Dual-enzymatical Cross-linked Gelatin Hydrogel Loaded With HUC-MSCs Improves Neural Function Repair Of Spinal Cord Injury In Mice

Spinal cord injury?SCI?is a neurologically damaged disease with high mortality and disability.The most serious effect of SCI is the loss of sensation and motor function below the plane of injury.The incidence of SCI is rising year by year,which brings a heavy burden to the patient'family and the whole society.Up to date,there is no effective treatment.SCI is mainly divided into primary and secondary injury based on the pathological process.Primary injury occurs tissue tearing and vascular structure destruction.Then,inflammatory cell infiltration,tissue edema,and oxygen free radical formation will cause more serious secondary injury.Most lesions are caused by the secondary injury,so the key is to reduce secondary injuries.Mesenchymal stem cells are a kind of pluripotent stem cells with high proliferation and differentiation ability.The transplantation of mesenchymal stem cells provides an effective strategy for the research of SCI.Human umbilical cord mesenchymal stem cells?hUC-MSCs?are a kind of multifunctional stem cells in neonatal umbilical cord tissue.hUC-MSCs are widely used in stem cell transplantation due to their convenient collection,low immunogenicity,high differentiation potential,and no ethical problem.Recently,stem cell transplantation has shown great potential in the treatment of SCI.However,therapeutic effect of stem cells transplantation is still limited by the low retention rate,low survival rate,and low differentiation efficiency after transplantation.These problems need to be solved urgently.A great number of studies have shown that hydrogel can improve the effect of stem cell transplantation.Therefore,construction of a hydrogel with good biocompatibility,degradability and injectability is beneficial to improve the effect of stem cell transplantation.Three-dimensional gelatin hydrogel has similar characteristics to extracellular matrix?ECM?,including high nutrient permeability,high water content,and adjustable mechanical properties.Injectable hydrogels are less invasive during implantation,can fill irregular defects and build bridges for damaged spinal cords,which exhibites great application potential in tissue recover and regeneration.However,the application of gelatin hydrogel has been limited due to its quick enzymatic degradation.It has been reported that grafting gelatin with 3-?4-hydroxy-phenyl?propionic acid?HPA?can improve its stability.In this study,galactose oxidase?Gal Ox?and horseradish peroxidase?HRP?were used to covalently crosslink the gelatin HPA polymers to form hydrogels.The dual-enzymatical cross-linked hydrogel can promote the survival,proliferation and neural differentiation of hUC-MSCs under 3D culture condition.In addition,the gelatin hydrogel loaded with hUC-MSCs can significantly improve the neural function repair of SCI in mice.ObjectiveIn this study,we intend to fabricate a new injectable gelatin-HPA?GH?hydrogel crosslinked by Gal Ox and HRP.The effects of GH hydrogel on stem cell survival,proliferation,and neural differentiation were measured in vitro;the effects of hUC-MSCs loaded with GH hydrogel on neural function repair in SCI mice were studied in vivo.MethodsPart I:Fabrication and Characterization of GH hydrogelGH was Synaptophysinthesized based on EDC/NHS coupling method,and then using the ¹H NMR and ultraviolet visible spectrophotometer to characterize the GH polymer.Gal Ox-2U and Gal Ox-4U GH hydrogel were prepared by dual-enzymatical cross-linking method.The gelation time of GH hydrogel was calculated by inverted tube method,the swelling ratio of hydrogel was calculated by freeze-drying method,the internal microstructure of hydrogel was characterized by scanning electron microscope?SEM?,the rheological behavior of hydrogel was evaluated by rheometer platform,and the stability of hydrogel was measured by degradation.Part II:Three-dimensional culture and neural differentiation of hUC-MSCs in vitroThe hydrogen peroxide?H₂O₂?content detection kit was used to calculated the H₂O₂ release concentration,and the CCK-8 kit was used to detect the toxic effect of H₂O₂ on hUC-MSCs at the same concentration.After culturing for 1,3 and 7 days,cell Live/Dead kit?Calcein-AM/PI?was used to observed the survival of hUC-MSCs in the hydrogel.Immunofluorescence?Ki67?was used to detect the proliferation of hUC-MSCs in the hydrogel.Immunofluorescence of?-?tublin,Synaptophysin?SYN?,and Neurofilament?NF-L?was used to analyze the effect of GH hydrogel on the neural differentiation of hUC-MSCs.Part III:GH hydrogel loaded with hUC-MSCs improves neural function repair of spinal cord injury in miceBefore application in vivo,the hemocompatibility and histocompatibility of hydrogel was tested by hemolysis test and HE staining.The modified Allen's method was used to strike the T9-T10 segment of C57 mice to construct SCI model.After 7days,the mice were random Ly divided into four groups:normal saline?NS?group,Scaffold group,hUC-MSCs group,and Scaffold+hUC-MSCs group.After treatment,the BMS scores of SCI mice were measured,and the improvement of hindlimb motor function was analyzed by footprint analysis.The changes of bodyweight,tail suspension test and sugar water preference test were used to evaluate the depression of the mice.Spinal cord water content was used to evaluate spinal edema.Di O was used to detect the retention of hUC-MSCs.q RT-PCR was used to detect the expression of neural differentiation and neurotrophic gene.Immunofluorescence was used to detect the expression of neural differentiation.Western blot was also used to detect the expression of neuronal regeneration,apoptosis and inflammatory protein.ResultPart ?:Fabrication and Characterization of GH hydrogelThe ¹H NMR signal of phenolic hydroxyl?6.7-7.1 ppm?in GH was significantly higher than that of gelatin alone,the UV absorption peak of phenolic hydroxyl in GH was also significantly higher than that of gelatin alone at 275 nm.GH hydrogels were transparent and could be moulded by the mold.The gelation time is within 5 minutes,and the water content is higher than 90%.The hydrogels had a porous network structure and the modulus curve was smooth and stable.The modulus of both hydrogel is within the range from 100 to 150 Pa,which is close to the rheological properties of neural tissue.The hydrogel of Gal Ox-4U can maintain stability for 14 days.Part ?:Three-dimensional culture and neural differentiation of hUC-MSCs in vitroBoth GH hydrogels are benefit to the survival,proliferation and differentiation of cells loaded within the hydrogel.The maximum concentration of H₂O₂released does not exceed 120?mol/L within 48 h,the CCK-8 results show that the concentration of H₂O₂ did not cause oxidative damage to cells.The hydrogel of Gal Ox-4U significantly promoted cell survival,proliferation and differentiation than Gal Ox-2U.After comprehensively comparing the various properties,we finally selected the Gal Ox-4U hydrogel for subsequent experiments.Part ?:GH hydrogel loaded with hUC-MSCs improves neural function repair of spinal cord injury in miceGH hydrogel has good hemocompatibility and histocompatibility without causing hemolysis and inflammatory response.The transplantation of GH hydrogel loaded with hUC-MSCs?Scaffold+hUC-MSCs group?increased BMS score,body weight,the retention of hUC-MSCs in lesion location,the expression of neural genes and related proteins.Improved hindlimb motility,relieved depression,and reduced spinal cord water content.Conclusion1.The dual-enzymatical cross-linked hydrogel has superior properties,such as injectability,fast gelation speed,high water content,biodegradability,and suitable rheological behavior close to nerve tissue.2.The hydrogel has good biocompatibility,and can promote the survival,proliferation and neural differentiation of hUC-MSCs under 3D culture.3.The hydrogel has good hemocompatibility and histocompatibility.The transplantation of hydrogel loaded with hUC-MSCs?Scaffold+hUC-MSCs group?can dramatically improve the hindlimb motor function,relieve depression,reduce inflammation and promot neural regeneration.