Effects of subgingival air-polishing with trehalose powder on oral biofilm during periodontal maintenance therapy: a randomized-controlled pilot study

Long-term success of periodontal treatment is crucially dependent on maintenance therapy with repeated subgingival instrumentation of deepened periodontal pockets [1]. Simultaneously, regular supportive periodontal therapy may reduce tooth loss over the years [2]. Since deeper periodontal pockets might not fully be eradicated during active periodontal therapy, repeated subgingival biofilm removal in those sites is essential during supportive periodontal treatment to disturb dysbiotic biofilms and to overcome inflammation [3, 4]. In this context the use of hand instruments as well as sonic scalers or air-polishing devices could be proven effective for subgingival instrumentation [5‐7]. Especially air-polishing has for several years become a promising alternative technique with equivalent clinical results and simultaneously proven lower pain sensation to the patient [5, 8, 9]. Air-polishing leads to an effective but gentle abrasion on the root surface by accelerating powder particles using compressed air. Here, powder properties play an important role to make abrasion more effective and simultaneously be easy on the hard and soft tissue in the periodontal pocket. So far two types of powders are suitable for subgingival air-polishing: glycine and erythritole [5, 6]. Trehalose is a new powder which has not been used in the context of air-polishing in a clinical trial before. It is a disaccharide, which consists of two α, α’- 1,1-glycosidically linked glucose molecules and is classified as a food additive in the USA as well as in Europe [10]. It shows particle diameters of 25–35 μm, has a pH of 6.4 and is highly water soluble (689 g/L) according to the manufacturer. There are indications that the use of air-polishing might be favorable concerning the removal of subgingival microbiota. Concerning reduction of bacterial counts immediately after subgingival instrumentation, a superiority of air-polishing over hand instrumentation could be proven in a clinical trial [7]. However, another investigation after twelve months could find no differences of the total bacterial count between air-polishing and sonic scaling compared to baseline [5]. In terms of suppression of selective periodontal pathogens, a clinical trial could show that 90 days after using air-polishing in the oral cavity including subgingival sites, oral mucous membranes and tongue mucosa, the total count of Porphyromonas gingivalis (P. gingivalis) was significantly lower than for the group that received only hand instrumentation at subgingival sites. For Tannerella forsythia (T. forsythia) however, bacterial counts were favorable for air-polishing after ten days returning to baseline values after 90 days [11]. Also, in patients without periodontitis, an adjunctive supragingival full-mouth-disinfection by air-polishing could lower counts of red complex bacteria like P. gingivalis and T. forsythia after nine days, but returning to baseline values after six and twelve weeks [12]. In a consensus conference in 2017 it was summarized, that subgingival air-polishing in periodontal pocket depths up to 9 mm is more effective in the removal of biofilm than hand instrumentation or (ultra-) sonic scalers [13].

The detection of subgingival microbiota has undergone a tremendous development over the years. Not least by decoding the oral microbiome, new dimensions of detection and investigation of disease specific clusters have emerged.

Besides the microbiological culture technique, FISH, PCR and Checkerboard DNA-DNA Hybridization are established research methods [14‐16]. Recently, next-generation high throughput sequencing has led to a revolution in the evaluation of oral biofilm. This method is characterized by high automatability and allows sequencing a large number of DNA samples in parallel [17]. However, the above-mentioned methods have the disadvantage, compared to the classical bacterial culture, of not distinguishing between living and dead bacteria. The culture technique is still the reference method to evaluate the survival of microorganisms when new chemical or mechanical eradication methods of the oral biofilm are being tested [18, 19]. Up to now, only other powders such as glycine and erythritol were used for microbiological evaluation of the efficiency of air polishing in periodontitis treatment. Moreover, the microbiological evaluation reported in literature considered only total bacterial counts or the differentiation of red complex members like Porphyromonas gingivalis and Tannerella forsythia. No comprehensive microbiological evaluation has been conducted by using the culture technique and identification by MALDI-TOF and 16S rRNA sequencing of unidentified isolates, so far.

Therefore, the aim of this study was to determine whether there is a difference in reduction of viable selected periodontal pathogens after subgingival treatment with air-polishing using trehalose powder compared to sonic scaling during supportive periodontal therapy over a time period of 3 and 6 months (primary endpoint). Secondary endpoints were the differences between groups concerning the relation of aerobic and anaerobic species, the distribution of other subgroups of species and the total number of different species at different time points.

This clinical trial respected the principles of the Declaration of Helsinki on human experimentation and was carried out in accordance with Good Clinical Practice. The Ethical Committee of the University Medical Center Freiburg verified this trial with a positive vote (EK No. 317/14). All enrolled participants signed an informed consent form as well as a data privacy statement. This report follows the criteria of the CONSORT statement [20].

For demographic data see Table 1.

This controlled randomized clinical trial investigated the effects of trehalose powder for the removal of subgingival biofilm in the context of supporting periodontal therapy over a six-month period. To the authors’ knowledge, trehalose powder was used for the first time in combination with air-polishing within a clinical trial. Furthermore, culture technique was used to draw a more differentiated picture of living and surviving bacteria in subgingival regions before and after air-polishing. So far, in similar studies this technique has been used only to determine the total bacterial load in the context of air-polishing [7, 27]. In the present study, however, the analysis of subgroups of different species as well as the analysis of aerobes and anaerobes was carried out. In contrast to this, other research groups have mostly used molecular genetic techniques and sequencing methods for the detection of previously selected bacterial species. This has the great disadvantage of not distinguishing living from dead bacteria and also limits the field of vision to a few species. The aim of the present work was to confirm the biofilm bacteria which survived both examined techniques of subgingival instrumentation. This was to evaluate the efficiency of air-polishing for the removal of the subgingival oral biofilm compared to the sonic treatment. Therefore, molecular genetic techniques such as real-time PCR, fluorescence in situ hybridization and high-throughput next generation sequencing methods have been omitted [28]. Nevertheless, such new culture independent technique would add new insight into the reestablished biofilms months after the treatment.

The results of this microbiological part of a larger study support the clinical findings [9]. Here, 10 out of 44 participants were examined for microbiological analysis. The results show that each method resulted in a significant reduction of bacteria after instrumentation and at different time points up to 6 months. No distinct differences between the two groups could be observed. However, the confidence intervals for the group differences indicate that larger studies are necessary to establish the equivalence of the two methods. The reduction of the total bacterial load after air-polishing shown in the present work agrees with the results of other studies. Moëne and co-workers [29] collected subgingival bacterial specimens two days prior to subgingival treatment with glycine powder and seven days post treatment. The authors reported a reduction in the absolute number of microorganisms, and moreover the number of six specific microorganisms (A. actinomycetemcomitans, F. nucleatum, P. gingivalis, P. intermedia, T. denticola, T. forsythia). However, the difference in reduction was not statistically significant compared to the use of hand instruments. In two different studies, both performed by the working group of Petersilka and co-workers, subgingival treatment was done on the buccal and lingual sites or on the interdental sites with glycine powder [7, 27]. Here, the total number of bacteria of the anaerobes before and immediately after treatment was determined. Both studies could show that a reduction in total bacterial counts after air-polishing treatment was achieved [7, 27]. Other authors have also shown that both therapies (sonic treatment and subgingival air- polishing with glycine powder) significantly reduced the periodontitis-associated species immediately and two days after treatment [6]. However, the authors did not determine the total bacterial counts, but only periodontal sites where different bacterial species had been detected were called positive sites. These increased again after 14 days and approached baseline levels before treatment. This is in accordance with the results of the present work, since after three months an increase in the total bacterial counts was observed independent of the treatment method. The observed increase in the number of total bacteria 3 months after treatment and the subsequent drop after six months may be due to the re-treatment of persistent periodontal pockets after three months. This post-treatment plays an important role in creating stable periodontal conditions and reduces tooth loss rate [30‐32]. An increase in bacterial counts after three months was also described in a study by Flemmig and co-workers [11]. The authors investigated the influence of the air-polishing treatment using glycine powder on two species of the red complex (P. gingivalis and T. forsythia). The bacterial count of both bacterial species was reduced immediately after treatment, but increased again after three months and approached baseline levels.

In this study the effects on Gram-positive anaerobic rods were especially noticeable in the air-polishing group in which a higher proportion of this bacterial group was detected directly after treatment than in the patient group treated with a sonic scaler. However, the numbers of bacterial species in the air-polishing group decreased again after six months compared to the 3-month value, and were lower than the counts after the sonic treatment, but not significantly. The percentage of all aerobes and all anaerobes in the present work was almost identical in both treatment groups before treatment. A difference in this distribution between the two treatments was observed at the final 6-month follow-up, but did not reach statistical significance (p > 0.05, Fig. 2).

The number of different species was compared between the groups as a measure of the diversity. Changing the diversity means an alteration of the biofilm ecology and balance which may lead to disturbing of the natural biofilm functions. Such alterations have been considered also in other treatment techniques such as antimicrobial photodynamic therapy [33].

Since both treatment methods are not bactericidal and have been used to just mechanically remove the subgingival biofilm for the reduction of bacterial counts and not to kill the bacteria, no significant differences of the microbial composition of the biofilm sample over the total period of the study could be expected. Indeed, only directly after treatment was the percentage of anaerobic Gram-positive rods significantly higher in the patients group treated with sonic scaler compared to the air-polishing group. This emphasizes again the comparability of both treatment methods as to the influence on the subgingival biofilm. Hence, the variations and differences in the percentages of the main bacterial groups depicted in Fig. 4 should be considered as a natural fluctuation of the regrown subgingival biofilm in both groups. A more detailed microbial analysis using the culture technique would not impact the outcome of the microbial results presented in this study. The effects of air-polishing using trehalose powder as well as sonic treatment on the biofilm structure should be further studied using confocal laser scanning microscopy on a biofilm model generated by total salivary bacteria ex vivo on human enamel samples. This would evaluate the impact of both methods on the destruction of the oral biofilm structure and would show the residues of the biofilm on the enamel surface. One could speculate, that due to the different aspects of the technical application of both methods, the impact on the morphological appearance of the biofilm residues might be different and should be taken into consideration. While sonic scaling might only affect the upper layers of the biofilm, air-polishing may cause deeper dispersion of the biofilm leading to a stronger destruction of the biofilm. However, this has to be evaluated in morphological studies as stated above.

The results of the present work suggest that air-polishing and sonic treatment have a comparable effect on the subgingival oral biofilm during supportive periodontal treatment. Moreover, the present study stressed the ability of trehalose powder to remove the subgingival biofilm mechanically. The microbiome of the regrown biofilm seems not to be affected. Yet, effects on the regrown subgingival biofilm should be studied in more detail by high throughput-sequencing in future studies.