Tsukanaka Masako, Yamamoto Koji, Fujibayashi Shunsuke, Pattanayak Deepak K, Matsushita Tomiharu, Kokubo Tadashi, Matsuda Shuichi, Akiyama Haruhiko
Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Shogoinkawahara-cho 54, Sakyo-ku, Kyoto, 606-8507, Japan.
J Bone Miner Metab. 2014 Nov;32(6):660-70. doi: 10.1007/s00774-013-0544-8. Epub 2013 Dec 6.
Stimulation of osteoblast proliferation and differentiation is important for the in vivo bone-bonding ability of biomaterials. Previous in vitro studies have used biochemical assays to analyze osteoblast-specific gene expression in cultured osteoblasts. In this study, we generated transgenic mice harboring a monomeric red fluorescent protein 1 transgene under the control of a 2.3-kb fragment of the Col1a1 promoter, which is active specifically in osteoblasts and osteocytes. We established a fluorescent primary osteoblast culture system to allow noninvasive observation of osteoblast proliferation and differentiation on opaque materials in vitro. We used this system to evaluate alkali- and heat-treated titanium, which has a strong bone-bonding ability in vivo, and we observed a rapid increase in fluorescence intensity and characteristic multifocal nodule formation. A cell proliferation assay and RT-PCR to examine osteoblast-specific gene expression showed increased osteoblast proliferation and differentiation consistent with the fluorescence observations. This mouse model allowed us to use fluorescence intensity to visualize and quantify in vivo newly formed bone around implanted materials in femurs. The use of these fluorescent osteoblasts is a promising method for simple screening of the bone-bonding ability of new materials.
刺激成骨细胞增殖和分化对于生物材料的体内骨结合能力很重要。先前的体外研究已使用生化分析来分析培养的成骨细胞中与成骨细胞特异性相关的基因表达。在本研究中，我们构建了转基因小鼠，其携带在Col1a1启动子的2.3 kb片段控制下的单体红色荧光蛋白1转基因，该启动子在成骨细胞和骨细胞中特异性活跃。我们建立了荧光原代成骨细胞培养系统，以便在体外对不透明材料上的成骨细胞增殖和分化进行无创观察。我们使用该系统评估了在体内具有强大骨结合能力的碱处理和热处理钛，并且观察到荧光强度迅速增加以及形成特征性的多灶性结节。细胞增殖分析和用于检测成骨细胞特异性基因表达的RT-PCR显示，成骨细胞增殖和分化增加，这与荧光观察结果一致。这种小鼠模型使我们能够利用荧光强度来可视化和量化股骨中植入材料周围的体内新形成骨。使用这些荧光成骨细胞是一种很有前景的方法，可用于简单筛选新材料的骨结合能力。
