骨髓基质细胞与交联明胶-聚羟基丁酸酯膜的生物相容性研究

目的研究生物材料交联明胶-聚羟基丁酸酯膜与骨髓基质细胞的生物相容性,探讨新型材料在骨组织工程中的应用前景。方法体外培养兔骨髓基质细胞,分别接种于G-PHB(交联明胶-聚羟基丁酸酯)、PHB(聚羟基丁酸酯)和G(交联明胶)材料膜片。采用MTT法检测细胞增殖活性,体视学方法检测细胞粘附能力,荧光双染法检测细胞完整性,扫描电镜观察细胞-材料界面。结果MTT检测发现G-PHB组增殖活性最强,而且表现为最佳的细胞粘附特性,与对照组比较差异有显著性意义。各组细胞完整性分析没有发现显著性差异。扫描电镜观察显示,G-PHB组细胞粘附及铺展良好,优于其他各组。结论交联后的生物降解膜材料G-PHB与BMSCs细胞的体外相容性明显优于单纯膜材料PHB和明胶,在骨组织工程学领域具有良好的研究价值和应用潜力。     

转基因细胞片复合PLGA支架修复兔软骨缺损的研究

细胞片技术是应用组织工程方法使培养细胞从培养表面分离而形成含有细胞外基质的一层完整片状结构,弥补了传统组织工程技术的不足,是获取种子细胞以及对种子细胞进行转移的一项新技术。为探讨体外生长分化因子-5(GDF5)基因转染修饰的BMSCs细胞片与GDF5转基因BMSCs负载的PLGA支架形成的共聚物修复兔甲状软骨缺损的效果,实验通过腺病毒转染GDF5基因至四代兔BMSCs,温度敏感性培养皿制备GDF5转基因细胞片并与负载有转染GDF5基因BMSCs的PLGA支架复合,移植至同种兔甲状软骨缺损处,分别于术后4、8周行大体观察和组织学检测其修复效果。实验分3组:(A)转基因细胞片包裹负载有转基因BMSCs的PLGA支架组;(B)负载有转基因BMSCs的PLGA支架组;(C)负载BMSCs的PLGA支架组。结果显示,体外成功收获了完整的GDF5转基因细胞片,Real time PCR检测到GDF5 mRNA的表达,行大体组织的II型胶原免疫组化和阿利新蓝染色显示:A组和B组均表达II型胶原和糖胺聚糖(GAG),但A组表达高于B组,有统计学意义(P0.05)。由此可得,转基因细胞片包裹负载转基因BMSCs PLGA支架较传统转基因BMSCs负载PLGA支架方法具有更加优越的成软骨能力,能更有效地促进软骨缺损的修复。     

骨髓间充质干细胞与聚3-羟基丁酸酯-co-4-羟基丁酸酯生物材料共培养细胞补片的初步研究

目的:研究聚3-羟基丁酸酯-co-4-羟基丁酸酯[P(3HB-co-4HB)]这种新型高分子材料与骨髓间充质干细胞(BMSCs)共培养,观察材料对干细胞的存活及增殖的影响,形成细胞补片的效果;从而找到一种适合BMSCs生长、增殖的高分子生物材料,作为治疗心肌梗死,软骨损伤等多种组织损伤疾病的修复方法之一。方法:取清洁级雄性健康BSL-C57小鼠作为实验对象,通过分离培养获得小鼠BMSCs,并进行流式细胞仪鉴定表面标志物。BMSCs培养至5代后,将BMSCs与P(3HB-co-4HB)制成的生物材料薄膜共培养,24h后固定进行电镜扫描,并用DAPI荧光染料染色处理,在荧光显微镜下观察并进行细胞计数,并描绘生长曲线。结果:BMSCs流式细胞术鉴定:CD34、CD45阴性,CD90弱阳性,CD73阳性。扫描电镜下,P(3HB-co-4HB)材料与BMSCs共培养形成的细胞补片,其表面细胞数量多,细胞状态正常。荧光显微镜下,对其细胞补片表面的细胞进行计数,并绘制生长曲线,显示表面细胞有逐渐增多的趋势。结论:P(3HB-co-4HB)材料与BMSCs共培养制成的细胞补片表面有细胞存活及增殖,由于P(3HB-co-4HB)材料本身具有良好的生物组织相容性及可降解等性质,所以该新型高分子材料可以作为干细胞治疗多种疾病的支架材料之一。     

PLGA的不同组成对支架材料性能的影响研究

研究PLGA的不同组成对支架材料的力学性能、降解性能和生物学性能的影响。采用溶液浇注/颗粒沥取法制备出不同组成的PLGA多孔支架,对支架的力学性能和降解速率进行考察,同时将人真皮成纤维细胞接种于不同组成的PLGA支架材料上,培养不同时间后,检测细胞的粘附率和增殖率,以及细胞产生的总胶原含量,并通过扫描电镜观察支架上的细胞形态。结果显示,随PLA比例的增加,支架的力学强度增加,降解速率降低,但都不是线性变化。70:30比例的支架,拉伸强度最高,而70:30和80:20两种比例的支架,其降解速率没有显著性差异。PLGA不同组成的支架,均具有良好的细胞相容性,成纤维细胞粘附率和增殖率在三种比例的支架上没有显著性差异,细胞在支架表面生长良好,分泌大量的细胞外基质,细胞基本铺满整个支架。本文研究发现,PLGA的组成对支架力学性能、降解性能和生物学性能有细小但显著的影响,这将对组织构建选用PLGA支架材料提供有益的帮助。     

静电纺丝的主要参数对ＰＬＧＡ纤维支架形貌和纤维直径的影响

目的以聚乳酸-羟基乙酸共聚物(PLGA)为材料,采用静电纺丝方法制备纤维支架,考察制备参数对纤维支架结构及纤维直径的影响规律。方法以四氢呋喃(THF)和N,N-二甲基甲酰胺(DMF)的混合液为溶剂,调节PLGA溶液浓度、流量及电场强度分别制备了具有不同表面形貌的纤维支架。通过扫描电镜(SEM)观察了纺丝溶液的浓度、流量及电场强度对纤维形貌和直径的影响。同时在制备的PLGA纤维支架上接种了人的真皮成纤维细胞,并对细胞在PLGA支架上的黏附和增殖情况进行了研究,从而来评价支架材料的细胞相容性。结论结果表明,随着纺丝溶液浓度的增加,纤维直径逐渐增大,纤维直径的分布也随之增大。随着流量的增加,纤维直径略有增大。随着电场强度的增大,纤维直径没有明显的变化。但是电压和浓度的增大有助于减少珠丝的产生。体外细胞培养实验证明,制备的PLGA纤维支架能支持细胞正常的黏附和增殖活动。     

自组装多肽纳米纤维支架诱导骨髓间充质干细胞定向分化

组织工程是现代修复重建医学领域的新思路,生物支架和种子细胞是组织工程两大关键要素。自组装多肽纳米纤维支架(SAPNS)是两亲性多肽(PAs)分子在一定条件下自组装成的一类具有三维网状结构的新型生物支架,其结构、生物功能、机械力学等特性类似天然细胞外基质(ECM),其内部经功能化修饰的抗原表位以高浓度呈递在纳米纤维表面并高效选择性地调控种子细胞生物学行为。种子细胞是组织成功再生的必需条件,骨髓间充质干细胞(BMSCs)因其良好的自我更新和多向分化潜能成为了组织工程最佳候选细胞。体外实验表明经特异功能化修饰的SAPNS在有/无辅助因子条件下可促进BMSCs黏附、增殖、迁移和定向分化,动物模型体内实验发现SAPNS结合BMSCs构建的组织工程移植物可修复缺损部位的组织结构和功能,故其在修复重建医学中有良好的应用前景。对SAPNS、自组装、BMSCs、SAPNS诱导BMSCs定向分化等方面进行了综述。     

组织工程中细胞与生物材料相互作用研究进展

种子细胞、生物材料和生长因子是组织工程三要素。生物材料模拟体内细胞外基质,为细胞提供良好的生长附着环境,维持细胞的活力和功能。材料表面的理化性质和表面改性分子直接影响细胞的粘附、增殖、迁移和分化等细胞行为,进而影响细胞功能和组织再生效果。材料表面修饰分子是细胞表面粘附和生长的直接接触位置,因此细胞与生物材料表面修饰分子的相互作用是组织工程的关键。文中重点介绍表面修饰分子对细胞表型及功能的影响,为组织工程关键问题的研究提供参考。     

骨髓间充质干细胞复合支架材料的骨组织工程研究进展

近年来,组织工程技术飞速发展,将种子细胞与支架材料相复合的骨组织工程研究已成为热点,并日趋走向成熟。这一全新的治疗方案将成为解决临床上各种原因造成的骨组织缺损的最有效途径之一。骨组织工程技术包括种子细胞、支架材料和生长因子三个方面。其中,BMSCs因具有多向分化能力、强大的增殖能力以及低免疫源性被认为是最理想的种子细胞,而支架材料的种类有很多种,目前对支架材料的选择也尚有分歧。如何找到理想的支架材料是骨组织工程研究中亟待解决的重要问题。本文就组织工程中与骨髓间充质干细胞(BMSCs)相复合的各类支架材料的研究现状进行综述,这些支架材料的研究将为骨组织工程支架材料的选择提供有效依据。     

丝素蛋白在组织工程中的应用及进展

组织工程是生物支架材料、种子细胞和生物活性因子的有机组合,其中支架材料为种子细胞的黏附载体,为细胞的生长增殖及新陈代谢提供适宜的微环境,并最终被生物体逐渐降解而被再生组织替代。支架材料为周围组织提供机械支持,并引导再生组织按照预定结构和方向生长。同时,各种生物活性物质可以加入支架材料中,比如各种生长因子以及抗体等,扩大了支架材料的应用范围。丝素蛋白具有可控且缓慢的生物降解性,突出的机械性能,良好的生物相容性,支持多种细胞的黏附、生长和分化增殖,已经用于血管、骨、软骨及神经组织等方面的组织工程研究。     

聚乙二醇对羟基丁酸和羟基己酸共聚物的共混改性研究

本研究以聚羟基脂肪酸酯家族中的新成员羟基丁酸和羟基己酸共聚物（PHBHHx）为基础,采用与聚乙二醇（PEG）共混的方法对其进行改性,研究结果证实：PHBHHx与PEG 共混物中比例为3:1及2:1时,两者完全物理相容。而PEG在共混物中比例升高时则导致相分离,成为部分相容体系。PEG掺入显著提高材料亲水性及表面自由能,使血管平滑肌细胞（RaSMCs）及血管内皮细胞（HUVECs）的细胞粘附及增殖大幅度提高,并且均具有一定的PEG含量依赖性。其中对RaSMCs的作用最为明显,RaSMCs能在PEG/PHBHHx比例为1:1的共混膜（E1X1）上持续增殖至融合,而HUVECs则呈粘附较差的类球形形貌,证实E1X1可以潜在应用于复合血管组织工程支架中的近内膜基材。     

Neurogenesis of bone marrow-derived mesenchymal stem cells onto β-mercaptoethanol-loaded PLGA film

Bone marrow-derived mesenchymal stem cells (BMSCs) are of particular interest in the field of tissue engineering because of their potential to differentiate into osteoblasts, chondrocytes, and neuronal cells. In order to promote the differentiation of BMSCs into specific cell types, appropriate scaffold biomaterials and bioactive molecules that can support the differentiation of BMSCs into specific cell types are needed. We hypothesized that β-mercaptoethanol (BME), which has been reported to induce the differentiation of BMSCs into neural-like cells, promotes BMSCs to differentiate into neural-like cells when BME is added to polymeric scaffolds containing the BMSCs. We fabricated biocompatible film shaped scaffolds composed of poly(lacti-co-glycolic) acid (PLGA) and various concentrations of BME to confirm that BME-promoted differentiation of BMSCs is concentration-dependent. Cell proliferation increased as the BME concentration in the films increased at the early stage, and the proliferation rate remained similar on the PLGA films for 3 weeks following the BMSC seeding. The expression of neuronal markers in differentiated BMSCs was assessed by RT-PCR. At 2- and 3-week time-points, mRNA expression of neurofilament and neuron specific enolase was significantly increased in PLGA/BME films containing 400 μM BME compared to PLGA films. Thus, we have identified BMSC-seeded PLGA/BME films with 200 μM and 400 μM BME as potentially useful candidates for neural tissue engineering applications by promoting BMSC proliferation and differentiation towards neural-like cells.     

Electrospun PLGA�Csilk fibroin�Ccollagen nanofibrous scaffolds for nerve tissue engineering

Electrospun nanofibrous scaffolds varying different materials are fabricated for tissue engineering. PLGA, silk fibroin, and collagen-derived scaffolds have been proved on good biocompatibility with neurons. However, no systematic studies have been performed to examine the PLGA-silk fibroin-collagen (PLGA-SF-COL) biocomposite fiber matrices for nerve tissue engineering. In this study, different weight ratio PLGA-SF-COL (50:25:25, 30:35:35) scaffolds were produced via electrospinning. The physical and mechanical properties were tested. The average fiber diameter ranged from 280 + 26 to 168 + 21 nm with high porosity and hydrophilicity; the tensile strength was 1.76 ± 0.32 and 1.25 ± 0.20 Mpa, respectively. The results demonstrated that electrospinning polymer blending is a simple and effective approach for fabricating novel biocomposite nanofibrous scaffolds. The properties of the scaffolds can be strongly influenced by the concentration of collagen and silk fibroin in the biocomposite. To assay the cytocompatibility, Schwann cells were seeded on the scaffolds; cell attachment, growth morphology, and proliferation were studied. SEM and MTT results confirmed that PLGA-SF-COL scaffolds particularly the one that contains 50% PLGA, 25% silk fibroin, and 25% collagen is more suitable for nerve tissue engineering compared to PLGA nanofibrous scaffolds.     

The biocompatibility of calcium phosphate cements containing alendronate-loaded PLGA microparticles in?vitro

The composite of poly-lactic-co-glycolic acid (PLGA) and calcium phosphate cements (CPC) are currently widely used in bone tissue engineering. However, the properties and biocompatibility of the alendronate-loaded PLGA/CPC (APC) porous scaffolds have not been characterized. APC scaffolds were prepared by a solid/oil/water emulsion solvent evaporation method. The morphology, porosity, and mechanical strength of the scaffolds were characterized. Bone marrow mesenchymal stem cells (BMSCs) from rabbit were cultured, expanded and seeded on the scaffolds, and the cell morphology, adhesion, proliferation, cell cycle and osteogenic differentiation of BMSCs were determined. The results showed that the APC scaffolds had a porosity of 67.43 ± 4.2% and pore size of 213 ± 95 µm. The compressive strength for APC was 5.79 ± 1.21 MPa, which was close to human cancellous bone. The scanning electron microscopy, cell counting kit-8 assay, flow cytometry and ALP activity revealed that the APC scaffolds had osteogenic potential on the BMSCs in vitro and exhibited excellent biocompatibility with engineered bone tissue. APC scaffolds exhibited excellent biocompatibility and osteogenesis potential and can potentially be used for bone tissue engineering.     

静电纺丝的主要参数对PLGA纤维支架形貌和纤维直径的影响

EST(expressed sequence tags,EST)是一段长约150～500bp基因表达的外源序列片段,是由大规模随机挑取的cDNA克隆测序得到的组织或细胞基因组的表达序列标签。一个EST代表生物某一时期的某种组织或细胞的一个表达基因。主要综述了EST技术的原理方法,哺乳动物早期胚胎研究的理论基础以及EST技术在早期胚胎研究方面的应用,并讨论了利用EST进行研究分析的发展趋势。     

Characterization of porous poly(D,L-lactic-co-glycolic acid) sponges fabricated by supercritical CO2 gas-foaming method as a scaffold for three-dimensional growth of Hep3B cells

This study presents the application of the porous poly(D,L-lactic-co-glycolic acid) (PLGA) sponges fabricated from an organic solvent free supercritical gas foaming technique. Two formulations of PLGA sponges with different co-polymer compositions (85:15 and 50:50) were fabricated as novel scaffolds to guide human hepatoma cell line, Hep3B cell growth in vitro. The PLGA sponges showed desirable biodegradability and exhibited uniform pore size distribution with moderate interconnectivity. It was observed in this study that cells cultured on PLGA sponges showed lower proliferation rate as compared to the control during 14 days of culture as measured by using total DNA and methylthiazol tetrazolium (MTT) assays. However, the cells cultured on the sponges tended to aggregate to form cell islets which were able to express better hepatic functions. The enzyme-linked immunosorbent assay (ELISA) results showed that the cell-sponge constructs secreted 1.5-3.0 times more albumin than the control when normalized to cellular content. In a similar fashion, its detoxification ability was also predominantly higher than that of the control as indicated by the ethoxyresorufin-O-deethylase (EROD) results. By comparing the cells growing on the two formulations of PLGA sponges, it was found that the PLGA 85:15 sponge exhibited better conductive and desirable environment for hep3B cells as justified by better cell infiltration, higher proliferation and hepatic function than the PLGA 50:50 sponge.     

BMSCs与PLGA三维生物支架复合修复喉软骨缺损的研究

目的 探讨骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）与聚乳酸/羟基乙酸共聚物（poly (lactide-co-glycolide),PLGA）三维生物支架在软骨源性形态发生蛋白1（cartilage-derived morphogenetic protein 1,CDMP1）和转化生长因子－β1（transforming growth factor-β1,TGF-β1）作用下向软骨细胞表型分化及体内修复喉软骨缺损的能力。方法 在体外高密度细胞悬液与PLGA共同构筑的三维立体培养体系下CDMP1和（或）TGF-β1联合诱导BMSCs向软骨细胞分化,观察诱导后细胞表型的表达;将培养体系移植入动物体内,从大体、组织学方面观察其对喉软骨缺损的修复效果。结果 诱导后的培养体系可表达特异性软骨基质Ⅱ型胶原和GAG;将培养体系移植入动物体内,可有效的修复喉软骨缺损。结论 BMSCs与PLGA三维生物支架在CDMP1和TGF-β1作用下所得组织工程化软骨可以有效的修复喉软骨缺损。     

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.     

Influence of collagen and chondroitin sulfate (CS) coatings on poly-(lactide-co-glycolide) (PLGA) on MG 63 osteoblast-like cells

Poly-(lactide-co-glycolide) (PLGA) is an FDA-approved biodegradable polymer which has been widely used as a scaffold for tissue engineering applications. Collagen has been used as a coating material for bone contact materials, but relatively little interest has focused on biomimetic coating of PLGA with extracellular matrix components such as collagen and the glycosaminoglycan chondroitin sulfate (CS). In this study, PLGA films were coated with collagen type I or collagen I with CS (collagen I/CS) to investigate the effect of CS on the behaviour of the osteoblastic cell line MG 63. Collagen I/CS coatings promoted a significant increase in cell number after 3 days (in comparison to PLGA) and after 7 days (in comparison to PLGA and collagen-coated PLGA). No influence of collagen I or collagen I/CS coatings on the spreading area after 1 day of culture was observed. However, the cells on collagen I/CS formed numerous filopodia and displayed well developed vinculin-containing focal adhesion plaques. Moreover, these cells contained a significantly higher concentration of osteocalcin, measured per mg of protein, than the cells on the pure collagen coating. Thus, it can be concluded that collagen I/CS coatings promote MG 63 cell proliferation, improve cell adhesion and enhance osteogenic cell differentiation.