title: Inhibition of microRNA-222 expression accelerates bone healing with enhancement of osteogenesis, chondrogenesis, and angiogenesis in a rat refractory fracture model.
abstract: 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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  Local administration of miR-222 inhibitor can accelerate bone healing by enhancing osteogenesis, chondrogenesis, and angiogenesis in the rat refractory model.
authors.affiliation: Department of Orthopedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. Electronic address: yoshizukamasaaki@yahoo.co.jp.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Yoshizuka
authors.affiliation: Department of Orthopedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Nakasa
authors.affiliation: Department of Orthopedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Kawanishi
authors.affiliation: Department of Orthopedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Hachisuka
authors.affiliation: Department of Orthopedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Taisuke
authors.initials: T
authors.lastname: Furuta
authors.affiliation: Department of Regenerative Medicine, Hiroshima University Hospital, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Miyaki
authors.affiliation: Department of Orthopedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Adachi
authors.affiliation: Department of Orthopedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Ochi
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000187:drug effects* / Q000235:genetics; D004195:Disease Models, Animal; D005264:Femoral Fractures / Q000188:drug therapy* / Q000601:surgery; D017102:Fracture Healing / Q000187:drug effects / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D015552:Injections, Intralesional; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000235:genetics*; D058990:Molecular Targeted Therapy; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000235:genetics; D010012:Osteogenesis / Q000187:drug effects* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012016:Reference Values; D014162:Transfection
chemical_list: D035683:MicroRNAs