CAJ | 학술논문

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.
목적 탐토이용연골세포제공적연골미배경유도골수기질세포(BMSC)재체외구건연골조직적가행성.방법 장분리출적저골수기질세포화연골세포진행체외배양,수집연골세포배양상청액,작위골수기질세포유도액종제2대개시진행유도분화.7 d후취출표본,면역조직화학검측연골특이성Ⅱ형효원표체,RT-PCR검측Ⅱ형효원화aggrecan적mRNA표체.체외분리배양적골수기질세포여연골세포,확증후량자이8∶2비례혼균,이5.0×107/ml적종농도접충우취간기을산/취유산(PGA/PLA)지가,이상동농도적단순연골세포화단순BMSC이급20%상술농도(1.0×107/ml)적단순연골세포작위대조조.표본우8주후취재,행대체관찰、습중、단백다당(GAGs)함량측정、조직학급면역조화등상관검측.결과 경유도후적골수기질세포적Ⅱ형효원면역조화검측양성,RT-PCR검측Ⅱ형효원화aggrecan mRNA정양성표체.혼합세포조급양성대조조체외배양8주후형성료단일성숙적연골조직,병보지료지가재료적대소화형상,량조신생연골재외관급조직학특정상야기본상동,면역조화결과 표명량조균대량표체연골특이성세포외기질Ⅱ형효원,공배양조적평균습중화단백다당(GAGs)함량균체도양성대조조적70%이상.이단순골수기질세포조부재국부형성료겁소량유치적연골양조직,차재료지가명현추축변형.저연골세포농도조수신생연골습중량능체양성대조조적30%,단재료지가명현추축변형,부재국부형성료불련속적연골조직,신생연골량명현소우공배양각조급양성대조조.결론 연골세포능재일정정도상제공연골형성적미배경,유효지유도BMSC향연골세포분화,병재체외형성조직공정화적연골조직.
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.