A Dual-enzymatically Cross-linked Injectable Gelatin Hydrogel Laden With BMSCs Promotes Nerve Function Repair In Mice With Traumatic Brain Injury

Traumatic brain injury（TBI）refers to the injury caused by violence on the head,most of the injuries are caused by external forces.TBI is divided into primary injury and secondary injury according to whether immediate injury occurs when violence acts on the head.Injury will cause serious damage to neurological function and often has a very high rate of disability and mortality.So far,there is no effective clinical treatment.Therefore,tissue regeneration and nerve repair after TBI are serious problems to be solved urgently.Studies have shown that the application of stem cell transplantation therapy can effectively relieve the lesions of neurons in the damaged parts and promote the repair of nerve function,bringing new hope for the treatment of TBI.Stem cell transplantation therapy can promote cell survival,proliferation,tissue regeneration and nerve function recovery in TBI model.Among many stem cells,Bone marrow derived mesenchymal stem cells（BMSCs）have the advantages of pluripotent differentiation potential,low immunogenicity and paracrine effect,which can differentiate into nerve cells,inhibit cell apoptosis and the formation of glial scar.However,stem cell transplantation therapy still has some shortcomings,such as low transplantation rate,survival rate and proliferation rate.Therefore,the key of improving the efficacy of stem cells is to prepare suitable biological scaffolds to efficiently load stem cells to the target area and improve the survival and proliferation of transplanted cells.In the study of tissue engineering,as the hydrolyzed product of collagen,gelatin has good biocompatibility and biodegradability,the introduction of phenol groups for in-situ modification to improve the injectability of hydrogel.The injectable hydrogels prepared by cross linking of Horseradish peroxidase（HRP）and Choline Oxidase（ChOx）are rich in many water-soluble polymers as nutrient substrates.The dual enzymatical cross-linking can adjust the gelation rate according to enzyme activity,and mild reaction condition has little adverse effect on cells.In addition,the gelatin hydrogel also has high water content and similar natural extracellular matrix（ECM）soft consistency of the network,can be used as tissue engineering and drug delivery of effective medium,at the same time,the superior physical and chemical properties of the gelatin hydrogel itself,such as mechanical properties,mechanical strength,etc.ObjectivesHerein,natural gelatin was used as raw material and modified with hydroxy propanoic acid（HPA）to get GH polymer（Gelatin-hydroxyphenyl）.A new type of injectable GH hydrogel was prepared by catalyzing the phenolic groups on GH covalent cross-linking with two enzymes（HRP/ChOx）.The physical and chemical properties of the hydrogel were characterized and detected.The hydrogel properties were optimized by continuously adjusting the ChOx enzyme activity to prepare GH hydrogel suitable for three-dimensional cell culture and good biocompatibility.The expression of BMSCs survival,proliferation,nerve differentiation and neurotrophic secretion were detected by GH hydrogel culture in vitro.In vivo experiments,we transplanted GH hydrogel loaded with BMSCs in situ to investigate its potential mechanisms on inflammation,neurotrophic secretion,apoptosis,nerve regeneration and functional repair in the injured site of TBI mice.MethodPart Ⅰ:Optimal preparation and characterization of GH hydrogelsThis mixed solution of DMF and H2O with a volume ratio of 2:3 was selected as the solvent to prepare GH polymer by EDC/NHS coupling reaction.The grafting efficiency of HPA was determined qualitatively and quantitatively by ¹H NMR and UV-Vis spectrophotometry.Through HRP/ChOx（HRP=1 U/m L;ChOx=0.25,0.5,1U/m L）was used to prepare injectable GH hydrogel.Scanning electron microscope（SEM）was used to observe and analyze the internal microstructure of GH hydrogels.Three groups of GH hydrogels,HRP_1UChOx_0.25U,HRP_1UChOx_0.5U and HRP_1UChOx_1U,were prepared,and the gelling time of the three groups of GH hydrogels was determined by inverted tube method.The moisture content of GH hydrogels in three groups was determined by freeze-drying method.The biodegradability of GH hydrogels in three groups under collagenase-specific enzymatic hydrolysis was determined by analytical balance weighing method.The mechanical properties of three groups of GH hydrogels were measured using a rotary rheometer.Part Ⅱ:Three-dimensional culture and neural differentiation of BMSCs in GH hydrogelDifferent concentrations of GH hydrogels were prepared by adjusting ChOx activity（0.25,0.5,1 U/m L）.After 24 h and 48 h,the cumulative production of H₂O₂was detected by the trace hydrogen peroxide（H₂O₂）detection kit.CCK-8 detected the effect of H₂O₂ accumulated in the same GH hydrogel extract on the activity of BMSCs;Three groups of GH hydrogels were prepared for BMSCs three-dimensional culture,and Calcein-AM/propidium iodide（PI）fluorescence labeling on day 1,3 and 5 was used to detect the cell compatibility of GH hydrogels.Ki67 immunofluorescence was used to detect the effect of GH hydrogel on the proliferation of BMSCs.β-III tubulin,Neurofilament-L（NFL）and NSE immunofluorescence was used to detect the effect of GH hydrogel on the neural differentiation of BMSCs.The effect of GH hydrogel on BMSCs neural differentiation and neurotrophic factor secretion was detected by Western blot.Part Ⅲ:GH hydrogel laden with BMSCs promotes neural function repair of traumatic brain injury in miceThe blood compatibility of GH hydrogel was tested by hemolysis experiment in fresh blood of C57BL/6 mice.HRP_1UChOx_0.25U hydrogel was injected subcutaneously into C57BL/6 mice.At 3,7,14 and 21 days,GH hydrogel and tissues around the injection site were peeled off,and hematoxylin and eosin（HE）staining was performed to observe the biocompatibility of inflammatory cells and tissues.The mice TBI model were established and randomly divided into NS group,GH group,MSC group and GH/MSC group.Seven days after the establishment of the model,orthotopic injection transplantation was performed,respectively.The animal experiments were divided into two batches:short-term（3,7 days）and long-term（35days）.After transplantation treatment,the body weight of mice in each group was recorded at 0,1,3,7,14,21,28 and 35 days.The motor nerve function recovery of TBI mice at 1,3,7,14,21,28,and 35 days was evaluated by modified neurological deficit score（m NSS）.Morris water maze test was conducted 28 to 33 days after transplantation to detect the recovery of spatial memory and learning ability of TBI mice.Sugar water preference test was conducted 28-34 days after transplantation treatment to detect the depression of mice.New object recognition experiment was carried out 34-35 days after transplantation treatment to detect the recognition and memory ability of mice.In the short-term experiment to detect the expression of inflammatory proteins in the injured sites of TBI mice,immunofluorescence staining（Arg-1,i NOS）were performed on frozen sections of the brain tissues and western blot（IL-10,IL-6）were tested by the tissue protein from damaged parts.Magnetic resonance imaging（MRI）of mouse brain tissue was used to analyze the defect volume in a long-term study.And evaluate the immunofluorescence staining in the brain tissue slices injury inflammation（Arg-1,i NOS）,nerve regeneration（Ki67,Neu N）protein expression and extraction brain tissue proteins were used for western blot test injury inflammation（IL-10,IL-6）,nerve nutrition（NGF,BDNF）and apoptosis（Bcl-2,Bax）and nerve regeneration（β-III tubulin,Neu N）protein expression.ResultsPart Ⅰ:Preparation and characterization of GH hydrogel1.Compared with gelatin alone,GH had obvious phenolic hydrogen spectrum peak（6.7-7.1 ppm）and UV absorption peak（275 nm）,which indicated that gelatin was successfully grafted with HPA,and the grafting rate of HPA was calculated as 23.27μmol/g..2.GH hydrogel is transparent and injectable,which can accurately fill the damaged area;The gelling time of GH hydrogels in the three groups was 4-11 min;The moisture content of hydrogels in the three groups was above 89%;The enzymatic hydrolysis rate of GH hydrogel slowed down with the increase of ChOx activity;The elastic modulus of GH hydrogels in the three groups were 101 Pa,418 Pa and 1457 Pa,respectively.Part Ⅱ:Three-dimensional culture and neural differentiation of BMSCs within GH hydrogel in vitro1.The H₂O₂ release of GH hydrogels increased with the increase of ChOx activity.The cumulative H₂O₂ release of HRP_1UChOx_0.25U,HRP_1UChOx_0.5U and HRP_1UChOx_1Uhydrogels within 48 h were 23.7,33.2 and 50.6μM,respectively.CCK-8 results showed that the H₂O₂ released by GH hydrogel did not cause significant oxidative damage to cells,and the survival rate was above 74%,showing good cell compatibility..2.The three groups of GH hydrogels were prepared with different concentrations of ChOx（0.25,0.5,1 U/m L）and BMSCs were cultured in vitro.It was observed that BMSCs survived well and were widely extended and cross-linked to form a network structure in HRP_1UChOx_0.25U.Part Ⅲ:GH Hydrogel laded with BMSCs promotes nerve function repair in TBI mice1.Hemolysis test showed that the hemolysis rate of GH hydrogels in the three groups was lower than 2%and HRP_1UChOx_0.25U was the lowest,indicating that GH hydrogels had good blood compatibility.Based on the above results,HRP_1UChOx_0.25Uhydrogel was selected for subsequent experiments.The results of subcutaneous injection of hydrogel and HE staining showed that the number of inflammatory invaded cells in the hydrogel decreased gradually over time,and no inflammatory reaction was found in the surrounding tissues,indicating good histocompatibility of the hydrogel.2.Successfully established the mice model of TBI,after transplant treatment,GH/MSC group increased body weight of mice,m NSS score lower,higher sucrose preference index,and discrimination index increased,Morris water maze experiment showed that mice escape latency shorten,purpose quadrant residence time and number of cross platform increases,these results indicated that transplantation therapy could promote the recovery of body weight and cognitive function in mice.4.After transplantation treatment,the stem cell transplantation group（MSC group,GH/MSC group）can regulate inflammation,neurotrophic secretion,apoptosis and nerve differentiation proteins,and promote brain tissue regeneration and nerve function repair.Conclusion1.The dual-enzymatically cross-linked hydrogel was optimized,which was soft in texture,able to shape according to mold,quick gelling time,high moisture content,good stability and mechanical strength close to brain tissue.The internal structure was porous network,which could provide a good microenvironment for the survival of cells.2.The GH hydrogel has good cytocompatibility.GH hydrogel loaded with BMSCs can significantly promote the survival,proliferation and neural differentiation of cells in three-dimensional culture in vitro.3.GH hydrogel has good blood and tissue compatibility,GH hydrogel load BMSCs transplantation in the treatment of TBI mice,can significantly reduce injury inflammation,promote the secretion of neurotrophic factors,reduce the nerve cell apoptosis,increase of survival and neural differentiation,promote weight gain in mice,cognitive function recovery and injury tissue regeneration.