Title: Efficacy of photodynamic therapy as an adjuctive in the treatment of dental caries: A systematic review.

Abstract:

Background: Mechanical removal followed by restoration with restorative materials has been considered as a standard treatment for dental caries. Incomplete removal of microorganisms with currently available procedures leads to a skew in research towards adjunctive antimicrobial procedures like antimicrobial photodynamic therapy (aPDT). Conflicting evidences are available in using PDT as antimicrobial therapy and its usage in treating dental caries.  So, in this review the focused question is to find the efficacy of aPDT in treatment of dental caries by reducing number of viable cariogenic bacteria.

Methods:  A systemic search in MEDLINE/PubMed, EMBASE, Scopus, ISI Web of knowledge, and Google-Scholar databases was done and a separate hand search was also conducted by two independent investigators (SP and MD) to address our focused question. Articles published up to July 2016 were considered. Terms used for search strategy were Photodynamic therapy, S. mutans, dental caries, clinical studies, randomized controlled trials, experimental studies, and combination of these terms. Data was extracted from prospective human studies and in-vitro studies without any language restrictions. Reviews, case reports, and letters to editors were excluded.

Results: Initially 104 references obtained with search strategy. Among them, 14 studies (9 experimental, 5 clinical studies) were included in this review based on the focused question. Though evidences from few studies are conflicting, majority of these studies reported that there is a reduction in viable cariogenic bacteria i.e S.Mutans after aPDT. Few studies have reported even reduction of other bacterial species count, Lactobacillus spcs and S. sanguinis,

Conclusion: Based on available evidences, aPDT is effective in reducing viable cariogenic bacterial count particularly S.mutans. Hence, aPDT can be used as an adjunctive treatment to regular restorative therapies. However, the parameters of photosensitizer and light sources should be standardized to apply aPDT as an adjunctive therapy to standard therapies of dental caries.

Introduction:

Dental caries is a disease of the mineralized tissues of teeth, namely enamel, dentin, and cementum, caused by the action of cariogenic bacteria (predominantly Streptococcus [S.] mutans) on fermentable carbohydrates. If left untreated, dental caries can cause demineralization of these mineralized structures and disintegration of their organic matrix(1). Between the years 2011 and 2012, the prevalence of dental caries among children (5 to 19 years old) and adults (20 to 44 years old) in the United States was 17.5% and 27.4%, respectively (2).

The standard treatment of dental caries involves the mechanical removal of the carious lesion followed by restoration of the tooth with a restorative material such as amalgam, composite resins, etc.(1). Studies have shown that cariogenic bacteria may either turn dormant or die when they are isolated from their source of nutrition (e.g. saliva) using a restoration of adequate integrity (3). However, as most of the existing restorative materials are ineffective in achieving long-standing sealing of the dental cavity, it is important to find adjunctive ways of disinfecting the affected dentin before final restoration for getting additive results.(4).

Several studies have shown the use of antimicrobial photodynamic therapy (aPDT) for the treatment of oral inflammatory conditions such as periodontitis, endodontic infections, and peri-implantitis.(5-9) Mechanism of action of aPDT involves the interaction between light and photosensitizer to inactivate cell functions.(10) A photosensitizer, when activated by light, produce reactive oxygen species and radicals which attacks bacterial cell membrane and kills the bacteria.(11) aPDT represents an adjunctive therapy to mechanical therapy in decontamination of oral bacteria, because it is a minimally invasive and nontoxic approach.(12) Results from in-vitro studies have shown that aPDT decreases cariogenic bacteria.(1, 13, 14) A study by Melo et al(15)showed a significant reduction in counts of S. mutansand Lactobacilli in carious lesions when treated by aPDT. Ricatto et al.(16) and Pereira et al.(17) showed a significant decrease in the viability of the planktonic Lactobacillus spp. and S. sanguinis respectively after treatment with aPDT. However, an in-vitro study by Teixeira et al (18) showed aPDT is ineffective in decreasing the viability of cariogenic bacteria.

Therefore, the purpose of the present study was to review the pertinent literature regarding efficacy of aPDT in management of dental caries as an adjunctive treatment by reducing viable count of cariogenic bacteria.

MATERIALS AND METHODS

Focused question

Based on guidelines of systematic review and meta-analysis (PRISMA)(19), a specific question was constructed. The addressed focused question was “Is aPDT effective in the treatment of dental caries by reducing the viability of cariogenic bacteria?”

Eligibility criteria

Eligibility criteria comprised of the following: (1) original studies; (2) experimental studies; (3) clinical studies; (4) intervention: treatment of dental caries using antimicrobial PDT; (5) bacterial profile in vitro after aPDT application. The exclusion criteria included; review papers, case reports, commentaries, interviews, and letters to the editors.

Search strategy

A search without language or time restrictions up to July 2016 was conducted in MEDLINE/PubMed, EMBASE, Scopus, ISI Web of knowledge, and Google-Scholar databases to address our focused question. A systematic approach to literature searching was used to identify the appropriate papers that report the efficacy of aPDT on cariogenic bacteria. Electronic database searches were performed using different combinations of Medical Subject Headings (MeSH) terms and free text words: 1. Photodynamic therapy; 2. S. mutans; 3. Dental caries; 4. Clinical studies; 5. Randomized controlled trials; 6. Experimental studies; and the combinations. Original studies were hand searched to find any articles that could have been missed during the initial search. Two authors (SP and FJ) performed hand search on following journals: Journal of Caries research, Journal of esthetic and restorative dentistry, Archives of oral biology, Journal of Dental Research, Lasers in Medical Science, Journal of Photochemistry and Photobiology, Photo-diagnosis and Photodynamic Therapy, and Journal of Research and Science. Any disagreements regarding study selection were resolved via discussion.

RESULTS:

All studies (1, 13-18, 20-26) included in our current review were performed at either universities or healthcare centers. Nine studies (16, 17, 20-26) were experimental (Table1) and 5 studies (1, 13-15, 18) had a clinical research design (Table 2). All experimental studies (16, 17, 20-26) were performed in an in vitro design.

Characteristics of experimental studies

All the experimental studies (16, 17, 20-26) included in our review were performed on cultured S. mutans isolates. Furthermore, studies by Ricatto et al(16) and Pereira et al(17) included isolates of  Lactobacillus and S. sanguinis respectively. (Table 3)

Laser and photosensitizer related parameters in experimental studies

A variety of light sources were used in the studies (16, 17, 20-26) included in our current review. Four studies (14, 17, 21, 22) used LED lights for aPDT. While, diode LASER(24), white light (26), High Potency Photopolymerizer (23) and halogen light (20) were used in test groups of other experimental studies. Light source with wavelengths ranging between 400 and 690 nm were used. The power output, energy fluency and duration of irradiation range between 30 mW and 3930 mW, 2.18 J cm−2 and 96 J cm−2 and 12 s and 180 s, respectively (Table1). In one study by Lee et al (20) information about wavelengths and power of light source remained unclear.

A variety of photosensitizers were used in experimental studies(16, 17, 20-26).  Two studies used Methylene Blue (MB)(67 to 100micro M), whereas, Toluidine Blue (TBO) (100microM), Erythrosine (20 microM), and Rose Bengal (2 microM) were used in studies by Bevilacqua et al(21), Lee et al(20), Leite et al(23) respectively. Studies by Paschoal et al(25, 26) compared the use of curcumin and TBO, whereas, studies by Costa et al(22) and Pereira et al(17) compared use of Erythrosine and Rose Bengal as photosensitizers in treatment of bacterial biofilms. (Table 1)

Outcomes of experimental studies

All the experimental studies(16, 17, 20-26) included in our current review shows the treatment of biofilms of patient mouth with aPDT has reported a significant decrease in the viability of the S. mutans when compared to control group. Also, studies by Ricatto et al(16) and Pereira et al(17) have showed a significant decrease in the viability of the Lactobacillus spp.and S. sanguinis respectively after treatment with aPDT. (Table 3)

Characteristics of clinical studies

Among the all the clinical studies (1, 13-15, 18) patients with deep dental caries were included in 3 studies(13-15), and other two studies(1, 18) included patients wearing acrylic palatal devices containing slabs of human dentine. The number of individuals ranged between 20 and 45 with age ranging between 3 and 38 years. The number of male and female participants ranged between 6 to 10 and 11 to 14, respectively. The number of teeth included in these studies (13-15) ranged from 10 and 100 teeth. Studies which used intra-oral devices loaded with human dentin slabs were included (1, 18). All the clinical studies have performed aPDT in test groups, however, no treatment was performed in the control groups (1, 13-15, 18). (Table 4)

Laser and photosensitizer related parameters in clinical studies

Three studies (1, 15, 18) used LED as their light source for aPDT, while Halogen light and diode LASER were used in test groups of other two clinical studies(13, 14) respectively. Four studies (13-15, 18)reported parameters of light source with wavelengths ranging between 550 nm and 700 nm. Power output and energy fluency ranging from 40 to 260 mill watts (mW) and 94 to 320 joules per square centimeters (J cm−2) respectively. In a study by Lima et al(1) information about wavelengths and power of light source remained unclear. (Table 2).

TBO and MB were used as photosensitizers in three (1, 15, 18) and two studies (13, 14), respectively. The concentration of TBO ranged between 5 and 100 microM. Three studies reported a pre-irradiation time of 5 minutes with their respective photosensitizer in the dark.(1, 13, 14)  (Table 2).

Outcomes of clinical studies

In four studies,(1, 13-15) treatment of microorganisms with aPDT has reported a significant decrease in the viability of the S. mutans; however, in one study(18), the viability of the S. mutans in both test and control groups were comparable even after aPDT. Interestingly in one study,(1) treatment for 10 min with energy fluency of 94 J cm−2 of LED irradiation had shown a significant effect on bacterial viability, even in the absence of any photosensitizer. Also, three studies (1, 13, 15) have shown significant decrease in the viability of the Lactobacilli after treatment with aPDT. (Table 4)

Discussion

This systematic review is based on the hypothesis that, aPDT is effective in the treatment of dental caries by reducing the viability of cariogenic bacteria like S. mutans. Results from 93% of studies(1, 13-17, 20-26) included in the present systematic review showed that aPDT showed significant reduction of microorganisms causing dental caries, however, only 7% of studies(18) showed an insignificant result in groups treated with or without aPDT. Even though results from the studies(1, 13-18, 20-26) appeared persuasive enough to conclude that aPDT exhibits antibacterial effects (against SM, LB, SS);  we observed a difference in the light source parameters/type and concentration/type of PS used in these studies.(1, 13-18, 20-26) For instance, experimental studies by Costa et al.,(22)Diniz et al.(24), Paschoal et al.(25) and Leite et al.(23) reported aPDT to exhibit antibacterial effects against S. mutans. In these studies (22-25) all the light source parameters, like the type of light (HCU, LED, LASER and WL respectively), the light wavelength (440 nm, 660 nm, 400-700nm, and 440-480nm, respectively), the energy fluency (95 J cm−2, 60 J cm−2, 42 J cm−2 and up to 96 J cm−2 ) and power output (200 mW, 40 mW, 3930mW) were inconsistent. Moreover, the duration of irradiation also varied among the studies (from 12.2 seconds up to 180 sec). Since most of the light source parameters varied between studies included in this review, it is difficult to accurately determine the parameters that would be most effective in treating oral bacteria causing dental caries.  However, two studies,(17, 22) showed consistency in light source parameters used.

In aPDT, the PS used has an ability to produce cytological agents to bring desired biologic effect.(27)   It has been reported that concentration of PS used in aPDT affects the overall antimicrobial efficacy.(28) Among the studies included in current review we have observed a discrepancy in the type of PS used. Approximately 57% studies used either TBO(1, 15, 18, 21) or MB(13, 14, 16, 24)  as PS, approximately 14% studies used either ER(20) or RB(23) and approximately 28% studies used two types, ER and RB(17, 22), CU and TB(25-27) as PS. We have also observed a difference in the concentration of PS used in these studies.(1, 13-18, 20-26). For example, in an experimental study(16), MB was used at a concentration of 100 microM whereas in a study a clinical study(13), the same PS was used at a concentration of 268 microM. However, due to absence of adequate clinical evidence, it is challenging to standardize the concentrations of PS that should be used for aPDT. It is also known that in a clinical setting, the concentration of PS used varies upon the severity of infection. Hence, further randomized controlled clinical trials are warranted to reach a consensus over the precise concentrations of PS used in treatment of dental caries.

Many in vitro studies have shown a great reduction in bacterial viability or even the fully elimination of cariogenic bacteria when organized in in-vitro biofilms (17, 20), bacteria embedded in collagen matrix (4, 29) or suspended in planktonic cultures (21, 22). However, when aPDT is tested under in vivo conditions by using dentin slabs (1,18, and 24) in oral environment and/or on natural teeth, the bacterial reduction is minimized. There might be many factors influencing the aPDT in oral environment. One assumption for this minimized in vitro bacterial reduction is, in carious tissue, the aPDT might be difficult because cariogenic bacteria are protected by a dentinal structure, which may limit the penetration of both the PS and the light(4). Furthermore, the interaction of light with dentinal structure is complex as a result of reflection and refraction at the surface of the intertubular and peritubular dentine.(15) Also, studies have shown that aPDT is more effective in monospecies biofilms when compared to multispecies biofilms.(30-32) Since most of the oral biofilms being contained multispecies microorganisms  the efficacy  of aPDT might not be in same way as shown in  in-vitro studies.

In a clinical setting the traditional treatment for deep dental caries is removal of all infected and affected dentin during caries excavation(33). However, by this approach there is always a chance of unnecessary removal of dentin tissue and an imminent risk of pulpal exposure, particularly in young patients.(34) With the advent of minimally invasive dentistry, the literature encourages conservative removal of carious tissue to promote the preservation affected dentin over the pulpal wall to avoid the pulpal exposure. By using adjunct aPDT immediate bacterial reduction can be obtained by dentin decontamination and this would further increase the chances of remineralization in the affected dentin. To date there is only one randomized clinical trial showing the decrease in cariogenic bacterial load in deep carious lesions treated by aPDT. Hence, based on the limited data available it is not possible to acknowledge the efficacy of aPDT in treatment of dental caries. Therefore, further randomized clinical trials are necessary to support the evidence of application of aPDT in treatment of dental caries.

Conclusions: According to evidences available, aPDT is effective in reducing viable cariogenic bacterial count. Further research is required in standardization of parameters of light and photosensitizer to acquire homogeneity between the studies and to use as an adjunctive antimicrobial treatment along with currently available standard treatments.