title: The effect of intra-articular injection of microRNA-210 on ligament healing in a rat model.
abstract: BACKGROUND
  It is known from clinical and experimental studies that the healing potential of the anterior cruciate ligament (ACL) is extremely poor and that early phases of ligament healing require an augmented blood supply. MicroRNA (miRNA) is a type of small, noncoding RNA that negatively regulates gene expression, and miRNA (miR)-210 is reported to be crucial for cell response to hypoxia, vascular endothelial growth factor (VEGF)-driven endothelial cell migration, and formation of capillary-like structures.
  
  
  OBJECTIVE
  The purpose of this study was to examine the effect of intra-articular injection of miRNA miR-210 on acceleration of ACL healing.
  
  
  METHODS
  Controlled laboratory study.
  
  
  METHODS
  Two experiments were performed in this study. The ACLs of 12-week-old male LEW/CrlCrlj rats were partially transected. First, the temporal expression change of miR-210 after ACL injury was analyzed using real-time polymerase chain reaction (PCR) on day zero, and 1, 2, and 4 weeks after injury (n = 5 at each time point). Next, intra-articular injection of double-stranded (ds) miR-210 with atelocollagen was performed soon after injury. The control group was injected with control small interfering RNA (siRNA). Four weeks after injection, biomechanical and histological assessments of samples stained with H&E as well as Masson trichrome, and immunohistochemistry for VEGF, fibroblast growth factor 2 (FGF2), isolectin B4, and collagen type I, were performed. Real-time PCR analysis was also performed for quantitative evaluation of miR-210, VEGF-A, and collagen type I.
  
  
  RESULTS
  Real-time PCR analysis revealed that miR-210 expression was decreased soon after injury but gradually increased thereafter. Histological analysis confirmed that the transected area was covered with healing tissue in the miR-210 group but remained devoid of any tissue in the control group 4 weeks after injury. Biomechanical analysis confirmed the improvement of biomechanical properties in the miR-210 group; the ultimate failure loads 4 weeks after injection were 30.5 ± 3.1 N in the miR-210 group and 22.8 ± 3.1 N in the control group (P < .05). Real-time PCR analysis showed that endogenous miR-210, VEGF, and collagen type I were highly expressed compared with controls, and immunohistochemistry for VEGF, FGF2, isolectin B4, and collagen type I showed that VEGF and FGF2 were highly upregulated, and there were abundant blood vessels and fibrotic deposition in the miR-210 group.
  
  
  CONCLUSIONS
  Injection of ds miR-210 was effective in promoting the healing of partially torn ACLs through enhancement of angiogenesis via upregulation of VEGF and FGF2.
  
  
  CONCLUSIONS
  It might represent a potential therapeutic approach for treatment of ACL injury.
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Shoji
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Nakasa
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Yamasaki
authors.forename: Akira
authors.initials: A
authors.lastname: Kodama
authors.forename: Shigeru
authors.initials: S
authors.lastname: Miyaki
authors.forename: Takuya
authors.initials: T
authors.lastname: Niimoto
authors.forename: Atsushi
authors.initials: A
authors.lastname: Okuhara
authors.forename: Naosuke
authors.initials: N
authors.lastname: Kamei
authors.forename: Nobuo
authors.initials: N
authors.lastname: Adachi
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Ochi
mesh_terms: D000818:Animals; D016118:Anterior Cruciate Ligament / Q000187:drug effects*; D004195:Disease Models, Animal; D007270:Injections, Intra-Articular; D036341:Intercellular Signaling Peptides and Proteins / Q000008:administration & dosage / Q000494:pharmacology*; D008297:Male; D035683:MicroRNAs / Q000008:administration & dosage / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D014945:Wound Healing / Q000187:drug effects*
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; C546388:MIRN210 microRNA, rat; D035683:MicroRNAs