Effect of sodium hypochlorite gel on bacteria associated with periodontal disease

Periodontitis is a bacterially induced chronic inflammatory disease, where an imbalance of the innate immune defense system markedly contributes to the destruction of the tooth-supporting tissue [1]. Microorganisms are organized in biofilms; the subgingival biofilms consist of hundreds of species [2, 3]. Among the bacteria more present in periodontitis than in periodontal health are Treponema denticola, Porphyromonas gingivalis, Tannerella forsythia, Filifactor alocis, and several others [4, 5].

Non-surgical removal of the microbial deposits, the subgingival debridement (instrumentation) is the standard in any cause-related periodontal therapy [6]. But a sole mechanical debridement may be insufficient in completely removing the causative biofilm [7] and may benefit from the use of an adjunctive antiseptic [8, 9]. The standard antiseptic in periodontal treatment is chlorhexidine digluconate (CHX) in a concentration of 0.12–0.2%, as a mouth wash for the home plaque control following periodontal treatment, but also to rinse the periodontal pocket right after mechanical debridement [10]. CHX shows good antibacterial properties and a long substantivity, although tooth surface discoloration is observed as side effect [11]. Further, it is cytotoxic to oral cells [12, 13], in vitro it may induce resistance in oral bacteria [13, 14], and it was found that CHX affects homoeostasis of oral microbiota and promotes selection of bacteria being resistant to common antibiotics in vivo [15].

An interesting alternative in periodontal therapy might be sodium hypochlorite (NaOCl)–based agents. They are a well-known irrigant in endodontic therapy [16]. In supportive periodontal therapy, NaOCl solution was tested and found as effective in reducing inflammation already in the early 1980s of the last century [17]. After a time of neglecting its positive properties in periodontal therapy, it was rediscovered about 10 years ago [18] when an amino acid (AA)–buffered slightly viscous NaOCl solution (AA-NaOCl gel) was introduced to the market. This formulation consists of two components, a diluted NaOCl solution and a viscous amino acid solution containing carboxymethyl cellulose as viscosity builder. The two components are mixed right before use in order to achieve a slightly viscous alkaline NaOCl solution for application in periodontal pockets. In vitro studies underlined the potential of the NaOCl gel. A dentine surface treated in vitro with AA-NaOCl gel and rinsed thereafter with sodium chloride solution did not affect cell viability of periodontal ligament fibroblasts, whereas cell adhesion and spreading were promoted [19]. In another in vitro study, we have found that AA-NaOCl gel was able to disaggregate biofilms [20] and that its principal mode of anti-biofilm action is not based on antibacterial activity.

Recently, clinical studies underlined a beneficial effect of adjunctive application of an AA-NaOCl gel in periodontal therapy [21, 22]. Combined with minimal invasive non-surgical periodontal therapy, the adjunctive use of the AA-NaOCl gel improved the clinical outcome with respect to the periodontal probing depth (PPD), clinical attachment loss, sites with PPD ≥ 5 mm, and bleeding on probing (BOP) positive sites [21]. In supportive periodontal therapy, the adjunctive use of AA-NaOCl gel was in favor to an application of a chlorhexidine or of a placebo gel regarding reduction of sites with BOP and pocket closure [22]. However, in peri-implant mucositis, the AA-NaOCl gel did not show significant additional benefit [23]. Here, it has to be noted that in both groups, a chlorhexidine gel was applied after instrumentation and no complete resolution of inflammation was reached [23].

The following in vitro study was aimed to answer the questions (i) how the activity of AA-NaOCl gel changes over time after mixing of the two components, (ii) how the concentration of NaOCl in the gel mixture affects its activity, (iii) if a lower pH of the gel mixture decreases its activity, and (iv) if the antibacterial and biofilm disaggregating activity remains when the second component (amino acids) is replaced by hyaluronic acid.

The densest stained biofilm was visible in the controls (Fig. 5a). The biofilm after applying HA (Fig. 5b) was less densely stained than the control. Sodium hypochlorite eliminated bacteria and seemed to degrade matrix of the biofilm (Fig. 5c). After adding AA-NaOCl gel (Fig. 5d) or HA/NaOCl (Fig. 5e), the biofilms were less stained than the control but more stained compared to NaOCl alone. No clear differences were observed between the AA-NaOCl gel and the HA/NaOCl mixture.

The purpose of the present in vitro study was to get deeper knowledge on the influence of different parameters on the anti-biofilm activity of an AA-NaOCl gel designated as an adjunctive in periodontal treatment. The studies’ parameters were the time after mixing before potential application, the pH, and the chemical composition of the formulation.

Periodontal disease is the biofilm-induced and host-mediated inflammation and destruction of the tooth surrounding tissue [26]. The crucial part of the therapy is the subgingival instrumentation, i.e., the mechanical removal of the supragingival biofilm and the mineralized deposits, to enable resolution of the inflammatory reaction and reestablishment of the adjunct tissues [9]. In the guideline of the European Federation of Periodontology however, the application of adjunctive antiseptics is not clearly recommended nor not recommend, but relied only on chlorhexidine. Nevertheless, CHX is not capable in degrading the extracellular matrix of the biofilm [27, 28]. Therefore, remnants still cover the surface and avoid reattachment of the tissue. In our study NaOCl 0.3% showed both a significant reduction of the total biofilm mass and a decrease in viability of the bacteria.

Before application, the AA-NaOCl gel has to be prepared by mixing. This raises the question for the best time-point which would also suit to the treatment protocol at all. To enable some flexibility in the application, the stability of the mixture is a precondition. The second component of the AA-NaOCl gel contains the amino acids glutamic acid, leucine, and lysine. In a recent study, NaOCl was mixed with different amino acids which generated N-chloro-amino acids [29]. Mixing glutamic acid or leucine with NaOCl produced two peaks, whereas the mixture with lysine resulted in a typical monochloramine peak [29]. The product of lysine with NaOCl was investigated further; it was moderately stable [29]. Our mixing time point experiments showed that the AA-NaOCl gel was capable of statistically significantly reducing cfu counts (numbers of the viable bacteria) up to 30 min after mixing. But only up to 20 min after mixing a log10 reduction above 2 was detectable. Equally, the NaOCl formulation reduced significantly biofilm metabolic activity up to 30 min after mixing. Based on these experimental data, it can be concluded that the formulation can be applied up to 30 min after mixing, preferentially within 20 min after mixing.

Sodium hypochlorite has the potential to cause toxic reactions due to its oxidizing capacity and its high pH [30]. Viabiltiy of human gingival fibroblasts decreased to about 35%, when 0.1% NaOCl solution was applied for 5 min [31]. But this should be also related to other antiseptics commonly used in dental therapy. Low concentrated NaOCl (0.05%) did not affect dental mesenchymal stem cell survival after 10 min, whereas low concentrated CHX (0.02%) caused a strong cytotoxicity [32]. Further, computational prediction did not find any mutagenic, tumorigenic irritant and reproductive toxicity [33].

Anyway it raised the question if the anti-biofilm activity can be kept when the concentration of NaOCl is reduced. Our results suggest that using a lower NaOCl concentration does not necessarily result in a reduced activity on biofilms. Lower NaOCl concentrations may be equally or even more active against an existing biofilm than the regularly used concentration. However, it has to mentioned that in this series in contrast to other experiments, the formulation containing regular NaOCl concentration (0.45%) did not result in a significant cfu reduction compared to control. This might be explained by the fact that the mixing of the AA containing gel component with the NaOCl component of different concentrations was done by pipetting and not by the “two-connected-syringe” system and following less effective. A concentration-dependent effect of NaOCl solutions was reported in dialysis catheters with lower concentrated NaOCl (0.005–0.1%) and a treatment time of 30 min [34].

The followed pH experiments indicated that a reduced pH results in a reduced activity of the NaOCl formulation on the biofilm and that a higher pH (pH 9 or above) is of importance for the activity of the NaOCl formulation. Our results contrast another study where NaOCl at pH 5 was more active than at pH 12 [35]. There, an in situ biofilm was created in root canals; bacterial cell viability was determined by live-dead staining [35]. A study on dialysis equipment did not find a clear influence of pH (8.5–11) on anti-biofilm activity [34].

As mentioned before, the AA-NaOCl gel is a mixture of two components. The AA-component provides viscosity for the formulation. It might generate monochloramines to a certain extent. Monochloramines can act as immunomodulators. They inhibited TNFα secretion in a murine macrophage cell line but reduced also dependent on the concentration of chloramines the viability of the cells [29]. In our recent in vitro study, the AA-component had direct antibacterial activity against some Gram-negative bacteria; it slightly inhibited biofilm formation but did not affect cfu counts of an existing biofilm [20]. Others tested the potential of D-amino acids (a mixture of three AA among them D-leucine) to inhibit biofilm formation of Enterococcus faecalis and found less biofilm formation, to a limited degree also in the presence of low concentrated NaOCl [36]. Nevertheless, there is the question if the AA component could be replaced by another one. Comparing the NaOCl component and the NaOCl gel formulation showed at least the same activity of the NaOCl alone as the gel formulation against biofilms [20]. In the present study, the second component was replaced by a cross-linked hyaluronic acid formulation. It functioned as a viscosity builder which may indicate that the HA was not degraded. Degraded hyaluronic acid loses its dynamic viscosity [37]. Hyaluronic acid is a molecule of interest for many treatment options in medicine as ophthalmology, rheumatology and dermatology [38]. Having a high molecular weight, it has anti-inflammatory and immunosuppressive properties; degraded and having lower molecular weight, it may stimulate an inflammatory response [39]. In periodontitis treatment, adjunctive hyaluronic acid was applied in non-surgical and surgical therapies; recently, a systematic review showed an additional benefit in the clinical outcome [40]. NaOCl combined with the HA formulation was at least as active as the AA-NaOCl-gel formulation in biofilm disaggregation. It is of interest to further study the combination, e.g., in different concentrations of the NaOCl and HA component each and also in different modes of application. Clinically a retrospective case series analysis underlined the potential of the combination when HA was used sequentially after the NaOCl gel application [41].

The present in vitro study focused on the anti-biofilm activity of a NaOCl gel formulation. Of clinical relevance is the finding that the AA-NaOCl gel formulation can be mixed up to 20 min prior to application. Further, the study indicates that the composition of the NaOCl gel formulation can be optimized. This study did not consider aspects of interaction of bacteria with host cells, adhesion of periodontal cells to dentin surfaces, cytotoxicity, or immunomodulation. This might be a limitation and should be investigated further together with a potential modification of the NaOCl gel.