Stem cell-based tissue engineering with silk biomaterials

Silks are naturally occurring polymers that have been used clinically as sutures for centuries. When naturally extruded from insects or worms, silk is composed of a filament core protein, termed fibroin, and a glue-like coating consisting of sericin proteins. In recent years, silk fibroin has been increasingly studied for new biomedical applications due to the biocompatibility, slow degradability and remarkable mechanical properties of the material. In addition, the ability to now control molecular structure and morphology through versatile processability and surface modification options have expanded the utility for this protein in a range of biomaterial and tissue-engineering applications. Silk fibroin in various formats (films, fibers, nets, meshes, membranes, yarns, and sponges) has been shown to support stem cell adhesion, proliferation, and differentiation in vitro and promote tissue repair in vivo. In particular, stem cell-based tissue engineering using 3D silk fibroin scaffolds has expanded the use of silk-based biomaterials as promising scaffolds for engineering a range of skeletal tissues like bone, ligament, and cartilage, as well as connective tissues like skin. To date fibroin from Bombyx mori silkworm has been the dominant source for silk-based biomaterials studied. However, silk fibroins from spiders and those formed via genetic engineering or the modification of native silk fibroin sequence chemistries are beginning to provide new options to further expand the utility of silk fibroin-based materials for medical applications.     

The effect of hyaluronic acid on silk fibroin conformation

The molecular conformation of silk fibroin drastically changes the physical properties of this biomaterial. Herein, we investigated the capacity of hyaluronic acid to modify the conformational transition of silk fibroin into its crystalline beta-sheet form. For this aim, matrices composed of these two polymers were prepared and studied. Instrumental analysis confirmed the presence of two intermixed phases: one of pure hyaluronic acid, and another consisting of a molecular dispersion of silk fibroin and hyaluronic acid. Studies performed with silk fibroin/hyaluronic acid matrices indicated that hyaluronic acid induces molecular transition of silk fibroin into a beta-sheet structure when incubated in water, and that it synergistically enhances beta-sheet formation together with methanol treatment. The enhancement of beta-sheet content observed for silk fibroin/hyaluronic acid matrices correlated with improved mechanical properties: blended matrices had higher compressive moduli and higher breaking strengths than pure silk fibroin matrices. These new properties, together with the capacity of silk fibroin/hyaluronic acid to form partially insoluble matrices without any treatment with organic solvents, make this blend composition an interesting material for biomedical applications.     

Macrophage responses to silk

Silk fibers have potential biomedical applications beyond their traditional use as sutures. The physical properties of silk fibers and films make it a promising candidate for tissue engineering scaffold applications, particularly where high mechanical loads or tensile forces are applied or in cases where low rates of degradation are desirable. A critical issue for biomaterial scaffolds is biocompatibility. The direct inflammatory potential of intact silk fibers as well as extracts was studied in an in vitro system. The results indicate that silk fibers are largely immunologically inert in short- and long-term culture with RAW 264.7 murine macrophage cells while insoluble fibroin particles induced significant TNF release. Soluble sericin proteins extracted from native silk fibers did not induce significant macrophage activation. While sericin did not activate macrophages by itself, it demonstrated a synergistic effect with bacterial lipopolysaccharide. The low level of inflammatory potential of silk fibers makes them promising candidates in future biomedical applications.     

丝生物材料处理及其在组织工程中应用研究现状

丝纤维是一种天然的共聚物,其作为手术缝线等已在临床上应用多年.丝纤维由位于中间的丝素蛋白和包裹丝素蛋白的丝胶蛋白构成.近年来,丝纤维材料由于生物相容性良好,降解缓慢,而且具有非常优异的机械性能,因而其可以作为一种新的生物医学支架材料获得广泛应用.而且由于技术手段的发展,能够对丝纤维材料进行多种加工和处理将其加工成多种形态的支架材料和进行表面修饰,并且通过遗传工程和基因工程进行裁切和生产重组的丝蛋白类似物,这使其在生物医学工程领域有广阔的应用前景.     

Silk-based biomaterials

Silk from the silkworm, Bombyx mori, has been used as biomedical suture material for centuries. The unique mechanical properties of these fibers provided important clinical repair options for many applications. During the past 20 years, some biocompatibility problems have been reported for silkworm silk; however, contamination from residual sericin (glue-like proteins) was the likely cause. More recent studies with well-defined silkworm silk fibers and films suggest that the core silk fibroin fibers exhibit comparable biocompatibility in vitro and in vivo with other commonly used biomaterials such as polylactic acid and collagen. Furthermore, the unique mechanical properties of the silk fibers, the diversity of side chain chemistries for 'decoration' with growth and adhesion factors, and the ability to genetically tailor the protein provide additional rationale for the exploration of this family of fibrous proteins for biomaterial applications. For example, in designing scaffolds for tissue engineering these properties are particularly relevant and recent results with bone and ligament formation in vitro support the potential role for this biomaterial in future applications. To date, studies with silks to address biomaterial and matrix scaffold needs have focused on silkworm silk. With the diversity of silk-like fibrous proteins from spiders and insects, a range of native or bioengineered variants can be expected for application to a diverse set of clinical needs.     

Silk fibroin/poly(vinyl alcohol) photocrosslinked hydrogels for delivery of macromolecular drugs

Hydrogels are three-dimensional polymer networks widely used in biomedical applications as drug delivery and tissue engineered scaffolds to effectively repair or replace damaged tissue. In this paper we demonstrate a newly synthesized cytocompatible and drug releasing photo-crosslinked hydrogel based on poly(vinyl alcohol) methacrylate and silk fibroin which possesses tailorable structural and biological properties. The initial silk fibroin content was 0%, 10%, 20%, 30%, 40% and 50% with respect to the weight of poly(vinyl alcohol) methacrylate. The prepared hydrogels were characterized with respect to morphology, crystallinity, stability, swelling, mass loss and cytotoxicity. FITC-dextrans of different molecular weights were chosen as model drugs molecules for release studies from the hydrogels. The hydrogels containing different silk fibroin percentages showed differences in pore size and distribution. X-ray diffraction analysis revealed that amorphous silk fibroin in poly(vinyl alcohol) methacrylate is crystallized to β-sheet secondary structure upon gelation. The sol fraction increased with increasing fibroin concentration in the co-polymer gel (from 18% to 45%), although the hydrogel extracts were non-cytotoxic. Similarly, the addition of silk fibroin increased water uptake by the gels (from 7% to 21%). FITC-dextran release from the hydrogels was dependent on the silk fibroin content and the molecular weight of encapsulated molecules. The study outlines a newer type of photo-crosslinked interpenetrating polymer network hydrogel that possess immense potential in drug delivery applications.     

Surface treatment of pure and PEG-4000 blended fibroin films and their characterizations as matrices for in vitro fibroblast culture

This study reports the effects of treatment with various concentrations of organic solvents for varying time points on matrices of fibroin, a silk protein isolated from the mulberry silkworm, Bombyx mori, which in native form has been extensively used in tissue engineering. Treatment of pure fibroin as well as polyethylene glycol- blended films with 90% organic solvent for 60 min induces optimal surface hydrophobicity and maximum conversion of the secondary structure from random coil to beta sheet. Long-term cell viability studies reveal that methanol and isopropanol-treated pure and blended films support cell adhesion, proliferation, and viability.     

Non-bioengineered silk gland fibroin micromolded matrices to study cell-surface interactions

Micropatterning/micromolding of protein molecules has played a significant role in developing biosensors, micro arrays, and tissue engineering devices for cellular investigations. Relevantly, there have been ample scopes for silk to be used as natural biomaterial in tissue engineering applications due to its attractive properties such as slow-controllable degradation, mechanical robustness, and inherent biocompatibility. In this paper, we report the fabrication of micromolded silk fibroin matrices, which have essentially been utilized to study cell-surface interactions. Fibroin protein has been isolated from the silk glands of nonmulberry Indian tropical tasar silkworms, Antheraea mylitta. The surface uniformity has been investigated using atomic force microscopy following the fabrication of silk micromolds. Subsequently, cellular interactions in terms of cell attachment, spreading, mitochondrial activity and proliferation have been studied in vitro using feline fibroblasts. Results have indicated a long term stability of patterns in micromolded silk matrices and negligible swelling. The versatility of described silk dissolution method coupled with ability to process large amount of silk protein into micromolded matrices and controllable surface topology may augment the desirability of silk fibroin as a natural biomaterial for bioengineering and biotechnological applications.     

壳聚糖改性丝素合金膜的制备及其相关性质研究

以无毒氧化葡萄糖醛作交联剂 ,采用溶液共混交联法制备壳聚糖改性丝素合金膜。用 FTIR、DSC表征其结构 ,测定其等电点、力学性能、不同 p H条件下的溶胀率和对模型药物 5 - Fu的渗透性。结果表明 :改性丝素合金膜中丝素和壳聚糖分子间存在着强烈的氢键相互作用及良好的相容性。改性膜的等电点对应的 p H值是 5 .35 ,而丝素膜的等电点是 4 .5。改性膜的力学性能优于单组分膜 ,当壳聚糖含量为 4 0 %～ 6 0 %时 ,具有最大的抗张强度和拉伸率 ,分别为 71.4～ 72 .7MPa和 2 .96～ 3.82 %。改性丝素合金膜对 5 - Fu的渗透量与壳聚糖的含量和时间成正相关关系 ,渗透系数随 p H值增大 (5→ 9)先逐渐减小然后略有增大 ,在 p H=7时最小。     

Paclitaxel-loaded silk fibroin nanospheres

Silk fibroin is a very promising biomedical material because of its renewability, nontoxicity, biocompatibility, and biodegradability. On the basis of a simple and mild method for the preparation of silk fibroin nanospheres with controllable size, the authors developed earlier, anti-cancer drug paclitaxel (PTX)-loaded silk fibroin nanospheres ranging from 270 to 520 nm were produced accordingly. The drug loading, encapsulation efficiency, and released property of PTX-loaded silk fibroin nanospheres are depended on the silk fibroin concentration and initial PTX-loading capacity. The maximum drug loading is about 6.9% and the release time of such a kind of nanospheres is over 9 days. The release time of PTX-loaded silk fibroin nanospheres can be as long as 2 weeks when the drug loading is about 3.0%. All these results imply that such a kind of biomacromolecule-based anti-cancer drug nanocarrier has a great potential for chemotherapy in clinical applications.     

Silk fibroin film from non-mulberry tropical tasar silkworms: A novel substrate for in vitro fibroblast culture

The silk protein fibroin, isolated from the cocoon of the domesticated mulberry silkworm, Bombyx mori, is used extensively in biomaterial design and in cell and tissue culture. We report here for the first time the potential application of fibroin obtained from the cocoon of non-mulberry tropical silkworm, Antheraea mylitta, as a substrate for in vitro cell culture. The mechanical strength of A. mylitta silk fibers indicates a stronger thread composition. The contact angle of A. mylitta fibroin films suggests that it has lower hydrophilicity and lower solubility in organic solvents compared to B. mori fibroin films. Retention of a secondary structure of fibroin in both A. mylitta and B. mori films is confirmed by Fourier transform infrared analysis. The adherence, growth and proliferation patterns of feline fibroblast cells on A. mylitta fibroin films suggest that this kind of film has a greater ability to support cell growth than B. mori fibroin films and is comparable to that of control. This study demonstrates that, as well as being non-toxic to dermal fibroblast cells, non-mulberry fibroin might be a useful alternative substrate to the more common B. mori fibroin for a variety of biomedical applications.     

Biospinning by silkworms: Silk fiber matrices for tissue engineering applications

The mechanism of biospinning of natural silk fibers has been an open issue for decades. In this report a natural bio-polymeric matrix based on biospun silk fibers obtained from Antheraea mylitta, a wild non-mulberry tropical tasar silkworm, is put forward for potential applications. This report deals with the conformational transitions of silk fibroin during the biospinning process and its potential to support cell adherence and proliferation. The silk fibers obtained were aligned into linear, mixed or random patterns forming interconnected, macroporous three-dimensional matrices. The matrices were morphologically and functionally characterized with respect to fiber diameter, crystallinity, mechanical strength and biocompatibility using feline fibroblast cells. Drawn silk fibers showed enhanced stability to protease treatment in comparison with naturally occurring native gland fibroin protein. A viability assay suggested biocompatibility of these matrices in vitro. Fluorescence and confocal microscopy indicated normal cell attachment, spreading and proliferation on these biospun silk matrices. The results provided evidence for the use of biospun silk matrices as natural, inexpensive and alternative substrata for tissue engineering applications.     

Silk Fibroin Processing and Thrombogenic Responses

Silkworm-derived fibroin, which constitutes the core of the silk filament, is an attractive protein–polymer for biomedical applications. Fibroin can also be processed into a variety of 2-D and 3-D formats to match morphological and structural features to specific applications. The focus of the present research was to correlate the structure of silk fibroin-derived biomaterials with plasma protein adsorption, platelet activation and inflammatory cell (THP-1 cell line) adhesion and activation. The amino-acid composition of the two types of silk studied influenced the crystallinity of the films, hydrophobicity, surface roughness and biological interactions. Protein adsorption was lower on samples with the higher crystallinity and hydrophobicity, in particular the chemotactic factors (C3a, C5a, C3b), while other proteins such as fibrinogen were comparable in terms of adsorption. As a consequence, platelets and immune cells responded differently to the various films obtained by following different processing protocols and stabilized by different methods (methanol or water vapour) in terms of their adherence, activation, and the secretion of inflammatory mediators by monocytes. The data presented here demonstrate that bioactivity can be influenced by changing the chemistry, such as the source of silk protein, or by the specific process used in the preparation of the materials used to assess biological responses.     

Silk protein fibroin from Antheraea mylitta for cardiac tissue engineering

The human heart cannot regenerate after an injury. Lost cardiomyocytes are replaced by scar tissue resulting in reduced cardiac function causing high morbidity and mortality. One possible solution to this problem is cardiac tissue engineering. Here, we have investigated the suitability of non-mulberry silk protein fibroin from Indian tropical tasar Antheraea mylitta as a scaffold for engineering a cardiac patch in vitro. We have tested cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication and contractility of 3-days postnatal rat cardiomyocytes on silk fibroin. Our data demonstrate that A. mylitta silk fibroin exhibits similar properties as fibronectin, a component of the natural matrix for cardiomyocytes. Comparison to mulberry Bombyx mori silk protein fibroin shows that A. mylitta silk fibroin is superior probably due to its RGD domains. 3D scaffolds can efficiently be loaded with cardiomyocytes resulting in contractile patches. In conclusion, our findings demonstrate that A. mylitta silk fibroin 3D scaffolds are suitable for the engineering of cardiac patches.     

Characterization and optimization of RGD-containing silk blends to support osteoblastic differentiation

The effect of blending two silk proteins, regenerated Bombyx mori fibroin and synthetic spidroin containing RGD, on silk film material structure (beta-sheet content) and properties (solubility), as well as on biological response (osteoblast adhesion, proliferation and differentiation) was investigated. Although the elasticity and strength of silks make them attractive candidates for bone, ligament, and cartilage tissue engineering applications, silk proteins generally lack bioactive peptides for enhancing cell functions. Thus, a synthetic spider silk, spidroin, containing two RGD cell adhesive sequences (RGD-spidroin) was engineered. RGD-spidroin was blended with different ratios of fibroin and spun coat into films on glass coverslips. beta-Sheet formation, contact angle, surface topography and RGD surface presentation were characterized and correlated with cell behavior. We found that the amount of beta-sheet formation was directly related to the RGD-spidroin content of the blends after annealing, with the pure RGD-spidroin demonstrating the highest amount of beta-sheet content. The increased beta-sheet content improved film stability under culture conditions. A new visualization technique demonstrated that the RGD presentation on the film surface was affected by both the RGD-spidroin content and annealing conditions. It was determined that 10mass% RGD-spidroin was necessary to improve film stability and to achieve osteoblast attachment and differentiation.     

Chondrogenic differentiation of rat MSCs on porous scaffolds of silk fibroin/chitosan blends

Adult bone marrow derived mesenchymal stem cells are undifferentiated, multipotential cells and have the potential to differentiate into multiple lineages like bone, cartilage or fat. In this study, polyelectrolyte complex silk fibroin/chitosan blended porous scaffolds were fabricated and examined for its ability to support in vitro chondrogenesis of mesenchymal stem cells. Silk fibroin matrices provide suitable substrate for cell attachment and proliferation while chitosan are promising biomaterial for cartilage repair due to it’s structurally resemblance with glycosaminoglycans. We compared the formation of cartilaginous tissue in the silk fibroin/chitosan blended scaffolds with rat mesenchymal stem cells and cultured in vitro for 3 weeks. Additionally, pure silk fibroin scaffolds of non-mulberry silkworm, Antheraea mylitta and mulberry silkworm, Bombyx mori were also utilized for comparative studies. The constructs were analyzed for cell attachment, proliferation, differentiation, histological and immunohistochemical evaluations. Silk fibroin/chitosan blended scaffolds supported the cell attachment and proliferation as indicated by SEM observation, Confocal microscopy and metabolic activities. Alcian Blue and Safranin O histochemistry and expression of collagen II indicated the maintenance of chondrogenic phenotype in the constructs after 3 weeks of culture. Glycosaminoglycans and collagen accumulated in all the scaffolds and was highest in silk fibroin/chitosan blended scaffolds and pure silk fibroin scaffolds of A. mylitta. Chondrogenic differentiation of MSCs in the silk fibroin/chitosan and pure silk fibroin scaffolds was evident by real-time PCR analysis for cartilage-specific ECM gene markers. The results represent silk fibroin/chitosan blended 3D scaffolds as suitable scaffold for mesenchymal stem cells-based cartilage repair.     

Potential of 3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan for in vitro cartilage tissue engineering

The use of cell-scaffold constructs is a promising tissue engineering approach to repair cartilage defects and to study cartilaginous tissue formation. In this study, silk fibroin/chitosan blended scaffolds were fabricated and studied for cartilage tissue engineering. Silk fibroin served as a substrate for cell adhesion and proliferation while chitosan has a structure similar to that of glycosaminoglycans, and shows promise for cartilage repair. We compared the formation of cartilaginous tissue in silk fibroin/chitosan blended scaffolds seeded with bovine chondrocytes and cultured in vitro for 2 weeks. The constructs were analyzed for cell viability, histology, extracellular matrix components glycosaminoglycan and collagen types I and II, and biomechanical properties. Silk fibroin/chitosan scaffolds supported cell attachment and growth, and chondrogenic phenotype as indicated by Alcian Blue histochemistry and relative expression of type II versus type I collagen. Glycosaminoglycan and collagen accumulated in all the scaffolds and was highest in the silk fibroin/chitosan (1:1) blended scaffolds. Static and dynamic stiffness at high frequencies was higher in cell-seeded constructs than non-seeded controls. The results suggest that silk/chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering.     

Behaviour of human mesenchymal stem cells on a polyelectrolyte-modified HEMA hydrogel for silk-based ligament tissue engineering

The aim of this study was to design a functional bio-engineered material to be used as scaffold for autologous mesenchymal stem cells in ligament tissue engineering. Polyelectrolyte modified HEMA hydrogel (HEMA-co-METAC), applied as coating on silk fibroin fibres, has been formulated in order to take advantage of the biocompatibility of the polyelectrolyte by increasing its mechanical properties with silk fibres. Human bone marrow mesenchymal stem cells behaviour on such reinforced polyelectrolyte has been studied by evaluating cell morphology, cell number, attachment, spreading and proliferation together with collagen matrix production and its mRNA expression. Silk fibroin fibres matrices with HEMA-co-METAC coating exhibited acceptable mechanical behaviour compared to the natural ligament, good human mesenchymal stem cell adhesion and with mRNA expression studies higher levels of collagen types I and III expression when compared to control cells on polystyrene. These data indicate high expression of mRNA for proteins responsible for the functional characteristics of the ligaments and suggest a potential for use of this biomaterial in ligament tissue-engineering applications.     

Rational design of a high-strength bone scaffold platform based on in situ hybridization of bacterial cellulose/nano-hydroxyapatite framework and silk fibroin reinforcing phase

Bacterial cellulose/hydroxyapatite (BC/HAp) composite had favourable bioaffinity but its poor mechanical strength limited its widespread applications in bone tissue engineering (BTE). Silk fibroin, which possesses special crystalline structure, has been widely used as organic reinforcing material, and different SFs have different amino acid sequences, which exhibit different bioaffinity and mechanical properties. In this regard, bacterial cellulose-Antheraea yamamai silk fibroin/hydroxyapatite (BC-AYSF/HAp), bacterial cellulose-Bombyx mori silk fibroin/hydroxyapatite (BC-BMSF/HAp), and BC/HAp nano-composites were synthesized via a novel in situ hybridization method. Compared with BC/HAp and BC-BMSF/HAp, the BC-AYSF/HAp exhibited better interpenetration, which may benefit for the transportation of nutrients and wastes, the adhesion of cells as well. Additionally, the BC-AYSF/HAp also presented superior thermal stability than the other two composites revealed by differential thermal analysis (DTA) and thermogravimetric analysis (TGA). Compression testing indicated that the mechanical strength of BC-BMSF/HAp was greatly reinforced compared with BC/HAp and was even a little higher than that of BC-AYSF/HAp. Tensile testing showed that BC-AYSF/HAp possesses extraordinary mechanical properties with a higher elastic modulus at low strain and higher fracture strength simultaneously than the other two composites. In vitro cell culture exhibited that MC3T3-E1 cells on the BC-AYSF/HAp membrane took on higher proliferative potential than those on the BC-BMSF/HAp membrane. These results suggested that compared with BC-BMSF/HAp, the BC-AYSF/HAp composite was more appropriate as an ideal bone scaffold platform or biomedical membrane to be used in BTE.     

Structural Characteristics and Properties of Silk Fibroin/Poly(lactic acid) Blend Films

This paper describes the preparation and characterization of blend films composed of regenerated silk fibroin (SF) and poly(lactic acid) (PLA). FT-IR and XRD of the SF/PLA blend films with different ratios indicated that the secondary structural transition of SF from Silk I to Silk II was induced upon blending with PLA. The effects of SF/PLA blend ratios on the mechanical and physical properties of the blend films were investigated. Compared to pure SF film, the mechanical and thermal properties of the blend films were improved, and surface hydrophilicity and swelling capacity decreased due to the secondary structural transition of SF to Silk II. Among the blend films with different ratios, the SF/PLA blend film with 7 wt% PLA content showed excellent mechanical properties. Meanwhile, the BSA adsorption amount on the blend film increased with the increase of PLA content. In vitro cell adhesion test showed that the blend film was a good matrix for the growth of L929 mouse fibroblast cells. Consequently, controlling the PLA content in the SF film can improve the mechanical and physical properties of the SF film and provide a promising opportunity to widen potential application of SF in the biomaterials field.