The Effects Of Surface Modified Titanium And Zirconia On Osteoblast Adhesion

Objective: Osseointegration has been the fundamental theory of modern implantology since 1960 s.Titanium?Ti?has been widely used as implant material due to its excellent mechanical and biocompatibilities.It has been found that surface modifications of titanium?Ti?could improve biological properties of titanium,and accelerate cell adhesion on the surface and osseointegration.However,because of its metal characteristics,there is a problem of the possibility of the grayish color of the titanium implant shining through the thin peri-implant mucosa or ion release,thus marring the entire aesthetic result.Zirconia has become a focus recently owing to high intensity,rigidity,corrosion resistance and ivory-like appearance of natural teeth,zirconia ceramic material can be a potential kind of dental implant material to instead of titanium.Zirconia and titanium were introduced into this paper,sandblasting and acid-etching were used for surface treatment,the surface topography of zirconia and titanium samples were determined by scanning electron microscope?SEM?,the surface elementary composition of zirconia and titanium samples were analyzed by energy spectrum analysis;osteoblast was cultured with zirconia and titanium samples,SEM,laser scanning confocal microscope?LSCM?,and MTT were used to observe the effection of different surface topography of zirconia and titanium on osteoblast initial adhension and proliferation,to provide experimental basis for ziaconia as dental implant materials.Methods:1 Preparation and measurement of titanium and zirconiaZirconia discs were prepared in 19 mm diameter and 1mm thick?30 sheets?,Ta2 discs were prepared in 19 mm diameter and 1mm thick?30sheets?.2 Experimental groupsExperimental samples were divided into 4 groups,15 sheets of each group,according to different surface treatment: A: rough zirconia,sandblasted with Al2O₃ and acid-etched with hydrofluoric acid and nitric acid;B: smooth zirconia;C: rough titanium,sandblasted with TiO2 and acid-etched with hydrochloric acid and sulfuric acid;D: smooth titanium.3 Surface roughness measurementThe surface roughness values for zirconia and titanium discs were measured by using TR200 roughness tester.4 Surface configurations identified by SEMZirconia and titanium discs were choosed randomly and observed by SEM after bonding and printing gold.5 Surface elements analysis with X-ray spectrum analyzerSurface elements of zirconia and titanium discs choosed randomly were analyzed by X-ray spectrum analyzer after bonding.6 Recovery and propagation of osteoblastCryopreservation cells were recovered,cultured at 37°C in humidified air containing 5% CO2,passaged at 90% cell confluence.7 Cell inoculationzirconia samples and titanium samples were put into 12-well culture plates after hyperthermia and high pressure disinfected respectively.Make the cell suspension with the densities of 1×10⁵ /ml and 2×10⁵ /ml by utilizing the 2nd³rd healthy osteoblast generations,the cells suspension was inoculated on the surfaces of zirconia and titanium of these 4 groups.8 Cellular morphology observed by SEMTwo of the zirconia and titanium disks were selected randomly after 1h,6h and 24 h of incubation.SEM was used to observe the cell morphology.9 Immunofluorescent staining for osteoblasts actinTwo of the zirconia and titanium disks were selected randomly after 1h,6h and 24 h of incubation,actin expression and distribution ofnosteoblasts was observed by immunofluorescent staining?Phalloidi?.10 The tetrazolium-based colorimetric assay?MTT test?was used for examining the proliferation of cells on each sample.Two of the zirconia and titanium disks were randomly selected after 1h,6h and 24 h of incubation.The MTT test assay was used for examining attachment of cells,the absorbance at 490 nm was measured using microplate reader.11 Statistical analysis:Statistical analysis was performed by SPSS21.0.All data were presented as Mean±Sd.The data of 1h and 6h was not normal distribution the data of 24 h was normal distribution but heterogeneity of variance.K-WH rank sum test was used.to compare differences in the group at each time,and differences between groups at the same time.two two compare between groups,significant level ?=0.05,P<0.05 was considered as the level of statistical significance.Results:1 Surface roughnessThe surface roughness of group A: 0.648±0.229?m;The surface roughness of group B: 0.450±0.053?m;The surface roughness of group C: 2.142±0.235?m;The surface roughness of group D: 0.674±0.152?m.2 SEMGroup A: there were approximate 0.2¹0?m cavities on the sheet surface,and the homogeneous cavities about 0.2⁰.8?m are predominantly.Group B: there were plain stripe-like structures on the surface.Group C: there were 10²0?m of first class cavities and 1⁵?m of second class cavities on the sheet surfaces.Group D: there were visible stripe-like structures on the surface.3 Surface elements analysis with X-ray spectrum analyzerNo heterogeneous elements were found on the surface of zirconia groups and titanium groups.4 Cellular morphology observed by SEMThe cells on group A and group C grow better,and more pseudopodia that embody into the surrounding cavities.But the cells on group B and group D surfaces mostly showed tiled stretching with larger areas and less pseudopodia.5 Immunofluorescent staining for osteoblasts actinThe actin expression of osteoblasts on the surface of group A and group C was hihger than that on group B and group D.There were more pseudopodia on group A and group C,cells formed a three-dimensional structure.Cells on the surface of group B and group D were flat,less pseudopodia.Osteoblasts showed long fusiform or polygon,cellular pseudopod touch each other to formed as network.Cells on the surface of group C showed less integral than that of group A.6 Absorbance values of osteoblasts cultivated on zirconia and titanium samples for 1h,6h,24 h.Absorbance values at 1h:Group A: 0.248±0.019,group B: 0.238±0.038,group C: 0.317±0.027,group D: 0.297±0.040;Absorbance values at 6h:Group A: 0.531±0.019,group B: 0.516±0.037,group C: 0.592±0.053,group D: 0.489±0.025;Absorbance values at 24h:Group A: 0.897±0.080,group B: 0.797±0.029,group C: 0.909±0.085,group D: 0.730±0.036.There were significant statistical differences within each group.Absorbance values at 1h:There were significant statistical differences between Group A and C,Group A and D,Group B and C,group B and D?P<0.05??There were no significant statistical differences between group A and B,group C and D?P>0.05?.Absorbance values at 6h:There were significant statistical differences between Group A and C,Group A and D,Group B and C Group B and D?P<0.05??There were no significant statistical differences between group A and B?P>0.05?.Absorbance values at 24h:There were significant statistical differences between Group A and B,Group A and D,Group B and C Group B and D,Group C and D?P<0.05??There were no significant statistical differences between group B and D,group C and D,group A and C?P>0.05?.Conclusion:1 Sandblasted and acid-etching treatment on zirconia and pure titanium surface can obtain better surface morphology.2 Zirconia is of similar biocompatibility with titanium,could promote osteoblasts initial adhesion.3 zirconia and pure titanium with sandblasted and acid-etching treatment could increase the expression of actin and promote early adhesion and proliferation of osteoblasts.