类人胶原蛋白-透明质酸血管支架的性能及生物相容性

将类人胶原蛋白与透明质酸按不同比例复合,控制透明质酸的终浓度(W/V)分别为0、0.01%、0.05%、0.1%,用京尼平交联,采用真空冷冻干燥方法构建出血管支架材料。通过扫描电镜、XPS分析、拉力测试、压力爆破实验、细胞毒性实验、血管支架细胞种植实验及小鼠皮下植入等方法对其表面超微结构、表面元素组成、力学性能、细胞毒性等级、细胞相容性、组织相容性进行了研究。结果表明:当透明质酸的含量为0.05%时,类人胶原蛋白-透明质酸支架的孔径比较均匀,孔隙率达94.38%,应力为(1000.8±7.9)kPa,爆破压力为(1058.6±8.2)kPa,细胞毒性实验合格,同时具有良好的细胞相容性、组织相容性及降解性能。     

III型类人胶原蛋白在大肠杆菌重组表达及发酵制备

【背景】胶原蛋白广泛应用于日用化工及生物医药中,相比传统方法,基因工程方法制备胶原蛋白具有避免病毒隐患、产量高等优点,逐步受到广泛关注。【目的】获得III型类人胶原蛋白基因,实现大肠杆菌中的异源表达。【方法】以人III型胶原蛋白α1链为模板,(Gly-X-Y)为最小研究单位,优选亲水性氨基酸,设计目标基因kit,构建重组大肠杆菌(Escherichia coli) pET-28a(+)-kit/BL21(DE3),并对其结构进行表征。【结果】类人胶原蛋白基因kit成功在大肠杆菌体系中表达,表达量约为0.53 g/L,7 L发酵罐上补料发酵后其最大表达量提高至3.02 g/L,亲和层析纯化类人胶原蛋白纯度约为91%,对其进行N端测序、氨基酸分析、质谱分析及圆二色谱分析,确定类人胶原蛋白成功表达。【结论】类人胶原蛋白的成功表达为未来规模化制备及其在日用化工及生物医药行业的应用奠定了基础。     

不同钙-醇溶解体系丝素蛋白的制备及表征研究

采用 ４种中性盐溶液 Ｃａ（ＮＯ３）２４Ｈ２Ｏ 甲醇、Ｃａ（ＮＯ３）２４Ｈ２Ｏ 乙醇、ＣａＣｌ２ 甲醇 水和 ＣａＣｌ２ 乙醇 水（摩尔比分别为 １∶２、１∶２、１∶２∶８、１∶２∶８）处理蚕丝纤维,透析后经冷冻干燥制成固体,利用ＳＤＳ ＰＡＧＥ、电镜扫描和红外光谱对制得的固体进行表征。ＳＤＳ ＰＡＧＥ结果表明：Ｃａ（ＮＯ３）２４Ｈ２Ｏ 醇体系降解丝素蛋白较 ＣａＣｌ２ 醇 水体系降解程度高;电镜扫描的结果表明 Ｃａ（ＮＯ３）２４Ｈ２Ｏ 甲醇和 ＣａＣｌ２ 乙醇 水溶解体系处理的丝素蛋白溶解比较完全,Ｃａ（ＮＯ３）２４Ｈ２Ｏ 甲醇处理的丝素蛋白冻干后为颗粒状,而 ＣａＣｌ２ 乙醇 水处理的丝素蛋白冻干后为片状。红外光谱的结果表明：４种溶液处理后的丝素蛋白构象均介于 β折叠和无规则卷曲之间,从而为丝素蛋白在药物缓释载体领域的应用提供了一定的理论依据。     

纳米丝素蛋白微球的制备及性能研究

以家蚕丝素蛋白为原料,基于丝素自组装理论,通过酶解-干燥-溶解法制备不同尺寸的丝素蛋白微球,制备出的微球具有良好的水不溶性和稳定的分散性。对微球的形貌和结构表征结果表明,用该方法制备的丝素蛋白微球为纳米微球,当酶的添加量为2%且蛋白自组装时间为4 h时,丝素蛋白微球的平均粒径最小,仅为(32±11)nm。红外光谱(FT-IR)和X射线衍射(XRD)结果显示,微球中β-折叠结构的多少决定了微球晶体的大小,β-折叠越多,微球中晶体的体积越大。通过调控丝素蛋白自组装过程,可以制备平均粒径在30～140 nm之间的纳米丝素蛋白微球,且不引入任何有机溶剂和无机溶剂,制备过程绿色环保,制备出的丝素蛋白微球安全无毒。     

丝素蛋白在电纺丝法构建组织工程支架中的应用进展

丝素蛋白是天然高分子纤维蛋白,具有良好的物理和机械力学性能及生物相容性,因而在组织工程领域有着广阔的应用前景。文中对丝素蛋白的化学组成、分子结构特点、提取方法以及利用静电纺丝技术在组织工程化支架构建中的应用作了概述。总结了丝素蛋白在用于组织工程材料上的性能和优势以及在人工血管、皮肤、骨组织等工程化支架方面的应用情况,探讨了丝素蛋白支架对细胞在其上生长、增殖和功能的影响,同时对丝素蛋白在组织工程化食道支架及其他再生医学上的应用前景进行了展望。     

原子力显微镜下丝素纤维及丝素蛋白的形态结构研究

利用原子力显微镜(AFM)、透射电镜(TEM)、圆二色谱仪(CD)等手段,研究了家蚕丝素纤维及丝素蛋白的形态结构,并尝试通过改变丝素蛋白溶液的酸碱性来观测其形态变化。结果表明,丝素纤维表面有许多沟槽和条纹,具有原纤结构特征;许多直径为20～50nm的圆形或椭圆形颗粒分子形成丝素蛋白的微观形态。在不同的酸碱条件下,球状颗粒分子具有不同的聚集方式,形成不同的微观形态。     

一种可止血的富血小板血浆壳聚糖/丝素蛋白多孔伤口敷料的制备及性能表征

为了进一步提高伤口敷料的止血性能,文中在生物相容性良好的壳聚糖溶液中引入含有多种生长因子的人源性富血小板血浆(Humanplatelet-richplasma,hPRP),并加入不同体积比例(1∶1、1∶3、3∶1、1∶0)的丝素蛋白溶液以提高材料的多孔性与止血性,通过冷冻干燥法制备不同配比的hPRP-壳聚糖/丝素蛋白敷料,并将纯壳聚糖敷料作为对照组,研究hPRP和丝素蛋白对敷料的止血性能的影响以及丝素蛋白对PRP中生长因子控制释放的影响。结果表明,在壳聚糖敷料中引入hPRP对敷料的止血性有所提高,但对敷料的多孔结构及吸水率无明显改善,若在hPRP-壳聚糖溶液中按照体积比为1∶1的比例加入丝素蛋白溶液,会得到具有较为均匀的多孔结构的敷料,敷料的孔隙率与吸水率分别可达到86.83%±3.84%与1 474%±114%,且该比例的敷料在快速止血性能上表现优异。此外,加入丝素蛋白与壳聚糖比例为1∶1的PRP敷料能有效减少PRP中生长因子在初始阶段的爆裂释放。因此,含hPRP的壳聚糖/丝素蛋白复合敷料有望成为一种能快速止血且能促进伤口愈合的新型伤口敷料。     

流动聚焦法制备丝素蛋白微球

[目的]旨在研究丝蛋白溶液的浓度、流量、是否载药对丝蛋白微球生成速率与粒径的影响.[方法]使用基于微加工技术制作的PDMS(聚二甲基硅氧烷)微流控芯片作为丝素蛋白微球的发生装置,通过调节微量注射泵改变丝素蛋白溶液的流量并使用高速摄影拍摄记录不同工况下微球的产生情况,最后使用Matlab进行数据处理分析.[结果]司盘80...     

元素衡算和代谢衡算在类人胶原蛋白表达期的应用研究

应用元素衡算和代谢衡算方法,建立用重组大肠杆菌发酵生产类人胶原蛋白表达期的数学模型,并利用非线性优化法对模型中的未知参数进行估算。结果表明该模型与重组大肠杆菌的生长、代谢动力学模型一致,且模型的关键计算参数αh、αp和mx分别为1.173 molo C-mol-1、293.814 molo C-mol-1和17.878 molo C-mol-1oh-1。该模型能较好地预测重组类人胶原蛋白表达期中的宏观反应速率,且在类人胶原蛋白发酵表达期,必须通过控制葡萄糖的补料速率来控制菌体的生长,当?=0.04 h-1时,类人胶原蛋白的比生成速率达最大值。     

丝素蛋白/壳聚糖复合材料在组织工程中应用的研究进展

丝素蛋白(silk fibroin,SF)和壳聚糖(chitosan,CS)具有良好的生物相容性和可降解性,然而单一组分的SF和CS支架材料的诸多缺点限制了其在组织工程研究中的应用。SF/CS复合材料克服单一组分SF和CS支架的缺点,具有力学性能优良、可塑性好、孔隙率及孔径可调和组分优势互补等特点。多种方法制备的SF/CS复合材料(微米/纳米颗粒、膜、纳米纤维、水凝胶和三维多孔支架)已用于骨、软骨、皮肤、神经、脂肪、心脏和角膜等组织工程或组织损伤修复的研究中。目前,国内外对于SF/CS复合材料在组织工程中应用的研究尚处于起步阶段。主要对SF/CS复合材料的特点、制备方法以及在多种组织工程中应用的研究现状进行了简要介绍。     

Fabrication and evaluation of poly(epsilon-caprolactone)/silk fibroin blend nanofibrous scaffold

In this study we investigated the blend electrospinning of poly(?‐caprolactone) (PCL) and silk fibroin (SF) to improve the biodegradability and biocompatibility of PCL‐based nanofibrous scaffolds. Optimal conditions to fabricate PCL/SF (50/50) blend nanofiber were established for electrospinning using formic acid as a cosolvent and three‐dimensional (3D) PCL/SF blend nanofibrous scaffolds were prepared by a modified electrospinning process using methanol coagulation bath. The physical properties of 2D PCL/SF blend nanofiber mats and 3D highly porous blend nanofibrous scaffolds were measured and compared. To evaluate cytocompatibility of the 3D blend scaffolds as compared to 3D PCL nanofibrous scaffold, normal human dermal fibroblasts were cultured. It is concluded that biodegradability and cytocompatibility could be improved for the 3D highly porous PCL/SF (50/50) blend nanofibrous scaffold prepared by blending PCL with SF in electrospinning. In addition to the blending of PCL and SF, the 3D structure and high porosity of electrospun nanofiber assemblies may also be important factors for enhancing the performance of scaffolds. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 265–275, 2012.     

The optimization of a scaffold for cartilage regeneration

Natural polymers offer various advantages in cartilage tissue engineering applications, thanks to their intrinsic bioactivity and adaptability, which can be exploited for the optimization of scaffold properties. In particular, silk fibroin has multifunctional features driven by the self-assembly of molecular subunits in appropriate environmental conditions. For these reasons, it was used in combination with hyaluronic acid to produce porous sponges for cartilage regeneration. The added amount of hyaluronic acid and the cross-linking with genipin modulated scaffold properties in a synergistic way, showing a strong inter-correlation among macroscopic and microscopic characteristics. Interestingly, hyaluronic acid affected silk fibroin conformation and induced a physical separation between the two material components in absence of genipin. Instead, this was prevented by the cross-linking reaction, resulting in a more interspersed network of protein and polysaccharide molecules partially resembling the structure of cartilage extracellular matrix. In addition, the systematic evaluation of sponge properties and how they can be modulated will represent a significant starting point for the interpretation of the complex outcomes driven by the scaffold in vitro and in vivo.     

Structural characteristics and biological performance of silk fibroin nanofiber containing microalgae spirulina extract

Silk fibroin (SF) nanofiber scaffold containing microalgae Spirulina extract were prepared by electrospinning and the performance and functionality of the scaffold were evaluated. The viscosity and conductivity of the dope solution of Spirulina containing SF were examined for electrospinability and we found that the morphological structure of SF nanofiber is affected by the concentration of Spirulina extract added. The platelet adhesion and coagulation time test confirmed that the Spirulina containing SF nanofiber scaffold had excellent ability to prevent blood clotting or antithrombogenicity that is comparable to heparin. Low cytotoxicity and excellent cell adhesion and proliferation were also observed for Sprulina containing SF nanofiber scaffold by methylthiazolyldiphenyl‐tetrazolium bromide assay and confocal fluorescence microscope using fibroblast and human umbilical vein endothelial cells. Based on these results, we believe SF nanofiber scaffold containing Spirulina extract has the potential to be used as tissue engineering scaffold that requires high hemocompatibility. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 307–318, 2014.     

Balanced electrostatic blending approach--an alternative to chemical crosslinking of Thai silk fibroin/gelatin scaffold

In tissue engineering, chemical crosslinking is widely used for conjugating two or more biomaterials to mainly control biodegradability and strength. For example, Thai silk fibroin/gelatin scaffold will offer mechanical strength from Thai silk fibroin and cell attraction from gelatin. However, chemical crosslinking requires crosslinking agent which could potentially pose negative impact from remaining trace amount of chemicals especially in medical application. Here we present an alternative approach to chemical crosslinking—a balance electrostatic blending approach. In this approach, two opposite charge biomaterials were selected for blending, with different ratios. Both materials were bound together with electrostatic force. The maximum binding was achieved when mixture electric potential approaches zero. In this work, we compared this approach with traditionally chemical crosslinking in terms of physical appearance, binding effectiveness, mechanical strength (in dry/wet conditions), in vitro biodegradation, and cell proliferation. We found that 50/50 weight ratio of Thai silk fibroin/gelatin scaffold had almost comparable properties to chemical crosslinked scaffold. It has similar appearance, binding effectiveness, and affinity for cell proliferation. For mechanical properties, even this approach yields lower dry compressive modulus compared with chemical crosslinking. But in wet condition, the compressive modulus from both methods is similar. However, the biodegradation time of non-crosslinked scaffolds is slightly faster than that of chemical crosslinked ones. These results demonstrate that a balance electrostatic approach is an alternative approach to chemical crosslinking when there is a concern of remaining trace amount of crosslinking agent in medical application.     

A novel composite scaffold for cardiac tissue engineering

Summary One approach to the engineering of functional cardiac tissue for basic studies and potential clinical use involves bioreactor cultivation of dissociated cells on a biomaterial scaffold. Our objective was to develop a scaffold that is (1) highly porous with large intereconnected pores (to facilitate mass transport), (2) hydrophilic (to enhance cell attachment), (3) structurally stable (to withstand the shearing forces during bioreactor cultivation), (4) degradable (to provide ultimate biocompatibility of the tissue graft), and (5) elastic (to enable transmission of contractile forces). The scaffold of choice was made as a composite of poly(Dl-lactide-co-caprolactone), poly(Dl-lactide-co-glycolide) (PLGA), and type I collagen, with open interconnected pores and the average void volume of 80±5%. Neonatal rat heart cells suspended in Matrigel were seeded into the scaffold at a physiologically high density (1.35×10⁸ cells/cm³) and cultivated for 8 d in cartridges perfused with culture medium or in orbitally mixed dishes (25 rpm); collagen sponge (Ultrafoam^⋆m) and PLGA sponge served as controls. Construct cellularity, presence of cardiac markers, and contractile properties were markedly improved in composite scaffolds as compared with both controls.     

A high molecular weight silk fibroin scaffold that resists degradation and promotes cell proliferation

The regulation of the biodegradation rate of 3D-regenerated silk fibroin scaffolds and the avoidance of premature collapse are important concerns for their effective applications in tissue engineering. In this study, bromelain, which is specific to sericin, was used to remove sericin from silk, and high molecular weight silk fibroin was obtained after the fibroin fibers were dissolved. Afterwards, a 3D scaffold was prepared via freeze-drying. The Sodium dodecyl sulfate–polyacrylamide gel electrophoresis results showed that the average molecular weight of the regenerated silk fibroin prepared by using the bromelain-degumming method was approximately 142.2 kDa, which was significantly higher than that of the control groups prepared by using the urea- and Na₂CO₃-degumming methods. The results of enzyme degradation in vitro showed that the biodegradation rate and internal three-dimensional structure collapse of the bromelain-degumming fibroin scaffolds were significantly slower than those of the two control scaffolds. The proliferation activity of human umbilical vein vascular endothelial cells inoculated in bromelain-degumming fibroin scaffolds was significantly higher than that of the control scaffolds. This study provides a novel preparation method for 3D-regenerated silk fibroin scaffolds that can effectively resist biodegradation, continuously guide cell growth, have good biocompatibility, and have the potential to be used for the regeneration of various connective tissues.     

酪氨酸酶催化氧化对丝素蛋白结构与性能的影响

采用酪氨酸酶对丝素蛋白催化氧化,考察了酶促氧化反应对丝素蛋白结构及丝素膜性能的影响。研究结果表明,酪氨酸酶可催化氧化丝素蛋白中酪氨酸残基生成多巴和多巴醌结构衍生物,并且两者含量随催化反应时间延长呈波动性变化;酶促反应后丝素蛋白中游离氨基含量下降,丝素风干膜断裂强度增加,表明酶促氧化中丝素大分子间发生自交联。XRD结果表明酪氨酸酶催化氧化对丝素蛋白二级结构有一定影响;SEM显示酶促改性可能影响丝素蛋白冷冻干燥膜多孔形态结构。