Cellular bioenergetic and migratory responses of human gingival fibroblasts to 940 nm diode laser photobiomodulation

Abstract

Wound healing in the oral cavity is challenged by continuous microbial exposure, mechanical loading, and fluctuating pH, making rapid and predictable soft tissue repair clinically relevant. Photobiomodulation (PBM) refers to the application of low-dose, non-ionizing light to modulate cellular behavior and support tissue repair. PBM effects have been documented not only in the red (600–700 nm) and near-infrared (780–1100 nm) ranges, but also, in more recent studies, using blue (~ 450 nm) and green (~ 532 nm) light, where measurable intracellular and extracellular matrix changes have been observed. This in vitro study examined how different energy densities of 940 nm diode laser PBM influence human gingival fibroblast (HGF) metabolism and migration, two key components of early wound repair. Commercially available human gingival fibroblasts were cultured as monolayers and irradiated with a 940 nm continuous-wave diode laser (Epic X, Biolase) at energy densities of 6.49, 8.84, and 14.92 J/cm²; non-irradiated cells served as controls. Metabolic activity (MTT assay), membrane integrity (LDH release), nitric oxide (NO) production (Griess assay), and intracellular ATP levels (CellTiter-Glo) were assessed up to 24 h after PBM. Cell migration and wound closure dynamics were quantified in a scratch model using digital holographic live-cell imaging. At 24 h, MTT analysis showed a modest increase in metabolic activity in PBM-treated cells, with the highest energy density (14.92 J/cm²) reaching approximately 19% above control. LDH release and NO levels remained similar to controls across all groups, indicating no detectable cytotoxicity or NO-mediated inflammatory response under the tested conditions. ATP quantification revealed time- and dose-dependent changes, with the largest increase (around 25% above control at 24 h) at 8.84 J/cm². Digital holography demonstrated that 6.49 J/cm² slightly increased migration speed and produced the fastest gap closure, whereas 14.92 J/cm² reduced migration velocity and delayed closure, consistent with a biphasic dose response. In this HGF monolayer wound model, short-exposure 940 nm PBM within a restricted fluence window (6.49–8.84 J/cm²) enhanced cellular energy metabolism without detectable cytotoxicity and selectively modulated fibroblast migration. Because these findings derive from a single-cell-line in vitro model, they should be viewed as experimentally defined irradiation parameters that can inform, but not replace, future in vivo and clinical studies on PBM-assisted oral soft tissue repair.

Introduction

Human gingival fibroblasts (HGFs) are indispensable for maintaining and restoring the structural integrity of the oral mucosa following injury. As the principal cell type of the gingival connective tissue, HGFs are responsible for synthesizing collagen, remodeling extracellular matrix (ECM), and coordinating immune responses during wound healing^1–3. In the context of oral surgery—including periodontal flap surgeries, mucogingival grafts, dental implant placements, apicoectomies, and third molar extractions—rapid and predictable soft-tissue regeneration is essential for minimizing complications and improving patient outcomes⁴. Fibroblasts contribute both mechanical support and biochemical regulation by migrating to the wound site, proliferating, and secreting ECM proteins and cytokines needed for remodeling².

Wound healing in the oral cavity proceeds through the classic cascade of hemostasis, inflammation, proliferation, and remodeling⁵. Unlike cutaneous wounds, the oral mucosa is continuously exposed to microbial load, mechanical forces, and fluctuations in pH and enzymatic activity, which increases the complexity of tissue repair⁶. Fibroblast behavior is influenced by oxygen tension, glucose availability, cytokine gradients, and intracellular signaling pathways such as TGF-β1, FAK, MAPK/ERK, and PI3K/AKT^7,8.

Photobiomodulation (PBM) refers to the application of low-dose, non-ionizing light to modulate cellular behavior and support tissue repair. PBM effects have been documented not only in the red (600–700 nm) and near-infrared (780–1100 nm) ranges⁹, but also, in more recent studies, using blue (~ 450 nm) and green (~ 532 nm) light, where measurable intracellular and extracellular matrix changes have been observed. Historically, PBM mechanisms were attributed primarily to cytochrome-c-oxidase (CcO) absorption in the mitochondrial respiratory chain. While CcO remains a major photoacceptor^10,11, contemporary photobiology recognizes additional pathways involving opsins, transient receptor potential (TRP) ion channels, redox modulation, and non-linear photobiological responses¹².

These primary photochemical events generate secondary mediators such as reactive oxygen species, nitric oxide, and calcium fluxes, influencing transcriptional programs related to proliferation, migration, and survival^13,14. PBM may also modulate interfacial water structuring, influencing ATP synthase rotation and ionic conductance.

Although the 940 nm diode laser is widely implemented in surgical dentistry for soft-tissue ablation, the PBM conditions used in this study differ fundamentally from surgical protocols. Surgical diode applications rely on initiated tips, pigment-dependent absorption, and photothermolysis at temperatures exceeding 100 °C. In contrast, PBM employs low irradiance, uninitiated fibers, and non-contact delivery, ensuring that pigment-dependent thermal mechanisms are not operative. Under these sub-ablative conditions, 940 nm irradiation induces photochemical effects without thermal damage, supporting its potential use for intraoperative and postoperative modulation of wound healing.

Near-infrared wavelengths between 800 and 980 nm have been shown to stimulate fibroblast proliferation, enhance collagen synthesis, and accelerate wound closure in both in vitro and in vivo models^15,16. These effects follow a biphasic dose–response pattern: low fluences promote activity, while higher doses may inhibit function due to excessive ROS accumulation or cellular stress.

When comparing diode wavelengths, it is essential to consider the inverse relationship between wavelength and photon energy (electron volts), as well as wavelength-dependent water absorption. The 980 nm wavelength has a higher water absorption coefficient than 810–940 nm, increasing the probability of thermal contribution at similar fluences^9,11. The 940 nm wavelength offers a balance between tissue penetration and reduced water absorption, making it suitable for non-thermal PBM interactions.

The biological outcomes of PBM depend on multiple parameters, including wavelength, fluence, irradiance, exposure time, and beam profile^17,18. Diode laser beams typically operate in the TEM₀₀ mode, characterized by a Gaussian spatial distribution in which the central region receives the highest photon density. This non-uniform irradiance profile becomes more pronounced at larger spot sizes and must be considered when designing PBM protocols¹⁸.

Adenosine triphosphate (ATP) production has emerged as a sensitive indicator of early regenerative capacity. PBM-induced changes in ATP, along with effects on metabolic activity (MTT), membrane integrity (LDH), nitric oxide signaling, and migration, provide a multifaceted view of fibroblast behavior relevant to wound repair^10,11,19.

Human gingival fibroblasts provide a clinically relevant model due to their accessibility, high proliferation rate, and reproducible behavior in wound healing assays²⁰. Digital holography enables quantitative, label-free monitoring of migration and gap-closure dynamics; however, as an in-vitro assay, it does not replicate the full complexity of in-vivo healing, including immune interactions, angiogenesis, and the optical scattering architecture of living tissue.

This study employed a standardized 1-second PBM exposure at 940 nm on gingival fibroblast monolayers, using fluences of 6.4, 8.84, and 14.92 J/cm² achieved by controlled adjustment of spot size. We assessed ATP output, metabolic activity, membrane integrity, nitric oxide signaling, and migration dynamics. The goal was to identify fluence ranges that promote regenerative behavior while maintaining cytocompatibility. Understanding how 940 nm PBM modulates fibroblast activity in a controlled model provides a foundation for refining laser-assisted soft-tissue management in oral surgery.

Materials and methods

Cell culture

Human gingival fibroblasts (hGF, CLS, Ref. 300703) were cultured in Dulbecco’s Modified Eagle Medium/ F-12 (DMEM/F12) supplemented with 5% fetal bovine serum (FBS) and 1% antibiotic, according to the manufacturer’s protocol. Cells were maintained at 37 °C in a 5% CO₂ humidified atmosphere. For all experiments, cells were seeded at a density of 4 × 10⁴ cells per well in 96-well plates and allowed to adhere overnight before laser irradiation.

Human gingival fibroblasts (hGF; CLS Cell Lines Service GmbH, Eppelheim, Germany; Ref. 300703) were used in this study.

The cell line was commercially obtained and maintained according to the supplier’s guidelines. Because only established cell lines were employed and no human or animal subjects were directly involved, institutional ethical approval was not required for this work.

Laser irradiation protocol

Photobiomodulation (PBM) was performed using the Epic X diode laser system (Biolase Inc., Foothill Ranch, CA, USA), operating in continuous wave (CW) mode at a wavelength of 940 nm. The laser energy was delivered via a standard surgical handpiece equipped with a single-use, uninitiated optical fiber tip (E3-4) with a core diameter of 400 μm. Prior to the experiments, the output power was calibrated using a power meter to ensure a stable delivery of 1.0 W across all treatment groups.

To ensure precise fluence delivery, the beam divergence was experimentally characterized rather than relying solely on manufacturer specifications. The half-angle divergence (θ) was measured to be approximately 12° (0.21 rad). Based on this geometry, the irradiation distance was adjusted to modulate the spot size and achieve the target energy densities (6.4, 8.84, and 14.92 J/cm²), while maintaining an output power of 1.0 W and exposure times of 1 s (for 6.4 and 8.84 J/cm²) or 3 s (for 14.92 J/cm²).

The spot diameter (D) at a given distance (d) was calculated using the formula: Spot diameter (D) = 2 × d × tan(θ) Where θ = 12°. The irradiated area (A) was subsequently derived as A = π × (D / 2)².

Example Calculation for 6.4 J/cm²: Target Fluence = 6.4 J/cm² Power = 1.0 W; Time = 1 s; Total Energy = 1.0 J Required Area (A) = Energy / Fluence = 1.0 J / 6.4 J/cm² ≈ 0.156 cm² Required Spot Diameter (D) = 2 × √(0.156 / π) ≈ 0.446 cm.

Using the experimentally verified half-angle of 12° (tan(12°) ≈ 0.2125): Required Distance (d) = D / (2 × tan(12°)) = 0.446 / (2 × 0.2125) ≈ 1.05 cm.

Thus, to achieve 6.4 J/cm², the fiber tip was positioned approximately 1.05 cm from the monolayer surface.

Spot sizes for all groups were pre-validated using a geometric verification.

All irradiations were performed under sterile conditions inside a biosafety cabinet, with the culture plates placed on a black, non-reflective surface to minimize light scattering and reflection. The laser was applied perpendicularly to the monolayer, ensuring uniform exposure across the wound area. No physical contact was made between the fiber tip and the culture wells, and care was taken to avoid overheating by limiting exposure strictly to 1 s.

Control wells were handled identically but received no laser exposure. It is critical to note that the diode laser beam emitted by the fiber optic tip exhibits a fundamental transverse electromagnetic mode (TEM₀₀), characterized by a Gaussian spatial distribution rather than a uniform “flat-top” profile¹. Consequently, the irradiance is non-homogeneous across the irradiated spot, delivering peak energy intensity at the central core (centroid) and diminishing exponentially toward the beam periphery. While the fluence values reported in this study (6.4, 8.84, and 14.92 J/cm²) represent the average radiant exposure over the calculated spot area—a standard approximation in PBM dosimetry—the cells located at the beam center are inevitably exposed to a higher photon density than those at the margins. As the working distance is increased to enlarge the spot size (as in the 6.4 J/cm² group), the peak central irradiance decreases, but the Gaussian distribution persists. To mitigate potential variability arising from this non-uniformity, the laser beam was consistently centered over the scratch wound, ensuring that the critical zone of cell migration received the maximal therapeutic dose.

All groups were immediately returned to the incubator after irradiation and maintained under standard culture conditions until endpoint analysis at 24 h.

A schematic illustration of the irradiation geometry and spot-size calculation used for fluence determination is provided in Fig. 1.

Fig. 1.

Schematic representation of the 940 nm diode laser irradiation setup used in this study. The diagram illustrates the fiber tip orientation, beam divergence, working distance, and resulting spot size on the cell monolayer. These geometric parameters were used to calculate the fluence delivered in each experimental group and to standardize the non-contact PBM protocol.

MTT assay

The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide) is a mono-tetrazolium salt featuring a positively charged quaternary tetrazole ring core with four nitrogen atoms, surrounded by three aromatic rings- including two phenyl groups and one thiazolyl ring. When reduced, MTT’s core tetrazole ring breaks down, forming a violet-blue, water-insoluble compound called formazan²¹.Due to its positive charge and lipophilic nature, MTT can penetrate cell membranes and the mitochondrial inner membrane of living cells. It is reduced to formazan by metabolically active cells. This redox reaction produces a colour change used to measure intracellular formazan levels, forming the basis of the MTT assay²².

Therefore, cell metabolic activity was assessed by the MTT assay (Biotium, Cat. No. 30006). Briefly, 100 µL of fresh culture medium and 10 µL of MTT reagent were added to each well and incubated for 2 h at 37 °C. Formazan crystals were solubilized in 200 µL isopropanol, and the absorbance was measured at 570 nm with a reference wavelength of 630 nm on a FLUOstar Omega microplate reader (BMG Labtech, Germany).

LDH assay

The LDH cytotoxicity test is a commonly applied assay to evaluate cytotoxicity of various causes and membrane integrity. It measures the amount of LDH, a stable enzyme found in the cytoplasm that leaks out when cells are damaged. This assay offers a quantitative assessment of cell lysis, making it a useful tool for determining cytotoxic effects²³.

The LDH Assay kit (MAK529) from Merck was used to quantify the marker of cytotoxicity, namely lactate dehydrogenase (LDH) - release in medium culture. After 24 h from the time of exposure, 100 µL of supernatant from each well was collected and incubated with assay reagent for 10 min at room temperature. The absorbance was read at 500 nm by the FLUOstar Omega microplate reader.

Griess assay

Nitric oxide (NO) functions as a signaling molecule regulating various biological processes in different cell types. It is produced by nitric oxide synthase (NOS) through redox reactions involving L-arginine. After production, NO has a very short half-life. Maintaining normal physiological levels of NO is essential for proper cellular function, but excessive NO can harm cells. NO is crucial for wound healing and antimicrobial defence, and it also plays a significant role in pathways that lead to cytokine production and release. Consequently, NO is used as a biomarker for inflammatory response initiation and oxidative stress in fibroblasts also²⁴.

Nitric oxide production was measured using the Griess Reagent System (Merck, MAK454). The reaction between supernatant and reagent in a 1:2 ratio lasted 10 min at a temperature of 60 °C. Absorbance was measured at 540 nm using the FLUOstar Omega microplate reader.

ATP assay

Intracellular ATP levels were measured by the CellTiter-Glo 2.0 Assay (Promega, Ref. G924A). Cells were incubated in a 1:1 mixture of culture medium and CellTiter-Glo reagent. Plates were shaken on a orbital shaker for 2 min and allowed to stabilize at room temperature for 10 min. Luminescence was read on the MyGlo plate reader (Promega), and results were processed on the Promega ProNect Data Platform.

Live-cell imaging and wound healing assay

Cell migration during wound healing was monitored by the Holomonitor M4 digital holographic microscope (Phase Holographic Imaging PHI AB, Lund, Sweden). The cells were seeded in 96-well plates and subjected to photobiomodulation at varying energy densities (6.49, 8.84, and 14.92 J/cm²), with non-irradiated cells serving as controls. A HoloLid (PHI AB) was placed on top of the plate for enhancing imaging quality and minimizing evaporation. Time-lapse imaging was performed every 30 min for 22 h. Throughout the experiment, the Holomonitor system was maintained in a standard humidified incubator at 37 °C and 5% CO₂ to mimic physiological conditions.

Statistical analysis

The results were presented as means ± standard deviations. All statistical analyses were performed using the Student’s t-test (Microsoft Excel). A p-value of less than 0.05 was considered statistically significant.

Results

MTT assay (metabolic activity)

The MTT assay performed 24 h post-irradiation revealed a slight increase in metabolic activity in the laser-treated gingival fibroblast monolayers compared to the non-irradiated control, as shown in Fig. 2. The highest energy density (14.92 J/cm²) showed the greatest effect, with approximately 19% increased cell metabolic activity compared to the control group. These findings suggest a dose-dependent photobiomodulatory effect of the 940 nm laser on the metabolic activity of gingival fibroblasts.

Fig. 2.

Human gingival fibroblast metabolic activity 24 h after treatment with 940 nm wavelength photobiomodulation laser at three energy densities (6.49 J/cm², 8.84 J/cm², and 14.92 J/cm²). Results are expressed relative to the control (non-irradiated cells) and are shown as mean ± SD (n = 3).

Lactate dehydrogenase release

Lactate dehydrogenase (LDH) release into the culture medium was measured 24 h after laser treatment as an indicator of membrane integrity. The LDH levels in all experimental groups were comparable to those of the control group, with no statistically significant differences, as shown in Fig. 3. This result demonstrates that the laser energies applied (6.49, 8.84, and 14.92 J/cm²) were insufficient to cause detectable cellular damage or compromise membrane integrity, indicating that the 940 nm laser is biocompatible under the experimental conditions.

Fig. 3.

LDH released from human gingival fibroblasts 24 h after exposure to 940 nm wavelength photobiomodulation laser at three energy densities (6.49 J/cm², 8.84 J/cm², and 14.92 J/cm²). Results are expressed relative to the control (non-irradiated cells) and are shown as mean ± SD (n = 3).

Nitric oxide production

The quantification of nitric oxide (NO) levels in the culture medium showed no statistically significant difference between the laser-irradiated samples and the control group after 24 h, as shown in Fig. 4. This suggests that the application of the 940 nm wavelength laser at the tested energy densities did not significantly affect NO production in gingival fibroblasts under the experimental conditions provided. These results indicate that the laser parameters used did not induce any NO-mediated inflammatory response after 24 h.

Fig. 4.

Nitric oxide released by human gingival fibroblasts 24 h after irradiation with 940 nm wavelength photobiomodulation laser at three energy densities (6.49 J/cm², 8.84 J/cm², and 14.92 J/cm²). Values are expressed relative to control (non-irradiated cells) and presented as mean ± SD (n = 3).

ATP quantification

Intracellular ATP assessment at three time points (30 min, 2 h, and 24 h) after laser treatment indicated time- and dose-dependent changes in cellular energy metabolism, as shown in Fig. 5. At an energy density of 14.92 J/cm², ATP content increased by approximately 10% over the control value at each of the three time points, suggesting ongoing improvement in mitochondrial function. In the 6.49 J/cm² group, no significant difference in ATP was observed at 30 min, an intermediate rise of about 9% at 2 h, and a statistically significant increase of approximately 15% at 24 h. Similarly, in the 8.84 J/cm² group, no change in ATP content was observed at 30 min, but it rose by approximately 12% at 2 h, reaching a maximum of about 25% above the control level at 24 h. These results confirm a cumulative and statistically significant increase in ATP, especially at 8.84 J/cm² after 24 h, demonstrating the dose-response photobiomodulatory effect of the 940 nm laser on gingival fibroblast metabolism.

Fig. 5.

ATP synthesized by human gingival fibroblasts 30 min, 2 h and 24 h following exposure to 940 nm wavelength photobiomodulation laser at three energy densities (6.49 J/cm², 8.84 J/cm², and 14.92 J/cm²). Results are normalized to the control (non-irradiated cells) and shown as mean ± SD (n = 3). * p < 0,05, ** p < 0,01 compared to the control.

Live-cell imaging and wound healing–holomonitor M4

Human gingival fibroblasts were live cell imaged continuously for 22 h with the Holomonitor M4 in a wound healing assay set up to assess the effect of photobiomodulation (PBM) on cell migration rate. Cell displacement analysis and tracking were performed to test the migration velocity at different energy densities of laser irradiation. For calculations of statistical analysis, periods when cells were contributing to wound closure (i.e., migrating into the gap) were utilized, as indicated by the slope with a red arrow in the representative plots (refer to Fig. 5). Stagnation periods when cells were not contributing to wound closure were excluded from velocity calculations so that they would be valid in the healing context. For each measured position in the well, an average migration speed was calculated from these selected intervals.

A global average was subsequently calculated from the individual means for each position. The results are shown in Table 1 for each tested energy density.

Table 1.

Average migration speed (µm/h) of human gingival fibroblasts during wound closure after photobiomodulation at different energy densities.

Treatment: laser irradiation	Speed (µM/H)	SEM	N
Control	21+/-1.9	1.9	6
ENERGY DENSITY: 6.49 J/cm2	23+/-0.77	0.77	8
ENERGY DENSITY: 8.84 J/cm2	21+/-0.6	0.6	6
ENERGY DENSITY: 14.92 J/cm2	16+/-1.48	1.48	6

According to Table 1, the control non-irradiated group had a migration speed mean of 21 ± 1.9 μm/h. Cells treated with an energy density of 6.49 J/cm² had marginally higher migration speed, which was 23 ± 0.77 μm/h. For 8.84 J/cm², the migration speed was the same as the control at 21 ± 0.6 μm/h. Notably, there was a slower speed of 16 ± 1.48 μm/h in the group receiving 14.92 J/cm², which reflects an inhibitory action with a higher energy density.

Based on the values presented in Table 1, the most representative migration plots were utilized to graphically represent the slope (indicated using a red arrow) that correlated with the phase where cells were migrating actively to close the gap width. The graphs (Fig. 6) visually confirm the quantitative data presented in Table 1, suggesting that the speed of cell migration is influenced by the density of energy applied in a dose-related manner. While a moderate energy density (6.49 J/cm²) slightly improved migration compared to the control, higher doses, and particularly 14.92 J/cm², were noted to inhibit migration. This would suggest a biphasic response with low-level photobiomodulation stimulating activity and high energy input being inhibitory in action.

Fig. 6.

Representative plots of cell migration demonstrating the phase of active displacement (indicated by red arrows) of gingival fibroblasts in 21 h and gap width closure over time in human gingival fibroblasts under different photobiomodulation energy densities; (a) Control – without irradiation; (b) 6,49 J/cm2 energy density; (c) 8,84 J/cm2 energy density; (d) 14,92 J/cm2 energy density.

Representative plots showing the dynamics of gap width closure during the 21-hour monitoring period are illustrated in Fig. 6. The initial wound area was approximately 300 μm for all samples. Gap closure in the 6.49 J/cm² group was observed around 11 h, but the 8.84 J/cm² group had closure around 13 h after treatment. The control and the 14.92 J/cm² group showed delayed closure, with the wound being fully closed around 19 h.

These graphs illustrate the effect of differential laser energy density on the rate of wound healing in vitro. Therefore, the fastest closure was realized in the 6.49 J/cm² group, implying an optimum stimulation effect at this energy density. In contrast, the control and the highest density group (14.92 J/cm²) had the longest closure times.

Phase holographic images of hGF cells using Holomonitor M4 are displayed in Fig. 7. Images correspond to groups exposed to 6.49 J/cm², 8.84 J/cm², or 14.92 J/cm² energy densities and non-irradiated. Notably, the 6.49 J/cm² group shows increased cell density and a more orderly distribution pattern compared to the other conditions. In contrast, the highest energy dose (14.92 J/cm²) presents the lowest cell density after 22 h. These findings support the dose-dependent effect of PBMT in that moderate laser doses may enhance cell migration and wound healing, while excessive energy may delay healing. Therefore, precise calibration of the laser parameters is crucial for therapeutic efficacy.

Fig. 7.

Representative Holomonitor M4 images of human gingival fibroblasts at different photobiomodulation energy densities (6.49 J/cm², 8.84 J/cm², and 14.92 J/cm²) and non-irradiated cells (control).

Discussion

These findings should be interpreted within the constraints of an in-vitro monolayer model, where extracellular matrix complexity, immune interactions, and three-dimensional architecture are not represented. Within this context, the present study investigated the biological effects of photobiomodulation (PBM) at 940 nm on human gingival fibroblasts (HGFs), assessing parameters relevant to early wound healing, including mitochondrial ATP output, cell metabolic activity, membrane integrity, inflammatory signaling, and migration dynamics.

The MTT assay revealed a modest, dose-dependent increase in metabolic activity in irradiated cells compared to non-irradiated controls, with the highest energy density (14.92 J/cm²) producing the greatest rise. PBM did not induce cytotoxicity at any tested dose, as shown by unchanged lactate dehydrogenase (LDH) release and stable nitric oxide (NO) levels, two sensitive indicators of membrane disruption and early inflammatory stress. These results support the biocompatibility of the irradiation parameters used. Comparable safety profiles have been reported in studies evaluating fibroblasts exposed to wavelengths between 830 nm and 970 nm at similar fluences^25,26.

Beyond viability, PBM produced clear metabolic effects. ATP measurements demonstrated a time- and dose-dependent elevation, with the most pronounced increase at 8.84 J/cm² after 24 h. Notably, the concurrent enhancement of migration at 6.4 J/cm² suggests that distinct fluence ranges may differentially support energy metabolism and motility. This observation aligns with the biphasic dose-response model described in the PBM literature, in which low fluences stimulate activity while higher doses may yield diminished or inhibitory effects due to ROS imbalance or calcium overload^17,27. Prior studies by Mvula et al.²⁸ similarly reported increased ATP production and proliferation at low to moderate fluences across near-infrared wavelengths.

Digital holographic analysis further indicated accelerated wound-gap closure at 6.4 J/cm², consistent with a PBM-mediated enhancement of gingival fibroblast motility. Migration is a central feature of the proliferative repair phase and depends on coordinated cytoskeletal reorganization, focal adhesion turnover, actomyosin dynamics, and matrix-remodeling pathways²⁹. The present findings agree with previous studies showing improved fibroblast migration under near-infrared PBM^30,31. The observation that migration was enhanced at a fluence slightly lower than that producing the highest ATP increase underscores the complexity of PBM-induced signaling and suggests that optimal migration does not necessarily coincide with maximal metabolic activation³².

Clinically, fibroblast-mediated wound repair is highly dependent on maintaining cellular viability and motility in inflamed or mechanically stressed environments. The ability of PBM to modulate these processes without inducing inflammation in vitro provides a rationale for further exploration in translational models, including clinical scenarios such as gingival graft healing where PBM has shown early benefits^33,34. However, whether such effects translate into measurable improvements in postoperative healing remains to be determined through appropriately designed in-vivo and clinical studies.

From a methodological standpoint, the standardized 1-second exposure used here—at a calibrated power setting of 1.0 W—minimized thermal confounding and reflects a reproducible approach suited for controlled comparisons. Adjusting fluence by varying spot size rather than exposure time facilitates consistency and may be useful in future translational studies, especially given that 940 nm diode platforms are already widely available in dental practice.

Comparisons between wavelengths require a nuanced understanding of their biophysical properties, specifically photon energy and chromophore absorption profiles. The energy delivered per photon (eV) is inversely proportional to the wavelength; consequently, 810 nm photons carry higher energy than those at 940–980 nm. Furthermore, the absorption coefficient of water varies significantly across this spectral range. The 980 nm wavelength coincides with a distinct water-absorption peak, leading to higher photon capture by intracellular water and a consequently greater potential for photothermal conversion compared to 940 nm–810 nm^35,36. This differential water absorption implies that 980 nm irradiation may induce localized thermal effects at lower fluences than 940 nm, potentially confounding pure PBM outcomes if dosimetry is not strictly controlled. By selecting 940 nm, we utilize a wavelength that balances effective penetration with a water-absorption profile that mitigates the risk of rapid bulk heating often associated with the 980 nm peak.

Furthermore, extrapolating these findings to the complex optical environment of oral soft tissues requires consideration of tissue-level photon propagation. Unlike the optically transparent culture medium, gingival tissue acts as a turbid medium in which light distribution is shaped by scattering, absorption, reflection, and internal remission. Variations in refractive index between healthy and inflamed tissues—often characterized by edema and altered collagen organization—further influence penetration depth and fluence distribution³⁷. Thus, while the surface parameters defined in this study provide a controlled baseline, clinical dosimetry may require adjustment to compensate for subsurface optical losses.

Several limitations must be acknowledged. The monolayer design does not capture the roles of immune cells, angiogenesis, extracellular matrix remodeling, or microbiological influences. The study also employed a single 24-hour time point, whereas healing unfolds over days to weeks. Additional markers—such as collagen deposition, integrin expression, or cytokine profiles—would enrich understanding of PBM-mediated regenerative pathways. More advanced models, including co-cultures, 3D scaffolds, and animal systems, will be essential to establish the relevance of these findings in vivo³⁸.

Despite these limitations, the present study identifies defined fluence ranges—6.4 and 8.84 J/cm²—that modulate fibroblast metabolism and migration without eliciting cytotoxic effects. These parameters represent experimental benchmarks that may inform future in-vivo and clinical research rather than finalized therapeutic protocols. Whether the cellular benefits observed here translate into improved wound-healing outcomes should be investigated in longitudinal and controlled clinical settings.

To our knowledge, this is the first investigation to apply digital holographic live-cell imaging with the Holomonitor system to track fibroblast behavior under 940 nm photobiomodulation, providing quantitative migration dynamics that are not available through conventional assays.

Conclusions

This study shows that short-exposure 940 nm photobiomodulation can modulate gingival fibroblast behavior under controlled in-vitro conditions. Sub-ablative irradiation supported ATP production and enhanced migration, while maintaining normal LDH and nitric oxide levels, indicating preserved membrane integrity and the absence of inflammatory activation.

Among the fluences tested, 6.4 J/cm² produced the most consistent combination of metabolic and migratory effects. These findings define experimental irradiation parameters that may guide the design of future in-vivo and clinical studies, although direct translation should be approached with caution.

It is important to recognize that the parameters used here—1.0 W, 1-second exposure, and distance-adjusted spot sizes—were optimized for a monolayer culture model. Established PBM safety consensus recommends careful monitoring of irradiance, typically below 500 mW/cm² in continuous-wave delivery, to avoid thermal contributions and biphasic inhibition³⁹.

Therefore, the exposure protocol applied in this study should be regarded as a controlled experimental baseline rather than a clinical guideline. Further research in three-dimensional models, animal studies, and well-designed clinical trials will be needed to determine how these cellular responses integrate into the broader wound-healing process in vivo.

Author contributions

Conceptualization, I.K.M, C.N. and A.R.; methodology, C.N, F.R, B.V ; software, A.P, A.C ; validation, I.K.M, S.P and M.I; formal analysis, C.N, B.V and F.R ; investigation, F.R., B.V, C.N, A.P ; resources, M.I, A.R and C.N.; data curation, A.C. and F.R ; writing—original draft preparation, C.N, B.V, A.P; writing—reviewand editing, A.R, I.K.M ; visualization, M.I. and S.P.; supervision, A.R., M.I. and S.P.;project administration, M.I. and S.P.; funding acquisition, S.P. All authors have read andagreed to the published version of the manuscript.

Data availability

The datasets generated and analysed during the present study are publicly available in the Zenodo repository at [https://doi.org/10.5281/zenodo.17377187](https:/doi.org/10.5281/zenodo.17377187) . The repository includes all raw experimental measurements and processed data used for statistical analysis and figure preparation.

Declarations

Competing interests

The authors declare no competing interests.