BACKGROUND: In the tooth regeneration and dental tissue engineering study, finding suitable stem cells are the central issues.OBJECTIVE: To isolate and culture child exfoliated deciduous tooth pulp stromal cells, and compare difference in dentin sialophosphoproteln expression prior to and following mineralization.DESIGN, TIME AND SETTING: The cell observational experiment was performed at the Central Laboratory of Daqing Oil Field General Hospital from December 2006 to December 2007.PARTICIPANTS: Dental pulp were obtained from exfoliated deciduous molar tooth of eight children aged from eight to ten, four males, four females. The possibility of infectious diseases, endocrine disease, dental caries and periodontal disease was excluded.METHODS: Deciduous teeth pulp stromal cells were obtained by tissue block adhesion method, and then cultured in DMEM with 10% fetal bovine serum for 14 days. Following digestion and passage, cells were incubated in DMEM/F12 supplemented with 50 pmol/L laevo-ascorbic acid, 10 nmol/L vitamin D3. 10 mmol/L Na β-glycerophosphate and 10-8 mol/L dexamethasone for another 14 days. Cells cultured in complete DMEM served as controls.MAIN OUTCOME MEASURES: Cell morphology and growth rule were observed under a microscope. Difference in dentin sialophesphoproteln expression was determined prior to and following mineralization using immunocytochemical staining.RESULTS: Dental pulp stromal cells from human exfoliated deciduous tooth, adhered to the wall, were polygon-shaped and characterized by large cell size and a relatively large nucleus and plenty cytoplasm after mineralization for 14 days. Over 80% cells were positive for dentin sialophosphoprotein. Non-induced cells were spindle, and only 2% cells were positive for dentin sialophosphoprotein.CONCLUSION: Odontogenic inductive culture might improve odontoblastic differentiation. It seems that human exfoliatod deciduous tooth pulp represents an new reservoir of adult stem cells with odontogenic potential.%背景:在牙组织工程和牙再生的研究中,寻找合适的种子细胞是当前的中心和热点.目的:分离培养儿童乳牙牙髓基质细胞,比较研究矿化诱导前后牙本质涎磷蛋白表达的差异.设计、时间及地点:观察性实验,于2006-12/2007-12在大庆油田总医院中心实验室完成.对象:选择8～10岁儿童8名,男4名,女4名,排除传染病及内分泌疾病史,乳牙排除牙体牙髓牙周疾病.牙髓取材于颌外门诊拔除的滞留乳磨牙.方法:通过组织块贴壁法获得乳牙牙髓基质细胞,并在含体积分数10%胎生血清的DMEM培养基中原代培养14 d,消化传代后用添加有10-8 mol/L地塞米松,50 μmol/L左旋抗坏血酸,10 nmol/L维生素D3,10 mmol/L β-磷酸甘油钠的DMEM/F12培养基诱导培养14 d,用不含添加成分的完全DMEM培养基培养作对照.主要观察指标:显微镜下观察细胞形态变化和生长规律.免疫细胞化学染色比较诱导前后牙本质涎磷蛋白的表达差异.结果:乳牙牙髓基质细胞呈成纤维细胞样贴壁生长,经矿化诱导14 d后多数细胞由梭形转变成多角形和柱型,胞核大而圆,B0%以卜细胞的细胞质牙本质涎磷蛋白表达阳性.而未经诱导的细胞仍为梭形,仅有2%的细胞牙本质涎磷蛋白染色呈阳性.结论:乳牙牙髓基质细胞经矿化诱导培养后具有向成牙本质细胞分化的潜能,可以尝试作为牙再生研究新的种子细胞来源.
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