| Spinal cord injury treatment has two major difficulties: First,the nervous system regeneration is weak;Second,the formation of scar tissue injury,inhibition of neuronal axonal regeneration.In view of the above difficulties,stem cell / tissue engineering scaffold implantation is one of the most promising treatments for repairing spinal cord injury.The main therapeutic principle of this strategy is:(1)to supplement the endogenous neural stem cells(NSCs)Nerve cells,and the formation of neural relay circuits(neural relay circuits);(2)improve the local microenvironment,activate the spinal cord internal regeneration mechanism to promote damage repair.In this study,we create a sustained delivery of SHH system,EMSCs were employed in this study as the seeded cells in to test the biocompatibility of SHH-GP-ASCSs.Then we transplanted the scaffolds into the lesioned area to repair the SCI in rats,and explore the mechanism.This scaffolds suggested that its potential for repairing SCI.Related research is as follows:1.Preparation of acellular spinal cord scaffolds(ASCSs)The fresh spinal cord was treated with chemical and physical methods to decellularization.The ASCSs were stained with HE and DAPI,the DNA extracted from the ASCSs tested by agarose gel with etdidium bromide.The results showed that no residual DNA in ASCSs.Histologic staining and immunohistochemistry showed that the ASCSs remained laminin,fibronectin and myelin.The microstructure was examined by scanning electron microscopy(SEM).2.Genipin-croslinked ASCSsUsing genipin to crosslinking ASCSs,and further explored the required time for cross-limking.The crosslinking index was measured at different times.The result showed that the cross-linking was saturated at 24 h.The in vitro degradation,moisturecontent and elastic modulus were determined,the results showed that no significant difference after GP-croslinking.3.Preparation of SHH sustained release ASCSsA suitable amount of SHH was added to different concentrations of genipin to pre-crosslink,and then the spinal cord decellularized scaffold was placed in cross-linking to prepare a biological composite scaffold(ASCSs-GP-SHH).The ELISA test of its sustained release of SHH showed that SHH can be slowly released for at least 4 weeks.And explored the optimal concentration of genipin in the construction of a sustained-release system.The results showed that genipin at 5mg/ml was the optimal concentration for the sustained-release system.4.Isolation and characterization of EMSCsThe EMSCs was isolated from nasal mucosa and cultured.Immunoflourescnet staining showed that almost all of EMSCs robustly expressed mesenchymal and neural crest stem cells markers,including CD144,CD44,HNK-1,nestin,vimentin and S100.We also found that EMSC can also produce neurotrophic factors,such as SHH,NT3,BDNF and so on.5.The EMSCs were seeded on SHH-GP-ASCSsThe cultured 3-5 generation EMSCs were co-cultured with the above biocomposite scaffolds.The MTT assay showed that the prepared biocomposite scaffold has good biocompatibility.The EMSCs were induced to differentiate into neuron-like cells on the scaffold.Immunofluorescence and Western Blot showed that EGCs expressed neuronal cell markers such as β-tubulin III,GAP43,MBP,and chAT after induction,and the expression of GFAP was significantly less than that of other groups.This indicates that the biocomposite scaffold not only induces neuron-like differentiation of EMSCs,but also inhibits the differentiation of EMSCs into glial cells.6.The mechanism of SHH-GP-ASCSs promoting the proliferation and differentiation of EMSCsThe protein of induced EMSCs was extracted and detected by Western Blot.The results showed that the induced EMSCs were expression of Integrin,FAK,Patched,Smoothen,Boc and SFKs.The results showed that the proliferation and differentiation of EMSCs were through the Integrins-FAK,Patched-Smoothened-Gli and BOC-SFKs pathway.7.To evaluate the effect of SHH-GP-ASCSs on the repair of SCI in ratsThe SHH-GP-ASCSs were transplanted into rat spinal cord injury area.After operation,the BBB score was used to evaluated the recovery after SCI.The results showed that SHH-GP-ASCSs could promote the function recovery after SCI.After 12 weeks,imunohistochemical staining and Western Blot results showed that in the SHH-GP-ASCSs groups the expression of Nestin,GAP43,NF200 and MBP were increased and GFAP decreased.The results showed that SHH-GP-ASCSs could repair the rat SCI by recruiting endogenous neural stem cells to the spinal cord injury and promoting it differentiated into nerve cells and inhibiting the glial scar formation. |
