CS-HEC-HA/GP Thermosensitive Hydrogel For Human Umbilical Cord MSC Transplant In Traumatic Brain Injury Therapy

Traumatic brain injury(TBI)is a nervous system diseases caused by external shock or puncture on the brain,along with high mortality and high morbidity characteristics.Presently,there is no effective treatment in clinic.TBI will leave a variety of complications,including motorial,cognitive,and social communication disorders,which bring great burden to the patients’ families and whole society.Stem cell transplantation therapy brings a hope to this problem.Human umbilical cord mesenchymal stem cells(hUC-MSCs)derived from human umbilical cord tissue,which has the characteristics of no ethical arguments,widely source,multi differentiation potential,and low immunogenicity,is considered as the perfect seed cells in tissue engineering.However,the low survival rate and insufficient quantity of transplanted stem cells still restrict the application of stem cell therapy.In order to improve the biological activity and therapeutic effect of stem cells,it is essential to create an appropriate microenvironment for supporting the growth and survival of transplanted stem cells.Tissue engineering is an emerging discipline by using cells and scaffold with good biocompatibility and biodegradable for transplantation therapy.The scaffold could mimic the extracellular microenvironment and provide a physical support for transplanted stem cells,therefore it was supposed to have the possibility to solve the problem of low survival rate and insufficient quantity of transplanted stem cells.Hydrogels possess a similar structure with extracellular matrix,good plasticity and absorbability.Hydrogels are one of the most commonly used neural scaffold materials.In this study,we fabricate a novel composite thermosensitive hydrogel CS-HEC-HA/GP,and loaded hUC-MSCs as an injectable scaffold aiming to treat TBI.The physicochemical and biological characters of this hydrogel,including gelation time,water content,degradation property,microtopography,rheological behavior,and biocompatibility were investigated in a series of tests.The therapeutic effect of CS-HEC-HA/GP thermosensitive hydrogel loaded with hUC-MSCs was also evaluated using a moderate TBI model in SD rats.To provide new strategies and methods for the treatment of TBI by stem cell transplantation.MethodsPart I: Fabrication and characterization of hydrogelsCS/GP,CS-HEC/GP and CS-HEC-HA/GP composite hydrogels were prepared by sol-gel method.Inverted tube test was used to detect the gelation time of hydrogels in each group.The moisture content and degradation rate of hydrogels were determined by freeze-drying method.Scanning electron microscope(SEM)was used to observe the internal morphology of these hydrogels.The elastic modulus of hydrogel was measured by rheometer platform.The immunophenotype of hUC-MSCs was identified by flow cytometry.Live/Dead fluorescence staining and CCK-8 was used to study the cellular survival,elongation,and proliferation.The three-dimensional distribution of hUC-MSCs within hydrogel was observed by confocal laser scanning microscope.Part II: The therapeutic effect of hydrogel loaded with hUC-MSCs on the TBI ratsCS-HEC-HA/GP hydrogels were transplanted subcutaneously into rats,and their injectable,biocompatibility and biodegradability were verified by observation,quality analysis and HE staining.The model of traumatic brain injury was established by Feeney’s weight-drop.The rats were randomly divided into Normal saline group(NS),scaffold group(Scaffold),hUC-MSCs group(MSC)and scaffold+ hUC-MSCs group(Scaffold+MSC).The neurological deficits of the rats were observed by mNSS.Novel object recognition test was used to evaluate the cognitive function recovery of rats.Sucrose preference test was used to detect the depression of TBI rats.Morris water maze test was used to evaluate the recovery of learning and memory function in rats.CV staining was used to observe the repair of damaged brain tissue.The survival and migration of hUC-MSCs,and the expression of mature neuron marker(NeuN)and proliferative cell specific marker(Ki67)in the DG region of brain were observed by immunofluorescence staining.The expression of NeuN and neurotrophic factor marker(BDNF)related genes in rat brain was detected by qRT-PCR.The expression of NeuN,BDNF and apoptosis-related protein(Bax)in rat brain was detected by Western Blot.ResultsPart I: Fabrication and characterization of hydrogelsThese chitosan composite hydrogels prepared in this study have a good temperature sensitivity.The gelation time of hydrogel at 37℃ in each group was within 10 min.The gelation time could be shortened by the increase the concentration of HEC or HA.The water content of all hydrogels was above 90% and increased with the increase of HEC or HA.The hydrogels in each group could be degraded in the artificial cerebrospinal fluid,and the degradation quality was about 30% on day28.There was no significant difference of the degradation rate between each hydrogel.Hydrogels presented a three dimensional porous network structure.Except for CS/GP hydrogel,the modulus of hydrogels in each group was ranged from 100 to 200 Pa.The modulus increased with the increase of oscillation frequency.The curve of elastic modulus was smooth and stable.The hUC-MSCs meet the identification criteria of mesenchymal stem cells by flow cytometry.The hUC-MSCs could be attached and elongated on the surface of CS-HEC/GP and CS-HEC-HA/GP hydrogels within 6 h.The biocompatibility of the hydrogel with 3% GP and 0.2% HA was significantly higher than that of other hydrogels.hUC-MSCs could uniformly distribute within hydrogels,and present a high survival and proliferate rate.The cellular proliferation rate in hydrogels with 3% GP and 0.2% HA was significantly higher than that of other groups.Part II: The therapeutic effect of hydrogel loaded with hUC-MSCs on the TBI ratsCS-HEC-HA/GP hydrogel has good injectability,biocompatibility and biodegradability.Compared with the control groups,the motor function,cognitive function,learning and memory function,depression of rats in the treatment groups were significantly improved,and the Scaffold+MSC group had a significantly better effect than the MSC group.Hydrogel scaffold could increases the retention and survival of hUC-MSCs in lesion location.Stem cell transplantation could significantly increase the expression level of NeuN,BDNF gene and protein in the brain of rats,reduce the expression of Bax protein,and increase the number of Ki67 positive cells.And the Scaffold+MSC group had a better effect than the MSC group.Conclusion1.CS-HEC-HA/GP composite hydrogel has good biocompatibility and biodegradability;2.CS-HEC-HA/GP composite scaffold loaded with hUC-MSCs makes a greater contribution to the recovery of motor,learning,and memory abilities compared with the injected pure hUC-MSCs and other control groups.In addition,the results of WB,qRT-PCR,and immunofluorescence staining suggested that hUC-MSCs laden CS-HEC-HA/GP hydrogel transplantation not only increases the retention and survival of hUC-MSCs in lesion location,but also makes a positive contribution to the survival/proliferation of endogenous nerve cell by nutrition supply and apoptosis suppression.