Bombyx mori derived scaffolds and their use in cartilage regeneration: a systematic review

Accepted Manuscript Bombyx mori derived Scaffolds and their use in Cartilage Regeneration: A Systematic Review Numan Fazal, Noreen Latief PII:

S1063-4584(18)31384-0

DOI:

10.1016/j.joca.2018.07.009

Reference:

YJOCA 4278

To appear in:

Osteoarthritis and Cartilage

Received Date: 13 April 2018 Revised Date:

5 July 2018

Accepted Date: 11 July 2018

Please cite this article as: Fazal N, Latief N, Bombyx mori derived Scaffolds and their use in Cartilage Regeneration: A Systematic Review, Osteoarthritis and Cartilage (2018), doi: 10.1016/ j.joca.2018.07.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Bombyx mori derived Scaffolds and their use in Cartilage Regeneration: A Systematic Review Numan Fazal 1, Noreen Latief 1* 1

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Abstract

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Centre of Excellence in Molecular Biology, University of the Punjab, Pakistan. Corresponding author: [email protected]

For the last two decades, silk has been extensively used as scaffolds in tissue engineering because of its remarkable properties. Unfortunately, the aneural property of cartilage limits its

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regenerative potential which can be achieved using tissue engineering approach. A lot of research has been published searching for the optimization of silk fibroin and its blends in order to get the best cartilage mimicking properties. However, according to our best knowledge, there is no systematic review available regarding the use of Bombyx mori derived biomaterials limited to cartilage related studies. This systematic review highlights the in vitro and in vivo work done for the past seven years on structural and functional properties of Bombyx mori derived

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biomaterials together with different parameters for cartilage regeneration. PubMed database was searched focusing on in vitro and in vivo studies using the search thread “silk fibroin” and “cartilage”. A total of 40 articles met the inclusion criteria. All the articles were deeply studied for cell types, scaffold types and animal models used along with study design and results. Five

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types of cells were used for in vitro while seven types of cells were used for in vivo studies. Three types of animal models were used for scaffold implantation purpose. Moreover, different

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types of scaffolds either seeded with cells or supplemented with various factors were explored and discussed in detail. Results suggest the suitability of silk as a better biomaterial because of its cartilage mimicking properties. Keywords: Silk fibroin, Scaffold, Cartilage regeneration, Biomaterials, Stem cells

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ACCEPTED MANUSCRIPT 1. Introduction

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Silk has been utilized as a surgical suture material for years and now it’s being

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extensively used as a biomaterial in various biomedical applications [1].Silk fibroin

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(SF) fibers are about 10–25µm in diameter and consist of two major proteins: (fibrous

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protein) and sericin (globular protein). Fibroin protein consists of a glycoprotein named

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P25 a light (26 kDa) and a heavy chain (325 kDa) in a ratio of 1:1, linked by a disulfide

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bond [2, 3]. Silk Fibroin mostly consist of serine, glycine and alanine amino acids that

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helps crystalline β-sheets formation within the silk fibres giving it an exceptional

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mechanical strength and hydrophobic domain structure. Silk can be obtained from many

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insects including spiders, flies, beetles, mites and scorpions but the most extensively

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studied and worldwide produced silk is obtained from Bombyx mori having diverse

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functions and biocompatibilities [4]. Commercial silk obtained from silkworms is

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categorized as mulberry and non-mulberry [5]. Bombyx mori silk serves as commercial

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mulberry while Antheraea assamensis (muga), Antheraea mylitta (tropical tasar),

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Antheraea pernyi (temperate oak tasar) and Samia ricini/Philosamia ricini (eri) serves

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as non-mulberry silk producers (Fig. 1) [6-8]. Due to good water vapour and oxygen

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permeability and low inflammatory response silk fibroin is known to be a suitable

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material for skeletal tissue engineering [9]. Moreover, SF offers exceptional benefits

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over conventional biomaterials as its cell adhesion and cell growth characteristics, low

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thrombogenicity, slow and controllable biodegradability, protease susceptibility, high

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tensile strength as well as flexibility makes SF highly biocompatible and ultimately a

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good natural scaffold for tissue engineering [10-14]. SF solution can be fabricated into

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films, particles, electrospun fibers, nets, sponges, hydrogels, and three-dimensional (3D)

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porous scaffolds [15-19]. They have been widely used in musculoskeletal, vascular,

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skin, hepatic, ligament/tendon and nerve tissue engineering [20-26]. Normal cartilage is an avascular tissue with an intercellular protein matrix

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reinforced by a three-dimensional (3-D) network of collagen fibrils [26]. This

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multilayered connective tissue is composed of chondrocytes that are dispersed in a thick

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extracellular matrix (ECM). The negatively charged glycosaminoglycans (GAGs)

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attracts water and provide a hydrophilic environment to the ECM that helps cartilage to

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experience a swelling pressure and ultimately provides tensile strength to the

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interspersed collagen network [27]. The aneural and alymphatic characteristics of

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cartilage limits the ability of its self-repair [28]. Untreated Injury of the cartilage may

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lead to swelling and pain, gradually leading to degenerative lesions, osteoarthritis or

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even disability [29].

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treatment of chondral defects but none of these prove vital in long-term complete

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treatment [30, 31]. A potential solution to these limitations can be achieved using tissue

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engineering approach [32]. A combination of patient’s own cells along with artificial

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scaffolds can be useful in engineering a biomimetic autologous construct for the

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treatment of osteochondral defects (Fig 2) [33, 34].

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Normally, conventional surgical procedures are applied for the

Cartilage tissue engineering is specifically applied to treat cartilage injury, with

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some key considerations: seeding cells, growth factors, scaffolds and their

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mechanobiology. Scaffolds selection play a key role in cartilage tissue engineering [35].

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Reports suggest that scaffolds having the chondrogenesis inducing ability are preferred

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for the repair of cartilage defects (Fig 3) [36]. Generally, an ideal scaffold should have

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good biocompatibility and biomechanical properties, suitable degradation time, and a

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three dimensional (3D) nanoporous structure [37]. Moreover, scaffold particularly used

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in cartilage regeneration should facilitate migration, has the ability to induce new

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ACCEPTED MANUSCRIPT extracellular matrix assembly and possesses appropriate mechanical properties to

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withstand the required physiological load [38]. The implanted scaffold seeded with

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cartilage regenerating cells can help in filling the wound and tissue repair to close the

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gap (Fig.4) [39, 40]. For seeding purpose, adipose derived mesenchymal stem cells

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(MSCs) based therapies are considered as a better choice for cartilage repair because of

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their remarkable regeneration properties[41, 42].

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In the recent years, silk proved to be a good choice to fill the tissue engineering

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gap because of its superior mechanical properties as compare to hydrogels or collagens.

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Moreover, silk promote cartilage deposition in a robust and homogeneous fashion

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because of its biocompatibility and slow biodegradation of silk fibroin materials related

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to collagen scaffolds [43]. Several articles have been published regarding the use and

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efficacy of silk scaffolds in cartilage tissue engineering by evaluating the

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biocompatibility, degradation rates, immune response towards silk scaffolds, blending

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and mechanical properties. This systematic review provides an insight to research

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progress in the field of biomaterials using silk scaffolds along with stem cells and their

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potential use in cartilage regeneration for the past seven years.

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2. Materials and Methods

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2.1 Search criteria

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A systematic review of the literature was done on the use of Bombyx mori derived

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scaffolds and their application in cartilage regeneration. PubMed database was searched

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focusing on in vitro and in vivo studies using the search thread “silk fibroin” and

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“cartilage”. Articles publishing date filter was set from 2011 to September, 2017.

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2.2 Inclusion and exclusion criteria Filtered articles were first screened by title and then abstract. Furthermore, Review

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articles, silk derived from other sources rather than Bombyx mori and articles other than

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English language were excluded. All the articles focusing on both Bombyx mori derived

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silk fibroin scaffolds and cartilage regeneration were included. References list of the

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articles were also screened to search for any possible article omitted in the study.

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3. Results

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Initial screening criteria resulted in a total of eighty-three articles. Forty articles were

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included in the study by further narrowing down the screening criteria mentioned above.

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Selected articles were further divided into two categories (in-vivo, in-vitro) based on

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their research approach. 27 out of 40 articles focused on in-vitro studies while 13

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articles (Articles = 12, Reports = 1) focused on in-vivo approach using different animal

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models (Fig. 5a). Brief details of the reviewed articles is listed in table 1 and table 2

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below.

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Three types of animal models (Rabbit, Mice, Sheep) were used for in vivo studies

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(Fig. 5b). Rabbits were used extensively as 10 of 13 studies focused on Rabbit models.

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Similarly, seven different types of cells were used in twelve in vivo studies (Cells =12,

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without cells = 1). Five studies out of 12 relied on bone marrow derived mesenchymal

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stem cells (Fig. 6a). Scaffolds were either surgically (n = 8) or subcutaneously (n = 5)

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implanted. Furthermore, five types of cells were used in-vitro (Fig. 6b). In-vitro studies

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were of diverse approaches ranging from simple to complex materials. Six studies used

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pure silk as a biomaterial. Sixteen studies revealed the effect of silk blended with a

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single material either in equal or in various ratios. Three studies used three materials

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blended together including silk fibroin and two studies, one focused on Silk-hyaluronic

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acid biphasic and other one trailered silk scaffold supplemented with Chondrogenic and

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Osteogenic induction medium.

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4. Discussion

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4.1 In-vitro studies

Millions of patients are suffering from cartilage defect nowadays caused by injury,

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trauma, and age-related degeneration [28]. Muscoskelatel disorders represent a global

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threat worldwide to healthy aging [44], ranked as second (21.3%) among years lived

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with disability (YLDs) globally after mental and behavioral problems (23.2%) [45]. As

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the current treatments for cartilage defects are unsatisfactory to restore the native

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cartilage, a different approach of scaffolding is used in cartilage tissue engineering in

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order to repair, maintain and improve tissue function [38, 46]. Silk as a natural product,

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exhibiting several properties mimicking cartilage, has been studied extensively in recent

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years.

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The articles studied in this systematic review highlight a variety of materials,

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techniques and approaches towards the goal of finding a better silk biomaterial. Silk has

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been used as a main component blended with several other materials in many ways:

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blended either in ratios with Hyaluronic acid [47], PLLA [48], Chitosan [49-52],

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cellulose [53], gelatin [52] or in various percentages with chitosan and hydroxyapatite

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[54], Hyaluronic acid [55] and Fibrin/ Hyaluronic acid [59]. Chitosan and blended with

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silk fibroin enhances β-sheet conformation and flexibility of the blended material [56,

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57]. Furthermore, the structural similarity of chitosan to that of glycosaminoglycans

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serves as a cartilage like properties to ensure better microenvironment while silk fibroin

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the control of tissue growth however, the immune response of the body should be

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remembered [58]. The Chondrocyte response towards scaffolds induced with pro-

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inflammatory cytokines (IL-1β and TNFα) studied by Kwon suggests that silk can be a

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better source regarding adaptability with the microenvironment, as chondrocytes seeded

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on silk were still able to produce enough extracellular matrix in a traumatic condition as

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compared to PLA [59]. Similar conditions (IL-1β and TNFα) were applied in another

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study conducted by Kwon, where different pore size of silk HIFP sponges revealed that

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the scaffolds with larger pore size supported high extracellular matrix production and

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low expression of cartilage matrix degradation genes[60]. Conversely, in some cases,

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cells seeded on scaffolds were supplemented with growth factors in order to enhance the

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extracellular matrix production [61-65]. Silk hydrogels reinforced with silk microfibers

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proved to be a good option instead of using silk alone as it gives extra mechanical

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strength and also provides microenvironment to chondrocytes in culture conditions [66].

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An enhanced chondrogenic differentiation potential of BM-MSCs was seen using silk

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fibroin/gelatin–chondroitin sulfate– hyaluronic acid (SF–GCH) scaffold [67]. In order

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to study the effect of multilayered osteochondral constructs on bone marrow stromal

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cells in vitro, a two chambered co-culture system was developed using silk as

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biomaterial. The chondrogenic, osteogenic and intermediate layer induction resulted in

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the formation of cartilage-like and bone-like tissues along with the intermediate

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osteochondral interface [68]. In a study conducted by Park and colleagues, biphasic

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scaffolding technique was used to achieve a similar intervertebral disc like structure in

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vitro. Silk protein was used for the Annulus fibrous (AF) and fibrin/hyaluronic acid

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(HA) gels for the NP region while porcine AF cells were seeded on AF region and

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chondrocytes on the NP region [69]. Salt-leached macro/microporous silk scaffolds

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ACCEPTED MANUSCRIPT (S16) and Silk nano-calcium phosphate scaffolds (SC16) were evaluated by Yan and

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colleagues and found that both scaffolds were non-cytotoxic, supported adhesion,

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proliferation and migration [70]. A comparative study of freeze dried and three

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dimensional silk scaffolds was performed revealing the better performance of three

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dimensional scaffolds [71]. Silk blends with various curcumin concentrations resulted in

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higher cell viability and better extracellular matrix production [72]. Similarly,

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Glutaraldehyde concentrations and freezing rates effects were checked by Nematollahi

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for tracheal regeneration. Freezing rate of 1 and 2°C/min and 0.8 wt% GA

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concentration proved to be suitable for cartilage regeneration [73]. Knitted silk scaffolds

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coated with poly 3-hydroxybutyrate and Chitosan were studied by Karbasi where Silk +

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poly (3-hydroxybutyrate + Chitosan Hybrid demonstrated better mechanical properties

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[74]. Kundanati studied the mechanical properties of Silk matrix hydrogel, Silk

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Network scaffold and Composite scaffolds [75]

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4.2 In-vivo studies

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In vitro evaluation of new biomaterials provide much information about their

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biomechanical properties, safety, efficiency and repair potential [76] however, the true

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assessment for their regenerative potential and immune response upon implantation

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requires animal models [77]. Moreover, in vivo animal studies are required by

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regulatory bodies for new devices before their translation into clinical practice [78].

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Three types of animal models were used for in vivo studies included in this

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review. Rabbits were mainly used because of their suitability as a practical model for

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early stages of therapy evaluation due to ease of handling, relative cost effectiveness,

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reasonable joint size for surgical procedures, spontaneous healing potential, sizable

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variation from human joint loading conditions and thin cartilage [79, 80]. Moreover, 7

ACCEPTED MANUSCRIPT cells used for in vivo analysis were chondrocytes, Mesenchymal stem cells,

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Mesenchymal progenitor cells and nucleus pulpus (NP) cells while one study did not

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use any cell source as the whole study focused on the scaffold load and inflammatory

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response towards scaffold. Bone marrow stem cells have been extensively used because

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of their easy isolation [81] adhesion to biomaterials [82] and differentiation potential

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when seeded on biomaterials [83].

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For in vivo evaluation of silk scaffolds, Deng observed improved cartilage

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regeneration capability of SF/CS scaffolds seeded with BMSC’s rather than scaffolds

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alone[84]. Similar results were obtained by Kazemnejad, where Scaffolds seeded with

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chondrocytes gave better results than scaffolds alone [85]. The study conducted by Zeng

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also confirmed the approach of seeding scaffolds with cells where NP cells with silk

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scaffolds were evaluated both in vitro and in vivo. NP cells seeded scaffolds provided

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better microenvironment, infiltration and ECM secretion [86]. The idea of using SFCS

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scaffolds mimicking tracheal construct seeded with rabbit chondrocytes showed

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cartilage-like structure in vivo, while gave better environment for cell proliferation, cell-

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cell interaction and differentiation in vitro [87]. Li observed abundant extracellular

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matrix deposition and formation of cartilage cells by seeding PMSC’s on SF scaffold

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[88]. A different approach of using bilayered scaffolds was performed by Yan where

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both cartilage regeneration in the top silk layer and subchondral bone ingrowth and

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angiogenesis in the bottom silk-nano-Calcium phosphate layer was observed [89].

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Biphasic scaffolds were evaluated by Ruan using silk fibroin, chitosan and nano-

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hydroxyapatite that exhibited two types of collagen expression [90]. Shen used knitted

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silk-collagen scaffold observing an increased ligament genes expression and adhesion

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of spindle shaped cells that helped restoring anterior cruciate ligament injury [91]. A

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targeted delivery approach using microsphere incorporated with TGF-β1was performed

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ACCEPTED MANUSCRIPT by Wang yielding enhanced cartilage regeneration [92]. Biphasic calcium phosphate

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ceramic (BCP), silk fibroin protein matrix (SFP) and collagen sponge (CS) constructs

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were assessed by Zheng and colleagues to find out the effect of material on

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chondrogenic induction and suggested that hydrogels are superior to porous materials in

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the induction of chondrogenic differentiation [93]. Sterodimas focused on the efficiency

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of mesenchymal progenitor cells that were grown on Silk-Alginate polymer and

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concluded that they are suitable for human ear mode[94]. Zhang used bi-layer collagen-

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silk scaffold in vivo and injected rabbit model with Parathyroid hormone-related protein

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(PTHrP) at different time periods, the Bilayered scaffold along with intra articular

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injections strategy was effective after 4-6 weeks of injury [95]. Gruchenberg lately

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checked the efficiency of silk scaffolds noticing that silk scaffolds can act like cartilage

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tissue as it withstands the load exerted with no inflammatory reactions [96]. Silk-

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Collagen scaffolds blended in a ratio of 7:3, incorporated with TGF-β1/PLGA

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microspheres showed greater biocompatibility by having good porosity, appropriate

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pore size and diameter and hence resulted in better distribution and adhesion of BMC’s.

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Moreover, the presence of TGF-β1/PLGA microspheres helped in promoting functional

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cartilage formation and working as a bridge through regeneration and integration

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between regenerated and surrounding cartilage [92].

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In vivo analysis reveals that blended/cross-linked scaffolds could be a good source for

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cartilage regeneration instead of pure silk scaffolds. Studies indicate that bio activated

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scaffolds improves the efficiency of cartilage regeneration and even the implanted

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scaffold can be induced through various supplements through injection process. The

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approach of multilayer scaffolds can provide better mimicking properties with a bottom

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layer in contact with bone and upper layer with neighboring cartilage. Furthermore, the

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implanted material and the adjacent cartilage.

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5. Conclusion

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In conclusion, studies on silk derived scaffolds showed promising results that confirms

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the suitability of silk as a better biomaterial because of its mimicking properties to that

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of cartilage. Silk scaffolds seeded with cells gave better results in all the studies as

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compare to silk alone. The approach of bilayered and trilayered scaffolds ensured

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improved cartilage mimicking properties. Moreover, the idea of injecting Parathyroid

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hormone-related protein and other growth factors at various time frames proved to be

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effective in treating defects. The increasing number of research papers clearly indicates

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the importance of silk derived biomaterials in tissue engineering. Nevertheless, the

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online data on silk scaffolds for cartilage regeneration reveals that almost less than a

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half work has been done in vivo so far as compare to in vitro. This leads to a gap

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between in vitro and in vivo research work and it can only be filled by focusing on in

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vivo studies in order to evaluate the better biomaterial for cartilage regeneration for

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clinical purposes.

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Abbreviations

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SF

Silk Fibroin

HA

Hyaluronic acid

PLLA

poly(L-lactic-acid)

CS

Chitosan

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ACCEPTED MANUSCRIPT Strontium-hardystonitegahnite

SF-GCH

Silk fibroin/gelatin–chondroitin sulfate–hyaluronic

HFIP

Hexafluoroisopropanol

CHS)

Chondroitin sulfate hyaluronate acid-silk fibroin scaffold

SF/G

Silk fibroin Gelatin

nHA

Nano-hydroxyapatite

PTHrP

Parathyroid hormone-related protein

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Author contributions

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Noreen Latief contributed to the study design and reviewed the final version of the

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manuscript while Numan Fazal contributed to data acquisition and paper write-up.

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Acknowledgment

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The authors are very grateful to Professor Habib Ahmad for his valuable suggestions for

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the technical improvement of the manuscript.

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Conflict of interest

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The authors declare no conflicts of interest regarding the publication of this paper

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Table 1. In vitro studies using Bombyx mori derived silk fibroin for cartilage regeneration.

S.NO

Author

Year Cell

source

/

Scaffold type used

Study design

RI PT

538

Type

Results

SC

1. Silk alone

2. Silk curcumin Do Kyung Kim [72]

2017

Rabbit

1. Silk Fibroin scaffolds

Chondrocytes

2. Silk/Curcumin blend

blend (0.5mg/ml)

M AN U

1

3. Silk curcumin

Curcumin blended with silk at 1mg/ml exhibited high extracellular matrix production and cell viability.

blend (1mg/ml)

TE D

4. Silk curcumin blend (2mg/ml)

Three freezing rates Tensile strength raised with increase in freezing rate and GA

Blended (0.5, 1, 2 °C/min) concentration. A high Glycosaminoglycans content was

EP

Silk-Chitosan

2

Zeinab Nematollahi1[73]

2017

Rabbit Chondrocytes

AC C

scaffolds with different Three glutaraldehyde observed at 2°C/min for 0.8wt% of GA concentration. freezing

rates

Glutaraldehyde

and (GA)

concentrations

(0, 0.4, 0.8 wt%)

concentrations

18

2016

Silk fibroin (SF)

umbilical cord

2.

SF-Hyaluronic

derived

acid (HA) scaffolds

mesenchymal

Chondrocytes

SF70 showed a consistent porous structure expressing

3. SF/HA (80:20 w/w)

chondrogenic markers.

1.

PLLA/SF nanofibers

EP

Rabbit

AC C

2016

2. SF/HA (90:10 w/w)

TE D

(HUMSCs)

Li Z [48]

The SF blended with HA scaffolds in particularly SF80 and

4. SF/HA (70:30 w/w)

stem cells

4

1. SF 100(Pure)

SC

Jaipaew [47]

1.

M AN U

3

Human

RI PT

ACCEPTED MANUSCRIPT

2.

PLLA/SF scaffold Unmodified

PLLA Scaffolds 1.

Silk fibroin + Chitosan (90:10 v/v)

19

The PLLA/SF scaffold supported chondrocytes growth than the unmodified PLLA scaffold.

ACCEPTED MANUSCRIPT

Vishwanath

2016

Silk

Fibroin

(SF)

blood derived

Chitosan (CS) Blends

+

2.

Chitosan (80:20

mesenchymal

V[49]

Silk fibroin +

v/v) 3.

other scaffolds.

Silk fibroin +

SC

stem cells

The SF/CS (80:20) blend ratio was found to be superior to

RI PT

5

Umbilical cord

Chitosan (70:30

M AN U

v/v) 4.

Silk fibroin +

Chitosan (60:40 v/v)

EP

TE D

5.

6

Kai Sun [71]

2016

Rat BMSCs

Chitosan (50:50 v/v) 1. Physical and

AC C

1. Silk fibroin/collagen 3D

Silk fibroin +

printed scaffolds

chemical

2. Silk Fibroin/Collagen

characterization of

freeze dried scaffolds

scaffolds

20

3D printed scaffolds showed better performance as compare to freeze dried scaffolds.

ACCEPTED MANUSCRIPT

2. Cells viability and

1. Control group for

2. Collagen/sodium alginate hydrogel (CAH) 3. Collagen sponge (CS) 4. Silk sponge (SK)

SK

and

co-cultured

scaffolds

exhibit

high

differentiation potential than control group.

M AN U

BMSCs

2. Inductive

group

for CH, CAH, CS and SK

3. Co-culture

TE D

2017

CH, CAH, CS and Induced

group

for CH, CAH, CS and SK

EP

Ling Zhang [65]

AC C

7

SC

1. Collagen hydrogel (CH)

RI PT

proliferation check

Evaluation of Structural and

FTIR results indicated the presence of all elements without

Mechanical properties

pollution. Tensile test suggest that microfibrous part of

21

ACCEPTED MANUSCRIPT

8

Saeed

Karbasi 2016

Nil

[74]

Knitted

coated

with

Substrate poly

hydroxybutyrate

scaffold could significantly affect mechanical properties of

of

(3-

1. Silk Subtrate

and

2. Silk poly (3-

nano part of the hybrid scaffold.

RI PT

Silk

hydroxybutyrat

SC

Chitosan

e hybrid

M AN U

3. Silk+ poly (3hydroxybutyrat e

+

Chitosan

TE D

Hybrid

Mechanical

Lakshminath Kundanati [75]

2016

Nil

Silk Scaffolds

1. Silk Network Composite scaffolds showed better results as compare to

AC C

9

EP

evaluation of

scaffold 2. Silk

matrix

hydrogel

22

matrix hydrogels and network scaffolds.

ACCEPTED MANUSCRIPT

3. Silk

scaffolds

RI PT

Composite

Zhou T [54]

2015

1.Chitosan/nano-

2. SF/HA(1:3wt%)

Rabbit

hydroxyapatite (HA)

3. SF/HA(2:2 wt%)

Chondrocytes

2.Chitosan and silk fibroin

4. SF/HA(3:1 wt%)

(SF)

All Cross-linked with

EP

hBM-MSC’s

AC C

2015

3. SHG-Silk Scaffold 4. Biphasic (Silk + SHG-

properties, supported growth and infiltration of cells.

2. SHG-Ceramic

2. SHG-Ceramic Scaffold Li JJ [63]

cartilage matrix in terms of compressive mechanical

1. Silk Scaffold

1. Silk Scaffold

11

Chitosan/SF/HA scaffolds partially mimicked the articular

tripolyphosphate

TE D

3.Chitosan/SF/HA

M AN U

10

SC

1. CS/HA (2%wt)

Scaffold 3. SHG-Silk Scaffold

Mechanical assessment of biphasic scaffold revealed the imitated

load-bearing

behavior

to

that

of

native

4. Biphasic Silk +

osteochondral tissue as well as matched the compressive

SHG-Silk Scaffold

properties.

Silk Scaffold)

23

ACCEPTED MANUSCRIPT

All groups

RI PT

supplemented with Chonrogenic and

SC

Osteogenic media 1.Silk fibroin Sawatjui N [67]

2015

BM-MSCs

2.Silk fibroin-gelatinchondroitin sulfate-

SF-GCH

scaffold

promoted

BM-MSCs

proliferation,

2. SF–GCH +Pellet chondrogenic differentiation and also provided a cartilage

M AN U

12

1. SF+ Pellet Culture

Culture

mimicking structure and environment.

hyaluronic acid (SF-GCH) scaffold

TE D

Chondrocytes response to

Kwon H [60]

2015

chondrocytes (BACs)

Silk HFIP scaffolds,

AC C

13

EP

Bovine articular

1. IL-1β

Silk hexafluoroisopropanol (HFIP) scaffolds with greater

2. TNFα

pore sizes, supported deposition of extracellular cartilage

Scaffold

with

pore matrix with lower expression of pro-inflammatory cytokines

sizes 1. 100–200 µm

24

(IL-1β) in articular chondrocytes.

ACCEPTED MANUSCRIPT

2. 300–400 µm

RI PT

3. 500–600 µm 4. 700–800 µm

Yodmuang

S 2014

[66]

Bovine

(SF+SF microfibers)

2. Agarose

Silk hydrogels reinforced with silk microfibers provided an

2. SF-Agarose Hydrogels

3. SF-Silk

hydrogels excellent structural and mechanical microenvironment to

M AN U

14

hydrogels 1. Silk

SC

1. SF-Silk

Chondrocytes

(SF+SF

chondrocytes in culture conditions as compared to silk

microfibers)

hydrogels alone.

TE D

4. SF-Agarose Hydrogels

Two ADSCs groups

AC C

EP

1. Chondrogenic

Adipose derived 15

Ding X [62]

2014

stem cells (ADSCs)

Silk

fibroin

hydroxyapatite trilayered scaffold

and

Induction

The trilayered and integrated osteochondral scaffolds

Medium(TGF-β1)

effectively supported cartilage and bone tissue generation in

blended 2. Osteogenic Induction

25

Vitro. Medium

ACCEPTED MANUSCRIPT

medium)

RI PT

(Dexamethasone

Human adipose Yan L P [70]

2014

tissue derived

scaffolds (S16)

stromal cells

2. Silk nano calcium

(hASCs)

phosphate scaffolds (SC16)

2014

BM-MSC’s

hyaluronate acid-silk

AC C

Sun L [61]

EP

Chondroitin sulfate 17

1. S16+ hASCs

S16 and SC16 were non-cytotoxic, supported cell adhesion,

2. SC16 + hASCs

proliferation and cell migration.

TE D

16

macro/microporous silk

M AN U

1.Salt-leached

SC

• Chondral Layer (SF) • Intermediate layer (SF/n-HA) • Bony Layer (SF/nHA)

fibroin scaffold (CHS)

Gene-modified

silk Mesenchymal stem cells (MSCs) seeded on scaffolds

cable-reinforced CHS proliferated, produced abundant collagen II, TGF-ߚ3 genes hybrid scaffold

and cartilage extracellular matrix (ECM) components.

1. Silk Scaffold + IL1β and TNFα

26

Pro-inflammatory cytokines were released faster by collagen

ACCEPTED MANUSCRIPT

Kwon H [59]

2013

Bovine Chondrocytes

response to

and silk scaffolds as compare to PLA scaffolds with higher

2. Collagen Scaffold

Chondrocytes

water uptake..

3. Polylactic acid PLA

2. Collagen

Scaffold+IL-1β and

SC

Scaffold

RI PT

18

1. Silk Scaffold

TNFα response to

M AN U

Chondrocytes

3. PLA Scaffold+IL1β and TNFα

Chomchalao [52]

2013

Articular chondrocytes

2. SF/C

AC C

19

Chondrocytes

EP

1. SF,

TE D

response

3. SF/G

1. Cell adhesion

SF/C and SF/G scaffolds promoted attachment and

2. Proliferation

proliferation of chondrocytes with more collagen II and

3. Production of

aggrecan than SF scaffold.

extracellular matrix (EMC)

27

ACCEPTED MANUSCRIPT

1. Cellulose Scaffold

20

Singh N [53]

2013

hBM-MSC’s

2. Cellulose

silk

Scaffolds (75:25)

factors upregulated chondrogenic marker genes SOX9,

3. Cellulose + Silk aggrecan, and type II collagen.

SC

Scaffolds

2. Cellulose + Silk Cellulose-Silk Scaffolds (75:25) devoid of specific growth

RI PT

1. Cellulose Scaffold

Scaffolds (50:50)

Rabbit bone marrow stromal

Silk sponge Scaffolds

cell

in BMSCs cultured scaffolds in Osteogenic and chondrogenic

chondrogenic

medium induced the formation of an osteochondral interface

medium

in a co-culture system.

2. BMSCs

cultured

scaffold

in

TE D

2013

EP

Chen K (a) [64]

scaffold

AC C

21

cultured

M AN U

1. BMSCs

osteogenic medium

3. Co culture of both scaffolds 1. Co-Culture

28

ACCEPTED MANUSCRIPT

Rabbit bone Chen K [97]

2016

marrow stromal

Silk sponge scaffolds

Co-culture system resulted in multilayered osteochondral

2. First:

Osteogenic constructs formation containing cartilage-like subchondral

layer

bone like tissue and an intermediate osteochondral interface.

cells (BMSCs)

SC

3. Second:

RI PT

22

diffusion chamber

Chondrogenic

M AN U

layer

4. Middle layer

1. Pure SF

TE D

2. Cross Linked

1. Silk fibroin (SF) 2012

None

2. Hyaluronic acid

EP

Foss C [55]

(HA)

AC C

23

SF/HA sponges

Physical separation of silk fibroin from hydrophilic

(with 1%

Hyaluronic acid resulted in un-cross-linked sponges, while a

WHA/WSF, 2%

homogeneous blend was observed in cross-linking sponges

WHA/WSF, and

preventing the physical separation phenomenon.

5% WHA/WSF ) 3. Not Cross Linked SF/HA sponges

29

ACCEPTED MANUSCRIPT

(with 1%

RI PT

WHA/WSF, 2%

WHA/WSF, and

1.

Silk

(1:1) Both silk fibroin/chitosan blended scaffolds, silk fibroin

1. Silk fibroin scaffolds

marrow cells

2. Silk fibroin + Chitosan

scaffolds (SFCS)

(Bobmyx mori) scaffolds and non-mulberry Antheraea

2.B. mori derived Silk mylitta fibroin

supported

attachment,

proliferation

and

scaffolds differentiation of rat BMSC’s with improved extracellular

(SFBM)

matrix deposition.

TE D

Kundu SC. [51]

Rat bone

3.A.mylitta

derived

Silk fibroin scaffolds

EP

N, 2012

AC C

Bhardwaj

fibroin/

M AN U

chitosan 24

SC

5% WHA/WSF )

from (SFAM) 1. Fibrin/ Hyaluronic acid (F/H)

30

gel

culture Lamellar scaffolds helped supporting annulus fibrous like tissue for over two weeks. A stimulated biphasic scaffold

ACCEPTED MANUSCRIPT

cells

+ Fibrin/ hyaluronic acid (HA)

silk)

nucleus pulpus tissue proved to be effective in total

RI PT

annulus fibrous

3. Fibrin/ Hyaluronic intervertebral disc formation.

2.Chondrocytes

acid having

gel

culture

1%

silk

M AN U

(F/H+ 1S)

4. Fibrin/ Hyaluronic acid

gel

culture

having 1.5% silk

TE D

2012

(F/H + 1.5S)

5. Fibrin/ Hyaluronic

EP

Park SH [69]

AC C

25

Biphasic Silk Fibroin (SF) 2. Silk gel only (2% resulted from the combination of annulus fibrous and

SC

1.Porcine

acid

gel

culture

having 2% silk (F/H + 2S) 1. F / H alone 2. 2% silk gel only,

31

Chondrogenic area expanded in all the groups particularly

ACCEPTED MANUSCRIPT

26

Park SH

2011

chondrocytes

Fibrin/HA

gel

culture 3. Fibrin / Hyaluronic

(F/H) + Silk Scaffolds

acid gel culture

the F/H + 1.5S. Moreover, strong mechanical properties were provided to nucleus pulpus tissue by silk mixed gels.

RI PT

Human

with 1% silk (F/H+

SC

1S)

4. Fibrin / Hyaluronic

M AN U

acid 1.5% (F/H + 1.5S)

5. Fibrin / Hyaluronic

27

Bhardwaj N [50]

2011

chondrocytes

SF Scaffold

AC C

Bovine

EP

TE D

acid 2% 2% (F/H

SF/CS scaffolds

+ 2S)

SF/CS scaffolds supported cell attachment, growth and

1. SF scaffold alone

chondrogenic phenotype With high Glycosaminoglycan and

2. SF / CS (1:1)

collagen accumulation in silk fibroin/chitosan (1:1) blended

3. SF / CS (2:1)

scaffolds

539

32

ACCEPTED MANUSCRIPT

S.NO

Table 2. In vivo studies using Bombyx mori derived silk fibroin for cartilage regeneration Author

Year

Animal Model

Defect Type

Delivery

Cells Type

bone

implantation

osteoid defect

BMSCs

TE D

Rabbit

Primary rabbit

EP

al [90]

et 2017

Surgical

fibroin/chitosan

Scaffold (SF/CS) 2. Silk fibroin/chitosan/ nanohydroxyapatite scaffold

3. Silk fibroin / chitosan- Silk fibroin / chitosan

33

Results

1. Silk

(SF/CS/nHA)

AC C

1 Ruan

Subchondral

M AN U

SC

Method

Study Design

RI PT

540

Bone

marrow

(BMSCs)

mesenchymal

showed

biphasic scaffolds.

good

stem

proliferation

cells on

ACCEPTED MANUSCRIPT

nano-

RI PT

hydroxyapatite

scaffold (SF / CS-

SC

SF/CS/nHA )

cylindrical defects of

Surgical

articular

implantation

cartilage

Osteochondral

Surgical

1. COL/SF 2. COL/SF

Collagen/Silk fibroin scaffolds carrying TGF-

marrow stromal

incorporated with

β1-incorporated PLGA microspheres increased

cells (BMSCs)

TGF-β1

cartilage tissue regeneration as compare to

TE D

Rabbit

EP

al [92]

et 2016

Rat Bone

AC C

2 Wang

M AN U

Thick

microsphere.

collagen/silk fibroin scaffolds alone.

The untreated defects (Control group) 1.

Fibrin glue +

chondrocytes (FGC) group Chondrocytes

34

2.Silk fibroin

Scaffolds with chondrocytes gave better results than scaffolds alone.

ACCEPTED MANUSCRIPT

3 Kazemnej

2016

Rabbit

defect

Implantation

(Autologous)

+

chondrocytes (SFC)

RI PT

ad et al

Scaffold

[85]

group

SC

3.Silk fibroin scaffold (SF) group

meniscectomy

Implantation

Nil

TE D

[96]

Sheep

1. Group 1 Scaffold )

Scaffold and sham groups revealed no

2. Group 2 Sham (sh)

significant

differences Moreover,

in

cartilage

silk

scaffold

3. Group 3 Partial

degeneration.

Meniscectomy (pm)

withstands the loads with no inflammatory

4. Group 4 Scaffold

reaction.

(sc6)

EP

erg et al

2015

Surgical

AC C

4 Gruchenb

Partial

M AN U

4. Untreated group.

Rabbit bone marrow

35

1. Pure silk scaffolds

Bilayered scaffold showed better integration

2. Silk-nano Calcium

with host, superior mechanical properties,

Phosphate scaffolds

stability and better osteogenic differentiation.

ACCEPTED MANUSCRIPT

Rabbit

[89]

Osteochondral

Subcutaneous

mesenchymal

3. Bilayered scaffolds

Cartilage regeneration observed in the top silk

defect

implantation

stromal cells

(Pure silk scaffolds

layer, ingrowth of subchondral bone and

+ Silk-nano Calcium

angiogenesis in the bottom silk-nanoCaP layer.

(RBMSCs)

RI PT

5 Yan et al 2014

Rabbit

Subcutaneous

dorsum

implantation

BMSCs

7 Zeng et al 2014

Nude mice

AC C

EP

al [93]

Pockets in

TE D

6 Zheng et 2014

M AN U

SC

Phosphate

Pockets in

Subcutaneous

scaffolds)

1. Biphasic calcium

BMSCs

showed

no

chondrogenic

phosphate ceramic

differentiation when encapsulated in SFP, BCP

(BCP),

and CS. Moreover, the diffusion chamber was

2. Silk fibroin protein

effective in preventing host immune rejection.

matrix (SFP) 3. Collagen sponge (CS) 1. Silk Scaffold+ NP in

Rabbit Nucleus pulposus (NP)

36

vitro evaluation 2. Silk Scaffold+ NP

Silk scaffolds showed high porosity, good inter connectivity

of

the

pores,

provide

an

appropriate microenvironment to support NP

ACCEPTED MANUSCRIPT

[86]

dorsum

implantation

cells

cells in vivo

cell growth, infiltration and ECM secretion.

SC

RI PT

evaluation

Anterior

Rabbit

[91]

Surgical

ligament (ACL)

implantation

Injury model

Rabbit Primary

M AN U

8 Shen et al 2014

cruciate

MSCs

9 Zhang et

2013

Rabbit

Osteochondral

injection of

Human

defects

PTHrP +

Chondrocytes

et 2013

Rabbit

Full thickness

1.

Silk Scaffold

2.

Knitted Silk-

1. In vitro=human

Collagen-silk scaffold implantation along with

chondrocytes + PTHrP Intra-articular injection of PTHrP proved 2.

In vivo= bi-

Implantation

scaffold + PTHrP

Surgical

after 2 months.

Collagen scaffold

layer collagen-silk

AC C

10 Deng

and adhesion into the scaffold was observed

Surgical

EP

al [95]

TE D

Intra-articular

An enhanced spindle-shaped cells migration

effective by inhibiting terminal differentiation and chondrogenesis enhancement.

Bone marrowderived mesenchymal

37

1. BMSCs + SF / CS scaffold group

SF/CS scaffold seeded with BMSCs enhanced cell proliferation and nearly repaired bone

ACCEPTED MANUSCRIPT

cartilage defect

implantation

stem cells (BMSCs)

2. SF / CS scaffold

defect as compare to control group.

alone group

RI PT

al [84]

3. Control group

s A, de 2013

(Prelimina

Immunocompet

Minor skin

Subcutaneous

ent Rabbit

incision

implantation

ry report)

Silk polymer +

Silk/alginate scaffolds maintained flexibility

progenitor cells

Alginate

and shape. Cartilage tissue formation revealed

(Ear Shaped

the suitability of auricular mesenchymal

biodegradable

progenitor cells in tissue-engineered human ear

12 Li [88]

et al 2012

Rabbit

Surgical

AC C

Full thickness

EP

TE D

[94]

Mesenchymal

M AN U

11 Faria J

SC

Sterodima

osteochondral

implantation

Placenta-

mixed lymphocyte

Human

derived

reactions effect on

responses.

PMSCs

PMSC/SF scaffold facilitate formation of

In vivo: PMSC/SF

cartilage cells and extracellular matrix

stem cells(PMSCs)

38

models.

In vitro: Allogeneic

mesenchymal

defect

scaffold)

biomaterial

PMSCs

inhibited

rabbit

T

cell

ACCEPTED MANUSCRIPT

RI PT

Complex

1. Silk

Fibroin

542 543 544

dorsum

implantation

Rabbit

M AN U

Surgical

chondrocytes

TE D

541

Pockets in

EP

[87]

Nude Mice

AC C

13 Zang et al 2011

blend SFCS scaffold provided 3-D environment for

SC

Chitosan

545

39

+

(SFCS)

chondrocyte proliferation, cell–cell contact,

2.Cell-Scaffold

differentiation under in vitro conditions and

construct (CS) Cell- promoted neo-chondrogenesis Scaffold

construct

wrapped

with

perichondrium (CSP)

ACCEPTED MANUSCRIPT Fig. 1. A schematic diagram representing five different types of commercially used Mulberry and Non-mulberry silkworms.

Fig. 2. Diagram showing the basic in vitro tissue engineering approach for cartilage

RI PT

regeneration.

Fig. 3. A graphical illustration of cells and scaffold interaction towards engineered tissue production

SC

Fig. 4. Diagram showing the basic in vivo tissue engineering approach for cartilage

M AN U

regeneration using silk scaffold.

Fig. 5. A schematic hierarchical diagram showing the articles selection process and animals model used (A). Articles selection process for systematic study (B). Animal models used in the study.

TE D

Fig. 6. A schematic pie charts showing the types and usage frequencies of cells used. (A) Types and frequencies of cells used in vivo, (B) Types and frequencies

AC C

EP

of cells used in vitro.

40

ACCEPTED MANUSCRIPT

Fig. 1. A schematic diagram representing five different types of commercially used Mulberry

RI PT

and Non-mulberry silkworms.

Fig. 2. Diagram showing the basic in vitro tissue engineering approach for cartilage regeneration.

Fig. 3. A graphical illustration of cells and scaffold interaction towards engineered tissue

M AN U

SC

production.

Fig. 4. Diagram showing the basic in vivo tissue engineering approach for cartilage regeneration using silk scaffold.

Fig. 5. A schematic hierarchical diagram showing the articles selection process and animals

EP

in the study.

TE D

model used (A). Articles selection process for systematic study (B). Animal models used

AC C

Fig. 6. A schematic pie charts showing the types/source and usage frequencies of cells used. (A) Types and frequencies of cells used in vivo, (B) Types and frequencies of cells used in vitro.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT