Experimental Study Of Biological Xenogeneic Bone Invivo And In Vitro

BACKGROUND AND SIGNIFICANCE:There is a high incidence of bone defects caused by trauma, cancer or metabolic diseases, it has been a challenging clinical problem of reconstruction of bone defects faced by orthopedic surgeons.Autogenous bone, allogeneic bone, heterogeneous bone and artificial bone were currently used in clinical. Cancellous bone autograft remains the optimum standard to which other bone substitutes must be compared, but the limited number of substitutes makes it difficult to achieve a wide range of bone defects. Allogeneic bone is commonly used in bone graft material, mainly used for repairing, filling bone defects, with fixed and supporting role, but limited its source, can not fully meet the needs of clinical graft. Xenograft bone material is not restricted sources, after appropriate treatment can be used as a bone graft substitute materials to repair bone defects, which can effectively solve the clinical transplantation problems of the shortage of autologous bone and allogeneic bone, but in order to achieve the similar clinical efficacy of autogenous bone graft the main problem is how to removal of immunity and reduce rejection. As a developing country with a large amount of population and a vast territory, casualties and infection or contamination bone defect cases have occurred caused by geological disasters, industrial accidents, traffic accidents. Therefore it’s high demand for bone defect material in the domestic market. Immune response will occur aftet transplatation of xenograft bone which has a rich source. There have been many different ways to solve their immune rejection, a variety of xenograft bone such as Kiel bone, Bio-OSS, RBX xenograft bone haved in clinical use now, the treatment effect is remarkable while the security has also been recognized.OBJECTIVE:In this study, two aspects of in vivo and in vitro experiments were involved.First, to evaluate the biocompatibility of xenogeneic bone and osteoblasts in vitro, so as to provide the experimental data for clinical application. We using the methods of osteoblasts co-cultured with extacts from bone-substitute material to assess the influence of SD rat osteoblasts on the activity of osteoblast proliferation, migration and cell cycle of xenogeneic cancellous bone combined with bone morphogenetic protein-2(bone morphogenetic protein BMP-2) through the process of supercritical CO2, ethylene oxide process and so on. Second, to evaluate the fusion of biological xenogeneic bone in a goat cervical spine model in vivo, provide an evidence of safety and effectiveness for further clinical application of biological xenogeneic cancellous bone.MATERIALS:Biological xenogeneic cancellous bone:provided by Guangdong GuanHao biological technology Co., LTD. preparated of U.S. Patented technology (US6106555A, US6231614B1). SD rat osteoblast cell line was cryopreserved by Guangzhou Military General Hospital Laboratory. Eighteen local adult goats and weights ranged from25to35kg provided by Guangdong GuanHao biological technology Co., LTD. Experimental Animal Center were selected.Chapter1:Study of the biocompatibility on xenogeneic bone and osteoblasts in vitroMETHODS:The SD rat osteoblasts were cultured in mediums with or without extracts,and the proliferation of osteoblasts was quantitively evaluated by3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyl tetrazolium bromide (MTT) colorimetry method at intervals of Id,3d,5d, and7d. Cell migration was observed by inverted phase contrast microscope by transwell assay, the cell cycle was detected by flow cytometry.Statiscal analysis was performed with SPSS20software package and the data were presented with(Mean+Standard Deviation). Comparisons between two groups were carried in the method of Student’s t test, being significant when p<0.05.RESULTS:1The activity of osteoblast proliferation Many cells had uniform configuration, large body, and long protuberances in both experimental group and control group cultured after1,3,5,7d observed under a microscope. The OD value dectected by the method of MTT react the number of cell proliferation indirectly, the OD value was increased in two groups with the prolongation of culture time. MTT study showed that extracts from xenogeneic bone had no statistical difference for the growth of osteoblasts contrast (without extracts) at different time points (p>0.05). The result showed that extracts from heterogeneous bone had no adverse effects on osteoblast proliferation, showed no significant cytotoxicity,thus explained that the heterogeneous bone has good cytocompatibility with osteoblast.2The migration of osteoblasts To value the migration of osteoblasts cultured in common medium and medium with extracts from heterogeneous bone by counting the average number of cells per field under high magnification by (×200) randomly selected five horizons wiht the Transwell experiment. Transmembrane cell number was13.40±2.51cells/field in the experiment group compared with8.00±1.87cells/field in the control group, there was significant difference,number of cell migration in the experiment group was more than the control group.3The cell cycle The cell cycle of osteoblasts was detected by flow cytometry cultured for24hours and analysis the result throuth Millipore software.The percentages of cell cycle of G0/G1, S, G2/M in experimental group were69.92±1.31%,3.83±0.31%,16.56±1.20%compared with71.61±1.69%,4.06±0.28%,17.24±0.94%in the control group. The statistical analysis showed no significant difference between the two groups,the cell cycle distribution in two groups were similar, The result showed that extracts from heterogeneous bone had no adverse effects on the cell cycle of osteoblasts.CONCLUSION:The xenogeneic cancellous bone combined with bone morphogenetic protein-2(bone morphogenetic protein BMP-2) through the process of supercritical CO2, ethylene oxide process etc. had no adverse effects on osteoblast proliferation and cell cycle, made the promotion of osteoblast migration, showed good cytocompatibility with osteoblast.Chapter2:Eeperimental study on application of biological xenogeneic bone in a goat model of anterior cervical interbody fusionMETHODS:Eighteen goats underwent C3-C4discectomy were randomely divided into3groups:group A (n=6):autologous tricortical iliac crest bone graft, groupB (n=6):PEEK cage with autogenous bone, groupC C n=6):PEEK cage with biological xenogeneic bone. Radiography was performed pre-and post-operatively and4,8,12and24weeks after operation, respectively. Discspace height(DSH)were measured in auterior, posterier and lateral view at the same time points. CT scanning were performed at12and24weeks after surgery to evaluate the efficacy of spinal fusion. At the same time3goats of each group were sacrificed and fused segments were harvested. All cervical fusion specimens were analyzed by general observation and histomorpholoy.Statiscal analysis was performed with SPSS20software package and the data were presented with(Mean±Standard Deviation). The overall results of DSH and fusion score from CT scans scores were analyze by the method of One-way ANOVA, Pairwise comparisons between every two groups were carried by the method of Bonferroni test, being significant whenp<0.05.RESULTS:1Result of general observation A goat died due to anesthesia-related complications after one day, then another goat was made up an alternative. All goats foodintaded and cultivated normally postoperative, the incision were mild swelling after surgery and disappeared5days postoperative. The incision heal well with no infection, exudate and rejection, etc.2The general observation of specimens The C3/4vertebrae were fusion largely with a lot of osteitis scab12weeks postoperative in group A. There were also a lot of osteitis scab in group B and group C while the combination of the cage and vertebrae was tightly. The C3/4vertebrae were bony fusion24weeks postoperative in group A, while the group B and group C achieved the same effect fusion. 3Imaging analysis3.1Detected the fusion through X-ray X-ray showed district obvious visible gap in all fusion segments of three groups, through no trabecular bone immediately after surgery.4weeks after the operation, the segments of fusion area has been increased density in group A, the fusion segments in group B and group C seem slightly fuzzy, lower density than group A, the soft tissue seem around the PEEK cage.12weeks after the operation, the fusion segments of fusion reached a higher density than before, and vertebral bone interface seem vague, with many trabecular bone. A low density area seem in the fusion segments of group B, with less trabecular bone. Compared with Group A, there was no significant difference in the density of fusion segments in Group C, while many trabecular bone were also be seem.24weeks after the operation, the density of fusion segments was significantly higher than before in group A, and normal bone boundaries disappeared. The density of fusion segments were slightly lower in group B than in group A, and there were a large number of trabecular bone, and the normal bone boundaries disappeared in both group B and group C, compared with group A, there was no significant difference in density. The measurement of DSH showed no diffenrence Preoperative and postoperative immediately in three groups.4,8,12and24weeks after operationthe the average DSH values of group B and group C were higher than group A (p<0.05), there were significant differences, while there were no significant differences between group B and group C(p>0.05).3.2CT scans and fusion score12weeks after surgery the gap between cage and vertebral body seem by CT scans was slightly blurred in group A, with many new bone formation and a rare bone connection. There were also many new bone formation while presented a lower blur in group B and group C.24weeks after surgery, new bone formation were seem in a vast majority of CT sections in group A, has formed a bony fusion between the cage and vertebral body, in groupB and group C, new bone formation and bony fusion were also observed in most of the CT sections, a large number of trabecular bone presented in the gap. The fusion score of group B and group C were lower than group A (p<0.05) at12weeks postoperative, while there were no significant differences between group B and group C(p>0.05).24weeks postoperative there were no significant differences in three groups(p>0.05).4Result of histological examination12weeks after surgery, the autologous iliac bone was wrapped by a lot of fibrous callus with many trabecular formated by new bone and capillaries in group A. In group B the surrounding of PEEK cages wrapped by fibrous callus with many new bone in the interface of cage, also observed many capillaries. In group C, we could also see the surrounding of PEEK cages wrapped by fibrous callus with many new bone and a lot of capillaries, parts of the biological xenogeneic cancellous bone were degraded.24weeks after surgery, the new bone trabecula between autologous iliac and vertebrae body has converted into mature bone trabecula in group A, and fibrous callus is replaced by mature bone bone callus, there has been bony fusion between autologous iliac and vertebrae body completely. In group B, the fibrous callus arounded the PEEK cage were replaced by bone callus with mature trabecular bone and bony fusion. In group C, the PEEK cage was surrounded by a large number of mature trabecular bone, the biological xenogeneic cancellous bone were replaced by normal bone completely with bony fusion.CONCLUSION:In cervical interbody fusion, compared with three cortical iliac bone graft PEEK cage combined with biological xenogeneic cancellous bone could maintain cervical lordosis better while obtained the same fusion effect and reduce the incidence of intervertebral collapse, also obtained the same fusion effect compared with PEEK cage combined with autologous bone, it may be the ideal interbody implant with a clinical broad application prospects.