Effects on gene expression of bone and cell-adhesive proteins by biomaterials

本研究旨在探讨成骨细胞与生物材料的黏附机制。首先，研究了生物材料的不同对成骨细胞与其黏附的影响。人骨肉瘤细胞系Saos2对羟基磷灰石的细胞粘附率与无血清孵育的钛细胞的粘附率相同，而对羟基磷灰石的细胞粘附率高于有血清培养的钛细胞。根据这些结果，我们推测血清中的蛋白质影响细胞与生物材料的黏附。将骨桥蛋白(OP)导入Saos2细胞。OP是骨基质中的一种非胶原性蛋白。据报道，OP在体外可增强成骨样细胞的黏附活性。我们研究了OP基因对成骨样细胞在钛表面黏附活性的影响。将C端带有标志标签的人OP基因亚克隆到逆转录病毒载体pBabe-Puro上。用含有OromyciiL的DMEM进行筛选，克隆表达OP-FLAG的Saos2细胞系(Saos2-OP)。RT-PCR法仅在Saos2-OP中观察到OP的表达，且仅在Saos2-OP中证实了OP包含标志标签的产生。在无血清DMEM中孵育12小时和24小时，Saos2-OP和亲本细胞在钛圆盘和聚苯乙烯盘上的贴壁细胞数差异有统计学意义，而在6小时后贴壁细胞数差异无统计学意义。扫描电子显微镜观察发现，Saos2-OP和Saos2的形态不同。与Saos2相比，Saos2-OP的形状更小、更厚，表面有小的球状结构，而Saos2-OP表面没有。这些结果表明，人骨桥蛋白是成骨细胞样细胞与钛黏附的有效增强剂，并且骨桥蛋白的转染影响了Saos2的形状。

The purpose of this study was to investigate the mechanism of adhesion of osteoblast to biomaterials. Firstly, the effect of the difference of the biomaterials on the adhesion of osteoblasts to them was investingated. Cell adhesion rate of a human osteosarcoma derived cell line (Saos2) to hydroxyapatite was same to that to titanium incubated without serum, whereas cell adhesion rate of Saos2 to hydroxyapatite was higher than that to titanium with serum. From these results, we speculated that the proteins within serum effected cell adhesion to biomaterials. We carried out the tranfection of osteopontin (OP) into Saos2. OP is a noncollagenous protein in bone matrix. It is reported that OP enhanced adhesive activity of osteoblast-like cells in vitro. We investigated the effect on adhesive activity of osteoblast-like cell to titanium by transfection of OP. Human OP cDNA given FLAG tag to C-terminus was subcloned to retrovirus vector (pBabe-puro). Selection was carried out with DMEM contained ouromyciiL Saos2 cell line that expressed OP-FLAG was cloned (Saos2-OP). Expression of OP was observed in only Saos2-OP by the RT-PCR and production of OP included FLAG tag was confirmed in only Saos2-OP. There was a significant difference between the numbers of Saos2-OP and parental cells attached to the titanium disc and polystyrene dish after 12 and 24 hours of incubation in serum-free DMEM, although there was no significance among the numbers of attached cells after 6 hours. Observation of SEM revealed the difference between the shapes of Saos2-OP and Saos2. The shape of Saos2-OP was smaller and thicker, compared with Saos2, and there were small global structure on the surface of Saos2-OP, but not on the surface of Saos2. These results suggested that human osteopontin is a potent enhancer of osteoblast-like cell adhesion to titanium, and that transfection of osteopontin influenced the shape of Saos2.