Establishment of a autologous cell transplantion method using mesenchymal stem cells for perio-dontal tissue and alveolus bone regeneration.

1.人骨髓细胞的分离和培养：骨髓细胞从志愿者的髂骨骨髓中分离。将人骨髓细胞铺板并在含有10%FBS的DMEM中培养。2.结缔组织生长因子（CTGF/CCN 2）促进hMSC在羟基磷灰石支架上的贴壁、迁移和存活将人骨髓细胞贴壁于多孔HA块上培养一周。将多孔HA/细胞复合物植入裸鼠皮下，分别加入CTGF（1 μ g）和蒸馏水（对照），4周后取植入物进行扫描电镜观察。扫描电镜观察显示，应用CTGF后，hBMSC样细胞在多孔HA支架内迁移并存活，而未应用CTGF时，支架内无活细胞。3. hMSC在钛片上的初始附着和增殖增强通过将钛片浸泡在1wt%的多聚磷酸溶液中24 h，实现多聚磷酸在钛片表面的吸附。钛片吸附多聚磷酸可促进hMSC的贴壁和增殖。4.成骨细胞基因表达对钛的影响将成骨细胞培养在钛片上，通过cDNA消减杂交技术寻找钛特异性基因。结果表明，钛表面对SOD-1、核糖体蛋白L19基因表达有明显抑制作用。

1.Isolation human bone marrow cells and cell culture.Bone marrow cells were isolated from human iliac bone marrow of volunteer. Human bone marrow cells were plated and cultured in DMEM containing 10% FBS. Culture medium was changed every 3 days, and their multipotent (osteoblastic and adipogenic) differentation abilities were confirmed2.Connective tissue growth factor (CTGF/CCN2) enhanced hMSC attachment, migration and survival in a hydroxyapatite scaffoldHuman bone marrow cells were incubated to attach onto porous HA blocks for a week. The porous HA/cells hybrids were implanted subcutaneously in nude mice (4 week-old) with CTGF (1ug) or distilled water (control).The implants were harvested after 4 weeks for SEM observation. SEM observation supported that hBMSC-like cells migrated and survived inside of the porous HA scaffold with CTGF application, while without CTGF, no viable cells were observed inside of the scaffold.3.hMSC initial attachment and proliferation enhanced on titaniumAdsorption of poly phosphoric acid to Ti disk surface was achieved by immersing Ti disk poly phosphoric acid solution (1 wt%) for 24 hours. Adsorption of polyphosphoric acid onto Ti disks enhanced attachment and proliferation of hMSC.4.Effect of gene expression of osteoblast on titaniumOsteoblast was cultured on titanium dish and searched a titanium specific gene by cDNA subtractive hybridization. It became clear on titanium that sod-1, gene expression of ribosomal protein L19 were restrained significantly.