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. Questions/purposes 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 TUNELpositive 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 -015-4172-6 Clinical Orthopaedics and Related Research ® A Publication of The Association of Bone and Joint Surgeons® 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...

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In Legg-Calvé-Perthes disease, loss of blood supply results in ischemic osteonecrosis of the femoral head (ONFH). Generally, macrophages play important roles in inflammatory responses to tissue necrosis, but their role in ONFH is not known. The purpose of this study was to determine the macrophage-inflammatory responses after ONFH and the receptor mechanisms involved in sensing the necrotic bone, using a piglet model of Legg-Calvé-Perthes disease. Induction of ONFH resulted in increased numbers of CD14 macrophages in the fibrovascular repair tissue compared with normal, as determined by immunohistochemistry. Quantitative real-time PCR analysis of macrophages isolated by laser capture microdissection showed significantly increased expression of proinflammatory cytokines IL-1β, tumor necrosis factor-α, and IL-6 in ONFH compared with normal. Because Toll-like receptors (TLRs) mediate macrophage-inflammatory responses in other inflammatory conditions, we determined their gene expression in macrophages and found significantly increased levels of TLR4 but not TLR2 and TLR9 in ONFH. Mechanistically, in vitro, bone marrow-derived macrophages treated with necrotic bone showed increased extracellular signal-regulated kinases 1/2 and Iκ kinase-α phosphorylation, increased proliferation, migration, and inflammatory cytokine expression, which were blocked by TLR4 inhibitor, TAK242, and by TLR4 ablation in macrophages using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease method. In conclusion, necrotic bone stimulates macrophage-inflammatory responses through TLR4 activation.

Legg-Calvé-Perthes disease (LCPD) is a childhood hip disorder of ischemic osteonecrosis of the femoral head. Hip joint synovitis is a common feature of LCPD, but the nature and pathophysiology of the synovitis remain unknown. The purpose of this study was to determine the chronicity of the synovitis and the inflammatory cytokines present in the synovial fluid at an active stage of LCPD. Serial MRI was performed on 28 patients. T2-weighted and gadolinium-enhanced MR images were used to assess synovial effusion and synovial enhancement (hyperemia) over time. A multiple-cytokine assay was used to determine the levels of 27 inflammatory cytokines and related factors present in the synovial fluid from 13 patients. MRI analysis showed fold increases of 5.0 AE 3.3 and 3.1 AE 2.1 in the synovial fluid volume in the affected hip compared to the unaffected hip at the initial and the last follow-up MRI, respectively. The mean duration between the initial and the last MRI was 17.7 AE 8.3 months. The volume of enhanced synovium on the contrast MRI was increased 16.5 AE 8.5 fold and 6.3 AE 5.6 fold in the affected hip compared to the unaffected hip at the initial MRI and the last follow-up MRI, respectively. In the synovial fluid of the affected hips, IL-6 protein levels were significantly increased (LCPD: 509 AE 519 pg/mL, non-LCPD: 19 AE 22 pg/mL; p ¼ 0.0005) on the multi-cytokine assay. Interestingly, IL-1b and TNF-a levels were not elevated. In the active stage of LCPD, chronic hip synovitis and significant elevation of IL-6 are produced in the synovial fluid. Further studies are warranted to investigate the role of IL-6 on the pathophysiology of synovitis in LCPD and how it affects bone healing.

In order to investigate the mechanisms of collapse in osteonecrosis of the femoral head, we examined which part of the femoral head was the key point of a collapse and whether a collapsed region was associated with the size of the necrotic lesion. Using 30 consecutive surgically removed femoral heads we retrospectively analysed whole serial cut sections, specimen photographs, specimen radiographs and histological sections. In all of the femoral heads, collapse consistently involved a fracture at the lateral boundary of the necrotic lesion. Histologically, the fractures occurred at the junction between the thickened trabeculae of the reparative zone and the necrotic bone trabeculae. When the medial boundary of the necrotic lesion was located lateral to the fovea of the femoral head, 18 of 19 femoral heads collapsed in the subchondral region. By contrast, when the medial boundary was located medial to the fovea, collapse in the subchondral region was observed in four of 11 femoral heads (p = 0.0011). We found that collapse began at the lateral boundary of the necrotic lesion and that the size of the necrotic lesion seemed to contribute to its distribution.

Both acetabular head index and band length ratio are important prognostic factors in the care of patients with subchondral insufficiency fractures of the femoral head.

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