The Effects Of Fibrin Scaffold On The Biological Characteristics Of Olfactory Ensheathing Cells Derived From Human Nasal Olfactory Mucosa

Objective:To establish an effective method for culture of olfactory ensheathing cells(OECs) derived from the adult human nasal olfactory mucosa and investigate the effects of fibrin scaffold on the biological charicteristics of the olfactory ensheathing cells.Methods:The nasal olfactory mucosa was harvested from the posterior part of the nasal septum and cultivated,purified by the method of the different rates of attachment among the various cells types.The morphological changes of cultured OECs were observed at different stages and identified by nerve growth factor receptor p75(NGFRp75),glial fibrillar acidic protein(GFAP) and S-100 immunofluorescence.Fibrinogen was mixed with medium in some proportionality,and added the fresh blood serum with equivalency.Then,the reticular structured fibrin scaffolds were preparation.Finally, the structure of the scaffold was observed by electron microscopy.The human olfactory ensheathing cells(hOECs) were cultured and seeded separately onto the fibrin(Fb) scaffolds as experimental group and the glass slides modified with poly-L-lysine (PLL) as control group.After cultured 14 days,the morph of the hOECs was observed by scanning electron microscopy.To investigate the effects of fibrin scaffold on the myelin associated proteins expression,the hOECs were immunofluorescence stained with antibodies against 2',3'-cyclic nucleotide-3'-phosphodiesterase(CNPase),myelin basic protein(MBP) and neurotrophins.The relative contents of proteins were detected by Western blotting.Results:The cultured mucosa cells presented mainly three morphological types:fusiform cell, bipolar cell and tripolar cell,and most of these cells were positive for NGFRp75,GFAP and S-100.The profile of fibrin scaffold appears translucent and soft spongy-like.Morphological observation by SEM showed that the scaffold formed a three-dimension(3-D) porous network structure.The hOECs grew on the fibrin scaffold showed ribbon-like features and arranged in alignment.Some hOECs have migrated into the scaffolds.The results of immunoluorescence staining and Western blotting indicated that the expressions of CNPase,MBP and neurotrophins of hOECs grew on the fibrin scaffold were higher than that in the control group.Conclusion:The olfactory ensheathing cells derived from nasal olfactory mucosa of adult human may be used as an autotransplant cells to repair the injured central nervous system.The profile of fibrin scaffold appears translucent and possesses flexibility.The fibrin scaffold possessed a 3-D porous network structure.Fibrin as a biomaterial has a bright perspective in construction of the tissue engineering scaffold to repair the injured spinal cord.The fibrin scaffold possesses a good biocompatibility with hOECs and could promote expressions of CNPase,MBP and neurotrophins in the hOECs seeded in the scaffold.The tissue engineering composed of hOECs and fibrin could be used for implantation to repair the injured spinal cord.