酸碱处理对多孔钛成骨细胞增殖和分化的影响

目的研究酸碱处理对多孔钛表面小鼠前成骨细胞MC3T3-E1增殖及分化的影响。 方法粉末冶金法制备多孔钛，酸蚀处理后，NaOH浸泡不同时间，得到酸碱处理多孔钛（AAPT）。制备AAPT试件73个，未处理多孔钛（NTPT）试件41个和未处理致密钛（NTDT）试件30个。采用扫面电镜观察表面形貌，BCA法检测蛋白吸附能力，筛选适宜的碱处理时间。对于NTPT及AAPT组试件，采用表面接触角分析仪与拉曼光谱仪分析表面水接触角和化学组成。对于NTDT、NTPT及AAPT组试件，CCK-8法检测细胞增殖活性，实时荧光定量聚合酶链反应检测碱性磷酸酶（ALP）及骨钙素（OCN）基因相对表达量。采用独立样本t检验和单因素方差分析进行统计分析，LSD-t检验进行两两比较。 结果12 h NaOH处理组（AA₁₂PT）纳米结构最为清晰，且蛋白吸附量（0.367 mg/ml）最高（F= 400.835，P<0.05），因此采用该组进行后续实验。AA₁₂PT组试件表面接触角（22.08°）显著小于NTPT组（93.7°），差异有统计学意义（F= 1.194，P<0.001）。AA₁₂PT组试件可见钛酸氢钠特征散射峰。接种3、5、7 d后，NTDT组的细胞增殖活性均高于AA₁₂PT组（F_{3 d}= 245.517、F_{5 d}= 102.442、F_{7 d}= 119.047，P<0.001），AA₁₂PT组均高于NTPT组（P_{3 d}= 0.005、P_{5 d}= 0.038、P_{7 d}= 0.010）。接种7 d后，AA₁₂PT与NTPT组的ALP基因和OCN基因相对表达量均高于NTDT组（F_ALP= 13.698，P_{ALP-AA1 2PT}< 0.001、P_ALP-NTPT= 0.002；F_OCN= 175.859，P_OCN<0.001）。接种14 d后，NTDT组的ALP基因及OCN基因相对表达量均高于NTPT组（F_ALP= 33.691，P_ALP= 0.045；F_OCN= 42.789，P_OCN= 0.044）。 结论酸碱处理可显著提高多孔钛材料的蛋白吸附量，促进成骨细胞的增殖、分化。

Proliferation and differentiation of MC3T3-E1 cells on acid-alkali treated porous titanium

ObjectiveTo evaluate the proliferation and differentiation of MC3T3-E1 cells on acid-alkali treated porous titanium. MethodsPorous titanium samples were fabricated by powder metallurgy process, then immersed in a mixed acid and NaOH solution in different periods to get acid-alkali-treated porous Ti (AAPT) . 73 AAPT samples, 41 non-treated porous Ti (NTPT) samples and 30 non-treated commercial dense titanium (NTDT) samples were made. The surface morphology was observed under a scanning electron microscopy. Protein adhesion was quantitatively analyzed by BCA method. The optimum treating time of alkali-treated in acid-alkali treatment was determined. For NTPT and AAPT, the contact angle and surface chemical structure were examined by an optical contact angle measuring device and a Raman spectrometer. CCK-8 and real-time fluorescence quantitative polymerase chain reaction were carried out to measure the proliferative activity and the gene expression of alkaline phosphatase (ALP) and osteocalcin (OCN) , respectively. The level of significance was determined by independent sample t test and an One-Way ANOVA followed by LSD-t test for a multiple comparison procedure. ResultsThe scanning electron micrographs showed the nano structure of 12 h alkali treatment (AA₁₂PT) was most distinct and obvious. And it had the highest protein adsorption (0.367 mg/ml) (F= 400.835, P<0.05) . So AA₁₂PT was chosen in the subsequent experiments. The contact angle on the surfaces of NTPT (93.7°) exhibited a statistically significantly higher in comparison to the contact angle of AA₁₂PT (22.08°) (F= 1.194, P<0.001) . The Raman spectroscopy indicated the presence of sodium hydrogen titanate on the surface of AA₁₂PT. MC3T3-E1 cells got better proliferation rate on NTDT than AA₁₂PT (F_{3 d}= 245.517, F_{5 d}= 102.442, F_{7 d}= 119.047, P<0.001) at day 3, 5 and 7. And AA₁₂PT enhanced cell proliferation significantly than NTPT (P_{3 d}= 0.005, P_{5 d}= 0.038, P_{7 d}= 0.010) . At day 7, the gene expression of ALP and OCN of AA₁₂PT and NTPT was higher than that of NTDT (F_ALP= 13.698, P_ALP-AA12PT<0.001, P_ALP-NTPT= 0.002; F_OCN= 175.859, P_OCN<0.001) . At day 14, the gene expression of ALP and OCN of NTDT was higher than that of NTPT (F_ALP= 33.691, P_ALP= 0.045; F_OCN= 42.789, P_OCN= 0.044) . ConclusionAcid-alkali treated porous titanium can significantly improve the amount of protein adsorption and promote proliferation and differentiation of osteoblasts.