Author: Prof. Paul Lambrechts Catholic University of Leuven, Faculty of Medicine, Department of Conservative Dentistry & Endodontics in collaboration with: Bart Huybrechts, Panagiotis Moisiadis, Lars Bergmans, Daniela Mattar, Wim Teughels, Martine Pauwels, Bart Van Meerbeek & Marc Quirynen, Belgium
Restorative dentistry is evolving from extension for prevention to adhesion for retention in order to maintain pulp vitality and reduce the dental countdown. From various sources (caries, leaking restorations, trauma, and microcracks), bacteria end up in the dentinal tubules and produce lactic acids, proteolytic enzymes and endotoxins. They infect and digest the surrounding dentin, and eventually can reach the dental pulp, possibly evolving into pulpitis and, in most cases, pulp necrosis.
Nowadays, treatment of dentinal caries involves assessing the level of tooth decay and removing the infected dentin in a minimally invasive manner to try to prevent endodontic treatment. When irreversible pulp damage has occurred, endodontic treatment should be minimally invasive as well, so as to not weaken the remaining tooth. Therefore, inactivation of all colonizing pathogens is the ultimate aim in dentistry, rather than removing all diseased tissue.
This is consistent with what is also happening in the medical field (killing etiological agents rather than treating symptoms). Caries should be diagnosed with smart detection systems like Diagnodent (KaVo) and treated with a combination of mechanical, physical and chemical methods to reach those sheltered microorganisms (Fig. 1a-f). This also includes the use of micro drills with micropreparation so as not to unnecessarily destroy teeth, as well as diamond-tipped sonics or ultrasonics in proximal areas. Magnification in surplus is mandatory to modern minimally invasive dentistry. Air abrasion technology is used to treat fissures and stained grooves (Fig. 2a-c). Lasers have some potential, but still present problems such as charring, cracking, crazing, thermal decomposition, collateral damage and carbonization of hard tooth tissues and even thermal deleterious effects to the surrounding tissues.
|Figs.1a-f Caries should be diagnosed with smart detection systems (such as Diagnodent, Kavo) and treated with a combination of mechanical, physical and chemical methods to reach sheltered microorganisms|
|Figs.2a-c Air abrasion techniques are used to treat fissures and stained grooves|
Recently, the photoactivated disinfection (PAD) concept has been introduced into modern dentistry. PAD is a unique combination of a photosensitizer solution and low-power laser light (Fig. 3a-c). The photosensitizer, which is mostly colored, adheres to or gets absorbed by microbial cells. In this perspective, the metaphor of microbial terrorist soldiers getting hit by paintball bullets is appropriate. To win the war against these terrorists, though, a bullet of paint will not be sufficient. The low-power laser will hit the marked targets and inactivate those microbial invaders.
Today, the fundamental mechanism of this "dual-cure" killing is not fully understood. Hypothetically, the photosensitizer binds to microbial cell walls or even enters cells. Laser light activates the photosensitizer and creates a cascade of energy transfer and variable chemical reactions in which singlet oxygen and free radicals play an important role. Thus, the results of this laser activation are cracks in the cell wall (implosion of cells), crucial protein degradement, organel demolishment, inactivation of their virulence factors (eg, toxins) and even DNA damage. The huge advantage of this action mechanism is that it's quite impossible for the organism to create resistance against it.
|Figs.3 a, b PAD combines a photosensitizer solution and low-power laser light. c. Low-power laser handpiece irradiating a mandibular premolar mounted in a glass bottle. This image shows the penetration capacity of low-power laser light|
Another advantage is the time aspect. Usually, sodium hypochlorite, antibiotics and other weapons against microbial threats need a lot of time to inactivate their targets. PAD needs a maximum of 150 seconds. And finally, PAD only works where it should: at the infected site, not elsewhere in the human body. From this perspective, the indications for PAD in the whole world of medicine are legio, regarding growing resistance of all types of bacteria against all types of antimicrobials nowadays.
In dentistry, this new concept is finding its way in carious dentin disinfection, in managing root canal infections and in reducing the bacterial load in periopockets.
Depending on dentin preparation techniques (drill, air abrasion, ultrasonics, laser, NiTi root canal rotaries), tooth substrate and dentin conditions will vary. A smear layer covers intact dentinal tubules; both can be invaded by microorganisms. To get back to the previous metaphor, the coronal or radicular dentinal tubules are the protective mountains in which terrorist combatants wait their turn while sheltered by the smear layer.
Figures 1a-f show an example in cariology of a molar with deep stained occlusal fissures. Removal of only denatured dentine (dentin where all structure has been lost) is necessary. After this stage, the area that is not denatured, though highly infected, could be treated with a PAD technique followed by remineralizing materials applied prior to restoring the tooth with materials that seal the cavity, such as glass ionomer cements.
Enterococcus faecalis is often seen as the true pathogenic nuisance in endodontics as a result of its high resistance to the endodontic treatment protocol and its high potential to invade dentinal tubules. When one removes the smear layer and opens the tubules, one opens the treasure chamber to one of the most hard-to-get microorganisms in endodontics. Thus, smear layer removal is crucial and even mandatory to endodontic treatment success.
Figures 4a-o show a step-by-step endodontic retreatment using the PAD procedures to disinfect root canals and anastomoses before final obturation.
|Figs.4a-g Step-by-step endodontic treatment using PAD to disinfect root canals and anastomoses before final obturation|
Certain photoactive agents are taken up by bacteria preferentially, with the agent residing in the proximity of or becoming attached to the cell wall. Some may even enter microorganisms. All bacteria have the potential to be targeted, though some combinations between certain sensitizers and certain organisms are more successful than others. Healthy human tissue will not be affected.
PAD techniques use low power lasers to elicit a photochemical reaction in a photosensitizer, which in turn exerts a lethal effect on particular cells, such as bacteria. PAD is basically a lethal laser photosensitization. Photosensitizers alone in the right doses are not toxic to bacteria. Low power (diode) laser energy in itself is, again, not particularly lethal to bacteria but is useful for photochemical activation of oxygen-releasing dyes. Singlet oxygen, a protoplasmic poison released from dyes, causes lethal membrane, organel and DNA damage to microorganisms.
|Figs.4 h-k Step-by-step endodontic treatment using PAD to disinfect root canals and anastomoses before final obturation|
|Figs.4 l-o Step-by-step endodontic treatment using PAD to disinfect root canals and anastomoses|
Several factors affect the results created using PAD. These include the type of dye used, the dye concentration, the dose of radiation applied and the species of microorganism involved.
Over 400 different photoactive dyes are known. Some of them are already used in dentistry:
PAD can be obtained with more than 400 photoactive substances, combined with different laser devices. Ten kinds of blue, purple and green dyes are the most effective and popular, mainly of the phenylmethane family. Blue seems to work the best and is best documented.
Much work has been done on PAD with TBO by the British research group of M. Wilson, J. Williams, G. Pearson, J. Dobson, S. Sarkar, T. Burns, J. Pratten, A. MacRobert, N. Komerik, M. Bhatti, and S. Bonsor, and by the Australian group of L. Walsh and M. Lee.
Several visible red semiconductor diode lasers are available (Fig. 3a):
Radiation doses are typically related to time, power, and energy density:
Virtually all microbial organisms can be inactivated by the right kind of PAD. In general, due to cell wall properties, Gram positive pathogens are more sensitive to PAD than their Gram negative counterparts. With PAD using TBO, this difference is marginal due to its cationic nature.
In the dental world, work has been done on periopathogens like Porphyromonas gingivalis, Actinobacillus actinomycetem comitans, Fusobacterium nucleatum, Streptococcus sanguinis, B. forsythus, Campylobacter rectus and Eikenella corrodens. All experiments showed significant kill rates. These rates were stronger in planktonic than in biofilm mode. There is a consistent lack of in vivo research.
S. sobrinus, Lactobacillus casei, Actinomyces viscosus, and Veillonella spp., main cariogenic microorganisms can also be inactivated by PAD. Even in biofilms on carious dentine, kill rates of S. mutans were significant. Again, these rates were stronger in planktonic than in biofilm mode. There is a consistent lack of in vivo research.
In the endodontic literature, E. faecalis, S. intermedius, F. nucleatum, P. micros and P. intermedia have been investigated regarding their sensitivity to PAD. All showed significant reductions in viable counts. Again, sufficient in vivo research lacks, but is underway.
Regarding the microbial nature and the locoregional character of oral diseases, the locoregional aspect of orodental pathology, upcoming therapy-resistant species, and decreasing efficiency of almost all antibiotics, orodental PAD applications are legio, including:
For disinfection purposes, PAD is effective for:
PAD can be used effectively in carious lesions and root canals since visible light transmits well across dentin (Fig. 3c).
The research group of Gavin Pearson (London) conducted a study of S. mutans in a planktonic environment. When exposed to PAD for 30s contact time with the dye and 60s exposure at 60 mW with the laser, 99% of the S. mutans was killed. Further in vivo studies on biofilms on carious dentin showed a similar, almost complete reduction of bacteria.
Different studies on cariogenic and endodontic species in biofilm modes suggested high potential for PAD as an adjunct in the routine disinfection protocol. Further research must be done to confirm or deny this thesis.
PAD using TBO sensitizer and low-power diode laser light has proven to be a safe combination. It is already commercially available in Europe, and American FDA approval for endodontic use is coming in the near future. Several safety issues have been examined:
All microbial assessments mentioned above were counts of viable microorganisms. This kind of research only takes into account the quantitative aspect, ie, the result of PAD. Other ways of assessing endodontic microbes could be very valuable.
Our team had another idea of visualizing the action of PAD. We used a Fe-ESEM (Field emission-Environmental Scanning Electron Microscope, Figs. 5a, b) to assess the qualitative rather than the quantitative aspect of lethal PAD. A Fe-ESEM is an SEM that allows us to examine microorganisms and microbial biofilms on a dentin substrate. It does not require high vacuum, dehydration and fixation of the sample, giving the opportunity to examine wet objects in their native state. This enables us to visualize bacteria in situ, embedded in their own extracellular matrix and in real time. The effects of different disinfective concepts (like sodium hypochlorite and PAD) can be clearly seen.
|Figs.5a,b A Field emission-Environmental Scanning Electron Microscope (Fe-ESEM) was used to assess the qualitative aspect of lethal PAD|
Figures 6a, b show a 1-day matured biofilm of Enterococcus faecalis that appears "out of focus." In fact the image is representational of a biofilm because it accurately shows how bacteria are embedded in their own juices – extracellular matrix proteins. Half an hour later, you could take another image and see how the bacteria have doubled. The reason that these images don't have the "classic" sharp look of SEM bacteria is that they are not "fixed" and dried.
Our research group had a special interest in the Enterococcus faecalis because it is sheltered in its biofilm in the dentinal tubules and bonded to the dentin by "adhesion interactions." This adhesive layer consists of extracellular polysaccharide matrices, strings and ropes that allow them to adhere to surfaces and to other bacteria.
One could ask whether it is a true pathogen or just a nuisance. The challenge is to first reach, second penetrate, and finally kill these clumps of colonies and biofilms with our irrigants and PAD. Publications of this ESEM research will be available soon.
|Figs.6a, b A 1-day microfilm of E. faecalis appears out of focus, but actually represents how bacteria are embedded in their own juices|
Recently a frightening paper appeared in the endodontic literature, illustrating dramatically the inability to eliminate microorganisms in a onevisit cleaning, shaping and obturation protocol. Nair's photomicrographs show sequential transverse sections at varying distances in the corono-apical direction from the apical portion of a necrotic non-instrumented and non-obturated distal root. As the images illustrate, the main canal contains microorganisms, the accessory canals are clogged with bacteria and both bacteria (filamentous and coccus forms) and yeast are clearly visible. In the apical area of newly obturated root canals, lots ofbacteria are left and hiding. This illustrates the need for more pronounced shaping for cleaning and disinfection protocols followed by a three-dimensional obturation.
Advances in the disinfective protocol are multifactorial: good aseptive control, proper cleaning of the canal which is only possible by proper shaping of it, good tissue resolving, smear layer degrading and bacteriotoxic irrigants at the right concentration and temperature supplemented with ultrasound-activated instruments and new technology like PAD. Finally, a three-dimensional obturation and a perfect coronal seal are mandatory to endodontic success.
A biofilm is a structured, heterogenous community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to inert or living surfaces.
The life cycle of a biofilm includes five phases: (1) attachment of bacteria to a substrate; (2) colony forming; (3) growing to a monolayer stage; (4) differentiating to a mature biofilm; and (5) detachment into clumps and/or a swarming phenomenon or "seeding dispersal."
A biofilm is a 3-D community of bacteria attached to a surface. It has the following characteristics:
Bacteria in biofilm mode are much more resistant and tolerant to antimicrobials compared to their planktonic counterparts because:
The multicellular characteristics of biofilms contribute to their survivability. Their resistance mechanisms are based upon:
One can think of biofilms as static, but they are not. These biofilm structures are viscoelastic.
Hydrodynamic forces - turbulent flow of liquids can lead to shear induced adherence failure, but only after a time. They are fairly resistant, so how much flow and penetration of irrigants do we need until they are finally dislodged?
|Figs.7a, b RinsEndo (Durr Dental, Germany)|
To dislodge a biofilm, we need active long-term agitation. The SonicCare tooth brush uses a similar principle: acoustic streaming and agitation to attempt to remove plaque biofilm. If we want to disturb any biofilm, we must do it actively.
Several devices can help us win the battle against biofilms. Ultrasound-driven instruments are just one example. Figures 7a, b show the RinsEndo device (Durr Dental, Bietigheirn Bissingen, Baden-Wurttemberg, Germany), which has a compressed air-driven dynamic root canal irrigation system for vigorous controlled flushing. It uses an alternating "flushing/suctioning" action to actively flush the root canal system. However, the needle insertion must be well controlled because it is an "active" rather than passive irrigation system.
Not only does NaOCl kill bacteria, it dissolves organic material and membranes. Concentration (0.5-5%), diffusion time, temperature and contact time are important.
NaOCl is a fairly corrosive liquid and cannot be applied for a long time when using the type of sensitive Fe-ESEM equipment used in our research. When 1 minute of NaOCl 2.5% was applied to a 2-day-old biofilm of S. anginosus, good viable count reduction was noted. Similar tests done with 6 day incubated biofilm of S. anginosus showed similar results. Likewise, similar tests done with 2 day incubated E. faecalis(2 min. NaOCl 2.5%) showed good but not complete results. Some E. faecalis still remain embedded deeper in the polysaccharide layer and can cause re-growth. In the case of those particular bacteria you need 15-20 minutes contact time in order to penetrate and dissolve the biofilm to its complete depth.
Figures 6a, b show a biofilm with E. faecalis, where exposure to the dye/laser PAD combination resulted in a substantial though not complete reduction of intact organisms. Hypothetically, this could be explained by the dye not penetrating to the full depth of the biofilm. Like NaOCl, the effectiveness depends on how well the dye diffuses into the biofilm and the contact and activation time. Ultimately, this all depends on how well you can deal with smear layer removal and have the TBO enter the tubules. The better you remove it, the better the penetrating power you have for PAD.
Studies of bacteria in biofilms subjected to PAD showed an 88% to 99% kill depending on the type of microorganism. If the original number of bacteria in the canal was 10 million (for example), a 99% reduction would still leave 100,000 bacteria in the tubules. If you do not entomb these remaining bacteria with a good endodontic seal, you may still get failure due to regrowth of these remaining bacteria. A failing coronal seal will also evolve into contamination of the obturation and ultimately into treatment failure.
Different bacteria also show different levels of susceptibility to PAD. That is because the complex structure of the biofilm means that different bacteria may be protected by being located in deeper layers of the biofilm, or have resisting properties because of their location and interaction with other organisms.
During endo treatment we need to neutralize microorganisms and their by-products. Finally sealing the tooth and preserving as much healthy tissue as possible will lead to treatment success. We need to do this successfully while at the same time working in a minimally invasive and effective manner. We all know that the ideal situation is complete cleaning and sealing of the canal system. The problem is the complex anatomy of the root canal system, which does not always allows us to do this.
Even most reknown endodontists fail to prepare all root canal surfaces. Prof Paul Lambrechts and Prof Lars Bergmans worked with Prof Pierre Machtou and Prof Clifford Ruddle. The latter two prepared a pre-scanned mandibular molar mesial root. After preparation, the roots were returned to Prof Lambrechts. Again they were scanned and matched to the images before instrumentation. Though they were nicely prepared and tapered, there were still anastomotic regions that could not be reached through thorough shaping. To reach these areas, additional tools are needed: active rinsing, ultrasonics, agitating devices and other methods.
Figures 8a-d show a horizontally sectioned mesial root with two canals, one each prepared with K3 and ProTapers. The canals were nicely centered and shaped but there were still lateral areas that were not encompassed by the preparations, as well as the interconnection of the isthmuses. Preparing the canals with this technique can actually push debris into the isthmus and lateral areas. This makes rinsing these isthmuses and lateral areas almost impossible. The bacteria can then multiply in these areas, every half an hour. That is a major problem.
Figs. 8a-d Horizontally sectioned mesial root with two canals, one each prepared with K3 and ProTaper files
So how do we deal with these organisms that have sheltered themselves in the dentin tubules and have culture reversal potential? Though debatable, Nair's study showed through apical surgery that a mandibular molar, presenting initially with primary apical periodontitis, still harbors lots of micro-organisms following thorough instrumentation, filling and then surgery of the root eight months after treatment.
A photomicrograph of the transverse section of the resected root apex showed incomplete obturation of both canals connected by a debris-filled wide isthmus that was just as wide as the prepared canals. TEMs of the bacterial masses in these isthmuses showed islands of "fibro-dentinal structures," the pulp and dentin debris that was pushed into the isthmus during preparation of the MB and ML canals. The bacteria are protected by this debris layer. TEM of a necrotic accessory canal in the area showed numerous filamentous and cocci bacteria embedded in an extracellular matrix.
Are we losing the fight? Do we feel uncomfortable seeing these results?
The problem has only one solution: optimizing the whole concept of a successful endodontic treatment, but most urgent, optimizing the disinfection protocol. Better cleaning of the root canal is only possible when irrigants can get to the right site, which means shaping in function of cleaning.
Besides shaping, smear layer management is very important. Without the removal of all dentin debris, a totally clean canal system is impossible. Active irrigation is mandatory to this aim. When the root canal system is as clean as possible, a perfect endodontic and coronal seal must be accomplished.
|Fig 9a,b Once the root canal preparation is finished and an ultrasonic instrument is used in a root canal system filled with NaOCI, a cloud of debris will appear. It must be aspirated and the canal filled again with NaOCI until the irrigant stays clear|
Fig. 10 a-c Clifford Ruddle's EndoActivator (patent pending) is a biofilm disruptor
Intensive in vitro analysis of the mandibular molar mesial root fill using Thermafil with computer reconstruction illustrates the ability to isolate where the carriers are located, where the sealer was located and how well the gutta-percha filled the isthmus (Fig. 11). This type of obturation technique can give excellent results, but only when performed in combination with a proper cleaning and shaping regimen. Bacteria can escape from the apex of the tooth, but we have our host defense system to help us. Our immune system has the ability to summon macrophages, neutrophils and PMNs to deal with those bacteria that may be exposed to it.
Fig. 11 Microfocus CT image of a root-filled molar. Thermafil has been used to obturate the root canal system three-dimensionally
Future developments in endodontics can be expected in optimization of three-dimensionally stable sealers. They provide superior flow and seal throughout the root canal system and also provide good adaptation and long-term stability.
Such a candidate for intratubular penetration is EndoRez from Ultradent. This is a UDMA resin filler, using a two-phase automixing tip with an injectable resin filler NaviTip. Its features include:
In an in vitro study of Endo Rez, we used a plastic carrier as a gutta-percha cone substitute to drive the EndoRez sealer into the radicular dentin tubulesof a prepared anterior maxillarycanal. The tooth was dissolved in 30% hydrochloric acid for 36 hours to remove the inorganic component. NaOCl 2.5% was used for 10 minutes to remove the organic component. What remains is the canal obturation with the filling material in place and dentinal resin tags, if they are present.
Photomicrographs showed thousands of spaghetti-like resin tags that had flopped over (they were no longer supported by the surrounding dentin, Fig. 12a). These tags were an astounding 1,000 to 1,500 microns long! They were completely polymerized and continuous. The resin tags perfectly correlated with the size and shape of the tubules, even down to the tubule branching. If there were any bacteria in these tubules, they would certainly have been entombed in the material. If you cannot kill the bacteria, entomb them by radial penetration (Fig. 12b).
|Fig. 12 a, b Entombing bacteria: After Endo Rez sealer was driven into the radicular dentinal tubules of a prepared maxillary canal, photomicrography show thousands of spaghetti-like resin tags flopped over, no longer supported by the surrounding dentin|
The drawback with resin-based systems, however, is the polymerization shrinkage. They shrink approximately 4% to 6%. This can result in severing of the material in the tubules from the core material and gap formation along the periphery. If this gap is re-infected, the case can fail. Therefore more work is needed to reduce the polymerization shrinkage so that this gap is minimized or eliminated.
Although extensive consistent evidence lacks, PAD seems to have high potential for disinfection of root canals. Mandatory to a benefit of adjunctive PAD treatment, all steps of the routine endodontic treatment should be followed carefully. PAD cannot replace shaping, cleaning, smear layer and biofilm management, but could go hand in hand with a perfect routine endodontic treatment to improve outcome. This is consistent with the concept of minimally invasive endodontics.
|Tolonium chloride in the root canal system of a mandibular molar. Note the laser tip (not irradiating) in a canal|
A complete list of references is available from the publisher.
©First published in ROOTS # 1, 2007