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Keywords:

  • bone;
  • fracture;
  • regeneration;
  • hypoxia-inducible factor 1α;
  • apoptosis

Abstract

HIF-1α activates genes under hypoxia and was hypothesized to regulate bone regeneration. Surprisingly, HIF-1α+/− fracture calluses are larger, stronger, and stiffer than HIF-1α+/+ calluses because of decreased apoptosis. These data identify apoptosis inhibition as a means to enhance bone regeneration.

Introduction: Bone regeneration subsequent to fracture involves the synergistic activation of multiple signaling pathways. Localized hypoxia after fracture activates hypoxia-inducible factor 1α (HIF-1α), leading to increased expression of HIF-1 target genes. We therefore hypothesized that HIF-1α is a key regulator of bone regeneration.

Materials and Methods: Fixed femoral fractures were generated in mice with partial HIF-1α deficiency (HIF-1α+/−) and wildtype littermates (HIF-1α+/+). Fracture calluses and intact contralateral femurs from postfracture days (PFDs) 21 and 28 (N = 5–10) were subjected to μCT evaluation and four-point bending to assess morphometric and mechanical properties. Molecular analyses were carried out on PFD 7, 10, and 14 samples (N = 3) to determine differential gene expression at both mRNA and protein levels. Finally, TUNEL staining was performed on PFD 14 samples (N = 2) to elucidate differential apoptosis.

Results: Surprisingly, fracture calluses from HIF-1α+/− mice exhibited greater mineralization and were larger, stronger, and stiffer. Microarray analyses focused on hypoxia-induced genes revealed differential expression (between genotypes) of several genes associated with the apoptotic pathway. Real-time PCR confirmed these results, showing higher expression of proapoptotic protein phosphatase 2a (PP2A) and lower expression of anti-apoptotic B-cell leukemia/lymphoma 2 (BCL2) in HIF-1α+/+ calluses. Subsequent TUNEL staining showed that HIF-1α+/+ calluses contained larger numbers of TUNEL+ chondrocytes and osteoblasts than HIF-1α+/− calluses.

Conclusions: We conclude that partial HIF-1α deficiency results in decreased chondrocytic and osteoblastic apoptosis, thereby allowing the development of larger, stiffer calluses and enhancing bone regeneration. Furthermore, apoptosis inhibition may be a promising target for developing new treatments to accelerate bone regeneration.