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

摘要

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 ±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 .%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，rOP－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（ MC3 T3－E1）中是否加入Ti－6Al－4V微粒和rOP－1，分为微粒组（5、10、15μg／mL Ti－6Al－4V）、处理组（微粒组加入200 ng／mL rOP－1）、阳性组（加入200 ng／mL rOP－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①rOP－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）；②rOP－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rOP－1后钙结节数量呈增多趋势。结论 Ti－6Al－4V微粒环境下，rOP－1无提高成骨细胞增殖能力，能提高细胞分化矿化能力，rOP－1可以作为潜在治疗关节假体无菌性松动一种方法。