聚乙二醇去细胞瓣复合支架联合人主动脉瓣膜间质细胞体外构建组织工程心脏瓣膜

目的:运用聚乙二醇(PEG)交联猪去细胞瓣,共价接枝GRGDSPC多肽(甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸-脯氨酸-半胱氨酸),并联合人主动脉瓣膜间质细胞(HAVICs)体外构建组织工程心脏瓣膜。方法:通过迈克尔加成反应,将去细胞瓣支架上引入的巯基与枝化状PEG末端丙烯酰基共价结合,使PEG交联去细胞瓣,并行生物力学检测。再利用PEG-去细胞瓣支架上未饱和的丙烯酰基与GRGDSPC多肽末端半胱氨酸的巯基发生迈克尔加成反应,对PEG-去细胞瓣支架行GRGDSPC多肽共价修饰,免疫荧光测定GRGDSPC多肽修饰效果。在RGD-PEG-去细胞瓣复合支架上种植HAVICs,缝制于生物反应器内,体外构建心脏瓣膜。接种2、4、8h后,通过细胞计数观察细胞粘附情况。体外培养10d后行形态学观察和DNA含量测定。结果:力学测试结果显示,PEG-去细胞瓣支架的最大抗张强度较单纯去细胞瓣支架明显提高(7.53±0.32)Mpa∶(5.65±0.28)Mpa],差异有统计学意义(P<0.05),与天然主动脉瓣的差异无统计学意义(P>0.05)。免疫荧光检测显示,GRGDSPC多肽可有效地与PEG-去细胞瓣共价接枝。不同时段细胞粘附计数及形态学观察提示,RGD-PEG-去细胞瓣复合支架明显促进HAVICs的粘附,并可在瓣膜表面形成连续单细胞层。DNA含量测定提示,RGD-PEG-去细胞瓣复合支架DNA含量明显增高(P<0.05)。结论:PEG交联去细胞瓣可明显改善瓣膜支架力学性能,GRGDSPC多肽接枝的PEG-去细胞瓣复合支架,可促进种子细胞的粘附,改善瓣膜支架生物学性能。

Fabrication of composite heart valves with decellularized valves modified by RGD peptide and human aortic valve interstitial cells

Objective:Decellularized valves were PEGylated and covalently modified by GRGDSPC peptides.Then human aortic valve interstitial cells(HAVICs) were seeded onto the valves in order to fabricate composite heart valves. Method:PEGylation of decellularized valves were completed by using a Michael-type addition reaction,which means that propylene acyl of branched polyethylene glycol was reacted with the thiol groups of decellularized valves.Biomechanical properties were compared between the decellularized valves,PEGylated decellularized valves and native aortic valves.PEGylated decellularized valves were covalently modified by GRGDSPC peptides through Michael-type addition reaction,in other words,the unsaturated propylene acyls of polyethylene glycol were reacted with thiol groups on cysteine residues of GRGDSPC peptides.The modified effect of GRGDSPC peptides was determined by immune fluorescence.HAVICs were seeded on PEGylated decellularized valves modified by GRGDSPC peptides.Then the valves were sutured into the bioreactor.Cell adhesion was tested respectively after 2,4 and 8 hours.After 10 days of culture,hematoxylin-eosin,scanning electron microscope and DNA content test were performed. Result:Mechanical tests showed: the maximal tensile strength of PEGylated decellularized valves was significantly higher than that of decellularized valves (7.53±0.32)Mpa vs(5.65±0.28)Mpa],and similar to that of native aortic valves(P>0.05).Immune fluorescence showed that: PEGylated decellularized valves could be effectively modified by GRGDSPC peptides through covalent bonding.Compared with PEGylated decellularized valves and decellularized valves,GRGDSPC peptides modified PEGylated decellularized valves significantly promoted the adhesion of HAVICs,and confluent monolayer cells could be formed on the surface of the valves.DNA content test showed that the DNA content of GRGDSPC peptides modified PEGy-lated decellularized valves was significantly higher(P<0.05). Conclusion:PEGylation can obviously improve the decellularized valves mechanical properties.GRGDSPC peptides modified PEGylated decellularized valves can promote the adhesion of HAVICs,and improve the valve biological properties.