A Novel Microsphere With Oxygen Delivery Capacity Improve The Hypoxic Microenvironment After Peripheral Nerve Injury

Peripheral nerve system(PNS)makes CNS accurately dominate our daily activities by connecting the spinal cord,brain with target organ.The treatment of peripheral nerve injury(PNI),especially lengthy peripheral nerve defects is challenging both in the field of regenerative medicine and daily clinical works.Althogh the autografting is still “the golden standard” of the treatment of lengthy peripheral nerve defects,it always be limited by the availability of donor nerve,the postoperative complications of donor sites and the severe immunologic rejection.As the great development of tissue engineering in the field of regenerative medicine,the preparation of artificial nerve scaffolds to bridge the lengthy nerve gaps has became the research trends at present.However,the limitation of key factor which effectively promote the nerve regeneration,the simple using of nerve scaffolds cannot provide the beneficial environment for the regrowth of axons.Recently,many nerve conduits with use of supportive cells were synthesized,which can provide guiding and supporting cues to improve the quality of regeneration after nerve injury.However,the low oxygen status was found in the conduits in the early period after the implantation,which directly leads the death or loss of function of the supportive cells,limited the regeneration and functional recovery after the surgery.In the previous study,we synthesized a hydrogel as the carrier of perfluorotributylamine(PFTBA)to improve the hypoxic microenvironment,and then enhance the survival of the olfactory ensheathing cells(OECs).However,with the deepening of the research,as the PFTBA consumed,the rapidly decreasing of the viability and activity of seed cells was found.The purpose of this study is to develop a novel bilyar microsphere by chitosan and PLGA as the carrier of PFTBA,then investigate the effects of this microspheres(MSs)on improve the survival of OECs under hypoxia in vitro and in vivo.The whole studies were divided into three parts:Part? The fabrication and physicochemical properties evaluation of Cs-PLGA microspheres with PFTBABackgrounds:In the early periods of the transplantation of seed cells,the survival of seed cells cannot be maintained because of the low oxygen level within conduits and the curative effects cannot achieve the expectation.In the previous study,we synthesized a hydrogel as the carrier of PFTBA to improve the hypoxic microenvironment,and then enhance the survival of the OECs.However,as the PFTBA consumed,the raplidly decreasing of the viability and activity of OECs was found.Therefore,to fabricate an ideal carrier for PFTBA is important.Objectives: To fabricate the Cs-PLGA microspheres with different density of PFTBA,and to verify their physicochemical properties and characteristics of oxygen release kinetics.Methods: Firstly,we use chitosan and PLGA as the core materials to fabricate the Cs-PLGA with different concentrations(5w/v%,10 w/v% and 20 w/v%)of PFTBA.The morphological appearance of OECs was observed by scanning electron microscope and immunofluorescent staining assay.After that,we evaluated the oxygen release kinetics of microspheres with different concentrations of PFTBA to preliminarily filtrate the appropriate content of PFTBA for the further research.In addition,we further fabricated the Cs MSs and PLGA MSs,then,introduced different kinds of MSs with 10% PFBTA into PCL nerve conduits to bridge the sciatic nerve defects in rats.Then,we detected the oxygen level of different groups each day after the 1st day of the transplantation to analyse the oxygen release kinetics of different kinds of microspheres.Result: Under the evaluation of scanning electron microsope(SEM)and immunofluorescent staining assay,we found that the Cs-PLGA microspheres have the round appearance and bilayer structure and the PFTBA was successfully wrapped in each MS.The mean diameter of PLGA MSs was 1.03-1.29?m;the mean diameter of chitosan MSs was 35.34-40.18?m.The degradation rate of the MSs achieved to nearly 60% after 12 weeks.In addition,the oxygen content of MSs with 10w/v% and 20w/v% PFTBA were significantly higher than that in MSs with 5w/v% PFTBA,and there are no differences in the comparison between these two groups.Furthermore,we found that the releasing rate of Cs MSs was higher than PLGA MSs,but the PLGA MSs can release oxygen for the longer time than Cs MSs.Conclusion: The Cs-PLGA MSs with PFTBA have obvious spherical bilayer structure with favourable mean diameters,degradation rate,and the PFTBA was wrapped in each MS.The oxygen in MSs can be released over 28 days.In addition,the Cs-PLGA MSs was more suitable than Cs MSs and PLGA MSs as an oxygen carrier in the further research.Part?The protective and supportive effects of Cs-PLGA MSs with PFTBA on OECs under hypoxia in vitroBackgrounds:As the rapidly development of the nerve tissue engineering,the scholars found that the introduction of OECs is capable of providing a beneficial microenvironment to improve the peripheral nerve regeneration by secreting various of neurotrophic factors.However,the researchers found that those cells which introduced into nerve conduits survived no longer than 8 weeks.In addition,it has also been reported that long-term cultured OECs failed on supporting regeneration and neuronal survival.Recently,many studies demonstrated that hypoxia might be one of the major causes for the death of cells in the early periods of transplantation.Therefore,it is a great challenge to improve the hypoxia within the conduits to enhance the survival of the seed cells.Objectives: To investigate the protective and supportive effects of Cs-PLGA MSs with different concentration of PFTBA on OECs under hypoxia in vitro.Methods: After the isolation and purification,all OECs were divided into 8 groups by different cultivating environments:(1)Normoxia group(Group A);(2)Normoxia+MSs with 5% PFBTA group(Group B);(3)Normoxia+MSs with 10% PFBTA group(Group C);(4)Normoxia+MSs with 20% PFBTA group(Group D);(5)Hypoxia group(Group E);(6)Hypoxia+MSs with 5% PFBTA group(Group E);(7)Hypoxia+MSs with 10% PFBTA group(Group F);(8)Hypoxia+MSs with 20% PFBTA group(Group F),and then cultivated for 24 and 48 h.The survival and cytoactive of OECs were evaluated by Flow-cytometry assay,Presto Blue assay and mmunofluorescent staining assay.The morphological appearance of OECs was observed by scanning electron microscope.The m RNA and secretion levels of NGF,BDNF and VEGF in Olfactory Ensheathing cells were assayed by RT-PCR and ELISA assay.Result: After 24 hours cultivated under hypoxia,the apoptosis of OECs occurred and the apoptotic ratio was increased gradually.Meanwhile,the viability of OECs decreased rapidly.The expression and secretion of NGF,BDNF and VEGF were also lower than those in normoxic condition.However,the Cs-PLGA MSs with PFTBA can significantly enhance the survival and viability of OECs under hypoxia,and the morphological appearances of OECs were similar as those under normoxia.Furthermore,the expression and secretion of neurotrophic factors were also up-regulated.Interestingly,the MSs with 10w/v% and 20w/v% PFTBA have shown more effectively influences on OECs under hypoxic condition than the MSs with 5w/v% PFTBA.In addition,there were no significant differences between 10w/v% PFTBA group and 20w/v% PFTBA group.Conclusion: The Cs-PLGA MSs with 10w/v% PFTBA were able to improve the survival and viability of OECs by improving the oxygen level in the hypoxic environment.And 10w/v% was the optimal content of PFTBA in MSs for the further research in vivo.Part ? OECs-MSs with PFTBA composite system for the promotion of sciatic nerve regeneration in ratsBackgrounds:It has been reported that the nerve conduits which were filled in seed cells showed better curative effects when they were used to bridge the lengthy nerve gaps.However,in the early periods after transplantation,the hypoxic condition within the conduits directly resulted in the death or loss of function of seed cells,and greatly limited the nerve regeneration and functional recovery after the treatment of nerve defects.In the previous study,we introduced the PFTBA hydrogel and OECs into nerve conduits to successfully bridge the 15mm-long sciatic nerve gap in rats.However,with the deepening of the research,we found that the PFTBA was released rapidly after transplantation because of the initial burst release of the hydrogel,so that the oxygen content in the inner portion of the conduits cannot maintained at a high level for a long time.Therefore,it is important to further optimize the carrier of PFTBA to increase the oxygen concentration in the early period and then maintain at a high level for a long time.Objectives: To investigate the efficacy of OECs-MSs with PFTBA composite system on improving the nerve regeneration and functional recovery after transplantation.Methods: The SD-rats were divided into 5 groups:(1)Autografting group(A group);(2)Conduits group(CF group);(3)Conduits filled with OECs group(CFO group);(4)Conduits filled with MSs group(CFM group);(5)Conduits filled with OECs and MSs group(CFOM group).And then,we used the PCL nerve conduits which were filled with different factors to bridge the 15mm-long sciatic nerve gap in rats.The effect of axons regeneration was evaluated by morphometry analysis,Fluoro-Gold retrograde tracing and immunofluorescence assay.The level of functional recovery was assessed by Behavioral analysis,Electrophysiological assessment and Histological analysis of target muscles.Result: The only use of OECs and Cs-PLGA MSs with PFTBA can improve the nerve regeneration and functional recovery,respectively,but the effects were inferior to those in Autogfrafting group.However,the OECs-(Cs-PLGA MSs with PFTBA)composite system can further promote the nerve regeneration and functional recovery after the transplantation,and there are no significant differences imparison with those in autografting group.Conclusion: The OECs-(Cs-PLGA MSs with PFTBA)composite system was capable of promoting the sciatic nerve regeneration and functional recovery by improving the hypoxia microenvironment then enhancing the survival and viability of seed cells after transplantation.