Biological Property And Characterization Of A Bone Sialopirotein Fragment Coated TiO2 Nanotube

ObjectiveTitanium(Ti)and titanium alloys are now widely used in dental implants and or-thopedic stents due to their advantageous mechanical properties,biocompatibility and stability.In recent years,a variety of titanium implant surface modifications have been developed to accelerate the osseointegration rate of implants.TiO2 is corrosion resistant and facilitates cell attachment and subsequent deposition of extracellular ma-trix.Recently,some progress has been made in the preparation of TiO2 nanotubes,and research related to its clinical application has been promoted.It is found that the sur-face roughness of TiO2 can be changed by modifying the surface of TiO2 nanotubes by different methods.A number of studies have shown that the adsorption of proteins or peptide coatings on the surface of the TiO2 implants can promote cell adhesion and growth.Among them,the peptide fragment of bone sialoprotein Phe-His-Arg-Arg-Ile-Lys-Ala(FHRRIKA)can promote the adhesion and self-renewal of human embryonic stem cells,and coating FHRRIKA on the metal surface can inducethe adhesion of me-senchymal stem cells and bone mineralization.In this study,in order to investigate the effects of FHRRIKA-functionalized TiO2 nanotube on the bioactivity of alveolar bone mesenchymal stem/progenitor cells,ex-plore the biological characteristics of heparin binding site interaction of TiO2 nano-tubes coated with osteophyte fragments and extracellular matrix,and the clinical sig-nificance of it for surface modification technology and development of novel dental implants,we first characterized FHRRIKA modified TiO2 nanotubes,and detected the biological behavior of the alveolar bone cells and gingival fibroblasts on the FHRRI-KA modified Ti02 nanotubes.Methods1.First,the alveolar bone cells and gingival fibroblasts from alveolar bone of the same volunteer were isolated and digested with collagenase I to prepare a single cell suspension and were cultured in vitro for 2 weeks in DMEM medium(about 3-5 gen-erations).After the generation,the cells were trypsinized and inoculated into 6-well plates to the fusion state,then the chemically defined osteogenic differentiation me-dium was added to induce osteogenic differentiation for 3 weeks.According to the kit instructions,TRIzol was used to extract total RNA of the two cells,and the expression levels of the osteogenic marker genes of both cells were detected by RT-PCR.The genes were RunX2,osteocalcin,osteopontin and bone sialoprotein.2.The thin Ti foil was treated by anodizing at 20 V for 3 hours to prepare TiO2 nanotube film with uniform diameter(100 nm).The TiO2 anodized nanotube layer was immersed in a 1×102mol/1(3-aminopropyl)-triethoxysilane(Sigma-Aldrich)so-lution in anhydrous hexane for 2 hours for silanization,followed by immobilization of the FHRRIKA peptides on TiO2 anodized nanotube layers in anhydrous DMF(1 mM)for 2 h.Then,the fuorescein isothiocyanate-conjugated FHRRIKA was synthesized and immobilized on the TiO2 nanotube layer under the same conditions,and the sur-face was imaged by a fluorescence microscope to detect whether the protein was suc-cessfully coated.Subsequently,the microstructure of the anodized nanotube layers and the immobilized peptides was observed by a sputter coater coating and a scanning electron microscopy(100,000× magnification),and the surface roughness of the pep-tide-coated TiO2 nanotube layer was observed by an MFP-3D-BIO Inverted Optical Atomic force microscopy system.The scan rate was 0.5 Hz and the scan area was about 1×1 μm2.Finally,the surface roughness of the nanotube layer was quantified using the NanoNavi Software.3.Alveolar bone cells and gingival fibroblasts were loaded onto the surface of FHRRIKA-modified anodized TiO2 nanotubes to serve as an experimental group.At the same time,nanotubes not modified with FHRRIKA peptide were used as a control group.After 4 hours of seeding,the following experiments were carried out:1)after the sample was washed with phosphate buffered saline,and fixed in 2.5%glutaralde-hyde,the sample were sputter-coated with gold,and the morphological features of the cells was observed by an FEI SIRION 200 system;2)similarly,after the cells were washed and fixed,they were blocked with 1%bovine serum albumin,and then were incubated with monoclonal anti-actin antibody(the primary antibody,1:2000),and rabbit anti-mouse fluorescein isothiocyanate-conjugated secondary antibody(1:500).The cell nuclei were stained by 4’,6-diamidino-2-phenylindole(DAPI),and finally the sample was mounted onto glass slides,and the actin of the cytoskeleton of the two groups of cells were subjected to confocal laser scanning microscopy for immunoflu-orescence detection.After washing the cells with phosphate buffered saline,the live cells were stained with calcein AM,and the dead cells were stained with ethidium homodimer,and the cell viability was determined by observing the ratio of living cells/dead cells under a fluorescence microscope.In addition,on the 7th and 14th day of cell culture,cells on the surface of FHRRIKA peptide-modified nanotubes were washed and lysed,and activity of alkaline phosphatase(ALP)was detected by activity assay.RT-PCR assay was performed for the expression levels of RunX2,osteocalcin,osteopontin and bone sialoprotein belonging to osteogenic marker genes were ana-lyzed to investigate cell differentiation.At the end of the experiment,the results were statistically analyzed using SPSS 19.0 software.Data are expressed as mean ± SEM.P<0.05 was used as the threshold,and the paired sample Student’s t test was used to compare each dependent variable.Results1.The donor homologous alveolar bone cells and gingival fibroblasts were suc-cessfully isolated and cultivated,and the cells displayed a spindle shape when cul-tured in DMEM medium.After 3 weeks of induction with the chemically defined os-teogenic medium,von Kossa staining revealed that mineralized matrix deposition was found only in alveolar bone cells,and mineralized nodules were detected in them,which were almost undetectable in gingival fibroblasts.Consistent with von Kossa staining,and the expression levels of osteogenic markers genes including Runx2,os-teocalcin,osteopontin and osteopontin were significantly higher in alveolar bone cells than those in gingival fibroblasts.2.Verification of the surface coating effects of the peptide coating revealed that the functionalized TiO2 surface showed peptide concentration-dependent fluorescence intensity,and the peptide concentration was stable at 1.6 mg/cm2,so we concluded that the FHRRIKA peptide can be stably coated on the surface of TiO2 anodized nano-tube layer.Scanning electron microscopy revealed that the FHRRIKA peptide modifi-cation did not change the surface topography of the TiO2 nanotube compared to the surface structure of the anodized TiO2 nanotubes.The diameters of the two sets of na-notubes were both about 100 nm.The results of atomic force microscopy showed that the functional modification of the peptide did not significantly change the surface roughness of the anodized nanotube layer.The uniformly ordered microstructure on the surface of the nanotube membrane can facilitate the binding of extracellular ma-trix proteins.3.Scanning electron microscopy showed that both alveolar bone cells and gin-gival fibroblasts can be successfully attached to FHRRIKA modified TiO2 anodized nanotube layers,but the number of adhered alveolar bone cells was approximately twice that of gingival fibroblasts.The FHRRIKA modification can selectively recruit alveolar bone cells.On the surface of unmodified Ti nanotubes,the cytoplasm was more condensed.On the FHRRIKA-modified surface,both alveolar bone cells and gingival fibroblasts formed a polymerized actin structure with a well-organized cy-toskeleton network,whereas a relatively disorganized cytoskeleton was observed in the cells on the unmodified nanotube surface.No morphological differences were ob-served between the two cell types on the modified and unmodified surfaces.The FHRRIKA-modified surface had a greater number of attached cells than the unmodi-fied surface,the modified surface showed preference for alveolar bone cells,approx-imately two times more alveolar bone cells than gingival fibroblasts were found to be adhered to the surface.This may be due to the FHRRIKA motif derived from bone salivary protein on osteoblasts.the alveolar bone cells on the FHRRIKA-modified surface had significantly higher alkaline phosphatase activity than those on the unmo-dified surface,whereas the alkaline phosphatase activity of gingival fibroblasts was barely detected on both modified and unmodified surfaces.Analysis of gene expres-sion of osteogenic markers revealed that the expression levels of osteogenic markers of alveolar bone cells increased significantly within 2 weeks of differentiation induc-tion,while the expression of osteogenic markers was hardly detected in gingival fi-broblasts.ConclusionOur results suggest that,in agreement with previous studies,alveolar bone cells have the potential to induce differentiation into bone,whereas gingival fibroblasts do not.In this study,we successfully coated the FHRRIKA peptide membrane to the sur-face of TiO2 nanotubes for the first time.FHRRIKA peptide modification can selec-tively recruit alveolar bone cells and enhance their adhesion to TiO2 nanotubes.FHRRIKA peptide coating of TiO2 anodized nanotube causes osteoinductive activity of alveolar bone cells,promotes osteoblast/progenitor cell differentiation and minera-lized matrix deposition,has the potential to promote tissue integration of TiO2 nano-tube implants,and provides innovative surface modification of TiO2 implants.There-fore,FHRRIKA coating has the potential to promote the integration between dental TiO2 nanomaterial implants and surrounding bonetissue and provides new ideas for surface modification of clinically designed implants.