丝素在骨组织工程中的应用及进展

蚕丝作为缝合线应用于临床已有多年历史,其中的丝素具有良好的生物相容性。丝素可制备成不同性状的新型生物材料如溶液、粉末、薄膜等,用其修复骨缺损的实验研究已相继开展。本文主婪对丝素在骨组织工程中修复骨缺损的应用及前景进行了综述。     

静电纺丝丝素蛋白水溶液制备无纺布

对不同条件下的丝素蛋白水溶液静电纺丝进行研究.在扫描电子显微镜(scanning electronic microscope,SEM)下观察了不同条件下制备出的丝素蛋白纤维的微观形貌,并用傅立叶变换红外光谱(Fourier transform infrared,FTIR)分析丝素蛋白在产物中的构象.结果 表明,丝素蛋白无纺布中的丝素蛋白以无规线团/silk Ⅰ为主,甲醇处理后,丝素蛋白无纺布中β折叠的含量明显增加.本研究为静电纺丝法制备丝素蛋白无纺布提供了新的途径,这种无纺布由于在制备过程中没有使用有机溶剂作静电纺丝溶剂,也没有共混其他聚合物,减少了溶剂残留并简化了制备过程,它将可被用于组织工程支架.     

Preparation and hemostatic property of low molecular weight silk fibroin

Effective hemorrhage control becomes increasingly significant in today’s military and civilian trauma, while the topical hemostats currently available in market still have various disadvantages. In this study, three low molecular weight silk fibroins (LMSF) were prepared through hydrolysis of silk fibroin in a ternary solvent system of CaCl₂/H₂O/EtOH solution at different hydrolysis temperatures. Fourier transform infrared spectroscopy analysis showed that the content of β sheet structure in the LMSF decreased with the increase in hydrolysis temperature. The results of thromboelastographic and activated partial thromboplastin time methods showed that the LMSF hydrolyzed at 50?°C can significantly strengthen the coagulation in blood and activate the intrinsic pathway of coagulation cascade. In the murine hepatic injury model, the LMSF hydrolyzed at 50?°C can promote the blood clotting and decrease the blood loss and bleeding time. Based on these results, it can be suggested that the developed LMSF has the excellent hemostatic effect and may be a promising material in clinical hemostatic application.     

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.     

Potential of biocompatible regenerated silk fibroin/sodium N-lauroyl sarcosinate hydrogels

Hydrogels are becoming widely used in biomaterial applications. The available methods for the preparation of these materials are continually growing. The gelation time (GT) of silk protein fibroin is difficult to control by physical methods. The cross-linkers used in available chemical techniques are likely to impact the biocompatibility of the resultant materials. In this paper, we demonstrate that the addition of sodium N-lauroyl sarcosinate (an amino-acid-based surfactant) accelerates the formation of hydrogels from fibroin. GT, turbidity variations, changes of viscoelasticity during the gelation process, and the mechanical properties of the products are measured. The secondary structure was probed by Fourier transform infrared spectroscopy, X-ray diffraction and the morphologies of the products were investigated by scanning electron microscopy. Transformations in the β-sheet content were monitored by the fluorescence of Thioflavine T and circular dichroism measurements. The relationship between the surface tension of sodium N-lauroyl sarcosinate and the GT was also explained. To investigate cell compatibility, fibroblast cells were seeded onto the surface of the hydrogels. The results indicate that the sodium N-lauroyl sarcosinate/fibroin GT can be controlled. This blend-hydrogel demonstrates excellent cell compatibility, good compression strength, and outstanding compression-recovery characteristics. Sodium N-lauroyl sarcosinate/silk fibroin hydrogels containing β-sheets have considerable potential as replacement materials in addressing the tissue defects involved with repair surgery.     

丝素蛋白/壳聚糖复合支架有良好的细胞相容性与渗透性

文题释义：丝素蛋白/壳聚糖复合支架：将壳聚糖溶于浓度为1%的冰乙酸,制备成质量浓度为35 g/L的壳聚糖溶液并与丝素蛋白溶液融合,将素蛋白与羧化壳聚糖按照体积比8∶2的比例混合,再将混合溶液注入48孔板中,每孔注入1 mL,最后通过冷冻干燥得到丝素蛋白/壳聚糖复合支架。 热重分析：是指在程序控制温度下测量待测样品的质量与温度变化关系的一种热分析技术,用来研究材料的热稳定性和组分。实验中将10 mg待检验的样品放于氮气环境下进行检测,测试温度升高控制范围为30-800 ℃,温度上升速度为10 ℃/min。 背景：丝素蛋白与壳聚糖为组织工程常用的支架材料,但二者单独应用均存在一定的不足,将两者混合使用可以互为改性,充分发挥优点,获取理想的复合支架材料。 目的：制备丝素蛋白/壳聚糖复合支架并对其进行性能测定。 方法：通过冷冻干燥方法制备丝素蛋白/壳聚糖复合支架,采用电镜扫描检测复合支架的形态结构,并进行热重分析、力学性能及细胞毒性检测。制备季铵化壳聚糖,利用核磁共振仪表征其核磁氢谱,Zeta电位仪检测其电位和粒径分布,凝胶电泳实验检测其保护DNA的情况,透射电镜观察其与DNA结合情况。 结果与结论：①扫描电镜显示丝素蛋白/壳聚糖复合支架具体良好的三维孔洞结构,孔径为50-100 μm;②热重分析显示当温度小于200 ℃时,丝素蛋白/壳聚糖复合支架的质量损失下降速度较低;当温度上升至200-500 ℃时,支架质量损失速度开始加快,损失量增多;在800 ℃时,复合支架的残余质量为38%;③丝素蛋白/壳聚糖复合支架的最大应变可以达到94.94%,最大承受应力为7.01 MPa;④CCK-8实验显示,丝素蛋白/壳聚糖复合支架对兔骨髓间充质干细胞没有细胞毒性,具有良好的细胞相容性;⑤核磁氢谱检测显示,季铵化壳聚糖的季铵化程度约为20%;⑥季铵化壳聚糖的粒径分布为(588.56±52.39) nm,季铵化壳聚糖颗粒的表面带正电荷,电位为(16.3±3.92) mV,有利于与DNA结合;⑦凝胶电泳实验显示,季铵化壳聚糖材料的比例越高,对DNA的包裹越好,当其与DNA的比例为1∶3时,对DNA达到包裹作用;⑧透射电镜显示,季铵化壳聚糖/DNA大部分的微粒呈实心圆形,微粒粒径差别较小,平均粒径约为200 nm;⑨结果表明,丝素蛋白/壳聚糖复合支架有良好的细胞相容性与细胞渗透性,利于细胞在支架间的生长。 ORCID: 0000-0002-2572-0229(章晓云) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

壳聚糖微球复合丝素基硫酸钙骨水泥的理化特性

背景：脊柱成形和脊柱后凸成形治疗中采用的硫酸钙骨水泥理化性质好,对人体无毒性作用,同时具有降解性能,但单独使用降解较快。 目的：研制具有载药缓释功能的壳聚糖微球丝素基硫酸钙骨水泥。 方法：采用三聚磷酸钠乳化交联法制备壳聚糖微球。采用浓度分别为3%,6%,9%的丝素溶液与CaSO₄•0.5H₂O混合,通过万能力学试验机确定骨水泥力学性能最佳时的丝素浓度,在此浓度下,按壳聚糖微球占CaSO₄•0.5H₂O的质量比分别为0.5%,1%,5%的比例制备壳聚糖微球丝素基硫酸钙骨水泥,测定其抗压强度,并通过X射线多晶衍射仪及傅里叶红外光谱明确达到最佳抗压强度组的骨水泥成分,电镜观察复合骨水泥中壳聚糖微球的形态。 结果与结论：当丝素溶液浓度为6%,壳聚糖微球含量为0.5%时,复合骨水泥的抗压强度最大,为  (39.17±1.96) MPa,此时复合骨水泥的初凝时间为(12.99±1.63) min,终凝时间为(21.55±0.54) min;骨水泥中主要晶相组成为硫酸钙,傅里叶红外光谱结果证实复合骨水泥中含有丝素及壳聚糖;复合骨水泥中的微球表面稍有皱缩,但球形仍然完整,未见明显破坏,可见在制备复合骨水泥的过程中微球能保持稳定而不被破坏。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

丝素蛋白材料生物相容性评价研究进展

丝素蛋白是一种具有优良理化特性的高分子生物材料,而且具有良好的生物相容性。主要对国内外关于丝素蛋白材料生物相容性评价的研究进展,尤其是其优异的生物功能性做了综述,并对下一步研究其生物相容性分子机制的发展趋势进行了探讨。     

Physiological pulsatile flow culture conditions to generate functional endothelium on a sulfated silk fibroin nanofibrous scaffold

Many studies have demonstrated that in vitro shear stress conditioning of endothelial cell-seeded small-diameter vascular grafts can improve cell retention and function. However, the laminar flow and pulsatile flow conditions which are commonly used in vascular tissue engineering and hemodynamic studies are quite different from the actual physiological pulsatile flow which is pulsatile in nature with typical pressure and flow waveforms. The actual physiological pulsatile flow leading to temporal and spatial variations of the wall shear stress may result in different phenotypes and functions of ECs. Thus, the aim of this study is to find out the best in vitro dynamic culture conditions to generate functional endothelium on sulfated silk fibroin nanofibrous scaffolds for small-diameter vascular tissue engineering. Rat aortic endothelial cells (RAECs) were seeded on sulfated silk fibroin nanofibrous scaffolds and cultured under three different patterns of flow conditioning, e.g., steady laminar flow (SLF), sinusoidal flow (SF), or physiological pulsatile flow (PPF) representative of a typical femoral distal pulse wave in vivo for up to 24 h. Cell morphology, cytoskeleton alignment, fibronectin assembly, apoptosis, and retention on the scaffolds were investigated and were compared between three different patterns of flow conditioning. The results showed that ECs responded differentially to different exposure time and different flow patterns. The actual PPF conditioning demonstrated excellent EC retention on sulfated silk fibroin scaffolds in comparison with SLF and SF, in addition to the alignment of cells in the direction of fluid flow, the formation of denser and regular F-actin microfilament bundles in the same direction, the assembly of thicker and highly crosslinked fibronectin, and the significant inhibition of cell apoptosis. Therefore, the actual PPF conditioning might contribute importantly to the generation of functional endothelium on a sulfated silk fibroin nanofibrous scaffold and thereby yield a thromboresistant luminal surface.     

Generation of bioactive nano-composite scaffold of nanobioglass/silk fibroin/carboxymethyl cellulose for bone tissue engineering

Abstract

The development of bone tissue construct through tissue engineering approach offers a great promise in meeting the increasing demand for repair and regeneration of damaged and/or diseased bone tissue. For the generation of bone tissue engineered construct, polymer-ceramic composite matrices with nanostructure architecture and mesenchymal stem cells (hMSCs) of human origin are of prime requirement. Keeping these in view, in the present work a novel electrospun nanofibrous silk fibroin (SF)/carboxymethyl cellulose (CMC)/nano-bioglass (nBG) composite scaffold that mimics native bone extracellular matrix with appropriate composition was designed and fabricated by free liquid surface electrospinning technique. The scaffold possesses desired morphological, structural, biodegradability, bioactivity, surface roughness and mechanical properties thereby exhibited an excellent platform to support the growth of cells. The in-vitro culture of hMSCs over the developed scaffold has shown adhesion, proliferation and viability of cells, thus facilitated cell-scaffold construct generation and further extracellular bone matrix formation through osteogenic differentiation as evident from alkaline phosphatase activity, biomineralization, immunostaining and Runx2/osteocalcin expression assessment. Thus, the developed hMSCs seeded scaffold construct might be suitable for bone tissue engineering applications.     

丝素蛋白的免疫学特性及细胞相容性研究进展

丝素蛋白作为一种具有良好生物相容性和低免疫原性的天然高分子材料在生物医学领域里有着广泛的应用价值。阐述了近年来对丝素蛋白在组织工程学研究中所涉及的免疫学方面及细胞相容性方面的研究概况。     

Fabrication and characterization of polyacrylamide/silk fibroin hydrogels for peripheral nerve regeneration

Various hydrogels have been used for repairing peripheral nerve injury; however, the silk fibroin (SF)-based hydrogels in peripheral nerve regeneration are still rarely reported. In this study, the SF/pAM hydrogels with different SF concentrations and ethanol treatment time were developed by solution blending and in situ radical polymerization. The physiochemical properties of composite hydrogels were measured, the cytotoxicity of hydrogels was evaluated by L929 fibroblasts, and the effect on peripheral nerve regeneration was evaluated via Schwann cells culture in vitro. The results showed that the physiochemical properties of SF/pAM hydrogels could be changed by varying SF concentration and ethanol treatment time, and the mechanical property was enhanced with increasing SF concentration, while the presence of SF in pAM hydrogels and ethanol treatment does not affect hydrogels structure in per se. All the composite hydrogels displayed no obvious cytotoxicity, while the SF/pAM composite hydrogels with 10% SF and 60-min ethanol treatment could obviously accelerate the attachment and proliferation of Schwann cells. Therefore, the SF/pAM composite hydrogels possessed the beneficial properties required for in situ cell scaffolding and may have potential application in peripheral nerve regeneration.     

复合骨碎补总黄酮的丝素蛋白/壳聚糖支架在兔关节软骨损伤中的应用研究

目的 以丝素蛋白/壳聚糖支架为载体将骨碎补总黄酮应用于兔软骨损伤局部,观察修复效果,为临床提供实验数据。方法 制备丝素蛋白/壳聚糖支架、骨碎补总黄酮缓释微球与负载骨碎补总黄酮缓释微球的丝素蛋白/壳聚糖支架,扫描电子显微镜下观察支架形貌,同时检测该支架的体外缓释能力。24只新西兰大白兔随机分3组,利用电钻在股骨滑车部位构建直径3.5 mm、深1.5 mm的软骨损伤模型,空白组软骨缺损处不植入任何材料,对照组植入单纯的丝素蛋白/壳聚糖支架,实验组植入负载骨碎补总黄酮缓释微球的丝素蛋白/壳聚糖支架,术后12周、24周行标本大体与组织学观察,RT-PCR检测修复组织Sox-9、II型胶原与聚集蛋白聚糖mRNA的表达量,Western blot检测软骨缺损部位II型胶原蛋白表达,分析软骨修复效果。结果 丝素蛋白/壳聚糖支架具有良好的三维孔隙结构,孔洞之间相互联通;制备的载药微球表面较光滑,为较规则的圆球形;载药微球均匀分散于丝素蛋白/壳聚糖支架基质中。丝素蛋白/壳聚糖支架可在体外持续稳定地释放骨碎补总黄酮,实验组软骨损伤修复效果优于对照组,对应的ICRS评分与Wakitani组织学评分高于对照组...     

Therapeutic efficacy of antibiotic-loaded gelatin microsphere/silk fibroin scaffolds in infected full-thickness burns

Despite advances in burn treatment, burn infection remains a major cause of morbidity and mortality. In this study, an antibacterial silk fibroin (SF) scaffold for burn treatment was designed; gelatin microspheres (GMs) were impregnated with the antibiotic gentamycin sulfate (GS), and the GS-impregnated GMs were then embedded in a SF matrix to fabricate GS/GM/SF scaffolds. The developed GS/GM/SF scaffolds could serve as a dermal regeneration template in full-thickness burns. The average pore size and porosity of the GS/GM/SF scaffolds were 40–80 μm and 85%, respectively. Furthermore, the drug release rate of the scaffolds was significantly slower than that of either GS/GM or GS/SF scaffolds. And the composite scaffold exhibited stronger antimicrobial activities against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Hence, we evaluated the wound-healing effects and antibacterial properties of the GS/GM/SF scaffolds in a rat full-thickness burn infection model. Over 21 days, the GS/GM/SF scaffolds not only significantly reduced burn infection by P. aeruginosa but also accelerated the regeneration of the dermis and exhibited higher epithelialization rates than did GS/SF and SF scaffolds. Thus, GS/GM/SF scaffolds are potentially effective for treatment of full-thickness infected burns, and GS/GM/SF scaffolds are a promising therapeutic tool for severely burned patients.     

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.     

Characterization of surface modified glycerol/silk fibroin film for application to corneal endothelial cell regeneration

Corneal endothelial cells (CEnCs) play a fundamental role in maintaining the transparency of the cornea. CEnCs lose their full proliferating capacity when tissue damages occur. The loss in proliferation rate is associated with corneal edema and decrease in visual acuity, leading in severe cases, to blindness. In these situations, a corneal transplant is usually needed to restore the original tissue functions. Tissue engineering is an efficient alternative for the production of implantable films, which can regenerate the tissue functions regulating at the same time the immune-response.

In this study, we proposed a stable and transparent film, composed of silk fibroin modified with glycerol (G/SF), as a potential substrate for corneal endothelial cells regeneration. Our results confirmed that G/SF films have a uniform structure, rougher surface and lower thickness respect to the SF film. In vitro tests show that G/SF films can induce a slight increase in CEnCs initial adhesion and proliferation rate if compared with the SF film. Morphology and gene expression evaluations demonstrated that the bioactive effects of silk fibroin were not affected by the presence of glycerol. For this reason, the G/SF films are suitable as CEnCs carrier and promising for the corneal damages treatments.     

3D printing process of oxidized nanocellulose and gelatin scaffold

For tissue engineering applications tissue scaffolds need to have a porous structure to meet the needs of cell proliferation/differentiation, vascularisation and sufficient mechanical strength for the specific tissue. Here we report the results of a study of the 3D printing process for composite materials based on oxidized nanocellulose and gelatin, that was optimised through measuring rheological properties of different batches of materials after different crosslinking times, simulation of the pneumatic extrusion process and 3D scaffolds fabrication with Solidworks Flow Simulation, observation of its porous structure by SEM, measurement of pressure-pull performance, and experiments aimed at finding out the vitro cytotoxicity and cell morphology. The materials printed are highly porous scaffolds with good mechanical properties.     

一种新的人工血管涂层及其实验研究

目的为克服目前使用的人工血管涂层材料性质普遍不稳定、不易提纯、交联用难且价格昂贵的缺陷,作者进行了人工血管丝素蛋白涂层的实验研究.材料和方法将丝素蛋白浸渍涂层涤纶人工血管内外壁并使用甲醛交联固定.通过血管壁渗水率、形态学以及力学性能等体外试验评价丝素蛋白涂层人工血管是否达到人工血管植入前标准.结果未涂层人工血管管壁渗水率通过丝素蛋白涂层降低了99%,达到植入时不漏血的目的.丝素蛋白涂层占重(117±22)mg/g显著低于较InterGardTM血管胶原涂层占重(302±23)mg/g(P＜0.05).结论丝素蛋白涂层人工血管可操控性好,渗水率低.同时在体外具备良好的生物降解性能.     

双层蛛丝蛋白血管支架的制备及其生物力学性能与细胞相容性研究

目的 应用静电纺丝法制备一种双层蛛丝蛋白血管支架,观察血管支架的微观结构,并研究其生物力学性能和细胞相容性。方法 配制纺丝液,通过静电纺丝,以旋转接收棒为收集装置,制备(pNSR16/PCL/CS)/(pNSR16/PCL/Gt)双层蛛丝蛋白血管支架。探讨质量分数和管壁厚度对血管支架孔隙率、爆破强度、拉伸性能、缝合强度和水渗透性的影响,并检测血管支架的细胞毒性和细胞黏附性能。结果 血管支架的微观结构为纤维随机分布的三维多孔网状,爆破强度、拉伸强度和缝合强度大小均与支架的质量分数和管壁厚度成正比,孔隙率、水渗透性和断裂伸长率大小与支架的质量分数和管壁厚度成反比。血管支架爆破强度的范围为43~183 kPa,高于生理血压;缝合强度高于0.19 N,符合体内移植要求;拉伸强度高于人体桡动脉血管,满足体内移植的要求;水渗透性为0.3~0.6 mL?min-1?cm-2。血管支架无细胞毒性,并有利于内皮细胞细胞黏附及增殖。结论 使用静电纺丝法制备的双层蛛丝蛋白血管支架是可行的,其优异的生物力学性能和生物相容性能表明其能应用于体外组织工程血管的构建,具有进一步应用于血管移植物研究的前景,为临床应用奠定了一定的基础。