Application Of Decellularized Valves Modified By Matrix Metalloproteinase-2 Degradable Poly-ethylene Glycol Hydrogel In Tissue Engineering Heart Valve

Objective: To synthesize and test two substrate peptides of matrix metalloproteinase (MMP).Methods : The MMP(W)X peptide with the sequence of Ac-CRD-GPQG↓IWGQ-DRC-NH2 was highly sensitive to MMP-2. The MMP(DF) X peptide with the sequence of Ac-CRD-GDQGIAGF-DRC-NH2。Ac-CRD-GPQG↓IWGQ-DRC-NH2 was insensitive to MMP-2. They were synthesized with the method of Fmoc solid phase and identified by the method of mass spectrum (MS) and high performance liquid chromatography (HPLC).Results: Analyzed by HPLC and MS,the purity of two peptides was at 99.06% and 99.29% respectively. And the molecular weights of them were at 1632.79/mol and 1553.69/mol respectively.Conclusion: The substrate peptides of MMP could be synthesized in vitro for tissue engineering. Objective: To construct a biodegradable compound hydrogel by four-armed polyethylene glycols (PEG) with the molecular weight of 20000 and matrix metalloproteinases (MMP) -2 substrate peptide.Methods: The experiments were randomly designed into three groups: MMP-2 sensitive compound PEG hydrogel, MMP-2 insensitive compound PEG hydrogel and simple PEG hydrogel. And the compound hydrogels were prepared by Michael addition reaction. Th observation of degradation in different mediums and test of the cell toxicity were done. Transforming growth factor (TGF) -β1 was embedded into hydrogel during production process and its release rate was calculated.Results: Compound hydrogel had excellent stability. The degradation rates of group A and B in phosphate buffer solution for four weeks were 68.36% and 64.23% respectively. There were no significantly difference between the two group and the control group (p>0.05). By adding MMP-2, the enzymatic degradation rate of group A reached 83.75%, but group B and C did not change significantly. No cell toxicity of compound hydrogel was detected by MTT test. TGF-β1 accumulative release rate for 7 days was 59.63%, 53.18% and 44.36% respectively(p>0.05). But by the addition of MMP-2, TGF-β1 release rate of group A became obviously faster.Conclusions: The MMP-2 enzymatically degradable PEG hydrogel had structure stability and no cell toxicity, and could effectively control the release of TGF-β1. It is a potential material to fabricate a new hybrid scaffold for tissue engineering heart valves. Objective: To construct a hybrid scaffold with matrix metalloproteinases-2 (MMP-2) enzymatically degradable PEG hydrogel and decellularized valves. And to measure the biological characteristics of the scaffold in tissue engineering heart valve.Methods: Decellularized valves were prepared by trypsinase and TritonX-100. Decellularized valves scaffolds were modified by MMP-2 sensitive PEG hydrogel with the help of a coupling reagent, 1-(3-Dimethylaminopropyl)-3-ethyl carbodimide (EDC) during the process of gelling. Hematoxylin-eosin staining and scanning electronic microscopy were performed. And the hydroxyproline content, cell DNA assay and biological mechanic properties of leaflets were measured.Results: Compared with the simple decelluarized valve scaffolds, the hybrid scaffolds were more compact in structures of extracellular matrix and perferable in the biological mechanics characteristics. And no significant changes were between them in DNA assay and hydroxyproline content.Conclusions: This kind of hybrid scaffolds had proper manifestation in the biological mechanic characteristics compared to the simple decelluarized valves. It is beneficial to the study of tissue engineering.