich plasma (PRP) or platelet-poor plasma (PPP)-immersed SF cocoon mats also have good biocompatibility, support cell growth, and accelerated wound healing when
Journal Pre-proof compared to PPP-SF and control (mats); the PRP-SF mats exhibited higher hydrophilicity compared to the cocoon and PPP-SF mats [137]. Muga fibroin electrospun mats supported L929 fibroblast growth and the delivery of gentamycin sulfate when compared to muga cast films [138]. Darshan et al. [139] fabricated Antheraea mylitta silk mats by degumming sericin from the cocoons and used repeated washing with sterile phosphate-buffered saline (PBS) for wound healing applications. The homogenous morphology of fibroin fibers, favorable mechanical
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properties and biocompatibility for epidermal cell primary culture in vitro was reported.
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Uniform nanofibrous SF mats were fabricated with average nanofiber diameters of 90 ± 0.021 nm
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and demonstrated biocompatibility for bone marrow derived-stromal cells [59], as were 3D bilayer
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skin substitutes made from electrospun SF layer (ESF) coated on decellularized amniotic
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membranes (AM) (Fig. 6c). The fabricated bilayer membranes improved mechanical properties and degradation rates compared to ESF and AM alone. ESF/AM artificial skin increased the
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secretion of pro-angiogenic factors (VEGFα and bFGF) from ADSCs in vitro. The biomechanical
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and biological results suggested ESF/AM artificial skin as a suitable skin substitute in various skin wounds, especially burns [68]. The potential of the ESF/AM bilayer membranes for treatment of
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scarless healing in a preclinical 3rd degree burn model in mice was demonstrated, with the best results in burn wounds implanted with bilayer membranes alone or in combination with ADSCs (Fig. 6d) [69]. The anti-hypertrophic scar property of this bilayer membrane was also proved in a rabbit ear model excisional wounds [140]. 4.2.2. SF blends SF has been used alone in the fabrication of skin substitutes, but it is preferred to blend SF with other materials to improve biological properties. SF blends have superior wound healing potential compared to SF alone [114, 116, 141]. The composition of SF with CTS in various form has a
Journal Pre-proof significant positive impact on biomechanical and biological properties of CTS/SF scaffolds. SF blends for wound healing applications were fabricated in fiber, film, sponge and other formats [116, 141-144]. 4.2.2.1. SF blend film/sponge SF blends in the form of films and sponges are useful for wound healing. Excisional wounds implanted with poly vinyl alcohol/CTS/silk fibroin (PVA/CTS/SF) healed faster than those
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implanted with CTS/SF, SF, PVA/CTS and CTS; SF sponges alone exhibited superior healing
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potential compared to CTS sponges [145]. Spunlaced SF (SSF) scaffolds were coated with a thin
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layer of CTS by hydrogen-bonding assembly to fabricate CTS/SSF 3D blend scaffolds with a mean
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pore size of 50–200 μm. This process improved the mechanical behavior, broad spectrum
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antibacterial activity, and in vivo healing compared to SSF and CTS alone [116]. Nonwoven CTS/SF films also exhibited haemocompatibility and cyto-compatibility as a promising skin
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substitutes [143]. In a multifunctional integrated strategy by Li et al. [27], bioactive glass (BG)
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was incorporated into CTS/SF scaffolds to fabricate composites of BG/CTS/SF with biocompatibility and useful mechanical properties, which promoted vascularization and mature
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blood vessel formation to accelerate wound healing in vivo. Bilayered SF/Gelatin (ratio 20:80) films were mixed with 1% sericin for a randomized clinical trial on 25 patients with split-thickness skin graft donor sites. The healing rate in wounds treated with SF/gelatin/sericin blended films was significantly faster than Bactigras, a commercially available wound dressing with less pain [115]. Accelerated neovascularization, reepithelialization, collagen deposition, wound contraction and healing were also observed in 2% isabgol/2% SF (Isabgol/SF 75/25) composites with a fibrous foam-like architecture. Isabgol (Isab, Psyllium husk) is a natural carbohydrate polymer in India with several health benefits [146].
Journal Pre-proof Sodium alginate (SA) was reported as a promising material for the fabrication of SF blends for wound dressings [114, 147, 148]. SA is cytobiocompatible and effects skin wound healing through wound exudate absorption [149, 150]. Wang et al. [114] fabricated a homogenous, cytocompatible and interconnected 3D 2% SF/2% SA porous composite by freeze drying (-20 to 80C) for wound healing applications. They demonstrated that freezing temperatures affected the pore diameters (54-532 μm) and porosity (66-94 %) of the final scaffold, as pore diameter and
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porosity decreased and increased with decrease of freezing temperature, respectively. Base on their
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findings, crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) provided a
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suitable degradation rate until new ECM and tissue formation progressed.
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Strontium (Sr) is a metal with antibacterial activity and wound healing potential. The incorporation
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of Sr in SF/SA met many characteristics for wound healing, such as water absorption, water vapor transmission rate, and mechanical properties for wound healing. It was also revealed that Sr (5
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mg/ml)/SF/SA had the best antibacterial activity, cyto-compatibility, promoted angiogenesis and
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wound healing potential in vitro [148]. A novel recombinant SF/elastin protein with silk and elastin blocks was developed and its wound healing potential in two forms (aqueous and sponge) was
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compared in vivo. Recombinant SF/elastin self-assembles from liquid to gel, and both forms improved granulation tissue formation. SF/elastin sponges absorbed wound exudate and formed a gel after application on the wound. The authors concluded that recombinant SF/elastin sponges showed better potential as wound dressings than the aqueous form [151]. Rameshbabu and colleagues showed wound dressing sponges made from SF ornamented with human placentaderived extracellular matrix (pECM). The placenta was decellularized and then solubilized by urea buffer. Different ratios of SF/pECM were prepared and freeze dried to fabricate porous sponges. A wide range of cytokines, growth factors and ECM proteins present in the placenta were retained
Journal Pre-proof after the decellularization process and during scaffold fabrication. SF/pECM improved adhesion, proliferation and migration of human foreskin fibroblasts (HFFs), human epidermal keratinocytes (HEKs) and human amniotic and membrane-derived stem cells (HAMSCs) with biocompatibility in vitro and in vivo. Implantation of SF/pECM into a full-thickness wound in rat showed a significant increase in re-epithelialization, wound closure, neovascularization, wound healing compared to SF sponges [152]. In another study, SF films were loaded with an immunomodulator
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peptide (neurotensin, NT) and stimulated by iontophoresis to modulate inflammatory responses
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and accelerate wound healing [153]. CTS/SF films were a useful system for delivery of ADSCs to
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the diabetic wound site in a rat model. The combination of ADSCs and CTS/F was suggested as
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an effective strategy in the management of diabetic wounds [154]. Panico et al. [155] revealed that the addition of glucose in SF film induced crystallization of SF. This increased the absorption
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capacity and flexibility of the SF films. Citrus pectin/SF sponge fabricated by freeze-drying
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method showed a promising wound dressing material due to high water uptake ability (800% in 24 h), low weight loss (21.3% in 40 days), suitable mechanical property and cyto-compatibility
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for fibroblast cells in vitro [156]. To mimic the natural skin extracellular matrix and structure,
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Yang et al. [157] recently fabricated a SF/HA/SA spongy composite by freeze drying method with around 92% in porosity and average pore size of 93 μm. SF/HA/SA composite indicated good physical stability, soft and elastic characteristics with cyto-biocompatibility for NIH-3T3 fibroblast cells in vitro. Implantation of SF/HA/SA composite to full-thickness burn wounds in rat model and histological observations suggest the potential of this composite as a wound dressing. Liu et al. [158] prepared a SF sponge by freeze drying method for controlled release of neurotensin-loaded gelatin microspheres for management of diabetic foot ulcers. Neurotensin, as a neuropeptide, act as an inflammatory modulator and leads to the release of cytokines and
Journal Pre-proof chemotaxis involved in immunomodulation responses, which accelerate neovascularization and wound healing [159, 160]. Neurotensin-loaded gelatin microspheres releasing SF scaffold showed average pore size and porosity of 40–80 μm and ∼85%, respectively. During 28 days posttreatment follow up, neurotensin-releasing scaffold exhibited good drug delivery system and wound dressing material. This accelerated the wound healing in diabetic foot ulcers through the increase of wound closure, fibroblast accumulation and tissue granulation at the wound site with
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minimal scar formation [158].
4.2.2.2. SF blend fibrous scaffolds
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The morphology, surface hydrophilicity, water-uptake capability, fibroblast proliferation and
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attachment of poly (3-hydroxybuty-rate-co-3-hydroxyvalerate) (PHBV) nanofibrous scaffold improved by the addition of ESF (50/50 ratio) and fabrication of PHBV/ESF nanofibrous scaffolds
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by co-electrospinning technique [144]. Shanmugam and Sundaramoorthy developed and
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characterized electrospun mats from Eri (nonmulberry silk) SF and poly-L-lactic acid (PLA), for
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skin wound healing applications. Pure ESF and ESF/PLA mats had an average fiber diameter of 320 nm and 502 nm, respectively. Water uptake and vapor transmission rates in the ESF/PLA were lower than that in the pure ESF mats. ESF showed higher hydrophilicity compared to ESF/PLA, with water contact angles of 23° and 79°, respectively. Both ESF and ESF/PLA were successfully loaded with tetracycline hydrochloride (TCH) and had effective antibacterial activity against Gram positive (S. aureus and S. epidermidis) and Gram negative bacteria (E. coli and P. aeruginosa). Both ESF and ESF/PLA accelerated wound healing compared to control wounds treated with gauze [161]. The addition of SF microfibers (mSF) to pure CTS membranes increased biomechanical properties in vitro and healing efficacy in vivo for the CTS/mSF compared to pure
Journal Pre-proof CTS membranes in full-thickness skin wounds in rats [141]. Zhang et al. [162]successfully fabricated porous mSF/poly(glycerol sebacate) (PGS) and CTS/PGS composite scaffolds with biocompatibility, cell adhesion and favorable degradation for skin tissue engineering applications [162]. Incorporating gold nanoparticles into ESF 3D mats improved degradation profiles, biomechanical
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behavior and elongation at break, similar to normal skin, which accelerates neovascularization and granulation tissue formation in full-thickness wound healing in rats [163]. 3D electrospun PCL
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containing various concentrations of SF particles, fabricated by cold-plate electrospinning and
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automatic magnetic agitation, also have potential as artificial skins. Using this custom-designed
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electrospinning technique, 3D porous mats with thicknesses up to 6 mm with interconnected
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macro-pores ranging from tens to hundreds of microns were successfully fabricated with a homogenous distribution of SF nanoparticles within the PCL mats. While both PCL and SF
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particle-incorporated PCL mats were biocompatible, faster wound healing and collagen deposition
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were shown with the PCL/SF mats with the higher content of SF particles [164]. The addition of SF to oxidized cellulose nanofibers (TOCN) significantly improved its mechanical and wound
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healing effectiveness. TOCN/SF 2% supported improved wound healing in critical sized rat skin excisional models compared to the TOCN nanofibers [165]. SF/chemically active PEGs (PEG-maleimide and PEG-thiol) were introduced as a cytobiocompatible and effective sealants with rapid cross-linking ability through chemical reactions [166]. SF as an anti-adhesion patch was suggested by Wang et al. [167], where various SF nanofibrous blends as ESF/ PVA, ESF/PEG, and ESF/polyethylene oxide (PEO) were prepared by single-spinneret electrospinning. The anti-adhesion properties of the blends were: PVA/ESF> pure ESF> ESF/PEG ~ESF/PEO. The pure ESF and PVA/ESF showed better collagen
Journal Pre-proof regeneration and wound healing compared to the other blends [167]. Selvaraj et al. [168] fabricated an antioxidant scaffold by incorporating Fenugreek, a natural antioxidant, into ESF. Fenugreek-incorporated ESF had better thermal and biomechanical properties, supported higher cell proliferation in vitro and increased re-epithelialization, collagen deposition and wound healing compared to ESF.
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Anti-inflammatory, anti-bacterial, and the wound healing potential of honey make it an interesting material for skin tissue engineering [169]. To improve the wound healing potential of SF,
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honey/ESF wound substitutes were successfully developed by electrospinning [128, 130]. Manuka
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honey (Apis mellifera) incorporated ESF mats exhibited improved water uptake and supported cell
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growth in comparison with ESF, and suggested as a dermal regeneration template [128].
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ESF loaded with various concentrations of Manuka honey by a green electrospinning technique showed smooth surface morphologies with high antibacterial activity [130]. This antibacterial
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activity depended on Manuka honey concentration in the materials. The honey was also
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biocompatible. The average fiber diameter increased with increase of Manuka honey concentration
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in the constructs and the systems showed wound healing potential in vivo. Pignatelli and coworkers introduced human platelet lysate-loaded ESF as a wound dressing for wound healing management. Human platelet lysate is a rich source of growth factors and cytokines involved in wound healing. ESF preserved and released this lysate to the wound site [170]. A separate study also showed that loading of PRP exosomes and a polysaccharide extracted from the rhizomes of Curcuma zedoaria in CST/SF hydrogel sponges resulted in wound dressings for diabetic wound management in rats [171]. SF/PVA/collagen I peptide composite was shown to be a good drug delivery system for localized delivery of S-Nitrosoglutathione, a drug involved in
Journal Pre-proof regulation of the microvascular blood supply through its vasodilator property and activation of VEGF [172], and for the management of ischemic chronic non-healing ulcers in vitro [173]. Inducing anisotropic features to the ESF scaffold can promote its biological behaviors such as cell migration, proliferation and adhesion in vitro, and tissue ingrowth, neovascularization, wound closure and in general wound healing in vivo [174]. In another study, the ESF successfully loaded with amniotic fluid and SA hydrogel to deliver amniotic fluid, a highly enriched fluid with multiple
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therapeutic agents, to the wound bed, promote cell proliferation, migration and wound healing.
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This bioactive dressing showed interesting biomechanical and biological results in vitro. This
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needs further animal study to validate its potential in wound healing in vivo [175].
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Recently, Miguel et al. [176] prepared a two-layered membrane using electrospinning method to
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mimic both epidermis and dermis layers of skin for skin tissue engineering applications. This asymmetric membrane was made from ESF/PCL, as top layer to mimic dense nature. The
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waterproof properties of the natural epidermis, and ESF/HA loaded with thymol (an herbal drug)
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to mimic the natural dermis structure. The electrospun asymmetric membrane showed high wettability, porosity, and the mechanical properties appropriate for the skin wound dressing. The
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release of thymol endowed the ESF/HA side of asymmetric membrane an antibacterial activity against S. aureus and P. aeruginosa with cyto-compatibility for human fibroblasts in vitro.
4.2.2.3. Other blend forms Hydrocolloids, hydrophilic polymers dispersed in water, are suitable for wound healing, especially for burns [177-179]. SF nanoparticles were mixed with CMC and styrene-isoprene-styrene (SIS) to fabricate SF-CMC hydrocolloid wound dressings. SF-CMC hydrocolloid exhibited structural
Journal Pre-proof stability and cyto-compatibility with suitable mechanical properties for wound healing. SF-CMC hydrocolloids also revealed improved burn wound closure and healing when compared to gauze and Neoderm®, a commercially available wound dressing [109]. Subcutaneous administration of microcarriers prepared from SF alone and in combination with gelatin for deep skin wounds in mice also had healing potential with infiltration of immune cells such as mononuclear cells,
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macrophages, and neutrophils without fibrosis [180]. Thangavel et al. [181] developed a hydrogel for wound healing, prepared from silk protein, L-
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proline (LP), a critical amino acid that is used for collagen synthesis by the human body, and CTS.
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SF/LP/CTS hydrogels displayed thermal stability, suitable surface morphology, in vitro cyto-
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compatibility for NIH 3T3s and antioxidant activity. The hydrogels provided sufficient moisture
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for the wound bed. In vitro degradation demonstrated suitable biodegradation rates of the hydrogels. The presence of LP in the fabricated hydrogels had a significant effect on in vitro cell
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viability, migration, proliferation and wound healing, when compared to CTS and SF/CTS
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hydrogels. Kawabata and colleagues [182] showed that the hydrogels fabricated from SF-elastin conjugates (containing 4 SF–like blocks, 7 elastin-like blocks, and 1 modified elastin block) had
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favorable mechanical properties for full-thickness skin wounds in diabetic mice. Incorporation of curcumin in SF hydrogels, fabricated under weak electric fields, was useful for wound dressings with sustained curcumin release, cyto-compatibility with immortalized human keratinocytes (HaCaT). Dhasmana et al. [183] indicated the synergetic effects of SF and acellular dermal matrix in vitro and in vivo. They decellularized goat skin and then dip-coated at different concentrations of SF aqueous solutions. They concluded that the acellular dermal matrix modified with SF showed better wound healing in vivo compared to un-modified matrix, and suggested this scaffold as a biocompatible and cost-effective wound dressing.
Journal Pre-proof There is an inherent self-assembly property between two different silk types at 37 ⁰C. Chouhan et al. [184] fabricated injectable SF hydrogel from two species of silks (BmSF and AaSF, ratio 1:1) and showed that their injectable hydrogel successfully self-assembled at 37 ⁰C. FTIR and X-ray diffraction revealed that the irreversible gelation was due to β-sheet structure formation. The fabricated injectable SF hydrogel supported the proliferation of primary HDF and improved the migration of keratinocytes and re-epithelialization in vitro. In vivo evaluations of injectable SF
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hydrogel on full-thickness third-degree burn wounds indicated the accelerated inflammation,
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proliferation and remodeling phases of wound healing processes when compared to collagen gel,
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as control
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5. Biofunctionalization
Biofunctionalization of SF with growth factors, cytokines and drugs is an effective method for the
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improvement of SF based wound dressings [185, 186]. Two different drug incorporation
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techniques (drug loading or coating) with EGF and silver sulfadiazine were used to incorporate
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the protein into various SF scaffolds, including silk films, lamellar porous silk films and ESF [186]. The functionalized SF scaffolds revealed improved re-epithelialization, wound closure, dermal proliferation and wound healing in a full-thickness excisional wound (8 mm) in mice compared to two commercially available wound dressings (air-permeable Tegaderm tape (3M) (-control) and Tegaderm Hydrocolloid dressing (3M) (+ control)). SF scaffolds (all forms) functionalized with both EGF/silver sulfadiazine (loading and coating methods) showed improved wound healing compared to the other groups [186].
Journal Pre-proof Tripeptide Arg‐Gly‐Asp (RGD), is the most common peptide motif responsible for cell adhesion. Proteins containing higher contents of RGD show improved cell adhesion and attachment. The addition of RGD nucleic acid sequences to the SF gene and the subequent secretion of RGDcontaining SF proteins by transgenic silkworm may be a promising strategy for the fabrication of functionalized materials with high cell adhesion a for human dermal fibroblasts. Transgenic RGDSF can accelerate re-epithelialization, wound closure, neovascularization and wound healing
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compared to SF [187].
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Although growth factors can be incorporated or loaded into SF scaffolds, burst release and rapid
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loss can occur depending on the specific factor and its associated chemistry, which reduces the
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sustained delivery to wound bed. To overcome this limitation, growth factor gene sequences can
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be added to the SF gene and the resulting growth factor-SF proteins can be secreted by transgenic silkworms. The gene sequence of EGF was added to the light chain of the SF gene to develop
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transgenic silkworms carrying EGF-SF conjugated gene sequences. The EGF/SF proteins secreted
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by the transgenic silkworms allow for the sustained presence of EGF in the scaffolds and improved
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fibroblasts proliferation (2.5-fold), to accelerate wound closure in vitro [188]. SF/gelatin electrospun mats were functionalized with astragaloside IV, a bioavailable ingredient from the root of Astragalus membranaceus with anti-inflammatory, antioxidant and anti-aging properties. The compound has been utilized for scar-free healing of deep partial-thickness burn wounds in rats. In vitro evaluations demonstrated promising cell adhesion and biocompatibility for immortalized human fibroblasts. In vivo experiments exhibit accelerated wound closure, neovascularization,
organized
collagen
formation
and
reduced
scar
formation.
SF/gelatin/astragaloside IV electrospun mats were useful topical delivery systems for burn wound healing [185].
Journal Pre-proof As described earlier, non-mulberry SF possesses additional cell-binding motifs (RGDs) compared to mulberry SF [16, 39, 43, 93]. Chouhan et al. [189] functionalized PVA/SF electrospun mats with human recombinant EGF, human recombinant bFGF and LL-37 antimicrobial peptides. SF proteins were extracted from mulberry SF (extracted from Bm) and non-mulberry SF (extracted from A. assama (AaSF) and P. ricini (PrSF)). All the electrospun mats had in vitro cytocompatibility with human dermal fibroblasts (HDF) and HaCaTs. In vitro cell migration scratch
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assays showed significantly increased migration when the cells were treated with a combination
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of EGF, bFGF and LL-37. The wound healing potential of mats in non-diabetic and diabetic full-
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thickness excisional wounds (12 mm) in a rabbit model demonstrated that the functionalized non-
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mulberry SF/PVA mats (PVA/Aa and PVA/Pr) supported faster granulation, wound closure, organized ECM formation, re-epithelialization and angiogenesis compared to controls,
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functionalized PVA and functionalized PVA/Mulberry SF (PVA/Bm) [189].
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Bienert et al. [190] genetically modified Bm larvae to produce functionalized silk with FGF, EGF,
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KGF, PDGF or VEGF growth factors. All of the functionalized silk membranes, as well as native Bm silk, had cyto-compatibility in vitro. EGF-, FGF- and VEGF-functionalized silk membranes
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showed better in vitro and in vivo (full-thickness excisional wounds in mice) wound healing compared to native, PDGF- and KGF-functionalized silk membranes. KGF- and VEGFfunctionalized silk membranes provided improved cell growth and migration support for keratinocyte, while the silk membranes functionalized with EGF and VEGF increased the adhesion of macrophages compared to the other scaffolds. Although the growth factor-functionalized silk membranes supported wound healing, VEGF-functionalized silk membranes showed slightly better wound healing potential.
Journal Pre-proof Liposomes were used to maintain bFGF content in SF hydrogels for sustained release. The bFGFencapsulated liposomes in SF hydrogels showed stable and sustained release of bFGF in mice in deep second-degree scald wound fluids and as a result, accelerated wound healing compared to SF hydrogels and bFGF-encapsulated liposomes [191]. Topical bioactive insulin was encapsulated in SF microparticles by coaxial electrospray for chronic wound healing. Insulin-functionalized SF microparticles were loaded into SF sponges to develop a skin wound dressing. The functionalized
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scaffolds showed sustained release of insulin up to 28 days. Insulin release from the SF scaffolds
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increased the migration of human keratinocytes and endothelial cells in vitro. In vivo examination
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of insulin-SF scaffolds on dorsal full-thickness diabetic wounds in rats demonstrated accelerated
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wound closure, neo-vascularization and healing compared to other experimental groups [192]. A 3D gelatin/sulfonated SF/FGF wound dressing fulfilled the biological needs for wound dressings.
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In this case, 3D gelatin grids were fabricated by 3D printing. The grid was embedded in sulfonated
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SF. FGF was incorporated into the gelatin/sulfonated SF composite through sulfonic acid groups. In vitro assays demonstrated favorable biomechanical and biological properties of the scaffolds,
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and in vivo applications of the gelatin/sulfonated SF/FGF scaffolds in full-thickness excisional
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wounds in rats showed accelerated granulation and healing. The presence of FGF stimulated the neo-vascularization in the wound bed [193].
6. Antibacterial SF derived scaffolds Post-wound infection leads to delayed wound healing, scar formation and cell and tissue death especially in burns [105]. Thus, antibacterial activity is a key need in wound dressings. Some materials, such as CTS, sericin and others, have intrinsic antibacterial activity [37, 194]. SF does
Journal Pre-proof not exhibit inherent antibacterial activity [59], thus many approaches have been developed to endow antibacterial activity to SF-derived scaffolds [147, 195-197]. Table 2 (a-b) summarizes in vitro (Table 2a) and in vivo (Table 2b) studies on antibacterial SF blends for infected skin wound healing applications. Several materials or drugs, such as heavy metals, antibiotics and antibacterial biomaterials were incorporated into SF to fabricate SF composites with antibacterial activity for various TERM applications [117, 118, 147, 196, 198]. SF biomaterials in various forms (films,
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microspheres, hydrogels, coatings) had utility as local antibiotic delivery systems for wound
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healing [148, 198]. Heavy metals, such as selenium, strontium and silver are common antibacterial
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agents [195, 199-201] used in the fabrication of antibacterial SF composites [148] in various
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formats, including films [202], gels [203], electrospun mats [204] and sponges [205].
6.1. Antibacterial SF fibrous scaffolds
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Most SF wound dressings with antibacterial activity are fabricated in fibrous forms. Several studies
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use Ag as a broad-spectrum and strong antibacterial agent to endow antibacterial activity to SF
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mats [206]. The antibacterial activity and toxicity against human cells is dependent on Ag concentration [199, 207, 208]. The adhesion and spreading of human epidermal keratinocytes and human epidermal fibroblasts on silver sulfadiazine–containing SF nanofibrous mat decreased with increase of silver sulfadiazine content in the mats. Optimal concentrations of Ag in wound dressings are critical, to balance high antibacterial activity and no toxicity for human cells [207]. Silk fibers were coated with silver nanocolloids for prevention of infections after surgery [198]. The coated silver nanoparticles (AgNPs) were prepared by two methods (in situ and ex situ). For in situ, the silk fibers were embedded in SF aqueous solutions containing silver nitrate, while for
Journal Pre-proof ex situ, the silk fibers were treated with colloidal AgNPs. Impregnation with AgNPs by both in situ and ex situ methods enhanced the thermal and mechanical properties of the silk fiber-AgNPs mats. The presence of the crystalline AgNPs coated on silk fibers was shown by X-ray diffraction. Both methods showed cyto-compatibility for 3T3 fibroblasts. Although all the mats exhibited strong antibacterial activity against P. aeruginosa and S. aureus, the silk fiber-AgNPs antibacterial mats fabricated by the in situ method displayed better antibacterial activity compared to the
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samples prepared ex situ. The higher antibacterial activity of silk fiber-AgNPs fabricated by in
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situ methods and containing a greater content of silver, compared with those scaffolds prepared ex
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situ, was attributed to the processing method and thus content of silver in the material [198].
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Babu et al. [209] demonstrated antibacterial activity of silver oxide (Ag2O) nanoparticles-
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embedded SF spun (Ag2O-SF spun) against the pathogens (S. aureus and Mycobacterium tuberculosis) and non-pathogenic (Escherichia coli) bacteria. They also showed cyto-
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compatibility of the Ag2O-SF spun mats for 3T3 fibroblasts and in vitro wound healing potential
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when tested by a scratch assay. Çalamak et al. [210] indicated that the incorporation of different concentrations of antibacterial polyethylenimine (PEI) in ESF generated antibacterial wound
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dressings with cyto-compatibility for L929 cancer cells[210]. The incorporation of antibiotics into SF fibers was also useful for the fabrication of antibacterial SF mat wound dressings. Ceftazidime (CTZ), a third generation cephalosporin antibiotic, was loaded into SF/gelatin and electrospun to fabricate antibacterial nanofibrous wound dressings. The CTZ/SF/gelatin material had antibacterial activity against P. aeruginosa and good cyto-compatibility in vitro [211]. The overuse of antibiotics can cause bacterial resistance, as the resistance to CTZ was reported in several studies [212]. Localized release from the above materials may be one option to address this problem.
In other approaches, antimicrobial peptides (AMP), strong and broad-spectrm
Journal Pre-proof antibacterial agents, were used to fabricate antibacterial SF mats [189, 196]. AMPs are natural antibacterial systems in eukaryotes and kill microorganisms (bacteria, virus, protozoa, and fungi) by disrupting the structure or function of microbial cell membranes. These peptides also inhibit ATP-dependent enzymes in cells [213]. AMP can negatively affect cell viability and even cause human cell death when used in high concentrations.
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Song et al. [196] immobilized KR12 (KRIVKRIKKWLR), an antimicrobial peptide, on ESF membranes, which were then tested for antibacterial and biological properties in vitro for wound
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healing. The ESF membranes were treated with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide
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hydrochloride (EDC) to activate carboxylic acid groups in the SF proteins. The activated
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carboxylic acid groups were bound to N-(2-aminoethyl) maleimide (AEM) linkers and then
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reacted with various concentrations of KR12 (ranging 50-500 µg/ml). The ESF/KR12 had antibacterial activity against S. aureus, S. epidermidis, E. coli and P. aeruginosa, while exhibited
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cyto-compatibility for HDF and keratinocytes in vitro, and suppressed expression of TNF-α from
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LPS-induced murine monocytes. ESF/PVA nanofibers were fabricated by electrospinning and were functionalized with the LL-37 antimicrobial peptide. ESF/LL37 showed sustained delivery
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of LL-37 with antibacterial activity against tS. epidermidis MTCC 435 and P. aeruginosa MTCC 1688, and biocompatibility and wound healing potential in vitro and in vivo [189]. Incorporation of ESF with EGF and ciprofloxacin showed controlled release of with antibacterial activity, accelerated re-epithelialization, neovascularization and wound healing in full-thickness excisional wounds in rabbits [214]. Lu et al. [215] fabricates a bi-layered SF/CTS mat by spraying CTS on SF fabric. They show that CTS form a thin layer with connective pores over the SF fabric. The bilayered SF/CTS mat exhibits strong antibacterial activity and no cytotoxicity with high potential
Journal Pre-proof wound healing. This is compared to commercially available foam adhesive dressing (TegadermTM) in full-thickness wounds in a rat model. Transgenic SF with antimicrobial peptides (Cecropin B or Moricin antimicrobial peptides) were used for experimentation as biomaterials [216, 217]. The transgene SF fibers exhibited high antibacterial activity against some bacteria such as E. coli, with excellent biocompatibility. This
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new antibacterial biomaterials for biomedical applications.
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strategy may be useful to endow intrinsic antibacterial activity to the spun SF fibers and provide
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Srivastava et al. [218] coated tasar ESF by AgNPs to fabricate an antibacterial wound dressing mat against P. aeruginosa, S. epidermidis, S. aureus and E. coli and cyto-compatibility for L929
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skin fibroblast. Recently, a promising antibacterial bilayered SF-based scaffold, a SF sponge layer
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covered by ESF loaded with silver sulfadiazine, was developed against S. aureus [219]. Most recently, Zheng et al. [220] incorporated different concentrations of nano-Cu2O (0.5, 1.0, and 5.0
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mg/mL) into poly(ethylene oxide) (PEO)–SF composite and electrospun to fabricate an
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antibacterial wound dressing mat. The decreased uniformity and increased hydrophilicity of mat
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morphology were observed with the increase of nano-Cu2O concentrations. As expected, antibacterial activity of the composite electrospun mat against E. coli and S. aureus bacteria increased with the increase of nano-Cu2O concentrations with no detectable cytotoxicity against pig iliac endothelial cells.
6.2. Antibacterial SF sponge Some antibacterial SF composites were fabricated in the sponge form for infected wound healing management. For example, incorporation of AgNPs into SF sponge showed to have favorable
Journal Pre-proof moisture permeability, satisfying tensile strength , water absorption capacity and stable Ag release with high antibacterial activity and wound healing potential in comparison with porcine acellular dermal matrix [205]. The use of two bactericidal agents of colloidal Ag and ciprofloxacin in SF/SA sponges was a promising strategy to fabricate a highly effective antibacterial wound dressing with favorable wound healing potential [147]. Liu et al. [142] developed a wettable CTS-SF/AgNPs sponge for infected skin wounds. They incorporated different concentrations of AgNPs into CTS-
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SF aqueous solutions, and pour into a container and freeze dried after freezing the casts at -20 C
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and -80 C to fabricate an antibacterial sponge. The sponge had high porosity with good wettability
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for providing moisture in the wound bed and appropriate mechanical property. The sponges also
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showed strong antibacterial activity against various bacteria such as S. aureus, E. coli, P. aeruginosa and Monilia albicans and cyto-compatibility for L929 cells, human fibroblasts and
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human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro. The antibacterial activity and
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wound healing potential of the CTS-SF/AgNPs sponges for the full-thickness wound in mice
[142].
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model infected with P. aeruginosa showed high antibacterial activity with no post wound infection
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Gentamicin sulfate (GS)-loaded SF/gelatin sponge fabricated by freeze drying method was introduced as another candidate for infected wound healing in vitro. The GS/SF/gelatin sponge indicated a decreased compressive modulus property with the increased content of SF and GS. It also exhibited enhanced water swelling capacity with the addition of GS. The sponges with higher content of SF demonstrated to have accumulative release of GS. The sponge had excellent antibacterial activity against S. aureus, S. epidermidis, M. luteus, B. cereus and P. aeruginosa. Only the GS/SF showed cytotoxicity effects for NHDF [221]. Incorporation of AgNPs in collagen/SF sponge fabricated by freeze drying technique also considered as a promising candidate
Journal Pre-proof for the treatment of infected wounds [222]. In the other two studies, chitin/SF 3D scaffolds were loaded with AgNPs and TiO2. These were suggested as effective wound dressings with cytocompatibility. They had also promising swelling, water uptake properties and antibacterial activities against E. coli, S. aureus, and Candida albicans. [223, 224].
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6.3. Antibacterial SF films
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Some antibacterial SF-derived wound dressing materials were fabricated in the form of films. For example, Alam et al. [202] developed a promising sustained drug release system by modifying the
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surface of SF/gelatin film with polyethylene glycol (PEG). The modified film was loaded with
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ciprofloxacin, a drug belongs to a group of antibiotic called fluoroquinolones, exhibited lower
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burst drug release compared to unmodified film. The modified SF/gelatin loaded with antibiotic showed an excellent drug release with high antibacterial activity and better wound healing
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potential in comparison of the unmodified antibiotic-loaded SF/gelatin film [202].
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Strontium is another additive choice with both antibacterial activity and wound healing potential.
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Strontium-loaded SF/SA blend film (Sr/SF/SA) had high antibacterial activity, cyto-compatibility and excellent wound healing potential in vitro. Among Sr/SF/SA films with various contents of Sr, the samples containing 5 mg/ml Sr exhibited the best result to meet the requirement for infected wound healing. Sr/SF/SA film also increased the secretion of important pro-angiogenic factors, VEGF and bFGF in vitro. This accelerated in vivo neovascularization [148]. Molybdenum diselenide (MoSe2) is an inorganic compound of molybdenum and selenium and has high antibacterial activity through its peroxidase-like activity. Activation of carboxylic acid groups of SF and functionalization with MoSe2 is an interesting strategy for endowing antibacterial activity
Journal Pre-proof to SF-derived wound dressing [119]. The addition of selenium nanoparticles in to ESF improved human dermal fibroblast metabolic activity and antibacterial behavior in vitro [201].
6.4. Antibacterial SF Gels Fabrication of antibacterial SF composites in gel form may be a promising strategy to achieve a
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cost-effective antibacterial ointment and excellent wound healing materials. It was reported that
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AgNPs-loaded SF gel had strong antibacterial activity against S. aureus and excellent wound healing potential in full-thickness wounds in the rat when compared to a commercially available
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wound healing gel (soframycin gel) [203]. It was also demonstrated that SF/AgNPs solution can
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be considered as a novel agent for treatment of biofilm-associated diseases, such as sinusitis. The
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sinusitis model was created in New Zealand White rabbits by inoculation with S. aureus. The solution with 384 mg/L AgNPs showed completely elimination of biofilms and repair of defected
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epithelium [225]. An SF hydrogel with improved mechanical properties was fabricated by using
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2-(N,N-dimethylamino) ethyl methacrylate as an excellent delivery system for controlled release
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of cefixime, belongs to a group of antibiotics called cephalosporin [226]. Their results indicated a high degree of water uptake and improved mechanical properties (tensile strength and elongation at break) compared to SF hydrogel without modification and high antibacterial activity.
7. Other biomedical applications of silk fibroins Universally known as textile material, silk threads have embarked on their journey towards other beneficial uses decades ago. Silk also remains a prominent component for several cosmetic products and medical sutures. SF, in particular, finds its niche in the world of biomedical science
Journal Pre-proof for its beneficial properties as a tunable yet biocompatible biomaterial. Besides wound healing and skin repair, fibroin has multiple biomedical applications in various types of TE including bone [227-229], neural [129, 230], cardiac [231, 232], ligament [233, 234], vascular [67, 235], muscle [236, 237], cornea [238, 239], dental [240, 241] , hepatic [242, 243], cartilage [244, 245], osteochondral [246, 247] and others (Fig. 2). Bioprinting techniques create multi-level 3D scaffolds, which can be modified or tailored as per the prerequisites for different TE needs [248].
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Combined with the delivery and sustained release of bioactive molecules (growth factors, drugs
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and antibiotics) in different formulations like microcapsules, nanoparticles, hydrogels, porous
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scaffolds, nanofibrous matrices and others [249-252] further expands the utility for silks. Other
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applications of silk include as food preservatives [253], optical devices [254] and electronics [255],
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among others.
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8. Benefits and concerns of using silks for biomedical applications
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Silk protein fibroin offers an impressive array of biomaterials, which can be applied in diverse
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areas of biomedical science. There are numerous desirable properties exhibited by this protein including ease of materials fabrication, mechanical strength, biocompatibility, flexibility, biodegradability, ease of chemical modification, non-cytotoxicity, no immune response, low production cost and available supplies of material. Although, silk is a natural biomaterial with no major adverse effects, there are still shortcomings associated with biomedical applications. The standardization of the production of cocoons starting from sericulture practices to biomedical grade cocoon materials needs special attention. Even handling of the raw materials after procurement, including storage, transportation and use in the laboratory by trained personnel are
Journal Pre-proof important factors to consider. The degumming process involved in the extraction of fibroin and regeneration from aqueous silk leads to a reduction in the mechanical strength of silk, which limits certain aspects involved in the fabrication process for matrices. Over the years, multiple and diversified animal studies have been reported for various applications of silks, and these validate the safe and effective use of silk [12, 13]. This reflects the promising characteristics of this product,
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but further clinical investigations are needed for specific biomedical products based on silks.
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9. Conclusions and future prospects
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Skin is the first line of defense as well as the most frequently damaged organ of the human body. Wound dressing materials and new techniques/ways to restore the lost or wounded structure of the
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skin have been pursued for many years. Most common clinically used allografts or autografts
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include certain limitations (immune response, restricted host tissue regeneration, lack of degradation), which lead to the need for artificial human skin by TERM. This review described
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the relevance of natural SF in this domain and the numerous categories of scaffolds fabricated and
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investigated. They aid in creating a skin substitute or dressing material. Silks (mainly silkworm silks) have exceptional physical, mechanical, chemical and biological properties, which permit the formation of different biomaterials. Silk protein fibroin is used in skin regeneration, and TERM applications. Films and fibrous matrices are most commonly used for these applications. Other kinds of matrices like sponges, hydrogels, hydrocolloids, microfibers, microcapsules, nanoparticles, bio-printed gel and others are also utilized effectively in partial or full thickness wounds. In order to augment their performance, added functional features like antibacterial activity, drugs and growth factors are included, as well as micro carriers or using genetically
Journal Pre-proof modified SF by creating /transgenic silkworms. Commonly practiced techniques are the inclusion of different inorganic or heavy metals (strontium, selenium, silver), antibiotics, antimicrobial peptides or establishing a transgenic SF having anti-microbial peptides to prevent infection during would healing. The fibroin matrices support the acceleration of cellular adhesion, wound contraction, re-epithelialization and angiogenesis, collagen formation with a reduced immune response. The prospects of using fibroin alone and its blends are quite exciting in wound healing
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due to the positive results achieved in vitro and in vivo. Genetically modified SF combined with
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emerging innovative methodologies may produce cost effective silk-based artificial skin with
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additional functional features. Successful wound healing with minimal scar formation and the
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present clinical trials using fibroin scaffolds further emphasize the utility of silk for the field of skin tissue engineering. The flexibility and ease of modification in silk scaffolds make it possible
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to create improved bi-layered skin-like constructs for clinical use. SF is an FDA approved material
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for some biomedical applications and some silk-based products for cosmetic (silk mask and gel) and medical applications (silk suture and Fibroheal
TM
Ag wound dressing) are already on the
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market. In addition, silk-based surgical meshes passed biocompatibility and safety testing for ISO
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10993. As stated earlier, some SF-based scaffolds such as SF and SF/gelatin/sericin films have successfully passed through preclinical and the randomized clinical trials for full-thickness skin wounds. It is expected that the electrospun SF approaches loaded with or without antibacterial agents may be the most effective SF based skin substitutes and subject to the future clinical trials. In summary, natural silk protein, fibroin has great prospects of expanding its niche in the field of skin tissue regeneration and repairing damaged skin.
Journal Pre-proof Acknowledgements SCK presently holds an European Research Area Chair and Full Professor position at 3B´s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Portugal, supported by the European Union Framework Programme for
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Research and Innovation HORIZON 2020 under grant agreement nº 668983 — FoReCaST.
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Authors’ contributions
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MG and SCK design the concept; MG wrote the paper along with SS; DLK and SCK reviewed
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during the preparation of the manuscript; DLK, AS, SCK and RLR revised the manuscript. All the
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Conflict of interests
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authors discussed, commented, revised and approved the final manuscript.
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Authors declare no conflict of interest.
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chitosan/poly (vinyl alcohol)/silk fibroin nanoparticles for wound dressings, International journal of biological macromolecules, 53 (2013) 88-92. [266] X. Sheng, L. Fan, C. He, K. Zhang, X. Mo, H. Wang, Vitamin E-loaded silk fibroin nanofibrous mats fabricated by green process for skin care application, International journal of biological macromolecules, 56 (2013) 49-56. [267] Z. Gu, H. Xie, C. Huang, L. Li, X. Yu, Preparation of chitosan/silk fibroin blending membrane fixed with alginate dialdehyde for wound dressing, International journal of biological macromolecules, 58 (2013) 121-126.
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+ sign indicates the important references for further consultation for an appropriate area (s) of work.
Journal Pre-proof Legends for figures (1 to 6) and write up for graphical abstract. Figure. 1. Illustration of the basic routes of tissue engineering and regenerative medicine. The cells are harvested from patients by biopsy and are grown in 2D culture under aseptic conditions. The cell culture medium is supplemented with growth factors and other specific features as required by the individual cell type. These cells are sub-cultured for the expansion of cells and are seeded onto 3D matrices for the construction of specific 3D tissues. These matrices/polymeric scaffolds may be natural, synthetic or blends
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and are fabricated in various material format such as hydrogels, porous sponges, nanofibrous mats,
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micro/nano particles, micro-/nano patterned surfaces, among others. These matrices may contain bioactive molecules to augment the proliferation of cells. For long-term culture, the matrices are grown in bioreactors
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transplanted to restore the damaged tissue in vivo.
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to support and maintain the cultures under aseptic conditions. The regenerated tissue in vitro may be
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Figure 2. Different applications of silk protein fibroin in tissue engineering and regenerative medicine. Silk provides flexibility to fabricate different kinds of matrices and thereby, finds use in multiple
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biomedical applications including tissue engineering targets like the regeneration and repair of damaged
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tissues. Apart from reconstructing injured areas, silk matrices also effectively deliver bioactive molecules including drugs and growth factors to the target site, as well as genes and cells, further enhancing utility in regenerative medicine.
Figure. 3. Arrays of silk protein fibroin matrices for tissue engineering and regenerative medicine. Silk as a biomaterial is often obtained from both mulberry and non-mulberry (shown above are some of the main cultivated silkworm species) silkworm silks. Depending on the silkworm species and availability, silk proteins may be isolated from the cocoons after degumming (removal of silk protein sericin) or from the mature 5th instar larvae (mainly from non-mulberry). Silk-based scaffolds are developed in various forms,
Journal Pre-proof which range from thin membranes/coatings to 3-D bio printed matrices to support organ specific requirements. Figure 4. The events of four overlapping phases of normal wound healing process during the first 30 days of post-wounding.
Figure. 5 (a-e). Silk fibroin film: from fabrication to animal and randomized clinical trial studies. (a)
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Schematic of the fabrication of transparent silk fibroin film. (b) Silk fibroin film with a thickness of 64.9 µm was much thinner than Suprathel (266 µm) and Sidaiyi (1789 µm). (c) Silk fibroin film exhibited better
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and faster wound healing compared to Suprathel and Sidaiyi during 90 days in porcine model [121]. (d)
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Improved wound closure in the wounds treated with SF film compared to Mapitel and the wounds with no
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treatment after 5, 10, and 15 days [122], (e) Hand burn wounds after 1 month treatment with Biobrane and
lP
Dressilk [123].
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Figure 6 (a-d). Silk fibroin fibrous scaffolds for skin wound healing (a) Electrospun silk fibroin scaffolds fabricated by cold-plate electrospinning methods (CPE) had higher porosity and pore sizes with
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controllable mat thickness and easy shaping compared to salt-leaching (SLE) and traditional
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electrospinning techniques (TE) [133]. (b) Silk fibrous mats were fabricated by treating the silkworm cocoons with various degumming process times (0-100 min). SEM micrographs showed the effects of degumming time on morphology and porosity [136]. (c) Electrospun silk fibroin (ESF) on amniotic membranes (AM), as well as cross sectional views of AM/ESF bilayer membranes, indicated successful the coating of AM with ESF [59]. (d) ESF/AM alone or seeded with adipose tissue-derived mesenchymal stem cells (ADSCs) exhibited better wound healing compared to AM and AM/MSCs in a mouse burn model [69].
Journal Pre-proof
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Graphical abstract
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Silkworm silk fibroin is a well-established natural protein in the realm of biomaterials with an array of matrices in its repository. These range from primary bio coating to state of the art,
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bioprinting en route for creating an advanced graft for diversified biomedical applications.
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Augmenting these matrices by incorporating functional traits like delivery of bioactive molecules/compounds (growth factor, drug, antibiotic, gene or cell) or conductivity make them smart matrices for skin tissue regeneration and skin repair.
Journal Pre-proof Table 1 (a-c). In vitro (a), in vivo (b) and clinical trials (c) carried out between 2000 and 2019 on different types of 2D and 3D SF or SF blends scaffolds/matrices for skin tissue regeneration, tissue engineering and regenerative medicine applications.
Table 1a: In vitro experimentations on SF or SF blends for skin tissue engineering applications. Design of
Major findings
experiment (s)
References
of
Material (s)
Bm fibroin peptides derived from digestion by
peptides
two important peptides present in N-terminal region
In vitro
[256]
-p
SF-derived
ro
chymotrypsin, VITTDSDGNE and NINDFDED, as
of SF protein promote human fibroblast growth,
lP
SF.
re
critical biological properties of N-terminal region of
Chitin/SF Nanofibrous mat made from various
In vitro
[257]
electrospinning of chitin and SF solution (chitin/SF blend); good cyto-compatibility for NHEK cells in 25
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nanofibrous mat
concentration of SF and chitin, and also simultaneous
na
Chitin/SF
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% chitin/75 % SF and chitin/SF blends. Conjugation using cyanuric chloride, higher adhesion, growth and viability of subcutaneous fibroblastic cells than pure SF, promoted adhesion of
Lactoseconjugated SF 2D films and 3D scaffolds
fibroblasts compared to myofibroblasts in lactoseIn vitro
conjugated SF film, loss of contractile phenotype of pre-differentiated myofibroblasts in lactoseconjugated SF substrates, suppressed myofibroblast differentiation cultured on 3D lactose-conjugated SF scaffolds and decreased host fibrotic reactions in lactose-conjugated SF scaffolds.
[258]
Journal Pre-proof Plasma treatment of ESF in presence of oxygen and Plasma-treated ESF mat
methane, decreased and increased hydrophilicity In vitro
after methane and oxygen plasma treatment,
[259]
respectively, good cellular activities in oxygen plasma-treated ESF for NHEK and NHEF cells. Successful incorporation of EGF into ESF
EGF-loaded In vitro
[260]
compatibility, accelerated wound closure and wound
nanofibrous mat
of
ESF
nanofibers, controlled release of EGF, good cyto-
healing in human skin-equivalents wound model in
ro
vitro.
transmission rate, biodegradation rate, nanofiber
In vitro
average diameter and morphology, suitable for full-
[131]
re
ESF mat
-p
SF concentration-dependent water vapor
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thickness wound dressing Increased mechanical properties with addition of SF,
nanofibrous mat
In vitro
antibacterial activity depends on type of bacteria,
[261]
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CTS/SF
better biocompatibility with promotion of growth and
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attachment of murine fibroblasts.
SF/PEGs sealant
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Cyto-biocompatible, strong and effective sealant, fast
In vitro
crosslinking (within seconds), better adhesive
[166]
property than the current leading PEG-based sealant, decreased swelling and longer degradation compared to PEG-based sealant. Nanofibers with average diameters of 385 ± 63 nm,
TiO2-loaded ESF mat
higher water content compared to commercially In vitro
available hydrocolloid wound dressing Tegasorb, excellent hemocompatibility and cyto-compatibility for L929 fibroblasts, strong antibacterial activity.
[262]
Journal Pre-proof Chemical interaction between chitosan and fibroin, increased flexibility, swelling ratio and CTS/SF film
In vitro
[263]
biodegradation along with the increase of CTS concentration, favorable cell adhesion and growth support for fibroblasts. Control release of vitamin C, good cyto-
loaded ESF
biocompatibility such as cell adhesion and growth for In vitro
[264]
mouse fibroblast L929 cells, increased expression of
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Vitamin C-
collagen type I alpha 1, glutathione peroxidase 1 and
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catalase and high anti-oxidative effects.
F nanofibrous
Intermolecular hydrogen bonding among CECTS, In vitro
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CECTS/PVA/S
PVA and SF and excellent cyto-compatibility for L929 cells.
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mat
[265]
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Changed morphology of nanofibers with different concentrations of vitamin E, ribbon-like and round
In vitro
[266]
of vitamin E, excellent water-resistant property, controllable and sustained release of vitamin E,
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loaded ESF mat
shape for lower (2%) and higher (8%) loading dose
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Vitamin E-
CTS/SF/ADA membrane
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promised cell adhesion, proliferation for mouse skin fibroblasts (L929) and anti-oxidant property.
In vitro
permeability, and excellent cell adhesion and
erol
[267]
proliferation for L929 fibroblasts. Good physicochemical and mechanical properties in
SF film/pectin/glyc
Promising stability, water absorption and vapor
In vitro
SF film containing 6% w/w pectin 12% w/w glycerol, promising cell adhesion and growth, and suitable for MSCs delivery.
[110]
Journal Pre-proof Surface modification with NaOH alkaline to form Nanofeatured SF membrane
nano-featured SF membrane, enhanced nano-
In vitro
topography, wettability and cellular function
[111]
compared to untreated SF scaffold. Increased plasticity and flexibility, improved elongation at break, increased water holding SF/dextrose film
In vitro
capacity, hydrophilicity and roughness, enhanced cell
[124]
of
attachment and proliferation and promising system
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for delivery of gentamicin.
Fabrication by cold-plate electrospinning method,
nanomatrix mat
higher porosity and pore size with controllable mat
In vitro
[133]
-p
ESF porous
thickness, easy shaping compared to SLE and
re
traditional electrospinning techniques.
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Incorporation of different contents of SF and fabrication of SF/BC with final ratios of 25/75 %,
scaffold
In vitro
50/50 % and 75%, higher water solubility and lower
na
SF/BC porous
uptake with the increase of SF content, non-cytotoxic
[268]
ur
and genotoxic for L929 fibroblasts, and improved
PHBV/ESF nanofibrous
Jo
cell adhesion with the increase of SF content to 50%.
In vitro
scaffold
Good surface hydrophilicity and water-uptake
[144]
capability, fibroblast cells proliferation and attachment in 50/50 ratio of PHBV/ESF. Average fiber diameter of 160 nm, better growth
Muga SF nanofibrous mat
In vitro
support for L929 fibroblast cells compared to muga SF film, better gentamycin sulphate release compared to muga SF film.
[138]
Journal Pre-proof Fabrication by freeze drying, homogenous, cytobiocompatible and interconnected 3D porous composite scaffold, freezing temperature-dependent SF/SA scaffold
pore diameter (54-532 μm) and porosity (66-94%),
In vitro
decreased pore diameter in lower freezing
[114]
temperature, Increased porosity in lower freezing temperature, suitable degradation rate and
of
crosslinking with EDC. Homogenous pore distribution, highest porosity (70%), water uptake and swelling ration in SLE
nanomatrix vs
compared to FD and SL, higher compressive strength
-p
fabricated by
of SL, similar compressive strength in FD and SLE,
In vitro
good biocompatibility of all three methods for NIH
[112]
re
3D SF scaffolds
ro
3D ESF porous
3T3 fibroblast cells, and favorable biomechanical and
methods
biological characteristics in ESF fabricated by SLE
lP
FD and SL
na
method for skin tissue engineering. Treatment with various rates of degumming
mats
In vitro
processes, favorable mechanical property, strong antibacterial activity, and good cyto-compatibility in
ur
Silk cocoon
[136]
Jo
vitro
SF/CTS/LP hydrogel
Promising thermal stability, favorable surface morphology, in vitro cyto-compatibility for NIH 3T3, antioxidant activity, suitable biodegradation rate of in
In vitro
vitro, capability to keep wounds moisture, better cell viability, migration, proliferation and wound healing of SF/CTS/LP in vitro compared to CTS and SF/CTS hydrogels.
[181]
Journal Pre-proof Improved mechanical and dynamic mechanical CTS/SF film
properties compared to nonwoven SF, good
In vitro
hemocompatibility, cyto-compatibility and
[143]
biodegradability. Promising water absorption, water vapor transmission rate and mechanical properties, antibacterial activity, cyto-compatibility for
In vitro
fibroblasts, better wound healing results in Sr/SF/SA
[148]
of
Sr/SF/SA film
film containing 5 mg/ml Sr, increased the secretion
ro
of VEGF and bFGF in vitro.
In vitro
and hydrophilicity, enhanced cell attachment and
re
film
elongation at break, increased water holding capacity [125]
proliferation and promising system for delivery of
lP
SF/Dextrose
-p
Favorable plasticity and flexibility, improved
gentamicin.
Antheraea mylitta silk mat
skin
na
and proliferation, and good biodegradation rate.
[162]
Homogenous morphology, favorable mechanical
In vitro
AM/ESF bilayered artificial
Good biocompatibility, promising cell attachment
ur
CTS/PGS
In vitro
Jo
SF/PGS and
In vitro
property, biocompatibility for epidermal cell primary culture, and thermal stability.
[139]
Electrospinning of SF on decellularized amniotic membrane, good cyto-biocompatibility and cell adhesion, mechanical property close to native skin, and angiogenic property.
[269]
Journal Pre-proof Non-covalent interactions between SF and NT, homogeneous NT distribution in SF film surface, sustained release of NT for up to 72h, decreased
Neurotensinloaded SF film stimulated by
production of interleukins from E. coli In vitro
[153]
liposaccharide-stimulated macrophages after exposure to NT-loaded SF film, immunomodulatory
iontophoresis
property, good biocompatibility for fibroblast, and bacteriostatic activity against gram-positive
of
microorganisms.
ro
Tunable amorphous structure and mechanical property, freeze drying at various temperatures of -20
scaffold
crystalline structure at freeze dry temperature > -9
In vitro
re
Amorphous SF
-p
⁰C to -5 ⁰C, water in-soluble behavior with ⁰C, water soluble with minimal crystalline region at
[270]
lP
freeze dry temperature > -20 ⁰C, potential method for tuning mechanical and biological properties of SF
na
scaffolds.
EGF/SF fusion
In vitro wound model
Jo
protein
ur
Development of EGF- light chain of SF conjugated
Curcuminloaded SF hydrogel
In vitro
gene sequences in transgenic silkworm, prevention of draining of EGF 2.5 fold increase in proliferation of
[188]
fibroblasts, and accelerated wound healing. Fabrication of electro-responsive SF hydrogel loaded with curcumin, 3D scaffold with 80% porosity and 100% swelling ratio, antibacterial activity, and good biocompatibility for HaCaT.
[271]
Journal Pre-proof Fabricated by freeze-drying, high porosity (83%) and Citrus pectin/SF sponge
interconnectivity with pore size around 120 µm, high In vitro
water uptake ability (800% in 24 h) and low weight
[156]
loss (21.3% in 40 days) and cyto-biocompatibility for fibroblast cells.
honey/ESF mat
biocompatibility with improved viability of primary human dermal fibroblasts. Better water uptake and cell growth compared to
In vitro
ESF, and enhanced human dermal fibroblast
Fabrication of ESF by electrospinning, dropping
re
amniotic fluid-SA hydrogel onto ESF, sustained and
In vitro
Fabricated by electrospinning, top layer (SF-PCL)
structure
mimicked the dense nature and waterproof properties
ur
In vitro
of the natural epidermis, down layer mimic natural
[176]
structure of dermis, antibacterial activity with cyto-
Jo
thymol)
na
proliferation, migration and spreading in vitro.
Two-layered
PCL/SF-HA-
[175]
lP
localized delivery of amniotic fluid, improved cell
hydrogel/ESF
membrane (SF-
[128]
adhesion, growth and infiltration.
Amniotic fluidSA
[170]
of
Manuka
In vitro
ro
mat
Controllable and sustained release of hPL, and good
-p
hPL-loaded ESF
biocompatibility for human fibroblasts. Induced crystallization of SF, and increased absorption capacity and flexibility and of the SF
Glucose/SF film
In vitro
films with the addition of glucose in SF film, cytobiocompatibility with positive effect in promoting the wound closure.
[155]
Journal Pre-proof SF/PVA/collage
Fabricated by electrospinning, sustained and localized
n I peptide
delivery of S-Nitrosoglutathione, randomly oriented In vitro
loaded with S-
fibers with high porosity, increased cell proliferation with the presence of collagen I peptide, cyto-
ne
biocompatibility with high cell adhesion property.
lP
re
-p
ro
of
Nitrosoglutathio
[173]
Major findings
ur
Design of experiment (s)
Jo
Material (s)
na
Table 1b: In vivo investigations with SF or SF blends for skin tissue engineering.
References
In vivo fullPVA/CTS/S
thickness
Good biocompatibility, flexibility and softness,
F sponge
excisional
increased vascular ingrowth, accelerated wound healing.
wound in rat
[272]
Journal Pre-proof Transparent, easily available and sterilizable, cost effective and transparent, faster healing time, greater
In vivo fullSF film
collagen regeneration and higher biocompatibility than
thickness wound
[273]
conventional hydrocolloid dressing, similar or slightly
in mice
better healing effects compared to lyophilized porcine dermis. Excellent cyto-compatibility for human ADSCs,
scaffold
of
[274]
compared to CTS/SF and control, promoted
ro
freeze-dried
epithelialization in ADSCs-seeded CTS/SF (75:25 ratio)
excisional fullthickness
differentiation of ADSCs into fibro-vascular,
wounds in mice
endothelial, and epithelial cells after culture on CTS/SF
-p
CTS/SF
accelerated wound closure, angiogenesis, re-
In vivo
re
scaffold.
freeze-dried
excisional fullthickness wounds in mice
epithelialization in ADSCs-seeded CTS/SF (75:25 ratio)
[274]
compared to CTS/SF and control, promoted differentiation of ADSCs into fibro-vascular, endothelial, and epithelial cells after culture on CTS/SF
ur
scaffold
accelerated wound closure, angiogenesis, re-
In vivo
na
CTS/SF
lP
Excellent cyto-compatibility for human ADSCs,
Jo
scaffold.
In vivo fullPorous SF
thickness
film
excisional wounds in rat
In vivo fullpHA/SF gel
thickness porcine wound
SF film with porous structure, increased re-
[275]
epithelialization, connective tissue thickness and neovascularization.
3D structure with furry texture and high porosity compared to SF and HA/SF gels, better reepithelialization, matrix formation and wound healing compared to SF and HA/SF gels.
[276]
Journal Pre-proof Recombina
In vivo deep
nt spider
second-degree
silk porous
burn wounds in
sheet
Recombinant silk proteins containing RGD (Arg-Gly-
[277]
Asp) motifs, increased bFGF expression, better wound healing and collagen synthesis compared to control.
rat
Cross-linking with genipin, decreased pore size,
porous scaffold
swelling ratios, degradation and release rates after crossIn vivo burn
linking, excellent cyto-biocompatibility for fibroblasts,
wounds
[278]
accelerated wound closure, re-epithelialization and
of
SF/elastin
wound healing compared to commercially available
ro
collagen base wound dressing Suprasorb C.
-p
Cross-linking with various concentration of GA, improved mechanical properties compared to SF
bi-layered wound dressing
re
controllable biodegradation rate depending cross-linking
thickness
lP
ericin/wax
without wax, homogeneous porous structure,
In vivo full-
degree, good biocompatibility for L929 mouse
excisional wounds in rat
[279]
fibroblasts, better wound closure, re-epithelialization,
na
SF/gelatin/s
collagen synthesis, and wound healing compared to commercially available wound dressing 3MTM
Jo
ur
Tegaderm.
SF/CS/hyal
In vivo full-
uronic acid
thickness
porous
excisional
scaffold
wounds in rat
A ternary scaffold with pore diameter and porosity of 95–248 µm and 88–93%, respectively, promoted cell adhesion, proliferation and survival, good neovascularization, and wound healing in animals implanted with SF/CS/HA(80/5/15 ratio).
[280]
Journal Pre-proof Functionalized with EGF and silver sulfadiazine by drug SF scaffolds (film, silk film, lamellar porous silk
loading or coating techniques, better in vivo reepithelialization, wound closure, dermal proliferation
In vivo full-
and wound healing rate for all functionalized SF
thickness
scaffolds and two incorporated techniques compared to
excisional
two commercially available wound dressings, better
wound in mice
wound healing for SF scaffolds (all forms)
ESF)
functionalized with both EGF/silver sulfadiazine compared to other groups.
of
film and
ro
SF fibers with different diameters (250-1200 nm),
Ex vivo full-
enhanced cell proliferation and migration in samples
thickness wound
with smaller fiber diameter, increased extracellular
healing model
re
matrix proteins in samples with smaller fiber diameter. Increased adhesion and proliferation of mouse
lP
In vivo fullthickness
fibroblasts (L929) compared to PLGA nanofibrous mat,
nanofibrous
excisional
speed up wound healing compared to commercially
mat
wound in
nanomatrix mat
Fabrication by salt-leaching electrospinning methods,
thickness
controllable pore size and morphology, well infiltration
excisional
of cells into ESF nanomatrix, superior healing potential
wound in rat In vivo
ESF mat
available wound dressing Comfeel.
ur
In vivo Full-
Jo
ESF
[281]
na
SF/PLGA
diabetic rat
[132]
-p
ESF mat
[186]
[127]
compared to Matriderm®. Homogenous morphology, excellent support of cell
subcutaneous
adhesion and proliferation of rBMCs and good in vivo
implant in rat
biocompatibility.
[59]
Journal Pre-proof Mean pore sizes of 50–200 μm, strong intermolecular In vivo fullCTS/SSF
hydrogen bonding, good biocompatibility, broad
thickness skin
spectrum antibacterial activity, improved mechanical
wound in rat
behavior, accelerated skin wound regeneration of rats
[116]
compared to SSF and CTS alone.
electrospun mat
properties for immortalized human fibroblast cells in
partial-thickness vitro, accelerated wound closure and neovascularization burn wound in
in vivo, organized collagen formation, reduced scar
rat
formation and excellent topical delivery system for burn wound healing.
Increased biomechanical property compared to CTS
In vivo full-
membrane, favorable biocompatibility, accelerated re-
re
CTS/mSF
[185]
of
e IV
In vivo deep
ro
astragalosid
Promising cell adhesion and good biocompatibility
-p
SF/Gelatin/
thickness skin
epithelialization, wound closure and wound healing
lP
wound in rat
[141]
compared to pure CTS membrane.
na
Average fiber diameter of 320 and 502 nm for pure ESF and ESF/PLA mats, respectively, lower water uptake and vapor transmission rate in ESF/PLA compared to
ur
mat
thickness in rat
Jo
ESF/PLA
In vivo full-
model
pure ESF mat, higher hydrophilicity of ESF compared
[161]
to ESF/PLA, high antibacterial activity of tetracycline hydrochloride-loaded ESF and ESF/PLA, accelerated wound healing in ESF and ESF/PLA groups compared to control wounds treated with gauze. Fibrous foam-like architecture, sufficient porosity and in
In vivo fullIsabgol/SF
vitro biodegradation, support NIH 3T3 fibroblast cells
thickness wound growth and adhesion, accelerated neovascularization, rein rat model
epithelialization, collagen deposition, wound contraction and healing in 2 % isabgol/2 % SF (Isabgol/SF 75/25).
[146]
Journal Pre-proof Excellent healing potential of both SF and SF/Gel SF and SF/Gelatin microcarrier s
microcarriers compared to control, increased infiltration
In vivo deep
of immune cells to wound area, minimal fibrosis
skin wound in
formation, increased expression of pro-inflammatory
mouse
[180]
cytokines TNF, IL-6 and IL-1β, and increased expression of chemokines CXCL1 and CXCL2.
d
In vivo burn
mechanical property for wound healing applications,
wound in rat
better burn wound closure and healing compared to gauze and Neoderm®.
BG/CTS/SF
Excellent biocompatibility, favorable mechanical
-p
In vivo full-
thickness wound property, vascularization and new blood vessel
Wet gold nanoparticle -loaded ESF mat
lP
Fabrication by a facile two-step freeze-drying
full-thickness
technology, increased dermal cell viability, proliferation
wound in rat
In vivo full-
thickness wound in rat
[134]
na
mat
In vivo dorsal
and migration, and accelerated in vivo wound healing.
ur
fibrous SF
formation, and accelerated wound healing.
Jo
/nano-
[27]
re
in rat Micro-
[109]
of
Hydrocolloi
Structural stability, good cyto-compatibility, suitable
ro
SF/CMC
Increased degradation profiles, improved biomechanical property, neovascularization and granulation tissue formation, and elongation at break values similar to normal skin.
[163]
Journal Pre-proof Fabrication by cold-plate electrospinning and automatic SF particleincorporate d PCL electrospun mats
magnet agitation system, 3D porous mats with thickness In vivo full-
up to 6 mm, interconnected macro-pores ranging from
thickness
tens to hundreds μm, homogenous distribution of SF
wounds in rat
nanoparticles within PCL mat, good biocompatibility,
model
higher wound healing rate and collagen deposition in
[164]
PCL/SF mat with higher content of SF particles, great
of
potential as artificial skin. Fabrication by SLE method, higher porosity compared
ro
wound closure, organized collagen matrix deposition,
model in rat
and decreased expression of pro-inflammatory
re
cytokines.
In vivo
retention capacity (440%), water vapor transmission rate (~2330 g m-2 day-1), sustained release of EGF for 3
excisional full-
na
ESF loaded
Good cyto-compatibility for HaCaT cells, high water
lP
Nonmulberry
with EGF
thickness wound days, high elasticity (~2.6 MPa), controllable and
and
in New Zealand white rabbit
[214]
sustained drug release, strong antibacterial activity, and accelerated re-epithelialization, neovascularization and
ur
ciprofloxaci
[113]
-p
ESF mat
to ESF nanosheets, accelerated re-epithelialization and
In vivo burn
wound healing.
Jo
n
In vivo fullSilkworm
thickness wound Good biocompatibility, antibacterial activity,
cocoon sol-
in wounds New
transparent, accelerated wound healing rate compared to
gel film
Zealand white
standard Mepitel® dressing.
rabbit
[122]
Journal Pre-proof Synthesis of growth factor-SF gene construct in transgenic silkworm by genetic engineering technique, secretion of growth factor-SF proteins from transgenic FGF-, EGF-
PDGF- and VEGFfunctionaliz ed silk
and in vivo wound healing potential of EGF-, FGF- or
In vivo full-
VEGF-functionalized silk membranes compared to
thickness
native, PDGF- and KGF-functionalized silk membranes,
excisional
[190]
accelerated keratinocyte cell growth and migration of
wounds in mice
KGF- and VEGF-functionalized silk membranes,
of
, KGF-,
silkworm, good biocompatibility in vitro, better in vitro
membrane
ro
increased adhesion of macrophage for EGF- and VEGFsilk membranes, and slightly better wound healing
-p
potential of VEGF-functionalized silk membrane
Fabrication by 3D bioprinting, favorable biomechanical
In vivo fullthickness
onated
excisional
SF/FGF
wound in rat
property, biocompatibility and cell adhesion for CFFs,
lP
Gelatin/sulf
increased cell migration in presence of FGF, accelerated
[193]
granulation, healing, and neo-vascularization in wound
na
3D
re
compared to other groups.
bed in presence of FGF.
ur
Comparison of two commercially burn wound
Dressilk vs Biobrane
Jo
dressings, Dressilk (PREVOR, France), made from
In vivo
superficial burns in 30 patients
silkworm silk, Biobrane (Smith and Nephew ltd, Hull, UK), a silicon based wound dressing coated with nylon mesh and porcine dermal collagen-derived peptides, both Dressilk and Biobrane provide a safe and effective microenvironment for burn wound healing, and also Dressilk is cost effective
[123]
Journal Pre-proof ESF/COL and CTS/COL nanofibrous
In vivo full-
Support proliferation and adhesion of fibroblasts and
thickness wound keratinocytes, good biocompatibility, and promote in rat
[135]
wound healing compared to gauze dressing.
mat Synthesis by green electrospinning technique, smooth
mat
excellent biocompatibility, increased average fiber
thickness wound
of
honey/ESF
surface morphology, high antibacterial activity,
In vivo full-
diameter with the increase of Manuka honey
in rat
[130]
concentration in construct, and accelerated wound
ro
Manuka
-p
healing.
Excellent biocompatibility and cell growth in PRP/SF
PRP/SF and PPP/SF compared to cocoon mat, enhanced
thickness wound
wound healing in PRP/SF and PPP/SF cocoon mat, and
in rabbit
[137]
better wound healing results in PRP/SF compared to
na
mats
re
PPP/SF
and PPP/SF cocoon mats, increased hydrophilicity in
In vivo full-
lP
PRP/SF and
PPP-SF and control.
ur
Fabrication by freeze drying, amorphous and β-sheet structures, pore radius and porosity ranging from 32.22
sponge
In vivo full-
Jo
SF/HA
μm to 290.76 μm and 74.1 % to 91.15 %, respectively,
thickness
good cyto-compatibility, improved fibroblast cell
wounds in rat
adhesion and proliferation compared to only SF,
[282]
accelerated vessel-like formation and wound healing compared to SF.
TOCN/SF nanofibrous mat
In vivo fullthickness excisional wound in rat
Improved mechanical and wound healing effectiveness compared to TOCN, good wound healing rate in TOCN/SF 2% compared to TOCN nanofibers.
[165]
Journal Pre-proof In vivo full
Potential antioxidant scaffold, better thermal and
Fenugreek-
thickness
biomechanical properties and cell proliferation in vitro
incorporate
excisional
compared to ESF, increased re-epithelialization,
d ESF mat
wounds in rat
collagen deposition and wound healing rate compared to
model
ESF. Encapsulation of insulin in SF microparticles by coaxial
InsulinIn vivo dorsal
ed SF
full-thickness
up to 28 days, increased migration of human keratinocyte and endothelial cells in vitro in insulin-
diabetic wound
[192]
functionalized SF scaffolds, accelerated wound closure,
ro
es-loaded
electrospraying technique, sustained release of insulin
of
functionaliz
microparticl
[168]
in rat
neo-vascularization and healing in insulin-SF scaffold
SF sponge
-p
compared to other experimental groups.
SF/Elastin protein
granulation tissue formation in both sponge and aqueous
lP
nt
In vivo fullthickness skin
forms, ability of wound exudate absorption and gel
wound in guinea formation after application on wound compared to
[151]
na
Recombina
re
Ability of self-assembly from liquid to gel, improved
pig
aqueous form, and better potential as wound dressing
encapsulate d liposome/S F hydrogel
SF solution
99.8 ± 0.5 nm diameter and −9.41 ± 0.10 mV zeta
In vivo deep
potential of bFGF-encapsulated liposome, sustained and
second-degree
controlled release of bFGF, stable and sustained release
scald wound in
of bFGF in wound fluid, accelerated wound healing
Jo
bFGF-
ur
compared to its aqueous form.
mouse
[191]
compared to SF hydrogel and bFGF-encapsulated liposome.
in vivo partial-
Increased viability and migration of NIH3T3 cells,
thickness
accelerated in vitro wound healing though NF-kB
excisional
signaling pathway and in vivo wound healing via NF-kB
wound in rat
regulated proteins.
[283]
Journal Pre-proof
Arg‐Gly‐ Asp motif-
thickness
containing
excisional
SF
Secretion by transgenic silkworm, accelerated migration
In vivo full-
and proliferation of NHDF compared to wild-type silkworm, good re-epithelialization, wound closure,
[187]
neovascularization, and wound healing compared to SF
wound in mouse
secreted by wild-type silkworm.
CTS/SF hydrogel
ZWP and
In vivo diabetic
collagen synthesis and wound healing in PRP-
of
loaded with
Good cyto-compatibility, higher neovascularization,
wound in rat
Exos/ZWP-loaded CTS/SF compared to PRP exosome or ZWP-loaded CTS/SF hydrogel sponge.
PRP
CTS/SF
ADSC
wound in rat
re
Promising delivery system for ADSCs, accelerated collagen synthesis, neovascularization and wound healing.
ur
seeded with
In vivo diabetic
[182]
wound healing rate compared to CMC gel.
Jo
scaffold
biocompatibility, and higher re-epithelialization and
wound in mouse
lP
hydrogel
Promising mechanical property, excellent
In vivo diabetic
na
conjugate
-p
exosomes SF-elastin
[171]
ro
sponge
[154]
Journal Pre-proof Decellularization and solubilization of placenta using SDS and urea buffer, respectively, preparation of different ratios of SF/pECM sponge, retained various In vivo full
cytokines, growth factors and ECM proteins after
SF/pECM
thickness
decellularization process and scaffold fabrication,
sponge
excisional
improved adhesion, proliferation and migration of HFFs,
wound in rat
HEKs and HAMSCs with favorable biocompatibility in
[152]
vitro and in vivo, and significant increase in re-
of
epithelialization, wound closure, neovascularization,
ro
wound healing compared to SF sponge.
Used SF from two different sources (mulberry and nonFunctionaliz
-p
mulberry), excellent in vitro biocompatibility for HDF In vivo diabetic
and HaCaT cells, increased cell migration after
EGF, bFGF
and not-diabetic
treatment with combination of EGF, bFGF and LL-37,
and LL-37
full-thickness
electrospun
excisional
lP
wound in rabbit
faster granulation, wound closure, organized ECM
[284]
formation, improved re-epithelialization and angiogenesis in functionalized non-mulberry SF/PVA
na
mat
re
ed PVA/SF/
mats compared to control, functionalized PVA and
layered artificial skin
In vivo 3rd burn
Jo
AM/ESF bi-
ur
functionalized PVA/mulberry SF.
wound on mouse
Decreased post-burn scar formation, accelerated neovascularization, wound closure and wound healing.
[69]
Journal Pre-proof ESF,
Fabrication by single-spinneret electrospinning
ESF/PVA,
technique, anti-adhesion property of the blends as
ESF/PEG,
In vivo trauma
following order: PVA/ESF> pure ESF> ESF/PEG
ESF/PEO
mouse model
~ESF/PEO, the highest collagen regeneration and
[167]
nonwoven
wound healing rate in ESF and PVA/ESF compared to
mat
others.
CTS/SF
ZWP and
In vivo diabetic
collagen synthesis and wound healing in PRP-
wound in rat
Exos/ZWP-loaded CTS/SF compared to PRP exosome or
ro
loaded with
Good cyto-compatibility, higher neovascularization, [171]
ZWP-loaded CTS/SF hydrogel sponge.
-p
sponge
of
hydrogel
PRP
re
exosomes
lP
Fabricated from two species of silks (AsSF and BmSF, ratio 1:1), irreversible gelation at 37 ⁰C due to β-sheet
degree burn wound in rat
structures formation, cyto-biocompatibility for primary HDF and improved migration of keratinocytes and re-
[184]
epithelialization in vitro, accelerated inflammation,
ur
SF hydrogel
In vivo third-
na
Injectable
Jo
proliferation and remodeling phases of wound healing
In vivo fullAnisotropic
thickness
ESF
excisional wound in rat
process in vivo. Fabrication by electrospinning, inducing anisotropic features by aligning the fibers in an electric field, improved
cyto-compatibility,
cell
migration
and
vascularization in vitro, accelerated wound closure and healing during 28 days follow up.
[174]
Journal Pre-proof Fabrication by freeze drying method, 92% porosity, 93 SF/HA/SA sponge
μm average pore diameter size, soft and elastic
In vivo full thickness burn
characteristics,
wound in rat
biocompatibility
good
physical
stability,
cyto-
for
NIH-3T3
fibroblast
cells,
[157]
accelerated burn wound healing during 21 follow up. Fabricated by freeze drying method, average pore size
Neurotensin
and porosity of 40–80 μm and ∼85%, respectively,
gelatin
excisional
microsphere
diabetic foot
s releasing
wound in rat
accelerated wound healing in diabetic foot ulcers through
of
In vivo
increased wound closure, fibroblast accumulation and tissue granulation at the wound site with minimal scar formation, suggest as drug delivery system and wound
SF sponge
-p
dressing.
Modification of acellular dermis by dip-coating at
goat
thickness
different concentrations of SF aqueous solutions,
acellular
excisional
synergetic effects of SF and acellular dermal matrix on
dermal
wound in albino
wound healing, improved wound healing in vivo
matrix
mice In vivo
lP
compared to un-modified matrix.
excisional
artificial
wound in rabbit
Jo
layered
skin
ear
[183]
na
AM/ESF bi-
re
In vivo Full-
ur
SF modified
[158]
ro
-loaded
Increased re-epithelialization, wound healing, decreased hypertrophic scar formation in the wounds treated with bilayer membrane compared to AM implanted wound.
[140]
Journal Pre-proof Table 1c: Clinical trials being carried out with SF or SF blends for skin tissue engineering.
Material (s)
Design of
Major findings
experiment (s)
References
Randomized Bilayered
trial on split-
Faster wound healing rate and less pain than Bactigras,
SF/Gelatin
thickness skin
a standard wound dressing.
[115]
of
graft donor site Roughness and transparency of SF film compared to Suprathel and Sidaiyi, two commercially available
thickness
wound dressing, as follows: Sidaiyi>Suprathel>SF
wound in
film and SF film>Suprathel> Sidaiyi, respectively,
-p
ro
In vivo full-
rabbit, porcine
re
waterproof, biocompatible, and effective barrier
and
against bacterial entrance, superior gas permeability
randomized
and fluid handling capacity than Suprathel and Sidaiyi,
single blind
SF film with thickness of 64.9 µm, thinner than
[121]
lP
SF film
na
clinical trial on Suprathel (266 µm) and Sidaiyi (1789 µm), better and 71 patients faster wound healing in both animal and clinical trial
Jo
ur
studies compared to Suprathel and Sidaiyi
Abbreviations used in the Table 1(a-c): A. assama silk fibroin (AaSF) ; Alginate dialdehyde (ADA); adipose tissue-derived mesenchymal stem cells (ADSCs); AM (amniotic membrane); Bacterial cellulose (BC); Bombyx mori (Bm); B. Mori silk fibroin (BMSF); N-carboxyethyl chitosan (CECTS); child foreskin fibroblasts (CFFs); carboxymethyl cellulose (CMC); Chondroitin sulfate (CS); Chitosan (CTS); 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC); epidermal growth factor (EGF); freeze-drying (FD); glutaraldehyde (GA); Immortalized human keratinocyte (HaCaT); human amniotic and membrane-derived stem cells (HAMSCs); Human dermal fibroblast (HDF); human epidermal keratinocytes (HEKs); human foreskin fibroblasts (HFFs); Human platelet lysate (hPL); N-(2-hydroxy)propyl-3-trimethyl ammonium chitosan chloride (HTCC); hyaluronic acid (HA); L-proline (LP); normal human epidermal fibroblasts (NHEF); normal human epidermal keratinocytes (NHEK); Neurotensin (NT); Sodium alginate (SA); salt-leaching (SL), spunlaced SF (SSF), Salt-leaching electrospinning (SLE),
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Jo
ur
na
lP
re
-p
ro
of
strontium (Sr); oxidized cellulose (TOCN); human placenta-derived extracellular matrix (pECM); polyethylene glycol (PEG); polyethylene oxide (PEO); Polarized Hydroxyapatite (pHA); poly-Llactic acid (PLA); poly(lactide-co-glycolic acid) (PLGA); platelet-poor plasma (PPP); platelet-rich plasma (PRP); P. ricini silk fibroin (PRSF); poly(vinyl alcohol) (PVA); Homogeneous polysaccharide extracted from the rhizomes of Curcuma zedoaria (ZWP)
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Tables 2 (a-b). In vitro and in vivo experimentations on antibacterial effects of SF or SF blend scaffolds prepared alone and/or adding with other bioactive ingredients.
Table 2a. In vitro experiments are being carried out on antibacterial effects of SF or SF blend scaffolds prepared alone and/or adding with other bioactive ingredients.
SF loaded with antibiotics
ro
ESF mat
[195]
Good cyto-compatibility and strong antibacterial activity against E. coli
[262]
-p
TiO2-loaded
High antibacterial activity against S. aureus and P. aeruginosa growth.
Controllable and sustained antibiotic release, good cyto-compatibility and strong antibacterial activity.
re
mat
References
[197]
lP
ESF/AgNPs
Major findings
of
Material (s)
na
Bionanotextile ESF incorporated with various concentrations of PEI PEI-loaded
(10, 20 and 30 % (w/w)), Further functionalization with sulphate
ESF mat
group, good cyto-compatibility for L929 fibroblasts cancer cell line,
[210]
ur
strong antibacterial activity against S. aureus and P. aeruginosa.
Jo
Fabrication of transgenic silkworm carrying SF-AMP conjugated acid Transgenic
nucleic sequences, secretion of SF-AMP proteins by transgenic
SF-AMP mat
silkworm, high antibacterial activity, low attachment property for bacteria, excellent biocompatibility, and intrinsic antibacterial activity.
[217]
Journal Pre-proof Coating of AgNPs on silk fibers by two in situ and ex situ methods, enhanced thermal and mechanical properties of silk fibers-AgNPs mat AgNPs-
in both fabrication methods, presence of the crystalline AgNPs on silk
loaded Silk
fibers, excellent biocompatibility of the mats fabricated by both
fibrous mat
methods for 3T3 fibroblasts, strong antibacterial activity against P.
[198]
aeruginosa and S. aureus, better antibacterial activity of the mats fabricated by in situ method compared to ex situ.
Vera mat
of
antibacterial agent, with silk fabric using 1, 2, 3, 4-
butanetetracarboxylic acid as cross-linker, good antibacterial behavior
[285]
ro
fabric/Aloe-
Chemical binding of various concentrations of Aloe-Vera, as an
in 15% Aloe-Vera/SF fabric.
-p
Silk
Immobilization of KR12 on ESF membrane, strong antibacterial activity against S. aureus, S. epidermidis, E. coli and P. aeruginosa,
nanofibrous
good biocompatibility for human dermal fibroblasts and keratinocytes
mat
in vitro, suppressed expression of TNF-α from LPS-induced murine
na
monocytes. ESF/Seleniu
Antibacterial activity against S. aureus and S. epidermidis, in vitro
nanoparticles
biocompatibility for human dermal fibroblasts.
Nanoparticles /ESF mat CTZ/SF/Gela tin nanofibrous mat
[204]
Jo
Selenium
ur
m
mat
[196]
lP
re
ESF/KR12
Excellent antibacterial activity against S. aureus, and cyto-compatible for human dermal fibroblast.
[201]
Increased average diameter of fibers with the increase of CTZ content, controllable and sustained release of CTZ, strong antibacterial activity against P. aeruginosa, and good biocompatibility.
[211]
Journal Pre-proof Colloidal Ag/Ciproflox
Gradual release of antibiotic and high antibacterial activity against S.
acin/ SF/SA
aureus and E. coli.
[147]
sponge Sr-loaded
High antibacterial activity, and excellent cyto-compatibility for
SF/SA film
fibroblasts.
hydrogel
methacrylate, excellent delivery system for controlled release of
of
loaded SF
Improved mechanical properties using 2-(N,N-dimethylamino) ethyl [226]
cefixime, high degree of water uptake, improved tensile strength and
ro
Cefixime-
[148]
elongation at break, high antibacterial activity.
-p
Decreased compressive modulus property with the increase of SF and
re
GS concentrations, increased water swelling and weight loss with addition of GS, accumulative release of GS in higher content of SF,
n sponge
excellent antibacterial activity against S. aureus, S. epidermidis, M.
[221]
lP
GS/SF/Gelati
except GS/SF.
na
luteus, B. cereus and P. aeruginosa, no cytotoxicity effects for NHDF
ur
Fabrication by freeze-drying method, high cyto-compatibility for HFFF2 cells, promising swelling and water uptake, good blood
NPs 3D
clotting, biodegradability and mechanical properties with high porosity,
sponge
and strong antimicrobial activity against E. coli, S. aureus, and
[223]
Jo
chitin/SF/Ag
Candida albicans.
Fabrication by freeze-drying method, good cyto-compatibility for chitin/SF/TiO 2
3D sponge
HFFF2 cells, promising swelling and water uptake (93%), good blood clotting, biodegradability and mechanical properties with high porosity (>90%), and strong antimicrobial activity against E. coli, S. aureus, and Candida albicans.
[286]
Journal Pre-proof Fabrication of transgenic silkworm carrying SF-AMP conjugated acid Transgenic
nucleic sequences, secretion of SF-AMP proteins by transgenic
SF/AMP mat
silkworm, good intrinsic antibacterial activity against E. coli, excellent
[216]
biocompatibility. Excellent antibacterial activity against pathogen bacteria such as S. Ag2O-loaded
aureus and M. tuberculosis, high antibacterial activity against non-
SF spun mat
pathogen (E. coli) bacteria, good biocompatibility of the Ag2O-SF spun
[209]
of
for 3T3 fibroblasts and excellent in vitro wound healing potential. Fabricated by electrospinning, antibacterial activity against P.
coated by
aeruginosa, S. epidermidis, S. aureus and E. coli, cyto-compatibility for
AgNPs
L929 skin fibroblast
scaffold
-p
A SF spongy layer covered by ESF loaded with silver sulfadiazine,
re
based
[218]
antibacterial activity against S. aureus.
[219]
lP
Bilayered SF-
ro
Tasar ESF
na
Fabricated by electrospinning, different concentrations of nano-Cu2O (0.5, 1.0, and 5.0 mg/mL), bead formation in higher concentration of nano-Cu2O, decreased uniformity. Showed increase hydrophilicity of
PEO–SF
mat morphology with the increase of nano-Cu2O concentrations,
electrospun
increased antibacterial activity of composite electrospun mat against E.
mat
coli and S. aureus bacteria with the increase of nano-Cu2O
[220]
Jo
ur
nano-Cu2O-
concentrations, no detectable cytotoxicity against pig iliac endothelial cells.
Table 2b. In vivo studies are being carried out on antibacterial effects of SF or SF blend scaffolds prepared alone and/or adding with other bioactive ingredients. Material (s)
Major findings
References
Journal Pre-proof
Sulfadiazin e silverloaded ESF mat
Final ESF mat containing various silver sulfadiazine contents (0.1, 0.5 and 1.0 wt %), decreased adhesion and spreading of human epidermal keratinocytes and epidermal fibroblasts with the increase of silver sulfadiazine concentration in mat and minimum cell adhesion and better wound healing in 1.0 wt % mat compared to 0.1 wt % and 0.5 wt%. Strong antibacterial activity against S. aureus, accelerated epidermal cell
AgNPs/SF
growth, excellent wound healing potential compared to a commercially
gel
available wound healing gel (soframycin gel).
ciprofloxaci
stable drug release with high antibacterial activity compared to unmodified film, and accelerated wound healing.
ciprofloxaci n mat
cells, strong antibacterial activity against E. coli, S. aureus, P, aeruginosa
ur
and
60% ciprofloxacin release within first 6 h, cyto-compatibility for HaCaT
and S. epidermidis and accelerated re-epithelialization, neovascularization
[214]
and wound healing.
Jo
with EGF
na
Non-
ESF loaded
[202]
lP
n sponge
mulberry
-p
Lower burst drug release compared to unmodified film, excellent and more
re
loaded with
ro
PEG
SF/Gelatin
[203]
of
Gelatin
modified-
[207]
High porosity with good wettability, appropriate mechanical property, Wetable
strong antibacterial activity against S. aureus, E. coli, P. aeruginosa and M.
CTS/SF/Ag
albicans and good cyto-compatibility for L929 cells, human fibroblasts and
NPs sponge
hUCMSCs in vitro and high antibacterial activity in vivo and no post wound infection.
[142]
Journal Pre-proof Col/SF/Ag
Increased neovascularization in vivo, accelerated re-epithelialization and
NPs sponge
wound healing and no post-injury infections in wound bed or edge.
MoSe2/SF nanosheet film
Two dimentional nanosheet film, high antibacterial activity, and great potential as wound disinfectant and wound healing.
Thin film formation of CTS on SF fabric by spraying, connective pores of
SF/CTS
CTS thin film over the SF fabric, strong antibacterial activity, no
mat
cytotoxicity and high potential wound healing compared to TegadermTM.
[215]
LL37
ro
epithelium in SF/AgNPs solution containing 384 mg/L AgNPs.
Sustained delivery of LL-37, excellent antibacterial activity against S. epidermidis and P. aeruginosa, biocompatibility and wound healing
[284]
potential in vitro and in vivo.
na
nanofibrous
[225]
-p
ESF/PVA/
activity in vitro, complete elimination of biofilms and repair of defected
re
solution
Good for treatment of biofilm-associated diseases, strong antibacterial
lP
SF/AgNPs
ur
Abbreviations used for Tables 2 (a-b): Silver oxide nanoparticles (Ag2O-SF); silver nanoparticles (AgNPs); antimicrobial peptide (AMP); Bacillus cereus (B. cereus); Collagen (COL); chitosan (CTS); ceftazidime (CTZ); electrospun silk fibroin (ESF); gentamicin sulfate
Jo
mat
[119]
of
Bi-layered
[222]
(GS); human caucasian foetal foreskin fibroblast (HFFF2); human umbilical cord mesenchymal stem cells (hUCMSCs); Monilia albicans (M. albicans); Micrococcus luteus (M. luteus); Molybdenum diselenide (MoSe2); Mycobacterium tuberculosis (M. tuberculosis); normal human dermal fibroblasts (NHDF); Pseudomonas aeruginosa (P. aeruginosa); polyethylenimine (PEI); poly(ethylene oxide) (PEO); poly(vinyl alcohol) (PVA); Sodium alginate (SA); Staphylococcus aureus (S. aureus); Staphylococcus epidermidis (S. epidermidis,); Strontium (Sr).
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6