| Part one Construction of Tissue Engineered Heart Valve UsingDifferent Seed CellsObjective: 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 bFGFObjective: 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 ScaffoldObjective: 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 bFGFObjective: 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. |
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