The Effect Of PFTBA-enriched Hydrogel On Promoting Peripheral Nerve Regeneration And Its Mechanisms

Peripheral nerve injury, especially nerve defect, is a worldwide challenge for currentclinical practice. Although nerve autografting is still considered as the "gold standard",this approach was limited by availability of donor nerves and postoperative complicationsof donor sites. Thus, tissue engineering nerve scaffolds have been considered as promisingalternatives to nerve autograft. However the inner portion of nerve scaffolds is underhypoxia condition due to large size and lower vascularization of scaffold. Manyresearchers suggested that low oxygen level in the inner portion of nerve scaffolds coulddecrease the viability of migratory SCs and functioning of newly regenerated tissue. Onthe other hand, insufficiency of oxygen supply within scaffold also could decrease theseeding cell viability and function. So it is important to increase the level of oxygen within the scaffold for improving axonal regeneration and functional recovery. Thus far,strategies to increase tissue oxygenation at the nerve repair site have generally focused onvascularization and hyperbaric oxygenation (HBO). However, the vascularization of nervescaffold need a long time and HBO treatment also has some limitations, includinginefficient supply in injured perineural or neural oxygen tension due to ischemia aftertrauma and the possibly negative effects of fluctuating oxygen tensions associated withinterval oxygenation chamber treatment schedules. For overcoming above drawbacks, it isnecessary to find a better strategy of increasing the level of oxygen within the nervescaffolds directly. In the present study, we fabricate chitosan-collagen conduit and asynthetic oxygen carrier (PFTBA)-fibrin hydrogel. Then we injected PFTBA-enrichedhydrogel supplemented with or without OECs into chitosan-collagen conduit, which wasused to bridge a12mm or15mm long nerve defect in vivo. We also investigate the effectof PFTBA on survival and function of OECs under hypoxia in vitro. The purpose ofcurrent study is to investigate the effects of PFTBA-enriched hydrogel on nerveregeneration, and explore the underlying mechanism of PFTBA in the treatment ofperipheral nerve defects. The whole studies were divided into four parts:Part Ⅰ: The Fabrication and corresponding properties evaluation of a novelCollagen-Chitosan conduitBackgrounds: The artificial nerve conduits have been demonstrated to be realisticalternatives to autologous nerve grafts for nerve regeneration in animal models andclinical experiments. To date, many nerve conduits have been approved by FDA and usedin clinics. However the wall of most conduit is sealed and non-biodegradable. Althoughabove desigh could protect regenerating axons from infiltrating scar tissue and provideregenerative microenvironment for the injured axons to regenerate, it also could block thetransportation of nutrients, oxygen and metabolic products, and needed a second operationto remove residual implant. Therefore, the fabrication of biological and biodegradableporous nerve conduit holds great potential in improving the outcome of nerve defect.Ojective: To fabricate the biodegradable Collagen-Chitosan conduit and further evaluatethe corresponding properties of above conduit.Methods: The collagen-chitosan conduits with different physical and chemical properties were fabricated in the present experiment. The inner structure of above conduits wasobserved under the scanning electronic microscope. Then a longitudinal comparisonanalysis was performed to determine the optimum parameters. Thereafter, the optimizedconduit filled with fibrin gel was implanted to examine the biological property in vivo.Results: With increasing of collagen component from0%to100%, the diameter ofmicropores decreased. The porosity of collagen-chitosan (4:1) nerve conduit was92.72±1.83%. In addition, the degradation rate of above conduit is moderate in vitro. Afterimplantation, collagen-chitosan (4:1) nerve conduit showed a good biocompatibility andcould pave a path for regenerating axons.Conclusion: The collagen-chitosan (4:1) nerve conduit has good physical, chemical andbiological properties and holds great potential in promoting the nerve regeneration.Part Ⅱ: The efficacy of nerve conduit filled with PFTBA-enriched hydrogel inbridging a12mm long sciatic nerve defect in rats and its mechanismsBackgrounds: Tissue-engineering nerve conduits have been demonstrated to be realisticalternatives in bridging large nerve defects. However, the low oxygen availability withinthe nerve conduits is still an obstacle for axonal regeneration. Many researchers suggestedthat increasing tissue oxygenation in the inner portion of conduits held the potential toimprove axonal regeneration and functional recovery after PNI. Most of previous workhas demonstrated that hyperbaric oxygenation (HBO) possess a beneficial effect on nerveregeneration. However, HBO treatment also has some limitations. For overcoming thesedrawbacks, one option is to increase the level of oxygen within the nerve conduits directlyby a synthetic oxygen carrier-PFTBA-enriched hydrogel. Combining aPFTBA-enriched-hydrogels mixture with tissue-engineered nerve conduits may enhanceaxonal regeneration and functional recovery after large nerve defects.Objective: To investigate the efficacy of the chitosan-collagen conduit filled withPFTBA-enriched fibrin matrix on axonal regeneration and functional recovery, andexplore the underlying mechanism of PFTBA in the treatment of peripheral nerve defects.Methods: The nerve conduit containing PFTBA-enriched hydrogel was used to bridge a12mm sciatic nerve defect in rats. The control rats were bridged with nerve conduits filledwith fibrin matrices without PFTBA. The effect of PFTBA-enriched hydrogel on axonal regeneration was examined using morphometric analysis, immunohistochemical stainingand FG retrograde labeling. The effect of PFTBA-enriched hydrogel on functionalrecovery was investigated by electrophysiological examination, behavioral study andmorphological appearance of target muscle. The underlying mechanism of PFTBA onnerve regeneration was analyzed by quantification of microvessel density, RT-PCR andWestern blot assay of BDNF, NGF, VEGF and S-100.Results: Although the beneficial effect of nerve conduits with PFTBA on nerveregeneration was still inferior to that of an autograft, we found that the nerve conduits withPFTBA showed better performance in promoting nerve regeneration than those withoutPFTBA. In addition, the mRNA and protein levels of BDNF, NGF and S-100were alsofound to be up-regulated in the nerve conduit by PFTBA. The above findings indicate thatPFTBA-enriched fibrin hydrogel is capable of enhancing survival and function ofmigrated SCs and further improving sciatic nerve regeneration.Conclusion: The PFTBA-enriched hydrogel is capable of improving the regenerativemicroenvironments within the nerve conduits for the injured axons by increasing theoxygen transfer directly, further enhancing survival and function of migrated SCs, henceimproving sciatic nerve regeneration.Part Ⅲ: The effect of PFTBA on olfactory ensheathing cell under hypoxia in vitroBackgrounds: With the development of nerve tissue engineering, the researchers foundthat a higher nerve functional recovery will be obtained by introducing the supportive cellsinto the nerves scaffolds. Amounts of studies have demonstrated that OlfactoryEnsheathing cell (OECs), as as a specialized glial cell, could enhance functional recoveryof the injured peripheral nerves. However insufficiency of oxygen supply within scaffoldcould decrease the viability and function of seeding OECs, and further reducing theoutcomes of sciatic nerve regeneration. So it is important to investigate the effect ofhypoxia on OECs, and find an effective method to improve the hypoxia environment inthe inner portion of nerve scaffolds.Objective: To investigate the effect of hypoxia on OECs and examine the protection ofPFTBA on OECs under hypoxia in vitro.Methods: After isolation and purification, Olfactory Ensheathing cells were divided into four groups: normal cultivated group (group A), normal cultivated+PFTBA group (groupB), hypoxic group (group C) and hypoxic+PFTBA group (group D), and then cultivatedfor12h and24h. According to the concentration of PFTBA, group B or D was divided into5%PFTBA group (group B1and D1) and10%PFTBA group (group B2and D2),respectively. The apoptotic ratio of Olfactory Ensheathing cells was examined by flowcytometry assay. The morphological appearance of Olfactory Ensheathing cells wasinvestigated by scanning electron microscopy examination. The activity of OlfactoryEnsheathing cells was characterized using DAPI staining and a MTT assay. The mRNA,protein and secretion levels of NGF and BDNF in Olfactory Ensheathing cells wereassayed by RT-PCR, Western blotting and ELISA assay.Results: Twelve hour after hypoxia, the apoptotic stress of olfactory ensheathing cells wasfound and apoptotic ratio was increased gradually. In addition, the activity of olfactoryensheathing cells decreased obviously, the expression and secretion of BDNF and NGFwere also significantly lower. Interestingly,10%PFTBA could significantly reduce thenumber of apoptosis cells, increase the activity of cells, and maintain the mRNA, proteinand secretion levels of NGF and BDNF in Olfactory Ensheathing cells.Conclusion: The10%concentration of PFTBA is capable of improving the hypoxiaenvironment in vitro, and further promoting the survival and functions of olfactoryensheathing cell under hypoxia.Part Ⅳ: The efficacy of nerve conduit filled with PFTBA-OECs enriched hydrogelin bridging a15mm long sciatic nerve defect in rats and its mechanismsBackgrounds: More and more researchers found that a higher nerve functional recoverywill be obtained by introducing the supportive cells into the nerves scaffolds. However theinner portion of nerve scaffolds is under hypoxia condition due to large size and lowervascularization of scaffold. Many researchers suggested that low oxygen level in the innerportion of nerve scaffolds could decrease the viability and functions of seeding cell, andfurther reducing the outcomes of sciatic nerve regeneration. So it is important to keepOECs with not only more even cell distribution, but also higher activity and function.Objective: To investigate the efficacy of the chitosan-collagen conduit filled withPFTBA-OECs enriched hydrogel on axonal regeneration and functional recovery, and explore the underlying mechanism of PFTBA in the treatment of peripheral nerve defects.Methods: The nerve conduit containing PFTBA-(GFP-expressing OECs) enrichedhydrogel was used to bridge a15mm sciatic nerve defect in rats. The control rats werebridged with nerve conduits filled with GFP-expressing OECs enriched hydrogel withoutPFTBA. The effect of PFTBA-OECs enriched hydrogel on axonal regeneration wasexamined using morphometric analysis and FG retrograde labeling. The effect ofPFTBA-OECs enriched hydrogel on functional recovery was investigated byelectrophysiological examination, behavioral study and morphological appearance oftarget muscle. The underlying mechanism of PFTBA on nerve regeneration was analyzedby quantification of microvessel density, quantification of GFP positive cells, and RT-PCRassay of BDNF, NGF, VEGF and S-100.Results: Although the beneficial effect of the combined PFTBA group on nerveregeneration was still inferior to that of an autograft, we found that the chitosan-collagenconduit filled with PFTBA-OECs enriched hydrogel showed better performance inpromoting nerve regeneration than those without PFTBA. In addition, PFTBAsignificantly increased the number of GFP-expressing OECs in the combined PFTBAgroup, and the mRNA levels of BDNF, NGF and S-100were also found to beup-regulated in the combined PFTBA group. These findings indicate that PFTBA iscapable of improving the survival and function of OECs under the hypoxic environment inthe early time after implantation, and further improving axonal regeneration and functionalrecovery.Conclusion: The PFTBA is capable of improving the hypoxia conditions in the early timeafter implantation, further enhancing survival and function of seeding OECs, henceimproving nerve regeneration.