Tosun T*, Külekçi G**, Balkanlı O**, Dişçi R***,Özdemir T*, Sandallı P*.

Department of *Oral Implantology, Faculty of Dentistry, University of Istanbul; Department of **Microbiology, Faculty of Dentistry, University of Istanbul; Department of ***Biostatistic and Public Health, Faculty of Medicine, University of Istanbul, Turkey.

Conventionally it is thought that the implant failures depend on two subjects:

1) exceeding occlusal loading, which is affected by the prosthetic reconstruction; 2) microbial attack, which depends on the peri-implant microflora and bacterial plaque. In this study it has been shown significant difference in partially edentulous versus totally edentulous patiens, regarding total bacterial count and Prevotella/Porphiromonas CFU’s. There was found also significant correlation between total bacterial count and Prevotella/Porphiromonas CFU’s. Thus oral hygen maintenance and patient motivation must be carried out more carefully in partially edentulous cases to avoid microbial attack risk. Although there is clear evidence that peri-implant microbiology is similar to the periodontal environment flora, as the number of study and the population of the subjects investigated are not enough to explain the relationships between peri-implant health or disease with the microorganisms it is needed to make more researchs in this area.

Key words: implants, dental plaque, microbiology, peri-implant, Prevotella/Porphiromonas

 

In the last decades osseointegrated dental implants have gained an important role in the treatment of edentulous patients with their very high success rates reported by several authors (1,3,4,14). Although these encouraging results, there are some problems with the implants,causing their failure, which appears with rapid loss of peri-implant bone. This phenomenon is still not explained exactly by the international research. Conventionally it is thought that the implant failures depend on two subjects:

1) exceeding occlusal loading, which is affected by the prosthetic reconstruction (20);

2) microbial attack, which depends on the peri-implant microflora and bacterial plaque (13).

The microbiologic studies on peri-implant flora investigate the etiologic role of the microorganisms on the failures (2,11,12,13,17). Although very little is known about the relationship of oral microflora and peri-implant mucosa, extensive information is available from periodontal literature, that may be extrapolated to the transmucosal implants. The similarities in morfology of the gingiva surrounding dental implants suggest that there may be some validity to a such comparison. There is sufficient evidence to inform that the microbiota of healthy and diseased peri-implant sulci are similar to that of the natural tooth crevice in health and desease. While such similarity may exist, some studies have shown significant differences between the microbiota at implants of partially edentulous and totally edentulous patients (2,6). This controversy is due to a few number of studies carried out to establish the microfloral ”profile” of the peri-implant sulcus. A correlation between the presence of pathogenic bacteria and peri-implant disease has not been demonstrated. Thus there is a need to continue to investigate the peri-implant microflora.

The aim of the present study was to investigate the peri-implant sulcus microbiota in patients treated with implant supported overdentures; and to find out the presence of oral pathogens in the salivary microflora; and evaluate the relationships between gingival health and the microflora.

 

MATERIAL-METHODS

The study included a group of six subjects, three males and three females, ranging age from 41 to 67 (average 55). Patients had totally 13 endosseous root form implants in the mandible. All the implans were located in the interforaminal area. Three of the subjects were totally edentulous on the both jaws, and each cases had two implants . The other three patients were partially edentulous on the upper jaw and totally edentulous on the lower jaw, and except one patient with three implants, all the cases had two implants. The patients were treated consecutively with endosseous implants. Surgical procedures were made by one surgeon (Sandallý P.). After successful healing period of three months, once achieved osseointegration, the patients had overdentures supported by Dolder bars, except one case with o-ring attachments on two implants.

The study has been initiated when the prosthesis were in function in a period of time ranging between 8 and 23 months. None of the patients has been administrated with antibiotics and had any peri-implant surgical treatment in the last 6 months, before microbiological sampling. The patients were evaluated regarding peri-implant soft tissue health using modified Plaque Index (Mombelli et al. 1987) (12) (see tab.1), modified Sulcus Bleeding Index (Mombelli et al. 1987) (12) (see tab.2), and Probing Depth were recorded in the mesial, buccal, lingual and distal sites of each implant using a calibrated plastic periodontal probe (PDT Perio Probe).

 

mP1I	Score 0:	No detection of plaque
	Score 1:	Plaque only recognized by running a probe across the smooth marginal surface of the implant.
	Score 2:	Plaque can be seen by the naked eye
	Score 3:	Abundance of soft matter

tab.1

 

MBI	Score 0:	No bleeding when a periodental probe is passed along the gingival margin adjacent to the implant.
	Score 1:	Isolated bleeding spots visible.
	Score 2:	Blood forms a confluent red line on margin.
	Score 3:	Heavy or profuse bleeding

tab.2

 

MICROBIOLOGICAL SAMPLING OF PERI-IMPLANT SULCI

After supragingival plaque removal by steril coton pellet, implant abutments were washed and rinsed gently by air spray, then were isolated by steril gauzes from the saliva and steril paper points were inserted gently into the peri-implant sulci in three most deep sites for each implant, and were left in place for 30 seconds.

Care was taken to obtain sampling with minimal or no contamination from microorganisms of the supragingival plaque and mucosal surfaces. Paper points taken from sulci were dropped into a vial containing 4.5 ml of reduced transport medium (RTF) (19), and dispersed with a Vortex mixer for 60 sec. Then the samples were serially dilluted with RTF in a 10 fold steps. Aliquotes of 0.1 ml of appropriate dillutions were incubated on CDC anaerobe blood agar containing trypticase-soy-agar with 5 g/lt of yeast extract, 5mg/ml of heamin, 0.1mg/ml vitamin K1 and 5% defibrinated sheep blood; for isolation of total bacteria counts. For the selection of Prevotella/Porphyromonas, CDC anaerobe blood agar with 40mg/ml kanamycin was used (21). Plates were incubated anaerobically for 7 days at 37 °C using an anaerobic system (Gas Generation Kit, Oxoid Ltd., Basingstoke, Hempshire England) and also cultivated aerobically.

 

MICROBIOLOGICAL SAMPLING OF SALIVA

Parafin stimulated saliva samples were collected in a sterile vial making the patients by chewing for 5 min.. For salivary yeast counts was used a medium of Sabouraud agar, Lactobacilli counts on Ragosa SL agar, Mutants Streptococci counts on MSB agar medium (5). Prevotella / Porphyromonas counts in the saliva were estimated on CDC anaerobe blood agar with kanamycin (21).

 

STATISTICAL ANALYSIS

Edentulous and partially edentulous patien’s assesments were considered as a unic group in order to investigate correlations between Prevotella/Porphiromonas CFU’s, total bacterial count CFU’s, PD, mPI and mSBI. For this group Spearman rank correlation test was used. Estimations of relationship between edentulous and partially edentulous patient’s Prevotella / Porphiromonas CFU and total bacterial count CFU was performed using Mann Whitney-U test.

 

RESULTS

Probing depth varied from 2.0 mm to 4.0 mm (2.5 mm±0.67, Median 2.0mm). mP1I revealed results between 0 and 3 with a mean of 1.417±1.165 and median 1.0. mSBI showed scores ranging from 0 to 3 (average 1.208±1.076, Median 1.5) (see tab.5).

One-tailed Spearman rank correlation test reveals that there is significant relation between increasing scores of Prevotella/Porphyromonas and total colonies (rs=0.4927, n=12, p=0.052). In partially edentulous patients biggest CFU rates was found with 1.6×108 around an implant which showed also 2.6×104Prevotella / Porphyromonas CFU score; but the highest Provotella / Porphyromonas rate was noticed around an other implant in the same patient.

There has been found a significance between mPI and mSBI results (rs=0.6847, n=12, p=0.007). There is not any statistically significant relation between probing depth and other parameters.

Total bacterial counts of totally and partially edentulous patients have statistically significant difference as partially edentulous cases have highest scores (two-tailed Mann Whitney-U; U=2.0, z=-2.5621, p=0.0104). Partially edentulous cases show also a significant difference in Prevotella/Porphyromonas CFU’s, using one-tailed Mann Whitney-U test, as Prevotella/Porphyromonas was detected only in two of six implants in totally edentulous cases (U=8.0, z=-1.7093, p=0.0437).

Salivary microflora detection results was not analysed statistically, due to few number of patients (tab.6).

 

Implant Number	Prevotella/ Porphyromonas (CFU)	Total Bacterial Count (CFU)	Probing Depth in mm. (PD)	Plaque Index (mPI)	Sulcus Bleeding Index (mSBI)
43	0	5.2×104	3	0	0
33	6×102	1.3×105	3	0	0.5
43	0	1.6×105	2	2	2
33	2×103	1×106	2	3	2
43	0	3×102	2	2	2
42	0	2×104	3	1	3
Mean	4.3×102	2.2×105	2.5	1.3	1.5
SD	8.0×102	3.8×105	0.5	1.2	1.1
Median	0	9.1×104	2.5	1.5	2

tab.3 Prevotella / Porphyromonas and total bacterial count in three totally edentulous patients.

 

Implant Number	Prevotella/ Porphyromonas (CFU)	Total Bacterial Count (CFU)	Probing Depth im mm. (PD)	Plaque Index (mPI)	Sulcus Bleeding Index (mSBI)
43	1.8×105	7×105	2	1	0
33	6×104	8×105	2	1	0
43	2.6×104	1.6×108	3	3	2
33	1.2×106	1.4×108	4	3	2
43	0	6.8×105	2	1	1
33	0	3.2×106	2	0	0
Mean	2.4×105	5.1×107	2.5	1.5	0.8
SD	4.7×105	7.6×107	0.8	1.2	0.9
Median	4.3×104	5×106	2	1	0.5

tab.4 Prevotella / Porphyromonas and total bacterial count in three partially edentulous patients.

 

 

	Prevotella/Porphyromonas (CFU)	TotalBacterial Count (CFU)	ProbingDepth im mm. (PD)	PlaqueIndex (mPI)	SulcusBleeding Index (mSBI)
Min	0	3×102	2	0	0
Max	1.2×106	1.6×108	4	3	3
Mean	1.2×105	2.6×107	2.5	1.4	1.2
SD	3.4×105	5.8×107	0.6	1.1	1.0
Median	3×102	7.5×105	2	1	1.5

tab.5 Minimum, maximum, mean, SD and median scores of totally and partially edentulous patients.

 

 

Yeast (CFU/ml)
				Number of Patient
	1	0.8×103
Totally Edentulous	2	–
	3	–
	1	5.6×103
Partially Edentulous	2	–
	3	4×103

 

Lactobacillus (CFU/ml)
				Number of Patient
	1	2.2×105
Totally Edentulous	2	1.6×105
	3	4.4×104
	1	1×106
Partially Edentulous	2	3.2×104
	3	2.3×105

 

Mutants Streptococci (CFU/ml)
				Number of Patient
	1	6×105
Totally Edentulous	2	1×106
	3	9×104
	1	4.3×106
Partially Edentulous	2	7×106
	3	2.7×105

 

Prevotella/Porphyromonas (CFU/ml)
				Number of Patient
	1	–
Totally Edentulous	2	–
	3	–
	1	0.2×103
Partially Edentulous	2	8.6×105
	3	1×104

tab6. CFU counts in salivary microflora.

 

 

DISCUSSION

The use of sulcus bleeding indexes was reported as an excellent objective evaluation of disease activity in periodontology (15,16). In present study there has been found correlation between peri-implant microbial plaque and sulcus bleeding rates. Parameters such as PI and SBI are effective to asses peri-implant soft tissue health.

Several studies (6) reveals bacterial reservoir role of natural teeth in the partially edentulous patients treated with implants. In this study it has been shown significant difference in partially edentulous versus totally edentulous patiens, regarding total bacterial count and Prevotella/Porphiromonas CFU’s. There was found also significant correlation between total bacterial count and Prevotella/Porphiromonas CFU’s. Thus oral hygen maintenance and patient motivation must be carried out more carefully in partially edentulous cases to avoid microbial attack risk.

Heimdahl et al.(9)in their study on ten totally edentulous patients treated with osseointegrated implants have investigated the effect of the implants on oral microflora. In salivary samples they could not isolate any S.mutans. S.sanguis was found only in two patients. It was concluded that oral implants have no effect on colony number of oral streptococci. Instead of this in our study it was found a high number of mutans streptococci.

A complete loss of teeth may eliminate some pathogenic bacteria for lack of a suitable habitat for colonization. The levels of mutans streptococci and lactobacilli in the saliva of edentulous mouths are very low, but return to the same or rise even to higher level than in dentate mouth when dentures are worn (7,18). Könenen et al.(10), found mutans streptococci and lactobacilli in 84% and of 92% the denture-wearing edentulous subjects respectively. Our results confirm the observations that dentures can serve as colonization sites for mutans streptococci and lactobacilli.

The findings of Könenen et al. (10), revealed common occurance of Prevotella / Porphyromonas, m.streptococci and lactobacilli in the edentulous mouths. In this study Prevotella / Porphyromonas were not found in three totally edentulous mouths. But they were recovered from all patients with partially edentulous jaws.

Mombelli et al.(12) on the study with osseointegrated hollow cylinder implants on totally edentulous patients have found high percent of Gram(+) facultatif microorganisms with successful implants. Around unsuccessful implants it was observed Prevotella intermedia more than Porphyromonas gingivalis, and there has not been found any Actinomyces actinomycetemcomitans. In our study also with the implants which demonstrated high mPI , mSBI and PD it was found black pigmented species.

 

CONCLUSION

Although there is clear evidence that peri-implant microbiology is similar to the periodontal environment flora as the number of study and the population of the subjects investigated are not enough to explain the relationships between peri-implant health or disease with the microorganisms it is needed to make more researchs in this area. This preliminary study aims to continue to collect data on the argument in order to evaluate long term results enlarging the population.

 

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