生物活性玻璃和牙本质浸提蛋白对人牙髓细胞的作用

目的：明确生物活性玻璃（bioactive glass, BG）和牙本质浸提蛋白（extracted dentin proteins, EDP）对人牙髓细胞增殖、成牙本质向分化、矿化的作用。方法： 采用酶消化法培养牙髓细胞，传代至第4代用于实验。分别用含BG-EDP浸提液、BG浸提液、EDP浸提液的达尔伯克必需基本培养基（Dulbecco’s minimum essential medium，DMEM）培养牙髓细胞，对照组为DMEM培养组；通过细胞活性噻唑蓝比色法检测细胞增殖能力,碱性磷酸酶（alkaline phosphatase，ALP）活性检测及real-time PCR检测细胞成牙本质向分化能力,茜素红钙化结节染色及氯化十六烷基吡啶半定量分析检测细胞矿化能力。结果： BG-EDP组3、7、9 d时（光密度值1.36±0.06、2.52±0.20 、2.72±0.29）能够增强人牙髓细胞增殖活性，同BG组（光密度值1.20±0.26、2.33±0.26、2.50±0.30）、EDP组（光密度值1.13±0.15、2.10±0.13、2.38±0.22）和对照组（光密度值0.84±0.17、1.84±0.18、1.95±0.19）比较差异具有统计学意义（P<0.05）。7 d时，BG-EDP组ALP活性与EDP组、对照组差异无统计学意义，分化相关基因（DSPP、DMP-1）表达无明显升高。14 d时，BG-EDP组ALP活性升高（56.67±1.83）， 显著高于EDP组（41.98±9.71）及对照组（30.82±6.70），P<0.05，但同BG组（56.29±6.20）相比差异无统计学意义（P>0.05）；BG EDP组DSPP表达量明显升高(5.79±1.94)，显著高于BG组(2.62±0.46)、EDP组(2.66±1.06)及对照组(1.84±0.76)，P<0.05；BG-EDP组DMP-1表达升高(3.87±1.87)，略高于BG组(1.89±0.90)、EDP组(2.38±1.04)和对照组 (2.25±0.93)，但差异无统计学意义（P>0.05）。诱导2周后，BG-EDP组形成较多矿化结节，氯化十六烷基吡啶半定量分析显示BG-EDP组钙化量最高（0.27±0.01）。结论： 同单纯的BG及EDP相比，BG-EDP复合后具有更强的促进人牙髓细胞的增殖、成牙本质向分化、矿化作用。

Effects of bioactive glass and extracted dentin proteins on human dental pulp cells

Objective:To investigate the proliferation,odontogenic differentiation and mineralization of human dental pulp cells (HDPCs) on bioactive glass(BG) and extracted dentin proteins(EDP).Me-thods: Primary HDPCs were isolated from third molars by enzyme digestion and were cultured in Dulbecco's minimum essential medium (DMEM).Then the 4th generation of HDPCs was cultured with DMEM,which contained BG-EDP,BG,and EDP,respectively.Meanwhile HDPCs were cultured in DMEM as control group.Proliferation of HDPCs was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT) colorimetric assay.Odontogenic differentiation was determined by alkaline phosphatase (ALP) activity assay and real-time PCR.Mineralization was investigated by Alizarin red staining and cetylpyridinium chloride (CPC) assay.Results: The proliferation of HDPCs was increased significantly in BG-EDP group on 3,7,and 9 d(optical density value:1.36±0.06,2.52±0.20,2.72±0.29) compared with BG(optical density value: 1.20±0.26,2.33±0.26,2.50±0.30),EDP(optical density value: 1.13±0.15,2.10±0.13,2.38±0.22) and control group(optical density va-lue: 0.84±0.17,1.84±0.18,1.95±0.19),P<0.05.After 7 days,ALP activity of BG-EDP group had no statistical difference compared with EDP group and control group;the expression of odontogenic differentiation genes (DSPP,DMP-1) showed no difference among all the groups(P>0.05).After 14 days,ALP activity of BG-EDP group (56.67±1.83) was significantly upregulated compared with EDP group (41.98±9.71) and control group (30.82±6.70),P<0.05,but had no statistical difference compared with BG group (56.29±6.20),P>0.05;DSPP gene expression was upregulated significantly in BG-EDP group (5.79±1.94) compared with the other groups (P<0.05);DMP-1 gene expression of BG-EDP group (3.87±1.87) increased but had no statistical difference compared with the other groups (P>0.05).The alizarin red staining showed more mineral nodules in BG-EDP group,the cetylpyridinium chloride semi-quantification presented higher calcification in BG-EDP group (0.27±0.01) compared with the other groups (P<0.05).Conclusion: Compared with either BG or EDP,BG-EDP significantly promotes the proliferation,odontogenic differentiation and mineralization of HDPCs.