Antimicrobial photodynamic therapy: A promise candidate for caries lesions treatment

Accepted Manuscript Title: Antimicrobial Photodynamic Therapy: a promise candidate for caries lesions treatment Author: Ivana M´arcia Alves Diniz Ivay Diniz Horta Cynthia Soares Azevedo Tha´ıs Regina Elmadjian Adriana Bona Matos Maria Regina Lorenzetti Simionato M´arcia Martins Marques PII: DOI: Reference:

S1572-1000(15)00041-1 http://dx.doi.org/doi:10.1016/j.pdpdt.2015.04.006 PDPDT 645

To appear in:

Photodiagnosis and Photodynamic Therapy

Received date: Revised date: Accepted date:

6-2-2015 20-3-2015 13-4-2015

Please cite this article as: Diniz IMA, Horta ID, Azevedo CS, Elmadjian TR, Matos AB, Simionato MRL, Marques MM, Antimicrobial Photodynamic Therapy: a promise candidate for caries lesions treatment, Photodiagnosis and Photodynamic Therapy (2015), http://dx.doi.org/10.1016/j.pdpdt.2015.04.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Antimicrobial photodynamic therapy: an effective and low-risk therapy for caries treatment Márcia

Alves

Diniz1

([email protected]),

([email protected]),

Cynthia

Ivay

Soares

Diniz

Horta2 Azevedo1

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Ivana

([email protected]), Thaís Regina Elmadjian1 ([email protected]),

Department of Restorative Dentistry, School of Dentistry, University of Sao Paulo

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1

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([email protected]), Márcia Martins Marques1*

Av. Prof. Lineu Prestes, 2227

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São Paulo, 05508-000, SP, Brazil 2

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Adriana Bona Matos1 ([email protected]), Maria Regina Lorenzetti Simionato3

School of Engineering, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG,

Av. Antônio Carlos, 6627

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Brazil

3

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Belo Horizonte, 31270-901, MG, Brazil

Department of Microbiology, Institute of Biomedical Sciences (ICB), University of Sao

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Paulo

Av. Prof. Lineu Prestes, 2415

São Paulo, 05508-000, SP, Brazil

*Márcia Martins Marques (corresponding author)

Department of Operative Dentistry, School of Dentistry, University of São Paulo Av. Prof. Lineu Prestes, 2227 São Paulo, 05508-000, SP, Brazil Phone: +55 11 3091-7839 ext. 213 [email protected]

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We assess an aPDT protocol on Streptococcus mutans grown on dentin slabs. We examine the aPDT effect on dental pulp cells having the dentin slabs as barriers.

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aPDT was able to effectively reduce the bacterial load of Streptococcus mutans. No cytotoxicity of aPDT products was observed regardless of the dentin slab

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thickness.

aPDT associating MB and laser may be a promise candidate for caries lesions

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treatment.

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Antimicrobial Photodynamic Therapy: a promise candidate for caries lesions treatment STRUCTURED ABSTRACT

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Background: Antibacterial photodynamic therapy (aPDT) is a promising adjunctive therapy to the treatment of caries lesions, mainly in the minimally invasive approach

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to preserve dental tissue and favor its repair. Here we analyzed both the efficacy of aPDT in reducing the bacterial load in cariogenic biofilms and the indirect effect of

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noxious components produced by aPDT on the viability of dental pulp cells.

Methods: The aPDT protocol was established using 0.025 g/mL methylene blue (MB)

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and 5 min pre-irradiation time. A continuous-wave diode laser (660nm, 0.04cm2 spot

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size, 40mW, 60s, 60J/cm2 and 2.4J) was used in punctual and distance modes to excite the MB. The protocol was first tested against Streptococcus mutans (U159) biofilms

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produced in 96-well microplates, and then evaluated on caries-like affected human

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dentin discs of three thicknesses. The number of colony forming units (CFU) was compared between groups. Discs were then assembled in metallic inserts to produce

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an artificial pulp chamber and allow investigation of the indirect effects of aPDT on dental pulp cells by the 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay. Data were analyzed using Student’s t test or one-way analysis of variance (ANOVA) followed by the Tukey’s test at a significance level of 5%. Results: Bacterial load reduction was observed in biofilms produced both in the microplates (p<0.05) and on the caries-like affected dentin discs (81.01% or mean reduction of log2 1.010±0.1548; p=0.0029). The cell viability of aPDT and control group was similar (p>0.05). Conclusions: aPDT may be considered a promise adjunctive therapy for deep carious lesions.

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Keywords: antimicrobial photodynamic therapy; methylene blue; diode lasers; dentin / pathology; dental caries.

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1. INTRODUCTION According to the concepts of minimally invasive dentistry, caries treatment is

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based on the removal of infected dentin and preservation of affected dentin, which is

capable of remineralization [1]. However, it is clinically difficult to ascertain the

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boundaries between infected and affected dentin. This challenge becomes even

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greater in the case of deep cavities, where the remaining thin layer of dentin is naturally humid and less rigid, owing to its proximity to the pulp chamber. As a result,

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affected dentin is often removed excessively. An alternative to the mechanical removal of microorganisms in the deepest portions of cavities would be the reduction

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or eradication of cariogenic pathogens in situ.

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Antibacterial photodynamic therapy (aPDT) is a good candidate for the treatment of deep dentin caries lesions. It could rapidly reduce the bacterial load of

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viable microorganisms present in dentin, thus favoring dental tissue repair. Moreover, the superficial and softened infected dentin tissue could be removed only partially, thus helping to preserve dental structure. Overall, aPDT may contribute to a more conservative approach for the caries lesions treatment. aPDT is a technique that relies on the use of a low-energy light source and a

dye, also termed photosensitizing agent (PS), for the production of reactive oxygen species (ROS), among which the singlet oxygen (1O2) stands out. The 1O2 is highly

electrophilic and can directly oxidize double bonds in biological molecules and macromolecules, thus playing an important role in cytotoxicity by causing cell damage and death [2].

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aPDT has shown promising results as an antimicrobial agent for cariogenic pathogens [3-12]. In fact, the effectiveness of aPDT in cariogenic biofilms produced in vivo has already been reported [8, 10]; however, there is no information about the

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effect of permeation through dentinal tubules of noxious components produced by aPDT on the dental pulp. Bearing this in mind, the aim of this study was to evaluate

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the efficacy of aPDT in reducing the bacterial load in cariogenic biofilm and to test

2. MATERIALS AND METHODS

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dentin discs with different thicknesses as barriers.

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the indirect effects of the aPDT protocol on cultured human dental pulp cells, using

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The study was approved by the Ethics Committee of the School of Dentistry,

Photosensitizer and antibacterial photodynamic therapy (aPDT)

protocol

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2.1.

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Universidade de São Paulo (CAE 09731313.0.0000.0075).

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The aPDT protocol applied was based on a previous publication [13]. Briefly,

a 0.050 g/mL stock aqueous solution of methylene blue (MB; Chimiolux, Aptivalux, Belo Horizonte, MG, Brazil) was diluted in a phosphate buffered solution (PBS) to obtain a final concentration of 0.025 g/mL (corresponding to approximately 67.5 µM of MB). This photosensitizer (PS) has maximum absorption at 660 nm [14]. The aPDT protocol was established as follows: the MB solution was applied to the cell cultures and after 5 min of pre-irradiation (PI) time (to allow dye uptake by the cells), the MB was removed from all wells. Cell cultures were then washed with PBS and submitted to laser irradiation using a continuous-wave InGaAlP diode laser (Twin Flex II, MM Optics, São Carlos, SP, Brazil). The parameters used were: 660 nm,

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40 mW, 0.04 cm2 spot size, in punctual and distance (1 cm) modes. The energy density used was 60 J/cm2, corresponding to 60 s of exposure time and 2.4 J of total

2.2.

Efficacy of aPDT in biofilms produced in microplates

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energy.

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The efficacy of the proposed aPDT protocol in reducing bacterial load was

tested against a biofilm composed of a Streptococcus mutans strain (UA159), with

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known cariogenicity, grown on microplates according to a method described in

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previous reports [15]. In brief, bacterial cultures were diluted 1:10 in tryptic soy broth (TSB; Difco, Becton Dickinson and Company-Sparks, Detroit, MI, USA) and

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spectrophotometrically adjusted to an OD600 of 0.3 (Biophotometer, Eppendorf, Westbury, NY, USA). Next, bacterial cultures were incubated for 3 h or until reaching

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an OD600 of 0.8. After the incubation period, bacterial cultures were centrifuged

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(5,000 g) for 5 min and resuspended in TSB supplemented with 5% sucrose, until an OD600 of 0.8 was achieved. Aliquots of bacterial cultures were then plated in 96-well

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microtitration plates (150 µl per well) in quadruplicate, and grown in a bacteriological incubator for 24 h at 37ºC until treatment was performed. The wells adjacent to the test wells were maintained empty to avoid overexposure of the cultures when performing laser irradiation. The experimental groups are described in Table 1. Immediately after aPDT treatment, 100 µl of the biofilm and its suspension were transferred to 10 mL of peptonated water to determine the amount of bacteria per milliliter. One hundred microliters of this dilution were then serially diluted in 900 µL of peptonated water until reaching the proportion of 1:10,000. From this final dilution, 25 µl of each sample were inoculated in tryptic soy agar (TSA) plates, in triplicate.

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Plates were incubated in a CO2 atmosphere at 37ºC, and colony-forming units (CFU) were counted after 48 h.

Efficacy of aPDT in biofilms produced on dentin disc surfaces

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2.3.

2.3.1 Dentin discs preparation

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Thirty-six freshly extracted third molars were donated by the Tooth Bank, School of Dentistry, University of São Paulo. The teeth were sectioned at the cement-

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enamel junction using a #3216 diamond bur (KG Sorensen, Cotia, SP, Brazil). The

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crowns were then sectioned transversely in the cervico-occlusal direction (IsometTM Low Speed Saw; Buehler, Lake Bluff, IL, USA) to obtain dentin discs with

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thicknesses of 0.5 (0.53 ± 0.035), 1.0 (1.0 ± 0.071) and 1.5 (1.53 ± 0.065) mm (n = 12 per group), measured with a digital caliper (Digimess, São Paulo, SP, Brazil). Dentin

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discs size had to present 10 (± 1) mm in diameter, in particular to fit in the metallic

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insert utilized for the cytotoxicity assay. The dentin discs were then polished serially with water-cooled abrasive discs (320-, 600-, and 1200-grit Al2O3 papers; Buehler).

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The smear layer was removed by soaking the dentin discs in a solution of EDTA (acid ethylenediaminetetraacetic) 0.5 M, pH 7.2, for 30 s. Next, dentin discs were sterilized with gamma radiation (25 kGy).

2.3.2 Production of caries-like affected dentin In order to simulate a clinical application of aPDT, caries-like lesions were

produced on the surface of the dentin discs according to a method described in a previously published studies [16, 17]. To this end, all dentin discs (n = 36) were bound with steel wire and acrylic resin and sterilized by gamma ray irradiation (25 kGy). For the demineralization challenge, biofilms of S. mutans UA159 were

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developed on the surface of the dentin discs in the presence of 200 mL of TSB, supplemented with 5% sucrose. The production of caries-like affected dentin was carried out for 7 days at 37oC, and the medium was renewed every 24 h by

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transferring the discs to another flask containing fresh medium.

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2.3.3 aPDT treatment efficacy

After performing the aforementioned aPDT protocol, the biofilm was scraped

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from the disc surface using a sterile cell scraper (Corning cell scraper, Sigma-Aldrich,

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St Louis, MO, USA), diluted in peptonated water, and 25 µl aliquots of the appropriate suspensions were inoculated in TSA plates, in triplicate. The cultures

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were incubated at 37oC for 48 h under 10% CO2, after which colony forming units

Cytotoxicity assay

2.4.1 Cell cultures

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2.4.

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were counted to compare the control with the aPDT groups.

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Dental pulp cells were obtained according to Gronthos et al. [18]. Only cells

from the 5th to 8th passages were used. Cells were cultured in DMEM (Dulbeccos’s

modified Eagle medium; Sigma-Aldrich), supplemented with 20% fetal bovine serum (GIBCO/Invitrogen, Grand Island, NY, USA) and 1% penicillin/streptomycin. A metallic insert designed to be used as an artificial pulp chamber (Figure 1) was placed in each of the 24 wells of the microtitration plates. Dental pulp cells were seeded into the artificial pulp chamber (1 x 104 cells/well) on the bottom of the metallic insert (38.48 mm2). Next, dentin discs were assembled in the metallic insert to simulate the dental pulp chamber roof in vitro. The dentin disc assembly was made in direct contact with the culture medium, without disturbing the cell monolayer. The

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treatments were performed after the cultures were incubated for 48 h at 37°C and 5% CO2, in a humidified atmosphere. Cell viability was assessed 48 h after the aPDT

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treatment.

2.4.2 Cell viability analysis (MTT reduction assay)

dimethylthiazol-2-yl)-diphenyltetrazolium

bromide

(MTT)

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Mitochondrial cell activity was assessed 48 h after aPDT, using the 3-(4,5assay

(Invitrogen,

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Molecular Probes, Eugene, OR, USA), according to the manufacturer’s instructions.

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The absorbance was read spectrophotometrically at 562 nm (Biotek II Biochrom Ltd.,

2.5.

Statistical analysis

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Eugendorf, Austria).

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The aPDT experiments performed in the microplates included four

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independent groups (no laser irradiation and no MB; 25 μg/mL MB alone; laser irradiation alone; and laser irradiation and 25 μg/mL MB), in 4 independent trials per

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group (n = 4). The data was transformed to a logarithmic scale and analyzed using one-way analysis of variance (ANOVA) followed by the Tukey’s test for multiple comparisons.

The aPDT experiments performed with caries-like affected dentin discs

included two independent groups (no laser irradiation and no MB; and laser irradiation and 25 μg/mL MB) in independent trials (n = 6). The data was transformed to a logarithmic scale and analyzed using Student’s t test. The aPDT experiments with dental pulp cells were conducted in 24-well microplates, and included six independent groups (no laser irradiation and no MB applied in 0.5, 1.0 and 1.5 mm dentin discs; and laser irradiation and 25 μg/mL MB

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applied in 0.5, 1.0 and 1.5 mm dentin discs). Four independent trials per group (n = 4) were considered. The data was analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons.

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All experiments were performed at least three times. The significance level of

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all comparisons was set at 5%.

3. RESULTS

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Figure 2 illustrates the bacterial load of the biofilms developed in microplates,

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for all of the experimental groups. No bacterial load reduction was observed in the groups treated solely with laser irradiation or solely with MB, in comparison with the

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control group. The aPDT protocol (MB+ / Laser+) was effective in reducing the bacterial load of the S. mutans UA159 biofilm (p < 0.05).

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The bacterial loads of the biofilms produced on the caries-like affected dentin

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discs, for the aPDT and the control groups, are shown in Figure 3. The aPDT protocol was able to significantly reduce the bacterial load on the surfaces of caries-like

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affected dentin discs in 81.01% (Mean reduction of log2 1.010±0.1548; p = 0.0029). The cell viability values for the dental pulp cells in the control group and for

those indirectly subjected to the aPDT protocol, according to the three thicknesses tested, are shown in Figure 4. Cell viability reductions were not observed in the aPDT groups, compared with the controls (p > 0.05), irrespective of dentin disc thickness.

4. DISCUSSION aPDT is a promising therapy for dental caries because it can rapidly reduce the bacterial load from the biofilm developed on dental tissue, thus reducing the excessive removal of affected dentin and favoring dental tissue repair. In this study, an aPDT

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protocol was tested in S. mutans biofilms produced in different substrates. aPDT was effective in reducing the bacterial load of biofilms produced both in microplates and on dentin disc surfaces. Moreover, the noxious species produced as a result of the

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aPDT reaction were unable to negatively affect dental pulp cell viability when carieslike affected dentin barriers were used, irrespective of dentin thickness. Our data

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demonstrates that aPDT is effective in killing cariogenic bacteria without being harmful to dental pulp cells.

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A variety of aPDT protocols have been described in the literature. For

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periodontal or endodontic purposes, aPDT has already proved safe and successful as an adjuvant therapy for eliminating specific pathogens [19-21]. Accordingly, a

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number of clinical trials have been performed to assess aPDT as a means of raising the predictability of periodontal or endodontic treatments [22-27]. However, little is

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known about the clinical suitability of aPDT in regard to caries lesions [8, 10]. aPDT

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could contribute to dental tissue repair by rapid elimination (or at least reduction) of cariogenic microorganisms, especially in deep cavities. It can be particularly

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attractive for patients being treated with ionizing irradiation, having autoimmune diseases or presenting hyposalivation from drug intake. These patients may suffer from caries lesions of sudden onset and rapid progression [28], and aPDT could help ensure low bacterial loads before cavity filling during the phase of oral environment stabilization.

To test the bacteria-killing ability of the aPDT protocol proposed here, its

effects were first assessed in biofilms grown in microplates. As expected, the application of a laser or MB alone had no effect on S. mutans viability, when compared with the control group. aPDT was effective in reducing bacterial loads in the biofilms produced in microplates, which is in accordance with the results of

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previous in vitro studies [6, 9, 29, 30]. Nevertheless, biofilms produced on caries-like affected dentin disc surfaces underwent a mean reduction of 81.01% in their bacterial loads after aPDT. This reduction of microorganisms on the dentin discs was lower

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than that observed in the 24-h biofilm produced in the microplates, which could be expected once biofilms grown on dentin may form more complex 3D structures [31],

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owing to the dissolution of mineralized tissue and damage to the collagen matrix. Moreover, bacteria may have been able to migrate into both the dentinal tubules and

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the exposed collagen matrix of intertubular dentin, after 7 days of cultivation on the

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dentin disc surfaces. Considering that photosensitization occurs predominantly in the outer layers of the biofilm or carious tissue [32], some microorganisms may have

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survived the therapy. Nevertheless, the aim of aPDT is not to sterilize dental tissue, but rather to reduce the bioburden in caries-affected dentin. Thus, the significant

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decrease observed in the amount of bacteria in the biofilms submitted to aPDT

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validates it as a promise adjunctive therapy for dental caries. Species such as Streptococcus mutans and Lactobacillus spp. are reported to

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be dominant in advanced caries [33-35], though other species (e.g. Veillonella, Bifidobacterium,

Propionibacterium,

low-pH

non–S.

mutans

streptococci,

Actinomyces and Atopobium) may also play important roles in caries production [36].

In this study, an S. mutans strain of known cariogenicity was used to produce caries-

like affected dentin discs. Although a single species does not represent the diversity of microorganisms involved in a disease, this monospecies model seems to be suitable to study dentin caries because it produces dentin surface demineralization [37]. Nevertheless, the dentin microbiome is totally different from the single specie setting tested here [36]. As such, the assessment of aPDT in randomized, controlled clinical trials is important to confirm its successful in vitro outcomes.

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In this light, only few studies show evidence of aPDT efficacy in vivo [8, 10]. Guglielmi et al. [8] applied aPDT (0.01% MB solution followed by laser irradiation at 660 nm, energy density of 320 J/cm2, and 100 mW for 90 s) in deep carious lesions.

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The therapy promoted a mean log reduction of 1.38 or 78.07% for S. mutans, 0.93 or 78.0% for Lactobacillus spp., and 0.91 or 76.03% for total viable bacteria. Although

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this study has shown aPDT efficacy in demineralized dentin, the authors did not report its effects on the dental pulp physiology.

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Dentin is considered a porous barrier, in which the tubular channels drive fluid

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movement through its structure [38]. Thus, it could be possible that cytotoxic substances produced as a result of aPDT may reach the dental pulp tissue through

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dentinal tubules. Our previous study showed that aPDT associated with 0.025 g/mL MB applied directly on dental pulp cells caused severe cytotoxicity [13]. In the

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present study, the indirect effect of aPDT on pulp cells was analyzed. In addition, we

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simulated the application of aPDT at the bottom of carious cavities in vivo by applying aPDT on the affected zone of caries-like dentin discs. We showed that,

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irrespective of dentin disc thickness, the indirect effect of aPDT was non-cytotoxic to dental pulp cells. This result encourages the clinical use of aPDT, even in deep carious lesions.

Reactive oxygen species are generated upon photosensitizer irradiation with

light of appropriate wavelength in the presence of oxygen. When the photosensitizer is photoactivated, it undergoes transition from a low-energy level to a higher-energy level called the triplet state [39]. As such, it may transfer its energy to any biomolecule or to molecular oxygen, resulting in the generation of cytotoxic species such as singlet oxygen, and other ROS. The free radicals produced, in turn, promote a

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bactericidal action by damaging of the cellular plasma membrane and/or damaging of the cell DNA [40]. Although the singlet oxygen is highly reactive, it has a short half-life of <0.04

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microseconds and may diffuse to a mean distance of only 100 nm [41]. For this reason, its efficacy is dependent of its close proximity of the target molecule [42]. In

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fact, MB uptake by bacterial cells is high when applied in water-based formulations,

but it is not able to deeply penetrate on dentin substrates [43]. Moreover,

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photosensitizers are more toxic against microbial species than against mammalian

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cells, and their photoactivated toxicity occurs much earlier in prokaryotic than in eukaryotic cells [44]. Finally, the power density of the laser is reduced by 50% when

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a dentin slice of 150-μm is interposed between the light source and the target, meaning its restricted action [45]. Therefore, the aPDT approach for carious lesions,

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even on thin dentin substrates, may be localized.

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Other possible harmful effects of aPDT on dentin have been searched for in the literature, such as a rise in dentin or intrapulpal temperature or a reduction in

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dentin hardness values. So far, it has been observed that the increase in surface temperature after aPDT is slight, and that there is no significant change in dentin surface hardness [37, 46] Although the degree of penetration of dyes and light in dentin is limited, even in a caries-like substrate [37, 44, 47], this does not rule out the possibility of a deeper penetration of substances leached from dyes into dentinal tubule fluid. Bearing this in mind, the results of the current study contribute to the related literature, in that they demonstrate that these putative noxious components derived from aPDT are not toxic to dental pulp cells. In conclusion, aPDT proved to be an effective therapy against S.mutans biofilms produced both in microplates and on dentin disc surfaces, irrespective of disc

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thickness. Moreover, microorganisms can be killed with no harm to dental pulp cells. One limitation of this study was the lack of simulation of the dentinal fluid flow produced by intrapulpal pressure, which may facilitate the penetration of the dye or

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free radicals generated as a result of aPDT. To test this hypothesis, aPDT could be further evaluated in an animal model. Taken together, our data show that aPDT is a

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promise adjunctive procedure for caries lesions treatment and may be considered a

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low-risk therapy for dental pulp cells.

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5. AKNOWLEDGEMENTS

This study was supported by FAPESP (Grants 2012/16552-0, 2013/03864-6) at

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MM´s lab. This financial agency has no involvement in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the

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decision to submit the paper for publication.

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Table 1 Experimental groups MB, Methylene blue; aPDT, Antibacterial Photodynamic Therapy. Treatment MB(-)/Laser(-)

MB

MB(+)/Laser(-)

Laser

MB(-)/Laser(+)

aPDT

MB(+)/Laser(+)

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Control

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Groups

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FIGURE CAPTIONS

Fig.1 Illustration of the artificial pulp chamber. (A) The opened device composed of

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an upper chamber on the top with a coil and a lower chamber. In between, there are two o-rings where the dentin discs are placed. (B, C) and a bottom chamber (D). The

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showing the slice of dentin positioned in between the O-rings.

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artificial pulp chamber closed. (C) Diagram of the device inside a cell culture well

Fig.2 Bacterial load of the biofilms developed in microplates for all of the

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experimental groups. * Significantly smaller than for all other groups (p < 0.05).

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Fig.3 Bacterial load of the biofilms developed on the caries-like affected dentin discs.

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** Significantly different (p = 0.0029)

Fig.4 Amount of viable cells, as assessed by the MTT assay, 48 h after application of

aPDT or otherwise (controls) on dentin discs of different thicknesses as barriers.

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