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

• DOI: 10.1002/jbm.a.37896
• Publication Date: 2025-03-14T09:39:25Z
• Authors: Asrar Elahi; Warwick Duncan; Kai Chun Li; Tanmoy Bhattacharjee; Dawn Coates

ABSTRACTBone 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.