微弧氧化技术与钛合金表面成骨细胞增殖及骨向分化能力

Micro-arc oxidation and osteoblast proliferation and osteogenic differentiation ability on titanium surface

背景:采用普通电化学法可在钛及其合金表面制备纳米级羟基磷灰石涂层,但该涂层吸收降解缓慢,需要8-12周的时间。而微弧氧化可在复杂表面形成均匀薄膜,有利于细胞黏附和骨组织长入。目的:探索微弧氧化羟基磷灰石涂层钛合金对成骨细胞增殖及骨向分化能力的影响。方法:采用电化学法与微弧氧化法分别制备羟基磷灰石涂层钛合金材料,检测两种材料表面的接触角。将成骨细胞系hFOB1.19接种于两组羟基磷灰石涂层钛合金材料表面,培养48 h时,采用扫描电镜观察细胞在材料上的形态变化;培养1,12,24,48,72h时,采用MTT法检测细胞增殖;培养1,3,5d时,比较两组材料表面细胞计数与碱性磷酸活性;培养第5天,采用WesternBlotting检测细胞内骨形态发生蛋白2和骨形态发生蛋白4表达。结果与结论:①微弧氧化组材料表面的接触角小于电化学组[(66.5±2.2)°,(52.8±2.1)°,P=0.0015)];②扫描电镜显示,电化学组成骨细胞形态不规则,胞体皱缩不饱满,与材料贴合较差;微弧氧化组细胞充分伸展,形态扁平,与材料贴合紧密;③微弧氧化组12-72 h的细胞增殖快于电化学组(P<0.05),培养3,5 d的细胞计数多于电化学组(P<0.05);④微弧氧化组培养1,3,5 d的细胞碱性磷酸酶活性高于电化学组(P<0.05);⑤微弧氧化组骨形态发生蛋白2和骨形态发生蛋白4表达均高于电化学组(P<0.05);⑥结果表明,微弧氧化羟基磷灰石涂层钛合金材料可促进成骨细胞的增殖及骨向分化能力。

BACKGROUND:Electrochemical method based nanoscale hydroxyapatite coating has slow degradation speed(8-12 weeks).Micro-arc oxidation based method could form homogeneous coating on complex surfaces,and this coating promotes cell adhesion and ingrowth in bone tissues.OBJECTIVE:To explore the effect of micro-arc oxidation hydroxyapatite coating titanium alloy on osteoblast proliferation and osteogenic differentiation ability.METHODS:Hydroxyapatite coating titanium alloy materials were prepared by electrochemical method and micro-arc oxidation method.The contact angles of the two materials were detected.Osteoblasts(hFOB1.19)were incubated on the hydroxyapatite coating titanium alloy materials for 48 hours.The morphological changes of osteoblasts on the materials were observed under scanning electron microscope.The cell proliferation was detected by MTT method at 1,12,24,48 and 72 hours.The cell count and alkaline phosphatase activity were detected at 1,3 and 5 days of culture.At 5 days,the expression levels of bone morphogenetic proteins 2 and 4 were detected by western blot assay.RESULTS AND CONCLUSION:(1)The contact angle in the micro-arc oxidation group was smaller than that in the electrochemical group[(66.5±2.2)°,(52.8±2.1)°,P=0.0015)].(2)Scanning electron microscope revealed that the osteoblasts in the electrochemical group had an irregular and shrunken shape,and adhered loosely to the material surface.The osteoblasts in the micro-arc oxidation had a fully outstretched and flat shape,and adhered tightly to the material surface.(3)From 12 to 72 hours,the cell proliferation in the micro-arc oxidation group was faster than that in the electrochemical group(P<0.05).At 3 and 5 days after culture,the cell proliferation in the micro-arc oxidation group was faster than that in the electrochemical group(P<0.05).(4)At 1,3 and 5 days,the alkaline phosphatase activity of osteoblasts in the micro-arc oxidation group was higher than that in the electrochemical group(P<0.05).(5)The expression levels of bone morphogenetic proteins 2 and 4 were significantly up-regulated in the micro-arc oxidation group compared with the electrochemical group(P<0.05).(6)These results indicate that micro-arc oxidation hydroxyapatite coated titanium alloy increases osteoblast proliferation and osteogenic differentiation ability.