Decellularized Bone Matrix (DecBM) Grafts for Calvaria Regeneration

Objectives: To define the optimal experimental parameters to decellularize bone matrix (DecBM) and evaluate the DecBM as an alternative graft for bone regeneration.
Methods: Rat calvaria were harvested and decellularized in a solution containing 0.5% sodium dodecyl sulfate (SDS) and 0.1% ammonium hydroxide by replacing every 36 hours for 3 weeks with constant shaking at RT. The completion of the decellularization was confirmed by Hematoxylin and Eosin (H&E) staining and DNA assays. After cytotoxic evaluation by live and dead assay, the effects of DecBM on mesenchymal stem cells’ (MSC) proliferation, osteogenic gene expression, and mineralization were investigated by MTS assay, real time PCR, and Alizarin Red S staining, respectively. After further evaluation of mechanical and biochemical characteristics of DecBM, new bone formation was analyzed with microCT, mineral apposition rate, and histomorphometry after 12 weeks of implantation of DecBM with MSCs in a critical sized defect in rat calvaria.
Results: Complete decellularization was shown through H&E staining and DNA measurements (<2.05 ng/mg). DecBM biocompatibility was indicated by culturing DecBM particles with MSCs. DecBM also displayed a conserved structure, mechanical strength (*p>0.05), and mineral content comparable to natural bone. After 12 weeks post-implantation, more new bone (43.24 ± 1.21 mm³) was formed around DecBM particles with MSCs than DecBM only (17.59 ± 2.58 mm³) and also merged with new bone between DecBM particles in the defect by MSCs.
Conclusions: DecBM displayed stimulatory effects on MSCs’ proliferation and osteogenic differentiation in vitro and in vivo. Effective bone regeneration was achieved and that DecBM can serve as a promising alternative for current biological grafts.