Original Research Article | Open Access

¹Department of Periodontics, Army Postgraduate Dental School, Postgraduate Dental College, Uniformed Services University, Fort Eisenhower, GA, USA
²Department of Clinical Investigation, Dwight David Eisenhower Army Medical Center, Fort Eisenhower, GA, USA

*Corresponding author: Thomas M Johnson, Department of Periodontics, Army Postgraduate Dental School, Postgraduate Dental College, Uniformed Services University, Fort Eisenhower, GA, USA

Abstract

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Objective: Biological effects of infrared laser energy at various exposure parameters have been characterized in previous in vitro and animal studies. However, the impact of pulse repetition rate (PRR) has not been evaluated in this context. The purpose of this investigation was to assess the influence of PRR on cytokine secretion from peripheral blood mononuclear cells (PBMCs) subjected to pulsed neodymium-doped yttrium aluminum garnet (Nd:YAG) laser energy.
Materials and Methods: Rat PBMCs were cultured in vitro then stimulated using a lipopolysaccharide concentration of 0 or 100 ng/ml. Cultures received Nd:YAG laser radiation (1064 nm, 5 W, 30 s) at PRR of 0 (untreated controls), 20, 30, 40, or 60 Hz. Concentrations of tumor necrosis factor-α (TNF-α), macrophage inflammatory protein (MIP)-1α, macrophage inflammatory protein (MIP)-2, monocyte chemoattractant protein-1 (MCP-1), interferon-gamma-induced protein (IP)-10, interleukin (IL)-6, and IL-10 were recorded using a magnetic microsphere immunoassay. The main effects of PRR and LPS stimulation on cytokine concentrations, and the interaction between PRR and LPS stimulation, were assessed using two-way analysis of variance. Bonferroni post hoc tests were used to identify pairwise differences between groups.
Results: The main effect of PRR was statistically significant for MIP-1α (P = 0.018), TNF-α (P = 0.025), MCP-1 (P < 0.001), MIP-2 (P = 0.013), and IL-6 (P = 0.031). Five of six pro-inflammatory cytokines exhibited significantly lower mean concentrations in laser-exposed compared with control cultures at one or more PRR. However, no statistically significant differences were found between PRR groups.
Conclusions: Under the described conditions, statistically significant differences in cytokine secretion were observed between laser-exposed and control cultures, consistent with prior reports. However, PRR appears to be an irrelevant factor in immunomodulation of PBMCs.
Keywords: Lasers; Inflammation; Monocytes; Cytokines; Low-Level Light Therapy; Lipopolysaccharides
Abbreviations: HMT: Host Modulation Therapy, PBM: Photobiomodulation, PRR: Pulse Repetition Rate, PBMCs: Peripheral Blood Mononuclear Cells, RPMI: Roswell Park Memorial Institute, FBS: Fetal Bovine Serum, LPS: Lipopolysaccharide, LPT: Laser Periodontal Therapy, SRP: Scaling and Root Planning

Introduction

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Periodontitis—which represents the most prevalent noncommunicable chronic inflammatory condition affecting humans—is a multifactorial disease resulting in severe periodontal tissue destruction and tooth loss in more than one billion individuals worldwide [1,2].

Putative periodontal pathogens within dental plaque represent the etiology of periodontitis [3-5]. However, most of the tissue destruction is not directly attributable to microorganisms or their products. Rather, loss of alveolar bone and clinical attachment derives primarily from the host inflammatory response [6]. Complex dental biofilms result in the emergence of a panoply of host-derived cytokines that mediate resorption of alveolar bone and loss of attachment [6-9].

Standard therapy for periodontitis includes professional mechanical plaque removal and establishment of an effective oral hygiene regimen that routinely disrupts biofilm development [1]. This approach has served as the mainstay of periodontal therapy for more than a century [10]. However, periodontists have also attempted to modify the inflammation and tissue destruction induced by etiological microorganisms—an approach termed “host modulation therapy” (HMT) [11]. HMT strategies include use of bone-sparing, antiproteinase, and anti-inflammatory agents [11,12]. These pharmacological interventions have had limited clinical applicability due in part to adverse effects of the medications. Electromagnetic radiation may represent a means of therapeutically altering host immunological function without risk of untoward medication-related effects [13].

It has long been recognized that electromagnetic radiation can interact with living tissues and that it is possible for some of these interactions to produce therapeutic effects [14-18]. Laser radiation is unique in that it consists of collimated, monochromatic Gaussian light beams that can be directed toward specific regions on the surfaces of living tissues [19]. Reduction in inflammation has been one of the most reproducible effects of laser photobiomodulation (PBM), a term encompassing photophysical and photochemical phenomena unrelated to thermal tissue responses [13,20-23]. Wavelengths in the red and near infrared spectral regions (600 through 1200 nm) have shown favorable anti-inflammatory effects [13]. Yamaura and colleagues exposed TNF-α-stimulated synoviocytes isolated from rheumatoid arthritis patients to infrared diode laser energy (810 nm), with fluence of 5 or 25 J/cm2 [24]. The authors reported dose-dependent reduction in mRNA and protein levels of TNF-α, IL-1β, and IL-8 [24]. Similarly, Hwang et al. compared IL-8 and IL-6 expression in cytokine-stimulated macrophages subjected to laser energy (405, 532, or 650 nm) at doses up to 1.6 J/cm2 [25]. All wavelengths significantly reduced IL-8 expression compared with controls; only the 405 nm wavelength produced statistically significant reduction in IL-6 expression [25]. In activated dendritic cells derived from the mouse femur, Chen and colleagues found reduced cell-surface markers of inflammation and IL-12 secretion in response to infrared diode laser irradiation (810 nm, 0.3 or 3 J/cm2) [26].

Researchers have also assessed the anti-inflammatory effects of lasers emitting in this segment of the electromagnetic spectrum using animal models. Safavi et al. assessed the influence of He-Ne laser irradiation (632.8 nm, 7.5 J/cm2) on IL-1β, interferon-Υ (IFN- Υ), and TNF-α expression in wounded gingiva of male Wistar rats [27]. The authors noted statistically significant reduction in IL-1β and IFN- Υ expression in the laser-irradiated groups compared with controls [27]. Aimbire and colleagues evaluated the effect of Ga-AsI-Al diode laser irradiation (650 nm, 5.2 J/cm2) on TNF-α concentrations in diaphragm muscle tissue from male Wistar rats [28]. The authors recorded statistically significant reduction in TNF-α concentrations in the laser-irradiated group compared with untreated controls [28]. Although existing evidence from in vitro and animal studies suggest a possible clinical role for laser irradiation in limiting inflammation, the ideal wavelength remains unidentified, and parameters such as fluence, irradiance, pulse duration, pulse repetition rate (PRR), exposure time, and number of exposures have not been optimized. The purpose of this investigation was to assess the influence of PRR on cytokine secretion levels in stimulated and unstimulated rat PBMC cultures subjected to near-infrared radiation from a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser.

Materials and Methods

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Cell Culture
Frozen Rat peripheral blood mononuclear cells (PBMCs) (IQ Biosciences, Berkeley, California, USA) were thawed and suspended in Roswell Park Memorial Institute (RPMI) medium, supplemented with 10% fetal bovine serum (FBS) penstrap. To remove cryoprotectant, cells were centrifuged for 7 minutes at 700 g, then transferred to 96-well plates (2 x 104 cells per well). Cells were stimulated with 0 or 100 ng/ml of lipopolysaccharide (LPS), and incubated for 24 hours at 37⸰C.

Irradiation Parameters
An Nd:YAG laser (1064 nm, Lightwalker AT, Fotona, Dallas, Texas, USA) was used to irradiate cultures, and a 320-micron optical fiber directed the laser beam perpendicularly to the plated cells at a distance of 1.8 mm. A standardized support system ensured a reliable and reproducible laser position. For each group, exposure time and power output remained constant at 30 seconds and 5 W, respectively. PRR was set at 0 (untreated controls), 20, 30, 40, or 60 Hz (Figure 1, Table 1). After irradiation, cells were returned to the incubator for one hour at 37˚ C.

  Table 1: Nd:YAG laser irradiation parameters by treatment group.

*Per pulse

Evaluation of Cytokine Concentrations
Supernatant was extracted and analyzed using a magnetic microsphere immunoassay (MAGPIX System, Luminex, Austin, Texas, USA) permitting quantification of IL-6, IL-10, MIP-1α, MIP-2, MCP-1, TNF-α, and IP-10. The sensitivities for these assays were 30.7, 2.7, 0.8, 9.0, 9.0, 1.9, and 1.4 pg/ml, respectively (Table 2). The analysis software (MAGPIX System, Luminex) processed assay images and determined cytokine/chemokine concentrations in pg/ml using standard curves.

  Table 2: Inflammatory mediators evaluated by magnetic microsphere immunoassay and the associated minimum detectable concentrations (MDCs)

Statistical Analysis
For each evaluated cytokine, a two-way analysis of variance was conducted to compare the main effects of LPS stimulation and PRR, and the interaction between these factors, on cytokine concentration. Bonferroni post hoc tests were used to elucidate statistically significant pairwise differences among PRR levels (0, 20, 30, 40, and 60 Hz). Differences were accepted as significant at an alpha level of 0.05.

Results

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Cytokine secretion by rat PBMCs was evaluated using magnetic microsphere immunoassay following exposure to LPS and treatment with directed Nd:YAG laser energy with PRRs ranging from 20 to 60 Hz (20, 30, 40, and 60 HZ) at constant pulse duration (100 μs), average power (5 W), and irradiation time (30 s). Thus, pulse energy ranged from 83 to 250 mJ, peak power ranged from 833 to 2500 W, and fluence ranged from 103 to 311 J/cm2 (Table 1). The degree to which LPS increased cytokine concentrations varied by cytokine, with the largest increases noted for IP-10, TNF-α, and MIP-2 (Figure 2). Notably, mean concentrations of five pro-inflammatory cytokines were significantly lower in laser-exposed versus control LPS-stimulated PBMC cultures (Table 3). These were the macrophage/monocyte-produced signaling molecule TNF-α (F (4, 110) = 2.90, P = 0.025), the chemotactic inflammatory cell recruiting protein MIP-1α (F (4, 110) = 3.13, P = 0.018), a related protein known as MIP-2 (F (4, 110) = 3.35, P = 0.013), the monocyte/macrophage chemoattractant protein MCP-1 (F (4, 110) = 10.51, P < 0.001), and the proinflammatory interleukin IL-6 (F (4, 110) = 2.76, P = 0.031). The model for IL-10 was not statistically significant (F(9, 110) = 1.67, P = 0.105), and the main effect of PRR was not statistically significant for IP-10 (F(4, 110) = 2.01, P = 0.098). The interaction term between LPS stimulation and PRR was significant for the MIP-1α (F(4, 110) = 3.39, P = .012) and TNF-α (F(4, 110) = 2.79, P = .030) models.

A trend for peak effect at PRRs of 20 Hz and 40 Hz was noted (Figure 2). Maximal reduction the in mean concentrations of IP-10 and MIP-2 was observed at PRR of 20 Hz, whereas maximal reduction in the mean concentrations of MIP-1α, MCP-1, IL-6, and IL-10 occurred at 40 Hz. It is important to note that these observations include some concentration differences that did not reach statistical significance.

All cytokines evaluated except IL-10 exhibited a trend for concentration reduction in laser-irradiated cultures compared with controls, with MIP1-α, TNF-α, MCP-1, MIP-2, and IL-6 each exhibiting a statistically significant decrease in concentration at one or more PRRs. Bonferroni post hoc tests identified the specific PRRs that resulted in statistically significant concentration differences compared with controls (Table 4). No statistically significant differences were noted between laser-irradiated cultures. Thus, PRR had no detectable influence on any cytokine concentration in this investigation.

  Table 3: Results of two-way factorial ANOVA for each cytokine evaluated

  Table 4: Bonferroni post hoc tests

Discussion

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Despite clinical advantages associated with laser periodontal therapy (LPT), the therapeutic use of pulsed Nd:YAG laser energy in the treatment of periodontitis remains a controversial topic within the field of periodontology [29-33]. Currently available evidence supports only modest clinical benefit beyond scaling and root planing (SRP) alone when

Nd:YAG lasers are used adjunctively with SRP [33]. High heterogeneity among studies and lack of controlled clinical research has hampered the ability of clinicians to draw conclusions regarding LPT efficacy and predictability [32,33]. It has been suggested that PBM-induced modulation of inflammation is a unique benefit of LPT among available periodontitis treatments [34]. However, it will be necessary to define specific laser energy parameters (pulse duration, PRR, average power, spot size, fluence, cumulative energy delivered) that produce immune modulation in a predictable and governable manner. Toward that objective, this study evaluated the impact of Nd:YAG laser PRR variation on the secretion of cytokines in unstimulated and LPS-stimulated rat PBMC cultures.

Prior studies have consistently reported statistically significant reductions in various inflammatory markers in response to PBM using devices that emit in the red and infrared portions of the electromagnetic spectrum [13,20-28]. Observations in the present study are consistent with these prior investigations. Only two evaluated cytokines did not exhibit a statistically significant change in concentration in any laser-exposed group—IP-10 and the potent anti-inflammatory cytokine IL-10. No previous investigation using a near infrared laser has directly assessed the influence of PRR on cytokine secretion. Under the described conditions, PRR exerted no statistically significant influence on levels of the evaluated mediators of inflammation.

The high number of technical parameters with potential to influence outcome measures represents a major challenge in conducting and interpreting research into the biological effects of lasers. It has been suggested that fluence (also called energy density) may be the parameter most appropriate for defining the “dose” applied [13]. For multiple outcome measures, biphasic dose responses have been reported. Over a range of PBM exposure levels, a response maximum is reached at some value. When the exposure is increased beyond that threshold, the observed positive response diminishes or vanishes. At even higher fluence values, the investigator may find a negative or inhibitory result [13]. It is likely that the results recorded in the present study were highly dependent upon the specific irradiation parameters applied and the target cell type. Although PRR had no apparent effect on cytokine concentrations in the present study, repetition rate could influence cytokine secretion when lower or higher fluence values are applied.

LPS, which is a constituent of gram-negative bacterial cell walls, was used to stimulate PBMC cell cultures in this in vitro study. The bacterial species that are frequently isolated together at sites exhibiting bone and attachment loss—Porphyrmonas gingivalis, Tannerella forsythia, and Treponema denticola—are facultative gram-negative anaerobes [3-5]. Thus, the LPS used in this study represented an attempt to mimic a stimulant that mononuclear cells encounter at periodontitis-affected sites. However, the model used in this study in no way replicates the complexity of the in vivo microenvironment. At periodontitis sites receiving LPT, laser energy interacts with a diverse set of bacterial species within the biofilm, host cells of the innate and adaptive immune systems, and numerous cell types within the sulcular epithelium, gingival connective tissue, alveolar bone, and periodontal ligament. Thus, the complex cascade of gene expression and intercellular signaling likely induced by LPT will be challenging to fully characterize [35-41].

Conclusions

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Under the described conditions, statistically significant differences in cytokine secretion were observed between laser-exposed and control cultures. However, findings of the present study do not support a correlation between PRR and cytokine concentrations. These observations may be highly specific for the target cell type utilized and the radiation parameters applied.
Author contributions: All authors have contributed substantially to conceptualization of this investigation, drafting the article, critical review, and editing. All authors have approved the final version of the manuscript.
Conflicts of interest: The authors report no financial, economic, or professional interests that may have influenced the design, execution, or presentation of this work.
Disclaimers: The views expressed in this manuscript are those of the authors and do not necessarily reflect the official policy of the United States Government, the Department of Defense, the Defense Health Agency, or Uniformed Services University of the Health Sciences.
Funding: The Defense Health Agency funded this research entirely. The authors received no extramural funding.

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