异体软骨细胞诱导骨髓基质细胞成软骨分化的量效关系

Dose-effect relationship between BMSCs and xenogenic chondrocytes for chondrogenesis

目的 探讨利用异体软骨细胞作为诱导因素，与骨髓基质细胞（BMSCs）共培养体外构建软骨复合物的可行性，以及两种细胞混合比例与构建软骨质量的量效关系。方法体外分别培养扩增BMSCs与异体软骨细胞，软骨细胞与BMSCs的混合比例分别为1：9（A组）、2：8（B组）、3：7（c组）、100％软骨细胞（D组）、100％BMSCs（E组），以5．0×10＾7／ml的细胞终浓度接种于聚羟基乙酸（PGA）材料支架上培养，各组标本均于体外培养6周后取材，通过大体观察、糖胺聚糖（GAG）含量测定、组织学以及免疫组织化学等方法检测组织工程化软骨形成的情况。结果各组细胞均与材料黏附良好，Ｃ、D两组标本基本保持原有的大小和形状，外观洁白、光滑类似软骨组织；组织学显示两组均有连续的软骨陷窝样结构形成，免疫组织化学显示有大量Ⅱ型胶原沉积。定量结果显示，C组的GAG含量达到D组的70％以上。其他各组培养物体积明显缩小，组织学及免疫组织化学显示软骨样组织形成不佳。结论异体软骨细胞与BMSC共培养可以构建出较好的软骨组织，表明软骨细胞对BMSCs发挥诱导作用，但是软骨细胞必须达到一定的数量要求。

Abstract： Objective To investigate the feasibility of constructing cartilages with co-culture of bone marrow derived stromal cells （BMSCs） and xenogenic chondrocytes in vitro, and the dose-effect relationship between quality of constructed cartilage and hybrid scale of two-type cells. Methods BMSCs and xenogenic chondrocytes were cultured and amplified in vitro, and then the two-type cells were mixed at different hybrid scale and then divided into five groups： Group A （1：9）, Group B （2： 8）, Group C （3： 7）, Group D （100% xeno- genic chondroeytes） and Group E （100% BMSCs）. The 5.0 x 10^7/ml cells as the final concentration were in- oculated on the PGA scaffolds, were culturing in vitro. The specimens in all groups were harvested at 6 weeks after culture, and gross observation, determination of GAG contents and immunohistochemical stain were per- formed to assay the engineered cartilages. Results The cells adhered PGA fibers well, and the constructs con- tained the original volume and shape in Group C and D, which showed white appearance and resemble carti- lage. Histological results showed continuous lacuna structure, immunohistochemieal stain revealed abundant deposition of type Ⅱ collagen. Determination of GAG content showed the constructs in Group C were about 70% of those in Group D, while, the constructs in other groups were smaller, histological and immunohisto- chemical stain revealed cartilage structure were not well. Conclusion Cartilage tissue can be well constructed by the co-culture of xenogenic chondrocytes and BMSCs in vitro, while, the xenogenic chondrocytes must meet the amount demand.