Ⅰ型胶原修饰的多孔材料聚乙醇酸-乳酸共聚物对兔骨髓间充质干细胞粘附和增殖及成骨细胞基因表达的影响

Effects of collagen Ⅰ coating on the porous poly-lactide-co-glycolid on adhesion,proliferation,and differentiation of mesenchymal stem cells

目的 探讨Ⅰ型胶原修饰多孔高分子材料聚乙醇酸 乳酸共聚物 (PLGA)对骨髓间充质干细胞 (MSC)粘附、增殖的影响及成骨细胞基因表达情况。方法 以密度梯度离心法获得成年兔骨髓间充质干细胞 ,在含 10 %小牛血清的RPMI 16 4 0为培养基条件下 ,以一定初始浓度分别接种于大小约为 0 3cm× 1 2cm× 2 0cm立体材料PLGA。其中 ,预先包被Ⅰ型胶原的PLGA作为实验组 ,未经Ⅰ型胶原修饰作为阴性对照组。分别于接种后 2、4、6和 8h收获细胞 ,通过检测 [3 H] 胸腺嘧啶核苷 ([3 H] TdR)的掺入率 ,反映细胞的粘附情况 ;检测细胞接种后 7、14、2 1d的 [3 H] TdR掺入率 ,了解细胞的增殖速率。应用RT PCR法检测细胞骨钙素 (OCN)、碱性磷酸酶 (ALP)、骨桥蛋白 (OPN)mRNA的表达 ,观察骨髓基质细胞的成骨细胞分化情况。并通过电镜 ,观察细胞在材料上的形态和贴附情况。结果 Ⅰ型胶原能明显促进MSC在高分子材料上的粘附 ,6h和 8h时间点与对照组相比 ,差异均有显著意义 (6h ,2 14 4cpm± 14 1cpmvs 1797cpm± 118cpm ,P =0 0 17;8h ,2 311cpm±113cpmvs 1891cpm± 10 3cpm ,P =0 0 10均P <0 0 5 )。Ⅰ型胶原也显著能促进MSC增殖 ,细胞培养第 7d ,实验组的cpm值为 10 2 1± 15 9,对照组 4 5 1± 6 7(t=- 7 330 ,P =0

Objective To investigate the effects of collagen Ⅰ on the adhesion, proliferation, and differentiation of MSCs on PLGA. Methods Collagen Ⅰ was added onto the surface of pores in pieces of 3-D porous poly-lactide-co-glycolid (PLGA) . Bone marrow-derived mesenchymal stem cells (MSCs) were obtained from New Zealand rabbits and were cultured for 3 generations,inoculated into the pores of PLGA pieces with the volume of 0.3 cm×1.2 cm ×2.0 cm,and then cultured in solution with -thymidine deoxyribose (TdR). PLGA pieces not coated by collagen Ⅰ were used as controls. The incorporation rate of -TdR was detected 2, 4, 6, and 8 hours, and 7, 14, and 21 days after culture, shown in count per minute (CPM) value, to determine the adhesion and proliferation of the MSCs. RT-POCR was used to examine the expressions of mRNA of the osteoblast markers: osteocalcin (OCN), alkaline phosphatase (ALP), and oesteopontin (OPN). Scanning electron microscopy (SEM) was used to observe the morphology of MSCs.Results The CPM value since 6 hours after culture between the experimental groupand control group began to be significantly different (both P<0.05) The CPM values 7, 14, and 21 days after culture between the experimental group and control groups (P<0.05 or P<0.01). OCN, ALP, and OPN mRNA were expressed in MSCs of the experimental group and only ALP mRNA was weakly expressed in the control group. SEM showed the distribution of spindle and polygonal cells in the pores of the 3-D PLGA pieces and distribution of cylindrical or round cells in the control group.Conclusion Collagen Ⅰ is effective in promoting the adhesion, proliferation, and differentiation of MSCs on PLGA.