S. V. van der Waal

The effects of hyperosmosis or high pH on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa: an in vitro study

AIM To investigate the effect of hyperosmotic hyperosmosis or alkaline stress on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa. METHODOLOGY Biofilms were grown on glass cover slips suspended in bacterial inoculate for 96 h, after which the cover slips with attached biofilms were immersed in brain heart infusion broth (BHI-broth) with 6 mol L(-1) sodium chloride (NaCl) representing the hyperosmotic group or Ca(OH)(2), pH 12.1, representing the alkaline group. Two per cent sodium hypochlorite and BHI- broth served as positive and negative controls, respectively. After treatment, the biofilms were washed, harvested and plated on blood-agar plates after serial dilution. The bactericidal effect was assessed by determining the colony-forming units (CFU). The effect on the biofilm mass was imaged with confocal laser scanning microscopy (CLSM). RESULTS Hyperosmosis reduced the CFU of both species significantly after 72 h (P < 0.0001). After 168 h, P. aeruginosa was eradicated and the E. faecalis reduction was more than 99%. High pH could not induce a significant bacterial reduction. CLSM revealed dense flocculation of the biofilms incubated in alkaline broth. CONCLUSION Hyperosmosis effectively reduced a dual-species biofilm of E. faecalis and P. aeruginosa, whilst high pH had limited bactericidal effect in this model.

Cytotoxicity, interaction with dentine and efficacy on multispecies biofilms of a modified salt solution intended for endodontic disinfection in a new in vitro biofilm model

AIM To investigate the cytotoxicity of a modified salt solution (MSS) and evaluate the antimicrobial properties of MSS on in vitro biofilm models. METHODOLOGY In a metabolic assay, fibroblasts derived from periodontal ligaments (PDL) of human extracted teeth were cultured and challenged with MSS or controls. Then, in active attachment biofilm models, the efficacy of MSS in the presence of dentine powder and in eliminating mature biofilms was investigated. In the dentine assay, a biofilm of Enterococcus faecalis was employed. For the final assay, microorganisms were retrieved from infected root canals and cultured to produce biofilms. After the treatments with MSS or the controls, the biofilms were collected, serially diluted and plated. The colony-forming units were counted. One-way anova was used to analyse the differences between the groups. A P < 0.05 was considered significant. RESULTS The PDL fibroblasts remained metabolically active after challenges with MSS. Dentine powder did not alter the efficacy of MSS (P > 0.05). In endodontic biofilms, the culturable bacteria were equally reduced by MSS, 2% chlorhexidine (CHX) or 2% sodium hypochlorite (NaOCl) (P > 0.05). CONCLUSIONS Modified salt solution is noncytotoxic in vitro and has good antimicrobial properties equal to CHX and NaOCl. Although the results are promising, ex vivo and in vivo studies are needed before its use as an interappointment root canal dressing can be considered.

Free available chlorine concentration in sodium hypochlorite solutions obtained from dental practices and intended for endodontic irrigation: are the expectations true?

OBJECTIVE Sodium hypochlorite (NaOCl) is an important tool in root canal disinfection although it is well known that the shelf-life of NaOCl is limited. In this study, NaOCl solutions that were collected from dental practices and were intended for endodontic irrigation were investigated to see whether they contained the expected concentration of free available chlorine. METHOD AND MATERIALS NaOCl solutions were collected from dental practices. The concentration of available chlorine per sample was determined with iodometric titration and the pH was measured. Each participating dentist completed a questionnaire that requested data on a range of issues relating to the assumed concentration of NaOCl and handling of the sample. RESULTS Eighty-four samples with questionnaires were received. NaOCl was purchased from supermarkets and drugstores (36%), dental suppliers (48%), or pharmacies (16%). The median expected concentration was 2% (n = 36). On average, 27% less available chlorine was measured than the dentist assumed was in the sample (P < .001). Fifteen percent of samples contained less than 1% available chlorine, which is needed for tissue dissolution and disinfection. The average pH was 11.5. CONCLUSION The greatest differences in concentrations were found in NaOCl sourced from supermarkets or drugstores. Future studies should elucidate the cause of this discrepancy. CLINICAL RELEVANCE In the meantime it is recommended to purchase NaOCl from professional suppliers, because this group showed the most reliable content of free available chlorine.

Does apical periodontitis have systemic consequences? The need for well-planned and carefully conducted clinical studies

Apical periodontitis, infection of the root canal system, may have systemic consequences. This proposal has been brought forward many times in dentistry literature but the general consensus is that there is no scientific basis for an association between endodontic infections and general health. This opinion paper argues that, in order to obtain such a scientific basis, or to rule out the issue all together, we need carefully designed longitudinal challenge model (that is, intervention) studies in which we follow specific biomarkers of inflammation. These biomarkers can be those that are currently being substantiated in chronic inflammation and low-grade inflammation studies in medicine and nutritional science, where the presence of these inflammatory disorders is linked to systemic outcomes. A list of suggested biomarkers has been included.

Comment on: methods of diagnosis and treatment in endodontics by SBU

Dear Editor, Recently, the Swedish Council on Health Technology Assessment (SBU) has evaluated the methods used by dentists to diagnose, prevent and treat infection and inflammation of the dental pulp. This resulted in a report ‘Methods of Diagnosis and Treatment in Endodontics’ that was published in 2010 in Swedish and in 2012 in English. One of the questions that the SBU addressed was: ‘Is there a risk that cases of acute and chronic infection originating in the dental pulp may give rise to pathological conditions in other organs?’ One of SBU’s conclusions was that the scientific basis was insufficient to assess the association between endodontic infections and disease conditions of other organs. When an association was found, the relationship was weak. Consequently, ‘there is a need to study the risk to general health when teeth with periapical inflammatory processes remain untreated’. Apical periodontitis (AP) is often considered as a minor chronic low-grade inflammation (Wu et al. 2006, Cotti et al. 2011, Gomes et al. 2013), but we, the undersigned, feel that with this description AP is not taken seriously. This terminology does not invite detailed research, because a low-grade problem could suggest that the benefit of investigation would be minimal. From histology and radiographic studies, we know that a minor lesion on a radiograph can be much bigger in vivo (Brynolf 1967) and that extensive disease of bone may be present when there is no evidence of it on a radiograph (Bender & Seltzer 1961). Moreover, the qualification of a low-grade inflammatory response may be erroneous. In medicine, a lowgrade inflammatory response is considered to be a metabolic disturbance, which results in the (temporary) expression of inflammatory mediators (IM). When a low-grade inflammation is present, no overt pathology such as vascular or tissue damage is seen. Examples of such low-grade inflammatory responses are stress, recent surgery or obesity (Calder et al. 2013). Even the transient increased expression of IMs after devouring a fatty or high-sugar containing meal is regarded as a low-grade inflammatory response. Apical periodontitis is a complex biological response of periapical tissues to pathogens originating from the root canal system. In general, the inflammatory response is a dynamic process that lasts from a few minutes to years, depending on the extent and type of the injury and the vascularity of the tissue. Inflammation involves circulatory changes, changes in vessel wall permeability, response of white blood cells and the release of soluble mediators through several pathways. The resolution of an inflammatory response is an active process with the release of anti-inflammatory and proresolving mediators (Serhan 2007). An inflammatory response begins with an acute phase, which can resolve when the initial trigger is eliminated. When elimination fails, the acute inflammatory response becomes chronic. With these descriptions of inflammatory responses, it is impossible to maintain the classification of AP as minor or low grade. Apical periodontitis is a chronic inflammatory response which like other chronic responses has the potential to exert systemic effects. During a chronic inflammation, IMs are continuously expressed because they are necessary to sustain the inflammatory response. These locally expressed IMs can spill over to the circulation where they can contribute to the development of comorbidity or aggravate existing pathology. Comorbidities have already been associated with chronic inflammation (Tonetti et al. 2007, Teeuw et al. 2010). Studies have focused on direct associations between AP and disease. This is regrettable, because the impact of a chronic inflammation on systemic health can be more subtle than AP being the direct cause of other illnesses. For the assessment of systemic effects of AP, many dependent variables such as the expression of cytokines, chemokines, immunoglobulins, other soluble IM, oxidative stress levels or variations in blood cellular markers have been used (Marton & Kiss 1992, Inchingolo et al. 2014). Because of this diversity, it is almost impossible to compare the data of various studies (Gomes et al. 2013). Furthermore, we wonder whether all markers that have been used in the past are suitable tools. For instance, immune globulins (IGs) have often been used as a biomarker of inflammation, and although it was possible to find a difference in IG-levels between healthy and AP subjects, no

The effects of hyperosmosis or high pH on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa

AIM To investigate the effect of hyperosmotic hyperosmosis or alkaline stress on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa. METHODOLOGY Biofilms were grown on glass cover slips suspended in bacterial inoculate for 96 h, after which the cover slips with attached biofilms were immersed in brain heart infusion broth (BHI-broth) with 6 mol L(-1) sodium chloride (NaCl) representing the hyperosmotic group or Ca(OH)(2), pH 12.1, representing the alkaline group. Two per cent sodium hypochlorite and BHI- broth served as positive and negative controls, respectively. After treatment, the biofilms were washed, harvested and plated on blood-agar plates after serial dilution. The bactericidal effect was assessed by determining the colony-forming units (CFU). The effect on the biofilm mass was imaged with confocal laser scanning microscopy (CLSM). RESULTS Hyperosmosis reduced the CFU of both species significantly after 72 h (P < 0.0001). After 168 h, P. aeruginosa was eradicated and the E. faecalis reduction was more than 99%. High pH could not induce a significant bacterial reduction. CLSM revealed dense flocculation of the biofilms incubated in alkaline broth. CONCLUSION Hyperosmosis effectively reduced a dual-species biofilm of E. faecalis and P. aeruginosa, whilst high pH had limited bactericidal effect in this model.

The effects of hyperosmosis or high pH on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa: an in vitro study: Effects of hyperosmosis or high pH on dual-species biofilm

AIM To investigate the effect of hyperosmotic hyperosmosis or alkaline stress on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa. METHODOLOGY Biofilms were grown on glass cover slips suspended in bacterial inoculate for 96 h, after which the cover slips with attached biofilms were immersed in brain heart infusion broth (BHI-broth) with 6 mol L(-1) sodium chloride (NaCl) representing the hyperosmotic group or Ca(OH)(2), pH 12.1, representing the alkaline group. Two per cent sodium hypochlorite and BHI- broth served as positive and negative controls, respectively. After treatment, the biofilms were washed, harvested and plated on blood-agar plates after serial dilution. The bactericidal effect was assessed by determining the colony-forming units (CFU). The effect on the biofilm mass was imaged with confocal laser scanning microscopy (CLSM). RESULTS Hyperosmosis reduced the CFU of both species significantly after 72 h (P < 0.0001). After 168 h, P. aeruginosa was eradicated and the E. faecalis reduction was more than 99%. High pH could not induce a significant bacterial reduction. CLSM revealed dense flocculation of the biofilms incubated in alkaline broth. CONCLUSION Hyperosmosis effectively reduced a dual-species biofilm of E. faecalis and P. aeruginosa, whilst high pH had limited bactericidal effect in this model.