Silklike materials constructed from sequences of Bombyx mori silk fibroin, fibronectin, and elastin

Two silklike proteins, [TGRGDSPAGG(GAGAGS)3AS]5 (FS5) and [TGRGDSPA-(GVPGV)2GG(GAGAGS)3AS]8 (FES8) were designed to demonstrate the superior performance as biomaterials of silklike proteins. The former protein consists of the crystalline domain sequence, (GAGAGS)n from Bombyx mori silk fibroin and cell-adhesive sequence TGRGDSPA coming from fibronectin-containing RGD triplet. The additional sequence (GVPGV)n from elastin was included in the latter protein. The considerably higher cell-adhesion activities of these proteins for NHDF and VERO cells were observed by comparing with those of silklike materials without RGD sequences and also the crystalline fraction of B. mori silk fibroin. This tendency was independent of the treatments, 4.5M LiClO4 or formic acid (FA), on silklike proteins. Their activities are also higher than those of commercial Fibronectin F for NHDF cell. Their structural characterization was studied using 13C solid-state NMR. Although the overlapped peaks in usual 13C CP/MAS NMR spectra make the detailed structural analysis difficult, the methyl resonance regions observed using dipolar dephasing NMR were very useful for the analysis. The presence of both random coil and beta-sheet structures was observed in these proteins clearly. The content of beta-sheet structure in both proteins increases after FA treatment when compared with the lyophilized samples. The production of electrospun nanofibers from their hexafluoroacetone solution was also tried. The silklike protein FES8 could prepare nonwoven silk fibers although FS5 could not.     

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.     

Osteoblast: Osteoclast co-cultures on silk fibroin, chitosan and PLLA films

This study investigates the growth of a co-culture of osteoblasts and osteoclasts on four different types of degradable biomaterials with bone tissue engineering potential. Single or co-cultures of osteoblasts and osteoclasts (used at a ratio of 1:100 osteoblast:osteoclasts) were cultured on vapour stabilised silk fibroin, methanol stabilised silk fibroin, chitosan and poly (l lactic acid) (PLLA) films for 10 days. Osteoclast differentiation was determined by tartrate resistant acid phosphatase (TRAP) staining, total cell number by a picogreen DNA assay, cell morphology by scanning electron microscopy (SEM) and the material topography by atomic force microscopy (AFM). Samples were also monitored for degradation by differential scanning calorimetry (DSC) and fourier transform infrared (FTIR). Results demonstrated that vapour stabilised silk fibroin, methanol stabilised silk fibroin and chitosan all support the growth of osteoblasts and osteoclasts in both single and co-cultures. PLLA showed poor osteoclast differentiation in both single and co-cultures but supported osteoblast attachment and proliferation. Both silk fibroin materials showed sign of early degradation in the ten-day period, but very little change was seen in chitosan and PLLA samples. This study indicates that this novel co-culture approach for bone tissue engineering may be possible if scaffolds are created from silk fibroin or chitosan.     

Enhanced cellular adhesion on titanium by silk functionalized with titanium binding and RGD peptides

Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. A quartz crystal microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived arginine–glycine–aspartic acid (RGD) peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by scanning electron microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell–biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required.     

Osteogenic differentiation of adipose derived stem cells on high and low aspect ratio micropatterns

Adipose derived stem cells (ADSCs) were cultured on collagen–silk fibroin films with microchannel and micropillar patterns to investigate the effects of cell morphology changes on osteogenic differentiation. Channel and pillar micropatterned films were prepared from collagen type I and silk fibroin. While higher ADSC proliferation profiles were obtained on micropillar blend film, microchannel blend films, however, caused twice higher aspect ratio and effective orientation of cells. Alkaline phosphatase activity of ADSCs was several times higher on microchannel surface when the measured activities were normalized to cell number. Effective deposition of collagen type I and mineral by the cells were observed for patterned and unpatterned films, and these extracellular matrix components were oriented along the axis of the microchannels. In conclusion, the use of collagen–fibroin blend film with microchannel topography increased the aspect ratio and alignment of cells significantly, and was also effective in the differentiation of ADSCs into osteogenic lineage     

Porous Silk Fibroin Film as a Transparent Carrier for Cultivated Corneal Epithelial Sheets

Biological carriers, such as the amniotic membrane and serum-derived fibrin, are currently used to deliver cultivated corneal epithelial sheets to the ocular surface. Such carriers require being transparent and allowing the diffusion of metabolites in order to maintain a healthy ocular surface. However, safety issues concerning biological agents encouraged the development of safer, biocompatible materials as cell carriers. We examined the application of porous silk fibroin films with high molecular permeability prepared by mixing silk fibroin and poly(ethylene glycol) (PEG), and then removal of PEG from the silk-PEG films. Molecular permeability of porous silk fibroin film is higher than untreated silk fibroin film. Epithelial cells were isolated from rabbit limbal epithelium, and seeded onto silk fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts. Stratified epithelial sheets successfully engineered on porous silk fibroin film expressed the cornea-specific cytokeratins K3 and K12, as well as the corneal epithelial marker pax6. Basement membrane components such as type-IV collagen and integrin β1 were expressed in the stratified epithelial sheets. Further more, colony-forming efficiency of dissociated cells was similar to primary corneal epithelial cells showing that progenitor cells were preserved. The biocompatibility of fibroin films was confirmed in rabbit corneas for up to 6 months. Porous silk fibroin film is a highly transparent, biocompatible material that may be useful as a carrier of cultivated epithelial sheets in the regeneration of corneal epithelium.     

Cell adhesion and proliferation on RGD-modified recombinant spider silk proteins

Due to the biocompatibility and biodegradability as well as the mechanical properties of the fibers, spider silk has become an attractive material for researchers regarding biomedical applications. In this study, the engineered recombinant spider silk protein eADF4(C16) was modified with the integrin recognition sequence RGD by a genetic (fusing the amino acid sequence GRGDSPG) as well as a chemical approach (using the cyclic peptide c(RGDfK)). Both modified silk proteins were processed into films, and thereafter characterized concerning secondary structure, water contact angle and surface roughness. No influence of the RGD-modifications on any of these film properties could be detected. However, attachment and proliferation of BALB/3T3 mouse fibroblasts were significantly improved on films made of the RGD-modified silk proteins. Interestingly, the genetically created hybrid protein (with a linear RGD sequence) showed similar or slightly better cell adhesion properties as the silk protein chemically modified with the cyclic RGD peptide.     

Preparation and characterization of nano-hydroxyapatite/silk fibroin porous scaffolds

Novel tissue engineering scaffold materials of nano-hydroxyapatite (nHA)/silk fibroin (SF) biocomposite were prepared by freeze-drying. The needle-like nHA crystals of about 10 nm in diameter by 50-80 nm in length, which were uniformly distributed in the porous nHA/SF scaffolds, were prepared by a co-precipitation method with a size. The as-prepared nHA/SF scaffolds showed good homogeneity, interconnected pores and high porosity. XRD and FT-IR analysis suggested that the silk fibroin was in beta-sheet structure, which usually provides outstanding mechanical properties for silk materials. In this work, composite scaffolds containing as high as 70% (w/w) nHA were prepared, which had excellent compressive modulus and strength, higher than the scaffolds at low nHA content level and other porous biodegradable polymeric scaffolds often considered in bone-related tissue engineering reported previously. The cell compatibility of composite scaffolds was evaluated through cell viability by MTT assay. All these results indicated that these nHA/SF scaffold materials may be a promising biomaterial for bone tissue engineering.     

丝素蛋白膜上褐藻糖胶的固定化及其体外抗凝血性能

以戊二醛为交联剂，将褐藻糖胶固定在NH3等离子体处理的丝素膜的表面上。用FTIR、UV分析了褐藻糖胶的结构；采用XPS分析了丝素膜表面元素；采用部分凝血活酶时间（APTT）、凝血酶原时间（PT）和凝血酶时间（TT）对其体外抗凝血活性作了初步的评价。丝素膜表面固定褐藻糖胶后，抗凝血性有了一定的提高。经稳定性测试，固定的褐藻糖胶仍具有一定的抗凝血性。     

Poly(epsilon-caprolactone-co-D,L-lactide) /silk fibroin composite materials: preparation and characterization

Poly(epsilon-caprolactone-co-D,L-lactide) copolymers with 10, 30, and 50% by weight of silk particles (size range: 5-250 microm) derived from Bombyx mori were blended in acetone solution. After evaporation of the solvent, the morphology, thermal behavior, and mechanical properties of the composites were examined. The composites were transparent and the silk fibroin particles were homogeneously distributed within the composite structure. The particles appeared as bright reflected images under the optical microscope, suggesting that they were in a crystalline state. DSC thermograms of the composites revealed that the glass transition of the matrix was at ca. -18 degrees C. Degradation of the silk fibroin occurred beyond 270 degrees C. The decomposition temperatures and degradation rate decreased with increasing silk fibroin content as revealed by TGA analysis. FTIR spectra of the composites showed absorption bands at 1730 and 1088 cm(-1) for the copolymer and at 3273 and 1617 cm(-1) for the silk fibroin. Although the characteristic lines of poly(epsilon-caprolactone-co-D,L-lactide) were independent of filler concentration. the absorption bands of the beta-sheet form of the silk fibroin increased slightly due to the interaction of silk fibroin with the copolymer.     

Corneal stromal bioequivalents secreted on patterned silk substrates

Emulating corneal stromal tissue is believed to be the most challenging step in bioengineering an artificial human cornea because of the difficulty in reproducing its highly ordered microstructure, the key to the robust biomechanical properties and optical transparency of this tissue. We conducted a comparative study to assess the feasibility of human corneal stromal stem cells (hCSSCs) and human corneal fibroblasts (hCFs) in the generation of human corneal stromal tissue on groove-patterned silk substrates. In serum-free keratocyte differentiation medium, hCSSCs successfully differentiated into keratocytes secreting multilayered lamellae with orthogonally-oriented collagen fibrils, in a pattern mimicking human corneal stromal tissue. The constructs were 90–100 μm thick, containing abundant cornea-specific extracellular matrix (ECM) components, including keratan sulfate, lumican, and keratocan. In contrast, hCFs tended to differentiate into myofibroblasts that deposited less organized collagen in a pattern resembling that of corneal scar tissue. RGD surface coupling coupling was an essential factor in enhancing cell attachment, orientation, proliferation, differentiation and ECM deposition on the silk substratum. These results demonstrated that an approach of combining hCSSCs with an RGD surface-coupled patterned silk film offers a powerful tool to develop highly ordered collagen fibril-based constructs for corneal regeneration and corneal stromal tissue repair.     

Immobilized sonic hedgehog N-terminal signaling domain enhances differentiation of bone marrow-derived mesenchymal stem cells

The signaling domain of Sonic hedgehog (Shh), a potent upstream regulator of cell fate that has been implicated in osteoblast differentiation from undifferentiated mesenchymal cells in its endogenous form, was investigated in an immobilized form as a means for accelerating differentiation of uncommitted cells to the osteoblast phenotype. A recombinant cysteine-modified N-terminal Shh (mShh) was synthesized, purified, and immobilized onto interpenetrating polymer network (IPN) surfaces also grafted with a bone sialoprotein-derived peptide containing the Arg-Gly-Asp (RGD) sequence (bsp-RGD (15)), at calculated densities of 2.42 and 10 pmol/cm2, respectively. The mitogenic effect of mShh was dependent on the mode of presentation, as surfaces with immobilized mShh and bsp-RGD (15) had no effect on the growth rate of rat bone marrow-derived mesenchymal stem cells (BMSCs), while soluble mShh enhanced cell growth compared to similar surface without mShh supplementation. In conjunction with media supplemented with bone morphogenetic protein-2 and -4, mShh and bsp-RGD (15)-grafted IPN surfaces enhanced the alkaline phosphatase activity of BMSCs compared with tissue culture polystyrene and bsp-RGD (15)-grafted IPN surfaces supplemented with soluble mShh, indicating enhanced osteoblast differentiation. The adhesive peptide bsp-RGD (15) was necessary for cell attachment and proliferation, as well as differentiation in response to immobilized mShh. The addition of immobilized Shh substantially improved the differentiation of uncommitted BMSCs to the osteoblast lineage, and therefore warrants further testing in vivo to examine the effect of the stated biomimetic system on peri-implant bone formation and implant fixation.     

Biomimetic surfaces for control of cell adhesion to facilitate bone formation

Cell adhesion to extracellular matrix ligands through integrin receptors plays a central role in bone formation and maintenance by anchoring cells and triggering signals that direct osteoblast proliferation and differentiation. Moreover, osteoblast adhesion to adsorbed, synthesized, or engineered extracellular ligands on synthetic surfaces is critical to numerous biomedical and biotechnological applications. Considerable research efforts have concentrated on the development of surfaces that promote osteoblast differentiation and bone formation. Emerging surface engineering approaches have focused on creating biomimetic substrates that target integrins to activate signaling pathways directing the osteoblast differentiation program. These initiatives generally rely on controlling the adsorption of extracellular matrix ligands or engineering synthetic supports presenting bioadhesive motifs from extracellular matrix proteins. These biomolecular approaches provide promising strategies for the engineering of robust biofunctional matrices that control cell adhesion and signaling and promote osteoblast proliferation, differentiation, and matrix mineralization.     

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.     

RGD抗体的研制和其在丝素膜中的应用

本研究应用双功能偶联剂小分子间苯二甲基二异氰酸盐(m—xylylen—diisocyanate，简称XDI)作桥梁，将短肽RGD(GLY—ARG—GLY-ASP—SER—PRO-LYS)粘附生长因子偶联到卵清白蛋白(Ovalbumin，简称OVA)载体上，对偶联物RGD-OVA经SDS-聚丙烯酰胺凝胶纯化和紫外光谱测定，确定RGD与OVA偶联比为11：1，用偶联物对兔子进行背部小剂量多点免疫注射，诱发出抗RGD抗体，利用得到的兔抗RGD抗体IgG包埋于丝素材料内，并借助抗原抗体的结合力，结合RGD在该丝素膜表面，对这种结合有RGD抗体的丝素膜进行培养血管内皮细胞(Endothelial cell，简称EC)的试验，结果表明，结合有RGD的丝素膜与其对照组相比，细胞数量有显著提高。     

Initial cell pre-cultivation can maximize ECM mineralization by human mesenchymal stem cells on silk fibroin scaffolds

Fast remineralization of bone defects by means of tissue engineering is one of many targets in orthopedic regeneration. This study investigated the influence of a range of pre-culture durations for human bone marrow derived mesenchymal stem cells (hMSC) before inducing differentiation into osteoblast-like cells. The aim was to find the conditions that lead to maximal extracellular matrix (ECM) mineralization, in terms of both amount and best distribution. Additionally, the influence of silk fibroin scaffold pore size on mineralization was assessed. The formation of mineralized ECM by hMSCs cultured in osteogenic medium on silk fibroin scaffolds was monitored and quantified for up to 72 days in culture using non-invasive time-lapse micro-computed tomography (micro-CT). ECM mineralization increased linearly 3 weeks after the beginning of the experiment with addition of differentiation medium. Biochemical end-point assays measured the amount of DNA, calcium deposits, alkaline phosphatase activity and cell metabolic activity to corroborate the hypothesis that an initial pre-culture period of hMSCs on silk fibroin scaffolds can accelerate mineralized ECM formation. According to the micro-CT analysis mineralization on silk fibroin scaffolds with pores of 112-224 μm diameter was most efficient with an initial cell pre-culture period of 9 days, showing 6.87±0.81× higher mineralization values during the whole cultivation period than without an initial cell pre-culture period.     

Directing osteogenesis of stem cells with hydroxyapatite precipitated electrospun eri–tasar silk fibroin nanofibrous scaffold

Stimulating stem cell differentiation without growth factor supplement offers a potent and cost-effective scaffold for tissue regeneration. We hypothesise that surface precipitation of nano-hydroxyapatite (nHAp) over blends of non-mulberry silk fibroin with better hydrophilicity and RGD amino acid sequences can direct the stem cell towards osteogenesis. This report focuses on the fabrication of a blended eri–tasar silk fibroin nanofibrous scaffold (ET) followed by nHAp deposition by a surface precipitation (alternate soaking in calcium and phosphate solution) method. Morphology, hydrophilicity, composition, and the thermal and mechanical properties of ET/nHAp were examined by field emission scanning electron microscopy, TEM, FT-IR, X-ray diffraction, TGA and contact angle measurement and compared with ET. The composite scaffold demonstrated improved thermal stability and surface hydrophilicity with an increase in stiffness and elastic modulus (778?±?2.4?N/m and 13.1?±?0.36?MPa) as compared to ET (160.6?±?1.34?N/m and 8.3?±?0.4?MPa). Mineralisation studies revealed an enhanced and more uniform surface deposition of HAp-like crystals, while significant differences in cellular viability and attachment were observed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and confocal microscopy study. The cell viability and expression of adhesion molecules (CD 44 and CD 29) are found to be optimum for subsequent stages of growth proliferation and differentiation. The rates of proliferation have been observed to decrease owing to the transition of MSC from a state of proliferation to a state of differentiation. The confirmation of improved osteogenic differentiation was finally verified through the alkaline phosphatase assay, pattern of gene expression related to osteogenic differentiation and morphological observations of differentiated cord blood human mesenchymal stem cells under fluorescence microscope. The results obtained showed the improved physicochemical and biological properties of the ET/nHAp scaffold for osteogenic differentiation without the addition of any growth factors.     

Production and characterization of a silk-like hybrid protein, based on the polyalanine region of Samia cynthia ricini silk fibroin and a cell adhesive region derived from fibronectin

There are a variety of silkworms and silk fibroins produced by them. Silks have many inherent suitable properties for biomaterials. In this paper, a novel silk-like hybrid protein, [DGG(A)(12)GGAASTGRGDSPAAS](5), which consists of polyalanine region of silk fibroin from a wild silkworm, Samia cynthia ricini, and cell adhesive region including Arg-Gly-Asp (RGD) sequence, derived from fibronectin, was designed and produced. The genes encoding the hybrid protein were constructed and expressed in Escherichia coli. The main conformation of the polyalanine region, that is, either alpha-helix or beta-sheet, could be easily controlled by treatment with different acidic solvents, trifluoroacetic acid or formic acid, respectively. This structural change was monitored with 13C CP/MAS NMR. Higher cell adhesive and growth activities of the hybrid protein compared with those of collagen were obtained.