Enhanced Survival Of Schwann Cells By A Synthetic Oxygen Carrier Promotes Sciatic Nerve Regeneration And Functional Recovery

The development of artificial nerve conduits as a promising alternative to autograft has been an intense focus of research interest in the peripheral nerve injury field.To further facilitate axon regeneration,supportive cells have been implanted into nerve scaffolds to provide biomolecules and hospitable microenvironment for regenerating axons.However,increasing studies have demonstrated that the biological activity of SCs seeded in nerve scaffolds decayed subsequently due to local hypoxic environment.In addition,slow vascularization and limited oxygen diffusion into the nerve grafts at the initial stage of transplantation lead to insufficient oxygen supply to SCs,which significantly limits their performance in promoting nerve regeneration.Thus,striving for a new strategy to increase oxygenation at the local injury site to maintain survival and function of SCs within the nerve scaffold holds the potential to improve axonal regeneration and functional recovery in the repair of nerve defects.To provide sustained oxygen for SCs before blood vessel ingrowth into scaffolds,one option is to increase the level of oxygen within the nerve scaffolds.Perfluorotributylamine(PFTBA),belongs to the family of perfluorocarbons(PFCs),has a high solubility of oxygen,which is 15-20 fold than that in water and 2-3 fold than that in blood.It has been proven that PFTBA has a linear relationship between oxygen partial pressure and oxygen concentration.Thus far,PFTBA has drawn great attention from researchers in the area of biomaterials for tissue restoration and regeneration.In the present study,two modalities of synthetic oxygen carrier were successfully established,which were capable of promoting functional recovery when they were applied together with nerve scaffold.The two modalities of ES were listed as follows:(1)We prepared nerve conduits filled with PFTBA-SC enriched fibrin hydrogel,which were used to bridge 15-mm-long sciatic nerve defects in rats.(2)We fabricated the core-shell structure using coaxial electrospinning technique,and developed a coaxial electrospun fiber scaffold containing PFTBA in the core and PCL in the shell.The scaffolds were used to bridge a 17-mm-long sciatic nerve defect in rats.We found that the PFTBA-enriched oxygen carrier was able to promote cell adhesion,migration,and proliferation under hypoxic conditions.The PFTBA-SC scaffold was capable of promoting axonal regeneration and remyelination of the regenerated axons.The present study enriches the cellular tissue engineering for nerve regeneration,and provides theoretical supports for the application of PFTBA in the treatment of critical nerve gaps.Part ?: The effect of synthetic oxygen carrier-enriched fibrin hydrogel on Schwann cells under hypoxia condition in vitroBackgrounds.Schwann cell,which plays a key role in peripheral nerve regeneration,is one of the most classic supportive cells in neural tissue engineering.However,the biological activity of SCs seeded in nerve scaffolds decays subsequently due to local hypoxia induced by ischemia.Objective.To investigate whether a synthetic oxygen carrier-enriched fibrin gel would provide a sustained oxygen release to cultured SCs in vitro for overcoming a temporary(48 h)oxygen deprivation.Methods.Perfluorotributylamine(PFTBA)-based oxygen carrying fibrin gel was prepared to provide oxygen for SCs under normoxic or hypoxic conditions.The dissolved oxygen within the culture media was measured by a blood-gas analyzer to quantify the time course of oxygen release from the PFTBA-enriched fibrin gel.SCs were cultured in the presence or absence of PFTBA-enriched fibrin gel under normoxic or hypoxic conditions.The tolerance of SCs to hypoxia was examined by a cell apoptosis assay.The growth of cells was characterized using S-100 staining and a CCK-8 assay.The migration of cells was examined using a Transwell chamber.The m RNA of brain-derived neurotrophic factor(BDNF),nerve growth factor(NGF),glial cell derived neurotrophic factor(GDNF),neural cell adhesion molecule(N-CAM)and vascular endothelial growth factor(VEGF)in SCs were assayed by RT-PCR.In addition,SCs cultured in 3D PFTBA-enriched hydrogel were characterized by Live/Dead staining and the m RNA levels of BDNF,NGF,GDNF,N-CAM and VEGF were assayed by RT-PCR.Results.The PFTBA-enriched fibrin hydrogel was able to promote cell adhesion,migration,and proliferation under hypoxic conditions.Interestingly,PFTBA applied through the fibrin hydrogel dramatically enhanced the m RNA of BDNF,NGF,GDNF,N-CAM and VEGF under hypoxic condition.Conclusion.This study showed that PFTBA-enriched fibrin hydrogel could provide a sustained oxygen release to cultured SCs for overcoming a temporary oxygen deprivation under hypoxic conditions.In addition,the use of PFTBA was capable of enhancing SCs survival and function under hypoxia condition in vitro.Part ?: Enhanced in vivo survival of Schwann cells by a synthetic oxygen carrier promotes sciatic nerve regeneration and functional recoveryBackgrounds.Local hypoxia in the early stages of peripheral nerve injury is a challenge for axonal regeneration.In addition,absence of a vascular system at the initial stage of transplantation makes it difficult to provide enough oxygen for SCs,which significantly limits their performance in supporting nerve regeneration.Objective.To investigate the possibility of enhanced in vivo survival of Schwann cells by a synthetic oxygen carrier could promote sciatic nerve regeneration and functional recovery.Methods.Perfluorotributylamine(PFTBA)-based oxygen carrying fibrin hydrogel was prepared and injected into Schwann cell(SC)-seeded collagen-chitosan conduits to increase oxygen supply to SCs within the conduits.The conduit containing PFTBA-SC gel was then applied to bridge a 15-mm sciatic nerve defect in rats.In the Vehicle group,the conduit was only filled with fibrin hydrogel;in the SC group,the conduit was filled with fibrin-SC mixture;in the PFTBA group,the conduit was filled with fibrin-PFTBA mixture;in the PFTBA + SC group,the conduit was filled with fibrin-PFTBA mixture and SCs.The effect of application of scaffold on axonal regeneration was examined using morphometric analysis and retrograde labeling.Their effect of functional recovery was investigated by electrophysiological study,behavioral study and histological appearance of target muscle.Results.Most of the GFP-labeled SCs initially seeded in the PFTBA hydrogel remained alive for approximately 28 days after in vivo implantation.The number of SCs was significantly higher in the PFTBA-SC scaffold than that in the SC scaffold without PFTBA.In addition,nerve regeneration and functional recovery were examined after nerve injury repair.The PFTBA-SC scaffold was capable of promoting axonal regeneration and remyelination of the regenerated axons.The PFTBA-SC scaffold was able to accelerate the recovery of motor and sensory function of the regenerating nerves.Electrophysiological analysis showed area under the curve of compound muscle action potential and nerve conduction velocity were also improved,and gastrocnemius muscle atrophy was partially reversed by PFTBA-SC scaffold.Furthermore,microvessel density analysis showed PFTBA-SC composites were beneficial for microvascular growth,which provided sustained oxygen for regenerating nerve in the later stages of nerve regeneration.Conclusion.Enhanced survival of SCs by PFTBA is capable of promoting sciatic nerve regeneration and functional recovery,which provides a new avenue for achieving better functional recovery in the treatment of peripheral nerve injuries.Part ?: Enhanced survival of Schwann cells by a synthetic oxygen carrier through coaxial electrospinning promotes sciatic nerve regeneration and functional recoveryBackground.Local ischemia and hypoxia are challenges for fabrication of cellular grafts and treatment of peripheral nerve injuries.Absence of a vascular system at the initial stage of transplantation makes it difficult to provide enough oxygen for cells,which significantly limits their performance in nerve regeneration.Objective.To investigate the possibility of enhanced survival of Schwann cells by a synthetic oxygen carrier through coaxial electrospinning promotes sciatic nerve regeneration and functional recovery.Methods.Perfluorotributylamine(PFTBA)-based oxygen carrying system was prepared through coaxial electrospinning technique.Schwann cells were cultured in 2D and 3D matrix based on electrospun fibers.Then,the system was applied to bridge a 17-mm sciatic nerve defect in rats.SCs-gel mixture without PFTBA was injected into the conduit without PFTBA to serve as Conduit+gel group.SCs-gel mixture without PFTBA was injected into the conduit with PFTBA to serve as PFTBA conduit+gel group.SCs-gel mixture with PFTBA was injected into the conduit without PFTBA to serve as Conduit+PFTBA gel group.The SC-gel mixture with PFTBA was injected into the conduit with PFTBA to serve as PFTBA conduit+PFTBA gel group.The effect of application of scaffold on nerve regeneration was examined using morphometric analysis and retrograde labeling.Their effect of functional recovery was investigated by behavioral study,electrophysiological study and histological appearance of target muscle.Results.In vitro study showed that PFTBA was capable of providing oxygen to SCs for their survival in 2D and 3D culture system in the hypoxic environment.In vivo study showed that most of the GFP-expressing SCs initially seeded in the PFTBA conduit remained alive for 14 days after implantation.The number of SCs was significantly higher in the PFTBA scaffold than that without PFTBA.Furthermore,we found that axons in PFTBA oxygen carrier scaffold exhibited better regeneration,remyelination and morphological appearance.In addition,electrophysiological and behavioral analyses showed PFTBA composites were beneficial for accelerating the recovery of motor and sensory function of the regenerating nerves.Conclusion.Based on coaxial electrospinning technique,PFTBA oxygen carrying system is capable of enhancing survival of SCs and promoting sciatic nerve regeneration and functional recovery,which provides a new strategy for fabricating cellular scaffolds to achieve better functional recovery in the treatment of peripheral nerve injuries.