蚕丝丝素纤维的制备及其与骨髓间充质干细胞相容性的研究

组织工程为韧带损伤修复提供了可行途径，但韧带修复对支架材料各方面性能要求都很高。在力学性能方面，不仅要求材料有一定的强度而且需要有良好的韧性。在满足力学性能的同时，支架材料还必须兼具优良的生物相容性。蚕丝作为一种天然生物蛋白质，由于其良好的力学性能显示了在组织工程方面应用的前景。但由于丝胶存在污染问题，因此脱胶成为蚕丝在医学领域应用的首要问题。本实验首先比较了三种脱胶试剂对蚕丝力学性质的影响，选择了影响最小的碳酸钠作为脱胶试剂，进而确定了碳酸钠脱胶的最佳条件为：试剂浓度0．40%，温度90℃，时间为1h。然后在脱胶后的丝紊纤维上种植了大鼠骨髓问充质干细胞（Rat bone marrow mesenchymal stem cells，rMSCs），通过扫描电镜（SEM）、荧光显微镜检测了丝素上细胞的生长情况，结果显示蚕丝具有良好的生物相容性，细胞亲和力。为蚕丝在韧带组织工程方面的进一步应用奠定了基础。     

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.     

Immobilization of L-asparaginase on the microparticles of the natural silk sericin protein and its characters

The natural silk sericin recovered from Bombyx mori silk waste by the degumming processing in the high-temperature and high-pressure is a macromolecular protein. Amino acid composition and molecular weight range of the sericin protein as a vector for enzyme immobilization were investigated. The silk sericin protein with different molecular mass from 50 to 200 kDa was poorly soluble microparticles with an average size of about 10 microm. Anti-leukemic enzyme L-asparaginase (L-ASNase) was covalently conjugated on the microparticles of the sericin protein. The immobilized L-ASNase on the natural support by cross-linking with glutaraldehyde maintained 62.5% of the original activity of the enzyme. The Km of sericin-conjugates was 8 times lower than that of native L-ASNase. The bioconjugation of L-ASNase widened the optimum reactive temperature range of the enzyme. The immobilized L-ASNase showed significantly higher stability when the temperature raised to 40-50 degrees C, it also showed preferable resistance to trypsin digestion as compared with native enzyme. The results are discussed regarding the possible explanations of sericin-induced enzyme stability, as well as the possible applications of immobilized L-ASNase research.     

Deposition of bone-like apatite on silk fiber in a solution that mimics extracellular fluid

The fabrication of apatite-organic polymer hybrids is one of several attractive methods for the development of biomaterials as a substitute for bone. Such materials have both bone-bonding ability and mechanical properties analogous to natural bone. The biomimetic process has focused attention on fabricating such hybrids, where bone-like apatite is deposited on an organic polymer surface in solutions that mimic physiological conditions. In this process, a bone-like apatite layer can be coated onto organic substrates either by using a simulated body fluid (SBF) with ion concentrations nearly equal to those of human extracellular fluid, or by using fluids that are supersaturated with respect to apatite at ambient conditions. In this study, we investigated the ability of natural silk and its related materials to facilitate apatite deposition under biomimetic conditions. Cloths made of raw silk or normal silk fibers were soaked in 1.5SBF, which has 1.5 times the ion concentration of SBF. Sericin film, which is made from an extract of degummed raw silk, was soaked in 1.5SBF. The cloth and the film soaked in 1.5SBF then were characterized by scanning electron microscopic (SEM) observation, energy dispersive X-ray microanalysis (EDX), and thin-film X-ray diffraction (TF-XRD). Apatite deposition was observed on the surface of cloth made from raw silk fiber after it was soaked in 1.5SBF, but it was not observed on cloth made from normal silk fibers. The apatite deposition on the raw silk fiber cloth was accelerated when the fibers were subjected to treatment with CaCl(2) solution at a concentration of at least 1 kmol/m(3) before immersion in 1.5SBF. Apatite deposition also was observed on the sericin film after the film was soaked in 1.5SBF for 7 days. These results indicate that apatite deposition on raw silk cloth is attributable to the catalytic effect of sericin because the surface of raw silk consists of sericin whereas that of normal silk contains fibroin. The deposition of the apatite and its crystal growth are accelerated by the presence of calcium ions on the sericin after treatment with CaCl(2) solution. Thus, sericin on natural silk fiber has the potential to facilitate apatite deposition and can be useful as a polymer material in the fabrication of hybrid materials analogous to bone through biomimetic processes.     

Advanced silk material spun by a transgenic silkworm promotes cell proliferation for biomedical application

Natural silk fiber spun by the silkworm Bombyx mori is widely used not only for textile materials, but also for biofunctional materials. In the present study, we genetically engineered an advanced silk material, named hSFSV, using a transgenic silkworm, in which the recombinant human acidic fibroblast growth factor (hFGF1) protein was specifically synthesized in the middle silk gland and secreted into the sericin layer to surround the silk fiber using our previously optimized sericin1 expression system. The content of the recombinant hFGF1 in the hSFSV silk was estimated to be approximate 0.07% of the cocoon shell weight. The mechanical properties of hSFSV raw silk fiber were enhanced slightly compared to those of the wild-type raw silk fiber, probably due to the presence of the recombinant of hFGF1 in the sericin layer. Remarkably, the hSFSV raw silk significantly stimulated the cell growth and proliferation of NIH/3T3 mouse embryonic fibroblast cells, suggesting that the mitogenic activity of recombinant hFGF1 was well maintained and functioned in the sericin layer of hSFSV raw silk. These results show that the genetically engineered raw silk hSFSV could be used directly as a fine biomedical material for mass application. In addition, the strategy whereby functional recombinant proteins are expressed in the sericin layer of silk might be used to create more genetically engineered silks with various biofunctions and 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.     

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。     

Silk fibroin electrogelation mechanisms

A silk fibroin gel system (e-gel), formed with weak electric fields, has potential utility in medical materials and devices. The mechanism of silk e-gel formation was studied to gain additional insight into the process and control of the material properties. Silk fibroin nanoparticles with sizes of tens of nanometers, composed of metastable conformations, were involved in e-gel formation. Under electric fields the nanoparticles rapidly assembled into larger nano- or microspheres with size range from tens of nanometers to several microns. Repulsive forces from the negative surface charge of the acidic groups on the protein were screened by the local decrease in solution pH in the vicinity of the positive electrode. By controlling the formation and content of silk fibroin nanoparticles e-gels could be formed even from low concentration silk fibroin solutions (1%). When e-gel formation was reversed to the solution state the aggregated nano- and microspheres dispersed into solution, a significant observation related to future applications for this process, such as drug delivery.     

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.     

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.     

犬骨髓基质细胞和蚕丝丝素的体外生物相容性

目的 探讨体外培养的犬骨髓基质细胞(BMSCs)与蚕丝丝素材料的生物相容性,寻找BMSCs组织工程化神经的支架材料.方法 通过差速贴壁法体外分离、培养犬骨髓基质细胞,与丝素共培养后,通过光镜(经免疫荧光染色)、扫描电镜观察细胞在丝素上黏附和生长情况.利用丝素浸出液培养BMSCs后,通过透射电镜观察细胞内部超微结构,用四甲基偶氮唑盐(MTT)法检测丝素、羟基磷灰石、有机锡浸出液和普通IMDM完全培养基培养细胞12、24、48、72h和7d的细胞活力,每组重复12次.流式细胞术检测丝素浸出液培养BMSCs的细胞周期及表型,实验重复3次.结果 通过光镜、扫描电镜观察,发现BMSCs 紧紧黏附于丝素材料,并沿着丝素纤维延伸,黏附于丝素纤维的细胞呈圆形、椭圆形及呈梭形.与普通IMDM完全培养基培养的细胞相比,透射电镜下可见丝素浸出液培养后的BMSCs内部结构未见异常;MTT检测丝素和羟基磷灰石浸出液对骨髓基质细胞的活力无显著性影响(P＞0.05);流式细胞术检测丝素浸出液对骨髓基质细胞周期和表型无明显影响.结论 蚕丝丝素材料与犬BMSCs生物相容性好,且未见丝素对BMSCs有毒性作用,可作为BMSCs组织工程化神经的支架材料.     

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.     

Biocompatibility evaluation of silk fibroin with peripheral nerve tissues and cells in vitro

Silk-based materials have been used in the field of bone or ligament tissue engineering. In order to explore the feasibility of using purified silk fibroin to construct artificial nerve grafts, it is necessary to evaluate the biocompatibility of silk fibroin material with peripheral nerve tissues and cells. We cultured rat dorsal root ganglia (DRG) on the substrate made up of silk fibroin fibers and observed the cell outgrowth from DRG during culture by using light and electron microscopy coupled with immunocytochemistry. On the other hand, we cultured Schwann cells from rat sciatic nerves in the silk fibroin extract fluid and examined the changes of Schwann cells after different times of culture. The results of light microscopy, MTT test and cell cycle analysis showed that Schwann cells cultured in the silk fibroin extract fluid showed no significant difference in their morphology, cell viability and proliferation as compared to that in plain L15 medium. Furthermore, no significant difference was found in expression of the factors secreted by Schwann cells, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and S-100, between Schwann cells cultured in the silk fibroin extraction fluid and in plain L15 medium by the aid of immunocytochemistry, RT-PCR and Western analysis. Collectively, these data indicate that silk fibroin has good biocompatibility with DRG and is also beneficial to the survival of Schwann cells without exerting any significant cytotoxic effects on their phenotype or functions, thus providing an experimental foundation for the development of silk fibroin as a candidate material for nerve tissue engineering applications.     

Macro/microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications

This study describes the developmental physicochemical properties of silk fibroin scaffolds derived from high-concentration aqueous silk fibroin solutions. The silk fibroin scaffolds were prepared with different initial concentrations (8, 10, 12 and 16%, in wt.%) and obtained by combining the salt-leaching and freeze-drying methodologies. The results indicated that the antiparallel β-pleated sheet (silk-II) conformation was present in the silk fibroin scaffolds. All the scaffolds possessed a macro/microporous structure. Homogeneous porosity distribution was achieved in all the groups of samples. As the silk fibroin concentration increased from 8 to 16%, the mean porosity decreased from 90.8 ± 0.9 to 79.8 ± 0.3% and the mean interconnectivity decreased from 97.4 ± 0.5 to 92.3 ± 1.3%. The mechanical properties of the scaffolds exhibited concentration dependence. The dry state compressive modulus increased from 0.81 ± 0.29 to 15.14 ± 1.70 MPa and the wet state dynamic storage modulus increased by around 20- to 30-fold at each testing frequency when the silk fibroin concentration increased from 8 to 16%. The water uptake ratio decreased with increasing silk fibroin concentration. The scaffolds present favorable stability as their structure integrity, morphology and mechanical properties were maintained after in vitro degradation for 30 days. Based on these results, the scaffolds developed in this study are proposed to be suitable for use in meniscus and cartilage tissue-engineered scaffolding.     

Preparation and characterization of silk fibroin as a biomaterial with potential for drug delivery

ABSTRACT: BACKGROUND: Degummed silk fibroin from Bombyx mori (silkworm) has potential carrier capabilities for drug delivery in humans; however, the processing methods have yet to be comparatively analyzed to determine the differential effects on the silk protein properties, including crystalline structure and activity. METHODS: In this study, we treated degummed silk with four kinds of calcium-alcohol solutions, and performed secondary structure measurements and enzyme activity test to distinguish the differences between the regenerated fibroins and degummed silk fibroin. RESULTS: Gel electrophoresis analysis revealed that Ca(NO3)2-methanol, Ca(NO3)2-ethanol, or CaCl2-methanol treatments produced more lower molecular weights of silk fibroin than CaCl2-ethanol. X-ray diffraction and Fourier-transform infrared spectroscopy showed that CaCl2-ethanol produced a crystalline structure with more silk I (alpha-form, type II beta-turn), while the other treatments produced more silk II (beta-form, anti-parallel beta-pleated sheet). Solid-State 13C cross polarization and magic angle spinning-nuclear magnetic resonance measurements suggested that regenerated fibroins from CaCl2-ethanol were nearly identical to degummed silk fibroin, while the other treatments produced fibroins with significantly different chemical shifts. Finally, enzyme activity test indicated that silk fibroins from CaCl2-ethanol had higher activity when linked to a known chemotherapeutic drug, L-asparaginase, than the fibroins from other treatments. CONCLUSIONS: Collectively, these results suggest that the CaCl2-ethanol processing method produces silk fibroin with biomaterial properties that are appropriate for drug delivery.     

Surface immobilization of antibody on silk fibroin through conformational transition

In recent studies silk fibroin has been explored as a new material platform for biosensors. Based on these developments, a procedure for the immobilization of antibodies on silk fibroin substrates was developed as a route to functionalizing these biosensor systems. By controlling the conformational transition of the silk fibroin, a primary antibody was immobilized and enriched at the surface of silk fibroin substrates under mild reaction conditions to maintain antibody function. Compared to chemical crosslinking, the immobilization efficiency in the present approach was increased significantly. This method, achieving high loading of antibody while retaining function, improves the feasibility of silk fibroin as a platform material for biosensor 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.     

Modification of sericin-free silk fibers for ligament tissue engineering application

Biomedical application of silk requires the removal of sericin that is the gumming material of native silk fibers. This is because sericin can elicit an adverse immune response after implantation in the human body. However, the removal of sericin causes the silk fiber to fray and weakens its structural property, making it very difficult to knit or braid them into a scaffold for ligament tissue engineering applications. The aim of this study was to replace sericin with gelatin using NDGA as a cross-linking agent to biomimic the natural structure of native silk fibers. The physical properties and biocompatibility of the modified and native silk fibers were compared by in vitro and in vivo models. The mechanical and swelling properties of sericin-free silk fibers were greatly increased after modification with gelatin. Both modified and native silk fibers were shown to be nontoxic by in vitro cytotoxicity tests. The in vivo study demonstrated that the modified silk fibers, after 4 weeks' subcutaneous implantation in rats, caused little or no inflammatory reaction as compared with native silk fibers. The superior mechanical properties and lower inflammatory potential of modified silk fibers make them a promising candidate for ligament tissue engineering applications.     

Sonication-induced gelation of silk fibroin for cell encapsulation

Purified native silk fibroin forms beta-sheet-rich, physically cross-linked, hydrogels from aqueous solution, in a process influenced by environmental parameters. Previously we reported gelation times of days to weeks for aqueous native silk protein solutions, with high ionic strength and temperature and low pH responsible for increasing gelation kinetics. Here we report a novel method to accelerate the process and control silk fibroin gelation through ultrasonication. Depending on the sonication parameters, including power output and time, along with silk fibroin concentration, gelation could be controlled from minutes to hours, allowing the post-sonication addition of cells prior to final gel setting. Mechanistically, ultrasonication initiated the formation of beta-sheets by alteration in hydrophobic hydration, thus accelerating the formation of physical cross-links responsible for gel stabilization. K(+) at physiological concentrations and low pH promoted gelation, which was not observed in the presence of Ca(2+). The hydrogels were assessed for mechanical properties and proteolytic degradation; reported values matched or exceeded other cell-encapsulating gel material systems. Human bone marrow derived mesenchymal stem cells (hMSCs) were successfully incorporated into these silk fibroin hydrogels after sonication, followed by rapid gelation and sustained cell function. Sonicated silk fibroin solutions at 4%, 8%, and 12% (w/v), followed by mixing in hMSCs, gelled within 0.5-2 h. The cells grew and proliferated in the 4% gels over 21 days, while survival was lower in the gels with higher protein content. Thus, sonication provides a useful new tool with which to initiate rapid sol-gel transitions, such as for cell encapsulation.