A Dual-enzymatically Cross-linked Injectable Gelatin Hydrogel Loaded With BMSC Improves Neurological Function Recovery Of Traumatic Brain Injury In Rats

Traumatic brain injury?TBI?is a major public health problem worldwide and a devastating event with high mortality and disability.After TBI,massive degeneration and necrosis of neurons occurred caused by primary and secondary damages,resulting in the deficit of neurological function.Neurological repair post-TBI remains to be a challenging clinical problem,minimizing the death of neurons and loss of neurological function is the main direct of research at present.Stem cell transplantation provides new idea and holds great promise for treating TBI.Mesenchymal stem cells?MSC?,administrated by either direct intracerebral injection or systemic injection after TBI,have improved remodelling brain tissues structure and neurological functional recovery in experimental animal models.Bone marrow-derived MSC?BMSC?appears to have many outstanding properties such as less immunogenicity,multiple differentiation potential,and fewer ethical problems than embryonic stem cells?ESC?.However,how to efficiently deliver BMSC to brain lesions remains a technical challenge in the treatment of TBI.Hence,it is pivotal to build neural scaffold which can deliver cells to the target area effectively in the cell-based therapy for TBI.Gelatin is a hydrolysis product of collagen which possesses numerous remarkable advantages for tissue engineering applications,including excellent biocompatibility,matrix metalloproteinase?MMP?mediated degradability,and retaining natural cell adhesion motifs?e.g.,Arg–Gly–Asp?RGD??.Phenol-rich gelatin polymer has been received considerable attention for the development of in situ forming injectable hydrogels enzymatically cross-linked by horseradish peroxidase?HRP?and glucose oxidase?GOX?due to their quick gelation rate,high specificity and mild reaction condition for cell and tissue.By injection or spray-based approaches,injectable hydrogels enable the minimally invasive implantation,simple encapsulation of cells and/or biomolecules,and accurate filling of any irregular tissue defects.Additionally,the 3D structure of gelatin hydrogel could provide physical cues for cellular attachment and growth in vitro and in vivo,and could transport the cells by encapsulating cells with their particular 3D porous structure.At the same time,the physical and chemical properties of gelatin hydrogels,such as water content,swelling behaviour and mechanical strength,will participate in regulating the biological functions of cells.These characteristics indicate that the dual-enzymatically crosslinked gelatin hydrogel has great potential for brain tissue engineering applications.ObjectivesIn this study,we intend to modify natural gelatin with hydroxybenzene propanoic acid?HPA?to obtain GH polymer?Gelatin-hydroxyphenyl?.GOX and HRP could catalyze the covalent cross-linking between phenol groups.We prepared injectable GH hydrogels by GOX and HRP,and then their physical and chemical properties were characterized.The GOX activity was adjusted to screen the best three-dimensional cell culture effect and biocompatibility.Orthotopic transplantation of GH hydrogel encapsulated with BMSC was used to treat TBI rats.The effects of GH/BMSC nerve scaffolds on neural repair in TBI rats and its underlying mechanism were systematically studied from the animal model and molecular level.MethodPart I:Preparation and characterization of GH hydrogelH₂O and DMF were mixed in a volume ratio of 3:2 as the solvent,and GH polymer was synthesized by an EDC/NHS coupling system.Nuclear magnetic hydrogen spectrum ¹H NMR and ultraviolet absorption UV-vis spectra were conducted to detect the modification of HPA qualitatively and quantitatively.GH hydrogels were prepared by GOX/HRP?GOX=0.1,0.2,0.4 U/m L;HRP=0.5 U/m L?dual enzyme catalytic system,and named GOX_0.1UHRP_0.5U,GOX_0.2UHRP_0.5U,GOX_0.4UHRP_0.5Uhydrogels respectively.Gelation time was determined by the tube tilt method;the degradation rate and water content of the gel were measured by freeze drying and weighing method.The biodegradability of the hydrogel was detected by the collagenase solution.The microscopic morphology of the GH hydrogels was observed by the scanning electron microscope?SEM?;the mechanical properties of the hydrogel was tested by the rotational rheometer.The GOX activity was adjusted?0.1?1 U/m L?to prepare GH hydrogels with different degrees of cross-linking for three-dimensional culture of BMSC,and AM-PI staining fluorescence photographs were taken on 1,3,and 7 days to test the cell viability.Micro Hydrogen Peroxide?H₂O₂?Assay Kit was used to detect the H₂O₂ content of GH hydrogels prepared with different GOX activities?0.1,0.2,0.4 U/m L?after gelation for24 h and 48 h.CCK-8 was used to detect the effect of H₂O₂ produced by GH hydrogels on BMSC activity.In order to evaluate the biocompatibility of the hydrogel,SD?Sprague Dawley?rats were injected subcutaneously with GH hydrogels,and HE staining was used to detect the inflammatory response of the tissue around the injection site at 3,7,and 14 days.Part II:Effect of GH hydrogel-encapsulated BMSC transplantation on nerve repair in TBI ratsThe moderate TBI model of SD rats was constructed by the modified Feeney free-falling method.The rats were randomly divided into Sham group,NS group,Scaffold group,BMSC group and Scaffold+BMSC group.Seven days after the model was established,different groups were transplanted accordingly.On 1,3,7,14,21,and 28days after transplantation,the rats were given a modified neurological deficit score?m NSS?to evaluate the motor function of TBI rats.The Morris water maze experiment was performed from day 23 to 28 after transplantation to evaluate the spatial memory and learning ability of TBI rats.After 28 days of treatment,all rats were sacrificed,and HE staining was used to quantitatively analyze the volume of brain tissue defects in each group.Nerve regeneration at rat dentate gyrus?DG region?was detected by immunofluorescence.Proteins and RNA at the injury site were extracted;Western Blot was used to detect the expression of neurotrophic?BDNF?,neuronal differentiation?Neu N,DCX,NSE?,and apoptosis?BAX?related protein.q RT-PCR was used to detect the expression of neurotrophic?NGF,BDNF,NT3?and neural differentiation?Neu N,?-?tublin,MAP2?related genes.ResultsPart I:Preparation and characterization of GH hydrogel1.Both the ¹H NMR and UV-vis spectra of GH polymer showed the characteristic signal of phenol group,while the spectra of gelatin alone did not show the characteristic peak of phenol group.These results indicating the successful synthesis of GH polymer,and the branch rate was 43.4?mol/g.2.GH hydrogels are injectable and expected to fill the lesions accurately.The microstructure of GH hydrogels is a three-dimensional loose network structure.The gelation time of the three groups of GH hydrogels is from 5 min to 11 min,elevated GOX activity leads to faster gelation;the water content of all three groups of hydrogels exceeds 90%.GH hydrogel enzymatic hydrolysis time increases with the increase of GOX activity,and all samples can stably exist in vitro in PBS for more than 14 days.The elastic modulus of GH hydrogel in different groups is directly proportional to GOX activity,which are 162 Pa,1287 Pa,and 3650 Pa.And the mechanical strength of GOX_0.1UHRP_0.5U hydrogels is similar to brain tissue,the differences between the three groups are significant?P<0.05?.3.GH hydrogels were prepared by GOX?0.1?1 U/m L?with different activities for three-dimensional culture of BMSC.BMSC had higher activity in GOX_0.1UHRP_0.5Uhydrogel and GOX_0.2UHRP_0.5U hydrogel,and when GOX was 0.1 U/m L,BMSC extended greatly in the hydrogel,indicating that GOX_0.1UHRP_0.5U hydrogel has the best cell compatibility.When GOX activity exceeds 0.4 U/m L,the cell activity in the hydrogel is decreases,and after 3 days,the cells die a lot,and the hydrogel shrinks.4.The release of H₂O₂ from GH hydrogel increased with the elevated of GOX activity.The cumulative release of H₂O₂ in GOX_0.1UHRP_0.5U,GOX_0.2UHRP_0.5U and GOX_0.4UHRP_0.5U hydrogel after 48 h was 180,290,470?M,respectively.CCK-8experiments suggested that H₂O₂ will injury cells badly when the concentration is reach500?M.Therefore,we chose GOX_0.1UHRP_0.5U hydrogel with good cytocompatibility for subsequent experiments.5.No pathological changes were observed in rats injected subcutaneously with GOX_0.1UHRP_0.5U hydrogel on the 3,7 and 14 day.Analysis of HE staining of tissues around the injection site showed no inflammatory response,indicating that GOX_0.1UHRP_0.5U hydrogel was biocompatible.Part II:Effect of GH hydrogel-encapsulated BMSC transplantation on nerve repair in TBI rats1.Moderate TBI model was successfully constructed by the free-fall strike method.Compared with the NS group and the Scaffold group,the m NSS score of the rats in the BMSC group and the Scaffold+BMSC group decreased significantly after 7 d,the number of experimental crossing platforms and the time stay at the target quadrant increased significantly?P<0.05?.These results indicate that the motor function,learning and memory ability,and cognitive function of the rats in the BMSC group and the Scaffold+BMSC group were improved,and the effects of the Scaffold+BMSC group were more significant.After 28 days of treatment,HE staining results showed that transplantation treatment in the Scaffold group,BMSC group,and Scaffold+BMSC group can significantly promote the regeneration of brain tissue at the injury site,and the effect of the Scaffold+BMSC group is more significant?P<0.05?.After 28 days of treatment,immunofluorescence results of frozen brain sections showed higher NSE,Neu N,and Ki67 expression in the DG region of the BMSC group and Scaffold+BMSC group.2.After 28 days of treatment,compared with the other groups,the expressions of neurotrophic factor?BDNF?and neuromarkers?NSE,DCX,Neu N?in the brain injury side of the Scaffold+BMSC group were significantly increased?P<0.05?,and the expression of apoptosis-related protein BAX was significantly down-regulated?P<0.05?.q RT-PCR results showed that compared with the NS group,the expression of neurotrophic factor-related genes?NGF,BNDF,NT3?in the Scaffold+BMSC group were all up-regulated,and the expression of NGF was significantly up-regulated?P<0.05?;compared with the NS group,the expression of neural markers?Neu N,?-III tubulin,MAP2?in the Scaffold+BMSC group was up-regulated,and the expression of MAP2 was significantly up-regulated?P<0.001?.Conclusion1.The interior of the GH hydrogel is a three-dimensional porous network structure.And it has good stability and high water content,which can provide a good living microenvironment for cells.The gelation time can meet the requirements of practical operation,and the mechanical strength is close to the brain tissue.For three-dimensional culture,GOX_0.1UHRP_0.5U hydrogel can promote encapsulated BMSC adhesion and proliferation,indicated it has better cell compatibility.GOX_0.1UHRP_0.5Uhydrogel can be degraded by organisms,when subcutaneous transplantation in rats it will not cause inflammatory reactions that suggest good biocompatibility.2.GH hydrogel-encapsulated BMSC transplantation in TBI rats can significantly improve the recovery of motor,learning and memory functions in rats,and promote tissue regeneration at the injured site.BMSC transplantation through GH hydrogel can inhibit neuronal apoptosis,increase the expression of neurotrophic factors at the injury site,and promote the survival and differentiation of endogenous neurons.