Gypsum-calcium carbonate-gelatine composite graft for bone repair: Biocompatibility and histological assessment in a rat model

Background Gypsum (calcium sulphate dihydrate; POP) is a resorbable synthetic bone substitute with favourable biocompatibility; however, its rapid degradation limits clinical effectiveness. A modified composite incorporating calcium carbonate and gelatine hydrogel (POPHD) was developed to improve degradation control and osteogenic performance. Objective This study aimed to evaluate and compare the biocompatibility and histological response of POP and POPHD in vitro and in vivo, with emphasis on cytotoxicity, soft-tissue response, and bone regeneration capacity. Methods Cytotoxicity was assessed using the MTT assay on NIH3T3 fibroblast cells after 24 h of exposure. Twenty male Wistar rats were used for in vivo evaluation, comprising primary dermal irritation testing, subcutaneous implantation (14 days), and femoral condyle implantation (3 and 30 days). Histological sections were stained with haematoxylin-eosin and Mallory's trichrome and evaluated using validated grading scales. Results Both materials demonstrated no cytotoxicity (cell viability >80%) and caused no skin irritation (PDII score 0). Subcutaneous implantation revealed comparable soft-tissue responses, though POPHD showed significantly greater angiogenesis (p = 0.003) and foreign body giant cell formation (p = 0.035), consistent with slower degradation. At 3 days post-femoral implantation, both groups showed similar early inflammatory responses. At 30 days, POPHD demonstrated superior bone formation quality and quantity (Score 3 vs Score 2) and enhanced angiogenic activity (p = 0.008) compared to POP. Conclusions Modification of gypsum with calcium carbonate-gelatine hydrogel preserved biocompatibility while reducing degradation rate and promoting more advanced bone regeneration, suggesting POPHD as a promising synthetic bone graft substitute warranting further long-term investigation.