不同直径羟基磷灰石纳米颗粒对成骨细胞代谢的影响

Effect of different nanophase hydroxyapatite particles on osteoblasts metabolism

目的:比较喷涂法制备的羟基磷灰石(HA)涂层表面2种不同直径纳米颗粒对成骨细胞增殖活性及功能代谢的影响。方法:将体外培养的乳鼠颅骨成骨细胞分别接种于具有2种直径范围纳米颗粒的HA涂层表面(I组:20- 40nm,Ⅱ组:70-100nm),并以无纳米颗粒HA涂层作对照,扫描电镜下观察涂层表面形态以及细胞贴附情况;采用MTT法、PNPP法和考马斯亮蓝蛋白含量检测法分别检测细胞的增殖活性、碱性磷酸酶(alkaline phosphatase,ALP)活性和细胞总蛋白含量,应用SPSS10．0统计软件包对数据进行单因素方差分析的q检验(SNK)。结果:细胞在HA涂层表面生长良好,MTT法测量显示,6d和8d时,Ⅰ组和Ⅱ组涂层表面细胞增殖活性具有显著性差异(P<0．05);ALP活性测量显示,5d和10d时,2组涂层表面细胞无显著差异(P>0．05),随着培养过程的进行,15d和20d时,Ⅰ组涂层表面细胞显著高于Ⅱ组涂层表面细胞(P<0．05);蛋白含量检测表明,Ⅰ组涂层表面细胞自5d至20d始终高于Ⅱ组涂层表面细胞(P<0．05)。结论:HA涂层表面纳米颗粒的直径可以影响成骨细胞的增殖活性和功能代谢,颗粒直径接近体内骨组织HA纳米晶体的涂层,生物相容性较好。

PURPOSE： To compare two different structural nanophase hydroxyapatite（HA） panicles on the proliferation activity and functions of osteoblasts. METHODS： Primary culture of osteoblasts from rat calvaria was established and then cultured on the coatings of different size of nano panicles （group Ⅰ：20-40nm,group Ⅱ ：70-100nm）, the HA coatings without nano-panicles was used as control group. Proliferation activity, protein content and synthesis of alkaline phosphatase （ALP） by osteoblasts were examined by MTT assay, coomassic brilliant blue method and PNPP test,and statistical significance was assessed by multiple comparison （q test, SNK） in one-way analysis of variance （ANOVA） with SPSS10.0 software package. RESULTS： Osteoblasts grew well on HA coatings. MTT assay demonstrated that there was significant difference between group Ⅰ and group Ⅱat 6^th day and 8^th day（P〈0.05）.At first half stage（5^th day and 10^th day） ALP activity test showed no significant difference between group Ⅰ and group Ⅱ（P〉0.05） and as the culture going on （15^th day and 20^th day）, there was significant difference between the two groups（P〈0.05）. Coomassic brilliant blue method showed that there was significant difference between group Ⅰ and group Ⅱ from 5^th day to 20^th day （P〈0.05）. CONCLUSION： The diameter of nano-panicles on the HA coatings could influence the proliferation activity and functions of the osteoblasts. The nano-panicles of similar size with HA crystal in vivo showed better cytocompatibility. Supported by Research Fund of Science and Technology Bureau of Shandong Province（Grant No.032040105）.