Inhibition of microRNA-222 expression accelerates bone healing with enhancement of osteogenesis, chondrogenesis, and angiogenesis in a rat refractory fracture model.

BACKGROUND: 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. METHOD: 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. RESULTS: 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. CONCLUSION: Local administration of miR-222 inhibitor can accelerate bone healing by enhancing osteogenesis, chondrogenesis, and angiogenesis in the rat refractory model.

The effect of administration of double stranded MicroRNA-210 on acceleration of Achilles tendon healing in a rat model.

BACKGROUND: Achilles tendons heal slower than other tissues, therefore requiring the developmnent of a strategy for accelerating the process. Vascular supply plays an important role in primary tendon healing, especially during the early healing phase. MicroRNA (miR)-210 has been reported as being crucial for angiogenesis, which is a key factor of tissue repair. We report herein that local injection of synthetic miR-210 into the injured Achilles tendon of a rat accelerated healing of the tendon. METHODS: Achilles tendons were transected and repaired via the Kessler suture technique in Sprague-Dawley rats. Then, double stranded (ds) miR-210 was injected into the repaired sites. The control group was injected with non-functioned dsRNA. At 2, 6 and 12 weeks, histological evaluations were performed. At two and six weeks, mechanical testing and angiogenesis were evaluated. Gene expression analysis using real-time polymerase chain reaction (PCR) and immunohistochemistry were performed at two weeks. RESULT: At two and six weeks, regular dense collagen tissue in the miR-210 group was observed and the diameter of collagen fiber in the miR-210 group was significantly higher than in the control. At two weeks, the ultimate failure load was significantly higher than in the control group, and expression of VEGF, FGF2 and type I collagen was upregulated. Abundant vessels in the miR-210 group were observed at two weeks, but there was no significant difference in vessel numbers between the two groups at six weeks. At 12 weeks, repaired Achilles tendons in the miR-210 group consisted of parallel and dense fibers, whereas wavy and loose fibers were still observed in the control group. CONCLUSION: The current study showed that single local injection of synthetic miR-210 promotes Achilles tendon healing in the early phase.

Acceleration of healing of the medial collateral ligament of the knee by local administration of synthetic microRNA-210 in a rat model.

BACKGROUND: Injury to the medial collateral ligament (MCL) of the knee joint is the most common ligament injury of the knee. Ligament healing generally takes a long time. Micro-ribonucleic acid (miRNA) is one of the noncoding RNAs and plays a crucial role in physiological function; miRNA (miR)-210 is known as a potent factor of angiogenesis, which is an important initiator of ligament healing. The purpose of this study is to examine the effect of local injection of double-stranded (ds) miR-210 on the healing of the MCL of rat knee joint. METHODS: MCLs of Sprague-Dawley rats were cut transversely. After the fascia and skin were sutured, dsmiR-210 or control dsRNA was injected into the injured site of MCL. At 2 weeks and 4 weeks, histological analysis and immunofluorescence staining of vascular endothelial growth factor, isolectin B4, collagen type 1, and Ki67 as well as a mechanical test were performed. Analysis of complementary deoxyribonucleic acid (cDNA) microarray data was performed at 1 week. RESULTS: Histological analysis showed that parallel fibres in the injured site were organised at 2 weeks and became thicker at 4 weeks in the miR-210-treated group, whereas the injured site in controls was filled with loose fibrous tissues and was thinner than that in the miR-210-treated group. The number of blood vessels in the miR-210-treated group was significantly higher than that in controls (p < 0.05), and vascular endothelial growth factor, Ki67, and collagen type 1 in the miR-210-treated group were intensely expressed in the repaired site as compared to the control group. The mechanical test indicated that the ultimate failure load in the miR-210-treated group was significantly higher than that in the control group at 2 weeks. The cDNA microarray analysis showed significant upregulation of genes related to cell proliferation and cell differentiation, and genes involved in negative regulation of apoptosis. CONCLUSION: This study showed that local injection of dsmiR-210 could accelerate MCL healing in rat, which is likely due to stimulation of angiogenesis at the healing site.

Intra-articular injection of synthetic microRNA-210 accelerates avascular meniscal healing in rat medial meniscal injured model.

INTRODUCTION: The important functions of the meniscus are shock absorption, passive stabilization and load transmission of the knee. Because of the avascularity of two-thirds of the meniscal center region, the treatment of tears in this area is hard. Recently, microRNAs have been proven to play an important role in the pathogenesis of diseases. We focused on microRNA (miR)-210, which plays a wide spectrum of roles comprising mitochondrial metabolism, angiogenesis, DNA repair and cell survival. This study aimed to investigate the effect of intra-articular injection of synthetic miR-210 on the injured meniscus in the avascular zone. METHODS: The middle segments of the medial meniscus of Spraque Dawley rats were incised longitudinally with a scalpel. An intra-articular injection of double-stranded (ds) miR-210 (for control group using control dsRNA) with atelocollagen was administered immediately after injury. Four weeks and 12 weeks after the injection, we conducted a histologic evaluation, immunohistochemical evaluation and Real-time PCR analysis. In vitro, the inner meniscus and synovial cells were isolated from rat knee joint, and were transfected with ds miR-210 or control dsRNA. Real-time PCR and immunohistochemical evaluations were performed. RESULTS: Twenty-four hours after the injection, FAM (Fluorescein amidite) labeled miR-210 was observed in the cells around the injured site. Four weeks after the injection, the injured site of the miR-210 group was filled with repaired tissue while that of the control was not repaired. In gene expression analysis of the meniscus, the expression of miR-210, Collagen type 2 alpha 1 (Col2a1), Vascular endothelial growth factor (VEGF), and Fibroblast growth factor-2 (FGF2) in the miR-210 group was significantly higher than that in the control. At 12 weeks, the intra-articular injection of miR-210 had healed the injured site of the meniscus and had prevented articular cartilage degeneration. In vitro, miR-210 upregulated Col2a1 expression in the meniscus cells and VEGF and FGF2 expression in the synovial cells. CONCLUSIONS: An intra-articular injection of ds miR-210 was effective in the healing of the damaged white zone meniscus through promotion of the collagen type 2 production from meniscus cells and through upregulated of VEGF and FGF2 from synovial cells.