Photodynamic antimicrobial chemotherapy for prevention and treatment of dental caries : a critical review

Introduction Photodynamic antimicrobial chemotherapy studies regarding dental caries have been present more frequently in the literature. However, photodynamic antimicrobial chemotherapy depends on the adjustment of variables such as the type of light source and, photosensitisers target microorganism; this makes it difficult to draw meaningful comparisons. The purpose of this paper was to provide a critical review related to this coadjuvant approach in the prevention and treatment of dental caries. Materials and Methods A database search was made via Medline/PubMed (keywords: photodynamic therapy and dental caries) and 33 articles were found. Results Twelve articles were included after using the filter tool, being excluded reviews and manuscripts reporting works not related to the studied area. Conclusion The manuscripts showed that photodynamic therapy presents optimal results against dental caries, even though better understanding of photodynamic antimicrobial chemotherapy and its components are necessary before the clinical application of this alternative modality in the dental practice. Photodynamic antimicrobial chemotherapy for prevention and treatment of dental caries: a critical review MA Paschoal1, S Duarte2*, L Santos-Pinto1* Introduction The human oral cavity is heavily colonised by a complex, relatively specific and highly interrelated range of microorganisms (as many as 1000 different species have been detected) collectively known as normal oral microflora1. A peculiarity of this environment is that most of the bacteria found here are present in complex aggregates (known as biofilms)2. Microbial biofilms are composed of microorganisms adhered both to each other and to dental surfaces (or interfaces) and embedded in an extracellular polymeric matrix, which includes water and nutrient channels3. A change in a key environmental factor will trigger a shift in the balance of the resident microflora, which will promote the emergence of acidogenic/aciduric bacteria. The constant accumulation of these kind of bacteria change the equilibrium towards dental demineralisation (dental caries lesions)4. The constituents of diet present an important role in the development of dental caries. Sucrose is considered the most cariogenic dietary carbohydrate, because it is fermentable and serves as a substrate for the synthesis of extracellular polysaccharides (EPS) and intracellular polysaccharides in cariogenic dental plaque4. In addition, the presence of EPS (mainly insoluble glucan) promote bacterial adherence to the tooth surface and contribute to the structural integrity of dental biofilms. Yet, there is a clear evidence showing that sucrose exposure and insoluble EPS lead to a more cariogenic biofilm5. Dental caries is among the most significant human chronicle infectious diseases and results in the progressive dissolution of enamel. With the disease progression, it can lead the underlying dentine compromising the vitality of the element and its fixation in the maxillomandibular complex6. Prevention of dental caries can be achieved by controlling the accumulation of dental plaque by mechanical removal7. In cases of insufficient biofilm disorganisation, the association with antimicrobial chemical agents, such as chlorhexidine may help in the decreasing of pathogenic bacteria levels7. Unfortunately, this preventive approach does not reach the population as a whole, allowing dental cavity formation. Treatment of the carious lesion involves the removal of infected dentine with posterior restoration of the affected tooth with any of the variety of materials, for example mercury amalgam, resin composite and glass ionomer cements8. Due to emergence of antibiotic resistant strains, alteration in taste, burning sensation, increase of calculus formation and staining of the teeth and restorative materials stimulated a search for alternative treatments9. Recently, approaches that might offer the possibility of efficient intraoral bacterial count reduction with minimum damage to systemic health (preventive approach) and avoid secondary caries development reducing the chance of material substitution and pulp inflammation as well (curative approach) are necessary. For these circumstances, photodynamic antimicrobial chemotherapy (PACT) offers the possibility of a novel modality to reduce pathogenic bacteria, and consequently, prevent against (new) dental caries lesions10. * Corresponding authors Emails: marcobpaschoal@hotmail.com; lspinto@foar.unesp.br; sd84@nyu.edu 1 Department of Pediatric Dentistry, Araraquara School of Dentistry, Univ Estadual Paulista (UNESP), Araraquara, SP, Brazil 2 Department of Basic Science and Craniofacial Biology, College of Dentistry, New York University (NYU), New York, NY, USA


Introduction
The human oral cavity is heavily colonised by a complex, relatively specific and highly interrelated range of microorganisms (as many as 1000 different species have been detected) collectively known as normal oral microflora 1 .A peculiarity of this environment is that most of the bacteria found here are present in complex aggregates (known as biofilms) 2 .Microbial biofilms are composed of microorganisms adhered both to each other and to dental surfaces (or interfaces) and embedded in an extracellular polymeric matrix, which includes water and nutrient channels 3 .A change in a key environmental factor will trigger a shift in the balance of the resident microflora, which will promote the emergence of acidogenic/aciduric bacteria.The constant accumulation of these kind of bacteria change the equilibrium towards dental demineralisation (dental caries lesions) 4 .
The constituents of diet present an important role in the development of dental caries.Sucrose is considered the most cariogenic dietary carbohydrate, because it is fermentable and serves as a substrate for the synthesis of extracellular polysaccharides (EPS) and intracellular polysaccharides in cariogenic dental plaque 4 .
In addition, the presence of EPS (mainly insoluble glucan) promote bacterial adherence to the tooth surface and contribute to the structural integrity of dental biofilms.Yet, there is a clear evidence showing that sucrose exposure and insoluble EPS lead to a more cariogenic biofilm 5 .
Dental caries is among the most significant human chronicle infectious diseases and results in the progressive dissolution of enamel.With the disease progression, it can lead the underlying dentine compromising the vitality of the element and its fixation in the maxillomandibular complex 6 .
Prevention of dental caries can be achieved by controlling the accumulation of dental plaque by mechanical removal 7 .In cases of insufficient biofilm disorganisation, the association with antimicrobial chemical agents, such as chlorhexidine may help in the decreasing of pathogenic bacteria levels 7 .Unfortunately, this preventive approach does not reach the population as a whole, allowing dental cavity formation.Treatment of the carious lesion involves the removal of infected dentine with posterior restoration of the affected tooth with any of the variety of materials, for example mercury amalgam, resin composite and glass ionomer cements 8 .Due to emergence of antibiotic resistant strains, alteration in taste, burning sensation, increase of calculus formation and staining of the teeth and restorative materials stimulated a search for alternative treatments 9 .
Recently, approaches that might offer the possibility of efficient intraoral bacterial count reduction with minimum damage to systemic health (preventive approach) and avoid secondary caries development reducing the chance of material substitution and pulp inflammation as well (curative approach) are necessary.For these circumstances, photodynamic antimicrobial chemotherapy (PACT) offers the possibility of a novel modality to reduce pathogenic bacteria, and consequently, prevent against (new) dental caries lesions 10 .
PACT is based on the combined use of a photosensitive drug (usually a dye) and an appropriate wavelength of visible light.Once exposed to light, the photosensitisers are activated to a short-lived excited state that then converts to a long-lived triplet state.This state may generate free radicals or superoxide ions resulting from hydrogen or electron transfer (type I) and can produce oxygen singlets or reactive oxygen species (ROS) (type II) (Figure 1).The abundant ROS generated reacts with the surrounding molecules and exerts a bactericidal effect on microorganisms 10 .
Overall, PACT has been extensively investigated for the treatment of several microorganisms, including bacterial oral pathogens, in both in vitro and clinical trials 11 .As a rapid, non-toxic and non-invasive antimicrobial approach, PACT emerges as a suitable process to reduce bacterial contamination, increasing the success of the treatment 12 .Furthermore, another advantage of PACT compared to antibiotics is that bacteria do not develop resistance to oxygen species, since there is no target specificity.As compared to chlorhexidine, PACT does not exert the reported sideeffects 12 .
Thus, based on the information highlighted above, the aim of this present work was to investigate the status of this coadjuvant approach, specifically in the prevention and treatment of dental caries, by revising critically the specific current literature.

Database search
An electronic search (executed on 17 October 2013) was performed in Medline/PubMed database using the following keywords (after consulting and adequacy to the controlled vocabulary descriptors MeSH): photodynamic therapy and dental caries.No limits regarding year of publication or type of study was made, in exception to published language (American/British English) and investigations that offer abstract and full text as well.

Results
The search resulted in 33 studies in which just 12 were included due to matching with the objective of the present investigation.These studies involved in vitro, in situ and in vivo experiments with a diversity of PACT protocols that were published between 2004 and 2012.Figure 2 describes a flux gram used to filter articles resulting in the number cited above.In addition, the main characteristics of these works are described in Table 1.

Discussion
The authors have referenced some of their own studies in this review.The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.
It has been known since the beginning of the last century that microorganisms can be killed by the combination of dyes and light.In 1900, Raab 13 reported the lethal effect of acridine and visible light on Paramecium caudatum and the  Decades of epidemiological, biochemical and animal studies reported that streptococci generally comprise the majority of dental plaque microorganisms and implicated Streptococcus mutans as the principal agent of dental caries 16 .In the included articles in this review, S. mutans was the most studied bacteria, since it is one of the microorganisms involved with the first stages of dental caries 17 .As the lesion progresses to deeper essential involvement of light and oxygen in the process was shortly thereafter demonstrated by von Tappeiner, who inserted the term 'photodynamic' 14 .However, the potential of PACT against microbial diseases was not exploited for several decades, especially for two basic reasons: (i) some well-known pathogens (specially gram-negative and protozoa) are poorly affected by most traditional photosensitising agents (e.g.xanthene, acridine dyes and porphyrins) and; (ii) the discovery of antibiotics raised the belief that microbiologically based diseases would have been gradually reduced to a level that no longer had a serious impact on human health. vitro and In situ-S.mutans Toluidine blue (100 ug/mL) Red LED 55 J/cm 2

Araújo et al. (2012) 30
In vitro-S.mutans and L. acidophilus Curcumin (0.75 and 1.5 g/L) Blue LED 5.7 J/cm 2 dentin, anaerobic species start to thrive and a transition takes place from predominantly facultative grampositive bacteria to strictly anaerobic gram-positive rods and cocci, and gram-negative rods.Thus, other specimens were also evaluated by the authors like Lactobacillus acidophilus, Streptococcus sobrinus and Streptococcus oralis [18][19][20] .
The effectiveness of PACT is based on some aspects: photosensitiser capability of interacting with the bacterial membrane, photosensitiser ability of penetration and action inside the cell, and ROS formation around the bacterial cell by illumination of the PS.In this review, all the selected papers evaluated and achieved optimal results against gram-positive bacteria (Table 1).In particular, gram-positive bacteria present at the outer wall (15-80 nm thick) containing up to 100 peptidoglican layers, which are intimately associated with lipotheicoic and negative charged teichuronic acids.This wall displays a relatively high degree of porosity, since various macromolecules, such as glycopeptides and polysaccharides were found to readily diffuse to the inner plasma membrane 21 .This fact allied to the absence of protective external membrane can explain the good PACT outcomes.
Seven of the 12 articles used photosensitisers of the phenotiazinium group (toluidine blue and methylene blue) [18][19][20][22][23][24][25] . This goup presents key characteristics that can explain their widespread use in PACT: capability of inactivating both gram-positive and gram-negative bacteria, their positively charged molecule, which may bind to the polyphosphates of the outer membrane and rapidly attracted by negatively charged mitochondria and inner organelles, high efficiency in production of ROS species and low costs 25 .
In the same way, anionic photosensitisers were used as well (erythrosine, radachlorin, phthalocyanines, rose bengal and photofrin) in five investigations 22,23,[26][27][28][29] .In these studies, low concentrations were able to photoinactivate planktonic suspensions, biofilm structures and dental plaque scraping of S. mutans with different light sources and exposure times.Costa et al. 26 achieved a log bacterial reduction of 5-6 when using rose bengal and erythrosine, whereas Ishiyama et al. 29 reduced in 1-3 of S. mutans bacterial suspension.Since these compounds are used as plaque disclosing agents and are activated with lights easily found at dental offices, it seems to be more appropriate for PACT application.Just one article used a natural photosensitiser (curcumin) presented an optimal bacterial reduction after proper light illumination exposure 30 .The authors cite easy manipulation, high rate of ROS formation and low costs as advantages of using these kind of photosensitisers 30 .
For efficiency of PACT, the dye must be activated by proper wavelengths from light sources.Thus, the basic requirement for lights is that they match the activation absorption spectrum of the photosensitiser and provide adequate dose of energy that are able to transit to a higher-energy triplet state 27 .
The literature presents three main classes of clinical PACT light sources: lasers, presents three main classes (LED) and halogen lamps.Laser has some advantages, such as monochromaticity and high efficiency (>90%) and high potency as well; however, they do have a high cost and requires a separate unit for each photosensitiser due to the different absorption wavelengths.On the other hand, the main advantages of LED over lasers are their low cost, portability, easy configuration arrays into different irradiation geometries and it demonstrated the same antimicrobial effects on S. mutans biofilm viability as stated by a recent publication 31 .Filtered halogen lamps have the advantage that they can be spectrally filtered to match any photosensitiser; however, they cannot be efficiently coupled into optical fibre bundles or liquid light guides.
The manuscripts included in this review (Table 1) contain publications using lasers and LED devices to activate different photosensitisers.Most of the papers employed red lights to activate phenotizinium dyes (toluidine blue and methylene blue) [18][19][20][22][23][24][25] , since the maximum absorbance of these components occur at 600-660 nm. Otherblue/green photosensitisers were porphyrin (photofrin) 28 and phthalocyanine derivatives 27 , which have the maximum absorbance for red light at 630 nm and 600-700 nm, respectively.These photosensitisers were reported to be successfully activated by compatible lights with their maximum absorbance.Other photosensitisers reported in the articles were red coloured, such as rose bengal 26,29 and erythrosin 26,29 , which absorb lights at 561 and 530 nm, respectively.For rose bengal, the employed light sources emitted wavelengths between 400 and 500 nm.The advantages employing these agents is that both photosensitisers (plaque disclosing agents) and light sources such as halogen lamps and LED at blue wavelength are present in the dental routine and can be used in PACT without requiring acquisition of new equipment 26,29 .
It is worth to highlight that, as another new modality of treatment, toxicity studies and more detailed in situ and animal models need to be used before clinical application; it can explain, in part, the fact that there are only two in vivo investigations 20,27 among the selected articles.
In summary, PACT will probably not replace classic therapy for dental caries.However, the photodynamic approach may improve, accelerate and lower the cost of dental treatment, with several advantages.It may be used in the future as a coadjuvant therapy for dental caries treatment, sterilising the dental surface during treatment.It may also have benefits on endodontic and periodontal treatments.

Figure 1 :
Figure 1: Schematic representation of PACT way of action.Source of light of specific wavelength is absorbed by a proper photosensitizer, which allows a transition form of low short-energy of oxygen to the excited long-excited singlet state.ROS and singlet oxygen formed are able to damage nucleic acid and plasmatic membrane with consequent microorganism death (photodamage or photosensitization effect).

Figure 2 :
Figure 2: Flux gram showing the result of articles search.