Researches On The Mechanism Of Painful Neuroma Prevention Using PDLLA/PRGD And Novel Nerve Conduit Materials

Although peripheral nerves have the ability to regenerate spontaneously,this automatic repair process would not be completed when a long distance defect was presented or target organs were missing,even worse,the terminal painful neuroma,as the complications of nerve damage,might be formed at the injury site and seriously affected the daily life of patients,which subsequently influenced their rehabilitation and brought a huge economic burden.For this clinical problem,many treatments have been developed,such as alternative therapy,analgesic medicine and tissue embedding surgery,however,none of them could achieve satisfying outcomes or prevent the relapse of neuroma.This problem was not effectively alleviated until the concept of nerve conduit capping method was introduced.The PDLLA/PRGD/?-TCP nerve conduit was synthesized in our lab,which has been successfully used to bridge the gap of rat sciatic nerve and no neuroma could be detected,so this conduit was supposed to be applicable for nerve capping technology.In this study,amputated rat limb models were initially established.Differences in autotomy scoring,inflammatory cell infiltration,scar formation,remyelination as well as the transcription level of related genes were compared for elucidating the possible mechanism of neuroma prevention.Additionally,graphene and functionalized PAMAM hydrogel were separately evaluated for improving the performance of nerve conduits,as they were considered to be a new potential material component and novel filler for nerve conduit.In the first part,biocompatibilities and degradation performances of composite nerve conduits with different combination of RGD peptides and?-TCP were evaluated.The changes on the mass loss and surface features demonstrated that?-TCP nanoparticles were beneficial for degradation,a milder pH value was obtained during those procedures.Compared to others,PDLLA/PRGD/?-TCP conduit exhibited the best performance throughout the cytotoxicity assay and cell live/dead staining,especially in the hiscompatibility testing,no abnormal tissue or excessive inflammatory cells accumulation were observed in the site of implantation,which were prevalent in the PDLLA group.Thus,the addition of RGD polypeptide and?-TCP nanoparticles enhances the biocompatibilities of the materials.In the previous research,PDLLA/PRGD/?-TCP nerve conduit was used to bridge 10 mm nerve gaps of rats,Ca²⁺,which was released with the degradtion of?-TCP nanoparticles,was found to have the ability of mediating axon growth,nerual cell migration and inducing nerve regeneration.However,such a bridge treatment could not be carried out in amputated nerves,as a result of the missing of distal stump.So the PDLLA/PRGD composite conduit was chosen in the following researches related to the amputated rat models.In the second part,PDLLA/PRGD nerve conduit was applied to cap the sciatic nerve terminals of SD rats.During the entire time of wound closure,no infection was noted in the experimental rats.Furthermore,a significantly lower average autotomy score was noticed in the conduit group,in which a similar trend of?-SMA transcription regulation was proved,this gene was considered to be a molecular indicator of painfulness in animal models.In addition,immunohistochemical analyses were employed to investigate the postsurgical inflammation response,macrophages and T cells were individually stained and counted.Results showed that the number of inflammatory cells in control group was higher than that in PDLLA/PRGD group.For further exploit,quantitative PCR was also carried out.As the genes of two critical inflammation factors,TNF-?and IL-1?,were chosen.Contrary to control rats,not only a lower expression but also a down regulation could be found in the PDLLA/PRGD conduit group.Masson and Sirius red staining technologies were also used for the assessment of scar formation,in the cross section image of control group,typical swollen and edema symptoms could be identified at the 2^nd week.Besides that,the regenerated connective tissues had invaded the spaces around nerves,followed by massive collagen deposition.Till the 8^th week,nerve fibers were tightly mingled with scar tissue and it was hard to be distinguished from each other.On the contrary,none of those phenomena appeared in the PDLLA/PRGD group,only the adjacent tissue was slightly edematous at the 2^nd week.Under the polarized light,collagen I and collagen III were discriminated with the help of Sirius red,it was illustrated that the differences on scarring were mainly contributed by the amount of collagen I,the content of collagen III in the two groups were nearly the same.Remyelination was the last aspect that we analyzed,involving in changes in microstructure of myelin sheath and transcript levels of corresponding genes.It was revealed that the diameter and thickness of myelin in PDLLA/PRGD group were larger than those in control,in which a lower unmyelinated/myelinated ratio was confirmed through statistical calculation.Total four myelination related genes were selected,their expression products were MPZ and MBP,the structure genes of myelin,MAG,as the marker of initiation of myelination,Krox20,the key participator of whole regulation network.In both groups,similar level of MAG was maintained at the 2^nd week and Krox20 was simultaneously increased,a dramatic down regulation of MPZ and MBP could be measured in the noncapped group.It was inferred that myelination was normally activated,but no sufficient structural genes were generated in control group.In conclusion,application of PDLLA/PRGD conduit could inhibit neuroma formation through the coordination of its physiological functions on inflammation,collagen deposition and remyelination.In the third part,the biocompatibility of graphene was preliminarily evaluated.As having a variety of excellent properties,this material was thought to be an ideal candidate for preparing medical devices.Among them,the outstanding conductivity would be indeed useful for nerve repair.Reduction of purchased graphite was the main synthesis routine for gaining graphene particles,of which average size was nearly 3087 nm.Hereafter,the reliability of graphene synthesis was confirmed via SEM,FTIR,ultraviolet/visible spectrum and XRD.Then,PC12 cell line was treated with graphene solutions containing different concentration for determining its cytotoxicity,and graphene concentration in the range of 0-100?g/mL did not show any adverse effect on cell morphology and activity.However the integrity of cell membrane will be disrupted,when it was higher than 20?g/mL,it was verified by the leakage of intracellular lactate dehydrogenase.Moreover,a significant increasement of reactive oxygen species in the cells could be detected in the cells,if the concentration was above 10?g/mL,which would inevitably lead the oxidative damage.Those experimental data revealing the concentration-dependent cytotoxicity would be the fundamental for graphene future application.The final part was an attemptation to develop a new kind of Poly?HEMA-GMA?-PAMAM hydrogel,the PAMAM dendritic macromolecules ranging from 0.5 to 4.5 generations were synthesized by addition and amylation reactions,in which propargylamine worked as the nucleus and methyl acrylate was the branched functional group.Poly?HEMA-GMA?backbones with different proportions were prepared by ATRP.As the beginning step,PAMAM dendritic polymers were clicked on the azido group,the successful preparation of macromolecules was confirmed via NMR,FTIR and GPC.After that,the hydrogel was crosslinked with glutaraldehyde.Being seeded with astrocytes on its surface,no significant cytotoxicity was found.And astrocytes grown on the hydrogel which was grafted with the 5^th generation of PAMAM dendron were able to preserve good morphology,all these results provided the preliminarily theoretical basis for the following research.