Application Of PDA-PLGA Cell Scaffold In The Treatment Of Diabetic Islet Cell Transplantation In Skeletal Muscle

Diabetes is a complex disorder of glucose and lipid metabolism,and its incidence is increasing.Progressive beta cell loss and damage are the central links in the development of diabetes.The current anti-diabetic drugs are not targeted at the islet ?-cell regeneration,they can only delay the progress of the disease and cannot fundamentally cure the disease.Although pancreatic transplantation can achieve the purpose of treating diabetes,in addition to the lack of donors,the problem of immune rejection after transplantation has also limited its widespread application.With the development of tissue engineering technology,islet and islet ? cell transplantation provide new strategies for the treatment of diabetes.In the process of islet cell transplantation,the choice of transplantation site and transplantation vector is the key to successful transplantation.Clinically,90% of islet transplantation is performed in the liver through the portal vein.However,many islet cells are lost during the transplantation process and cannot function for a long time after transplantation.At present,intramuscular transplantation has shown good clinical application prospects.Skeletal muscle can meet the microenvironment requirements of islet cells and facilitate the monitoring of insulin-secreting cells after transplantation.However,transplantation of clusters of insulin-secreting cells can lead to local hypoxia in the clusters and affect cell viability.Therefore,the preparation of degradable cell scaffolds that can provide a metabolic environment and proliferation space for cell growth has become the key to islet cell transplantation.The cell scaffold has an extremely important role as a cell transplant carrier,and it can provide a place for seed cells to grow and exchange materials.Electrospinning is currently the most commonly used method for stent preparation.Its operation is simple.The prepared stent has a very high specific surface area and flexible surface properties.The stent structure can provide sufficient space for cell adhesion,reproduction,and differentiation.In terms of scaffold materials,poly(lactic-co-glycolic acid)(PLGA)is the most widely used biodegradable polymer material in biomedical fields,and has been widely used in drug delivery devices and tissue engineering scaffolds.In order to make the scaffolds have good surface hydrophilicity and histocompatibility,polydopamine(PDA)coating is usually used to functionally modify the surface of the scaffolds in order to construct a cell transplantation carrier with better overall performance.However,no comprehensive research has been conducted on the safety evaluation of PDA-coated PLGA(PDA-PLGA)scaffolds and its application to in vivo transplantation of islet cells.In this project,the PLGA cell scaffold will be prepared by electrospinning method.Based on this,the surface of the scaffold will be modified by polydopamine coating treatment,and characterize it.Through co-cultivation of cells and scaffolds,PDA-PLGA cell scaffolds were evaluated for in vitro safety,and transplanted under the abdominal skin,abdominal wall and skeletal muscles of rats,respectively,to evaluate the in vivo safety of the scaffolds.Finally,the islet cells were inoculated on the PDA-PLGA cell scaffold,and the transplantation treatment of islet cells was performed on the skeletal muscle of the diabetic rat to explore its therapeutic effect on diabetes.The following is the research content of this thesis.1.Preparation of PDA-PLGA nanofiber scaffold.This experiment uses PLGA with different molecular weights to prepare PDA-PLGA nanofiber scaffolds through electrospinning technology and dopamine coating treatment.Scanning electron microscope(SEM)was used to compare the fiber morphology and structure,to test the mechanical properties,water contact angle and degradation performance of cell scaffolds with different molecular weights.The nanofiber scaffolds suitable for in vivo transplantation were screened.The results showed that compared with conventional PLGA scaffolds After coating with polydopamine,the hydrophilicity and tensile properties of fiber scaffolds are significantly improved,and the degradation time is extended for a certain period of time.Compared with low-molecular-weight fiber scaffolds,high-molecular-weightPDA-PLGA fiber scaffolds have more Good mechanical properties and longer degradation time.The results show that high molecular weight PDA-PLGA fiber scaffolds are more suitable as carriers for cell transplantation.2.Safety evaluation of PDA-PLGA cell scaffolds at different transplant sites.This experiment is evaluated at both the cell level and the animal level.At the cell level,umbilical vein endothelial cells(HUVEC),macrophages(RAW264.7),etc.are co-cultured with the sterilized fiber scaffold to detect the cells on the scaffold.At the animal level,fibrous scaffolds were transplanted into the rat's abdominal subcutaneous,abdominal cavity wall,and skeletal muscle at three different positions.The serum biochemical indicators of the rat,the lactic acid level of the stent degradation product,and HE and Masson staining of each organ and tissue was performed to evaluate the safety of the PDA-PLGA cell scaffold.The results show that the PDA-PLGA cell scaffold can promote cell growth,and the scaffold transplantation under the abdominal subcutaneous,abdominal wall and skeletal muscle sites in rats has shown good biological safety,indicating that PDA-PLGA cell scaffolds are used in tissue engineering.The field has good application prospects.At the same time,stent transplantation in skeletal muscle has less effect on serum biochemical indicators of rats and is conducive to angiogenesis,indicating that skeletal muscle has certain development potential as a transplant site.3.The application of PDA-PLGA scaffold in the treatment of islet cell transplantation.First,rat pancreatic islet ? cells(RINm5f)were co-cultured with sterilized PDA-PLGA cell scaffolds,and the secretory function of islet cells was detected by a glucose-stimulated insulin secretion test(GSIS).Supernatants of insulin-containing cells in the co-culture system were administered to skeletal muscle cells(C2C12)for co-culture to detect changes in sugar consumption and sugar uptake of skeletal muscle cells.At the animal level,first a rat model of type 1 diabetes was established by injection of streptozotocin(STZ),RINm5 f cells were seeded on a PDA-PLGA cell scaffold,and transplanted in the skeletal muscle of diabetic rats.Fasting blood glucose changes,and insulin immunofluorescence of tissue sections at different timepoints,to explore the therapeutic effect of islet cell and scaffold complex on diabetes.The results showed that the PDA-PLGA cell scaffold had no effect on the secretion function of islet cells.The PDA-PLGA cell scaffold carrying RINm5 f cells transplanted in the skeletal muscle of diabetic rats could reduce blood glucose in rats and had a certain therapeutic effect on diabetes,but 14 After a few days,the treatment effect began to weaken.At 28 days,insulin fluorescence weakened significantly,and the hypoglycemic effect basically disappeared.In summary,this topic clarified that 1.PDA coating can effectively improve the hydrophilicity and mechanical properties of PLGA electrospinning stents,slow down the degradation rate,and large molecular weight PLGA is more suitable for use in vivo than small molecular weight.Transplantation;2.PDA coating can promote cell adhesion and proliferation on the surface of the stent.Stent transplantation under the abdominal skin,abdominal wall and skeletal muscle has no effect on the growth of rats.After 14 days,there is basically no significant difference in the examination indicators of each group,and the effect of skeletal muscle transplantation on serum biochemical indicators in the early stage of transplantation Smaller,PDA-PLGA cell scaffold has better biological safety;3.PDA-PLGA cell scaffold has no effect on the survival and secretion function of islet cells,and the islet cell-scaffold complex has a significant effect on rat skeletal muscle transplantation.Diabetes has a significant therapeutic effect,and skeletal muscle may be a good transplant site for diabetes treatment through islet cell transplantation.