Miao Chun-Lei, Duan Peng, Mu Shao-Chun, Tang Sheng-Jian
Hospital of Plastic Surgery, Weifang Medical College, Weifang 261041, China.
Zhonghua Zheng Xing Wai Ke Za Zhi. 2011 Mar;27(2):113-8.
To investigate the feasibility of chondrogenesis in vitro with bone marrow stromal cells (BMSCs) induced by the co-cultured chondrocytes.
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 x 10(7)/ml. The cartilage cells and BMSCs were also inoculated separately at the same concentration as the positive and negative control. Pure cartilage cells at 20% of the above mentioned concentration (1.0 x 10(7)/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.
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.
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.
探讨共培养软骨细胞诱导骨髓基质细胞（BMSCs）体外软骨形成的可行性。
从猪体内分离并培养BMSCs和软骨细胞。软骨细胞的上清液用作第二代BMSCs的诱导液。7天后，取样进行免疫组织化学和逆转录-聚合酶链反应（RT-PCR），以检测特异性II型软骨胶原、II型胶原和聚集蛋白聚糖mRNA的表达。将培养的BMSCs和软骨细胞按8:2（BMSC:软骨细胞）的比例混合，并以5.0×10⁷/ml的终浓度接种到聚乙醇酸/聚乳酸（PGA/PLA）支架中。软骨细胞和BMSCs也分别以与阳性和阴性对照相同的浓度接种。以上述浓度20%（1.0×10⁷/ml）的纯软骨细胞作为低浓度软骨细胞对照组。8周后收集样本。进行大体观察、湿重、糖胺聚糖（GAGs）测定以及组织学和免疫组织化学检查。
诱导后的BMSCs中II型胶原、II型胶原和聚集蛋白聚糖mRNA的表达呈阳性。在共培养组和阳性对照组中，体外培养8周后形成了纯成熟软骨，支架的大小和形状得以维持。两组新形成的软骨在外观和组织学特性上几乎相同。免疫组织化学结果表明，两组的软骨细胞均大量表达软骨特异性II型胶原。共培养组的平均湿重和GAG含量达到阳性对照组的70%以上。纯BMSC组仅在局部区域形成极少量未成熟软骨组织，且支架明显收缩变形。虽然低浓度软骨细胞组新生成软骨组织的湿重达到阳性对照组的30%，但支架明显收缩变形。仅形成局部和不连续的软骨组织，新形成的软骨量明显少于共培养组和阳性对照组。
软骨细胞可为软骨形成提供微环境，还能有效诱导BMSCs分化为软骨细胞并在体外形成组织工程软骨。
