Unleashing the Hidden Potential: The Dynamic Duo of Antimicrobial Photodynamic Therapy and Photobiomodulation: A Spectrophotometric Study

Abstract

Background: Despite intensive research, the ideal protocol applied to maximize the overall benefits of antimicrobial photodynamic therapy (aPDT) remains unexplored. Evidence exists that following aPDT, the diffused light beyond the photosensitizer can exert a secondary therapeutic effect known as photobiomodulation (PBM), which stimulates the healing of the surrounding tissues. Therefore, the aim of this study was to examine the attenuation properties of five different photosensitizers activated by their corresponding laser wavelengths. Methods: The illumination of various concentrations of chosen photosensitizers, curcumin, methylene blue, toluidine blue, indocyanine green and a methylene blue derivative, irradiated by their respective laser wavelengths (445 nm, 635 nm, 660 nm and 808 nm) was explored via a spectrophotometric analysis. The onward transmitted light intensities for each combination of a photosensitizer and laser wavelength were assessed. The attenuation percentages observed were statistically evaluated using an analysis-of-variance (ANOVA) model. A Tukey’s post hoc test was performed to determine the significance of differences between individual group mean values. Results: With the exception of toluidine blue illuminated by an 808 nm laser, which showed the lowest intensity loss, all the other photosensitizers presented an attenuation range of 63% to 99%. Conclusions: At appropriate concentrations, all the examined photosensitizers may allow the passage of sufficient wavelength-dependent light transmission. Calculated fluences are proposed to achieve secondary, beneficial PBM effects.

1. Introduction

To date, the inappropriate and often excessive use of antibiotics has resulted in an increase in the prevalence of highly resistant pathogens. This phenomenon is considered a major threat extending to public health worldwide [1,2]. Consequently, recent urgent calls to investigate new strategies for defeating the multi-antibiotic resistant microbial strains have emerged [3].

Photodynamic therapy (PDT) can serve as an effective alternative approach through its ability to inactivate resistant bacteria [4]. It can be described as a non-thermal photochemical reaction, where a non-toxic dye (photosensitizer—PS) is excited by light of an appropriate wavelength [5]. A long-lived triplet state of the PS is then produced; this can interact with molecular oxygen, leading to the generation of reactive oxygen species (ROS), including singlet oxygen (¹O₂), which may provoke cytotoxic effects coupled with the damage of critical biomolecules such as proteins [5]. Each of the three essential components (photosensitizer, light and oxygen) are quite harmless by themselves, but their combination in this manner gives rise to the generation of aggressively reactive and cytotoxic ROS, which exert damaging effects at both molecular and cellular levels [6]. PDT is characterized by a dual selectivity, which is based on (a) differences in bound photosensitizer concentrations between target and normal tissues, and (b) the degree of spatial confinement of the light applied onto the target area [7].

Various terms have been used to describe this treatment, and these include photo-activated chemotherapy (PACT), light-activated disinfection (LAD), photodynamic disinfection (PDD) and photoactivated disinfection (PAD) [8]. For antimicrobial purposes implemented in dental therapy procedures, and to avoid confusion with photodynamic therapy performed on tumours, antimicrobial photodynamic therapy (aPDT) is now regarded as the most suitable term [9].

Although more than a century has passed since the discovery of photodynamic therapy [10], this treatment is still not widely used. The main reason for this may be attributed to the multifactorial mechanisms of photomedicine. The mechanism of action of this modality correlates with several influencing factors, such as the photosensitizer applied, the light delivery system and the oxygenation level of the target tissue. A recent systematic review concluded that “no safe recommendation on aPDT protocols can be extrapolated for clinical use at this point in time” [11]. As such, the ideal protocol applied to maximize the benefits of aPDT remains largely unexplored. It has been suggested that tissue healing following photodynamic treatment is significantly improved. This can be explained by the fact that although the photosensitizer is topically administered to the target tissue, the delivered light scatters and diffuses beyond the area of interest. Consequently, a secondary therapeutic effect known as photobiomodulation (PBM) is exerted, which enhances the healing and repair of the surrounding tissues [12,13].

PBM is considered to be an emerging therapy, with more than 50 years of intensive research supporting its deployment [14]. Indeed, in PBM, cells or tissues are basically exposed to light (lasers or LEDs) of low fluence, specifically within the 400–1000 nm wavelength range [15,16]. The photon energy is transferred to specific chromophores and absorptive tissue molecules, such as cytochrome c oxidase in mitochondria and calcium channels in plasma membranes [17,18]. PBM has been shown to increase adenosine triphosphate (ATP), nitric oxide (NO^●) and reactive oxygen species (ROS) production [19], promote DNA synthesis, accelerate tissue repair and induce stem cell proliferation and differentiation, through downstream effects [20].

As stated by Abrahamse and Hamblin, “light therapy has many characteristics that can accurately be described as a ‘Janus’ effect”. One consideration of this indicates that the combination of light with the correct overall dose of a photosensitizer (PDT) can eliminate microorganisms, cancers and unwanted tissues. However, a second consideration, which represents the “other side of ‘Janus’ face”, is that light alone delivered at a low irradiance and fluence (PBM) can heal and regenerate damaged tissues. Therefore, it can be suggested that photodynamic therapy is the “killer”, and that photobiomodulation serves as the “healer” [21].

Hence, examining the optical properties of the photosensitizer and the target tissue is of the utmost importance in order to achieve a predictable treatment, and this aspect should be borne in mind when determining the photodynamic effects of these treatments [22]. An incident light beam impacting on matter will undergo reflection, refraction, absorption and scattering before exiting it as transmitted light. Both scattering and absorption reduce the light energy, leading to beam attenuation, and therefore the optical penetration depth (distance at which the light intensity reduces to 37% of its initial value) will be affected [22,23].

Knowledge of the light distribution is vital for the calculation of laser parameters applied during aPDT in order to maximize the favourable effects both on the target (photosensitizer) and surrounding tissues. The transmitted light passing through the photosensitizer may lead to beneficial PBM effects. In general, spectrophotometric measurement of the transmitted light through samples is widely considered to be a reliable method commonly performed in different studies [24,25]. To the best of our knowledge, to date, there has not been any study evaluating the light attenuation through various photosensitizers. Hence, in this study, the light intensity losses of five photosensitizers (curcumin, methylene blue, toluidine blue, indocyanine green and a methylene blue derivative) illuminated by their corresponding laser wavelengths (445 nm, 635 nm, 660 nm and 808 nm) were examined, using a high-resolution spectrophotometric analysis. The applied wavelength range (445–808 nm) falls within the aforementioned limits of PBM [15]. Following this analysis, calculated fluences are proposed in order to achieve secondary beneficial PBM effects on the surrounding tissues in clinical practice.

2. Materials and Methods

2.1. Photosensitizers and Laser Sources

For these experiments, the examined photosensitizers and their respective concentrations are based on ones that have been clinically explored [11,26]. The analysed photosensitizers with their respective concentrations and mixing procedures are indicated below:

• Methylene blue (Methylene Blue, Sigma-Aldrich Merck KGaA, Darmstadt, Germany) at 0.05, 0.10 and 10.00 mg/mL (w/v), respectively, in sterile distilled water.
• Toluidine blue gel (ready to use syringe) (BluLase PDT Gel, Schneider-Dental, Pilsach, Germany). Information regarding the concentration present and the preparation is not provided by the manufacturer.
• Indocyanine green (Emundo^®, A.R.C. laser GmbH, Nurnberg, Germany) at 1.00 mg/mL and 0.10 mg/mL (w/v) in sterile distilled water.
• For the higher concentration, a single tablet containing 1.00 mg was dissolved in a 1.00 mL volume of sterile distilled water, according to the manufacturer’s instructions. For the lower concentration, one tablet of 1.00 mg was dissolved in 10.00 mL of sterile distilled water.
• Curcumin (UltraCur+Pro^®, Weber Μedical GmbH, Lauenförde, Germany) at 5.00 mg/mL; according to the manufacturer, this product is water-soluble, which resolves the obstacle of low water solubility [8]. For this purpose, one tablet of 60 mg was dissolved in 12.00 mL of sterile distilled water.
• A methylene blue derivative (ready to use syringe) (Photolase^® Europe Ltd., Hamburg, Germany). Information concerning the concentration present and the preparation of this solution was not provided by the manufacturer.

Regarding the laser devices and parameters, the following systems and parameters were used:

• a 445 nm wavelength device (SiroLaser Blue, Sirona Dental Systems GmbH, Bensheim, Germany), at 150 mW of power output and with an applicator tip of a 6 mm diameter.
• a 635 nm wavelength device (Klas-DX/LX162, Konftec Corporation, New Taipei City, Taiwan), at 150 mW of power output and with an applicator tip of a 6 mm diameter.
• a 660 nm wavelength device (Klas-DX/LX161, Konftec Corporation, New Taipei City, Taiwan), at 150 mW of power output and with an applicator tip of a 6 mm diameter.
• a 808 nm wavelength device (Klas-DX/LX182, Konftec Corporation, New Taipei City, Taiwan), at 150 mW of power output and with an applicator tip of a 6 mm diameter.

The power output of each laser was calibrated before use with a power meter (Thorlabs PM160T-HP Thorlabs GmbH, Bergkirchen, Germany). The specific power of 150 mW was selected for all lasers, since this was the lowest value attainable by the SiroLaser Blue laser device. Moreover, this value is within the range of 100–200 mW, which is usually applied clinically as well as in in vitro investigations [11].

To perform the experiments, the following combinations of photosensitizers and laser wavelengths were implemented; these were based on the absorption maxima of each photosensitizer [27] and the laser wavelengths available:

• Curcumin + 445 nm;
• Methylene blue + 660 nm;
• Toluidine blue + 635 nm;
• Toluidine blue + 808 nm (as declared by the manufacturer) [28];
• Indocyanine green + 808 nm;
• Methylene blue derivative + 660 nm;
• Methylene blue derivative + 808 nm (as declared by the manufacturer) [29].

All the information regarding the photosensitizers and their respective manufacturers, concentrations and laser parameters applied in the experiment are provided in

Table 1. Photosensitizers and their respective manufacturers, concentrations and laser parameters applied in the experiments conducted.

Photosensitizers	Brand Name/Manufacturer	Concentration (mg/mL)	Laser Wavelength (nm)	Power Output (mW)	Tip Diameter (mm)
Curcumin	Ultracur+Pro®, Weber Μedical GmbH, Lauenförde, Germany	5	445	150	6
Methylene blue	Methylene Blue, Sigma-Aldrich Merck KGaA, Darmstadt, Germany	0.05	660	150	6
Methylene blue	Methylene Blue, Sigma-Aldrich Merck KGaA, Darmstadt, Germany	0.10	660	150	6
Methylene blue	Methylene Blue, Sigma-Aldrich Merck KGaA, Darmstadt, Germany	10.00	660	150	6
Methylene blue derivative	Photolase®, Europe Ltd., Hamburg, Germany	unknown	660	150	6
Methylene blue derivative	Photolase® Europe Ltd., Hamburg, Germany	unknown	808	150	6
Toluidine blue	BluLase PDT Gel, Schneider-Dental, Pilsach Germany	unknown	635	150	6
Toluidine blue	BluLase PDT Gel, Schneider-Dental, Pilsach Germany	unknown	808	150	6
Indocyanine green	Emundo®, A.R.C. laser GmbH, Nurnberg, Germany	0.10	808	150	6
Indocyanine green	Emundo®, A.R.C. laser GmbH, Nurnberg, Germany	1.00	808	150	6

.

2.2. Testing Procedure

The experimental procedure was conducted on a vibration-free optical table at a stable room temperature of 18 °C, at the Laser Laboratory of the Institute for Plasma Physics and Lasers of the Hellenic Mediterranean University of Rethymno, Greece. The experimental set-up and the different optical elements used for performing the spectral measurements are indicated in Figure 1.

In order to achieve a uniform area of thickness for each component, a 1 mm layer of thickness quartz glass cuvette was applied (Hellma Macro-cuvette 100-QX, Hellma GmbH, Müllheim, Germany). A new and clear cuvette was used for each photosensitizer in order to ensure its transparency.

All photosensitizers examined were freshly mixed prior to implementation of the analysis. After attenuating the intensity from the laser source (LS), the central part of the output was selected by an iris diaphragm (I), which controlled the intensity, shape and divergence of the beam to interact with the examined sample. The light beam was focused with a biconvex lens (F) of a focal length of f = 15 cm. To avoid undesired scattering, a mask was applied before light reached the sample. As noted above, the sample holder was a specially designed cuvette, which contained the photosensitizer at a spatially uniform thickness of 1 mm, since this is a previously described optimal path length for spectrophotometric studies that allows accurate measurements [30].

After passing through the cuvette, the light pathway was further controlled with another mask. Prior to reaching the sensor fibre, the light intensity was moderated if needed, with different attenuator filters. Finally, the transmitted beam was directed to the optical sensor fibre (QP100-2-VIS-BX, Ocean Optics, Largo, FL, USA), which was connected to a high-resolution spectrophotometer (HR4000CG-UV-NIR, Ocean Optics, Largo, FL, USA). The spectrophotometer monitored absorbance within the wavelength range of 400–1000 nm.

For every measurement made, a reference transmission spectrum was recorded for each laser wavelength without any photosensitizer, i.e., for the cuvette alone. Then, the transmission spectrum of each examined photosensitizer activated by its respective laser wavelength, as explained above, was recorded at an integration time of 15 s. For each group, test and control, 3 measurements were performed. As such, the mean of n = 3 replicate measurements was obtained.

The recorded intensities were stored on a PC for each measurement, visualised through “Oceanview” software (Ocean Optics, Largo, FL, USA) and made available for further analyses of the light intensity modifications.

2.3. Data Collection

Attenuation was measured by evaluating the light intensity losses through each tested photosensitizer activated by its specific laser wavelength value. This measurement is expressed relative to the light intensity observed in the absence of the respective photosensitizer, and the results are expressed as a percentage.

This was computed according to the formula provided in Equation (1), where I = intensity and ps = photosensitizer.

100[(Ips on cuvette) − I(cuvette alone)/I(cuvette alone)]

(1)

Photo-optical data for each photosensitizer were transferred to and further analysed using a Microsoft Excel software module, version 16.71 for a Mac (Microsoft Corporation, Redmond, Washington, DC, USA). The proportional intensity change values were attributed to attenuation of the light that underwent absorption, reflection and scattering through each photosensitizer sample.

2.4. Statistical Analysis

The statistical significance of differences observed between the attenuation percentages was determined with a one-way completely randomized design analysis-of-variance (ANOVA) model. Since percentage data do not conform to a normal distribution, data were arcsine (proportion)-transformed prior to the analysis. Although a test for normality (Shapiro–Wilk test) found that the raw (untransformed) dataset did conform to a normal distribution (p > 0.05), that for heteroscedasticity (i.e., heterogeneous intra-sample variances) using a Levene’s test was very close to statistical significance (p = 0.059), and therefore the applied arcsine (proportion) transformation was fully justified.

A post hoc analysis was conducted using a Tukey’s highest significant difference (hsd) test. A p value of 0.05 was selected as the significance criterion value.

The statistical analysis was performed using the Modelling Data and Describing Data modules of XLSTAT2020 software version 22.4 (Addinsoft, New York, NY, USA; www.xlstat.com, accessed on 7 May 2023).

Since preliminary examinations of the precision (repeatability) of the % light attenuating values of the photosensitizer/wavelength combination treatments revealed that they were very highly reproducible, a prospective power calculation for a repeat sample size (n) per treatment was not conducted. Indeed, typical coefficients of variation for replicate samples in these different groups ranged from as low as 0.01 to only 0.07%. However, a retrospective sample size and power calculation of the complete dataset acquired was performed using the power and size calculator GIGA Calculator tool (Webfocus llc, Sofia, Bulgaria, www.gigacalculator.com, accessed on 19 June 2023) [31] for differences between the mean values of the 10 examined groups for comparison. For the purpose of determining the sample size, various data parameters were considered. These included the smallest possible difference of mean % attenuation observed in our study, specifically in the ICG1 + 808 and PL + 808 groups, which had mean attenuation levels of 89.98% and 88.74%, respectively. The standard deviation was estimated from the fundamental error term of the ANOVA model applied. Additionally, a minimum detectable effect (MDE) of 1.00% attenuation, a Type I error rate (α) of 0.50% and a power (1-β) of 95% were taken into account. Based on these considerations and the extremely high reproducibility of the repeat absorbance measurements made as explained above and according to the abovementioned software used, the primary outcome of the sample size requirement was determined to be only n = 2 per group. Therefore, our selection of n = 3 replicates per group was found to be more than acceptable. The α value selected for this calculation corresponded to a Bonferroni-corrected significance level of 5% (i.e., 5%/10 = 0.50% only).

3. Results

The light transmission values measured for each examined photosensitizer and its respective reference without the product (control reference sample), expressed in arbitrary units, were further analysed using Equation (1) to generate the attenuation percentage of each photosensitizer with the above specified concentrations (mg/mL) applied. The data extracted are presented in Figure 2 and Figure 3.

The methylene blue derivative activated by a 660 nm laser source and methylene blue itself at the highest added concentration of 10 mg/mL (w/v) gave rise to the highest attenuation percentages observed, i.e., 99.10% and 94.50%, respectively. Conversely, the lowest percentage attenuation observed was that with toluidine blue activated by an 808 nm laser.

Regarding methylene blue and indocyanine green, which are the two photosensitizers applied at different concentrations, it is evident that intensity losses are elevated based on increasing its concentration.

In view of the very high level of reproducibility between the replicate attenuation values determined, ANOVA performed on the arcsine-transformed dataset revealed that there were extremely highly significant differences between all photosensitizer/wavelength treatment groups applied (p = 5.73 × 10⁻⁵⁴). Moreover, the post hoc ANOVA analysis using the Tukey’s (hsd) test confirmed that all possible comparisons between these treatment groups were all very highly significant, the largest one observed being that between the PL + 660 nm and the TB + 808 nm combinations. Indeed, with the exception of the PL + 808 vs. MB0.1 + 660 comparison, which had a p value of 2.51 × 10⁻⁴, all these two-sample comparisons had p values of <10⁻⁶. Since the 95% confidence intervals (CIs) for these mean values were very narrow and not visible when displayed in Figure 3, they are listed below in

Table 2. Arcsine-transformed mean values and their 95% CIs for all photosensitizer/laser wavelength treatment combinations featured in this study. Abbreviations: CUR, Curcumin; MB, Methylene blue; PL, Photolase (methylene blue derivative); TB, Toluidine blue; ICG, Indocyanine green.

Group	Mean	Lower Bound (95%)	Upper Bound (95%)
CUR + 445	0.880	0.879	0.881
ICG0.1 + 808	0.683	0.682	0.683
ICG1 + 808	1.119	1.118	1.120
MB0.05 + 660	0.705	0.704	0.706
MB0.1 + 660	1.096	1.095	1.096
MB10 + 660	1.238	1.237	1.239
PL + 660	1.437	1.436	1.438
PL + 808	1.092	1.091	1.092
TB + 635	0.940	0.939	0.941
TB + 808	0.118	0.117	0.119

for reference purposes.

4. Discussion

In the current experimental study, the attenuation properties of five different photosensitizers were evaluated. The laser wavelengths examined were 445 nm, 635 nm, 660 nm and 808 nm. These wavelengths, and their corresponding light sources, were selected since they represent those which are most commonly employed in aPDT dental applications. Except for toluidine blue illuminated by an 808 nm laser showing the lowest intensity loss, all the others gave attenuation ranges of 63% to 99%.

It is evident that the concentration of the photosensitizer clearly affects the results. Higher concentrations of methylene blue and indocyanine green revealed no measurable light intensity after passing through the tested photosensitizer. This can be explained by the two merged laws, specifically the Bouguer–Lambert and Beer’s law. Indeed, the applied formula is provided in Equation (2).

log I₀/I_d = A = ε ∗ c ∗ d

(2)

where I₀ is the initial intensity of the light, I_d is the intensity after travelling the distance d in the medium, A is the absorbance, ε is the extinction coefficient of the medium at a selected wavelength, c is the molar concentration and d is the travelling distance in the medium or light path length [32].

Additionally, higher concentrations (c) of the applied photosensitizer can limit the efficacy of the treatment through “photobleaching”. This phenomenon mainly occurs when the generated singlet oxygen chemically reacts with the photosensitizer, and hence hinders any further photodynamic processes [33,34].

Besides added concentration, another parameter that determines the absorbance value is, of course, the wavelength-dependent molar extinction coefficient (ε) of each photosensitizing agent. These values should be noted, since photosensitizers with higher values of this parameter can dramatically increase absorbance, a phenomenon affecting light penetration and leading to “optical shielding” at their corresponding wavelengths of maximum absorption [23]. This phenomenon refers to light blocking attributable to high superficial absorbance, and hence inhibition of the light from reaching deeper layers of the target tissue involved [35].

Regarding the light path length (d), in this experimental set-up, a 1-mm path-length cuvette was used for all evaluations; this is a previously described optimal path length for spectrophotometric studies to allow accurate measurements [30]. Additionally, this is the approximate thickness of the photosensitizer typically applied in clinical dental procedures [9]. Therefore, this parameter can be considered as a constant in the experiments conducted.

As noted in the Introduction, photodynamic therapy is based on the combination of three elements: the photosensitizer involved, light source and available oxygen level [5]. Therefore, the nature of each photosensitizer, the light pathway into the surrounding tissues of the oral cavity and the presence, concentration and availability of oxygen (for ¹O₂ generation) should be thoroughly examined.

4.1. Photosensitizers

The clinical systemic use of methylene blue in humans has been examined for more than 100 years with outstanding safety records [12]. Indeed, in aPDT dental applications, it is considered the most studied photosensitizer presenting positive treatment outcomes [11]. It is both a cationic and hydrophilic compound, i.e., an amphipathic species, of a low molecular mass. In view of its positive charge, it can bind both to the lipopolysaccharides present at the outer membrane of Gram-negative bacteria and to the teichuronic acid residues of the outer membrane of Gram-positive bacteria [8,11]. Its maximal absorption band (λ_max. value) is located at 660–665 nm [6,27].

Toluidine blue has received clinical approval to be used as an adjunct in dentistry [12]. It is a low-molecular-mass cationic agent, with amphipathic characteristics in view of its hydrophilic portion. As documented above regarding its cationic charge, it can bind both to Gram-positive and Gram-negative bacteria [8,11]. Its peak absorption band is centred at a wavelength of 635 nm [36].

Regarding indocyanine green (ICG), its use for medical applications has been approved by the United States Food and Drug Administration (FDA) [9]. Its absorbance can be critically affected by the dissolving medium, its concentration and binding equilibria to plasma proteins [37,38]. However, its self-organization through van der Waals forces and hydrophobic interactions can lead to the generation of aggregates, which may result in dramatic changes in its optical properties [39]. Its peak absorption spectral band is located at 780–810 nm [37,39], and its respective fluorescence maximum is at 820 nm [39]. Furthermore, ICG presents a special characteristic concerning its mechanism of action, which is predominately based on photothermal (80%) rather than photochemical (20%) phenomena [40]. Specifically, at concentrations below 30 μM (equivalent to 0.023 mg/mL), the treatment outcome will solely depend on ¹O₂ generation, while at higher values (>0.023 mg/mL), both photodynamic and photothermal effects will take place [41].

As for curcumin, it has been shown to exhibit therapeutic properties, including anti-inflammatory, photosensitization, chemopreventative and chemotherapeutic effects. Notably, as a natural product, it represents a biologically safe substance even at high doses of up to 12 g/kg per day. It is activated by blue wavelengths, including a peak absorption within the 420–435 nm range [42,43].

A special reference should be made of the methylene blue derivative (Photolase) photosensitizer. For this agent, the manufacturer declares that a presumably chemical modification to the original authentic molecule can lead to a “long-waved flank”, which results in optical activation at a wavelength of 810 nm [29]. This process may be attributed to the bathochromic shift of methylene blue, where the λ_max value shifts to longer wavelengths, since a decreased energy gap between the ground and excited states of the photosensitizer is observed [44].

4.2. Light Pathway

After passing through the photosensitizer, light will diffuse into the surrounding tissues, which, relative to common clinical applications, are the gingiva, bone and/or dentin. As such, the optical properties of these, which are wavelength-dependent, should be thoroughly examined and addressed. Specifically, in the 350–800 nm wavelength range, water absorption is negligible for all structures. On the contrary, in gingival tissue, absorption bands of oxyhaemoglobin can be observed at 415 nm, 542 nm and 576 nm [45]. From 650 nm to 800 nm, scattering is the predominant interactive outcome. For the dentin and bone, the spectrum is mainly described by the scattering effects of hydroxyapatite and collagen [45,46,47,48].

The above optical effects (absorption and scattering) will determine the light penetration depth of these tissues. This term plays a pivotal role concerning the correct dose required during phototherapy [49]; the wavelengths of our interest are 445 nm, 635 nm, 660 nm and 808 nm. According to Selifonov et al. [45], the penetration depth of human gingiva will range from 1 mm (at 450 nm), 4.6 mm (at 650 nm) and up to 6.9 mm (at 800 nm). As for dentin, these indices will be 8.9 mm (at 450 nm), 18.7 mm (at 650 nm) and 55 mm (at 800 nm). Regarding the bone, what is particularly notable is the knowledge that it represents similar characteristics in terms of its composition to dentin [46,49]; however, it is slightly softer than dentin [49]. Therefore, it can be assumed that these indices will be quite similar.

An undesired effect concerning the light pathway during photodynamic therapy is the presence of competitive absorbers of the photosensitizers. Haemoglobin, which is the main absorber, and other proteins can decrease the effectiveness of the performed therapy [4,34]. These highly absorbing molecules, especially within the visible region, may lead to local deviations from the calculated light distribution. Furthermore, “unevenly distributed” absorbers can give rise to local “cold spots” for the fluence rate [34].

An additional fact that may cause complications regarding the irradiance applied is heterogeneously distributed light scattering, which is expected to occur at the boundaries of different tissues, leading to either “hot” or “cold” spots. Moreover, the effect of scattering can decrease light penetration in such tissues [50].

4.3. Oxygenation

Adequate oxygenation of the target tissue is fundamental for inducing and propagating oxidative damage to microorganisms [51]. Apart from the surrounding environmental conditions, variations in O₂ levels can be attributed to the irradiation parameters employed. Various studies that investigated O₂ diffusion during photodynamic therapies confirmed that oxygen depletion is correlated with the applied irradiance, resulting in reduced treatment efficacies [52,53,54,55,56,57,58,59]. Indeed, not only high fluence rates but also the amount of photosensitizer (via photobleaching) can lead to the consumption of the available O₂ in the first few seconds of irradiation. To overcome this issue, a low fluence and longer treatment times, to ensure that sufficient doses are received, are preferrable [50]. A promising method to enhance reoxygenation of tissues is known as “light fractionation”. Dark intervals of a few seconds administered at intervals of only a few seconds (as an “on–off” procedure) allow tissues to be reoxygenated, and thus contribute to enhancing the immune response and treatment outcomes [60].

4.4. PBM

This spectrophotometric study revealed that all the examined photosensitizers applied at appropriate concentrations can allow light transmission. As a result, in clinical conditions, light will diffuse into the surrounding tissues, leading to possible PBM effects [12,13,61,62].

According to the multi-phasic response in PBM, photobiostimulation is observed within the range of 2–10 J/cm² of the applied fluence, where, specifically at 5 J/cm², the optimal effect can be achieved [14]. Considering each photosensitizer’s intensity loss percentage and the respective calculated transmission percentage, the fluence range of 2–10 J/cm² and the 5 J/cm² value where optimal PBM effects may be observed, a subsequent fluence range can be calculated to deliver beneficial PBM effects in the surrounding tissues when photodynamic therapy is performed.

The following formulas in Equations (3) and (4) are applied for this purpose:

Fluence range calculated = (2–10 J/cm²)/transmission (%)

(3)

Optimal fluence calculated = (5 J/cm²)/transmission (%)

(4)

Information regarding the photosensitizers, laser wavelength applied, respective intensity loss and transmission, fluence range calculated and optimal fluence calculated are provided in

Table 3. Information regarding the photosensitizers, laser wavelength applied, respective intensity loss and transmission, fluence range calculated and optimal fluence calculated. Abbreviations: CUR, Curcumin; MB, Methylene blue; PL, Photolase (methylene blue derivative); TB, Toluidine blue; ICG, Indocyanine green.

Photosensitizers (Concentration in mg/mL)	Laser Wavelength (nm)	Intensity Loss (%)	Transmission (%)	Fluence Range Calculated (J/cm2)	Optimal Fluence Calculated (J/cm2)
Curcumin (5.00)	445	77.06	22.94	8.72–43.59	21.80
MB (0.05)	660	64.80	35.20	5.68–28.41	14.20
MB (0.10)	660	88.92	11.08	18.05–90.25	45.12
MB (10.00)	660	94.50	5.50	36.36–181.81	90.90
PL (unknown)	660	99.10	0.90	222.22–1111.11	555.55
TB (unknown)	635	80.74	19.26	10.38–51.92	25.96
ICG (0.10)	808	63.08	36.92	5.42–27.08	13.54
ICG (1.00)	808	89.98	10.02	19.96–99.80	49.90
PL (unknown)	808	88.74	11.26	17.76–88.81	44.40
TB (unknown)	808	11.79	88.21	2.27–11.34	5.67

.

The most commonly applied fluences for performing PDT, which correspond to photochemical reactions, range from 0.1 to 200 J/cm². Concerning irradiance (fluence rate), this value should be below 300 mW/cm², since detrimental tissue heating may occur beyond this upper limit [63]. Hence, Photolase illuminated by a 660 nm laser, which exhibits this dramatically high attenuation percentage (99.1%), cannot combine beneficial PBM effects along with PDT. It should also be noted that the manufacturer suggests irradiation with an 808 nm laser device [29,64].

An innovating concept that should be borne in mind, when performing PBM, is the “photon fluence”, which can be characterized as the wavelength-adjusted energy density, where the photon energy of each wavelength (in eV) plays a critical role in determining the nature of light–biological tissue interactions. Therefore, to achieve a more accurate light dose, the new formula of photon fluence should be applied, as indicated below [65]:

Photon fluence = irradiance ∗ time ∗ photon energy

(5)

To summarize, in order for the clinician to maximise the positive treatment outcomes prior to performing such therapies, the following considerations should be taken into account:

• The photosensitizer’s characteristics, such as its concentration and wavelength-dependent extinction coefficient.
• The precise nature of the surrounding tissues that will determine the light penetration depth.
• The tissue oxygenation status that will be enhanced by applying low fluences with longer irradiation times, along with some “time-off” intervals.
• The new concept of “photon fluence”, where the individual wavelength-specific photon energy (in eV) is considered.

As mentioned in the Introduction [1,2], the excessive and inappropriate use of antibiotics has resulted in the development of highly resistant microbiota. Consequently, aPDT has emerged as an alternative treatment [4]. Besides its primary antimicrobial effect, aPDT has been observed to have a secondary beneficial impact on the healing of the surrounding tissues in the form of PBM [12,13]. The following approaches are commonly employed alongside or as alternatives to antibiotics in the management of oral health issues. Anti-inflammatory and analgesic drugs help alleviate pain and reduce inflammation in oral conditions [66,67]. Antimicrobial mouthwashes and oral gels are used to target and control oral microbial infections [68,69]. Ozone has antimicrobial properties and is capable of eliminating bacteria, viruses and fungi and its clinical efficacy and safety in different dental procedures are still being evaluated [70]. Acupuncture and homeopathy may be used to provide pain relief and promote overall well-being [71,72]. Platelet-rich fibrin (PRF) and stem cells are being explored for their potential in accelerating wound healing and tissue regeneration in oral and maxillofacial surgery [73,74]. Additionally, some recently introduced compounds, such as probiotics [75], lysates [76] and postbiotics [77], have been demonstrated as having a significant influence on an oral environment, leading to modified clinical and microbiological parameters in patients.

Since this is a laboratory-based experimental study, and the laboratory conditions are totally different than in the oral environment, caution should be exercised before extrapolating results to in vivo patient therapy. As previously mentioned, in natural clinical conditions, several factors can significantly influence the treatment outcome, such as the presence of competitive absorbers of each photosensitizer, the optical properties of the surrounding tissues (such as the gingiva, bone and/or dentin) and the availability of oxygen in the treatment environment. As such, the present findings of this study cannot be reliably replicated in clinical conditions or used to interpret therapy outcomes [78].

Consequently, the energy densities calculated and proposed in this study are only theoretical. The treatment outcome, when applying these fluences, should be further investigated through well-designed in vivo studies, comparing against the treatment alternatives noted above and conventional methods. This research will aim to explore the efficacy of the examined photosensitizers activated by these theoretical fluences in combining the beneficial effects of photobiomodulation (PBM) in the surrounding tissues when antimicrobial photodynamic therapy (aPDT) is performed at a specific target.

5. Conclusions

This study focused on the light intensity losses through five photosensitizers of various concentrations, which were activated by their corresponding laser wavelengths. Within the conditions of our study, and with the exception of toluidine blue illuminated by an 808 nm laser that showed the lowest intensity loss, obtained results revealed that for a path length of 1 mm, all the others gave attenuation ranges of 63 to 99%. Calculated fluences for each photosensitizer, respective to its applied concentration and laser wavelength, have been proposed in order for a clinician to be able to exploit the secondary beneficial PBM effects following aPDT.

Further studies applying the calculated and proposed fluences for each employed photosensitizer should be performed in order to assess the combined favourable effects of photomedicine.