| Objective:In the middle of the 20th century,the Swedish scientists Branemark advanced bone combined theory,which laid a theoretical basis and promote the development for oral implantology,and implant restoration became the first choice for tooth replacement in clinical practice.different materials,different structure were designed for implant and implant surface to increase the implant bone union,and the roughened treatments is the main method for surface treatment.As the most mature processing method,it could promote the adhesion of osteoblast,and accelerate bone integration.But certain failure rate exists,inflammation around the implant is regarded as an important factor which lead to the failure of planting（13-18）.bacteria adhesion on the implantation surface is crucial for inflammation around the implant.Bacteria adhesion increased the plaque formation probability,And coarsening of implant surface,influence the bacteria adhesion and proliferation implant surface.Compared with titanium implants,zirconia with excellent properties,has become an advanced research hotspot.In this study,To Explore the effect of different materials and surface treatment on bacteria adhesion and proliferation,zirconia and titanium with Sand blasting-double acid surface treatment were used.Scanning electron microscopy（SEM）was used to observe the material surface morphology,energy spectrum analysis was used to detect the elements of materials.porphyromonas gingivalis and Prevotella intermedia inoculation on the planting material.Colony forming unit count method,SEM,MTT method,and crystal violet staining assay were used to observe the bacteria adhesion and proliferation.The results could provide theoretical basis for the further application of implanting.Methods:1 Preparation and measurement of titanium and zirconiaZirconia discs were prepared in 19mm diameter and 1mm thick（46 sheets）,Ta2 titanium discs were prepared in 19mm diameter and 1mm thick（46 sheets）.2 Experimental groupsExperimental samples were divided into 4 groups,18 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 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 Bacteria recovery,activationStandard strains of thawing,Porphyromonas gingivalis（Pg）,Prevotella intermedia（Pi）were inoculated on strengthen the brucella medium,in anaerobic culture box containing 10%CO2,10%H2,80%N2,37℃,48h,Optical microscope to observe bacteria to check out the pure consecutive batches to the third generation as experiment with strains of bacterial strains.7 Bacteria adhesionAmong Porphyromonas gingivalis（Pg）,Prevotella intermedia（Pi）inoculated separately to each of the surface of the specimen.8 Tablet colony counting method for calculating the number of bacteria on the surface of the materialIn training time for 24 h,72 h,120 h,the fluid of PBS elution was not attached to the bacteria on titanium plate and then get the bacteria on the titanium plate by ultrasonic vibration suspension,quickly absorb lOul bacteria liquid with times than dilution（dilution ratiol:103）,quickly absorb liquid mixing of bacteria 50ul on strengthen the brucella medium surface,evenly coated in the anaerobic environment to cultivate 120h,according to the number of colonies and dilution ratio,bacteria number calculation.9 Determined by MTT method to detect all kinds of bacteria on the surface of the material.Two kinds of bacteria inoculated respectively within 24 h,72 h,120 h,randomly taking out Group A and Group B inoculated bacteria of zirconia and Group C and D of bacterial pure titanium plate 6 pieces of each,determined by MTT method to detect each sample surface bacteria at 550 nm absorbance（OD）value.Each group sets up three parallel groups.10 Crystal violet staining method to detect all kinds of bacteria on the surface of the materialTwo kinds of bacteria inoculated respectively within 24 h,72 h,120 h,randomly taking out group A,B inoculated bacteria of zirconia and C,D of two groups of bacterial pure titanium plate 6 pieces of each,1%glutaraldehyde fixation after using crystal violet stain,33%acetic acid after decolorizing,testing each sample surface bacteria at 590 nm absorbance（OD）value.Each group set up three parallel groups.11 Scanning electron microscopy（sem）10 testing all kinds of bacteria on the surface of the materialTraining time for 24 h,72 h,120 h,PBS wash,1%glutaraldehyde fixation,dehydration,ethanol gradient critical point drying,ion coating machine spray gold,scanning electron microscope the specimen surface bacteria adhesion.12 Statistical analysisStatistical analysis was performed by SPSS21.Tablet colony counting method,determined by MTT staining method,crystal violet staining method with completely random design analysis of variance,the set of comparison between the SNK-q test All data were presented as Mean±Sd.significant level α=0.05,P<0.05 was considered as the level of statistical significance.Results:1 Surface roughnessThe surface roughness of group A:0.648±0.229μm;The surface roughness of group B:0.450±0.053μm;The surface roughness of group C:2.142±0.235μm;The surface roughness of group D:0.674±0.152μm.2 SEMGroup A:there were approximate 0.2～10μm cavities on the sheet,surface,and the homogeneous cavities about 0.2～0.8μm are predominantly.Group B:there were plain stripe-like structures on the surface.Group C:there were 10～20μm of first class cavities and 1～5μm 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 Tablet colony counting method for calculating the number of bacteria on the surface of the material1)Two groups of sample surface bacteria culture 24 h,72 h,120 h,the number of bacteria on the culture plate with mean:1-1)Porphyromonas gingivalis（Pg）（ATCC33277）24h,A(10-3):45.33±9.48,B(10-3):27.83±7.08,C(10-3):31.83±10.61,D(10-3):21.67±5.4672h,A(10-3):90.67±6.47,B(10-3):78.50±6.80,C(10-3):93.67±11.76 D(10-3):66.5±9.65120h,A(10-3):241.33±32.83,B(10-3):202.33±20.55,C(10-3):273.83±32.05,D(10-3):208.17±19.771-2)Prevotella intermedia（Pi）（ATCC15033）24h,A(10-3):38.67±13,19,B(10-3):29.53±14.04,C(10-3):58.83±11.86 D(10-3):25.33±11.3172h,A(10-3):96.83士 14.88,B(10-3):56.33±25.01,C(10-3):96.5± 18.00 D(10-3):58.67±15.24120h,A(10-3):406.83±45.81,B(10-3):145.33±37.33,C(10-3):323.67±43.80,D(10-3):183.17±20.80.There were significant statistical differences within each group（p<0.05）2)Comparison between each group the same time point group2-1)Porphyromonas gingivalis（Pg）（ATCC33277）24h:the number of bacteria adhesion than smooth rough zirconia group zirconium oxide and smooth pure titanium group,fewer than coarse pure titanium group;Coarse pure titanium group of bacterial adhesion number more than smooth pure titanium group,group A and group B,group A and group C,group A and group D,group C and group D colony number difference was statistically significant（P<0.05）.Smooth zirconia group（group B）adhesive bacteria and coarse pure titanium group（group C）,and smooth pure titanium group（group D）the number of colonies no statistically significant difference（P>0.05）,but group B than in group C and group D bacteria adhesion number mean less.72h:Rough zirconia set the number of bacteria adhesion than smooth zirconium oxide and smooth pure titanium group;Smooth zirconia set of adhesive bacteria than less coarse pure titanium and smooth pure titanium group;Coarse pure titanium group of bacteria adhesion amount is more than smooth pure titanium group,group A and group B,group A and group D and group B and group C,group B and group D,group C and group D colony number difference was statistically significant（P<0.05）.Rough zirconia group（group A）and coarse pure titanium group（group C）adhesive bacteria number difference was not statistically significant（P>0.05）,but in group A than in group C bacteria adhesion number mean less.120h:Rough zirconia group of bacteria adhesion number than smooth zirconium oxide and smooth pure titanium group,fewer than coarse pure titanium group;Smooth zirconia than coarse pure titanium group less number;Coarse pure titanium group of bacteria adhesion amount is more than smooth pure titanium group,group A and group B,group A and group C,group A and group D,group B and group C,group C and group D colony number difference was statistically significant（P<0.05）.Smooth zirconia group（group B）and smooth pure titanium group（group D）the number of colonies no statistically significant difference（P>0.05）.But the number of group B than in group D bacteria adhesion mean less.2-2)Prevotella intermedia（Pi）（ATCC15033）24h:Rough zirconia set the number of bacteria adhesion less than coarse pure titanium group;Smooth zirconia than coarse pure titanium group less number;Coarse pure titanium group the number of bacteria adhesion than smooth pure titanium group,group A and group C,group B and group C,group C and group D colony number difference was statistically significant（P<0.05）.Rough zirconia group（group A）number of bacterial adhesion and smooth zirconia（group B）,smooth pure titanium group（group D）;Smooth zirconia（group B）and smooth pure titanium group（group D）colony number difference was not statistically significant（P>0.05）.But group B than in group A and group D than in group A and group B A small amount mean group D bacteria adhesion.72h:Rough zirconia set the number of bacteria adhesion than smooth zirconium oxide and smooth pure titanium group;Smooth zirconia group adhere to the amount of bacteria than less coarse pure titanium group;Coarse pure titanium group the number of bacteria adhesion is relatively smooth and pure titanium,group A and group B,group A and group D and group B and group C,group C and group D colony number difference was statistically significant（P<0.05）.Rough zirconia group（group A）and coarse pure titanium group（group C）.The number of bacteria adhesion,smooth zirconia group（group B）and smooth pure titanium group（group D）difference was not statistically significant（P>0.05）,but in group A than in group C,group B than in group D the number of bacteria adhesion mean less.120h:Rough zirconia set the number of bacteria adhesion than smooth zirconium oxide and smooth pure titanium group,fewer than coarse pure titanium group;Smooth zirconia than coarse pure titanium group less number;Coarse pure titanium group the number of bacteria adhesion is relatively smooth and pure titanium,group A and group B,group A and group C,group A and group D,group B and group C,group C and group D colony number difference was statistically significant（P<0.05）.Smooth zirconia group（group B）and smooth pure titanium group（group D）the number of colonies no statistically significant difference（P>0.05）.But the number of group B than in group D bacteria adhesion mean less.5 Determined by MTT method to detect all kinds of bacteria on the surface of the material1)Groups of sample surface bacteria inoculation training 24 h,72 h,120 h after the absorbance value respectively1-1)Porphyromonas gingivalis（Pg）（ATCC33277）Absorbance values at 24h:Group A:0.628±0.09,group B:0.184±0.01,group C:0.77±0.15,group D:0.22 ± 0.02;Absorbance values at 72h:Group A:1.08±0、10,group B:0.61 ±0.10,group C:1.17±0.12,group D:0.89 ± 0.24;Absorbance values at 120h:Group A:2.02±0.51,group B:1.07±0.20,group C:2.26±0.03,group D:1.69 ± 0.62.There were significant statistical differences within each group.1-2)Prevotella intermedia（Pi）（ATCC15033）Absorbance values at 24h:Group A:0.62±0.15,group B:0.19±0.04,group C:0.85±0.18,group D:0.22 ± 0.02Absorbance values at 72h:Group A:0.95±0.13,group B:0.69±0.09,group C:1.24±0.02,group D:0.72 ±0.11Absorbance values at 120h:Group A:1.92±0.12,group B:1.15±0.06,group C:2.18±0.05,group D:1.35 ± 0.17.There were significant statistical differences within each group.2)Comparison between each group the same time point group2-1)Porphyromonas gingivalis（Pg）（ATCC33277）24h:Rough zirconia set of absorbance values than smooth zirconium oxide and smooth pure titanium group;Smooth zirconia group absorbance value than coarse pure titanium and smooth pure titanium group is small;Coarse pure titanium group absorbance value is larger than smooth pure titanium groups,group A and group B,group A and group D and group B and group C,group B and group D,group C and group D absorbance value was statistically significant（P<0.05）.Rough zirconia group（group A）and coarse pure titanium group（group C）absorbance value difference was not statistically significant（P>0.05）,but in group A than in group C absorbance values mean little.72h:The same as 24 h.120h:Rough zirconia group absorbance value is larger than smooth zirconia group;Smooth zirconia than coarse pure titanium group numerical;Coarse pure titanium group absorbance value is larger than smooth pure titanium groups,group A and group B,group B and group C,group C and group D absorbance value difference was statistically significant（P<0.05）.Rough zirconia group（group A）absorbance value and coarse pure titanium group（group B）,smooth pure titanium group（group D）;Smooth zirconia（group B）and smooth pure titanium group（group D）value of the absorbance difference was not statistically significant（P>0.05）.But in group A than in group C,group D than in group A and group B A group D absorbance values mean little.2-2)Prevotella intermedia（Pi）（ATCC15033）24h:Rough zirconia set of absorbance values than smooth zirconium oxide and smooth pure titanium group;Smooth zirconia set of absorbance value is smaller than coarse pure titanium group;Coarse pure titanium group absorbance value is larger than smooth pure titanium groups,group A and group B,group A and group D and group B and group C,group C and group D absorbance value difference was statistically significant（P<0.05）.Rough zirconia group（group A）and coarse pure titanium group（group C）absorbance value,smooth zirconia group（group B）and smooth pure titanium group（group D）the value of the absorbance difference was not statistically significant（P>0.05）,but in group A than in group C,group B than in group D absorbance values mean little.72h:Rough zirconia set of absorbance values than smooth zirconium oxide and smooth pure titanium group is large,relatively coarse pure titanium group is small;Smooth zirconia set of absorbance value relatively coarse pure titanium group is small;Coarse pure titanium group absorbance value is larger than smooth pure titanium groups,group A and group B,group A and group C,group A and group D,group B and group C,group C and group D absorbance value difference was statistically significant（P<0.05）.Smooth zirconia group（group B）and smooth pure titanium group（group D）of the value of the absorbance difference was not statistically significant（P>0.05）.But the group B than in group D absorbance values mean little.120h:The same as 72 h.6 Crystal violet staining method to detect all kinds of bacteria on the surface of the material1)Groups of sample surface bacteria inoculation training 24 h,72 h,120 h after the absorbance value respectively1-1)Porphyromonas gingivalis（Pg）（ATCC33277）24h:Group A:1.34±0.07,group B:0.59±0.04,group C:1.38±0.32,group D:1.07±0.15;72h:Group A:1.60±0.29,group B:0.66±0.05,group C:1.95±0.33,group D:1.22 ± 0.29;120h:Group A:1.92±0.44,group B:1.07±0.15,group C:2.13±0.25,group D:1.29±0.08There were significant statistical differences within each group.1-2)Prevotella intermedia（Pi）（ATCC15033）24h:Group A:1.43±0.19,group B:0.60±0.11,group C:1.62±0.31,group D:1.07±0.0972h:Group A:1.95±0.08,group B:0.73±0.08,group C:2.10±0.14,group D:1.24 ± 0.22120h:Group A:2.01±0.25,group B:1.22±0.05,group C:2.58±0.25,group D:1.28±0.34.There were significant statistical differences within each group.2)Comparison between each group the same time point group2-1)Porphyromonas gingivalis（Pg）（ATCC33277）24h:Rough zirconia set of absorbance values than smooth zirconium oxide and smooth pure titanium group;Smooth zirconia set of absorbance value is smaller than smooth and rough pure titanium group,group A and group B,group A and group D and group B and group C,group B and group D colony number variations was statistically significant（P<0.05）.Rough zirconia group（group A）and coarse pure titanium group（group C）absorbance value,smooth pure titanium group（group D）and coarse pure titanium group（group C）absorbance value difference was not statistically significant（P>0.05）,but in group A than in group C,group D than in group C absorbance values mean little.72h:Rough zirconia group absorbance value is larger than smooth zirconia group;Smooth zirconia set of absorbance value is smaller than coarse pure titanium,smooth pure titanium group,group A and group B,group B and group C,group B and group D absorbance rooted difference was statistically significant（P<0.05）.Rough zirconia group（group A）and coarse pure titanium group（group C）,smooth pure titanium group（D）,smooth pure titanium（C）and coarse pure titanium group（D）the value of the absorbance difference was not statistically significant（P>0.05）,but in group A than in group C,group D than in group A than in group C,group D absorbance values mean little.120h:Rough zirconia set of absorbance values than smooth zirconium oxide and smooth pure titanium group;Smooth zirconia set of absorbance value is coarser pure titanium and smooth pure titanium group is small;Coarse pure titanium group absorbance value is larger than smooth pure titanium groups,group A and group B,group A and group D,group B and group C,group B and group D and group C and group D absorbance value was statistically significant（P<0.05）.Rough zirconia group（group A）and coarse pure titanium group（group C）the value of the absorbance difference was not statistically significant（P>0.05）,but in group A than in group C absorbance values mean little.2-2)Prevotella intermedia（Pi）（ATCC15033）24h:Rough zirconia set of absorbance values than smooth zirconium oxide and smooth pure titanium group;Smooth zirconia set of absorbance values than coarse pure titanium and smooth pure titanium group is small;Coarse pure titanium group absorbance value is larger than smooth pure titanium groups,group A and group B,group A and group D,group B and group C,group B and group D and group C and group D absorbance value was statistically significant（P<0.05）.Rough zirconia group（group A）and coarse pure titanium group（group C）the value of the absorbance difference was not statistically significant（P>0.05）,but in group A than in group C absorbance values mean little.72h:The same as 24 h.120h:Rough zirconia set of absorbance values than smooth zirconium oxide and smooth pure titanium group is large,relatively coarse pure titanium group is small;Smooth zirconia set of absorbance value relatively coarse pure titanium group is small;Coarse pure titanium absorbance value is relatively smooth and pure titanium groups,group A and group B,group A and group C,group A and group D,group B and group C,group C and group D the absorbance of the value of the difference was statistically significant（P<0.05）.Smooth zirconia group（group B）and smooth pure titanium group（group D）of the value of the absorbance difference was not statistically significant（P>0.05）.But the group B than in group D absorbance values mean little.7 Scanning electron microscopy（sem）tests of various kinds of bacteria on the surface of the materialAt 24 hours,Porphyromonas gingivalis（Pg）and Prevotella intermedia（Pi）were less adherent to the surface of the two materials.The results showed that the number of smooth zirconia and rough titanium groups was large and dense at 72h and 120h,and the number of bacteria on rough surface was geometric growth,bacteria gathered into a group,was a large area of multi-level growth.Conclusion:1 zirconium oxide and pure titanium material surface after roughening treatment increased the gum porphyrin,universal among bacteria bacterium in the material surface adhesion.2 zirconia compared with pure titanium,have reduce gingival porphyrin,universal among bacteria bacterium adhesion properties.3 after roughening treatment of zirconium oxide surface is lower than that of pure titanium surface roughness,but its surface morphology change increases the gum porphyrin,universal among bacteria bacterium in the material surface adhesion. |
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