Naturally derived proteins and glycosaminoglycan scaffolds for tissue engineering applications

Accepted Manuscript Naturally derived proteins and glycosaminoglycan scaffolds for tissue engineering applications

Nehar Celikkin, Chiara Rinoldi, Marco Costantini, Marcella Trombetta, Alberto Rainer, Wojciech Swieszkowski PII: DOI: Reference:

S0928-4931(16)32246-9 doi: 10.1016/j.msec.2017.04.016 MSC 7837

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

21 November 2016 2 April 2017 3 April 2017

Please cite this article as: Nehar Celikkin, Chiara Rinoldi, Marco Costantini, Marcella Trombetta, Alberto Rainer, Wojciech Swieszkowski , Naturally derived proteins and glycosaminoglycan scaffolds for tissue engineering applications. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.04.016

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ACCEPTED MANUSCRIPT Naturally Derived Proteins and Glycosaminoglycan Scaffolds for Tissue Engineering Applications Nehar Celikkin1,†, Chiara Rinoldi1,†, Marco Costantini2, Marcella Trombetta2, Alberto Rainer2, Wojciech Swieszkowski1*. University of Technology, Faculty of Material Science and Engineering, 141 Woloska str., 02507, Warsaw, Poland. 2Tissue Engineering Unit, Department of Engineering, Università Campus Bio-Medico di Roma, Via Alvaro del Portillo 21, 00128 Rome, Italy

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1 Warsaw

† These authors contributed equally to this work

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Tissue engineering (TE) aims to mimic the complex environment where organogenesis takes place via fabricating advanced materials in vitro, which can recapitulate organ or tissue functions. Cells, three-dimensional scaffolds and signaling factors are the three main and essential components of tissue engineering. Over the years, materials and processes have become more and more sophisticated, allowing researchers to precisely tailor the final chemical, mechanical, structural and biological features of the designed scaffolds. In this review, we will pose the attention on two specific classes of naturally derived polymers: fibrous proteins and glycosaminoglycans (GAGs). These materials hold great promise for advances in the field of regenerative medicine as i) they generally undergo a fast remodeling in vivo favoring neovascularization and functional cells organization and ii) they elicit a negligible immune reaction preventing severe inflammatory response, both critical requirements for a successful integration of engineered scaffolds with the host tissue. We will discuss the recent achievements attained in the field of regenerative medicine by using proteins and GAGs, their merits and disadvantages and the ongoing challenges to move the current concepts to practical clinical application.

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Keywords: Natural polymers, hydrogel scaffolds, glycosaminoglycans (GAGs), fibrous proteins, regenerative medicine * Corresponding Authors: Prof. Wojciech Swieszkowski, PhD Faculty of Material Science and Engineering, Warsaw University of Technology, 141 Woloska str., 02-507, Warsaw, Poland. E-mail: [email protected]

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ACCEPTED MANUSCRIPT 1.Introduction The term Tissue Engineering - as it is nowadays used - was firstly introduced in 1988 by Professor Robert Nerem at “UCLA Symposia on Molecular and Cellular Biology”. During the symposium, the participants discussed about a comprehensive definition for this emerging science and they finally agreed on the following: “Tissue Engineering (TE) is the application of

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the principles and methods of engineering and life sciences toward the fundamental understanding of structure-function relationships in normal and pathologic mammalian tissue and the development of biological substitutes to restore, maintain or improve function in defective or lost tissue due to different disease conditions” [1]. This definition laid the

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foundations of a new broad and multidisciplinary science that in the last three decades has grown enormously in complexity and nowadays requires a close collaboration among

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researchers with backgrounds in medicine, biology, chemistry, materials science, engineering, and physics [2]. Although TE has its foundation in many domains, at the very beginning

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biomaterials science gave the largest contribution to the creation and development of this field. In fact, several innovative techniques and approaches to process natural and synthetic

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biopolymers, metals and ceramics were rapidly developed allowing researchers to create 3D

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porous scaffolds to be tested as scaffolds for in vitro regeneration of tissues after cell seeding [3]. These new technologies were essential for the development of TE as they provided the

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first tools to researchers to move from 2D to 3D cell cultures. The possibility of culturing cells in 3D represented a milestone as finally researchers could abandon Petri dishes for 3D supports that could reproduce more accurately the environment in which cells grow, spread and differentiate. Among the available materials, naturally derived polymers

such

as

proteins—collagen,

fibrin,

fibroin,

gelatin,

elastin,

etc.—and

polysaccharides—alginate, hyaluronic acid, cellulose, chitosan, etc.—have always gained great attention

among

researchers

community

thanks

to

their

high

biocompatibility,

biodegradability and possibility to undergo fast remodeling in vivo [4–10]. Furthermore, 2

ACCEPTED MANUSCRIPT naturally derived polymers can be chemically modified thanks to the high number of easily reactive functional groups present along their backbone—mostly primary amine and carboxylic acid groups. Up to date, naturally derived polymers have been modified by the introduction of a large variety of chemical moieties, resulting in derivatives with improved adhesion, biodegradability and crosslinkability properties. An additional advantage offered by

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naturally derived polymers is the possibility to resuspend living cells in their solution and to process them in the form of hydrogels [10–13]. Cells encapsulation in such materials is particularly attracting in regenerative medicine and tissue engineering applications as they represent an ideal biomimetic environment in which cells can migrate and self-organize to

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form a functional micro/mini tissue.

In this review, we will present an overview of the most recent and advanced achievements

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attained in the field of tissue engineering (TE) and regenerative medicine (RM) by using fibrous proteins and glycosaminoglycans (GAGs), their advantages and demerits and the

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ongoing challenges to move the current concepts to practical clinical application. The

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presented review will be structured in two main sections: the first will discuss about fibrous proteins, while the second will focus on glycosaminoglycans as biomaterials for the

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production of biomedical scaffolds [14]. In each section, we will describe the achievements in

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TE and RM for a selection of polymers that belongs to each category. We will analyze in details their source of production, the possibility to be processed with the most popular and advanced technologies (i.e., 3D printing and bioprinting, microfluidics, electrospinning, etc.), their degradation mechanisms in vivo, their current clinical applications and the possibility to translate some of the recent research achievements into practical clinical application in the near, mid- and long-term future.

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ACCEPTED MANUSCRIPT 2. Fibrous proteins as 3D scaffold material Fibrous proteins are one of the major examples of nature’s ability in optimizing protein-based biomaterials with functional features for survival, mechanical support, or energy conversion. Fibrous proteins are the result of genetic blueprints, in which the final complex architectures are achieved via a self-assembly of tailored amino acids sequences.

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One of the most popular biomaterial design strategies consists of mimicking either the morphology or the chemical composition of the extracellular matrix (ECM). The ECM is a complex network of biomacromolecules including fibrous proteins and GAGs. In order to process biomaterials into engineered constructs that would eventually recapitulate some

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features of ECM morphology and/or its chemical composition, several methods have been developed and successfully tested so far. A schematic overview of the most common

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fabrication technologies, resulting scaffold morphologies and related cell culture strategies is

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shown in Figure 1.

Figure 1. Schematic overview of the most common strategies employed to process fibrous protein and GAG solutions (aqueous or in organic solvent) into engineered constructs for tissue engineering applications. 4

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The high number of accessible functional groups on protein backbone for modification and targeting ligands make proteins a great candidate for a variety of tissue engineering applications. Furthermore, the Arg-Gly-Asp (RGD), Ile-Lys-Val-Ala-Val (IKVAV), Tyr-Ile-GlySer-Arg (YIGSR) sequences (among the others), present in fibrous proteins, modulate and

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enhance cell adhesion. Among all proteins, collagen type I, gelatin—the denatured version of collagen—elastin and silk fibroin are the most commonly used scaffold materials. Collagens are the most abundant proteins of the ECM in vertebrates and play an important role in maintaining the biological and structural integrity of ECM. Up to date, 29 different collagen types have been found and classified based on their structure. The primary structure

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of collagen is a triple-stranded helix consisting of collagen polypeptide-alpha chains [15].

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Collagens can self-assemble and, depending on the chemical composition, can form strong, long crosslinked fibrils [16]. Collagen fibrils are resistant to tensile forces and hence are major

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constituents of connective tissue, skin, tendon, blood vessel, cornea, cartilage, and bone [17]. Collagen is a highly versatile material as it can be processed in numerous physical forms.

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Collagen-based materials can be fabricated into injectable gels, films, meshes, and fibers for

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biomedical application purposes including 3D cell culture, vascular and cardiovascular grafts [18,19], bone [20] and cartilage repair [21], skin grafts [22,23], corneal defects [24–26],

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peripheral nerve repair [27], spinal cord, and traumatic brain injury repair [28]. Gelatin is a natural biopolymer derived either by partial acidic (gelatin type A) or alkaline (gelatin type B) hydrolysis of collagen derived mainly from porcine or bovine skin, bones and tendons. The differences in collagen sources [29] and preparation techniques affect gelatin properties such as solubility, transparency, color, odor, taste, gelation temperature, amino acid composition and molecular weight distribution [30]. One of the biggest advantages of using gelatin consists in its high solubility in water at physiological temperature, allowing gelatin to be more easily processed in cell-friendly conditions compared to collagen. 5

ACCEPTED MANUSCRIPT It is well known that the chemical composition of ECM varies from one tissue to another, according to the biomechanical role that it has to exert. For example, the ECM of vascular and connective tissues is represented by a network of very elastic fibers as they have to bear constant cyclic stresses. These fibers provide ideal mechanical properties thanks to their ability to elongate and recoil after stretch. Elastic fibers are generally composed of cross-

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linked elastin surrounded by a variety of fibrous proteins. Elastin plays a critical role in the structural integrity of several tissues including skin, blood vessels, and other organs, and its structural dysfunctions might result in scarring, unusual wound contraction, loss of elasticity, leaky blood vessels, and loss of elastic properties of the skin [28].

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Biocompatible fibrous proteins can also be extracted from other natural sources. The major example is fibroin, a scleroprotein industrially obtained from silkworm silk after a

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degumming process. Fibroin has several major advantages over other protein based biomaterials, including low immunogenicity, extraordinary mechanical properties, low cost of

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production and purification, and slow degradation rate with long-term retention of strength.

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Thanks to the aforementioned very attractive characteristics, fibroin has been tested in vitro

2.1 Collagen

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and in vivo for the repair and regeneration of several tissues, especially load bearing tissues.

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Collagen is one of the main components of connective tissue. Its features of bioactivity, biodegradability, and cell-adhesive properties make it a highly preferable material in tissue engineering. The fundamental structural unit of collagen is a long (~ 300 nm), thin (~ 1.5 nm in diameter) right-handed triple helix composed of three left-handed single helices (two α1 and one α2 chains, Figure 2) [31].

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Figure 2. a,b) Collagen fundamental structural unit composed of three left-handed helices assembled to form a single right-handed helix. c) Typical collagen amino acid sequence: the glycine residue in the X-Y-Gly repeat is invariant in natural collagen with the most common triplet being Pro-Hyp-Gly (10.5%). Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the

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attachment of cells and matrix molecules. There are specific sites in collagens to which cells

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can attach, either directly or through protein intermediaries [32]. Often the interaction is indirect and mediated by matrix glycoproteins, however cells also express receptors, which

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can directly bind to the triple helical domains in collagens. Integrins are the largest family of cell adhesion receptors and integrin-type receptors are composed of one α and one β subunit—two transmembrane proteins that are non-covalently linked together. α1, α2, α10, and α11, are considered to form the subgroup of the vertebrate collagen receptor integrins [32,33]. Different integrin sub groups can be expressed by same cell types and they may have complementary or contrary effects in signaling. Thus, cellular response to collagens is dependent on the dominance of the receptors. Other members of collagen receptor family are Discoidin domain receptors 1 and 2 (DDR1 and DDR2) which are two closely related receptor 7

ACCEPTED MANUSCRIPT tyrosine kinases that are known to bind to collagens and act in an integrin-independent manner. There are also plasma membrane proteins that have been reported to bind to collagens. Glycoprotein VI (GPVI, p62) is a member of the paired immunoglobulin-like receptor family which is an important collagen receptor on platelets. Ligand binding to GPVI activates a series of signaling events regulating the activity of other receptors and the function

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of platelets during thrombosis.

The native collagen fiber is extremely resistant to proteolytic degradation at physiological pH for instance in adult rats the half-life values of collagenous structures has been determined beyond one year. However, under certain physiological conditions,

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previously stable collagen fibers can be rapidly removed by specific enzymes which appear to operate in a selective and conservative manner [34].

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Matrix metalloproteases (MMPs) or matrixins are a subfamily of metalloproteases, which consist of 23 distinct proteases in humans. MMPs are largely excreted proteins with

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several conserved domains. All MMPs contain the catalytic domain, which is shielded off in the

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inactive form of the enzyme by the prodomain. This propeptide interacts with the catalytic region through a conserved cysteine residue and the Zn2+ ion in the catalytic pocket. MMP-1,

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MMP-8 and MMP-13 are the water soluble MMPs which are responsible of enzymatic

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degradation of collagens [35].

Collagen can be easily shaped into different forms such as hydrogels [36–38], 3D sponges [39–41], micro/nano-fibers [42–44], and membranes [45,46]. As a consequence, collagen has been tested for a wide range of tissue engineering applications, such as osteochondral [21], cardio-vascular [18,19,47], skin [22,23], corneal [24–26], and urogenital [48] repair, besides its use in nerve guidance [23,49], wound dressing [50], and drug delivery systems [49,51] (Table 1). According to the architecture and the regeneration process of the aimed tissue, collagen is processed with different techniques. As an example, collagen vitrigel

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ACCEPTED MANUSCRIPT membranes have been studied by Tada et al. to accelerate the tracheal epithelium regeneration. Vitrigel is produced by rehydration after the vitrification of a traditional collagen hydrogel. The collagen vitrigel has been prepared in three steps involving gelation, vitrification, and rehydration processes to convert the vitrified material into a thin and transparent gel membrane with enhanced strength [52]. The study has shown that collagen

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vitrigel membranes promoted epithelial tissue growth more than collagen sponges due to improved strength and surface smoothness [53]. In another study, hybrid structures were prepared by infiltration of collagen hydrogel into commercial collagen sponges. Hybrid scaffolds were investigated about their effect on periodontal wound healing for class II

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furcation defects, showing early periodontal tissue formation due to an early exchange of space between the degrading scaffold and the periodontal cells [54]. Apart from the promising

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research in epithelial and periodontal tissue growth, collagen scaffolds have also been used in nerve tissue engineering and as nerve guides. It has been reported that the transplantation of

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hMSCs with commercially available collagen sponges enhanced the effect of hMSCs on axonal

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sprouting of corticospinal tract fibers into the denervated side of the spinal cord after traumatic brain injury [55]. Moreover, collagen scaffolds can also be conjugated with growth

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factors and drugs to improve the regeneration/integration of the engineered tissue. Ito et al.

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reported that b-FGF-impregnated collagen-gelatin sponges accelerated the regeneration of adipose tissue compared to pristine collagen sponges. The sustained release of the b-FGF from collagen-gelatin sponges has been demonstrated to induce angiogenesis and adipose stem cell proliferation [56]. Besides fibers, sponges, or hydrogels, a different technique to design 3D collagen structures has been proposed by Alekseeva et al. [57]. The technique, namely plastic compression (PC), involves rapid expulsion of water from cell-seeded collagen gels, in a single axis of flow. Cheema et al. suggested a rapid fabrication of living tissue models via building up layers of cell-collagen constructs with this technique. The authors demonstrated that PC

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ACCEPTED MANUSCRIPT collagen scaffolds showed collagen levels matching those of native bone, skin, and bladder tissues, and presented similar cellular responses [58]. Collagen is a suitable scaffold material for producing highly porous scaffolds and has been shown to support growth and function of many cell types. It can be manufactured into devices that are adhesive and can be sutured [59,60]. High porosity of collagen constructs provides volume for matrix accumulation and it

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appears that the fine collagen fibers can even integrate into the expanding neo-ECM [61]. Despite its favorable characteristics, for the regeneration of load-bearing tissues, such as bone, high mechanical strength is required to avoid scaffold fracture and to maintain the scaffold integrity during bone healing. To this aim, researchers have considered reinforcing

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hydrogels with a second, stiff phase such as carbon nanotubes [62,63] or HAp nanoparticles [64,65]. Silva et al. proposed a collagen/nanotube composite scaffold for bone tissue

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engineering thanks to its biofunctionality, bioactivity and higher tensile strength compared to pristine collagen scaffold [66]. Another common nano structure used to increase the

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mechanical strength of collagen scaffolds is represented by nano hydroxyapatite (nHAp)

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particles. Cunniffe et al. have shown that the suspension of nHAp into collagen scaffolds ensue an 18 fold increase in the tensile strength. Furthermore, the study showed that the tensile

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strength of the scaffolds increased with the increased concentration of nHAp particles without

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having a significant effect on scaffold porosity [64].

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ACCEPTED MANUSCRIPT Table 1. Current strategies of collagen-based materials in tissue engineering. Targeted organ/ tissue

Scaffold fabrication technique

Reference

 Kumar et al. [67]

Keratocyte (isolated from bovine eyes) -

 Highly condensed collagen membranes which mimic the native environment of cornea

 Guo et al. [25]

 Scaffolds led to simultaneous disruption of wound contraction, reduced scar formation, and enhanced induced regeneration

 Soller et al. [23]

-

 ECM mimicking hybrid material which amplified the regeneration capacity of growth factors

 Lee et al. [68]

HDF

 Efficient removal of excess crosslinker (glutaraldehyde)

 Liu et al. [22]

In vitro:  High cell attachment  Good cell proliferation In vivo (mice & rabbit model):  Not inducing cytotoxic environment  Less fatty scar tissue In vitro:  High seeding efficiency  High expression of cell-tocell junction and contractile proteins when

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 Scaffolds supported cell growth and cell attachment  Resistant to the chronic wound environment

Kirk et al. [50]

SMC

 Ahn et al. [48]

In vitro:  Monolayer UC-MSC have a poor ability to differentiate into adipocytes  Higher osteogenic marker expression in BM-MSC than in UC-MSC

UC-MSC, BM-MSC

 Combination of cell sheet engineering and electrospinning technology  Expression strong cell-tocell junction and contractile proteins  Anatomical origin of MSCs has affected the differentiation and cellular behaviour  Collagenous matrix, UCMSC exceeded BM-MSC in ECM synthesis, suggesting different mechanisms for the formation of bone.

Human platelets in human rich plasma

Fibrous collagen matrix

In vitro:  Good cell proliferation and attachment

Freeze drying – chemical crosslinkin g

Collagen type I

Highly porous tubular structure

Bone

Freeze dryingchemical crosslinkin g

Collagen type I – heparin binding peptide

Porous sponge

Skin

Freeze drying – chemical crosslinkin g

Collagen type I chitosan

Highly porous sponge

In vivo (rat model):  Reduction in capsule thickness was accompanied by reduction in number of contractile cells  down regulation of cytokines TGF-β1 and TGFβ2  down regulation in expression of the major myofibroblast marker (αSMA) and upregulation of TGF-β3 In vivo:  BMP-2 promoted effective bridging of the defect  The combination of Collagen type I+BMP2+HBP+Heperan Sulfate significantly induced bone formation In vitro:  Good cytocompatibility  Accelerated cell infiltration and proliferation

Wound dressing

Freeze drying – chemical crosslinkin g

Collagen type I – hyaluronic acid

Cardio vascular

Electrospinning

Poly(ϵcaprolacto ne) (PCL) -collagen type I

Electrospu n tubular matrix

Adipose / bone

Thermal crosslinkin g

Collagen

Hydrogel

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In vitro:  Low platelet adhesion In vivo (rat model):  perfusion- fixed interposed grafts showed maintenance of graft patency and lack of aneurismal dilation

Porous sponge

Remarks

 Similar mechanical properties to the native vascular tissue

Multilayered, rolled tubular structures

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Nerve

Cells

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Vitrification

In vivo/in vitro

Collagen type ILYBS 10 (elastinlike protein polymer) Collagen type I

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Corneal

Scaffold morphology

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Layer-bylayer

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Cardiovascular

Scaffold composition

 Schneider et al. .[69]

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ACCEPTED MANUSCRIPT 2.2. Gelatin Gelatin is a natural polymer obtained from the hydrolysis of collagen. Its low immunogenicity, fast resorbability, and in vivo remodeling make gelatin an attractive candidate for biomedical applications [70]. Even though gelatin is a denaturated form of collagen, neither collagen receptor integrins nor discoidin domain receptors (DDRs) can recognize gelatin. However,

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gelatin contains RGD motifs, typically recognized by another subset of receptors, namely integrin type fibronectin receptors. Fibronectin binding to gelatin is very sensitive to pH, to ionic strength and charged amino acyl side chains of fibronectin. Fibronectin is a large dimeric glycoprotein containing modular domains that mediate self-assembly, binding to cell surface

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receptors, and interactions with gelatin. A range of biochemical studies defined a 42-kDa domain containing six modules, 6FnI1–2FnII7–9FnI, to be the gelatin-binding domain (GBD), due

fragments of the GBD, 6FnI1–2FnI and

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to its ability to strongly and specifically bind to gelatin. Moreover two more separate 8–9FnI,

have also been shown to independently bind

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gelatin, but with a decreased affinity [71].

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The extensive use of gelatin in tissue engineering is due to its fast resorbability and remodeling characteristics in vivo. Just like collagens, gelatin is also catabolized by matrix

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metalloproteases. MMP-2 (gelatinase A, 72-kDa type IV collagenase) is one of the two

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described human gelatinases in the MMP family, named for their ability to proteolytically degrade gelatin. MMP-2 differs from other MMPs in that the catalytic domain contains cysteine-rich inserts that resemble the collagen-binding regions of the type II repeats in fibronectin. MMP-9 is another gelatinase (gelatinase B, 92-kDa type IV collagenase) which first was discovered in neutrophils. MMP-9 activation can be mediated by removal of the prodomain by serine proteases or other MMPs, or with a direct response to oxidative stress that disrupts the cysteine switch. While a considerable overlap exists in the substrates degraded by MMP-2 and MMP-9, MMP-9 is incapable of direct proteolysis of collagen I [35].

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ACCEPTED MANUSCRIPT Another attractive characteristic of gelatin consists of, its high solubility in warm water (> 25 °C) while it forms a transparent gel at lower temperatures. Due to its thermoresponsive behavior, gelatin scaffolds, without any further derivatization or crosslinking, are not stable for in vitro or in vivo culturing. For this reason, numerous crosslinking strategies have been developed to stabilize

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gelatin scaffolds. Gomes et al. have investigated the effect of different crosslinking techniques on the mechanical, structural, and cytocompatible characteristics of electrospun gelatin fibers [72]. In their study, they have chosen glutaraldehyde (GTA), genipin, and dehydrothermal (DHT) treatment for crosslinking gelatin fibers. DHT resulted in non-stable constructs, while

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genipin-crosslinked fibers showed reduced proliferation at early time points. Fibers crosslinked with GTA vapors were the most stable and suitable for TE applications in

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combination with fibroblasts. Another cytocompatible gelatin crosslinking technique consists in using transglutaminases (TGase), a family of naturally occurring enzymes found in almost

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all living organisms and in a variety of tissues. TGase plays a role in a wide variety of cellular

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functions, such as blood clotting and liver detoxification. As a cell adhesion protein, TGase provides increased cell attachment compared to the other methods [73]. Moreover, TGase

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functions by covalently binding the ɛ-amino group of a lysine residue and a γ-carboxamide

manner [74] .

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group of glutamine to create intra- and inter-molecular covalent links in a cytocompatible

The use of gelatin scaffolds sprawls into wide range of TE applications such as bone regeneration [75–78], cardio-vascular and skeletal muscle tissue engineering [79–81], drug delivery systems [82–84], and hepatic tissue engineering [85] (Table 2). As an example, gelatin scaffolds were incorporated with magnesium calcium phosphate (MCP) and BMP-2 to study the effect of these components on in vivo bone formation [76]. The study showed that the addition of MCP improved the osteoinductivity of

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ACCEPTED MANUSCRIPT gelatin porous sponges. Furthermore, the integration of BMP-2 significantly improved the osteoinductive properties of gelatin-MCP scaffolds. In another study, 3D nanofibril gelatin scaffolds were shown to shorten the nodular aggregation of osteoblasts, collagen type I formation, and calcification time compared to the 2D culture of osteoblasts harvested from newborn mice calvaria [78].

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Besides the use of gelatin in bone tissue applications, Li et al. have used gelatin-silk fibroin scaffolds to create a biomimetic 3D native vascular tree for hepatic tissue engineering. They showed that the branched and channeled design of the scaffolds improved the mass transport through the scaffold as well as the viability of harvested primary hepatocytes from

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female Sprague Dawley rats [85].

Another attractive use of gelatin consists in creating photo-responsive hydrogel

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scaffolds [86–88]. In several works, researchers synthesized the methacryloyl derivative of gelatin (GelMA) by simply reacting methacrylic anhydride with gelatin [87,89]. The photo-

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responsive GelMA offers notable advantages over gelatin such as, i) stability at physical

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temperature after cell-friendly UV-crosslinking, ii) in situ gel formation and potential use in minimally invasive operations, iii) possibility to encapsulate and deliver cells in one step.

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Therefore, methacrylate functionalized gelatin has already been abundantly studied for wide

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variety of tissue engineering [90–95], stereolithography and 3D bioprinting applications [87,96]. Furthermore, GelMA was also studied in composites to be qualified for different TE applications. As an example, Ahadian et al. have shown that incorporating CNTs into GelMA scaffolds not only enhances scaffold flexibility and strength, but also the electrical conductivity which is a key feature for cardiac muscle regeneration. Their study shows that adding CNTs into gelatin methacryloyl (GelMA) hydrogel scaffolds increased their Young’s modulus and electrical conductivity without significantly affecting gel porosity [97]. Besides

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ACCEPTED MANUSCRIPT CNTs, nHAp particles have also been added to gelatin scaffolds to increase the mechanical strength for osteochondral tissue engineering [98,99]. Table 2. Current strategies of gelatin-based materials in tissue engineering. Targeted organ/ tissue

Scaffold fabrication technique

Adipose

Scaffold composition

Scaffold morphology

Particle leaching – Chemical Crosslinking

Gelatin – Alginate Beads

3D Porous hydrogel

Bone

Freeze Drying – DHT Crosslinking

Magnesium /Calcium Phosphate – Gelatin ( +BMP-2)

Porous sponge

Mesench ymal

Chemical Crosllinking - Freeze Drying

Gelatin

Cardiac

Electrospinn ingChemical Crosslinking

Cornea

In vivo/in vitro

Cells

Remarks

 Extracellular environment may also more closely mimic the native extracellular niche of adipose tissue

 Phull et al. [100]

-

 BMP-2 addition to the scaffolds significantly increased bone healing  Sustained release of BMP-2 from scaffolds has been obtained

 Hussain et al. [76]

BMMSCs

 Increased crosslinker concentration led to confined scaffold inner architecture  The composition of gelatin – crosslinker in the scaffolds regulated the mechanical and pore architecture  The PSG-Gelatin aligned fiber demonstrated higher tensile modulus and lower elongation in dry state  CM showed high and synchronized beating rate with aligned PGS-gelatin blend scaffolds  Higher transmittance than acellularized cornea for visible light  Highly porous membranes  Biocompatible thin gelatin membranes for corneal tissue engineering

 McAndrews et al. [101]

ADSC

 The scaffolds improved and promoted cell attachment, spreading, proliferation and osteogenic differentiation of rat ADSCs in vitro  The combination of the scaffolds and ADSCs enhanced osteogenesis at mice and rat models  biomimetic GelMA scaffolds could promote osteoblastic differentiation and bone formation

 Fang et al. [90]

Hepatoc ytes

 A hepatic vasculature tree mold was fabricated with PDMS with high precision 3D printing  Highly permeability was achieved  biomimetic strategy could be potentially used to regenerate vascularized tissue

 Li et al. [85]

ADSC

Porous sponge

In vitro:  Mechanical properties and sponge architecture affected the differential potential of MSCs

Gelatin – PGS (poly(glyce rol sebacate)

Aligned and random electrospu n mats

In vitro:  High cell viability  CM and CF were able to sense the aligned or anisotropic topology of scaffolds  Elongated cells through the aligned blend scaffolds

Chemical Crosslinking

Heparin – Gelatin (+bFGF)

Thin membrane

Bone

Thermally induced phase separation/ chemical crosslinking

Gelatin Methacryla te

Hepatic

Molding

Gelatin – Silk Fibroin

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In vitro:  Improved cell proliferation & attachment  The scaffold provided a porosity and pore size large enough to support differentiation of ADSCs into adipocytes. In vivo (rat cranial defect):  No inflammatory response  Good integration with old bone

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In vitro:  Supports HCEC growth  Proper morphology for HCEC and ECM synthesis In vivo (mice & rabbit model):  No trigger of inflammatory process  Slow in vivo degradation

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Porous Hydrogels

Highly porous 3D tubular scaffolds

In vitro:  Cell viability close to 100%  Increased ALP activity and mineralized nodules compared to 2D culture In vivo ( nude mice & rat calvarial defect):  Ectopic bone formation and high osteogenic marker expression was observed in subcutaneous nude mice model.  Significantly higher new bone formation was observed in the presence of the scaffold In vitro:  High cell viability  High cell proliferation

Reference

CM, CF

Human corneal endoth elial cells

 Kharaziha et al. [102]

 Niu et al. [103]

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ACCEPTED MANUSCRIPT 2.3 Elastin Elastin is an ECM protein providing elasticity to tissues which are exposed to cyclic stress. As a result, elastin is the most abundant ECM protein in tissues where elasticity is of major importance, like in blood vessels, elastic ligaments, lung, cartilage, and skin. Elastin is an insoluble protein with a half-life of 70 years, making elastin one of the most stable proteins

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known. Elastin is derived from tropoelastin which self-assembles under physiological conditions and initiates coacervation [104]. It is well established that the elastic behavior of elastin is of entropic origin. However, despite several decades of efforts, this phenomenon is not fully understood, as the exact structure of the elastin network still remains elusive. Hence,

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elastin structure is generally represented as an elastomeric material in which tropoelastin precursor chains are connected one each other via cross-links composed of polyfunctional

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amino acids, namely desmosine and isodesmosine (Figure 3).

Figure 3. Elastomeric behavior of elastin protein. (left) Elastin relaxed structure in which tropoelastin chains (blue) are randomly folded and kept together by desmosine and isodesmosine crosslinks (red). By applying an external load, tropoelastin chains can rapidly stretch (right). This stretching/relaxation mechanism is fully reversible.

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ACCEPTED MANUSCRIPT Integrins are crucial determinants in the course of cell survival and tissue integrity. It has been observed that many cellular responses to growth factors are reliant on integrinmediated cell adhesion to ligands found within the ECM. Integrins traditionally recognize their ligands via an RGD motif, however, elastin was excluded as a potential ligand for integrins, on the basis that it does not contain RGD sequences. There are several non-RGD

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proteins that have been identified as ligands for integrins, such as the widely expressed integrin α5β3 [105]. Integrin α5β3 binds to the C-terminus (Domain 36) of a commonly found isoform of human tropoelastin in a single-site saturable manner in the presence of Mn2+ ion. Few studies have suggested that G protein-coupled receptors are able to mediate the effects of

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elastin peptides on cells. The focal attachment of elastin to cells was attributed to a 120 kDa glycoprotein called elastonectin [106], an otherwise unidentified protein that is expressed by

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skin fibroblasts.

The degradation process of elastins is conducted by elastases which present differing

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substrate specificities and catalytic mechanisms. In fact, enzymes with elastolytic activity can

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be found in most of the major proteolytic families, including serine, thiol, aspartic, and metallo enzymes. Despite the differences in catalytic mechanisms, all these elastases share a common

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specificity for cleaving peptide bonds associated with hydrophobic or aromatic amino acids.

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Moreover recently, metalloproteinases secreted by mammalian cells (of the known MMPs, the 92- and 72-kDa gelatinases, human macrophage metalloelastases (HME), and matrilysin) have also been shown to have elastolytic activity and degrade insoluble elastin [107]. As mentioned above, elastin has major importance in biomechanical response of tissues subjected to cyclic stresses. Weisbecker et al. studied the major role of elastin in thoracic aorta softening. According to their study, when the tissue was treated with collagenase, tissue did not show any softening under quasi-cyclic loading. However, when they used elastase for enzymatic degradation of elastin under same loading conditions, tissue

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ACCEPTED MANUSCRIPT lost its integrity [108]. As this stand, the presence of elastin should not be underestimated for tissues under cyclic stress. Therefore, TE applications for skin, vascular grafts, and tendons put great attention in the use of elastin (Table 3). Naturally derived elastin is commercially available in insoluble elastin, tropoelastin or hydrolyzed elastin forms. Furthermore, recombinant tropoelastin or tropoelastin fragments

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can be synthesized for various TE applications [109]. As an example, photocrosslinkable elastin-like-polypeptide (pELP) hydrogels have been suggested as a potential sealant for soft, flexible tissue injuries like the ones in blood vessels, skin, lung, or cardiac tissue, thanks to their in vitro cytocompatibility and in vivo biocompatibility [110].

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In another study, elastin-collagen freeze dried scaffolds have been reported as potential 3D constructs for skin [111], vascular constructs [112–115] , lung tissue engineering

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[116], or as a cardiac patch for cell delivery [117–119]. Researchers showed that elastin addition to collagen scaffolds significantly improved the viscoelastic response during creep

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and cyclical strain recovery of the 3D scaffolds, without affecting pore size and architecture.

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The study also asserts that, even though the addition of elastin decreased the stiffness of pure collagen scaffolds, it enhanced the cyclical strain recovery and reduced the creep strain [118].

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Tropoelastin-collagen hybrid material has also been proposed for skin substitutes. Rnjak-

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Kovacina et al. blended tropoelastin and collagen with the aim of recapitulating protein composition of native skin dermis, with particular emphasis on increasing cell proliferation while maintaining scaffold elasticity [120]. The study showed that blending elastin and pure collagen improves the viscoelastic properties of scaffolds. Elastin-collagen blends have been also investigated for alveolar bone formation. Amruthwar et al. showed that the addition of elastin like polypeptide (ELP) to collagen scaffolds significantly increased the Young’s modulus, tensile strength, and toughness of the

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ACCEPTED MANUSCRIPT scaffolds. The study also revealed that ELP addition did not cause any negative effect on cell viability, ALP activity, or osteogenic differentiation [121]. Elastin-based scaffolds have a great potential in soft tissue engineering thanks to elastin self-assembling properties and viscoelastic character. However, for each TE application it is crucial to pinpoint the specific characteristics of elastin component which

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need to be addressed.

Table 3. Current strategies of elastin-based materials in tissue engineering. Targeted organ/ tissue Bone

Scaffold fabrication technique Thermal Crosslinking

Scaffold compositio n Elastin like peptide Collagen

Cardiac

UV crosslinking – Molding

Methacrylat ed tropoelasti n (MeTro)

Porous Hydrogel

Lung

Thermal Crosslinking

ElastinCollagen

Porous Hydrogel

Dermal

Electrospin ning

Elastin Collagen

Electrospun mesh

Vascular

Enzymatic crosslinking (Transgluta minase)– Freeze Drying

Human elastin-like polypeptides (HELP)

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In vitro:  High cell attachment  Gene expressions confirmed hydrogels were suitable for bone TE In vitro:  Great proliferation spreading and elongation of cardiomyocytes (CM)

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Remarks

Reference

MC3T3

 Suitable TE constructs with suitable mechanical properties for bone TE

 Amruthwar [121]

CM

 CMs were aligned long micropatterned MeTro surfaces  Tissue constructs mimicked the natural myocardium  Matching mechanical properties to the single alveolar wall was achieved

 Annabi et al. [119]

 Dunphy [116]

35FL

Dermal fibrobla sts

 Increased collagen concentration with decreased elastin content reduced the porosity  Good candidate for dermal substitutes

 RnjakKovacina [111]

In vitro:  Good cytocompatibility

HUVEC

 The produced HELP-based matrices can be proposed as non-conventional scaffolds for vascular tissue regeneration.

 Bozzini [113]

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Porous hydrogel

Cells

In vitro:  The presence of cells increased the stiffness of constructs  The cells were elongated and fully adhered In vitro:  Good cell attachment, filtration and proliferation In vivo (mice model):  In subcutaneous implantation angiogenesis was obtained

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Porous hydrogel

In vivo/in vitro

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2.4 Fibroin

Scaffold morphology

Fibroin (FIB) is another scleroprotein broadly used for the production of scaffolds in TE. It is mainly produced from silkworm silk after a purification process aimed to remove sericin – the other main protein composing silk. Among all the possible sources, of note is the silk produced by Bombyx mori, a member of the Bombycidae family. The primary structure of fibroin mainly consists of a recurrent amino acid sequence composed of glycine, serine, and alanine. Such structure evolves into highly packed beta 19

ACCEPTED MANUSCRIPT pleated sheets that form a crystalline-like secondary structure (stabilized by inter-chain

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hydrogen bonding), responsible for its remarkable physical properties [122] (Figure 4).

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Figure 4. a) Most recurrent amino acid sequence in fibroin and b) schematic representation of beta-pleated sheet crystal domains present in fibroin. Fibroin has several major advantages compared to other protein-based biomaterials derived

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from tissues of allogeneic or xenogeneic origin, as they might trigger an inflammation reaction when implanted in vivo.

Besides fibroin superior in vivo performance, cell interaction with silk fibroin substrates were first evaluated on 2D cell culture in terms of attachment and proliferation. Minoura et al. studied the attachment and proliferation characteristic of L929 fibroblasts on various silk fibroins obtained from two different silkworms, B. mori (domestic silkworms) and Antheraea pernyi (wild-type silkworms). The results indicated stronger cell adhesion on films formed by silk fibroins from A. pernyi, the wild-type silkworm, than those from B. mori domestic 20

ACCEPTED MANUSCRIPT silkworms due to the presence of the tripeptide Arg(R)-Gly(G)-Asp(D) in the silk fibroin amino acid sequence from the wild silkworms. However, even if the initial attachment of cells to the B. mori silk fibroin was lower than the A. pernyi silk fibroin, both fibroin structures have shown similar results to collagen in terms of cell attachment and growth kinetics [123,124]. The cost of extraction and purification of other protein-based biomaterials is generally higher

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compared to fibroin due to the strict protein isolation and purification protocols. In contrast, fibroin purification is routinely carried out using a simple alkali- or enzyme-based degumming procedure to remove sericin from silk fibers. From a structural point of view, silk fibroin is composed of three units: a heavy chain (H) and a light chain (L), which are bound

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together by a disulfide bond, and a non-covalently linked 25 kDa glycoprotein (P25). The extraordinary mechanical properties of fibroin are the result of a precise control at the nano

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scale over the size, number, distribution, and orientation of crystalline and non-crystalline domains [125].

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Another feature that should be taken into consideration regarding the use of fibroin in TE is

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its degradation characteristics. Li et al. analyzed the enzymatic degradation of fibroin

[126].

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reporting that it can be catabolized via α-chymotrypsin, collagenase IA, and protease XIV

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A great advantage of fibroin is represented by its water solubility in its α-helical and random coil forms. The possibility to process fibroin as a water solution in mild manufacturing and cell-friendly conditions allowed fibroin to be shaped into different forms such as films [127– 129], wet-spun fibers [130,131], hydrogels [132–136], 3D sponge-like scaffolds [137–144], micro- and nanoparticles [145–147], and 3D printed fiber meshes [148–150] (Table 4). Thanks to its unique mechanical properties, fibroin has been exploited to develop scaffolds for load bearing tissue engineering targeting bone, cartilage and ligament tissues. Scaffolds targeting bone tissue should provide proper matrix toughness and promote matrix deposition

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ACCEPTED MANUSCRIPT and calcification (Table 4). Meinel et al. [139] have proposed a method for the fabrication of 3D porous sponge-like scaffolds made of silk fibroin through a salt-leaching method for the regeneration of critical size bone defects. In this study, they seeded such 3D porous scaffolds with a Matrigel suspension of bone-marrow derived mesenchymal stromal cells (BM-MSCs). Constructs were cultured in osteogenic conditions in vitro for 5 weeks and then implanted in

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mouse circular calvarial defects for another 5 weeks, demonstrating that the quality of engineered bone resembled was comparable to the native bones in terms of organic and inorganic components and structural morphology. In another study, Kim et al. [151] demonstrated that fibroin nanofiber mesh fabricated via electrospinning technique could

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guide bone regeneration in vivo. After 12 weeks of culture in rabbit model, they demonstrated that FIB membranes induced new bone formation with a complete healing of the induced

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calvarial defects. Following a similar approach, Li et al. [152] fabricated fibroin membranes containing bone morphogenetic protein 2 (BMP-2) and/or nano-hydroxyapatite particles

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(nHAp) in order to favor in vitro differentiation of BM-MSCs. After 31 days of culture, the

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authors demonstrated that the fabricated composite meshes better supported differentiation of BM-MSCs with respect to control scaffolds, enhancing the mineralization of the constructs

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and the transcription of relevant genes involved in osteogenesis.

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Fibroin has been also tested for cartilage regeneration in form of 3D porous sponge-like scaffolds [153–155]. Vishwanath et al. prepared fibroin/chitosan porous scaffolds via freezedrying for cartilage regeneration purposes. They evaluated in vitro the performances of such porous scaffolds with MSCs after 21 days of culture in chondrogenic conditions. The authors found that the fibroin/chitosan blend ratio affected the chondrogenic differentiation of MSCs reaching the best performances in the case of a blend ratio of 80:20 (w/w). Furthermore, the scaffolds possessed desired morphological pore size, porosity, compressive strength and degradation properties, making them a valid candidate for cartilage regeneration. In a very

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ACCEPTED MANUSCRIPT interesting work, Saha et al. [154] fabricated 3D porous scaffolds with fibroin extracted from Bombyx mori and from Antheraea mylitta through an optimized freeze-drying procedure. In this work, they evaluated the capability of FIB scaffolds obtained from the two different sources to support chondrogenic or osteogenic differentiation of BM-MSCs. Interestingly, they found that scaffolds produced with FIB extracted from A. mylitta supported greater the

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chondrogenic differentiation of BM-MSCs, while those produced with FIB from B. mori better promoted their osteogenic differentiation. Thus, they fabricated multi-layered constructs containing additionally BMP-2 and Transforming Growth Factor 3 (TGF-3) and implanted them into an osteochondral defect in rat patella-femoral groove, showing a good neo-tissue

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ingrowth and host tissue integration.

In a recent work, Chen et al. [155] proposed a method for the production of highly regular

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porous scaffolds based on an indirect additive manufacturing technique. They demonstrated that it is possible to create regular microchannels within a FIB porous scaffold with a

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thermoplastic sacrificial mold built by an inkjet 3D printer. The obtained scaffolds were then

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tested in vitro and in vivo with porcine derived chondrocytes. In both cases, FIB-based scaffolds supported chondrocyte proliferation and phenotype maintenance. Furthermore, FIB

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scaffolds did not degrade appreciably during the culturing period (12 weeks), thus

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representing a good candidate for long term in vivo tests. Among the possible applications, FIB in form of electrospun membranes or casted film represents an ideal material for wound dressing and skin regeneration. Jeong et al. [156] studied the behavior of O2 and CH4 plasma treated FIB electrospun membranes. They demonstrated that O2 plasma treated membranes were more hydrophilic compared to those untreated or treated with CH4 and promoted better adhesion and cellular activities of keratinocytes (NHEK) and fibroblasts (NHEF). In another study, Sugihara et al. [157] prepared FIB films for full-thickness skin wounds regeneration. In this study, they compared

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ACCEPTED MANUSCRIPT in vivo the performance of such film with that obtained from two commercially available products – namely Alloask D and DuoActive dressings. 2 weeks after dermatotomy, FIB film greatly outperformed the other two products in terms of neo-epithelization. Furthermore, in contrast with the other two materials, FIB film prevented edema formation and inflammatory reactions. Authors concluded that FIB films are valid candidate for wounds

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regeneration and may be clinically useful for wound treatment.

Despite the aforementioned advantages, fibroin scaffolds are still not indicated for load bearing TE applications due to their low mechanical strengths. Many groups suggested that the incorporation of nano-ceramic structures into fibroin scaffolds may enhance the

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mechanical strength of the scaffolds. As an example, Kim et al. enhanced the mechanical properties of electrospun fibroin scaffolds by uniformly dispersing hydroxyapatite (HAp)

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nanoparticles within fibroin nanofibers. The mechanical testing on fibroin-HAp scaffolds revealed that the tensile strength, yield strength and Young’s modulus increased with

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increasing the HAp concentration until a threshold 20% w/w. However, for HAp

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concentration above 20% w/w, the polymer chain networks within SF nanofibers were disrupted, weakening the mechanical strength [158]. Kim et al. demonstrated also that the

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addition of titanium oxide nanoparticles (TiO2 NPs) into fibroin scaffolds not only resulted in

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an improvement of mechanical properties of silk fibroin scaffolds, but also led to a decrease of the swelling ratio of scaffolds and to an improved viability for osteoblasts and fibroblast [159].

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ACCEPTED MANUSCRIPT Table 4. Current strategies of fibroin-based materials in tissue engineering. Scaffold morphology

Salt leaching

FIB/Matrige l

3D Porous hydrogel

Bone

Electrospin ning

FIB

Porous membrane

Bone

Electrospin ning

FIB/BMP-2, FIB/BMP2/nHAp

Porous membrane

Cartilage

Freezedrying

FIB, FIB/Chitosa n

3D spongelike scaffold

Cartilage

Indirect additive manufactur ing (AM)

FIB

Porous scaffold with regular micro/mac ro structure

Osteoch ondral

Freezedrying

mulberry FIB from Bombyx mori/nonmulberry FIB from Antheraea mylitta

Skin

Electrospin ning

FIB

Porous membrane

Skin

Film casting

FIB

Film

Cells

In vitro:  great cell proliferation  good calcium deposition In vivo:  proper matrix deposition  good remodeling In vitro:  Good cell proliferation and osteocalcin production  Increase ALP expression In vivo (rabbit):  No inflammatory response  Good integration with old bone In vitro:  The fabricated membrane efficiently delivers labile cytokines and other components  The incorporation of BMP-2 and/or nHAp into the scaffolds enhanced bone formation significantly after 31 days of culture. In vitro:  Good cell proliferation  SF/CS (80:20) best blend ratio

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In vitro:  High cell viability  Proper morphology of chondrocytes, distribution of cells and ECM synthesis In vivo (mice model):  No trigger of inflammatory process  Col II was more prominent in wider pores In vitro:  Good cell proliferation and differentiation towards chondrocytes and osteoblasts after 4/8 weeks of culture In vivo:  Multi-layered constructs composed of mulberry and non-mulberry derived FIB containing BMP-2 and TGF-3 support osteochondral In vitro:  The adhesion of NHEK and NHEF to FIB electrospun membrane depends to the type of plasma treatment, exhibiting better performances for O2 plasma treatment  The O2 plasma-treated FIB nanofibers showed a significantly higher level of NHEK cell attachment In vivo (mice model):  14 days after dermatotomy, 93% of silk film area was epithelized

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3D spongelike scaffold

Remarks

Reference

BMMSCs

 possibility to heal critical size bone defects

 Meinel et al. [139]

MC3T3E1

 In vivo formation of a bone bridge between FIB membrane and old bone  Good mechanical stability

 Kim et al. [151]

BMMSCs

 BMP-2 survived the aqueous-based electrospinning process in bioactive form  BMP-2 supported higher calcium deposition and enhanced transcript levels of bone-specific markers

 Li et al. [152]

MSCs

 Pore size could be controlled by tuning chitosan concentration  High porosity 70-93% and high swelling properties of the constructs

 Vishwanath et al. [153]

Porcine chondro cytes

 GAG and Col II per cell basis showed a significant increase with time  Precise control over final morphology

 Chen et al. [155]

BMMSCs

 Slow and even pre-cooling at -20°C led to more uniform porous interconnected structures with defined pore sizes  Depending on the FIB source, the constructs possessed intrinsic capability for chondrogenic or osteogenic differentiation  O2 plasma treatment made the electrospun membrane more cell adhesive

 Saha et al. [154]

 FIB films significantly reduced edema formation in the wounds  FIB films did not cause inflammatory response as demonstrated by low neutrophil-lymphocyte infiltration.

 Sugihara et al. [157]

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Bone

In vivo/in vitro

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Scaffold composition

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Scaffold fabrication technique

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Targeted organ/ tissue

NHEK, NHEF

--

 Jeong et al. [156]

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ACCEPTED MANUSCRIPT 3. Glycosaminoglycans (GAGs) for the fabrication of 3D scaffolds Besides fibrous proteins, the ECM is also composed of proteoglycans. The basic proteoglycan unit consists of a protein with one or more covalently attached glycosaminoglycans (GAGs). Due to their lubricant properties, GAGs have the function of absorbing shocks and preserving the integrity and structure of the ECM [160]. GAGs are long, unbranched polysaccharides with

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a repeating disaccharide unit, consisting of an amino sugar (N-acetylglucosamine or Nacetylgalactosamine) along with a uronic acid (glucuronic or iduronic acid) or galactose [14]. Hyaluroran (Fig. 5a) [161,162], chondroitin sulfate (Fig. 5b) [163,164], dermatan sulfate [165,166], heparin [167,168], and heparan sulfate [169,170] are the five members of the GAG

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family that are mostly used for biomedical applications.

Figure 5. Examples of GAGs: structure of hyaluronan (a) and chondroitin sulfate (4-sulfation) (b). Because of their natural and physiological characteristics, they are largely used as a scaffold material in tissue regeneration, showing the capability of undergoing a controlled degradation without cytotoxic by-products and promoting cell viability [171]. GAGs are mostly used in form of 3D hydrogels due to their easy processability and swelling properties. GAG hydrogels can easily adapt to any defect size or shape, adhere and integrate with the surrounding tissue. 26

ACCEPTED MANUSCRIPT The surface features, swelling, permeability and mechanical properties of GAGs hydrogels can be tuned and optimized via different crosslinking strategies [161]. However, the most crucial characteristic of hydrogels is their injectability, which allows a homogenous cell distribution inside the final construct, easy incorporation of growth factors, and the use of less invasive surgical procedures.

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GAGs derived polymers are mostly used in the cartilage tissue regeneration due to its structural similarities with cartilage tissue (accounting for 25-30 wt% GAGs, in dry weight), as well as the required mechanical, swelling, and lubrication properties [14,171]. However, in the last years GAGs have also been used as scaffolds for tissue regeneration in orthopedic

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[172], neurological [173], ophthalmology [174], cardiac [175], and dermal [176] fields.

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3.1 Hyaluronan

Hyaluronan (HA) is a GAG with a repetitive disaccharide unit, made of glucuronic acid (GlcUA)

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and N-acetylglucosamine linked via alternating β-1,3 and β-1,4 anhydroglycosidic bonds

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[171]. It has been proven that hyaluronic acid potentially interacts with several cell transmembrane receptors such as CD44, CD54, and CD168, TSG6, GHAP, LYVE-1, ICAM-1, and

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RHAMM [177]. These interactions generally result in the activation of cascade signals which

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affect cell attachment, migration, proliferation and morphogenesis. Inflammation and wound healing processes are also influenced by the presence of HA [178]. HA degradation process is mostly enzymatic, however cell related mechanism plays an important role as well. The enzymatic degradation process is operated by three enzymes present in the body: hyaluronidase, β-D-glucuronidase, and β-N-acetyl-hexosaminidase. The molecular weight of HA is drastically decreased by the enzymes resulting in oligosaccharide fragments. Moreover, it has been reported that these degradation products might promote angiogenesis [179].

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ACCEPTED MANUSCRIPT HA is mostly involved in tissue hydration [180], a key compound in the ECM of cartilage [14], as well as a major constituent of the eye vitreous and aqueous humor [173]. Moreover, it is an important constituent of the synovial fluid, contributing to its elasticity and viscosity. HA acts as a fluid shock absorber while maintaining the structural and functional characteristics of the cartilage matrix. Therefore, it induces proteoglycan synthesis and

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aggregation [160]. HA also modulates the inflammatory response, provides the associated tissues with tensile strength, and promotes cell adhesion and growth [14]. In tissue engineering, HA is most commonly used in the form of injectable hydrogel as it can be easily loaded with drugs or cells, for instance, and delivered to the target defect independently of its

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shape [181].

The in situ gel forming strategy offers many practical advantages such as i) non-invasive

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surgery, ii) stable hydrogel structure, and iii) the possibility of incorporating bioactive molecules, cells, or drugs [161].

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In addition to the hydrogel form, HA has been used for the fabrication of nanofiber meshes

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[182], nanosphere/nanoparticles [183], 3D sponge-like scaffolds [184], and scaffold coatings [185] (Table 5).

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A very important feature characterizing hyaluronan is its molecular weight: in fact, molecular

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weight deeply affects rheological properties and induces distinct biological responses in vivo, for example in terms of angiogenesis [186]. Low/medium molecular weight hyaluronans (10-700 kDa) show good viscoelasticity, moisture retention, mucoadhesive properties, low mechanical strength, and rapid in vivo bioresorption [108,120]. Due to these characteristics, they are employed in syringe injection therapies, regenerative applications in ophthalmology [173,174], orthopedics [172,185], wound healing [181,187], and cosmetic fields.

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ACCEPTED MANUSCRIPT In particular, the intra-articular injection of hyaluronan into osteoarthritic joints is claimed to restore the viscoelasticity of the synovial fluid, augment the flow of joint fluid, normalize endogenous hyaluronan synthesis, inhibit hyaluronan degradation, relieve pain, and improve joint functions [14]. On the other side, high molecular weight hyaluronans, in the range 700–6000 kDa, are more

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often employed in cartilage tissue regeneration, due to their greater viscoelasticity, intraarticular dwell-time and for the stimulation of chondrocyte proliferation [171]. Recently, pure HA scaffolds have been used for bone [36] and cartilage [188] regeneration applications, and in corneal endothelial cell (CEC) sheet transplantation [174].

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Cui et al. studied the cellular infiltration and migration in a highly porous (pore diameter 20÷ 200 m) HA hydrogel synthesized by hydrazone bond crosslinking. The HA scaffold showed

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good equilibrium swelling ratio and degradation properties. Besides, the authors claimed that the fabricated HA scaffold was a promising support to mimic the nature of bone ECM due to

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its biocompatibility and osteoinductive properties [36].

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Moreover, due to its biological stability, glucose permeability, and high water content, pure HA hydrogels favor CEC homeostasis, and are currently used for corneal regeneration

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application. Lai et al. proposed nitrogen gas injection in a HA solution in order to increase the

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porosity of the structure and to avoid skin ingrowth [173]. The in vivo studies demonstrated that the cell sheets could promote the regeneration of corneal endothelium and recover the transparency and minimize the corneal edema. HA is often used in combination with other natural or synthetic polymers. For example, many research groups produced biomedical scaffolds made by hyaluronan and chitosan due to chitosan biocompatibility, non-cytotoxicity and cell-adhesive properties[189,190]. It has been shown that the addition of chitosan in the HA structure increased collagen-II production, aggrecan and proteoglycan gene expression in the presence of chondrocytes [191]. For these

29

ACCEPTED MANUSCRIPT reasons, HA/chitosan scaffolds have been mostly used in cartilage [192] and periodontal tissue regeneration [193]. Therefore, the combination of HA with ceramic materials such as hydroxyapatite has also been exploited in the frame of bone regeneration due to its superior mineralization ability [194]. In other studies, addition of collagen in the preparation of the HA scaffolds was pursued in

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order to mimic the native ECM. Kontturi et al. proposed a scaffold made of type II collagen/HA chemically crosslinked using poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4SPEG) and loaded with TGF-β. They demonstrated that the hydrogels were stable and enhanced cell viability and maintained the chondrocyte cell differentiation. They concluded that the scaffold

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could be applied in cartilage tissue regeneration application as a chondrocyte delivery vehicle [161].

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Scaffolds made of HA-gelatin hydrogels are also used in tissue engineering due to their great biological properties [175,195]. Gaetani et al. produced a hyaluronic acid/gelatin 3D-printed

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patch (HA/GEL) loaded with human cardiac-derived progenitor cells (hCMPCs) for cardiac

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tissue engineering. The HA/GEL/hCMPCs construct was printed and implanted in a mouse model of myocardial infarction. The study shows, HA/GEL 3D printed constructs enhanced

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the hCMPC delivery to myocardial infarction area by promoting hCMPC attachment and

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proliferation. It has also been shown that in the presence of 3D printed patch, the hCMPCs retained their cardiogenic phenotype in vitro up to 30 days and were able to differentiate in the appropriate condition [175]. Centola et al. used a hyaluronan-fibrin scaffold obtained by freeze-drying and loaded with an anti-vascular endothelial growth factor (VEGF) antibody (Bevacizumab) as a vehicle for the xenotransplantation of human nasal chondrocytes in an ectopic murine model. The authors demonstrated that the synergistic effect of the chondrosupportive environment and the suppression of VEGF signaling promoted long-term neo-cartilage stability [196]. Furthermore,

30

ACCEPTED MANUSCRIPT scaffolds made of HA blended with synthetic polymers are used for regeneration of hard tissues. Chen et al. demonstrated the osteoconductivity of poly(ϵ-caprolactone) (PCL) scaffolds coated with hyaluronic acid and tricalcium phosphate (HA/TCP). In this study, they indicated that the coating improved the proliferation and osteogenic differentiation of hMSCs, showing a good distribution and homogeneous cell matrix and calcium deposition [185].

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The encapsulation of nanoparticles into hydrogel structures has recently gained the researcher attention to create hybrid materials. Different kinds of nanoparticles have been loaded in HA hydrogels to overcome some of their disadvantages, such as poor mechanical properties, high swelling profile, and too rapid degradation, which can hamper their use for et al. proposed a method for a fast, homogeneous and

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some biomedical applications.

efficient calcium phosphate (CP) particle incorporation into HA hydrogels. The

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nanocomposite hydrogel showed a significant improvement of the mechanical properties (~ 4-fold increase) and a slower degradation rate compared with pure HA hydrogel. Moreover, in

et al. considered the encapsulation of silica particles in

PT

scaffolds [197]. Therefore,

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vivo and in vitro studies reported that the addition of CP improved the bio-stability of the

the HA hydrogels, demonstrating that concentrations higher than 2% could create a silica

CE

network in within the HA network. This led to decreased swelling properties and enhanced

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mechanical characteristics [198]. The incorporation of graphene oxide particles in the HA construct was also investigated. Improvement of the mechanical properties and pHresponsive drug release profile were reported by

et al. [199]. In another study, Chen at

al. proposed HA scaffolds in the form of fibers. Silver nanoparticles were incorporated into electrospun HA/PCL nanofibrous structures. The scaffolds were obtained via co-axial electrospinning technique. The use of silver nanoparticles was exploited to avoid post-surgery bacterial infection and peritendinous adhesions. In vitro and in vivo studies showed that the combined effect of HA and silver nanoparticles inhibited fibroblast attachment and

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ACCEPTED MANUSCRIPT proliferation and improved lubrication, proving that the scaffolds could be potentially used for the prevention of peritendinous adhesion [182].

Table 5. Current strategies of hyaluronan-based materials in tissue engineering. Targeted organ/ tissue

Scaffold fabrication technique

Scaffold composition

Scaffold morphology

In vivo/in vitro

Cells

 Cui et al. [36]

MSCs

 Uniform cellular matrix and calcium deposition

 Chen et al. [185]

In vitro:  Viability > 90% In vivo (rat model):  New bone formation around 78%  Tissue-in growth within the defect site In vitro:  Viability > 90%  High ATP1A1 expression In vivo (rabbit model):  Poor tissue reconstruction

3T3

 Defect site covered  Potential bone substitute

 Subramaniam et al. [200]

CEC

 High pre-freezing temperature leads larger pore size and high porosity  Corneal endothelial cytocompatibility and cell delivery performance

 Lai et al. [174]

In vitro:  High ATP1A1 expression In vivo (rabbit model):  Regeneration of corneal endothelium  Restoring corneal transparency In vitro:  Superior aggrecan and collagen II expression, GAG quantity, and proteoglycan staining under hypoxia condition

CEC

 Reconstruction of the posterior corneal surface  Improvement endothelial tissue function

 Lai et al. [173]

BMSCs

 Hypoxia conditions enhance chondrogenesis  HA scaffolds support the creation of hyaline-like cartilaginous tissue

 Bornes et al. [188]

Hydrogel

In vitro:  Cell encapsulation  Maintaining of cell phenotype

Chondr ocytes

 the seeding density of chondracytes in the scaffolds affects the matrix production (GAG, Collagen II) as well as the gene expression ( Collagen I, II and SOX 9 ) Hydrogel:  Injectability,  Stability  Possibility of bioactive factors incorporation  Maintenance and promotion of viability and chondrocytic properties  PBS uptake >95%  Degradation temperatures did not change after sterilization  High cell migration  Reduction in adverse remodeling  Preservation of cardiac performance

 Ramesh et al. [192]

Hyaluronic acid

Porous hydrogel

In vitro:  Enhanced cell proliferation

Bone

3D FDM/coati ng by soaking

PCL/hyaluro nic acid/β tricalcium phosphate

Porous

In vitro:  Enhanced proliferation  ALP and COLI expression and osteogenic differentiation

Bone

Slurry

Hyaluronic acid/ calcium sulfate/ Hydroxyapat ite

Porous

Corneal

Chemical Crosslinkin g

Hyaluronic acid

Porous hydrogel

Corneal

Chemical Crosslinkin g + gas foaming

Hyaluronic acid

Porous hydrogel

Cartilage

biopsy punch on sheets of esterified HA (HYAFF) Chemical crosslinkin g

Hyaluronic acid

Porous non-woven mesh

NU

MA

ED

PT

CE

AC

MC3T3

SC RI PT

Chemical Crosslinkin g

Hyaluronic acid/ chitosan

Reference

 Good equilibrium swelling ratio  Good degradation properties

Bone

Cartilage

Remarks

Cartilage

Chemical crosslinkin g

Hyaluronic acid/collage n II

Hydrogel

In vitro:  Viability of encapsulated cells >90%  GAGs and collagen II expression

Chondr ocytes + TGFβ1

Periodo ntal

Chemical crosslinkin g

Hyaluronic acid/ chitosan

Hydrogel

In vitro:  High cell viability  High CD44 expression

NIH/3T 3 + MG63

Cardiac

3D printing

hyaluronic acid/gelatin

Hydrogel

In vitro:  Cell attachment and proliferation  Cell cardiogenic phenotype maintaining In vivo (mouse model):  Increase in cardiac and

hCMPCs

 Kontturi et al. [161]

 Miranda et al. [180]

 Gaetani et al. [175]

32

ACCEPTED MANUSCRIPT vascular differentiation Hydrogel

--

--

 Viscoelastic properties comparable with the human vocal fold tissue

 Kazemirad et al. [195]

Hyaluronic acid/polyca proctone + silver nanoparticle s hyaluronic acid/calcium phosphate nanoparticle s

Fibrous

In vitro:  Cell viability > 80%  Low cell attachment and proliferation In vivo (rabbit model):  Peritendinous anti-adhesion

human foreskin fibrobla sts (Hs68)

 Anti-adhesion barrier  Lubrication  Anti-bacterial properties

 Chen et al. [182]

Hydrogel

In vitro:  Cell attachment and proliferation  Cell viability > 80% In vivo:  Enhancement of biostability

L929 fibrobla sts

 Mechanical properties improvement  Slower degradation rate

 Jeong et al. [197]

L929 fibrobla sts

 Decrease of swelling properties  Mechanical properties improvement

 Vallés-Lluch et al. [198]

3T3

 Mechanical properties improvement  pH responsive drug release

 Song et al. .[199]

Tissue engineer ing

Chemical crosslinkin g

Tissue engineer ing

Chemical crosslinkin g

hyaluronic acid/silica nanoparticle s

Hydrogel

In vitro:  Cell attachment and proliferation  Cell viability > 80%

Tissue engineer ing/drug delivery

Enzymatic crosslinkin g

hyaluronic acid/graphe ne oxide particles

Hydrogel

In vitro:  Cell viability > 80%

NU

Peritend inous

SC RI PT

hyaluronic acid/gelatin

Chemical crosslinkin g electrospin ning

MA

Vocal fold

3.2 Chondroitin sulfate

ED

Chondroitin sulfate (ChS) is a sulfated glycosaminoglycan (GAG) composed of a linear chain of N-acetylgalactosamine and glucuronic acid [14]. ChS chains actively interact with several

PT

superficial cell sites influencing cell morphogenesis, adhesion, division and proliferation

CE

[201]. Chondroitin sulfate plays an important role in growth factor signals including hepatic growth factor/scatter factor and heparin-binding growth factors and has the ability to bind

AC

with neural cell adhesive molecules (Ng-CAM and N-CAM) guaranteeing an excellent adhesion and spreading [202]. Moreover, it promotes the development of the central nervous system (CNS) and wound healing [203]. The interaction with cells also leads to ChS degradation, with an initial phase involving cleavage of the protein core of the proteoglycan [204]. In the human body, ChS is an important structural component of the cartilage tissue. Due to its negative polarity, ChS provides resistance to compression by retaining water within the tissue matrix [205]. It also stimulates the synthesis of proteoglycans and decreases the catabolic activity of chondrocytes by inhibiting the synthesis of proteolytic enzymes [160,206]. 33

ACCEPTED MANUSCRIPT The polyanionic nature of chondroitin sulfate makes it an excellent candidate for electrostatic bond with polycations [207,208] and growth factors [209]. 3D scaffolds made by ChS provide cell adhesion, differentiation, and proliferation, favoring GAG expression and collagen deposition [210]. Moreover, ChS promotes chondrogenic gene expression [211], leads bone defect regeneration [210], supports osteoarthritis treatments [206], and favors wound

SC RI PT

healing process [176] (Table 6).

In tissue engineering, ChS is often used in the form of hydrogels [207] or nanoparticles [205] for drug delivery applications.

Chitosan, as a naturally derived cationic polymer, is often electrostatically combined with ChS.

NU

It has been proven that the addition of chitosan to ChS led to a better chondrogenic activity [207]. For these reasons, 3D ChS/chitosan natural scaffolds have been produced for cartilage

MA

regeneration [211] and, lately, for bone [160] and nerve [209] regeneration as well. Silva et al. designed a layer-by layer chitosan/ChS scaffold for cartilage regeneration. The 3D

ED

structure showed high porosity, viscoelastic properties and 300% water uptake capacity.

PT

Furthermore, in vitro studies have demonstrated that chitosan/ChS scaffolds supported the phenotype maintenance of bovine chondrocytes, and the chondrogenic differentiation of

CE

multipotent bone marrow derived stromal cells. The production of chondrogenic matrix

AC

started after 14 days of cell culture and the use of hypoxia and mechanical stimulation were proposed to accelerate chondrogenesis [207]. Association of ChS in the HA structure is a well known blend because of its ability to promote chondrocyte synthesis of hyaluronan, glucosamine and collagen type II, while inhibiting ECM degrading enzymes. Moreover, the addition of ChS to HA improves HA mechanical properties and increases its degradation time in vivo [208,211]. For these reasons, the ChS/HA combination has been proposed for cartilage tissue engineering [212]. Furthermore, ChS and

34

ACCEPTED MANUSCRIPT HA blends are used in skin regeneration, due to its capability to favor angiogenesis during wound healing process and enhance scaffold vascularization [176]. Yan et al. proposed a new 3D scaffold for dermal regeneration made of silk fibroin (FIB)/chondroitin sulfate (ChS)/hyaluronic acid (HA). The scaffolds showed very high porosity (88-93%) with pore size between 95 and 248 m, which promoted cell adhesion,

SC RI PT

migration and proliferation. Moreover, the structures were implanted in a rat model for dermal regeneration purposes in order to investigated the wound healing process. The obtained results showed that the addition of ChS on the FIB/HA scaffolds improved angiogenesis and collagen deposition promoting dermis regeneration in vivo [208].

NU

Furthermore, in order to lead the nucleus pulposus [213] and nerve [209] regeneration, ChS was blended with synthetic polymers to produce 3D hydrogel or microspheres composite

Xu

et

al.

proposed

MA

scaffolds. These blends showed suitable mechanical properties and swelling characteristics. Poly(D,L-lactide)/Chondroitin

Sulfate/Chitosan

scaffolds

with

ED

incorporation of nerve growth factor (PDLLA/ChS/CS/NGF) for peripheral nerve defect

PT

regeneration. The scaffolds showed appropriate mechanical properties besides non-cytotoxic degradation byproducts. In vivo studies based on rat animal model, demonstrated a functional

CE

reconstruction of the damaged nerves [209].

AC

Nair et al. developed and validated a composite hydrogel made of chitosan– poly(hydroxybutyrate-co-valerate) with ChS nanoparticles for nucleus pulposus tissue engineering. The scaffolds were stable in PBS and showed proper water uptake ability and adequate viscoelastic characteristics. Moreover, in vitro studies demonstrated the addition of ChS nanoparticles significantly improved the viability and chondrogenic differentiation of MSCs [205]. Recently, Costantini et al. have fabricated 3D porous hydrogel scaffolds loaded with BM-MSCs with a blend of photocurable ChS and gelatin through a 3D bioprinting approach. They

35

ACCEPTED MANUSCRIPT demonstrated that the addition of ChS to gelatin notably favored the expression of collagen type II and aggrecan, limiting the expression of collagen type I and X, thus obtaining a more functional differentiation of MSCs towards articular cartilage [214]. In order to produce scaffolds for bone [210] or osteochondral applications with suitable mechanical properties, composite engineered materials were designed blending ChS with

SC RI PT

ceramic [210,215] or metallic [160] particles.

Gupta et al. incorporated tricalcium phosphate and chondroitin sulfate in poly (D,L-lactic-coglycolic acid, PLGA) microsphere-based scaffolds. The addition of TCP and ChS into PLGA microspheres enhanced cell differentiation and proliferation. Due to the suitable pore size and

NU

geometry, the scaffolds promoted glycosaminoglycan (GAG) expression and collagen production, increased calcium content and supported osteogenesis [210].

MA

In a different study, Venkatesan et al. developed a tri-component scaffold made of chitosan, hydroxyapatite and ChS for bone tissue regeneration. The scaffold pores appeared very well

ED

connected with pore size between 60 and 400 μm. The authors demonstrated that the

PT

combination of chitosan and ChS induced cell proliferation and alkaline phosphatase activity,

engineering [215].

CE

concluding that the proposed scaffold could be a potential candidate for bone tissue

AC

In another study by Alex et al., metallic particles such as TiO2 were encapsulated in the CS hydrogel structure. They demonstrated the improvement of the mechanical properties as well as the antibacterial, bioactive and biocompatible characteristics of the proposed scaffold [216]. Table 6. Current strategies of chondroitin sulfate-based materials in tissue engineering. Targeted organ/ tissue

Bone

Scaffold fabrication technique

Freezedrying

Scaffold composition

Scaffold morphology

Chondroitin sulfate/Chit osan Ti02

Hydrogel

In vivo/in vitro

In vitro:  Cell viability >80%  Calcium and phosphate deposition

Cells

MG-63

Remarks

 Bioactivity  Antibacterial  Adequate mechanical properties

Reference

 Alex et al. .[216]

36

ACCEPTED MANUSCRIPT Freezedrying

chondroitin sulfate/ chitosan/ hydroxyapat ite

Porous

In vitro:  Cell proliferation  alkaline phosphatase activity, collagen I expression

MG-63

 pore size 60 to 400 μm  controlled biodegradation

 Venkatesan et al. [215]

Bonecartilage interface

Raw material approach

Chondroitin sulfate/ tricalcium phosphate/ PLGA

Microspher es

In vitro:  Mitogenic effect  Glycosaminoglycan and collagen expression  Calcium deposition

BMSCs

 Favoring of osteogenesis  Enhancing neo-tissue formation

 Gupta et al. [210]

Cartilage

Layer-bylayer

Chondroitin sulphate and chitosan

Multilayered

In vitro:  Cell adhesion and proliferation  GAGs expression

Bovine chondro cytes and hMSCs

 High porosity  Water uptake 300%  Maintaining of chondrogenic phenotype  hMSCs chondrogenic differentiation

 Silva et al. [207]

Cartilage

Chemical Crosslinkin g

Chondroitin sulfate/ Hyaluronic acid

Porous hydrogel

In vitro:  Cell viability  Cell proliferation

ASCs

 High water uptake capacity  Pore size 20–150 μm  Chondrogenic potential

 Dinescu et al. [211]

Cartilage

3D printing

Chondroitin sulfate/ gelatin/hyal uronic acid

Hydrogel

In vitro:  High cell viability (85 ÷ 90%) chondrogenic differentiation

BMSCs

 High printing resolution (≈100 μm)  Robustness and accuracy of method

 Costantini et al. [214]

Skin

Freezedrying

Chondroitin sulfate/gela tin hyaluronic acid

Porous sponge

rHFSCs + VEGF

 Pore diameter of 133.23 ± 43.36 μm  Partial revascularization  Wound healing

 Quan et al. [217]

Dermal

Freezedrying

Chondroitin sulfate/ silk fibroin /hyaluronic acid + NGF

Porous

In vitro:  Cell adhesion and proliferation  CK19, integrin α6 and β1 expression In vivo (rat model): Blood vessel formation In vitro:  Maintaining of cell morphology  Cell adhesion and proliferation In vivo (rat model):  Angiogenesis  Collagen expression

L929

 Pore interconnection  Water retention  Stimulation of the woundhealing process

 Yan et al. [218]

Nerve

layer-bylayerElectroStaticassembly Chemical crosslinkin g

Chondroitin sulfate/PDL LA/Chitosan

In vivo (rat model):  No connective tissues formation

--

 Non-toxic degradation products  Functional recovery

 Xu et al. [219]

Chondroitin sulfate/ poly(Nisopropylacr ylamide)

Hydrogel

In vitro:  No cytotoxicity

HEK

 Proper compressive modulus  Adhesive tensile strength 0.4-1 kPa

 Wiltsey et al. [220]

Electrostati c crosslinkin g

chondroitin sulfate/ chitosan/PH BV

Hydrogel + nanoparticl es

In vitro:  Viability and adhesion  Chondrogenic differentiation

ADMSCs

 Water uptake capability  Viscoelasticity  Proper shear modulus and stress relaxation

 Nair et al. [221]

Nucleus pulposus

NU

MA

ED PT Multilayered

CE

AC

Nucleus pulposus

SC RI PT

Bone

3.3 Heparin Heparin is a natural negatively charged glycosaminoglycan, which has a linear backbone containing sulfonic, carboxylic and sulfanilamide groups. Its most interesting features are the anticoagulation activity and the affinity with various growth factors (GFs) such as basic 37

ACCEPTED MANUSCRIPT fibroblast growth factor (bFGF), VEGF, and BMP [222,223]. Heparin is well known for its capability to control fibroblast and endothelial biological activity influencing cell mitosis, migration and differentiation. It also modulates angiogenesis and autocrine cell proliferation [224]. However, it is subject to denaturation by enzymatic degradation which decreases its function [225].

SC RI PT

It has been demonstrated that the disaccharidic unit of heparin affects the interactions with proteins and cells. In particular, heparin N-sulphation and 3–O-sulphation play an important role in the anticoagulant properties, while heparin 2–O-sulfated iduronic acid residues and 6– O-sulfated glucosamine residues are fundamental for FGF signal transduction. Heparin also

NU

influences the osteogenic activity due to its ability in controlling the BMP expression [226]. The anticoagulation activity can be described by the interaction of heparin with the protein

MA

antithrombin III. The modification of the antithrombin III conformation results in binding with thrombin and coagulation factor Xa and in serine proteases inhibition, thus avoiding the

ED

blood coagulation [227]. This peculiar characteristic of heparin makes it an interesting

PT

candidate for improving the hemocompatibility while preventing thrombogenicity of the biomaterials used for biomedical application (Table 7). Heparin coating on medical devices,

CE

such as extracorporeal circuits for cardiopulmonary bypass (CPB) and stents have already

AC

been successfully achieved [228]. However, heparin anticoagulation properties are also used in vascular and liver tissue engineering. Heparin surface modification, covalent immobilizing, and layer-by-layer coating have been developed in order to inhibit blood coagulation on the scaffold surface. Gong et al. designed a vascular graft composed of decellularized aortic tissue reinforced by PCL electrospun nanofibers, using heparin to create a coating for the inner surface of the graft in order to increase its hemocompatibility [229].

38

ACCEPTED MANUSCRIPT In a similar manner, several groups working on liver regeneration modified decellularized liver matrices with heparin to inhibit the thrombogenicity of the matrix. Layer-by-layer, selfassembly, covalent binding via multi-point attachment, or end-point attachment techniques were also investigated for heparin immobilization. Bruinsma et al. demonstrated that layerby-layer heparin deposition significantly decreased thrombosis phenomena and reduced the

SC RI PT

blood flow resistance, while supporting re-cellularization without altering the hepatocellular function [227]. Bao et al. demonstrated that heparin end-point attachment method outperformed compared to other deposition methods with showing higher deposition efficiency. Scaffolds coated with this technique revealed to be highly bio and hemocompatible,

NU

non-thrombogenic and able to assist hepatocyte and endothelial cell proliferation [230]. Finally, You et al. investigated the effect of heparin-based hydrogel as coating. The possibility

MA

of loading cells such as fibroblasts in the hydrogel matrix, allowed the graft to improve hepatocyte function, increasing albumin expression and cell junction formation [231].

ED

Heparin has been also considered as a carrier for drug delivery systems, thanks to its ability of

PT

non-covalently binding to different bioactive molecules such as growth factors (GFs), protecting them from degradation and even enhancing their binding to cell surface receptors

CE

[232]. Lee et al. proved that heparin-conjugated carrier systems might have clinical relevance

AC

as they could retain low concentration of GFs in the scaffold, avoiding undesired effects related to GF abundance such as adipose tissue formation [233]. Because of these properties, researchers started to use heparin to modify the surface of biomedical scaffolds for tissue healing and regeneration, controlling the release and the efficiency of GFs [234]. Kim et al. loaded BMP-2 in heparin modified porous poly(lactide-co-glycolide) microspheres for bone regeneration purposes. The results showed significantly higher ALP activity, calcium deposition, and osteocalcin and osteopontin mRNA expression [234]. Fernández-Muiños et al.

39

ACCEPTED MANUSCRIPT ., instead, designed a scaffold made of self-assembling peptide RAD16-I and heparin sodium salt loaded with VEGF165. They demonstrated that the graft enhanced the expression of collagen type II and proteoglycans [232], proposing it as a suitable candidate for cartilage engineering. Long et al. incorporated bFGF in the heparin coating of electrospun PCL scaffolds, showing improved human foreskin fibroblast proliferation in vitro, however they couldn’t

SC RI PT

observe any statistically significant ligament regeneration in a rat model of ligament reconstruction [223].

Spadaccio et al. used electrospinning to incorporate heparin into poly(L-lactide) (PLLA) fibers that were seeded with BM-MSCs. Sustained release of heparin increased cell proliferation and

NU

induced a shift toward CD31 positivity [235].

Lately, the synergetic effect of heparin combined with metallic [236], ceramic [237], and

MA

magnetic [238] nanoparticles has been explored. It has been demonstrated that particle addition increases the mechanical properties of heparin constructs while heparin guarantees

ED

the antithrombogenicity and the control release of angiogenic factors. Knaack et al. proposed

PT

a collagen/hydroxyapatite nanocomposite scaffold for bone regeneration, investigating different methods of heparin and VEGFs encapsulation. The mineralized scaffolds showed

CE

efficient VEGF binding and a controlled growth factor release, enhancing the vascularization

AC

of the construct [237].

Table 7. Current strategies of heparin-based materials in tissue engineering. Targeted organ/ tissue

Bone

Bone

Scaffold fabrication technique

Layer-bylayer

Freezedrying

Scaffold composition

Scaffold morphology

Heparin/ chitosan

Multilayered

Heparin/fib rin/ ErhBMP-2

Hydrogel

In vivo/in vitro

In vitro:  Cytocompatibility  Alkaline phosphatase activity  nuclear factor kappa-B ligand expression In vivo (mouse model):  rhBMP-2 controlled release 

Cells

Remarks

ASCs

 Osteoprogenitor cell phenotype is maintained

--

 Mineralized tissue formation  Low adipose tissue formation

Reference

 Romero et al. [239]

 Lee et al. [240]

40

ACCEPTED MANUSCRIPT Heparin/PL GA/BMP-2

Porous microspher es

In vitro:  ALP activity  calcium deposition OCN and OPN expression

Heparin/col lagen/hydro xyapatite

Bilayered

In vitro:  Cell proliferation

Heparin/ decellulariz ed liver matrix

Bilayered

Liver

End-point attachment

Heparin/ decellulariz ed liver matrix

Multilayered

In vitro:  albumin production  urea production In vivo (rat model):  no clots  low thrombosis level In vitro:  anticoagulant ability  hemocompatibility In vivo (pig model):  no thrombosis evidence

Liver

Lithograph y +crosslinki ng

Heparin/3T 3TCPS/poly urethane

topographi cally patterned + coating

In vitro:  albumin expression  cell junction

Cartilage / vascular

Selfassembly

Hydrogel

Vascular

layer-bylayer assembly

Peptide RAD16-I and heparin sodium salt. Heparin/de cellularized vascular matrix/PCL

Multilayered

Ligamen t

Electrospin ning + crosslinkin g

Heparin/PC L/bFGF

Coated electrospu n

 Osteogenic differentiation

 Kim et al. [168]

HDME C

 Heparin stable bounds  Controlled released of GFs  Favoring vascularization

 Knaack et al. .[237]

 Recellularization  Maintaining of hepatocellular function  Unsuccessful transplantation

 Bruinsma et al. [227]

Hepatoc ytes

 Heparin immobilization  HA scaffolds support the creation of hyaline-like cartilaginous tissue

 Bao et al. [241]

Hepatoc ytes

 Pitches of 400nm allow high hepatocyte functionality

 You et al. [242]

In vitro:  collagen II expression  proteoglycans expression

ADSC and HUVECs

 Proper mechanical properties  Chondrogenic differentiation

 FernándezMuiños et al. [243]

In vitro:  biocompatibility In vivo (rat model):  angiogenesis  anticoagulation In vitro:  cell proliferation In vivo (rat model):  cell infiltration aligned collagen fiber

Plateletrich plasma

 No vasodilation or aneurysm formation

 Gong et al. [244]

HFFs

 Maximum failure load: 30% of the native ACL.  bFGF linear release

 Leong et al. [245]

NU

Hepatoc ytes and HUVECs

MA

Liver

ED

Bone

MG-63

SC RI PT

Fluidic method + electrostati c interaction Freezedrying and chemical crosslinkin g layer-bylayer assembly

PT

Bone

4. Conclusion

CE

In this review, we have attempted to give an overview about the recent achievements and use

AC

of fibrous proteins and GAGs in tissue engineering and biomedical applications. Thanks to the significant technological strides of the last decade, nowadays researchers can design and process natural biopolymers into different scaffold forms through very sophisticated techniques, which allow precise tailoring of the final chemical, mechanical, structural and biological features of the constructed scaffolds. The achieved accuracy and reproducibility have permitted better understanding how to design the optimal microenvironments for cell growth, spreading and differentiation. However, to date a thorough knowledge of all processes involved in organogenesis is still missing. Trying to mimic and replicate nature is 41

ACCEPTED MANUSCRIPT one of the biggest challenges that mankind has always faced and, probably, will represent a major challenge during the current century. Besides the applications of engineered constructs for organ/tissue reconstruction, naturally derived biomaterials are nowadays tested in combination with bioactive molecules, therapeutic drugs, and cells for the treatment of age-associated chronic diseases such as

SC RI PT

cardiovascular diseases, arthritis, osteoporosis etc. and infectious diseases such as HIV, tuberculosis, infectious gastrointestinal diseases. Most likely, with the projections of population growth forecasting a two-fold increase by 2050, addressing these problems represents a hot topic for both academia and pharmaceutical companies. This, in turn, will

NU

guide biomaterials scientists in the development of a new generation of smart biomaterials for the treatment of the world most pressing healthcare problems.

MA

In conclusion, we believe that natural biopolymers such as GAGs and fibrous proteins and their derivatives will continue to play a key role not only in the field of tissue engineering, but

ED

also for the development of innovative drug delivery systems, biosensors, environmental-

PT

friendly packaging materials, cosmetics, and biomedical products with increasing clinical

CE

applications.

AC

5. Acknowledgements

N.C. would like to thank for the funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013/ under REA grant agreement No. 607868 (iTERM). C.R. would like to thank for the funding from the National Centre

for

Research

and

Development

(STRATEGMED1/233224/10/NCBR/2014)

and

in

the

frame

NewJoint

of project

START

project

(Pol-Nor/202

132/68/2013).

42

ACCEPTED MANUSCRIPT

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[1]

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[241] J. Bao, Q. Wu, J. Sun, Y. Zhou, Y. Wang, X. Jiang, et al., Hemocompatibility improvement of perfusion-decellularized clinical-scale liver scaffold through heparin immobilization., Sci. Rep. 5 (2015) 10756. doi:10.1038/srep10756. [242] J. You, V.K. Raghunathan, K.J. Son, D. Patel, A. Haque, C.J. Murphy, et al., Impact of Nanotopography, Heparin Hydrogel Microstructures, and Encapsulated Fibroblasts on Phenotype of Primary Hepatocytes., ACS Appl. Mater. Interfaces. (2014). doi:10.1021/am504614e. [243] T. Fernández-Muiños, L. Recha-Sancho, P. López-Chicón, C. Castells-Sala, A. Mata, C.E. Semino, Bimolecular based heparin and self-assembling hydrogel for tissue engineering applications, Acta Biomater. 16 (2015) 35–48. doi:10.1016/j.actbio.2015.01.008. [244] W. Gong, D. Lei, S. Li, P. Huang, Q. Qi, Y. Sun, et al., Hybrid small-diameter vascular grafts: Antiexpansion effect of electrospun poly ε-caprolactone on heparin-coated decellularized matrices, Biomaterials. 76 (2016) 359–370. doi:10.1016/j.biomaterials.2015.10.066. [245] N.L. Leong, A. Arshi, N. Kabir, A. Nazemi, F. a Petrigliano, B.M. Wu, et al., In vitro and in vivo evaluation of heparin mediated growth factor release from tissue-engineered constructs for 60

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anterior cruciate ligament reconstruction., J. Orthop. Res. 33 (2015) 229–36. doi:10.1002/jor.22757.

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The most significant proteins and GAGs for tissue engineering and regenerative medicine are reviewed. Technological advances enable enhanced design of natural biopolymeric scaffolds. Scaffolds based on fibrous proteins and GAGs promote functional cell organization and differentiation both in vitro and in vivo. The use of bioactive molecules and nanoparticles in combination with fibrous proteins and GAGs is discussed.

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