Preparation Of PRGD/PDLLA/β-TCP/NGF Composites Nerve Conduit And Its Application In Peripheral Nerve Injury

Peripheral nerve injury is one of the common traumas which results in severe sensory-motor deficits in patients.The nerve conduit is a new therapy method for peripheral nerve damage with significant limitations,which can not achieve the same effect as that of nerve autograft.Because nerve regeneration needs suitable microenvironment and proper biological factors which can promote nerve regeneration,a novel RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid)-alt-(L-lysine)]}/poly(d,l-lactic acid)/β-tricalcium phosphate/nerve growth factor(PRGD/PDLLA/β-TCP/NGF) composite nerve conduit was prepared, moreover,the structure and properties of which were characterized.And the biocompatibility of which was evaluated by cell experiments in vitro and animal experiments in vivo.The results support the experimental data for the clinical treatment of peripheral nerve injury.In this paper,A new polymer RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid)-alt-(L-lysine)]}(PRGD) was synthesized.The process for preparing PRGD involved with four steps:firstly, (3S)-3-[4-(benzyloxycarbonylamino)butyl]morpholine-2,5-dione(BMD) was synthesized by bromoacetyl bromide and N^ε-(benzyloxycarbonyl)-L-lysine.Secondly, poly {(lactic acid)-co-[(glycolic acid)-alt-(N^ε-benzyloxycarbonyl-L-lysine)]}(PLGLZ) was obtained by copolymerization of D,L-lactide and BMD.Then,poly{(lactic acid)-co-[(glycolic acid)-alt-(L-lysine)]}(PLGL) was synthesized by catalytic hydrogenation of PLGLZ.Finally,PLGL was modified with RGD peptide.The structures of PRGD and its precursors were characterized by FT-IR,¹H NMR,¹³C NMR,MS,GPC,Amino acid analysis(AAA).The results showed that:BMD was successfully synthesized.This monomer was easily copolymerized with D,L-lactide at wide range of mole fractions in the feed.The selective removal of the benzyloxy carbonyl group from the PLGLZ copolymer was carried out by catalytic hydrogenation.After deprotection,the PLGL copolymer was modified with RGD peptide at a concentration of 6.9～18.8μmol/g.RGD peptide could modify PLGL at wide range of mole fractions in the feed.According to study the preparation process of the composite membrane and factors in influence membrane performance,the composite membrane was prepared using ethyl acetate as solvent under 20℃and 40%humidity by solvent volatilization method.The range of molecular weight of PDLLA was selected as 1.0×10⁵～1.5×10⁵. The grain size ofβ-TCP power is less than 500nm.The content ratio of PDLLA, PRGD,β-TCP and NGF was fixed respectively at 85%,10%,5%and 0.0006%.The structure and properties of the composite membrane were characterized.The results showed that:211.6ng/mL NGF was released from the composite membrane in the first 4 hours,and the releasing rate was slower after.The hydrophilicity of the composite materials had been greatly improved comparing with PDLLA.The tensile strength of the composite membrane was 5.7±0.23 MPa.The elongation at break of the composite membrane was 1631.34%±228.66%.The ring rigidity of the composite conduit was 168.93±20.24 KPa.The compression set of the composite conduit was 15.85%±0.69%.The pH value of degradation medium,weight loss ratio of the composite membrane and the variation of molecular weight were determinated and the variation of the morphology and microstructure of the composite membrane was observed during the degradation in vitro.The results showed that:β-TCP can neutralize the acidity which produced by degradation of PDLLA and PRGD,availably adjust the pH value of degradation medium to keep at neuter.Adding PRGD andβ-TCP can promote the composite membrane to degradate,so PRGD/PDLLA/β-TCP/NGF composite materials has better biodegradation properties than PDLLA.After cultivating RSC96 schwann cells on the surface of the materials,the cell activity and the surface feature of the RSC96 schwann cells were detected by the MTT experiments and scanning electron microscope.The experiments in vitro of RSC96 schwann cells and materials showed that PRGD/PDLLA/β-TCP/NGF composite materials has better cell affinity than PDLLA,which can promote the RSC96 schwann cells adhesion,proliferation and growth on the surface of materials.The PDLLA conduit,PRGD/PDLLA/β-TCP composite conduit, PRGD/PDLLA/β-TCP/NGF composite conduit and autonerve were transplanted to repair the 10mm rat sciatic nerve defect.Functional recovery of the sciatic nerve regeneration was evaluated at 3 months after operation through gross observation, triceps weight,electrophysiology,histology and axon morphologic assessment.The results showed that:(1) Regenerated nerve fibers of the PRGD/PDLLA/β-TCP composite conduit group was significantly superior to that of the PDLLA conduit group in histological and axon morphologic index.The PRGD/PDLLA/β-TCP composite conduit is more beneficial to nerve regeneration than PDLLA conduit. (2) The biodegradable PRGD/PDLLA/β-TCP/NGF composite conduit had good compatibility with rat tissue and it could effectively promote nerve regeneration after bridging sciatic nerve defect of rats.The effect was as good as that of autograft, significantly superior to the PRGD/PDLLA/β-TCP composite conduit group and PDLLA conduit group.PRGD/PDLLA/β-TCP/NGF composite conduit was a potential ideal conduit.We hope it will be applied clinically with farther study to relieve pains of patients.