| Background:For the surface modification of titanium implants,traditional strategies focus on further exploring new surface micromorphology and improving the physical attachment of osteoblasts on the surface of biomaterials.However,in addition to optimizing physical cell attachment,adhesion mediated signaling pathways should be emphasized and regulated to facilitate early osseointegration.Sandblasted,Large-grit,Acid-etched(SLA)technology is the most classical and widely used method for surface modification of titanium implants.Osteoblasts adhere to the implant surface indirectly through integrin.Integrin can sense the stimulation of the micro-morphology of the extracellular surface,transfer it into the cells,and initiate and regulate intracellular signaling pathways.YAP/TAZ can transfer from cytoplasm to nucleus,co-activate Runx2 through transcription,and regulate the expression of osteogenic genes.In our previous research work,it has been proved that the surface micromorphology of SLA titanium affects the intracellular localization and expression of YAP/TAZ in osteoblasts,thereby promotes the differentiation of osteoblasts.Integrin can regulate YAP/TAZ through Ras analogue GTPases or by regulating cytoskeleton tension.Purposes:On the basis of our previous research,SLA titanium plates and Polished titanium plates were prepared to observe the expression of integrin αl,α2 and β1 inMC3T3-E1 cells on the surface of the two kinds of titanium plates.Furthermore,the gene silencing of integrin αl,α2 and β1 was performed on SLA titanium plates,and the expression of YAP/TAZ and osteogenic differentiation marker genes was observed.Combined with the previous experimental results,it was determined whether the micro-morphology of SLA could further regulate the differentiation of osteoblasts through the mediation of YAP/TAZ by integrin al,a2 and β1.Thus,the molecular regulation mechanism of osseointegration on SLA titanium surface was further explained,which provides a theoretical basis for further improvement of implant surface modification technology.Methods:1.SLA titanium plates and Polished titanium plates were prepared.The surface micromorphology of the two kinds of titanium plates was observed,and the average surface roughness Ra was measured.2.The proliferation activity of MC3T3-E1 cells on the surface of two kinds of titanium plates was detected by CCK-8 technique,and the osteogenic differentiation ability of MC3T3-E1 cells on the surface of two kinds of titanium plates was measured by ALP activity determination.3.The expression of integrin al,a2 and β1 in MC3T3-E1 cells on the surface of SLA titanium plates was detected by qRT-PCR and Western blot.4.MC3T3-E1 cells were cultured on the surface of SLA titanium plates.Integrin al,ca2 and β1 were silenced by siRNA transfection technique,and the silencing efficiency was measured.5.On the surface of SLA titanium plates,the expression of YAP and TAZ in MC3T3-E1 cells decreased significantly at 3,7 and 14 days after silencing integrin subunits(P<0.05),especially at 7 and 14 days after silencing integrin subunits(P<0.05).6.The expression of osteogenic differentiation marker genes such as Runx2,in MC3T3-E1 cells on the surface of SLA titanium disc was detected by qRT-PCR and Western blot after silencing the integrin α1,α2 and β1 for 3,7 and 14 days.Results:1.Polished titanium plates surface was smooth,showing silver-white metallic luster;SLA titanium plates surface was rough,without obvious metallic luster,showing uniform gray.Scanning electron microscopy(SEM)showed that the surface of the polished titanium plates was smooth,and there were many scratches,which interlaced with each other and had different directions.The optical profiler showed that the average surface roughness of SLA titanium plates was much higher than that of polished titanium plates(P<0.05).2.CCk8 assay showed that MC3T3-E1 cells proliferated well on the surface of two kinds of titanium plates,and the number of polished titanium plates was slightly higher than that of SLA titanium plates.The results of ALP semi-quantitative kit showed that the osteogenic differentiation activity of MC3T3-E1 cells on SLA titanium plates surface was significantly higher than that on polished titanium plates surface(P<0.05).3.The results of qRT-PCR and Western blot showed that integrin al,a2 and β1 in MC3T3-E1 cells on the surface of SLA titanium plates were highly expressed at the level of mRNA and protein(P<0.05).4.After silencing integrin αl,α2 and β1 in MC3T3-E1 cells,the expression of integrin al,a2 and β1 decreased significantly at the level of mRNA and protein(P<0.05),and the silencing efficiency was about 70%.5.On the surface of SLA titanium plates,the expression of YAP and TAZ in MC3T3-E1 cells decreased significantly at 3,7 and 14 days after silencing integrin al,α2 and β1(P<0.05),especially at 7 and 14 days after silencing integrin αl,α2 andβ1(P<0.05).6.On the surface of SLA titanium plates,the expression of osteogenic marker genes Runx2,BSP and OCN in MC3T3-E1 cells at 3,7 and 14 days after integrin αl,a2 and β1 silencing significantly decreased at the level of mRNA;the expression of Runx2 decreased more significantly at 7 and 14 days than at 3 days;the expression of BSP decreased most significantly at 14 days;the expression of OCN also decreased most significantly at 14 days(P＜0.05).Western blot results showed that the expression levels of Coll and Runx2 decreased after 3 days of silence.The protein levels of Runx2,ALP and BSP decreased significantly after 7 days of silence,and the decrease of ALP was the most obvious.The protein levels of osteogenic indicators Runx2,ALP,BSP and OCN were also significantly decreased after 14 days of silence,and the decrease of OCN was the most obvious,and the decrease of BSP was more significant than that of 7 days.Conclusion:1.Compared with the polished titanium plates,the surface micromorphology of SLA titanium plates facilitates osteoblast differentiation.2.On the surface of SLA titanium micromorphology,the expression of integrin al,α2 and β1 in MC3T3-E1 cells increased.3.Under the effect of SLA titanium micromorphology,integrin αl,α2 and β1 mediated the high expression and nuclear transfer of YAP/TAZ,regulated the expression of osteogenic genes,and promoted the osteogenic differentiation of MC3T3-E1 cells. |
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