Repair Of Spinal Cord Injury By Decellularized Spinal Cord/GelMA Composite Scaffold Combined With MenSCs In Rats

ObjectiveThis study investigates the use of a decellularised spinal cord（d SC）/gelatin methacryloyl（GelMA）composite scaffold combined with menstrual blood-derived mesenchymal stem cells（MenSCs）to repair spinal cord injury（SCI）in rats.The study will elucidate the regulation of MenSCs by d SC/GelMA hydrogel composite scaffold in vitro;in vivo transplantation promotes axonal regeneration and improvement of limb motor function in SCI rats.This project will provide a new strategy for repairing SCI and lay a new theoretical and practical foundation for translation to the clinic.Methods1.In vitro screening of GelMA hydrogels with suitable physical properties by polymerization and stability.2.Decellularized matrix（dECM）was obtained by sequentially shaking rat thoracic spinal cord segments in 1% Triton^TM X-100/0.01 M PBS and 1% sodium deoxycholate/0.01 M PBS.The extent of decellularization was evaluated by hematoxylin&eosin（H&E）staining,residual DNA detection and scanning electron microscopy（SEM）.3.Decellularized scaffolds are lyophilized and ground into powder.d SC can be transformed into decellularized spinal cord extracellular matrix-gel（DSCG）by pepsin digestion,p H neutralization and ion equilibration.4.The composite scaffolds were made by mixing DSCG solution with GelMA hydrogel in the ratio of 1:9（1D9G）,2:8（2D8G）,3:7（3D7G）and comparing the biomechanical properties of the three groups of composite scaffolds: water retention,contact angle,degradation rate,porosity and Young’s modulus.5.MenSCs were mixed with different ratios of composite scaffolds and cultured in 3D.Live/dead cell staining,phalloidin staining to detect the survival and migration of MenSCs in 3D culture.Proliferation kits Ed U and CCK8 to detect the proliferation of MenSCs.Immunofluorescence assay（Immunofluorescence,IF）to detect the differentiation ability of MenSCs.6.The composite scaffold most suitable for the growth of MenSCs was transplanted to the injury of the total transection SCI rat model,and the functional recovery of SCI rats was assessed by IF and motor function.Results1.5% GelMA hydrogel light for 18 s is more suitable for culturing cells.2.The results of H&E staining,residual DNA detection and SEM all showed almost complete removal of cells from the d ECM scaffold.3.Compared to 1D9G and 2D8G composite scaffolds,3D7G has better water retention,hydrophilicity and greater softness.For SCI studies,3D7 G composite scaffolds are superior in terms of biological properties.4.Live/dead cell staining,phalloidin staining,Ed U cell proliferation assay and CCK8 assay showed that MenSCs had the highest survival rate,the highest cell fusion rate and faster proliferation in the 3D7 G composite scaffold.IF assay showed that MenSCs had a higher percentage of differentiation towards neurons in culture in the3D7G composite scaffold.5.In vivo experiments showed that 3D7G scaffolds encapsulating MenSCs transplanted in a rat model of total transection SCI promoted an increase in the number of Tuj1-positive cells,a decrease in GFAP expression,a decrease in inflammatory response,and the growth of axons as well as the restoration of motor function.Conclusions1.DSCG/GelMA can be used as an ideal scaffold material for repairing SCI rats,providing a good microenvironment for MenSCs to survive,migrate,proliferate and differentiate to neurons.2.The 3D7G composite scaffold is biocompatible and co-cultures with MenSCs in vitro to promote cell survival,migration,proliferation and differentiation to neurons.3.The 3D7G composite scaffold combined with MenSCs was transplanted in a rat model of total transection of SCI to build a nerve regeneration "bridge" in the injured area,providing support and guidance for nerve regeneration and promoting the recovery of motor function in SCI rats,providing an important strategy for the repair of SCI.