HIF-1alpha has been shown to be a central mediator of cellular response to hypoxia. The role it plays after ischemic injury to the immature femoral head is unknown. The purpose of this study was to determine the region of the femoral head affected by hypoxia following ischemic injury to the immature femoral head and to determine the site of HIF-1alpha activation and revascularization. We hypothesize that the epiphyseal cartilage, rather than the bony epiphysis, is the site of HIF-1alpha activation following ischemic osteonecrosis and that the epiphyseal cartilage plays an important role in the revascularization process.
Materials and methods: Femoral head osteonecrosis was surgically induced in 56 immature pigs. Hypoxyprobe staining, cell viability assay, HIF-1alpha western blot, RT-qPCR of HIF-1alpha, VEGF, VEGFR2, and PECAM, and micro-CT assessments of microfil-infused femoral heads were performed.
Results: Severe hypoxia was present in the bony epiphysis and the lower part of the epiphyseal cartilage following ischemia. In the bony epiphysis, extensive cell death and tissue necrosis was observed with degradation of proteins and RNAs which precluded further analysis. In the epiphyseal cartilage, the loss of cell viability was limited to its deep layer with the remainder of the cartilage remaining viable. Furthermore, the cartilage from the ischemic side showed a significant increase in HIF-1alpha protein level and HIF-1alpha expression. VEGF expression in the cartilage was dramatically and significantly increased at 24 h, 2 and 4 weeks (p<0.05 for all) with 5 to 10 fold increase being observed on the ischemic side compared to the normal side. PECAM and VEGFR2 expressions in the cartilage were both significantly decreased at 24 h but returned to the normal levels by 2 and 4 weeks, respectively. Micro-CT showed revascularization of the cartilage on the ischemic side with the vessel volume/total volume equaling the normal side by 4 weeks.
Conclusions: Acute ischemic injury to the immature femoral head induced severe hypoxia and cell death in the bony epiphysis and the deep layer of the epiphyseal cartilage. Viable chondrocytes in the superficial layer of the epiphyseal cartilage showed HIF-1alpha activation and VEGF upregulation with subsequent revascularization occurring in the cartilage.