胶原基组织工程支架对成纤维生长因子的控制释放与组织再生功能

目的：通过将碱性成纤维细胞生长因子(basic fibroblast growth factor bFGF)与胶原基组织工程支架进行复合，实现对bFGF的控制释放，从而更有效的实现组织再生功能。方法：采用ELISA酶联免疫试剂盒对溶出的bFGF进行定量分析，考察支架对bFGF的短期控制释放过程。结果：bFGF可以通过静电作用与胶原基支架结合，均匀分布在多孔三维支架中，48h后，bFGF的溶出浓度趋于恒定，经过甲醛交联的支架对bFGF的结合量较高；bFGF的结合量受到壳聚糖的含量和交联方法的影响。结论：胶原基组织工程支架能够对生长因子在较长时间内实现控制释放，促进组织的再生修复。     

血管内皮生长因子/纳米晶胶原基骨缓释系统与人骨髓间充质干细胞的体外生物相容性

背景:利用载体或缓释系统负载生长因子,既能保护生长因子的生物活性,又可以使生长因子缓慢释放,从而持续促进细胞生长及组织修复再生,是目前控制释放载体材料应用研究的方向之一.目的:观察血管内皮生长因子/纳米晶胶原基骨缓释系统的体外缓释效果及与种子细胞的生物相容性.方法:人骨髓间充质干细胞经体外诱导培养、扩增后种植于血管内皮生长因子+肝素/纤维连接蛋白+纳米晶胶原基骨支架(实验组)和单纯的纳米晶胶原基骨支架(对照组)行体外培养.测定缓释支架材料上血管内皮生长因子的释放量和持续时间;种植细胞后细胞的黏附率;培养3,7,10,14 d时支架材料中细胞数、碱性磷酸酶活性以及细胞在材料上的生长状况.结果与结论:①该缓释支架具有一定的血管内皮生长因子缓释效果,持续时间可达14 d.②第3代人骨髓间充质干细胞经成骨诱导培养,可表达成骨细胞表型:碱性磷酸酶细胞化学染色、Ⅰ型胶原免疫荧光染色均为阳性.③体外实验显示该缓释支架与入骨髓间充质干细胞具有优良的生物相容性:相同时间点实验组的细胞黏附率、支架上细胞数量及细胞碱性磷酸酶活性均明显高于对照组;扫描电镜发现两组材料上均有细胞生长,但实验组的细胞生长状况明显好于对照组.     

组织工程细胞支架及其相关技术研究

细胞、生物材料制备的细胞支架,以及组织和器官的构建是组织工程的三大要素.理想的细胞支架应具有一定的生物降解速度、良好的细胞亲和性、呈特定的三维多孔结构,且含有可控制释放的生长因子.本文介绍了在组织工程细胞支架用生物材料合成和改性、细胞支架构建、细胞支架表面改性、生长因子控制释放以及所研制的细胞支架在皮肤、骨、软骨、神经修复等组织工程研究方面的最新进展和相关技术.     

组织工程神经控释系统的理论研究与临床应用

自体神经移植虽然是神经缺损修复的金标准,却由于受到取材的限制,组织工程神经因而成为最好的替代选择.组织工程神经控释系统是通过载有生长因子的组织工程神经支架在局部持续不断释放生长因子,在损伤局部形成促进神经再生的微环境,促进神经轴突再生.目前组织工程神经控释系统所用的材料和方法多种多样,每种材料和方法都有自身的优缺点,寻找合适的材料和方法控释生长因子,达到最好的神经修复效果,是组织工程神经控释系统研究的主要方向.文章主要探讨近年来一些组织工程神经控释材料在应用方法和技术上取得的新进展,并对其进行归纳总结,按照控释原理进行创新性的分类.     

成年兔雪旺细胞与医用组织引导再生胶原膜联合培养后植入体内的实验研究

目的：探索用组织工程方法构建人工神经的可行性。方法：应用医用组织引导再生胶原膜作为支架与成年兔雪旺细胞培养2周,以雪旺细胞胶原膜复合物的形式修复自体神经缺损,8周后对所获得的组织工程神经移植物进行形态学研究。结果：成年兔雪旺细胞在医用组织引导再生胶原膜上生长良好并均匀分布于支架表面,形成bungner带,且基质分泌旺盛,神经已通过移植物长入远端,胶原膜已吸收,结论：雪旺细胞可以在医用组织引导再生胶原膜上得到扩增,形成的三维立体结构具有人工神经的基本特性,本研究为组织工程方法修复长段神经缺损打下基础。     

组织工程软骨研究中各种生长因子的协同作用和最佳效应

组织工程技术的核心是将种子细胞接种到三维立体支架上,形成两者的复合体,复合体在体外培育一段时间后,进行体内移植以进行受损组织或器官的修复和替换。近年来的研究发现,生长因子对细胞的生长、增殖以及组织工程化人体组织的构建均有重要作用。这些生长因子可通过传输信号来调节细胞活动。但它们的半衰期很短,可是通过生长因子与生物材料结合则可以延长这些因子的生物活性,并能控制其释放。目前,在合成性的支架材料中复合生物活性因子,已被认为是组织工程可能的应用方法之一。     

组织工程支架

组织工程的目的是培养生物替代品来补救或替换损伤和老化的器官和组织。一种比较有前途的方法就是在可降解支架上移植细胞，支架的首要目标是在一个三维空间实现细胞外基质的功能，关键问题是支架中有能满足要求的生物信号，这样细胞反应的每一个方面：细胞黏附、扩散、分裂和显型都能够得到控制直至一个新的组织的形成。该文对如何控制支架的内部结构，如何控制和调节支架中生长因子的释放和如何将细胞植入支架等方面的新技术研究进展进行综述。     

细胞移植及髓核组织工程重建修复椎间盘退变

背景:通过细胞组织工程将细胞或细胞支架复合体植入退变缺损的椎间盘内,使退变的椎间盘再生可能是治疗椎间盘退变性疾病最为理想的方法.目的:评价组织工程髓核体内移植抑制腰椎间盘退变的临床疗效及应用前景.方法:由第一作者检索1990/2010 PubMed数据、中国知网及万方数据库有关组织工程髓核、组织工程材料及骨髓间充质干细胞治疗腰椎间盘退变方面的文献.结果与结论:目前常用的细胞支架主要包括胶原支架、琼脂糖支架、藻酸盐支架、聚乙醇酸支架与壳聚糖支架以及复合材料等.通过自体椎间盘细胞或间充质干细胞结合基因技术筛选种子细胞,进行细胞和/或细胞支架复合体移植恢复或再生相关细胞外基质的合成,通过逆转和修复椎间盘细胞病理性改变,为退变的椎间盘组织和功能恢复提供了全新的治疗策略.     

胰岛素样生长因子1与转化生长因子β2促进组织工程软骨构建的体外实验

背景:软骨组织的再生能力差,软骨组织工程能利用较少的细胞、支架材料和细胞因子对缺损进行修复.目的:观察胰岛素样生长因子1 与转化生长因子β2 联合应用对组织工程软骨形成的影响.方法:用酶消化法获取人软骨细胞,将培养的细胞以4×109 L-1 的细胞浓度接种在藻酸钙凝珠支架上,分别加入200 μg/L 胰岛素样生长因子1 和(或)1 μg/L 转化生长因子β2 进行立体培养.于培养的第3,5,7,9,1,13 天,进行细胞计数,观察软骨细胞的增殖情况.培养2 周后,进行大体形态观察和阿尔新蓝-过碘酸雪夫氏染色(AB-PAS)及抗Ⅱ型胶原免疫组织化学染色.结果与结论:细胞计数及免疫组织化学染色显示,胰岛素样生长因子1 和转化生长因子β2 均能促进软骨细胞增殖和软骨相关基质黏多糖及Ⅱ型胶原的分泌,其中胰岛素样生长因子1 的作用主要体现在促细胞增殖方面,而转化生长因子β2 的作用主要体现在促进软骨相关基质形成方面,二者联合应用具有促进组织工程软骨形成的协同作用.     

组织工程软骨研究中各种生长因子的协同作用和最佳效应

组织工程技术的核心是将种子细胞接种到三维立体支架上，形成两者的复合体，复合体在体外培育一段时间后，进行体内移植以进行受损组织或器官的修复和替换。近年来的研究发现，生长因子对细胞的生长、增殖以及组织工程化人体组织的构建均有重要作用。这些生长因子可通过传输信号来调节细胞活动。但它们的半衰期很短，可是通过生长因子与生物材料结合则可以延长这些因子的生物活性，并能控制其释放。目前，在合成性的支架材料中复合生物活性因子，已被认为是组织工程可能的应用方法之一。     

In situ adipogenesis in fat tissue augmented by collagen scaffold with gelatin microspheres containing basic fibroblast growth factor

In situ adipose tissue regeneration in fat tissue by collagen sponges and gelatin microspheres containing basic fibroblast growth factor (bFGF) was investigated. A minced collagen sponge scaffold (1 ml) was incorporated with microspheres containing 10 µg bFGF and administered into a defect of rabbit fat tissues. Adipogenesis at the administered site was evaluated histologically. The adipose tissue regeneration induced by the administration of mixed collagen scaffold and microspheres containing bFGF was significantly stronger than that of either collagen scaffold alone or microspheres containing bFGF alone. The histological area of in situ adipogenesis by the mixed collagen scaffold and microspheres containing bFGF was enhanced over time by repeated administration. It is concluded that the repeated administration of collagen scaffold and microspheres containing bFGF is a promising way to achieve adipose tissue regeneration inside inherent fat tissue. This technique might be applicable for the reconstruction of volume contour deformities by trauma or surgical interventions of adipose tissue in a minimally invasive manner. Copyright © 2009 John Wiley & Sons, Ltd.     

壳聚糖-明胶-碱性成纤维细胞生长因子三维大孔支架对骨髓间充质干细胞增殖的影响

背景:干细胞的分化潜能与培养条件有密切关系,改变支架材料的表面特性,三维结构,增加生长因子均可实现对干细胞增殖分化的控制.目的:制备适合骨髓间充质干细胞附着生长的、具有最佳孔隙率和孔隙结构的药物缓释组织工程支架--三维大孔支架,提供能促进多能干细胞生长的微环境.设计:重复测量设计.单位:广州中医药大学基础医学院解剖教研室.材料:实验所用健康成年 SD 大鼠由广州中医药大学实验动物中心提供.壳聚糖、碱性成纤维细胞生长因子购自Sigma公司.方法:实验于2003-03/2006-12主要在广州中医药大学基础医学院解剖教研室完成.采用冷冻干燥的方法,用不同比例的壳聚糖.碱性成纤维细胞生长因子-明胶依次混匀,通过控制冷冻、复温和干燥时间处理使其具有最佳孔隙率和孔结构,制备具缓释碱性成纤维细胞生长因子功能的三维大孔支架.取SD大鼠股骨和胫骨骨髓,分离、培养骨髓间充质干细胞并移植于缓释碱性成纤维细胞生长因子的三维大孔支架上进行三维培养,与无碱性成纤维细胞生长因子的支架对照.实验过程中对动物的处置符合动物伦理学标准.主要观察指标:用ELISA和扫描电镜观察支架的三维结构和缓释性能,用苏木精-伊红染色、MTT、细胞计数及扫描电镜方法观察缓释碱性成纤维细胞生长因子的三维大孔支架对骨髓间充质干细胞生长状态和细胞活力的影响.结果:含有碱性成纤维细胞生长因子的三维大孔支架有碱性成纤维细胞生长因子缓释性能,孔隙尺寸与不含碱性成纤维细胞生长因子的支架三维结构相比,差异无显著性意义(P＞0.05).含有碱性成纤维细胞生长因子的三维大孔支架能提高在支架上立体培养的骨髓间充质干细胞存活率,促进骨髓间充质干细胞黏附、增殖和活力,与不含碱性成纤维细胞生长因子的支架相比,差异有显著性意义(P＜0.05).结论:含有碱性成纤维细胞生长因子的三维大孔支架能缓释碱性成纤维细胞生长因子,有利于在支架上立体培养的骨髓间充质干细胞存活,为其在组织工程中的应用打下基础.     

聚乳酸类缓释型支架材料的研究与应用意义

目的聚乳酸是具有良好的生物相容性和生物降解特性的聚合物,是美国食品和药品管理局(FDA)认可的一类可植入体内的生物材料.细胞因子在组织工程骨构建中的促进成骨、血管化等作用越来越受到广泛关注.但外源性细胞因子在体内半衰期较短,不能在骨修复局部聚集和保持相对较高浓度.主要分析聚乳酸及其衍生化合物材料可作为多肽及蛋白质类药物的载体,通过其自身降解来调节药物释放,用于骨组织工程研究的研究现状与应用前景.资料来源应用计算机检索Medline 1993/2004文章,检索词为"polylactic acid,bone morphogenetic protein,vascular endothelial growth factor,TGF-β",限定语言English;检索中国期刊网1993/2004医药卫生类文章,限定语言种类中文.资料选择纳入标准①有关聚乳酸及其衍生物构建骨组织工程支架的研究.②聚乳酸类支架负载细胞因子研究.排除标准①陈旧文献.②重复研究.资料提炼共收集到96篇与聚乳酸组织工程支架相关文献,其中23篇纳入引用. 资料综合①聚乳酸/聚乳酸和聚羟基乙酸的共聚物是一种良好的可生物降解的控释骨架,且具有支架和缓释的双重作用.聚乳酸是最早作为骨、软骨组织工程的支架材料,也是目前运用最广泛的骨组织工程材.②骨形态发生蛋白是应用于骨组织工程最多的因子,负载、缓释骨形态发生蛋白的聚乳酸类支架研究最为深入,未见血管内皮生长因子负载于聚乳酸类支架材料研究.结论对于可缓释多种因子的聚乳酸类支架材料的开发与应用已经成为骨组织工程支架新的热点.     

转染碱性成纤维生长因子基因的骨髓间充质干细胞在珊瑚骨表面生长状况

背景:颌面骨缺损是临床上常遇到的问题,寻找理想的种子细胞同支架材料复合构建组织工程化人工骨成为该类疾病治疗的发展趋势.目的:将转染碱性成纤维生长因子基因的骨髓间充质干细胞与珊瑚骨的复合培养,观察转染碱性成纤维生长因子基凶的骨髓间充质干细胞在珊瑚支架材料上生长状况.设计、时间及单位:骨组织工程实验,于2006-0312008-06 在中山大学口腔医学研究所完成.材料:选用海南省浅海滩产石头状滨珊瑚为原料,将其制成8mm×8mm×2mm的珊瑚人工骨小块.方法:采用密度梯度离心法分离新西兰大白兔骨髓间充质干细胞,采用贴壁筛选法对分离出的骨髓间充质下细胞进行纯化,利用脂质体转染bFGF-pcDNA3到骨髓间充质干细胞.取生长良好的转染碱性成纤维生长因子基因骨髓间充质干细胞和未转染的骨髓间充质干细胞,分别接种于不同珊瑚表面.主要观察指标:MTT法观察细胞-支架联合培养骨髓间充质干细胞的增殖情况和利用扫描电镜观察珊瑚支架上细胞的生长状况.结果:MTT法检测显示细胞-支架联合培养转染组细胞与联合培养未转染组细胞增殖相比差异有显著性意义(P＜0.05),联合培养转染组细胞生长增殖强于未转染组细胞.而联合培养的转染组细胞同单纯培养转染组细胞增殖相比差异无显著性意义(P＞0.05).扫描电镜观察显示复合骨髓间充质干细胞细胞贴附在珊瑚上,并在材料上完全铺展,形态多样,细胞向孔内长入或跨越微孔表面,部分区域有细胞外基质形成.结论:转染碱性成纤维生长因子基因的骨髓间充质干细胞在珊瑚支架材料上生长状况较未转染组好,珊瑚人工骨不影响骨髓间充质干细胞的增殖,可以作为骨髓间充质干细胞支架材料构建组织工程骨.     

Collagen scaffolds for nonviral IGF-1 gene delivery in articular cartilage tissue engineering

This study investigated the use of a type II collagen-glycosaminoglycan (CG) scaffold as a nonviral gene delivery vehicle for facilitating gene transfer to seeded adult articular chondrocytes to produce an elevated, prolonged and local expression of insulin-like growth factor (IGF)-1 for enhancing cartilage regeneration. Gene-supplemented CG (GSCG) scaffolds were synthesized by two methods: (1) soaking a pre-cross-linked CG scaffold in a plasmid solution followed by a freeze-drying process, and (2) chemically cross-linking the plasmid DNA to the scaffold. Two different plasmid solutions were also compared: (1) naked plasmid IGF-1 alone, and (2) plasmid IGF-1 with a lipid transfection reagent. Plasmid release studies revealed that cross-linking the plasmid to the CG scaffold prevented passive bolus release of plasmid and resulted in vector release controlled by scaffold degradation. In chondrocyte-seeded GSCG scaffolds, prolonged and elevated IGF-1 expression was enhanced by using the cross-linking method of plasmid incorporation along with the addition of the transfection reagent. The sustained level of IGF-1 overexpression resulted in significantly higher amounts of tissue formation, chondrocyte-like cells, GAG accumulation, and type II collagen production, compared to control scaffolds. These findings demonstrate that CG scaffolds can serve as nonviral gene delivery vehicles of microgram amounts of IGF-1 plasmid (<10 microg per scaffold) to provide a locally sustained therapeutic level of overexpressed IGF-1, resulting in enhanced cartilage formation.     

Evaluation of a novel collagen–gelatin scaffold for achieving the sustained release of basic fibroblast growth factor in a diabetic mouse model

The objective of this study was to evaluate the ability of a scaffold, collagen–gelatin sponge (CGS), to release basic fibroblast growth factor (bFGF) in a sustained manner, using a pressure‐induced decubitus ulcer model involving genetically diabetic mice. We confirmed that CGSs impregnated with a bFGF concentration of up to 50 µg/cm² were able to sustain the release of bFGF throughout their biodegradation. We prepared decubitus ulcers on diabetic mice. After debriding the ulcers, we implanted CGSs (diameter 8 mm) impregnated with normal saline solution (NSS) or bFGF solution (7, 14, 28 or 50 µg/cm²). At 1 and 2 weeks after implantation, the mice were sacrificed and tissue specimens were obtained. The wound area, neoepithelium length and numbers and total area of newly formed capillaries were evaluated. The CGSs impregnated with NSS became infected and degraded, whereas the CGSs impregnated with 7 or 14 µg/cm² bFGF displayed accelerated dermis‐like tissue formation and the CGSs impregnated with 14 µg/cm² bFGF produced significant improvements in the remaining wound area, neoepithelium length and numbers and total area of newly formed capillaries compared with the NSS group. No significant difference was observed between the NSS and 50 µg/cm² bFGF groups. CGSs impregnated with 7–14 µg/cm² bFGF accelerated wound healing, and an excess amount of bFGF did not increase the wound‐healing efficacy of the CGSs. Our CGS is a scaffold that can release positively charged growth factors such as bFGF in a sustained manner and shows promise as a scaffold for skin regeneration. Copyright © 2012 John Wiley & Sons, Ltd.     

采用反求工程和快速原型技术精确定制骨组织工程支架

背景:组织工程基本原理是从患者获取组织,经体外培养扩增种子细胞,接种在支架内,通过支架引导形成三维外形的组织,最后植入该患者体内以替代病损组织的功能,以后随着新生血管长入,支架逐步溶解,新生组织最终与周围组织完全融合。目的:探讨应用反求工程和快速原型技术定制个体特异的解剖外形骨组织工程支架的可行性,克服常规制作方法的缺陷。设计:定制个体特异的解剖外形骨组织工程支架方法。单位:解放军广州军区广州总医院全军创伤骨科中心。材料:支架CAD设计在广东省机械研究所CAD培训中心完成,支架快速成型制作在广东省龙创域公司完成,快速成型工艺为立体光固化工艺,采用材料为光敏树脂。方法:实验于2004-10/2005-01在广州军区广州总医院全军创伤骨科中心完成。按反求工程的基本原理,采用医学CT/MRI扫描获取患者骨骼的分层图像信息,采用计算机辅助技术进行三维重建和曲面重构建立骨骼感兴趣区域的解剖模型,并在建立的解剖模型的外形轮廓内进行支架内部结构的计算机辅助设计,建立其计算机辅助设计模型,最后采用快速成型工艺精确制作骨组织工程支架的原型。主要观察指标:①CT/MRI扫描三维重建与解剖建模结果。②个体化组织工程支架的内部结构设计结果。③个体化组织工程支架快速成型制作。结果:①经CT/MRI图像三维重建,建立了骨关节解剖模型。②计算机辅助设计软件设计支架内部结构成功地建立了骨组织工程支架的实体模型。③骨组织工程支架CAD模型指导快速成型工艺成功地制作了个体化解剖外形的支架,制作的骨组织工程支架内部结构非常精细,具有高孔隙率和较好的孔隙相互连通性能。结论:采用反求工程和先进制造技术,可以任意制作个体化解剖外形的组织工程支架;在所有快速成型工艺中,以立体光固化成型精度最高,表面光滑、成型质量最好。     

The influence of sustained dual‐factor presentation on the expansion and differentiation of neural progenitors in affinity‐binding alginate scaffolds

Biomaterials capable of controlling the release of multiple growth factors (GFs) could potentially promote the integration of co‐transplanted neural progenitor cells (NPCs) and stimulate the plasticity and regenerability of the lesioned spinal cord. As a first step towards the employment of such a vehicle for cell therapy, this study examined the capability of an alginate–sulphate/alginate scaffold, able to capture and rigorously control the release of GFs, to promote the expansion and lineage differentiation of NPCs in vitro. Epidermal growth factor (EGF) and fibroblast growth factor‐2 (bFGF) were affinity‐bound to alginate–sulphate (200 ng/scaffold) and the bioconjugates were mixed with partially calcium‐crosslinked alginate. NPCs isolated from 18 day‐old rat embryo brains and seeded into the scaffold during preparation were found to proliferate and differentiate within the vehicle. A continuous release of both bFGF and EGF was noted for a period of 21 days. The concentrations of released GFs were sufficient to promote extensive NPC proliferation at initial cultivation times; the number of neurospheres in the scaffold was twice the number found in the 2D cultures supplemented with 20 ng/ml each factor every 3 days. Between days 10–14, when the GF concentrations had substantially declined, extensive cell migration from the neurospheres as well as lineage differentiation were noted in the scaffold; immunocytochemical analyses confirmed the presence of neurons, astrocytes and oligodendrocytes.The scaffold has a potential to serve as cell delivery vehicle, with proven capability to promote cell retention and expansion, while enabling NPC lineage differentiation in situ. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of fibrin derivatives for controlled release of heparin-binding growth factors.

The goal of this work was to develop a growth factor delivery system for use in wound healing that would provide localized release of heparin-binding growth factors in a biomimetic manner, such that release occurs primarily in response to cell-associated enzymatic activity during healing. A key element of the drug delivery system was a bi-domain peptide with an N-terminal transglutaminase substrate and a C-terminal heparin-binding domain, based on antithrombin III. The bi-domain peptide was covalently cross-linked to fibrin matrices during coagulation by the transglutaminase activity of factor XIIIa and served to immobilize heparin electrostatically to the matrix, which in turn immobilized the heparin-binding growth factor and slowed its passive release from the matrix. Basic fibroblast growth factor (bFGF) was considered as an example of a heparin-binding growth factor, and cell culture experimentation was performed in the context of peripheral nerve regeneration. A mathematical model was developed to determine the conditions where passive release of bFGF would be slow, such that active release could dominate. These conditions were tested in an assay of neurite extension from dorsal root ganglia to determine the ability of the delivery system to release bioactive growth factor in response to cell-mediated processes. The results demonstrated that bFGF, immobilized within fibrin containing a 500-fold molar excess of immobilized heparin relative to bFGF, enhanced neurite extension by up to about 100% relative to unmodified fibrin. A variety of control experiments demonstrate that all components of the release system are necessary and that the bi-domain peptide must be covalently bound to the fibrin matrix. The results thus suggest that these matrices could serve as therapeutic materials to enhance peripheral nerve regeneration through nerve guide tubes and may have more general usefulness in tissue engineering.