Study On The Preparation And Biological Properties Of Smart Hydrogel-decellularized Scaffold

PartⅡThe preparation of poly (ethylene glycol) hydrogeland the research of its matrix metalloproteinase enzymatic release of vascular endothelial growth factorChapter one The preparation and identification of four branched propylene acylation poly (ethylene glycol) (4branched-PEG-DA)Objective:To prepare four branched propylene acylation poly (ethylene glycol) (4branched-PEG-DA) by grafted polymerization method, and identify the chemical structure and properties are in accordance with the experiment design and requirementsMethods:The linear polyethylene glycol (PEG) monomers (Molecular weight 20, OOODa) as raw materials, through the four-step synthesis method, four acryloyl functional groups were introduced at the monomer ends. By detections of infrared spectrometry (IR),1H-nuclear magnetic resonance (1H-NMR), high performance liquid chromatography(HPLC), Matrix-assisted laser desorption/ionization(MALDI) and gel filtration chromatography (GFC), the polymer products are in accordance with the experimental design to meet the follow-up requirements, in molecular structure, substitution of functional groups, final production rate, molecular weight and molecular weight uniformity. Results:By IR and 1HNMR detection, our 4branched-PEG-DA was confirmed the molecular structure matched the design and the substitution of functional groups was 96.3%; HPLC showed that the purity of the product was 98.4%; MALDI indicated the actual average molecular weight of the product matched the design; GFC results showed that the product Poly-dispersity value was 1.02, proved molecular weight was uniform.Conclusion:4branched-PEG-DA prepared by the grafted polymerization method is in accordance with the design of experiment and meets the requirements of follow-up, in aspects of molecular structure, functional groups substitution, final production rate, molecular weight and molecular weight uniformity.Chapter two The research of matrix metalloproteinase enzymatic release of vascular endothelial growth factor in poly (ethylene glycol) hydrogelObjective:To prepare poly (ethylene glycol) hydrogel released vascular endothelial growth factor by matrixmetalloproteinase (VEGF-MMP peptide-PEG gel),and observe the morphology and their enzymatic controlled release effect for the subsequent experiments.Methods:Using 9-fluorenyl methoxy carbonyl (Fmoc) solid phase peptide synthesis, we synthesized matrix metalloproteinase (MMP) substrate peptide containing thiol groups (-SH). After 4branched-PEG-DA and vascular endothelial growth factor 165 (VEGF-165) covalently bounded, according to Michael addition reaction, the combination of MMP substrate peptide containing-SH generated the poly (ethylene glycol) hydrogel loading VEGF (VEGF-MMP peptide-PEG gel) Observation of morphology and microstructure of VEGF-MMP peptide-PEG gel were fulfilled by camera and scanning electron microscopy. Use of enzyme-linked immunosorbent assay (ELISA), to reveal the relationship between matrix metalloproteinase 2 (MMP-2) and VEGF-MMP peptide-PEG gel.Results:At room temperature, the VEGF-MMP peptide-PEG gel was jelly-like. The scanning electron microscopy showed the microstructure of the hydrogel was reticular. The VEGF-MMP peptide-PEG gel sensitive to MMP (VEGF-MMP (W) X-PEG gel) was 0.251±0.101um in surface pore average diameter and 10.1±1.21% of porosity. ELISA showed that VEGF-165 encapsulation efficiency of VEGF-MMP peptide-PEG gel was 84.33±1.18%. The collapsed phase and ELISA results showed that under different concentrations of MMP-2, VEGF-MMP (W) X-PEG gel possessed a stable VEGF release rates.Conclusion:VEGF-MMP (W) X-PEG gel prepared by Michael addition reaction possessed a stable enzymatic controlled release with MMP-2, which to meet the further experiment requirements.PartⅡThe preparation of VEGF controlled release hydrogel-decellularized scaffold and the research of its biocompatibility in vitroChapter one The preparation and identification of VEGF controlled release hydrogel-decellularized scaffoldObjective:To prepare VEGF controlled release hydrogel- decellularized scaffold, and identify the structure by the morphology and immunofluorescence staining.Methods:Decellularized valve modified with Sulfo-NHS-SS-Biotin was identified by immunofluorescence staining. According to avidin-biotin combination, biotinylated VEGF controlled release hydrogel (VEGF-bio-MMP (W) X-PEG gel) and decellularized scaffold were compound, and identified by HE staining and scanning electron microscopy.Results:The decellularized scaffold modified with biotin was stained by avidin-Cy3, while fluorescence microscopy showed the valve matrix was red. Morphological observation showed that the hydrogel-decellularized scaffold possessed decellularized matrix structure andreticular microstructure of the hydrogel.Conclusion:VEGF controlled release hydrogel-decellularized scaffold was successfully prepared in accordance with the design of experiment.Chapter two The research of biocompatibility of VEGF controlled release hydrogel-decellularized scaffold in vitroObjective:To study the biocompatibility of VEGF controlled release hydrogel-decellularized scaffold in vitro, according to GB/T1688 national standards.Methods:Refer to GB/T1688 national standards, the hybrid scaffold, for blood interaction experiments, was studied on the influence to platelet activation, clotting time, hemolysis; the hybrid scaffold and its extract, for cytotoxic experiment, were studied on the influence to cell proliferation and apoptosis.Results:The scanning electron microscopy and flow cytometry testing, confirmed that the hybrid scaffold can reduce the decellularized influence to platelet adhesion and activation; PT/APTT/INR test showed that the hybrid scaffold had no significant effect on the clotting time; indirect and direct hemolytic experiments confirmed that the hybrid scaffold hemolysis rate was lower than the the GB/T16886 national standards; by cell proliferation and apoptosis detection, the hybrid scaffold and its extract were proved that had no significant effect on MRC-5 human embryonic lung cells.Conclusion:Refer to GB/T1688 national standards, VEGF controlled release hydrogel-decellularized scaffold possessed fine biocompatibility.PARTⅢThe preparation of Anti CD34-VEGF controlled release hydrogel-decellularized scaffold and the research of its biological properties in vivoChapter one The preparation and identification of Anti CD34-VEGF controlled release hydrogel-decellularized scaffoldObjective:To prepare Anti CD34-VEGF controlled release hydrogel-decellularized scaffold (smart hydrogel-decellularized scaffold), and identify the structure by the immunofluorescence staining.Methods:decellularized valve was modified with Sulfo-NHS-SS- Biotin. According to avidin-biotin combination, biotinylated VEGF controlled release hydrogel (VEGF-bio-MMP (W) X-PEG gel) and decellularized scaffold were compound. In the same way, the biotinylated Anti CD34 antibody and hydrogel-decellularized scaffold were compound, and identified by immunofluorescence staining.Results:Under fluorescence microscopy, hydrogel-decellularized scaffold combined with Anti CD34 antibody showed that the valve matrix was green while stained by anti-IgG-FITC.Conclusion:The smart hydrogel-decellularized scaffold was successfully prepared in accordance with the design of experiment.Chapter two The research of biological properties of smart hydrogel-decellularized scaffold in vivoObjective:To establish a rabbit carotid artery valve pipeline transplantation model, and evaluate the biological properties of smart hydrogel-decellularized scaffold in vivo.Methods:Using a modified "Cuff" method, to establish rabbit carotid artery valve pipeline transplantation model. To determine the animal model is successful by the Color Doppler Ultrasound detecting the pipes are open or not in postoperative 24 hours and 7 days. After 24 hours, take out the pipes to evaluate the adhesion to CD34+ cells by histomorphology analysis. After 7 days, take out the pipes to assess the degree of endothelialization by histomorphology and eNOS mRNA quantitative analysis.Results:The rabbit carotid artery valve pipeline transplantationmodel were successful, by the ultrasound results showing that the transplanted pipes were open. After 24 hours, the corresponding test results showed that our scaffold had an obvious adhesion to the CD34+ cells. After 7 days, the corresponding test results showed that the degree of endothelialization of our scaffold were superior to the controls.Conclusion:smart hydrogel-decellularized scaffold possessed excellent biological properties.