Approach to get mineralized tissue using pulp cells

本研究的目的是通过简单的技术从人牙髓组织中获得多能细胞。为了得到MILs，我们根据细胞表面抗原的指示分离细胞。利用荧光激活细胞分选仪(FACS)和磁性细胞分选系统(MACS)，我们做了以下实验：(1)无菌采集人牙髓组织，允许其生长，继续生长，然后用FACS进行分析。我们使用CD73、74、106、146、166、271和Stro-1抗原。结果表明，除CD166外，其余6种抗原的表达水平均较低，但20%的CD166染色细胞呈阳性表达。(2)将除CD73外的6种抗原与上述抗原偶联，并应用于MACS系统。我们继续培养抗原阳性细胞，并将其分化为成骨细胞。结果显示，CD74和106阳性细胞的茜素红染色强度低于CD74和106阴性细胞。CD146和Stro-1阳性细胞与CD146和Stro-1阴性细胞显示相同的茜素红染色水平。CD166和271阳性细胞较CD166和271阴性细胞有明显的茜素红染色。CD166和271阳性细胞显示出较高的碱性磷酸酶活性。CD166和271阳性细胞的茜素红染色明显高于未筛选的牙髓细胞，尤其是CD166阳性细胞，说明MACS系统用于CD166是一种从人牙髓中筛选高容量成骨细胞的有效方法。

The purpose of this study was to get pluripotent cells from human pulp tissues by simple technique. To get the mils, we separated cells by the indication for cell surface antigens. Using Fluorescence Activated Cell Sorter (FACS) and Magnetic Cell Sorting (MACS) systems, we have done the following experiments(1) We collected human pulp tissues aseptically, allow for them by outgrowth, continue to growth, then analysis them by FACS. We used CD73, 74, 106, 146, 166, 271, and STRO-1 antigen. As the results, the expression level of six antigen except CD166 were quite low, however positive expression were detected by 20% of CD166 stained cells.(2) We conjugated the cells to the above antigen except CD73, and apply them to MACS system. We continued to growth the antigen positive cells, and differentiated them to osteoblasts. As the results, Alizarin red staining levels were lower in CD74 and 106 positive cells than in CD74 and 106 negative cells. CD146 and STRO-1 positive cells show same Alizarin red staining levels compared with CD146 and STRO-1 negative cells. CD166 and 271 positive cells show obvious strong Alizarin red staining compared with CD166 and 271 negative cells. These CD166 and 271 positive cells show high activity of Alkaline phosphates. Alizarin red staining of CD166 and 271 positive cells were obviously higher than non selected pulp cells, especially in CD166 positive cells.Taken together from these results show that MACS system utilized for CD166 is a useful method to sort out high capacity of osteogenic cells from human pulp.