Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Into Insulin-producing Cells Via Biological Scaffolds

Objectives:As an in vitro cell culture system,organoid has a spatial structure similar to that of its corresponding organ,which can reproduce some functions of the corresponding organ.In recent years,studies on organoid tissue engineering have provided engineering basis and new ideas for the treatment of type 1 diabetes.As a carrier of cell growth and differentiation,biomaterial scaffolds can simulate extracellular matrix and provide space for cell growth.Good biocompatibility is the property of suitable tissue engineering scaffold materials.Electrostatic spinning technology has micron or nanometer spinning structure,which has great potential as tissue engineering scaffolds.The modified material method of polydopamine can form polydopamine layer on a variety of polymer,metal and metal oxide materials,to adsorb the peptide molecules,and maintain the active conformation of the loaded protein.Bone marrow mesenchymal stem cells,under the stimulation of appropriate growth factors and induced environment,can differentiate and present the functional characteristics of insulin-secreting cells.Exendin-4 is a long-acting glucagon-like peptide receptor agonist that promotes differentiation of mesenchymal stem cells into insulin-secreting cells.In this study,electrospinning scaffolds of poly(L-lactic-acid)/gelatin was coated with Exendin-4 via polydopamine(p DA),and used for differentiation of BM-MSCs into insulin-producing cells(IPCs)by a two-stage protocol.The characterizations of coated PLLA/G and functions of the inducted IPCs were evaluated.Gene and protein expression of islet-associated cell differentiation markers was detected to demonstrate the presence of IPCs.The aim of this study is to provide the experimental basis for tissue engineering system,and provide a new idea of diabetes treatment.Research methods:In this study,the electrostatic spinning technology was used to prepare the gelatin polylactic acid electrostatic spinning fiber membrane scaffold(PLLA/G membrane).The dopamine-modified gelatin polylactic acid electrostatic spinning(PLLA/ g-p DA membrane)was prepared by the surface polydopamine(p DA)modified adhesion method,and then the gelatin polylactic acid electrostatic spinning fiber membrane(PLLA/ G-p DA-Exendin-4 membrane)loaded with Exendin-4 was prepared.The chemical composition of PLLA/G-p DA-Exendin-4 membrane was analyzed by FTIR.The physical and chemical properties of three kinds of scaffold materials(PLLA/G membrane,PLLA/ G-p DA membrane,PLLA/G-p DA-Exendin-4 membrane)were evaluated by SEM and contact Angle measurement.In order to evaluate the cell proliferation and compatibility of Exendin-4 loaded scaffolds,and to further induce differentiation,primary bone marrow mesenchymal stem cells were isolated and cultured in this study,and their phenotypes were detected by flow cytometry to identify their purity.In vitro,bone marrow mesenchymal stem cells were inoculated with Exendin-4 loaded electrostatic spinning membrane scaffold,the microscopic morphology of cells on the surface of the material was observed by scanning electron microscopy,the cell proliferation level was detected by MTT method,and the biocompatibility of each spinning membrane was analyzed.The results showed that Exendin-4 loaded electrostatic spinning membrane had good biocompatibility and cell proliferation.In this study,bone marrow mesenchymal stem cells(BMSCs)were inoculated with Exendin-4 gelatin polylactic acid(PLLA)electrospinning membrane,and the widely used three-step method was selected at home and abroad to induce the insulin-secreting differentiation of BMSCs inoculated with composite membrane materials.By dithiazone staining,RT-q PCR,immunofluorescence and ELISA,the expressions of insulin,c-peptide,PDX-1,pax-4,GLUT2 and other key insulin-related genes and proteins in the obtained cells were detected.Results:In this experiment,The FTIR spectra of the scaffolds were performed to determine the p DA coating and Exendin-4 incorporation.All of the variations of the FTIR spectra further conformed that p DA was fixed on the surfaces of PLLA/G-p DA,and Exendin-4 was loaded on PLLA/G-p DA-PLLA/G-p DA-Exendin-4 scaffolds.The result of adsorption of PLLA/G-p DA membrane and PLLA/G membrane of BSA showed that,the accumulated adsorption of PLLA/G-p DA membrane was significantly increased at the reaction time of 120 minutes,so almost all the Exendin-4 peptides in each hole could be loaded on PLLA/G-p DA-Exendin-4 membrane.The results of contact Angle measurement showed that the hydrophilic ability of PLLA/G-p DA-Exendin-4 membrane was stronger than that of PLLA/ g-p DA membrane,while the hydrophilic ability of PLLA/G-p DA membrane was weaker.It can be speculated that PLLA/G-p DA-Exendin-4 structure is more hydrophilic than pure PLLA/G membrane and more conducive to cell surface adhesion.Bone marrow mesenchymal stem cells were cultured with Exendin-4 gelatin polylactic acid electrospinning composite membrane.The results of SEM and fluorescence staining showed that the bone marrow mesenchymal stem cells grew flat and dispersed on PLLA/G-p DA-Exendin-4 membrane,PLLA/G-p DA membrane and PLLA/G membrane.The cell growth morphology of the three types of spinning membranes were basically the same,and the PLLA/G-p DA-Exendin-4 membrane on the surface could provide a good scaffold environment for the bone marrow mesenchymal stem cells to adhere to.MTT colorimetric method was used to determine the cell proliferation ability of bone marrow mesenchymal stem cells in PLLA/G-p DA-Exendin-4 membrane,PLLA/G-p DA membrane and PLLA/G membrane.The results showed that both PLLA/G-p DA-Exendin-4 membrane and PLLA/G membrane could support adhesion and proliferation bone marrow mesenchymal stem cell.The results indicated that the scaffolds had good cell compatibility.Compared with PLLA/G film determined by MTT results showed PLLA/G-p DA-Exendin-4 membrane to a certain extent can promote bone marrow mesenchymal stem cell proliferation.On the contrary,MTT results showed that the cell proliferation level of PLLA/G-p DA scaffold cells was lower than that of PLLA/G membrane.Above results are consistent with the results of contact Angle measurement.The qualitative staining results of dithiazone showed that insulin mass expressed by induced cells were stained reddish-brown by dithiazone,which indirectly showed that the induced cells could produce insulin particles which were rich in zinc ions.It can be speculated that BMSCs differentiated cells induced by three-step method in vitro could have the function of insulin expression,so this method was applied to the induction of bone marrow mesenchymal stem cell differentiation with Exendin-4-loaded electrostatic spinning membrane.Real-time quantitative PCR results showed that,compared with the conventional stent-free induced Insulin-producing cells,the electrospinning membrane loaded with Exendin-4 had a certain promoting effect on the induced differentiation of BMSCs cells secreting insulin,which may be related to the increased expression of PDX-1,PAX-4,glut2.Stenting loaded with Exendin-4 may continue to activate the expression of PDX-1 gene in BMSCs cells,thus promoting the expression of important genes of insulin,PDX-1,PAX-4,GLUT2 and other cells,thus differentiating toward islet like cells.The results of immunofluorescence detection and ELISA further verified that the relative fluorescence intensity of insulin,C-peptide and PDX-1 in Insulin-producing cells induced by PLLA/G-p DA-Exendin-4 membrane was significantly higher than that induced by insulin-producing cells without scaffold and third passage BMSCs without induced rat BMSCs cells(all P < 0.05).According to the results of the qualitative detection and semi-quantitative determination compared to conventional non-scaffolds induced insulin-producing cells,in could be speculated that PLLA/G-p DA-Exendin-4 membrane was preferable carrier for induced IPCs derived from rat BMSCs.Conclusion:(1)Exendin-4 could be loaded on PLLA/G-p DA-Exendin-4 membrane with high adsorption rate via p DA modification method.(2)PLLA/G-p DA-Exendin-4 membrane structure was more hydrophilic than PLLA/G membrane,which was more conducive to cell surface adhesion.(3)The results of scanning electron microscopy and fluorescence staining showed that bone marrow mesenchymal stem cells were able to adhere to and spread on PLLA/G-p DA-Exendin-4 membrane.(4)MTT results showed that PLLA/G-p DA-Exendin-4 membrane could promote the proliferation of bone marrow mesenchymal stem cells to a certain extent.(5)Compared with conventional non-scaffold induced insulin-producing cells,PLLA/G-p DA-Exendin-4 membrane could improve the level of insulin expression of insulin-producing cells differentiated from BMSCs.The results of this study demonstrated that PLLA/G-p DA-Exendin-4 nanofibrous scaffolds was preferable carrier induced IPCs derived from rat BMSCs.