CS/PVA凝胶负载基因转染的BMSCs移植修复兔关节软骨缺损的研究
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摘要
第一部分温敏型壳聚糖/聚乙烯醇水凝胶的制备及生物相容性研究
目的：研究温敏型壳聚糖/聚乙烯醇水凝胶的制备条件以及不同壳聚糖与聚乙烯醇比例制成凝胶的强度、空间结构、红外光谱特征及平衡溶胀度、体外降解率,并评估凝胶的生物相容性。
方法：将2%壳聚糖盐酸溶液调至PH中性后与不同比例的聚乙烯醇混合后,观察不同样本的凝胶化时间及凝胶的强度,红外光分析仪观察凝胶的红外光谱特征、扫描电镜观察凝胶冷冻干燥后的空间结构并检测不同凝胶样本的平衡溶胀度和降解率。并通过致敏实验,全身毒性反应,细胞毒性反应及肌肉植入实验,评价凝胶的生物相容性。
结果：不同比例壳聚糖和聚乙烯醇凝胶样本的强度,空间结构以及溶胀度测试结果表明,随着聚乙烯醇在凝胶中比例增加,样本的凝胶化时间延长,强度增加,网络结构致密,溶胀度变小。致敏实验证明凝胶对SD大鼠皮肤无刺激,过量注射大白兔无全身毒性反应,细胞毒性0-Ⅰ级,在材料植入肌肉后早期有炎性浸润及纤维包膜形成,随植入时间延长,炎性反应消失,包膜变薄,植入12w后见血管长入,凝胶部分降解,证实凝胶材料具有良好的组织相容性。
结论：壳聚糖/聚乙烯醇复合水凝胶具有温度敏感特性,互穿网络结构和更好的机械强度,细胞毒性低,组织相容性好,符合组织工程支架材料的要求。
第二部分Ad-hTGF-β1体外诱导兔BMSCs向软骨细胞分化
目的：通过Ad-TGF-β1转染兔BMSCs,诱导BMSCs向软骨细胞定向分化。
方法：体外分离培养兔骨髓间充质干细胞,观察细胞生长状态并绘制生长曲线。第三代BMSCs经过Ad-hTGF-β1转染后,观察细胞形态变化,并通过转染后不同时间点甲苯胺蓝染色观察细胞内糖胺聚糖的表达。并在转染后1w、2w、3w、4w不同时间点使用RT-PCR方法检测hTGF-β1和Aggrecan mRNA在细胞内的表达情况,在转染后2w提取细胞蛋白,使用Western blot方法分析hTGF-β1, CollagenII蛋白的表达,并在不同时间段进行Ⅱ型胶原免疫组化检测细胞浆内Ⅱ型胶原的表达。
结果：兔BMSCs经Ad-hTGFβ-1转染后,早期出现细胞坏死、漂浮,3d后恢复正常。转染2w后甲苯胺蓝染色阳性,并持续表达。RT-PCR表明转染后1-4w后BMSCs均表达Aggrecan和hTGFβ-1的mRNA,随培养时间延长Aggrecan表达量增加,hTGFβ-1表达量递减,未转染组均表达阴性。Western blot;检测到转染后2w细胞内的hTGFβ-1和Ⅱ型胶原蛋白表达,空载病毒转染组和未转染组阴性。转染后的细胞Ⅱ型胶原免疫组化阳性,随培养时间增加,阳性率增加。
结论：经Ad-hTGFβ-1转染的BMSCs在体外培养过程中可表达软骨细胞表型。
第三部分CS/PVA凝胶负载Ad-TGF-β1转染的BMSCs移植修复兔关节软骨缺损
目的：观察温敏型CS/PVA凝胶负载Ad-hTGFβ-1转染的BMSCs移植修复兔关节软骨缺损的实验效果。
方法：体外分离培养兔BMSCs,以CS/PVA凝胶作为三维培养支架,激光共聚焦显微镜下观察其增殖活性。并以构建成功的Ad-hTGF-β1载体转染体外培养的BMSCs,在转染1周后以细胞免疫荧光方法检测Ad-hTGF-β1的表达效率。用24只成年新西兰大白兔制造关节软骨缺损模型,双侧后肢均用于实验,动物模型分为4组(A、B、C、D组),各组动物6只。A组：凝胶复合转染BMSCs修复组；B组：凝胶复合未转染BMSCs修复组；C组：凝胶修复组；D组：空白对照组。分别于术后4周、8周及16周各处死实验动物8只,取缺损部位及周围正常软骨组织,进行大体标本观察及染色组织学观察。通过术后16周大体标本和组织学染色结果评价各组修复效果,按照Wakitani关节软骨组织评分,对各组修复效果进行统计学分析,以P<0.05有统计学意义。
结果：兔BMSCs在CS/PVA凝胶中保持较高的增殖活性。免疫组化证实Ad-hTGF-β1转染BMSC的阳性率为85.4%。术后4周大体标本观察：A、B、C三组缺损部位被瓷白色凝胶填充,填充物与周围股骨关节面结合紧密,关节表面光滑平整,填充组织与正常关节软骨分界线明显,D组软骨缺损部位为陈旧性淤血块填充。术后8周大体标本观察见A组及B组缺损部位边缘与正常软骨分界模糊不清,缺损区组织形似软骨,色泽与周围软骨组织接近。C组表面光滑平整,与周围软骨结合牢靠,颜色与周围软骨区别较大,界限清楚。D组缺损底部及周围有部分肉芽填充。术后16周取材见A组关节软骨缺损部位为软骨样组织填充,关节面平整,与正常关节软骨界限不清,缺损区外观质地同其他各组有明显差别。术后16周A组组织学观察见再生的软骨组织细胞排列及细胞密度与正常软骨相似,番红O染色阳性,二型胶原免疫组化阳性,Wakitani评分同其它各组相比差异有显著性(P<0.05)。
结论：CS／PVA凝胶作为一种温敏型可注射支架材料,其负载hTGFβ-1转染的BMSCs移植可用于兔关节软骨缺损修复。
PartⅠ:The preparation and biocompatibility of injectable thermo-sensitive chitosan/Poly(vinyl alcohol) hydrogel
Objective:To investigate the preparation conditions of thermosensitive chitosan/ PVA(Polyvinyl alcohol) hydrogel and evaluate the mechanical properties,spatial structure,infrared spectral characteristics and swelling degree,degradation rate and gel biocompatibility of these hydrogen.
Methods:2% chitosan hydrochloride solution was transferred to a neutral PH and mixed with different proportions of PVA,then the gelation time and strength were observed,infrared spectral characteristics were analysised by infrared analyzers,spatial structures were observed under a scanning electron microscopy (SEM) after freeze-drying, the equilibrium swelling degree and the degradation rate of these different samples were tested.Then through sensitization experiments, systemic toxicity, cell toxicity and muscle implantation test to evaluate the biocompatibility of them.
Results:The different ratios of chitosan and polyvinyl alcohol gels showed that with a larger proportion of PVA in the gel,the gelaton time were extended,the strength were increased, the network structure became dense,swelling degree turned smaller.Gel had no simulations to the SD rats skin through sensitization experiments,rabbits without systemic toxicity after excessive injection,the cytotoxicity were between grade 0 toⅠ.there were inflammatory infiltration and fibrous capsule formation of the early days after the gel implanted into muscle,with the time prolonged,inflammatory reaction disappeared and capsule thinning.The blood vessels grew into the gel and the gel were seen part of degradation after 12w implantation.All these results were certificated that the gel had good histocompatibility.
Conclusion:Chitosan/PVA composite hydrogel with a thermosensitive characteristics,interpenetrating network structure,better mechanical strength,low cytotoxicity and good biocompatibility,It complys with the requirement of tissue engineering scaffold material.
Part II:Rabbit BMSCs differentiation into chondrocytes induced by Ad-TGF-β1 in vitro
Objective:To induce bone marrow mesenchymal stem cells differentiation into chondrogenic by Ad-TGF-β1 transfection.
Methods:Rabbit bone marrow mesenchymal stem cells were isolated and cultured in vitro,to observe the cell's growth state and growth curve.The morphological changes of the third passage of MSCs was observed after transfectection by Ad-hTGF-β1,and through toluidine blue staining detecting the expression of glycosaminoglycan in the cells after transfected at different time.RT-PCR methods to detect hTGF-β1 and Aggrecan mRNA expression in cells after transfection 1w,2w,3w,4w at different time points.Using Western blot method to analysis hTGF-β1 and Collagen II expression 2w after transfection and type II collagen immunohistochemistry to defect the type II collagen expression in the transfected cells.
Results:There were some cells nesrosis at the early after transfection,then returned to normal in about 3d, toluidine blue staining were positive 2w after transfection and continue to express with the time.RT-PCR showed that mRNA of hTGFβ-1 and Aggrecan were expressed in the transfected cells at different time points,the expression of Aggrecan were increased and that of hTGF-β1 were reduced with the time.HTGF-β1 and Type II collagen protein were defected by Western blot methods in the transfected cells after 2 weeks and they were both negative in the empty virus infection and untransfected group.Type II collagen of transfected cells were positive by immunohistochemical staining and positive rate increased with the time.
Conclusion:Bone marrow mesenchymal stem cells could express the process of chondrocyte phenotype by Ad-hTGFβ-1 transfected in vitro.
Part III:Experiment study of rabbit articiular cartilage defects repaired by thermosensitive CS/PVA composite hydrogel engineered hTGF-β1 transfected bone marrow mesenchymal stem cells
Objective:To investigate the experiment effects of rabbit joint articular cartilage defects repaired by thermosensitive CS/PVA composite hydrogel engineered hTGFβ-1 transfected bone marrow mesenchymal stem cells.
Methods:Bone marrow mesenchymal stem cells were isolated and cultured in vitro.The CS/PVA composite hydrogel were prepared as a three-dimensional scaffolds and to observe the activity of proliferation under laser confocal microscope The mesenchymal stem cells were tranfected by Ad-hTGF-β1 in vitro and the the positive rate of transfection was defected by cell immunofluorescence methods after Ad-hTGF-β1 transfected for one week.24 adult New Zealand white rabbits with full articular cartilage defects were randomly divided into four groups,each group had 6 animals,both hind limbs were used in the experiment.Group A:to repair the defects with hydrogel combined with transfected cells;Group B:to repair the defects with hydrogel combined with untransfected cells;Group C:to repair the defects with hydrogel only;Group D:blank control group.24 animals were sacrificed after 4 weeks,8 weeks and 16 weeks.The defect site and the surrounding normal cartilage tissue were obtained for gross observation and histological staining.Specimens and histological observation were used to evaluate the repairment effect after 16 weeks according to Wakitani's score,which had statistically significant when P<0.05.
Results:Rabbit BMSCs showed a high activity of proliferation.in the CS/PVA gel,when transfected by Ad-hTGF-β1,the positive rate was about 85.4% by cell immunofluorescence methods.Gross observation after 4 weeks,the defect sites of group A,B and C were filled with porcelain white gel and the filler combined with the surrounding articular surface of the femur closely. The articular surface were smooth and the filler had a significant boundaries with the normal tissure.The cartilage defect sites of group D were filled with old blood stasis block.After 8 weeks,the defect edge of group and group B were unclear with the normal tissure,the shape of cartilage defects were closely to the surrounding cartilage.the texture and color of group C had a widely difference with the cartilage around.and that of group D were filled with granulation in and around the bottom of the defect. After 16 weeks the defects of group A were repaired by cartilage-like tissue,the surface of these articulars were smooth and could not distinguish the normal articular cartilage and the defects.The cell arrangement and densities of regenerated cartilage were similar to normal cartilage,Safranin O staining were positive and typeⅡcollagen immunohistochemistry were positive.There was a significant difference according to Wakitani's score compaired with other groups.
Conciusion:CS/PVA gel is a kind of thermosensitive injectable scaffold.Rabbit articiular cartilage defects could be repaired by CS/PVA hydrogel engineered hTGF-β1 transfected bone marrow mesenchymal stem cells.
目的：研究温敏型壳聚糖/聚乙烯醇水凝胶的制备条件以及不同壳聚糖与聚乙烯醇比例制成凝胶的强度、空间结构、红外光谱特征及平衡溶胀度、体外降解率,并评估凝胶的生物相容性。
方法：将2%壳聚糖盐酸溶液调至PH中性后与不同比例的聚乙烯醇混合后,观察不同样本的凝胶化时间及凝胶的强度,红外光分析仪观察凝胶的红外光谱特征、扫描电镜观察凝胶冷冻干燥后的空间结构并检测不同凝胶样本的平衡溶胀度和降解率。并通过致敏实验,全身毒性反应,细胞毒性反应及肌肉植入实验,评价凝胶的生物相容性。
结果：不同比例壳聚糖和聚乙烯醇凝胶样本的强度,空间结构以及溶胀度测试结果表明,随着聚乙烯醇在凝胶中比例增加,样本的凝胶化时间延长,强度增加,网络结构致密,溶胀度变小。致敏实验证明凝胶对SD大鼠皮肤无刺激,过量注射大白兔无全身毒性反应,细胞毒性0-Ⅰ级,在材料植入肌肉后早期有炎性浸润及纤维包膜形成,随植入时间延长,炎性反应消失,包膜变薄,植入12w后见血管长入,凝胶部分降解,证实凝胶材料具有良好的组织相容性。
结论：壳聚糖/聚乙烯醇复合水凝胶具有温度敏感特性,互穿网络结构和更好的机械强度,细胞毒性低,组织相容性好,符合组织工程支架材料的要求。
第二部分Ad-hTGF-β1体外诱导兔BMSCs向软骨细胞分化
目的：通过Ad-TGF-β1转染兔BMSCs,诱导BMSCs向软骨细胞定向分化。
方法：体外分离培养兔骨髓间充质干细胞,观察细胞生长状态并绘制生长曲线。第三代BMSCs经过Ad-hTGF-β1转染后,观察细胞形态变化,并通过转染后不同时间点甲苯胺蓝染色观察细胞内糖胺聚糖的表达。并在转染后1w、2w、3w、4w不同时间点使用RT-PCR方法检测hTGF-β1和Aggrecan mRNA在细胞内的表达情况,在转染后2w提取细胞蛋白,使用Western blot方法分析hTGF-β1, CollagenII蛋白的表达,并在不同时间段进行Ⅱ型胶原免疫组化检测细胞浆内Ⅱ型胶原的表达。
结果：兔BMSCs经Ad-hTGFβ-1转染后,早期出现细胞坏死、漂浮,3d后恢复正常。转染2w后甲苯胺蓝染色阳性,并持续表达。RT-PCR表明转染后1-4w后BMSCs均表达Aggrecan和hTGFβ-1的mRNA,随培养时间延长Aggrecan表达量增加,hTGFβ-1表达量递减,未转染组均表达阴性。Western blot;检测到转染后2w细胞内的hTGFβ-1和Ⅱ型胶原蛋白表达,空载病毒转染组和未转染组阴性。转染后的细胞Ⅱ型胶原免疫组化阳性,随培养时间增加,阳性率增加。
结论：经Ad-hTGFβ-1转染的BMSCs在体外培养过程中可表达软骨细胞表型。
第三部分CS/PVA凝胶负载Ad-TGF-β1转染的BMSCs移植修复兔关节软骨缺损
目的：观察温敏型CS/PVA凝胶负载Ad-hTGFβ-1转染的BMSCs移植修复兔关节软骨缺损的实验效果。
方法：体外分离培养兔BMSCs,以CS/PVA凝胶作为三维培养支架,激光共聚焦显微镜下观察其增殖活性。并以构建成功的Ad-hTGF-β1载体转染体外培养的BMSCs,在转染1周后以细胞免疫荧光方法检测Ad-hTGF-β1的表达效率。用24只成年新西兰大白兔制造关节软骨缺损模型,双侧后肢均用于实验,动物模型分为4组(A、B、C、D组),各组动物6只。A组：凝胶复合转染BMSCs修复组；B组：凝胶复合未转染BMSCs修复组；C组：凝胶修复组；D组：空白对照组。分别于术后4周、8周及16周各处死实验动物8只,取缺损部位及周围正常软骨组织,进行大体标本观察及染色组织学观察。通过术后16周大体标本和组织学染色结果评价各组修复效果,按照Wakitani关节软骨组织评分,对各组修复效果进行统计学分析,以P<0.05有统计学意义。
结果：兔BMSCs在CS/PVA凝胶中保持较高的增殖活性。免疫组化证实Ad-hTGF-β1转染BMSC的阳性率为85.4%。术后4周大体标本观察：A、B、C三组缺损部位被瓷白色凝胶填充,填充物与周围股骨关节面结合紧密,关节表面光滑平整,填充组织与正常关节软骨分界线明显,D组软骨缺损部位为陈旧性淤血块填充。术后8周大体标本观察见A组及B组缺损部位边缘与正常软骨分界模糊不清,缺损区组织形似软骨,色泽与周围软骨组织接近。C组表面光滑平整,与周围软骨结合牢靠,颜色与周围软骨区别较大,界限清楚。D组缺损底部及周围有部分肉芽填充。术后16周取材见A组关节软骨缺损部位为软骨样组织填充,关节面平整,与正常关节软骨界限不清,缺损区外观质地同其他各组有明显差别。术后16周A组组织学观察见再生的软骨组织细胞排列及细胞密度与正常软骨相似,番红O染色阳性,二型胶原免疫组化阳性,Wakitani评分同其它各组相比差异有显著性(P<0.05)。
结论：CS／PVA凝胶作为一种温敏型可注射支架材料,其负载hTGFβ-1转染的BMSCs移植可用于兔关节软骨缺损修复。
PartⅠ:The preparation and biocompatibility of injectable thermo-sensitive chitosan/Poly(vinyl alcohol) hydrogel
Objective:To investigate the preparation conditions of thermosensitive chitosan/ PVA(Polyvinyl alcohol) hydrogel and evaluate the mechanical properties,spatial structure,infrared spectral characteristics and swelling degree,degradation rate and gel biocompatibility of these hydrogen.
Methods:2% chitosan hydrochloride solution was transferred to a neutral PH and mixed with different proportions of PVA,then the gelation time and strength were observed,infrared spectral characteristics were analysised by infrared analyzers,spatial structures were observed under a scanning electron microscopy (SEM) after freeze-drying, the equilibrium swelling degree and the degradation rate of these different samples were tested.Then through sensitization experiments, systemic toxicity, cell toxicity and muscle implantation test to evaluate the biocompatibility of them.
Results:The different ratios of chitosan and polyvinyl alcohol gels showed that with a larger proportion of PVA in the gel,the gelaton time were extended,the strength were increased, the network structure became dense,swelling degree turned smaller.Gel had no simulations to the SD rats skin through sensitization experiments,rabbits without systemic toxicity after excessive injection,the cytotoxicity were between grade 0 toⅠ.there were inflammatory infiltration and fibrous capsule formation of the early days after the gel implanted into muscle,with the time prolonged,inflammatory reaction disappeared and capsule thinning.The blood vessels grew into the gel and the gel were seen part of degradation after 12w implantation.All these results were certificated that the gel had good histocompatibility.
Conclusion:Chitosan/PVA composite hydrogel with a thermosensitive characteristics,interpenetrating network structure,better mechanical strength,low cytotoxicity and good biocompatibility,It complys with the requirement of tissue engineering scaffold material.
Part II:Rabbit BMSCs differentiation into chondrocytes induced by Ad-TGF-β1 in vitro
Objective:To induce bone marrow mesenchymal stem cells differentiation into chondrogenic by Ad-TGF-β1 transfection.
Methods:Rabbit bone marrow mesenchymal stem cells were isolated and cultured in vitro,to observe the cell's growth state and growth curve.The morphological changes of the third passage of MSCs was observed after transfectection by Ad-hTGF-β1,and through toluidine blue staining detecting the expression of glycosaminoglycan in the cells after transfected at different time.RT-PCR methods to detect hTGF-β1 and Aggrecan mRNA expression in cells after transfection 1w,2w,3w,4w at different time points.Using Western blot method to analysis hTGF-β1 and Collagen II expression 2w after transfection and type II collagen immunohistochemistry to defect the type II collagen expression in the transfected cells.
Results:There were some cells nesrosis at the early after transfection,then returned to normal in about 3d, toluidine blue staining were positive 2w after transfection and continue to express with the time.RT-PCR showed that mRNA of hTGFβ-1 and Aggrecan were expressed in the transfected cells at different time points,the expression of Aggrecan were increased and that of hTGF-β1 were reduced with the time.HTGF-β1 and Type II collagen protein were defected by Western blot methods in the transfected cells after 2 weeks and they were both negative in the empty virus infection and untransfected group.Type II collagen of transfected cells were positive by immunohistochemical staining and positive rate increased with the time.
Conclusion:Bone marrow mesenchymal stem cells could express the process of chondrocyte phenotype by Ad-hTGFβ-1 transfected in vitro.
Part III:Experiment study of rabbit articiular cartilage defects repaired by thermosensitive CS/PVA composite hydrogel engineered hTGF-β1 transfected bone marrow mesenchymal stem cells
Objective:To investigate the experiment effects of rabbit joint articular cartilage defects repaired by thermosensitive CS/PVA composite hydrogel engineered hTGFβ-1 transfected bone marrow mesenchymal stem cells.
Methods:Bone marrow mesenchymal stem cells were isolated and cultured in vitro.The CS/PVA composite hydrogel were prepared as a three-dimensional scaffolds and to observe the activity of proliferation under laser confocal microscope The mesenchymal stem cells were tranfected by Ad-hTGF-β1 in vitro and the the positive rate of transfection was defected by cell immunofluorescence methods after Ad-hTGF-β1 transfected for one week.24 adult New Zealand white rabbits with full articular cartilage defects were randomly divided into four groups,each group had 6 animals,both hind limbs were used in the experiment.Group A:to repair the defects with hydrogel combined with transfected cells;Group B:to repair the defects with hydrogel combined with untransfected cells;Group C:to repair the defects with hydrogel only;Group D:blank control group.24 animals were sacrificed after 4 weeks,8 weeks and 16 weeks.The defect site and the surrounding normal cartilage tissue were obtained for gross observation and histological staining.Specimens and histological observation were used to evaluate the repairment effect after 16 weeks according to Wakitani's score,which had statistically significant when P<0.05.
Results:Rabbit BMSCs showed a high activity of proliferation.in the CS/PVA gel,when transfected by Ad-hTGF-β1,the positive rate was about 85.4% by cell immunofluorescence methods.Gross observation after 4 weeks,the defect sites of group A,B and C were filled with porcelain white gel and the filler combined with the surrounding articular surface of the femur closely. The articular surface were smooth and the filler had a significant boundaries with the normal tissure.The cartilage defect sites of group D were filled with old blood stasis block.After 8 weeks,the defect edge of group and group B were unclear with the normal tissure,the shape of cartilage defects were closely to the surrounding cartilage.the texture and color of group C had a widely difference with the cartilage around.and that of group D were filled with granulation in and around the bottom of the defect. After 16 weeks the defects of group A were repaired by cartilage-like tissue,the surface of these articulars were smooth and could not distinguish the normal articular cartilage and the defects.The cell arrangement and densities of regenerated cartilage were similar to normal cartilage,Safranin O staining were positive and typeⅡcollagen immunohistochemistry were positive.There was a significant difference according to Wakitani's score compaired with other groups.
Conciusion:CS/PVA gel is a kind of thermosensitive injectable scaffold.Rabbit articiular cartilage defects could be repaired by CS/PVA hydrogel engineered hTGF-β1 transfected bone marrow mesenchymal stem cells.
引文
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