转化生长因子β1促进生物稳定性聚氨酯支架表面软骨组织生成
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摘要
目的评估以生物稳定性聚氨酯(polyurethane PU)为中心支架,应用转化生长因子β1(TGFβ1)促进支架表面残耳软骨细胞生长,制备软骨组织替代物的可行性。
内容实验采用弹性聚氨酯(polyurethane PU)材料作为中心支架；以体外扩增获得的残耳软骨细胞作种子细胞；以TGFβ1促进软骨组织生长。将细胞-支架复合后,诱导培育形成软骨组织-支架复合物。
方法
取残耳软骨组织,胶原酶消化法获得软骨细胞,体外传代培养及生物学特性研究。
体外实验将第5代软骨细胞接种于支架表面,实验组使用TGFβ1诱导,分别于第1、2、3周取材进行大体观察,组织学和免疫组化检测。应用2x3析因设计处理湿重数值。
体内实验实验组使用含有TGFβ1的完全培养基,对照组使用普通完全培养基,体外培养1周后,接种于裸鼠皮下。分别于第4、8、12周取材进行大体观察,组织学,免疫组化,RT-PCR和扫描电镜检查。使用SPSS16.0统计软件处理湿重数值。
结果原代和第1-5代残耳软骨细胞生长旺盛。细胞在聚氨酯支架表面形成软骨组织,向支架内迁移并分泌基质。组织学检测显示基质内有糖胺多糖及Ⅱ型胶原形成,RT-PCR检测到各实验组SOX9、RUNX2、Aggrecan、CollagenX基因表达水平均高于所对应的阳性对照组,扫描电镜显示细胞在支架表面及孔隙内生长。统计显示体内实验中实验组和对照组获得的软骨组织湿重具有显著差异。
结论残耳软骨细胞能够形成包裹聚氨酯支架的软骨组织层,TGFβ1有促进与聚氨酯支架复合的软骨组织生长的作用。
Objective:The aim of the present study was to evaluate the quality of candidate product based on human-microtia-derived cells and polyurethane scaffold and to identify the necessity of Transforming Growth Factorβ1.
Contents:Human misshapen auricular chondrocytes from microtia were embedded in elastic polyurethane scaffold and treated with transforming growth factorβ1 to form candidate for cartilage.
Methods:The human misshapen auricular chondrocytes from microtia were isolated and cultured to observe morphological changes, growth curves. The second passage chondrocytes were identified by immunohistochemistry with collagen type II monoclonal antibody.
In vitro the fifth passage chondrocytes were embedded in polyurethane scaffold and cultured under the conditions:#1 without growth factors (control);#2 with 10ng/ml of TGFβ1 (test). After 1,2 and 3 weeks, morphological examination were then studied.The specimens were processed hard tissue sections. Sections were cut and performed with histology assessments. After testing the data for normal distribution and equal variance, differences between the groups were analyzed by the Univariate Analysis of Variance. Statistical significance was accepted for a value of p<0.05.
The fifth passage chondrocytes were directly seeded into polyurethane scaffold and cultured under the conditions:#1 without growth factors (control);#2 with 10ng/ml of TGFβ1(test) for 7 days. Then the chondrocyte-polyurethane constructs were implanted into Balb/c nude mice subcutaneously. After 4,8 and 12 weeks follow-up, the cartilage-scaffold constructs in nude mice were taken out for morphological examination, expressions of collagen typeII, collagen typeⅪ, RunX2, SOX9 and aggrecan in cartilage with RT-PCR, histology assessments and scanning electron microscopy(SEM). After testing the data for normal distribution and equal variance, differences between the groups were analyzed by the Wilcoxon Signed Ranks. Statistical significance was accepted for a value of p< 0.05.
Results:The growth curve showed that the proliferation of chondrocytes from the first to the fifth passage was powerful. Scanning electron microscopy (SEM) pictures showed that cartilage grew well both inside and outside of the scaffold. Histology slices from the test groups showed a better three-dimensional cell growth and extensive cell distribution inside the scaffolds. The result of RT-PCR showed expression of SOX9, RUNX2, Aggrecan and CollagenX higher in experimental group when compare to corresponding control groups. The wet weight results of cartilage obtained from the mice showed significant difference (p<0.05)between the groups under the conditions:#1 without growth factors(control);#2 with lOng/ml of TGF 0 1(test).
Conclusion:Biological stabile polyurethane scaffold represent a promising new type of scaffold for the reconstruction of cartilage. TGFβ1 can accelerate the formation of cartilage on the polyurethane scaffold.
内容实验采用弹性聚氨酯(polyurethane PU)材料作为中心支架；以体外扩增获得的残耳软骨细胞作种子细胞；以TGFβ1促进软骨组织生长。将细胞-支架复合后,诱导培育形成软骨组织-支架复合物。
方法
取残耳软骨组织,胶原酶消化法获得软骨细胞,体外传代培养及生物学特性研究。
体外实验将第5代软骨细胞接种于支架表面,实验组使用TGFβ1诱导,分别于第1、2、3周取材进行大体观察,组织学和免疫组化检测。应用2x3析因设计处理湿重数值。
体内实验实验组使用含有TGFβ1的完全培养基,对照组使用普通完全培养基,体外培养1周后,接种于裸鼠皮下。分别于第4、8、12周取材进行大体观察,组织学,免疫组化,RT-PCR和扫描电镜检查。使用SPSS16.0统计软件处理湿重数值。
结果原代和第1-5代残耳软骨细胞生长旺盛。细胞在聚氨酯支架表面形成软骨组织,向支架内迁移并分泌基质。组织学检测显示基质内有糖胺多糖及Ⅱ型胶原形成,RT-PCR检测到各实验组SOX9、RUNX2、Aggrecan、CollagenX基因表达水平均高于所对应的阳性对照组,扫描电镜显示细胞在支架表面及孔隙内生长。统计显示体内实验中实验组和对照组获得的软骨组织湿重具有显著差异。
结论残耳软骨细胞能够形成包裹聚氨酯支架的软骨组织层,TGFβ1有促进与聚氨酯支架复合的软骨组织生长的作用。
Objective:The aim of the present study was to evaluate the quality of candidate product based on human-microtia-derived cells and polyurethane scaffold and to identify the necessity of Transforming Growth Factorβ1.
Contents:Human misshapen auricular chondrocytes from microtia were embedded in elastic polyurethane scaffold and treated with transforming growth factorβ1 to form candidate for cartilage.
Methods:The human misshapen auricular chondrocytes from microtia were isolated and cultured to observe morphological changes, growth curves. The second passage chondrocytes were identified by immunohistochemistry with collagen type II monoclonal antibody.
In vitro the fifth passage chondrocytes were embedded in polyurethane scaffold and cultured under the conditions:#1 without growth factors (control);#2 with 10ng/ml of TGFβ1 (test). After 1,2 and 3 weeks, morphological examination were then studied.The specimens were processed hard tissue sections. Sections were cut and performed with histology assessments. After testing the data for normal distribution and equal variance, differences between the groups were analyzed by the Univariate Analysis of Variance. Statistical significance was accepted for a value of p<0.05.
The fifth passage chondrocytes were directly seeded into polyurethane scaffold and cultured under the conditions:#1 without growth factors (control);#2 with 10ng/ml of TGFβ1(test) for 7 days. Then the chondrocyte-polyurethane constructs were implanted into Balb/c nude mice subcutaneously. After 4,8 and 12 weeks follow-up, the cartilage-scaffold constructs in nude mice were taken out for morphological examination, expressions of collagen typeII, collagen typeⅪ, RunX2, SOX9 and aggrecan in cartilage with RT-PCR, histology assessments and scanning electron microscopy(SEM). After testing the data for normal distribution and equal variance, differences between the groups were analyzed by the Wilcoxon Signed Ranks. Statistical significance was accepted for a value of p< 0.05.
Results:The growth curve showed that the proliferation of chondrocytes from the first to the fifth passage was powerful. Scanning electron microscopy (SEM) pictures showed that cartilage grew well both inside and outside of the scaffold. Histology slices from the test groups showed a better three-dimensional cell growth and extensive cell distribution inside the scaffolds. The result of RT-PCR showed expression of SOX9, RUNX2, Aggrecan and CollagenX higher in experimental group when compare to corresponding control groups. The wet weight results of cartilage obtained from the mice showed significant difference (p<0.05)between the groups under the conditions:#1 without growth factors(control);#2 with lOng/ml of TGF 0 1(test).
Conclusion:Biological stabile polyurethane scaffold represent a promising new type of scaffold for the reconstruction of cartilage. TGFβ1 can accelerate the formation of cartilage on the polyurethane scaffold.
引文
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[3]张之智,胡金明,赵鹏.关节镜下微骨折术修复膝关节软骨缺损[J].中国骨与关节损伤杂志,2008,23(7)：578-579.
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[5]司徒镇强,吴军正.细胞培养[M].西安市北大街85号：世界图书出版西安公司,2008：26-27.
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[11]Song Y, Wennink J W H, Kamphuis M M J. Effective seeding of smooth muscle cells into tubular poly(trimethylene carbonate) scaffolds for vascular tissue engineering[J].J Biomed Mater Res,2010,95A:440-446.
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[13]Corey J M, Gertz C C, Wang B. The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons[J].Acta Biomater,2008,4(4):863-875.
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[18]Ajili S H, Ebrahimi N G, Soleimani M. Polyurethane/polycaprolactane blend with shape memory effect as a proposed material for cardiovascular implants[J]. Acta Biomaterialia,2009,5:1519-1530.
[19]陈昊东,赵剑豪,曾戎.聚碳酸亚丙酯/壳聚糖纳米纤维复合三维多孔支架的构建与性能[J].中国组织工程研究与临床康复,2010,14(21)：3873-3877.
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