骨髓基质干细胞和去细胞猪瓣膜支架构建组织工程瓣膜的实验研究
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摘要
第一部分 组织工程心脏瓣膜支架的制备
1 不同组织工程瓣膜制作方法的比较
目的 探讨去垢剂和胰蛋白酶对于猪主动脉瓣膜去细胞过程的影响,研究组织工程心脏瓣膜生物支架的最佳制备方法。
方法 将猪新鲜主动脉瓣膜分为3组,组1用1.5mol/L氯化钠震荡12小时和0.5%十二烷基硫酸钠(SDS)溶液处理30分钟,组2用0.25%的曲拉通X100、0.25%的脱氧胆酸钠、0.02%的EDTA、RNase I 200mg╱L和DNase I 20mg╱L溶液,恒温37℃持续震荡48小时,组3用0.05%的胰蛋白酶、0.02%的EDTA溶液,恒温37℃持续震荡24小时以及PBS持续震荡清洗24小时等处理,去除内皮细胞和间质细胞,常规苏木精伊红和Masson染色,光镜和电子扫描显微镜检查,评价去细胞效果。DAPI细胞核染色,免疫荧光显微镜观察,评价细胞核去除效果。
结果 NaCl-SDS对瓣膜超微结构损伤小,外观改变不明显,但去细胞不彻底,HE切片仍然见大量细胞核;胰蛋白酶法去细胞彻底,对结构损害大,胶原纤维可见明显断裂,曲拉通脱氧胆酸钠法未见细胞核,能有效去除细胞成分,对瓣膜结构损伤较小,扫描电镜可见基底膜。DAPI细胞核染色发现NaCl-SDS法大量DNA残留,胰蛋白酶法和曲拉通脱氧胆酸钠法未见DNA。
结论 曲拉通脱氧胆酸钠法可以有效去除细胞成分,对瓣膜结构影响较小,是一种有效去细胞的方法。
2 组织工程心脏瓣膜支架制作方法的改进及其生物学特性
目的 研究去细胞组织工程心脏瓣膜支架制作方法的改进及其生物学特性。
方法 以胰蛋白酶处理瓣膜30分钟后,再以曲拉通脱氧胆酸钠核酸酶处理48小时,进行扫描电镜和组织学检查。测定瓣膜去细胞前后的含水量,细胞的可溶性蛋白含量,以岛津力学检测仪检测瓣膜的力学特性,瓣膜浸润提取液对细胞生长的影响。将去细胞瓣叶植入新西兰种大白兔皮下,2、4、8、12周取材以检测瓣叶的组织相容性,以商用生物瓣膜为对照组。
Part One1. Tissue-engineered heart valve leaflets: comparison of different decellularization procedures of porcine heart valvesObjective Tissue engineering of heart valves should avoid the disadvantages of conventional prostheses. To determine the most effective method of producing the acellularized porcine heart valve leaflets, we compared pathological findings of the porcine heart valve leaflets produced by three methods: NaCl-SDS, trypsin and Triton -X100, sodium deoxycholate with DNAase 200mg/L, RNAase 20mg/L.treatment and further analyzed the biological character of the best one.Methods Aortic valve leaflet were harvested from pigs in slaughterhouse of Shanghai city. They were immediately immersed in 4℃ PBS solution. After washed at room temperature in povidone-iodine-solution and sterile PBS solution. Leaflets were assigned in one of the three preparation methods for decellularization. And then light and electron microscopic analyses were performed.Results HE stain showed that cells were almost absent in the leaflet treated with trypsin, and Triton-X100, while cells were partly present in the leaflets treated by NaCl-SDS. Scanning electronic microscopic revealed trypsin removed cells completely but caused strong structural alterations. Treatment with Triton-X100 achieved both complete decellularization and preservation of the matrix structure. Conclusion Techniques of decellularization are highly variable in efficiency and matrix preservation and satisfactory results were achieved in our study with Triton-X100, sodium deoxycholate and DNAase, RNAase. 2. Study on the biological properties of decellularized tissue-engineered porcine heart valve leafletsObjective To study the biological properties of decellularized tissue-engineeredporcine heart valve leaflets.Methods The harvesting and immediate treatment of porcine heart valve leaflets
was as same as above. Then the valve weight, protein content, stress strain, destroying stress, were examined. Cell toxicity and biocompatibility were also evaluated. Decellularized tissue-engineered porcine heart valve leaflets were implanted in rabbit subcutaneous, and animals were sacrificed after 2,4,8,12 weeks. Results Compared with that before the cell removal, valve weight in the decellularized aortic valve leaflets was increased significantly, but there were no significant difference in stress-strain, destroying stress between normal valve leaflets and decellularized. Cell toxicity was not significant. Biocompatibility study demonstrated that valve leaflets were absorbed gradually, Microscopic inspection of implantation site revealed no pathological inflammatory response. Conclusion The decellularized porcine heart valve leaflets have stable biological properties and can be used as an ideal tissue engineering valve scaffold. Part TwoHuman mesenchymal stem cells —a new cell source for heart valve tissue engineering.Objective Human mesenchymal stem cells (MSC) exhibit the potential to differentiate into multiple cell-lineages and can be easily obtained clinically. This study was designed to assess the feasibility of using human bone marrow-derived mesenchymal stem cells as a kind of source to construct tissue engineering heart valve.Methods Bone marrow was aspirated from the sternum of surgical field. Cells were isolated using Ficoll gradient, cultured, and characterized based on immunofluorescent staining flowcytometry, histology, and immunohistochemistry. Interstitial cells (IC) derived from the human rheumatic aortic heart valves and smooth muscle cells(SMC) from umbilical cord were cultured in vitro as contrast. Results Isolated MSC demonstrated fibroblast-like morphology. Phenotype analysis revealed positive signals for a-smooth muscle actin and vimentin. Flowcytometry of MSC pre-seeding was negative for CD31, CD14, positive for a-smooth muscle actin and vimentin. The mean fluorescence intensity ratio (MFIR) of MSC > IC and SMC was 4.9、 4.5、 4.2 and 2.7、 3.1 and 2.4 respectively. Immunohistology showed production of collagen I and collagen III. Conclusions Mesenchymal stem cells can be isolated noninvasively from the
sternum demonstrated myofibroblast-like character, bone marrow may be a potentialsource of cells for tissue engineering heart valves.Part ThreeIn vitro experience with tissue engineered heart valve on deeellularized porcine heart aortic valveObjective To evaluate the feasibility of tissue engineered heart valve on deeellularized porcine aortic valves in vitro.Methods A detergent and enzymatic extraction process has been developed to remove cellular components from porcine aortic valve, glutaraldehyde fixed heart valve was set as control group. MSC were seeded on the deeellularized valves in experiment group, Their growth condition and relationship with deeellularized porcine heart valve leaflets were observed.Results SEM show that a larger amount of cells attached on scaffolds and demonstrated viable secretionally active myofibrolasts and confluent homogenous tissue surface. The cells had covered a larger area of scaffold with a lot of matrix synthesized. The HE stain showed large amount of cells growing on the scaffolds and some cells were found in the central part of scaffolds. There was no cell growth on glutaraldehyde fixed heart valve.Conclusion It is possible to seed MSC on deeellularized porcine heart valve leaflets to create a viable tissue-engineered heart valve leaflets. Part FourExperiment study on constructing heart valve use marrow stromal cells and deeellularized porcine valve by tissue engineering approach Objective The objective of this study was to investigate the feasibility of creating tissue engineered heart valves from human mesenchymal stem cells (MSC) as an alternative cell source and deeellularized porcine heart valve leaflets to obtain viable constructs in nude mice.Methods MSC were isolated, expanded in vitro and labeled with fluorescent tracer DAPI. Porcine heart valves were deeellularized by Triton, RNAse and DNAse, reseeded and cultured under static conditions 7 days. Then cell-scaffold constructs
1 不同组织工程瓣膜制作方法的比较
目的 探讨去垢剂和胰蛋白酶对于猪主动脉瓣膜去细胞过程的影响,研究组织工程心脏瓣膜生物支架的最佳制备方法。
方法 将猪新鲜主动脉瓣膜分为3组,组1用1.5mol/L氯化钠震荡12小时和0.5%十二烷基硫酸钠(SDS)溶液处理30分钟,组2用0.25%的曲拉通X100、0.25%的脱氧胆酸钠、0.02%的EDTA、RNase I 200mg╱L和DNase I 20mg╱L溶液,恒温37℃持续震荡48小时,组3用0.05%的胰蛋白酶、0.02%的EDTA溶液,恒温37℃持续震荡24小时以及PBS持续震荡清洗24小时等处理,去除内皮细胞和间质细胞,常规苏木精伊红和Masson染色,光镜和电子扫描显微镜检查,评价去细胞效果。DAPI细胞核染色,免疫荧光显微镜观察,评价细胞核去除效果。
结果 NaCl-SDS对瓣膜超微结构损伤小,外观改变不明显,但去细胞不彻底,HE切片仍然见大量细胞核;胰蛋白酶法去细胞彻底,对结构损害大,胶原纤维可见明显断裂,曲拉通脱氧胆酸钠法未见细胞核,能有效去除细胞成分,对瓣膜结构损伤较小,扫描电镜可见基底膜。DAPI细胞核染色发现NaCl-SDS法大量DNA残留,胰蛋白酶法和曲拉通脱氧胆酸钠法未见DNA。
结论 曲拉通脱氧胆酸钠法可以有效去除细胞成分,对瓣膜结构影响较小,是一种有效去细胞的方法。
2 组织工程心脏瓣膜支架制作方法的改进及其生物学特性
目的 研究去细胞组织工程心脏瓣膜支架制作方法的改进及其生物学特性。
方法 以胰蛋白酶处理瓣膜30分钟后,再以曲拉通脱氧胆酸钠核酸酶处理48小时,进行扫描电镜和组织学检查。测定瓣膜去细胞前后的含水量,细胞的可溶性蛋白含量,以岛津力学检测仪检测瓣膜的力学特性,瓣膜浸润提取液对细胞生长的影响。将去细胞瓣叶植入新西兰种大白兔皮下,2、4、8、12周取材以检测瓣叶的组织相容性,以商用生物瓣膜为对照组。
Part One1. Tissue-engineered heart valve leaflets: comparison of different decellularization procedures of porcine heart valvesObjective Tissue engineering of heart valves should avoid the disadvantages of conventional prostheses. To determine the most effective method of producing the acellularized porcine heart valve leaflets, we compared pathological findings of the porcine heart valve leaflets produced by three methods: NaCl-SDS, trypsin and Triton -X100, sodium deoxycholate with DNAase 200mg/L, RNAase 20mg/L.treatment and further analyzed the biological character of the best one.Methods Aortic valve leaflet were harvested from pigs in slaughterhouse of Shanghai city. They were immediately immersed in 4℃ PBS solution. After washed at room temperature in povidone-iodine-solution and sterile PBS solution. Leaflets were assigned in one of the three preparation methods for decellularization. And then light and electron microscopic analyses were performed.Results HE stain showed that cells were almost absent in the leaflet treated with trypsin, and Triton-X100, while cells were partly present in the leaflets treated by NaCl-SDS. Scanning electronic microscopic revealed trypsin removed cells completely but caused strong structural alterations. Treatment with Triton-X100 achieved both complete decellularization and preservation of the matrix structure. Conclusion Techniques of decellularization are highly variable in efficiency and matrix preservation and satisfactory results were achieved in our study with Triton-X100, sodium deoxycholate and DNAase, RNAase. 2. Study on the biological properties of decellularized tissue-engineered porcine heart valve leafletsObjective To study the biological properties of decellularized tissue-engineeredporcine heart valve leaflets.Methods The harvesting and immediate treatment of porcine heart valve leaflets
was as same as above. Then the valve weight, protein content, stress strain, destroying stress, were examined. Cell toxicity and biocompatibility were also evaluated. Decellularized tissue-engineered porcine heart valve leaflets were implanted in rabbit subcutaneous, and animals were sacrificed after 2,4,8,12 weeks. Results Compared with that before the cell removal, valve weight in the decellularized aortic valve leaflets was increased significantly, but there were no significant difference in stress-strain, destroying stress between normal valve leaflets and decellularized. Cell toxicity was not significant. Biocompatibility study demonstrated that valve leaflets were absorbed gradually, Microscopic inspection of implantation site revealed no pathological inflammatory response. Conclusion The decellularized porcine heart valve leaflets have stable biological properties and can be used as an ideal tissue engineering valve scaffold. Part TwoHuman mesenchymal stem cells —a new cell source for heart valve tissue engineering.Objective Human mesenchymal stem cells (MSC) exhibit the potential to differentiate into multiple cell-lineages and can be easily obtained clinically. This study was designed to assess the feasibility of using human bone marrow-derived mesenchymal stem cells as a kind of source to construct tissue engineering heart valve.Methods Bone marrow was aspirated from the sternum of surgical field. Cells were isolated using Ficoll gradient, cultured, and characterized based on immunofluorescent staining flowcytometry, histology, and immunohistochemistry. Interstitial cells (IC) derived from the human rheumatic aortic heart valves and smooth muscle cells(SMC) from umbilical cord were cultured in vitro as contrast. Results Isolated MSC demonstrated fibroblast-like morphology. Phenotype analysis revealed positive signals for a-smooth muscle actin and vimentin. Flowcytometry of MSC pre-seeding was negative for CD31, CD14, positive for a-smooth muscle actin and vimentin. The mean fluorescence intensity ratio (MFIR) of MSC > IC and SMC was 4.9、 4.5、 4.2 and 2.7、 3.1 and 2.4 respectively. Immunohistology showed production of collagen I and collagen III. Conclusions Mesenchymal stem cells can be isolated noninvasively from the
sternum demonstrated myofibroblast-like character, bone marrow may be a potentialsource of cells for tissue engineering heart valves.Part ThreeIn vitro experience with tissue engineered heart valve on deeellularized porcine heart aortic valveObjective To evaluate the feasibility of tissue engineered heart valve on deeellularized porcine aortic valves in vitro.Methods A detergent and enzymatic extraction process has been developed to remove cellular components from porcine aortic valve, glutaraldehyde fixed heart valve was set as control group. MSC were seeded on the deeellularized valves in experiment group, Their growth condition and relationship with deeellularized porcine heart valve leaflets were observed.Results SEM show that a larger amount of cells attached on scaffolds and demonstrated viable secretionally active myofibrolasts and confluent homogenous tissue surface. The cells had covered a larger area of scaffold with a lot of matrix synthesized. The HE stain showed large amount of cells growing on the scaffolds and some cells were found in the central part of scaffolds. There was no cell growth on glutaraldehyde fixed heart valve.Conclusion It is possible to seed MSC on deeellularized porcine heart valve leaflets to create a viable tissue-engineered heart valve leaflets. Part FourExperiment study on constructing heart valve use marrow stromal cells and deeellularized porcine valve by tissue engineering approach Objective The objective of this study was to investigate the feasibility of creating tissue engineered heart valves from human mesenchymal stem cells (MSC) as an alternative cell source and deeellularized porcine heart valve leaflets to obtain viable constructs in nude mice.Methods MSC were isolated, expanded in vitro and labeled with fluorescent tracer DAPI. Porcine heart valves were deeellularized by Triton, RNAse and DNAse, reseeded and cultured under static conditions 7 days. Then cell-scaffold constructs
引文
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2. Cebotari S, Mertsching H, Kallenbach K, et al. Construction of autologous human heart valves based on an acellular allograft matrix. Circulation, 2002,106: SI63-68.
3. Kasimir MT, Rieder E, Seebacher G, et al. Comparison of different decellularization procedures of porcine heart valves. Int J Artif Organs, 2003, 26: 421-427.
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