| Spinal cord injury(SCI)is one of the most serious injuries to the central nervous system(CNS),and it is difficult to achieve effective neural recovery only for a single factor.Stem cells from human exfoliated deciduous teeth(SHED)are derived from neural crest and show strong neurotropic characteristics.SHED-derived exosomes show the function of their mother cells,that is,the ability of microenvironment regulation and the therapeutic potential of replacing lost cells.Systemic exosome therapy is limited by the low retention rate of the lesion site,while local injection of exosomes is difficult to obtain effective retention and sustained effect at the lesion site.Therefore,there is an urgent need to construct an exosome delivery device to achieve large retention,sustained release and efficient integration of exosomes at the lesion site in SCI.1.Culture and identification of seed cellsIn this study,morphology of SHED are observed by optical microscope,surface marker proteins of SHED were detected by flow cytometry,proliferation potential of SHED was detected by plate cloning experiment,and the osteogenic and adipogenic differentiation potential of SHED was detected by osteogenic and adipogenic differentiation experiments.Results showed that SHED expressed marker proteins CD44,CD73,CD90 and CD 105 of stem cells,but did not express negative marker protein-HLA.SHED have strong proliferation potential and the potential to differentiate into osteoblasts and adipocytes.In addition,in this study,SHED were induced to differentiate into nerves by induction experiments in vitro.The proliferation of cells before and after induction differentiation was detected by CCK8 experiment,and the expression of cell skeleton and nerve marker protein before and after induction differentiation was detected by immunofluorescence.The results showed that the proliferation ability of SHED was significantly weakened after induction of neural differentiation.The cell morphology changed,the protrusions increased and the protrusions became longer.The cells expressed more MAP2,a nerve marker protein.2.Preparation and characterization of spinal cord assemblies(SCA)In this study,the proliferation of cells cultured with different concentrations of ascorbic acid(AA)was detected by CCK8 assay,the mechanical properties of SCA were detected by atomic force microscopy,the structure and morphology of SC A were detected by HE,and the distribution of two kinds of cells in SCA was detected by celltracker CMFDA and CM-DiI staining.Collagen distribution and expression of nerve marker proteins β3-tubulin,MAP2,Nestin,S100,CGRP and MBP in SCA were detected by immunofluorescence.The results showed that the optimal AA concentration of SHED was 120 μg/mL.The mechanical properties of SCA are similar to those of spinal cord tissue.SCA are kind of porous three-dimensional structure;The two types of functional cells exist in large quantities in SCA and are scattered.Collagen I and collagenⅢ were expressed in large quantities in SCA,and their distribution was nestlike.Neuromarker proteins β3-tubulin,MAP2,Nestin,S100,CGRP and MBP are expressed in SCA in large quantities.3.Neurotrophic characteristics of spinal cord assemblies-derived exosomes(SCA-Exo)In this study,exosomes were extracted by overspeed centrifugal method,exosome morphology was observed by transmission electron microscopy,exosome size was detected by particle size analysis,exosome marker protein was detected by WB,exosome miRNA was detected by transcriptomic sequencing,and exosome protein was detected by proteomic sequencing.The results showed that exosomes were double membrane and concave disk structure.The average particle size of SCA-Exo and SHED aggregates-derived exosomes(SA-Exo)was 73.79 nm and 73.68 nm respectively.Exosomes expressed CD9,CD81 and TSG101,but did not express Calnexin.Transcriptome sequencing results showed that compared with SA-Exo,there were 96 different miRNAs in SCA-Exo,among which 55 miRNAs were up-regulated and 41 miRNAs were down-regulated.GO enrichment showed that differential miRNAs were enriched in aspects of "nervous system development","angiogenesis","neuron differentiation","Wnt signaling pathway","axonogenesis" and "myelination".KEGG pathway showed that differential miRNAs were enriched in the "VEGF signaling pathway","axon guidance","neurotrophin signaling pathway" and other signaling pathways.Proteome sequencing results showed that compared with SA-Exo,there were 267 differentially expressed proteins in SCA-Exo,among which 226 proteins were up-regulated and 41 proteins were down-regulated.GO enrichment analysis showed that differential proteins were enriched in "nerve development","negative regulation of intrinsic apoptotic signaling pathway" and "VEGF-activated receptor activity ".KEGG pathway analysis showed that differential proteins were enriched in "Wnt signaling pathway","neurotrophin signaling pathway","axon guidance" and other signaling pathways.4.The therapeutic effect of SCA on SCI ratsIn this study,SCI models were constructed by total spinal cord transection operation,and weight changes of rats in each group were compared by body weight test.The recovery of sensory function of hind limbs was detected by Von Frey filament test,the recovery of autonomic urination was detected by bladder histology,and the recovery of motor function of hind limbs was detected by BBB score.The results showed that the hindlimb sensation of SCA rats recovered earlier,20 days after surgery,20%of the rats in the Ctrl group recovered,80%of the rats in the SA group recovered,and all the rats in the SCA group recovered.The spontaneous urination of SCA rats was resumed earlier,the bladder folds of SCA rats were obvious,and the bladder volume and weight of SCA rats were smaller than those of other groups.At 60 days after surgery,the BBB scores of the Ctrl group,SA group and SCA group were 2.60±0.89,7.60±1.14 and 11.60±1.14,respectively.The hind limb motor function of SCA rats recovered earlier and better.5.Study on the repair mechanism of SCA in SCI ratsIn this study,protein immunofluorescence staining was used to detect the expressions of NF,GFAP,MBP and CGRP in the spinal cord tissues of rats in each group,and statistical analysis was conducted to compare the cystic area of the spinal tissue and the differences in the expression of the above proteins among the groups.HE staining was used to detect whether there was tissue damage in major organs.The results showed that compared with the other groups,SCA group had smaller cystic area of spinal cord tissue,higher expressions of NF,MBP and CGRP,and less positive expression of GFAP.SCA have no side effects and are safe biological filling materials.In summary,we designed a bioactive scaffold with spinal cord-like structure-SCA.SCA maximally mimic the developing spinal cord microenvironment and promote cell survival,proliferation and differentiation.SCA are also naturally stable structures for the sustained release of exosomes.In addition,SCA loaded with both SHED and iSHED functional cells can continuously secrete exosomes in a stable extracellular matrix microenvironment.SCA-Exo contain a variety of neurotrophic factors and have strong neurotrophic properties.SCA-Exo can effectively promote neuronal regeneration,axon extension and inhibit glial scar formation by acting on target cells.The continuous action of SCA-Exo improved the pathological microenvironment and promoted in situ central neuroplasticity.Applying SCA to SCI rat showed significant efficacy in the recovery of sensory and motor functions.Thus,SCA therapy is an effective treatment strategy for SCI based on neurotrophic exosome delivery and also provides valuable insight into the pathological microenvironment regulation of CNS diseases and tissue injury. |
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