hin Oral tpl Res 1984 165-171 Printed in Deamark Al righ rearoed ICAL ORAL CLINICAL ORAL IMPLANTS RESEARCH 199K 0975-7161 The microflora adjacent to osseointegrated implants supporting maxillary removable prostheses ‘Smedberg J-I. Svenstiter G, Edwardsson $. The microflora adjacent to | J. Smedberg’, osseointegrated implants supporting maxillary removable prostheses. | G. Svensatter’, S. Edwardsson? Department of Prosthetic Dentistry, Public Dental Service, St Erik Hospital ‘Stockholm, "Department of Oral Microbiology, Faculty of Odontology, Lund University, Malm, Swoden Clin Oral Impl Res 1993: 4: 165-171, © Munksgaard, 1993 The study examines the effect of maxillary prosthetic appliances on the composition of the microflora in the peri-implant sulcus, Two groups of patients participated. One group consisted of 18 people with removable prostheses, and a second group of 9 people with fixed prostheses was chosen to maich them in age. sex and function period of their prosthetic appliances. One implant site per patient was chosen for microbiological sampling, and the samples were taken on 2 separate occasions from all } the subjects, with a 3-month interval. From those with removable pros- theses, a further sample was collected by scraping a squared area from | the mucosal side of the prosthesis. The prevalence of black-pigmented | Porphyromonas: Prevotella, Actinobacllus actinomycetemeomitans, mutans streptococci. lactobacilli, enteric bacteria and yeasts was analysed using blood-agar and selective agar media. The results show that the prevalence ‘was significantly higher for Lactobacillus, Prevotella spp. and yeasts in subjects with removable prostheses than in subjects with fixed prostheses. No significant difference was registered in the pattern of microbial compo- sition in subjects with the removable prosthesis when the peri-implant sulcus plaque and the biofilm on the corresponding mucosal side of their prosthesis were examined. The insertion of a removable reconstruction to cover the area of the osseointegrated implants gave rise toa progressive change in the | peri-implant plaque towards a more aciduric microflora, | Key words: osseointegration titanium implant ~ microbiology fixed prostheses ~ removable prostheses: Jan-Ivan Smedberg. Department ‘of Prosthetic Dentistry, Public Dental Service, ‘St Exik Hospital, Flemingatan 22, '$-112 82 Stockholm, Sweden Accepted for publication 9 August 1993, Osseointegrated dental implants ad modum Brane- mark used as abutments for fixed or removable prosthetic appliances have a high long-term fixture success rate (Adell et al. 1981). Microbiological studies have demonstrated that the composition of the microbiota in both the healthy and diseased peri-implant sulcus is similar to that found in the healthy and diseased dentate sulcus (Adell et al. 1986, Mombelli et al. 1987. Apse et al. 1988, Becker et al. 1990, Quirynen & Listgarten 1990). The mi- croflora of the teeth in partially edentulous people with implants seems to serve as reservoirs for mi- crobial colonization of the titanium implants in people with conventional fixed appliances (Quiry- nen & Listgarten 1990) The microbial aggregation on the fitting surface of removable dentures consists of a complex flora with mainly gram-positive bacteria and yeasts (Budtz-Jérgensen et al. 1983, Theilade & Budtz~ Jorgensen 1988). Particularly in people with den- ture stomatitis in various stages, an acidic environ- ment is created that favors the growth of aciduric microorganisms such as lactobacilli and yeasts (Ol- sen & Birkeland 1975). Less is known about whether such a localized and more enclosed en- vironment also influences the composition of the peri-implant sulcus plaque flora in people with re- movable prosthetic reconstructions, The aim of the study therefore was to analyze the composition of the microflora normally present in the peri-implant sulcus plaque in people with removable prostheses on osseointegrated implants ad modum Brinemark. A study was also made of the composition of the aggregated microbial bio-Smedberg et al. film taken from a corresponding site on the remov- able prosthesis in the same patient. Patients with conventional prostheses fixed to the implants were included as a comparison group. Material and methods Subjects Twenty-seven people (12 men and 15 women) with a median age of 65 years (range 42-76) were se- lected for the study. All the subjects in the study considered themselves to be in good general health At least I year prior to the present examination, all 18 people with removable prostheses had re- ceived their prosthetic appliances on osseointe- grated implants ad modum Branemark (Table 1). The removable prosthetic appliance were made ac- cording to a particular design, normally using a tapered cast and milled bar connected to and splin- ting the fixtures (Lothigius et al. 1991). The detach- able part of the reconstruction is a horseshoe shaped Co-Cr reinforced overdenture, retained to the bar by 4 attachments (Ceka Revax", Ceka Center, Antwerp, Belgium). Table 1. Age, sex, dentition inthe manclble and the function time of the maxilary gtosthetc appliances. tot: B, fixed partial denture , crown, P, removable partial denture; 0, osseintegrated prosthesis, Denttion in. Function time Patent Ago == Sex mandible (monte) Removable prosthesis 1 6 u o 25 2 a u BP 18 3 5 F TG 19 4 6 F 8 18 5 82 F TP 25 6 52 4 TP 18 7 2 4 8 18 8 59 “ 0 23 9 n F ate 7 10 56 F 6 19 "1 3 F TP 19 12 2 Mu 8 2% 3 53 F 0 19 14 51 F 8 19 15 59 4 T 2 16 57 F re 2 7 82 F TP 13 18 1 M 1 13 Fixed prosthesis 1 60 u le 17 2 6 M 1 19 3 n F Gt 13 4 88 F 0 % 5 2 F T 18 6 58 M uP 2 7 69 M 18 FA 8 85 M T 19 9 6 F B 4 10 55 F BP 18 The clinical variables studied among these people are described in detail in a separate report (Smedberg et al. 1993). The presence of stomatitis in the supporting mucosal tissue of the removable prosthesis was registered according to Bergendahl (1982) with 0=normal pink and pale mucosa, 1 slightly erythematous mucosa, 2=moderate ery- thematous mucosa and 3= pronounced erythema- tous mucosa. The Plaque Index (PI) and Bleeding Index (BI) used implied visual registration of plaque or no plaque and bleeding or no bleeding. The marginal bleeding was registered while a peri- odontal probe (CP-12, Hu-Friedy, Chicago, TL, USA) was held horizontally and pressed down on the marginal tissue. Nine people with conventional screw-retained fixed prostheses were included as a reference group. These people were matched with 9 people with re- movable prostheses as to age, sex. period of function of their prosthetic appliance and the dentition in the opposing jaw (Table 1). All patients had lost the ma- jority of their teeth due to periodontitis, but a pre- requisite for all patients in the study was that the present periodontal situation should be under con- trol, ‘None of the people had used antibioties during the 2-month period preceding the sampling oc- casions, On the sampling day, no oral hygiene pro- cedures were allowed prior to the collection of the material. Before the microbiological sampling, the stimulated salivary secretion rate was estimated as described by Heintze et al. (1983), Microbiological sampling In each person, one peri-implant sulcus site was chosen for the microbial sampling, Microbial samples were collected from a total of 27 sites, all on the buccal surface of one of the medial fixtures in the frontal region. A prerequisite for site selection was a peri-implant sulcus depth of less than 3 mm. Furthermore, only healthy sites or those showing just a very mild inflammatory reaction, with some redness of the marginal tissue, were accepted for sampling. Two samples were taken from each site, with a 3-month interval, using 3 paper points per sample. Each point was placed in the sulcus for 10 s and then transferred to 2.0 ml of reduced transport fluid (RTF, Loesche et al. 1972) with glass beads and without EDTA (Fig. I). From people with a remov- able prosthesis another sample was also taken from the mucosal side of the removable prosthesis in the region corresponding to the fixture from which the peri-implant sulcus sample was taken (Fig. 2). An area (approximately 0.25 cm?) was scraped with a sterile curette and the collected material was trans-Fig. 1. Microbiological sampling from the per nplant suleus ferred to 2.0 ml of RTF, All samples were processed at the laboratory within 24h. Microbiological analysis Al inoculation procedures were carried out in an anaerobic chamber. The samples were homo- genized with a mechanical mixer for 30 s and inocu- lated from appropriate dilutions onto the media given in Table 2. After incubation, the media were analyzed using a dissection microscope (magnifi- cation 16-40 times). Confirmatory tests were per- formed as described below. The total number of anaerobically and aerobically cultivable micro- organisms were counted on Brucella blood agar and blood agar respectively. Colony-forming units (CFU) on Brucella agar resembling black-pig- menting Porphyromonas! Prevotella (Shah & Col- lins 1988, 1990) were Gram-stained, isolated and tested for the presence of trypsin-like activity and the production of indole, fermentation of lactose and to fluoresce red under long-wave UV light (Slots 1982, Shalt & Collins 1990, Johnson & Hold- eman 1983, Tanner et al. 1986). In doubtful cases, the pattern of metabolic end products and reac- tions in the Rapid ANA System (Innovative Diag- nostic System, Atlanta, GA, USA) were analyzed (Fischer et al. 1994). CFU exhibiting typical mor- phology for Actinobacillus actinomycetemeomitans on TSBY-agar were Gram-stained and tested for the presence of catalase activity. Colonies with a doubtful morphology were isolated and tested as described by Holm et al. (1987). CFU with doubt- ful morphology on MSB-agar were Gram-stained, isolated and their capacity to ferment mannitol was studied. On MacConkey agar CFU, consisting of gram-negative rods, were considered as “enteric bacteria”, CFU on Sabouraud agar, being blasto- Microflora of osseointegrated implants Fig 2, Microbiological sampling from the mucosal side of re ‘movable side of prostheses (Group B) spores in Gram-stained smears, were identified as yeasts The number of CFU on blood agar incubated aerobically and on the selected media was ex- pressed as a percentage of the total number of CFU on the Brucella blood agar. Statistical methods In all analyses, the mean values of the 2 samples collected on different occasions were used. All cul- tures with no detectable growth were taken as zero when calculating the mean value, The median and range were measured for each variable in the fol- lowing groups. “Table 2, Cuture media, cultivation procedures and identitieation Couture median and cultivation procedures Identification Brucella blood agar (Becton & Dickinson, Gockeys- Total cultivable vile, MO. USA) with 5% human erythrocytees, 0.5% anaerobic count leked human erytracytes and 5 mil menadione, 7 days anaerobicaly Blood agar 5-7 days aerobicaly (Holdeman et al Total cultivable 1977) aerobic count TSBV-agar 3 days in N, and 5% C0,{ Slots 1982) Actinobacilus ‘2ctinomyeetem- comitans MSB-agat 2 days in Np and CO, (Gold et al 1973) Mutans strepto- Selective lactobacil agar (ico Laboratates, Deri, Lactobes Mi, USA) 4 days aerobically, pouted pate technique MacConkey agar (Dic) 2 days aerobically Enteric bacteria ‘Sabouraudcextrose ager (Dc) 4 days aerabicaly Yeasts 187Smedberg et al. Table 3, Median values per sample and ranges expressed in "iogvalues and isolation frequency ofthe analyzed microbial groups in the pertimplant sulous in subjects with fixed prosthesis (samples) and removable prathesis (R-samples) samp Isolation frequency Median Range “otal cutvable aerobic count 598 495-708, 590 491-850 Total culivablo anaerobic count Bat 499-752 620 601-872 Porphyromonas gingivals one NO 09 ND Prevotella intermedia 518 NO nD-670 49 NO NDT Prevotella metaninogenca 48 ND ND-A57 og ND ‘Actinobeclus actinomycetemcamitans ane ND ND-S1t 19 NO -ND-552 Mutans steptococs igre 338 ND-533 819 257 _ND-6.33, Lactobacl 1718 57° NO-Sas 99 170 100-419 Entec bacteria, 98 090 ND-4.16, 319 ND ND-O13. Yeasts 1318 zat 519 it NO224 ND-5.13 * P<0.05. NO: not detected R-samples: the microflora of peri-implant sulcus plaque in people with removable pros- theses. the microflora of the mucosal side of the removable prosthesis in the corre- sponding region to the fixtures the microflora from peri-implant sul- cus plaque in people with fixed pros- theses. The composition of the microflora in the samples were compared and subjected to nonparametric statistical testing (Mann-Whitney U-test). B-samples: F-samples ‘The mean values for the stimulated salivary flow in people with removable prostheses were 1.9 ml/ min (0.7 min; 4.3 max) and, in people with fixed prostheses, 1.4 ml/min (0.6 min; 2.6 max). In 5 of 18 people with a removable prosthesis, the gingival tissue supporting the prosthesis was affected by stomatitis, especially in the areas of the saddle extensions. Four people had stomatitis grade | and I person had stomatitis grade 2. No local inflam- mation was observed around the fixtures. The median values of the total anaerobic and aerobic cultivable flora were similar in both the R- and F-samples (Table 3). The ratio between anaer- obic and aerobic growth was approximately 3 to | for the R-samples and 2 to | for the F-samples. Por- phyromonas gingivalis was not detected in any of the samples. Prevotella intermedia and Prevotella mel- aninogenica were both found in approximately one quarter of the R-samples and the corresponding B- samples. In the F-samples, P intermedia was ob- served in 1 of 9 samples. Taken together, the iso- lation frequency of both Prevotella spp. was higher (P<0.05) in the R-samples than that in the F- samples (median values of 1.55 versus not detect- able). The occurrence of 4. actinomycetemcomitans 168 and mutans streptococci was similiar in the two groups. The isolation frequency of lactobacilli and yeasts was higher (P< 0,05) in the R-samples than in the F-samples. Similar patterns were obtained when the calculations were based on the percentage of the total cultivable flora (Fig. 3). Even higher median values were found for lactobacilli (113.5 x 10%) and yeasts (6.4 x 10°) in people with stomatitis in group R, where the prevalence of yeasts was 4 times higher than in people with healthy mucosa (1.6 x 104) and that of lactobacilli (38.3 x 10°) 3 times higher. How- ever, the number of people with stomatitis is limited, and therefore it is difficult to draw any firm con- clusions from this result. The relative number of the studied microbial groups was compared. No significant differences ‘were observed in the composition of the microflora in the peri-implant sulcus plaque in subjects with the removable prosthesis (R-samples) and the cor- responding mucosal side of their prosthesis (B- samples, Fig. 3). Prevotella spp. were, however, detected in fewer B-samples than in R-samples (6 and 9, Fig. 3). In line with previous observations on the microbial composition of the peri-implant sulcus in partly edentate people showing a healthy or a slightly inflamed area (Lekholm et al. 1986, Apse et al 1989, Ong et al. 1992), we observed P. intermedia and P. melaninogenica infrequently and in low num- bers. This was particularly the case among the people with a fixed prosthesis (F-samples). As pre- viously suggested (Apse et al. 1989, Quirynen & Listgarten 1990), the teeth might have served as a reservoir for these microorganisms. The incidence of these 2 species taken together was, however, significantly higher in the peri-implant sulcus of people with a removable prosthesis (R-samples)0.01 0.001 FRB FRB FRB FRB ekigmeing testi mans ent suspooses | | Pete Fig. 3. Comparison of the microbiological plaque in F=. R- and B-samples expressed as a percentage of total cultivable flora Median values are indicated, * Number of subjects with nonde- tectable levels of the microbial group. ND = not detected. F the microflora from peri-implant sulcus plaque in persons with Fixed prostheses. R=the microflora of peri-implant sulcus plaque in persons with removable prostheses. B= the microflora, of the mucosal side of the removable prosthesis in the corte sponding region to the fixtures, than in that of the people with a fixed prosthesis (F-samples). The occurrence of Prevorella spp. is usually high in mature plaque with an anaerobic environment (Marsh & Martin 1992). This might have been the case in the implant plaque of people with a removable prosthesis. since these people also exhibited higher PI and BI scores than those of the F-group (Smedberg et al. 1993). In the samples from the denture base (B-samples), P_ intermedia and P melaninogenica were recovered from ap- proximately one third of the samples. A possible contributing factor to the presence of Prevotella spp. but not of P gingivalis, particularly in the R- and B-samples, might have been the fact that 2 intermedia as opposed to P. gingivalis is able to grow in an acidic environment (Hamilton et al 1989, Takahashi & Schachtele 1990), like that pres- ent on the mucosal side of dentures (Olsen & Birke- land 1975; Zgraggen & Graft 1975). A. actinomycetemcomitans was only observed in some of the R- and F-samples, and no differences Microflora of osseointegrated implants were found in its frequency in the sulcus samples from either group. This microorganism was un- detectable in the samples from the denture base. The latter finding corresponds to the observations by Theilade et al. (1983, 1988), who reported that A. actinomycetemcomitans was rarely present, and to Kéndnen et al. (1991), who were unable to detect, it in samples from denture plaque. Thus, although A, actinomycetemcomitans can be considered part of the microflora in the tooth sulcus and on the mucosa of the bucca and tongue (Zambon et al 1983), it does not seem able to establish itself in denture plaque. Even if the number of people with denture stomatitis in this study is limited, our findings of higher numbers of lactobacilli and yeasts in this, group of patients than of those without stomatitis agree with the findings of Theilade & Budtz~ Jorgensen (1988) and Cummings et al. (1990). Evidence suggesting that the microbial flora of the peri-implant sulcus is influenced by the local en- vironment created by the removable prosthesis comes from the analyses of the prevalence of lacto- bacilli and yeasts in the 3 groups of samples. Apart from being isolated more frequently in the two R- and the two B-samples at the both samplings oc- casions, lactobacilli and yeasts were also found in significantly higher numbers in the R-samples than in the F-samples. So the tightly covering prosthesis seems to create an acidic environment (Olsen & Birkeland 1975; Zgraggen & Graf 1975), encourag- ingaciduric lactobacilliand yeasts to establish them- selves in the peri-implant sulcus, where they are not normally found (Lekholm et al. 1986, Mombelli et al. 1988, Mombelli & Mericske-Stern 1990), The ecology of the gingival crevice is influenced by the anatomy of the site as well as the flow and the properties of the gingival crevicular uid. which, among others things, gives rise to pH rang- ing from 6.9 to 7.4-7.8 due to proteolysis, es- pecially during disease. Normally this environment favors high numbers of gram-negative anaerobes in particular, compared with those of other habi- tats in the oral cavity (Marsh & Martin 1992) Thus, our results indicate that such a distinet habi- tat, with its specific microbial community, might be influenced and changed by factors in the immediate environment created by the removable prosthesis Only a limited amount of saliva will be able to enter the area. This seems to give rise to a progress- ive change in the peri-implant flora towards acidur- ic microorganisms. Yeasts, considered significant pathogens in denture stomatitis (Theilade & Budtz~ Jorgensen 1988), will be part of that flora. P in- termedia, associated with periodontal diseases, which might be regarded as an aciduric micro- organism because of its capacity to grow at low 169Smedberg et al pH (Takahashi & Schachtele 1990), also constitutes a significant part of the flora around the abutment, in people with a removable prosthesis. The import- ance of these changes for the development of peri implantitis remains to be elucidated. However, our findings emphasize the importance of oral hygiene and the need to design the maxillary prosthesis around the abutments in such a way that saliva will have free entrance. Acknowledgements We would like to acknowledge Ulla-Britt Larsson, Gertie Nilsson and Elisabeth Petzelius for their contribution to this study Cette étude examine effet de prothéses amovibles supérieures sur la composition de la flore sous-gingivale paro-implantaire, Deux groupes de patients ont participé a cette étude. Un groupe consistat en 18 personnes avec prothése amovible et un second deneuf porteurde prothese fixe. Les deux groupes ont&téassortis suivant Mage, le sexe et la période d'utilisation de leur prothése Un site implantaire par patient a étéchoisi pour échantillonnage mierobien. Ces échantillons ont été prélevés & deux occasions chez ous lessujets trois mois d'intervalle. Pour ceux quiavaient ‘une prothése amovible, un échantillon supplémentaire a t€ co: leeté en gratiant une zane touchant la muqueuse dees prothéses. La fréquence globale des Porphyromonas! Prevotella pigmentés noirs, de Actinobacillus actinomycetemcomitans, des streploco- ques mutans, des lactobacilles, des bacteres entériques et des le- vures a été analysée en utilisant des milieux d'agar au sang et G'ager sélectionnés. Les résultats ont monteé que la fréquence slobale était significativement (P <0 05) supérieure pour le Lac- Tobacillus, les sous-espces de Prevorella et les levures chez les sujets qui avaient une prothése amovible compare d celle enre- aistrée chez les patients qui étaient porteurs d'une prothése fixe. Parcontre. aucune difference significativen’aétéenregistrée dans la composition microbienne lorsque la plaque sous-gingivale paro-implantaire et le biofilms su la 2one dela prothese corres ondante a la muqueuse étaient examinés. Linsertion d'une re construction amovible pour recouvrir la zone des implants ostéo- intégrés s'accompagne dune plaque paro-implantaire de plus en plus aeidurique. Zusammenfassung Die Arbeit untersucht den Einfluss der Bauart von Oberkieferre konstruktionen auf die Zusammensetzung der Mikroflora im pe- riimplantaren Sulcus. Es nahmen zwei Gruppen von Patienten teil, Eine bestand aus 18 Patienten mit abnehmbaren Prothesen, die andere aus 9 Patienten mit fest zementierten Arbeiten. Die beiden Gruppen wurden bezoglich Alter, Geschlecht und funk- tioneller Belastung der Rekonstruktion verglichen und ausge- Wwahlt. Eine Stelle mit Implantaten pro Patient wurde zur mikro- biologischen Probenentnahme ausgewahlt, Die Probenentnah- te fand zweimal mit einem Interval von 3 Monaten statt. Von den Patienten mit abnehmbaren Rekonsteuktionen wurde 2u- svzich eine Probe von der Unterseite der Prothese entnommen. Die Pravalenz von schwarzpigmentierenden Porphyromonas’ Prevotella, Actinobacillus actinomycetemcomitans, Streptococcus ‘mucans, Lactobacillen, Enterobacterien und Hefepilzen wurden ‘mit Blutagar und selektiven Agarmedien untersucht. Die Resul tate eigten, dass die Privalenz fir Lacobacilus, Prevotella Spe cies und Hefepilzen bei Patienten mit abnehmbaren Prothesen 70 signiikant héher war als bei Patienten mit zementierten Rekon- struktionen, Wurde die periimplantire Plaque aus dem Sulcus mit desjenigen von der unmittelbar benachbarten Unterseite der Prothese abgekratzten Plague verglichen, konnten keine signif kanten Unterschiede festgestelit werden. Die Eingliderung einer abnehmbaren Prothese. die cine Abdeckung der Region mit dem osseointegrierten Implantat zur Folge hatte, bewirkle eine Wei terentwieklung der Plague hin zu einer mehr und mehr sdurepro: duzierenden Mikrofiors Flestudio examina el efecto producido por aplicaciones proteti> cas maxilares en Ia composicién de la microflora del sulcus periimplantario, Participaron dos grupos de pacientes. Se esco {10 un grupo formado por 18 personas con protesis removible, Yun segundo grupo de 9 personas con protess fia agrupando: ls por edades, sexo y periodo de functionamiento de sus aplica- ciones proteticas. Se esogid un emplazamiento implantario por paciente para muestreo microbiologico y las muestras fueron tomadas en dos ocasiones separadas en todos los sujetos, con un intervalo de tres meses. Para aquellos que portaban protesis removible, se tomo tna muestra més, rascando un area cuadra- da del lado mucoso de la protess. Se analizo la prevalencia de Porphyromonas! Prevotella pigmentadas de negro, Actinoba cillus actinomycetemcomitans, strepiococo mutans, lactobacilo, bacterias entericas y levaduras usando agar-sangre y medios selectivos de agar, Los resultados indicaron que la prevalencia Fue significativamente mas alta para Lactobacillus, Prevotella ssp y levaduras en sujetos con protesis removible comparados Con sujetos con protess fija. No se registro ninguna diferencia Significativa en el patron de la composicion microbiana en sujetos con protesis removible cuando se examind la placa del surco periimplantario y la biopelicula en el lado mucoso correspondiente de la protesis. La insereion de una reconstruc- ‘sion removible para cubrir el area del implante osteointegrado id lugar @ un cambio progresivo en la placa periimplantaria hacia una microflora mas aciduria, References Adell, R.. Lekholm, U.. Rockler, B. & Brinemark. P-l. (1981) A 15.years study of osseointegrated implants in treatment of the edentulous jaw. 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