| Chapter 1:Preparation of Porcine Bone and Experimental Study on its Biological PropertiesObjective:To develop a feasible method to prepare deantigenated porcine bone through enzyme treatment,which has no effect on the osteoinductivity and mechanic properties of the xenogeneic bone graft produced thereby.Methods:(1)Enzyme treatment of porcine bone:Xenogeneic bone was prepared from flesh porcine bone by a series treatment includingα-Galactosidase of different activity digesting for 8 or 16 hours at 37℃,washing,freezedrying and irradiation at a does of 25kGy.(2)Determination of theα-Gal(α-gal epitopes):The concentration ofα-Gal within porcine bone tissue were tested by ELISA in order to choose a suitable processing way for deantigenation.(3)Crosslinking of glutaraldehyde with the poricine bone:Crosslinking of glutaraldehyde with the poricine bone prepared by the selected group was performed and concentration ofα-Gal was defined and compared with that of fresh porcine bone,enzyme treated porcine bone and human bone.The processing programe then was decided according to the testing result.(3) Morphological observation:Gross observation and SEM observation were performed and diameters of the caves of the porcine cancellous bone were measured.(4)Mechanic properties testing:The compressive properties of porcine bone at different stage of processing were tested to evaluate the effects of different treatment on its mechanic property.Sstatistical analysis was performed with SPSS11.5 software package and the data were presented with(Mean±Standard Deviation).The data were tested with variance analysis,being significant when P＜0.05.Results:(1) Enzyme treatment of porcine bone:The OD value of different groups in ELISA test were significantly different.The selected enzyme treatment was digesting byα-Galactosidase with an activity of 30U/ml for 8 hours at 37℃.(2) Crosslinking of glutaraldehyde with the poricine bone:No statistic significance was found among the OD values of cross-linked porcine bone and human bone,while there were significant difference between fresh porcine bone and other groups. According to the results,the finally processing methods wasα-Galactosidase of a 30U/ml activity digesting for 8 hours.(3) Morphological observation:The porous structure of prepared porcine cancellous bone was similar to that of human cancellous bone.The diameters of the caves were between 150μm -600μm,while the diameters of small pore in the trabecula were between 10μm-100μm.(4) Mechanic testing of porcine bone:Strength and elastic module of different groups had no statistical difference (P＜0.05) for the compressive test,indicating that different processing methods had no significant effect on the mechanical property of the porcine bone.Conclusion:The antigen of porcine bone was removed effectively byα-Galactosidasse digesting,while the morphological structure and mechanic properties of the porcine bone remainedChapter 2 Biological Evaluation of the Prepared Porcine Bone GraftObjective:To evaluate the biocompatibility of the prepared porcine bone by in vitro cytotoxity experiment and in vivo animal experiment.Methods:(1) Culture of rabbit BMSCs:Rabbit BMSCs were obtained as the experimental cells and the activity and content of CD44 surface antigen of the BMSCs were tested.(2)In vitro cytotoxity experiment:The leaching liquor of porcine bone was made by immersing the bone into L-DMEM medium with a scale of 10ml medium per gram bone materials at 37℃for 72 hours in a sterilized condition and L-DMEM medium which contained 10%FBS served as the control medium.200μl of BMSCs suspension with a cell concentration of 1×105/ml were seeded to the and cultured for 24 hours.Then the culture medium was removed and 200μl leaching liquor or control culturing medium were added according to groups respectively. Then cells were cultured at 37℃and 5%CO2.The medium were exchanged every 3 days according to groups.Every 24 hours,5 wells of each group were selected and 20μl 5mg/ml MTT solution was added to each well.150μl DMSO was added after cultured for 4 hours.The 96-well plate was slightly shaken for about 10 minutes and the OD value at 490 nm was determined.(3)Co-culturing of the porcine bone and rabbit BMSCs:The prepared porcine bone,sized 5mm×5mm×5mm,were placed in the 12-well plate and 150μl BMSCs suspension of a concentration of 1×105/ml was seeded to the surface of the porcine bone and 10%FBS L-DMEM was added to immerse the porcine bone.Inverted phase contrast microscope observation and SEM observation were performed at 1,2,3 weeks.(4)Biological evaluation in vivo:18 Wistar mice,weighted 150-180g,were anesthetized.Subcutaneous and intramuscular implantation of prepared porcine bone and human bone pieces was performed.6 of the mice were killed each time respectively at 1,4,12 weeks and gross observation and histological observation were performed.Results:(1) Culture of rabbit BMSCs:The cultured BMSCs were in a good condition.About 95%of F1 to F3 generation cells was active.93.9%of F1 generation,95.1%of F2 generation and 95.8%of F3 generation cells contained CD44 surface antigen.(2) In vitro cytotoxity experiment:The OD values of study group and control group at different time had no significant difference.(3) Co-culturing of porcine bone and BMSCs:Postoperatively 1-3 weeks,Polygon shaped and fusiform shaped BMSCs with a well condition adhered tightly to the surface of the porcine bone.Lots of pseudopodia from BMSCs reached the surface of the porcine bone.Conclusion:The enzyme degested porcine bone had no obviously effect on the growth of BMSCs and showed good cell affinity when co-cultured with BMSCs. The local implantation also showed the prepared porcine bone contains good tissue compatibility.The porcine bone contained good biocompatibility and could be safe for clinic use.Chapter 3 Study on the Osteoinductivity of the Prepared Porcine Bone GraftObjective:To evaluate the osteoinductivity of the prepared porcine graft by histological observation and ALP activity determination in an ectopic bone formation model,using immuno-suppressed Wistar rats as experimental animal.Methods:Samples of study group were enzyme-treated porcine bone, demineralized with 0.6N hydrochloric acid.The control group samples were the demineralized porcine bone,which were treated with autoclave for 30 minutes to destroy the activity of bone formation material.All samples were freezedried and irradiated at a does of 20kGy.Dexamethasone was intraperitoneally injected into Wistar rats in order to suppress their imunno-response.Intramuscular bone transplantation was performed by placing the demineralized porcine bone and control samples between bluntly dissected muscle groups of hind limbs of the rats. Histological observation was performed at different time post-operatively.ALP activity was tested postoperatively to determine the osteoinductivity of the demineralized porcine bone implants.Result:The study group:Mesenchymal cells migrated into the absorpted lacuna and chondrocytes could be seen within the porcine bone implants at 3 weeks postoperatively;more cartilage presented at 4 weeks;adult cartilage was formed within the implants at 5 weeks;at 6 weeks,bone formation was more active and lots of cartilage within the implants and osteoid in the absorbed lacuna could be seen.No bone formation could be found in the control group 3-6 weeks postoperatively.ALP activity of the experimental group was obviously higher than that of the control group 3-6 weeks postoperatively.Conclusion:The prepared porcine bone contained good osteoinductivity and could be a potential bone substitute for clinic implantation. Chapter 4 Experimental Study of the Prepared Porcine Bone for Bone Reparation in a Radial Segmental Defect of RabbitObjective:To investigate the reparation of segmental bone defect by the prepared porcine bone and provide experimental reference for potential clinic use.Methods 24 rabbits,weighted 2-2.5kg,were anesthetized and a 12mm-long bone defect was made by cutting the middle shaft of both side radii.Grafts were implanted according to groups:(A)Control group:no implantation was performed to the defect;(B) Xenogeneic bone group:the prepared porcine bone were implanted;(C) Allogeneic bone group:rabbit allogeneic cancellous bone were implanted;(D) Autograft group:the cut bone were implanted to the opposite side radius.8 animals were sacrificed(4 graft for each group were obtained) for each time respectively at 4, 8,12 weeks postoperatively.Generally and X-ray,histological observation were performed to evaluate the reparation of the segmental defect..Results:Gross observation:All the animals were in good status and no detectable complications were found.X-ray observation:Postoperatively 4 weeks, little callus was formed and the defect was clear in the control group;bone defect was filled with shadow with a lower density than that of the rabbit radius and ulna,small quantity of callus was formed in the xenogeneic bone group;allogeneic bone group was similar to the porcine bone group;a little callus was formed in auto graft group. 8 weeks post-operationally,bone defect was clear and small quantity of callus was formed in control group;bone defect was filled with a lower density shadow and the bridge area was not very sharp in xenogeneic bone group;callus was formed and high-density shadow could be seen along the ulna in allogeneic bone group;the auto graft and the rabbit radius was connected with blurry shadow.12 weeks postoperatively,callus was formed in the end of the radius and the marrow cavity was blocked and bone non-union was formed in the control group;the defect was filled with low-density shadow and the bridge area was blurry in porcine bone group and allogeneic bone group;recanalization of the marrow cavity was formed between the graft and the radius in auto graft group.Score of X-ray of xenogeneic bone group ahc no significant difference while the difference of the xenogeneic bone group with control group or autograft group are both significant.Histological observation:the defect of control group was filled with connective tissue without active bone formation.postoperatively 4 weeks,bone defect of control group was filled with fibrous tissue which contained lots of lymphocytes;bone defect was filled with connective tissue and lots of lymphocytes could be seen in porcine bone group and rabbit allogeneic bone group;chondrocytes and osteoid grown between the radius and the auto graft in auto graft group.Postoperatively 8 weeks,the defect of control group was filled with fibrous tissue which contained lymphocytes;bone absorption and cement line could be seen in the xenogeneic bone group and rabbit allogeneic bone group;the autograft was connected with the rabbit radius shaft with active bone formation and absoption within the graft.Postoperatively 12 weeks,the defect of control group were filled with connective tissue;small quantity of new bone formation could be seen in xenogeneic bone group and rabbit allogeneic bone group; the autograft was mainly substituted by newly formed bone with active bone formation.Conclusion The prepared porcine xenogeneic bone cobtained a cure effect similar to that of rabbit allogeneic bone in a rabbit radial segmental defect model, hence it may be a potential good bone substitute for bone implantation. |
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