纳米羟基磷灰石/丝素蛋白复合支架材料的降解特性及生物相容性研究

应用共混法制备了纳米羟基磷灰石/丝素蛋白复合支架材料, 通过体外降解和细胞培养实验研究了复合支架材料的降解特性和生物相容性. 体外降解实验结果显示, 复合支架材料具有稳定的降解能力; 在降解过程中, 羟基磷灰石由于与降解液发生钙、磷等离子的交换, 使其结晶得到了进一步生长和完善. 利用细胞计数法、四甲基偶氮唑盐(MTT)比色法和碱性磷酸酶(ALP)活性测定等分析了复合支架材料的生物相容性, 结果表明, MG63细胞在复合支架材料上具有良好的粘附、增殖能力, 并可引起早期的骨分化. 因此, 纳米羟基磷灰石/丝素蛋白复合支架作为骨组织工程的支架材料具有良好的应用前景.     

羟基磷灰石/丝素蛋白复合纤维的制备及其矿化研究

在以静电纺丝法制备羟基磷灰石(HAP)/丝素蛋白(SF)复合纤维的前提下, 采用同轴共纺法获得了以HAP为“芯”、SF为“皮”的双组分电纺纤维(电纺膜), 并通过扫描和透射电镜、红外光谱以及X射线衍射等手段对电纺纤维进行了表征. 结果表明, HAP均存在于上述两种方法制备的纳米纤维中, 但同轴共纺法不仅可以避免HAP/SF共混电纺时pH值对丝素蛋白结构的影响, 并且能大大提高电纺膜中HAP的含量. 同时, 我们还分别以SF纤维、HAP/SF复合纤维和HAP/SF“皮-芯”纤维作为有机基质, 对羟基磷灰石在其上的矿化过程进行了探索, 结果表明含较多羟基磷灰石的HAP/SF“皮-芯”纤维更有利于矿化的进行.     

Preparation and characterization of new materials based on silk fibroin,chitosan and nanohydroxyapatite

Abstract

In this paper, a series of porous nanohydroxyapatite/silk fibroin/chitosan (nHA/SF/CTS) scaffolds were successfully prepared using the freeze-drying method. The biomaterials were characterized by attenuated total reflection Fourier transform infrared spectroscopy, and mechanical testing and thermogravimetric analysis. Moreover, studies of porosity, pore size, swelling properties and in vitro degradation test were performed. Research has proved that micro-structure, porosity, water adsorption and compressive strength were greatly affected by the components’ concentration, in particular the content of silk fibroin. SEM observations showed that the scaffolds of nHA/SF/CTS are highly porous, with pore size in wide range from 25 to 300?µm which is suitable for cell growth. nHA/SF/CTS scaffolds have sufficient mechanical integrity to resist handling during implantation and in vivo loading. Both, the compressive modulus and compressive strength of the scaffold, decrease with the increase in silk fibroin content.     

Bioinspired Unidirectional Silk Fibroin–Silver Compound Nanowire Composite Scaffold via Interface‐Mediated In Situ Synthesis

Bioinspired unidirectional porous materials have emerged as a unique class of scaffolds for the fabrication of macroscopic nanomaterial assemblies. However, these scaffolds usually serve simply as mechanical carriers to support various building blocks. Here, we report that the unidirectional silk fibroin scaffold can not only act as a carrier, but also serve as a controllable multiscale reactor to achieve the in situ synthesis of a Ag₃PO₄ nanowire network anchored to ordered channels. Both the silk fibroin matrix and the interface play important roles in the nucleation and growth of the Ag₃PO₄ nanowires. This unidirectional composite scaffold can be used for efficient water disinfection. Furthermore, the facile chemical transformation of Ag₃PO₄ in the composite scaffold into Ag₂S provided an analogous unidirectional composite silk scaffold that displays both efficient solar water evaporation effect and antibacterial activity. It is expected that this method can be extended to fabricate a series of silk‐based unidirectional composite scaffolds with varying functionalities.     

Green Process to Prepare Silk Fibroin/Gelatin Biomaterial Scaffolds

A new all‐aqueous and green process is described to form three‐dimensional porous silk fibroin matrices with control of structural and morphological features. Silk‐based scaffolds are prepared using lyophilization. Gelatin is added to the aqueous silk fibroin solution to change the silk fibroin conformation and silk fibroin–water interactions through adjusting the hydrophilic interactions in silk fibroin–gelatin–water systems to restrain the formation of separate sheet like structures in the material, resulting in a more homogenous structure. Water annealing is used to generate insolubility in the silk fibroin–gelatin scaffold system, thereby avoiding the use of organic solvents such as methanol to lock in the β‐sheet structure. The adjusting of the concentration of gelatin, as well as the concentration of silk fibroin, leads to control of morphological and functional properties of the scaffolds. The scaffolds were homogeneous in terms of interconnected pores, with pore sizes ranging from 100 to 600 µm, depending on the concentration of silk fibroin used in the process. At the same time, the morphology of the scaffolds changed from lamellar sheets to porous structures based on the increase in gelatin content. Compared with salt‐leaching aqueous‐derived scaffolds and hexafluoroisopropanol (HFIP)‐derived scaffolds, these freeze‐dried scaffolds had a lower content of β‐sheet, resulting in more hydrophilic features. Most of gelatin was entrapped in the silk fibroin–gelatin scaffolds, without the burst release in PBS solution. During in vitro cell culture, these silk fibroin–gelatin scaffolds had improved cell‐compatibility than salt‐leaching silk fibroin scaffolds. This new process provides useful silk fibroin‐based scaffold systems for use in tissue engineering. Furthermore, the whole process is green, including all‐aqueous, room temperature and pressure, and without the use of toxic chemicals or solvents, offering new ways to load bioactive drugs or growth factors into the process.

     

纳米TiO_2改性蚕丝丝素蛋白膜的研究

用溶胶凝胶法制备纳米TiO2改性再生蚕丝丝素蛋白膜.该丝素膜机械强度提高,在水中溶失率下降.UV、AFM、SEM测试的结果表明,丝素中纳米TiO2粒径约为80 nm,纳米粒子在丝素膜中分布均匀;XRD、FTIR测试结果表明,纳米TiO2的加入,可使改性丝素膜的结晶结构从SilkⅠ向SilkⅡ转化,其结晶度也随之提高;TGA测试表明改性丝素膜的热转变温度比纯丝素膜有所改变.     

蚕丝纤维/丝蛋白复合材料力学性能的优化

在制备具有良好力学性能的蚕丝纤维/丝蛋白复合材料的基础上,采用力学测试、扫描电镜以及广角X-射线衍射等手段,考察了溴化锂预处理和甲醇后处理这两种方法对蚕丝纤维/丝蛋白界面性能的影响.力学测试的结果表明,在相同纤维含量(如20 wt％)的情况下,采用6 mol L-1溴化锂对定向排列的蚕丝纤维预处理10 min,所得的蚕...     

肝素化丝素支架作为骨形态发生蛋白-2缓释载体的研究

骨形态发生蛋白-2(BMP-2)的缓释载体一直是骨组织工程中的研究热点.本研究通过化学改性制备了两种肝素化丝素支架,并浸渍吸附BMP-2,研究了BMP-2在不同丝素支架样品上的吸附能力、体外释放性能及其对人骨肉瘤细胞MG-63碱性磷酸酶活性(ALP)的影响.结果表明,肝素化丝素支架对BMP-2具有较强的吸附能力,并能保持其体外缓慢释放性能;MG-63细胞在肝素化支架上生长状态良好,并具有显著的增殖能力,负载BMP-2后的肝素化支架能显著促进MG-63细胞的分化.因此,肝素化丝素支架是一种较理想的BMP-2缓释载体.     

Silk fibroin microfibers and chitosan modified poly (glycerol sebacate) composite scaffolds for skin tissue engineering

Porous mSF/PGS and CS/PGS composite scaffolds were prepared by the combination of poly(glycerol sebacate) (PGS) with silk fibroin microfibers (mSF) and chitosan (CS) as modifiers through particulate leaching and freeze-drying techniques. Both mSF/PGS and CS/PGS scaffolds show highly interconnected and open porous structures, and the crosslink density and water absorption of PGS were obviously enhanced by the modifiers. Moreover, the silk fibroin microfiber and chitosan can slow down and control the degradation rate of PGS. The biocompatibility of these porous PGS based composite scaffolds for skin tissue engineering was evaluated by cell culture experiments, and the results indicate of the good attachment, proliferation and deep penetration of cells into these composite scaffolds.     

Chinese Oak Tasar Silkworm Antheraea pernyi Silk Proteins: Current Strategies and Future Perspectives for Biomedical Applications

Chinese nonmulberry temperate oak tasar/tussah, Antheraea pernyi (Ap) silk is a natural biopolymer that has attracted considerable attention as a biomaterial. The proteinaceous components of Ap silk proteins, namely fibroin and sericin may represent an alternative over mulberry Bombyx mori silk proteins. In fact, the silk fibroin (SF) of Ap is rich in Arginyl‐Glycyl‐Aspartic acid (RGD) peptides, which facilitate the adhesion and proliferation of various cell types. The possibility of processing Ap silk proteins into different distinct 2D‐ and 3D‐based matrices is described in earlier studies, such as membranes, nanofibers, scaffolds, and micro/nanoparticles, contributing to a different rate of degradation, mechanical properties, and biological performance useful for various biomedical applications. This review summarizes the current advances and developments on nonmulberry Chinese oak tasar silk protein (fibroin and sericin)‐based biomaterials and their potential uses in tissue engineering, regenerative medicine, and therapeutic delivery strategies.     

Silk Fibroin Combined with Electrospinning as a Promising Strategy for Tissue Regeneration

The development of tissue engineering scaffolds is of great significance for the repair and regeneration of damaged tissues and organs. Silk fibroin (SF) is a natural protein polymer with good biocompatibility, biodegradability, excellent physical and mechanical properties and processability, making it an ideal universal tissue engineering scaffold material. Nanofibers prepared by electrospinning have attracted extensive attention in the field of tissue engineering due to their excellent mechanical properties, high specific surface area, and similar morphology as to extracellular matrix (ECM). The combination of silk fibroin and electrospinning is a promising strategy for the preparation of tissue engineering scaffolds. In this review, the research progress of electrospun silk fibroin nanofibers in the regeneration of skin, vascular, bone, neural, tendons, cardiac, periodontal, ocular and other tissues is discussed in detail.     

Incorporation of two‐dimensional nanomaterials into silk fibroin nanofibers for cardiac tissue engineering

Mimicking the extracellular matrix to have a similar nanofibrous structure regarding electrical conductivity and mechanical properties would be highly beneficial for cardiac tissue engineering. The molybdenum disulfide, MoS₂, and reduced graphene oxide, rGO, nanosheets are two‐dimensional nanomaterials which can be considered as great candidates for enhancing the electrical and mechanical properties of biological scaffolds for cardiac tissue engineering applications. In this study, MoS₂ and rGO nanosheets were synthesized and incorporated into silk fibroin nanofibers, SF, via electrospinning method. Then, the human iPSCs transfected with TBX‐18 gene, TBX18‐hiPSCs, were seeded on these scaffolds for in vitro studies. The MoS₂ and rGO nanosheets were studied by Raman spectroscopy. After incorporation of the nanosheets into SF nanofibers, the associated characterizations were carried out including scanning electron microscopy, transmission electron microscopy, water contact angle, and mechanical test. Furthermore, SF, SF/MoS₂, and SF/rGO scaffolds were used for in vitro studies. Herein, the scaffolds exhibited acceptable biocompatibility and considerable attachment to TBX18‐hiPSCs confirmed by 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyl tetrazolium bromide, MTT, assay, and scanning electron microscopy. Also, the real‐time PCR and immunostaining studies confirmed the maturity and upregulation of cardiac functional genes, including GATA‐4, c‐TnT, and α‐MHC in the SF/MoS₂ and SF/rGO scaffolds compared with the bare SF one. Therefore, the reinforcement of these SF‐based scaffolds with MoS₂ and rGO endues them as a suitable candidate for cardiac tissue engineering.     

Electroactive 3D Scaffolds Based on Silk Fibroin and Water‐Borne Polyaniline for Skeletal Muscle Tissue Engineering

Silk fibroin (SF) with good biocompatibility and degradability has great potential for tissue engineering. However, the SF based scaffolds lack the electroactivity to regulate the myogenic differentiation for the regeneration of muscle tissue, which is sensitive to electrical signal. Herein, a series of electroactive biodegradable scaffolds based on SF and water‐soluble conductive poly(aniline‐co‐N‐(4‐sulfophenyl) aniline) (PASA) via a green method for skeletal muscle tissue engineering are designed. SF/PASA scaffolds are prepared by vortex of aqueous solution of SF and PASA under physiological condition. Murine‐derived L929 fibroblast and C2C12 myoblast cells are used to evaluate cytotoxicity of SF/PASA scaffolds. Moreover, myogenic differentiation of C2C12 cells is investigated by analyzing the morphology of myotubes and related gene expression. These results suggest that electroactive SF/PASA scaffolds with a suitable microenvironment, which can enhance the myogenic differentiation of C2C12 cells, have a great potential for skeletal muscle regeneration.     

Preparation and characterization of chitin whisker-reinforced silk fibroin nanocomposite sponges

For highly porous form such as sponges or scaffolds, the induction of the β-sheet formation of silk fibroin to make the water-stable materials usually results in their high shrinkage leading to a difficulty in controlling shape and size of materials. Thus, the objective of this study was to improve dimensional stability of silk fibroin sponge by incorporating chitin whiskers as nanofiller. Chitin whiskers exhibited the average length and width of 427 and 43 nm, respectively. Nanocomposite sponges at chitin whiskers to silk fibroin weight ratio (C/S ratio) of 0, 1/8, 2/8, or 4/8 were prepared by using a freeze-drying technique. The dispersion of chitin whiskers embedded in the silk fibroin matrix was found to be homogeneous. The presence of chitin whiskers embedded into silk fibroin sponge not only improved its dimensional stability but also enhanced its compression strength. Regardless of the chitin whisker content, SEM micrographs showed that all samples possessed an interconnected pore network with an average pore size of 150 μm. To investigate the feasibility of the nanocomposites for tissue engineering applications, L929 cells were seeded onto their surfaces, the results indicated that silk fibroin sponges both with and without chitin whiskers were cytocompatible. Moreover, when compared to the neat silk fibroin sponge, the incorporation of chitin whiskers into the silk fibroin matrix was found to promote cell spreading.     

THE PREPARATION AND STUDY ON THE NANO-TiO2/SILK FIBROIN COMPOSITE FILMS BY THE SOL-GEL METHOD

Based on the sol-gel technique using butyl titanate as oxide precursor, the regenerated SF (silk fibroin)/nano-TiO2 composite films were synthesized. Different amounts of butyl titanate to SF were used to verify this effect on the characteristics of the formed materials. Samples were characterized by thermogravimetric analysis, X-ray diffractometry, UV, AFM and FT-IR spectroscopy.The experimental results reveal that, compared to the pure silk fibroin films, the mechanical strength of these regenerated SF/nano-TiO2 composite films were increased and the dissolubility in water of SF/nano-TiO2 composite films in aqueous solution were decreased. The diameter of nano-TiO2 particle films was about 80nm through UV and AFM. The nano-TiO2 particles were well dispersed in the regenerated silk fibroin. It was found that the crystal structures of the composite films were transited from typical Silk Ⅰ to typical Silk Ⅱ by the XRD and FTIR. Furthermore, the crystallinity of the composite films was obviously improved. Through the TGA, it was demonstrated that the heat transition temperature of composite films was also enhanced.     

纳米TiO2/丝素复合膜的制备

探讨了纳米TiO2在丝素溶液中的分散条件,在此基础上制备了纳米TiO2/丝素复合膜,并用原子力显微镜(AFM)和扫描电子能谱(EDS)对复合膜进行了表征。实验结果表明,制膜方法较为合理。当以有机物A为分散剂,纳米TiO2含量为1/1000时,其以粒径50 nm左右均匀地分散于复合膜中。     

Tunable Interfacial Properties in Silk Ionomer Microcapsules with Tailored Multilayer Interactions

Microencapsulation techniques represent a critical step in realizing highly controlled transport of functional materials in multiphase systems. The first demonstration of microcapsules prepared from minimally grafted silk ionomers (silk fibroin modified with cationic/anionic charge groups) are presented here. These tailored biomacromolecules have shown significantly increased biocompatibility over traditional polyelectrolytes and heavily grafted silk ionomers, but the low grafting density had previously limited attempts to fabricate stable microcapsules. In addition, the first microcapsules from polyethylene‐glycol‐grafted silk ionomers are fabricated and the corresponding impact on microcapsule behavior is demonstrated. The materials are shown to exhibit pH‐responsive properties, with the microcapsules demonstrating an approx. tenfold decrease in stiffness and an approx. threefold change in diffusion coefficient when moving from acidic to basic buffer. Finally, the effect of assembly conditions of the microcapsules are shown to play a large role in determining final properties, with microcapsules prepared in acidic buffers showing lower roughness, stiffness, and an inversion in transport behavior (i.e., permeability decreases at higher pH).     

Physical characteristics and properties of waterborne polyurethane materials reinforced with silk fibroin powder

Degummed silk filament was pulverized with a home‐made machine to obtain silk fibroin (SF) powder, and the structure, morphology, and particle size of the SF powder were investigated. The individual spherical particles and aggregates with different morphology of silk fibroin coexisted in water. A waterborne polyurethane (WPU) aqueous dispersion was blended with the SF powder to prepare novel blended materials with improved physical properties. The average particle size and zeta potential of the WPU/SF aqueous dispersions were characterized. The result showed that the WPU/SF dispersion with higher SF content exhibited a less negative zeta potential and a larger average particle size. Furthermore, the effect of SF content on the morphology, miscibility, and mechanical properties of the resulting blended films was studied by scanning electron microscopy, wide‐angle X‐ray diffraction, dynamic mechanical thermal analysis, and tensile testing. The films showed an improved Young's modulus and tensile strength from 0.3 to 33.8 MPa, and 0.6 to 5.2 MPa, respectively, with the increasing of SF up to a content of 26 wt %. The negative charges in the periphery and the small particle size made a good effort on dispersing SF powder into the WPU matrix as small aggregates, and the SF powder led to the efficient strengthening of WPU materials. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 940–950, 2010     

THE PREPARATION AND STUDY ON THE NANO-TiO_2/SILK FIBROIN COMPOSITE FILMS BY THE SOL-GEL METHOD

1. INTRODUCTIONSilk is a well described natural fiber produced by the silkworm, B. mori, which has been used traditionally in the form of thread in textiles for thousands of years. This silk contains a fibrous protein that forms the thread core and glueli…     

Generation of Haploid Spermatids on Silk Fibroin-Alginate-Laminin-Based Porous 3D Scaffolds

In vitro production of sperm is a desirable idea for fertility preservation in azoospermic men and prepubertal boys suffering from cancer. In this study, a biocompatible porous scaffold based on a triad mixture of silk fibroin (SF), alginate (Alg), and laminin (LM) is developed to facilitate the differentiation of mouse spermatogonia stem cells (SSCs). Following SF extraction, the content is analyzed by SDS-PAGE and stable porous 3D scaffolds are successfully prepared by merely Alg, SF, and a combination of Alg-SF, or Alg-SF-LM through freeze-drying. Then, the biomimetic scaffolds are characterized regarding the structural and biological properties, water absorption capacity, biocompatibility, biodegradability, and mechanical behavior. Neonatal mice testicular cells are seeded on three-dimensional scaffolds and their differentiation efficiency is evaluated using real-time PCR, flow cytometry, immunohistochemistry. Blend matrices showed uniform porous microstructures with interconnected networks, which maintained long-term stability and mechanical properties better than homogenous structures. Molecular analysis of the cells after 21 days of culture showed that the expression of differentiation-related proteins in cells that are developed in composite scaffolds is significantly higher than in other groups. The application of a composite system can lead to the differentiation of SSCs, paving the way for a novel infertility treatment landscape in the future.