A Dual-enzymatically Cross-linked Hyaluronic Acid Hydrogel Loaded With BMSC And NGF Improve Neurological Function Recovery Of Traumatic Brain Injury In Mice

Traumatic brain injury(TBI)is a kind of severe central nerve injury disease,which has a high incidence in the world and a high mortality and disability rate.After the occurrence of TBI,the pathological manifestations are complex,mainly divided into primary injury and secondary injury,which will cause a large number of neuron loss,brain tissue damage,blood brain barrier destruction,accompanied by severe inflammatory reaction,and eventually lead to serious neurological dysfunction.The main research direction of TBI treatment is to alleviate the harm of secondary injury and promote the recovery of nerve function after injury.Stem cells have high proliferative activity and differentiation potential.Stem cell-based transplantation,as a new therapeutic method and strategy,provides new hope for promoting nerve repair after TBI.Bone marrow derived mesenchymal stem cells(BMSC)have many advantages,such as wide source,mature isolation technology,low immunogenicity and multidirectional differentiation potential.Although stem cells have shown great potential as a cell therapy in the treatment of nerve injury,the major challenges in stem cell therapy are the loss and low survival rate of stem cells at the transplant site.The use of tissue engineering technology to construct biological scaffolds to deliver stem cells to the damaged area is expected to improve the survival efficiency after stem cell transplantation.Injectable hydrogel scaffolds have been widely used in tissue engineering because of their good biocompatibility,biodegradability and injectability.Hyaluronic acid is one of the major components of the extracellular matrix,which is involved in a variety of cell life processes,including movement,angiogenesis and signal transduction.Hyaluronic acid hydrogels have high water content,loose porous structure and high nutrient permeability.As a scaffold material,it can improve the microenvironment of stem cell survival,promote the survival and proliferation of exogenous stem cells in the injured site,and ultimately improve the therapeutic effect of stem cells.Nerve growth factor(NGF)can promote the growth,development,differentiation and maturation of central and peripheral neurons,maintain the normal function of the nervous system,and speed up the repair of the injured nervous system.Nerve growth factor can stimulate the survival and maturation of neurons during the development of the peripheral nervous system,and has a certain protective effect on neurons in the brain.In basic research and clinical treatment,it has been used as a therapeutic agent for the recovery and maintenance of nerve function.The in-situ injectable hydrogels catalyzed by Galactose oxidase(Gal Ox)and Horseradish peroxidase(HRP)have the advantages of fast gelling speed,high catalytic specificity and mild reaction conditions.Injectable hydrogels can encapsulate cells and bioactive molecules for precise filling of irregular tissue defects through minimally invasive implants.The three-dimensional porous structure of hyaluronic acid hydrogels can provide a physical environment for the attachment and growth of stem cells and facilitate the inclusion of stem cells and growth factors for transplantation therapy.Meanwhile,the physical and chemical properties of hydrogels are also involved in regulating the biological functions of stem cells,such as water content,swelling rate and mechanical strength.These properties indicate the potential of hyaluronic acid hydrogels catalyzed by two enzymes in brain tissue engineering.ObjectivesIn this study,we use the Tyramine(Tyr)to modify hyaluronic acid to obtain HT(Hyaluronic acid-Tyramine)polymer,an injectable HT hydrogel was prepared by covalently cross-linking the phenol groups on HT with double enzyme catalysis(Gal Ox/HRP),and its chemical and physical properties were characterized,select the best effect of cell three-dimensional culture and biological compatibility of HT hydrogel parameters.Subsequently,HT hydrogel(HT/BMSC/NGF)coated with BMSC and NGF was orthotopic transplanted to TBI mice,and the therapeutic effect of HT/BMSC/NGF scaffold on TBI mice and its potential molecular mechanisms were systematically studied through the behavioral and molecular experiments.MethodPart Ⅰ:Preparation and characterization of HT hydrogelHT copolymer was synthesized by grafting Tyr covalently onto HA polymer chain using EDC/NHS system.~1H NMR hydrogen spectrum and UV-vis spectra were conducted to detect the HT grafting rate qualitatively and quantitatively.Gal Ox/HRP(Gal Ox=1U/m L;HRP=1U/m L)were used to prepare different concentrations of HT hydrogels,which were named as 0.5%HT,1%HT and 1.5%HT hydrogels respectively.The gelation time of hydrogel was measured by inversion method.The water content,degradation rate and biodegradability of HT hydrogel was determined by freeze drying and weighing method.The microscopic pore structure of the HT hydrogels was characterized by the scanning electron microscopy(SEM),the rotational rheometer was used to test the mechanical properties of the HT hydrogels.CCK-8 was used to test the impact of HT hydrogel extract on the activity of BMSC.The survival of BMSC in HT hydrogel on 1,3 and 5 days was detected by three-dimensional culture technique and fluorescence labeling(Calcein-AM/PI).Ki67 was used to detect the proliferation activity of BMSC in HT hydrogel.Then 0.5%HT was added with different concentrations of NGF(50 ng/m L NGF,100 ng/m L NGF,150ng/m L NGF,200 ng/m L NGF),and the effects of HT/NGF hydrogel extracts on BMSC activity were detected by CCK-8.The effect of NGF on BMSC survival was detected by three-dimensional culture and fluorescence labeling.Ki67 was used to detect the effect of NGF on BMSC proliferation in hydrogel.Part Ⅱ:Effect on nerve repair in TBI mice of HT hydrogel-loaded BMSC and NGF transplantationThe hemolysis test,subcutaneous injection and HE staining test were used to detect the blood compatibility and histocompatibility of the hydrogel.The modified Feeney free-falling method was used to construct moderate TBI model of C57 mice.Then,the mice were transplanted after the model establishment seven days,divided into NS group,HT+NGF group,HT+BMSC group and HT+NGF+BMSC group randomly.After transplantation 1,3,7,14,21,and 28 days,the motor function of TBI mice were evaluated by the modified neurological severity score(m NSS).From day23 to 28 post-transplantation,the Morris water maze experiment was used to evaluate the learning ability of TBI mice.The mice were sacrificed for sampling after 28 days of treatment,and Image J was used to count the area of brain tissue defects in each group.Western blot was used to study the expression of proteins related with inflammation(IL-6),apoptosis(Bax,Bcl-2),neurotrophin(BDNF)and neural differentiation(NFL,NSE,Neu N).Neural regeneration of mouse dentate gyrus(DG region)was detected by immunofluorescence staining.ResultsPart Ⅰ:Preparation and characterization of HT hydrogel1.From the results of UV-vis spectra and 1H NMR spectra,the characteristic signal of phenolic group was observed in HT polymer,while not observed in hyaluronic acid.Data showed that the Tyr was successfully grafted onto HA polymer,and the grafting rate was 69.29μmol/g.2.The injectable HT hydrogel has a three-dimensional network structure and can fill the damaged site accurately.The gelation time of HT hydrogels(0.5%HT,1%HT,1.5%HT)is from 4 min to 8 min.The water content of the three groups of hydrogels was about 98%.All HT hydrogel samples were more stable more than 28 days in PBS,and the enzymatic hydrolysis rate slowed down with the increase of HT content.The swelling rate of HT hydrogel increased with the increase of HT concentration.The swelling rate of 0.5%HT was smaller and the gel structure was maintained well.The elastic modulus of HT hydrogels in the three groups was close to 100 Pa,and the mechanical strength was similar to that of brain tissue.3.CCK-8 was used to detect the effect of different concentrations(0.5%HT,1%HT,1.5%HT)of HT hydrogel extracts on BMSC activity.The results showed that the hydrogels in each group had good cytocompatibility and the cell survival rate was more than 80%.The results of three-dimensional culture showed that BMSCs had high activity in the three groups of hydrogels.When the concentration of HT was 0.5%,BMSCs had more extension in the hydrogels.Because the swelling rate of 0.5%HT hydrogels was small,the density of cells contained in the hydrogels was relatively large,which facilitated the interaction between cells.Ki67 immunofluorescence assay showed that all three groups of hydrogels could promote the survival and proliferation of BMSCs encapsulated in them.4.CCK-8 results showed that NGF had a proliferation effect on BMSCs on the first day.Ki67 immunofluorescence assay showed that NGF treatment could enhance the proliferation of BMSCs in hydrogel.Part Ⅱ:Effect on nerve repair in TBI mice of HT hydrogel-loaded BMSC and NGF transplantation1.The results of hemolysis test showed that the hemolysis rate of HT hydrogel was between 0.14%and 0.41%,far less than 2%,and it had good blood compatibility.The results of subcutaneous injection and HE staining showed that no pathological changes were observed at 3,7 and 14 day after the injection of 0.5%HT hydrogel,and no obvious inflammatory reaction was found in the tissues around the injection site,indicating that the 0.5%HT hydrogel had good biocompatibility.2.The moderate mouse TBI model was successfully established;Compared with NS group and HT+NGF group,the m NSS score of stem cell transplantation group(HT+BMSC group,HT+NGF+BMSC group)was significantly decreased on 21 and28 days,and the number of water maze crossing platform and destination quadrant residence time were significantly increased from 23 to 28 days(P<0.05).These results showed that the neuromotor ability,cognitive and memory function of HT+BMSC and HT+NGF+BMSC mice were significantly improved,and the therapeutic effect was more significant in HT+NGF+BMSC group.After 28 days of treatment,Image J showed that the HT+NGF group,HT+BMSC group and HT+NGF+BMSC group could significantly reduce the area of brain tissue injury,and the effect was more significant in HT+NGF+BMSC group.3.Compared with the other groups after treatment 28 days,the expression of inflammatory promoter protein IL-6 was down-regulated significantly(P<0.05),the expression of apoptosis promoter protein Bax was down-regulated significantly(P<0.05)and the expression of apoptotic inhibitory protein Bcl-2 was up-regulated(P<0.05),the expression of neurotrophic factor(BDNF)and nerve marker(NFL,NSE,Neu N)in HT+NGF+BMSC group were increased significantly(P<0.05),indicating that BMSCs and NGF loaded with HT hydrogel improved the brain microenvironment after TBI,increased the expression level of neuron-related proteins,and promoted the survival and proliferation of neurons.After 28 days of treatment,the immunofluorescence results showed HT+BMSC group and HT+NGF+BMSC group increase the Neu N and Ki67 expression in DG region,indicating that HT hydrogel encapsulation of BMSC and NGF significantly promoted the survival and proliferation of neurons in DG region of mice hippocampus.Conclusion1.HT injectable hydrogel has high water content and good stability,which can provide for the survival of cells a good microenvironment.The cell compatibility of0.5%w/v HT hydrogel was the best,and the adhesion and proliferation of BMSC could be promoted under three-dimensional culture condition,and the addition of NGF had better effect on the survival and proliferation of BMSC.2.HT hydrogel loaded BMSC and NGF plays transplantation in the treatment of TBI mice can improve the motor function and memory ability significantly,inhibit the apoptosis of neurons,promote the neuronal survival and regeneration,increase the expression of neurotrophic factors,so as to promote nerve repair and regeneration.