Study On Conductive Biodegradable Polyester/CNT Composite Fibers For Nerve Regeneration

With the development of studies on tissue engineering,conductive nerve conduits have been widely investigated using conductive materials such as polypyrrole,polyaniline and carbon nanotubes(CNTs),but these materials are usually unable to be biodegraded.It is simple to fabricate conductive conduits using biodegradable biomaterials and CNTs for in vivo applications,however,an important issue closely in relation to the applications is the in vivo fate of the non-degradable CNTs,which is still unclear to the current and needs further studies.In this thesis,a kind of fluorescent-labeled multiwalled CNTs(MWCNTs-FITC)was prepared and composited with biodegradable poly(lactide-co-glycolide)(PLGA)for the purpose.Briefly,the MWCNTs-FITC was synthesized through the combination of atom-transfer radical polymerization(ATRP)and fluorescein-isothiocyanate-isomer-I(FITC)labeling.PLGA@MWCNTs-FITC composite fibrous membranes with fibers being parallel-aligned were prepared via electrospinning and rolled into multi-channel conduits with the assistant of acupuncture pins as the channel template.These conduits,displaying fluorescence,were then applied to repair the 8-mm-defect of rat's sciatic nerve,in which,the location of the MWCNTs-FITC could be observed using confocal laser scanning microscope(CLSM)and small animal imager alongside the PLGA degradation and nerve regeneration.The main works of this thesis are summarized as follows:(1)Firstly,MWCNTs were grafted with poly(glycerol methacrylate)via ATRP and amino groups were introduced via reacting with ethanediamine,which provided binding sites for FITC to obtain the fluorescent MWCNTs-FITC.Parallel-aligned PLGA@MWCNTs-FITC composite fibers were then electrospun from PLGA solution containing MWCNTs-FITC using a rolling rod as the collector.Under scanning electron microscope(SEM)and transmission electron microscope(TEM),the MWCNTs were observed distributing homogeneously along fiber direction without showing obvious aggregation at the content of 3 wt.%MWCNTs-FITC.Under fluorescent spectrophotometer(FS)and CLSM,the MWCNTs-FITC were illustrated by green fluorescence.The conductivity of the PLGA@MWCNTs-FITC composite fibrous membrane reached 1×10-4 S/cm.The PLGA@MWCNTs-FITC composite fibrous membranes didn't show substantial cytotoxicity,which was proofed by cell proliferation,live/dead staining and cell morphology.They supported the attachment,proliferation and further mature of RSC96 cells,and were able to guide the cells elongating along with the orientation of the fibers.(2)Utilizing acupuncture pins as the channel template,the fibrous membranes were rolled into multichannel nerve conduits,and the uniform distribution of multichannel structure could be identified by macroscopic,SEM and CLSM observation.After being implanted under the skin of nude mice for six weeks,strong fluorescence of the conduits could still be observed,which suggested that the fluorescence of the labeled MWCNTs was relatively stable in vivo.In addition,the results of HE staining showed that the conduits could effectively defend the cells'invasion,which provided the possibility for the application of the conduits in repairing nerve defects.(3)The nerve regeneration experiment was conducted using the animal model of 8-mm defects in rat's sciatic nerve by suturing the fluorescent multichannel nerve conduits between the two stubs.Six and twelve weeks after the surgery,the results of walking track analysis and electrophysiological test showed that the fluorescent multi-channel nerve conduits exhibited stronger effect to facilitate the regeneration of sciatic nerve than that of PLGA multichannel conduits as comparison.More significantly,the location and the distribution of MWCNTs were systematically studied by both HE staining and CLSM observation.From HE staining images,only those CNTs aggregates with relatively large size could be observed clearly,while the fluorescent images captured by CLSM could reveal the existence of CNTs with much smaller size.It was identified that fluorescent CNTs still mainly remained in the fiber matrix 12 weeks post surgery,because the degradation and weight loss of the conduit were not so significant at the time point.At 24 weeks post surgery,numerous CNTs could be observed having leaked out from the conduit materials and formed aggregates alongside the PLGA degradation.However,the majority of the introduced MWCNTs-FITC stayed relatively stable within the regenerated nerve tissue,because no fluorescent signal was able to be identified for liver,kidney and spleen tissues,as well as,for muscle tissues adjacent to the regenerated nerve.These findings indicated that the possibility of MWCNTs immigrating into the surrounding tissue and the blood system was not high,probably due to the aggregation.Based on these studies,it is proved that MWCNTs intend to gather up and remain in the regenerated tissue in the form of aggregates,which are difficult to immigrate into the surrounding tissues or blood system without rising significant problems such as cell cytotoxicity and biocompatibility.Thus,the present study provides a useful characterization method for MWCNTs and provides the guidance for the potential in vivo application of MWCNTs.