Deep-freezing and decontamination strategy for a large autologous bone graft with presentation of the osteogenic potential of resident osteoblasts.

BACKGROUND: Musculoskeletal injuries resulting from high-impact trauma often lead to extensive bone defects that require effective reconstruction to prevent limb amputation. However, contamination of large autologous bone grafts in open fractures remains a major clinical challenge. This study aimed to establish a reliable protocol for decontamination of massive autologous bone graft and to assess the viability and regenerative potential of osteoblasts from frozen bone, including the effects of deep freezing and the cryoprotectant dimethyl sulfoxide (DMSO). METHODS: A 28 cm femoral segment, extruded during a high-energy road traffic accident, was stored at - 80 °C for 15 days before microbiological analysis revealed contamination. The bone was thawed, decontaminated, and successfully reimplanted. To validate this protocol, femoral head fragments were experimentally contaminated with various microbial strains and treated using the same procedure. To evaluate how low-temperature storage (15 and 30 days, with or without DMSO) impacts osteoblasts, cells were isolated from both fresh and frozen bone and analyzed using a panel of cellular and molecular biology techniques, including histology, flow cytometry, immunofluorescence, gene expression analysis, and real-time imaging. RESULTS: The decontamination protocol effectively eliminated microbial contamination from both the clinical autograft and experimentally treated femoral head fragments, with minimal antibiotic residues. Histological analysis confirmed preservation of osteoblasts, osteocytes, and chondrocytes in frozen bone. Osteoblasts from frozen bone retained proliferative and differentiation capacities comparable to those from fresh bone, with no evidence of mitotic chromosomal instability. Cryopreservation with DMSO-containing media slightly increased population doubling time. Expression of RUNX2 was highest in fresh and 15-day frozen bone, while reduced in 30-day samples. No significant differences were observed between DMSO and non-DMSO groups regarding osteogenic potential. ALPL and COL1A1 expression significantly increased after 14 days of osteogenic induction. Time-lapse imaging demonstrated active osteoblast migration and division, and the presence of extracellular vesicles and nanotubes indicated functional cellular communication. CONCLUSIONS: This proof-of-concept study demonstrates that large autologous bone grafts can be frozen and subsequently decontaminated while retaining viable osteoblasts with high proliferative and regenerative potential. By combining a unique and successful clinical case with controlled experimental analyses, the study offers translational relevance, and the findings on osteoblast viability after freezing are promising.