Development of a decellularized porcine bone graft by supercritical carbon dioxide extraction technology for bone regeneration

A series of novel decellularized porcine collagen bone graft (DPB) materials in a variety of shapes and sizes were developed by the supercritical carbon dioxide (SCCO2) extraction technique. The complete decellularization of DPB was confirmed by hematoxylin and eosin staining, 4,6-diamidino-2-phenylindole (DAPI) staining, and residual DNA analysis. The native intact collagen remained in the DPB after the SCCO2 process was confirmed by Masson trichrome staining. The physicochemical characteristics of DPB were investigated by scanning electron microscopy and x-ray diffraction. The cytotoxicity and biocompatibility tests according to ISO10993 and its efficacy for bone regeneration in osteochondral defects in rabbits were evaluated. The rabbit pyrogen test confirmed DPB was non-toxic. In vitro and in vivo biocompatibility tests of the DPB did not show any toxic or mutagenic effects. The bone regeneration potential of the DPB presented no significant histological differences compared to commercially available deproteinized bovine bone. In conclusion, DPB produced by SCCO2 exhibited similar chemical characteristics to human bone, no toxicity, good biocompatibility, and enhanced bone regeneration in rabbits comparable to that of deproteinized bovine bone. Results from this study could shed light on the potential application of the SCCO2 extraction technique to generate a native decellularized scaffold for bone tissue regeneration in human clinical trials.