多孔钛的骨传导性及其孔隙结构对MC3T3-E1细胞早期分化的影响

Osteoconduction and influence of porosity and pore size of porous titanium on the early stage differentiation of MC3T3-E1 cells

目的探讨多孔钛的骨传导性及其孔隙结构对小鼠前成骨细胞MC3T3-E1早期分化的影响。方法碳酸氢铵造孔剂结合粉末冶金法制备6组由不同平均孔隙率及不同平均孔径组合的多孔钛试样,即AI:(43.1±0.7)%和(154.8±11.9)μm;AII:(40.9±1.5)%和(295.6±8.5)μm;AIII:(44.3±1.1)%和(560.4±25.6)μm;BI:(53.3±1.2)%和(191.6±3.7)μm;BII:(51.7±2.7)%和(303.8±8.2)μm;BIII:(49.9±3.9)%和(583.1±21.7)μm,Ta2级商业致密纯钛作为对照组;每组3个试样。MC3T3-E1细胞接种于24孔板3 h贴壁后置入多孔钛试样,培养3 d和5 d后激光扫描共聚焦显微镜(LSCM)观察经FITC微丝蛋白荧光探针标记的细胞。MC3T3-E1细胞接种于已置入试样的24孔板,培养7 d和14 d后测定碱性磷酸酶(alkaline phosphatase,ALP)活性,评价细胞的早期分化能力;培养21 d后茜素红染色观察细胞晚期矿化结节。结果 MC3T3-E1细胞贴壁后,5 d可长入多孔钛孔内及表面。7 d和14 d多孔钛组ALP活性显著高于对照组(P<0.05);其中BI组在14 d的ALP活性显著高于其它各组(P<0.05)。21 d多孔钛试样表面及孔隙内均可见钙结节形成。结论在本实验条件下,多孔钛具有一定的骨传导性;平均孔隙率为53.3%且平均孔径为191.6μm的多孔钛利于MC3T3-E1细胞的早期分化。

Objective To Evaluate the osteoconduction and the influence of porosity and pore size of porous titanium on the early stage osteoblast differentiation of mice preosteoblast MC3T3-E1 cells.Methods Six groups of porous titanium disk-shape specimens with different porosity ratios and pore sizes [AⅠ：（43.1 ± 0.7）％,（154.8 ± 11.9） μm; AⅡ：（40.9 ± 1.5）％,（295.6 ± 8.5） μm; AⅢ：（44.3 ± 1.1）％,（560.4 ± 25.6） μm; BⅠ：（53.3 ±± 1.2）％,（191.6±3.7） μm; BⅡ：（51.7±2.7）％,（303.8±8.2） μm; BⅢ：（49.9±3.9）％,（583.1 ±21.7） μm] were fabricated by powder metallurgy process using NH4HCO3 powder as space holder,and commercially pure titanium （CpT） Ta2 disk-shape specimens （C group） were made as control group.Cell migration ability was measured by putting porous titanium disks into cell culture medium with adhered MC3T3-E1 in it.After 3 and 5 days,cytoskeleton was labelled with fluorescence probe and observed under Laser Scanning Confocal Microscope （LSCM） to observe whether cells migrated onto the disks and into pores.After 7 and 14 days,the alkaline phosphatase （ALP） activity of MC3T3-E1 seeded on different disks was detected with ALP kit.After 21 days,calcium nodules were stained with Alizarin Red S and observed under a stereomicroscope.Results MC3T3-E1 cells could migrate onto the disks and into pores 5 days after being seeded,.After 7 and 14 days,MC3T3-E1 cells on porous titanium disks showed higher ALP activity than those on CpT disks （P ＜ 0.05）.At 14 days,cells on disks of BⅠ group showed higher ALP activity than those on disks of any other group （P ＜ 0.05）.Alizarin Red S staining results showed that calcium nodules successfully formed both on surfaces and into pores of porous titanium disks.Conclusions Porous titanium showed osteoconduction in the present study.Porous titanium with an average pore size of 191.6 μ m and a porosity of 53.3％ could better promote the early stage osteoblast differentiation of MC3T3-E1 cells.