Novel Cytokine-loaded PCL Hydrogel Scaffold Composite For Spinal Cord Injury Repair

Background Spinal cord injury(SCI)is a serious public health problem.There are approximately 250,000 to 500,000 new cases recorded worldwide each year.Once triggered by an external physical impact,such as fall,car accidents,sports injuries,and violence,a series of complex pathological changes will occur around the injury,which may seriously hinder axonal regeneration and functional recovery.These pathological changes includes ischemia,glial and neuronal necrosis,the release of the free radical,and immune cells infiltration in the acute and subacute phases,glial scars,cystic cavity formation,lack of neurotrophic stimulation or permissive substrates in the chronic phase.This not only brings catastrophic physical and psychological trauma to patients,but also brings a heavy economic burden on society and families due to their long-term hospitalization,poor rehabilitation results and excessive dependence on care.Over the past century,progress in SCI mechanisms research has promoted the development of the clinical management.The use of the high dose methylprednisolone,surgical interventions and rehabilitative protocols have decreased the morbidity and improved the functional outcomes of SCI patients.Despite all this,the curative effect is far from satisfactory.Especially in the repair of injured spinal cord,there is still no good strategy.Neuroprotective and neuroregenerative therapies have been translated from preclinical studies into clinical trials.However,no randomized clinical trial has demonstrated the efficacy of a repair strategy for improving functional recovery from SCI at present.and recent advances in biological and engineering strategies have provided us with a promising alternative.So far,various biocompatible materials such as collagen,hyaluronic acid,chitosan,and polylactic acid-glycolic acid have been used in the research of spinal cord repair.However,considering the complicated pathological condition,a comprehensive treatment based on the pathophysiological mechanism seems to be more effective.Objective In recent years,research on the repair of spinal cord injury has almost reached consensus.Studies have shown that a single intervention method has limited effectiveness in the SCI repair.If we want to achieve better results,we should aim at the pathophysiology of SCI and conduct corresponding interventions at different stages.These necessary conditions include:(1)stimulation of the intrinsic regenerative capacity of neurons;(2)supporting substrates required for neuron axon regeneration and extension;(3)chemical induction factors required for further growth;(4)postoperative rehabilitation training.Guided by this theory,we tried to use Poly-ε-caprolacton(PCL)as a primer for directional axonal growth,and verified the effect of PCL on PC12 cell growth in vitro.Fibroblast growth factor 2(FGF2)and epidermal growth factor(EGF)are used to induce peripheral cells to secrete laminin and other substrates which are needed to support axon growth.Glial-derived neurotrophic factor(GDNF)was used as a chemical to further induce the axon regeneration.Hydrogels serve as a carrier for these growth factors.Hydrogels involving these three types of cytokines are evenly filled around polycaprolactone(PCL)nanospinning,and the physicochemical properties of the scaffold complex are examined in vitro.The scaffold complex was transplanted to the defect site of transected spinal cord injury rats in an attempt to repair the spinal cord injury.Methods 1 Construction and study on characteristics of the scaffold complexes: 1.1 Identification of PCL nanospinning and study of physicochemical properties of PCL hydrogel: A hydrogel loaded with cytokines was synthesized with PCL to prepare a copolymerized complex.After preparation of these complexes,nuclear magnetic spectroscopy and infrared spectroscopy were used to identify PCL.Then the microstructure of PCL,hydrogel and scaffold complexes was observed by scanning electron microscopy.the BET specific surface area of the PEG hydrogel was measured by a fully automatic specific surface area and porosity analyzer.Mechanical properties of the hydrogel and PCL/hydrogel scaffold complexes were carried out using universal tensile machine.Compression and cyclic compression tests were performed on cylindrical samples.1.2 Study on the release kinetics of scaffold complexes:The cumulative release of cytokines by scaffold complexes were quantified by ELISA kits.2 PC12 cells and primary neurons were co-cultured respectively with PCL nanospinning: After sterilizing the PCL,the PCL was fixed in the cell culture dish.Primary neurons and PC12 cells were inoculated onto the surface of PCL in vitro,and the absorbance value(OD value)was measured by CCK-8 methods to detect the cell compatibility of PCL to the nervous system.The expression of MAP2 in PC12 cells was observed by immunofluorescence,and adhering and directional growing cells were counted.3 Scaffold complexes transplantation for repair of transected SCI in rats: Subsequently,30 female Sprague-Dawley rats were randomly divided into spinal cord injury control group(10 rats),hydrogel/cytokines group(10 rats)and PCL/hydrogel/ cytokines group(10 rats),which were transected by microsurgery.The T9 spinal cord tissue was removed to 2 mm,and animal models of spinal cord transection injury were established.The hydrogel/cytokines and PCL/hydrogel/cytokines were transplanted to the spinal cord injury site.The hindlimb motor function of the rats was evaluated by BBB score 1 day after the successful modeling and once every week for 8 weeks.Two months after modeling,rats were perfused,and The injured spinal cord tissue was sectioned in a coronal plane.The tissue growth of the injury site was observed by HE staining,and the axon regeneration of neurons(MAP2 positive cells)and the production of laminin substrates were observed via immunofluorescence,in order to investigate the repair effect of the scaffold composite on spinal cord injury.Results 1 Construction and study on characteristics of the scaffold complexes: 1.1 Nuclear magnetic spectroscopy and infrared spectroscopy were used to identify them When the scaffold complexes were identified by nuclear magnetic spectroscopy and infrared spectroscopy,we found that they have the characteristic peak values of PCL.Scanning electron microscopy showed that PCL nanospinning was a rough fiber-like structure with a fiber diameter of about 200 μm.The measurement results of the pore size of the hydrogel by the automatic specific surface and porosity analyzer BET found that the BET specific surface area and pore size of the hydrogel were 7.7254 m2 g-1 and 16.66?,respectively.Previous studies have shown that,hydrogels with similar BET specific surface area have good adsorption capacity.After measuring the compression modulus of PCL hydrogel,it was found that when the compression deformation of hydrogel is 20%-30%,the pressure change range is 0.83-2.40 Kpa,and the PCL/hydrogel complex pressure change range is 2.75-5.07 Kpa.Although the two are quite different,they are still close to the deformation modulus of normal spinal cord tissue.1.2 Study on the release kinetics of scaffold complexes: PCL/hydrogel complex has excellent sustained-release effect on FGF?,EGF and GDNF.When the experiments were processed to 21 days,the cumulative release amount was close to 25%.The sustained and slow release of these cytokines was beneficial to stimulate surrounding cells to secrete substrates that support the growth of neuron axons and to further chemically induce axons.2 PC12 cells and primary neurons were co-cultured respectively with PCL nanospinning: CCK-8 assay illustrated that there was no significant difference between the PCL group and the control group about the absorbance at 450 nm.Florescence images of the cells cultured on complexes were shown that compared with the control group,pure PCL could promote neurites adhesion and directional growth.In addition,we quantified directional growth cells in different groups.We found that the number of directional growth cells in the PCL group was significantly higher than that in the control group.3 Scaffold complexes transplantation for repair of transected SCI in rats: The vivo experiments showed that the behavioral assessment(BBB score)of the PCL/hydrogel/cytokines group was significantly higher than that of the control group and the hydrogel/cytokines group(P ? 0.05)from the 7th week after the intervention of the scaffold complexes.The results of the HE staining showed that at the injury site,the control group and the hydrogel/cytokines group showed obvious tissue voids and disorganized tissues.However,the tissue in PCL/hydrogel/cytokines group was relatively ordered,and directional along the PCL.The vivo tissue immunofluorescence results showed that at the injury site,the axon of the PCL/hydrogel/cytokines group(MAP2 positive cells)grew along the direction of the PCL fiber bundle and it was significantly longer than the injury control group and hydrogel/cytokines group(P? 0.05).In addition,the quantitative analysis indicated that the density of regenerated laminin in the PCL/hydrogel/cytokines group was significantly higher than that in control group and hydrogel/cytokines group(p < 0.05).And they got a common label with the regenerated axons.Conclusion In summary,we had successfully constructed a PCL–based hydrogel complex system,in which PCL provided physical support for axonal regeneration,FGF2 and EGF were added to the hydrogel to increase axon growth-supportive substrates(such as laminin),and GDNF was added to further chemoattract propriospinal axons.The vitro experiments showed that single PCL could promote neurites adhesion and directional growth.With the help these cytokines and hydrogel,the scaffold complex provided a favorable biological microenvironment for cell survival and growth.After implantation in the transected spinal cord tissue,the the scaffold complex promoted the axon’s directional regeneration in the conduit,thus promoting the recovery of motor function after SCI.Meanwhile,this composite also promoted the production of laminin which played an important role in the axon growth-supportive substrates.Meanwhile,it laid the theoretical foundation for our further research.