体外培养成骨细胞对自体骨髓间充质干细胞定向成骨分化的影响

Effects of osteoblast culture in vitro on osteogenetic activity of bone marrow mesenchymal stem cells

目的观察体外培养成骨细胞对自体骨髓间充质干细胞(BMSC)增殖和定向成骨分化的影响,探讨此诱导方法作为骨组织工程种子细胞来源方法的可行性。方法应用密度梯度离心联合贴壁筛选法从兔股骨骨髓中分离、纯化BMSC,取第3代BMSC常规培养设为空白对照组,利用含有钙化诱导剂的DMEM培养基诱导培养第3代BMSC,设为钙化对照组。取第3代成骨细胞,1∶1的比例和第3代BMSC共同培养,设为实验组。碱性磷酸酶(ALP)染色及钙结节染色鉴定诱导结果,ALP定量测定观察共同培养中成骨细胞对BMSC诱导影响。总蛋白定量测定观测诱导成骨细胞的分化活性。结果成骨细胞与BMSC共同培养24h后,在倒置光学显微镜下可见两种细胞形态混杂生长,共同培养5d后,细胞形态趋于一致,多呈长梭形。诱导培养5d后,实验组与钙化对照组ALP染色均为阳性。诱导培养21d,两组钙结节茜素红染色均呈阳性,而BMSC空白对照组并未见钙结节形成。ALP定量测定,实验组于3、5、7、9d均低于钙化对照组,且差异均具有统计学意义。实验组与空白对照组ALP定量测定,在4个时间段差异也均具有统计学意义。总蛋白定量测定显示实验组于第3天,A值高于钙化对照组,差异具有显著统计学意义(P<0.01),而第5天、第7天、第9天时却低于钙化对照组,差异具有显著统计学意义(P<0.01)。实验组于各时间段均高于空白对照组,差异亦具有统计学意义。结论成骨细胞与BMSC共同培养应用于BMSC的成骨诱导,从诱导效率和诱导后成骨细胞的分化活性方面,都能证实该共同培养方法的有效性。但与钙化诱导方法相比,共同培养的诱导效果差于钙化诱导方法。

Objective To observe the effect of the osteoblast on osteoblastic induction of bone marrow mesenchymal stem cells （BMSC） in vitro and investigate the feasibility of the method as the seed cells source of bone tissue engineering. Methods The BMSC were cultured by used density gradient centrifugation and adherence screening from rabbit flank bone. The 3rd generation of BMSC by convention culturing were used as the blank control group. The 3rd generation of BMSC cultivated by the calcifying DMEM medium were used as calcify control group. The co-cultivated the 3rd generation of osteoblasts and the 3rd generation of MSC in the ratio of 1 ： 1 as the experimental group. The induction result cells was assessed by alkaline phosphatase（ALP） staining and calcium node staining. The effect of osteoblast on osteogenetic activity of BMSC was tested by ALP quantitative assay and total protein quantitative assay. Results The morphology of BMSC was observed by the inverted phase contrast microscope after co-culture the osteoblast and BMSC in 24 hours. The ALP staining of the calcifying control group and the experimental group showed positive reaction after 5 days. After cultured 21 days, the calcium node staining of the calcifying control group and the experimental group showed positive reaction. The calcium node was not found in the blank control group. The data value of ALP quantitative assay of the blank control group was lower than that in the calcifying control group in the 3,5,7,9 days and there were significant differences （P 〈 0.01） between them. The significant difference was shown between the experimental group and the blank control group. Total protein quantitative assay showed that there were significant differences in 3rd day between the calcifying control group and the blank control group （P 〈 0.01）. In every stage of cuhuring there was significant difference between the experimental group and the blank control group （P 〈 0.01）. Conclusion The technique ofosteoblasts and BMSC co-culture is efficacy shown by the induction efficiency and the osteogenetic activity, but these effects in calcifying induction technique is superior to that in co-culture induction.