Preparation And Mechanical Properties Study Of A Hybrid Scaffold By Branched Polyethyl Glycol Cross-linking Porcine Decellularized Aortic Leaflets

PartⅠSynthesis and Identification of branched PEG derivativesObjective:Synthesis and characterization of two different branched PEG derivatives with the terminal group of N-hydroxy succinimide (-NHS) or diacrylate (-DA). The product yield and purity were assessed.Methods:The linear PEG3400 polymer was utilized as raw material, and four N-hydroxysuccinimides or diacrylates were introduced into the terminal of the polymer by graft copolymerization. IR,1H MRI and 13C MRI were applied to character the structure of the products.Results:The chemical structure of the products was accurate. The yield of PEG-NHS and PEG-DA were 91.5% and 90.4%, and the purity of PEG-NHS and PEG-DA were 94.6% and 92.8%, respectively.Conclusion:The linear PEG3400 could be converted into branched PEG-NHS or PEG-DA dervatives, and achieved functionalization. PartⅡCross-linking and optimization of branched PEG to the decellularized porcine aortic leafletsChapter 1 Preparation and sulfhydryl modification of the decellularized porcine aortic leafletsObjective:To introduce sulfhydryl into the decellularized porcine aortic leaflets, and optimize the reaction conditions.Methods:The fresh porcine aortic valves were digested by 0.05%trypsin/0.02%EDTA and 1% TritonX-100. HE stain and SEM were used to assess the effects of decellularization procedure. The sulfhydryl was introduced into the decellularized aortic valves (DAVs) by SATA. The reaction conditions, such as concentration of SATA, pH, temperature and reaction time, were optimized by orthogonal design.Results:The decellularization procedure removed the valular endothelial cells (VECs) and interstitial cells (VICs) successfully, and preserved the extracellular matrix (ECM) well. The optimized conditions were summarized as follow:the concentration of SATA was 2 mg/mL, the pH was 7.4-7.8, the temperature was 37℃, and the reaction time was 2 hours.Conclusion:The decellularization procedure we utilized could prepare ideal DAVs. The DAVs could be modified effectively by SATA.Chapter 2 Comparison of PEG-NHS cross-linking amino group and PEG-DA cross-linking sulfhydryl groupObjective:Compare the reactivity difference of two kind of cross-linking methods. Assess the effects of PEG-NHS and PEG-DA crosslinking on the mechanical property of the DAVs.Methods:PEG-NHS with the molecular weight of 3400Da,8000Da and 12000Da were allowed to react with free amino group (-NH2) of non-treated DAVs, meanwhile PEG-DA with the molecular weight of 3400Da,8000Da and 12000Da ware allowed to react with DAVs modified by sulfhydryl (-SH). The cross-linking degrees at 2h,4h and 6h were calculated and compared. The tensile test was used to asssess the tensile strength of each group.Results:The cross-linking degrees at 2h,4h and 6h of PEG-DA group were significantly higher when compared with those of PEG-NHS groups at above time points (P<0.05). With the increase of molecular weight of PEG-NHS and PEG-DA, the tensile strength of each group showed an increasing trend, but the PEG-DA groups were remarkably higher than those of PEG-NHS groups (P<0.05).Conclusion:Compared with the reaction of PEG-NHS cross-linking amino group, the reaction between PEG-DA and sulfhydryl had higher reactivity, and can enhance the mechanical property of the DAVs.Chapter 3 Effects of molar weight of PEG-DA on the mechanical properties of the decellularized porcine aortic valvesObjective:To investigate the effects of polyethylene glycol with different molar weight crosslinking on the mechanical properties of the DAVs.Methods:A total of eighty DAVs which were introduced sulfhydryl groups were randomly assigned into five groups, and allowed to react with branched PEG-DA with the molecular weight of 3.4,8,12,20 and 40kDa. The mechanical properties were obtained by uniaxial planar tensile testing.Result:The tensile strength enhanced from 5.95±0.44MPa to 8.05±0.15MPa with molecular weight of PEG-DA increasing to 20kDa. However, when the molecular weight of PEG-DA increased to 40kDa, the tensile strength decreased remarkably. The young's modulus of each group had a positive correlation with the molecular weight of PEG-DA, and increased sharply to 45.68±2.74MPa when the molecular weight of PEG-DA increased to 40kDa. The difference between PEGDA-20kDa group and other groups was significant (P<0.05).Conclusion:The molecular weight of PEG-DA could affect the mechanical properties of the DAVs. Branched PEG-DA with the molecular weight of 20kDa could significantly improve the mechanical behaviors of the DAVs, and may be a promising polymer cross-linker which can be used in the modification of the DAVs for tissue engineering.Chapter 4 Optimization of PEG20000-DA cross-linking the decellularized porcine aortic leafletsObjective:To optimize the reaction conditions of PEG20000-DA cross-linking the DAVs.Methods:The reaction conditions, such as molar ratio of -DA and -SH, concentration of PEG20000-DA, reaction time, were optimized by orthogonal design.Results:when the molar ratio of -Da and -SH increased from 5:1 to 20:1, the cross-linking degree significantly increased. However, when the molar ratio further increased to 40:1, the cross-linking degree did not remarkably increase. When the concentration of PEG was lOmg/mL, the cross-linking degree reached maximum. The cross-linking achieved plateau phase when the reaction time was 8hrs, and prolonging the reaction time did not appear helpful to the cross-linking effectiveness.Conclusion:The optimized reaction conditions were summarized as follow:the molar ratio of -DA and -SH was 20:1, the concentration was 10mg/mL, and the reaction time was 8 hours.Part III PEG20000-DA cross-linking the DAVs constructs hybrid scaffold for tissue engineering heart valveChapter 1 Effects of PEG20000-DA cross-linking on the physical characters and mechanical properties of DAVsObjective:To evaluate the effects of PEG20000-DAcross-linking on the physical and mechanical properties of the decellularized porcine aortic valves. Methods:Eighty fresh native porcine aortic valves were randomly assigned into four groups:native group (also termed control group), decellularized aortic valves (DAVs group), PEG20000-DA cross-linking the DAVs group (PEG group), and glutaraldehyde-fixed the DAVs (GA group). Each group has twenty samples. The tissue thickness, porosity, and water content were investigated. Tensile test was utilized to assess the tensile strength and the young's modulus at circumferential and radial directions. Bending test was carried out to evaluate the flexural modulus.Results:The tissue thickness, porosity and water content of PEG group were remarkably decreased when compared with those of other groups (P<0.05). The tensile strengths of PEG group at circumferential and radial directions were 7.55±0.11MPa and 4.27±0.20MPa, respectively. The young's moduluses of PEG group at circumferential and radial directions were 35.6±2.48MPa and 19.11±0.68MPa, respectively. The difference between PEG group and others was significant (P<0.05). Flexural modulus of PEG group at the stress of 0.5MPa was 10.92Ma, which was much higher than that of other groups (P<0.05).Conclusion:PEG20000-DA cross-linking could change the physical characters of the DAVs, and improve their mechanical properties.Chapter 2 Effects of PEG20000-DA cross-linking on the structure and conformation stability of the decellularized aortic valvesObjective:To investigate the effects of PEG20000-DA cross-linking on the structure and conformation stability of the DAVs.Methods:Differential scanning calorimeter (DSC) was taken to determinate the thermal shrinkage temperature (Ts) of the PEG20000-DA cross-linked DAVs (PEG group). Resistance to enzymatic degradation was tested by 40u/ml I type collagenase D-hanks solution. The effects of PEG20000-DA cross-linking on the triple helix of collagen protein were researched by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR).Results:The Ts of PEG group is 75.16±0.69℃, and significantly higher than that of the DAVs (P<0.05). The enzymatic degradation rates at 6h and 12 h of PEG group were 17.52±1.69% and 48.83±2.67%, which were much lower when compared with the DAVs. The CD and FTIR test showed that PEG20000-DA cross-linking did not change the characteristic absorption peak of collagen protein of the DAVs.Conclusion:PEG20000-DA cross-linking enhanced the structural stability, but did not result in bad effects on the conformation stability of the DAVs.Chapter 3 Effect of PEG20000-DA cross-linking on the anti-calcification ability of the decellularized aortic valvesObjective:To assess the anti-calcification ability of the PEG20000-DA cross-linked DAVs in rabbit subcutaneous model.Methods:Glutaraldehyde fixed native aortic valves (GA-NAV group), glutaraldehyde fixed decellularized aortic valves (GA-DAV group) and PEG20000-DA cross-linked decellularized aortic valves (PEG group) were implanted subcutaneous in rabbits. Six samples were taken out for calcium staining and quantitative analysis at two weeks, four weeks and eight weeks.Results:Calcium staining found that GA-NAV group appeared severe calcification with the time increasing, GA-DAV group also occurred calcification, but much lower than GA-NAV group. PEG group did not appear obvious calcification. The calcium quantitative analysis showed that calcium contents in GA-NAV group at two weeks, four weeks and eight weeks were 22.39±1.79μg/mg,93.05±6.08μg/mg and 174.97±8.34μg/mg, respectively. Calcium contents in GA-DAV group were 5.57±2.11μg/mg,31.15±5.16μg/mg and 72.54±6.50μg/mg, respectively. Calcium contents in PEG group were 0.46±0.07μg/mg,0.68±0.10μg/mg and 1.92±0.25μg/mg, respectively. The difference between each group was significant (P<0.05).Conclusion:PEG20000-DA cross-linking can remarkably inhabit the calcification of the decellularized aortic valves in rabbit subcutaneous model.