title: Development of a mouse model of ischemic osteonecrosis.
abstract: BACKGROUND
  Availability of a reliable mouse model of ischemic osteonecrosis could accelerate the development of novel therapeutic strategies to stimulate bone healing after ischemic osteonecrosis; however, no mouse model of ischemic osteonecrosis is currently available.
  
  
  OBJECTIVE
  To develop a surgical mouse model of ischemic osteonecrosis, we asked, (1) if the blood vessels that contribute to the blood supply of the distal femoral epiphysis are cauterized, can we generate an osteonecrosis mouse model; (2) what are the histologic changes observed in this mouse model, and (3) what are the morphologic changes in the model.
  
  
  METHODS
  We performed microangiography to identify blood vessels supplying the distal femoral epiphysis in mice, and four vessels were cauterized using microsurgical techniques to induce ischemic osteonecrosis. Histologic assessment of cell death in the trabecular bone was performed using terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) and counting the empty lacunae in three serial sections. Quantitation of osteoclast and osteoblast numbers was performed using image analysis software. Morphologic assessments of the distal femoral epiphysis for deformity and for trabecular bone parameters were performed using micro-CT.
  
  
  RESULTS
  We identified four blood vessels about the knee that had to be cauterized to induce total ischemic osteonecrosis of the distal femoral epiphysis. Qualitative assessment of histologic sections of the epiphysis showed a loss of nuclear staining of marrow cells, disorganized marrow structure, and necrotic blood vessels at 1 week. By 2 weeks, vascular tissue invasion of the necrotic marrow space was observed with a progressive increase in infiltration of the necrotic marrow space with the vascular tissue at 4 and 6 weeks. TUNEL staining showed extensive cell death in the marrow and trabecular bone 24 hours after the induction of ischemia. The mean percent of TUNEL-positive osteocytes in the trabecular bone increased from 2% ± 1% in the control group to a peak of 98% ± 3% in the ischemic group 1 week after induction of ischemia (mean difference, 96%; 95% CI, 81%-111%; p < 0.0001). The mean percent of empty lacunae increased from 1% ± 1% in the control group to a peak of 78% ± 15% in the ischemic group at 4 weeks (mean difference, 77%; 95% CI, 56%-97%; p < 0.0001). Quantitative analysis showed that the mean number of osteoclasts per bone surface was decreased in the ischemic group at 1, 2, and 4 weeks (p < 0.0001, < 0.0001, and p = 0.02, respectively) compared with the control group. The mean number of osteoclasts increased to a level similar to that of the control group at 6 weeks (p = 0.23). The numbers of osteoblasts per bone surface were decreased in the ischemic group at 1, 2 and 4 weeks (p < 0.0001 for each) compared with the numbers in the control group. The mean number of osteoblasts also increased to a level similar to that of the control group at 6 weeks (p = 0.91). Mean bone volume percent assessed by micro-CT was lower in the ischemic group compared with the control group from 2 to 6 weeks. The mean differences in the percent bone volume between the control and ischemic groups at 2, 4, and 6 weeks were 5.5% (95% CI, 0.9%-10.2%; p = 0.01), 5.3% (95% CI, 0.6%-9.9%; p = 0.02), and 6.0% (95% CI, 1.1%-10.9%; p = 0.008), respectively. A deformity of the distal femoral epiphysis was observed at 6 weeks with the mean epiphyseal height to width ratio of 0.74 ± 0.03 in the control group compared with 0.66 ± 0.06 in the ischemic group (mean difference, 0.08; 95% CI, 0.00-0.16; p = 0.03).
  
  
  CONCLUSIONS
  We developed a novel mouse model of ischemic osteonecrosis that produced extensive cell death in the distal femoral epiphysis which developed a deformity with time.
  
  
  CONCLUSIONS
  The new mouse model may be a useful tool to test potential therapeutic strategies to improve bone healing after ischemic osteonecrosis.
authors: Center of Excellence in Hip Disorders, Texas Scottish Rite Hospital for Children, 2222 Welborn Street, Dallas, TX, 75219, USA.
authors: Nobuhiro
authors: N
authors: Kamiya
authors: Ryosuke
authors: R
authors: Yamaguchi
authors: Olumide
authors: O
authors: Aruwajoye
authors: Naga Suresh
authors: NS
authors: Adapala
authors: Harry K W
authors: HK
authors: Kim
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D004838:Epiphyses / Q000098:blood supply; D005269:Femur / Q000098:blood supply / Q000473:pathology*; D006614:Hindlimb / Q000098:blood supply; D007091:Image Processing, Computer-Assisted; D020287:In Situ Nick-End Labeling; D007511:Ischemia / Q000473:pathology; D008297:Male; D008807:Mice, Inbred BALB C; D010020:Osteonecrosis / Q000473:pathology*; D014057:Tomography, X-Ray Computed