RESUMO
BACKGROUND: In recent years, there have been many studies on the use of supercritical fluids for biological material treatment in countries outside China. However, little is reported on application of supercritical fluids to bone tissue extraction, in particular in China. OBJECTIVE: To evaluate the effectiveness of supercritical carbon dioxide extraction technology in the treatment of porcine femur cancellous bone and its effect on bone biological properties. METHODS: Porcine femoral bone blocks that were subjected to supercritical carbon dioxide extraction (study group) or not (control group) were prepared to determine bone mineral density, microstructure, maximum compressive strength, elastic modulus, bone tissue composition, collagen content and perform histological analysis. Bone marrow mesenchymal stem cells (BMSCs) were inoculated into two groups of bone blocks, and cultured for 1 day. The microporous structure of trabecular bone and cell adhesion and growth in bone material-cell composite were observed by scanning electron microscopy. The two groups of bone blocks were implanted subcutaneously in SD rats. The inflammatory reaction of subcutaneous tissue was observed histologically at 1, 2 and 4 weeks after surgery. The experimental protocol had been approved by the Animal Ethics Committee of Chinese PLA General Hospital, China. RESULTS AND CONCLUSION: There were no significant differences in pore size, bone mineral density, maximum compressive strength, elastic modulus and collagen content between the study and control groups (P>0.05). Scanning electron microscopy showed that in the control group, the material pores had poor connectivity and there was soft tissue residue; in the study group, material pores were connected to each other and the structure was intact. Fourier transform infrared spectroscopy and X-ray diffraction analysis showed that the two groups of bone tissue materials had similar absorption and diffraction peaks. Thermogravimetric analysis showed that supercritical carbon dioxide extraction could reduce water content in bone tissue. Hematoxylin-eosin staining showed that there were no soft tissue residues in the bone, and the cell residues in the bone pit were significantly reduced in the study group, while soft tissue and cell residues were observed in the control group. Sirius red staining and modified Masson staining showed that the structure of bone collagen in the study group was intact, the cytoplasmic components reduced, and the cytoplasmic components in the control group remained significantly. Scanning electron microscopy showed that there was no obvious cell adhesion in the control group, but cell adhesion growth was obvious in the study group. Perivascular inflammatory response in the bone tissue implantation region was obviously weaker in the study group than in the study group. These results suggest that supercritical carbon dioxide extraction technology is an effective and environment-friendly bone tissue processing technology. It can effectively remove porcine cancellous bone cells and soft tissue without affecting its collagen structure and content and mechanical properties, retaining intact bone pore structure, increasing cell adhesion and growth, and effectively reducing inflammatory rejection.