1.

Introduction

Dental caries remains one of the most prevalent chronic diseases in humans, and it is an important cause of oral pain and dental loss, which lead to school and work absenteeism, affecting individuals’ daily activities, and emotional stability.¹ Dental caries is caused by specific pathogenic microorganisms that metabolize carbohydrates ingested to form acids, deficient oral hygiene habits, and high-sugar diets.²

Growing evidence indicates that one-step incomplete excavation seems suitable to treat deep caries lesions.³ The partial removal of carious tissue and the subsequent restoration of the dental element are sufficient to reduce the caries microbiota.⁴ Protocols for ultraconservative caries removal, such as Carisolv™⁵ and Papacarie™,⁶ have been used as an alternative method for complementing the effects of manual excavation, thus reducing cariogenic microbiota.

Several studies have shown that oral bacteria are susceptible to antimicrobial photodynamic therapy (aPDT).^7,8 aPDT has recently been studied as a coadjuvant therapy against microorganisms of dental caries, which suggests that it might be useful as adjunctive therapy to current deep carious lesion.⁹10.11.12.13.^–14 Therefore, the aim of this study was to perform a systematic review and meta-analysis to evaluate the effect of aPDT as a coadjuvant therapy in reducing microorganisms in deep carious lesions.

2.

Methods

This systematic review was registered in the PROSPERO database (PROSPERO registry number: CRD42015029891) and was conducted following the PRISMA statements.¹⁵

2.2.

Strategy for Identification and Selection of the Studies

A broad search for articles was conducted, and only articles published before March 29, 2016, were considered for review. The selection process is described in Fig. 1. The following databases were used: Pubmed, Web of Science (WOS), Scopus, Cochrane Library, and Lilacs. The gray literature was also consulted trough Opensigle. The MeSH terms “Photochemotherapy” and “Dental caries” were used. MeSH synonyms, related terms, and free terms were included. These keywords were selected from DeCS—Health Sciences Descriptors developed and from the Medical Subject Headings (MeSH) of the US National Library of Medicine. The terms were combined to refine the search results (the combination of these search descriptors is shown in Table 1). The titles and abstracts of the identified articles were independently evaluated by two researchers (POO and LAA) to determine whether they met the inclusion criteria for the review. The electronic search was supplemented by a manual search of the reference list from the included articles.

Fig. 1

Flowchart for process of article search and selection using the preferred reporting items for systematic reviews (PRISMA) framework.

Table 1

Electronic database used and search strategy.

Database	Search strategy
PubMed	#1 (“Photochemotherapy” [MeSH Terms] OR “Photochemotherapy” [Title/Abstract] OR “Photodynamic Therapies” [Title/Abstract] OR “Photodynamic Therapy” [Title/Abstract] OR “TFD” [Title/Abstract])
	#2 (“Dental Caries” [MeSH Terms] OR “Dental Caries” [Title/Abstract] OR “Dental Decay” [Title/Abstract] OR “Carious Dentin” [Title/Abstract] OR “Carious Dentins” [Title/Abstract])
	#1 and #2
Scopus	(TITLE-ABS-KEY (Photochemotherapy) OR TITLE-ABS-KEY (Photodynamic Therapies) OR TITLE-ABS-KEY (Photodynamic Therapy OR TITLE-ABS-KEY (TFD))
	(TITLE-ABS-KEY (Dental Caries) OR TITLE-ABS-KEY (Dental Decay) OR TITLE-ABS-KEY (Carious Dentin) OR TITLE-ABS-KEY (Carious Dentins))
	#1 and #2
WOS	#1 TS=(“Photochemotherapy” OR “Photodynamic Therapies” OR “Photodynamic Therapy” OR “TFD”)
	#2 TS=(“Dental Caries” “OR “Dental Decay” OR “Carious Dentin” OR “Carious Dentins”)
	#1 and #2
Lilacs	(ab:(Photochemotherapy)) OR (ab:(Photodynamic Therapies)) OR (ab:(Photodynamic Therapy)) OR (ab:(TFD)) AND (ab:(Dental Caries)) OR (ab:(Dental Decay)) OR (ab:(Carious Dentin)) OR (ab:(Carious Dentins))
Cochrane Library	#1 (Dental Caries:ti,ab,kw or Dental Decay:ti,ab,kw or Carious Dentin:ti,ab,kw or Carious Dentins:ti,ab,kw)
	#2 (Photochemotherapy:ti,ab,kw or Photodynamic Therapy:ti,ab,kw or Photodynamic Therapies:ti,ab,kw or TFD:ti,ab,kw)
	#1 and #2

3.

Results

3.2.

Quality Assessment of Risk of Bias

Figure 2 demonstrates the evaluation of the inner methodological risk of bias, according to the Cochrane Collaboration’s tool for assessing risk of bias. Melo et al.’s¹³ study presented the best classification. In general, the results and exclusion criteria were well described in all selected papers. The risk of bias was more frequent in allocation, randomization, and sample size calculation.

Fig. 2

Quality assessment of the selected studies (the cochrane collaboration tool for assessing risk of bias). Yes (+)—low risk of bias; No (-)—high risk of bias. The cells shaded in gray present higher the risk of bias.

3.6.

Meta-Analysis

Two articles were not included in the meta-analysis: one article⁹ due the fact that there was missing data, and we did not achieve contact with the authors, and the other¹⁴ only used real-time PCR to analyze the microorganism reduction. The four included studies evaluated the total viable bacteria count.¹⁰11.12.^–13 Three studies^10,12,13 evaluated Lactobacillus spp. counts and two studies^10,13 evaluated mutans streptococci counts. Forest plots are presented in Fig. 2. The studies showed low heterogeneity in the total viable bacteria counts analysis and in the mutans streptococci counts analysis ($I^2=0\%$). The Forest plots (Fig. 3) demonstrated that all of the meta-analyses presented a significant difference before and after aPDT. For the total viable bacteria count analyses, mean difference and 95% confidence interval was 1.32 [1.13, 1.51]; ($p<0.00001$). Mean difference and 95% confidence interval for mutans streptococci counts were 1.63 [1.05, 2.20]; ($p<0.00001$) and were 1.50 [1.22, 1.79]; ($p<0.00001$) for Lactobacillus spp.

Fig. 3

Mean and the standard deviation (Log $10\mathrm{CFU}/\mathrm{ml}$) comparison before and after aPDT regarding established subgroups: (a) total viable bacteria; (b) mutans streptococci; and (c) Lactobacillus spp. counts.

4.

Discussion

aPDT has been gaining attention in research on alternative antimicrobial approaches, and studies have shown that aPDT has such properties.^18,12,19 aPDT is effective in killing oral microorganisms present in dentine caries that are produced in situ and in vitro and may be useful in minimally invasive dentistry.^20,21 Assuming that the in vitro and in situ techniques are effective, we suggest the following question: Is aPDT an effective coadjuvant therapy to reduce microorganisms in deep carious lesions? To answer this question, a systematic review and meta-analysis were carried out.

Systematic reviews and meta-analyses are important tools and are commonly used for scientific evidence of health practices. They are likely to be used with increasing frequency as current initiatives to share clinical trial data gain momentum and may be particularly important in reviewing controversial therapeutic areas.¹⁵ These types of studies also provide possible recommendations for future studies, evaluate the applied research methods, and provide a summary of evidence related to a specific intervention strategy.

Our systematic review and meta-analysis of the included studies clearly supported the hypothesis that aPDT is an effective coadjuvant tool in reducing microorganisms in the treatment of deep carious lesions. A significant reduction in microorganism counts was observed in all analysis, which highlights this method as a useful minimally invasive tool for dental clinicians.

The included studies used different clinical protocols. Longo et al.,¹¹ Araújo et al.,¹² and Steiner-Oliveira et al.¹⁴ did not describe the location point where the irradiation was carried out. Araújo et al.¹² held a 1-min irradiation, divided into two irradiations of 30 s. However, it is not described whether or not irradiation was performed at a single point or two points in the cavity. The same author made an analysis of superficial and deep dentin, but the result when comparing to control was statistically different when the dentin was directly irradiated or not directly irradiated. Thus, it is suggested that new studies should evaluate if the irradiation at more than one point in the cavity results in a greater reduction of microorganisms or, if a single point of irradiation in the cavity is enough. The single irradiation point would be very beneficial, especially for treatment of primary teeth in pediatrics, because the shorter the chair time, the better. Furthermore, three of the six studies did not mention the removal of excess PS. Longo et al.¹¹ and Melo et al.¹³ removed the excess before the irradiation, whereas Steiner-Oliveira et al.¹⁴ carried out the irradiation first, followed by the removal of PS excess.

In some studies, the reported data were confusing. Longo et al.¹¹ used adults and children in their sample, however, they did not report the findings in each age group separately. Araújo et al.¹² used a sample of children aged from 3 to 9-years old, however, it was not clear what type of teeth were studied since the children in this age group can present deciduous or permanent molars. This lack of information in both studies can raise questions such as what kind of teeth is aPDT more effective for, deciduous or permanent? Could the primary dentition respond to the treatment differently than the permanent dentition, and thus have different results? Based on these questions, we recommend future studies to confirm or refute these hypotheses.

The six evaluated studies presented potential risk of bias. Only Melo et al.¹³ followed all checklist recommendations reported by the CONSORT statement (consolidated standards of reporting trials).²² Only three studies supported their findings on sample size calculation.^10,13,14 From the six selected and analyzed studies, four underwent randomization.^9,11,13,14 Regarding the blinding of participants (single blind), Longo et al.,¹¹ Melo et al.,¹³ and Steiner-Oliveira et al.¹⁴ followed this CONSORT recommendation for conducting microbiology analyses using blind counting of codified labeled samples (double blind) ensuring the absence of biased results.

From analyzing the aPDT parameters, we observed that the authors used different parameters, such as PS, light source, and dosimetry. Borges et al.⁹ and Melo et al.,¹³ used red light-emitting diode (LED) as a light source. Steiner-Oliveira et al.¹⁴ also used LED as a light source but only in the group of PS TBO (LEDTB). The light source is selected according to the type of PSs. In terms of costs, LED present advantages when compared with the laser sources. A difference in irradiation time was also used. However, all studies achieved positive results in reducing bacteria even while using different application times. This observation suggests that a protocol with a shorter exposure period would be highly advantageous. In the case of pediatric care, this would be a wonderful advantage as shortened chair time is a priority.

Despite the fact that the different aPDT parameters have been applied in different ways, all of the studies reached a reduction of most of the tested microorganisms, thus confirming the effectiveness of the use of aPDT as an adjunctive in the treatment of deep caries. However, among the observed reductions, some studies showed significant reductions and others did not, which may have been influenced by different parameters of aPDT. Guglielmi et al.¹⁰ and Araújo et al.¹² used MB as a PS in their clinical protocol, and both placed the PS in contact with the teeth for 5 min before irradiation. Although these authors used different light sources, their results were similar.

Borges et al.⁹ and Melo et al.,¹³ in turn, used the same PS (TBO), the same light source, red LED, and the same dose of $94\mathrm{J}/\mathrm{c}{\mathrm{m}}^2$, but they used different output powers. Borges et al.⁹ used the output power of 40 mW, whereas Melo et al.¹³ used 150 mW. Thus, these different parameters used in the aPDT protocol may have influenced the difference in results for the species present in total microorganisms and for Lactobacillus. Both of them achieved reduction, however, only Melo et al.¹³ had a significant reduction.

Several PSs are used to achieve the antimicrobial effect during the application of aPDT, but MB and TBO have been the most commonly tested option in in vivo and in situ studies.^20,2324.^–25 In this context, it is important to analyze the absorption spectrum of the PS and the emission spectra of the light source to have an efficient photodynamic action. Guglielmi et al.¹⁰ and Steiner-Oliveira et al.,¹⁴ in one of their experimental groups, used MB and red low power LASER light source with a wavelength of 660 nm that is coincident with the absorption band of MB (610 to 660 nm).²⁶ Araújo et al.¹² also used MB, however, a halogen curing light unit was used, which emits white light that has an emission spectrum ranging between 500 and 800 nm. Melo et al.¹³ and Borges et al.⁹ used TBO and LED sources that provided the emission spectrum within the characteristic absorption range for this PS (590 to 630 nm).²⁶ Longo et al.,¹¹ in turn, used AlClPc and a red laser of 660 nm, which is coincident with the electromagnetic spectrum, in which the phthalocyanine group absorbs light (660 to 700 nm).²⁶ However, Steiner-Oliveira et al.¹⁴ tested two PS s, TBO with LED and MB with a low-power laser. This study demonstrated that all therapies, including the control group, reduced the number of all tested microorganisms except for S. sobrinus, and no statistical differences were observed among the protocols. In turn, there are no differences between the LED and low-power laser parameters. For these authors, the main goal of aPDT is to perform a conservative treatment of deep caries lesions to reduce the number of microorganisms in the remaining affected dentin to avoid the need for endodontic treatment.

The preirradiation time of 5 min appears to be an important detail to reach the antibacterial effect of aPDT PS in regards to maintaining the inside bacteria, and this time enables greater absorption of light.²⁷ Borges et al.⁹ showed that the use of a laser without a PS, and PS s in the absence of a light source, result in a minimal reduction in the amount of bacteria, without a significant effect on the reduction of microorganisms.

With regard to the volume of dentin analyzed, only Borges et al.⁹ and Melo et al.¹³ provided the dentin weight information. The weighing of each dentin can promote the standardization of the number of CFU/mg of dentin collected, and thus enables better comparisons. Based on this, we recommend that future studies weigh the dentin and evaluate the amount of CFU/mg of dentin that is analyzed.

Of all of the studies that used CFU as their counting method and to which we had access to the data, the study of Araújo et al.¹² stood out for achieving the highest log reduction value.

This systematic review has grouped the data found in the selected studies, describing the parameters of the aPDT, and the species that each of the studies assessed. It was observed that, in the studies included in this review, the vast majority of bacteria investigated were of the Gram-positive genus, which are microorganisms likely to be affected by aPDT. This can be explained on the basis of the link mechanism of cell membrane, in a function of the structural variations in their cell walls. Gram-negative bacteria have an outer membrane complex that includes two lipid bilayers that serve as a physical and functional barrier between the cells and the environment, whereas Gram-positive cells are more sensitive and have a relatively permeable thickness of membrane.²⁸

While analyzing the aPDT parameters used in the selected studies, we observed that different culture media and atmospheres for incubating the microorganisms were used. In relation to the way samples were transported to the laboratory for CFU analyses, Guglielmi et al.¹⁰ used the VMGA III, a transport media, Longo et al.¹¹ selected BHI medium, whereas Araújo et al.¹² performed immediate processing. Borges et al.⁹ and Melo et al.¹³ did not provide this information. All specimens must be promptly transported to the laboratory for CFU counts, preferably within 2 h. If processing is delayed, specimens collected for detection of bacterial agents may be stored under specified conditions.²⁹ Generally, transport media provide a nonnutrient source that sustains the viability of both aerobic and anaerobic organisms without allowing significant growth.³⁰ The BHI medium, used by Longo et al.¹¹ as a transport media, is a nutritive base that is used to cultivate a wide variety of organisms.³¹ This medium can overestimate the number of CFU.

The samples that were analyzed by quantitative real-time PCR were placed in microtubes and stored at $-80°\mathrm{C}$ until used¹² or placed in microtubes containing $100\mu \mathrm{L}$ of TE buffer and stored at $-20°\mathrm{C}$ until used.¹⁴

Although the results of these studies are encouraging, more in vivo studies are necessary to solve doubts regarding parameters such as irradiation time and whether the use of multiple irradiation points improves the use of this therapy in clinical settings.

5.

Conclusion

Our study demonstrated that aPDT is an effective coadjuvant therapy to reduce microorganisms in deep carious lesions.