β-TcP/pluronicF-127复合支架结合BMSCs修复兔关节骨软骨缺损的实验研究
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摘要
第一部分：凋亡相关基因在骨关节炎软骨中的表达及意义目的:研究骨关节炎中细胞凋亡情况，凋亡相关基因(FAS、BCL-2、caspases3)等在骨关节炎中的表达及意义，从而在一定程度阐明骨关节炎的发病机理。方法:通过Hulth法建立兔关节炎模型取得软骨标本，免疫组化S-P法检测凋亡相关基因(FAS、BCL-2、caspases3)在骨性关节炎软骨细胞中的表达情况，TUNEL法检测骨性关节炎软骨细胞凋亡情况，并探讨凋亡相关基因(FAS、BCL-2、caspases3)的表达与软骨细胞凋亡程度之间的相关性。结果:术后8周时关节软骨呈现典型的0A病理特征。实验组和对照组关节软骨细胞凋亡指数的差异具有显著性(P<0.05);在软骨细胞中FAS蛋白表达实验组阳性率高于对照组，差异具有显著性(P<0.05);实验组关节软骨中软骨细胞凋亡程度与FAS蛋白的表达呈正相关，差异具有显著性(P<0.05)；在关节软骨细胞中bcl-2蛋白的表达实验组阳性率低于对照组，但差异没有显著性(P>0.05);实验组关节软骨中软骨细胞凋亡程度与bcl-2蛋白的表达呈负相关，差异具有显著性(P<0.05); caspase-3在实验组的软骨细胞阳性率高于对照组，差异具有显著性(P<0.01);实验组关节软骨中软骨细胞凋亡程度与caspase3的表达呈正相关，差异具有显著性(p<0.05);结论:1.Hulth法可制作较好的骨关节炎动物模型。2.关节软骨细胞的凋亡明显增加可能是关节炎形成的重要原因之一。3.骨关节炎中FAS,BCL-2,caspase3等凋亡相关基因的表达与细胞凋亡程度具有明显相关性，表明表达其与骨关节炎有密切关系。
第二部分兔骨髓间充质干细胞的分离、培养、鉴定及体外诱导分化
目的:探索兔骨髓间充质干细胞的分离、培养、鉴定及体外诱导分化的方法，研究其体外增殖，分化能力。探讨其作为骨组织工程种子细胞的可行性和应用价值。方法:采用密度梯度离心与全骨髓培养结合的方法分离、纯化兔骨髓间充质干细胞，体外培养;以流式细胞仪检测BMSCs的表面抗原表达;在相差显微镜下观察细胞形态并绘制生长曲线;分别体外诱导第3代细胞向软骨细胞和骨细胞分化，对诱导分化为骨细胞进行碱性磷酸酶染色、VonKossa染色和I型胶原和骨钙素免疫组化染色检测鉴定，对诱导分化为软骨细胞进行甲苯胺蓝染色、番红O染色和Ⅱ型胶原免疫组化染色检测鉴定。结果:经密度梯度离心结合全骨髓培养法分离所得到的细胞形态均一，呈长梭形或纺锤形。BMSCs增殖能力活跃,流式细胞仪检测BMSCs显示CD105,CD44,表达阳性，CD34和CD45表达为阴性。成骨诱导培养后，可见碱性磷酸酶染色、VonKossa染色、I型胶原和骨钙素免疫组化染色阳性;向软骨细胞诱导分化后，细胞甲苯胺蓝染色、番红0染色和Ⅱ型胶原免疫组化染色为阳性。结论:密度梯度分离法与全骨髓培养法结合能理想的分离、纯化BMSCs; BMSCs在体外具有良好的增殖潜能，BMSCs在诱导剂作用下能表现出成骨细胞和软骨细胞生物学特性，是组织工程理想的种子细胞。
第三部分BMscs/β-TcP/pluronicF-127复合支架修复兔关节软骨缺损的实验研究
目的:研究β-TcP/pluronicF-127复合支架结合BMSCs修复兔膝关节骨软骨缺损的效果，探讨β-TcP/pluronicF-127复合支架做为组织工程支架承载BMSCs修复骨软骨缺损的可行性。方法:分离培养兔自体MSCs，将36只健康新西兰大白兔并随机分为三组(A组、B组、C组)，于股骨髁关节面制备关节软骨缺损模型(每孔直径5mm、深度4-5mm)。A组、B组关节缺损处分别植入BMscs/β-TcP/pluronicF-127.、BMscs/β-TcP复合支架，C组为空白组不作处理。术后3、6个月分别取材，进行缺损区组织学、组织化学和免疫组织化学分析。A组、B组和正常关节标本进行生物力学测试。结果:A组能形成丰富的透明软骨样修复组织，与周围组织整合良好;B组以不成熟透明软骨;C组无修复组织。总的修复效果A组最理想，C组最差。生物力学测试得出: B组和C组标本的蠕变时间和应力松弛时候较正常关节均缩短，杨氏模量较正常关节偏小，但差异均无统计学意义。结论: BMSCs是良好的组织工程种子细胞，β-TcP/pluronicF-127复合支架是良好的生物工程支架,能承载BMSCs至缺损区修复骨软骨缺损缺损，修复效果理想。BMscs/β-TcP/pluronicF-127复合支架材料构建组织工程软骨，是—种修复关节软骨缺损行之有效的方法。
Part Ⅰ Significance and expression of apoptosis control genes in OAcartilage
objective：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 the
expressions 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 vitro
objective: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 OsteoChondralDefects
Objective: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.
第二部分兔骨髓间充质干细胞的分离、培养、鉴定及体外诱导分化
目的:探索兔骨髓间充质干细胞的分离、培养、鉴定及体外诱导分化的方法，研究其体外增殖，分化能力。探讨其作为骨组织工程种子细胞的可行性和应用价值。方法:采用密度梯度离心与全骨髓培养结合的方法分离、纯化兔骨髓间充质干细胞，体外培养;以流式细胞仪检测BMSCs的表面抗原表达;在相差显微镜下观察细胞形态并绘制生长曲线;分别体外诱导第3代细胞向软骨细胞和骨细胞分化，对诱导分化为骨细胞进行碱性磷酸酶染色、VonKossa染色和I型胶原和骨钙素免疫组化染色检测鉴定，对诱导分化为软骨细胞进行甲苯胺蓝染色、番红O染色和Ⅱ型胶原免疫组化染色检测鉴定。结果:经密度梯度离心结合全骨髓培养法分离所得到的细胞形态均一，呈长梭形或纺锤形。BMSCs增殖能力活跃,流式细胞仪检测BMSCs显示CD105,CD44,表达阳性，CD34和CD45表达为阴性。成骨诱导培养后，可见碱性磷酸酶染色、VonKossa染色、I型胶原和骨钙素免疫组化染色阳性;向软骨细胞诱导分化后，细胞甲苯胺蓝染色、番红0染色和Ⅱ型胶原免疫组化染色为阳性。结论:密度梯度分离法与全骨髓培养法结合能理想的分离、纯化BMSCs; BMSCs在体外具有良好的增殖潜能，BMSCs在诱导剂作用下能表现出成骨细胞和软骨细胞生物学特性，是组织工程理想的种子细胞。
第三部分BMscs/β-TcP/pluronicF-127复合支架修复兔关节软骨缺损的实验研究
目的:研究β-TcP/pluronicF-127复合支架结合BMSCs修复兔膝关节骨软骨缺损的效果，探讨β-TcP/pluronicF-127复合支架做为组织工程支架承载BMSCs修复骨软骨缺损的可行性。方法:分离培养兔自体MSCs，将36只健康新西兰大白兔并随机分为三组(A组、B组、C组)，于股骨髁关节面制备关节软骨缺损模型(每孔直径5mm、深度4-5mm)。A组、B组关节缺损处分别植入BMscs/β-TcP/pluronicF-127.、BMscs/β-TcP复合支架，C组为空白组不作处理。术后3、6个月分别取材，进行缺损区组织学、组织化学和免疫组织化学分析。A组、B组和正常关节标本进行生物力学测试。结果:A组能形成丰富的透明软骨样修复组织，与周围组织整合良好;B组以不成熟透明软骨;C组无修复组织。总的修复效果A组最理想，C组最差。生物力学测试得出: B组和C组标本的蠕变时间和应力松弛时候较正常关节均缩短，杨氏模量较正常关节偏小，但差异均无统计学意义。结论: BMSCs是良好的组织工程种子细胞，β-TcP/pluronicF-127复合支架是良好的生物工程支架,能承载BMSCs至缺损区修复骨软骨缺损缺损，修复效果理想。BMscs/β-TcP/pluronicF-127复合支架材料构建组织工程软骨，是—种修复关节软骨缺损行之有效的方法。
Part Ⅰ Significance and expression of apoptosis control genes in OAcartilage
objective：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 the
expressions 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 vitro
objective: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 OsteoChondralDefects
Objective: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.
引文
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