间充质干细胞联合复合支架构建组织工程瓣膜的研究
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摘要
第一部分组织工程瓣膜种子细胞的研究
目的：采用间充质干细胞(MSCs)构建组织工程心脏瓣膜(TEHV)，并对MSCs的细胞生物学，及所构建TEHV的生物学和生物力学性能进行研究。
方法：分离培养大鼠MSCs和肌成纤维细胞，进行细胞鉴定后分别种植于去细胞猪主动脉瓣叶支架上，然后在体外培养14d构建TEHV，并分别做为实验组A和B。对MSCs的细胞生物学，及两组TEHV的生物学和生物力学性能进行检测。
结果：MSCs表达CD29(94．82％)和CD44(93．59％)。免疫组化和荧光染色检测显示MSCs的Vimentin染色均为阳性，部分MSCs的a-SMA染色阳性。实验组B的a-SMA、MMP-13和TIMP-1 mRNA表达均高于实验组A(P＜0．05)，而LOX蛋白含量和LOX mRNA表达均低于实验组A(P＜0．05)。两组TEHV的形态学结构相似。实验组A支架上MSCs的Vimentin染色均为阳性，部分MSCs的a-SMA染色阳性。两组TEHV的DNA和羟脯氨酸含量，最大负荷、最大应力、最大应变和弹性模量的差异无统计学意义(P＞0．05)。
结论在采用MSCs体外构建TEHV过程中，其细胞生物学性能与肌成纤维细胞相比存在差异，而两者所构建TEHV的生物学和生物力学性能相当。这说明MSCs构建的TEHV并没有在性能方面体现出明显的优势。有必要通过其他方法，对MSCs构建的TEHV性能加以改善。
第二部分bFGF调控组织工程瓣膜构建的研究
目的：探讨碱性成纤维细胞生长因子(bFGF)对MSCs构建的TEHV的生物学和生物力学性能的影响。
方法：分离培养大鼠MSCs，种植于去细胞瓣叶支架上。将支架置于含10ng／ml bFGF的培养液中，培养14d构建的TEHV做为实验组A。实验组B除培养液中不添加bFGF外，其余同实验组A。实验组C为大鼠肌成纤维细胞构建的TEHV。对MSCs的细胞生物学，及各组TEHV的生物学和生物力学性能进行检测。
结果：实验组A和C的a-SMA、MMP-13和TIMP-1 mRNA表达的差异无统计学意义(P＞0．05)，但均高于实验组B(P＜0．05)。实验组A和C的LOX蛋白含量和LOX mRNA表达的差异无统计学意义(P＞0．05)，但均低于实验组B(P＜0．05)。实验组A支架的再细胞化程度明显优于实验组B和C；DNA和羟脯氨酸含量高于实验组B和C(P＜0．05)。各组TEHV生物力学性能的差异无统计学意义(P＞0．05)。
结论：在采用MSCs联合bFGF体外构建TEHV过程中，通过bFGF的调控作用，可以使MSCs的表型特性、基质金属蛋白酶及其抑制物的表达增强，达到与肌成纤维细胞相当的水平；可以使MSCs的LOX蛋白和基因表达降低，达到与肌成纤维细胞相当的水平；可以提高TEHV的生物学性能。但bFGF改善TEHV生物力学性能的作用不明显。
第三部分组织工程瓣膜复合支架的研究
目的：采用静电纺丝技术制备复合支架，然后在支架上种植MSCs构建TEHV，并研究复合支架对TEHV的生物学和生物力学性能的影响。
方法：分离培养大鼠MSCs和肌成纤维细胞。实验组A为猪主动脉瓣叶经去细胞处理后，在其表面涂铺采用静电纺丝技术制备的聚(3—羟基丁酸酯—co—4—羟基丁酸酯)(P3／4HB)纤维膜，构建复合支架。实验组B和C为去细胞猪主动脉瓣叶支架。在实验组A和B的支架上种植MSCs，在实验组C的支架上种植肌成纤维细胞，各组均在体外培养14d构建TEHV，并进行生物学和生物力学检测。
结果：复合支架的整个P3／4HB纤维膜结构均一，平均直径是156±3nm，并且与去细胞瓣叶支架表面紧密结合。各组支架的再细胞化程度相当，DNA和羟脯氨酸含量差异无统计学意义(P＞0．05)。实验组A与实验组B、C相比，最大负荷、最大应力和弹性膜量明显上升(P＜0．05)，而最大应变的差异无统计学意义(P＞0．05)。
结论：复合支架能明显增强TEHV的生物力学性能，而其对TEHV生物学性能的改善作用不明显。
第四部分间充质干细胞联合bFGF复合支架构建组织工程瓣膜的研究
目的：采用MSCs联合bFGF复合支架构建TEHV，以改善TEHV的生物学和生物力学性能。
方法：分离培养大鼠MSCs。实验组A为猪主动脉瓣叶经去细胞处理后，在其表面涂铺采用缓释和静电纺丝技术制备的含有bFGF壳聚糖纳米粒的P3／4HB纤维膜，构建复合支架(bFGF)。实验组B为在去细胞瓣叶表面涂铺P3／4HB纤维膜，构建复合支架。实验组C和D为去细胞瓣叶支架。然后在各组支架上种植MSCs，实验组C在含10ng／ml bFGF的培养液中培养，其余各组均在不含bFGF的培养液中培养。各组均在体外培养14d构建TEHV。各组TEHV进行生物学和生物力学检测。
结果：复合支架(bFGF)的载药量为0．00077％，包封率为88．1％，累积释放度为1d约16．3％，5d约42．4％，10d约51．1％和15d约53．9％。复合支架(bFGF)的整个bFGF壳聚糖纳米粒P3／4HB纤维膜结构均一，并且与去细胞瓣叶支架表面紧密结合。实验组A和C支架的再细胞化程度明显优于实验组B和D，DNA和羟脯氨酸含量明显高于实验组B和D(P＜0．05)。实验组A和B与实验组C和D相比，最大负荷、最大应力和弹性膜量明显上升(P＜0．05)，而最大应变的差异无统计学意义(P＞0．05)。
结论：采用MSCs联合复合支架(bFGF)构建TEHV，能明显改善和增强TEHV的生物学和生物力学性能。复合支架(bFGF)的制备为构建有再生活性和良好功能人工心脏瓣膜替代物提供了新方法和新思路。
Part one Construction of Tissue Engineered Heart Valve UsingDifferent Seed Cells
Objective: Tissue engineered heart valve (TEHV) was constructed using mesenchymalstem cells (MSCs) as seed cells. The cell biology of MSCs, and thebiological and biomechanical properties of the TEHV were investigated.
Methods: MSCs and myofibroblasts were isolated and cultured. After cell identification,cells were seeded onto decellularized aortic valve leaflet scaffold and culturedfor 14 days to constuct TEHV in group A and B. The cell biology of MSCs,and the biological and biomechanical properties of the TEHV were examed.
Results: MSCs expressed CD29 (94.82%) and CD44 (93.59%). Immunocytochemistryshowed the expression of vimentin by all MSCs and the expression of a-SMAby some MSCs. The expression of a-SMA, MMP-13 and TIMP-1 mRNAwere significantly inceased in group B, compared with lower values of groupA (P＜0.05). However, the content of LOX and expression of LOX mRNAwere inceased in group A, compared with lower values of group B (P＜0.05).In the scaffolds of group A, all MSCs expressed vimentin and some MSCs expressed a-SMA. TEHV in two groups showed simliar morphologcicalstructure. And the differences of DNA and hydroxyproline contents,Max-load, Max-stress, elastic modulus and Max-strain between two groupshave no statistical significance (P＞0.05).
Conclusion: During the process of TEHV construction using MSCs as seed cells in vitro,the differences of cell biology between MSCs and myofibroblasts werepresent. But the biological and biomechanical properties of TEHV usingMSCs or myofibroblasts as seed cells were similar. And it is necessary toimprove the properties of TEHV using other methods.
Part two Construction of Tissue Engineered Heart ValveUsing bFGF
Objective: The biological and biomechanical properties of TEHV constructed by MSCsand bFGF were investigated.
Methods: MSCs were isolated and cultured. Then MSCs were seeded onto decellularizedaortic valve leaflet scaffold. TEHV were cutlured with DMEM contained 10ng/ml bFGF in vitro for 14 d in group A. In group B, TEHV were cutluredwith DMEM only for 14 d. In group C, TEHV were constructed usingmyofibroblasts as seed cells. The cell biology of MSCs, and the biologicaland biomechanical properties of the TEHV were examed.
Results: Group A and C showed comparable expression of a-SMA, MMP-13 andTIMP-1 mRNA (P＞0.05). However the mRNA expression weresignificantly inceased in group A and C, compared with lower values ofgroup B (P＜0.05). Group A and C showed comparable content of LOX andexpression of LOX mRNA (P＞0.05). However the content of LOX and expression of LOX mRNA were inceased in group B, compared with lowervalues of group A and C (P＜0.05). Recellularization of scaffolds in group Awas significantly improved compared to group B and C. And the DNA andhydroxyproline contents were inceased in group A, compared with lowervalues of group B and C (P＜0.05). And Max-load, Max-stress, elasticmodulus and Max-strain of TEHVs between each group have no statisticalsignificance (P＞0.05).
Conclusion: During the process of TEHV construction in vitro, when regulated by bFGF,the expression of phenotype of MSCs was equal to myofibroblasts. Theexpression of matrix metalloproteinase and tissue inhibitor ofmetalloproteinase of MSCs was equal to myofibroblasts. The expression ofLOX of MSCs was equal to myofibroblasts. And the biological propertiesof TEHV can be improved. But the biomechanical properties of TEHV cannot be improved by bFGF.
Part three Construction of Tissue Engineered Heart ValveUsing Hybrid Scaffold
Objective: The objective of this study was to fabricate hybrid scaffolds using anelectrospinning technique. Then TEHVs were engineered by seeding MSCsonto the scaffolds. The effects of the hybrid scaffolds on the biological andbiomechanical properties of TEHVs were investigated.
Methods: MSCs and myofibroblasts were obtained from rats and cultured. In group A,porcine aortic heart valve leaflets were decellularized, coated withpoly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3/4HB) using anelectrospinning technique. Group B and C were decellularized valve leafletsscaffolds. In group A and B, scaffolds were reseeded by MSCs and culturedover a time period of 14 days. In group C, scaffolds were reseeded by myofibroblasts and cultured over an equivalent time period. Specimens ofeach group were examined biologically and biomechanically.
Results: The average diameter of the P3/4HB fibers was 156±3 nm. Electrospunmembrane of the P3/4HB firmly combined with the surface of thedecellularized valve leaflets and showed uniform fibers and microstructure.Recellularization was comparable to the specimens in each group. And thespecimens of each group revealed comparable amouts of cell mass and4-hydroxyproline (P＞0.05). However, the specimens in group A showed asignificantly increase of Max-load, Max-stress and elastic modulus, comparedto group B and C (P＜0.05). And the differences of Max-strain between threegroups were considered no statistical significance (P＞0.05).
Conclusion: This study demonstrated the superiority of the hybrid scaffolds to increase thebiomechanical properties of TEHV. And compared to the decellularized valveleaflet scaffolds, the hybrid scaffolds showed similar effects on theproliferation of MSCs and formation of extracellular matrix.
Part four Construction of Tissue Engineered Heart ValveUsing Mesenchymal Stem Cells and Hybrid Scaffold with bFGF
Objective: TEHVs were constructed using MSCs and hybrid scaffold with bFGF. Andthe biological and biomechanical properties of the TEHV wereinvestigated.
Methods: MSCs were obtained from rats and cultured, in group A, porcine aortic heartvalve leaflets were decellularized, coated with bFGF/chitosan/P3/4HBusing slow release and electrospinning technique. In group B,decellularized heart valve leaflets coated with P3/4HB using anelectrospinning technique. In group C and D, the scaffolds weredecellularized heart valve leaflets. The scaffolds in each group werereseeded by MSCs. Scaffolds in group C were cultured in DMEMsupplemented with 10 ng/ml bFGF. However, scaffolds in other groupswere cultured in DMEM without bFGF. Scaffolds in each group were cultured over a time period of 14 d to construct TEHVs. Specimens of eachgroup were examined biologically and biomechanically.
Results: The protein-loading capacity of the hybrid valve scaffolds (bFGF) and theirassociation efficiency of were 0.00077% and 88.1%. In vitro release ofbFGF showed that the extent of release was 16.3% at 1d, 42.4% at 5d,51.1% at 10d and 53.9% at 15d. The electrospun membrane ofbFGF/chitosan/P3/4HB firmly combined with the surface of thedecellularized valve leaflets and showed uniform fibers and microstructure.In group A and C, recellularization of the scaffolds was significantlyimproved compared to group B and D. And biochemical analysis revealedan increase of cell mass and the content of 4-hydroxyproline compared togroup B and D (P＜0.05). In group A and B, the specimens showed asignificantly increase of Max-load, Max-stress and elastic modulus,compared to group C and D (P＜0.05), and the differences of Max-strainwere considered no statistical significance (P＞0.05).
Conclusion: This study demonstrated the superiority of the hybrid scaffold with bFGFseeded by MSCs to enhance the biological and biomechanical properties ofTEHV. Hybrid scaffold with bFGF could be useful for the generation ofviable, functional heart valve prostheses.
目的：采用间充质干细胞(MSCs)构建组织工程心脏瓣膜(TEHV)，并对MSCs的细胞生物学，及所构建TEHV的生物学和生物力学性能进行研究。
方法：分离培养大鼠MSCs和肌成纤维细胞，进行细胞鉴定后分别种植于去细胞猪主动脉瓣叶支架上，然后在体外培养14d构建TEHV，并分别做为实验组A和B。对MSCs的细胞生物学，及两组TEHV的生物学和生物力学性能进行检测。
结果：MSCs表达CD29(94．82％)和CD44(93．59％)。免疫组化和荧光染色检测显示MSCs的Vimentin染色均为阳性，部分MSCs的a-SMA染色阳性。实验组B的a-SMA、MMP-13和TIMP-1 mRNA表达均高于实验组A(P＜0．05)，而LOX蛋白含量和LOX mRNA表达均低于实验组A(P＜0．05)。两组TEHV的形态学结构相似。实验组A支架上MSCs的Vimentin染色均为阳性，部分MSCs的a-SMA染色阳性。两组TEHV的DNA和羟脯氨酸含量，最大负荷、最大应力、最大应变和弹性模量的差异无统计学意义(P＞0．05)。
结论在采用MSCs体外构建TEHV过程中，其细胞生物学性能与肌成纤维细胞相比存在差异，而两者所构建TEHV的生物学和生物力学性能相当。这说明MSCs构建的TEHV并没有在性能方面体现出明显的优势。有必要通过其他方法，对MSCs构建的TEHV性能加以改善。
第二部分bFGF调控组织工程瓣膜构建的研究
目的：探讨碱性成纤维细胞生长因子(bFGF)对MSCs构建的TEHV的生物学和生物力学性能的影响。
方法：分离培养大鼠MSCs，种植于去细胞瓣叶支架上。将支架置于含10ng／ml bFGF的培养液中，培养14d构建的TEHV做为实验组A。实验组B除培养液中不添加bFGF外，其余同实验组A。实验组C为大鼠肌成纤维细胞构建的TEHV。对MSCs的细胞生物学，及各组TEHV的生物学和生物力学性能进行检测。
结果：实验组A和C的a-SMA、MMP-13和TIMP-1 mRNA表达的差异无统计学意义(P＞0．05)，但均高于实验组B(P＜0．05)。实验组A和C的LOX蛋白含量和LOX mRNA表达的差异无统计学意义(P＞0．05)，但均低于实验组B(P＜0．05)。实验组A支架的再细胞化程度明显优于实验组B和C；DNA和羟脯氨酸含量高于实验组B和C(P＜0．05)。各组TEHV生物力学性能的差异无统计学意义(P＞0．05)。
结论：在采用MSCs联合bFGF体外构建TEHV过程中，通过bFGF的调控作用，可以使MSCs的表型特性、基质金属蛋白酶及其抑制物的表达增强，达到与肌成纤维细胞相当的水平；可以使MSCs的LOX蛋白和基因表达降低，达到与肌成纤维细胞相当的水平；可以提高TEHV的生物学性能。但bFGF改善TEHV生物力学性能的作用不明显。
第三部分组织工程瓣膜复合支架的研究
目的：采用静电纺丝技术制备复合支架，然后在支架上种植MSCs构建TEHV，并研究复合支架对TEHV的生物学和生物力学性能的影响。
方法：分离培养大鼠MSCs和肌成纤维细胞。实验组A为猪主动脉瓣叶经去细胞处理后，在其表面涂铺采用静电纺丝技术制备的聚(3—羟基丁酸酯—co—4—羟基丁酸酯)(P3／4HB)纤维膜，构建复合支架。实验组B和C为去细胞猪主动脉瓣叶支架。在实验组A和B的支架上种植MSCs，在实验组C的支架上种植肌成纤维细胞，各组均在体外培养14d构建TEHV，并进行生物学和生物力学检测。
结果：复合支架的整个P3／4HB纤维膜结构均一，平均直径是156±3nm，并且与去细胞瓣叶支架表面紧密结合。各组支架的再细胞化程度相当，DNA和羟脯氨酸含量差异无统计学意义(P＞0．05)。实验组A与实验组B、C相比，最大负荷、最大应力和弹性膜量明显上升(P＜0．05)，而最大应变的差异无统计学意义(P＞0．05)。
结论：复合支架能明显增强TEHV的生物力学性能，而其对TEHV生物学性能的改善作用不明显。
第四部分间充质干细胞联合bFGF复合支架构建组织工程瓣膜的研究
目的：采用MSCs联合bFGF复合支架构建TEHV，以改善TEHV的生物学和生物力学性能。
方法：分离培养大鼠MSCs。实验组A为猪主动脉瓣叶经去细胞处理后，在其表面涂铺采用缓释和静电纺丝技术制备的含有bFGF壳聚糖纳米粒的P3／4HB纤维膜，构建复合支架(bFGF)。实验组B为在去细胞瓣叶表面涂铺P3／4HB纤维膜，构建复合支架。实验组C和D为去细胞瓣叶支架。然后在各组支架上种植MSCs，实验组C在含10ng／ml bFGF的培养液中培养，其余各组均在不含bFGF的培养液中培养。各组均在体外培养14d构建TEHV。各组TEHV进行生物学和生物力学检测。
结果：复合支架(bFGF)的载药量为0．00077％，包封率为88．1％，累积释放度为1d约16．3％，5d约42．4％，10d约51．1％和15d约53．9％。复合支架(bFGF)的整个bFGF壳聚糖纳米粒P3／4HB纤维膜结构均一，并且与去细胞瓣叶支架表面紧密结合。实验组A和C支架的再细胞化程度明显优于实验组B和D，DNA和羟脯氨酸含量明显高于实验组B和D(P＜0．05)。实验组A和B与实验组C和D相比，最大负荷、最大应力和弹性膜量明显上升(P＜0．05)，而最大应变的差异无统计学意义(P＞0．05)。
结论：采用MSCs联合复合支架(bFGF)构建TEHV，能明显改善和增强TEHV的生物学和生物力学性能。复合支架(bFGF)的制备为构建有再生活性和良好功能人工心脏瓣膜替代物提供了新方法和新思路。
Part one Construction of Tissue Engineered Heart Valve UsingDifferent Seed Cells
Objective: Tissue engineered heart valve (TEHV) was constructed using mesenchymalstem cells (MSCs) as seed cells. The cell biology of MSCs, and thebiological and biomechanical properties of the TEHV were investigated.
Methods: MSCs and myofibroblasts were isolated and cultured. After cell identification,cells were seeded onto decellularized aortic valve leaflet scaffold and culturedfor 14 days to constuct TEHV in group A and B. The cell biology of MSCs,and the biological and biomechanical properties of the TEHV were examed.
Results: MSCs expressed CD29 (94.82%) and CD44 (93.59%). Immunocytochemistryshowed the expression of vimentin by all MSCs and the expression of a-SMAby some MSCs. The expression of a-SMA, MMP-13 and TIMP-1 mRNAwere significantly inceased in group B, compared with lower values of groupA (P＜0.05). However, the content of LOX and expression of LOX mRNAwere inceased in group A, compared with lower values of group B (P＜0.05).In the scaffolds of group A, all MSCs expressed vimentin and some MSCs expressed a-SMA. TEHV in two groups showed simliar morphologcicalstructure. And the differences of DNA and hydroxyproline contents,Max-load, Max-stress, elastic modulus and Max-strain between two groupshave no statistical significance (P＞0.05).
Conclusion: During the process of TEHV construction using MSCs as seed cells in vitro,the differences of cell biology between MSCs and myofibroblasts werepresent. But the biological and biomechanical properties of TEHV usingMSCs or myofibroblasts as seed cells were similar. And it is necessary toimprove the properties of TEHV using other methods.
Part two Construction of Tissue Engineered Heart ValveUsing bFGF
Objective: The biological and biomechanical properties of TEHV constructed by MSCsand bFGF were investigated.
Methods: MSCs were isolated and cultured. Then MSCs were seeded onto decellularizedaortic valve leaflet scaffold. TEHV were cutlured with DMEM contained 10ng/ml bFGF in vitro for 14 d in group A. In group B, TEHV were cutluredwith DMEM only for 14 d. In group C, TEHV were constructed usingmyofibroblasts as seed cells. The cell biology of MSCs, and the biologicaland biomechanical properties of the TEHV were examed.
Results: Group A and C showed comparable expression of a-SMA, MMP-13 andTIMP-1 mRNA (P＞0.05). However the mRNA expression weresignificantly inceased in group A and C, compared with lower values ofgroup B (P＜0.05). Group A and C showed comparable content of LOX andexpression of LOX mRNA (P＞0.05). However the content of LOX and expression of LOX mRNA were inceased in group B, compared with lowervalues of group A and C (P＜0.05). Recellularization of scaffolds in group Awas significantly improved compared to group B and C. And the DNA andhydroxyproline contents were inceased in group A, compared with lowervalues of group B and C (P＜0.05). And Max-load, Max-stress, elasticmodulus and Max-strain of TEHVs between each group have no statisticalsignificance (P＞0.05).
Conclusion: During the process of TEHV construction in vitro, when regulated by bFGF,the expression of phenotype of MSCs was equal to myofibroblasts. Theexpression of matrix metalloproteinase and tissue inhibitor ofmetalloproteinase of MSCs was equal to myofibroblasts. The expression ofLOX of MSCs was equal to myofibroblasts. And the biological propertiesof TEHV can be improved. But the biomechanical properties of TEHV cannot be improved by bFGF.
Part three Construction of Tissue Engineered Heart ValveUsing Hybrid Scaffold
Objective: The objective of this study was to fabricate hybrid scaffolds using anelectrospinning technique. Then TEHVs were engineered by seeding MSCsonto the scaffolds. The effects of the hybrid scaffolds on the biological andbiomechanical properties of TEHVs were investigated.
Methods: MSCs and myofibroblasts were obtained from rats and cultured. In group A,porcine aortic heart valve leaflets were decellularized, coated withpoly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3/4HB) using anelectrospinning technique. Group B and C were decellularized valve leafletsscaffolds. In group A and B, scaffolds were reseeded by MSCs and culturedover a time period of 14 days. In group C, scaffolds were reseeded by myofibroblasts and cultured over an equivalent time period. Specimens ofeach group were examined biologically and biomechanically.
Results: The average diameter of the P3/4HB fibers was 156±3 nm. Electrospunmembrane of the P3/4HB firmly combined with the surface of thedecellularized valve leaflets and showed uniform fibers and microstructure.Recellularization was comparable to the specimens in each group. And thespecimens of each group revealed comparable amouts of cell mass and4-hydroxyproline (P＞0.05). However, the specimens in group A showed asignificantly increase of Max-load, Max-stress and elastic modulus, comparedto group B and C (P＜0.05). And the differences of Max-strain between threegroups were considered no statistical significance (P＞0.05).
Conclusion: This study demonstrated the superiority of the hybrid scaffolds to increase thebiomechanical properties of TEHV. And compared to the decellularized valveleaflet scaffolds, the hybrid scaffolds showed similar effects on theproliferation of MSCs and formation of extracellular matrix.
Part four Construction of Tissue Engineered Heart ValveUsing Mesenchymal Stem Cells and Hybrid Scaffold with bFGF
Objective: TEHVs were constructed using MSCs and hybrid scaffold with bFGF. Andthe biological and biomechanical properties of the TEHV wereinvestigated.
Methods: MSCs were obtained from rats and cultured, in group A, porcine aortic heartvalve leaflets were decellularized, coated with bFGF/chitosan/P3/4HBusing slow release and electrospinning technique. In group B,decellularized heart valve leaflets coated with P3/4HB using anelectrospinning technique. In group C and D, the scaffolds weredecellularized heart valve leaflets. The scaffolds in each group werereseeded by MSCs. Scaffolds in group C were cultured in DMEMsupplemented with 10 ng/ml bFGF. However, scaffolds in other groupswere cultured in DMEM without bFGF. Scaffolds in each group were cultured over a time period of 14 d to construct TEHVs. Specimens of eachgroup were examined biologically and biomechanically.
Results: The protein-loading capacity of the hybrid valve scaffolds (bFGF) and theirassociation efficiency of were 0.00077% and 88.1%. In vitro release ofbFGF showed that the extent of release was 16.3% at 1d, 42.4% at 5d,51.1% at 10d and 53.9% at 15d. The electrospun membrane ofbFGF/chitosan/P3/4HB firmly combined with the surface of thedecellularized valve leaflets and showed uniform fibers and microstructure.In group A and C, recellularization of the scaffolds was significantlyimproved compared to group B and D. And biochemical analysis revealedan increase of cell mass and the content of 4-hydroxyproline compared togroup B and D (P＜0.05). In group A and B, the specimens showed asignificantly increase of Max-load, Max-stress and elastic modulus,compared to group C and D (P＜0.05), and the differences of Max-strainwere considered no statistical significance (P＞0.05).
Conclusion: This study demonstrated the superiority of the hybrid scaffold with bFGFseeded by MSCs to enhance the biological and biomechanical properties ofTEHV. Hybrid scaffold with bFGF could be useful for the generation ofviable, functional heart valve prostheses.
引文
1．董念国，史嘉玮，胡平．组织工程心脏瓣膜支架的研究现状和发展趋势．中华实验外科杂志，2007，24(3)：261—262．
2. Mol A, Rubbens MP, Stekelenburg M, et al. Living heart valve and small-diameter artery substitutes-an emerging field for intellectual property development. Recent Pat Biotechnol, 2008,2(1): 1-9.
3. Hong H, Dong GN, Shi WJ, et al. Fabrication of biomatrix/polymer hybrid scaffold for heart valve tissue engineering in vitro. ASAIO J, 2008 Nov-Dec, 54(6): 627-632.
4. Schoen FJ. Evolving concepts of cardiac valve dynamics: the continuum of development,functional structure, pathobiology, and tissue engineering. Circulation, 2008, 118(18):1864-1880.
5. Del Gaudio C, Bianco A, Grigioni M. Electrospun bioresorbable trileaflet heart valve prosthesis for tissue engineering: in vitro functional assessment of a pulmonary cardiac valve design. Ann Ist Super Sanita, 2008, 44(2): 178-186.
6. Kidane AG, Burriesci G, Cornejo P, et al. Current developments and future prospects for heart valve replacement therapy. J Biomed Mater Res B Appl Biomater, 2009, 88(1): 290-303.
7. Migneco F, Hollister SJ, Birla RK. Tissue-engineered heart valve prostheses: 'state of the heart'.Regen Med, 2008, 3(3): 399-419.
8. Hayashida K, Kanda K, Oie T, et al. "In vivo tissue-engineered" valved conduit with designed molds and laser processed scaffold. J Cardiovasc Nurs, 2008, 23(1): 61-64.
9. Schmidt D, Mol A, Kelm JM, et al. In vitro heart valve tissue engineering. Methods Mol Med, 2007,140: 319-330.
10．孙晓宁，赵强，徐德民，等．骨髓间充质干细胞和猪去细胞瓣膜支架构建组织工程瓣膜．复旦学报(医学版)，2007，2：183—186．
11. Sacks MS. Biomechanics of engineered heart valve tissues. Conf Proc IEEE Eng Med Biol Soc,2006, 1: 853-854.
12. Schaefermeier PK, Cabeza N, Besser JC, et al. Potential cell sources for tissue engineering of heart valves in comparison with human pulmonary valve cells. ASAIO J, 2009 Jan-Feb, 55(1): 86-92.
13. Hare JM, Chaparro SV. Cardiac regeneration and stem cell therapy. Curr Opin Organ Transplant,2008, 13(5): 536-542
14. Breymann C, Schmidt D, Hoerstrup SP. Umbilical cord cells as a source of cardiovascular tissue engineering. Stem Cell Rev, 2006, 2(2): 87-92.
15. Batten P, Sarathchandra P, Antoniw JW, et al. Human mesenchymal stem cells induce T cell anergy and downregulate T cell allo-responses via the TH2 pathway: relevance to tissue engineering human heart valves. Tissue Eng, 2006, 12(8): 2263-2273.
16. Sutherland FW, Perry TE, Yu Y, et al. From stem cells to viable autologous semilunar heart valve. Circulation, 2005,111(21): 2783-2791.
17. Nolite SV, Xu W, Rennekampff HO, et al. Divesity of fibroblasts-a review on implications for skin tissue engineering. Cells Tissues, Organs, 2008,187(3): 165-176.
18. Habibovic P, de Groot K. Osteoinductive biomaterials-properties and relevance in bone repair. J Tissue Eng Regen Med, 2007 Jan-Feb, 1(1): 25-32.
19. Macri L, Silverstein D, Clark RA. Growth factor binding to the pericellular matrix and its importance in tissue engineering. Adv Drug Deliv Rev, 2007, 59(13): 1366-1381.
20. Narine K, De Wever O, Van Valckenborgh D, et al. Growth factor modulation of fibroblast proliferation, differentiation, and invasion: implications for tissue valve engineering. Tissue Eng, 2006, 12(10): 2707-2716.
21. Denys H, Derycke L, Hendrix A, et al. Differential impact of TGF-beta and EGF on fibroblast differentiation and invasion reciprocally promotes colon cancer cell invasion. Cancer Lett, 2008, 266(2): 263-274.
22. Fried M, Capetandes A. The effect of enantiomers of beta-agonists on myofibroblast-derived vascular endothelial growth factor and other matrix components in the presence of dust-mite extract. Allergy Asthma Proc, 2008, 29(2): 182-188.
23. Seliktar D, Zisch AH, Lutolf MP, et al. MMP-2 sensitive, VEGF-bearing bioactive hydrogels for promotion of vascular healing. J Biomed Mater Res A, 2004, 68(4): 704-716.
24. Mola A, van Lieshouta MI, Dam-de Veen CG, et al. Fibrin as a cell carrier in cardiovascular tissue engineering applications. Biomaterials, 2005, 26: 3113-3121.
25. Nakanishi Y, Chen G, Komuro H, et al. Tissue-engineered urinary bladder wall using PLGA mesh-collagen hybrid scaffolds: a comparison study of collagen sponge and gel as a scaffold. J Pediatr Surg, 2003, 38: 1781-1784.
26. Kim BS, Putnam AJ, Kulik TJ, et al. Optimizing seeding and culture methods to engineer smooth muscle tissue on biodegradable polymer matrices. Biotechnol Bioeng, 1998, 57: 46-54.
27. Jeong SI, Kim SH, Kim YH, et al. Manufacture of elastic biodegradable PLCL scaffolds for mechanoactive vascular tissue-engineering. J Biomat Sci Polym Ed, 2004, 15: 645-660.
28. Jeong SI, Kim BS, Kang SW, et al. In vivo biocompatibilty and degradation behavior of elastic poly(L-lactide-co-e-caprolactone) scaffolds. Biomaterials, 2004, 25(28): 5939-5946.
29. Knight RL, Wilcox HE, Korossis SA, et al. The use of acellular matrices for the tissue engineering of cardiac valves. Proc Inst Mech Eng [H], 2008, 222: 129-143.
30. Stamm C, Khosravi A, Grabow N, et al. Biomatrix/polymer composite material for heart valve tissue engineering. Ann Thorac Surg, 2004, 78(6): 2084-2092; discussion 2092-2093.
1. Perry TE, Kaushal S, Sutherland FW, et al. Thoracic surgery directors association award: Bone marrow as a cell source for tissue engineering heart valves. Ann Thorac Surg, 2003, 75(3):761-767.
2. Hwang WS, Ryu YJ, Park JH, et al.Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science, 2004, 303(5664): 1669-1674.
3. Hoerstrup .SP, Kadner A, Melnitchouk S, et al. Tissue engineering of functional trileaflet heart valves from human marrow stromal cells. Circulation, 2002, 106(12 Suppl 1): 143-150.
4．董念国，孙宗全，傅平，等．聚二乙醇酸与Degrapol对肌成纤维细胞粘附、生长的影响．华中科技大学学报(医学版)，2003，32：71-73．
5．洪吴，董念国．组织工程心脏瓣膜的研究进展．临床心血管病杂志，2006，22(7)：442-444．
6. Maish MS, Hoffman-Kim D, Krueger PM, et al. Tricuspid valve biopsy: a potential source of cardiac myofibroblast cells for tissue-engineered cardiac valves. J Heart Valve Dis, 2003, 12:264-269.
7. Schenke-Layland K, Opitz F, Gross M, et al. Complete dynamic repopulation of decellularized heart valves by application of defined physical signals-an in vitro study. Cardiovascular Research,2003, 60: 497-509.
8．董念国，叶晓峰，史嘉玮，等．组织工程瓣膜天然支架去细胞方法的比较．中华实验外科杂志，2005，22：377．
9. Sutherland F W H, Perry T E, Ying Y, et al. From stem cells to viable autologous semilunar heart valve. Circulation, 2005, 111: 2783-2791.
10. Kim SS, Lim SH, Cho SW, et al. Tissue engineering of heart valves by recellularization of glutaraldehyde-fixed porcine valves using bone marrow-derived cells. Artificial Organs, 2006, 30(7): 554-567:
11. Hoerstrup S P, Kadner A, Melnitchouk S, et al. Tissue engineering of functional trileaflet heart valves from human marrow stromal cells. Circulation, 2002, 106: 143-150.
12．洪吴，董念国，史嘉玮．Amplex Red荧光法检测组织工程瓣膜赖氨酰氧化酶活性．中国胸心血管外科临床杂志，2007，14(1)：27-30．
13. Steinhoff G; Stock UA, Karim N, et al.Tissue Engineering of Pulmonary Heart Valves on Allogenic Acellular Matrix Conduits : In Vivo Restoration of Valve Tissue. Circulation, 2000, 102:50-55
14．史嘉玮，董念国，孙宗全，等．RGD肽联合转化生长因子-β_1对组织工程瓣膜构建的影响．中华医学杂志，2006，86：2074-2077．
15．董念国，孙宗全，付平，等．组织工程心脏瓣膜构建的研究．中华实验外科杂志，2002，19(1)：88．
16．韩啸，蒋雄刚，徐鹏，等．聚乙二醇和胰蛋白酶用于猪动脉管道去细胞效果比较．华中科技大学学报(医学版)，2007，36(4)：442—444．
17．董念国，史嘉玮，胡平．组织工程心脏瓣膜支架的研究现状和发展趋势．中华实验外科杂志，2007，24(3)：261—262．
18. Kidane AG, Burriesci G; Cornejo P, et al. Current developments and future prospects for heart valve replacement therapy. J Biomed Mater Res B Appl Biomater, 2009, 88(1): 290-303.
19. Migneco F, Hollister SJ, Birla RK. Tissue-engineered heart valve prostheses: 'state of the heart'.Regen Med, 2008, 3(3): 399-419.
20. Schmidt D, Mol A, Kelm JM, Hoerstrup SP. In vitro heart valve tissue engineering. Methods Mol Med, 2007, 140: 319-330.
21. Blevins TL, Carroll JL, Raza AM, et al. Phenotypic characterization of isolated valvular interstitial cell subpopulations. J Heart Valve Dis, 2006, 15(6): 815-822.
22. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science, 1998, 282(5391): 1145-1147.
23. Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007, 318(5858): 1917-1920.
24. Henderson JK, Draper JS, Baillie HS, et al. Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. Stem Cells, 2002, 20(4):329-337.
25. Sacks MS. Biomechanics of engineered heart valve tissues. Conf Proc IEEE Eng Med Biol Soc,2006, 1: 853- 854.
26. Pierret C, Friedrichsen P. Stem cells and society: an undergraduate course exploring the intersections among science, religion, and law. CBE Life Sci Educ, 2009 Spring, 8(1): 79-87.
27. Grivennikov IA. Embryonic stem cells and the problem of directed differentiation. Biochemistry(Mosc), 2008, 73(13): 1438-1452.
28. Schaefermeier PK, Cabeza N, Besser JC, et al. Potential cell sources for tissue engineering of heart valves in comparison with human pulmonary valve cells. ASAIO J, 2009 Jan-Feb, 55(1): 86-92.
29. Hare JM, Chaparro SV. Cardiac regeneration and stem cell therapy. Curr Opin Organ Transplant,2008, 13(5): 536-542
30. Breymann C, Schmidt D, Hoerstrup SP. Umbilical cord cells as a source of cardiovascular tissue engineering. Stem Cell Rev, 2006, 2(2): 87-92.
31. Batten P, Sarathchandra P, Antoniw JW, et al. Human mesenchymal stem cells induce T cell anergy and downregulate T cell allo-responses via the TH2 pathway: relevance to tissue engineering human heart valves. Tissue Eng, 2006, 12(8): 2263-2273.
32. Kadner A, Hoerstrup S P, Zund G, et al. A new source for cardiovascular tissue engineering: human bone marrow stromal cells. European Journal of Cardio-thoracic Surgery, 2002, 21: 1055-1060.
33．孙晓宁，赵强，徐德民，等．骨髓间充质干细胞和猪去细胞瓣膜支架构建组织工程瓣膜．复旦学报(医学版)，2007，34：183—186．
34. Vincentelli A, Wautot F, Juthier F, et al. In vivo autologous recellularization of a tissue-engineered heart valve: Are bone marrow mesenchymal stem cells the best candidates? J Thorac Cardiovasc Surg, 2007, 134: 424-432.
35. Hong H, Dong GN, Shi WJ, et al. Fabrication of biomatrix/polymer hybrid scaffold for heart valve tissue engineering in vitro. ASAIO J, 2008 Nov-Dec, 54(6): 627-632.
36. Cushing MC, Liao JT, Anseth KS. Activation of valvular interstitial cells is mediated by transforming growth factor-β1 interactions with matrix molecules. Matrix Biology, 2005, 24:428-437.
37. Rabkin-Aikawa E, Farber M, Aikawa M, et al. Dynamic and reversible changes of interstitial cell phenotype during remodeling of cardiac valves. J Heart Valve Dis, 2004, 13(5) : 841-847.
38. Itoh Y, Nagase H. Matrix metalloproteinases in cancer. Essays Biochem JT-Essays in biochemistry, 2002, 38: 21-36.
39. Ohkawa K, Fujii K, Nishida A, et al. Lysyl oxidase-catalyzed cross-linking and insolubilization reactions of Lys-containing polypeptides and synthetic adhesive proteins. Biomacromolecules, 2001, 2(3): 773-779.
40. Zhao Y, Gao S, Chou IN, et al. Inhibition of the expression of lysyl oxidase and its substrates in cadmium- resistant rat fetal lung fibroblasts. Toxicological Sciences, 2006, 90 (2): 478-489.
41. Smith-Mungo LI, Kagan HM. Lysyt Oxidase: properties, regulation and multiple functions in biology. Matrix Biol, 1998,16(7): 387-398.
42. Schoen FJ. Evolving concepts of cardiac valve dynamics: the continuum of development, functional structure, pathobiology, and tissue engineering. Circulation, 2008,118(18): 1864-1880.
43. Heinrich AC, Patel SA, Reddy BY, et al. Multi- and inter-disciplinary science in personalized delivery of stem cells for tissue repair. Curr Stem Cell Res Ther, 2009, 4(1): 16-22.
44. Pittenger MF, Mackay AM, Jaiswal SC, et al. Multilineage potential of mesenchymal stem cell. Science, 1999, 284(5411): 143-147.
45. Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical users. Exp Hematol, 2000,28(8): 875-884.
46. Schoen J F. New frontiers in the pathology and therapy of heart valve disease: 2006 Society for Cardiovascular Pathology, Distinguished Achievement Award Lecture, United States-Canadian Academy of Pathology, Atlanta, GA, February 12, 2006. Cardiovascular Pathology, 2006, 15(5): 271-279.
1. Cetin M, Aktas Y, Vural I, et al. Preparation and in vitro evaluation of bFGF-loaded chitosan nanoparticles. Drug Delivery, 2007, 14(8): 525-529.
2. Amidi M, Romeijn SG., Borchard G, et al. Preparation and characterization of protein-loaded N-trimethyl chitosan nanoparticles as nasal delivery system. J Controll Rel, 2006, 111: 107-116.
3. Aktas Y, Andrieux K, Alonso MJ, et al. Preparation and in vitro evaluation of chitosan nanoparticles containing a caspase inhibitor. Int J Pharm, 2005, 298: 378-383.
4. Calvo P, Remu(?)n(?)an-L(?)pez C, Vila-Jato JL, et al. Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. J Appl Polym Sci, 1997, 63: 125-132.
5. Berthold A, Cremer K, Kreuter J. Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model for antiinflammatory drugs. J Controll Rel, 1996, 39: 17-25.
6. Cetin M, Capan Y, Vural I, et al. Preparation and characterization of bFGF and BSA-loaded microspheres. J Drug Del Sci Tech, 2005, 15: 371-375.
7. Mol A, Rubbens MP, Stekelenburg M, et al. Living heart valve and small-diameter artery substitutes-an emerging field for intellectual property development. Recent Pat Biotechnol, 2008, 2(1): 1-9.
8. Hong H, Dong GN, Shi WJ, et al. Fabrication of biomatrix/polymer hybrid scaffold for heart valve tissue engineering in vitro. ASAIO J, 2008 Nov-Dec, 54(6): 627-632.
9. Schoen FJ. Evolving concepts of cardiac valve dynamics: the continuum of development, functional structure, pathobiology, and tissue engineering. Circulation, 2008,118(18): 1864-1880.
10. Stock UA, Wiederschain D, Kilroy SM, et al. Dynamics of extracellular matrix production and turnover in tissue engineered cardiovascular structures. J Cell Biochem, 2001, 81: 220-228.
11. Schenke-Layland K, Opitz F, Gross M, et al. Complete dynamic repopuiation of decellularized heart valves by application of defined physical signals-an in vitro study. Cardiovascular Research, 2003, 60: 497-509.
12. Sheridan MH, Shea LD, Peters MC, et al. Bioabsorbable polymer scaffolds for tissue engineering capable of sustained growth factor delivery. J Control Release, 2000, 64: 91-102.
13. Lee KY, Peters MC, Anderson KW, et al. Controlled growth factor release from synthetic extracellular matrices. Nature, 2000, 408: 998-1000.
14. Lutolf MP, Lauer-Fields JL, Schmoekel HG, et al. Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: Engineering cell-invasion characteristics. PNAS, 2003, 100(9): 5413-5418.
15. Edelman ER, Mathiowitz E, Langer R. et al. Con:trolled and modulated release of basic fibroblast growth factor. Biomaterials, 1991, 12: 619-626.
16. Cai DZ, Zeng C, Quan DP, et al. Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-β1 delivery for cartilage tissue engineering.Chinese Medical Journal, 2007, 120: 197-203.
17．董念国，史嘉玮，胡平．组织工程心脏瓣膜支架的研究现状和发展趋势．中华实验外科杂志，2007，24(3)：261—262．
18. Stamm C, Khosravi A, Grabow N, et al. Biomatrix/polymer composite material for heart valve tissue engineering. Ann Thorac Surg, 2004, 78(6): 2084-2092: discussion 2092-2093.
1．宋强，董念国．心脏组织工程研究新进展．临床心血管病杂志，2003，4：251—252．
2. Shinoka T, Shun-Tim D, Ma PX, et al. Creation of viable pulmonary artery autograft through tissue engineering. J Thorac Cardiovasc Surg, 1998, 115: 536-545.
3. Zund G ,Ye Q, H0erstrup SP, et al. Tissue engineering in cardiovascular surgery: MTF, a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshes. Eur J Cardiothorac Surg, 1999, 15: 519-524.
4. Sodian R, Sperling JS, Martin DP, et al. Fabrication of trileaflet heart valve scaffold from a polyhydroxylkanoate biopolyyester for use in tissue engineering. Tissue Eng, 2000, 6: 183-188.
5. Sodian R, Hoerstrup SP, Sperling JS, et al. Evaluation of biodegradable, three-dimensional matrices for tissue engineering of heart valve. ASAIO J, 2000, 46: 107-110.
6．董念国，孙宗全，付平等．聚二乙醇酸与Degrapol对肌成纤维细胞粘附、生长的影响．华中科技大学学报(医学版)，2003，1：71—73．
7．董念国，孙宗全，付平等．组织工程心脏瓣膜构建的研究．中华实验外科杂志，2002，1：88．
8. Hoerstrup SP, Sodian R, Daebritz S, et al. Functional living trileaflet heart valves grown in vitro.Circulation, 2000, 102(19 suppl 3): Ⅲ44-Ⅲ49.
9．胡平．组织工程用外科植入物-高分子材料的加工、改性及应用．中国医疗器械信息，2006，12(7)：13-21．
10．叶晓峰，董念国，孙宗全．组织工程瓣膜天然支架去细胞方法的比较．中华实验外科杂志，2005，22(3)：377．
11．叶晓峰，董念国，孙宗全，等．不同预处理对去细胞瓣细胞黏附率的影响．临床心血管病杂志，2005，21(6)：367-369．
12. Steinhoff G, Stock U, Karim N, et al. Tissue engineering of pulmonary heart valves on allogenic acelluler matrix conduits in vivo restoration of valve tissue. Circulation, 2000, 102(19 suppl3): Ⅲ50-Ⅲ55.
13. Ye Q, Zund G, Benediket P, et al. Fibrin gel as a three dimeusional matrix in cordiovascular tissue engineering. Eur J Cardiothorac Surg, 2000, 17: 587-591.
14．赵东锷，汪钢，俞世强，等．应用胶原膜支架构建组织工程心脏瓣膜的初步研究．中华胸心血管外科杂志，2002，5：257-260．
15. Lutolf MP, Lauer-Fields JL, Schmoekel HG, et al. Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: Engineering cell-invasion characteristics. PNAS, 2003,100(9): 5413-5418.
16. Stamm C, Khosravi A, Grabow N, et al. Biomatrix/polymer composite material for heart valve tissue engineering. Ann Thorac Surg, 2004, 78(6): 2084-2092; discussion 2092-2093.
17. Hong H, Dong GN, Shi WJ, et al. Fabrication of biomatrix/polymer hybrid scaffold for heart valve tissue engineering in vitro. ASAIO J, 2008 Nov-Dec, 54(6): 627-632.
18. Wilcox HE, Korossis SA, Booth C, et al. Biocompatibility and recellularization potential of an acellular porcine heart valve matrix. J Heart Valve Dis, 2005, 14(2): 228-36; discussion 236-237.
19. Paranya G, Vineberg S, Dvorin E, et al. Aortic valve endothelial cells undergo transforming growth β-factor mediated and non-β-factor mediated transdifferentiation in vitro. Ame J Patho, 2001, 159: 1335-1343.
20. Dohmen PM, Ozaki S, Verbeken E, et al. Tissue engineering of an auto-xenograft pulmonary heart valve. Asian Cardiovasc Thorac Ann, 2002, 10: 25-30.
21. Cebotari S, Mertsching H, Kallenback K, et al. Construction of autologous human heart valves based on an acellular allograft matrix. Circulation, 2002, 106(suppl 1): 63-68.
22. Kasimir MT, Weigel G, Sharma J,et al. The decellularized porcine heart valve matrix in tissue engineering: platelet adhesion and activation. Thromb Haemost, 2005, 94(3): 562-567.
23. Toshihiko S, Hongbing H, Hisataka Y, et al. Human enclothelial progenitor cell-seeded hybrid graft:Proliferative and antithrom bogenic potentials in vitro and(?) fabrication processing. Tissue Eng, 2003, 9:127-136.
24. Ozawa T, Donald A, Mickle G, et al. Optimal biomaterial for creation of autologous cardiac grafts. Circulation, 2002, 106(12 suppl 1): 1176-1182.
25. Hare JM, Chaparro SV. Cardiac regeneration and stem cell therapy. Curr Opin Organ Transplant, 2008, 13(5): 536-542
26. Breymann C, Schmidt D, Hoerstrup SP. Umbilical cord cells as a source of cardiovascular tissue engineering. Stem Cell Rev, 2006, 2(2): 87-92.
27. Batten P, Sarathchandra P, Antoniw JW, et al. Human mesenchymal stem cells induce T cell anergy and downregulate T cell allo-responses via the TH2 pathway: relevance to tissue engineering human heart valves. Tissue Eng, 2006, 12(8): 2263-2273.
28. Kadmer A, Hoerstrup SP, Zund G, et al. A new source for cardiovasculor tissue engineering : Human bone marrow stromal cells. Eur Cardio Thorac Surg, 2002, 21:1055-1060.
29. Perry TE, Kaushal S, Sutherland FWH, et al. Bone marrow as a cell a source for tissue engineering heart vahes. Ann Thorac Surg, 2003, 75: 761-767.
30. Knight RL, Booth C, Wilcox HE, et al. Tissue engineering of cardiac valves: re-seeding of acellular porcine aortic valve matrices with human mesenchymal progenitor cells. J Heart Valve Dis, 2005, 14(6): 806-813.
31. Dreger SA, Thomas P, Sachlos E, et al. Potential for synthesis and degradation of extracellular matrix proteins by valve interstitial cells seeded onto collagen scaffolds. Tissue Eng, 2006, 12: 2533-2540.
32. Hoerstrup SP, Kadner A, Breymann C, et al. Living, autologous pulmonary artery conduits tissue engineered from human umbilical cord cells. Ann thorac Surg, 2002, 74(1): 46-56.
33. Ralf S, Simon P, Hoerstrup SP, et al. Tissue Engineering of heart valves:in vitro experience. Ann Thorac Surg, 2002, 70: 140-144.
34. Lichtenberg A, Cebotari S, Tudorache I, et al. Flow-dependent re-endothelialization of tissue-engineered heart valves. J Heart Valve Dis, 2006,15(2): 287-293; discussion 293-294.
2. Mol A, Rubbens MP, Stekelenburg M, et al. Living heart valve and small-diameter artery substitutes-an emerging field for intellectual property development. Recent Pat Biotechnol, 2008,2(1): 1-9.
3. Hong H, Dong GN, Shi WJ, et al. Fabrication of biomatrix/polymer hybrid scaffold for heart valve tissue engineering in vitro. ASAIO J, 2008 Nov-Dec, 54(6): 627-632.
4. Schoen FJ. Evolving concepts of cardiac valve dynamics: the continuum of development,functional structure, pathobiology, and tissue engineering. Circulation, 2008, 118(18):1864-1880.
5. Del Gaudio C, Bianco A, Grigioni M. Electrospun bioresorbable trileaflet heart valve prosthesis for tissue engineering: in vitro functional assessment of a pulmonary cardiac valve design. Ann Ist Super Sanita, 2008, 44(2): 178-186.
6. Kidane AG, Burriesci G, Cornejo P, et al. Current developments and future prospects for heart valve replacement therapy. J Biomed Mater Res B Appl Biomater, 2009, 88(1): 290-303.
7. Migneco F, Hollister SJ, Birla RK. Tissue-engineered heart valve prostheses: 'state of the heart'.Regen Med, 2008, 3(3): 399-419.
8. Hayashida K, Kanda K, Oie T, et al. "In vivo tissue-engineered" valved conduit with designed molds and laser processed scaffold. J Cardiovasc Nurs, 2008, 23(1): 61-64.
9. Schmidt D, Mol A, Kelm JM, et al. In vitro heart valve tissue engineering. Methods Mol Med, 2007,140: 319-330.
10．孙晓宁，赵强，徐德民，等．骨髓间充质干细胞和猪去细胞瓣膜支架构建组织工程瓣膜．复旦学报(医学版)，2007，2：183—186．
11. Sacks MS. Biomechanics of engineered heart valve tissues. Conf Proc IEEE Eng Med Biol Soc,2006, 1: 853-854.
12. Schaefermeier PK, Cabeza N, Besser JC, et al. Potential cell sources for tissue engineering of heart valves in comparison with human pulmonary valve cells. ASAIO J, 2009 Jan-Feb, 55(1): 86-92.
13. Hare JM, Chaparro SV. Cardiac regeneration and stem cell therapy. Curr Opin Organ Transplant,2008, 13(5): 536-542
14. Breymann C, Schmidt D, Hoerstrup SP. Umbilical cord cells as a source of cardiovascular tissue engineering. Stem Cell Rev, 2006, 2(2): 87-92.
15. Batten P, Sarathchandra P, Antoniw JW, et al. Human mesenchymal stem cells induce T cell anergy and downregulate T cell allo-responses via the TH2 pathway: relevance to tissue engineering human heart valves. Tissue Eng, 2006, 12(8): 2263-2273.
16. Sutherland FW, Perry TE, Yu Y, et al. From stem cells to viable autologous semilunar heart valve. Circulation, 2005,111(21): 2783-2791.
17. Nolite SV, Xu W, Rennekampff HO, et al. Divesity of fibroblasts-a review on implications for skin tissue engineering. Cells Tissues, Organs, 2008,187(3): 165-176.
18. Habibovic P, de Groot K. Osteoinductive biomaterials-properties and relevance in bone repair. J Tissue Eng Regen Med, 2007 Jan-Feb, 1(1): 25-32.
19. Macri L, Silverstein D, Clark RA. Growth factor binding to the pericellular matrix and its importance in tissue engineering. Adv Drug Deliv Rev, 2007, 59(13): 1366-1381.
20. Narine K, De Wever O, Van Valckenborgh D, et al. Growth factor modulation of fibroblast proliferation, differentiation, and invasion: implications for tissue valve engineering. Tissue Eng, 2006, 12(10): 2707-2716.
21. Denys H, Derycke L, Hendrix A, et al. Differential impact of TGF-beta and EGF on fibroblast differentiation and invasion reciprocally promotes colon cancer cell invasion. Cancer Lett, 2008, 266(2): 263-274.
22. Fried M, Capetandes A. The effect of enantiomers of beta-agonists on myofibroblast-derived vascular endothelial growth factor and other matrix components in the presence of dust-mite extract. Allergy Asthma Proc, 2008, 29(2): 182-188.
23. Seliktar D, Zisch AH, Lutolf MP, et al. MMP-2 sensitive, VEGF-bearing bioactive hydrogels for promotion of vascular healing. J Biomed Mater Res A, 2004, 68(4): 704-716.
24. Mola A, van Lieshouta MI, Dam-de Veen CG, et al. Fibrin as a cell carrier in cardiovascular tissue engineering applications. Biomaterials, 2005, 26: 3113-3121.
25. Nakanishi Y, Chen G, Komuro H, et al. Tissue-engineered urinary bladder wall using PLGA mesh-collagen hybrid scaffolds: a comparison study of collagen sponge and gel as a scaffold. J Pediatr Surg, 2003, 38: 1781-1784.
26. Kim BS, Putnam AJ, Kulik TJ, et al. Optimizing seeding and culture methods to engineer smooth muscle tissue on biodegradable polymer matrices. Biotechnol Bioeng, 1998, 57: 46-54.
27. Jeong SI, Kim SH, Kim YH, et al. Manufacture of elastic biodegradable PLCL scaffolds for mechanoactive vascular tissue-engineering. J Biomat Sci Polym Ed, 2004, 15: 645-660.
28. Jeong SI, Kim BS, Kang SW, et al. In vivo biocompatibilty and degradation behavior of elastic poly(L-lactide-co-e-caprolactone) scaffolds. Biomaterials, 2004, 25(28): 5939-5946.
29. Knight RL, Wilcox HE, Korossis SA, et al. The use of acellular matrices for the tissue engineering of cardiac valves. Proc Inst Mech Eng [H], 2008, 222: 129-143.
30. Stamm C, Khosravi A, Grabow N, et al. Biomatrix/polymer composite material for heart valve tissue engineering. Ann Thorac Surg, 2004, 78(6): 2084-2092; discussion 2092-2093.
1. Perry TE, Kaushal S, Sutherland FW, et al. Thoracic surgery directors association award: Bone marrow as a cell source for tissue engineering heart valves. Ann Thorac Surg, 2003, 75(3):761-767.
2. Hwang WS, Ryu YJ, Park JH, et al.Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science, 2004, 303(5664): 1669-1674.
3. Hoerstrup .SP, Kadner A, Melnitchouk S, et al. Tissue engineering of functional trileaflet heart valves from human marrow stromal cells. Circulation, 2002, 106(12 Suppl 1): 143-150.
4．董念国，孙宗全，傅平，等．聚二乙醇酸与Degrapol对肌成纤维细胞粘附、生长的影响．华中科技大学学报(医学版)，2003，32：71-73．
5．洪吴，董念国．组织工程心脏瓣膜的研究进展．临床心血管病杂志，2006，22(7)：442-444．
6. Maish MS, Hoffman-Kim D, Krueger PM, et al. Tricuspid valve biopsy: a potential source of cardiac myofibroblast cells for tissue-engineered cardiac valves. J Heart Valve Dis, 2003, 12:264-269.
7. Schenke-Layland K, Opitz F, Gross M, et al. Complete dynamic repopulation of decellularized heart valves by application of defined physical signals-an in vitro study. Cardiovascular Research,2003, 60: 497-509.
8．董念国，叶晓峰，史嘉玮，等．组织工程瓣膜天然支架去细胞方法的比较．中华实验外科杂志，2005，22：377．
9. Sutherland F W H, Perry T E, Ying Y, et al. From stem cells to viable autologous semilunar heart valve. Circulation, 2005, 111: 2783-2791.
10. Kim SS, Lim SH, Cho SW, et al. Tissue engineering of heart valves by recellularization of glutaraldehyde-fixed porcine valves using bone marrow-derived cells. Artificial Organs, 2006, 30(7): 554-567:
11. Hoerstrup S P, Kadner A, Melnitchouk S, et al. Tissue engineering of functional trileaflet heart valves from human marrow stromal cells. Circulation, 2002, 106: 143-150.
12．洪吴，董念国，史嘉玮．Amplex Red荧光法检测组织工程瓣膜赖氨酰氧化酶活性．中国胸心血管外科临床杂志，2007，14(1)：27-30．
13. Steinhoff G; Stock UA, Karim N, et al.Tissue Engineering of Pulmonary Heart Valves on Allogenic Acellular Matrix Conduits : In Vivo Restoration of Valve Tissue. Circulation, 2000, 102:50-55
14．史嘉玮，董念国，孙宗全，等．RGD肽联合转化生长因子-β_1对组织工程瓣膜构建的影响．中华医学杂志，2006，86：2074-2077．
15．董念国，孙宗全，付平，等．组织工程心脏瓣膜构建的研究．中华实验外科杂志，2002，19(1)：88．
16．韩啸，蒋雄刚，徐鹏，等．聚乙二醇和胰蛋白酶用于猪动脉管道去细胞效果比较．华中科技大学学报(医学版)，2007，36(4)：442—444．
17．董念国，史嘉玮，胡平．组织工程心脏瓣膜支架的研究现状和发展趋势．中华实验外科杂志，2007，24(3)：261—262．
18. Kidane AG, Burriesci G; Cornejo P, et al. Current developments and future prospects for heart valve replacement therapy. J Biomed Mater Res B Appl Biomater, 2009, 88(1): 290-303.
19. Migneco F, Hollister SJ, Birla RK. Tissue-engineered heart valve prostheses: 'state of the heart'.Regen Med, 2008, 3(3): 399-419.
20. Schmidt D, Mol A, Kelm JM, Hoerstrup SP. In vitro heart valve tissue engineering. Methods Mol Med, 2007, 140: 319-330.
21. Blevins TL, Carroll JL, Raza AM, et al. Phenotypic characterization of isolated valvular interstitial cell subpopulations. J Heart Valve Dis, 2006, 15(6): 815-822.
22. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science, 1998, 282(5391): 1145-1147.
23. Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007, 318(5858): 1917-1920.
24. Henderson JK, Draper JS, Baillie HS, et al. Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. Stem Cells, 2002, 20(4):329-337.
25. Sacks MS. Biomechanics of engineered heart valve tissues. Conf Proc IEEE Eng Med Biol Soc,2006, 1: 853- 854.
26. Pierret C, Friedrichsen P. Stem cells and society: an undergraduate course exploring the intersections among science, religion, and law. CBE Life Sci Educ, 2009 Spring, 8(1): 79-87.
27. Grivennikov IA. Embryonic stem cells and the problem of directed differentiation. Biochemistry(Mosc), 2008, 73(13): 1438-1452.
28. Schaefermeier PK, Cabeza N, Besser JC, et al. Potential cell sources for tissue engineering of heart valves in comparison with human pulmonary valve cells. ASAIO J, 2009 Jan-Feb, 55(1): 86-92.
29. Hare JM, Chaparro SV. Cardiac regeneration and stem cell therapy. Curr Opin Organ Transplant,2008, 13(5): 536-542
30. Breymann C, Schmidt D, Hoerstrup SP. Umbilical cord cells as a source of cardiovascular tissue engineering. Stem Cell Rev, 2006, 2(2): 87-92.
31. Batten P, Sarathchandra P, Antoniw JW, et al. Human mesenchymal stem cells induce T cell anergy and downregulate T cell allo-responses via the TH2 pathway: relevance to tissue engineering human heart valves. Tissue Eng, 2006, 12(8): 2263-2273.
32. Kadner A, Hoerstrup S P, Zund G, et al. A new source for cardiovascular tissue engineering: human bone marrow stromal cells. European Journal of Cardio-thoracic Surgery, 2002, 21: 1055-1060.
33．孙晓宁，赵强，徐德民，等．骨髓间充质干细胞和猪去细胞瓣膜支架构建组织工程瓣膜．复旦学报(医学版)，2007，34：183—186．
34. Vincentelli A, Wautot F, Juthier F, et al. In vivo autologous recellularization of a tissue-engineered heart valve: Are bone marrow mesenchymal stem cells the best candidates? J Thorac Cardiovasc Surg, 2007, 134: 424-432.
35. Hong H, Dong GN, Shi WJ, et al. Fabrication of biomatrix/polymer hybrid scaffold for heart valve tissue engineering in vitro. ASAIO J, 2008 Nov-Dec, 54(6): 627-632.
36. Cushing MC, Liao JT, Anseth KS. Activation of valvular interstitial cells is mediated by transforming growth factor-β1 interactions with matrix molecules. Matrix Biology, 2005, 24:428-437.
37. Rabkin-Aikawa E, Farber M, Aikawa M, et al. Dynamic and reversible changes of interstitial cell phenotype during remodeling of cardiac valves. J Heart Valve Dis, 2004, 13(5) : 841-847.
38. Itoh Y, Nagase H. Matrix metalloproteinases in cancer. Essays Biochem JT-Essays in biochemistry, 2002, 38: 21-36.
39. Ohkawa K, Fujii K, Nishida A, et al. Lysyl oxidase-catalyzed cross-linking and insolubilization reactions of Lys-containing polypeptides and synthetic adhesive proteins. Biomacromolecules, 2001, 2(3): 773-779.
40. Zhao Y, Gao S, Chou IN, et al. Inhibition of the expression of lysyl oxidase and its substrates in cadmium- resistant rat fetal lung fibroblasts. Toxicological Sciences, 2006, 90 (2): 478-489.
41. Smith-Mungo LI, Kagan HM. Lysyt Oxidase: properties, regulation and multiple functions in biology. Matrix Biol, 1998,16(7): 387-398.
42. Schoen FJ. Evolving concepts of cardiac valve dynamics: the continuum of development, functional structure, pathobiology, and tissue engineering. Circulation, 2008,118(18): 1864-1880.
43. Heinrich AC, Patel SA, Reddy BY, et al. Multi- and inter-disciplinary science in personalized delivery of stem cells for tissue repair. Curr Stem Cell Res Ther, 2009, 4(1): 16-22.
44. Pittenger MF, Mackay AM, Jaiswal SC, et al. Multilineage potential of mesenchymal stem cell. Science, 1999, 284(5411): 143-147.
45. Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical users. Exp Hematol, 2000,28(8): 875-884.
46. Schoen J F. New frontiers in the pathology and therapy of heart valve disease: 2006 Society for Cardiovascular Pathology, Distinguished Achievement Award Lecture, United States-Canadian Academy of Pathology, Atlanta, GA, February 12, 2006. Cardiovascular Pathology, 2006, 15(5): 271-279.
1. Cetin M, Aktas Y, Vural I, et al. Preparation and in vitro evaluation of bFGF-loaded chitosan nanoparticles. Drug Delivery, 2007, 14(8): 525-529.
2. Amidi M, Romeijn SG., Borchard G, et al. Preparation and characterization of protein-loaded N-trimethyl chitosan nanoparticles as nasal delivery system. J Controll Rel, 2006, 111: 107-116.
3. Aktas Y, Andrieux K, Alonso MJ, et al. Preparation and in vitro evaluation of chitosan nanoparticles containing a caspase inhibitor. Int J Pharm, 2005, 298: 378-383.
4. Calvo P, Remu(?)n(?)an-L(?)pez C, Vila-Jato JL, et al. Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. J Appl Polym Sci, 1997, 63: 125-132.
5. Berthold A, Cremer K, Kreuter J. Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model for antiinflammatory drugs. J Controll Rel, 1996, 39: 17-25.
6. Cetin M, Capan Y, Vural I, et al. Preparation and characterization of bFGF and BSA-loaded microspheres. J Drug Del Sci Tech, 2005, 15: 371-375.
7. Mol A, Rubbens MP, Stekelenburg M, et al. Living heart valve and small-diameter artery substitutes-an emerging field for intellectual property development. Recent Pat Biotechnol, 2008, 2(1): 1-9.
8. Hong H, Dong GN, Shi WJ, et al. Fabrication of biomatrix/polymer hybrid scaffold for heart valve tissue engineering in vitro. ASAIO J, 2008 Nov-Dec, 54(6): 627-632.
9. Schoen FJ. Evolving concepts of cardiac valve dynamics: the continuum of development, functional structure, pathobiology, and tissue engineering. Circulation, 2008,118(18): 1864-1880.
10. Stock UA, Wiederschain D, Kilroy SM, et al. Dynamics of extracellular matrix production and turnover in tissue engineered cardiovascular structures. J Cell Biochem, 2001, 81: 220-228.
11. Schenke-Layland K, Opitz F, Gross M, et al. Complete dynamic repopuiation of decellularized heart valves by application of defined physical signals-an in vitro study. Cardiovascular Research, 2003, 60: 497-509.
12. Sheridan MH, Shea LD, Peters MC, et al. Bioabsorbable polymer scaffolds for tissue engineering capable of sustained growth factor delivery. J Control Release, 2000, 64: 91-102.
13. Lee KY, Peters MC, Anderson KW, et al. Controlled growth factor release from synthetic extracellular matrices. Nature, 2000, 408: 998-1000.
14. Lutolf MP, Lauer-Fields JL, Schmoekel HG, et al. Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: Engineering cell-invasion characteristics. PNAS, 2003, 100(9): 5413-5418.
15. Edelman ER, Mathiowitz E, Langer R. et al. Con:trolled and modulated release of basic fibroblast growth factor. Biomaterials, 1991, 12: 619-626.
16. Cai DZ, Zeng C, Quan DP, et al. Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-β1 delivery for cartilage tissue engineering.Chinese Medical Journal, 2007, 120: 197-203.
17．董念国，史嘉玮，胡平．组织工程心脏瓣膜支架的研究现状和发展趋势．中华实验外科杂志，2007，24(3)：261—262．
18. Stamm C, Khosravi A, Grabow N, et al. Biomatrix/polymer composite material for heart valve tissue engineering. Ann Thorac Surg, 2004, 78(6): 2084-2092: discussion 2092-2093.
1．宋强，董念国．心脏组织工程研究新进展．临床心血管病杂志，2003，4：251—252．
2. Shinoka T, Shun-Tim D, Ma PX, et al. Creation of viable pulmonary artery autograft through tissue engineering. J Thorac Cardiovasc Surg, 1998, 115: 536-545.
3. Zund G ,Ye Q, H0erstrup SP, et al. Tissue engineering in cardiovascular surgery: MTF, a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshes. Eur J Cardiothorac Surg, 1999, 15: 519-524.
4. Sodian R, Sperling JS, Martin DP, et al. Fabrication of trileaflet heart valve scaffold from a polyhydroxylkanoate biopolyyester for use in tissue engineering. Tissue Eng, 2000, 6: 183-188.
5. Sodian R, Hoerstrup SP, Sperling JS, et al. Evaluation of biodegradable, three-dimensional matrices for tissue engineering of heart valve. ASAIO J, 2000, 46: 107-110.
6．董念国，孙宗全，付平等．聚二乙醇酸与Degrapol对肌成纤维细胞粘附、生长的影响．华中科技大学学报(医学版)，2003，1：71—73．
7．董念国，孙宗全，付平等．组织工程心脏瓣膜构建的研究．中华实验外科杂志，2002，1：88．
8. Hoerstrup SP, Sodian R, Daebritz S, et al. Functional living trileaflet heart valves grown in vitro.Circulation, 2000, 102(19 suppl 3): Ⅲ44-Ⅲ49.
9．胡平．组织工程用外科植入物-高分子材料的加工、改性及应用．中国医疗器械信息，2006，12(7)：13-21．
10．叶晓峰，董念国，孙宗全．组织工程瓣膜天然支架去细胞方法的比较．中华实验外科杂志，2005，22(3)：377．
11．叶晓峰，董念国，孙宗全，等．不同预处理对去细胞瓣细胞黏附率的影响．临床心血管病杂志，2005，21(6)：367-369．
12. Steinhoff G, Stock U, Karim N, et al. Tissue engineering of pulmonary heart valves on allogenic acelluler matrix conduits in vivo restoration of valve tissue. Circulation, 2000, 102(19 suppl3): Ⅲ50-Ⅲ55.
13. Ye Q, Zund G, Benediket P, et al. Fibrin gel as a three dimeusional matrix in cordiovascular tissue engineering. Eur J Cardiothorac Surg, 2000, 17: 587-591.
14．赵东锷，汪钢，俞世强，等．应用胶原膜支架构建组织工程心脏瓣膜的初步研究．中华胸心血管外科杂志，2002，5：257-260．
15. Lutolf MP, Lauer-Fields JL, Schmoekel HG, et al. Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: Engineering cell-invasion characteristics. PNAS, 2003,100(9): 5413-5418.
16. Stamm C, Khosravi A, Grabow N, et al. Biomatrix/polymer composite material for heart valve tissue engineering. Ann Thorac Surg, 2004, 78(6): 2084-2092; discussion 2092-2093.
17. Hong H, Dong GN, Shi WJ, et al. Fabrication of biomatrix/polymer hybrid scaffold for heart valve tissue engineering in vitro. ASAIO J, 2008 Nov-Dec, 54(6): 627-632.
18. Wilcox HE, Korossis SA, Booth C, et al. Biocompatibility and recellularization potential of an acellular porcine heart valve matrix. J Heart Valve Dis, 2005, 14(2): 228-36; discussion 236-237.
19. Paranya G, Vineberg S, Dvorin E, et al. Aortic valve endothelial cells undergo transforming growth β-factor mediated and non-β-factor mediated transdifferentiation in vitro. Ame J Patho, 2001, 159: 1335-1343.
20. Dohmen PM, Ozaki S, Verbeken E, et al. Tissue engineering of an auto-xenograft pulmonary heart valve. Asian Cardiovasc Thorac Ann, 2002, 10: 25-30.
21. Cebotari S, Mertsching H, Kallenback K, et al. Construction of autologous human heart valves based on an acellular allograft matrix. Circulation, 2002, 106(suppl 1): 63-68.
22. Kasimir MT, Weigel G, Sharma J,et al. The decellularized porcine heart valve matrix in tissue engineering: platelet adhesion and activation. Thromb Haemost, 2005, 94(3): 562-567.
23. Toshihiko S, Hongbing H, Hisataka Y, et al. Human enclothelial progenitor cell-seeded hybrid graft:Proliferative and antithrom bogenic potentials in vitro and(?) fabrication processing. Tissue Eng, 2003, 9:127-136.
24. Ozawa T, Donald A, Mickle G, et al. Optimal biomaterial for creation of autologous cardiac grafts. Circulation, 2002, 106(12 suppl 1): 1176-1182.
25. Hare JM, Chaparro SV. Cardiac regeneration and stem cell therapy. Curr Opin Organ Transplant, 2008, 13(5): 536-542
26. Breymann C, Schmidt D, Hoerstrup SP. Umbilical cord cells as a source of cardiovascular tissue engineering. Stem Cell Rev, 2006, 2(2): 87-92.
27. Batten P, Sarathchandra P, Antoniw JW, et al. Human mesenchymal stem cells induce T cell anergy and downregulate T cell allo-responses via the TH2 pathway: relevance to tissue engineering human heart valves. Tissue Eng, 2006, 12(8): 2263-2273.
28. Kadmer A, Hoerstrup SP, Zund G, et al. A new source for cardiovasculor tissue engineering : Human bone marrow stromal cells. Eur Cardio Thorac Surg, 2002, 21:1055-1060.
29. Perry TE, Kaushal S, Sutherland FWH, et al. Bone marrow as a cell a source for tissue engineering heart vahes. Ann Thorac Surg, 2003, 75: 761-767.
30. Knight RL, Booth C, Wilcox HE, et al. Tissue engineering of cardiac valves: re-seeding of acellular porcine aortic valve matrices with human mesenchymal progenitor cells. J Heart Valve Dis, 2005, 14(6): 806-813.
31. Dreger SA, Thomas P, Sachlos E, et al. Potential for synthesis and degradation of extracellular matrix proteins by valve interstitial cells seeded onto collagen scaffolds. Tissue Eng, 2006, 12: 2533-2540.
32. Hoerstrup SP, Kadner A, Breymann C, et al. Living, autologous pulmonary artery conduits tissue engineered from human umbilical cord cells. Ann thorac Surg, 2002, 74(1): 46-56.
33. Ralf S, Simon P, Hoerstrup SP, et al. Tissue Engineering of heart valves:in vitro experience. Ann Thorac Surg, 2002, 70: 140-144.
34. Lichtenberg A, Cebotari S, Tudorache I, et al. Flow-dependent re-endothelialization of tissue-engineered heart valves. J Heart Valve Dis, 2006,15(2): 287-293; discussion 293-294.