The experimental study for the repair of the osteochondral defect by Mesenchymal stem cells using cell sorter

近十年来，人们对间充质干细胞（mesenchymal stem cells, MSCs）的治疗进行了大量研究。大多数临床报告，特别是来自皮肤科医生的报告，都将骨髓贴壁细胞（BMACs）描述为MSCs。BMACs一般采用Dexter’s法或Percoll梯度法分离，用人血清、胎牛血清和细胞因子培养，增加干细胞数量。随后，它们被移植到患者的骨骼、关节软骨或肌腱组织中。在过去的10年里，有报道称很少有msc可以从骨髓细胞中获得；例如，仅从10^6-10^7个骨髓细胞中获得4个间充质干细胞。此外，最近也有报道BMAC移植不成功的病例。这些病例的失败归因于培养和取样的方法或缺乏趋化因素，但是否移植真正的MSCs的问题很少受到关注。关于BMAC培养中MSCs的数量是否增加，以及BMAC的异质性尚未通过检测细胞表面标记物来分析。因此，临床医生之前可能移植了比实际干细胞更多的成纤维细胞或其他细胞。体外培养骨髓细胞（BMACs）已广泛应用于骨和软骨再生治疗。本实验采用荧光活化细胞分选仪（FACS）直接根据细胞表面形成模式从BMACs中分离间充质干细胞（MSC）。该间充质干细胞群体具有向成骨细胞、软骨细胞和脂肪细胞分化的高潜力，而BMACs（非间充质干细胞群体）中的残余细胞仅分化为成骨细胞和脂肪细胞两种谱系。一般来说，当细胞不通过颗粒培养时，不会发生均匀的软骨分化。纯化的间充质干细胞可通过单层培养皿培养产生软骨样基质。在培养的BMACs中，这些多电位细胞的比例小于5%。MSCs在与漂浮细胞分离后3-4周内增殖达到17倍，此后不再增加。基于我们的研究结果，当需要对软骨进行有效的组织修复时，我们必须考虑使用FACS对分离的MSCs进行有效利用的方法。少

Over the past decade, a large number of studies on treatment with mesenchymal stem cells(MSCs) have been conducted. Most clinical reports, especially those from dermatologists, have described bone marrow-adherent cells (BMACs) as MSCs. BMACs are generally separated using Dexter's method or the Percoll gradient method, and are cultured with human serum, fetal bovine serum and cytokines to increase the number of stem cells. Subsequently, they are transplanted to patients' bones, articular cartilage or tendious tissue. During the last 10 years it has been reported that few MSCs can be harvested from bone marrow cells ; for example, only four MSCs were obtained from 10^6-10^7 bone marrow cells. Moreover, unsuccessful cases of BMAC transplantation have been reported recently. The failure in these cases was attributed to the methods of culture and sampling or a lack of chemotactic factors, but the issue of whether real MSCs were transplanted was given little attention. There have been no stu … More dies of whether the number of MSCs increases in BMAC cultures and the heterogeneity of BMACs has not yet been analyzed by examining cell-surface markers. Therefore, clinicians might have previously transplanted many more fibroblasts or other cells than actual stem cells. Cultured bore marrow cells (BMACs) have been commonly used in bone and cartilage regeneration therapy. Here we isolated mesenchymal stem cells (MSC) from BMACs by fluorescence activated cell sorter (FACS) directly according to cell surface maker patterns. This MSC population had a high potential to differentiate into osteoblasts, chondrocytes and lipocytes, while the residual cells in BMACs (non-MSC population) differentiate into only two lineages, osteoblasts and lipocytes. Generally, homogeneous chondrogenic defferentiation never happens when cells are not cultured through pellet cultures. However, purified MSCs could produce cartilage-like matrix through monolayer dish culture. The percentage of these multipotential cells was less than 5% in cultured BMACs. MSCs proliferated up to 17-fold in 3-4 weeks after separation from floating cells and did not increase thereafter. Based on our results, when effective tissue repair of the cartilage is required, we must consider the way on their effective use of isolated MSCs using FACS. Less