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. 2025 Oct 4;25:1528. doi: 10.1186/s12903-025-06764-y

Investigation of the effect of photobiomodulation therapy with different wavelengths on nerve regeneration: an experimental study

Esengul Sen 1,5,✉, Mehmet Emin Onger 2, Hatice Duran 3, Yunus Balel 4
PMCID: PMC12497345  PMID: 41046269

Abstract

Background

This study aims to evaluate the effects of photobiomodulation therapy (PBMT) applied with laser (light amplification by stimulated emission of radiation) and laser-LED light on nerve healing in crush-type inferior alveolar nerve (IAN) injuries.

Methods

Twenty-four rats were divided into 4 groups: Sham/control (n = 8), P1 (n = 8), and P2 (n = 8). The right IANs of the rats were exposed, and crushed nerve injuries were done with micro forceps for 30 s. P1 group received PMBT (a combination of 904 nm, 22 mW GaAlAs infrared laser, and 650 nm, 10 mW InGaAlP LEDs, 9 J) for 14 sessions, once every two days. The P2 group received a 940 nm InGaAsP diode laser in the same manner. The sham-control group received a placebo treatment with the laser probe turned off. On the 28th post-operative day, samples were taken from the damaged nerve areas and analyzed by histomorphometric methods.

Results

Axon number, axon area, and myelin thickness were analyzed to assess the IAN regeneration. The maximum number of axons (4438.2) was observed in the sham-control group. The axon area (8.63 µm2) and myelin sheath thickness (2.21 ± 0.30 μm) were measured the most in the control group. No significant difference was found when the axon area/myelin thickness ratio was analyzed between the groups (p>0.05). While P1 and P2 did not differ significantly, both outperformed the sham-control group in axon number and axon area (p≤0.05).

Conclusions

The analyses showed that PBMT positively affected the regeneration and maturation of the crush-injured inferior alveolar nerve. These findings support further investigation into PBMT’s potential clinical role in accelerating nerve repair after crush injuries.

Keywords: Photobiomodulation, LEDs, Laser, Rat, Mandibular nerve, Nerve regeneration

Background

The inferior alveolar nerve (IAN) provides innervation of the teeth in the lower jaw, lip, and chin tip [1]. Various injuries may occur in the IAN after oral and maxillofacial surgical procedures. Changes in the sense of taste, paresthesia, and dysesthesia may occur in the tongue, gingiva, jaw, and lip as a sequel to the damage of the IAN, a sensory nerve [2–4]. These complications of surgery can cause significant problems for the patient. They may cause permanent psychological issues, and the occurrence of these complications may create legal problems for the physician as well as psychological issues.

Different methods are used to treat nerve damage with varying success rates. Non-invasive and invasive procedures are used for treatments. Cryotherapy, acupuncture, ozone therapy (OT), transcutaneous electrical nerve stimulation (TENS), percutaneous electrical nerve stimulation (PENS), and photobiomodulation therapy (PBMT) are the methods that are frequently preferred and extensively researched in the literature [5–8].

Photobiomodulation (PBM) is a process that uses non-ionizing visible and near-infrared (NIR) wavelengths to induce photophysical and photochemical events without thermal damage. When applied to the tissue, lasers, light-emitting diodes (LEDs), and broadband light with filters elicit biological responses. These effects form the basis of PBMT [9, 10]. There are several cellular and molecular aspects to the function of PBM in the healing of nerve damage. Possible mechanisms of photobiomodulation in nerve regeneration include reducing prostaglandins, increasing axonal diameter, myelin thickness, Schwann cell numbers, and synthesizing growth factors [11–14].

Lasers (light amplification by stimulated emission of radiation) form the majority of PBMTs. The therapeutic effects of lasers have been reported since the 1960 s, but LEDs have had a shorter history in literature [15, 16].

LEDs can offer promising results due to their wide beam and cost-effectiveness. A review found that LED and Laser had similar biological effects in skin wounds, such as decreased inflammatory cells, increased fibroblast proliferation and collagen synthesis, stimulation of angiogenesis, and granulation tissue formation [17]. However, with similar biological effects, the studies showed that LED lights can be just as effective as laser lights. Cellular response to photostimulation is not related to specific properties of laser light, such as coherence [18].

The sciatic nerve model is a well-known photobiomodulation (PBM) study subject for peripheral nerve injury, but IAN and the lingual nerve are of distinct clinical significance in the oral and maxillofacial regions [8, 14, 19, 20]. Unlike mixed (sensory-motor) nerves such as the sciatic nerve, the IAN is purely sensory [21, 22], making its regenerative response particularly relevant in clinical scenarios involving dental trauma, surgical iatrogenesis, or fractures. The IAN’s susceptibility to injury during third molar extractions, dental implant surgery or orthognathic surgery highlights the need for targeted therapies.

Despite this, PBM studies on IAN regeneration remain scarce [23–26], with existing literature dominated by heterogeneous protocols (varying wavelengths, doses, and application times). Moreover, the specific pathophysiology of the IAN based on sensation may result in different regenerative outcomes compared to nerves that include motor function. This makes it difficult to make assumptions based on data from sciatic nerves. Because of these reasons, IAN was chosen for this experimental model.

To our knowledge, no study compares laser and laser-LED-based PBM on the regeneration of IAN crush injury in an experimental animal model. This study hypothesized that laser-LED-based PBM had a positive effect on IAN regeneration. This study aimed to compare the impact of PBMTs applied with laser and laser-LED-based devices in nerve healing in crush-type IAN injury using histomorphometry methods. The primary purpose of this study was to show the effect of photobiomodulation at different wavelengths on the IAN recovery.

Materials and methods

Animals

The protocol of this study was approved by the Tokat Gaziosmanpasa University Animal Experiments Local Ethics Committee with the decision number 51879863-42. It was supported by Tokat Gaziosmanpasa University Scientific Research Projects Commission Presidency with project number 2020/48. This animal study followed the National Institutes of Health guidelines for the care and use of laboratory animals. All Wistar Albino rats used in the experiments were purchased from Tokat Gaziosmanpasa University Experimental Medicine Research Unit, and all experiments were performed in the same unit.

Sample size

To calculate the sample size, a pilot study was conducted with 8 rats, with 2 rats in each group. Using G*Power Version 3.1.9.6 software, the effect size was determined to be 1.1890706 based on the mean G Ratio results obtained from the pilot study groups. After setting the alpha value to 0.05 and a statistical 95%, a power analysis revealed that a minimum of 20 subjects were required to conduct the study.

A total of 24 Wistar Albino rats were used in the study, 8 of which weighed approximately 300–350 g for each group. 10 days before the start of the study, the rats were kept in a temperature and humidity-controlled environment with feed and water resources. Rats were divided into 4 groups: Sham-Control Group, Control group, P1 group, and P2 group. Rats in the control group received no surgical procedure or treatment. The left side of the rats was used in this group. The sham-control group underwent IAN crush-nerve injury surgery but received no treatment.

Animals were divided into groups using a computer-based random order generator (https://www.graphpad.com/quickcalcs/randomize). Four different investigators were involved for each animal: a first investigator (HD) administered the treatment based on the randomization table. This investigator was the only one to know which group to treat. A second investigator (ES) performed the surgical procedure. A third investigator (YB) performed PBMT. Finally, a fourth investigator (MEO) performed the histological analysis. Groups were coded before statistical analysis. Statistical analysis was made blindly.

Surgical procedure and nerve injury model

Rats were first administered general anesthesia by intraperitoneal injection of 75 mg/kg ketamine hydrochloride (Ketalar, Eczacıbaşı, Turkey) and 5 mg/kg xylazine hydrochloride (Rompun, Bayer, Germany). The experimental animal was fixed on the table in a suitable position for the procedures. The surgical steps are shown in Fig. 1. Exposure of the nerve was performed as previously described by Kassab et al. [27]. The nerves on the right side were crush-injured, and no operation was performed on the left side.

Fig. 1.

Fig. 1

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Operational stages. a Skin incision. b Exposing the bone and view of the IAN trace. c Exposing the IAN. d Compressing the IAN with micro forceps. e Suturing subcutaneous tissues. f Suturing the skin

Three horizontal and one vertical lines were planned on the rat’s face. Its horizontal line extends from the outer canthus of the eye to the tragus. The 2nd horizontal line extends parallel to the 1st line as the extension of the commissura labiorum. The 3rd line is where the incision will be made; It lies equidistant and parallel to the 1st and 2nd lines. A skin incision was made after local anesthesia (Ultracain-DS; Hoechst Marion Roussel, Istanbul, Turkey) was applied to the area with an articaine solution containing epinephrine. After the skin incision, it was advanced until it met the masseteric fascia and made contact with the bone with the blunt tip of the straight scissors. The masseter muscle fibers were advanced with scissors to reach the 1 st and 2nd lines (4th line). The masseter muscle was retracted with suitable retractors, exposing the mandibular ramus region. The IAN line was also observed as a translucency extending from the condyle to the bony prominence along the ramus. The outer bone cortex region was removed with a 1 mm diameter round carbide bur under saline irrigation so that 2–3 mm IAN was visible. The exposed IAN was compressed with micro forceps (D 103.00; Bahadir, Samsun, Turkey) for 30 s. Subcutaneous and skin tissues were primarily sutured in layers with 4/0 sutures (Vicryl, Ethicon, Brussels, Belgium). Rats were given anti-inflammatory drugs for 4 days.

PBMT procedure

PBMT was applied to the experimental groups in the first session immediately after the surgery, every 48 h for 14 sessions [28, 29] with 9 J energy on the damaged area, with the probe noncontact with the skin, perpendicular to the injury area. To minimize potential sources of bias during PBMT application, a trained technician not involved in treatment allocation restrained all rats gently in a standardized position by hand. The treatment administrator (investigator HD), who was the only individual aware of group assignments, followed a fixed application protocol to ensure consistency across all animals. A 22 mW GaAlAs infrared laser with a wavelength of 904 nm and a combination of 10 mW InGaAlP LEDs with a wavelength of 650 nm, GRR Laser (GRR 2000, Ankara, Turkey) were applied to the P1 group with an intraoral probe. The intraoral probe contains 1 red laser and 4 LEDs and has a 10 mm prob diameter. The device transfers 9 J of energy during a 1-minute application. The device features are summarized in Table 1. Biolase Diode Laser (Biolase Technology, Inc., Irvine, CA) was applied to the P2 group with a wavelength of 940 nm InGaAsP semiconductor diode, maximum 10 W, 12 V, 9 J, continuous mode emitting. To provide a placebo effect in the sham group, the red light of the laser used in the P2 group was applied, but no exposure was made. The device features are summarized in Table 1.

Table 1.

Features of the PBMT devices used

Laser- LED-based PBMT
Parameters Laser-Led Device (P1 group) Laser Device (P2 Group)
Manufacturer GRR Laser Medical Company, Ankara/Turkey EPIC 10
Type GaAlAs Infrared Laser InGaAIP LED InGaAs Semi-conductor diode
Peak wavelength (nm) 904 nm* 650 nm* 940 ± 10 nm*
Peak power (mW) 5mW-200mW 10 W
Prob diameter (mm) 10 mm 15 mm
Frequency Continuous output Continuous output Continuous output
Radiation time (second) 60 60 63
Energy (J) 9 J* 9 J*
Fluence (J/cm2) 9 J/cm2 9 J/cm2
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*nm means nanometer, J means Joule

On the 28th post-operative day, rats were anesthetized with a mixture of 5 mg/kg xylazine hydrochloride and 75 mg/kg ketamine hydrochloride to minimize possible nerve damage. Samples were taken from the damaged nerve areas in the same manner as we did previously. The rats were euthanized by administering an additional dose of xylazine hydrochloride (10 mg/kg) and ketamine hydrochloride (200 mg/kg) for the high dose to prevent them from suffering. Histological analyses were performed in the laboratories of the Ondokuz Mayıs University, Faculty of Medicine, Department of Histology and Embryology.

Histological and stereological analysis

After the rats were euthanized, inferior alveolar nerves were obtained and fixed with glutaraldehyde. Following post fixation for one week, samples were embedded in resin blocks and made ready for sectioning. Sections were taken using a microtome (Thermo Scientific Shandon, USA) with a thickness of 0.5 μm and were stained with toluidine blue for light microscopic examination. Stereological approaches were used to evaluate relevant parameters. The myelinated axon area and the total number of myelinated axons were estimated using the fractionator method, whereas myelin sheet thickness was analyzed using the nucleator method [30]. Stereoinvestigator 9.0 software was used in the stereology analysis system at Ondokuz Mayıs University Faculty of Medicine, Department of Histology and Embryology.

The stereology analysis system is a camera-attached microscope system (Olympus BX43, Center Valley, PA). Nerve samples were analyzed at x100 magnification. An unbiased counting frame (25 × 25 µm2) was used to calculate the number of myelinated axons (Fig. 2).

Fig. 2.

Fig. 2

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a During the stereological analysis of myelinated axons, while myelinated axons intersecting the inclusive edge (green) within the counting frame were counted, the axons intersecting the excluding margin (red) were not counted. The diameter of the axon corresponding to the upper left corner of each counting frame was measured in the vertical and horizontal planes. b The mean value was calculated by measuring the myelin sheath thickness in four regions, as shown in the fig. (Modified from [30])

Statistical analysis

The obtained data were analyzed using the statistical package program (SPSS 21, IBM Corp., Armonk, NY, USA). The One Way ANOVA (Tukey Post-Hoc Test) test was used to compare the groups, and a significance level of 0.05 (p) was used.

Results

Despite removing the inferior alveolar nerve from the region and the extreme care taken during histological analysis, excluding three nerves from each group was deemed appropriate due to damage. Axon number, axon area, and myelin thickness values obtained after histological analysis are shown in Table 2.

Table 2.

Mean values of axon number, axon area, and myelin thickness by groups

Axon Count Axon Area (µm2) Myelin Sheath Thickness (µm)
Mean± Std. Dev. Mean± Std. Dev. Mean± Std. Dev.
Control (n=5) 2263.0±268.36 8.63±0.59 2.21±0.30
Sham-Control (n=5) 4438.20± 573.55 6.66±0.37 1.99±0.20
P1 (n=5) 2613.20± 130.51 8.05±1.22 1.85±0.29
P2 (n=5) 2588.40±148.23 8.18±0.05 1.65±0.39
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Std. Dev. means standard deviation

Axon count

The maximum number of axons was observed in the sham group (4438.2,± 573.5) (Table 2 ). There was a statistically significant difference between the sham and other groups (p ≤ 0.05). No statistically significant difference was found between P1 and P2 groups (Fig. 3).

Fig. 3.

Fig. 3

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Representation of axon numbers according to the data obtained from each group as a result of statistical analysis. ns shows statistically non- significant differences, and * shows statistically significant differences

Axon area

Axon area was the highest in the control group (8.63 ± 0.59) and the lowest in the sham group (6.66 ± 0.37)(Table 2). A statistically significant difference was found between the sham and other groups (p ≤ 0.05). Although the axon area is wider in the P2 group, no statistically significant difference was found between the P1 and P2 groups (Fig. 4).

Fig. 4.

Fig. 4

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Representation of the axon area according to the data obtained from each group as a result of the statistical analysis. ns shows statistically non-significant differences, and * shows statistically significant differences

Myelin sheath thickness

Myelin sheath thickness was measured the most in the control group (2.21 ± 0.30)(Table 2). There was no statistically significant difference between PBM groups (p = 0.073) (Fig. 5).

Fig. 5.

Fig. 5

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Representation of myelin thickness according to the data obtained from each group as a result of statistical analysis. ns shows statistically non-significant differences, and * shows statistically significant differences

Axon area/myelin thickness

No statistically significant difference was found when the axon area/myelin thickness ratio was analyzed between the groups (p ≥ 0.05). This ratio shows that axonal and myelin sheath growth is within physiological limits.

Discussion

This study investigated the effect of PBMT applied with laser (940 nm λ InGaAsP Semiconductor diode) and laser-LED light (904 nm λ GaAlAs infrared laser + 650 nm λ InGaAlP LEDs) on nerve healing in crush-type IAN injury. No statistically significant difference was found between these two photobiomodulation groups regarding axon number, axon area, and myelin thickness. However, there was a statistically significant difference in axon area and axon number in the photobiomodulation-applied groups compared to the sham-control group.

In vitro studies have shown that PBMT causes significant neurite sprouting and outgrowth in cultured neuronal cells [31], as well as Schwann cell proliferation [32]. Additionally, it has been proposed that PBMT may promote neuronal recovery from injury by modifying mitochondrial oxidative metabolism [33] and directing neuronal growth cones in vitro. This may be due to the interaction with cytoplasmic proteins and, specifically, the stimulation of actin polymerization at the leading axon edge [34]. A possible molecular explanation was provided by demonstrating an increase in growth-associated protein-43 immunoreactivity in the early stages of rat sciatic nerve regeneration after PBMT, and another study showed that application of PBMT upregulates calcitonin gene-related peptide mRNA expression in facial motor nuclei after axotomy [35]. Considering these cellular effects of PBMT, this study was designed to compare devices with different wavelengths in a rat IAN crush injury model.

Applying it in varying combinations by combining different photobiomodulation settings can significantly increase their synergistic potential in nerve regeneration [36, 37]. Martins et al. [38] applied a GaAs (910 nm wavelength, 6 J/cm2 energy density, 70 mW output power) laser in IAN crush-style nerve damage for 10 sessions once every 2 days. They found that the axon diameter and myelin sheath thickness were higher than in the non-laser group. They reported that it positively affects the treatment of nerve damage. Following nerve injury, the growth of numerous new axonal sprouts from the proximal to distal region is observed as an indicator of regenerative processes. These nascent axons are characterized by their smaller diameters and thinner myelin sheaths, with sprouts that extend toward the distal end, eventually entering a maturation phase. Excessive axons may be related to a worse functional assessment. Maturation is evidenced by an increase in axonal area and the thickening of the myelin sheath. Over the course of chronic regeneration, it is anticipated that the regenerated nerve fibers will attain the axon count, average axonal diameter, and average myelin sheath thickness comparable to those observed in healthy nerve fibers [30, 39]. Larger diameter of the axons suggests improved nerve function, accelerated regeneration, a reduced inflammatory response, and less swelling of paranodal areas [29]. When the results of this study are compared with our research, they are similar in axon diameter. Myelin sheath thickness was similar in all groups, but slightly higher in the control group compared to the P2 group (p = 0.047). This can be attributed to the short time assessment of the study. In a study of LED treatment of a rat median nerve immediately after a crush injury with (630 nm, 9 J cm-2, 300 mW, and 0.3 W cm-2), the authors found that myelin sheath thickness in the LED group was greater than in the injury group. It has been found to be beneficial in terms of median nerve regeneration and muscle recovery [40]. Myelin sheath thickness provides information about the maturity of nerve fibers rather than recovery. These results show that PBM has the potential for the recovery and maturation of injured peripheral nerves. Diker et al. [41] applied photobiomodulation with 660 nm and 808 nm different wavelength GaAlAs laser to the crush-style nerve damage model in IAN in rats. They reported that the 808 nm wavelength laser was more effective in nerve healing. In their study, Wang et al. [20] investigated the effects of an 808-nm GaAlAs laser with different energy levels (3 J/cm2, 8 J/cm2, 15 J/cm2) on nerve regeneration in crush-type nerve damage created in the rat sciatic nerve and reported that photobiomodulation increased myelin sheath thickness and GAP43 expression levels [20]. Similarly, in other studies [42–44], photobiomodulation has been shown to be effective in the healing of crush-type nerve damage in rats. The results of this study show that photobiomodulation is effective in nerve damage regeneration, as demonstrated in our study.

Laser types, wavelengths, and doses vary in the clinical applications of photobiomodulation. The most commonly Visible light (400–700 nm) and near-infrared (NIR) light (700–1100 nm) are the most commonly preferred wavelengths for PBMT. Wavelength affects laser radiation’s penetration into tissue [45]. It was challenging to say which wavelength was effective in the treatment, and different wavelengths are used in clinical studies in the literature. Hudson et al. reported that 808 nm of light penetrates as much as 54% deeper than 980 nm of light in bovine tissue [46]. Gasperini et al. [47] showed that combined 660-nm and 789-nm wavelength PBM can accelerate the healing of IAN after bilateral sagittal split osteotomies. Guarini et al. [48] reported that PBM with 810-nm GaAlAs laser irradiation improved neural recovery of IAN 2 years after BSSO. In a study investigating lip paresthesia after sagittal split ramus osteotomy, the authors applied the same PBMT devices used in our research. They concluded that the PBMT groups showed better results than the medication group, and the clinical results of the two-wavelength PBMT group were better than the other groups [49]. Due to their optical penetration distance, lasers with 600–700 nm wavelengths are preferred for superficial tissues and 780–950 nm for deeper ones [29]. To increase the success rate of the treatment, the laser power must be effectively transmitted to the target layer of the tissue without significant absorption in other layers [45]. This study aimed to increase the penetration by adding LEDs with a wavelength of 650 nm to the InGaAlP infrared laser with a wavelength of 904 nm used in the P1 group. The results of this study showed that the chosen laser parameters were effective in IAN regeneration. Although there were no statistically significant differences between the groups using different wavelengths in this study, the axon number and myelin thickness were higher in the P1 group than in the P2 group. This situation can be evaluated as LED-laser-based PBMT is effective in neuronal maturation.

Ozturk et al. investigated different diode laser treatment protocols for the regeneration of IAN injuries. They applied indium gallium arsenide phosphide (InGaAsP) diode laser with a wavelength of 976 nm for 10-session and 20-session treatment protocols. The results showed that the 20-session treatment protocol had the highest axon number than the 10-session groups [23]. In our study, 14 sessions of photobiomodulation therapy were preferred, and the duration of the therapy was found to be sufficient and adequate [28, 29].

In the literature, limited studies investigated the effect of LED PBMT on nerve regeneration. Rohringer et al. investigated the impact of PBMT by LEDs of different wavelengths on endothelial cells in vitro. The authors treated human umbilical vein endothelial cells with either 475 nm, 516 nm, or 635 nm light. It was found that green light was more potent in stimulating the proliferation and migration of endothelial cells than red-light therapy [50].

There were some possible limitations in this study. Wavelength is not the only parameter that determines the effectiveness of PBM. Variables such as power, dose, and laser type are other factors that determine the effectiveness of PPBM. The fact that the laser used in the P1 group is a GaAlAs laser and the laser used in the P2 group is an InGaAsP laser may affect the results obtained. The different designs of the application probe used in the P1 and P2 groups may cause various levels of distribution of the laser on the tissue. In addition, most of the studies in the literature did not clearly define the necessary important laser irradiation parameters such as dose, power, duration, and application method. This makes it difficult to compare different studies and may be responsible for conflicting results. The other limitation of this study is the lack of molecular assays for the IAN. In future studies, it would be more beneficial to add a more detailed assessment of the nerve recovery.

Conclusion

In conclusion, when the findings of our study were evaluated, it was observed that PBM had a short-term positive effect on the healing of inferior alveolar nerve damage. Studies examining the long-term nerve regeneration of LED-laser-based PBMT are needed.

Acknowledgements

Thanks to Tokat Gaziosmanpasa University Scientific Research Fund.

Clinical trial number

This study was presented at the Turkish Association of Oral and Maxillofacial Surgery 28th International Scientific Congress.

Thanks to the Tokat Gaziosmanpasa University Scientific Research Fund.

Abbreviations

IAN

Inferior alveolar nerve

OT

Ozone therapy

TENS

Transcutaneous electrical nerve stimulation

PENS

Percutaneous electrical nerve stimulation

PBM

Photobiostimulation

NIR

Non-ionizing visible and near infrared

PBMT

Photobiostimulation Therapy

LEDs

Light-emitting diodes

LASER

Light amplification by stimulated emission of radiation

InGaAlP

Indium Gallium Aluminum Phosphorus

InGaAsP

Indium Gallium Arsenide Phosphide

GaAs

Gallium Arsenide

GaAlAs

Gallium Aluminum Arsenide

Authors’ contributions

ES contributed to the conceptualization, writing - original draft preparation, and funding acquisition. HD contributed to the methodology. ES and YB performed the experiments. MEO performed the formal analysis and supervised the work. All authors have read and approved the final manuscript.

Funding

This study was funded by Tokat Gaziosmanpasa University (Project Number 2020/48).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The protocol of this study was approved by Tokat Gaziosmanpasa University Animal Experiments Local Ethics Committee with the decision number 51879863-42.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Data Availability Statement

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