| Objective:To investigate the effect of 3-Aminopropyltriethoxysilane(APTES)combined with graphene oxide(GO)coating on the surface of pure titanium on the proliferation of rat Schwann cells(SCs)and the expression of nerve regeneration related factors.Methods:GO coatings with different mass concentrations(0.25,0.5,0.75,1.0mg/m L)were prepared on the surface of titanium(Ti)by covalent bonding of amino groups of APTES.The microstructure,composition and contact angle of the coatings were characterized by scanning electron microscopy(SEM),Raman spectroscopy and contact angle meter.Schwann cells were cultured in vitro,and the cell morphology was observed under an inverted microscope.The purity of SCs(RSC96 cell line)was identified by immunofluorescence staining of central nervous system specific protein S100-β and glial fibrillary acidic protein GFAP.SCs were inoculated on smooth titanium sheet(SS)(control group),silane coupling agent modified titanium sheet(experimental group,Ti-APTES group)and silane coupling agent combined with different mass concentrations of graphene oxide coated titanium sheet(experimental group,0.25-Ti-APTES-GO group,0.5-Ti-APTES-GO group,0.75-TiAPTES-GO group,1.0-Ti-APTES-GO group).The proliferation activity of SCs on titanium sheets in different treatment groups was detected by CCK-8 method.Realtime fluorescence quantitative PCR(q RT-PCR)and Western blotting(WB)were used to detect the expression of nerve growth factor(NGF)and glial cell derived neurotrophic factor(GDNF)in SCs cultured on titanium plates of different treatment groups on the fifth day.Results:1.Scanning electron microscopy observation of silane coupling agent composite graphene oxide coating on the surface of titanium sheet,with the increase of GO mass concentration,the cloud-like protrusions are more dense;the surface of the Ti-APTES group showed long rod-like structure protrusions,and the surface of the SS group was relatively flat.2.Raman spectroscopy detected two graphene characteristic peaks on the surface of Ti-APTES-GO group,namely D peak and G peak,while SS group and Ti-APTES group showed a straight line.3.Compared with the SS group,the contact angle of the Ti-APTES group was the smallest,while the contact angle of the TiAPTES-GO group decreased with the increase of the mass concentration of graphene oxide,the static water contact angle decreased and the hydrophilicity was better.4.Under the microscope,the morphology of SCs cells was normal and the growth was good;immunofluorescence staining identified high purity of cell lines.CCK-8 test results showed that compared with SS group,high concentration 0.75-Ti-APTES-GO group and 1.0-Ti-APTES-GO group showed inhibitory effect on SCs.5.q RT-PCR results showed that the expression levels of NGF and GDNF genes in the 0.5-TiAPTES-GO group were the highest(P<0.05).6.WB results showed that the expression of NGF protein in 0.5-Ti-APTES-GO group was higher than that in SS group and 0.25-Ti-APTES-GO group(P<0.05),while there was no significant difference in NGF protein expression between 0.5-Ti-APTES-GO group and TiAPTES group(P>0.05).In addition,the protein expression of GDNF in the 0.5-TiAPTES-GO group was also the highest(P<0.05).Conclusions:Under the conditions of this study,the graphene oxide coating with 0.5 mg/m L silane coupling agent on the surface of pure titanium has no obvious cytotoxicity,and can better promote the expression of nerve growth factor and glial cell-derived neurotrophic factor of SCs on the surface.It provides a theoretical basis for the coating to improve the bone perception ability around the implant. |
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