Decellularization of Massive Bone Allografts By Perfusion: A New Protocol for Tissue Engineering

In terms of large bone defect reconstructions, massive bone allografts may sometimes be the only solution. However, they are still burdened with a high postoperative complication rate. Our hypothesis is that the immunogenicity of residual cells in the graft is involved in this issue. Decellularization by perfusion might therefore be the answer to process and create more biologically effective massive bone allografts. Seventy-two porcine bones were used to characterize the efficiency of our sodium hydroxide-based decellularization protocol. A sequence of solvent perfusion through each nutrient artery was set up to ensure the complete decellularization of whole long bones. Qualitative (histology and immunohistochemistry [IHC]) and quantitative (fluoroscopic absorbance and enzyme-linked immunosorbent assay) evaluations were performed to assess the decellularization and the preservation of the extracellular matrix in the bone grafts. Cytotoxicity and compatibility were also tested. Comparatively to nontreated bones, our experiments showed a very high decellularization quality, demonstrating that perfusion is mandatory to achieve an entire decellularization. Moreover, results showed a good preservation of the bone composition and microarchitecture, Haversian systems and vascular network included. This protocol reduces the human leukocyte antigen antigenic load of the graft by >50%. The majority of measured growth factors is still present in the same amount in the decellularized bones compared to the nontreated bones. Histology and IHC show that the bones were cell compatible, noncytotoxic, and capable of inducing osteoblastic differentiation of mesenchymal stem cells. Our decellularization/perfusion protocol allowed to create decellularized long bone graft models, thanks to their inner vascular network, ready for in vivo implantation or to be further used as seeding matrices.