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粘附机制。首先，研究了生物材料的差异对成骨细胞粘附的影响。人骨肉瘤细胞系Saos 2与无血清培养的羟基磷灰石和钛的粘附率相同，而与有血清培养的钛的粘附率高于与羟基磷灰石的粘附率。根据这些结果，我们推测血清中的蛋白质影响细胞对生物材料的粘附。我们将骨桥蛋白（osteopontin，OP）转染到Saos 2细胞中。OP是骨基质中的一种非胶原蛋白。有报道称OP在体外可增强成骨样细胞的粘附活性。将人OP cDNA克隆到逆转录病毒载体pBabe-puro上，构建成骨样细胞（osteoblastlikecells，OP），观察OP转染对成骨样细胞与钛粘附活性的影响。用含有尿霉素的DMEM进行选择，克隆表达OP-FLAG的Saos 2细胞系（Saos 2-OP）。通过RT-PCR仅在Saos 2-OP中观察到OP的表达，并且仅在Saos 2-OP中确认了包括FLAG标签的OP的产生。在无血清DMEM中孵育12和24小时后，附着于钛盘和聚苯乙烯盘的Saos 2-OP和亲本细胞的数量之间存在显著差异，尽管在6小时后附着的细胞的数量之间没有显著性。扫描电镜观察显示Saos 2-OP与Saos 2的形态不同。与Saos 2相比，Saos 2-OP的形状更小、更厚，表面有小的球形结构，而Saos 2表面没有。这些结果表明，人骨桥蛋白是成骨细胞样细胞粘附钛的有效增强剂，骨桥蛋白的转染影响Saos 2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.