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中华口腔医学研究杂志(电子版) ›› 2015, Vol. 09 ›› Issue (03) : 193 -199. doi: 10.3877/cma.j.issn.1674-1366.2015.03.003

所属专题: 文献

基础研究

多孔纯钛孔隙率及孔隙尺寸对蛋白吸附及成骨细胞分化的影响
刘帅1, 李湘霞1, 杨越2, 赵克1,()   
  1. 1. 510055 广州,中山大学光华口腔医学院·附属口腔医院;广东省口腔医学重点实验室
    2. 518020 深圳,暨南大学第二临床医学院·深圳市人民医院口腔医学中心
  • 收稿日期:2014-12-25 出版日期:2015-06-01
  • 通信作者: 赵克

Protein adhesion and osteoblast differentiation on porous titanium with different porosity and pore size

Shuai Liu1, Xiangxia Li1, Yue Yang2, Ke Zhao1,()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
    2. The Second Clinical Medical College of Jinan University, Stomatology Center of Shenzhen People′s Hospital, Shenzhen 518020, China
  • Received:2014-12-25 Published:2015-06-01
  • Corresponding author: Ke Zhao
  • About author:
    Corresponding author: Zhao Ke, Email:
引用本文:

刘帅, 李湘霞, 杨越, 赵克. 多孔纯钛孔隙率及孔隙尺寸对蛋白吸附及成骨细胞分化的影响[J]. 中华口腔医学研究杂志(电子版), 2015, 09(03): 193-199.

Shuai Liu, Xiangxia Li, Yue Yang, Ke Zhao. Protein adhesion and osteoblast differentiation on porous titanium with different porosity and pore size[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2015, 09(03): 193-199.

目的

从分子生物学角度研究筛选利于蛋白质吸附、成骨细胞分化的合适孔隙结构,为多孔钛材料作为牙种植体提供研究基础及制备参数。

方法

筛选不同粒径(Ⅰ组:0~200 μm、Ⅱ组:200~400 μm、Ⅲ组:400~600 μm)的NH4HCO3造孔剂颗粒,按照不同的质量分数(A组:20%、B组:30%)与钛粉均匀混合,粉末冶金法制备6组多孔纯钛试件,不添加造孔剂制备致密纯钛试件作为对照组;每组10个试件。蛋白定量试剂盒测定蛋白质吸附量以评价多孔纯钛试件的蛋白吸附能力,实时荧光定量聚合酶链反应(PCR)检测Runt相关转录因子2(Runx2)、碱性磷酸酶(ALP)及骨钙素(OCN)基因表达量以评价成骨细胞分化水平。

结果

多孔纯钛的蛋白质吸附量高于致密纯钛(P < 0.05),其中小平均孔隙尺寸及高平均孔隙率组较其余多孔纯钛组吸附更多蛋白质(P < 0.05)。多孔纯钛的孔隙率和孔隙尺寸对成骨细胞分化的影响具有交互效应。成骨细胞培养第7天,仅AI组Runx2基因表达量高于对照组(P < 0.05);培养第14天,多孔纯钛各组Runx2基因的表达量均低于对照组(P < 0.05)。成骨细胞培养7、14 d后,多孔纯钛各组ALP基因的表达量高于对照组(培养第7天,BⅢ组除外;培养第14天,AⅠ、AⅢ组除外)(P < 0.05)。成骨细胞培养第7天,AⅠ、BⅠ组OCN基因的表达量较对照组及其他平均孔隙尺寸组高(P < 0.05);成骨培养第14天,仅BⅠ组OCN基因的表达量高于对照组(P < 0.05)。

结论

本实验条件下,孔隙结构提高了纯钛试件的蛋白质吸附量,且平均孔隙率为53.3%、平均孔径为191.6 μm的多孔钛试件利于小鼠前成骨细胞MC3T3-E1的成骨向分化。

Objective

To evaluate the effect of porosity and pore size of the porous titanium on the protein adhesion and osteoblast differentiation from the perspective of molecular biology, thus to provide theoretical evidence for clinical application of porous titanium and to guide the optimal designer of porous titanium.

Methods

Through controlling particle sizes (Ⅰ: 0~200 μm, Ⅱ: 200~400 μm, Ⅲ: 400~600 μm) and weight ratios (A: 20%, B: 30%) of NH4HCO3, six groups of porous titanium samples with different porosities and pore sizes were fabricated by powder metallurgy process, dense titanium were made as control group, 10 specimens per group. The adhesive proteins were tested with protein quantification kit to evaluate the protein adsorption capacity of porous titanium. The gene expression of Runt transcription factor 2 (Runx2) , alkaline phosphatase (ALP) , and osteocalcin (OCN) of MC3T3-E1 cells were tested by real-time fluorescence quantitative polymerase chain reaction (PCR) .

Results

All porous titanium groups showed higher protein adsorption than control group (P < 0.05) . The higher the mean porosity is and the smaller the mean pore size is, the more serum proteins adhere to compared with the other porous titanium groups (P < 0.05) . The porosity and pore size have interaction effects on differentiation of osteoblasts. Osteoblasts only expressed higher Runx2 gene on group AⅠ than control group at day 7 (P < 0.05) . While cells on control group expressed significantly higher levels of transcript for Runx2 than the cells on all porous Ti disks at day 14 (P < 0.05) . The gene expression of ALP is significantly higher compared with control group in all porous Ti disks on 7 and 14 day (P < 0.05) , BⅢ at day 7 and AⅠ, AⅢ at day 14 is excluded. For gene expression of OCN, on group AⅠ and BⅠ are higher than on dense Ti at day 7 (P < 0.05) ; on group BⅠ is higher than on dense Ti at day 14 (P < 0.05) .

Conclusions

All porous titanium groups shows higher protein adsorption than control group. The porous titanium with mean porosity of 53.3% and mean pore size of 191.6 μm facilitates the osteoblastic differentiation of mouse osteoblast precursor cells MC3T3-E1.

表1 各组试件平均孔隙率和平均孔隙尺寸情况( ± s
表2 基因的引物序列
图1 各组多孔纯钛试件及致密纯钛试件的蛋白质吸附量
图2 成骨细胞培养7、14 d后各组多孔纯钛试件及致密纯钛试件Runx2基因的相对表达量
图3 成骨细胞培养7、14 d后各组多孔纯钛试件及致密纯钛试件ALP基因的相对表达量
图4 成骨细胞培养7、14 d后各组多孔纯钛试件及致密纯钛试件OCN基因的相对表达量
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