materials Article Antibacterial Effect of Endodontic Disinfections on Enterococcus Faecalis in Dental Root Canals—An In-Vitro Model Study Stefan Kranz * , André Guellmar, Franziska Braeutigam, Silke Tonndorf-Martini, Markus Heyder, Markus Reise and Bernd Sigusch Department of Conservative Dentistry and Periodontology, Jena University Hospital, Friedrich-Schiller-University, An der Alten Post 4, 07743 Jena, Germany; [email protected] (A.G.); [email protected] (F.B.); [email protected] (S.T.-M.); [email protected] (M.H.); [email protected] (M.R.); [email protected] (B.S.) * Correspondence: [email protected] Abstract: Enterococcus faecalis (E. faecalis) is rather unsusceptible to many root canal disinfections which often cause a therapeutic problem. Therefore, the present in vitro study observed the efficiency of different endodontic antiseptics in their capability to suppress E. faecalis, especially inside dentinal tubules. Prior to any testing, root canals of extracted third human molars were inoculated with E. faecalis for 48 h. Antiseptic dressings with chloramine-T or calcium hydroxide (CaOH) for 24 h or irrigations with 1.3% sodium hypochlorite (NaOCl) were applied with n = 10 in each group. As control irrigation with normal saline was used. All treated canals were manually enlarged from Citation: Kranz, S.; Guellmar, A.; size ISO 50 to 110 and the ablated dentin debris was subjected to microbial culture analysis. Bacterial Braeutigam, F.; Tonndorf-Martini, S.; colonization of the dentinal tubules up to 300 µm was verified by scanning electron microscopy Heyder, M.; Reise, M.; Sigusch, B. and histological sample preparation. Application of crystalline chloramine-T caused total bacterial Antibacterial Effect of Endodontic suppression inside the dentinal tubules. Dressings with CaOH showed only minor effects. Irrigation Disinfections on Enterococcus with NaOCl caused total eradication of bacteria adhering to the root canal walls, but also failed to Faecalis in Dental Root Canals—An completely suppress E. faecalis inside the dentinal tubules. The study showed that chloramine-T is of In-Vitro Model Study. Materials 2021, strong antiseptic activity and also efficient in suppressing E. faecalis inside dentinal tubules. 14, 2427. https://doi.org/10.3390/ ma14092427 Keywords: chloramine-T; Enterococcus faecalis; temporary dressing; calcium hydroxide; sodium Academic Editor: Tonino Traini hypochlorite; intracanal disinfection; root canal treatment; endodontic treatment Received: 21 March 2021 Accepted: 1 May 2021 Published: 7 May 2021 1. Introduction Chemo-mechanical root canal preparation involves the removal of pathogens by Publisher’s Note: MDPI stays neutral mechanical shaping of the root canal walls accompanied by an alternating irrigation with regard to jurisdictional claims in with disinfection fluids. The treatment of infected dental root canals by high quality published maps and institutional affil- disinfection is crucial to ensure healing of the apical region and long-term success of an iations. endodontic therapy. In this regard, pathogens that have survived inside dentinal tubules or in inaccessible areas of the root canal system might cause re-infection and treatment failure [1,2]. In those cases, it was shown that in post-treatment endodontic flare-ups besides Copyright: © 2021 by the authors. Streptococci, Lactobacilli, Actinomyces and fungi, the Gram-positive bacterial species Licensee MDPI, Basel, Switzerland. Enterococcus faecalis (E. faecalis) is often detected [3,4]. Investigations have shown that in This article is an open access article cases of secondary root canal infections the prevalence of E. faecalis can reach values up to distributed under the terms and 70% [5–8]. conditions of the Creative Commons E. faecalis is also known to be rather unsusceptible towards many disinfectants com- Attribution (CC BY) license (https:// monly applied during antiseptic root canal treatment which include chlorhexidine [9,10], creativecommons.org/licenses/by/ sodium hypochlorite [11,12] and calcium hydroxide [13,14]. Even exposure to clindamycin, 4.0/). Materials 2021, 14, 2427. https://doi.org/10.3390/ma14092427 https://www.mdpi.com/journal/materials Materials 2021, 14, 2427 2 of 12 tetracycline, erythromycin or treatment with Lugol’s iodine solution as well as mineral trioxide aggregate was not efficient in suppressing the species completely [15–17]. Unlike others, E. faecalis is also able to penetrate far into the dentinal tubules which ensures protection from antiseptic measures, too [2]. In order to overcome the lack of inefficient intracanal disinfection, new chemical com- pounds and methods are constantly under investigation for their suitability in endodontic antiseptic treatment [18–23]. Surprisingly, the already known disinfectant N-chloro tosylamide sodium salt (chloramine-T) shows promising characteristics [24]. Chloramine-T is of high chlorination and oxidation power and proofs sufficient antimicrobial behavior towards a wide variety of microorganisms including bacteria, fungi, yeasts and also human pathogenic viruses such as coxsackie, parainfluenza, adeno-, polio- and corona-viruses [25–27]. In contrast to sodium hypochlorite (NaOCl) which is still considered the “gold stan- dard” in chemo-mechanical root canal preparation, chloramine-T is also of less cytotoxic- ity [24,27,28]. Although chloramine-T is a well-known disinfectant, studies investigating the bacteri- cidal effect on endodontic pathogens are still rather rare. Especially for pathogens such as E. faecalis which are able to colonize dentinal tubules, application of chloramine-T by means of a temporary dressing might be of potential interest. Therefore, the present in vitro study was aimed on investigating the antibacterial effect of chloramine-T, CaOH and NaOCl in their capability to suppress E. faecalis inside the dental root canal system and especially inside dentinal tubules. 2. Materials and Methods 2.1. Cultivation of Enterococcus Faecalis In the present in vitro study the Gram-positive bacterial species Enterococcus faecalis (ATCC 29212) was used. The strain was grown under anaerobic standard conditions (80% N2, 10% CO2, and 10% H2) in 10 mL Schaedler fluid media (Oxoid Ltd., Hamp- shire, UK) for 3 h. Subsequently, bacteria were harvested by centrifugation (4000 rpm, 8 min) and washed twice with PBS. For inoculation, bacterial solutions were arranged by re-suspending the bacterial pellet in Schaedler fluid media (OD546nm of 0.5 which corresponds to 108 CFU/mL). 2.2. Preparation of the Dental Root Canals A total of 50 extracted non-decayed human wisdom teeth with round and straight roots were collected and stored in normal saline at 4 ◦C until use. The study was approved by the Ethics Committee of the Faculty of Medicine, University Hospital Jena, Germany (#2019-1401-Material). Informed consent of each patient was given. Prior to any treatment, soft tissue remands were removed from the external root surfaces with curettes (Hu-Friedy, Frankfurt am Main, Germany). Subsequently, the crowns were removed using a water-cooled cut-off wheel (Horico Dental, Berlin Germany) to receive final root lengths of 10 mm. The pulp tissue was removed and the root canals were manually enlarged with files (Densply Detrey GmbH, Konstanz, Germany) up to size ISO 50. For irrigation normal saline was used. To ensure a leak-proof outer insolation, the root surfaces were coated with glass ionomer cement (Ketac Bond, 3M ESPE, Neuss, Germany). For more convenient handling, the prepared root specimens were embedded in Flexitime impression putty material (Heraeus Kulzer GmbH, Hanau, Germany). In Figure1 the enlargement of the main root canal with endodontic files and irrigation with normal saline is shown. 2.3. Inoculation of the Root Canals with E. faecalis Prior to inoculation, all canals were irrigated with 2 mL Calcinase (lege artis Pharma GmbH & Co KG, Dettenhausen, Germany) for 180 s and afterwards rinsed with 3 mL Materials 2021, 14, 2427 3 of 12 distilled water each. Subsequently, all specimens were autoclaved in a moist chamber for 20 min at 121 ◦C. Figure 1. The picture shows root specimens embedded in putty material: (a) enlargement of the main root canal with endodontic files; (b) irrigation of the shaped canal with normal saline. After sterilization, all root canals were inoculated with 100 µL of the E. faecalis suspen- sion (OD546nm of 0.5) for 48 h under anaerobic standard conditions (80% N2, 10% CO2, and 10% H2). To avoid drying of the samples, all specimens were moistened with Schaedler fluid media after 24 h of incubation. 2.4. Microscopic Evaluation of the Infected Root Specimens After 48 h bacterial colonization of the dentinal tubules was observed by scanning electron microscopy. Therefore, the infected specimens were prepared as described at Kneist et al. [29]. In brief, after infection with E. faecalis the roots were fractured and fixed with 2.5% glutaraldehyde/cacodylate buffer for 24 h, washed three times in cacodylate buffer and subsequently dehydrated for 30–60 min by sequential washes in 30% to 100% graded ethanol. After critical-point drying, the specimens were sputtered with gold and observed using a scanning electron microscope (LEO 1450VP, Zeiss, Oberkochen, Germany) at 20–20,000× magnification. In addition, remands of the fractured and fixed specimens were subjected to histologi- cal evaluation, too. Therefore, the fragments were additionally decalcified in 10%