CO₂-releasing hydrogels promote bone healing by activating transcription factor IRF1 to regulate HOXA2

Bone repair is a complex repair process involving various factors. Carbon dioxide (CO₂) therapy has been shown to accelerate fracture repair and bone healing. The present study aimed to explore the effects of CO₂-releasing hydrogels on osteoblast formation and the involvement of interferon regulatory factor-1 (IRF1) and homeobox A2 (HOXA2) during this process. The effects of CO₂-releasing hydrogels on MC3T3-E1 cells were evaluated using a CCK-8 assay for cell proliferation, Western blotting for protein expression, and alizarin red S staining for osteogenic capacity. The relationship between IRF1 and HOXA2 was assessed by bioinformatics analysis, dual-luciferase reporter assays, and chromatin immunoprecipitation assays. Co-transfection experiments were conducted to investigate the impact of HOXA2 overexpression on osteoblast formation under IRF1 knockdown conditions. A mouse bone defect model was established to validate the effects of CO₂-releasing hydrogels and the involvement of IRF1 and HOXA2 in vivo. CO₂-releasing hydrogels positively regulated MC3T3-E1 cell proliferation, bone formation-related protein expression, ALP activity, and osteogenic capacity. IRF1 was significantly upregulated in hydrogel-treated cells and demonstrated a pivotal role in osteoblastogenesis. IRF1 targeted and regulated the expression of HOXA2. Importantly, overexpression of HOXA2 mitigated the effects of IRF1 knockdown on osteoblast formation. In the in vivo mouse model, CO₂-releasing hydrogels accelerated bone healing by promoting collagen deposition, bone mineralization, and upregulating bone formation-related factors. This study highlights the significant potential of CO₂-releasing hydrogels and unveiled the regulatory effects of IRF1 and HOXA2 in promoting osteoblast differentiation and bone healing. These findings may facilitate the development of therapeutic strategies aimed at enhancing bone regeneration and promoting effective healing.