Spinal Microvascular Endothelial Cell-derived Drug-laden Exosomes Combined With Aligned Scaffolds For The Treatment Of Spinal Cord Injury

Background:Spinal cord injury(SCI)refers to the damage to the structure and function of the spinal cord caused by trauma and disease,and is a kind of central nervous system injury with a high disability rate.It has a serious impact on the quality of life of patients and adds a heavy economic burden to families and society.The low regenerative capacity of the CNS,coupled with the extremely complex local unfavorable microenvironment,further inhibits nerve regeneration and hinders post-injury repair.Therefore,the treatment of spinal cord injury remains a worldwide challenge.For acute spinal cord injury,currently,the common clinical treatment is high-dose methylprednisolone shock combined with surgical decompression therapy.However,the results are not ideal,and high-dose methylprednisolone treatment is often accompanied by significant side effects(such as pulmonary embolism,gastrointestinal hemorrhage infection,and other complications).Although stem cells in combination with biomaterials have shown great potential in the treatment of SCI,they are limited by the ethical restrictions,immunogenicity,and tumorigenic risk associated with stem cell therapy.Meanwhile,more and more studies have shown that Exosome(Exo)can be used as an alternative to cell therapy for SCI because Exo can produce therapeutic effects comparable to cell transplantation.Moreover,Exo as a natural carrier can carry small molecules across the blood-spinal cord barrier to achieve slow release of drugs in the spinal cord injury area,showing good therapeutic effects and great potential for translational applications.However,the characteristics and functions of Exosomes depend on their sources,and different sources of Exo may produce differences in the treatment of spinal cord injury.Exosomes involved in current SCI studies are mostly derived from mesenchymal stem cells,neural stem cells,etc.Therefore,there is still a need to explore and develop more exosomes of cellular origin to improve the repair of spinal cord injury.The neurovascular unit(NVU)is mainly composed of neurons,blood-spinal cord barrier,and extracellular matrix,and is the basic functional unit of the central nervous system.Spinal microvascular endothelial cells are important cells that constitute the blood-spinal cord barrier and play an important role in the exchange of substances in the central nervous system and in maintaining the stability of the internal environment.After spinal cord injury,vascular damage and disruption of the blood-spinal cord barrier can lead to spinal cord hemorrhage,edema,oxidative stress and excessive inflammatory response,resulting in neuronal necrosis and apoptosis.The poor microenvironment in the injury area inhibits neurovascular regeneration and promotes the differentiation of endogenous neural stem cells to astrocytes to form scar,which can be reduced by paclitaxel by differentiating them to neurons.In addition,the transplantation of exogenous endothelial cells can improve the quantity and quality of blood vessels in the injured area and improve the local microenvironment of ischemia and hypoxia to promote the recovery of motor function.Objective:The tissue engineering technology of stent materials combined with drugs,biological factors,and stem cells has shown great potential in the repair of spinal cord injury.However,how to avoid local body fluid washout resulting in short residence time of drugs and factors loaded by scaffold materials is the key to construct effective drug carriers.Since,exosomes have nanoscale dimensions,can pass the blood-spinal cord barrier,and are rich in m RNA,micro RNA,proteins,lipid molecules and other contents that can be used for intercellular material transfer and information exchange,they can be used as natural carriers of nanomedicines.The results of our previous study showed that Spinal Cord Microvascular Endo-thelial Cells(SCMECs)combined with biomaterials transplantation could significantly improve the vascular microenvironment in rats with spinal cord injury,therefore,SCMECs can be considered as seed cells to extract exosomes for the treatment of spinal cord injury.The group’s previous study showed that paclitaxel could promote the differentiation of endogenous neural stem cells to neurons and promote the recovery of motor function in SCI rats.Based on this,this paper proposes to design a multifunctional scaffold loaded with paclitaxel in combination with spinal microvascular endothelium-derived exosomes(SCMECs-Exo)aligned GelMA/PCL electrostatic spinning,aiming to investigate the ability of the multifunctional scaffold to promote neurovascular regeneration,inhibit inflammation and scarring and improve the efficacy of SCI functional recovery.Method:The aligned GelMA/PCL electrostatic spinning films were prepared by electrostatic spinning technique.Scanning electron microscope(SEM),universal material testing machine,and rheometer were used to characterize the aligned GelMA/PCL electrostatic spinning membrane and GelMA hydrogel,and the adhesion of neural stem cell(NSC)on the aligned GelMA/PCL electrostatic spinning membrane was observed using SEM.The extracted NSCs and SCMECs were identified and observed using inverted microscopy,flow cytometry and laser confocal microscopy.SCMECs-Exo were extracted using an ultracentrifuge and co-incubated with Ptx.Subsequently,SCMECs-Exo and SCMECs-Exo-Ptx were identified using transmission electron microscopy(TEM),Malvern particle size meter,and Malvern potentiostat.The encapsulation rate and release of SCMECs-Exo were detected by high performance liquid chromatography(HPLC).For in vitro experiments,the effects of different concentrations of SCMECs-Exo(200,400,800 μg/ml)on NSC proliferation were firstly assessed by CCK 8,and subsequently,the effects of different concentrations of SCMECs-Exo(50,100,200μg/ml)on NSC proliferation and differentiation were assessed by laser confocal microscopy.In vivo experiments were performed using a rat whole transection SCI model.First,the promotional effect of different levels of SCMECs-Exo(50,100,200μg)on neurovascular regeneration and the inhibitory effect on inflammation were assessed in a short-term group(10 d),grouped as follows: scaffold + exo-50 group,scaffold + exo-100 group,and scaffold + exo-200 group.At 10 d postoperatively,the material was taken after perfusion,embedding,sectioning and immunofluorescence staining.Vascular regeneration was assessed by RECA,CD 31,and v WF staining;nerve regeneration was assessed by Tuj-1 staining;and inflammation inhibition was assessed by CD 68 staining.The above experiments demonstrated that 100 μg SCMECs-Exo promoted neurovascular regeneration and inhibited inflammation best.Based on the above results,follow-up experiments were performed,and the early experimental groups were as follows: control group,scaffold group,scaffold + PTX group,scaffold + exo-100 group,scaffold + exo-100 + PTX group;late experimental groups were grouped as follows: control group,scaffold group,scaffold + PTX group,scaffold + exo-100 group,scaffold + exo-100 + PTX group.After modeling,the recovery of hind limbs of SCI rats was continuously assessed by BBB score from 2 w to 8 w.At 8 w,hind limb muscle strength test,oblique test and gait analysis experiment were performed to assess the recovery of hind limb muscle strength and coordination in SCI rats.Subsequently,the rats were taken by cardiac perfusion and stained by extraspinal images,spinal H&E,Masson,Nissl and LFB staining and spinal immunofluorescence staining(RECA,CD 31,Nestin,Tuj-1,MAP 2,5-HT,Ch At,TH,NF,MBP,Olig 2,Syn,CD 4,CD 8,CD 86,CD 206.Iba-1,CS-56,Fibronectin,Laminin,etc.),external bladder imaging,H&E and Masson staining were used to systematically evaluate the therapeutic effects of multifunctional stent SCI,and the related mechanisms were investigated by high-throughput sequencing,Western Blot method and qPCR.Meanwhile,the safety of multifunctional stents was evaluated by hematological results and H&E staining of heart,liver,spleen,lung and kidney.Results:An aligned GelMA/PCL electrostatic spinning membrane was prepared using an electrostatic spinning method with a fiber diameter of 108.82 ± 60.09 nm,and the microstructure was not altered by the dropwise addition of GelMA hydrogel to the fiber membrane.In order to simulate the spinal cord structure,the fiber membrane was made into a scaffold with a diameter of 2 mm and a length of 4 mm.The SEM showed that the central pipe diameter of the scaffold was 857 μm,and the inter-fiber membrane spacing was 363 ± 288 μm.The tensile stress-strain curve was performed,and the results showed that the maximum fracture strength of the aligned scaffold was 722.49 k Pa,the fracture energy was 192.56 KJ/m2,and the maximum elongation was 62.52 %.was 62.52 %,and its Young’s modulus was 0.91 ± 0.06 k Pa,which was similar to that of neural tissue(1-10 k Pa).According to the compressive stress-strain curve,its elastic modulus was 0.04 ± 0.01 k Pa,which was similar in size to the modulus promoting the differentiation of NSC to neurons(0.001-0.1 k Pa).The GelMA hydrogel covered on the surface of the fibrous membrane had a gelation time of 113 s and an elastic modulus of 313.3 Pa.The extracted NSCs were homogeneous in morphology and stained positive for Nestin and SOX 2.The microvascular endothelial cells were "pavement-like" under light microscopy,stained positive for CD 31 and negative for α-SMA,and the flow pattern results showed positive for v WF and negative for Desmin.SCMECs-Exo had a diameter of 142.2 ± 1.4 nm,a zeta potential of-29.56 m V,and expressed CD 9,CD 63,and TSG 101;no significant changes occurred after drug loading(diameter of 131 nm,zeta potential of-21.31 m V,and expressed CD 9,CD 63,and TSG 101).In vitro experiments,CCK 8,proliferation and differentiation assays demonstrated that 100 μg SCMECs-Exo had the best effect on NSC proliferation and differentiation.In vivo experiments,the results were similar,with the highest percentage of fluorescence area positive for RECA,CD 31,v WF and Tuj-1 in the scaffold + exo-100 group and a low percentage positive for CD 68 staining.In the early experimental group,the scaffold + exo-100 + PTX group had the highest percentage of positive fluorescence area for CD 31,RECA,Nestin,Tuj-1 and CD 206,and Nestin had high co-localization staining with Ki 67,while the percentage of positive staining for CD 4,CD 8,CD 86 and Iba 1 was low;in the late experimental group,the scaffold + exo-100 + PTX group had the highest percentage of positive fluorescence area for RECA,MAP 2,5-HT,Ch At,TH,NF,MBP,Olig 2,Syn,CD 206,and RECA had high co-localization staining with Ki 67,while CD 86,Iba 1,CS-56,Fibronectin,and Laminin staining had a low percentage of positive staining.nissl staining showed the best neuronal morphology in the scaffold + exo-100 + PTX group,and LFB staining showed similar results.h&e staining showed that the scaffold + exo-100 + PTX group had the smallest cavity area,and Masson staining showed that it had the least collagen fiber deposition.External bladder images,H&E staining and Masson staining,had similar results,i.e.,scaffold +exo-100 + PTX group effectively reduced intravesical hypertrophy and myofibrosis.The scaffold + exo-100 + PTX group performed best in BBB score,gait analysis,oblique test and muscle strength test in spinal cord injured rats.The mechanism of scaffold + exo-100 + PTX multifunctional scaffold to promote neurovascular regeneration was further investigated.The results of high-throughput sequencing analysis demonstrated that SCMECs-Exo may act through the wnt/β-catenin pathway,and the expression contents of β-catenin,Wnt 1,and Wnt 3a were examined by Western Blot method and qPCR,which showed that the scaffold + exo-100 + PTX group had the highest β-catenin,Wnt 1,and Wnt 3a expression.Finally,the safety of scaffold + exo-100 + PTX multifunctional scaffold was evaluated.From the hematological results,we can see that Ptx has an effect on leukocytes,lymphocytes,and neutrophils,but the side effects of Ptx can be mitigated to some extent by using SCMECs-Exo for the slow release of Ptx.From the results of H&E staining of the viscera,no significant differences were seen between the groups.Conclusions:Aligned GelMA/PCL electrospun scaffolds combined with Ptx-loaded SCMECsExo exhibited the ability to inhibit inflammation and promote neurovascular regeneration in SCI treatment,and improved muscle strength and motor function in the hind limbs of rats with spinal cord injury.This provides a new therapeutic strategy for modulating the inflammatory microenvironment of SCI to achieve dual neuro-vascular protection.