Photodynamic therapy: a targeted therapy in periodontics

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1 Australian Dental Journal 2009; 54:(1 Suppl): S102 S109 doi: /j x Photodynamic therapy: a targeted therapy in periodontics M Raghavendra,* A Koregol,* S Bhola* *PMNM Dental College and Hospital, Bagalkot, Karnataka, India. ABSTRACT The oral cavity is colonized by a large number and highly diversified communities of micro-organisms. Bacterial biofilm present on tooth or root surface is a major cause of gingivitis and periodontitis. Chemical antimicrobial agents are widely used in prophylactic and therapeutic regimens for dental plaque related diseases, which are among the most common human infections. As these agents are difficult to maintain at therapeutic concentrations in the oral cavity and can be rendered ineffective by resistance development in target organisms, there is a need for an alternative antimicrobial approach. A novel approach, photodynamic therapy (PDT), could be a solution to these problems. Lethal photosensitization of many bacteria, both Gram positive and Gram negative was found in many studies. The advantage of this new approach includes rapid bacterial elimination, minimal chance of resistance development and safety of adjacent host tissue and normal microflora. Thus, the available knowledge of photodynamic therapy should encourage a more clinically oriented application of this technique. Keywords: Antimicrobial chemotherapy, periodontal disease microbiology, peri-implantitis, phototherapy, scaling and root planing. Abbreviations and acronyms: BD = benzoporphyrin derivative; LED = light-emitting diodes; PDT = photodynamic therapy; ROS = reactive oxygen species. INTRODUCTION The accumulation of biofilm on the tooth surfaces or tooth-gingival interfaces results in dental caries or periodontal diseases. Periodontitis is the most prevalent disease in the oral cavity caused by Gram negative obligate anaerobes. The current treatment regimen involves mechanical debridement and this may be augmented with antibiotic therapy. 1,2 Antimicrobial agents further suppress the periodontal pathogens, increasing the benefits of conventional mechanical therapy. However, the emergence of resistant microorganisms and a shift in the microflora after extended use, limits the use of antimicrobials. 3 Photodynamic therapy (PDT) can be defined as eradication of target cells by reactive oxygen species produced by means of a photosensitizing compound and light of an appropriate wavelength. 4 It could provide an alternative for targeting microbes directly at the site of infection, thus overcoming the problems associated with antimicrobials. Photodynamic action describes a process in which light, after being absorbed by dyes, sensitizes organisms for visible light induced cell damage. Allison et al. described PDT as a therapy that is truly the marriage of a drug and a light. 5 Access of the photosensitizer and light to the lesion S102 presents no great difficulty. Application of PDT to periodontal infection could prove to be a valuable adjunct to mechanical procedures, if the photosensitizer has broad spectrum activity against bacterial pathogens and selectivity for prokaryotic cells. Photodynamic therapy At the beginning of the last century, researchers found that microbes became susceptible to visible light mixed with a photosensitising compound. Raab et al. first showed the killing of protozoa Paramecium caudatum in the presence of acridine orange when irradiated with light in the visible range of spectrum. This combination of two non-toxic elements dye and light in an oxygenated environment induces damage and total destruction of micro-organisms. 4 In 1904, Jodlbaner and Von Tappeiner coined the term photodynamic to describe oxygen-dependent chemical reactions induced by photosensitization which could inactivate bacteria. 6 In 1978, Daugherty et al. successfully applied this novel technique for the treatment of different cancers. 7 It was thought that a common feature between tumour cells and micro-organisms was high proliferation and an active metabolism. Since micro-organisms are able to accumulate different photosensitizers, it is believed that ª 2009 Australian Dental Association

2 Photodynamic therapy in periodontics photodynamic inactivation of them might be effective. 8 Due to the limited activity of porphyrin containing photosensitizers towards Gram negative bacteria, special positively charged photosensitizers have been developed which could easily penetrate the bacterial membranes. 9,10 The effect of cyanide photosensitizer on both Gram negative and Gram positive species has also been studied. 11 PDT, as a novel approach in medicine, was first approved by the US Food and Drug Administration (FDA) in 1999 to treat pre-cancerous skin lesions of the face or scalp. Ongoing investigations demonstrated practical usefulness of photosensitization in the broad field of different sciences including virology, microbiology, immunology and dermatology. Mechanism of action PDT involves three components: photosensitizer, light and oxygen. When a photosensitizer is irradiated with light of specific wavelength it undergoes a transition from a low-energy ground state to an excited singlet state. Subsequently, the photosensitizer may decay back to its ground state, with emission of fluorescence, or may undergo a transition to a higher-energy triplet state. The triplet state can react with endogenous oxygen to produce singlet oxygen and other radical species, causing a rapid and selective destruction of the target tissue (Fig 1). This utilization of oxygen in the production of reactive oxygen species is known as photochemical oxygen consumption. The triplet-state photosensitizer reacts with biomolecules by two mechanisms. The Type I reaction involves electron hydrogen transfer directly from the photosensitizer, producing ions or electron hydrogen removal from a substance molecules to form free radicals. These radicals react rapidly with oxygen resulting in the production of highly reactive oxygen species (superoxide, hydroxyl radicals, hydrogen peroxide). The Type II reaction produces electronically excited and singlet oxygen. These two reactions indicate the mechanisms of tissue cell damage which is dependent on both oxygen tension and photosensitizer concentration. 12 PDT produces cytotoxic effects on subcellular organelles and molecules. Its effects are targeted on mitochondria, lysosomes, cell membranes and nuclei of tumor cells. Photosensitizer induces apoptosis in mitochondria and necrosis in lysosomes and cell membranes. 13 Photosensitizers PDT uses several photoactive components. An ideal photosensitizer should be non-toxic and activated upon illumination. In general, the optimal photosensitizer should have a number of photo-physical, chemical and biological characteristics (Table 1). 12 In addition, for treatment of periodontal infections, the photosensitizer should bind with bacteria and plaque without causing any cosmetic issues, such as unwanted staining of gingiva and other soft tissues. Furthermore, it should be acceptable to patients and personnel and easily access pathogens present in deeper periodontal pockets. Types of photosensitizers Chemically, many photosensitizers belong to dyes and porphyrin-chlorine groups. A variety of photosensitizers 14 include: (1) Dyes: tricyclic dyes with different meso-atoms methylene blue, toludine blue O and acridine orange; and phthalocyanines aluminum disulphonated phthalocyanine and cationic Zn(II)- phthalocyanine; (2) Chlorines: chlorine e6, stannous (IV) chlorine e6, chlorine e6-2.5 N-methyl-d-glucamine (BLC1010), polylysine and polyethyleneimine conjugates of chlorine e6; (3) Porphyrines: haematoporphyrin HCl, photofrin and 5-aminolevulinic acid (ALA), benzoporphyrin derivative (BPD); (4) Xanthenes: erythrosine; and (5) Monoterpene: azulene. Light sources A laser or visible light source is used to activate the photosensitizer. Early laser systems were complex and expensive. Subsequently, diode laser systems that were easy to handle, portable and cost-effective were Table 1. Optimal properties of a photosensitizer Fig 1. Mechanism of action of PDT. Photosensitizer (PS) upon irradiation with light at appropriate wavelength undergoes transition into singlet and triplet state. It reacts with endogenous oxygen to form reactive species and highly reactive species causing cell death. 1 Highly selective 2 Low toxicity and fast elimination from skin and epithelium 3 Absorption peaks in the low-loss transmission window of biological tissues 4 Optimum ratio of the fluorescence quantum yield to the interconversion quantum yield 5 High quantum yield of singlet oxygen production in vivo 6 High solubility in water, injection solutions and blood substitutes 7 Storage and application light stability ª 2009 Australian Dental Association S103

3 M Raghavendra et al. developed. More recently, non-laser light sources are used, such as light-emitting diodes (LED) that are less expensive, small, lightweight and highly flexible. Photosensitizers can also be activated by low power visible light at a specific wavelength. Human tissues transmit red light efficiently at wavelengths of 630 nm and 700 nm and these correspond to light penetration depths from 5 mm to 15 mm respectively. 15,16 The use of a visible light source is beneficial in visualizing the target area, localization of the photoinactivation without damaging host tissue and presenting little damage to the operator. 17 Activation of the photosensitizer is dependent on the total light dose, the dose rates, the depth of light penetration and the localization of target area. Sources of light delivery vary depending on the location and morphology of the lesion. The light should be uniform and should deliver precise calculation of the delivered dose. Fibre-optic catheters with terminal cylindrical diffusers or lenses are often used. The tip of the fibre can be formed into various shapes allowing for diffusion in all directions or for focus. Currently, the use of fibre optics is very expensive and not FDA approved. Only diffusing fibres (1 5 cm) are commercially available. 18 Modern fibre-optic systems and different types of endoscopes can deliver light more accurately to the target lesion. Custom-sized and custom-shaped fibres are needed to achieve more homogenous illumination. 19,20 Overall, the light must penetrate as far as possible into the tissues and not produce thermal effects. Photodynamic therapy and periodontitis Biofilm in oral cavity causes two of the most common diseases, dental caries and periodontal diseases. An effective approach of periodontal therapy is to change the local environment to suppress the growth of periodontal pathogens. Micro-organisms in gelatinous matrix (glycocalyx) are less accessible to antibiotics. Using antimicrobial agents to treat periodontitis without disruption of the biofilm ultimately results in treatment failures. It is difficult to maintain therapeutic concentrations at the target sites and target organisms can develop resistance to drugs. This resistance is minimized by using PDT. Polysaccharides present in extracellular matrix of oral biofilm are highly sensitive to singlet oxygen and susceptible to photodamage. Breaking the biofilm may inhibit plasmid exchange involved in transfer of antibiotic resistance and disrupt colonization. 21 PDT is even effective against antibioticresistant bacteria. Antioxidant enzymes produced by bacteria may protect against some oxygen radicals, but not against singlet oxygen. 22 Photodynamic antimicrobial chemotherapy could be an ideal complement to conventional scaling and root S104 planing. It employs a quick and simple protocol that allows the clinician to kill bacteria, inactivate virulence factors left behind after scaling and root planing. It is used during initial and maintenance therapy for the treatment of periodontitis. The activity of PDT against periodontopathic bacteria has been reported in vitro and in vivo for a range of photosensitizers (Tables 2 and 3) During inflammation there is venous stagnation and reduced oxygen consumption by tissues. This decrease in oxygen level and change in ph may enhance the growth of anaerobic species. In such cases, PDT may improve tissue blood flow in the microcirculatory system and reduce venous congestion in gingival tissues. 40 Furthermore, PDT may increase oxygenation of gingival tissues by per cent. This in turn decreases the time and speed of oxygen delivery and utilization, thus normalizing oxygen metabolism in periodontal tissues. 40 The susceptibility to destruction of bacteria by PDT is different between Gram positive and Gram negative species. Gram positive bacteria are more susceptible to photoinactivation than Gram negative bacteria. The structural variations in their cytoplasmic membrane are responsible for the enhanced susceptibility of Gram positive bacteria to binding to photosensitizers. In Gram positive bacteria, the relatively porous outer cytoplasmic membrane, peptidoglycans, and lipoteichoic acid outside the cytoplasmic layer allow the neutral or anionic photosensitizer to bind efficiently to diffuse into sensitive sites. In Gram negative bacteria, the structure of the outer membrane is more complex, forming a physical and functional barrier between the cell and its environment, thereby making it difficult for the photosensitizer to gain access into internal target sites. 41 However, this diffusion may be enhanced by: (1) linking the sensitizer to a polycationic molecule (poly-l-lysine-chlorine, polymyxin B nonapeptide). These weaken the intermolecular interactions of the lipopolysaccharide constituents, disorganize the structure, and render it permeable to drugs by enabling them to cross the outer membrane; 42 (2) use of membrane active agents (treatment with tris-edta), which release lipopolysaccharide or the induction of competence with sensitized pathogen; 43 and (3) conjugating the sensitizer to monoclonal antibodies that bind to cell-surfacespecific antigens. 44 The selective uptake of photosensitizers by bacteria can be enhanced by conjugation with various peptides. For example, Poly-L-lysine (pl) chlorine e6 conjugates kill P. gingivalis without affecting the viability of epithelial cells. The polycationic lysine polypeptide is responsible for the initial binding of the photosensitizer to bacteria due its structural similarity to antimicrobial peptides causing cell lysis. 32 Linking the toludine blue O to a monoclonal ª 2009 Australian Dental Association

4 Photodynamic therapy in periodontics Table 2. Photodynamic therapy on plaque biofilm in in vitro studies Photosensitizer Light (nm*) laser source Periodontal pathogens Toludine blue O Methylene blue Aluminum disulphonated phthalocyanine 633 nm Helium Neon Streptococcus sanguinis Porphyromonas gingivalis Fusobacterium nucleatum Actinomyces actinomycetemcomitans Hematoporphyrin HCl LED Streptococcus sanguinis Aluminum disulphonated phthalocyanine 24 Chlorine e6-pentalysin conjugate 25 Toluidine blue 26 Red light (662) A. actinomycetemcomitans Fusobacterium. nucleatum, Porphyromonas gingivalis, Campylobacter rectus, Eikenella corrodens Streptococcus sanguis Toludine blue O (25 um) 27 Red light (4.4 J) Porphyromonas gingivalis Porphycene Polylysine Conjugates (10 lm**) 28 Visible light Prevotella intermedia, Fusobacterium nucleatum, Peptostreptococcus micros Actinobacillus actinomycetemcomitans Methylene blue nm Diode laser A. actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus sanguis Helium Neon red-filtered Porphyromonas gingivalis Xenon lamp 5-aminolevulinic acid LED Pseudomonas aeruginosa Poly-L-lysine chlorin e6 conjugates 32 Red light diode (671) Actinomyces viscosus Porphyromonas gingivalis Chlorine e6, BLC 1010, BLC Diode (662) Fusobacterium nucleatum, Porphyromonas gingivalis, Capnocytophaga gingivalis Endogenous porphyrins 34 Blue light (380 to 520) Prevotella intermedia, P. nigrescens, P. melaninogenica, P. gingivalis Toludine blue O (1 mg ml) 35 Diode laser (635), 12 J cm 2 Leptotrichia buccalis, Vignal s bacillus, Fusobacterium, Actinomycetes, Chain coccus Streptococcus, Veillonella, etc. Toluidine blue O (12.5 lg ml) 30 Light-emitting diodes. *nm = nanometer. ** = micromolar concentration. J = Joules. Table 3. Photodynamic therapy on plaque biofilms in vivo studies Photosensitizer Light (nm*) laser source Periodontal pathogens Toludine blue O (50 lg ml) 36 nucleatum He-Ne (632), 30 seconds Porphyromonas gingivalis, Fusobacterium Toludine blue O LED Porphyromonas gingivalis in rats Chlorine e6 BLC LED Porphyromonas gingivalis, Fusobacterium nucleatum. Phenothiazine chloride (10 mg ml) nm & 60 mw cm 2 SRP vs. PDT, Non-significant results *nm = nanometer. Light-emitting diodes. SRP = scaling and root planing. PDT = photodynamic therapy. antibody has been shown to inactivate the lipopolysaccharide of P. gingivalis. 44 Hence, conjugated photosensitizers are beneficial in targeting bacteria or particular virulence factors without damaging epithelial cells. The roles of virulence factors in pathogenesis of periodontal diseases are well documented. Lipopolysaccharide possess a wide spectrum of immunological and endotoxin activities. It can cause activation of macrophages, production of interleukin-1, release of prostaglandin E 2, the local Schwartzman reaction and is a potent stimulator of bone resorption in inflammatory periodontal diseases. Endotoxin on root surface inhibits fibre reattachment on cementum. PDT has another advantage in inactivating virulence factors secreted by micro-organisms. Following exposure of P. gingivalis to low-energy He-Ne laser (632 nm) and TBO (25 um ml), the activity of lipopolysaccharide and IL-1 secretion from human peripheral mononuclear cells exposed to such treatment were significantly reduced. 45 In addition, there was a substantial, lightdose dependent decrease in the proteolytic activity (94 per cent) of P. gingivalis. 46 Such effects may be of benefit in the treatment of infections due to these organisms. There are two basic mechanisms that have been proposed to account for the lethal damage caused to bacteria by PDT: (1) DNA damage and (2) damage to the cytoplasmic membrane, allowing leakage of cellular contents or inactivation of membrane transport systems and enzymes. 41 Breaks in both single- and doublestranded DNA, and the disappearance of the plasmid ª 2009 Australian Dental Association S105

5 M Raghavendra et al. supercoiled fraction have been detected in both Gram positive and Gram negative species after PDT with a wide range of photosensitizer structural types. Although DNA damage occurs, it may not be the prime cause of bacterial cell death. The alteration of cytoplasmic membrane proteins, disturbance of cell-wall synthesis and the appearance of a multi-lamellar structure near the septum of dividing cells, along with loss of potassium ions from the cells may be other possible ways of bacterial death. 47,48 It has been hypothesized that photosensitizers that operate chiefly via Type I mechanisms penetrate the outer membrane of Gram negative bacteria, while the Type II photosensitizers penetrate the outer membrane of Gram positive bacteria more efficiently. 41 The bactericidal activity of PDT depends on various factors. The surface charge of the photosensitizer determines its binding with the cell membrane. The electrostatic interaction between the positively charged surface of photosensitizer and the negatively charged membrane of the bacteria can affect bacterial killing. In an in vitro experiment, the polycationic conjugation of c e6 molecules to pl (5 lm concentration) on P. gingivalis produced 99% killing and on A. viscosus >99.99% after one minute of incubation after exposure to red light for 10 minutes in a concentration-dependent manner. The polycationic charged polypeptide, lysine, is probably responsible for the initial binding to bacteria. 32 But even non-cationic compounds photosensitizers, such as porphycene polylysine conjugates, are used in the inactivation of both Gram positive and Gram negative bacteria provided they are bound to a polylysine moiety. 28,49 The environmental conditions surrounding bacteria may influence the efficient binding of photosensitizers. In vitro studies have shown that blood agar culture media, hemin content and the ph of the medium used may inhibit the binding of photosensitizer with pathogens. Blood contained in the culture media adsorbs the part of laser light, hemin competes with the photosensitizer binding sites and bacterial metabolic by-products alters the ph of the medium. All of these alter the binding of the photosensitizers to the target sites, resulting in less binding and reduced photoinactivation. 10,33,50 However, the black-pigmented species, such as P. gingivalis, Prevotella intermedia and Prevotella nigrescens, are more susceptible to elimination by lethal photosensitization. Intracellularly, they accumulate various amounts of different porphyrin molecules (P. intermedia 267 ng mg, P. nigrescens 47 ng mg, P. melaninogenica 41 ng mg and P. gingivalis 2.2 ng mg), together with varying amounts of iron-free protoporphyrin IX. These photosensitive porphyrins absorb visible light at different wavelength and different energy level and enhance the killing effect. 51 S106 Various in vitro studies have shown that periodontal micro-organisms are killed more than 4 5 times at micromolar concentration after incubation times as short as 5 10 minutes and irradiation under mild experimental conditions, such as fluence rates around 50 mw cm 2 and irradiation times shorter than 15 minutes. 52 Whether PDT can be clinically implemented as a successful anti-infectious procedure in periodontal diseases is not clear because of a lack of controlled clinical studies. However, the clinical applicability of PDT in treatment of periodontitis has been tested in the non-surgical management of aggressive periodontitis. PDT and non-surgical periodontal treatment show similar clinical outcomes at three months evaluation 39 without difference in crevicular TNF-a and RANKL concentrations at 30 days interval. 53 In another study, only bleeding on probing was significantly decreased when compared to other parameters at six months interval. 54 However, it has also been reported that PDT adjunctive to non-surgical periodontal treatment enhances the clinical outcomes. 55 In this threemonth split-mouth study, gingival bleeding, probing depth, gingival recession, attachment level and gingival crevicular fluid flow rates were significantly decreased showing higher impact of PDT on the treated sites. 55 Moreover, a reduction in gingival recession compared to non-surgical periodontal therapy in aggressive periodontitis patients has been reported. 39 From the above, it is clear that further clinical trials are required for definitive assessment of PDT in the field of periodontal therapy. Effect on mucosal tissues The epithelium at the dentogingival area acts as a primary barrier for invasion of noxious stimuli. It may also act as a barrier to the penetration of photosensitizers. The thick layer of stratified keratinized gingival epithelium acts as a barrier for diffusion of water-soluble photosensitizers. Sulcular epithelium shows increased penetration due to its non-keratinization. Bacterial penetration into epithelial cells and connective tissue is very important in periodontitis. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans can infiltrate through the epithelial barrier into the periodontal tissues; elimination of these is therefore possible by PDT. Uptake by epithelial cells depends on incubation time; after application of photosensitizer till its activation by light. Most of the studies used relatively short incubation time. This reduces the total uptake of the cells and binding at the plasma membrane rather than internalized. Thus, reactive oxygen species generated at the surface are very much less likely to be able to diffuse to a more sensitive intracellular location than those in the case of bacteria. This may be the reason why these cells are unharmed. 32 ª 2009 Australian Dental Association

6 Photodynamic therapy in periodontics Table 4. Advantages of PDT in periodontal treatment Development of resistance to the PDT is less as singlet oxygen and other free reactive oxygen species interact with several cell structures and different metabolic pathway. As PDT is non-invasive local therapy, following application of a sensitizer, a light source delivered into the target area precisely via a fibre optic cable, so disturbances of the microflora at other sites would not occur and damage to the adjacent host tissues can be avoided. PDT offers thorough irrigation and elimination of pathogens in inaccessible areas of periodontal pocket within short span of time, thus beneficial to both operator and the patient. The risk of bacteraemia after periodontal debridement can be minimized. There is no need to prescribe antibiotics, therefore the possibilty of side effects is avoided. There is no need to anaesthetize the area and destruction of bacteria is achieved in a very short period (<60 seconds). The possibility of adverse effects on host tissues has often been raised as a possible disadvantage of the use of PDT for the treatment of infectious diseases. PDT for periodontal treatment in vivo would require a therapeutic regimen where bacteria are killed without damaging adjacent tissue. In vitro and in vivo animal models suggest that this may not be a problem, since the photosensitizer concentrations and light energy doses necessary to kill the infecting organism have little effect on adjacent host tissues. 56 In vivo animal studies using toludine blue dye reported no adverse changes on epithelium and underlying connective tissue. 37 Oral fibroblasts were unaffected (in vitro). 26 A combination of methylene blue (100 microg m L) and visible light (42 mw cm) on cutaneous keratinocytes showed cells were killed times more slowly. 57 Some studies have reported adverse effects on oral tissues and salivary glands. BPD, a hydrophobic chlorine-like porphyrin derivative, is very phototoxic on oral epithelial cells (HCPC-1) in vitro due to its penetration into the cellular membrane quickly and localize in an intracellular site that is very sensitive to photodamage. 32 Gingival ulceration, muscle necrosis and necrotizing sialometaplasia of salivary glands were observed in rabbits following systemic administration of disulfonated phthalocynanine (5 mg kg and 20 J at 675 nm). 58 Haematoporphyrin derivative resulted in vesicle formation on tongue with oedema, cellular infiltration and reduction in number of vessels but muscle fibres remained intact. 59 Clinically, it is important to ensure that the effect of sensitizers on epithelial cells and connective tissues is minimal. The depth of penetration into epithelium is dependent on the type of photosensitizer and incubation time. Presently, the effects of photosensitizer are not supportive in the treatment of periodontal diseases. Only few photosensitizers were shown to have no adverse effects on oral and gingival tissues. It is recommended that more studies using topically applied photosensitizer are required to be used in clinical practice. CONCLUSIONS This new strategy of using PDT is less traumatic and quicker in the treatment of periodontal diseases but it is still in the experimental stage of development and testing. Development of new photosensitizers, more efficient light delivery systems and further clinical studies are required to establish the optimum treatment parameters for PDT. Photodynamic therapy in vitro studies have shown greater (>95%) reduction in micro-organisms. Clinical trials are also encouraging. In addition to reducing clinical parameters in periimplantitis cases, there is some evidence that PDT will also inactivate virulence factors of periodontal pathogens, enhancing post-treatment outcomes. 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8 Photodynamic therapy in periodontics 54. Chondros P, Nikolidakis D, Christodoulides N, Rössler R, Gutknecht N, Sculean A. Photodynamic therapy as adjunct to non-surgical periodontal treatment in patients on periodontal maintenance: a randomized controlled clinical trial. Lasers Med Sci 2009;24: Braun A, Dehn C, Krause F, Jepsen S. Short-term clinical effects of adjunctive antimicrobial photodynamic therapy in periodontal treatment: a randomized clinical trial. J Clin Periodontol 2008; 35: Kömerik N, Curnow A, MacRobert AJ, Hopper C, Speight PM, Wilson M. Fluorescence biodistribution and photosensitizing activity of toluidine blue O on rat buccal mucosa. Lasers Med Sci 2002;17: Zeina B, Greenman J, Corry D, Purcell WM. Cytotoxic effects of antimicrobial photodynamic therapy on keratinocytes in vitro. Br J Dermatol 2002;146: Meyer M, Speight P, Bown SG. A study of the effects of photodynamic therapy on the tissues of the rabbit jaw. Br J Cancer 1991;64: Pe MB, Sano K, Inokuchi T. Effects of photodynamic therapy in the normal mouse tongue. J Oral Maxillofac Surg 1993;51: Address for correspondence: Dr M Raghavendra PMNM Dental College and Hospital Bagalkot, Karnataka India dr_raghum2000@yahoo.com ª 2009 Australian Dental Association S109