二氧化锆梯度复合羟基磷灰石生物材料对大鼠成骨细胞体外活性的影响

Effect of graded composite zirconia-hydroxyapatite on viability of rat osteoblast cells cultured in vitro

目的探讨二氧化锆(ZrO_2)梯度复合羟基磷灰石(HAP)生物陶瓷材料体外对原代大鼠成骨细胞活性的影响。方法以纯ZrO_2材料为对照,测量两种材料的表面粗糙度,并用扫描电镜、X射线衍射仪对复合材料进行分析；体外将新生SD大鼠颅骨分离培养的成骨细胞直接于两种材料上培养；用对硝基酚磷酸盐法测定成骨细胞碱性磷酸酶(ALP)活性；酶联免疫(EHSA)法检测成骨细胞分泌白细胞介素(IL)-6和转化生长因子(TGF)-β的水平；RT-PCR法检测成骨细胞TGF-β的mRNA水平；扫描电镜观察成骨细胞与两种材料的体外复合情况；用复合材料的浸提液进行细胞毒性实验,计算相对增殖率(relativeproliferationrate,RGR)及毒性分级。结果梯度复合材料的表面粗糙度明显高于对照材料(P＜0．01)；梯度复合材料组成骨细胞不同时间点的ALP活性显著高于对照组；培养2～4 d,两组细胞IL-6和TGF-β的分泌水平差异也有统计学意义(P＜0．05,0．01)；两组在TGF-βmRNA水平差异也有统计学意义(P＜0．01)；14 d后,梯度复合材料表面的成骨细胞骨化明显。四甲基偶氮唑盐(MTT)比色法检测,阴性对照组与材料组的毒性级分别为0级和Ⅰ级,差异无统计学意义(P＞0．05)。结论ZrO_2梯度复合HAP生物材料明显促进体外培养的大鼠成骨细胞增殖和分化,具有较好的生物相容性。

Objective To evaluate the effect ot a novel orthopedic biomaterial, graded composite zirconia （ZrO2） hydroxyapatite （HAP） on activity of rat osteoblast cells （OB） cuhured in vitro. Methods The pure zirconia material was used as control to measure surface roughness of the composite material that was examined by using scanning electron microscope （SEM） and X-ray diffractometer （XRD）. The rat osteoblast cells were cultured on the two materials. Alkali phosphatase （ALP） of the two groups was measured and ELISA was used to detect IL-6 and TGF-βconcentration of the supernatant of OB cells. Tumor growth factor-β （TGF-β） mRNA was detected by RT-PCR. SEM was used to observe OB cells on the two materials. The extract of the composite material was used for a cytotoxicity test to calculate the relative proliferation rate （RGR） and classify the toxicity. Results The surface roughness of the gradual composite materials was significantly higher than that of the control materials （ P 〈 0.01 ）. The ALP of the gradual material group was markedly higher than that of the control group at different intervals. There was significant difference of the IL-6 and TGF-βconcentrations 2-4 days after culture between two groups （P 〈0.05, P 〈 0.01 ）. The mRNA level of TGF-β of the two OB groups also showed marked statistical difference （P 〈 0.01 ）. The ossification of the OB cells on the composed material was marked after 14 days. The MTT color experiments showed no statistic significance between materials group and negative group, with the toxicity at level Ⅰ and 0 （ P 〉 0.05 ）. Conclusion Graded composite ZrO2 HAP can significantly promote proliferation and differentiation of OB cells cultured in vitro and has good biocompatibility.