Experimental Study Of Restoration Of Osteochondra Defect Of Rabbit With β-TcP/Pluronicf-127Composite Scaffold Combined With BMSCs

Part Ⅰ Significance and expression of apoptosis control genes in OAcartilageobjective：To investigate the expression and significance of apoptosis control genes(FAS、BCL-2、caspases3) in articular Cartilage of osteoarthritic animal model and study therelation of theexpressions with apoptosis of articular chondroeytes cell.Method:Specimens of thearticular cartilage obtained from osteoarthritic animal model established through Hulthmethod in rabbits. Immunohistochemistry was used to detect the expression of apoptosiscontrol genes(FAS、BCL-2、caspases3) in articular cartilage of animal model.TUNELassay was used to detect the apoptosis of the articular chondrocytes cell.The relation of theexpression of apoptosis control genes(FAS、BCL-2、caspases3) with apoptosis of articularchondrocytes cell was studied.results: After eight weeks in operation， the tyPicalhistoPathologic character of aPoPtosis morphology was seen in the articular cartilage.There was statistically significant difference of Al between the osteoarthritic and normalcartilage grouPs(P<0.05). The Positive exPression rate of Fas in osteoarthriticchondrocytes were higher than that in normal chondrocytes，and there was statisticallysignificant difference between chondrocytes of osteoarthritic and normal groups(P<0.05)，there was a Positive correlation between degrees of the exPression of FAS andchondrocytes apoptosis in osteoarthritic articular cartilage(P<0.05);The Positive expressionrate of Bcl-2Protein in osteoarthritic chondrocytes was lower than that in normal chondrocytes，but there was no statistically significant difference between the twogroups(P>0.05). There was a negative correlation between degree of the expression ofBcl-2and chondrocytes apoptosis osteoarthritic articular cartilage (P<0.05); The Positiveexpression rate of caspase3in osteoarthritic chondrocytes were higher than that in normalchondrocytes，and there were statistieally significant difference between the two groups(P<0.01).There was a Positive correlation between degrees of the exPression of caspase3and chondrocytes apoptosis in osteoarthritic articular cartilage(P<0.05).Conclusion:1.Hulth method can establishes a ideal model of osteoarthritis.2.The apoptosisof chondrocytes in OA was higher than that in normal. It might be one of the importantcausations of OA.3．There was a significant correlation between degrees of the exPressionof FAS,BCL-2,caspase3and chondrocytes apoptosis in osteoarthritic articular cartilage.Itdemonstrated that the abnormal expression of FAS,BCL-2,caspase3closely related to thebiological behaveior of OA. PartⅡ.The isolation，cultivation，identification and differentiation ofrabbit bone mesenchymal stem cells in vitroobjective:To exPlore the method of isolating，cultivating and identifieation of rabbit bonemesenchymal stem cells (BMSCs)，and study their capacity of Proliferation and Potentialof differentiation in vitro. To investigate potential applicability of the MSCs as the seedcells in tissue engineering.Method:Rabbit BMSCs of bone marrow was isolated by densitygradient centrifugation and bone marrow culture，cultured in vitro. hMSCs were analyzedby the flow cytometry to detect the surface antigens. Cellular morphologies were observedunder phase-contrast microscope and the growth curve was drawn accordingly.The3rdPassage cell was induced osteogenesis and chondrogenesis respectively，then alkalinePhosPhatase and VonKossa staining and immunocytochemical stain of collegenⅠ and osteocalcin were used to examined the osteogenesis cells and toluidin blue and safranin ostaining and immunocytochemical stain of collegen Ⅱwere used to examined thechondrogenesis cells.Results: The morphous of BMSCs obtained by density gradientcentrifugation and bone marrow culture are spindle-shaped or Polygon-shape uniformly，The MSCs proliferated rapidly in in vitro culture. Flow cytometry analysis showed that invitro expanded hMSCs expressed mesenchymal cell marker, including CD105、CD44、anddidn’t express CD34、CD45. The staining of the alkaline PhosPhatase, VonLKossa，collegen Ⅰa nd osteocalcin were positive after osteoinduction. VonLKossa staining showedthe calcium nodule. For chondrogenesis cells，the toluidin blue Staining、safranln o stainingand Collegen Ⅱi mmunocytochemical stain was Positive.Conclusion:Density gradientcentrifugation combined with bone marrow culture can isolate and purify the BMSCsideally. BMSCs have satisfactory Proliferation Potentiality in vitro，Under the effect ofinducer，BMSCs can represent specific cyte PhenotyPe of chondrocytes or osteocytes，which are ideal seed cells of tissue engineering. Part Ⅲ. Experimental Study on the Repairing Effect of BMscs/β-TcP/pluronicF-127Composite Scaffold on Rabbit Articular OsteoChondralDefectsObjective:To investigate the reparation effect of osteochondral defect withβ-TcP/pluronicF-127Composite Scaffold combined with BMSCs. ExPlore the feasibilitythatβ-TcP/pluronicF-127Composite Scaffold，loaded with BMSCs，work as tissueengineering scaffold to restore the osteochondral defect.Method: MSCs were isolated andexpanded from autologous rabbit bone marrow.36Newzealand White Rabbits wererandomly divided into three grouPs(A，B，C group). Full-thickness articular osteochondraldefects(5mm in diameter and4-5mm in depth) were created mechanically in the femoral trochlea of the rabbits. BMscs/β-TcP/pluronicF-127、BMscs/β-TcP Composite Scaffoldwere respectively transplanted into the defects of A group and B group，the C groupgroup is blank and underwent no special treatment. Samples were extracted3and6monthsafter operation for histological,histo-chemical and immuno-histo-chemical analysis.Biomechanics test was Proeeed to examine the sample of A grouP，B grouP and normaljoint.Result:The specimens harvested from A group demonstrated a hyaline Cartilageformation， the new formed cartilage integrated satisfactorily with Adjacent normalcartilage;The B group demonstrated a immature Hyaline cartilage. The C grouPdemonstrated no significant repairing effect. In the Whole，the restore effect of A grouP isthe best，and C grouP is the worst. Biomechanics test show: The creep time and stressrelaxation time of A group and B group is shorter than the normal joint，and young，smodulus is smaller than the normal joint but there is no significance difference betweenthem. Conclusion:The BMSCs are satisfactory seed cells of tissue engineering，Theβ-TcP/pluronicF-127Composite Scaffold is a satisfactory scaffold，which can carry BMSCs tothe defect area and restore the osteochondral defect. The restore effect is ideal，Tissueengineering cartilage using BMscs/β-TcP/pluronicF-127composite Scaffold may be aPromising way for the treatment of osteochondral defects.