Double Functional Cationic Polymer Modified Cerium Oxide Nanoparticles Gelatin/Hyaluronic Hydrogel Scaffold System Treat Complete Spinal Cord Injury

Background:Spinal cord injury(SCI)is a disorder or loss of sensory,motor and vegetative nerve function,often with a poor prognosis,leading to paralysis or even death.the average age of SCI patients is less than 40 years,and most of them are bedridden for the rest of their lives,with the consequent high cost of treatment and care placing a huge burden on their families and society.At the same time,there are a large number of new SCI patients in China every year(according to statistics,the incidence of SCI in China is 40 per million people per year),so the development of effective treatments for SCI needs to be urgently addressed,and so far spinal cord injury repair is still a worldclass clinical challenge.Following spinal cord injury,a large number of nerve cells die and are lost within minutes to hours.Secondary damage following primary injury,including inflammatory responses,apoptosis,vascular abnormalities,glutamate excitotoxicity,free radical formation and lipid peroxidation,leads to secondary death of neuronal cells and glial cells,and glial and fibrous scar formation,resulting in permanent functional degeneration.Of these,the inflammatory response is thought to be a key component of secondary injury.Large amounts of pro-inflammatory factors such as tumour necrosis factor(TNF),interleukin-1(IL-1)and IL-6 are released in the injury area,which further induce the accumulation of chemotactic inflammatory cells and exacerbate the local inflammatory response.A large number of damage-associated molecular patterns(DAMPs)are also attempted after primary and secondary injury and are released into the blood and tissue fluid,where they are directly or indirectly involved in various inflammatory responses and blood acid complications.In addition,reactive oxygen species(ROS)in the microenvironment are significantly increased during the inflammatory response to SCI,synergistically exacerbating neuronal cell death,axonal degeneration and motor dysfunction.In recent years,various strategies,including cell transplantation,nanomedicines and biotissue engineering,have been applied to the treatment of SCI.Among them,nanomaterials can effectively scavenge DAMPs and ROS after SCI and modulate local microenvironmental changes;bio-scaffold materials can provide mild conditions for SCI repair.Objective:Hydrogel systems based on gelatin(GL)and hyaluronic acid(HA)of natural material origin have good biocompatibility and can fill the physical defects after injury,promote the migration and maturation of endogenous neural stem cells after SCI,and provide a matrix system for the delivery of bioactive molecules,drugs and cells.The construction of a hydrogel system that mimics the physiological spinal cord environment with stable delivery of active factors,drugs and cells is therefore fundamental to the treatment of total transverse spinal cord injury lesions.The repair of SCI is dependent on the spinal cord injury microenvironment,therefore,in order to more effectively promote neural network reconstruction and functional recovery after SCI,the post-injury microenvironment needs to be improved and reshaped in such a way that it not only reduces the level of neuronal cell death but also promotes neural regeneration.Therefore,in this study,we propose to construct cerium oxide nanomaterials with dual functions of neuroprotection and tissue regeneration to modify the post-injury inflammatory microenvironment in order to improve the therapeutic repair of spinal cord injury.The cationic polymer modifiedcerium oxide nanoparticles(cCONPs)can scavenge injury-related pattern molecules and reactive oxygen species from the microenvironment,reduce the level of inflammatory response and demonstrate neuroprotective effects.In addition,the cationic polymer modified-cerium oxide nanoparticles were used to deliver and slowly release brain-derived neurotrophic factor(BDNF),which promotes axonal growth and nerve regeneration,to better promote nerve injury repair and tissue regeneration while regulating the inflammatory microenvironment.Methods:1.Firstly,an optimized gelatin-hyaluronic acid visible light cross-linked hybrid hydrogel system was constructed.Scanning electron microscopy(SEM),infrared Fourier spectroscopy analysis and swelling experiments were used to characterize the constructed GL/HA hydrogels.The therapeutic effects were observed by in situ transplantation of the optimised GL/HA hydrogels into the T9 total transection spinal cord injury rat model.The groups were: CON group,GL/HA mixed hydrogel ratio 10/0group,GL/HA mixed hydrogel ratio 9/1 group,GL/HA mixed hydrogel ratio 8/2 group,GL/HA mixed hydrogel ratio 7/3 group.Two time points were taken at postoperative day 10 and 60,and immunofluorescence staining technique was used to focus on the effects of transplantation treatment on endogenous neurogenesis,migration,maturation and differentiation,modulation of inflammatory response after injury,and glial scar formation.The recovery of motor function in SCI rats was assessed using BBB score,motor electrophysiological testing and oblique trigger test.To comprehensively evaluate the effectiveness of GL/HA hydrogel system for the treatment of complete SCI and to provide a stable base material system for the subsequent bifunctional nanomaterials.2.To design and prepare a bifunctional nanomaterial system with both ROS and DAMPs scavenging and slowrelease cytokines.The cationic polymer modified-CONPs(cCONPs)were obtained by hydrothermal synthesis of CONPs and modification of cationic polymers(PAMAM-G3)on the surface,followed by loading of cytokines BDNF by physical interaction and encapsulation of the nanoparticles in GL/HA hydrogels.The GL/cCONPs-BDNF bifunctional nano-hydrogel system described in this project was obtained.Dynamic light scattering(DLS),transmission electron microscopy(TEM)and scanning electron microscopy(SEM)were used to characterise the size and potential of the cCONPs nanoparticles.In vitro assays,firstly,the ability of different concentrations of cCONPs nanoparticles to scavenge oxygen radicals,bind to DAMPs and slow release of BDNF was examined using elisa assay.Next,the biocompatibility of the GL/cCONPs system was examined using cell live-dead staining assays.The protective effect of the GL/cCONPs system on cells and the reduction of intracellular ROS levels were then tested in H2O2-simulated peroxidative stress conditions.Finally,the inhibitory effect of the GL/cCONPs system on the inflammatory response under simulated injury conditions was investigated by PCR and IF staining.In the in vivo experimental part,the same T9 total transection SCI rat model was applied in four groups(CON group,GL group,GL/cCONPs group and GL/cCONPsBDNF group).The efficacy of the GL/cCONPs-BDNF bifunctional hydrogel scaffold system for the treatment of SCI was evaluated by taking samples at 10 and 60 days postoperatively.IF staining and multi-factor antibody microarray techniques were used to observe the microenvironmental changes,nerve regeneration,axonal extension,scar formation and vascular regeneration in the injured area after transplantation of GL/cCONPs-BDNF system,respectively.In addition,the safety of the treatment system was assessed by H&E staining of vital organs.Finally,the recovery of motor function was assessed by evaluating the motor function of SCI rats.Results:1.A visible light cross-linked GL/HA hybrid hydrogel system was constructed and optimised.It has good biocompatibility and fills the defect in the spinal cord injury area by in situ gel formation.We found that the addition of HA to the GL hydrogel reduced the post-SCI inflammatory response and inhibited scar formation in a concentrationdependent manner.Furthermore,the GL/HA ratio of 9/1 and 8/2 significantly promoted the migration and occurrence of endogenous NSCs and enhanced neural differentiation maturation and axonal regeneration.The treatment method of GL/HA hybrid hydrogel transplantation promoted the recovery of motor function in rats with T9 total transection spinal cord injury.2.Polymeric cation modified cCONPs nanoparticles with a diameter size of243.29 nm were prepared.In vitro experiments showed that the GL/cCONPs system was not significantly toxic to NSCs and raw 264.7 cells at a concentration of 50 mg/mL.cCONPs nanoparticles had good ability to scavenge ROS,bind free DAMPs and protect against cell damage under H2O2-induced injury conditions.Reduced inflammatory response induced by DAMPs mimicry.And cCONPs-BDNF nanoparticles are able to stabilize long-lasting and slow-release BDNF factors.In a total transection SCI injury model,treatment by GL/cCONPs-BDNF transplantation was able to promote recovery of motor function in rats.In contrast to the CON group,the GL/cCONPs system improved the microenvironment by modulating the post-injury inflammatory response;GL/cCONPs-BDNF promoted the regeneration and maturation of damaged endogenous neurons through the long-acting slow release of BDNF factors;and inhibited long-term scar formation.Conclusion:In summary,we successfully constructed and optimized a gelatin-hyaluronic acid hydrogel system,and on this basis,we constructed a bifunctional cerium oxide nanoparticle hydrogel system with both neuroprotective effects and promotion of tissue regeneration and repair,which significantly reduced the inflammatory response in the damaged area by scavenging ROS and DAMPs and improved the post-injury microenvironment.At the same time,the bifunctional material system was used to release growth factors with long-lasting effects to give nutritional support to the injured area,promote neural differentiation,regeneration and maturation,and ultimately promote motor kinetic recovery after SCI injury,providing a new idea for SCI treatment.