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Shared cellular characteristics of dysfunctional stromal cell differentiation underpin impaired healing in two models of perturbed fracture healing

ABSTRACT: 10% of the fifteen million bone fractures each year that occur in the US fail to heal, which leads to significant loss of function, morbidity, and mortality. However, very little is known about the cellular and molecular mechanisms underpinning inferior healing. Two perturbed fracture models, surgically induced ischemia and the genetic deletion of the self-antigen CD47, were studied to identify unifying cellular and molecular mechanisms. C57Bl/6 wild-type (WT) mice with intact vasculature (WT Intact), WT mice with induced ischemia (WT Ischemic), and CD47-null mice with intact vasculature (CD47-KO) underwent transverse tibial fractures. Single-cell RNA-sequencing (scRNAseq) was performed during the inflammatory stage (4 days post-fracture) and the stromal stage (7 days post fracture) to assess transcriptomic alterations in perturbed fracture calluses.

ORGANISM(S): Mus musculus

PROVIDER: GSE292578 | GEO | 2026/01/10

REPOSITORIES: GEO

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Project description:HIF-1a activates genes under hypoxia and was hypothesized to regulate bone regeneration. Surprisingly, HET HIF-1a fracture calluses are larger, stronger and stiffer than WT HIF-1a calluses due to 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 following fracture activates hypoxia-inducible factor 1 alpha (HIF-1a) leading to increased expression of HIF-1 target genes. We therefore hypothesized that HIF-1a is a key regulator of bone regeneration Materials and Methods: Fixed femoral fractures were generated in mice with partial HIF-1a deficiency (HET HIF-1a) and wild type littermates (WT HIF-1a). Fracture calluses and intact contralateral femurs from post fracture day (PFD) 21 and 28 (N=5-10) were subjected to MicroCT evaluation and 4-point bending in order 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 HET HIF-1a mice exhibit greater mineralization and are 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, demonstrating higher expression of pro-apoptotic PP2A and lower expression of anti-apoptotic BCL2 in WT HIF-1a calluses. Subsequent TUNEL staining demonstrates that WT HIF-1a calluses contain larger numbers of TUNEL positive chondrocytes and osteoblasts than HET HIF-1a calluses. Conclusions: We conclude that partial HIF-1a 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. Keywords: Bone, Fracture, Apoptosis, Hypoxia, Microarray

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Shared cellular characteristics of dysfunctional stromal cell differentiation underpin impaired healing in two models of perturbed fracture healing

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