Uklanjanje mikroorganizama, upalno promijenjenog i nekrotičnog sadržaja iz endodontskog prostora ključno je za uspjeh endodontskog tretmana. Anatomska građa endodontskog prostora je izrazito kompleksna, stoga sama mehanička obrada nije dovoljna. Potrebna su nam kemijska sredstva koja djeluju na dijelove korijenskog kanala nedostupne endodontskim instrumentima. Takva sredstva, osim što trebaju imati antimikrobni učinak, trebaju biti i biološki sigurna. Svrha ovoga istraživanja bila je utvrditi ima li poliheksametilen bigvanid (PHMB) u koncentraciji od 0,2% antimikrobni učinak na rezistentne mikroorganizme specifične za endodontski prostor zuba unutar nezrelog (48 sati) i zrelog biofilma (4 tjedna), usporediti rezultate s djelovanjem 2,5%-tnog natrijevog hipoklorita (NaOCl) te ispitati citotoksični učinak PHMB-a i NaOCl-a. Zatim je napravljeno istraživanje na mješovitoj kulturi mikroorganizama unutar četiri tjedna starog plaka u kojoj je umjesto P. aeruginosa odabran Staphylococcus epidermidis. Rezultati su uspoređivani s rezultatima dobivenim nakon tretiranja zubi 2,5%-tnim NaOCl-om i 0,2%-tnim klorheksidinom (CHX). Ispitivanje citotoksičnosti PHMB-a i NaOCl-a provodilo se uz pomoć spektrofotometrijskog MTT testa na fibroblastima kineskog hrčka V-79. Rezultati u ovom radu pokazali su da 0,2%-tni PHMB ima jednako antimikrobno djelovanje na E. faecalis, P. aeruginosa i S. epidermidis kao i 2,5%-tni NaOCl, ali bolje djeluje na C. albicans. Obje otopine imaju statistički značajno bolje antimikrobno djelovanje od 0,2%-tnog CHX-a. Rezultati biološkog ispitivanja (citotoksičnost) pokazuju da su oba sredstva citotoksična s tim da PHMB uzrokuje nekrozu, a NaOCl apoptozu V-79 stanica. Za eventualnu kliničku primjenu PHMB-a potrebna su daljnja in vitro i in vivo ispitivanja.
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The purpose One of the primary objectives in the root canal treatment is to reduce the microbial population in the root canals of infected teeth. This is usually accomplished by mechanical preparation along with the use of irrigant solutions. Antimicrobial irrigant solutions may reach canal ramifications and inaccesible areas and permeate completely through dentinal tubules. This is even more important if we consider the penetration depth of some very resistant and small bacteria such as Enterococcus faecalis which can penetrate dentinal tubules to the depth of 1483.33m (nutrient-rich aerobic condition) or 620m (nutrient-deprived anaerobic conditions). It is present as mushroom shaped microcolonies. The most frequently used irrigant in the treatment of infected root canals is sodium hypochlorite (NaOCl). It has a solvent activity for both necrotic and vital tissues, but at the same time has a cytotoxic effect when injected in the periapical tissues, leaves a bad smell and taste, has a corosive potential and may cause allergic reactions. Polyhexamethylene biguanide (PHMB), active component of Bigvasan IB10 (Arch Chemicals. Inc. UK), (C8H19N5) biocide of biguanidine family, has been developed as a disinfectant for surfaces, objects, instruments. It is also used in wound treatment, promoting wound healing, in mouthwash formulations and in soft lenses care solutions. Enterococcus faecalis, Pseudomonas aeruginosa, Candida albicans and Staphylococcus epidermidis are considered to be the most resistant species in infected root canals and are often associated with endodontic treatment failures. The aim of this study was to test ex vivo the effectiveness of 0.2% PHMB and compare it with the effectiveness of 2.5% NaOCl in the elimination of monocultures of resistant microorganisms such as E. faecalis, P. aeruginosa and C. albicans. Our interest also was the effectiveness of those components including 0.2 % chlorhexidine (CHX) on mixed four-week culture of E. faecalis, S. epidermidis and C. albicans. The third part of this thesis was to determine whether PHMB and NaOCl are cytotoxic to cells, and if so, which type of cell death they induce. Ivana Medvedec Mikić Doktorski rad Materials and methods Single-rooted human teeth, extracted for orthodontic or periodontal reasons, were collected. The crowns and the apical parts were removed, the root canals were enlarged with Hedström files to the size 40. Overnight broth cultures of E. faecalis (ATCC 51299), P. aeruginosa (ATCC27853) and C. albicans (clinical isolate) were prepared in tryptic soy broth (Triptic Soy Broth, Difco), specimens for P. aeruginosa, E. faecalis and C. albicans were immerged into 2 ml of broth culture. After the incubation, the root canals were irrigated with saline, and the dentine samples were collected from the inner root canal walls with Hedström files size 50 (sample A). The specimens were treated with 0.2% PHMB, 2.5% NaOCl or with 0.2% CHX. Sample A presented negative control (rinsed only with saline) and sample B presented results after irrigation with 0.2% PHMB and 2.5 % NaOCl. The samples were subcultured on three blood agar plates each. After 48 h incubation at 37 C, visible colonies from appropriated dilutions were counted and the average colony forming units per milliliter (CFU/mL) was calculated and log10-transformed for each sample. The same procedure was done with four-week old culture of E. faecalis, S. epidermidis and C. albicans and the samples were subcultured on selective agars. To determine the cytotoxicity of PHMB and NaOCl, MTT test was used. Two different concentrations of each solution and saline as control were tested on chinese hamster fibroblasts V-79. The concentrations tested on microorganisms were 0.2% and 0.1% for PHMB and 2.5% and 1.25% for NaOCl. In order to reproduce the clinical conditions, contact time was a few seconds, five and ten minutes. Since the cytotoxic assay showed that both, NaOCl and PHMB, caused the cell death in V-79 cells, the following experiment was made to investigate the type of cell death that was induced by those compounds. The loss of membrane integrity was examined by measuring the massive influx of trypan blue in the cells in order to determine if necrosis has occurred. The cells that have lost the membrane integrity, i. e. necrotic cells, were clearly stained blue. The induction of apoptosis was determined by observing the morphological features of the cells treated with NaOCl. The cells were stained with DNA intercalators, acridine orange (AO) and ethidium bromide (EtBr). Acridine orange enters both, the living and the dead cells. Ethidium bromide does not penetrate through the cell membrane of the living Ivana Medvedec Mikić Doktorski rad cells, only through the damaged membrane of the dead cells. The red color of ethidium bromide prevails over green acridine orange so that the dead cells fluoresce red. Thus, simultaneous staining of the cells with acridine orange and ethidium bromide allows distinguishing between the living and the dead cells. Results In the study of unmature biofilm after the treatment with PHMB, P. aeruginosa was eradicated from all the specimens, E. faecalis was also reduced and C. albicans was not grown in seven out of eleven specimens. NaOCl showed weaker results on E. faecalis and P. aeruginosa, especially in the case of C. albicans. As for four-week old culture of E. faecalis, S. epidermidis and C. Albicans, PHMB and NaOCl showed similar results in the case of E. faecalis. CHX showed weaker results on all microorganisms, especially on S. epidermidis. In the case of C. Albicans, the input values were significantly lower than in the bacteria tested, therefore the results should be taken with caution. From the results obtained it is possible to conclude that PHMB shows better antimicrobial effect compared to CHX in the case of both bacteria and the results are comparable to those obtained by the treatment with NaOCl. The obtained results show that these compounds after a few seconds, 5 or 10 minutes of incubation with 0.1% and 0,2% PHMB or 1.25% and 2.5% NaOCl destroyed most of the cells. The types of cell death that were induced with NaOCl and PHMB were different. After rinsing the cells with 0.2% and 0.1% PHMB, all the cells were dead due to the necrosis. After the treatment with 1.25 or 2.5% NaOCl all the cells were dead due to the apoptosis. Significance The results of this study indicate that PHMB in concentration of 0.2% has good antimicrobial effects on microorganisms in immature and mature biofilm. The results are comparable to those obtained by the treatment with NaOCl. Both solutions showed statistically significant better results compared to CHX. Both solutions are cytotoxic to chinese hamster fibroblasts V-79, with a difference in the type of cell death; PHMB caused the necrosis and NaOCl the apoptosis of the cell. Promising antimicrobial results of PHMB suggest further experiments to introduce the PHMB as an endodontic irrigant.