Yoshizuka Masaaki, Nakasa Tomoyuki, Kawanishi Yoshitaka, Hachisuka Susumu, Furuta Taisuke, Miyaki Shigeru, Adachi Nobuo, Ochi Mitsuo
Department of Orthopedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
Department of Orthopedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
J Orthop Sci. 2016 Nov;21(6):852-858. doi: 10.1016/j.jos.2016.07.021. Epub 2016 Aug 18.
It is difficult to achieve bone union in case of non-union with non-invasive techniques. MicroRNAs (miRNAs) are short, non-coding RNAs that act as repressors of gene expression at the level of post-transcriptional regulation. This study focuses on microRNA (miR)-222 as it is known to be a negative modulator of angiogenesis, an essential component of fracture healing. The purpose of this study was to analyze the effects of miR-222 on osteogenic and chondrogenic differentiation in human mesenchymal stromal cell (MSC)s in vitro, and to determine whether local administration of miR-222 inhibitor into the fracture site could achieve bone union in vivo.
miR-222 expression in human bone marrow mesenchymal stem cells (hMSCs), and osteogenic differentiation in hMSCs, were investigated. The gain or loss of miR-222 function was examined, in order to assess the effects of miR-222 on osteogenic and chondrogenic differentiation in hMSCs. A femoral transverse fracture was completed in rats, and the periosteum at the fracture site was cauterized. Then, either an miR-222 inhibitor or an miR-222 mimics, mixed with atelocollagen, was administered into the fracture site. A non-functional inhibitor negative control was administered to the control group. At 2, 4, 6, and 8 weeks, radiographs of the fractured femurs were obtained. Immunohistochemistry was performed at 2 weeks to evaluate the capillary density. At 8 weeks, micro-computed tomography (μCT) imaging analysis and histological evaluations were performed.
The expression of miR-222 significantly decreased as osteogenic differentiation of hMSCs proceeded. Inhibition of miR-222 promoted osteogenic differentiation, and over expression of miR-222 inhibited osteogenic differentiation in hMSCs, which was confirmed by measuring expression of Runx2, collagen type 1A1 (COL1A1), and osteocalcin. Inhibition of miR-222 promoted chondrogenic differentiation in hMSCs, which was confirmed by measuring expression of collagen type II (COL2A1), aggrican, and SOX9. Bone union at the fracture site was achieved in only the groups treated with the miR-222 inhibitor, confirmed by radiographic, μCT and histological evaluation at 8 weeks after administration. Immunohistochemistry showed that capillary density in the miR-222 inhibitor group was significantly higher than that in the control group and in the miR-222 mimics group.
Local administration of miR-222 inhibitor can accelerate bone healing by enhancing osteogenesis, chondrogenesis, and angiogenesis in the rat refractory model.
对于骨不连病例，采用非侵入性技术实现骨愈合较为困难。微小RNA（miRNA）是短链非编码RNA，在转录后水平作为基因表达的抑制因子发挥作用。本研究聚焦于miR-222，因为已知其是血管生成的负性调节因子，而血管生成是骨折愈合的重要组成部分。本研究的目的是分析miR-222对人骨髓间充质干细胞（MSC）体外成骨和软骨分化的影响，并确定向骨折部位局部给予miR-222抑制剂是否能在体内实现骨愈合。
研究了人骨髓间充质干细胞（hMSC）中miR-222的表达以及hMSC中的成骨分化。检测miR-222功能的获得或缺失，以评估miR-222对hMSC成骨和软骨分化的影响。对大鼠造成股骨横行骨折，并烧灼骨折部位的骨膜。然后，将miR-222抑制剂或miR-222模拟物与去端胶原蛋白混合后注入骨折部位。对照组给予无功能的抑制剂阴性对照。在第2、4、6和8周，获取骨折股骨的X线片。在第2周进行免疫组织化学检测以评估毛细血管密度。在第8周，进行微计算机断层扫描（μCT）成像分析和组织学评估。
随着hMSC成骨分化的进行，miR-222的表达显著降低。抑制miR-222可促进hMSC的成骨分化，而miR-222的过表达则抑制hMSC的成骨分化，这通过检测Runx2、1A1型胶原蛋白（COL1A1）和骨钙素的表达得以证实。抑制miR-222可促进hMSC的软骨分化，这通过检测II型胶原蛋白（COL2A1）、聚集蛋白聚糖和SOX9的表达得以证实。仅在给予miR-222抑制剂治疗的组中实现了骨折部位的骨愈合，给药后8周的X线、μCT和组织学评估证实了这一点。免疫组织化学显示，miR-222抑制剂组的毛细血管密度显著高于对照组和miR-222模拟物组。
在大鼠难治性模型中，局部给予miR-222抑制剂可通过增强成骨、软骨生成和血管生成来加速骨愈合。
