Advanced Silk Fibroin Biomaterials for Cartilage Regeneration - ACS

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Advanced Silk Fibroin Biomaterials for Cartilage Regeneration Gu Cheng, Zahra Davoudi, Xin Xing, Xin Yu, Xin Cheng, Zubing Li, Hongbing Deng, and Qun Wang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00150 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 8, 2018

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ACS Biomaterials Science & Engineering

Advanced Silk Fibroin Biomaterials for Cartilage Regeneration Gu Cheng a, Zahra Davoudi c, Xin Xing a, Xin Yu a, Xin Cheng a, Zubing Li a, *, Hongbing Deng b, *, Qun Wang c, *

a

Department of Oral and Maxillofacial Surgery, Hospital of Stomatology & The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine, Ministry of Education, Wuhan University, Wuhan 430079, China

b

Hubei International Scientific and Technological Cooperation Base of Sustainable Resource and Energy, School of Resource and Environmental Science, Wuhan University, Wuhan 430079, China

c

Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50014, USA

*Corresponding authors: E-mail: [email protected], Tel/Fax: 86-027-87686217; (Z. Li) E-mail: [email protected]; [email protected], Tel/Fax: 86-027-68778501 (H. Deng) E-mail: [email protected], Tel/Fax: 1-515-2944218; (Q. Wang)

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Abstract Silk fibroin is regarded as a natural fibrous protein with tunable mechanical properties, acceptable biocompatibility and the favorable capability of enhancing attachment, proliferation and differentiation of chondrocytes. These properties make it suitable for the fabrication of scaffold and a broad range of silk fibroin-based biomaterials for cartilage regenerative therapy which can heal functional cartilage without scar tissue. It can be used as a single material for making different kinds of scaffold or as a composite with other types of biomaterials. Together with some growth factors, silk fibroin scaffolds can form a support for the growth and differentiation of seeding cells, such as chondrogenic lineage cells and mesenchymal stem cells. The recent advancements of silk fibroin in cartilage regeneration is summarized in this review. Furthermore, the manufacture methods of silk fibroin materials and their applications in the regeneration of cartilage were also discussed.

Keywords: silk fibroin; cartilage regeneration; fabrication methods; scaffolds.

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1. Introduction The regeneration of damaged cartilage is limited due to the poor capability of the self-repair. Cartilage defects and inability of cartilage to regaining its original organization and functionality will eventually lead to osteoarthritis

1-2

. Therefore,

repair strategies for damaged articular cartilage are important3-6. The contemporary treatments in clinics include arthroscopic debridement/lavage, microfracture/drilling, autologous chondrocyte transplantation (ACT) and other procedures7-9. None of these treatment options can help the patient to recover completely and the problems accompany those including limitations of donors, healing complication of the donor site, lack of enough endurance of the implants, limited mobility and low rate of success of transplantations have not been resolved 10-14. As traditional treatments for cartilage injury have some disadvantages in clinic, cartilage regenerative medicine for the treatment of cartilage injuries and damages have been widely studied as a promising alternative therapy

15-16

. Cartilage is an

avascular, aneural and alymphatic tissue surrounded by a dense extracellular matrix (ECM) 17-18, 19-21. Mimicking the natural ECM is a prominent criterion in the selection of an ideal scaffold for cartilage repair. The ECM was comprised of water (70–80%), collagen (50–75%), and glycosoaminoglycans (15–30%)

10, 22-23

. Chondrocytes secrete matrix, and in turn the

ECM directs the cellular behaviors with the aid of integrin–ligand interactions and provides mechanical support for cells and tissue. Furthermore, glycosoaminoglycans content is an important mark for cartilage formation as they help linking cells to the ECM and the neighboring cells 24. The ECM is spatially organized on multiple length scales, resulting in an extra mechanical function which could resist to compressive loads with low friction articulation. In total, the distribution of the ECM is vital for the maintenance and health of cartilage tissue 25. Silk fibroin, as a natural protein, has been fabricated into various kinds of constructs for production of cartilaginous ECM26-27. Two main proteins are forming the silk fibroin skeleton, sericin, the outer-layer coating protein and fibroin, the inner-layer protein 28. Silk fibroin contains 18 different kinds of amino acids 29. Many 3



studies have shown that fabrication of silk fibroin-based scaffolds with diverse structures yields favorable biocompatibility in addition to the tunable degradation rate and mechanical properties

30

. Compared to other biological materials, fibroin was

shown to have predictable proteolytic degradation by changing the fibroin diameter, the failure strength, the number of cycles to failure, and mass31. By controlling the drying rate, Lu et.al reduced the content of silk II (β-sheet) and attained water-insoluble silk scaffolds with faster degradation compared to the scaffolds fabricated by methanol and water annealing. This property would expand the biomedical functionality of the silk fibroin-based scaffolds due to the desirable higher rate of in vivo degradation in some applications 32. Furthermore, silk fibroin also has advantage of minimal inflammatory reaction33. Compared to silk, inflammatory reaction of collagen or even poly lactic acid scaffolds was more pronounced in vitro and in vivo, and thus it is presumed that silk is more biocompatible than collagen or even poly lactic acid 34. The dedifferentiation of chondrocytes during the process of in vitro expansion raises a challenge for cartilage regeneration. However, the three dimensional scaffolds made from silk fibroin could reverse this process and promote the expression of the cartilaginous ECM35. Consideration the advantages of silk fibroin, it can be used alone or as a composite with other materials for scaffold fabrication to trigger cartilage regeneration. An ideal source of seed cells with chondrogenic potential is equally important with a scaffold in cartilage tissue engineering. The cellular functions were also affected by physical and mechanical characteristics of scaffolds, such as topography, tensile strength and stiffness. Three main kinds of chondrogenic cells for cartilage tissue engineering, the undifferentiated MSCs, the mature chondrocytes isolated from animal or human articular cartilage, and the progenitor chondrocyte-like (ATDC5) cell line were discussed in this review 36-37. Chondrocytes can directly secrete cartilage associated ECM factors without external induction 38, and they are commonly isolated from articular cartilage tissues. However, decrease of chondrocyte phenotype and the cease of chondrogenic markers producing during in vivo expansion hinders its clinical application. Consequently, 4


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ideal scaffolds for cartilage tissue engineering should keep the chondrogenic phenotype of chondrocytes and promote the expression of chondrogenic markers, such as collagen type II, glycosaminoglycans and aggrecan 39. A recent study showed that an injectable silk fibroin-based composite biomaterial with the unique structure can maintained the morphology of porcine chondrocytes and enhanced cellular viability

40

. Another study demonstrated that the scaffolds fabricated from both A.

assamensis silk and P. ricini silk could both enhance the metabolic ability of porcine chondrocytes. Moreover, chondrocytes attached onto the A. assamensis silk scaffold expressed more collagen type II and sulphated glycosaminoglycans when compared with those of P. ricini silk, which suggested that silk fibroin-based scaffolds fabricated by different methods have different influences on the biological activities of chondrocytes

41

. Based on the results of the above-mentioned studies, it can be

concluded that chondrocytes are suitable candidates for cartilage tissue engineering. MSCs, with capability of differentiating into chondrocytes, are easier to be isolated than chondrocytes

42

. MSCs are often incorporated into the silk fibroin

scaffolds for cartilage tissue engineering, offering many advantages as chondrocytes 43

. Chondrogenic differentiation makers of MSCs can be upregulated on silk

fibroin/collagen composite mats in comparison with those of the collagen mats. This study proved that the chondrogenic differentiation of MSCs could be stimulated by silk fibroin 44. Jaipaew seeded human MSCs derived from umbilical cord blood onto the silk fibroin-based scaffold and found that the scaffold provided mechanical and morphological stimuli for human MSCs with cartilage regeneration enhancement 45. Another candidate for cartilage tissue engineering is ATDC5 cells. Kundu et al. inoculated ATDC5 cells into the silk fibroin-based sponges which fabricated from Samia ricini cocoons and demonstrated that ATDC5 cells-seeded sponges possessed good mechanical strength and flexibility. Interestingly, the ATDC5 cells-seeded silk sponges not only enhanced growth of ATDC5 cells, but also promoted matrix production46, suggesting that combination of ATDC5 cells with these sponges might be a proper choice of scaffolds for cartilage regeneration. In tissue engineering, growth factors can be added to the polymeric scaffold 5



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during the scaffold fabrication or after that. Seeding cells into the silk fibroin scaffolds, together with some growth factors may play a supernumerary promoting role on regeneration of cartilage. It is generally known that Insulin-like Growth Factor-1 (IGF-1) contributes a lot in cartilage anabolism and maintaining the cartilage phenotype

47-48

. Uebersax et al. fabricated IGF-1-loaded silk fibroin scaffolds and

found out that the IGF-1-loaded scaffolds have a better promotion effect on human MSCs chondrogenesis in comparison with the controls with no IGF-1 loading

49-50

.

Transforming Growth Factor-beta 3 (TGF-β3) enhances chondrogenesis and cartilage regeneration, and cartilage formation triggered by TGF-β3 is of great importance for the development of embryo51-52. Mechano Growth Factor (MGF) is an alternative of IGF-1. Luo 53 found that incorporation of MGF and TGF-β3 into silk fibroin scaffolds significantly promoted migration of stem cells to the defect sites, and in turn enhanced cartilage formation compared to the TGF-β3-loaded scaffolds. Thus, it can be regarded as an innovative technology for cartilage repair. Varghese et al found out that chondroitin sulphate was able to promote chondrogenic transformation of bone MSCs54. However, it was shown in a different study that the chondroitin sulphate crosslinking into silk fibroin scaffold could not statistically alter the measured collagen or glycosaminoglycan content, but resulted in the amplified stiffness of the scaffold

55

. Thus, silk fibroin could increase the contents of collagen and

glycosaminoglycan. It was demonstrated that chondroitin sulphate-conjugated silk constructs could promote cellular metabolic activity, and enhance the chondrogenic re-differentiation potential with significantly improved mechanical properties over silk-only constructs. Furthermore, they found that the interaction of chondroitin sulphate with cellular surface Annexin 6 receptors results in triggering the signaling cascade through PKC-α, activation of MEK-ERK pathway and regulation of integrin expression. Stimulation of WNT-5A pathway by integrin activates the p38-MAPK pathway. Combination of these cascades leads to an enhanced production of ECM proteins and glycosoaminoglycans 56.

2. Silk fibroin in cartilage tissue engineering 6


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2.1 Fabrication methods of silk fibroin-based biomaterials There are many methods to fabricate silk fibroin or silk fibroin-based composites for cartilage regeneration, such as freeze-drying, salt leaching, gas foaming and electrospinning. Various forms of silk fibroin-based biomaterials using those methods can be fabricated and applied for cartilage regeneration including mat/films, particles, fibers, hydrogels, porous sponge scaffolds (Figure 1 and Table 1) 57-63. 2.1.1 Freezing–drying The freeze-drying method is considered as a conventional process to obtain porous structure of scaffold by freezing and drying [Figure 2]. Hydrogel or porous sponge scaffolds of silk fibroin can be formed through this method

64

, and the pore

microstructures (pore size, pore interconnectivity and porosity) can be effectively controlled by changing the freezing regime like adjusting the cooling temperatures or silk fibroin concentration. However, there will be residual organic solvent in the scaffold and the strength of the scaffolds is not high enough for cartilage regeneration using this method of fabrication. In a recent study, a new silk fibroin 3D architecture with lamellar structure was fabricated using the freeze-drying technique. Human MSC attached, proliferated and produced ECM on the patterned surface of the lamellar morphology. Additionally, gelatin/silk fibroin microspheres made by freeze-drying method can form a release delivery system and be used for cartilage protection or repair in the cartilage tissue engineering 65-66. 2.1.2 Salt leaching Sodium chloride granules，paraffin wax particles，or sucrose particles are utilized as the porogenic agents for the salt leaching method, resulting in 3D porous sponge scaffolds with controlled high porosity, large surface and pore size that can be applied in different kinds of tissue engineering scaffolds [Figure 3(A)]

67-68

. Makaya et al.

showed that the pore size of fibroin scaffolds was enlarged, and the cartilage formation was improved with the use of salt leaching technique. Comparing with the scaffold with smaller pores, those with larger pores have an enhanced effect on involvement and ECM deposition of chondrocytes

67

. In another study, a newly

developed sucrose/hexafluoroisopropanol silk fibroin scaffolds showed more tunable 7



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morphology in comparison with the salt/water scaffold, due to the slower degradation and tighter deposition. It became a better candidate for cartilage tissue engineering. The high-strength salt/water scaffold, however, is more suitable for the bone substitutes fabrication. The microfiber-reinforced silk hydrogel scaffold fabricated by salt leaching method showed an improved mechanical property and a superior chondrocyte response on silk fibroin-silk hydrogels 16. 2.1.3 Gas foaming Gas foaming is a kind of method to create an interconnected pore structure with high porosity, which facilitate migration of chondrogenic cells. The principle of this technique for pore creation is rapid pressure relief around the liquefied polymer, which will results in thermokinetics instability69. Varying the amount of gas in polymer can control both pore structure and porosity of the fabricated constructs. Nazarov et al. employed ammonium bicarbonate to act as a porogens and fabricated a cartilaginous scaffold by gas forming[Figure 3(B)]

70

. Compared to the

scaffolds made by other techniques such as salt leaching, this fabricated scaffold had a higher degree of interconnectivity and compressive strength. For instance, in scaffolds fabricated by salt leaching technique, both closed and interconnected pores were present which led to an uneven distribution of compressive forces and collapse of scaffolds. Furthermore, a higher interconnectivity may result in higher connections among cells, and it facilitates the formation of 3D matrix. 2.1.4 Electrospinning Electrospinning is regarded as a convenient and efficient way to fabricate scaffolds with high porosity in tissue engineering

71-75

. Silk fibroin-based mats or

fibers could be formed by electrospinning. However, the electrospinning mats fabricated from silk fibroin are not suitable to construct a tissue-engineered cartilage due to the morphological difference between a two-dimensional membrane and a three-dimensional cartilage tissue. 2.1.5 Hydrogels The hydrogels have excellent physiological characteristics with the properties which are like the natural ECM. Different kinds of hydrogels such as alginate, 8


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chitosan, hyaluronic acid, collagen type II and silk fibroin have been frequently prepared for cartilage tissue engineering. Among those, silk fibroin exhibited controllable degradation rates and had superior mechanical strength compared to the other hydrogels. Park et al. fabricated a sonicated silk hydrogel for the regeneration of cartilage and proved that the addition of silk lowered the degradation of the composite fibrin/HA hydrogels while maintaining growth of chondrogenic cells76. Moreover, silk fibroin hydrogels also can be used as a carrier system with growth factors and seed cells incorporated. A hepatocyte growth factor-loaded silk fibroin/collagen hydrogel was fabricated by Kamp et al. Under the influence of hepatocyte growth factor, various kinds of stem cells, including chondrogenic precursor cells could be recruited to defect sites 77. The silk fibroin/ polyethylene-glycol-diacrylated composite hydrogel was fabricated and used as a three dimensional cell-carrier system. The stem cells embedded in the composite hydrogel exhibited an excellent metabolic activity

78

.

Inspired by this study, chondrogenic cells could also be incorporated into silk fibroin-based hydrogels.

2.2. Silk fibroin-based composite scaffolds for cartilage regeneration Sericin removal during the course of silk fibroin scaffolds fabrication, results in weaker in vivo inflammatory responses of silk fibroin products compared to the collagen or polylactide. This property is regarded as the major advantage of silk fibroin applied in cartilage tissue engineering

79-80

. Furthermore, excellent

biocompatibility enables silk as one of the most suitable materials to fabricate high-quality cartilage tissue 81. In practical applications, silk fibroin is often used with other biomaterials for the study of cartilage regeneration. 2.2.1 Silk fibroin The scaffolds fabricated by silk fibroin provided morphological cues for the maintainence of chondrocyte phenotypes and promoted the expression of cartilaginous ECM

41

. The adjustable properties of these scaffolds enable better

communication between biomaterials and cells68. Talukdar et al. seeded chondrocytes with different densities onto the 3D silk fibroin scaffolds, and it was found that the 9



cells/scaffold compounds with the highest density of chondrocytes expressed compressive stiffness and modulus several times more than those of the empty scaffolds with no cells. It indicates that high density of chondrocytes improves the regeneration of cartilage82. Reinhart-King et al. studied how matrix mechanics influence the stability of cell contacts. The results suggested that matrix stiffness determined the contacting length of cells which can be detected by adjacent cells83. Bhardwaj et al. have also fabricated silk fibroin-based scaffolds with large aperture, high degree of porosity, excellent biocompatibility and thermal properties, which promoted the proliferation and attachment of chondrocytes and also improved the secretion and deposition of cartilaginous ECM 41. To explore the underlying mechanism, Otaka et al. observed aggregation and migration of chondrocytes on the silk fibroin scaffolds and found that cells attached to the fibroin expressed a higher proximity with other cells compared to those attached to the silk fibroin with beta-sheet structures and RGD amino sequences (ProNectin). It indicates that the fibroin substrate could promote the aggregation of chondrocytes. Moreover, silk fibroin constructs seeded with a high density of chondrocytes exhibited a phenotype more closely resembling the natural cartilage tissue in physiological conditions 84. 2.2.2 Silk fibroin combined with other materials (Silk fibroin composite scaffolds) The ideal scaffolds for cartilage tissue engineering should possess a high degree of elasticity and sufficient mechanical strengths to maintain the shape of cartilage tissue

85

. As a natural fibrous protein, silk fibroin possessed numerous biological

properties such as biocompatibility, excellent physical properties, and biodegradation 86

. However, several disadvantages such as large brittleness and relatively

hydrophobicity limits its applications in tissue engineering 81, 87. Blending silk fibroin and other natural polymers can overcome these shortcomings and improve the property of silk fibroin-based materials for cartilage tissue engineering. 2.2.2.1 Silk fibroin/chitosan Chitosan is also a non-toxic biomaterial and can be enzymatically degraded in 10


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human body 88-89. Moreover, glycosaminoglycan residues existed in chitosan structure can assist supporting the growth of cartilage tissue and the structural similarity of chitosan to various glycosaminoglycans of the articular cartilage is a potential advantage for using chitosan in cartilage regenerative therapies. The importance of this property of chitosan relies on the ability of glycosaminoglycans to adjust the chondrocytes morphology, differentiation, and functions 90. Incorporation of chitosan into the silk fibroin-based scaffolds could improve the elasticity and flexibility of the scaffolds for cartilage tissue engineering. Production of silk fibroin/chitosan composite scaffolds by incorporation of chitosan into silk fibroin can neutralize the mechanical properties by forming hydrogen bond between chitosan and silk fibroin. This results in enhanced mechanical properties of silk fibroin scaffolds, and also improved adhesion and proliferation of seed cells. A recent study demonstrated that incorporation of silk fibroin into chitosan scaffold reduced the degradation time of the composite scaffolds containing chitosan

91

. The

growth and attachment of bovine chondrocytes were also supported by silk fibroin/chitosan scaffold with 1 to 1 ratio, and higher glycosaminoglycan and collagen content was detected after in vitro culture for 2 weeks 92. Furthermore, the mechanical properties of the thermosensitive silk fibroin/chitosan hydrogels were significantly optimized by introducing of silk fibers into the composite chitosan fabricated from. The proliferation and viability of the cells, as well as collagen type II and glycosaminoglycans production were also augmented 93. The same promoting function was found in vivo. To repair the cartilage defects, Deng et.al seeded bone MSCs in a 3D silk fibroin/chitosan scaffold and implanted it inside the rabbit knee [Figure 4(A)]. Bone MSCs-seeded silk fibroin/chitosan scaffolds showed higher modified Wakitani scores compared to other groups. Thus, they considered silk fibroin/chitosan scaffolds as a good niche for the growth of stem cells and cartilaginous matrix production 94. Overall, incorporation of chitosan could improve the degradability, mechanical properties of the silk fibroin-based composite scaffold, facilitate the adherent and proliferation of the chondrogenic cells, and enhance the expression of cartilaginous 11



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matrix. Thus, it is safely concluded that the silk fibroin/chitosan composite scaffolds would be a promising candidate for cartilage regeneration. 2.2.2.2 Silk fibroin/collagen Collagen, one of the major components of ECM, reveals an excellent biocompatibility accompanied by lower immunogenicity, cell adhesion, and proper biodegradability. Furthermore, its specific interactions with biological molecules facilitate ingrowth of seed cells into scaffolds

95-96

. The incorporation of collagen

increased the biocompatibility of the composite scaffold made by silk fibroin and collagen, and the composite scaffold displayed an operative improvement in the characteristics of each component 97. Furthermore, introduction of collagen provided the silk fibroin/collagen composite scaffold with excellent ability of cell affinity. The prepared scaffold enhanced healing of cartilage tissue and integration after implantation into the full-thickness articular cartilage defects of rabbits 98. Ghezzi fabricated dense collagen-silk fibroin-dense collagen electrospun mats using layer by layer method and seeded MSCs onto the scaffold. Growth and chondrogenic differentiation of MSCs were compared with those seeded on the dense collagen or silk fibroin scaffolds. The results suggested that the chondrogenic differentiation markers and cartilage matrix were upregulated on dense collagen-silk fibroin-dense collagen mats in comparison with those of the dense collagen mats

99

.

The higher mechanical stiffness of dense collagen-silk fibroin-dense collagen mats compared to dense collagen may be attributed to the involvement of silk fibroin content to improve the poor strength of collagen. On the other hand, adding collagen into silk fibroin-based scaffold promoted the chondrogenic differentiation of chondrocytes and enhanced the production of cartilaginous matrix. 2.2.2.3 Silk fibroin/poly L-lactic-acid Poly L-lactic-acid, a synthetic biomaterial, was already approved by the FDA for clinical usage

100

. With excellent mechanical strengths and adjustable degradability

time, incorporation of poly L-lactic-acid may construct a silk fibroin/poly L-lactic-acid composite scaffold with suitable degradability and functional mechanical characteristics approaching those of native cartilage 101. 12


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Nevertheless, the break down products of poly L-lactic-acid was acid and induced aseptic inflammation. Moreover, the hydrophobic nature of poly L-lactic-acid inhibits the nutrition penetration. Silk fibroin biomaterials, however, have little inflammation reactions and present hydrophilic property after removing the sericin from the raw material. Therefore, combination of poly L-lactic-acid and silk fibroin might increase the hydrophilicity of the poly L-lactic-acid-based scaffolds, facilitating attachment of chondrogenic cells 102. 2.2.2.4 Silk Fibroin/agarose Agarose is extracted from algae and routinely fabricated as the hydrogel biomaterial in most situations. The introduction of agarose into the silk fibroin-based hydrogel also improved the mechanical properties of the composite scaffold 64. However, agarose is not biodegradable and can cause immunogenic responses when it is implanted into the body. Thus, blending biodegradable and non-immunogenic silk fibroin with agarose may also enhance the biocompatibility of the silk fibroin/agarose composite scaffold. The previous study showed that silk fibroin hydrogel provided a neutral and hydrophilic environment. This environment supported the spherical chondrocyte phenotype, that is similar to or to some extent better than that of agarose 103. Singh for the first time blended silk fibroin and agarose to fabricate a hydrogel for cartilage tissue engineering. The results demonstrated that both the growth of chondrocytes and the production of cartilaginous ECM were promoted by silk fibroin/agarose blended scaffolds compared to those of pure agarose hydrogels. TNF-α expression of chondrocytes located in the blended scaffolds was similar with that of tissue culture plate [Figure 4(B)]

64

. So, the blended hydrogels

minimized the immune response by blending with non-immunogenic silk fibroin and also retained the desired properties as native articular cartilage. However, the proper ratios of silk fibroin and agarose used for the cartilage regeneration should be optimized meticulously. 2.2.2.5 Silk fibroin/ hyaluronic acid Hyaluronic acid, with high viscosity and good swelling property, can retain a certain amount of water and has positive effects for cells attachment, ECM expression 13



and nutrients transportation

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104-105

. These properties allowed hyaluronic acid to be an

excellent candidate for construction of a tissue-engineered cartilage. Blending of hyaluronic acid with silk could keep balance of water within the silk fibroin/hyaluronic acid scaffold and maintain survival of chondrogenic cells. Jaipaew et.al combined silk fibroin with hyaluronic acid using salt leaching preparation method and obtained the silk fibroin/hyaluronic acid composite scaffold with high swelling ratio and water uptake. It could be inferred that introduction of hyaluronic acid might help to form an elastic structure in the silk fibroin/hyaluronic acid composite scaffold

45

. Sawatjui et.al also incorporated hyaluronic acid into the silk

fibroin-based scaffolds and proved that the growth and chondrogenesis ability of bone MSCs were promoted in the fabricated silk fibroin/hyaluronic acid composite scaffold compared to silk fibroin scaffold. Moreover, an increase in the sulfated glycosaminoglycan production was also observed in cells adhered to the fibroin/hyaluronic acid composite scaffold 106. Also, the weak properties of hyaluronic acid like instability and limited mechanical properties may be improved107, by blending it with other polymers, such as silk fibroin that has superior mechanical properties compared to hyaluronic acid. Foss et.al prepared a 3D silk fibroin/hyaluronic acid scaffold by salt leaching technique with the goal of maintaining the phenotype of chondrocytes. The results demonstrated that the degree of crosslinking in silk fibroin/hyaluronic acid composite scaffold was smaller than that of silk fibroin scaffold without hyaluronic acid incorporated 108. As a natural component in native cartilage, hyaluronic acid may contribute to the cartilage regeneration more directly. Incorporation of hyaluronic acid facilitated the survival of chondrogenic cells and production of cartilaginous matrix

108

. Moreover,

the instability and rapid degeneration of hyaluronic acid can be moderated by silk fibroin with higher stability, better mechanical properties and slower degeneration rate. Therefore, it is more suitable to apply the combination of those two in cartilage tissue engineering. 2.2.2.6 Silk fibroin/cellulose Cellulose is a hydrophilic polymer with biodegradability, easy-defoaming and 14


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chelating abilities. With incorporation of cellulose, silk fibroin/cellulose showed a reinforced mechanical strength and a slow degradation time compared with those of silk scaffold

109

. Another study also demonstrated that blending of silk fibroin and

cellulose generated a composite scaffold with an improved mechanical property 110. Cellulose also has the disadvantages of excessive stiffness and relative long degradation time. Introduction of silk fibroin with relative high elasticity can overcome the disadvantages of cellulose and bring advantageous properties of the both materials at the same scaffold

111

. Singh established a specific mixture of

cellulose and silk fibroin to stimulate MSC chondrogenic maturation with no need to any chondrogenic growth factors stimulation. This scaffold was introduced as an implantable device for triggering cartilage repair by endogenous stem cells. By mixing with silk fibroin, a significant decrease in extravagant tensile strength of cellulose was observed. However, this kind of blended scaffold still needs to be further investigated in vivo 112. 2.2.3. Silk fibroin-based scaffolds for cells delivery The repair of cartilage defects cannot achieve a very satisfactory result due to its limited ability of regeneration. Furthermore, the lack of cells and blood supply within the cartilage also impair the regeneration ability of the native cartilage. Therefore, cell-preloading

therapies,

in

which

three

dimensional

constructs

carrying

chondrogenic cells can be served as a cells niche, might solve this problem. With high swelling ability and similar physical properties to native cartilage, silk fibroin-based hydrogel can maintain the integrity of three dimensional polymeric structure, and has been regarded as a candidate for cell delivery 113. Silk fibroin-based hydrogel preloading with cells is fabricated by mixing cells with the aquenous silk fibroin solution. After gelation of the silk hydrogel, cells were authored and encapsulated in the three dimensional hydrogel

114-115

. In this

cells-preloading system, silk fibroin-based hydrogel plays a vital role in protection of the chondrogenic cells and provided new options to facilitate cartilage regeneration 116

. Coaxial electrospinning is a technology which has been developed to fabricate 15



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nanofibers with core-shell structure and encapsulate bioactive growth factors into the core. This technology can also be used to generate the silk fibroin-based coaxial nanofibrous mats with living cells incorporated into the core solution of nanofibers. The cells-containing coaxial nanofibers have been attained by cells suspension flows through the internal tube and the solution of silk-based composite polymer flows through the external tube with confluence at coaxial spinneret before ejection

117

.

However, two dimensional shape of the coaxial nanofibrous mat compromise its usage in cartilage tissue engineering.

3. Sterilization methods used for the silk fibroin-based scaffolds Sterilization of the tissue-engineered scaffolds is the critical step before culturing the cells in vitro and performing animal experiments in vivo, as microbial contaminants within scaffolds should be inactivated before such operations. However, sterilization methods might change the properties and structure of the scaffold prepared by silk fibroin, especially in protein or polymer forms. These properties include topography, morphology, crystallinity, tensile strength and stiffness, which greatly influenced the attachment and proliferation of cells within the silk fibroin-scaffolds

118

. Therefore, only a limit amount of methods were suitable for

sterilization of the silk fibroin-based scaffolds 119. 3.1 Irradiation Irradiation with ultraviolet light and gamma ray were the routinely used sterilization methods. Among them, ultraviolet light cannot penetrate into the deep regions of scaffolds. So, it is not suitable for sterilization of three dimensional scaffolds, but rather for electrospunning membranous ones 120. Under the treatment of gamma irradiation, the random coil in silk fibroin may translate into β-sheet, which reducing the degradation time of scaffolds

121

. Other

characteristics of Bombyx mori silk fibroin films, however, were not significantly affected by irradiation of gamma ray

122

. Consequently, gamma-irradiation is

suggested as a suitable sterilization method as gamma ray can penetrate into the deep area of the bulk silk scaffolds. 16


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3.2 Heat Heat sterilization includes autoclaving and dry-heating. Whether the structural and mechanical properties of the silk fibroin-based scaffolds are affected after heat sterilization depends on fabrication techniques and the sources of silk protein. A previous study demonstrated that sterilization of autoclaving did not change the structure of the silk fibroin scaffolds fabricated from B. mori L. cocoons 119. Also, the morphology and structure of fibers fabricated from spider silk proteins could be retained after the treatment of autoclaving in water

123

. However, most studies

demonstrated that autoclaving has limit negative effects on the morphological and mechanical properties of the silk fibroin-based scaffold. Garry et al. proved that tensile strength of the sutures fabricated from silk was slightly reduced. But the stiffness was not changed by thermal sterilizations, including both autoclaving and dry-heating. The reason accounted for this phenomenon is that proteinaceous component in silk sutures was denatured by heat

124

.

Furthermore, autoclaving in a

humid state reduces the mean diameter of pores and porosity of the porous silk scaffolds

119

, and increase gelation time of the aqueous silk fibroin solution

121

. To

summarized, autoclaving in a dry state is an appropriate sterilization method for the silk fibroin-based scaffolds. 3.3 Ethylene oxide Ethylene oxide is a routinely used chemical agent for sterilization of biomedical instruments or constructs. However, sterilization by ethylene oxide leaved harmful residues within scaffolds

119

. In consideration of the difficulty in clearance of

chemical residues, ethylene oxide sterilization is not recommended if there are other options. 3.4 Ethanol treatment 60%-80% ethanol is easily accessible and a most commonly used agent for sterilization

119

. The scaffolds treated by aqueous ethanol still possessed good

biocompatibility as the ethanol disinfectant did not negatively affect morphological or mechanical characteristics of the scaffolds

119

. Although aqueous ethanol cannot

eliminate all the microbiological contaminant, such as hydrophilic viruses and fungal 17



Page 18 of 38

spores, it is recommended to use this sterilization method in most situations.

4. Conclusion and outlook Silk fibroin-based scaffold has similar compressive module to the native cartilage, and its controlled degradation rate and excellent biocompatibility made it a suitable candidate for cartilage regeneration. Silk fibroin plays a vital role in cartilage regeneration, but other novel technologies, such as two-dimensional nanomaterials have been added to it to improve its functionality and overcome its defects. The high ratio of surface to volume and ultrathin structure of two-dimensional nanomaterials has been shown to reinforce the mechanical properties of various kinds of polymers

125

. Some two-dimensional nanomaterials with the ability of delivering

growth factors were also utilized to fabricate scaffolds with a control releasing function

126

. These unique advantages are absent in the three dimensional bulk

scaffolds that allows two-dimensional nanomaterials to be a suitable candidate for biomedical applications, including tissue engineering and control releasing of bioactive factors 127-128. Consequently, future researches should be focused on the reinforcement of silk fibroin-based scaffolds with the two-dimensional nanomaterials. It is safely deduced that incorporation of the two-dimensional nanomaterials may help to improve the mechanical stiffness of silk fibroin-based scaffolds and deliver the chondrogenic grow factors to the specific sites for cartilage regeneration. Thus, using this composite scaffold may enhance the silk fibroin application in cartilage tissue engineering, and as a result increase the cartilage regeneration more efficiently. Acknowledgements This work was supported by the National High Technology Research and Development Program of China (863 program, No. 2015AA020313) and the National Key

Research

and

Development

Program

of

China

(Grant

Numbers

2016YFB0303303) and the Natural Science Foundation of China (No.81470718 and 81771051). There is no conflict of interest among authors. Dr. Wang is grateful for the support from Crohn’s & Colitis Foundation of America (CCFA) Career Award (No. 18


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348137), PhRMA Foundation Research Starter Award (No. RSGTMT17), and McGee-Wagner Interdisciplinary Research Foundation.

19



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78. Ciocci, M.; Cacciotti, I.; Seliktar, D.; Melino, S., Injectable silk fibroin hydrogels functionalized with microspheres as adult stem cells-carrier systems. Int. J. Biol. Macromol. 2018, 108, 960. DOI: 10.1016/j.ijbiomac.2017.11.013 79. Kundu, B.; Kurland, N. E.; Yadavalli, V. K.; Kundu, S. C., Isolation and processing of silk proteins for biomedical applications. International Journal of Biological Macromolecules 2014, 70 (8), 70-77. DOI: 10.1016/j.ijbiomac.2014.06.022 80. Koh, L. D.; Cheng, Y.; Teng, C. P.; Khin, Y. W.; Loh, X. J.; Tee, S. Y.; Low, M.; Ye, E.; Yu, H. D.; Zhang, Y. W., Structures, mechanical properties and applications of silk fibroin materials. Progress in Polymer Science 2015, 46, 86-110. DOI: 10.1016/j.progpolymsci.2015.02.001 81. Yan, L. P.; Oliveira, J. M.; Oliveira, A. L.; Caridade, S. G.; Mano, J. F.; Reis, R. L., Macro/microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications. Acta Biomaterialia 2012, 8 (1), 289-301. DOI: 10.1016/j.actbio.2011.09.037 82. Talukdar, S.; Nguyen, Q. T.; Chen, A. C.; Sah, R. L.; Kundu, S. C., Effect of initial cell seeding density on 3D-engineered silk fibroin scaffolds for articular cartilage tissue engineering. Biomaterials 2011, 32 (34), 8927. DOI: 10.1016/j.biomaterials.2011.08.027 83. Reinhart-King, C. A.; Dembo, M.; Hammer, D. A., Cell-Cell Mechanical Communication through Compliant Substrates. Biophysical Journal 2008, 95 (12), 6044. DOI: 10.1529/biophysj.107.127662 84. Otaka, A.; Takahashi, K.; Isshiki, K.; Kambe, Y.; Kojima, K.; Tamada, Y.; Tomita, N. How do chondrocytes aggregate on fibroin substrate, Engineering in Medicine and Biology Society, 2013, 405-408. DOI:10.1109/EMBC.2013.6609522 85. Lee, J. M.; Sultan, M. T.; Kim, S. H.; Kumar, V.; Yeon, Y. K.; Lee, O. J.; Park, C. H., Artificial Auricular Cartilage Using Silk Fibroin and Polyvinyl Alcohol Hydrogel. International Journal of Molecular Sciences 2017, 18 (8), 1707. DOI: 10.3390/ijms18081707 86. Li, H.; Fan, J.; Sun, L.; Liu, X.; Cheng, P.; Fan, H., Functional regeneration of ligament-bone interface using a triphasic silk-based graft. Biomaterials 2016, 106, 180. DOI: 10.1016/j.biomaterials.2016.08.012 87. Bhumiratana, S.; Grayson, W. L.; Castaneda, A.; Rockwood, D. N.; Gil, E. S.; Kaplan, D. L.; Vunjaknovakovic, G., Nucleation and growth of mineralized bone matrix on silk-hydroxyapatite composite scaffolds. Biomaterials 2011, 32 (11), 2812-2820. DOI: 10.1016/j.biomaterials.2010.12.058 88. Ding, F.; Deng, H.; Du, Y.; Shi, X.; Wang, Q., Emerging chitin and chitosan nanofibrous materials for biomedical applications. Nanoscale 2014, 6 (16), 9477-9493. DOI: 10.1039/c4nr02814g 89. Yang, Y.; Wang, S.; Wang, Y.; Wang, X.; Wang, Q.; Chen, M., Advances in self-assembled chitosan nanomaterials for drug delivery. Biotechnology advances 2014, 32 (7), 1301-1316. DOI: 10.1016/j.biotechadv.2014.07.007 90. Altman, A. M.; Yan, Y.; Matthias, N.; Bai, X.; Rios, C.; Mathur, A. B.; Song, Y. H.; Alt, E. U., IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. Stem Cells 2009, 27 (1), 250. DOI: 10.1634/stemcells.2008-0178

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Figure Captions Fig.1 Silk based materials. The summarized topics include two parts: forms and fabrication 65

methods. Reproduced with permission from American Chemical Society;

16

.Copyright 2014 Springer;

. Copyright 2015 Elsevier;

67

64

.Copyright 2016

.Copyright 2009 Elsevier.;

55

.Copyright

2009 Elsevier.

Fig.2 Silk fibroin scaffolds fabricated by freezing-drying method. (a) Schematic diagram illustrating the formation process of SF scaffolds during the freezing-drying method. (b) Optical images of SF hydrogel.AG, agarose; AG-BM, Agarose blended with B. mori silk fibroin; AG-AA, Agarose blended with A. assamensis silk fibroin. Reproduced with permission from 64. Copyright 2016 American Chemical Society. (c) Two-photon excited fluorescence (TPEF) images of SF lamellar scaffolds. (d) SEM images of the cross section of the lamellar scaffold after cultured for 3 weeks with hMSCs. Reproduced with permission from

66

. Copyright 2012 Elsevier. (e) SEM image of

GA-crosslinked G/SF microspheres. (f) and (g) Cumulative release of curcumin and piperine in the GA-crosslinked G/SF microspheres (30/70, 50/50, 70/30, and 100/0). C represents curcumin and P represents piperine. Reproduced with permission from 65. Copyright 2014 Springer.

Fig.3 Silk fibroin porous scaffolds fabricated by salt leaching and gas foaming method. (A) Silk fibroin porous scaffolds fabricated by salt leaching. (a) Schematic diagram illustrating the formation process of silk fibroin porous scaffolds during the salt leaching method. (b-i) Evaluation of the of Su/H and Sa/W fibroin scaffolds by optical pictures (b,f) and SEM images (c,g), Safranine O staining in 8 week

in

vitro

culture

(d,h)

and

immunohistochemical

staining

of

collagen

II

(e,i).Su/H:sucrose/hexafluoroisopropanol fibroin scaffolds; Sa/W: salt/water fibroin scaffolds. Reproduced with permission from

67

. Copyright 2009 Elsevier. (B) Silk fibroin scaffolds fabricated

by gas foaming method. (a) Schematic diagram illustrating the formation process of silk fibroin scaffolds during the gas foaming method. (b) and (c) SEM images of the porous 3D silk fibroin scaffolds of NH4HCO3: silk (10:1 wt %). b: inner; c: outer. Reproduced with permission from

70

.

Copyright 2004 American Chemical Society.

Fig.4 Applications of blended silk fibroin materials in cartilage TE. (A) SF/CS scaffold. Evaluation of the SF/CS scaffold by optical images (a) and SEM images (b). (c)-(h) The effects of the SF/CS 31



scaffold to repair cartilage defects in the rabbit knee at 12 weeks. (c, e, g). Gross observation of cartilage defects at 12 weeks of surgery(d, f, h). Hematoxylin–eosin staining,×100;.NC: normal tissues, RC: repaired tissues. Reproduced with permission from 94. Copyright 2013 Springer. (B) SF/Agarose scaffold. (a) Optical iappearance of the hydrogels. (b)-(d)FESEM images of the hydrogels. Scale bar=200µm. AG: agarose ; AG-BM: Agarose blended with B.mori silk fibroin; AG-AA: Agarose blended with A.assamensis silk fibroin. Reproduced with permission from 64. Copyright 2016 American Chemical Society.

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Tables Table 1 Types of Silk fibroin formats in cartilage TE Forms

Ref

Films

17, 30, 32, 34, 41, 61

Fibers

31, 100, 111, 130

3D scaffolds

45, 53, 60, 68, 80, 82, 84, 91-93, 107-108, 129, 131-133

Hydrogels

16, 64, 103, 134

Sponges

40, 46, 93, 96, 129, 135

33



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Fig.1 Silk based materials. The summarized topics include two parts: forms and fabrication methods. (a) was reproduced with permission from

57

. Copyright 2010 Mary Ann Liebert, Inc; (b)

65

was reproduced with permission from .Copyright 2014 Springer. (c) was reproduced with permission from

64

.Copyright 2016 American Chemical Society. (d) was reproduced with permission from

Copyright 2015 Elsevier. (e) and (f) were reproduced with permission from Elsevier.

34


67

16

.

. Copyright 2009

Page 35 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig.2 Silk fibroin scaffolds fabricated by freezing-drying method. (a)Schematic diagram illustrating the formation process of SF scaffolds during the freezing-drying method. (b) Optical images of SF hydrogel.AG, agarose; AG-BM, Agarose blended with B. mori silk fibroin; AG-AA, Agarose blended with A. assamensis silk fibroin. Reproduced with permission from

64

. Copyright 2016 American

Chemical Society. (c) Two-photon excited fluorescence (TPEF) images of SF lamellar scaffolds. (d) SEM images of the cross section of the lamellar scaffold after cultured for 3 weeks with hMSCs. Reproduced with permission from 66. Copyright 2012 Elsevier. (e) SEM image of GA-crosslinked G/SF microspheres. (f) and (g) Cumulative release of curcumin and piperine in the GA-crosslinked G/SF microspheres (30/70, 50/50, 70/30, and 100/0). C represents curcumin and P represents piperine. Reproduced with permission from 65. Copyright 2014 Springer.

35



Page 36 of 38

Fig.3 Silk fibroin porous scaffolds fabricated by salt leaching and gas foaming method. (A) Silk fibroin porous scaffolds fabricated by salt leaching. (a) Schematic diagram illustrating the formation process of silk fibroin porous scaffolds during the salt leaching method. (b-i) Evaluation of the of Su/H and Sa/W fibroin scaffolds by optical pictures (b, f) and SEM images (c, g), Safranine O staining in 8 week

in

vitro

culture

(d,

h)

and

i).Su/H:sucrose/hexafluoroisopropanol Reproduced with permission from

67

immunohistochemical

fibroin

scaffolds;

Sa/W:

staining

of

salt/water

collagen fibroin

II

(e,

scaffolds.

. Copyright 2009 Elsevier. (B) Silk fibroin scaffolds fabricated

by gas foaming method. (a) Schematic diagram illustrating the formation process of silk fibroin scaffolds during the gas foaming method. (b) and (c) SEM images of the porous 3D silk fibroin scaffolds of NH4HCO3: silk (10:1 wt %). b: inner; c: outer. Reproduced with permission from Copyright 2004 American Chemical Society.

36


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Fig.4 Applications of blended silk fibroin materials in cartilage TE. (A) SF/CS scaffold. Evaluation of the SF/CS scaffold by optical images (a) and SEM images (b). (c)-(h) The effects of the SF/CS scaffold to repair cartilage defects in the rabbit knee at 12 weeks. (c, e, g). Gross observation of cartilage defects at 12 weeks of surgery(d, f, h). Hematoxylin–eosin staining,×100;.NC: normal tissues, RC: repaired tissues. Reproduced with permission from

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. Copyright 2013 Springer. (B) SF/Agarose

scaffold. (a) Optical iappearance of the hydrogels. (b)-(d)FESEM images of the hydrogels. Scale bar=200µm. AG: agarose ; AG-BM: Agarose blended with B.mori silk fibroin; AG-AA: Agarose blended with A.assamensis silk fibroin. Reproduced with permission from 64. Copyright 2016 American Chemical Society.

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Title: Advanced Silk Fibroin Biomaterials for Cartilage Regeneration Authors: Gu Cheng a, Zahra Davoudi c, Xin Xing a, Xin Yu a, Xin Cheng a, Zubing Li a, *, Hongbing Deng b, *, Qun Wang c, *

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Advanced Silk Fibroin Biomaterials for Cartilage Regeneration - ACS

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