Preparation And Property Study Of Nerve Growth Factor Sustained-release Gradient Nerve Conduit

A NGF sustained release nerve conduit was prepared to repair peripheral nervedefects. Nerve growth factor (NGF) was compound to the carrier material through thelayer by layer electrostatic self-assembly and coupled cross-linking technology. Polylactic acid (PDLLA) used as the base and adding beta three calcium phosphate(β-TCP) to prepare porous substrates. Through the ion electrostatic adsorption ofhyaluronic acid (HA) and chitosan (CHS), self-assembly multilayer construction wasmade, then introducing positive charge on the surface of the substrate; NGF throughcrosslinking agent EDC cross coupling to self-assembly layer; finally the polylacticacid/beta three calcium phosphoric acid/hyaluronic acid/chitosan/nerve growth factor(PDLLA/β-TCP/HA/CHS/NGF) porous gradient nerve conduit was made and severalmethods were used to characterize and evaluate the performance of the nerve conduit.By exploring the influence on preparation, such as self-assembly layers, time,temperature, pH, concentration and assembly ratio to determine the best conditionsfor preparing PDLLA/β-TCP/HA/CHS/NGF nerve conduit. The results showed thatthe optimum preparation conditions was each layer assembled30min, controlledtemperature at30℃and pH value was3.8, HA/CHS concentration ratio was3:1.Fourier transform infrared spectroscopy (ATR-FTIR), X-ray diffraction (XRD),scanning electron microscopy (SEM), computer-controlled electronic universaltesting machine and the contact angle measurement machine were used tocharacterize and evaluate the structure, mechanical properties and hydrophilicproperties of the nerve conduit. The results showed that the sustained release NGFgradient pore nerve conduit was designed to meet the requirements of tissueengineering materials and would be able to promote the repair and regeneration ofperipheral nerve injury.In vitro degradation testing of nerve conduit was carried out to determinemedium pH value changes, weight loss rate during the process of degradation and toobserve the structure changes after the degradation by scanning electron microscope.Results showed that the added β-tricalcium phosphate could be adjusted andmaintained pH value at a neutral of the medium, as polylactic acid degradation theproduct was acidic and would accelerate the degradation of nerve conduits. In the week12, the weight loss was about48.3%and with the time increasing β-tricalciumphosphate eventually caused disintegration of the nerve conduit.The content of NGF was measured by enzyme linked immunosorbent assay(ELISA) to study the release property of the nerve conduit. The results showed thatNGF could release with the continuous degradation of the assembly layer, releaseperiod was about30days, the cumulative release amount was about90%whichimplied it had good release property and the NGF could promote PC12axon growthand elongation which implied NGF in extracts of nerve conduit was still retains itsbiological activity.The bone marrow mesenchymal stem cells (BMMSC), adrenal pheo-chromocytoma (PC12), Schwann cells (RSC96) were co-cultured with PDLLA,PDLLA/β-TCP, PDLLA/HA/CHS, PDLLA/β-TCP/HA/CHS/NGF nerve conduit todetermine the biological compatibility by CCK-8. And observe the surface adhesioncell number by immunofluorescence staining to calculate the adhesion rate. Theresults show that we could see the growth state of cells on the surface,PDLLA/β-TCP/HA/CHS/NGF nerve conduit had low toxicity and good cell adhesion,PC12axon elongation experiment showed the release of NGF could induce nerve cellaxon elongation; the rate reached54%, which proved that the nerve conduit had goodcell compatibility.