Three-dimensional Bioprinting PTX-loaded Dual-sensitive Micelles Hydrogel Scaffold For Repairing Spinal Cord Injury

【Objective】Preparation of 3D printed porous SilMA hydrogels to explore the physical properties of hydrogel scaffolds.Paclitaxel nanoparticles(PTX-NPs)with p H/temperature sensitive properties were synthesized by small molecule fragments,and the effects of different concentrations of PTX-NPs on the differentiation ability of neural stem cells(NSCs)were investigated.NSCs and PTX-NPs were co-loaded with3 D printed porous SilMA hydrogel to form a composite scaffold,and to investigate the effect of the scaffold on the repair of spinal cord injury(SCI)in rats.【Methods】The 3D printed SilMA hydrogel scaffolds and p H/temperature dual-sensitive PTX-NPs were characterized to explore their physical and chemical properties.In vitro,we investigated the effect of different concentrations of paclitaxel-loaded micellar nanoparticles on the differentiation ability of NSCs.The effects of different concentrations of paclitaxel-loaded sensitive micelles on NSCs differentiation were investigated by extracting myelin-associated proteins to simulate the inhibitory microenvironment.Protein blotting analysis was performed to investigate the signaling mechanism of paclitaxel dual-sensitive micelles nanoparticles in promoting NSCs differentiation.In vivo,the recovery of hind limb mobility in rats after SCI was explored at different time points by constructing a SCI hemisection model and implanting different groups of scaffolds into the injury.At the eighth week,immunofluorescence staining of SCI sections was used to explore the neural regeneration under the influence of drugs.【Results】The 3D printed hydrogel scaffold is porous and provides a supportive environment for NSCs while being able to act as a neural regeneration conduit to promote neural regeneration.Paclitaxel micelles are PH/temperature sensitive,enabling smart and responsive drug release while improving the bioavailability of hydrophobic paclitaxel in vivo.In vitro,10 ng/ml paclitaxel micelles can better promote the differentiation of NSCs into neurons and reduce the formation of astrocytes.The 3D SilMA hydrogel scaffold containing paclitaxel micelles showed more neuronal expression and less astrocyte expression when the drug content was 50 ng.Similarly,the SilMA hydrogel scaffold loaded with 50 ng of paclitaxel showed more differentiation of NSCs toward neurons in a simulated inhibitory microenvironment.In protein blotting analysis,paclitaxel was able to promote NSCs differentiation toward neurons and reduce astrocyte regeneration through MAPK/ERK signaling pathway.In vivo,tissue immunofluorescence sections showed that the SCI-SilMA-NSCs-PTX group was able to significantly promote better differentiation of NSCs into neurons while reducing astrocyte production in the injury area,reducing fibrous scar formation and promoting regeneration of motor-associated neurons compared to other groups.Behavioral assay BBB scores showed that the hind limb activity of rats in SCI-SilMA-NSCs-PTX group was significantly improved.【Conclusion】In this study,we synthesized p H/temperature sensitive paclitaxel micelles nanoparticles and constructed 3D SilMA hydrogel scaffolds loaded with paclitaxel micelles.The composite scaffold combined with NSCs was applied in a SCI model,and in vivo experiments confirmed that the drug-loaded composite porous scaffold combined with NSCs significantly promoted the regeneration of neurons and axons at the lesion and reduced the formation of astrocytes and glial scars.Ultimately,these favorable conditions promoted the recovery of motor function in rats.3D SilMA hydrogel scaffolds loaded with paclitaxel microgel bundles combined with NSCs provide an effective therapeutic strategy for repair of SCI.