contaminated. Removing necrotic tissue, debris, and bioÞlms from the untouched areas can only be done on chemical means. Sodium hypochlorite is the only irrigant that can dissolve organic matter and (in high concentration) detach bioÞlms. Therefore, sufÞcient use of sodium hypochlorite is important in order to obtain maximal cleaning effect in the whole canal.

Models to Study Cleaning of Isthmus Areas

Modern instruments and instrumentation techniques are able of reaching all irregularities of the root canal system. Therefore, dentists must irrigate the uninstrumented areas to remove debris and bioÞlms. Isthmus areas (connections between two root canals in the same root) in posterior teeth are challenging to irrigate, which may result in survival of microorganisms and only partial removal of dentin debris and tissue remnants. The incidence of canal isthmi varies depending on the type of tooth [76], root level [6], and age [77]. In one study the prevalence of isthmi ranged from 17 to 50 % in the apical 5 mm of the mesial root of mandibular Þrst molars, with the highest prevalence at the 3-mm level [78]. Another study [77] showed that the highest prevalence of isthmus in mandibular Þrst molars is 4Ð6 mm from the apex.

Use of micro-CT in endodontic research has made it easier to study the effects of instrumentation and irrigation in the root canal system. Recently, a method was presented to quantitatively assess accumulation and removal of inorganic debris in molar teeth instrumentation and irrigation [79, 80]. However, limitations of the micro-CT include that it only can be used on extracted teeth and that it can detect inorganic but not organic matter. Consequently, the chemical effects on soft tissues by NaOCl cannot be measured. A study evaluated the packing of hard tissue debris into isthmus areas of mesial roots of mandibular molars using rotary ProTaper instruments without any irrigation [79]. It showed that ca. 30 % of the original canal system was Þlled with hard tissue debris after preparation. The study emphasized that debris accumulation can

be an undesired consequence of instrumentation. Such packed debris may have a negative impact on the sealability of root canals and reduce the effectiveness of disinfection. Even copious irrigation during and after instrumentation was not able to prevent or remove the debris packed into the isthmus area between the main root canals [80]. Thus, despite rigorous irrigation, the accumulation of dentin debris seems to occur and restrict cleaning and disinfecting the areas blocked by the debris (Fig. 4.2).

In an in vivo situation, the canal is like a closed-end channel, which often results in gas entrapment and a vapor lock effect at its apical end [84Ð86] during irrigation [12, 81Ð89]. Studies designed to simulate a closed root canal system have demonstrated incomplete debridement from the apical part of the canal walls with the use of a syringe delivery technique [90Ð92]. Johnson et al. [93] compared debridement efÞcacies of a sonic irrigation technique (Vibringe; Cavex Holland BV, Haarlem, the Netherlands) with side-vented needle irrigation (SNI) in the mesiobuccal root of maxillary Þrst molars using a closed canal model. The tooth selection in this study was that the mesiodistal isthmus width of completely patent isthmi or partially obliterated isthmi had to be less than one-quarter of the diameter of the unshaped canals along the canal levels (i.e., 1Ð2.8 mm from the anatomical apex) from which histological sections would eventually be prepared after completed chemomechanical preparation. Histological sections showed that neither technique could completely remove the debris from the canal or isthmi. A signiÞcant difference between the two methods was only identiÞed between the canals and the isthmi. Both instrumented canal spaces and uninstrumented isthmus regions are cleared of soft tissue debris to the same extent using the sonic irrigation device or the conventional SNI technique.

The presence of a complex, variable, multispecies bioÞlm was recently demonstrated in the entire length of the isthmus of a tooth, which had initially been treated 10 years earlier and then retreated 2 years later [94]. Gram-positive and Gram-negative organisms were both detected. In light of the well-documented challenges in

Fig. 4.2 Micro-computed tomographic cross sections of mesial root canals of four mandibular molars treated with rotary NiTi instruments (aÐd). The cross sections are shown before instrumentation (left) and after instrumentation (right). Note the presence of accumulated hard tissue debris in the ribbonshaped isthmus area after instrumentation (the four cross sections on the right)

a

b

c

d

obtaining the desired cleanliness, this area can have a negative impact on the long-term prognosis of non-surgical endodontic treatment.

Dentin Canals

The bulk of root dentin is traversed by the dentin canal (dentinal tubules). Bacteria have been shown to be present in dentinal tubules in most teeth with apical periodontitis [95Ð97]. Several different approaches have been used to study the effect of irrigation on microbes inside the dentin canals. ¯rstavik and Haapasalo [98] investigated the effect of endodontic irrigants and locally used antibacterial agents in standardized bovine dentin blocks infected with test bacteria. The authors

reported that bacteria colonized the main root canal lumen and dentin canals. E. faecalis infected the entire length of the tubules, whereas Escherichia coli penetrated approximately 600 μm. Some other studies have shown that bacteria can penetrate dentinal tubules to depths of 200 μm or more [99, 100] (Fig. 4.3). Mechanical cleaning/disinfection means the removal of some of the infected root canal wall dentin. However, complete uniform enlargement of a root canal by 200 μm is not achieved with any of the contemporary instruments [101, 102]. Berutti et al. [103], using bacterial culture from dentin samples, showed that irrigating the canal with sodium hypochlorite (after removing the smear layer) rendered the dentinal tubules bacteria-free only to a depth of 130 μm from the canal lumen.

Berber et al. [54] investigated the efÞcacy of 0.5, 2.5, and 5.25 % sodium hypochlorite as intracanal irrigants associated with hand and rotary instrumentation techniques against E. faecalis within root canals and dentinal tubules. The samples collected from the root canals with paper points were obtained just after biomechanical preparation in order to evaluate the chemicomechanical action immediately after the instrumentation. The dentin samples were obtained using burs of different diameters in order to evaluate the presence of bacterial cells inside the dentinal tubules following the biomechanical procedures. The samples obtained with each bur were placed into brainÐheart infusion (BHI) broth, incubated at 37 ¡C, and plated onto BHI agar. The results indicated that instrumentation and irrigation with saline only removed more than 95 % of the bacterial cells from the root canal. At all depths of the root canals and for all techniques used, 5.25 % NaOCl was shown to be the most effective irrigant solution tested when dentinal tubules were analyzed, followed by 2.5 % NaOCl. No differences between the different hypochlorite concentrations in cleaning the main root canals were found. Although dentin in most teeth with apical periodontitis is infected by bacteria invading from the main root canal, histological sections stained with the Brown and Brenn method and

SEM studies have both shown that bacteria are found only in a few dentinal tubules even after a prolonged period of incubation [98, 104]. Such a low level of dentin infection makes it difÞcult to reliably measure the effects of disinfecting agents by culture or by confocal laser scanning microscopy (CLSM). Therefore, a dentin model that allows predictable, dense, and deep penetration of bacteria would be most useful for the study of endodontic disinfection [100, 105]. Recently, a standardized three-dimensional in vitro model for quantitative assessment of bacterial viability in dentin by CLSM after infection and disinfection of the dentinal tubules was developed [64].

The effect of concentration, time of exposure, and temperature on the penetration of NaOCl into dentinal tubules was recently studied [106]. The depth of penetration of NaOCl was determined by the bleaching of the stain and measured by light microscopy. The results showed that the ability of sodium hypochlorite to penetrate dentinal tubules was dependent on time, concentration, and temperature, but the relative effect of the three factors was much smaller than expected. For instance, penetration after 20-min exposure was only twice (not ten times) as much as after 2-min exposure, and the differences between penetration by 1 and 6 % NaOCl were rather small (Fig. 4.4). Maximum penetration of

300 μm was seen when 6 % sodium hypochlorite was used for 20 min at 45 ¡C in coronal and midroot dentin.

Several studies have reported that dentin weakens the antibacterial effectiveness of calcium hydroxide, iodine potassium iodide, and sodium hypochlorite [32, 33]. The survival of the bacteria could therefore also be attributed to their invasion into the dentinal tubules where they are better protected from endodontic medicaments than in the main canal. This may be caused by the difÞculty of the solutions to penetrate into the tubules, inactivation of the medicaments by dentin, or the microbial biomass in the tubules [33]. During chemomechanical preparation of the root canal, use of chelating agents and acids results in selective removal of inorganic dentin components, exposing collagen Þbers. Portenier et al. [34] studied the potential inhibitory effect of bovine dentin matrix (collagen), demineralized dentin powder (treated with EDTA or citric acid), and skin collagen on the antibacterial activity of 0.02 % CHX and 0.1/0.2 % iodine potassium iodide (IPI) solution. Dentin matrix (3 % w/v), which mostly consists of puriÞed dentin collagen, was a potent inhibitor of both CHX and IPI, with most E. faecalis cells surviving after 24 h of incubation with the medicaments in the given concentrations. Dentin matrix was a slightly less effective inhibitor of

IPI than dentin, but on CHX its effect was stronger than that of dentin. This is in accordance with earlier reports which have shown that IPI was more susceptible to dentin than to organic compounds, whereas the opposite was true for CHX [32, 33]. When EDTA or citric acid was Þrst used to dissolve the apatite, dentin inhibited the activity of CHX more than untreated dentin powder but less than puriÞed dentin matrix. No difference was detected between EDTA and citric acid treatment [34]. When IPI was tested, demineralized dentin (pretreated with EDTA or citric acid) showed no inhibitory activity. It can be speculated that rinsing with EDTA or citric acid before irrigation with disinfecting agents might weaken the effect of CHX but strengthen the effect of IPI. Comparative experiments have indicated that skin collagen is a weaker inhibitor of IPI and CHX than dentin matrix [34]. Together with the observation that dentin treated with EDTA or citric acid caused inhibition that was stronger than with skin collagen but weaker than with dentin matrix, this indicates that there are important differences between type I collagen products obtained from different sources and through different production and puriÞcation methods. In summary, dentin is a complex chemical and anatomical environment that needs to be carefully considered when designing studies looking at the effects of irrigation.