Biocompatibility Study Of Biological Xenogeneic Bone By Cytology And Immunology Methods

BACKGROUND AND SIGNIFICANCE:Bone grafting represents a frequently performed procedure to repair bone defect and enhance bone regeneration in a variety of conditions in orthopaedic as well as oral and maxillofacial surgery. There are many kinds of bone graft substitutes including autografting, allografting, xenografting, synthetic and composite bone grafting had been utilized in clinical orthopedics at present. It is well-known that cancellous bone autograft is the optimum standard to repair bone defect, but the limited resource makes it difficult to repair relative wide range of bone defects. Allogeneic bone is usually used for bone defect, mainly used for repairing and filling bone defects, but limited its source too, can not fully meet the needs of clinical graft. Xenograft bone material are rich in sources, after appropriate treatment method, it can be used as suitable 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 the main problem of xenograft is how to removal of immunity materials and reduce rejection reaction and save it’s osteoconduction and osteoinduction in order to achieve the similar clinical efficacy of autogenous bone graft. As a developing country with a large amount of population, bone defect cases usually happened by geological disasters, industrial accidents or traffic accidents. Therefore there is high demand for bone-graft material. Immune rejection response will occur after transplatation of xenograft bone. There have been many different method to solve immune rejection by many year’s research, a variety of xenograft bones such as Kiel bone, Bio-OSS, RBX xenograft bone have been use in clinical now, the treatment effect were satisfactory and the security has also been confirmed.OBJECTIVE:In this study, we evaluation the biocompatibility between rat osteoblast cells and xenogeneic bone by the method of cytology and immunology experiments. Firstly, we evaluated the biocompatibility of xenogeneic bone and osteoblasts in vitro using the methods of osteoblasts co-cultured with xenograft bone material directly. Then we assessed the influence of SD rate osteoblasts on the activity of osteoblast proliferation, apoptosis and biocompatibility by methods of hemolysis test, transwell test, MTT and the ANPP. Secondly, we established goat cervical spine model by implantating fusion device made of heterogeneous bone and observed the fusion results by imaging and morphology. Then we evaluated the immune rejection by measuring the content of CD4+/IL-2+, CD4+/IL-4+, CD4+and CD8+T lymphocyte cell in the blood at different time point by flow cytometry in order to observe the immune rejection and provide an evidence of safety and effectiveness for further clinical application of biological xenogeneic bone.MATERIALS:Biological xenogeneic cancellous bone:provided by Guangdong GuanHao Biotechnology Company, made by U.S. Patented technology (US6106555A, US6231614B1); Biological fusion device made of heterogeneous bone:provided by Guangdong GuanHao Biotechnology Company. SD rat osteoblast cell line was cryo-preserved by Guangzhou Military General Hospital Laboratory. Eighteen local adult goats and weights ranged from 25 to 35kg provided by Experimental Animal Center were selected Affiliated to Guangdong GuanHao Biotechnology Company.Chapter oneBiocompatibility study of biological xenogeneic bone by cytology methodMETHODS:There were three groups in this experiment. There were experimental group, positive control group, and nagetive control group. Experimental group means the SD rat osteoblasts were cultured with antigen cross-linking xenogeneic bone particles; Positive control group means the SD rat osteoblasts were cultured in H-DMEM mediums with antigen non-crosslinking xenogeneic bone particles; Nagetive control group means SD rat osteoblasts were cultured in H-DMEM mediums alone without any xenogeneic bone particles. The proliferation of osteoblasts was quantitively evaluated by 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyl tetrazolium bromide (MTT) colorimetry method at intervals of Id,3d,5d, and 7d. The result of MTT was used to evaluate the cell toxicity and biological compatibility of xenogeneic bone. Three days later, osteogenesis induced liquid was mixed into mediums, The content of ALP (alkaline phosphatase) was measured by meathod of PNPP (pnitrophenylphosphate) at intervals of 3d, 1w,2wand 3w. The content of ALP used to evaluate the impact on osteogenesis ability of xenogeneic bone. Osteoblast cell cycle and apoptosis were measured by flow cytometry instrument in order to evaluate the impact on Osteoblast cell cycle and apoptosis. Acute hemolysis test between xenogeneic bone and venous blood in West Atlantic white rabbit was use to observe the biocompatibility between xenogeneic bone and blood. There are four groups in transwell test. There are Al group (xenogeneic bone particles after antigen cross-linking), A2 group (matched group to A1), Blgroup (xenogeneic bone particles without antigen cross-linking) and B2 group (matched group to B1). Cell migration were observed by microscope and the osteoblast number of which traversed the film were determination in order to evaluate the migration ability of osteoblast.Statiscal analysis was performed with SPSS20.0 software package and the data were presented with (Mean+Standard Deviation). Comparisonsbetween two groups were carried in the method of analysis of variance and LSDt-test or Tamhane multiple comparison test, being significant when p<0.05.RESULTS:1 The activity of osteoblast proliferation The osteoblast had good configuration in both experimental group and control group cultured after 1,3,5,7 d observed under a microscope. The OD value measured by the method of MTT reflect the number of cell proliferation indirectly, the OD value was increased in three groups with the prolongation of culture time. MTT study showed that all of the three groups had no statistical difference for the growth of osteoblasts at different time points (p>0.05).The result showed that heterogeneous bone had no adverse effects on osteoblast proliferation, showed no significant cytotoxicity, thus explained that this xenograft bone has good cytocompatibility with osteoblast cell.2 The OD value of ALP measured by the method of PNPP The osteoblast had good configuration in all of the three experimental group three days after mixed with osteogenesis induced liquid. The OD value of ALP measured by PNPP method reflect the osteogenesis capacity of cell indirectly. The OD value was increased in all of the three groups with the prolongation of culture time. At the time of 3day and 1w, there was no significant difference when comparison among all of the three groups (P>0.05). The cross-linked bone groups had significant difference with both control group and non-crosslinked xenograft bone groups at 2w and 3w time points (p< 0.05). the non-crosslinked xenograft bone groups had significant difference with control group at 2w and 3w time points (p< 0.05).3 The cell cycle The cell cycle of osteoblasts was detected by flow cytometry and the result was analyzed through Millipore software. The percentages of cell cycle in G0/G1,S, G2/M were respectively 70.10±1.1%,3.82±0.02%,16.56±0.02% in group A; 70.10±0.20%,3.82±0.02%,16.56±0.01% in group B;70.20±0.04%, 3.86±0.02%,16.52±0.05% in group C. The statistical analysis showed no significant difference among all of the three groups (P>0.05).The cell cycle distribution in three groups were similar, The result showed that xenograft bone had no adverse effects on the cell cycle of osteoblasts cell.4 The migration of osteoblasts To evaluate the migration of osteoblasts cell which cultured in common medium and with two kinds of xenograft bone by counting the average number of cells per field under high magnification by (×200) and randomly selected five horizons by Transwell test. Traversed membrane cell number was 13.42±0.02 cells/field in experiment groupAl,8.00±0.02 cells/field in control groupB1; 13.39±0.02 cells/field in experiment groupA2,8.00±0.07 cells/field in control groupB2. Statiscal analysis was performed with SPSS20.0 software package and the data were presented with (Mean+Standard Deviation). Comparisons between two groups were carried in the method of analysis of variance and Tamhane multiple comparison test, being significant when (P<0.05). There was significant difference number of cell migration between the experiment group and control groupB1 (P<0.05). The number of experiment groupA1 were far more than the control group. There was significant difference number of cell migration between the experiment groupA2and control groupB2 (P<0.05). There was no significant difference number of cell migration between experiment groupAl and experiment groupA2 (P>0.05).5 Cell apoptosis experimental The apoptosis of osteoblasts was detected by flow cytometry and the results were analyzed through Guava Nexin software. The percentages of cell apoptosis of groupA, groupB an groupC respectively was 0.87±0.01%,0.88±0.03%,0.91±0.01% at 24h time point and was 2.16±0.03%, 2.20±0.01%,6.19±0.06% at 48h time point. The statistical analysis showed no significant difference among the three groups at 24h time point (P>0.05). But at 48h, The non-crosslinked bone groups had significant difference with both control group and cross-linked xenograft bone groups (P< 0.05). The result showed that xenograft bone had no adverse effects on the cell cycle of osteoblasts in 24hours, however, at 48hours, the non-crosslinked xenograft bone can promote osteoblast apoptosis obvious.6 Acute hemolysis test The supernatant were transparent in both two xenogeneic bone groups and the same to the negative control group. No hemolysis phenomenon occurred in two experiment groups. The supernatant was rosy which means red blood cell rupture dissolve completely in positive control group. Hemolysis rate was 1.02% in cross-linked xenograft bone group,1.13% in non-crosslinked xenograft bone group. There was no significant difference between both two experiment groups and negative group (P>0.05).Conclusion:1, Biological xenogeneic bone had benefit effects on the osteogenesis migration capacity of SD rat osteoblasts when cultured with it in vitro and the cross-linked xenograft had more significant effects than no- ncrosslinked bone.2, Xenograft bone had no adverse effects on the cell cycle of osteoblasts in 24hours when cultured with it in vitro, however, at 48hours, the non-crosslinked xenograft bone can promote osteoblast apoptosis obvious but the crosslinked xenograft bone had no harm affect at the same time.3, Biological xenogeneic bone had no harmful effects on the proliferation activity and cell cycle of SD rat osteoblasts when cultured with it in vitro and does not cause hemolysis reaction which means good biocompatibility.Chapter twoBiocompatibility study of biological xenogeneic bone by immunology methodMETHODS:Eighteen adult locally goats were randomely divided into three groups. GroupA (n=6):autologous tricortical iliac crest bone was implanted into goats and fixed by plate. GroupB (n=6):xenogeneic bone cage was implanted into goats and fixed by plate. GroupC:(n=6):no surgery was performed as control group. Radiography was performed pre-operatively and post-operatively and 12,24 weeks after operation respectively to observe the situation of internal fixation and evaluate the efficacy of spinal fusion. Three goats of each group were sacrificed at 12weeks and 24weeks after surgery and cervical vertebra fused segments were harvested, and removed the soft tissue, plate and screws. All cervical fusion specimens were analyzed by general observation and histomorpholoy study via HE staining. Extraction of 20 ml blood at the time of 1w,2w,4w and 8w and the blood was used to measure the content of CD4+/IL-2+, CD4+/IL-4+, CD4+ and CD8+Tcell by the way of FCM (flow cytometry) in order to evaluate immune rejection of xenogeneic bone.Statiscal analysis was performed with SPSS20.0 software package and the data were presented with (Mean+Standard Deviation). Comparisonsbetween two groups were carried in the method of analysis of variance and LSDt-test, being significant when P<0.05.1 Result of general observation A goat died due to anesthesia-related complications after one day, another goat was made up Instead of it. All goats were gave normal food and wild grazing postoperative, the incision were mild swelling in parts of goats after surgery and disappeared 5 days postoperatively. All the incisions heal well with no infection on the end.2 The general observation of specimens The C3/4 vertebrae were fusion largely with bone scab 12 weeks postoperatively in group A, but in group B, C3/4 vertebrae were not fusion and only a little callus formed. The C3/4 vertebrae were bony fusion completely 24 weeks postoperative in group A, but for group B, the C3/4 vertebrae were not fusion well yet.3 The result of CD4+ and CD8+T cell At 1 week, there were no significant difference among the three groups in the content of CD4+ and CD8+ Tcell (P>0.05). At 2 weeks, there were no significant difference between autologous bone group and negative control group (P>0.05). There were significant difference between autologous bone group and xenogeneic bone cage groups in the content of CD4+ and CD8+Tcell (P<0.05). There were significant difference between negative control group and xenogeneic bone cage groups in the content of CD4+ and CD8+Tcell (P< 0.05). At 4 weeks, there were no significant difference between autologous bone group and negative control group (P>0.05). There were significant difference between autologous bone group and xenogeneic bone cage groups in the content of CD4+and CD8+Tcell (P<0.05). There were significant difference between negative control group and xenogeneic bone cage groups in the content of CD4+and CD8+Tcell (P<0.05). At 8 weeks, there were no significant difference between autologous bone group and negative control group (P>0.05). There were no significant difference between autologous bone group and xenogeneic bone cage groups in the content of CD4+and CD8+Tcell (P>0.05). There were no significant difference between negative control group and xenogeneic bone cage groups in the content of CD4+ and CD8+Tcell (P>0.05).4 The result of CD4+/IL-2+ and CD4+/IL-4+T cell At 1 week, there were no significant difference among the three groups in the content of CD4+/IL-2+and CD4+/IL-4+T cell (P>0.05). At 2 weeks, there were significant difference between autologous bone group and negative control group (P<0.05). There were significant difference between negative control group and xenogeneic bone cage groups in the content of CD4+/IL-2+ and CD4+/IL-4+T cell (P<0.05). There were no significant difference between autologous bone group and xenogeneic bone cage groups in the content of CD4+/IL-2+ and CD4+/IL-4+T cell (P>0.05). At 4 weeks, there were significant difference between autologous bone group and negative control group in the content of CD4+/IL-2+ and CD4+/IL-4+T cell (P<0.05). There were significant difference between autologous bone group and xenogeneic bone cage groups in the content of CD4+/IL-2+and CD4+/IL-4+T cell (P<0.05). There were significant difference between negative control group and xenogeneic bone cage groups in the content of CD4+/IL-2+and CD4+/IL-4+T cell (P<0.05). At 8 weeks, there were no significant difference between autologous bone group and negative control group in the content of CD4+/IL-2+and CD4+/IL-4+T cell (P>0.05). There were no significant difference between autologous bone group and xenogeneic bone cage groups in the content of CD4+/IL-2+ and CD4+/IL-4+T cell (P>0.05). There were no significant difference between negative control group and xenogeneic bone cage groups CD4+/IL-2+ and CD4+/IL-4+T cell (P>0.05).Conclusion1, Xenogeneic bone cage can cause immune rejection in the early days when transplanted into goat and gave rise to the increase of CD4+, CD8+Tcell and CD4+/IL-2+,CD4+/IL-4+T cell, which reached climax at 4eeks and gradually returned to normal at 8 weeks.2, Surgical trauma and pain induced goat stress reaction which make the CD4+/IL-2+ and CD4+/IL-4+T cell number increasing, this effect immediately began after opertation and the cell number reach to highest at about 2 weeks after surgery and maintain to postoperative 4 weeks, this stress response, however, cannot cause the CD4+ and CD8+T cell increase.