Preparation of decellularized bone graft material with supercritical carbon dioxide extraction technique.

Objectives: To evaluate the immunogenicity and osteogenic ability of animal-derived bone graft material decellularized with supercritical carbon dioxide.

Methods: Porcine femurs were randomly divided into two groups after preliminary treatment, and decellularized with conventional method (conventional control group) or supercritical carbon dioxide (experimental group). Clearance rate of galactose-α-1, 3-galactose (α-Gal) and residual DNA of the two groups were analyzed to assess the immunogenicity of the xenogenic materials. Clearance rate of α-Gal was determined by enzyme-linked immunosorbent assay and residual DNA was detected by fluorescence method. Nine SPF-grade male athymic nude mice of 6 weeks old were randomly divided into experimental group, conventional control group and positive control group. Samples were implanted over biceps femoris muscle of athymic nude mice, the explants were collected 4 weeks post implantation, hematoxylin and eosin (HE) staining and immunohistochemistry were applied to determine the osteogenic ability and bone tissue-associated protein expressions of the implants.

Results: The clearance rates of α-Gal antigen in the experimental group and the conventional control group were (99.09±0.26)% and (30.18±2.02)%, respectively (t=58.67, P<0.01). The residual DNA of the experimental group, the conventional control group and the positive control group were (13.49±0.07) ng/mg, (15.20±0.21) ng/mg and (14.70±0.17) ng/mg, the residual DNA in the experimental group was significantly lower than that in the conventional control group (t=－13.41, P<0.01) and the positive control group (t=－11.30, P<0.01). HE staining showed that multiple bone formation centers with active osteogenesis and rich bone marrow were observed in experimental group 4 weeks after implantation, only a small number of bone formation centers were observed in the conventional control group and the positive control group, with no obvious osteoblasts present. Immunohistochemistry results indicated that the expressions of alkaline phosphatase, Runt-related transcription factor 2, typeⅠcollagen and osteocalcin in the experimental group showed an increasing trend compared with those in the conventional control group and the positive control group.

Conclusions: Compared with clinically used demineralized bone matrix and bone graft material decellularized with conventional method, bone graft material decellularized with supercritical carbon dioxide exhibits lower immunogenicity and better osteogenic ability.