Study On Biological Properties Of The Composite PEG-crosslinked Decellularized Valve With Multi-signal

Part one:Construction and in vitro biocompatibility of the composite PEG-crosslinked decellularized valveObjective:To construct a composite material by polyethylene glycol (PEG) crosslinking of decellularized valves, and evaluate the biocompatibility of the composite material in vitro.Methods:First, branched PEG with the functional group of propylene acyl was synthesized, and thiol was introduced into decellularized valves. Then the composite valve was constructed by the Michael addition reaction between propylene acyl and thiol. Second, the effects of PEG crosslinking on biomechanical properties of decellularized valve were evaluated. For assessing the biocompatibility, cell proliferation rates of the human umbilical vein endothelial cells (HUVECs) which were exposed to the extract of composite material were detected by CCK-8 assay.Results:The PEG-crosslinking reaction for the construction of composite material has a mild conditions and a high efficiency; The tensile strength of the composite materials was obviously higher than that of the decellularized ones(P<0.05), and there was no significant difference in tensile strength between composite materials and natural valves(P>0.05). The toxicity of composite material were classified Grade 0, and no significant difference of toxicity was observed between the composite material group and the negative control group; HUVECs treated with culture medium containing extracts of composite material show great cytoactivity and normal morphology.Conclusions:A noncytotoxic composite material can be obtained by PEG crosslinking with the decellularized valves, and the tensile strength of the composite material is similar to the natural one.Part two:Construction of the composite PEG-crosslinked decellularized valve with multi-signalChapter one:Construction of the composite valve conjugated with RGD peptideObjective:To evaluate the feasibility and effectiveness of conjugation of RGD (Arg-Gly-Asp) peptide on the composite PEG-crosslinked decellularized valve and optimize the reaction condition.Methods:The composite PEG-crosslinked decellularized valve was reacted with 1ml GRGDSPC peptides solution of different concentrations (1.5mg/ml, 1mg/ml, 0.5mg/ml,0.25mg/ml) at 37℃. The conjugated effect was evaluated quantitatively by spectrophotometer at different time points (1h,2h, 4h), and the qualitative tests were performed by immunofluorescence. The decellularized valves were set as negative control.Results:The GRGDSPC peptides could be conjugated effectively with the PEG-composite valve, and the quality of conjugated GRGDSPC peptides reaches a climax amount of (0.79±0.01)mg when the concentration of GRGDSPC peptides solution was lmg/ml and the reaction time was 2h.Conclusion:The construction of RGD-composite material can be achieved by grafting GRGDSPC peptide on the composite PEG-crosslinked decellularized valve in vitro.Chapter two Construction of the composite valve conjugated with VEGFObjective:To evaluate the feasibility and effectiveness of conjugation of VEGF on composite PEG-crosslinked decellularized valve and optimize the reaction condition.Methods:The composite PEG-crosslinked decellularized valve was reacted with lml VEGF solution of different concentrations (500pg/ml, 1000pg/ml,2000pg/ml) at 37℃. The quality of conjugated VEGF was measured by ELISA at different time points (1h,2h,4h,12h), and the qualitative tests were performed by immunofluorescence. The decellularized valves were set as negative control.Results:The VEGF could be conjugated effectively on the composite valve by PEG, and the maximum quality of conjugated VEGF was (896.87±3.27)pg when the concentration of VEGF solution was 1000pg/ml and reaction time was 4h.Conclusion:The construction of VEGF-composite material can be achieved by grafting VEGF on the composite PEG-crosslinked decellularized valve in vitro.Chapter three:Construction of the composite PEG-crosslinked decellularized valve with multi-signalObjective:To assess the probability and effectiveness of conjugation of RGD peptide and VEGF on composite PEG-crosslinked decellularized valve simultaneously.Methods:The composite PEG-crosslinked decellularized valve were reacted with lml reaction mixture solution (including lmg/ml GRGDSPC peptide and 1000pg/ml VEGF) at 37℃for 4h. Then the immunofluorescence was taken to make a qualitative evaluation of the conjugation effects. The decellularized valves were set as negative control.Results:The GRGDSPC peptides and VEGF could be conjugated effectively onto composite material by PEG.Conclusion:The construction of composite PEG-crosslinked decellularized valve with multi-signal (RGD and VEGF) can be achieved by conjugating the RGD peptide and VEGF on the composite PEG-crosslinked decellularized valve in vitro. Part three:Study on biological properties of the composite PEG-crosslinked decellularized valve with multi-signalChapter one:The isolation, Culture and identification of EPCsObjective:To provide human endothelial progenitor cells (EPCs) for the further study by isolation, culture and identification of EPCs from human umbilical cord blood.Methods:Fresh umbilical blood was collected and density gradient centrifugated to isolate mononuclear cells (MNCs). The cells then were cultured in the medium supplemented with VEGF and bFGF. Morphology, immunofluorescence and flow cytometry were applied to confirm that the attached cells were EPCs. The proliferation and migration capability of EPCs were compared with HUVECs.Results:As the time went on, the shape of the attached cells changed from small round to spindle. the typical colonies were gradually formed and the cobblestone-like morphology as a specific characteristic of mature endothelial cells was observed; after been cultured for 7 days, more than 90% of the attached cells express both Dil-acLDL and FITC-UEA-Ⅰ. Flow cytometric analysis showed that the positive staining rate of the attached cells for VEGFR2,CD34 and CD133 were (77.35±4.86)%, (52.42±6.64)% and (19.36±2.14)% respectively, and at the 28th day, the positive staining rate of attached cells for VEGFR2 and CD34 were (81.06±7.31)% and (7.62±3.14)% respectively while CD133 can not been stained. Compared with HUVECs, EPCs have more potent potential of proliferation and migration (P<0.05).Conclusion:EPCs can be isolated successfully from human umbilical cord blood by a simple, economical and feasible method composed of density gradient centrifugation and adherent filtration. EPCs can be introduced to endothelial cells and have great capacity of proliferation and migration, which makes it an ideal seed cells. Chapter two:Measurement on biological properties of the composite PEG-crosslinked decellularized valve with multi-signalObjective:To investigate the effect of composite valve with multi-signal on the adhesion and proliferation of EPCs, and provide the basis for the further construction of TEHVMethods:The experiment were assigned into 5 groups:Group A were decellularized valves, Group B were composite PEG-crosslinked decellularized valves, Group C were VEGF-composite valve, Group D were RGD-composite valve and Group E were multi-signal composite valve(n=8). EPCs were seeded onto the valve of different groups, the cells number and counts per minute(cpm) were detected by cell counting and thymidine(3H-TdR) up-take method at 2h,4h,8h respectively after seeding to estimate the adhesion amount of EPCs in different groups. the cells number and cpm of each group were detected using cell counting and thymidine(3H-TdR) up-take method at 2d,4d,8d respectively after seeding to evaluate the proliferation of EPCs. Hematoxylin and eosin (HE) stain and scanning electron microscopy (SEM) were performed to give a morphology evidence of the growth of EPCs at 8d after seeding. Finally the expression of t-PA and eNOS of group E were measured by RT-PCR to assess the antithrombotic function of the multi-signal composite valve.Results:(1) After been seeded for 2h,4h and 8h, the results of cell count and cpm in each group were as the following:Group D>Group E>Group C>Group A>Group B, but there was no significant difference between Group A and Group B(P> 0.05), there was also no significant difference between Group D and Group E at the 8h after the seeding(P>0.05), but the differences in the other groups were significant at the other time points(P<0.05), (2) At the 2d,4d and 8d after been seeded, the results of cell count and cpm in each group were as the following: Group A was always higher than Group B(P>0.05), the cell number and cpm in group C,Group D and Group E were significantly higher than those in Group A and Group B (P<0.05) at the time point. At the 2d after the seeding, the cell number and cpm in Group E was higher than those in Group C (P<0.05), the cell number and cpm in Group D was higher than those in Group E(P>0.05); at the 4d after the seeding, the cell number and cpm in Group E higher those in group D(P<0.05), the cell number and cpm in Group D was higher than those in Group C(P<0.05); at the 8d after the seeding, the cell number and cpm in Group E was higher than those in Group D(P<0.05), the cell number and cpm in Group D was higher than those in Group C(P>0.05). (3) the results of HE and SEM indicated that the confluent and compact monolayer could be formed on the surface of Group E compared with other groups at 8d after the seeding, and the expression level of t-PA and eNOS gene in this neo-endothelium were very similar to those in HUVECs.Conclusion:The RGD peptides and VEGF conjugated on the composite PEG-crosslinked decellularized valve can achieve a synergistic promotion on the adhesion and proliferation of EPCs seeded on the composite valve, which is useful for the construction of tissue engineering heart valves.