关节腔内骨髓间充质干细胞与同种异体骨的共培养

Bone marrow mesenchymal stem cells co-cultured with allogenic bone in the articular cavity

背景:构建组织工程软骨的时候,同种异体骨与骨髓间充质干细胞是常用的支架材料和种子细胞,以往大多采用体外培养的方式。膝关节内游离体可以长期存在于关节腔中,并维持一定的软骨组织学特性。因此,关节腔可能为软骨细胞的生长和发育提供良好的环境条件。目的:探讨在关节腔内骨髓间充质干细胞与同种异体骨复合培养的效果。方法:纳入5只新西兰新生白兔进行骨髓间充质干细胞分离与培养,利用1只成年新西兰白兔制备同种异体骨,进行骨髓间充质干细胞与同种异体骨复合。实验分组:腔内培养组将骨髓间充质干细胞与同种异体骨复合物培养于动物关节腔内,正常对照组设为同腔内正常软骨,体外培养组实施常规体外培养。培养4,8,12周进行组织学苏木精-伊红染色观察及Ⅱ型胶原免疫组织化学染色观察。结果与结论:1培养12周进行苏木精-伊红染色和观察,正常对照组细胞质和软骨基质出现红染,细胞核出现蓝染,软骨细胞按照一定的方向呈紧密状有序排列。腔内培养组细胞质和软骨基质出现红染,细胞核出现蓝染,支架材料基本吸收。软骨细胞长入支架之中,细胞按照一定应力方向排列,且形态变小。体外培养组软骨细胞出现大量增殖,但呈无序状排列;2经免疫组织化学染色和观察,计数可得,随着培养时间的推移,腔内培养组的A值呈现出不断上升的情况,体外培养组和正常对照组均未出现明显的改变。且培养4,8,12周,正常对照组和腔内培养组的A值均显著高于体外培养组(P<0.05)。培养4,8周,腔内培养组的A值均显著低于正常对照组(P<0.05)。但培养12周,腔内培养组与正常对照组A值差异无显著性意义(P>0.05);3实验结果表明,在体外和关节腔内环境下,均可以利用同种异体骨与骨髓间充质干细胞复合培养获得组织工程软骨,且关节腔内培养可以获得更好的培养效果。

BACKGROUND： Bone marrow mesenchymal stem cells and allogenic bones are commonly used as seed cells and scaffolds, respectively, for constructing tissue-engineered cartilage through in vitro co-culture. The loose body of the knee joint can survive in the articular cavity for a long time, and maintain certain characteristics of cartilage tissues. Therefore, the articular cavity can provide a good environment for the growth and development of chondrocytes. OBJECTIVE： To investigate the effect of bone marrow mesenchymal stem cells co-cultured with allogenic bone in the articular cavity. METHODS： Bone marrow mesenchymal stem cells were isolated from five newborn New Zealand white rabbits. One adult New Zealand rabbit was enrolled to prepare allogenic bone for co-culture with bone marrow mesenchymal stem cells. Afterwards, bone marrow mesenchymal stem cells and allogenic bone composites were cultured in the articular cavity（intracavitary culture group） or in vitro as in vitro culture group, respectively; the normal cartilage tissues grew in the articular cavity as control group. Cells were observed by hematoxylin-eosin staining and type II collagen immunohistochemistry staining at 4, 8 and12 weeks of culture. RESULTS AND CONCLUSION： At 12 weeks culture, hematoxylin-eosin staining showed： in the control group, chondrocytes arranged tightly and directionally with red stained cytoplasm and cartilage matrix as well as blue nuclei; in the intracavitary culture group, the scaffold was mostly absorbed and chondrocytes grew into the scaffold in a certain direction with smaller shape, while cytoplasm and cartilage matrix were red stained, blue nuclei appeared; in the in vitro culture group, abundant chondrocytes proliferated in a disordered arrangement. Immunohistochemistry staining showed： the absorbance（A） values in the intracavitary culture group showed a continuous increase, but no obvious change was in the other two groups. Moreover, at 4, 8 and 12 weeks of culture, A values in the control group and intracavitary culture group were significantly higher than that in the in vitro culture group（P〈0.05）; at 4 and 8 weeks, A value in the intracavitary culture group was significantly lower than that in the control group（P〈0.05）, but at 12 weeks, there was no significant difference in A value between the two groups（P〉0.05）. These results suggest that the tissue-engineered cartilage can be constructed by bone marrow mesenchymal stem cells co-cultured with allogenic bone under in vitro and in vivo environment, especially in the articular cavity.