Department of Oral Implantology and Regenerative Dental Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
J Cell Mol Med. 2018 Feb;22(2):1127-1137. doi: 10.1111/jcmm.13366. Epub 2017 Oct 24.
Bisphosphonates impair function of osteoclasts and prevent bone resorption, the mechanism of which has been studied extensively. However, the possible effects of bisphosphonates on chondroblast differentiation and calcium deposition by osteoblasts have only been demonstrated recently. Moreover, cells from monocytic lineage are capable of stimulating osteoblast proliferation. Hence, susceptibility of osteoblasts to various factors requires further investigation. A primary culture of bone marrow-derived stromal cells was treated with liposomal clodronate (0.1, 0.5, or 1.0 mg/ml) or conditioned medium from liposomal clodronate. Liposomal clodronate (0.25 mg) was injected into mouse femur for in vivo experiments. The effects of liposomal clodronate were examined by alkaline phosphatase staining and/or activity assay, and real-time RT-PCR was used for studying the effect on osteogenic gene expression. Administration of liposomal clodronate to bone marrow-derived mesenchymal stromal cell culture enhanced alkaline phosphatase activity and mRNA levels of Runx2 and Dlx5. In addition, conditioned medium from liposomal clodronate also stimulated osteogenic characteristics similar to those of observed in vitro, and the number of exosomes in the conditioned medium was highest when pre-treated with liposomal clodronate. Western blot analysis revealed the presence of RANK proteins in exosomes collected from conditioned medium of liposomal clodronate. Identical observations were obtained in vivo, as liposomal clodronate-injected mouse femur showed increased alkaline phosphatase activity and Runx2 and Dlx5 mRNA expressions, even though the numbers of monocytes and macrophages were reduced. In conclusion, osteoblast differentiation was promoted via soluble RANK-containing exosomes in response to clodronates.
双膦酸盐会损害破骨细胞的功能并防止骨吸收，其机制已得到广泛研究。然而，双膦酸盐对成软骨细胞分化和破骨细胞钙沉积的可能影响最近才被证明。此外，单核细胞谱系的细胞能够刺激成骨细胞增殖。因此，成骨细胞对各种因素的敏感性需要进一步研究。用脂质体氯膦酸盐（0.1、0.5 或 1.0mg/ml）或脂质体氯膦酸盐的条件培养基处理骨髓源性基质细胞的原代培养物。将脂质体氯膦酸盐（0.25mg）注射到小鼠股骨中进行体内实验。通过碱性磷酸酶染色和/或活性测定检查脂质体氯膦酸盐的作用，并通过实时 RT-PCR 研究对成骨基因表达的影响。向骨髓源性间充质基质细胞培养物中给予脂质体氯膦酸盐可增强碱性磷酸酶活性和 Runx2 和 Dlx5 的 mRNA 水平。此外，脂质体氯膦酸盐的条件培养基也刺激了类似于体外观察到的成骨特征，并且在用脂质体氯膦酸盐预处理时，条件培养基中的外泌体数量最高。Western blot 分析显示，从脂质体氯膦酸盐条件培养基中收集的外泌体中存在 RANK 蛋白。在体内也得到了相同的观察结果，因为注射了脂质体氯膦酸盐的小鼠股骨显示碱性磷酸酶活性和 Runx2 和 Dlx5 mRNA 表达增加，尽管单核细胞和巨噬细胞的数量减少。总之，破骨细胞通过对氯膦酸盐的反应促进了成骨细胞分化。
