Porphyromonas Gingivalis in Different Peri-Implant Conditions: a Pilot Cross - Sectional Study

Abstract

Objective

To assess the potential correlation between clinical peri-implant parameters and the presence of Porphyromonas gingivalis in different peri-implant conditions.

Material and methods

The study included 30 patients from the Department of Periodontology and Oral Medicine divided into three equal groups, defined according to the following diagnoses: peri-implantitis (PI), peri-implant mucositis (PM), and peri-implant health (HI). Clinical parameters such as peri-implant probing depth (PPD), bleeding on probing (BOP), suppuration on probing (SUP), and plaque index (PI) were recorded. The samples of peri-implant crevicular fluid were collected and relative levels of Porphyromonas gingivalis were analyzed using Real-Time PCR (Reverse transcriptase – real-time polymerase chain reaction).

Results

The mean patient age was 51.33 ± 12.61 years. The mean value for Porphyromonas ginigvalis relative level was higher in the PI group (14.80 ±31.51) compared to PM and HI group (0.48 ± 1.34 and 0.06 ± 0.12, respectively). This parameter significantly differed between PI and HI (p=0.012), as well as PI and PM (p=0.049). The mean whole mouth probing pocket depth (PPD) in the PI group (4.02mm ± 0.77mm) was also significantly greater compared to the other two groups (3.39mm ± 0.52mm in PM and 1.97mm ± 0.54mm in the HI group. The SUP in the PI (mean value of 18%) group was more frequent than in PM (mean value of 14%) while the SUP was not detected in the HI group. The correlation between clinical parameters and relative levels of Porphyromonas gingivalis was not significant.

Conclusions

The findings of this research indicate higher relative levels of Porphyromonas gingivalis in peri-implant lesions, especially in peri-implantitis.

Introduction

The outcomes of dental implant treatment are evaluated by means of survival and success rate. While survival rate means that dental implant is still present in the mouth, the success rate additionally assumes the absence of any type of biological and/or mechanical complications, alongside with accomplished function and esthetics (1). Although nowadays high implant survival and success rates could be achieved in even challenging clinical scenarios (2), long-term success rates of dental implant treatment could be compromised by the inflammatory processes in the surrounding peri-implant tissues. Inflammation around dental implants causes biological complications that manifest in the following two conditions: peri-implant mucositis and peri-implantitis (3, 4).

Peri-implant mucositis represents the reversible inflammation of peri-implant soft tissues (Figure 1), while the clinical signs of peri-implantitis are characterized by soft tissue inflammation, accompanied by progressive peri-implant marginal bone loss (Figure 2) (5, 6). Peri-implant mucositis is regarded as the predecessor of peri-implantitis, since, if not treated, it can easily progress into peri-implantitis.

Figure 1.

Clinical photography combined with x-ray represents inflammation of peri-implant soft tissue in peri-implant mucositis without bone loss.

Figure 2.

Clinical photography combined with CBCT slices represents a bone loss in peri-implantitis.

Even though different potential inducers of inflammatory reaction in peri-implant compartment were described (7), the main etiological factor causing biological complications is the bacterial biofilm in the peri-implant area. Importantly, periodontal pathogens persisted for a long period in the oral cavity of edentulous subjects with a history of periodontitis, even in the absence of other hard subgingival surfaces in the mouth (8). Additionally, the fast microbial colonization of the implant surface after the installation has been demonstrated, showing a similar composition of bacterial species at implant and tooth sites three months later (9). It should also be mentioned that periodontitis and peri-implantitis exhibit some similarities concerning the etiology, pathogenesis, risk factors, diagnosis, and treatment (10). Nonetheless, there are differences, mostly in terms of disease progression and response to treatment. Peri-implantitis shows a non-linear and aggressive progression, developing faster than the periodontal disease (11).

As mentioned above, the main etiological factor in both peri-implant diseases is oral biofilm. Its formation starts around 30 minutes after the implant placement, and within two weeks the polymicrobial flora harboring healthy implants becomes similar to the flora surrounding natural teeth in the same mouth (12).

In peri-implantitis, the biofilm structure changes towards higher counts of more pathogenic microorganisms such as Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans (13).

Among all these microorganisms, Porphiromonas gingivalis is the most common pathogen belonging to the red complex defined by Socransky (14). It is a Gram-negative, anaerobic, and asaccharolytic rod referred to be the keystone pathogen, which causes dysbiosis due to its ability to alter normal microbiota. This microorganism expresses virulence factors such as capsules, fimbriae, and proteases (gingipains). Capsule and fimbriae take place in facilitating colonization by adhering to periodontal epithelial cells, with the capsule helping co-aggregation with other pathogens and inducing anti-phagocytic activity. Gingipains are responsible for resistance to the host defense mechanisms (15-18).

Even today, the pathogenesis of peri-implantitis is not completely understood, the bacterial peri-implant flora has not yet been fully studied, and the treatment guidelines have not been established. With regard to the currently available information in the literature, the objectives of this research were to evaluate the potential correlation between clinical peri-implant parameters and the presence of Porphyromonas gingivalis.

Material and methods

Patient population

This cross-sectional pilot study included patients recruited at the Department of Periodontology and Oral Medicine, School of Dental Medicine, University of Belgrade. The present research was approved by the Ethics Committee of the School of Dental Medicine, prior to study initiation (no 36/15). The study was conducted in full accordance with the 2013 revision of the Helsinki Declaration of 1975.

The subjects included in the study were systemically healthy individuals at least 18 years of age, non-smokers or light smokers (up to 10 cigarettes per day). The patients who were enrolled earlier had received dental implants at the Department of Periodontology and Oral Medicine, after which they were coming for routine check-ups or spontaneous visits. All participants included had dental implants for at least one year in function. All types of titanium endosteal implants and prosthetic reconstructions have been included. Zirconium, pterygoid and zygomatic implants have not been considered. In case more than one implant was present, only the dental implant with the most severe clinical and radiological presentation was included.

The exclusion criteria were pregnancy or lactation, systematic diseases or drug therapy that affected the periodontium and bone metabolism, periodontal/peri-implant therapy in the last 6 months, local/ systematic usage of antimicrobial agents in the last 6 months, and usage of anti-plaque mouth wash in the last month.

Patients were divided into three groups according to the Classification of peri-implant conditions and diseases: peri-implantitis (PI), peri-implant mucositis (PM), and peri-implant health (HI).

The diagnosis of peri‐implant health required: absence of clinical signs of inflammation; the absence of bleeding and/or suppuration on gentle probing; no increase in probing depth compared to previous examinations; the absence of bone loss beyond crestal bone level changes resulting from initial bone remodeling (19).

The diagnosis of peri‐implant mucositis required: the presence of bleeding and/or suppuration on gentle probing with or without increased probing depth compared to previous examinations; the absence of continuing bone loss (observed on a radiograph): the absence of loss beyond crestal bone level changes resulting from initial bone remodeling (5) (Figure 1).

The diagnosis of peri‐implantitis required: the presence of bleeding and/or suppuration on gentle probing; an increased probing depth compared to previous examinations; the presence of bone loss beyond crestal bone level changes resulting from initial bone remodeling.

In the absence of previous examination, the data diagnosis of peri‐implantitis was based on: the presence of bleeding and/or suppuration on gentle probing; probing depths of ≥6 mm; bone levels ≥3 mm apical of the most coronal portion of the intraosseous part of the implant (6) (Figure 2).

Clinical parameters and microbiological sampling

During the same appointment, after the accurate diagnosis had been made, the following clinical parameters were recorded and analyzed: peri-implant probing depth (PPD), bleeding on probing (BOP), suppuration on probing (SUP), and plaque index (PI).

Clinical parameters were measured at six sites per implant using a periodontal probe (mesio-buccal, mid-buccal, distobuccal, mesio-lingual, mid-lingual, and disto-lingual). These parameters were recorded in periodontal charts.

During the same visit, after the clinical examination, the selected implant site was isolated using cotton rolls in order to perform the microbiological sampling. Sampling was conducted by means of 3 paper points (size 30) that were inserted into the peri-implant sulcus/pocket until resistance was met, and left for a period of 30 seconds. The procedure was repeated to obtain a control sample. Paper points were stored inside sterile Eppendorf tubes that contained RNA stabilization solution (RNAlater Stabilization Solution, Thermo Fisher Scientific), then refrigerated overnight (2–8°C), after which the solution was removed and the samples were stored at −80°C. Further analyses were made in the Laboratory for Basic research, School of Dental Medicine, University of Belgrade, by means of Real-Time PCR (Reverse transcriptase – real-time polymerase chain reaction). The method utilized specific primers and implied multiplication of the bacterial genome. In separated tubes, the human genome was also multiplicated, as a control group. The results of this analysis were reported as relative levels of microorganisms- RQ value (relative quantity).

Statistical analysis

The statistical package (SPSS software package, version 22.0; SPSS Inc.) was used for all data analyses. Descriptive statistics was presented as mean ± standard deviation, minimum and maximum. Comparisons between every two groups individually were performed by the Mann-Whitney test. The Spearman's rank correlation coefficient (ρ) was used to assess the relationship between clinical parameters and relative levels of Porphiromonas gingivalis. The significance was set at p<0.05.

Results

The present study included 30 patients with 30 inserted dental implants. Patient groups (PI, PM, HI) were balanced and each group consisted of 10 subjects. Subjects aged between 30 and 73 years, with mean patient age of 51.33 ± 12.61 years. The mean, standard deviation (SD), minimum (min), and maximum (max) values of clinical parameters for the assessed groups are presented in Table 1.

Table 1. Mean, standard deviation, minimum and maximum values for examined clinical parameters.

	PPD			BOP			SUP			PI
	Mean±SD	min	max	Mean±SD	min	max	Mean±SD	min	max	Mean±SD	min	max
PI	4.02±0.77	2.70	5.30	0.67±0.36	0.00	1.00	0.18±0.29	0.00	0.83	0.50±0.36	0.00	1.00
PM	3.39±0.52	3.00	4.50	0.93±0.16	0.50	1.00	0.14±0.15	0.00	0.50	0.65±0.33	0.00	1.00
HI	1.97±0.54	1.30	3.00	0.05±0.11	0.00	0.33	0.00±0.00	0.00	0.00	0.02±0.05	0.00	0.17

Mean PPD was the greatest in the PI group compared to the other two groups, as shown in Table 1. This parameter was significantly different among all three groups. The p values for examined clinical parameters among all groups are presented in Table 2; each group was compared with the other two groups.

Table 2 P. values for examined clinical parameters among groups. The significance was set at p<0.05.

	PPD	BOP	SUP	PI
PI/PM	p=0.036	p=0.049	p=0.806	p=0.354
PI/HI	p<0.001	p=0.001	p=0.031	p=0.001
PM/HI	p<0.001	p<0.001	p=0.005	p<0.001

The BOP and PI were the greatest in the PM group (Table 1). A significant difference in BOP was obtained between all groups, while in the PI parameter statistical significance was expressed between groups PI and HI and PM and HI (Table 2).

The SUP was more frequent in the PI group than in the PM group (Table 1), while it was not detected in the HI group. This parameter did not show a statistically significant difference regarding the appearance between PI and PM groups (Table 2).

The relative level of Porphyromonas gingivalis was the highest in the PI group (Figure 3). P-value significantly differed among groups PI and HI and PI and PM groups, as it is shown in Table 3, as previously, each group was compared with other two groups. No statistically significant correlations have been noted between the examined clinical peri-implant parameters and the relative level of Porphyromonas gingivalis (Spearman's rank correlation coefficient, p>0.05) (Table 4).

Figure 3.

Mean values for the relative level of Porphyromonas gingivalis in groups.

Table 3 P. values for the relative level of Phorphyromonas gingivalis among groups. The significance was set at p<0.05.

	Porphyromonas gingivalis
PI/PM	p=0.049
PI/HI	p=0.012
PM/HI	p=0.470

Table 4. Correlations between the examined clinical peri-implant parameters and relative levels of Porphyromonas gingivalis.

		PPD	BOP	SUP	PI
Porphyromonas gingivalis	rho	0.229	0.293	0.332	0.131
	p value	0.223	0.116	0.073	0.491

rho - Spearman’s correlation coefficient

Discussion

The findings of the present cross-sectional pilot study indicated that values of recorded clinical parameters were mostly higher in the PI group than in the other two groups. These results are in accordance with available literature data. The study by Ramanauskaite et al indicated that analyzed peri-implant clinical parameters such as PPD, BOP, and SUP were associated with the severity of peri-implant diseases. In addition, this study showed higher PPD and BOP in peri-implant mucositis and peri-implantitis compared to healthy sites, while SUP was detected in the PI group only (20). More recent research from Monje et al (21), revealed that only PPD significantly differed between PI and PM, while BOP showed a significant difference only when compared between PM and PI to HI groups. PPD exhibited a similar trend in the present and all the previous studies, likewise the conclusion by Monje et al, stating that particularly this parameter might distinguish diagnoses between peri-implant conditions (21). However, it should not be forgotten that the diagnosis of these conditions was obtained by summation of all clinical parameters.

Furthermore, the present study observed higher BOP values in PM than in PI. Additionally, SUP was detected in both PI and PM groups, with greater values in PI, however without significant differences between PI and PM. These findings are not in full accordance with the literature data where BOP exhibited greater values in PI than PM, while SUP was observed only in PI (20). One could hypothesize that the observations noted in the present study might be a consequence of a rather small sample size.

Moreover, this research revealed significantly higher relative levels of Porphyromonas gingivalis in PI compared to the other two groups. Similarly, Cortelii et al reported comparable findings where Porphyromonas gingivalis and red complex species were more frequent in PI than in HI (22). Additionally, a novel review article also confirmed that Gram-negative anaerobic species were involved in the pathogenesis of the periodontal disease, with Porphyromonas gingivalis being one of the bacteria with the highest virulence (23). Furthermore, the present study did not find a statistically significant correlation between recorded clinical parameters and relative levels of Porphyromonas gingivalis, with greater mean values in Porphyromonas gingivalis levels as the disease progressed, from PM to PI. This result is not in accordance with the available literature, since Rutar et al, stated that PPD was in positive correlation with spirochete counts, total anaerobic cultivable bacterial counts, and frequency of Porphyromonas gingivalis (24). In addition, the recent report revealed the highest correlation values between PPD and bacterial load parameters of Porphyromonas gingivalis (25). However, another novel research also did not find a significant correlation between submucosal microbial dysbiosis and certain clinical parameters (26). As stated before, the present cross-sectional study was pilot research comprising a small number of patients and inserted implants (only 30). Therefore, it has to be emphasized that all the observations made in this study should be interpreted with caution since a larger cohort of patients is needed to confirm or contradict the above-mentioned results.

Conclusions

Within the limitations of this pilot study, it can be concluded that peri-implant diseases, especially peri-implantitis, are characterized by higher relative levels of Porphyromonas gingivalis. Clinical parameters in these groups of patients are in correlation with the severity and the stage of the peri-implant disease. If further research continues on a larger patient sample size, the correlation between clinical parameters and relative levels of Porphyromonas gingivalis might be detected.