Clinical and microbiological characteristics of peri-implantitis cases: a retrospective multicentre study

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1 G. Charalampakis Å. Leonhardt P. Rabe G. Dahlén Clinical and microbiological characteristics of peri-implantitis cases: a retrospective multicentre study Authors affiliations: G. Charalampakis, G. Dahlén, Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Sweden Å. Leonhardt, Student Clinic, Institute of Odontology, Public Dental Health Service, Västra Götaland, Sweden P. Rabe, Periodontology, Division of Specialist Dental Care in Halland, County Hospital, Halmstad, Sweden Corresponding author: Georgios Charalampakis Department of Oral Microbiology and Immunology, Institute of Odontology Sahlgrenska Academy, University of Gothenburg Box 450, Gothenburg , Sweden Tel.: Fax: georgios.charalampakis@odontologi.gu.se Key words: culture, DNA DNA hybridization, implant surface, multi-centre, osseointegration, peri-implantitis, retrospective Abstract Objectives: The aim of this study was to follow patient cases retrospectively in a longitudinal manner from the time of implant placement to the time they were diagnosed with peri-implant disease, and to identify associated clinical and microbiological features of peri-implant disease. Material and methods: A total of 281 patient cases were chosen from the archives of the Oral Microbiological Diagnostic Laboratory, Gothenburg, Sweden, based on bacterial samples taken from diseased implants. A form was designed and filled in separately for each case including data on patient, implant and disease profile. Results: Most cases were severe peri-implantitis cases (91.4%). In 41.3% of the patients, periimplantitis was developed early, already after having implants in function less than 4 years. The type of implant surface was significantly associated with the time in years implants were in function, before disease was developed (P < 0.05). The microbiological results by both culture and checkerboard analysis, although failed to fully correspond to the severity of the disease in terms of magnitude, proved to show that peri-implantitis is a polymicrobial anaerobic infection with increased number of AGNB (aerobic Gram-negative bacilli) in 18.6% of the patients. Conclusions: Peri-implantitis is a biological complication of implants in function that poses a threat to their long-term survival. It may develop earlier around implants with rough surfaces and it may represent a true infection. Microbiological sampling methods should be improved and uniformed so as to fully unveil the microbiological profile of the disease. Date: Accepted 23 May 2011 To cite this article: Charalampakis G, Leonhardt Å, Rabe P, Dahlén G. Clinical and microbiological characteristics of peri-implantitis cases: a retrospective multicentre study. Clin. Oral Impl. Res. 23, 2012, doi: /j x Osseointegration, that is, establishment and maintenance of bone-to-implant contact, although met with doubt and criticism among clinicians initially, became an evidence-based concept in clinical practice to restore full and partial edentulism. Replacing missing teeth with titanium oral implants has nowadays become a routine and widespread procedure. Several longitudinal studies have reported high survival rates of the implants in function, ranging from 90% to 95% over a period up to 20 years (Lekholm et al. 2006; Astrand et al. 2008; Kim et al. 2008) and have encouraged clinicians to proceed with this type of rehabilitation. However, biological complications do occur around dental implants, both early and late (Berglundh et al. 2002; Alsaadi et al. 2008a, 2008b). The early ones take place at the time up to abutment connection and are easier to diagnose because of the lack of osseointegration. Late implant failures result from the inability to maintain osseointegration. The most prominent and insidious complication around dental implants, emerging at a later stage is peri-implantitis. Peri-implantitis around dental implants is termed in line with periodontitis around teeth and is defined as an inflammatory lesion of bacterial aetiology that, in addition to mucosal inflammation, is characterized by loss of supporting bone (Albrektsson & Isidor 1994; Zitzmann & Berglundh 2008). Peri-implantitis was considered to be a rare phenomenon and early reports on its prevalence revealed low figures ranging from 5% to 10% (Mombelli & Lang 1998; Bragger et al. 2001). Recently published data indicate that as many as 16% (Roos-Jansaker et al. 2006a) up to 28% (Fransson et al. 2005) of the patients with implants show peri-implantitis on one or more implants after 5 10 years. In another recent study (Fransson et al. 2009), it was demonstrated that two implants out of five 2011 John Wiley & Sons A/S 1045

2 (40%) in each subject with peri-implantitis were affected in cross-arch rehabilitations supported on dental implants. The discrepancy in prevalence figures between early and recent reports is attributed to underdiagnosis of the problem in the past. Clinicians had, for many years, as endpoint of their clinical focus, the establishment of osseointegration and no potential biological complications following osseointegration. Moreover, complications were considered mostly technical and any bone loss around implants, depicted accidentally on x-rays, was attributed to functional overload. This single-minded explanatory model for bone loss around implants, based on technical rather than biological considerations, stemmed from the fact that the implant was first inserted in edentulous patients. Bacteria were not considered a threat in the absence of teeth and their replacement by pristine surfaces of an implant. Bacteria have nowadays been identified as colonizing implant surfaces in partially as well as in fully edentulous patients in a fashion similar to teeth. Tannerella forsythia, Porphyromonas gingivalis, Treponema denticola, Prevotella nigrescens, Prevotella intermedia, Fusobacterium nucleatum, Campylobacter spp., Parvimonas micra (previously designated as Peptostreptococcus micros) and Aggregatibacter actinomycetemcomitans have been detected in peri-implantitis sites both in fully (Adell et al. 1986; Mombelli et al. 1988; Bower et al. 1989; Hultin et al. 2002; Quirynen et al. 2005; Devides & Franco 2006) and partially edentulous patients (Mombelli et al. 1987; Leonhardt et al. 1999; Botero et al. 2005; Shibli et al. 2008; Tabanella et al. 2009). Microorganisms that are less frequently associated with periodontitis, such as Staphylococcus spp., enterics and Candida spp., have also been found in peri-implant infections (Alcoforado et al. 1991; Leonhardt et al. 1999; Renvert et al. 2008; Salvi et al. 2008). The role of the bacteria in the peri-implantitis lesions remains essentially unknown. The aim of this retrospective study was to follow patient cases in a longitudinal manner from the time of implant placement to the time they were diagnosed with peri-implant disease and identify associated clinical and microbiological features of peri-implant disease. Material and methods The basis for this retrospective study consisted of a pool of patients with peri-implantitis from whom microbial samples had been harvested around diseased implants and sent for microbial analysis to the Oral Microbiological Diagnostic Laboratory, Microbiology, Institute of Odontology, Sahlgrenska Academy, Gothenburg, Sweden. A total of 301 patient cases were consecutively selected from the archives of the Oral Microbiological Diagnostic Laboratory between January 2005 and January The patients stemmed from 16 public dental health service clinics and 14 private dental clinics all around Sweden. Twenty-five centres were identified according to the geographical region. A form was designed and filled in separately for each case including data on patient, implant and disease profile. One of the authors (G. Charalampakis), who had no affiliation to any of the centres, visited the centres with the greatest representation. Another author (P. Rabe), based in Halmstad, was the clinician responsible for the majority of bacterial samples taken. In centres where the cases were very few, it was decided to request the periodontologists/general dentists responsible for these patients to fill in the respective forms and send them to the Department of Oral Microbiology. Twenty forms were not completed by the various clinicians and we ended up with a material of 281 cases. The following variables were collected from clinical and radiographic examination both from paper patient files and from the database and recorded in the individualized form to the extent it was made possible: age, gender, medical history (with focus on cancer, cardiovascular disease, diabetes, osteoporosis, rheumatoid arthritis, depression coupled with associated medication and therapy), smoking habit (current smoker, previous smoker, never smoked, snuff), smoking dose (heavy, moderate, light smoker), periodontal condition (healthy, gingivitis, previous periodontitis, current periodontitis), number of teeth, number of implants, type of implants, implant position (arch and region), level of oral hygiene, extent and severity of disease. The baseline for this retrospective examination was the time clinicians recorded pathology and decided to proceed with microbial sampling around dental implants. Clinical considerations Various definitions and clinical thresholds were decided for reasons of consistency before the investigation was initiated and the respective clinicians who completed the forms were informed. Specifically: Systemic health: Patients taking medication for diseases other than the ones described in the medical history, that is, asthma, high blood pressure without associated cardiovascular disease were considered systemically healthy. Level of oral hygiene performance: Suboptimal level of oral hygiene was set at O Leary s Plaque Index above 20%, implying more than 20% of all sites with visible plaque on consecutive recall visits. Dose of smoking: Light smoking was considered 1 9 cigarettes/day, moderate cigarettes/day and heavy smoking >15 cigarettes/day. Region in the mouth: Anterior region was considered the region between incisors and canines, whereas posterior, the premolar molar region. Previous periodontal disease: Some marginal bone loss, but no suppuration or repeated Bleeding on Probing (BoP) and probing pocket depth (PPD) 4 mm. Definition of peri-implantitis: Presence of suppuration and/or BoP with PPD 5 mm and radiographic images of marginal bone loss 1.8 mm (or three threads of implants with implant pitch 0.6 mm) after 1 year of implants in function. Severity of peri-implantitis: Presence of pus and/or BoP with PPD 7mmonat least one aspect of the implant. If probing measurement had not been recorded, radiographic images had been considered and marginal bone loss >1/3 implant length after 1 year of implants in function was assigned as severe peri-implantitis. Any other case with lower threshold was regarded as mild peri-implantitis. Extent of peri-implantitis: Localized if up to 30% of the implants were affected, whereas generalized when >30% of the implants were affected. Disease development: The time span for disease to occur. Early disease development was synonymous to having implants in function less than 4 years, and late disease development to having implants in function for more than 6 years before disease was developed. Microbiological considerations Microbial samples that were sent to the Oral Microbiological Diagnostic Laboratory were processed for the type of microbiological analysis requested by the clinicians. The main concern for the clinician was to identify the pathogens associated with the 1046 Clin. Oral Impl. Res. 23, 2012 / John Wiley & Sons A/S

3 peri-implant infection so as to choose the most effective antibiotic regimen. The sample sites around diseased implants were isolated with cotton rolls and supragingival plaque was removed with sterile cotton pellets. One to three sterile paper points/site (Johnson & Johnson, East Windsor, NJ, USA) were inserted to the depth of the peri-implant pocket and kept in place for 15 s. Microbial samples scheduled for culture were then transferred aseptically to vials, containing 3.3 ml VMGA III (Dahlen et al. 1993). The samples were transported and processed in the laboratory. After mixing a volume of 0.1 ml of the concentrated transport medium to 1 : 100 and 1 : 10,000 times dilution in VMGA III, bacteria were plated onto the surface of an enriched Brucella blood agar plate (BBL; Microbiological System, Cockeysville, MD, USA). The agar plates were incubated anaerobically in jars using the hydrogen combustion method (Möller & Möller 1961) at 37 C for 6 8 days for calculating the total viable count (TVC). Porphyromonas gingivalis was distinguished from P. intermedia/p. nigrescens by its haemagglutinating activity and lack of auto-fluorescence in UV light (Slots & Genco 1979; Slots & Reynolds 1982). Blood agar (Difco), Staphylococcus agar (Difco), Enterococcus agar (BBL) and tryptic soy serum bacitracin vancomycin agar plates (BBL) were inoculated and incubated for 2 and 5 days, respectively, at 37 C in air with 10% CO 2. Special attention was given to Staphylococcus aureus, Staphylococcus epidermidis, enterococci and aerobic Gram-negative bacilli (AGNB). Staphylococcus aureus was distinguished from S. epidermidis by performing DNase test on special DNA agar plate (Difco). The plates were examined for typical colony morphology and the specific bacteria were registered as percentage of TVC. Microbial samples scheduled for checkerboard DNA DNA hybridization were placed in sterile Eppendorf tubes and analysed according to the respective methodology (Socransky et al. 1994), as modified by Papapanou et al. (1997). They were transferred to 100 ll TE buffer (10 mm Tris HCl, 1 mm EDTA, ph 7.6) and 100 ll 0.5 M NaOH was added and the suspensions boiled for 5 min. After boiling, 800 ll 5 M ammonium acetate was added to each tube and the samples were processed according to standardized procedures. The hybrids formed between the bacterial DNA and the probes were detected by application of an antidigoxigenin antibody conjugated with alkaline phosphatase and incubation with a chemiluminiscent substitute (CSPD; Boehringer-Mannheim, Phoenix, AZ, USA). The obtained chemiluminiscent signals were transformed into a scale of scores from 0 to 5 according to Papapanou et al. (1997). The cut-off of score 1 was chosen to contrast colonized vs. non-colonized sites, and that of score 3 was chosen to contrast heavily colonized (score 3 or more) vs. non-colonized (score 0) and less heavily colonized sites (scores 1 and 2). In culture, we used five different scales to frame the magnitude of bacteria (A. actinomycetemcomitans, P. gingivalis, P. intermedia/p. nigrescens, S. aureus, S. epidermidis, enterococci, AGNB) and fungi as proportions of TVC. The cut-off points for this semiquantification were based on a previously published study (Dahlen et al. 1982): nondetectable colonies, 0; very sparse growth for colonies, <0.1% TVC; sparse growth for colonies, 0.1 1%; moderate growth for colonies, 1 10%; and heavy growth for colonies, >10%. It is also important to mention that there was a small change in the panel of the targeted bacteria used in the DNA DNA checkerboard hybridization since 2006, as performed at the Oral Microbiological Diagnostic Laboratory. The bacterial species Eikenella corrodens, Selenomonas noxia and Streptococcus intermedia were substituted for Porphyromonas endodontalis, Filifactor alocis and Prevotella tannerae, respectively (Dahlen & Leonhardt 2006). Statistical analysis Both descriptive statistics and statistical analyses were performed with the statistical package PASW Statistics 18.0 (SPSS Inc., Chicago, IL, USA). Variables are presented as absolute and relative frequencies (%). The statistical computational unit was at the subject level. Clinical recordings and microbial samples taken at site level were pooled to a mean value per patient. In all analyses, data were not normally distributed and nonparametric tests were performed. Chi-squared tests were applied to study associations between categorical variables. Kruskal Wallis tests were used to study differences between the groups of a categorical variable with regard to a dependent continuous variable. Six follow-up Mann Whitney (MW) U tests were performed between pairs of groups to find out which groups are statistically significant from one another. Results were regarded statistically significant at P < However, for the multiple MW tests, we applied a Bonferroni correction and thus used a stricter alpha level of 0.05/6 = for determining significance. Results Baseline patient characteristics (Table 1) Of the patients, 61.6% were women and the prevailing age interval was years (70.5%). Most patients visited public dental centres (80.1%). One third of the cases (33.5%) stem from one centre, based in Halmstad. One critical question is whether patients from this clinic had different features from the rest of the clinics, which would have influenced the results. Separate statistical tests have been performed for this centre, but patient, disease and implant characteristics did not deviate from the overall results (data not shown). Thus, this centre was not considered to be an outlier; 44.1% of the patients were considered non-healthy and one or more diseases were recorded. More details with regard to diseases and associated medication are presented in Table 1; 38.4% were current cigarette smokers and 41.3% never smoked. For 93 cigarette smokers, the dose of smoking was retrieved from the patient file and the information is presented in Table 1. Baseline dental status and periodontal conditions (Table 2) In all, 14.2% were edentulous in both jaws, but had extensive implant-supported reconstructions. Oral hygiene was considered good (plaque scores < 20%) in 88.3% of the patients. Only 2.1% of the dentate patients were considered periodontally healthy; 54.4% were diagnosed with current periodontitis at the time peri-implantitis was diagnosed around dental implants; 20.3% had a history of periodontitis and 8.5% had gingivitis. Baseline implant treatment characteristics (Table 3) Most patients (52%) had relatively extensive implant-supported reconstructions with four to six implants. Single tooth replacement occurred in 7.1% and the majority of these cases (65%) were front teeth replacements; 56.6% of the patients had implants placed in the maxilla, 34.5% in the mandible and 8.9% in both arches. Replacement of missing teeth with implants in both anterior and posterior regions occurred in 45.9% of the patients. With regard to implant type, 40.2% had Nobel Biocare implants (Nobel Biocare, Gothenburg, Sweden), 22% Straumann (ITI, Basel, Switzerland) and 16% Astra-Tech (Astra, Mölndal, Sweden). One patient case was restored with 3i (Biomet, Palm Beach Gardens, FL, USA). Implant surface was also recorded and is presented in Table 3. The use of antibiotic 2011 John Wiley & Sons A/S 1047 Clin. Oral Impl. Res. 23, 2012 /

4 Table 1. Baseline patient characteristics Variables Subcategory N * % Age Gender Male Female Centre Public Private Medical condition Healthy Non-healthy Unclear Diseases Arthritis Cancer Cardiovascular diseases Diabetes Depression Osteoporosis Intervention Radiation Medication Prophylaxis Biphosphonates Calcium blockers Corticosteroids Cytotoxic Insulin Smoking habit Current smoker Previous smoker Never smoked Snuff Unclear Smoking dose Heavy (>15 cig/day) Moderate (10 15 cig/day) Light (1 9 cig/day) Unclear * Number of subjects in absolute count. Percentage of subjects. Table 2. Baseline dental status and periodontal conditions Variables Subcategory N * % Edentulism Dentate Fully edentulous Unclear Oral hygiene Good Suboptimal Periodontal conditions Healthy Gingivitis History of periodontitis Current periodontitis Unclear * Number of subjects in absolute count. Percentage of subjects. prophylaxis during surgery was obvious in 107 patients (38%) and the most common regimen was Penicillin V (2 g, 1 h) prior to surgery. In one case (0.4%), it was clearly stated that no antibiotics were administered for prophylaxis. In a few cases, we could retrieve information with regard to fenestration, narrow zone of keratinized mucosa and thick prostheses. The results are shown in Table 3. Baseline peri-implant disease characteristics (Table 4) Only three patients (1.1%) had mucositis, that is, inflammation of the mucosa around dental implants without accompanying bone loss and increased PPD. The remaining 278 patients (98.9%) had peri-implantitis; 82.9% were diagnosed as generalized and 17.1% as localized peri-implantitis cases. Suppuration and BoP were constant findings in the clinical recordings around the diseased dental implants. A craterous form of bone loss, of varying width around these implants was also repeatedly detected on periapical follow-up x-rays. Severe peri-implantitis was diagnosed in 91.4% and mild peri-implantitis in 6.8% of the patients. For 262 cases, it was also possible to know when the implants were installed and when the initial signs of disease were detected. In 41.3% of the patients, periimplantitis was developed early, already after having implants in function less than 4 years. In 46 patients (16.6%), peri-implantitis was diagnosed at a later time point after the actual development of the disease. The type of implant surface proved to be statistically significantly associated with the timing of disease development. The 3i implant was excluded from the statistical test because of very low representation. Non-parametric Kruskal Wallis tests were performed and revealed a statistically significant difference between implant surface and disease development with chi-squared (degrees of freedom, number of cases): χ 2 (5, 214) = , P = MW U-tests were applied to assess which implant surfaces were significantly associated with the time span of disease development. Astra TiOBlast and Straumann TPS were not found to be statistically significantly associated with disease development with respective P-values 0.168, > Nobel TiUnite and Astra Osseospeed proved to be significantly associated with early disease development with respective P-values and Straumann SLA was significantly associated with moderate and late disease development (P = 0.008) and Nobel MK II with late development (P = 0.000). Associations between implant surface and disease development are depicted in Fig. 1. Microbiological characteristics (Tables 5 and 6) Microbial samples that were taken around the diseased implants were processed for the culture or checkerboard DNA DNA hybridization method or both. Most centres had a clear predilection towards either the traditional technique, that is, culture or the molecular technique DNA DNA checkerboard hybridization. A total of 139 patient cases (49.5%) were analysed by culture, 120 patient cases (42.7%) by checkerboard and 22 (7.8%) by both techniques. Microbiological results were underpowered in terms of magnitude for both culture and checkerboard analysis. We were dealing mostly with severe peri-implantitis, which implies that we would expect high number of bacteria to survive and thrive in the deepened pockets around the diseased dental implants. Culture analysis showed that P. intermedia/p. nigrescens were the most representative bacteria in magnitude as found in moderately heavy and heavy growth in 27.3% of all cases. Staphylococcus epidermi Clin. Oral Impl. Res. 23, 2012 / John Wiley & Sons A/S

5 Table 3. Baseline implant treatment characteristics Variables Subcategory N * % Number of implants Single > Arch Maxilla Mandible Both Region Anterior Posterior Both Type of implants Nobel Biocare Straumann Astra-Tech i Unclear Implant surface Nobel turned surface Nobel TiUnite Straumann TPS Straumann SLA Astra TiOBlast Astra Osseospeed Unclear Antibiotic prophylaxis Yes during installation No Unclear Fenestration Yes Unclear Narrow zone of Yes keratinized mucosa Unclear Thick prosthesis Yes Unclear * Number of subjects in absolute count. Percentage of subjects. 25% 20% 15% 10% 5% 0% Nobel turned surface Nobel TiUnite Straumann TPS Straumann SLA Astra TiOBlast Astra Osseospeed <4 years 4 6 years >6 years Fig. 1. Implant surface in relation to peri-implantitis development. The Y-axis is percent of subjects. Table 7 presents the pattern of detectability of species separately for culture and checkerboard analysis. Culture was unable to detect any of the targeted species in 18.6% of the cases, whereas checkerboard only in 0.7%. On the other hand, culture and checkerboard analyses, although used different scales, proved to detect moderately heavy/ heavy amounts of bacteria in 54.7% and 65% of the cases, respectively. Table 4. Baseline characteristics of peri-implant diseases Variables Subcategory N * % Peri-implant disease Mucositis Peri-implantitis Peri-implantitis extent Generalized Localized Peri-implantitis severity Severe Mild Unclear Development Early (<4 years) years Late (>6 years) Unclear * Number of subjects in absolute count. Percentage of subjects dis was more prevalent (28.5%) compared with S. aureus (7.9%). Aggregatibacter actinomycetemcomitans could not be identified in 10 of 161 culture cases because of overgrowth of other bacteria (especially nonlactose-fermenting Gram-negative rods, e.g. Pseudomonas spp. and Klebsiella spp). It was detected in moderately heavy/heavy growth in 6% of the patients. Fungi and enterococci were seldom found. AGNB were found in moderately heavy/heavy growth in 18.6% of the cases. Detailed data from culture analysis are presented in Table 5. With regard to checkerboard analysis of 142 cases, T. forsythia was the most prevalent with score 3, corresponding to 10 5 cells, as detected in 37.3% of the cases. It was followed by T. denticola, which had a similarly high score of 3 in 31% of the cases. Porphyromonas gingivalis was less prevalent compared with P. intermedia and even less compared with P. nigrescens and P. endodontalis. Selenomonas noxia and A. actinomycetemcomitans were the least representative bacteria. Table 6 summarizes the aforesaid results. Discussion The patient sample in this study represents a group of the Swedish population with periimplant disease. The majority of the patients were above middle age (>60), which per se increases the risk of facing general health problems and needing medications. Some of the patients were severely diseased with cancer, severe cardiovascular disease, rheumatoid arthritis explaining why they were referred to specialist hospital departments or university clinics both for dental implant installation and for treatment of biological complications around dental implants. Oral hygiene was generally recorded as good with plaque scores below 20% in 88.3% of the patients, which does not correspond to the high percentage of patients with active periodontal and peri-implant disease. We are not aware if plaque disclosing solution was used during the recordings. Moreover, we lack information with regard to the degree of compliance these patients had at the ordinary dentists before being referred to the specialist centres. However, we expect suppuration and bleeding to stem from the deepest part of the pocket irrespective of the condition at the mucosal margin and the good oral hygiene John Wiley & Sons A/S 1049 Clin. Oral Impl. Res. 23, 2012 /

6 Table 5. Presence and counts of bacteria as detected by culture analysis Scale Bacteria Not detected Very sparse Sparse Moderately heavy Heavy Aggregatibacter actinomycetemcomitans Porphyromonas gingivalis Prevotella intermedia/prevotella nigrescens Staphylococcus aureus Staphylococcus epidermidis Enterococci AGNB Fungi All values are indicative of percentage of subjects. AGNB, aerobic Gram-negative bacilli. Table 6. Presence and counts of bacteria as detected by checkerboard analysis Bacteria 0 1, 2 3, 4, 5 Porphyromonas gingivalis Prevotella intermedia Prevotella nigrescens Tannerella forsythia Aggregatibacter actinomycetemcomitans Fusobacterium nucleatum Treponema denticola Parvimonas micra Campylobacter rectus Eikenella corrodens * Selenomonas noxia * Streptococcus intermedia * Porphyromonas endodontalis * Filifactor alocis * Prevotella tannerae * Score All values are indicative of percentage of subjects. * Reduced absolute count of subjects because of change of the panel. Table 7. Pattern of detectability of species for culture and checkerboard analysis Microbial analysis Subcategory N * % Culture Not detected Detected at moderately heavy/heavy amounts Checkerboard Not detected Detected at moderately heavy/heavy amounts * Number of subjects in absolute count. Percentage of subjects. All species with %TVC = 0. At least one species with %TVC > 1%. All species with score 0. k At least one species with score 3. TVC, total viable count. An inevitable weakness of this study is the lack of control group, that is, patients with no peri-implant disease. A control group would have made it possible to compare periimplant diseased and non-diseased patients around dental implants and better identify risk factors for disease initiation and progression. However, this problem lies in the basis of the study design, as we focused on patients who were diagnosed with peri-implant disease and had been referred for microbial analysis for further investigation. Interestingly, severe peri-implantitis was also dominant (91.4%) in the sample. Thus, all patients presented true pathology, far from threshold dilemmas about being peri-implantitis or not. Another weakness of the study is the heterogeneity, associated with the multicentre retrospective design. The clinicians who sent the samples are from various clinics in Sweden and are mostly periodontologists with a special interest in microbiology. The data are inevitably influenced by the individual clinician sending the sample for analysis. However, this study has selected cases consecutively from the everyday clinical practice to illustrate the clinical and microbiological profile of peri-implantitis. The alarming high percentage of patients (54.4%), with active periodontitis at the time of diagnosis of peri-imlantitis, is worth noting. It is likely that the presence of periodontitis-associated microorganisms for an extended period of time explains the higher incidence of peri-implantitis in periodontitis-susceptible patients. A plethora of studies have proved that in partially edentulous patients, periodontal pathogens may be transmitted from teeth to implants, implying that periodontal pockets may serve as reservoirs for bacterial colonization around implants (Lekholm et al. 1986; Apse et al. 1989; Leonhardt et al. 1992, 1993; Mombelli et al. 1995; Hultin et al. 2002; De Boever & De Boever 2006; Quirynen et al. 2006). In addition, 23.8% of the subjects had a history of periodontitis. All systematic reviews, having addressed the question whether patients with a history of periodontitis have an increased risk of peri-implant disease (Van der Weijden et al. 2005; Schou et al. 2006; Karoussis et al. 2007; Quirynen et al. 2007; Ong et al. 2008; Schou 2008), have come up with positive answers. Thus, if patients suffered from periodontitis at the time of implant placement, they could be considered prone to develop peri-implantitis. Placing implants in a periodontitis patient without preceding successful periodontal treatment must, based on existing evidence, be considered maltreatment. Our retrospective analysis revealed that some of the diseased implants were associated with fenestrations at the time of surgery, narrow zone of keratinized mucosa and thick prostheses. Fenestration was reported in 10 cases during implant placement and 1050 Clin. Oral Impl. Res. 23, 2012 / John Wiley & Sons A/S

7 this dehiscence may act as a quicker pathway for bacteria to induce peri-implantitis at a later time point. Narrow zone of keratinized mucosa has been associated with periimplantitis (Roos-Jansaker et al. 2006b), whereas increased width of keratinized mucosa around implants is associated with lower mean alveolar bone loss and improved indices of soft tissue health (Bouri et al. 2008; Zigdon & Machtei 2008; Kim et al. 2009). In our material, 18 patients were reported to present narrow zone of keratinized mucosa around diseased implants. In such cases, the insufficient keratinized mucosa in the vicinity of implants could be a potential contributing factor. The insufficiency does not necessarily mediate, but may induce adverse effects on the hygiene management and soft tissue health condition. Similarly, thick design of bridgework over implants with practically no space between implants and/or at the mucosal margin eliminates access for cleaning and optimal oral hygiene. A recent study (Serino & Strom 2009) points to plaque as a risk factor for peri-implantitis because of inaccessible prosthetic rehabilitation. In our material, 9 of 32 patients with thick prostheses also had suboptimal plaque control. In 46 patients (17.3%), peri-implantitis was diagnosed 2 3 years after the actual development of the disease. Looking at peri-apical radiographs from different time intervals, there was obvious progressive bone loss long before the lesions were diagnosed as peri-implantitis. This verifies the belief that peri-implantitis was not always reported as a pathological entity at the time of development. Clinicians were reluctant to probe around dental implants, sticking to the historical dogma that probing around implants should be avoided. However, disease had occurred earlier in time in these cases, which later were diagnosed as peri-implantitis cases. Identifying the actual development of the disease and contributing factors is important extra information about the disease pattern. Implant surface may be such a contributing factor to disease development. Straumann TPS and Astra TiOblast, which are both moderately roughened surfaces with a blasted appearance, were not found to be statistically significantly associated with disease development. On the other hand, Straumann SLA and Astra Osseospeed, both chemically modified surfaces, were related to disease development. Nobel TiUnite was statistically significantly associated with early disease development, whereas the turned Brånemark surface, with late disease development. A plausible scenario is that bacteria are not well protected in smooth compared with rough surfaces and are not favoured to express increased virulence and in turn provoke early damage to the host. TiUnite surface is a highly crystalline and phosphateenriched micro-structured surface with open pores in the low micrometre range. This craterous surface probably allows a more direct invasion of the pathological microflora through the pores. This might explain the early disease development with this type of implant surface in our results. No patient with TiUnite or Osseospeed surface developed disease at a late time span (>6 years) probably because these implants represent newer surfaces compared with the rest, such as those launched in the market after the year 2000, making it less probable to have such implants with so many years of function included in our study. However, implant surface is only one aspect of implant design. Other aspects (e.g. size and type of abutment) could not unfortunately be retrieved from our data for further discussion. The aforesaid associations between disease development and implant surface should be interpreted with caution additionally because of the retrospective analysis of the data. These findings need further investigation in randomized prospective clinical trials. The microbiological results stem from two types of analysis, the traditional culture and the molecular technique, checkerboard. We used different thresholds of magnitude for both techniques so as to describe not just the presence/absence of the associated periimplant pathogens but also a measure of growth and increased number. Although we would expect bacteria to be a lot more representative in terms of magnitude in the deepened peri-implant pockets, we did not observe optimal correlations with the clinical results. Culture and checkerboard analysis failed to map out an increased number of pathogens as expected in the severe peri-implantitis cases and gave essentially underpowered results. Underpowered results do not imply that microbiology has underpowered role in the pathogenesis of peri-implantitis, but that we face a clear inability to frame the associated microbiota. This may be explained partly by the difficulty to reach the deepest part of the peri-implant pocket with paper points because of the complexity of the implant design and presence of threads as well as because of the design of the bridgework. Thick forms of prosthesis would interfere with the probing procedure and the metal probe let alone with the microbial sampling and the less rigid paper point. Clinicians should take effort to remove the prosthetic superstructures before taking bacterial samples around diseased implants. Moreover, part of the role of the clinician is to modify the bridgework in a way that would facilitate the patient to locate the implant necks and perform optimal cleaning at the mucosal margins. The variance in clinical and microbiological findings may also be explained by the presence of bacteria not only in the periimplant crevicular fluid but also in the periimplant tissues. Invasion of bacteria in the peri-implant tissues would imply not only that peri-implantitis is a true infection but also that associated bacteria would not be captured with the current microbiological sampling techniques. Both methods of microbial analysis (culture and checkerboard) have different scales, and results were analysed statistically separately for each method (Tables 5 and 6). However, A. actinomycetemcomitans was detected at moderately heavy/heavy growth by culture only in 6% of the patients and at scores 3, 4 or 5 by checkerboard analysis in 4.2% of the patients. This finding that A. actinomycetemcomitans was not representative in peri-implantitis lesions is in accordance with previous studies (Salcetti et al. 1997; Listgarten & Lai 1999; Shibli et al. 2008). Prevotella intermedia/p. nigrescens were the most frequently detected species at moderately heavy/ heavy growth in samples analysed by culture (27.3%). AGNB, which consist of enteric rods and non-lactose-fermenting Gram-negative rods, for example, Pseudomonas spp. and Klebsiella spp., were also detected at moderately heavy/heavy growth in increased number of patients, analysed by culture (18.6%). Staphylococcus aureus has been discussed in the aetiopathogenesis of peri-implantitis (Renvert et al. 2008; Salvi et al. 2008), but was not representative in our material, not detected in 91.9% of the cases. Tannerella forsythia was the most frequently detected species at increased magnitude (score 3) in samples analysed by checkerboard (37.3%). Certain authors (Socransky et al. 1998, 2004) have tried to associate a triad of bacteria (P. gingivalis, T. denticola and T. forsythia), the so-called red complex, with disease progression. Grouping bacteria in such a fashion may not fully reflect the descriptive microbiological pattern of the disease in all individual cases. Prevotella nigrescens, which is not included in the red complex group, is per se in our material more prevalent compared with red complex in the maximum scores (score 2011 John Wiley & Sons A/S 1051 Clin. Oral Impl. Res. 23, 2012 /

8 3). All anaerobic bacteria included in the checkerboard panel have the potential to be in increased number, but the species names may vary from case to case. Any of the species included in the checkerboard panel is enough to be associated with disease if it has a score 3. In our material, red complex represents only 23.9% of the cases, whereas any species represents almost three times more cases (65%). Thus, clinicians should not focus so much on specific bacteria or groups of bacteria. Peri-implantitis seems to be mostly a polymicrobial endogenous subepithelial infection and not a specific monoinfection. In some cases, where AGNB are prevalent, we could consider peri-implantitis to be an exogenous opportunistic infection. The clinician should know what to expect from each microbial analysis. Culture allows the detection of bacteria not included in the checkerboard panel (enterococci, AGNB) as well as fungi. However, from the plethora of obligatory anaerobic bacteria that thrive in the deepened peri-implant pocket, culture detects in a practically simplified way only the black-pigmented P. intermedia/p. nigrescens and P. gingivalis that lacks auto-fluorescence in UV light (Slots & Genco 1979; Slots & Reynolds 1982). This explains why culture proved to be negative in 18.6% of the cases. On the other hand, checkerboard as presented in the current panel targets a lot more anaerobic bacteria and it is reasonable to understand why it gave negative result in only one case. It is also important to stress that culture detected at least one species at moderately heavy/heavy growth in 54.7% of the patients. Checkerboard detected at least one species with score 3, 4 or 5 in 65% of the patients. The latter results, although not comparable, show that the microbiological results did not fully reflect the clinical reality in terms of magnitude, but still were able to identify disease in a high percentage of patients. Both methods were not positive for all cases, which might imply that sampling per se is a greater problem than the methods of bacterial disclosure. To conclude, peri-implantitis is a biological complication of implants in function that poses a threat and may lead to implant loss. Osseointegration alone is not sufficient to ensure longterm implant survival. With the widespread use of implants, clinicians are bound to run into cases of peri-implantitis more and more often and should be alert. Peri-implantitis lesion may represent a true infection and may develop earlier around implants with rough surfaces. Microbiological sampling methods should be improved and uniformed so as to fully unveil the microbiological profile of the disease. The current study has weaknesses and limitations because of its retrospective design and the potential confounders that could not be adjusted. Thus, results should be interpreted with caution and firm clinical conclusions may not be directly inferred. However, this material represents the largest clinical and microbiological analysis of patients with peri-implantitis to date. Its multicentre design provides useful information compiled from various centres, which could be further investigated in future prospective trials. Acknowledgements: We extend our thanks to people from all centres for their advice and generous help to access all possible patient data. With regard to public dental and university clinics, we thank Prof. Sten Isaksson, Dr Lars-Åke Johansson, Dr Hadar Hallström and Dr Helena Nilsson in Halmstad, Dr Gunnar Olsson in Falun, Dr Catrine Isehed and Dr Anders Holmlund in Gävle and Hudiksvall, Dr Kåre Buhlin in Huddinge, Dr Christer Slotte in Jönköping, Dr Gunnar Heden in Karlstad, Dr Lena Jonsson and Dr Anders Olsson in Kiruna and Luleå, Dr Anders Teiwik and Dr Nils Ravald in Linköping, Dr Carin Starkhammar- Johansson in Motala, Dr Lilian Runstad and Dr Lottie Adler in Kista, Dr Göran Palm and clinic co-ordinator Ingrid Ekstedt in Ystad, Dr Margareta Fredriksson in Västerås and Dr Björn Cassel in Gothenburg. Moreover, with regard to private dental clinics, we thank Elmborgs Dental Clinics in Högsby, Oskarshamn, Kalmar, Ljungby, Mönsterås, dental hygienist Linette Engström in Högsby, dental hygienist Siv Brunnberg in Gothenburg, Dr Peter Ingemarsson in Gothenburg, Dr Annika Skoglund in Helsingborg, Huddingeimplantat Clinic in Huddinge, Dr Anders Skoglund in Karlstad, Dr Anna Cynkier Wertheim in Malmö, Dr Jan Fornell in Stockholm, Dr Sven Christer- Nilsson in Östersund and Dr Tommy Valtersson in Nybro. We sincerely thank Dr Lars Laurell for critical reading of the manuscript and statistician Tommy Johnsson. Dr Torgny Alstad and Dr Aris Seferiadis are also greatly acknowledged for statistical advice. The study was funded by the Oral Microbiological Diagnostic Laboratory, Gothenburg, Sweden, and by grants from TUA Research, Gothenburg, Sweden. Conflict of interest: The authors declare that there are no conflicts of interest in this study. 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