软骨细胞诱导骨髓基质细胞体内软骨形成

目的探讨软骨细胞在体内非软骨形成部位促进骨髓基质细胞(BMSCs)向软骨分化并形成软骨的可行性。方法猪BMSCs与软骨细胞按一定比例(6∶4或7∶3)混匀,取2.5×107个混合细胞悬浮于0.5ml30%Pluronic溶液后注射到裸鼠皮下(n=6)。相同数量的单纯软骨细胞或BMSCs同样方法注射,分别作为阳性对照及阴性对照,0.75×107个软骨细胞同样注射作为低浓度软骨细胞对照。各组均8周后取材检测。结果混合细胞组及阳性对照组均形成了成熟的软骨。组织学可见成熟软骨陷窝、异染基质及Ⅱ型胶原表达。两组新生软骨糖胺多糖(GAG)含量差异无统计学意义(P>0.05),两混合组平均湿重分别为(320±48)mg和(294±37)mg,均达到阳性对照组70%以上。BMSCs组仅形成了纤维性组织,低浓度软骨细胞组在局部形成了少量软骨,但新生软骨平均湿重低于阳性对照的30%。结论上述结果提示软骨细胞能诱导BMSCs在体内非软骨形成部位向软骨分化并形成软骨组织。     

组织工程化软骨分泌的可溶性因子对骨髓基质干细胞诱导作用的实验研究

目的 探讨组织工程化软骨分泌的可溶性因子是否能够单独诱导骨髓基质干细胞(bone marrow stromai cells,BMSCs)软骨分化.方法 体外培养扩增猪BMSCs、猪关节软骨细胞以及皮肤成纤维细胞,以5.0×107/ml的细胞终浓度分别接种于聚羟基乙酸/聚乳酸(PGA/PLA)支架,应用隔离池进行隔离共培养.以软骨细胞-材料复合物与BMSCs-材料复合物隔离共培养为实验组,以皮肤成纤维细胞-材料复合物与BMSCs-材料复合物隔离共培养为对照组1,以单纯BMSCs-材料复合物为对照组2.各组标本均于体外培养8周后取材,通过大体观察,组织学,以及免疫组织化学,RT-PCR等方法对新生组织进行评价.结果 隔离共培养8周后,实验组软骨细胞材料-复合物诱导的BMSCs-材料复合物形成的组织略有缩小,外观类似软骨组织,组织学检测见软骨陷窝样结构,SafraninO染色可见软骨特异性基质分泌,免疫组化显示有大量Ⅱ型胶原沉积,RT-PCR检测组织表达Ⅱ型胶原、Ⅸ型胶原、COMP、Sox9等软骨特异性基因,提示形成了较成熟软骨样组织;而对照组成纤维细胞材料复合物诱导的BMSCs-材料复合物和未经任何诱导的BMSCs-材料复合物形成的组织淡黄色,明显缩小、变薄、质地较软,组织学检测均未形成软骨陷窝样结构,主要为纤维性成分,各种软骨特异性相关检测均为阴性.结论 软骨细胞分泌的可溶性因子能够单独诱导BMSCs软骨分化,可能是软骨细胞形成的微环境中发挥诱导作用的主要因素.     

组织工程化软骨分泌的可溶性因子对骨髓基质干细胞诱导作用的实验研究

目的 探讨组织工程化软骨分泌的可溶性因子是否能够单独诱导骨髓基质干细胞(bone marrow stromai cells,BMSCs)软骨分化.方法 体外培养扩增猪BMSCs、猪关节软骨细胞以及皮肤成纤维细胞,以5.0×107/ml的细胞终浓度分别接种于聚羟基乙酸/聚乳酸(PGA/PLA)支架,应用隔离池进行隔离共培养.以软骨细胞-材料复合物与BMSCs-材料复合物隔离共培养为实验组,以皮肤成纤维细胞-材料复合物与BMSCs-材料复合物隔离共培养为对照组1,以单纯BMSCs-材料复合物为对照组2.各组标本均于体外培养8周后取材,通过大体观察,组织学,以及免疫组织化学,RT-PCR等方法对新生组织进行评价.结果 隔离共培养8周后,实验组软骨细胞材料-复合物诱导的BMSCs-材料复合物形成的组织略有缩小,外观类似软骨组织,组织学检测见软骨陷窝样结构,SafraninO染色可见软骨特异性基质分泌,免疫组化显示有大量Ⅱ型胶原沉积,RT-PCR检测组织表达Ⅱ型胶原、Ⅸ型胶原、COMP、Sox9等软骨特异性基因,提示形成了较成熟软骨样组织;而对照组成纤维细胞材料复合物诱导的BMSCs-材料复合物和未经任何诱导的BMSCs-材料复合物形成的组织淡黄色,明显缩小、变薄、质地较软,组织学检测均未形成软骨陷窝样结构,主要为纤维性成分,各种软骨特异性相关检测均为阴性.结论 软骨细胞分泌的可溶性因子能够单独诱导BMSCs软骨分化,可能是软骨细胞形成的微环境中发挥诱导作用的主要因素.     

组织工程化软骨分泌的可溶性因子对骨髓基质干细胞诱导作用的实验研究

目的 探讨组织工程化软骨分泌的可溶性因子是否能够单独诱导骨髓基质干细胞(bone marrow stromai cells,BMSCs)软骨分化.方法 体外培养扩增猪BMSCs、猪关节软骨细胞以及皮肤成纤维细胞,以5.0×107/ml的细胞终浓度分别接种于聚羟基乙酸/聚乳酸(PGA/PLA)支架,应用隔离池进行隔离共培养.以软骨细胞-材料复合物与BMSCs-材料复合物隔离共培养为实验组,以皮肤成纤维细胞-材料复合物与BMSCs-材料复合物隔离共培养为对照组1,以单纯BMSCs-材料复合物为对照组2.各组标本均于体外培养8周后取材,通过大体观察,组织学,以及免疫组织化学,RT-PCR等方法对新生组织进行评价.结果 隔离共培养8周后,实验组软骨细胞材料-复合物诱导的BMSCs-材料复合物形成的组织略有缩小,外观类似软骨组织,组织学检测见软骨陷窝样结构,SafraninO染色可见软骨特异性基质分泌,免疫组化显示有大量Ⅱ型胶原沉积,RT-PCR检测组织表达Ⅱ型胶原、Ⅸ型胶原、COMP、Sox9等软骨特异性基因,提示形成了较成熟软骨样组织;而对照组成纤维细胞材料复合物诱导的BMSCs-材料复合物和未经任何诱导的BMSCs-材料复合物形成的组织淡黄色,明显缩小、变薄、质地较软,组织学检测均未形成软骨陷窝样结构,主要为纤维性成分,各种软骨特异性相关检测均为阴性.结论 软骨细胞分泌的可溶性因子能够单独诱导BMSCs软骨分化,可能是软骨细胞形成的微环境中发挥诱导作用的主要因素.     

犬骨髓基质干细胞体外定向分化为软骨细胞

目的 体外诱导犬骨髓基质干细胞(BMSCs)定向分化为软骨细胞，探讨体外诱导成软骨的方法和条件。方法 自犬肋骨取骨髓2～3ml，体外行原代和传代培养扩增，顺序加入碱性成纤维细胞生长因子(bFGF)和转化生长因子β1(TGF-β1)，以培养瓶内较高细胞浓度培养，诱导BMSCs分化为软骨细胞。甲苯胺蓝、阿新蓝染色检测软骨基质的分泌，免疫组织化学染色检测软骨特异性Ⅱ型胶原表达。结果 诱导的软骨样细胞甲苯氨蓝异染性、阿新蓝染色阳性；Ⅱ型胶原免疫组织化学检测阳性。结论 应用bFGF和TGF-β1体外可以诱导犬BMSCs分化为软骨细胞，诱导的软骨细胞可作为软骨组织工程较理想的种子细胞。     

CDMP-1促进成人骨髓间充质干细胞成软骨分化的研究

目的 探讨软骨源性形态发生蛋白1(CDMP-1)诱导成人骨髓间充质干细胞(BMSCs)向软骨细胞分化的可行性及最佳诱导浓度.方法 分离培养BMSCs,免疫荧光法检测BMSCs表面CD34,CD45,CD105表达.采用含0、10、50、100ng/ml CDMP-1的软骨诱导液诱导培养BMSCs 21 d,倒置显微镜观察细胞形态变化;RT-PCR检测不同浓度CDMP-1组细胞Ⅱ型胶原和Aggrccan表达:免疫组化检测Ⅱ型胶原表达;番红O和阿利新蓝染色检测蛋白多糖表达.结果 成人BMSCs呈梭形漩涡状生长,CD44、CD105表达阳性,CD34呈阴性表达.CDMP-1诱导7 d后细胞形态逐渐由长梭形向多边形、多角形转化.不同浓度CDMP-1诱导21 d,Ⅱ型胶原表达量各组间差异均具有统计学意义(P<0.05);Aggrecan的表达CM组和10 ng组之间差异无统计学意义(P>0.05),其他各组间差异均具有统计学意义(P<0.05).免疫组化检测Ⅱ型胶原表达阳性,阿利新蓝和番红O染色均为阳性.结论 含100 ng/mJ CDMP-1软骨诱导液能有效促进成人BMSCs向软骨表型分化.     

三种亚型软骨组织来源干细胞的分离培养及鉴定

目的分离培养猪不同亚型软骨组织(弹性软骨、透明软骨以及纤维软骨)来源干细胞并鉴定,为软骨组织工程提供理想种子细胞。方法利用纤维连接蛋白黏附法分别从猪耳软骨、关节软骨以及椎间盘软骨中分离培养干细胞,并进行传代。倒置相差显微镜下观察细胞形态变化,流式细胞术鉴定细胞表面抗原表达水平(阳性标志物CD29、CD90及阴性标志物CD34、CD45),单克隆形成实验鉴定软骨干细胞单克隆形成能力。三向诱导分化鉴定软骨来源干细胞的成软骨、成骨及成脂多向分化潜能。RT-PCR检测成骨(Ⅰ型胶原、Ⅹ型胶原)、成软骨[蛋白聚糖(Aggrecan)、II型胶原]、成脂[脂联素(Adiponectin)、脂肪酸合成酶(fatty acid synthase,FAS)]相关基因表达,并以猪BMSCs作为对照。结果通过纤维连接蛋白黏附法分别从耳软骨(弹性软骨)、关节软骨(透明软骨)、椎间盘软骨(纤维软骨)分选出一群细胞,细胞高表达干细胞表面阳性标志物CD29、CD90,几乎不表达干细胞表面阴性标志物CD34、CD45。经过体外2周培养,单个细胞均能形成细胞克隆。三向诱导分化显示软骨来源的干细胞具备成软骨、成骨和成脂分化能力。RT-PCR结果显示,成骨诱导后关节和椎间盘来源软骨干细胞的Ⅰ、Ⅹ型胶原基因相对表达量明显高于BMSCs(P0.05),耳软骨来源干细胞与BMSCs比较差异无统计学意义(P0.05);成软骨诱导后,3种亚型软骨组织来源干细胞Aggrecan、Ⅱ型胶原基因相对表达量均高于BMSCs(P0.05);成脂诱导后,3种来源软骨干细胞Adiponectin及FAS基因相对表达量均低于BMSCs,但比较差异无统计学意义(P0.05)。结论不同亚型的猪软骨组织中均存在软骨干细胞,具有干细胞的典型特征。     

基于永生化软骨细胞和骨髓基质干细胞的工程化软骨形成的实验研究

目的 :探讨hTERT转染的永生化软骨细胞和骨髓基质干细胞 (MSCs)裸鼠体内形成软骨的能力。方法 :通过真核表达载体 ,把人端粒酶逆转录酶基因转入兔髁状突软骨细胞 ,筛选并挑选阳性克隆进行扩增培养 ;取兔骨髓 ,密度梯度离心和培养 ,经诱导、扩增后与支架材料 β 磷酸三钙 (β TCP)复合 ,构建细胞 β TCP复合体 ,体外培育 1～ 2d后 ,植入裸鼠体内。通过粘多糖 (GAG)含量和Ⅱ型胶原的表达来检测 3和 6个月复合体软骨形成情况。结果 :两种细胞和 β TCP复合体在裸鼠体内均能形成软骨样组织 ;6个月 ,工程化软骨GAG含量和Ⅱ型胶原的表达均低于正常软骨组织 ,但无显著性差异 (P >0 .0 5 )。结论 :hTERT转染的永生化软骨细胞和骨髓基质干细胞均具有良好的软骨形成能力 ,在软骨组织工程修复软骨缺损方面具有重要的应用前景。     

高截面纵横比容器和微载体技术在胎儿骨髓基质干细胞向软骨细胞转化中的应用

目的利用体外细胞培养方法,进行胎儿骨髓基质干细胞(BMSCs)向软骨细胞的诱导分化,以有效获得表型正常的软骨组织工程功能细胞。方法从胎儿骨髓血中提取BMSCs,体外扩增培养3代,流式细胞仪鉴定胎儿BMSCs的纯度后,1.2×107个细胞与Cytodex-3微载体充分混匀,放入含无血清完全培养基的高截面纵横比容器(HARV,旋转式细胞培养系统的一种)中诱导培养,7、14d取材,用RT-PCR检测细胞内Ⅱ型前胶原mRNA的表达,同时以培养瓶诱导培养作为对照。结果HARV诱导培养组在培养7、14d检测出细胞内Ⅱ型前胶原mRNA的表达,而培养瓶诱导培养组则未检出。结论HARV诱导培养具有使胎儿BMSCs向软骨细胞转化的能力。     

生长分化因子-5诱导小鼠骨髓基质干细胞向软骨分化过程中对缝隙连接蛋白43表达的影响

目的探讨生长分化因子-5(GDF-5)在小鼠骨髓基质干细胞(BMSCs)向软骨分化过程中对缝隙连接蛋白43(Cx43)表达的影响。方法体外培养小鼠BMSCs,贴壁细胞传代,取第3代细胞,加入地塞米松、VitC、胰岛素和GDF-5诱导培养,72 h后免疫细胞化学和阿尔辛蓝染色分别检测软骨细胞特异性Ⅱ型胶原和蛋白多糖的表达；在诱导24、48和72 h后进行如下检测：MTT法测定GDF-5对小鼠BMSCs增殖的影响；RT-PCR、Western blotting和免疫细胞化学法检测GDF-5对Cx43蛋白表达的影响。结果MTT结果显示不同时间GDF-5对小鼠BMSCs的增殖无影响；RT-PCR、Western blotting和免疫细胞化学结果表明诱导后不同时间均有Cx43 mRNA和蛋白的表达；诱导72 h免疫细胞化学显示有Ⅱ型胶原蛋白的表达,阿尔辛蓝染色阳性,有蛋白多糖基质的分泌。结论GDF-5可以通过上调缝隙连接蛋白Cx43的表达来促进小鼠BM- SCs向软骨方向的分化。     

Characterization of proteoglycan production and processing by chondrocytes and BMSCs in tissue engineered constructs

OBJECTIVE: The goal of this study was to characterize the proteoglycan (PG) production and processing by bone marrow stromal cells (BMSCs) within a tissue engineered construct. METHODS: Bovine BMSCs and articular chondrocytes (ACs) were isolated from an immature calf, seeded into agarose gels, and cultured up to 32 days in the presence of TGF-beta1. The localization of various PGs was examined by immunofluorescence and histological staining. The role of proteolytic enzymes in construct development was further investigated by examining the effects of aggrecanase and MMP inhibitors on PG accumulation, aggrecan processing, and construct mechanics. RESULTS: BMSCs developed a matrix rich in sulfated-glycosaminoglycans (sGAG) and full-length aggrecan, but had low levels of versican. The BMSC constructs had less collagen II and aggrecan compared to the AC constructs cultured under identical conditions. AC constructs also had high levels of pericellular collagen VI, while BMSCs had a pericellular matrix containing little collagen VI and greater levels of decorin, biglycan, and fibronectin. Treatment with the aggrecanase inhibitor did not affect sGAG accumulation or the dynamic moduli of the BMSC constructs. The MMP inhibitor slightly but significantly inhibited sGAG accumulation and lowered the dynamic moduli of BMSC constructs. CONCLUSIONS: The results of this preliminary study indicate that long-term culture of BMSCs with TGF-beta1 promotes the development of an aggrecan-rich matrix characteristic of native articular cartilage; however, BMSCs accumulate significantly lower levels of sGAG and assemble distinct pericellular microenvironments compared to ACs. PG turnover does not appear to play a major role in the development of tissue engineered cartilage constructs by BMSCs.     

软骨细胞与骨髓基质细胞共培养体外构建软骨组织的实验研究

目的 探讨利用软骨细胞提供的软骨微环境诱导骨髓基质细胞(BMSC)在体外构建软骨组织的可行性.方法 将分离出的猪骨髓基质细胞和软骨细胞进行体外培养,收集软骨细胞培养上清液,作为骨髓基质细胞诱导液从第2代开始进行诱导分化.7 d后取出标本,免疫组织化学检测软骨特异性Ⅱ型胶原表达,RT-PCR检测Ⅱ型胶原和aggrecan的mRNA表达.体外分离培养的骨髓基质细胞与软骨细胞,扩增后两者以8∶2比例混匀,以5.0×107/ml的终浓度接种于聚羟基乙酸/聚乳酸(PGA/PLA)支架,以相同浓度的单纯软骨细胞和单纯BMSC以及20%上述浓度(1.0×107/ml)的单纯软骨细胞作为对照组.标本于8周后取材,行大体观察、湿重、蛋白多糖(GAGs)含量测定、组织学及免疫组化等相关检测.结果 经诱导后的骨髓基质细胞的Ⅱ型胶原免疫组化检测阳性,RT-PCR检测Ⅱ型胶原和aggrecan mRNA呈阳性表达.混合细胞组及阳性对照组体外培养8周后形成了单一成熟的软骨组织,并保持了支架材料的大小和形状,两组新生软骨在外观及组织学特征上也基本相同,免疫组化结果 表明两组均大量表达软骨特异性细胞外基质Ⅱ型胶原,共培养组的平均湿重和蛋白多糖(GAGs)含量均达到阳性对照组的70%以上.而单纯骨髓基质细胞组仅在局部形成了极少量幼稚的软骨样组织,且材料支架明显皱缩变形.低软骨细胞浓度组虽新生软骨湿重量能达阳性对照组的30%,但材料支架明显皱缩变形,仅在局部形成了不连续的软骨组织,新生软骨量明显少于共培养各组及阳性对照组.结论 软骨细胞能在一定程度上提供软骨形成的微环境,有效地诱导BMSC向软骨细胞分化,并在体外形成组织工程化的软骨组织.

Abstract：

Objective To investigate the feasibility of chondrogenesis in vitro with bone marrow stromal cells (BMSCs) induced by the co-cultured chondrocytes. Methods The BMSCs and chondrocytes were separated from pig and cultured. The supernatant of chondrocytes was used as the inducing solution for BMSCs from the 2nd generation. 7 days later, samples were taken and underwent immunohistochemistry and RT-PCR for detection of the expression of specific type Ⅱ cartilage collagen,type Ⅱ collagen and aggrecan mRNA. The cultured BMSCs and chondrocytes were mixed at a ratio of 8:2(BMSC: cartilage cell) and were inoculated into a polyglycolic acid/polylactic acid (PGA/PLA) scaffold at the final concentration of 5.0 × 107/ml. The cartilage cells and BMSCs were also inoculated seperately at the same concentration as the positive and negative control. Pure cartilage cells at 20% of the abovementioned concentration (1.0 × 107/ml) were used as the low concentration cartilage cell control group. Samples were collected 8 weeks later. General observations, wet weight, glycosaminoglycans (GAGs) determination and histological and immunohistochemistry examinations were performed. Results The expression of type Ⅱ collagen, type Ⅱ collagen and aggrecan mRNA were positive in induced BMSCs.In the co-cultured group and the positive control group, pure mature cartilage was formed after 8 weeks of culture in vitro, and the size and shape of the scaffold were maintained. The newly formed cartilage in the two groups were almost the same in appearance and histological properties. The immunohistochemistry results indicated that the cartilage cells of the two groups all expressed ample cartilage-specific type Ⅱ collagen. The average wet weight and GAG content in the co-cultured group reached more than 70% of those in positive control group. Only an extremely small amount of immature cartilage tissues was formed in local regions in pure BMSC group, and the scaffold was obviously shrunk and deformed. Although the wet weight of newly generated cartilage tissue in the low concentration cartilage cell group reached 30% of that in positive control group, the scaffold was obviously shrunken and deformed. Only regional and discontinuous cartilage tissues were formed, and the amount of newly formed cartilage was obviously less than that in the co-culture group and the positive control group. Conclusions Chondrocytes can provide a micro-environment for the formation of cartilage, and also effectively induce BMSC to differentiate into chondrocytes and form tissue-engineered cartilage in vitro.     

氧预处理的BMSCs联合支架材料修复骨缺损的研究进展

骨髓间充质干细胞在一定的条件下可以分化为具有特定功能的细胞系,作为种子细胞参与骨缺损的修复,显示了良好的应用前景.氧预处理是种子细胞在种植到支架材料前,为改善细胞生存和适应能力所采取的措施,旨在减少或防止种子细胞丢失.本文就经过氧预处理的骨髓间充质干细胞联合支架材料修复骨缺损的研究进展进行综述.     

同种异体软骨细胞与自体骨髓基质干细胞混合共培养构建软骨皮下移植的可行性研究

背景：软骨组织工程的种子细胞问题是目前研究的热点和难点,如何找到一种既能够避免对自体软骨进行取材又能够达到稳定软骨构建目的的方法呢？本研究尝试利用少量同种异体羊软骨细胞作为软骨诱导微环境提供者,与扩增后的羊自体BMSC混合共培养并植入皮下环境,探讨利用同种异体软骨细胞共培养构建软骨皮下移植的可行性。方法：本实验对山羊软骨细胞和BMSC分别进行取材和分离培养扩增,并将以上细胞分为以下四组进行混合并接种在PGA支架材料上：A组：100%自体软骨细胞;B组：30%自体软骨细胞＋70%自体BMSCs;C组：30%同种异体软骨细胞＋70%自体BMSCs;D组：100%同种异体软骨细胞。经过体外构建6周后植入羊皮下进行体内构建12周,对所形成的组织块进行大体观察和组织学染色等评价。结果：自体软骨细胞组和自体软骨细胞混合自体BMSC组皮下移植后可见成熟软骨组织形成,但同种异体软骨细胞参与的两组（包括同种异体软骨细胞混合自体BMSC的实验组和单纯异体软骨细胞组）在皮下环境中都因为较强的免疫反应未能形成软骨组织。结论：同种异体软骨细胞以及PGA支架材料的存在对于组织工程软骨在羊皮下环境的构建有负面影响。     

骨髓间充质干细胞移植促进移植气管上皮再生

目的 探讨经静脉移植的骨髓间充质干细胞(BMSCs)对深低温处理的同种异体气管移植后的存活和上皮再生作用.方法 用深低温冻储2周和6周的大鼠气管进行同种异体气管移植后,将PKH-26标记后的3～5代BMSCs经鼠尾静脉移植入受体内,通过观察局部PKH-26荧光检测和供体气管的组织学、上皮爬行和再生情况,评价BMSC对移植气管的新生上皮再生作用.结果 骨髓间充质干细胞移植并深低温冻储后的移植气管结构完整,组织学检查管腔内为假复层纤毛柱状上皮所覆盖,软骨无变性坏死;术后均能长期存活.PKH-26;标记的BMSCs在受损气管组织定植后呈现红色荧光.结论 BMSCs可迁移并修复受损组织,并通过促进移植气管上皮再生,从而促进气管的损伤恢复.

Abstract：

Objective To investigate the role of BMSCg on enhancing the implant survival and bacheal epithelium regeneration. Methods After transplanted with cryopreserved 2 weeks and 6 weeks allocraft, PHK-26 labeled 3-5 passage BMSCs were injected into the recipient rats via tail vein. Rats in the control groups were injected with the same amount of PBS.We observed the histology of the transplanted trachea including epithelium growth and regeneration, and the PKH-26 fluorescence levels at the para-anastomotic trachea to evaluate the role of BMSC transplantation on the epithelium regeneration. Results Rats from BMSCs injection group survived a long period. Histological observation showed that the tracheal lumen was covered by psudo-striated ciliated columnar epithelium. The cartilage structure was intact. There are no signs of denaturation and necrosis. In the PBS injection group, epithelium regeneration is better in PBS-6-week group than PBS-2-week group. The longest survival time in PBS-6-week group was 32 days, whereas it was 10 days in PBS-2-week group. In BMSCs injection group, rats in BMSC-6-week groups survived longer than 8-week group(12 rats were terminated at 1 week, 4 weeks and 8weeks as planned). There was one rat who survived and were terminated at the designated 8 weeks time point (there were 8regenerated epithelium was similar in the two BMSC transplanted groups. PKH-26 labeled BMSCs migrated to the implant site and showed red fluorescence, with most red fluorescence shown at the anastomotic part. Conclusion BMSCs can migrate to the impaired tissue to repair it. BMSCs may exert their reparation function via enhancing epithelium regeneration.     

大鼠组织提取液对体外培养骨髓基质细胞的影响

目的研究大鼠脑组织成份和其它组织提取液对体外培养骨髓基质细胞(bonemarrowstromalcells,BMSC)的影响。方法提取大鼠全脑、灰质、白质、垂体、肌肉、肝脏等组织液,进行原代骨髓基质细胞培养,观察并测定其ALP染色阳性率。以空白培养液作对照,观察大鼠各组织提取液对骨髓基质细胞增殖,分化,矿化的作用。结果(1)各组织提取液对原代骨髓基质细胞培养后,其ALP染色阳性率以灰质和全脑组最高,其次是白质和肌肉组。(2)除肝脏组外其余各组织提取液对BMSC均有不同程度的增殖刺激作用,以灰质最强,其它依次为肌肉、白质、全脑与血清组,肝脏组则显示对BMSC增殖有抑制作用。(3)肝脏组对BMSC具有分化抑制作用,其它各组均显示分化刺激作用,增加程度依次为灰质、全脑、白质、肌肉与血清。(4)灰质组的矿化结节形成率最高,其它依次为全脑、白质、肌肉与血清。结论大鼠脑组织中的灰质提取液能显著刺激原代骨髓基质细胞增殖,且向成骨细胞诱导分化,并且在二代以后的BMSC培养中,表现出最强的刺激增殖、向成骨细胞分化和体外矿化的能力。     

以海藻酸三维支架构建骨髓间充质干细胞源性生物人工肝组织的实验研究

目的 以骨髓间质干细胞(bone mesenchymal stem cell,BMSC)为种子细胞,以海藻酸三维支架为载体,研究两者的相容性及其构建生物人工肝组织的可能性.方法 将大鼠BMSC接种于海藻酸三维支架上,采用倒置相差显微镜、扫描电镜分别检测BMSC的黏附、生长与增殖情况,评价两者的相容性;同时以表皮生长因子(EGF)、肝细胞生长因子(HGF)、成纤维生长因子4(FGF-4)等细胞因子混合液诱导BMSC向肝细胞分化,检测培养液内尿素氮和白蛋白含量,RT-PCR检测培养细胞ALB和AFP基因的表达,糖原染色及免疫荧光法检测细胞糖原合成功能和CK-18的表达. 结果大鼠BMSC在海藻酸三维支架上能正常地黏附、生长和增殖;于海藻酸三维支架内加入诱导液作用于BMSC后,培养液内尿素和ALB的含量逐渐上升,RT-PCR检测到培养细胞有AFP和ALB基因的表达,同时被诱导的细胞具有糖原储存的功能并表达CK-18.结论 海藻酸三维支架与大鼠BMSC具有良好的相容性;以EGF、HGF、FGF-4等细胞因子混合液为诱导因子,能在海藻酸三维支架上成功诱导BMSC向肝细胞分化;海藻酸三维支架和BMSC可望成为肝脏组织工程理想的种子细胞和细胞载体.     

Tissue engineering of tendons and ligaments by human bone marrow stromal cells in a liquid fibrin matrix in immunodeficient rats: Results of a histologic study

Introduction The original complex structure and mechanical properties are not fully restored after ligament and tendon injuries. Due to their high proliferation rate and differentiation potential, Bone Marrow Stromal Cells (BMSC) are considered to be an ideal cell source for tissue engineering to optimize the healing process. Ideal matrices for tissue engineering of ligaments and tendons should allow for homogenous cell seeding and offer sufficient stability. Material and methods A mixture of human BMSC and liquid fibrin glue was injected into a standardized full-thickness window defect of the patellar tendon of immunodeficient rats (BMSC group). The histology of the tissue was analysed 10 and 20 days postoperatively and compared to four control groups. These groups consisted of a cohort with a mixture of human fibroblasts and fibrin glue, fibrin glue without cells, a defect group without treatment, and a group with uninjured patellar tendon tissue. Results Tendon defects in the BMSC group revealed dense collagen fibres and spindle-shaped cells, which were mainly orientated along the loading axis. Histologic sections of the control groups, especially of untreated defects and of defects filled with fibrin glue only, showed irregular patterns of cell distribution, irregular formed cell nucleoli and less tissue maturation. Compared to healthy tendon tissue, higher numbers of cells and less intense matrix staining was observed in the BMSC group. No ectopic bone or cartilage formation was observed in any specimen. Conclusions Injection of human BMSC in a fibrin glue matrix appears to lead to more mature tissue formation with more regular patterns of cell distribution. Advantages of this “in-vivo” tissue engineering approach are a homogenous cell-matrix mixture in a well-known and approved biological matrix, and simple, minimally-invasive application by injection.     

Fetal tracheal augmentation with cartilage engineered from bone marrow-derived mesenchymal progenitor cells

Background/Purpose: The authors have described previously the use of engineered fetal cartilage in a large animal model of fetal tracheal repair. This study was aimed at comparing cartilage engineered from bone marrow-derived stromal cells (BMSC) to native and engineered cartilage, in this model.Methods: Ovine BMSC were expanded in vitro, seeded onto biodegradable scaffolds, and maintained in transforming growth factor beta 1 (TGF-β1)-supplemented medium for 3 months (group I). Identical scaffolds were seeded with fetal chondrocytes (group II). All constructs were analyzed in vitro, implanted into fetal tracheas, and harvested after birth for further analysis.Results: There were no differences in survival between the groups. All BMSC-based constructs exhibited chondrogenic differentiation. Matrix analyses in vitro showed that both groups had similar levels of glycosaminoglycans (GAG) and type II collagen (C-II), but lower levels of elastin when compared with native fetal cartilage. Yet, compared with group II, group I had higher levels of GAG, equal levels of C-II, and lower levels of elastin. However, remodeling resulted in no differences between the 2 groups in any of these variables in vivo.Conclusions: The bone marrow may be a useful cell source for cartilage engineering aimed at the surgical repair of severe congenital tracheal anomalies, such as tracheal atresia and agenesis, in utero.     

Type IV collagen induces podocytic features in bone marrow stromal stem cells in vitro

Bone marrow-derived stromal stem cells (BMSC) can differentiate along a variety of mesenchymal lines, including mesangial cells. For determining whether BMSC can be induced to differentiate along podocytic lines in vitro, canine BMSC were cultured on plastic, type I collagen, and NC1 hexamers of type IV collagen from normal and Alport canine glomerular basement membrane. Results were compared with a mouse podocyte cell line. In the case of the podocyte line, differentiation occurred on all three matrices as indicated by the expression of synaptopodin and CD2-associated protein (CD2AP) and organization of myosin heavy chain IIA into a linear pattern. BMSC proliferated equally well on all matrices, but cells that were grown on type IV collagen NC1 hexamers became larger and stellate. Evidence for podocytic differentiation occurred on all three collagen matrices as indicated by the redistribution of myosin IIA to a linear pattern and expression of synaptopodin, CD2AP, and alpha-actinin. A punctate distribution of CD2AP was seen only in cells that were grown on normal and Alport glomerular basement membrane NC1 hexamers. Differentiated podocytes expressed the alpha1, alpha2, and alpha5 chains of type IV collagen but at higher levels in cells that were grown on NC1 hexamers. Similar results were obtained in BMSC for the alpha1 and alpha2 chains only. The alpha3, alpha4, and alpha6 chains were never detected in the podocyte line or BMSC. These results indicate that BMSC undergo a degree of podocytic differentiation in vitro and greater when grown on type IV collagen NC1 hexamers than type I collagen. Alport and normal NC1 hexamers seem equally permissive to BMSC growth and differentiation, suggesting that these processes are not influenced specifically by the alpha3/alpha4/alpha5 network. BMSC may be useful in the development of stem cell-based reconstitution of glomeruli that are damaged by disease and for gene therapy of genetic glomerular diseases such as Alport syndrome.