Material properties of bone in the femoral head treated with ibandronate and BMP-2 following ischemic osteonecrosis

Bone morphogenetic protein (BMP)-2 and ibandronate (IB) decrease the femoral head deformity following ischemic osteonecrosis of the femoral head (ONFH). The purpose of this study was to determine the effects of BMP-2 and IB on the mineral content and nanoindentation properties of the bone following ONFH. ONFH was surgically induced in a femoral head of piglets.

Search for a reliable model for bisphosphonate-related osteonecrosis of the jaw: establishment of a model in pigs and description of its histomorphometric characteristics

The pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ) remains unknown, and the development of a reliable experimental model would help to improve our understanding of it. 

In vivo monitoring of activated macrophages and neutrophils in response to ischemic osteonecrosis in a mouse model


Matthew C. Phipps, YiHui Huang, Ryosuke Yamaguchi, Nobuhiro Kamiy, Naga S. Adapala, Liping Tang, and Harry K. W. Kim


Ischemic osteonecrosis (IO) is caused by disruption of the blood supply to bone. It is a debilitating condition with pathological healing characterized by excessive bone resorption and delayed osteogenesis. Although the majority of research has focused on the role of osteoblasts and osteoclasts in the disease progression, we hypothesize that innate immune cells, macrophages and neutrophils, play a significant role. With the recent development of real-time imaging probes for neutrophils and macrophages, the purpose of this study was to investigate the kinetic immune cell response in a mouse model of IO. Our results show that induction of IO leads to a significant accumulation of activated neutrophils and macrophages at the affected tissue by 48 h after surgery. Additionally, the accumulation of these immune cells remained elevated in comparison to sham controls for up to 6 weeks, indicative of chronic inflammation. Immunohistochemistry confirmed the immune cell infiltration into the necrotic bone marrow and the increased presence of TNFα-positive cells, demonstrating, for the first time, a direct response of these cells to ischemia induced necrotic bone. These new findings support a hypothesis that IO is an osteoimmunologic condition where innate immune cells play a significant role in the chronic inflammation.

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Development of a Mouse Model of Ischemic Osteonecrosis


Nobuhiro Kamiya MD, PhD, Ryosuke Yamaguchi MD, PhD, Olumide Aruwajoye MS, Naga Suresh Adapala PhD, Harry K. W. Kim MD, MS


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 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).

Conclusion 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.

Clinical Relevance The new mouse model may be a useful tool to test potential therapeutic strategies to improve bone healing after ischemic osteonecrosis.

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Loss of HIF-1α in the Notochord Results in Cell Death and Complete Disappearance of the Nucleus Pulposus


Christophe Merceron, Laura Mangiavini, Alexander Robling, Tremika LeShan Wilson, Amato J. Giaccia, Irving M. Shapiro, Ernestina Schipani, Makarand V. Risbud


The intervertebral disc (IVD) is one of the largest avascular organs in vertebrates. The nucleus pulposus (NP), a highly hydrated and proteoglycan-enriched tissue, forms the inner portion of the IVD. The NP is surrounded by a multi-lamellar fibrocartilaginous structure, the annulus fibrosus (AF). This structure is covered superior and inferior side by cartilaginous endplates (CEP). The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome. The hypoxia inducible factor-1α (HIF-1α) is expressed in NP cells but its function in NP development and homeostasis is largely unknown. We thus conditionally deleted HIF-1α in notochordal cells and investigated how loss of this transcription factor impacts NP formation and homeostasis at E15.5, birth, 1 and 4 months of age, respectively. Histological analysis, cell lineage studies, and TUNEL assay were performed. Morphologic changes of the mutant NP cells were identified as early as E15.5, followed, postnatally, by the progressive disappearance and replacement of the NP with a novel tissue that resembles fibrocartilage. Notably, lineage studies and TUNEL assay unequivocally proved that NP cells did not transdifferentiate into chondrocyte-like cells but they rather underwent massive cell death, and were completely replaced by a cell population belonging to a lineage distinct from the notochordal one. Finally, to evaluate the functional consequences of HIF-1α deletion in the NP, biomechanical testing of mutant IVD was performed. Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress. These findings are similar to the changes usually observed during human IVD degeneration. Our study thus demonstrates that HIF-1α is essential for NP development and homeostasis, and it raises the intriguing possibility that this transcription factor could be involved in IVD degeneration in humans.

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Local Administration of Bone Morphogenetic Protein-2 and Bisphosphonate During Non-Weight-Bearing Treatment of Ischemic Osteonecrosis of the Femoral Head


Harry K.W. Kim, MD, MS; Olumide Aruwajoye, MS; Justin Du ; Nobuhiro Kamiya, MD, PhD


Background: Non-weight-bearing decreases the femoral head deformity but increases bone resorption without increasing bone formation in an experimental animal model of Legg-Calvé-Perthes disease. We sought to determine if local administration of bone morphogenetic protein (BMP)-2 with or without bisphosphonate can increase the bone formation during the non-weight-bearing treatment in the large animal model of Legg-Calvé-Perthes disease.

Methods: Eighteen piglets were surgically induced with femoral head ischemia. Immediately following the surgery, all animals received an above-the-knee amputation to enforce local non-weight-bearing (NWB). One to two weeks later, six animals received local BMP-2 to the necrotic head (BMP group), six received local BMP-2 and ibandronate (BMP+IB group), and the remaining six received no treatment (NWB group). All animals were killed at eight weeks after the induction of ischemia. Radiographic, microcomputed tomography (micro-CT), and histomorphometric assessments were performed.

Results: Radiographic assessment showed that the femoral heads in the NWB, BMP, and BMP+IB groups had a decrease of 20%, 14%, and 10%, respectively, in their mean epiphyseal quotient in comparison with the normal control group. Micro-CT analyses showed significantly higher femoral head bone volume in the BMP+IB group than in the BMP group (p = 0.02) and the NWB group (p < 0.001). BMP+IB and BMP groups had a significantly higher trabecular number (p < 0.01) and lower trabecular separation (p < 0.02) than the NWB group. In addition, the osteoclast number per bone surface was significantly lower in the BMP+IB group compared with the NWB group. Calcein labeling showed significantly higher bone formation in the BMP and BMP+IB groups than in the NWB group (p < 0.05). Heterotopic ossification was found in the capsule of four hips in the BMP+IB group but not in the BMP group.

Conclusions: Administration of BMP-2 with bisphosphonate best decreased bone resorption and increased new bone formation during non-weight-bearing treatment of ischemic osteonecrosis in a pig model, but heterotopic ossification is a concern.

Clinical Relevance: This preclinical study provides new evidence that BMP-2 with bisphosphonate can effectively prevent the extreme bone loss associated with the non-weight-bearing treatment and increase new bone formation in the femoral head in this animal model of ischemic osteonecrosis.

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