Supercritical <scp>CO₂</scp> With Enzymatic Posttreatment Enhances Mechanical and Biological Properties of Cancellous Bovine Bone Block Grafts

Bone loss resulting in large bony defects presents a significant challenge for surgeons. In cases requiring reconstruction, bone "block" grafts that have the key attributes of both physical robustness and biocompatibility are required to facilitate bone healing and regeneration. Current technologies employed for the development of block grafts often result in constructs with suboptimal strength and integration. This study aimed to develop a bovine-derived bone block graft using the process of supercritical fluid (SCF) extraction to maintain mechanical strength and biocompatibility. Bone blocks were prepared from the condyles of bovine femurs. After optimization, the blocks were divided into six groups; Group 1: Raw bone, Group 2: SCF-CO₂, Groups 3: SCF-CO₂-H₂O₂, and Group 4: SCF-CO₂-H₂O₂ + Pepsin. Characterization of the constructs included analysis of organic material (thermogravimetric analysis, TGA), crystallinity using x-ray diffraction (XRD), surface topography with scanning electron microscopy (SEM), and chemical composition using Fourier-transform infrared (FTIR) spectroscopy. Mechanical strength was assessed using compression testing, and clinically relevant handling was investigated with a bench-top drill test. Biological testing was carried out in vitro using human bone marrow-derived mesenchymal stem cells (hBMSCs). The SCF-treated bone blocks showed promising results with enhanced mechanical strength (raw bone [mean = 23.01 8.9 MPa], SCF-CO₂-H₂O₂ [mean = 48.9 ± 11.6 MPa], p < 0.0001) reduced organic content (raw bone = 17.6%, SCF-CO₂-H₂O₂ + Pepsin = 12.4%), and significantly higher hBMSCs' metabolic activity on the SCF-CO₂ and SCF-CO₂ + H₂O₂ compared to Bio-Oss at 24, 48, 72, and 96 h (p < 0.05). SEM photomicrographs showed reduced debris in trabecular structures with open pores after SCF-CO₂ treatment, especially in SCF-CO₂-H₂O₂ + Pepsin blocks. Moreover, the bench-top clinical handling test demonstrated the ease of block fixation with surgical screws. Overall, the SCF-CO₂ and posttreatments of bovine block grafts showed potential for clinical application.