| Spinal cord injury(SCI)is a central nervous system injury that can cause severe motor,sensory,and autonomic dysfunction.Since damaged neurons are difficult to regenerate and repair,there has been currently no effective treatment for spinal cord injury.With the development of biotechnology and clinical medicine,cell transplantation has become a potential method to promote the repair of SCI.Transplanted cells can promote spinal cord injury repair through a variety of mechanisms,including neuroprotection,immunomodulation,axon regeneration,neuronal relay formation and myelin regeneration.Due to the adverse microenvironment of the damaged part,the survival and maturation of the transplanted cells become an urgent problem in the clinical treatment of SCI.Scaffolds and cells are the two major components of tissue engineering.In recent years,biological.materials have been widely used in the repair of nervous system damage to improve the microenvironment and promote the survival of transplanted cells.Therefore,a biological scaffold that provides structural and functional support for transplanted cells is beneficial for effective repair of nerve damage.Studies have shown that silk fibroin fibers that are transplanted to the site can improve the damaged microenvironment and promote nerve fiber regeneration without causing an inflammatory reaction,which is an ideal choice for tissue engineering in neuroregenerative medicine.Previous studies in our laboratory showed that silk fibroin fibers had good biocompatibility to neuronal cells and provided abundant adhesion sites for cells.Designing it into a porous three-dimensional(3D)structure contributes to nerve cell migration,proliferation,differentiation,and nerve fiber regeneration at the site of injury.The three-dimensional silk fibrous(3D-SF)used in this has a large amount of fibrous porous structure and has a larger specific surface area.Dermal fibroblasts have become excellent seed cells for reprogramming due to their advantages such as widely distributed,abundant,convenient for autologous transplantation,and strong proliferative ability in vitro.Rat dermal fibroblasts(RDFs)were obtained by tissue mass culture and purified by the passage.Cellular immunofluorescence staining was performed and the results showed that the cells were positive for S100A4 and Vimentin.The RDFs were seeded into the 3D-SF scaffold.The fluorescence staining of the cytoskeleton F-actin and Hoechst 33258 revealed that the RDFs grew along the direction of the scaffold fibers and had a spindle-shaped stereoscopic morphology.Cell proliferation was detected by CCK-8 assay,indicating that 3D-SF provided more adhesion sites and proliferation space for RDFs,and the number of cells in culture continued to increase within 7 days-culture.Flow cytometry was used to detect the cytotoxicity of 3D-SF scaffolds to RDFs,and there was no significant difference in the survival rate of cells grown on 3D-SF compared with those cultured in PDL-coated matrix,suggesting that 3D-SF did not cause cell apoptosis.To sum up,3D-SF has good biocompatibility and is a good choice for cell transplantation.At present,stem cells with neuronal differentiation potential are used for transplantation to repair central nervous system damage.However,most of the stem cells transplanted to the lesion differentiated into astrocytes and oligodendrocytes rather than functional neurons,leading to poor therapeutic effect.Recent studies have shown that direct reprogramming of somatic cells into neurons by multiple methods such as the introduction of exogenous transcription factors,induction of small molecule compounds,and transduction of miRNAs,which can be regarded as a new method to obtain a large number of functional cells transplanted for the treatment of central nervous system injury.Among them,small molecule-induced reprogramming has become the current research hotspots due to their advantages such as high transdifferentiation efficiency,short induction period,and avoiding the risk of exogenous gene insertion.Their biological effects can be precisely controlled by different concentrations and combinations,and are often rapid and reversible.Numerous studies have shown that simple small molecule compounds can reprogram fibroblasts into neurons of different subtypes.In conclusion,small molecule compound-induced reprogramming has great potential for clinical application.In order to screen chemical methods that efficiently mediate the transformation of dermal fibroblasts into neurons,we reviewed a large number of literature and preliminarily identified potential candidate molecules.Seven small molecules were obtained as the main functional molecules by pre-experimental screening,respectively CHIR99021,Forskolin,LDN193189,SB431542,SP600125,VPA and Y27632,which were abbreviated as CFLSSVY.They can modulate signaling pathways that play a key role in neural development and neural differentiation,or regulate histone methylation and acetylation modifications to improve reprogramming efficiency.After induced differentiation of RDFs by CFLSSVY for 7 days,cellular immunofluorescence staining showed that Tubb3-positive chemically induced neurons(CiNs)were obtained.In order to explore whether these seven small molecules are necessary for reprogramming,we reduced the small molecules on the basis of CFLSSVY.Immunofluorescence staining revealed that the reduction of different small molecules was also able to induce reprogramming of RDFs into neurons.Among them,only three small molecules CFV(CHIR99021,Forskolin,VPA)can initiate transdifferentiation,indicating that these three small molecules play a key role in the process of inducing RDFs reprogramming,which is necessary for cell fate transformation.Compared with other combinations,the reprogramming efficiency induced by CFLSSVY is the highest(Tubb3 positive rate is up to 80%),and the CiNs morphology was more typical and stereoscopic.CFLSSVY was finally used as the best small molecule combination for inducing reprogramming of RDFs.In order to promote the survival and maturation of neurons,we optimized the culture program.Studies have shown that CHIR99021,Forskolin and SP600125(CFS)can promote neuronal survival and maturation.After induction for 7 days,other small molecules were removed,and only CFS culture was retained until 14 days.A large number of neurons were NeuN positive,indicating that these cells were mature neurons.After CFS culture for 28 days,most of the CiNs were Synapsin-1 positive,indicating the expression of the synaptic protein,which implied the functional neurons.In summary,the chemical culture protocol that we screened and identified to efficient induction of RDFs reprogramming to mature CiNs laid a foundation for the treatment of SCI by cell transplantationSo far,small molecule-induced differentiation of fibroblasts into neurons has progressed,but its application in the repair of nerve injury in vivo has rarely been reported.Therefore,we used 3D-SF as the scaffold,and transformed the RDFs on the scaffold into mature neurons using the identified small molecule induction protocol,marking that neural networks consisted of 3D-SF and CiNs was successfully constructed.In order to study the repair effect of neural networks,SCI model of rats was established by removing 2 mm of spinal cord from thoracic T9～T10 segment.After the surgery,the neural networks were immediately transplanted to the spinal cord injury of rats,and the observation was carried out for 8 weeks.Histological level detection showed that the transplantation of neural networks effectively reduced the damage cavities and collagen deposition,and promoted nerve fiber regeneration.We detected hindlimb motor function by BBB score and 45°grid climbing experiment and found that transplantation of neural networks significantly promoted the recovery of hindlimb motor function in rats.This study confirmed that transplantation of neural networks consisting of dermal fibroblast-reprogrammed neurons and silk fibrous scaffold significantly promoted nerve regeneration and functional recovery at the site of injury,which provided a new strategy for repairing central nervous system injury including SCI. |
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