重组成骨蛋白-1在钛合金微粒环境下对MC3T3-E1成骨细胞增殖、分化、矿化的影响

Effect of rop-1 on the proliferation,diferentiation,and mineralizatio of titanium alloy particles-induced MC3T3-E1 osteoblastic cells

目的在钛-6铝-4钒(Ti-6Al-4V)合金微粒环境下观察重组成骨蛋白-1(recombinant OP-1,r OP-1)对成骨细胞的影响,为防治关节假体无菌性松动提供新的治疗途径。方法根据小鼠颅顶骨前成骨细胞亚克隆14(MC3T3-E1)中是否加入Ti-6Al-4V微粒和r OP-1,分为微粒组(5、10、15μg/m L Ti-6Al-4V)、处理组(微粒组加入200 ng/m L r OP-1)、阳性组(加入200ng/m L r OP-1)和对照组,检测各组24、72、120 h MC3T3-E1细胞增殖能力、72 h碱性磷酸酶(akaline phosphatase,AKP)、骨钙素(osteocalcin,OCN)和骨桥蛋白(osteopontin,OPN)mRNA的表达,及120 h成骨细胞的矿化能力。结果 1r OP-1无促进Ti-6Al-4V微粒环境下成骨细胞增殖能力,与微粒组比较,差异无统计学意义(P>0.05);2r OP-1可提高成骨细胞分化,与对照组比较差异有统计学意义(P<0.05);同时逆转Ti-6Al-4V微粒抑制成骨细胞分化,与微粒组比较差异有统计学意义(P<0.05);3茜素红S染色后Ti-6Al-4V微粒钙结节数量随着浓度增加逐渐降低,和微粒组比较,加入r OP-1后钙结节数量呈增多趋势。结论 Ti-6Al-4V微粒环境下,r OP-1无提高成骨细胞增殖能力,能提高细胞分化矿化能力,r OP-1可以作为潜在治疗关节假体无菌性松动一种方法。

Objective Our results has been shown that titanium alloy particles （Ti-6Al-4V） inhibited RUNX2 mRNA and-proteinexpression of osteoblasts .In order to improve the boneformation on osteoblasts induced-Ti-6Al-4V, we observed the effect of rOP-1 on the proliferation, diferentiation, andmineralizatio of Ti-6Al-4V induce osteoblast, to provide a new treatment method of artifi-cial joint aseptic loosening .Methods Cell counting Kit8 test used to assay the cell proliferation after 24 h, 72 h and 120 h-incubation with Ti-6Al-4V ＆#177;rOP-1, respectively.Furthermore, alkaline phosphatase （AKP）, osteocalcin （OCN） and osteopontin （OPN） mRNA level were detected by reverse transcription-polymerase chain reaction （RT-PCR）, respectively, at the time point of 72 h.Cell bio-mineralization was assessed through alizarin red S staining after 120 h.Results ①Compared with Ti-6Al-4V group, after 24 h, 72 h and 120 h-incubation with Ti-6Al-4V＋rOP-1, the proliferation of osteoblast did not increase significance with comparison of the Ti -6Al-4V group （P〉0.05）;②compared with Ti-6Al-4V group, AKP, OCN and OPN mRNA level were increased by 72 h-exposure to Ti-6Al-4V＋rOP-1, respectively （P〈0.05）.rOP-1can stimulate osteoblast differentiation and mineralization , compared with Ti-6Al-4V group, there was statistically significance （ P〈0.05）;③compared with Ti-6Al-4V group, alizarin red staining demonstrated that the quantity of calcium nodules were markedly increased in the Ti-6Al-4V＋rOP-1 group at hours 120.Conclusion Our result indi-cate that rOP-1 can stimulate differentiation and mineralization , while the proliferation remained unchanged on osteoblast with Ti-6Al-4V particles.We suppose that rOP-1 may be a therapeutic candidate for the prevention of artificial joint aseptic loosening .