WHICH WAVELENGTH OF LASER YIELD THE MOST SUCCESSFUL RESULTS IN MRONJ TREATMENT? AN EXPERIMENTAL STUDY

doi:10.21203/rs.3.rs-3338450/v1

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WHICH WAVELENGTH OF LASER YIELD THE MOST SUCCESSFUL RESULTS IN MRONJ TREATMENT? AN EXPERIMENTAL STUDY

https://doi.org/10.21203/rs.3.rs-3338450/v1

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Background: This study aims to investigate the effectiveness of lasers at various wavelengths in treating medication-related osteonecrosis of the jaw (MRONJ) using biochemical, clinical scoring, micro CT analysis, and histopathological methods. Methods: In our study, there were 6 groups, including one SHAM group, one CONTROL group, and four experimental groups, with 8 rats in each individual group. Each rat received antiresorptive drug intraperitoneally for 4 weeks and then extracted the left second molar in the mandible. All animals were sacrificed at the end of the 12th week. In the experimental groups, lasers at wavelengths of 405nm, 445nm, 660nm, and 808nm were applied to the animals. In all groups, parameters such as serum vitamin D levels, bone density and bone volume at the extraction site, new bone formation, dead bone count, inflammatory cell count, and epithelial regeneration were examined. Additionally, clinical scoring was conducted after sacrifice. Statistical analyses were performed with the NCSS (Number Cruncher Statistical System) 2007 Statistical Software (Utah, USA) package program. The results were evaluated at the p<0.05 significance level. Results: According to the results obtained from our study, new bone formation in all experimental groups was significantly higher than in the SHAM and CONTROL groups. Furthermore, the 660nm and 808nm wavelengths increased considerably serum vitamin D levels. The most successful outcomes were observed in clinical scoring, dead bone count, epithelial cell regeneration, and bone density in the 660nm and 808nm wavelength groups. Conclusions: The combined use of lasers at 660nm and 808nm wavelengths may yield successful results in treating MRONJ.

Photobiomodulation therapy

Laser therapy

Medication-related osteonecrosis of the jaw

Zoledronate

Vitamin D

Medication-related osteonecrosis of the jaws (MRONJ) represents a severe adverse drug reaction characterized by progressive bone deterioration affecting the maxilla and mandible in patients undergoing treatment for conditions such as osteoporosis, osteopenia, cancer, hypercalcemia of malignancy, bone metastases from solid tumors, or lytic lesions associated with multiple myeloma. The primary culprits responsible for MRONJ are antiresorptive agents, encompassing both nitrogen-containing and non-containing bisphosphonates (BPs), as well as receptor activators of nuclear factor kappa-B ligand (RANK-L) inhibitors. Additionally, antiangiogenic agents like bevacizumab, sunitinib, and sorafenib have been implicated in MRONJ development [1]–[3].

While the exact pathophysiology of MRONJ remains still not fully understood, it is postulated that a combination of factors contributes to its onset. These factors may include inflammation or infection, invasive dental procedures involving bone such as tooth extraction or minor trauma, compromised immunity, vitamin D deficiency, excessive inhibition of bone resorption, angiogenesis suppression, and altered bone remodeling [4]–[6].

MRONJ usually clinically presents with gingival ulcers in the region of exposed bone. As the disease progresses, it leads to severe pain, purulent drainage, intraoral or extraoral fistula, impaired oroantral communication, and pathologic bone fracture [7]. Several procedures on MRONJ management have been performed since its first description in 2003 [7], [8]. However, the treatment strategy needs to be revised, with no consensus regarding non-surgical versus surgical approaches. Non-surgical management of MRONJ includes optimization of oral health by reinforcement of oral hygiene with regular brushing and antimicrobial mouth rinse, frequent dental checks, and antibiotic therapy. In contrast, surgical management includes removing necrotic bone area(s) [8].

Recent research results have emphasized adjuvant therapeutic approaches, including hyperbaric oxygen, low-level laser therapy (LLLT), and platelet-rich plasma. These investigations have illuminated the potential advantages of incorporating LLLT to complement conventional medical treatments, particularly in the early stages of medication-related osteonecrosis of the jaws (MRONJ). LLLT exhibits superiority over antimicrobial mouthwashes and antibiotic therapy in these initial stages due to its capacity to modulate cellular metabolism, enhance wound healing processes, and alleviate pain.

Conversely, for patients in advanced stages of MRONJ, minimally invasive surgical interventions, such as laser surgery, emerge as a more favorable alternative than traditional surgical approaches. The positive impact of laser irradiation on MRONJ treatment is attributed to its bio-stimulatory effects on the organic bone matrix and osteoblasts. This includes elevating the mitotic index and stimulating osteoblastic cell proliferation, differentiation, and activity [9]–[11].

Laser biostimulation (or biomodulation) has many applications, primarily in wound healing and pain management. Given the current lack of evidence supporting the use of laser-assisted interventions to MRONJ, we have examined the effects of different wavelength diode lasers on soft and hard tissue healing in experimental MRONJ model in rats based on evaluations of biochemical markers, radiological findings, clinical assessment, and histopathological findings [11]–[13].

1. Animal Handling

A total of 48 male Sprague-Dawley rats (Aziz Sancar Institute of Experimental Medicine, Istanbul, Turkey), 12 weeks old and weighing 250 ± 20 g, were used in the study. The rats were housed in a temperature, humidity, light-controlled room (23 ± 3 ̊C; 55 ± 15%; 12h light, dark cycle); standard laboratory chow and tap water supplied ad libitum. The experimental protocol of this study was reviewed and approved by the Istanbul University Animal Experiments Local Ethics Committee. All methods are reported per ARRIVE guidelines [14].

2. Study Design and Surgical Procedure:

MRONJ formation process in rats:

After all animals adapted to the laboratory temperature and humidity, 8 rats were randomly assigned to a sham group. The remaining 40 rats received 0.06 mg/kg zoledronic acid (ZOL, diluted in phosphate-buffered saline, pH 7.4) by intraperitoneal injections. Weekly repeat doses were administered for 4 weeks to develop bisphosphonate-related jaw osteonecrosis in this manner.

After a total of four ZOL injections, the rats were anesthetized with 0.5 mL/kg intramuscular injection of ketamine (100 mg/mL). The mandibular left first molars of all animals were extracted. After wiping the wound surfaces with polyvinylpyrrolidone (PVP) antiseptic solution, a single dose of tramadol (2 mg/kg) and cefazolin (50 mg/kg) injections were administered for two days postoperatively to prevent postoperative infection and pain shock. The rats in the sham group did not undergo any procedure other than regular intraperitoneal injection of the same volume of saline and mandibular first molar extraction [15](Table 1).

Table 1

Study groups, wavelengths, and animal numbers.
Groups	Central Wavelength	Number Of Test Subjects (n)
Sham	n/a	8
Control	n/a	8
Laser 1	405nm	8
Laser 2	445nm	8
Laser 3	660nm	8
Laser 4	808nm	8

3. Laser Procedure

The rats were randomly divided into five groups: a control and 4 experimental groups (8 rats each). No laser was applied to the rats in the control and sham groups. Low-level laser (LLL) at different wavelengths was performed twice a week for 16 seconds for four weeks to a single standard point created in the socket of the extracted tooth with the laser tip positioned parallel to the long axis using the following irradiation parameters: 480 mW/cm2 optical power intensity, 6 J/cm2 irradiation dose and 120 mW power in continuous operation mode. Consequently, 48 J/cm2 energy was applied per rat by performing 1.5 J/point LLL for 16 seconds in 8 sessions. LLL applications were performed on experimental group rats as follows: 405 nm for Group 1, 445 nm for Group 2, 660 nm for Group 3, and 808 nm for Group 4.

InGaN (Indium Gallium Nitride)-based diode laser for 405 nm/445 nm applications, AlGaInP (Aluminium Gallium Indium Phosphide)-based diode laser for 660 nm application, GaAlAs (Gallium Aluminium Arsenide)-based diode laser for 808 nm application were used with spot size of 0. 25cm2 (Kale CNC Ltd. Istanbul Turkey). Full technical specs of the lasers used are given in Table 2. The wavelengths, the power, and the application dosage chosen for laser irritation were determined by examining the physical properties of the lasers and tissues. The power of the laser was calculated as a minimum of 145mw so that the reactions we tried to simulate could take place without tissue damage and heat in the tissues. However, a minimum threshold of 105 mw is needed to achieve the desired effects. These two values were the main window while determining the power of lasers, so we preferred 120mw in our experiments.

Table 2

Laser Technical Parameters
	Laser 1	Laser 2	Laser 3	Laser 4
Central Wavelength	405nm	445nm	660nm	808nm
Wavelength Accuracy	± 2nm	± 2nm	± 2nm	± 4nm
Laser Structure	InGaN	InGaN	AlGaInP	GaAlAs
Type of Energy Source	Diode	Diode	Diode	Diode
Beam Delivery Method	Hand Probe	Hand Probe	Hand Probe	Hand Probe
Beam Profile	Gaussian	Gaussian	Gaussian	Gaussian
Average Beam Power	120mw	120mw	120mw	120mw
Aperture	5mm x 5mm	5mm x 5mm	5mm x 5mm	5mm x 5mm
Operation Mode	Continuous Wave	Continuous Wave	Continuous Wave	Continuous Wave
Beam Shape	Square	Square	Square	Square
Illumination in the Diaphragm	480mw/cm²	480mw/cm²	480mw/cm²	480mw/cm²
Area of Application	0.25cm²	0.25cm²	0.25cm²	0.25cm²
Laser irradiation Dose	6Joule/cm²	6Joule/cm²	6Joule/cm²	6Joule/cm²
Irradiation Period (Single Session)	16 Second	16 Second	16 Second	16 Second
Energy Delivered in one Session	1.5 Joule	1.5 Joule	1.5 Joule	1.5 Joule
Total Number of Sessions	8	8	8	8
Session Frequency	Week / 2	Week / 2	Week / 2	Week / 2
Total Energy Intensity	48Joule/cm²	48Joule/cm²	48 Joule/cm²	48Joule/cm²
Total Application Time	128 Second	128 Second	128 Second	128 Second
Total Net Energy Delivered	12 Joule	12 Joule	12 Joule	12 Joule

4. Biochemical Analysis

At the end of the 12th week, rats were anesthetized with ether, and heart blood samples were taken and placed in yellow-capped gel tubes. It was centrifuged at X1500 g for 10 minutes, and serum samples were stored at -80°C until analysis.

Vitamin D levels in these samples were measured in a Biotech brand µQuant model ELISA system at Istanbul Medical Faculty Biochemistry Department Research Laboratory with a commercially available ELISA kit, following the manufacturer's instructions (USCN Life Science Inc, Wuhan, China).

5. Sacrification

Following blood collections for biochemical analysis at the end of the 12th week of the study), all animals were anesthetized using intraperitoneal ketamine hydrochloride (50 mg/kg) and xylazine (10 mg/kg), then a sacrification process was carried out.

6. Clinical Evaluation

After the animals were sacrificed, their mandibles were dissected. They were all photographed for macroscopic examination and subjected to clinical scoring. In clinical scoring, 0 points for well-healed sockets, 1 point for hyperemic mucosa around the extraction site, 2 points for impaired soft tissue integrity and exposed bone, and 3 points for the presence of abscess and suppuration given(Fig. 1). Two blinded observers have conducted the entire clinical assessments according to these criteria.

7. Bone Scan Analysis with Micro Computed Tomography (Micro Ct)

The specimens were meticulously prepared for scanning in a SkyScan 1174v2 computed tomography apparatus located in Kontich, Belgium. Each sample underwent scanning with a 0.25 mm aluminum filter, utilizing a voltage of 50 kVp, a current of 800 µA, and a power setting of 40 W. The scan parameters were finely tuned to optimize an exposure time of 3500 ms, achieve a resolution of 1024x1304 pixels, and employ a zoom factor of 30.51 µm. Each sample underwent a complete 360-degree rotation with angular increments of 1.00 degrees, requiring approximately 50 minutes for completion. (Fig. 2)

Following the scanning procedure, a total of 361 raw images were generated in the TIFF format, and these images were subjected to reconstruction utilizing the NRecon software (Version 1.6.10.2). Subsequently, approximately 800 sections were extracted in BMP format, as depicted in Fig. 2. These sections were imported into the CTan software (Version 1.16.4.1+), facilitating the execution of densitometric and morphometric measurements to establish quantitative parameters and create visual models.

To delineate the boundaries of the region to be measured, a semi-automatic circular Region of Interest (ROI) was delineated in the horizontal plane on the image slices, as illustrated in Fig. 3. A threshold value was ascertained within the histogram to differentiate between the voxels representing the sample under examination and those representing the surrounding air, as exemplified in Fig. 4. Specifically, black voxels were assigned a value of 0, signifying the minimum intensity, while white voxels were assigned a value of 255, representing the maximum intensity.

Volumetric ratios were subsequently computed separately using the designated ROIs and the threshold data. In order to generate three-dimensional modeling images of the specimens, the data were transferred to the CTVol software (Version 2.3.2.0). A calcium hydroxyapatite (CaHA) calibration bar (phantom) was employed for the analysis, featuring calcium densities of 0.25 g/mm3 and 0.75 g/mm3. The software CTAn (Version 1.16.4.1+) was used to calculate measurements of bone mineral density (expressed in g/cm3) and bone volume (in cm3) within the specified region.

8. Histopathological Analysis

After micro CT analysis, the mandibles of the rats were separated and fixed in 10% formalin solution. Samples were decalcified in formic acid buffered with citrate for 48 hours and then embedded in paraffin. 5 µm-thick serial sections were taken from the region around the extraction socket in the longitudinal plane and stained with hematoxylin and eosin (HE) for histopathological examination. For each sample, three sections were examined for new bone formation, dead bone areas (without viable osteocytes), inflammatory cell infiltration, and epithelial cell regeneration under a light microscope at ×100 magnification. The examination process was carried out by an experienced pathologist who was unaware of the study protocol.

9. Statistical Analysis

This study conducted statistical analyses using the NCSS (Number Cruncher Statistical System) 2007 Statistical Software package program based in Utah, USA.

In the data assessment process, in addition to employing descriptive statistical techniques such as mean, standard deviation, median, and interquartile range, an assessment of variable distribution was carried out using the Shapiro–Wilk normality test. One-way analysis of variance (ANOVA) was utilized to compare groups for variables that exhibited a normal distribution, while subgroup comparisons were conducted using the Tukey multiple comparison test. The chi-square test was employed to compare categorical data. The significance level for evaluating results was set at p < 0.05.

Only three animals died due to excessive weight loss, so the experimental procedure was successfully completed by replacing the deceased ones. Biochemical, clinical, radiological, and histological findings are as follows.

1. Biochemical Findings

A statistically significant difference was observed between mean vitamin D levels in sham, control, 405 nm, 445 nm, 660 nm, and 808 nm groups (p = 0.0001) (p = 0.002). Serum vitamin D levels of the 660 nm group were significantly higher than sham, control, 405 nm, 445 nm, and 808 nm groups (p = 0.006, p = 0.0001). Mean vitamin D levels in the sham group were significantly higher than control, 405 nm, and 445 nm groups (p = 0.008, p = 0.0001). The level of the 808 nm group was considerably higher than the control, 405 nm, and 445 nm groups (p = 0.025, p = 0.0001). No statistically significant difference was observed between the other groups (p > 0.05). The results of the serum vitamin D levels are given in Fig. 5.

2. Clinical Findings in Subjects

The subjects who underwent tooth extraction after repeat doses of zoledronic acid were examined regarding wound healing and divided into four groups (Table 3). Proper healing was seen in 20 subjects, while the hyperemic mucosal area was seen in 19 rats. Exposed bone area was seen in 7 subjects as only 2 remaining animals had abscess and suppuration.

Table 3

Clinical findings.
	No Symptoms	Hyperemic Mucosa	Exposed Bone	Abscess/Suppuration
Sham (1–8)	7	1	0	0
Control (9–16)	0	2	4	2
405nm (17–24)	1	5	2	0
445nm (25–32)	0	7	1	0
660nm (33–40)	7	1	0	0
808nm (41–48)	5	3	0	0
Total	20	19	7	2

A statistically significant difference was observed between the healing score distributions of the sham, control, 405 nm, 445 nm, 660 nm, and 808 nm groups (p = 0.0001).

The normal healing presence distributions of sham, 660 nm, and 808 nm groups were higher than those of control, 405 nm, and 445 nm groups. Abscess distributions of the control group were higher than those of the sham, 405 nm, 445 nm, 660 nm, and 808 nm groups. The distribution of hyperemic tissue presence in 405 nm and 445 nm groups was higher than in sham, control, 660 nm, and 808 nm groups (Fig. 6, Table 4,5,6).

Table 4

Statistical evaluation of clinical findings (Chi-Square test)
Clinical Scoring	No Symptoms		Hyperemic Mucosa		Exposed Bone		Abscess/ Suppuration		p
Sham	7	87,50%	1	12,50%	0	0,00%	0	0,00%	0,0001
Control	0	0,00%	2	25,00%	4	50,00%	2	25,00%
405 nm	1	12,50%	5	62,50%	2	25,00%	0	0,00%
445 nm	0	0,00%	7	87,50%	1	12,50%	0	0,00%
660 nm	7	87,50%	1	12,50%	0	0,00%	0	0,00%
808 nm	5	62,50%	3	37,50%	0	0,00%	0	0,00%

3. Radiological Findings

Micro CT examinations revealed a statistically significant difference between the bone mineral density means of sham, control, 405 nm, 445 nm, 660 nm, and 808 nm groups (p = 0.0001). The bone mineral density mean of the 660 nm group was significantly higher than the other five groups(p = 0.002, p = 0.0001). The mean of the 808 nm group was substantially higher than the sham, control, and 405 nm groups (p = 0.007, p = 0.0001). No statistically significant difference was observed between the other groups (p > 0.05) (Fig. 7).

Furthermore, study groups exhibited significant differences in the mean volumes of bone(p = 0.0001). The mean bone volume of the sham group was statistically significantly higher than the other groups except 660 nm(p = 0.0001). The mean bone volumes of 660 nm and control groups were significantly higher than 405 nm, 445 nm, and 808 nm groups (p = 0.002, p = 0.0001). A significant difference in the mean bone volume of the 808 nm group was found, which presented a higher mean bone volume than the control and 405 nm groups (p = 0.001, p = 0.0001). The 445 nm group was also significantly higher than the control group according to mean bone volume (p = 0.013). No statistically significant difference was observed between the other groups (p > 0.05) (Fig. 8).

4. Histological Findings

A statistically significant difference was observed between new bone formation, dead bone areas, epithelial cell regeneration, and inflammation cell rates of sham, control, 405 nm, 445 nm, 660 nm, and 808 nm groups (p = 0.0001). The sham group's mean new bone formation rate was statistically significantly lower than the mean rates of the other groups except the control group(p = 0.0001). In addition, the control group's rate was statistically significantly lower than the mean rates of the 405 nm, 445 nm, 660 nm, and 808 nm groups (p = 0.001, p = 0.0001). No statistically significant difference was observed between the other groups (p > 0.05) (Fig. 9,10).

Regarding the number of dead bones, the mean value of the 445 nm groups was statistically significantly higher than the mean values of the other groups except the control group (p = 0.007, p = 0.001). The control group's mean value was statistically significantly higher than the other groups, except for the 445 nm group(p = 0.021, p = 0.001). The rates of the 405 nm and 880 nm groups were significantly higher than the sham and 660 nm groups (p = 0.002, p = 0.001) (p = 0,020, p = 0,007). Additionally, while no statistically significant difference was observed between the other groups (p > 0.05) (Fig. 11,12).

Regarding the epithelial cell regeneration, the mean values in the 880 nm and 660 nm groups were significantly higher than in the sham, control, 405 nm, and 445 nm groups (p = 0,001). No statistically significant difference was observed between the other groups (p > 0.05) (Fig. 12,13).

The mean inflammation cell count of the 880 nm group was significantly higher than the mean values of the other groups except the control group(p = 0.036, p = 0.002). Furthermore, the mean value of the 660 nm group was found to be significantly lower than the mean values of the control, 405 nm, and 445 nm groups (p = 0.001), while no statistically significant difference was observed between the other groups (p > 0.05) (Fig. 14,15)

Table 5

One-Way Analysis of Variance
	Vitamin D	New Bone Formation	Bone Mineral Density	Mean Bone Volume	Epithelial Cell Regeneration	Inflammation Cell Count	Dead Bone Count
Sham	67,58 ± 15,75	2,49 ± 0,40	1,55 ± 0,02	25,02 ± 1,77	3,139 ± 0,492	0,066 ± 0,013	0,169 ± 0,077
Control	34,29 ± 3,25	2,94 ± 1,39	1,50 ± 0,05	14,37 ± 1,81	2,939 ± 0,542	0,073 ± 0,042	0,541 ± 0,167
405 nm	36,72 ± 7,58	4,65 ± 0,68	1,53 ± 0,03	15,84 ± 0,66	3,125 ± 0,503	0,078 ± 0,082	0,378 ± 0,105
445 nm	46,52 ± 14,22	5,44 ± 0,45	1,59 ± 0,09	17,70 ± 1,73	2,603 ± 0,873	0,059 ± 0,019	0,595 ± 0,144
660 nm	89,24 ± 14,43	4,65 ± 0,50	1,82 ± 0,09	23,98 ± 1,05	5,431 ± 0,183	0,019 ± 0,006	0,108 ± 0,044
808 nm	65,16 ± 7,74	4,67 ± 0,55	1,68 ± 0,09	20,03 ± 3,29	6,451 ± 1,757	0,099 ± 0,044	0,315 ± 0,175
p*	0,0001	0,0001	0,0001	0,0001	0,0001	0,001	0,0001

Table 6

Tukey Multiple Comparison Test
Tukey Multiple Comparison Test	Vitamin D	Bone Mineral Density	Mean Bone Volume	New Bone Formation	Dead Bone Count	Epithelial Cell Regeneration	Inflammation Cell Count
Sham / Control	0,0001	0,697	0,0001	0,831	0,001	0,372	0,563
Sham / 405 nm	0,0001	0,976	0,0001	0,0001	0,002	0,875	0,753
Sham / 445 nm	0,008	0,873	0,0001	0,0001	0,001	0,074	0,293
Sham / 660 nm	0,006	0,0001	0,882	0,0001	0,244	0,001	0,001
Sham / 808 nm	0,998	0,007	0,0001	0,0001	0,02	0,001	0,036
Control / 405 nm	0,998	0,979	0,644	0,0001	0,031	0,431	0,494
Control / 445 nm	0,289	0,129	0,013	0,0001	0,43	0,345	0,713
Control / 660 nm	0,0001	0,0001	0,0001	0,001	0,001	0,001	0,003
Control / 808 nm	0,0001	0,0001	0,0001	0,0001	0,021	0,001	0,188
405 nm / 445 nm	0,531	0,445	0,382	0,297	0,007	0,074	0,916
405 nm / 660 nm	0,0001	0,0001	0,0001	0,999	0,001	0,001	0,023
405 nm / 808 nm	0,0001	0,001	0,001	0,999	0,207	0,001	0,103
445 nm / 660 nm	0,0001	0,0001	0,0001	0,287	0,001	0,001	0,001
445 nm / 808 nm	0,025	0,119	0,164	0,319	0,009	0,001	0,021
660 nm / 808 nm	0,002	0,002	0,002	0,999	0,007	0,093	0,002

The treatment of MRONJ remains a topic of ongoing debate within the medical world. Two main approaches are generally considered for treating MRONJ: interventional (surgical) and conservative strategies. The traditional approach aims to alleviate the patient's symptoms using various antibiotics, pain relievers, and mouthwashes. Conversely, surgical treatment aims to remove the necrotic area before it further enlarges.

While conservative treatment can successfully alleviate or eliminate symptoms in some patients, it may be insufficient in others. On the other hand, in some instances, surgical interventions can exacerbate the clinical course of the disease. This dilemma has prompted clinicians to explore alternative approaches that could assist in deciding between surgical or conservative treatment [16], [17].

Among these adjunctive therapies, specific treatments have gained prominence, including the application of bone marrow stem cells (BMSC), platelet-rich plasma (PRP) treatments, hyperbaric oxygen (HBO) therapy, and laser applications [18].

Upon reviewing the existing literature, clinical and experimental studies investigating lasers' effectiveness in treating medication-related osteonecrosis of the jaw (MRONJ) have been identified. Published clinical studies have reported successful outcomes in supporting surgical and medical treatment when lasers were retrospectively used [19]. In a retrospective study by Vescovi et al., combined medical treatment and Low-Level Laser Therapy (LLLT) resulted in higher success rates than patients receiving only medical treatment. Additionally, combining traditional surgery and LLLT yielded better results than conventional surgery alone [20]. Furthermore, a meta-analysis conducted by Momesso et al. reported that laser surgery achieved a success rate of 90% in MRONJ patients. In comparison, the combination of classical surgical treatment and LLLT achieved a 73.6% success rate. Solely relying on surgical treatment resulted in a success rate of 69%, while patients receiving only medical treatment had a notably low success rate of 18% [21].

Moreover, research has explored the effects of lasers on osteoblast cell proliferation and migration, vascularization, and inflammatory cells and has consistently yielded positive and significant results in various in vivo and in vitro settings [22]–[25]. However, comparative studies examining the effects of lasers on MRONJ treatment across different wavelengths are outside our current knowledge. With this background in mind, our study aims to investigate the effects of lasers at four different wavelengths on both hard and soft tissues in treating MRONJ. We will conduct histopathological, radiological, biochemical, and clinical assessments to evaluate the impact of lasers on MRONJ treatment comprehensively.

In our study, it is possible to assert that the clinical assessment conducted on animals before their sacrifice confirms the efficacy of our MRONJ induction protocol. Specifically, following the protocol established in a study by Zandi and colleagues [15], animals in the CONTROL group received intraperitoneal (IP) zoledronic acid for four weeks, followed by tooth extraction. Subsequently, these animals were observed for eight weeks, during which MRONJ typically develops. After 8 weeks, clinical observations in the CONTROL group revealed that 4 out of 8 animals exhibited exposed bone at the extraction site, 2 showed abscess formation during palpation, and 2 displayed areas of disrupted hyperemic mucosa around the extraction socket.

The observations in the SHAM group, where 7 animals exhibited routine healing, further validate the effectiveness of our protocol. Additionally, the substantial disparity in the amount of necrotic bone observed between the CONTROL and SHAM groups, along with significant differences in serum vitamin D levels and average bone volume, suggests the successful induction of MRONJ in the animals. Moreover, while examining the clinical scoring values, it becomes evident that groups utilizing lasers with wavelengths of 660nm and 808nm exhibited results similar to the SHAM group. Contrarily, an analysis of clinical scores suggests that the 405nm and 445nm lasers may not be sufficiently effective for clinical MRONJ treatment. In summary, our study results support the effectiveness of the MRONJ induction protocol, with apparent differences between the CONTROL and SHAM groups. Furthermore, specific laser wavelengths appear more promising in treating MRONJ based on their clinical scoring results. However, the 405nm and 445nm lasers may require further investigation or alternative applications in MRONJ treatment due to their less favorable clinical outcomes.

Based on the results of our study, it is clear that lasers with wavelengths of 660nm and 808nm substantially impacted increasing serum vitamin D levels in rats afflicted with MRONJ. Notably, the 660nm laser surpassed vitamin D levels observed in the SHAM group. However, the precise mechanism underlying the influence of lasers on serum vitamin D levels remains a topic necessitating additional investigation. Likewise, a separate study [26] utilizing a 630nm wavelength laser observed a similar rise in serum vitamin D levels as in our study. Nevertheless, this particular study focused solely on a single wavelength and did not explore the effects of different wavelengths. Given our study's outcomes, it is reasonable to speculate that lasers operating at 660nm and 808nm wavelengths may stimulate vitamin D synthesis.

In recent years, there has been an increase in research examining the connection between vitamin D levels and MRONJ treatment [27]. These investigations have indicated that individuals using antiresorptive medications who have low serum vitamin D levels are at a heightened risk of developing MRONJ and exhibit a positive correlation between vitamin D levels and the severity of the disease [28], [29]. Consequently, considering the notable increase in vitamin D levels within the 660nm and 808nm groups, where the most significant improvements were observed in our study, it is conceivable to propose that vitamin D exerts beneficial effects on MRONJ treatment. One of the remarkable findings from our research is the marked elevation in serum vitamin D levels achieved through applying lasers with specific wavelengths in the context of MRONJ treatment. Further investigations in this domain are warranted better to understand these findings' implications and potential advantages.

In the micro CT analysis of bone density, it is observed that the 660nm group exhibits the highest values. The 808nm wavelength also significantly surpasses the bone density of the SHAM, CONTROL, and 405nm groups. Furthermore, the measurements of average bone volume in the 660nm group are nearly similar to those of the SHAM group. These findings are consistent with the results of clinical scoring and vitamin D levels. This alignment between the micro CT results, clinical scoring, and vitamin D measurements suggests a strong correlation between the laser wavelengths and their effects on bone density and overall outcomes in MRONJ treatment.

In our study, when examining histological sections, we observe significantly higher values for new bone formation in all laser groups compared to the CONTROL and SHAM groups. This finding aligns with existing literature that indicates lasers can enhance osteoblast proliferation and differentiation in cell culture studies [30]–[33]. Moreover, experimental studies on animal models have reported that laser applications stimulate new bone formation [34], [35].

Based on our results, the laser that had the most pronounced effect on new bone formation was the 445nm wavelength laser, so the 445nm wavelength laser stands out positively among the laser groups. However, the 445nm group exhibited the highest values when examining the amount of dead bone. These suggest that while the 445nm wavelength laser has a particularly positive impact on new bone formation, its effect on dead bone quantity should also be considered in the overall evaluation of laser efficacy for MRONJ treatment. Further research is needed to explore the balance between new bone formation and dead bone quantity in different laser wavelengths.

The results indicate that the 660nm group had the lowest amount of dead bone. In this group, the results were even lower than those of the SHAM group, and in the 808nm group, the results were lower than those of the CONTROL group. We do not have any studies explaining why the 660 nm laser reduces the amount of dead bone. However, when we look at the survey conducted by Tani et al. [36] on osteoblast stem cells using lasers with wavelengths of 405, 635, and 808 nm, we understand that only the 635 nm laser increased the production of vinculin protein in osteoblast cells. Vinculin is an essential protein involved in cell-matrix and cell-cell adhesion, and the increase in vinculin activity leads to the increased attachment of osteoblast cells to the inorganic matrix of the bone and each other [37]. This could explain why the amount of dead bone observed in the group using a 650 nm laser in our study was significantly lower compared to the control group.

When considering both the amount of dead bone and the parameter of new bone formation together, the use of 660nm and 808nm wavelengths in MRONJ treatment may lead to successful clinical outcomes.

The group with the least observed inflammatory cell counts was the where 660nm wavelength was applied. This observation supports our study's new bone formation, micro CT, and clinical scoring findings and is also consistent with previous research exploring lasers' effects on inflammation. In an experimental study conducted by Honmura et al.[38], the anti-inflammatory efficacy of diode lasers with a wavelength of 780 nm was demonstrated by measuring TNF-α and IL-1 expression. Huang et al. [39] evaluated the cytological effects of LLLT on bone inflammatory cells in an in vivo setting using a 920 nm laser. They found that the inflammatory markers iNOS, TNF-α, and IL-1 were suppressed in the LLLT-treated groups.

According to a study by Choi et al., lasers with a wavelength of 635 nm were found to suppress inflammation by reducing the release of cytokines that cause inflammation in cells [40]. Lopes-Martins et al. [41]also conducted an experimental study. They found that using a 650 nm laser significantly decreased total leukocyte and neutrophil counts in all experimental groups compared to the control group. In our research, the group with the least observed inflammatory cells was the 660 nm laser group. This finding is consistent with the studies conducted by Lopes-Martins et al. and Choi et al.

In studies investigating the effect of lasers on epithelial regeneration, the effectiveness of lasers with a wavelength of 660 nm and 808 nm is generally mentioned. In line with the data obtained in the Amaroli A[42] and Topaloglu N[43], our study also showed a limited increase in epithelial regeneration in the group where a 660 nm laser was used. Still, the main effect was observed in the group with an 808 nm laser. Additionally, based on the data obtained from our study, the 445 nm laser has a negative effect on epithelial regeneration as it lowers the values even below those of the control group, suggesting a negative impact on an active step in the regeneration mechanism.

One of the most significant limitations of our study is the relatively low number of experimental animals used due to ethical considerations. Additionally, in our research, while we compared the wavelengths of lasers, the dosages used were the same in each group, preventing us from exploring the effects of different doses. Therefore, conducting a study that examines the impact of different doses, especially using the 660nm and 808nm wavelengths, would be highly valuable.

Based on the results of our study, both the 660nm and 808nm wavelengths have positive effects in MRONJ treatment. Since the 660nm wavelength appears more effective in bone tissue, while the 808nm wavelength has more prominent effects in soft tissue, combining both wavelengths may hold promise in MRONJ treatment.

However, the mechanisms through which lasers affect vitamin D levels in the blood and the role of blood vitamin D levels in the development and progression of MRONJ warrant further investigation. Therefore, more research is needed to elucidate laser therapy's mechanisms and potential clinical applications in MRONJ treatment.

ACKNOWLEDGMENTS

Funding: This work was partially supported by the TUBITAK ARDEB SBAG TURKEY [217S985, 2018]

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No competing interests reported.

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WHICH WAVELENGTH OF LASER YIELD THE MOST SUCCESSFUL RESULTS IN MRONJ TREATMENT? AN EXPERIMENTAL STUDY

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Abstract

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1. INTRODUCTION

2. MATERIALS AND METHODS

1. Animal Handling

2. Study Design and Surgical Procedure:

3. Laser Procedure

4. Biochemical Analysis

5. Sacrification

6. Clinical Evaluation

7. Bone Scan Analysis with Micro Computed Tomography (Micro Ct)

8. Histopathological Analysis

9. Statistical Analysis

3. RESULTS

1. Biochemical Findings

2. Clinical Findings in Subjects

3. Radiological Findings

4. Histological Findings

4. DISCUSSION

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