A Novel Approach Via Surface Modification Of Degradable Polymers With Adhesive DOPA-IGF-1 For Neural Tissue Engineering

According to the national center for spinal cord injury statistics(NSCISC),The annual incidence of SCI has been reported to be 40 cases per million worldwide each year,mostly due to traffic accidents,followed by violent injuries,falls and sports injuries.The microenvironment at a spinal cord injury site is complicated,and more than one process needs to be regulated in order for neurons and axonal regrowth to occur.Not only should hindering factors,such as gliosis or inflammation,be minimized,but the controlled release of growth factors should be sustained.In order to solve this problem,neural tissue engineering has attracted more and more attention in recent years.PLGA is a kind of the most promising degradable materials in tissue engineering because the degradation rate of the copolymer and the physical and chemical properties can be adjusted.However,PLGA has little advantages in hydrophilicity and histocompatibility.Thus,how to improve the bioactivity of PLGA is a difficulty to be solved.With growth factors and cells,neural scaffolds implanted into the injured spinal cord can promote nerve regeneration and functional recovery.In the preliminary work,we synthesized DOPA-IGF-1.When pH was regulated to 8.5,the oxidation reaction of catechol groups of DOPA was considered to resulted in chemical cross-linking;it demonstrated that this DOPA-IGF-1 would lead to the formation of a protein layer on the surface of titanium,enhanced the NIH3T3 adhesion,and increased the cell growth activity.IGF-1 is a 70 amino acids polypeptide.IGF-1 can promote the neurons regeneration,the growth of synapses,cells proliferation and inhibit the apoptosis of neurons.IGF-1 can also promote the proliferation、migration、secretion and differentiation of mesenchymal stem cells.Therefore,DOPA-IGF-1 was modified on PLGA membrane surface to improve the biological activity of PLGA surface for providing a functional biological material for spinal cord injury repairation.Object:1: DOPA-IGF-1 was modified on PLGA surface to prepare DOPA-IGF-1 @PLGA membrane,which was compared with cIGF-1 to observe the biological activity of hUCMSCs cultured on DOPA-IGF-1@PLGA membrane.2.DOPA-IGF-1@PLGA+hUCMSCs membrane was prepared by conbining hUCMSCs and DOPA-IGF-1@PLGA,and its role was observed in hemisection spinal cord injury of SD rats.Method:1: SDS-PAGE and western blot were used to verify the target protein YKYKYIGF-1(Y-IGF-1).For obtaining the DOPA-IGF-1,the tyrosinase hydroxylation turns Y into DOPA.The adsorption capacity of cIGF-1、Y-IGF-1、DOPA-IGF-1 on PLGA films was observed by ELISA.AFM,XPS and Contact Angle Detection were used to observe the changes of DOPA-IGF-1@PLGA films surface physical and chemical properties.2: The biological effect of hUCMSCs cultured on cIGF-1@PLGA 、Y-IGF-1@PLGA and DOPA-IGF-1@PLGA films was observed.HUCMSCs proliferation was observed by CCK-8 and the optimal concentration of DOPA-IGF-1 was determined.Cells adhesion and growth were observed by phalloidin staining.qRT-PCR and western blot assay were used to detect the genes expression and paracrine activity of hUCMSCs cultured on different PLGA membranes.3: PC12 cells were investigated for the functionality of the paracrine activity of hUCMSCs.PC12 cells were cultured in hUCMSCs culture medium by centrifugation.The axon growth of PC12 cells in acellular medium(CM)was detected by optical microscope,the quantitative analysis of axon growth of PC12 cells was detedted by ImageJ.Gene expression of PC12 cells differentiation was detected by qRT-PCR.4: DOPA-IGF-1@PLGA+hUCMSCs and PC12 cells were co-cultured by transwell method to observe the synergistic effect between DOPA-IGF-1 and hUCMSCs.The growth of PC12 cells axon was detected by microscope and quantitative analysis was measured by ImageJ.PC12 cells differentiation was observed by immunofluorescence MAP2 staining.5: A hemisection spinal cord injury of SD rats was established to further verify the synergistic effect between DOPA-IGF-1 and hUCMSCs on DOPA-IGF-1@ PLGA+ hUCMSCs films.BBB score and EMG were used to evaluate motor function and neuroelectrophysiology,respectively.Histologically,the recovery of injured spinal cord was observed by HE staining and immunofluorescence.Result:1: In this study,the target protein Y-IGF-1 was obtained through the amplification,expression and purification of escherichia coli,and the adhesive growth factor DOPA-IGF-1 was obtained through the hydroxylation of tyrosinase.The PLGA membrane was incubated in cIGF-1、Y-IGF-1、DOPA-IGF-1 protein solutions for 12 h.ELISA,AFM and XPS experiments fully confirmed that DOPA-IGF-1 was effectively adhered on the PLGA material surface.The protein content and the roughness of the membrane of the modified DOPA-IGF-1@PLGA membrane were increased.The hydrophilicity of the DOPA-IGF-1@PLGA membrane was increased by the Contact Angle experiment.2: Cell experiment verified the hUCMSCs biological effect cultured on cIGF-1@PLGA、Y-IGF-1@PLGA and DOPA-IGF-1@PLGA membranes.We found that DOPA-IGF-1@PLGA had a significant proliferation effect on hUCMSCs at 50ng/mL through CCK-8.The cells adhesion was observed on the DOPA-IGF-1@PLGA membrane surface.Expression levels of NGF,BDNF and VEGF were increased under the action of DOPA-IGF-1@PLGA by qPR-PCR.Western blot experiments confirmed that DOPA-IGF-1@PLGA promoted NGF protein expression and paracrine function of hUCMSCs.3: We tested the paracrine function of hUCMSCs using PC12 cells in different culture mediums(CMs).PC12 cells were cultured in each CMs after centrifugation.Through the microscope,ImageJ and PC12 axon-related genes,we found that DOPA-IGF-1@PLGA promoted the paracrine effect of hUCMSCs,and the growth factor secreted into the CMs promoted the growth and differentiation of PC12 cells.4: We explored the synergistic effect between DOPA-IGF-1 and hUCMSCs on DOPA-IGF-1@PLGA+hUCMSCs membrane through cell experiments,to preliminarily explore the combined synergistic effects of DOPA-IGF-1 and hUCMSCs.By observing the growth of PC12 cells axon and immunofluorescence,it was found that the efficacy and duration of DOPA-IGF-1@PLGA+hUCMSCs membrane were stronger than other experimental groups.5: DOPA-IGF-1@PLGA+hUCMSCs membrane was transplanted into the injuried spinal cord site by spinal cord hemisection experiment of rats.Through the results of BBB score and EMG,we found that DOPA-IGF-1@PLGA+hUCMSCs membrane promoted the restoration of motor function and the conduction of neural electrical signals in rats.By HE staining and immunofluorescence staining,we found that DOPA-IGF-1@PLGA+hUCMSCs membrane promoted the growth of neurons,reduced the inflammatory response,and regulated the local microenvironment of SCI.Conclusion:1: DOPA-IGF-1 exhibited strong binding ability to PLGA films compared with commercial IGF-1 and Y-IGF-1 and enhanced the biocompatibility of PLGA membrane surface.DOPA-IGF-1@PLGA films promoted the biological effect of hUCMSCs.2: DOPA-IGF-1 and hUCMSCs play a synergistic effect in the process of spinal cord repairation in rats,and DOPA-IGF-1@PLGA+hUCMSCs membrane can significantly promote the recovery of spinal cord function in rats.