Construction And Application Of Tissue-engineered Nerve Grafts Using A Scaffold-independent And Injectable Drug Delivery System Based On Thermosensitive Hydrogels

Current interventions for bridging peripheral nerve defects are limited.Nerve autografting has been considered as gold standard since 1940s,but it has limitations such as donor site morbidity.Alternatively,tubular nerve conduits have been developed to substitute autografts.A few of them have been approved by the Food and Drug Administration(FDA)since 1999,but they have yet to gain widespread use due to their suboptimal functional outcomes.To improve the performance of these hollow conduits,researchers have explored various filler materials that provide topographical cues.Recently,increasing attention has been paid to the addition of acellular and/or cellular cues via materials and tissue-engineering techniques,establishing a new research field,namely peripheral nerve tissue engineering.Tissue-engineered nerve grafts(TENGs)with acellular cues,especially neurotrophic factors(NTFs),are more clinically attractive because they have a simpler path to the bedside and are more appropriate for treating acute injuries when autologous cell seeding is impractical.Over the past 30 years,although a number of TENGs that can controllably release NTFs have been fabricated and perform better than blank conduits,none of them extends beyond the laboratory bench.This imbalance between scientific innovation and translation to patient benefit prompts us to rethink the existing design concepts of TENGs based on a translational perspective.At present,drug delivery strategy is typically applied by combining with the structure of conduits.NTFs can be incorporated into scaffold materials(either wall or fillers)during conduit fabrication through blending,adsorption,affinity,covalent link,coatings,electrospinning,microparticle incorporation,etc.However,these approaches may hinder the clinical translation and commercialization of TENGs.First,NTFs are initially dissolved to impregnate carriers or react with binding mediators,followed by drying process.Maintaining the bioactivity of these liable proteins during processing and storage may be challenging.Second,a single device may not have the flexibility of delivering multiple types of drugs because different drugs require different linkers.Third,the materials used for nerve scaffolds possess strict biomechanical and biocompatible requirements,thus endowing them with additional function of drug delivery can lead to a more complex and higher cost of fabrication process.To overcome these limitations as well as to facilitate the clinical translation of TENGs with acellular cues,we set our sights on new biomaterial that can function as an independent drug delivery system(DDS)without the need of conduit modification and binding mediators.Specifically,injectable thermoresponsive hydrogels can serve as promising candidates.Being liquefied at room temperature,they can be easily loaded with drugs and implanted through a syringe.Upon heating to body temperature,they can gel within minutes and form a drug-releasing depot.Besides,the incorporation of NTFs into TENGs can bypass complex pre-loading and display longer storage stability when injected at the time of surgery.However,no study has yet examined such applications.Therefore,the present study designed a chitosan scaffold filled with thermosensitive hydrogels based on methoxy-poly(ethylene glycol)-b-poly(?-ethyl-L-glutamate)(mPEG-PELG)to deliver nerve growth factor(NGF),which can gel in situ in the lumen upon injection and function as a field-installed conduit-filler.Synthesis of mPEG-PELG and characterization:mPEG-PELG was synthesized through ring-opening polymerization of?-ethyl-L-glutamate N-carboxyanhydride(ELG-NCA)by utilizing amino-terminated mPEG(mPEG-NH2)as a macroinitiator.First,mPEG-NH2 was dissolved in toluene,and the excess water was removed by azeotropic distillation.Subsequently,anhydrous DMF and ELG-NCA were added into the flask.The reaction mixture was then stirred at 30°C for 3 days under a nitrogen atmosphere.After stirring,the solution was precipitated with chilled diethyl ether.The obtained products were washed twice with diethyl ether and dried to constant weight in a vacuum at room temperature.~1H NMR spectra were recorded on a Bruker AV 400 NMR spectrometer in trifluoroacetic acid-d(CF3COOD).Number-and weight-average molecular weights(Mn,Mw)and polydispersity indexes(PDI=Mw/Mn)were determined by gel permeation chromatography(GPC).The sol-gel phase transition behaviors of the mPEG-PELG copolymer solutions in phosphate buffered saline(PBS,pH 7.4)were determined by a vial inverting approach with a temperature increment of 1°C per step.Rheology experiments of the mPEG-PELG hydrogels with or without NGF were performed on an MCR 301 Rheometer.The formed hydrogels could be degraded incubated with proteinase K,and the block copolymers did not show cytotoxicity in vitro.NGF dosing,in vitro release testing and bioactivity testIn order to optimize the loading dose of NGF for its release of 1–10 ng/mL,an in vitro release test was conducted by loading 50,100,and 150 ng of NGF into mPEG-PELG aqueous solution(100?L,8.0 wt%).The hydrogels with 1.0 ng/?L NGF released an average concentration of 13.87 ng/mL at day 1 and dropped off to below 10 ng/mL at day 2.During the 28 days of incubation,NGF concentration plateaued to approximately 1.5 ng/mL,and approximately 81.4%of NGF were released from the hydrogels.Hence,this formulation was selected for further experiments,as it could maintain stable concentrations within optimal therapeutic ranges during the entire course of treatment.The bioactivities of released NGF were evaluated with regard to the induction of neurite outgrowth from PC12 cells and the activation of downstream MAPK/ERK pathway.PC12 cells were exposed to the release medium collected on day 1,2,4,7,10,14,21 and 28.The results indicated that the released NGFs exerted their abilities to induce ERK phosphorylation and promote neurite outgrowth.Therapeutic effects of this NGF-loaded TENGs based on mPEG-PELGThe in vivo study adopted this scaffold-independent drug delivery system based on mPEG-PELG.A novel TENG,a chitosan scaffold filled with nerve growth factor(NGF)-loaded mPEG-PELG that gel in the lumen upon injection during surgery and function as a drug-releasing conduit-filler,was designed.All rats were randomly divided into 5 groups:(i)Scaffold group,(ii)Scaffold+mPEG-PELG group,(iii)Scaffold+NGF/IM group,(iv)Scaffold+NGF/mPEG-PELG group,and Autograft group.In Scaffold group,the defect was bridged by a plain chitosan scaffold.In Scaffold+mPEG-PELG group,the defect was bridged using a chitosan conduit,and 100?L of mPEG-PELG solution was added into the lumen through a 1 mL syringe to form gel in situ.In Scaffold+NGF/IM group,the defect was bridged by a chitosan scaffold,and 2.0?g/kg of NGF was administered daily via intramuscular injection(IM).In Scaffold+NGF/mPEG-PELG group,the defect was bridged using a chitosan scaffold,and 100?L of NGF-loaded mPEG-PELG solution was added into the lumen through a 1 mL syringe to form gel in situ for 10 min.In Autograft group,the defect was bridged with the segments of transected nerves.The effects of Scaffold+NGF/mPEG-PELG on nerve regeneration with respect to axon outgrowth,remyelination,motor endplate reinnervation,electrophysiological and functional recovery were assessed in vivo.Notably,mPEG-PELG alone did not hamper nerve regeneration,as evidenced by the similar results obtained from Scaffold and Scaffold+mPEG-PELG groups.Also,TENG(Scaffold+NGF/mPEG-PELG group)achieved outstanding performances relatively close to autografts.Conclusions:The results demonstrated that NGF-loaded mPEG-PELG controllably and sustainably released bioactive NGF for 28 days.When bridging a 10 mm rat sciatic nerve gap,the morphological,electrophysiological,and functional analyses revealed that NGF-releasing TENG(Scaffold+NGF/mPEG-PELG)achieved superior regenerative outcomes compared to plain scaffolds and those combined with systemic delivery of NGF(daily intramuscular injection),and its effects were relatively similar to autografts.This study has proposed a TENG using thermosensitive hydrogels as an injectable implant to controllably release NGF,which has promising therapeutic potential and translatability.Such TENGs obviate the need for conduit modification,complex preloading or binding mediators,therefore they allow the ease of drug switching in clinical practice and greatly simplify the manufacturing process due to the independent preparation of drug delivery system.Collectively,this preclinical study introduced a first step towards the development of a novel drug-releasing TENG that exerts both scientific and translational advantages.