转化生长因子β_1基因修饰的间充质干细胞/仿生基质材料的体外复合培养

目的 探讨转化生长因子β_1(TGF-β_1)基因转染对间充质干细胞(MSCs)增殖分化的影响,评价涂覆多聚赖氨酸(PLYS)的聚DL乳酸(PDLLA) 仿生基质材料能否作为关节软骨组织工程学研究的支架材料。方法 将组织工程学与分子生物学有机结合,体外将具有多重生物学效应的TGF-β_1基因转入关节软骨组织工程首选种子细胞--间充质干细胞（MSCs）,并与表面涂覆PLYS的PDLLA可降解三维多孔基质材料体外复合培养。以单纯空载体转染MSCs为对照,通过扫描电镜等方法观察种子细胞的粘附、增殖及分化等情况。结果 基质材料孔隙率为88%,其中孔径为150～200μm的有效孔占80%。所有细胞均能很好地粘附于基质材料上,其中TGF-β_1基因修饰的MSCs增殖分化活性明显优于对照组。结论 利用分子组织工程学原理可使TGF-β_1持续高效发挥作用,使提高关节软骨缺损的修复质量和远期疗效成为可能。涂覆PLYS的PDLLA仿生基质材料不仅具有良好的生物相容性和结构相容性,而且具有更好的表面相容性和生物学活性,在一定程度上解决了细胞-基质材料之间存在的界面不相容问题,是关节软骨缺损修复的良好基质材料。     

TGF—β1基因转染间充质干细胞的量效关系及安全性研究

为探讨能获得最佳转染效率和促增殖分化效应的TGF－β1基因转染条件，剂量及基因治疗的安全性，体外将TGF－β1基因转入骨膜源间充质干细胞（MSCs) , 用水貂肺上皮细胞生长抑制法，透射电镜及软琼脂培养等方法检测，结果发现：转基因ＭＳＣs表达基因TGF-β1具有生物学活性，当３μ１脂质体介导１μgＴＧＦ－β１基因转染时，能获得最佳转染结合成为分子组织工程学，为高质量地修复关节软骨损损奠定了良好的基础。     

转化生长因子β1基因对骨髓基质干细胞增殖分化的影响

目的 观察转化生长因子β1（TGF－β1）基因能否转入骨髓基质干细胞（MSCs)，并探讨经TGF－β1基因转染后的MSCs增殖和分化情况。方法 取新西兰雄性大耳白兔的骨髓，得骨髓源MSCs体外培养。将重组TGF－β1和空载pcD-NA3基因分别转染MSCs后，免疫组化（SABC）法检测TGF－β1转染是否成功，用透射电镜和流式细胞仪观察两组MSCs的增殖和分化情况。结果 SABC法证明TGF－β1瞬间转染成功；转染48h后，实验组MSCs增殖较对照组显著；实验组MSCs具有一定的分化趋势。结论 重组TGF－β1基因转入骨髓源MSCs的方法是可行的，瞬间TGF－β1转染对MSCs的增殖和分化有显著促进作用。     

生长因子诱导骨髓间充质干细胞成软骨分化的研究

目的 建立一种体外分离兔骨髓间充质干细胞（MSCs)的方法，诱导其表达软骨细胞表型。方法 抽取兔骨髓，经密度梯度离心和粘附分离得到MSCs。用生长因子诱导，观察细胞的增殖情况和软骨特异性基质的表达水平。结果 ①纤维粘连蛋白促进MSCs贴壁，可提高细胞分离率；②TGF－β1，IGF－I和维生素C结合可促进MSCs增殖，表达软骨特异性的Ⅱ型前胶原mRNA和基质蛋白多糖成分。结论 ①密度梯度离心结合粘附分离可提高MSCs的分离效率。②TGF－β，IGF－I和维生素C结合诱导可促进MSCs增殖，表达软骨细胞表型。     

TGF-β1基因转染间充质干细胞的量效关系及安全性研究

为探讨能获得最佳转染效率和促增殖分化效应的TGF-β1基因转染条件、剂量及基因治疗的安全性,体外将TGF-β1基因转入骨膜源间充质干细胞(MSCs),用水貂肺上皮细胞生长抑制法、透射电镜及软琼脂培养等方法检测.结果发现转基因MSCs表达的TGF-β1具有生物学活性.当3μl脂质体介导1μgTGF-β1基因转染时,能获得最佳转染效率和促MSCs增殖及向成软骨方向定向分化效应;转基因细胞无恶性转化倾向.从而把组织工程学与分子生物学有机结合成为分子组织工程学,为高质量地修复关节软骨缺损奠定了良好的基础.     

TGF-β_1基因转染间充质干细胞的量效关系及安全性研究

为探讨能获得最佳转染效率和促增殖分化效应的 TGF-β1 基因转染条件、剂量及基因治疗的安全性 ,体外将TGF-β1 基因转入骨膜源间充质干细胞 ( MSCs) ,用水貂肺上皮细胞生长抑制法、透射电镜及软琼脂培养等方法检测。结果发现 :转基因 MSCs表达的 TGF-β1 具有生物学活性。当 3μl脂质体介导 1μg TGF-β1 基因转染时 ,能获得最佳转染效率和促 MSCs增殖及向成软骨方向定向分化效应 ;转基因细胞无恶性转化倾向。从而把组织工程学与分子生物学有机结合成为分子组织工程学 ,为高质量地修复关节软骨缺损奠定了良好的基础。     

脂质体介导胰岛素样生长因子-1基因修饰骨髓间充质干细胞成软骨分化

目的脂质体介导IGF-1目的基因转染骨髓间充质干细胞（MSCS），并研究对MSCS成软骨分化的特异性细胞外基质的影响。方法以密度梯度离心结合全骨髓培养法获得纯化骨髓间充质干细胞（MSCs），经流式细胞仪检测，细胞表面标志CD29和CD44表达阳性，但是CD14和CD45表达阴性。采用脂质体介导法将重组真核表达质粒pcDNA3．1-IGF-1转染MSCs，对转染后骨髓间充质干细胞的Ⅱ型胶原（ColⅡ）的表达行Western blot检测。结果获得纯度较高的兔骨髓间充质干细胞（MSCs）；脂质体为介导将全长兔IGF-1目的基因导入MSCs，Western blot鉴定转染成功，转染后的MSCs较未转染MSCs成软骨分化特异性细胞外基质ColⅡ、蛋白表达明显增强。结论以脂质体介导法可以将IGF-1目的基因转染骨髓间充质干细胞，转染后成软骨分化的特异性细胞外基质增多。     

脂质体介导转化生长因子-β1基因修饰骨髓间充质干细胞成软骨分化

目的 脂质体介导TGF-β1目的基因转染骨髓间充质干细胞(MSC)，研究对MSC成软骨分化的特异性细胞外基质的影响。方法 以梯度离心结合换液法获得并纯化骨髓间充质干细胞(MSCs)，采用脂质体介导法将重组真核表达质粒pcDNA3-TGF-β1转染MSCs，Rr-PCR和Western blot鉴定后，对转染后骨髓间充质干细胞的Ⅱ型胶原(ColⅡ)、纤维连接蛋白(FIN)的表达行RT-PCR和Western blot检测。结果 获得纯度较高的成年中国小型猪骨髓间充质干细胞(MSCs)；脂质体为介导将全长人TGF-β1目的基因导入MSC，经RT-PCR和Western blot鉴定转染成功，转染后的MSC较未转染MSC成软骨分化特异性细胞外基质ColⅡ、FNmRNA和蛋白表达明显增强。结论以脂质体介导法可以将TGF-β1目的基因转染骨髓间充质干细胞，转染后成软骨分化的特异性细胞外基质增多。     

兔骨髓间充质干细胞定向诱导移植修复关节软骨的实验研究

目的：通过诱导兔骨髓间充质干细胞（MSCs）,观察干细胞向软骨细胞的分化效果。方法：纯化兔骨髓间充质干细胞;用含转化生长因子β1的诱导液培养骨髓间充质干细胞;以MTT测定诱导细胞的增殖活性;免疫组化检测Ⅱ型胶原蛋白的表达;诱导后的细胞移植于白兔膝关节内,X线摄片观察软骨修复形成情况。结果：诱导液可有效诱导MSCs向软骨细胞表型转化,且骨髓间充质干细胞向软骨细胞分化所需的特异性细胞外基质——Ⅱ型胶原明显升高,移植后效果明显。结论：兔骨髓间充质干细胞定向诱导后移植修复关节软骨是有效的。因此骨髓间充质干细胞有可能成为软骨组织工程较理想的种子细胞来源。     

hIGF-1基因修饰骨髓基质干细胞复合PLGA移植修复关节软骨缺损

目的：研究人胰岛素样生长因子-1（hIGF-1）基因修饰的骨髓基质干细胞（MSCs）复合PLGA材料移植对关节软骨缺损的修复作用.方法：从质粒pcDNA3.1-hIGF-1中扩增出hIGF-1基因,亚克隆后酶切出目的片段插入pIRES2-EGFP中,构建成真核表达载体pIRES2-EGFP-hIGF-1,鉴定后转染兔MSCs并检测hIGF-1在其中的表达;将转染细胞复合PLGA支架材料立体培养后植入实验性兔关节软骨缺损处,不同时间取材,检测对软骨缺损修复的效果.结果：成功构建了GFP标记的真核载体pIRES2-EGFP-hIGF-1,并在靶细胞中得到理想表达;hIGF1基因修饰的MSCs复合支架材料移植明显促进了缺损软骨的修复质量,组织学评分与对照组相比有统计学差异（P＜0.05）.结论：IGF-1是软骨组织工程种子细胞MSCs基因修饰的理想候选基因,以之修饰的MSCs作为种子细胞可以明显提高组织工程方法对软骨缺损的修复效果.     

干细胞及其仿生基质微环境在关节软骨再生修复中的作用综述

关节软骨由于无神经、无血管的特性,一旦损伤,其自我修复十分困难.干细胞技术的发展为关节软骨再生提供了新的希望.当前,不同来源的干细胞及多种应用方式在关节软骨修复中展现出不同程度的治疗效果.然而,干细胞对其所处的微环境均有极强的敏感性,这使得越来越多的研究者开始关注通过生物功能性支架构建的仿生微环境来调控干细胞,进而加速...     

负载TGF-β1微球的壳聚糖-丝素支架复合骨髓间充质干细胞修复兔关节软骨缺损的实验研究

目的研制负载转化生长因子β1（transforming growth factor—β1,TGF—B。）壳聚糖微球的壳聚糖-丝素支架复合骨髓间充质干细胞（mesenchymal stem cells,MSCs）体外构建组织工程软骨,观察将其移植修复兔膝关节软骨缺损的效果。方法通过密度梯度离心法及贴壁培养法分离纯化MSCs,以抗兔CD14、CD44等单抗采用流式细胞仪进行MSCs表面抗原鉴定,获得纯化的MSCs。采用乳化交联法获得包裹TGF—B,壳聚糖缓释微球,并将该微球负载在冷冻干燥获得的壳聚糖一丝素支架上。将培养的MSCs种植到支架上,体外构建组织工程软骨,移植到兔关节软骨缺损处。移植空白支架的为实验对照组,移植体外构建的组织工程软骨为实验组。主要观察指标,流式检测的MSCs细胞表面标记情况,微球的形态,支架与细胞共培养后电镜下情况,支架移植后的兔关节修复情况及组织学检查。结果流式检测MSCs第4代及第9代细胞的CD14及CD44表面抗原的表达情况发现两代MSCsCD14表达基本呈阴性,CD44表达呈阳性,符合间充质干细胞的特性。扫描电镜观察所制备的微球基本呈球形,表面光滑,直径约为1μm,大小较均一,分散度好。细胞在壳聚糖-丝素三维支架上生长状态良好,电镜观察,可见支架孔隙内有细胞黏附生长。移植治疗后发现实验组修复明显好于对照组,实验组术后3个月实验组已经有较硬的类软骨组织填充修复,切片显示有软骨细胞规则排列,甲苯胺蓝染色为阳性,而对照组仅为纤维组织修复,切片显示为纤维组织或纤维软骨组织修复,软骨细胞排列紊乱,甲苯胺蓝染色阴性或弱阳性。结论负载TGF—β1壳聚糖微球的壳聚糖-丝素支架复合骨髓间充质干细胞体外复合培养后移植治疗修复兔膝关节软骨缺损,具有较好的疗效。     

Expression of Transforming Growth Factor β1 in Mesenchymal Stem Cells: Potential Utility in Molecular Tissue Engineering for Osteochondral Repair

Summary: The feasibility of using gene therapy to treat full-thickness articular cartilage defects was investigated with respect to the transfection and expression of exogenous transforming growth factor (TGF)-β1 genes in bone marrow-derived mesenchymal stem cells (MSCs) in vitro. The full-length rat TGF-β1 cDNA was transfected to MSCs mediated by lipofectamine and then selected with G418,a synthetic neomycin analog. The transient and stable expression of TGF-β1 by MSCs was detected by using immunohistochemical staining. The lipofectamine-mediated gene therapy efficiently trans fected MSCs in vitro with the TGF-β1 gene causing a marked up-regulation in TGF-β1 expression as compared with the vector-transfected control groups, and the increased expression persisted for at least 4 weeks after selected with G418. It was suggested that bone marrow-derived MSCs were sus ceptible to in vitro lipofectamine mediated TGF-β1 gene transfer and that transgene expression persist-ed for at least 4 weeks. Having successfully combined the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology, an innovative concept, I.e.molecular tissue engineering, are put forward for the first time. As a new branch of tissue engineer-ing, it represents both a new area and an important trend in research. Using this technique, we have a new powerful tool with which: (1) to modify the functional biology of articular tissue repair along defined pathways of growth and differentiation and (2) to affect a better repair of full-thickness artic ular cartilage defects that occur as a result of injury and osteoarthritis.     

Molecular tissue engineering: Applications for modulation of mesenchymal stem cells proliferation by transforming growth factor β1 gene transfer

Summary The effect of transforming growth factor β₁ (TGF-β₁) gene transfection on the proliferation of bone marrow-derived mesenchymal stem cells (MSCs) and the mechanism was investigated to provide basis for accelerating articular cartilage repairing using molecular tissue engineering technology. TGF-β₁ gene at different doses was transduced into the rat bone marrow-derived MSCs to examine the effects of TGF-β₁ gene transfection on MSCs DNA synthesis, cell cycle kinetics and the expression of proliferating cell nuclear antigen (PCNA). The results showed that 3 μl lipofectaminemediated 1 μg TGF-β₁ gene transfection could effectively promote the proliferation of MSCs best; Under this condition (DNA/Lipofectamine= 1μg/3μl) flow cytometry and immunohistochemical analyses revealed a significant increase in the³H incorporation, DNA content in S phase and the expression of PCNA. Transfection of gene encoding TGF-β₁ could induce the cells at G0/G1 phase to S1 phase, modulate the replication of DNA through the enhancement of the PCNA expression, increase the content of DNA at S1 phase and promote the proliferation of MSCs. This new molecular tissue engineering approach could be of potential benefit to enhance the repair of damaged articular cartilage, especially those caused by degenerative joint diseases. This project was supported by a grant from National Natural Science Foundation of China (No. 30170270).     

Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-β1 delivery for cartilage tissue engineering

Background Natural articular cartilage has a limited capacity for spontaneous regeneration. Controlled release of transforming growth factor-β(1) (TGF-β(1)) to cartilage defects can enhance chondrogenesis. In this study, we assessed the feasibility of using biodegradable chitosan microspheres as carriers for controlled TGF-β(1) delivery and the effect of released TGF-β(1) on the chondrogenic potential of chondrocytes.Methods Chitosan scaffolds and chitosan microspheres loaded with TGF-β(1) were prepared by the freeze-drying and the emulsion-crosslinking method respectively. In vitro drug release kinetics, as measured by enzyme-linked immunosorbent assay, was monitored for 7 days. Lysozyme degradation was performed for 4 weeks to detect in vitro degradability of the scaffolds and the microspheres. Rabbit chondrocytes were seeded on the scaffolds containing TGF-β(1) microspheres and incubated in vitro for 3 weeks. Histological examination and type II collagen immunohistochemical staining was performed to evaluate the effects of released TGF-β(1) on cell adhesivity, proliferation and synthesis of the extracellular matrix.Results TGF-β(1) was encapsulated into chitosan microspheres and the encapsulation efficiency of TGF-β(1) was high (90.1%). During 4 weeks of incubation in lysozyme solution for in vitro degradation, the mass of both the scaffolds and the microspheres decreased continuously and significant morphological changes was noticed. From the release experiments, it was found that TGF-β(1) could be released from the microspheres in a multiphasic fashion including an initial burst phase, a slow linear release phase and a plateau phase. The release amount of TGF-β(1) was 37.4%, 50.7%, 61.3%, and 63.5% for 1, 3, 5, and 7 days respectively. At 21 days after cultivation, type II collagen immunohistochemical staining was performed. The mean percentage of positive cells for collagen type II in control group (32.7%±10.4%) was significantly lower than that in the controlled TGF-β(1) release group (92.4%±4.8%, P<0.05). Both the proliferation rate and production of collagen type II in the transforming growth factor-β(1) microsphere incorporated scaffolds were significantly higher than those in the scaffolds without microspheres, indicating that the activity of TGF-β(1) was retained during microsphere fabrication and after growth factor release.Conclusion Chitosan microspheres can serve as delivery vehicles for controlled release of TGF-β(1), and the released growth factor can augment chondrocytes proliferation and synthesis of extracellular matrix. Chitosan scaffolds incorporated with chitosan microspheres loaded with TGF-β(1) possess a promising potential to be applied for controlled cytokine delivery and cartilage tissue engineering.     

Expression of transforming growth factor β<Subscript>1</Subscript> in mesenchymal stem cells: Potential utility in molecular tissue engineering for osteochondral repair

Summary The feasibility of using gene therapy to treat full-thickness articular cartilage defects was investigated with respect to the transfection and expression of exogenous transforming growth factor (TGF)-β₁ genes in bone marrow-derived mesenchymal stem cells (MSCs)in vitro. The full-length rat TGF-β₁ cDNA was transfected to MSCs mediated by lipofectamine and then selected with G418, a synthetic neomycin analog. The transient and stable expression of TGF-β₁ by MSCs was detected by using immunohistochemical staining. The lipofectamine-mediated gene therapy efficiently transfected MSCsin vitro with the TGF-β₁ gene causing a marked up-regulation in TGF-β₁ expression as compared with the vector-transfected control groups, and the increased expression persisted for at least 4 weeks after selected with G418. It was suggested that bone marrow-derived MSCs were susceptible toin vitro lipofectamine mediated TGF-β₁ gene transfer and that transgene expression persisted for at least 4 weeks. Having successfully combined the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology, an innovative concept, i.e. molecular tissue engineering, are put forward for the first time. As a new branch of tissue engineering, it represents both a new area and an important trend in research. Using this technique, we have a new powerful tool with which: (1) to modify the functional biology of articular tissue repair along defined pathways of growth and differentiation and (2) to affect a better repair of full-thickness articular cartilage defects that occur as a result of injury and osteoarthritis. GUO Xiaodong, male, born in 1970, Doctor in Charge     

Development and potential of a biomimetic chitosan/type Ⅱ collagen scaffold for cartilage tissue engineering

Background Damaged articular cartilage has very limited capacity for spontaneous healing. Tissue engineering provides a new hope for functional cartilage repair. Creation of an appropriate cell carrier is one of the critical steps for successful tissue engineering. With the supposition that a biomimetic construct might promise to generate better effects, we developed a novel composite scaffold and investigated its potential for cartilage tissue engineering.Methods Chitosan of 88% deacetylation was prepared via a modified base reaction procedure. A freeze-drying process was employed to fabricate a three-dimensional composite scaffold consisting of chitosan and type Ⅱcollagen. The scaffold was treated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. Ultrastructure and tensile strength of the matrix were carried out to assess its physico-chemical properties. After subcutaneous implantation in rabbits, its in vivo biocompatibility and degradability of the scaffold were determined. Its capacity to sustain chondrocyte growth and biosynthesis was evaluated through cell-scaffold co-culture in vitro. Results The fabricated composite matrix was porous and sponge-like with interconnected pores measuring from 100－250 μm in diameter. After cross-linking, the scaffold displayed enhanced tensile strength. Subcutaneous implantation results indicated the composite matrix was biocompatible and biodegradable. In intro cell-scaffold culture showed the scaffold sustained chondrocyte proliferation and differentiation, and maintained the spheric chondrocytic phenotype. As indicated by immunohistochemical staining, the chondrocytes synthesized type Ⅱ collagen. Conclusions Chitosan and type Ⅱ collagen can be well blended and developed into a porous 3-D biomimetic matrix. Results of physico-chemical and biological tests suggest the composite matrix satisfies the constraints specified for a tissue-engineered construct and may be used as a chondrocyte carrier for cartilage tissue engineering.