| Backgroud and objectiveSpinal cord injury(SCI)is a common spinal surgery disease,which causes a series of pathological changes such as neuron loss and axon rupture in the spinal cord.This is a disaster for patients,because it can lead to sensory,motor and autonomic dysfunction below the level of injury,and even cause dysfunction of multiple organs such as respiration,urinary tract and digestive system.Currently,the commonly used clinical treatment methods include drug therapy,surgical therapy and rehabilitation therapy,but none of these methods can achieve the reconstruction of spinal cord structure,promote neuronal regeneration and restore axon conduction function.Tissue engineering techniques effectively combine scaffolds,seed cells and cytokines,bringing new possibilities for recovery from spinal cord injury.Previous studies have suggested that neuro-differentiated mesenchymal stem cells and scaffolds that continuously release cytokines can better promote spinal cord functional recovery.In this study,we first designed a novel membrane scaffold that can be applied to the spinal cord,which is attached to the surface of preneurally differentiated BMSCs and can continuously release NGF,and then transplanted it into acute SCI rats to evaluate the therapeutic effect of this combined therapy.Methods1.In vitro experimentBMSCs from SD rats obtained by whole bone marrow adherents were identified by flow cytometry,and then the BMSCs were induced into neuron-like cells by combined induction of multiple cytokines,and the neural cell markers were detected by immunofluorescence staining to confirm the feasibility of the neural induction scheme.The novel membrane scaffolds were divided into blank scaffolds without NGF and experimental scaffolds with sustainable NGF release.BMSCs induced by neuronal differentiation for 6 days were implanted on the two scaffolds,and the survival rate of the cells on the two scaffolds was detected by cck-8 method at 12h,24h,and 48h.Neuro-differentiated BMSCs were implanted on both scaffolds for 6 days,followed by further induction for 12 days.The biocompatibility between preneurally differentiated BMSCs and the novel thin film scaffolds was observed by scanning electron microscopy and immunofluorescence staining.The key proteins in the TrkA/ERK pathway were detected by western blot analysis,and the possible mechanism of the difference between the two scaffents in inducing BMSCs to differentiate into nerve cells was preliminarily explored.2.in vivo experimentAcute SCI model in rats was prepared by ALLEN’s strike method and treated with tissue engineering technology.The transplanted cells included BMSCs and preneurally differentiated BMSCs,and the scaffolds included experimental scaffolds and blank scaffolds.Group intervention was performed according to experimental needs.After surgery,the recovery of SCI rats’ motor function was continuously observed by BBB exercise score and inclined grid test for 8 weeks.At 1,4 and 8 weeks after surgery,H&E staining/Nissner staining,immunofluorescence staining and nerve electrophysiological detection were used to observe the regeneration of neurons and axons,axon remyelination,cavity and glial scar formation and axon nerve conduction in the spinal cord injury tissues of rats in each group.Results1.In vitro experimentThe attached cells isolated by whole bone marrow attachment method showed relatively uniform distribution of spindle shape,and flow cytometry showed negative CD45 and CD34,and positive CD90 and CD44,which confirmed that these attached cells were BMSCs with high purity.BMSCs were induced by a combination of multiple cytokines,and the structure of BMSCs was like nerve cells,and the cell body shrank and formed slender synapses.Further immunofluorescence staining detection found that about 50%of the cells were nestin positive,and the positive rate of MAP2 was about 70%.CCK-8 assay showed that there was no significant change in the cell survival rate of BMSCs induced by neurogenic differentiation in the two scaffolds compared with the BMSCs without scaffolds.BMSCs were inoculated on the two scaffolds for 6 days after induction for 12 days.Scanning electron microscopy showed that some cell bodies shrank and synapses were elongated.Immunofluorescence staining showed that all the cells were positive for F-Actin.The positive rate of Nestin,MAP2 and GFAP in the cells on the experimental scaffold was about 60%,70%and 10%,respectively.The positive rate of Nestin,MAP2 and GFAP in the cells on the blank scaffold was about 50%,50%and 20-25%,respectively.And the difference between the two was statistically significant.These results suggest that there is a good biocompatibility between preneurally differentiated BMSCs and novel thin film scaffolds.Under the same induction scheme,the proportion of neurons in the experimental scaffold group was significantly higher than that in the blank scaffold group.Phosphorylated TrkA and ERK,the key proteins in TrkA/ERK pathway,were detected by western blot analysis.The results showed that the cell content of the experimental scaffold group was significantly higher than that of the blank scaffold group,and the difference was statistically significant.2.in vivo experimentObservation at 1,4 and 8 weeks after surgery showed that all the grafts were in good position.According to the BBB motor score and the inclined grid test,the motor function and the coordination of the anterior and posterior limbs were significantly better in the combined group of preneurally differentiated BMSCs and sustainably released NGF scaffold than in the other groups.The results of H&E staining and Nissner staining showed that the spinal cord structure of the combined group was relatively intact,with more nissner bodies and fewer cavities,while the spinal cord structure of the other groups was chaotic,the nerve cells were extensively disintegrated,the number of Nissner bodies was reduced and more large cavities were present.The positive rate of GFAP in the spinal cord of rats in the combined group was the lowest and significantly lower than that in other surgery groups,while the positive rate of MAP2,NeuN and MBP in neurons was the highest and significantly higher than that in other surgery groups.Golgi staining showed that the density of dendritic spines in the spinal cord of rats in the combined group was the highest and significantly higher than that in the other groups.The amplitude of SEP and MEP in the combined group was higher than that in the other groups,and the latency was shorter than that in the other groups.In the above observation indicators,the differences between the combined group and the other surgical groups were statistically significant.Conclusion1.BMSCs with high purity can be obtained by the whole bone marrow adherence method.In vitro,the combined induction method of multiple cytokines can successfully induce BMSCs into neuronal cells.2.The new film scaffold has good biocompatibility with pre-neurally differentiated BMSCs.3.In the presence of NGF and chitosan,BMSCs can be induced into neurons.In this process,NGF plays an important role,and it is preliminatively speculated that the mechanism may be realized through the TrkA/ERK pathway.4.Compared with the treatment plan of single transplantation of sustained release NGF scaffold or pre-neural differentiation of BMSCs,the treatment plan of pre-neural differentiation of BMSCs combined with sustained release of NGF film scafold can better promote the recovery of neurological function of SCI rats. |
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