Preparation And Evaluation Of A PH Sensitive Injectable Hydrogel For Nerve Growth Factor Sustained Release

Peripheral nerve injury is a common clinical disease.The current research is focused on nerve conduits(NCs)to repair the damaged nerve.However,the empty NCs often show inferior recovery outcome due to the lack of the bioactive component belonging to the microenvironment substances of peripheral nerve.In this study,in order to obtain the fillers in the lumen of hollow NCs to improve the microenvironment of nerve regeneration.From the design of the biomimetic molecular of the extracellular matrix material,the similar extracellular matrix component chitosan(CS)and extracellular matrix component hyaluronic acid(HA)were selected to prepare a biocompatible and biodegradable pH-induced CS-HA injectable hydrogel via amidation reaction.The properties of CS-HA injectable hydrogel were studied.Then the release behaviors of nerve growth factor(NGF)and the in vitro biocompatibilities of CS-HA injectable hydrogel were investigated after NGF was encapsulated into CS-HA injectable hydrogel.Moreover,the ability of CS-HA/NGF hydrogel fillers in PDLLA/?-TCP NCs to improve functional recovery in rats model of peripheral nerve injury was investigated.The vial inversion method were used to measure the gelation time of CS-HA injectable hydrogel.The structure and morphology of hydrogel were characterized by fourier transform infrared spectroscopy(FTIR)and scanning electron microscopy(SEM).Computer-controlled electronic universal testing machine was employed to investigate the mechanical property of hydrogel.The swelling and degradation behavior of hydrogel was determined by weighing method.The results showed the hydrogel can complete the gelation process within 50 s to 6 mins by changing the composition of the hydrogel under the effect of physiological pH to meet the need of injection.It was fabricated with an interconnected porous and a controllable pore diameter.The porous structure is conducive to the growth of cells and the adsorption of NGF.Mechanical tests showed that it had favourable compression modulus.The hydrogel with a pH sensitive property when the pH ranged from 2.0 to 9.0,and can be degraded more than 70% within 8 weeks in vitro which was suitable the regeneration of peripheral nerve.The formulation of CS-HA injectable hydrogel was optimized by using BSA as a model drug.Then the NGF release behaviors were investigated after NGF was encapsulated into the optimized CS-HA injectable hydrogel by the method of ELISA.The results showed when the mass ratio of CS/HA was approaching 3:1,the amount of cumulative release increased.The release period of NGF was about 28 days,the cumulative release rate was about 79.47%.The results demonstrated its potential as a carrier to encapsulate and sustained release NGF for the repair of peripheral nerve defect.The RSC96 rat schwann(RSC96)cells,bone marrow mesenchymal stem cells(BMMSCs),and pheochromocytoma line 12(PC12)cells were co-cultured with CS-HA/NGF hydrogel.The method of CCK-8 was selected to evaluate the cytotoxicity of the hydrogel,the immunofluorescence staining and scanning electron micrograph were employed to study the adhesion and proliferation of cells on the surface of the hydrogel,the PC12 cells were used for the bioactivity test of the released NGF.The results suggested CS-HA/NGF hydrogel was suited for the growth of neuronal cells.The PC12 cells could differentiate into neuronal phenotype after co-cultured with the sustained release liquid.Which indicated the good ability of hydrogel to protect the activity of NGF.The PDLLA/?-TCP NCs,PDLLA/?-TCP/CS-HA NCs,PDLLA/?-TCP/CS-HA/ NGF NCs and autologous nerve were used in the repair of rat sciatic nerve defect of 10 mm.The morphology and histology of postoperative rats were analyzed after 1 and 3 mouth.The results of experiments showed that the added CS-HA/NGF hydrogels had a significant positive effect on the axon regeneration and myelination compared to those in the PDLLA/?-TCP NCs,and similar to the autograft group.Which can effectively promote the regeneration of peripheral nerve damage.The results showed that the CS-HA/NGF injectable hydrogels can effectively promote the regeneration of peripheral nerve.It would be a promising candidate for neural tissue engineering...