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细胞培养。从志愿者髂骨髓中分离骨髓细胞。人骨髓细胞在含10%胎牛血清的DMEM中培养。每3天更换一次培养基，证实其多能性（成骨细胞和成脂细胞）分化能力2。结缔组织生长因子（CTGF/CCN2）增强hMSC在羟基磷灰石支架中的附着、迁移和存活人骨髓细胞在多孔HA块上孵育一周。用CTGF （1ug）或蒸馏水（对照）皮下植入裸鼠（4周龄）多孔HA/细胞杂交体。4周后收获种植体进行扫描电镜观察。2 . SEM观察支持hbmsc样细胞在CTGF作用下在多孔HA支架内迁移存活，而不加CTGF作用时，支架内未观察到活细胞。钛盘聚磷酸溶液（1 wt%）浸泡24小时，实现了hMSC在钛盘表面的初始附着和增殖。钛盘吸附多磷酸增强了hmsc的附着和增殖。成骨细胞基因表达对钛的影响在钛培养皿上培养成骨细胞，通过cDNA减法杂交寻找钛特异性基因。在钛上发现，核糖体蛋白L19基因sod-1的表达明显受到抑制。

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.