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The treatment of severe musculoskeletal injuries, such as loss of bone tissue and consolidation disorders, requires bone transplantation, and the success of this bone reconstruction depends on the grafts transplant's osteogenic, osteoconductive, and osteoinductive properties. Although the gold standard is autograft, it is limited by availability, morbidity, and infection risk. Despite their low capacity for osteoinduction and osteogenesis, decellularized bone allografts have been used in the search for alternative therapeutic strategies to improve bone regeneration. Considering that bone marrow stromal cells (BMSCs) are responsible for the maintenance of bone turnover throughout life, we believe that associating BMSCs with allograft could produce a material that is biologically similar to autologous bone graft. For this reason, this study evaluated the osteogenic potential of bone allograft cellularized with BMSCs. First, BMSC was characterized and allograft decellularization was confirmed by histology, scanning electron microscopy, and DNA quantification. Subsequently, the BMSCs and allografts were associated and evaluated for adhesion, proliferation, and in vitro and in vivo osteogenic potential. We demonstrated that, after 2 hours, BMSCs had already adhered to the surface of allografts and remained viable for 14 days. In vitro osteogenic assays indicated increased osteogenic potential of allografts compared with beta-tricalcium phosphate (β-TCP). In vivo transplantation assays in immunodeficient mice confirmed the allograft's potential to induce bone formation, with significantly better results than β-TCP. Finally, our results indicate that allograft can provide structural support for BMSC adhesion, offering a favorable microenvironment for cell survival and differentiation and inducing new bone formation. Taken together, our data indicate that this rapid methodology for cellularization of allograft with BMSCs might be a new therapeutic alternative in regenerative medicine and bone bioengineering., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to disclose., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)