| Objective:Peripheral nerve regeneration and functional recovery with long distance defect has remain been a major clinical challenge.Autograft has been the gold standard for this kind of injury in clinical treatment.However,the application of this therapy is greatly limited by the lack of donator,the decrease of the function of donor target organs and the poor matching of nerve function.Herein,it is particularly significant to design and construct a class of biomaterials that are beneficial to nerve regeneration and functional recovery and can replace autologous nerve transplantation.Therefore,Utilizing polyurethane(PU)’s good biocompatibility,biodegradability and multifunctional active site,combining with the gastrodin’s anti-inflammatory,anti-oxidative stress,neuroprotective effects and polyhydroxyl chemical structure,this study is devoted to design a kind of polyurethane conduit functionalized by gastrodin to optimize and provide a beneficial regenerative microenvironment for injured nerves,and to evaluate its degradation and gastrodin sustained-release performance as well as the regulatory on biological behavior in vitro and in vivo.Methods:Gastrodin sustained-release PU conduit was prepared by in-situ polymerization and dipping method.PU prepolymer was synthesized by in-situ polymerization with polycaprolactone(PCL2000),isophorone diisocyanate(IPDI)and lysine ethyl dihydrochloride(Lys-OEt·2HCl)as chain extender.Gastrodin with different mass fractions(0%,1%,5%)were loaded onto the polymers and named as PU,1Gastrodin/PU,and 5Gastrodin/PU,respectively.The polymer and its twice mass NaCl(particle size less than 50μm)were dissolved by stirring in 1,4-dioxane ring to make the hole.The conduit with interconnected micropores was prepared by dipping the dissolved polymer with 1.28 mm diameter stainless steel wire,drying and washing for desalination.The conduit was degraded in 0.1M NaOH solution and the release of gastrodin was detected by HPLC.Schwann cells(SCs)viability was evaluated by Live/Dead kit,and the bioactivity of the conduit was estimated by the gene expression of cytokines such as BDNF and GDNF by real-time quantitative polymerase chain reaction(RT-qPCR).The anti-inflammatory was evaluated by the gene expression of tumor necrosis factor α(TNF-α)and inducible nitric oxide synthase(iNOS)using RAW264.7.The SD rat model of sciatic nerve defect was bridged with the conduit in vivo,and the biological activity was evaluated by behavioral,electrophysiological and morphological tests.Results:The results of SEM show that the conduit has a uniformly distributed and interconnected porous structure.In the degradation experiment,the degradation rate increased with the increase of Gastrodin content,and the 5Gastrodin/PU group showed sustained and stable release of Gastrodin as the degradation proceeded.After co-culture of SCs with the materials,the cell activity of the 5Gastrodin/PU group was significantly higher than the 1Gastrodin/PU and PU groups,and the degree of SCs myelination in 5Gastrodin/PU group was significantly increased,which was also supported by the significantly decreased expression of NCAM in this group.In addition,the expression of BDNF and GDNF in 5Gastrodin/PU group was also significantly higher than other groups.Furthermore,after co-culture with RAW264.7,the expressions of TNF-α and iNOS were significantly inhibited in 5Gastrodin/PU,indicating that the sustained release of Gastrodin from 5Gastrodin/PU material had a more effective anti-inflammatory effect.After 10 mm sciatic nerve defect model of SD rats was bridged by conduit,the results of behavioral,electrophysiological and morphological methods were analyzed for nerve regeneration.The results showed that the injured nerves were recovered to varying degrees,and the autograft was still significantly better than the experimental group.At the same time,the results also showed that the number of regenerated nerves,microvascular formation and functional recovery in the 5Gastrodin/PU group were more similar to the autograft group.Conclusion:Gastrodin can be polymerized successfully to PU biomaterials,and can be released continuously with the degradation of materials.The released Gastrodin from 5Gastrodin/PU can not only promote the expression of GDNF and BDNF,but also inhibit the expression of inflammatory cytokines TNF-α and iNOS.The conduits also promote the myelination of SCs and the differentiation of nerve cells,especially in the 5Gastrodin/PU group.Therefore,a theoretical basis is provided for a good microenvironment of nerve injury repair.The implantation of 5Gastrodin/PU into sciatic nerve defect in SD rats significantly enhances the recovery of motor function and regeneration of nerve tissue,making it a strong candidate material for the repair of peripheral nerve injury. |
