The Preparation And Applied Basic Researches On Cell/Scaffold Three-dimensional Composition For Accelerating Diabetic Ulcer Healing

Objective:In this study,we selected the natural macromolecule hydrogel material as three-dimensional cell scaffold to load transgenic cells which were Chinese hamster ovary cells(CHO) that could express platelet-derived growth factor (PDGF-BB) highly,in order to build cell/scaffold three-dimensional composition. And applied it to skin ulcers of diabetic rats model to evaluate the accelerating healing function.Methods:Used the transgenic techniques,transfected the PDGF-BB gene into CHO by the specific vector and used zeocin to gain a cell strain which expressing interest protein steadily,identified the interest protein by fluorescence microscope and immunocytochemistry,detected the protein quantity by ELISA,.Choosed natural macromolecule material chitosan and modified it by NAC to synthesize disulfide bond chitosan which sulfydryl contents is 321.4μmol/g,adopted 10 g/L concentrations to dissolve the compound into deionized water generating hydrogel,tested it's cytotoxicity by typan blue and MTT;used it loading transgenic CHO as 3D cell scaffolds to construct cell/scaffold composition.Observated the pore size and the cells' distribution of the water generating hydrogel in the cell/material composites by the general morphology,microscopy and scanning electron microscope. Evaluated the PDGF-BB expression of this composition by ELISA.Through systemic allergic experiments,the skin sensitization test,intradermal test and the intravenous injection of sub-acute toxicological experiments to evaluate the animal biocompatibility of the hydrogel material.Injected streptozoein(STZ) through caudal vein to prepare diabetic rat models and used hole puncher to prepare diabetic ulcer models,then took the cell/scaffold composition on the surface of the diabetic animal skin ulcer to evaluate the healing function through calculating the ulcer healing rate and watching the materials HE slides from the rats' ulcer wounds, otherwise watched the immunohistochemistry of PDGF on the ulcer wounds. Evaluated the expression of PDGF in the granulation tissue by immunohistochemistry.Results:ELISA results showed that the transgenic CHO cells can high-throughput expression of rhPDGF-BB,and the chitosan hydrogel composition without any negative impacts,as well as trypan blue exclusion method and MTT method showed the hydrogel material had good biocompatibility.After it loading transgenic CHO as 3D cell scaffolds to construct cell/scaffold composition,scanning electron microscopy showed that the hydrogel became network structure which had relatively uniform pore size and good cell adhesion.The Wistar rats all becomed the diabetic models,the cell/scaffold composition could accerlate the model animal ulcer healing significantly(P＜0.05). Immunohistochemistry results showed that each group PDGF expression at different times no statistical significant difference(P＞0.05).Conclusion:Succeeded constructed the enhanced green fluorescent protein eucaryotic expression vector carrying rhPDGF-BB,transfected eukaryocyte and gained a cell strain which expressing interest protein steadily.The natural macromolecule chitosan hydrogel scaffolds have well biocompatibility,apertures well-distributed,be good to cells growth.The hydrogel composed 3D cell scaffolds loading transgenic cell constructed cell/ scaffold composition,this atificial composition accelerated the diabetic animal skin ulcer healing clearly,to establish foundation for therapy of the diabetic foot skin ulcer.