Establishment Of Two Modalities Of Regenerative Microenvironments And Their Efficacy In Repairing Large Nerve Defects In Rats

Peripheral nerve injury (PNI) is a common casualty and frequently resultsfrom trauma and accidents, greatly compromising the life quality of patients andhaving a significant socioeconomic impact. Despite the intrinsic growth capacityof neuronal cell body following PNI, functionally successful nerve regenerationis generally disappointing, especially in a large peripheral nerve defect wherecontinuity of the epineurium is disrupted. Recovery after this injury is hardlywithout surgical intervention. For the reconstruction of a large nerve defect(>5cm in human), a healthy nerve is needed to be obtained as the nerve autograft,which is considered the therapeutic gold standard of bridging a large nerve defect.However, nerve autografting has some disadvantages, including limitedavailability of donor grafts and postoperative complications of donor sites, such as scarring and neuroma formation. In the recent decades, techniques of tissueengineering (TE) and regenerative medicine (RM) have been widely applied infabricating tissue-engineered nerve grafts (TENGs) for bridging large peripheralnerve defects, which has formed an important branch of tissue engineering,namely, the neural tissue engineering (NTE).After PNI, the intrinsic growth capacity within the neuronal cell body isactivated and the proximal stump of the peripheral nerve begins regeneration. Thestrength of nerve autografting lies in its efficacy in establishing of a regenerativemicroenvironment (RME) for promoting axonal regeneration and penetratingthrough the whole length of the nerve autograft, namely, the endoneurialmicrostructure which provides regenerative axons with physical and molecularguiding cues, while Schwann cells (SCs) and neurotrophic factors which provideneurotropic and neurotrophic support for axonal regrowth. In addition, adequatelyvascularizing nerve grafts are equally important to provide sufficient diffusion ofoxygen, nutrients, and waste products for supporting and maintaining viablenerve tissues. Also，electrical stimulation (ES) holds the potential to activate thegrowth capacity within the neuronal cell body, guiding the outgrowth of axonalgrowth cones, hence improving nerve regeneration. Therefore, there-establishment of RME plays a pivotal role during the process of nerveregeneration and functional recovery.Up to present, joint application of TE and RM to fabricate a TENG andfunctionalizing the TENG by introducing SCs and/or NTFs holds the potential toimprove peripheral nerve regeneration and make it a beneficial candidate fornerve autografts. However, most nerve scaffolds are hollow tubes made fromboth synthetic and natural materials, which provide macroguidance toregenerating axons by confining axonal regeneration within the conduit and loosely directing regeneration toward the distal stump. The lack of organizedmicrostructure results in regeneration characterized by random clusters ofregenerating nerve fibers surrounded by extracellular matrix tissue. Nervescaffold with innerstructures mimicking the basal lamina microstructures inautografts is rarely reported, and so is the case with establishing RME withinscaffolds of this kind. In the present study, a collagen-chitosan scaffold withlongitudinal oriented micro-channels (CCH) was fabricated, and was thenfunctionalized by vascularizing the scaffold with omentum or seeding SCs intothe scaffold. The two modalities of RME were listed as following:⑴.Autologous omentum was harvested to wrap around the CCH nerve scaffold toenrich the vascularization of the scaffold, so as to provide sufficient diffusion ofoxygen, nutrients, and waste products for supporting and maintaining viablenerve tissues.⑵. SCs were seeded into the3D microstructure of the CCHscaffold to mimic the RME of nerve autografts, so as to providing neurotropicand neurotrophic support for axonal regeneration. The current study not onlyenriches and develops the research contents of NTE, but also provides theoreticalsupports for reconstruction of large nerve defects. The whole study was mainlydivided into three parts as following:PartⅠ: Fabrication of CCH Nerve Scaffold and Measurement of RelatedPropertiesBackground Tissue-engineered nerve scaffolds hold great potential inbridging large peripheral nerve defects. However, most nerve scaffolds arehollow tubes and lack the microstructure within nerve autografts, frequentlyleading to suboptimal nerve regeneration and unsatisfied functional recovery. In the recent years, extensive attention has been paid to fabricate nerve scaffoldswith innerstructures imitating the basal lamina microchannels in nerve autografts.We have developed a collagen-chitosan nerve scaffold with longitudinallyoriented microchannels, which resembles the dimensions of the basal laminamicro-channels in nerve autografts, and found it effective in guiding axonaloutgrowth linearly and continuously.Objective To Fabricate CCH nerve scaffold for further investigation and tomeasure the related properties.Methods The CCH scaffold was prepared according to a “unidirectionalfreezing” method which has been described previously. The CCH scaffoldconsisted of collagen and chitosan was then cross-linked by genipin. Themicrostructures of the CCH scaffold was viewed with a scanning electronmicroscope (SEM). The porosity, the degeneration rate and the swelling rate werealso evaluated.Results The microstructure of the CCH scaffold resembled the basal laminamicro-channels of nerve autografts. The CCH scaffold showed longitudinallyoriented micro-channels, which were arranged in a honeycomb-like pattern in thecross plane. In addition, the scaffold exhibited a higher porosity rate, thedegeneration rate and the swelling rate of cross-linked scaffold were significantlylower than those of uncross-linked scaffold.Conclusion The CCH scaffold is relatively easy to prepare, handle, store,and sterilize. Furthermore, the scaffold holds optical properties, and thelongitudinally oriented micro-channels in the scaffold are capable of guiding thelinear growth of regenerated axons. PartⅡ: Omentum-Wrapped Scaffold with Longitudinally OrientedMicro-Channels Promotes Axonal Regeneration and FunctionalRecoveryBackground Insufficient vascularization of nerve scaffolds limits neuraltissues survival and regeneration, which hampers the successful implantation andclinical application of nerve scaffolds. The great omentum possesses a highvascularization capacity and enhances regeneration and maturation of tissues andconstructs to which it is applied. However, combined application of nervescaffolds and omentum on axonal regeneration and functional recovery in thetreatment of large peripheral nerve defects has rarely been investigated thus far.Objective To investigate the feasibility of combined usage of omentum andCCH scaffold for reconstruction of large peripheral nerve defects.Methods A CCH scaffold was used to bridge a15-mm-long sciatic nervedefect in rats, and an omentum flap was then secured to the proximal and thedistal nerve sites by3perineural10/0sutures. The axonal regeneration andfunctional recovery were examined by a combination of walking track analysis,electrophysiological assessment, Fluorogold (FG) retrograde tracing, as well asmorphometric analyses to both regenerated nerves and target muscles.Findings The results demonstrated that axonal regeneration and functionalrecovery were in the similar range between the omentum-wrapping group and theautograft group, which were significantly better than those in the scaffold alonegroup. Further investigation showed that the protein levels of vascular endothelialgrowth factor (VEGF), brain-derived neurotrophic factor (BDNF) and nervegrowth factor (NGF) were significantly higher in the omentum-wrapping group than those in the scaffold alone group in the early weeks after surgery.Conclusion These findings indicate that the omentum-wrapped scaffold iscapable of enhancing axonal regeneration and functional recovery. The beneficialeffect of omentum-wrapping on nerve regeneration might be related with theproteins produced by omentum.PartⅢ: Schwann Cell-Seeded Scaffold with Longitudinally OrientedMicro-Channels for Reconstruction of Sciatic Nerve in RatsBackground The strength of nerve autografting lies in its efficacy inestablishing a RME for promoting axonal regeneration and penetrating throughthe whole length of the nerve autograft, namely, the endoneurial microstructurewhich provides regenerative axons with physical and molecular guiding cues, andSCs and NTFs which provide neurotrophic support for axonal outgrowth. TENGshave produced promising results in bridging large peripheral nerve defects, whichmakes it possible to avoid sacrificing healthy nerves as nerve autografts.However, most TENGs lack the microstructure and the permissive environmentwithin nerve autografts, thus frequently leading to suboptimal nerve regenerationand unsatisfied functional recovery.Objective To investigate the efficacy of the SCs-seeded scaffold forpromoting nerve regeneration and functional recovery.Methods In the present study, SCs were introduced into the CCH scaffoldin vitro, and the adhesion, migration and proliferation of SCs within the scaffoldwere observed at the predefined time points (3d,1w and2w). Furthermore, theSCs-seeded scaffold was used to bridge a15-mm-long sciatic nerve defect in rats,with an attempt to provide a more permissive environment for axonal outgrowth. Rats with nerve autografts or L-CCH scaffolds alone were taken as positivecontrol or negative control, respectively. Both morphological analysis andfunctional assessment were employed to evaluate axonal regeneration andfunctional recovery.Findings The findings showed that SCs adhered and migrated into theL-CCH scaffold and displayed a longitudinal arrangement in vitro. Axonalregeneration as well as functional recovery was in the similar range between theSCs-seeded scaffold and the autograft groups, which were superior to those in theL-CCH scaffold alone group.Conclusion These findings indicate that the SCs-seeded CCH scaffold,which resembles the microstructure as well as the permissive environment ofnerve autografts, holds great promise in nerve regeneration therapies.