Objective To develop a deantigenated porcine bone graft through enzyme treatment and to study the biomechanical properties and osteoinductivity of the xenogeneic bone graft. Methods Deantigenated xenogeneic bone was prepared from porcine bone by a series treatment including α-galactosidase (α-Gal) treatment, freezedrying and irradiation at a does of 25 kGy. Samples were divided into 4 groups according to the treatment methods: fresh bone (group A); deantigenated bone (group B); deantigenated and freezedried bone (group C); deantigenated-freezedried-irradiated bone(group D). SEM observation to group B samples was performed and the diameters of the caves of the porcine cancellous bone were measured. The α-Gal contents of samples of groups A, B, D were measured by ELISA. The effects of different treatments on porcine bone mechanic properties were evaluated through compressive test of 4 groups. The prepared porcine cortical bone were demineral ized and ectopically implanted into muscle groups of hind l imbs of Wistar rats as experimental group, and the demineral ized cortical bone which were inacitviated by high temperature and pressure were implanted as control group. Histological observation was performed and ALP activity was tested at different time postoperatively to investigate the osteoinductivity of the xenogeneic bone implants. Results The porous structure of prepared porcine cancellous bone was similar to that of human cancellous bone. The diameters of the caves were between 150-600 μm. The A value of the groups A, B, D was 0.358 ± 0.027, 0.191 ± 0.011, 0.191 ± 0.009, respectively, with statistically differences between groups (P lt; 0.05). While the biomechanical properties among 4 groups had no statistically difference (P gt; 0.05). Histological observation showed mesenchymal cells immigrated into cartilage and converted into chondrocytes at 3 weeks postoperatively. Cartil iage was formed at 4 weeks and osteoid and more adult cartilage was formed at 6 weeks within the implants of experimental group, while there was no bone formation in control group. ALP activity of experimental group at different times were obviously higher than that of the control group (P lt; 0.05). Conclusion The main xenogeneic antigen of the porcine bone was removed while the mechanic properties and osteoinductivity remained, thus it may be a suitable bone substitute for cl inical use.
Objective To study the influence of the immersed time by hydrogen dioxide on the characteristics of bovine cancellous bone granules in various periods. Methods Ten 24-month-old Qinchuan bovine, male or female, weighing 150-170 g, were selected. Cancellous bone granules from metaphysic of bovine long bone were cut into cubes of 5 mm × 5 mm ×5 mm and immersed by 8.8 mol/L hydrogen dioxide for 0, 12, 24, 36, 48, 60 and 72 hours separately. Determination of ash, scanning electron microscope, X-ray energy spectrum and micro CT were used to investigate the changes of composition, structure and qual ity of bone. Results With the immersed time increasing, the contents of organics in the bone cancellous were reduced gradually, and obviously decreased during the periods of 0 to 24 hours and 60 to 72 hours (P lt; 0.05). The contents of calcium and phosphorus decreased gradually, they could not be detected almost after 60 days (P lt; 0.05). Bone mineral density and bone mineral content were decreased obviously after 60 hours (P lt; 0.05). The bone trabecula became sl immer and trabecular spacing became larger. Conclusion Hydrogen dioxide can be used to remove the antigen in xenogeneic bone; however as the time increasing (more than 60 hours) the composition and structure will be damaged. Thus it is important to control the immersed time for maintaining the biological characteristics of xenogeneic bone substitute as well as el iminating antigen by hydrogen dioxide.
ObjectiveTo evaluate the physical and chemical properties, immunogenicity, and osteogenesis of two antigen-extracted xenogeneic bone scaffolds—decalcified bone matrix (DBM) and calcined bone.MethodsBy removing the inorganic and organic components of adult pig femus, xenogeneic DBM and calcined bone were prepared respectively. The density and pH value of the two materials were measured and calculated, the material morphology and pore diameter were observed by scanning electron microscope, and the surface contact angle was measured by automatic contact angle measuring instrument. The safety, osteogenic activity, and immunogenicity of the two materials were evaluated by cytotoxicity test, osteoblast proliferation test, DNA residue test, and human peripheral blood lymphocyte proliferation test. The two materials were implanted into the 5 mm full-thickness skull defect of 6-week-old male Sprague Dawley rats (the blank control group was not implanted with materials). The materials were taken at 4 and 8 weeks after operation, the repair effect of the materials on the rat skull was observed and evaluated by gross observation, Micro-CT scanning, and HE staining observation.ResultsCompared with calcined bone, DBM has lower density and poor hydrophilicity; the pH value of the two materials was 5.5-6.1, and the pore diameter was 160-800 μm. The two materials were non-cytotoxic and could promote the proliferation of osteoblasts. The absorbance (A) values of osteoblast proliferation at 1, 4, and 7 days in the DBM group were significantly higher than those in the calcined bone group (P<0.05). The DNA residues of the two materials were much lower than 50 ng/mg dry weight, and neither of them could stimulate the proliferation and differentiation of human peripheral blood lymphocytes. The results of animal experiments in vivo showed that the bone volume/total volume (BV/TV) in DBM group and calcined bone group were significantly higher than that in blank control group at 4 weeks after operation (P<0.05), and that in calcined bone group was significantly higher than that in DBM group (P<0.05); at 8 weeks after operation, there was no significant difference in BV/TV between groups (P>0.05). HE staining showed that at 4 and 8 weeks after operation, the defect in the blank control group was filled with fibrous connective tissue, the defect was obvious, and no bone growth was found; the defect in DBM group and calcined bone group had been repaired to varying degrees, and a large number of new bone formation could be seen. The material degradability of DBM group was better than that of calcined bone group.ConclusionThe physical and chemical properties and degradability of the two kinds of xenogeneic bone scaffolds were slightly different, both of them have no immunogenicity and can promote the repair and reconstruction of skull defects in rats.