Bacterial Adhesion on Titanium Nitride-coated and Uncoated Implants: An In Vivo Human Study

ORAL IMPLANTOLOGYTuesday Mar 11 2003 02:02 PMAllen Press x DTPro SystemRESEARCHBACTERIAL ADHESION ON TITANIUM NITRIDECOATEDAND UNCOATED IMPLANTS: AN INVIVO HUMAN STUDYAntonio Scarano, DDSMaurizio Piattelli, MD, DDSGiuseppe Vrespa, MD, DDSSergio Caputi, MD, DDSAdriano Piattelli, MD, DDSKEY WORDSBacterial adhesionDental implantsImplant surfaceTitanium nitrideAntonio Scarano, DDS, is a research fellowat the Dental School, University of Chieti,Italy.Maurizio Piattelli, MD, DDS, is an associateprofessor at the Dental School, University ofChieti, Italy.Giuseppe Vrespa, MD, DDS, is a visitingprofessor at the Dental School, University ofMilano, Italy.Sergio Caputi, MD, DDS, is a professor ofprosthetic dentistry at the Dental School,University of Chieti, Italy.Adriano Piattelli, MD, DDS, is a professorof oral pathology and medicine at the DentalSchool, University of Chieti, Italy. Addresscorrespondence to Dr Piattelli at Via F.Sciucchi 63, Chieti, Italy 66100 (e-mail:[email protected]).80 Vol. XXIX/No. Two/2003Titanium nitride (TiN) has been used in many fields as a surgical instrumentcoating that makes the surgical materials more resistant to wear and corrosion.The aim of the present study was an in vivo evaluation of the bacterial adhesionto TiN-coated (test) and uncoated (control) titanium implants. Six patients agedbetween 21 and 25 years and in excellent systemic health participated in thestudy. All of the participants gave their informed consent. The participants wereselected on the basis of good periodontal health and no signs of mouth breathing.In each of the 6 participants, a removable acrylic device was adapted to themolarpremolarregion of each quadrant of the jaws. One 4 3 13 mm titanium implantwas glued to the buccal aspect of each device. The plasma spray covered 11.5mm of the body of the implant, whereas the neck was machined titanium. Testimplants were glued to the right devices and control implants were glued to theleft devices. After 24 hours, the implants were removed from each device andprocessed for scanning electron microscopy for evaluation of the machinedportion of the implant covered by bacteria. A total of 24 implants were used inthis study, 12 test and 12 control. Surface characterization of the machinedportion of the neck of the implant was performed on an additional 10 implants(5 test and 5 control). On test implants the implant surface covered by bacteriawas significantly lower compared with that of control implants (P 5 .0001). Thesurface roughness was similar in both groups. TiN surfaces showed a significantreduction of the presence of bacteria, and this fact could probably be importantin the decrease of the inflammation of the peri-implant soft tissues.INTRODUCTIONorim 29_106 Mp_80File # 06emdental implants depends on the integrationof the biomaterial with thesetissues. The maintenance of a healthyconnective tissue-implant interface isbelieved to be of critical importance tolong-term implant survival.1,2 It hasntal implants are surroundedby 3 differenttissues: epithelium, fibrocollagenoussoft connectivetissue, and bone, andthe long-term stability ofFIGURES 1-4. FIGURE 1. Test implant; no rods or filamentous bacteria are present (original magnification 33.350). FIGURE 2. Test implant;few cocci (arrows) are present (original magnification 35.000). FIGURE 3. Test implant; single cocci are present (original magnification310.000). FIGURE 4. Test implant; at higher magnification, salivary proteins (arrows) and bacteria (large arrow) are present (originalmagnification 320.000).ORAL IMPLANTOLOGYTuesday Mar 11 2003 02:02 PMAllen Press x DTPro Systembeen hypothesized that a certain widthof the peri-implant tissues is requiredto enable a proper epithelial-connectivetissue attachment. If this soft tissuewidth is not satisfactory, bone resorptionwill occur.3-6Although the success rate of dentalimplants is very high, there are stillfailures, some of which may be relatedto an absence of attachment betweenthe gingival connective tissue and theimplant. The fibers attached to the implantserve as a barrier to epithelial migration,and thus impede bacterial invasion.7 One investigation of the effectsof newly formed plaque on the periimplantand periodontal tissues in beagledogs showed that the plaque accumulationat both tooth and implantproduced a similar inflammatory lesion.8 The microflora around implantsis similar to that of natural teeth,9 andmicrobial pathogens associated withperiodontitis may also contribute toimplant failures.10 This assumptionwas confirmed by a study that foundthat tissue destruction was more pronouncedaround implants than teethafter a 6-week application of cotton ligaturesfollowed by a 1-month period ofplaque accumulation.11 The destructionextended into the bony tissue adjacentto the implants, but not the teeth. Onemonth after removal of the ligatures,remission and encapsulation of the in-flammatory process was observed inthe periodontal tissues surroundingthe teeth, but only rarely at the imorim29_106 Mp_81File # 06emAntonio Scarano et alplants. These results demonstrated thatthe quality and quantity of plaque adhesionon the implant surface is importantin the long-term success ofdental implants.The initial event in the pathogenesisof most bacterial diseases is the adhesionof bacteria to the implant surface.Therefore, preventing bacterialadhesion to either the intraoral hard orsoft tissues is of utmost importance. Astrong correlation was reported in vivobetween the number of bacteria adheringto the periodontal epithelium andits degree of inflammation.12 Bacterialadherence to implants is considered tobe an important event in the pathogenesisof peri-implant disease. The implantsurfaces are normally in contactJournal of Oral Implantology 81FIGURES 5-8. FIGURE 5. Control implant; few cocci are present (arrows; original magnification 33.550). FIGURE 6. Control implant; a largeportion of the area is covered by salivary proteins (original magnification 35.000). FIGURE 7. Control implant; at higher magnification,salivary proteins (arrows) and cocci (large arrows) are present (original magnification 310.000). FIGURE 8. Control implant; many coloniesof microorganisms are present (original magnification 320.000).ORAL IMPLANTOLOGYTuesday Mar 11 2003 02:02 PMAllen Press x DTPro SystemBACTERIAL ADHESIONS ON IMPLANTSwith oral fluids, including saliva andgingival crevicular fluid, which is a serumtransudate. The mechanisms bywhich oral bacteria adhere to solid surfacesare not fully understood. In theoral cavity, the dental implant surfacesare covered by an acquired pellicle,which is formed by adsorption of salivarycomponents to the surface of thetransgingival abutment or healing implantcomponents. Oral bacteria mustthen interact with these salivary componentsin order to adhere to the surface.Important factors for the accumulationof dental plaque to implant surfacesare surface characteristics andchemical composition of implant surfaces.The effect of surface roughness82 Vol. XXIX/No. Two/2003on bacterial adherence is complex.Coating the titanium alloy implantswith titanium nitride (TiN) by themethod of physical vapor deposition(PVD) produces a stable layer on theimplant surface that changes the chemicalcomposition. PVD is the most commonmethod of depositing TiN on orthopedicimplants, and TiN is formedby the reaction of pure titanium andnitrogen gas in a vapor phase beforedeposition.13,14Reports have characterized TiN ashaving a very high chemical inertness,low friction coefficient, and good biocompatibility.15-17 In the medical field,TiN has been used to reduce the attritioncoefficients and increase the hardnessand corrosion resistance of surfacorim29_106 Mp_82File # 06emes, thus reducing the interaction of biologicalliquids with the metal bases.18The aim of the present study was toevaluate in vivo the bacterial adhesionto TiN-coated and uncoated implants.MATERIALS AND METHODSSix patients between 21 and 25 years ofage and in excellent systemic healthparticipated in the study. All patientsgave their informed consent, and theprotocol was approved by the ethicscommittee of our university. The participantswere selected on the basis ofgood periodontal health and no signsof mouth breathing. One week prior tothe beginning of the study, supragingivalplaque and calculus were professionallyremoved, oral hygiene proce-TABLESD2.011.9of the machined surface covered bybacteria.Scanning electron microscopeAfter removal, the implants were putin 2.5% glutaraldheyde in 0.1 m sodiumcacodylate buffer pH 7.4 for 4hours. They were then dehydratedwith increasing concentrations of ethanolsolutions (50%, 70%, 90%, and100%) and left for 12 hours in 113 Freon(trichlorotrifluoroethane) as a transitionfluid to critical point drying(CPD) Bomb Polaron. The chamberswere finally glued to aluminum stubsand coated with 20 to 30 nm of gold.The membrane surface facing both theinner and the outer rooms was examinedwith a Leo 435VP scanning electronmicroscope (Leo) operating at 20to 30 kV with tilt angles ranging from108C to 458C. SEM evaluations wereperformed by 3 independent observerswho expressed an estimate of bacterialamount on the coronal machined portionof the implants.The percentage of surface coveredby bacteria was calculated using a lightmicroscope (Laborlux S, Leitz, Wetzlar,Germany) connected to a high-resolutionvideo camera (3CCD, JVC KYF55B,JVC Professional Products, Milan,Italy) and interfaced to a monitorand PC (Intel Pentium III 1200 MMX,Intel Ireland Ltd, Kildare, Ireland).This optical system was associatedwith a digitizing pad (Matrix VisionGmbH, Oppenweiler, Germany) and ahistometry software package with image-capturing capabilities (Image-ProPlus 4.5, Media Cybernetics Inc, Immaginiand Computer Snc, Milano, Italy).ORAL IMPLANTOLOGYTuesday Mar 11 2003 02:02 PMAllen Press x DTPro SystemStatistical evaluation of the percentage of implant surface covered by bacteriaMean13.121.3TiN-coatedUncoated* Significant at 95% (according to the ANOVA test).dures were established, and ideal gingivalhealth conditions were obtainedin all volunteers (Loe and Silness GingivalIndex 5 0). None of the subjectshad used mouth rinses or had takenantibiotics during the previous 6months.In each of the 6 participants, a removableacrylic device was adapted tothe molar-premolar region of eachquadrant of the jaws. The devices wereself-retaining and did not require etchingor bonding to the teeth surfaces.One 4 3 13 mm titanium implant (PrimaryHealing Implant, San VittoreOlona, Milan, Italy) was glued to thebuccal aspect of each device. TiN-coatedimplants (test) were glued to theright devices and uncoated implants(control) were glued to the left devices.The plasma spray covered 11.5 mm ofthe body of the implant, whereas theneck was machined titanium. A total of24 implants (12 test and 12 control)were used in this study.The surface roughness was evaluatedwith a Leo 435 VP scanning electronmicroscope (Leo, Cambridge, UK)and a Mitutoyo Surftest 211 Profilometer(Mitutoyo Corp, Tokyo, Japan)with an average of 3 readings performedfor each surface. The arithmeticalmean of surface roughness of everymeasurement within the total distance(roughness average [Ra]) was assessed.An additional 10 implants (5 test and5 control) were analyzed. Two areas of200 mm in diameter were evaluated foreach implant surface. Neither cleaningprocedures nor agents for chemicalplaque control were applied to the implantsfor the complete duration of thetest period. After 24 hours, all implantswere removed from each device andprocessed for scanning electron microscopyfor evaluation of the portionThe differences in the percentages ofsurface covered by bacteria in the 2P-value SE.0001*.0001*0.580.54Statistical evaluationorim 29_106 Mp_83File # 06emAntonio Scarano et algroups were evaluated with the analysisof variance (ANOVA). The percentageof implant surface covered bybacteria was expressed as a mean 6SD and SE. Statistically significant differenceswere set at P , .05.RESULTSSurface characterizationGrooves and ridges, typically producedduring the manufacturing, werepresent in both types of surfaces. Thesurface roughness (Ra) was 0.79 mm forthe test implants and 0.76 mm for thecontrol implants. No changes in theoriginal texture of the surface was producedby the PVD coating.Test implantsIn many areas, no bacteria or salivaryproteins were observed (Figure 1). Inother areas, only small colonies of afew cocci were found (Figures 2 and 3).Polymorphous aggregates of microorganismswere present, consistingmainly in cocci and short rods (Figure4). No calcification of the bacteria wasobserved. The area covered by bacteriawas 13.1% 6 2.01 (Table).Control implantsA plaque was observed, consisting in afew cocci and a higher proportion ofrods and filamentous-shaped bacteria(Figure 5). A thin and regular layer ofcocci was found in many areas of thesurface. Salivary proteins, in contactwith the implant surface, were foundin a large portion of the surface (Figure6). At higher magnifications, salivaryproteins, cocci, and many colonies ofmicroorganism were found (Figures 7and 8). The area covered by bacteriawas 21.3% 6 1.9% (Table).Statistical evaluationThe implant surface covered by bacteriaon test implants was significantlylower than that of control implants (P5 .0001; Table).DISCUSSIONThe present study has shown that statisticallysignificant differences exist inJournal of Oral Implantology 83ORAL IMPLANTOLOGYTuesday Mar 11 2003 02:02 PMAllen Press x DTPro SystemBACTERIAL ADHESIONS ON IMPLANTSbacterial adhesion between TiN-coatedand uncoated implants. Surface propertiesof transgingival implant componentsare important determinants inbacterial adhesion. It has been shownthat rougher surfaces increased bacterialadhesion.19 The influence of surfaceroughness on bacterial adhesion andcolonization of transgingival abutmentsand healing implant componentshave been demonstrated in vitro20and in vivo21 studies, using bothmicrobiological22 and morphologicalmethods.Bacterial adherence to the implantsurface is considered to be an importantevent in the pathogenesis of bacterialinfections and implant failure.This adherence is associated with thedevelopment of inflammation of theperi-implant soft tissues, crestal boneloss, and peri-implant pathology. Theinitial, nonspecific adherence isthought to involve nonspecific processesmediated by physico-chemical interactionssuch as hydrophobic interactions23and electrostatic interactions.24The specific adherence is mediated byextracellular polysaccharides and lectin-like substances.25 In the oral cavity,teeth and dental implant surfaces(transgingival abutment or healing implantcomponents) are covered by anacquired pellicle formed by adsorptionof salivary components to the surfaceof the teeth, implants, and restorations.Oral bacteria must then interact withthese salivary components in order toadhere to the surface. A correlation betweenplaque accumulation and progressivebone loss around implants hasbeen reported in experimental26 andclinical studies.27 Mombelli et al28 evaluatedthe microbiota associated withsuccessful or failing implants and suggestedthat ''peri-implantitis'' shouldbe regarded as a site-specific infectionwith microbial features similar tochronic periodontitis. Possible causativefactors for crestal bone loss observedin the first year of function includebacterial adhesion on implantsurfaces, surgical trauma, occlusaloverload, presence of a microgap be-84 Vol. XXIX/No. Two/2003tween implant and abutment, necessityof the presence of a biologic width, implantcrestal module, etc.29 Studies inhumans and animals have demonstratedthat the gingiva and the peri-implantmucosa (PiM) respond to denovo plaque formation with overt in-flammation (eg, increased migration ofleukocytes through the junctional [barrier]epithelium and the establishmentof a leukocyte-rich lesion [ICT] in theconnective tissue lateral to the barrierepithelium).30 Bacterial infection is oneof the primary reasons for implant failureafter osseointegration.31The results of the present studyshow that the implants coated withTiN presented a minor quantity of thesurface covered by bacteria present inthe oral cavity. The result was producedby the different surface compositionand not from differences in surfaceroughness, because the roughnesswas similar in test and control implants.Our results confirm those reportedin another study.32 In conclusion,TiN-coated surfaces showed asignificant reduction of the presence ofbacteria, and this fact could be importantfor the health of the peri-implantsoft tissues.ACKNOWLEDGMENTSThis work was partially supported bythe National Research Council (CNR),Rome, Italy; and by the Ministry of Education,University, and Research(MIUR), Rome, Italy. A special acknowledgmentand thanks to Mr MarcelloPiccirilli for his assistance withthe morphological data, and to Dr LuigiCiavarelli Macozzi for his technicalassistance in the use of the scanningelectron microscope.REFERENCES1. Hoshaw SJ, Brunki JB, CochranGVD. Mechanical loading of Branemarkimplants affects interfacial bonemodeling and remodeling. Int J OralMaxillofac Implants. 1994;9:345-360.2. Adell R, Lekholm U, Rockler B,Branemark PI. A 15-year study of osseointegratedimplants in the treatorim29_106 Mp_84File # 06emment of the edentulous jaw. Int J OralSurg. 1981;10:387-416.3. Abrahamsson I, Berglundh T,Wennstrom J, Lindhe J. The peri-implanthard and soft tissues at differentimplant systems: a comparative studyin the dog. Clin Oral Implant Res. 1996;7:212-219.4. Abrahamsson I, Berglundh T,Glantz PO, Lindhe J. The mucosal attachmentat different abutments: an experimentalstudy in dogs. J Clin Periodontol.1998;25:721-727.5. Abrahamsson I, Berglundh T,Lindhe J. Soft tissue response to plaqueformation at different implant systems:a comparative study in the dog. ClinOral Implant Res. 1998;9:73-79.6. Berglundh T, Lindhe J. Dimensionof the peri-implant mucosa: biologicalwidth revisited. J Clin Periodontol.1996:23;971-973.7. Ruggeri A, Franchi M, Trisi P,Piattelli A. Histologic and ultrastructuralfindings of gingival circular ligamentsurrounding osseointegratednon-submerged loaded titanium implants.Int J Oral Maxillofac Implants.1994;9:636-643.8. Berglundh T, Lindhe J, MarinelloC, Ericsson I, Liljenberg B. Soft tissuereaction to de novo plaque formationon implants and teeth. Clin Oral ImplantRes. 1992;3:1-8.9. Apse P, Ellen RP, Overall CM,Zarb GA. Microbiota and crevicularfluid collagenase activity in the osseointegrateddental implant sulcus: acomparison of sites in edentulous andpartially edentulous patients. J PeriodontRes. 1989;24:96-105.10. Ong ES, Newman HN, WilsonM, Bulman JS. The occurrence of periodontitis-related microorganisms in relationto titanium implants. J Periodontol.1992;63:200-205.11. Lindhe J, Berglundh T, EricssonI, Liljenberg B, Marinello C. Experimentalbreakdown of periimplant andperiodontal tissues: a study in the beagledog. Clin Oral Implant Res. 1992;3:9-13.12. Vaahtoniemi L, Raisanen S,Stenfors L-E. Attachment of bacteria toORAL IMPLANTOLOGYTuesday Mar 11 2003 02:02 PMAllen Press x DTPro Systemoral epithelial cells in vivo: a possiblecorrelation to gingival health status. JPeriodont Res. 1993; 28:308-311.13. Pappas MJ, Makris G, BuechelFF. Titanium nitride ceramic filmagainst polyethylene: a 48 million cyclewear test. Clin Orthop. 1995;317:64-70.14. Peterson CD, Hillberry BM,Heck DA. Component wear of totalknee prostheses using Ti-6Al-4V, titaniumnitride coated Ti-6Al-4V, and cobalt-chromium-molybdenum femoralcomponents. J Biomed Mater Res. 1988;22:887-903.15. Scarano A, Piattelli M, VrespaG, Petrone G, Iezzi G, Piattelli A. Bonehealing around titanium and titaniumnitride (TiN) coated dental implantswith 3 different surfaces: an experimentalstudy in rats. Clin Implant DentRelat Res. In press.16. Dion I, Baquey C, Candelon B,Monteites JR. Hemocompatibility of titaniumnitride. Int J Artif Organs. 1992;15:617-621.17. Galante JO, Lemons J, Spector18. Harman MK, Banks SA, HodgeWA. Wear analysis of a retrieved hipimplant with titanium nitride coating.J Arthroplast. 1997;12:938-945.19. Rimondini L, Fare S, BrambillaE, et al. The effect of surface roughnessWJ, Walker ML, Apostolid A. Adsorp- 31. Becker W, Becker BE, NewmanM, Wilson PD Jr, Wright TM. The bio- tion of streptococcus mutans on chem- MG, Nyman S. Clinical and microbiologiceffects of implant materials. J Or- ically treated hydroxyapatite. J Dent logic findings that may contribute tothop Res. 1991;9:760-775. dental implant failure. Int J Oral MaxillofacImplants. 1990;5:31-38.orim 29_106 Mp_85File # 06emAntonio Scarano et alI, Liljenberg B, Marinello C. Experimentalbreakdown of peri-implant andperiodontal tissues. A study in the beagledog. Clin Oral Implant Res. 1992;3:9-16.27. Becker W, Becker BE, NewmanMG, Nyman S. Clinical and microbiologicfindings that may contribute todental implant failure. Int J Oral MaxillofacImplants. 1990;5:31-38.28. Mombelli A, Van Oosten MA,Schurch E, Lang NP. The microbiota associatedwith successful or failing osseointegratedtitanium implants. OralMicrobiol Immunol. 1987;2:145-151.29. Oh T-J, Yoon J, Misch CE, WangH-L. The causes of early implant boneloss: myth or Science? J Periodontol.2002;73:322-333.30. Pontoriero R, Tonelli MP, CarnevaleG, Mombelli A, Nyman SR,Lang NP. Experimentally induced periimplantmucositis: a clinical study inhumans. Clin Oral Implant Res. 1994;5:254-259.32. Grossner-Schreiber B, GriepentrogM, Haustein I, et al. Plaque formationon surface modified dental implants:an in vitro study. Clin Oral ImplantRes. 2001;12:543-551.Journal of Oral Implantology 85on early in vivo plaque colonization ontitanium. J Periodontol. 1997;68:556-562.20. Drake DR, Paul J, Keller JC. Primarybacterial colonization of implantsurfaces. Int J Oral Maxillofac Implants.1999;14:226-232.21. Gatewood RR, Cobb CM, KilloyWJ. Microbial colonization on naturaltooth structure compared withsmooth and plasma-sprayed dental implantsurfaces. Clin Oral Implant Res.1993;4:53-64.22. Bollen CM, Papaioanno W, VanEldere J, Schepers E, Quirynen M, vanSteenberghe D. The influence of abutmentsurface roughness on plaque accumulationand peri-implant mucositis.Clin Oral Implant Res. 1996;7:201-211.23. Nesbit WE, Doyle RJ, TaylorKG. Hydrophobic interaction and theadherence of streptococcus sanguis tohydroxylapatite. Infect Immun. 1982;38:637-644.24. O'Brien NJ, Fan PL, LoescheRes. 1978;2:910-914.25. Schilling KM, Bowen WH. Glucanssynthesized in situ in experimentalsalivary pellicle function as specificbinding sites for streptococcus mutans.Infect Immun. 1992;60:284-294.26. Lindhe J, Berglundh T, Ericsson