Study On Construction And Calcification Mechanism Of Tissue Engineering Heart Valves Fabricated With Composite Scaffolds In A Bioreactor System

Part one:Construction of the composite PEG-crosslinked decellularized valve conjugated with RGD peptideChapter one:Construction of PEG-crosslinked decellularized valveObjective:To construct a composite decellularized valve scaffold crosslinked by polyethylene glycol (PEG).Methods:Branched PEG with functional group of propylene acyl was synthesized. Using the SATA method, sulfhydryl group was added to decellularized valve. The composite scaffold material was constructed through the Michael addition reaction. The effects of PEG crosslinking on biomechanical properties were evaluated.Results:The PEG-crosslinking reaction required a mild condition and had a high efficiency. The tensile strength of the PEG-crosslinked decellularized valve was higher than that of the decellularized valves(P<0.05). There was no significant difference in comparison with natural valves(P>0.05).Conclusion:PEG-crosslinked decellularized valve could be obtained through the Michael addition reaction, and its tensile strength was comparable to that of the natural valves. Chapter two:Construction of the composite PEG-crosslinked decellularized valve conjugated with RGD peptideObjective:To construct the composite PEG-crosslinked decellularized valve conjugated with RGD peptide.Methods:PEG-crosslinked decellularized valve was underwent Michael addition reaction with lml GRGDSPC peptides solution at 37℃. The effect of GRGDSPC peptides modification was quantitatively determined by spectrophotometer. Immunofluorescence was also performed to test the modification effect.Results:The GRGDSPC peptides could be effectively conjugated with the PEG-crosslinked decellularized valve as determined by the immunofluorescence test.Conclusion:Construction of the composite PEG-crosslinked decellularized valve conjugated with RGD peptide could be accomplished in vitro by Michael addition reaction between GRGDSPC peptide and PEG-crosslinked decellularized valve. Part two:Construction of the bioreactor systemObjective:To construct a bioreactor system and evaluate its hydrodynamics and biocompatibility in vitro.Methods:Bioreactor system was composed of a peristaltic pulp, a flow chamber, silica gel tubes and a fluid reservoir. PEG-crosslinked decellularized valves conjugated with RGD peptide were implanted into the flow chamber to test the hydrodynamics and biocompatibility of the bioreactor in vitro.Results:Pulsating flow could be generated by the peristaltic pulp of the bioreactor system in order to effectively simulate the hemodynamics environment in vivo. Different levels of shear stress could be accomplished by precisely regulation of the flow rate. Conclusion:The bioreactor system was successfully constructed in vitro. Different levels of shear stress could be accomplished by precisely regulation of the flow rate.Part three:Construction and calcification mechanism of tissue engineering heart valves fabricated with composite scaffolds in a bioreactor system Chapter one:Isolation, Culture and identification of VICsObjective:To obtain human valvular interstitial cells (VICs) by isolation, culture and identification of VICs from human aortic valves.Methods:Valvular interstitial cells were isolated from human aortic valves by collagenase digestion. Valvular interstitial cells were cultivated in culture medium containing 10% fetal bovine serum. Immunofluorescence was used to identify the purities of cell population.Results:Collagenase digestion method could successfully isolate valvular interstitial cells from aortic valves in vitro. Spindle-shape primary cultures of VICs formed a monolayer and were radiated arranged. They formed overlapped layers as their numbers increased. Compacted cellular monolayer was formed during 5th-7th day. Valvular interstitial cells were positively stained for both a-SMA and vimentin.Conclusion:Collagenase digestion was a simple and feasible method by which valvular interstitial cells could be isolated from human aortic valves. VICs had a great capacity of proliferation which made it an ideal kind of seed cells.Chapter two:Construction of tissue engineering heart valves with composite scaffolds in the bioreactor systemObjective:To construct tissue engineering heart valves in the bioreactor by seeding valvular interstitial cells onto the PEG-crosslinked decellularized valves conjugated with RGD peptide.Methods:Valvular interstitial cells were seeded onto the PEG-crosslinked decellularized valves conjugated with RGD peptide. Then the valves were sutured into the bioreactor system to construct tissue engineering heart valves. Cytometry was used to detect cell adhesion at 2 hours,4 hours and 8 hours respectively after seeding. Both scanning electron microscopy and hematoxylin and eosin stain were performed at 10th day after seeding. DN A content test were also performed then.Results:Compared with PEG-crosslinked decellularized valves and decellularized valves, PEG-crosslinked decellularized valves conjugated with RGD peptide significantly promoted the adhesion of VICs. A confluent monolayer cells could be formed on the surface of the valves. DNA content test showed that the DNA content of PEG-crosslinked decellularized valves conjugated with RGD peptide was significantly higher. (25.98±2.45μg/valve vs.19.61Π1.94μg/valve vs.18.40±1.89μg/valve, P<0.05)Conclusion:Modification of PEG-crosslinked decellularized valves by GRGDSPC peptides could promote the adhesion of valvular interstitial cells seeded on valves in the bioreactor system and thereby enhanced the biological performance of the tissue engineering heart valves. Chapter three:Induction the calcification of tissue engineering heart valves by blocking the Notch-1 signal pathwayObjective:To induct the calcification of tissue engineering heart valves in the bioreactor system by blocking the Notch-1 signal pathway of valvular interstitial cells seeded on the PEG-crosslinked decellularized valves conjugated with RGD peptide.Methods:Cells from three to eight passages were treated with the y-secretase inhibitor DAPT for 3 weeks. The culture medium with DAPT was changed every 3 to 4 days. Negative control group was treated with normal culture medium whereas positive control group was treated with calcification culture medium. After the different treatment, VICs were seeded on the PEG-crosslinked decellularized valves conjugated with RGD peptide. Then the valves were sutured into the bioreactor for 10 days. Calcium deposit was stained by alizarin red staining. Real time reverse transcription PCR was used to detect the mRNA expression of a-SMA, osteocalcin and Cbfa1.Protein levels of osteocalcin, osteopontin and Cbfal was measured by western blot.Results:Calcification deposit was higher in experimental group than in negative control group. Real time PCR indicated that mRNA expression of a-SMA, osteocalcin and Cbfal was significantly higher than those of negative control group (P<0.05).Western blot suggested that protein levels of osteocalcin, osteopontin and Cbfal were much higher in experimental group than those of negative control group (P<0.05).Conclusion:Induction the calcification of tissue engineering heart valves could be successfully accomplished by blocking the Notch-1 signal pathway with theγ-secretase inhibitor DAPT.