A Novel Approach For Preparing Tissue Engineering Decellularized Valve Scaffold With Poly(Ethylene Glycol)-poly(?-caprolactone)

Objective:The objective of this study was to explore the feasibility of preparing a decellularized valve scaffold with Methoxy poly(ethylene glycol)-poly(?-caprolactone)(MPEG-PCL).MPEG-PCL was used as a decellularization agent to explore its decellularization effect on porcine aortic valve(PAV).We evaluated its decellularization activity versus two commonly used agents(Triton X-100 and sodium deoxycholate(SD))in terms of histological morphology,content of valve-related components,biocompatibility and mechanical properties.Metholds:Ring-opening polymerization was applied to synthesize MPEG-PCL,which was subsequently characterized with fourier transform infrared spectroscopy(FTIR)and proton nuclear magnetic resonance(1H-NMR).Fresh PAVs were subjected to decellularization treatment using MPEG-PCL at different concentrations.The optimal decellularization concentration was determined based on histological morphology.The PAVs were treated with MPEG-PCL at the empirically determined optimal concentration,Triton X-100 or sodium deoxycholate(A: MPEG-PCL,B: Triton X-100,C: SD,D: Native valve).The decellularization effects of these three agents were assessed using hematoxylin-eosin(H&E)staining,Masson's trichrome stain and scanning electronic microscopy(SEM)from the perspective of histological morphology.The decellularization effects of the different groups were evaluated by measuring the contents of the valve-related components.Valves in different groups were tested for biocompatibility and mechanical properties;cytotoxicity testing determined the toxicity of different decellularized valves.Results:Fourier transform infrared spectroscopy(FTIR)revealed that the synthesized copolymer exhibited stretching vibration peaks for the MPEG block and PCL block(3443.05 cm-1,1728.66 cm-1,1109.81 cm-1);proton nuclear magnetic resonance(1H-NMR)spectrum showed that the synthesized copolymer exhibited proton peaks for the MPEG block and PCL block(d 1.38,1.65,2.31,4.06,3.64 ppm).The molecular weight of the copolymer was about 4000 D.Light microscopy revealed that 1% MPEG-PCL fully decellularized the valve cells,and the valve fiber structure remained intact.Versus untreated normal valves,the amount of residual DNA in the decellularization groups treated with 1% MPEG-PCL,Triton X-100 and SD were significantly reduced(A: 5.00 ± 0.11 ng/mg vs.B: 21.26 ± 0.84 ng/mg vs.C: 16.31 ±0.83 ng/mg vs.D: 336.94 ± 9.33 ng/mg)(P<0.05).The elastin contents in the valves of all decellularization groups were also significantly reduced(A: 202.43 ± 22.70ng/mg vs.B: 226.82 ± 25.21 ng/mg vs.C: 209.43 ± 17.54 ng/mg vs.D: 326.39 ±43.74 ng/mg).However,the water content and collagen content in none of the decellularization groups were significantly different.In all decellularization groups,some degree of platelet adhesion was observed,but the hemolysis rates(HR)of all groups were significantly smaller versus the normal group.Cytotoxicity testing showed that MPEG-PCL was non-cytotoxic,and the cell proliferation rates of the MPEG-PCL group were significantly higher than that of Triton X-100 and SD groups at both 24 h and 48 h(24 h / 48 h: A: 97.16 ± 1.66% / 98.30 ± 1.28% vs.B: 87.17 ±0.41% / 86.79 ± 3.10% vs.C: 77.22 ± 1.96% / 72.90 ± 2.75% vs.D: 97.70 ± 1.41% /97.72 ± 0.72%).For every decellularization group,the relevant mechanical properties of the valve scaffolds along circumferential and radial directions were not significantly different from that of native normal valves.Conclusions:MPEG-PCL(1%)can be used to prepare decellularized porcine aortic valves.Its decellularization effect is superior to that of Triton X-100 and SD at the same concentration.MPEG-PCL is non-cytotoxic and can completely remove cells from the valve while maintaining an intact extracellular matrix(ECM)ultrastructure.Thus,MPEG-PCL is a new agent for preparing decellularized biological scaffolds for tissue engineered heart valve.