Effects on Dentin Bonding

Schmidlin et al. [13] showed that, despite a possible retention of surface and subsurface oxiderelated substances during high-dose ozone application, shear bond strength was not impaired. Magni et al. [14] indicated that ozone gas did not compromise the mechanical properties of the adhesives. Cadenaro et al. [15] demonstrated that using ozone gas to disinfect the cavity before placing a restoration there had no influence on immediate enamel and dentin bond strength. Cehreli et al. [16] revealed that pretreatment with ozone improved the marginal sealing ability of the fissure sealants. Bojar et al. [17] showed that ozone therapy improved shear bond strength of two root canal sealers (AH26 and EZ-Fill). Gurgan et al. [18] showed that ozone treatment did not impair the shear bond strength of two self-etch adhesives (Clearfil SE Bond and Clearfil Tri-S Bond) used to coronal and radicular dentin. According to Arslan et al. [19] ozone did not significantly affect the dentin bond strength of a siloranebased resin composite, Filtek Supreme. Garcia et al. [20] revealed that ozone gas and ozonated water had no deleterious effects on bond strengths and interfaces. Bitter et al. [21] showed that adhesion of the self-adhesive resin cement RelyX Unicem was significantly reduced after using gaseous ozone. According to Rodriguez et al. [22] ozone decreased the microtensile bond strength of a dentin-compos- ite resin interface. Dalkilic et al. [23] indicated that ozone reduced the initial microtensile bond strength.

In dental surgery, ozonated water was used to promote hemostasis, enhance local oxygen supply, and inhibit bacterial proliferation [4]. One study was found to evaluate the effect of ozone gas in oral and maxillofacial surgery, where ozone therapy was found to be beneficial for the treatment of refractory osteomyelitis in the head and neck in addition to treatment with antibiotics, surgery, and hyperbaric oxygen [4].

Ozone gas in a concentration of ~4 g m3 (HealOzone; KaVo, Biberach, Germany) is already being used clinically for endodontic treatment. The following summarizes the information available to date (July 2014) of the use of ozone in endodontics [24].

Effect of Ozone on Dentin

Hypersensitivity

Dentin hypersensitivity (DH) is characterized by a short, sharp pain arising from exposed dentin in response to stimuli that are typically thermal, evaporative, tactile, osmotic, or chemical and cannot be ascribed to any other form of dental defect or pathology [25]. The application of ozone as a treatment of dentin hypersensitivity was described more than 50 years ago [26]. Dähnhardt et al. [27] assessed the effect of treatment with gaseous ozone on DH. Findings revealed no significant reduction in pain compared to the placebo group. More recently, in an 8-week, three-visit, triple-blinded, randomized controlled clinical trial with two HealOzone machines (ozone/air), Azarpazhooh et al. [28] confirmed the findings of Dähnhardt et al. [27]. Another study investigated the effect of ozone, with or without the use of desensitizing agents, on the patency and occlusion of simulated hypersensitive dentin. Results indicated that the combined use of ozone/fluoride resulted in a significantly higher percentage of tubular occlusion than fluoride desensitizer alone. However, no significant difference was found between oxalate desensitizer and the combined use of ozone/ oxalate [29]. It has been demonstrated that ozonated olive oil as a monotherapy was not efficient in reducing postsurgical root dentin hypersensitivity. However, using it in combination with a mineral wash containing calcium sodium phosphosilicate had a positive impact on the reversal of postsurgical root dentin hypersensitivity [30].

Antibacterial Activity

Biofilm is a mode of bacterial growth in which dynamic communities of interacting sessile cells are irreversibly attached to a solid surface, as well as each other, and are embedded in a selfmade matrix of extracellular polymeric substances [31]. A microbial biofilm is considered a community when it meets the following criteria: possesses the ability to self-organize (autopoiesis), resists environmental perturbations (homeostasis), is more effective in association than in isolation (synergy), and responds to environmental changes as a unit rather than as single individuals (communality) [31].

A systematic review of the applications of ozone in dentistry showed that there was some evidence that ozone (in both gaseous or aqueous phases) was a potentially effective disinfectant agent for removing the biofilms and related microorganisms such as Legionella pneumophila, Mycobacterium spp., Pseudomonas aeruginosa, and Candida spp. from dental unit water systems and was an effective bactericidal agent for removing S. mutans, methicillin-resistant

Staphylococcus aureus, Candida albicans, and

E. faecalis from dentures [32]. In endodontics, so far, four in vitro studies investigated the bactericidal effect of ozone as compared to 2.5 % sodium hypochlorite, the standard irrigation solution in endodontics. The results of this outcome are controversial.

While Nagayoshi et al. [33] found nearly the same antimicrobial activity against E. faecalis and S. mutans and a lower level of cytotoxicity of ozonated water as compared to 2.5 % NaOCl, in a study by Hems et al. [34] NaOCl was found to be superior to ozonated water in killing E. faecalis in broth culture and in biofilms, while gaseous ozone had no effect on the E. faecalis biofilms. Muller et al. [35] has also found 5 % NaOCl superior to gaseous ozone in eliminating microorganisms organized in a cariogenic biofilm. Moreover, a recent study has found that irrigating infected human root canals with ozonated water, 2.5 % NaOCl, and 2 % chlorhexidine and the application of gaseous ozone for 20 min were not sufficient to

inactivate E. faecalis [36]. The antibacterial effectiveness of ozone has been revealed in several other studies [37–45].

Antifungal Activity

Fungi constitute a small part of the oral microbiota. The largest proportion of the fungal microbiota is made up of Candida species. Candida (C.) albicans is the fungal species most commonly detected in the oral cavity of both healthy and medically compromised individuals [46]. The incidence of C. albicans in the oral cavity has been reported to be 30–45 % in healthy adults [47, 48] and 95 % in patients infected with human immunodeficiency virus [46]. Studies using culturing, molecular genetics, and in situ electron microscopy methods have demonstrated that fungi are not common members of the microbiota associated with primary endodontic infections [46, 49]. However, they seem to be more common in the root canals of root-filled teeth in which the treatment has failed [46, 49]. Cardoso et al. [39] evaluated the effectiveness of ozonated water in the elimination of C. albicans from root canals and found that it reduced the number of C. albicans cells immediately; however, it showed no residual activity. Huth et al. [41] showed that highly concentrated gaseous and aqueous ozone was dose-, strain-, and timedependently effective against C. albicans in suspension and the biofilm test model.

Ozone and Endotoxin

Gram-negative microorganisms not only have different virulent factors and produce toxic products and subproducts in apical and periapical tissues but also contain endotoxin in their cell walls [38, 50, 51]. Endotoxin, which consists of lipopolysaccharides (LPSs), is liberated during bacterial cell multiplication or death and is responsible for a series of important biological effects [38]. Its action on macrophages initiates the release of a series of inflammatory, bioactive, chemical mediators or cytokines such as tumor necrosis

factor and interleukins 1, 6, and 8 [38]. There are very few studies on the effect of ozone on endotoxin. Cardoso et al. [27] showed that ozonated water did not neutralize endotoxin. Furthermore, Noguchi et al. [39] indicated that ozonated water had the ability to improve LPS-induced inflammatory responses and the suppression of odontoblastic properties of KN-3 cells (a rat odontoblastic cell line) through direct inhibition of LPS.

Conclusion

Despite the promising in vitro evidence, the clinical application of ozone in dentistry (so far used in the management of dental and root caries) has not achieved a strong level of efficacy and cost-effectiveness. More well-designed and conducted double-blind randomized clinical trials with adequate sample size, limited or no loss to follow-up, and carefully standardized methods of measurement and analyses are needed to evaluate the possible use of ozone as a treatment modality in dentistry.

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Root canal debridement and disinfection control are two of the main steps in root canal therapy [1, 2]. Control of bacterial load from an infected root

D.E. Jaramillo, DDS

Department of Endodontics,

University of Texas Health Science Center

at Houston, School of Dentistry,

7500 Cambridge St. Suite 6415,

Houston, TX 77054, USA

e-mail: David.E.Janamillo@Uth.tmc.edu

canal before obturation is necessary to have a more predictable outcome [3]. Bacteria will be present as biofilm colonies and will be responsible to establish disease and infection [4, 5]. Inside the root canal, it will be attached to the canal walls, well within dentinal tubules, fins, lateral canals, and foramina [6]. In a different study, Nair [7] found the presence of bacteria within these areas such as the root canal, fins, webs, isthmuses, etc., even after cleaning, shaping, and

filling of the root canal system. When bacteria colonize the root canal system, it becomes very hard to effectively remove it from these inaccessible areas.

During root canal therapy, the endodontist faces all types of complications, one of which is the root canal morphology. There are several studies where several authors Hess [8], Weine [9], Pineda [10], and Vertucci [11, 12] have verified the complexity of the root canal system. Root canals can present difficulty with accessibility, and in some areas of the root canal system, accessibility by instrumentation, irrigation, or even intra-canal medication is not possible. Because of this inaccessibility, different irrigation techniques have been proposed in order to obtain better disinfection rates.

Access to these areas is basically impossible for hand and/or rotary instruments [13], intracanal medications [14, 15], or through a conventional irrigation technique. Several techniques have been developed for the irrigation of the root canal system. One of the most effective has been the passive ultrasonic irrigation technique described by Weller [16] and van der Sluis [17]. Once the root canal has been shaped, the irrigation solution will flow better inside the root canal and an ultrasonically activated wire can vibrate and produce an acoustic action. Ahmad [18] said the streaming produced will help free canal walls of debris and biofilm from the surfaces. The irrigation solution is used to reach inaccessible areas; however, the streaming might not be strong enough to remove the debris, smear layer, or even biofilm.

Schwalow and Townes following Einstein’s theory of simulated emission described the principles of microwave amplification by stimulated emission of radiation. After the development of laser (light amplification by stimulated emission of radiation), it was introduced to dentistry in 1965 by Stern [19]. Today lasers widely used in dentistry include diodes, Nd:YAG, erbium, and even CO2 which produces radiation in both the near and far infrared electromagnet spectrum [20].

Several authors Saks [21], Klein [22], and McGuff [23] had demonstrated a good effect using lasers against microorganisms. In the mid-

1980s, some areas of dentistry started to explore the use of the laser, primarily CO2 laser in periodontal therapy, oral surgery, and endodontics. Pini [24] using the excimer laser was successful in removing organic tissue from inside the root canals.

In an effort to accomplish a better seal of the apical constriction, Weichman et al. [25, 26] used a neodymium-yttrium-aluminum-garnet (Nd:YAG) from both inside and outside of the apical foramen unsuccessfully. Dederich [27] found a reduced permeability on the canal walls once it had been irradiated with Nd:YAG lasers due the melting and thermal ablation of the laser beam on the dentin surface. Levy [28] compared the cleaning and shaping of Nd:YAG laser to conventional files. He reported no increased of temperature in the outer surface of the root. The shape (taper) of the canals was equal, and according to his grading, he found smoother and cleaner root canal surface in the laser group. Kantola [29] found higher levels of calcium and phosphorous after applying the CO2 laser which he attributed to the increase in organic content resulting after burning off of the organic component by the laser energy.

Gordon [30], using an in vitro model, found the use of Er,Cr:YSGG (erbium, chromium: yttrium-scandium-gallium-garnet) laser to have a good antimicrobial effect on dentital tubules infected with E. faecalis. The FDA has approved this type of laser to clean, shape, and enlarge the root canal as well as for its use in osseous, apical, and periodontal surgery. This laser frequency is highly absorbed by water and as such has a significant impact on the bacterial cell itself. This laser works by penetrating into the dentin surface by several factors. The wavelength of the Er,Cr:YSGG laser (2.78 μm) is absorbed by dentin due to the presence of hydroxide and interstitial water (chromophores of this wavelength). Each laser pulse is composed of 150 μs duration, and each one of these pulses is responsible for the penetration of its energy about 3 μm into the water. The penetration of water and the collapse of water vapor formed can penetrate as deep as 1,000 μm or more into the dentin tubules. This is known as a

micropulse-induced sequential absorption. The expansion and collapse of water vapor will produce acoustic waves that will be strong enough to disrupt intratubular bacteria. The author found the largest reductions in bacterial load (CFU) have occurred when the laser was used in the absence of a water spray. An increase in the outer surface of the root temperature of 2.6 °C was documented. The heat generated from these settings creates deleterious effect on the root canal surface, i.e., charring, necrosis, and melting of dentin.

There are many different types of lasers and wavelengths used in dentistry to perform endodontic procedures today. They all function primarily by direct radiation of light energy to tooth surfaces by way of thermal reaction. Low-level laser therapy (LLLT) is a noninvasive and simple technique mainly used for different regenerative medicine procedures. In endodontics, the use of this low-energy laser has been introduced with satisfactory results. Erbium lasers are solid-state lasers whose lasing medium is primarily erbium doped. Er:YAG lasers typically emit light with a wavelength of 2,940 nm, which is infrared light. Unlike Nd:YAG lasers, the output of an Er:YAG laser is strongly absorbed by water. This unique characteristic leads to new applications in root canal therapy such as LAI. Farges [31] found an increase of temperature with Nd:YAG lasers up to 7.2 °C. Folwaczny [32] stated good antibacterial effect of Nd:YAG laser due to radiation energy. But the increased in temperature could be an undesirable effect. A very important fact to be seriously considered in this study was the sampling technique they described in this paper. The NaOCl solution used as irrigation solution was not inactivated after the procedure, which could lead to inaccurate results in the microbiology aspect of this study.

According to Peters [33], 35 % of the root canal wall surface remained untouched by instruments after the cleaning and shaping phase of the root canal therapy. This is important data that has been misinterpreted and misunderstood in a large number of papers and presentations. The results were obtained by mathematical operation, by

doing an average of 3 root canals, instead of doing it canal by canal. There is a big difference in between the root canal areas left untouched on buccals and palatal canal. While buccals canals were touched in an average of 85 %, the palatal canal remained untouched 80 %. This would indicate that the root canal cleaning and shaping is totally dependent on the root canal morphology itself.

Kerekes and Tronstad [34] studied the morphology and found it hard to standardized a root canal preparation protocol due to the diversity and differences of root canal morphology. They found that 1 mm from the apex sizes varied from mesio-buccal canal to be as large as a number 40 hand file. The distal-buccal canal was equivalent to a number 60 hand instrument at the 3 mm level, and the palatal canal had even greater differences at the 1 mm level varying from 0.15 to 3.4 mm. The most rounded area found was present at the 1–3 mm level. According to these findings, it is more evident why some canals showed more untouched areas after root canal instrumentation. Wu [35] observed and confirmed oval root canals areas to be left untouched and unfilled depending on the obturation technique used. Tatsuta [36] found the presence of calcospherites (predentin) (Fig. 13.1) in the untouched areas of the root canal. These areas are an excellent niche for necrotic pulp tissue and bacteria to hide away

Fig. 13.1 SEM image of predentin area. It is evident the presence of bacteria hiding in these areas that remained untouched and unaltered after the cleaning, shaping, and conventional (needle) irrigation of the root canal system