- Email: [email protected]
Comparison of Single Versus Multiple Low-Level Laser Applications on Bone Formation in Extraction Socket Healing in Rabbits (Histologic and Histomorphometric Study)
Comparison of Single Versus Multiple Low-Level Laser Applications on Bone Formation in Extraction Socket Healing in Rabbits (Histologic and Histomorphometric Study) Nesma Mohamed Khalil, BDS, MSc, PhD,* and Marwa G. Noureldin, BDS, MSc, PhDy Purpose:
This study evaluated the effect of low-level laser therapy (LLLT) on bone healing after tooth extraction in healthy rabbits and compared the effect between single and multiple doses of laser therapy.
Materials and Methods:
Thirty-six New Zealand white male rabbits were randomly divided into 3 equal groups: a control (C) group, a single laser (SL) group, and a multiple laser (ML) group. The mandibular right first premolar was extracted. The SL group received a single dose of diode laser immediately after extraction. The ML group received a dose immediately after extraction and then every 72 hours for 12 days. The C group extraction sites were left untreated by laser. Eighteen animals were sacrificed at each of the experimental periods 3 and 6 weeks after extraction. The sockets were removed from the harvested mandibles and prepared for light microscopic examination and histomorphometric analysis.
Results:
The SL and ML groups showed more bone formation and rapid maturation compared with the C group at 3 and 6 weeks postoperatively. At 6 weeks, the SL group showed the formation of compact bone. Furthermore, the ML group exhibited well-vascularized bone marrow spaces. Histomorphometric analysis showed an increase in the percentage of newly formed bone in the SL and ML groups compared with the C group. Moreover, the difference in the percentage of newly formed between the SL and ML groups was not statistically relevant.
Conclusion:
This rabbit model showed that single or multiple diode laser applications can be used to enhance bone formation after tooth extraction. Ó 2019 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 77:1760-1768, 2019
Tooth extraction is the most frequently performed surgical procedure in dentistry. Healing after extraction is a normal straightforward process that occurs in most cases. However, problems can occur and lead to a longer healing period that can be associated with discomfort to the patient.1
A blood clot is an essential step for the subsequent phases of tissue healing. Cells, such as fibroblasts, endothelial cells, macrophages, and osteoblasts, are attracted to the blood clot. These cells are fundamental for the development and maturation of tissue until complete healing is achieved.2
Received from Faculty of Dentistry, Alexandria University,
sity, Champolion Street, Azarita, Alexandria, Egypt; e-mail: nesma_
Alexandria, Egypt. *Lecturer, Department of Oral Biology.
[email protected] Received February 23 2019
yLecturer, Department of Oral and Maxillofacial Surgery.
Accepted March 27 2019
Conflict of Interest Disclosures: Neither author has any relevant
Ó 2019 American Association of Oral and Maxillofacial Surgeons
financial relationship(s) with a commercial interest.
0278-2391/19/30363-5
Address correspondence and reprint requests to Dr Khalil:
https://doi.org/10.1016/j.joms.2019.03.037
Department of Oral Biology, Faculty of Dentistry, Alexandria Univer-
1760
1761
KHALIL AND NOURELDIN
Several studies have described different methods to accelerate the repair process, including mechanical stimulation,3,4 low-intensity ultrasound,5,6 7,8 electromagnetic material, biological growth factors,9 and low-level laser therapy (LLLT).10,11 LLLT can biologically modulate and quicken the healing process by stimulating cell proliferation, vascularization, and bone formation.12 However, the suitable amount of energy required to provide notable new tissue formation with a higher quality of organization within a shorter period remains the most important question and has yet to be answered.13 The outcome of LLLT varies according to treatment parameters, which are power, power density, wavelength, energy, energy density, beam profile, duration, and frequency of treatment.14 The aim of this study was to evaluate the effect of LLLT on bone healing after tooth extraction in healthy rabbits and to compare the effect between single and multiple doses of laser.
Materials and Methods Thirty-six New Zealand white male rabbits (age range, 8 to 10 months; weight, 2 to 2.5 kg) were randomly divided into 3 equal groups (12 rabbits each): a control (C) group, a single laser (SL) group,
and a multiple laser (ML) group. This study was conducted according to the rules of research on experimental animals and approved by the Faculty of Dentistry at the Alexandria University (Alexandria, Egypt; institutional review board number 00010556IORG 0008839). According to MacNeill et al,15 42 days are needed for complete bone healing in rabbits. Therefore, in the present study, bone healing was assessed 3 and 6 weeks after extraction to investigate the first and last stages of bone healing. TOOTH EXTRACTION
The animals received an intramuscular injection of ketamine hydrochloride (35 mg/kg; Rotexmedica, Trittau, Germany) plus xylazine (5 mg/kg; Xyla-ject; Adwia Pharmaceuticals SAE, Cairo, Egypt). After onset of local anesthesia, the lower right first premolars were extracted. A straight elevator was used to luxate the tooth, and then the tooth was carefully rotated and extracted using forceps (Fig 1). The tip of the elevator was pressed against the apical part of the socket to break down the apical germinal tissue to halt tooth regrowth.16 To decrease pain, diclofenac sodium (75 mg/3 mL; Voltaren; GSK, Brentford, UK) was injected intramuscularly. After surgery, the antibiotic
FIGURE 1. Surgical phase. A, Extraction socket. B, Extracted tooth. Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
1762 cefotaxime sodium (500 mg; Cefotax; Atlantic Pharmaceuticals, Inc, Atlanta, GA) was injected for 4 days. LASER APPLICATION
A diode laser device was used in this study (Lambda SpA, Brendola, Italy). Laser parameters were calibrated as 980-nm wavelength, 0.5-W power output, 60-second duration, and continuous wave mode. For the SL and ML groups, the diode laser tip was held in close contact to the socket (Fig 2), allowing all walls to be lasered.16 The SL group received a single dose immediately after extraction. The ML group received a dose immediately after extraction and then every 72 hours for 12 days. Extraction sites in the C group were left untreated by laser. HISTOLOGIC ANALYSIS
Eighteen rabbits (6 from each group) were euthanized with an overdose of ketamine 3 and 6 weeks postoperatively. The mandibles were obtained and split into 2 halves. A saw was used to obtain the socket area of the right mandibular first premolar. Specimens were fixed in 10% neutral buffered formalin, washed, and then decalcified in 10% trichloroacetic acid. After washing, the specimens were dehydrated in ascending grades of alcohol, cleared in xylene, and infiltrated and embedded in paraffin wax. Sections 4 to 5 mm thick were cut buccolingually from the paraffin blocks using a rotary microtome and stained with hematoxylin and eosin.17 Light microscopic examination of the sections was conducted by a calibrated examiner who was blinded to the experimental groups.
FIGURE 2. Laser application to the socket after tooth extraction. Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
LASER FOR BONE FORMATION IN EXTRACTION SOCKET HISTOMORPHOMETRIC ANALYSIS
Histomorphometric evaluation of the newly formed bone was performed using ImageJ 1.46r (National Institutes of Health, Bethesda, MD). Three buccolingual sections from each specimen were obtained at different standardized depths. For each section, a photograph was taken at the apical third of the socket18,19 using the same magnification (100). On each photograph, the total surface area was measured. Then, the bone marrow spaces were outlined using the brush tool from the tool bar. Afterward, all bone marrow spaces were selected using the wand tracing tool and their surface area was measured. To obtain the surface area occupied only by bone, the bone marrow surface area was subtracted from the total area. The same procedure was repeated for the other 2 sections and the mean was obtained. The percentage of newly formed bone was calculated as follows20: % newly formed bone = (bone surface area/total surface area) 100 The same procedure was repeated for each of the 6 samples in each group. The results obtained were expressed as mean and standard deviation. Analysis of variance was used to compare all groups. The significance level was set at .05, and P values less than .05 were statistically significant. SPSS 20.0 (IBM Corp, Armonk, NY) was used for statistical analysis.
Results HISTOLOGIC RESULTS
After 3 Weeks Histologic examination of specimens obtained from the C group showed limited new bone formation radiating from the socket wall. The bone consisted of spicules containing large, numerous, and haphazardly arranged osteocytic lacunae covered by osteoblasts. Some osteoclasts also were seen lining some parts of the bone trabeculae. The bone marrow spaces appeared well vascularized (Fig 3). The SL group exhibited greater bone formation. The bone appeared more mature, with relatively thick cancellous bone trabeculae, which contained numerous parallel resting lines indicating successive bone formation (Fig 4). Similar to the SL group, the ML group exhibited more numerous and mature bony trabeculae compared with the C group. Wellvascularized bone marrow spaces were visible (Fig 5). After 6 Weeks Light microscopic examination of the 6-week specimens showed the formation of more bone compared with the 3-week specimens of the C, SL, and ML groups. In the C group, the socket
1763
KHALIL AND NOURELDIN
FIGURE 3. Light micrographs of socket healing in the control group after 3 weeks. A, Newly formed bone spicules (arrows) can be seen extending from the socket wall for a limited distance toward the center of the socket and surrounding vascularized bone marrow spaces (stars). The central part of the defect contains dense fibrous tissue (arrowheads). B, Higher magnification of the inset in A showing the structure of the bony spicules, which contain numerous osteocytes (short arrows) and are lined by osteoblasts (long arrows). Areas of active bone formation are noted by the high density of bone-forming cells (arrowheads). Upper inset, Plump osteoblasts (arrows). Lower inset, Osteoclast cells on the endosteal surface of bone trabeculae (arrows) (hematoxylin and eosin stain; magnification, 40 in A, 100 in B, 400 in insets). Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
was filled with cancellous bone trabeculae, which appeared relatively thin and disconnected, that surrounded well-vascularized bone marrow spaces. Moreover, the osteocytic lacunae were haphazardly
arranged (Fig 6). In contrast, in the SL and ML groups, more bone formation was observed, which appeared more organized than in the C group. In the SL group, compact and cancellous bone types
FIGURE 4. Light micrographs of the healing socket at 3 weeks in the single laser group. A, A larger amount of newly formed bone is seen extending from the socket wall toward its center. The socket exhibits thin newly formed bony spicules (solid stars), and the deeper portions contain denser and more mature bone trabeculae (hollow stars), which contain numerous resting lines (arrows). Blood vessels containing red blood corpuscles are seen in the bone marrow spaces (arrowheads). B, Higher magnification of the inset in A showing the structure of the woven bone. A line of demarcation (arrows) between the old and newly formed bone is visible. In the deeper areas of the socket, dense fibrous tissue is visible (arrowheads). Inset, Osteoblasts (short arrows) lining the bone surface, osteoclasts (long arrows), and large, irregularly arranged osteocytic lacunae (arrowheads) (hematoxylin and eosin stain; magnification, 40 in A, 100 in B, 400 in insets). Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
1764
LASER FOR BONE FORMATION IN EXTRACTION SOCKET
FIGURE 5. Light micrographs of the healing socket at 3 weeks in the multiple laser group. A, Newly formed bone with different trabecular thicknesses. Some trabeculae appear relatively thin (solid stars), whereas others appear relatively thick (hollow stars). B, Higher magnification of the inset in A showing the structure of the cancellous bone trabeculae surrounding well-vascularized bone marrow spaces (arrows). A line of demarcation is visible between the old and newly formed bone (arrowheads) (hematoxylin and eosin stain; magnification, 40 in A, 100 in B). Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
were seen. The compact bone consisted of small osteons composed of a centrally located Haversian canal and concentric bone lamellae. Relatively plump osteoblasts were seen lining the bone trabeculae (Fig 7). Moreover, in the ML group, thick cancellous bone trabeculae were seen traversing the socket area and contained regularly distributed osteocytes. Bone marrow spaces were well vascularized. Remodeling of bone was evident by the presence of some osteoclasts in their lacunae (Fig 8).
HISTOMORPHOMETRIC ANALYSIS
The percentage of bone surface area in the different groups is presented in Table 1. At 3 weeks, the SL and ML groups showed an increase in the percentage of bone surface area compared with the C group; however, the difference between the SL and C groups was not significant (P = .108). At 6 weeks, there was a statistically significant increase in the percentage of bone surface area in the SL and ML groups compared with the C group (P = .008 and .019, respectively). In addition, no statistically significant difference between the SL and
FIGURE 6. Light micrographs of the socket healing at 6 weeks in the control group. A, Cancellous bone trabeculae fill the defect area especially at the central part of the socket (arrows), whereas other parts appear with relatively wide marrow spaces (arrowheads). B, Higher magnification of the inset in A showing the organization of the bone trabeculae, which appear relatively irregular and discontinuous. Wellvascularized bone marrow spaces (arrows) are seen. Inset, Flattened osteoblasts lining bone surface (arrows) and a relatively irregular arrangement of osteocytes (arrowheads) (hematoxylin and eosin stain; magnification, 40 in A, 100 in B, 400 in inset). Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
1765
KHALIL AND NOURELDIN
FIGURE 7. Light micrographs of the healing socket at 6 weeks in the single laser group. A, Formation of cancellous (solid stars) and compact bone (arrows) filling the socket. B, Higher magnification of the inset in A showing the structure of the compact bone, which consists of many small osteons (arrows). Inset, Continuous layer of osteoblasts (arrows) can be seen lining the bone trabeculae, which appear more voluminous than those seen in control group (hematoxylin and eosin stain; magnification, 40 in A, 100 in B, 400 in inset). Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
ML groups was noted at 3 and 6 weeks postoperatively (P = .873 and .896, respectively).
Discussion LLLT can involve multiple laser applications, which require more time and higher costs compared with a single laser application. Most previous studies investigated the effect of multiple applications of low-level laser on bone healing.21-25 Few studies investigated the effect of a single laser application on bone
healing.26,27 Therefore, the present study was conducted to assess and compare the effect of a single dose versus multiple doses of diode laser on bone repair after tooth extraction in rabbits and whether a single laser application could be sufficient to provide similar effects as multiple laser applications. This was determined histologically and histomorphometrically. The rabbit model was chosen because it is simple to handle compared with rat and mouse models. Moreover, the Haversian systems of rabbits are similar to
FIGURE 8. Light micrographs of the healing socket at 6 weeks in the multiple laser group. A, Formation of relatively thick and interconnected cancellous bone trabeculae (solid stars) occupying the socket area and surrounding marrow spaces of different sizes and obvious vascularity (arrows). B, Higher magnification of the inset in A showing the organization of the bony trabeculae with relatively regularly distributed osteocytes (short arrows) and osteoblasts (long arrows) lining the outer surface of the trabeculae. Numerous blood vessels are visible (arrowheads). Inset, Osteoclasts (arrows) in Howship lacunae (hematoxylin and eosin stain; magnification, 40 in A, 100 in B, 400 in inset). Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
1766
LASER FOR BONE FORMATION IN EXTRACTION SOCKET
Table 1. COMPARISON AMONG THE 3 STUDIED GROUPS ACCORDING TO PERCENTAGE OF BONE SURFACE AREA
Control Group (n = 6) 3 wk Significance among groups 6 wk Significance among groups P0
57.9 8.1 P1 = .108, P2 = .043*, P3 = .873 68.7 4.8 P1 = .008*, P2 = .019*, P3 = .896 .018*
Single Laser Group (n = 6)
Multiple Laser Group (n = 6)
F Value
P Value
66.0 5.6
67.8 5.3
4.045*
.039*
80.0 4.4
78.6 7.1
7.483*
.006*
.001*
.014*
Note: Analysis of variance was used to compare F values, and pairwise comparison between 2 groups was performed using the Tukey post hoc test. The P value compared the 3 study groups. Abbreviations: P0, P value by Student t test for 3 versus 6 weeks; P1, P value for control versus single-laser group; P2, P value for control versus multiple-laser group; P3, P value for single-laser versus multiple-laser group. * Statistically significant at P # .05. Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
those of humans, which is a vital advantage in terms of using the outcomes obtained with such animals for understanding human bone healing.28 LLLT involves the use of low levels of red and near infrared light for therapeutic purposes. It also is called ‘‘cold laser’’ because it does not produce heat in the penetrated tissues. The wavelength range of LLLT is 600 to 1,070 nm.29 Shorter wavelengths (<700 nm) are used for superficial tissues, and longer wavelengths have deeper penetration and a greater biological stimulatory effect.29-33 Therefore, in this study the wavelength of the diode laser used was 980 nm to ensure better penetration of the socket after extraction. In the present work, results of the single and multiple diode laser applications showed enhanced new bone formation and rapid maturation after tooth extraction. In the C group after 3 weeks, the bone consisted mainly of woven bone. However, in the SL and ML groups, mature bone was seen, which showed more thick cancellous bone trabeculae. In addition, at 6 weeks the osteoblasts lining the bone trabeculae in the SL group appeared more voluminous compared with the C group. This was in agreement with the report by Mirdan27 who used a single 980-nm dose of diode laser on rabbit dental sockets and concluded that there was stimulation and facilitation of the bone healing process in addition to its effect on coagulation of the blood, which provided a dressing for the wound site. This enhancement of new bone formation can be explained by the stimulatory effect of laser on the function of osteoblasts. Several studies found that LLLT was effective in the stimulation of osteoblastic
differentiation.34-36 Furthermore, laser can increase the expression of osteocalcin and several growth factors, including transforming growth factor-b1 and bone morphogenetic protein-2 from osteoblasts.37 In addition, histologic observations of the present work showed that LLLT at 6 weeks led to enhanced bone formation, which exhibited regularly distributed osteocytic lacunae, compared with the C group, which exhibited haphazardly arranged osteocytes. This is in accordance with the work of Tas Deynek and Ramoglu25 who found that laser application to expanded sutures in rats led to marked increases in osteoblasts and osteocytes compared with the control group. The effect of LLLT on cells is known as biostimulation. When the light reaches the cell, it is absorbed by a certain chromophore inside the mitochondria.29 This leads to excitation of electrons,38 controls reactive oxygen species production, increases the production of adenosine triphosphate,39 and upregulates the production of transcription factors.40 These transcription factors are key molecules for the regulation of other cellular activities, including the proliferation, differentiation, and secretion of growth factors and cytokines.30 Contrary to the present results, Bouvet-Gerbettaz et al41 and Coombe et al42 found that laser had no beneficial effect on the proliferation and differentiation of osteoblasts. Moreover, Atasoy et al23 studied the effect of multiple LLLT irradiation on tibia bone defects in rats and found no relevant difference in the number of osteoblasts, osteocytes, and osteoclasts and new blood vessel formation between the control and laser-irradiated groups. These conflicting
1767
KHALIL AND NOURELDIN
outcomes of LLLT application on bone repair could have been caused by the variation of the physical parameters used by different researchers. The present histologic observations showed the presence of some osteoclasts on the endostea surface of bone trabeculae in the SL group at 3 weeks and in the ML group at 6 weeks. Schell et al43 reported that osteoclasts play an important role in all phases of bone healing. These cells not only resorb bone but also maintain the structural integrity of bone and its physiologic remodeling throughout life. Furthermore, Cackowski et al44 proved that osteoclasts can stimulate angiogenesis through the secretion of matrix metalloproteinase-9 (MMP-9). In adults, MMP-9 is mainly produced by osteoclasts and to a lesser extent by cells such as osteoblasts, macrophages, and neutrophils.45-47 One of the suggested mechanisms for the role of MMP-9 in enhancing angiogenesis is that it can release growth factors such as vascular endothelial growth factor from the bone matrix.48-50 Light microscopic observations of the ML group in the present study showed well-vascularized bone marrow spaces at 3 and 6 weeks and in the SL group at 3 weeks. Good vascularity is important during bone healing. This finding corresponds to the results of Tim et al51 who investigated the effect of LLLT (830 nm) on angiogenic genes in rat tibia bone defects. They concluded that laser increased the expression of angiogenic genes, such as angiopoietin-2 and -4, platelet-derived growth factor, and fibroblast growth factors-2 and -14, especially during the early stages of bone healing. Moreover, Garavello et al52 reported on the dual action of low-level laser on angiogenesis in tibia bone defect healing in rats. They found an increase in the number of blood vessels in the irradiated group after 1 week; however, after 2 weeks there was a notable decrease in the blood vessel count. In the authors’ opinion, this could be attributed to the shorter wavelength used in that study (633 nm), resulting in insufficient laser penetration. In the present study, histologic qualitative analysis showed the formation of compact bone in the SL group at 6 weeks. This indicates the rapid and enhanced bone remodeling and structural organization of the formed bone inside the socket after laser irradiation. Histomorphometric analysis of the present work was accomplished using ImageJ 1.46r software. Histomorphometry is used to evaluate bone healing and provide a numerical assessment to facilitate comparison among different groups.18 ImageJ has many advantages and is considered a reliable and inexpensive software for the evaluation of changes in different tissue structures.53 The present histomorphometric analysis confirmed the light microscopic observations, in which an increase in the percentage of bone surface
area was noted in the SL and ML groups compared with the C group at the 2 observational periods. However, the increase was not relevant in the SL group at 3 weeks. In addition, no relevant difference was detected between the SL and ML groups at the 2 observational periods. Furthermore, at 6 weeks, the SL group showed a larger percentage of bone surface area compared with the ML group (80 and 78.6%, respectively), but the difference was not significant (P = .896). These results suggest that the single laser application can be effective in enhancing bone formation compared with the multiple laser application. These findings corroborate those of Pretel et al26 who concluded that the application of diode laser at a single dose directly to surgically created bone defects in rats abbreviated the bone healing process and was effective in accelerating bone repair compared with non-lasered surgical wounds. However, Ng et al13 compared single versus multiple laser applications on the repair of the medial collateral ligament and stated that multiple laser treatments produced better outcomes compared with a single treatment using similar parameters. In conclusion, based on the present results, laser irradiation after tooth extraction can be effective in enhancing bone formation in the rabbit model. This can be attributed to its stimulatory effect on boneforming cells and enhanced angiogenesis. Furthermore, the single laser application can be as effective as multiple laser applications. This could be an important advantage in clinical practice to save time and lower the cost of several laser irradiations.
References 1. Blum IR: Contemporary views on dry socket (alveolar osteitis): A clinical appraisal of standardization, aetiopathogenesis and management: A critical review. Int J Oral Maxillofac Surg 31:309, 2002 2. Okamoto T, Okamoto R, Alves Rezende MC, Gabrielli MF: Interference of the blood clot on granulation tissue formation after tooth extraction. Histomorphological study in rats. Braz Dent J 5:85, 1994 3. Rubin CT, Lanyon LE: Regulation of bone mass by mechanical strain magnitude. Calcif Tissue Int 37:411, 1985 4. Turner CH, Forwood MR, Rho JY, Yoshikawa T: Mechanical loading thresholds for lamellar and woven bone formation. J Bone Miner Res 9:87, 1994 5. Hasuike A, Sato S, Udagawa A, et al: In vivo bone regenerative effect of low-intensity pulsed ultrasound in rat calvarial defects. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 111:e12, 2011 6. Fındık Y, Baykul T: Effects of low-intensity pulsed ultrasound on autogenous bone graft healing. Oral Surg Oral Med Oral Pathol Oral Radiol 117:e255, 2014 7. Kooistra BW, Jain A, Hanson BP: Electrical stimulation: Nonunions. Indian J Orthop 43:149, 2009 8. Adie S, Harris IA, Naylor JM, et al: Pulsed electromagnetic field stimulation for acute tibial shaft fractures: A multicenter, double-blind, randomized trial. J Bone Joint Surg Am 93:1569, 2011 9. McCarthy TL, Ji C, Centrella M: Links among growth factors, hormones, and nuclear factors with essential roles in bone formation. Crit Rev Oral Biol 11:409, 2000
1768
LASER FOR BONE FORMATION IN EXTRACTION SOCKET
10. Kazancioglu HO, Ezirganli S, Aydin MS: Effects of laser and ozone therapies on bone healing in the calvarial defects. J Craniofac Surg 24:2141, 2013 11. Nagata MJH, Santinoni CS, Pola NM, et al: Bone marrow aspirate combined with low-level laser therapy: A new therapeutic approach to enhance bone healing. J Photochem Photobiol B 121:6, 2013 12. Kawasaki K, Shimizu N: Effects of low-energy laser irradiation on bone remodeling during experimental tooth movement in rats. Lasers Surg Med 26:282, 2000 13. Ng GYF, Fung DTC, Leung MCP, Guo X: Ultrastructural comparison of medial collateral ligament repair after single or multiple applications of GaAlAs laser in rats. Lasers Surg Med 35: 317, 2004 14. Woodruff LD, Bounkeo JM, Brannon WM, et al: The efficacy of laser therapy in wound repair: A meta-analysis of the literature. Photomed Laser Surg 22:241, 2004 15. MacNeill SR, Cobb CM, Rapley JW, et al: In vivo comparison of synthetic osseous graft materials. A preliminary study. J Clin Periodontol 26:239, 1999 16. Hamad SA, Naif JS, Abdullah MA: Effect of diode laser on healing of tooth extraction socket: An experimental study in rabbits. J Maxillofac Oral Surg 15:308, 2016 17. Feldman AT, Wolfe D: Tissue processing and hematoxylin and eosin staining. Methods Mol Biol 1180:31, 2014 18. Gorustovich AA, Steimetz T, Nielsen FH, Guglielmotti MB: Histomorphometric study of alveolar bone healing in rats fed a borondeficient diet. Anat Rec 291:441, 2008 19. Steimetz T, Gorustovich AA, Collet AM, et al: Histologic and histomorphometric study of bone repair under acute Trypanosoma cruzi infection in rats. Acta Odontol Latinoam 25:193, 2012 20. Chen J, He Y, Shan C, et al: Topical combined application of dexamethasone, vitamin C, and b-sodium glycerophosphate for healing the extraction socket in rabbits. Int J Oral Maxillofac Surg 44:1317, 2015 21. Son J, Kim Y-B, Ge Z, et al: Bone healing effects of diode laser (808 nm) on a rat tibial fracture model. Vivo 26:703, 2012 22. do Nascimento MF, de Almeida BM, Cunha JLS, et al: Improvement of bone repair in diabetic rats subjected to ƛ780 nm lowlevel laser therapy. Acta Cir Bras 30:660, 2015 23. Atasoy KT, Korkmaz YT, Odaci E, Hanci H: The efficacy of lowlevel 940 nm laser therapy with different energy intensities on bone healing. Braz Oral Res 31:e7, 2017 24. Gurler G, Gursoy B: Investigation of effects of low level laser therapy in distraction osteogenesis. J Stomatol Oral Maxillofac Surg 119:469, 2018 25. Tas Deynek G, Ramoglu SI: Effects of different settings for 940 nm diode laser on expanded suture in rats. Angle Orthod; https://doi.org/10.2319/052318-392.1, January 2, 2019 [Epub ahead of print] 26. Pretel H, Lizarelli RFZ, Ramalho LTO: Effect of low-level laser therapy on bone repair: Histological study in rats. Lasers Surg Med 39:788, 2007 27. Mirdan BM: A 980nm diode laser clot formation of the rabbit’s dental sockets after teeth extraction. Iraqi J Laser 11: 37, 2018 28. Nunamaker DM: Experimental models of fracture repair. Clin Orthop 355(suppl):S56, 1998 29. Chung H, Dai T, Sharma SK, et al: The nuts and bolts of low-level laser (light) therapy. Ann Biomed Eng 40:516, 2012 30. Karu TI, Kolyakov SF: Exact action spectra for cellular responses relevant to phototherapy. Photomed Laser Surg 23:355, 2005 31. Medina-Huertas R, Manzano-Moreno FJ, De Luna-Bertos E, et al: The effects of low-level diode laser irradiation on differentiation, antigenic profile, and phagocytic capacity of osteoblast-like cells (MG-63). Lasers Med Sci 29:1479, 2014 32. Park JJ, Kang KL: Effect of 980-nm GaAlAs diode laser irradiation on healing of extraction sockets in streptozotocin-induced diabetic rats: A pilot study. Lasers Med Sci 27:223, 2012
33. Pires-Oliveira DA, Oliveira RF, Amadei SU, et al: Laser 904 nm action on bone repair in rats with osteoporosis. Osteoporos Int 21: 2109, 2010 34. Petri AD, Teixeira LN, Crippa GE, et al: Effects of low-level laser therapy on human osteoblastic cells grown on titanium. Braz Dent J 21:491, 2010 35. Soleimani M, Abbasnia E, Fathi M, et al: The effects of low-level laser irradiation on differentiation and proliferation of human bone marrow mesenchymal stem cells into neurons and osteoblasts—An in vitro study. Lasers Med Sci 27:423, 2012 36. Saygun I, Nizam N, Ural AU, et al: Low-level laser irradiation affects the release of basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), and receptor of IGF-I (IGFBP3) from osteoblasts. Photomed Laser Surg 30:149, 2012 37. Pyo S-J, Song W-W, Kim I-R, et al: Low-level laser therapy induces the expressions of BMP-2, osteocalcin, and TGF-b1 in hypoxiccultured human osteoblasts. Lasers Med Sci 28:543, 2013 38. Sutherland JC: Biological effects of polychromatic light. Photochem Photobiol 76:164, 2002 39. Karu T: Primary and secondary mechanisms of action of visible to near-IR radiation on cells. J Photochem Photobiol B 49:1, 1999 40. Chen AC-H, Arany PR, Huang Y-Y, et al: Low-level laser therapy activates NF-kB via generation of reactive oxygen species in mouse embryonic fibroblasts. PLoS One 6:e22453, 2011 41. Bouvet-Gerbettaz S, Merigo E, Rocca J-P, et al: Effects of low-level laser therapy on proliferation and differentiation of murine bone marrow cells into osteoblasts and osteoclasts. Lasers Surg Med 41:291, 2009 42. Coombe AR, Ho CT, Darendeliler MA, et al: The effects of low level laser irradiation on osteoblastic cells. Clin Orthod Res 4: 3, 2001 43. Schell H, Lienau J, Epari DR, et al: Osteoclastic activity begins early and increases over the course of bone healing. Bone 38: 547, 2006 44. Cackowski FC, Anderson JL, Patrene KD, et al: Osteoclasts are important for bone angiogenesis. Blood 115:140, 2010 45. Colnot C, Thompson Z, Miclau T, et al: Altered fracture repair in the absence of MMP9. Development 130:4123, 2003 46. Haeusler G, Walter I, Helmreich M, Egerbacher M: Localization of matrix metalloproteinases (MMPs), their tissue inhibitors, and vascular endothelial growth factor (VEGF) in growth plates of children and adolescents indicates a role for MMPs in human postnatal growth and skeletal maturation. Calcif Tissue Int 76: 326, 2005 47. Vu TH, Shipley JM, Bergers G, et al: MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93:411, 1998 48. Bergers G, Brekken R, McMahon G, et al: Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol 2:737, 2000 49. Engsig MT, Chen QJ, Vu TH, et al: Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J Cell Biol 151:879, 2000 50. Ishibashi O, Niwa S, Kadoyama K, Inui T: MMP-9 antisense oligodeoxynucleotide exerts an inhibitory effect on osteoclastic bone resorption by suppressing cell migration. Life Sci 79: 1657, 2006 51. Tim CR, Bossini PS, Kido HW, et al: Effects of low level laser therapy on inflammatory and angiogenic gene expression during the process of bone healing: A microarray analysis. J Photochem Photobiol B 154:8, 2016 52. Garavello I, Baranauskas V, da Cruz-H€ ofling MA: The effects of low laser irradiation on angiogenesis in injured rat tibiae. Histol Histopathol 19:43, 2004 53. Egan KP, Brennan TA, Pignolo RJ: Bone histomorphometry using free and commonly available software. Histopathology 61: 1168, 2012
This study evaluated the effect of low-level laser therapy (LLLT) on bone healing after tooth extraction in healthy rabbits and compared the effect between single and multiple doses of laser therapy.
Materials and Methods:
Thirty-six New Zealand white male rabbits were randomly divided into 3 equal groups: a control (C) group, a single laser (SL) group, and a multiple laser (ML) group. The mandibular right first premolar was extracted. The SL group received a single dose of diode laser immediately after extraction. The ML group received a dose immediately after extraction and then every 72 hours for 12 days. The C group extraction sites were left untreated by laser. Eighteen animals were sacrificed at each of the experimental periods 3 and 6 weeks after extraction. The sockets were removed from the harvested mandibles and prepared for light microscopic examination and histomorphometric analysis.
Results:
The SL and ML groups showed more bone formation and rapid maturation compared with the C group at 3 and 6 weeks postoperatively. At 6 weeks, the SL group showed the formation of compact bone. Furthermore, the ML group exhibited well-vascularized bone marrow spaces. Histomorphometric analysis showed an increase in the percentage of newly formed bone in the SL and ML groups compared with the C group. Moreover, the difference in the percentage of newly formed between the SL and ML groups was not statistically relevant.
Conclusion:
This rabbit model showed that single or multiple diode laser applications can be used to enhance bone formation after tooth extraction. Ó 2019 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 77:1760-1768, 2019
Tooth extraction is the most frequently performed surgical procedure in dentistry. Healing after extraction is a normal straightforward process that occurs in most cases. However, problems can occur and lead to a longer healing period that can be associated with discomfort to the patient.1
A blood clot is an essential step for the subsequent phases of tissue healing. Cells, such as fibroblasts, endothelial cells, macrophages, and osteoblasts, are attracted to the blood clot. These cells are fundamental for the development and maturation of tissue until complete healing is achieved.2
Received from Faculty of Dentistry, Alexandria University,
sity, Champolion Street, Azarita, Alexandria, Egypt; e-mail: nesma_
Alexandria, Egypt. *Lecturer, Department of Oral Biology.
[email protected] Received February 23 2019
yLecturer, Department of Oral and Maxillofacial Surgery.
Accepted March 27 2019
Conflict of Interest Disclosures: Neither author has any relevant
Ó 2019 American Association of Oral and Maxillofacial Surgeons
financial relationship(s) with a commercial interest.
0278-2391/19/30363-5
Address correspondence and reprint requests to Dr Khalil:
https://doi.org/10.1016/j.joms.2019.03.037
Department of Oral Biology, Faculty of Dentistry, Alexandria Univer-
1760
1761
KHALIL AND NOURELDIN
Several studies have described different methods to accelerate the repair process, including mechanical stimulation,3,4 low-intensity ultrasound,5,6 7,8 electromagnetic material, biological growth factors,9 and low-level laser therapy (LLLT).10,11 LLLT can biologically modulate and quicken the healing process by stimulating cell proliferation, vascularization, and bone formation.12 However, the suitable amount of energy required to provide notable new tissue formation with a higher quality of organization within a shorter period remains the most important question and has yet to be answered.13 The outcome of LLLT varies according to treatment parameters, which are power, power density, wavelength, energy, energy density, beam profile, duration, and frequency of treatment.14 The aim of this study was to evaluate the effect of LLLT on bone healing after tooth extraction in healthy rabbits and to compare the effect between single and multiple doses of laser.
Materials and Methods Thirty-six New Zealand white male rabbits (age range, 8 to 10 months; weight, 2 to 2.5 kg) were randomly divided into 3 equal groups (12 rabbits each): a control (C) group, a single laser (SL) group,
and a multiple laser (ML) group. This study was conducted according to the rules of research on experimental animals and approved by the Faculty of Dentistry at the Alexandria University (Alexandria, Egypt; institutional review board number 00010556IORG 0008839). According to MacNeill et al,15 42 days are needed for complete bone healing in rabbits. Therefore, in the present study, bone healing was assessed 3 and 6 weeks after extraction to investigate the first and last stages of bone healing. TOOTH EXTRACTION
The animals received an intramuscular injection of ketamine hydrochloride (35 mg/kg; Rotexmedica, Trittau, Germany) plus xylazine (5 mg/kg; Xyla-ject; Adwia Pharmaceuticals SAE, Cairo, Egypt). After onset of local anesthesia, the lower right first premolars were extracted. A straight elevator was used to luxate the tooth, and then the tooth was carefully rotated and extracted using forceps (Fig 1). The tip of the elevator was pressed against the apical part of the socket to break down the apical germinal tissue to halt tooth regrowth.16 To decrease pain, diclofenac sodium (75 mg/3 mL; Voltaren; GSK, Brentford, UK) was injected intramuscularly. After surgery, the antibiotic
FIGURE 1. Surgical phase. A, Extraction socket. B, Extracted tooth. Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
1762 cefotaxime sodium (500 mg; Cefotax; Atlantic Pharmaceuticals, Inc, Atlanta, GA) was injected for 4 days. LASER APPLICATION
A diode laser device was used in this study (Lambda SpA, Brendola, Italy). Laser parameters were calibrated as 980-nm wavelength, 0.5-W power output, 60-second duration, and continuous wave mode. For the SL and ML groups, the diode laser tip was held in close contact to the socket (Fig 2), allowing all walls to be lasered.16 The SL group received a single dose immediately after extraction. The ML group received a dose immediately after extraction and then every 72 hours for 12 days. Extraction sites in the C group were left untreated by laser. HISTOLOGIC ANALYSIS
Eighteen rabbits (6 from each group) were euthanized with an overdose of ketamine 3 and 6 weeks postoperatively. The mandibles were obtained and split into 2 halves. A saw was used to obtain the socket area of the right mandibular first premolar. Specimens were fixed in 10% neutral buffered formalin, washed, and then decalcified in 10% trichloroacetic acid. After washing, the specimens were dehydrated in ascending grades of alcohol, cleared in xylene, and infiltrated and embedded in paraffin wax. Sections 4 to 5 mm thick were cut buccolingually from the paraffin blocks using a rotary microtome and stained with hematoxylin and eosin.17 Light microscopic examination of the sections was conducted by a calibrated examiner who was blinded to the experimental groups.
FIGURE 2. Laser application to the socket after tooth extraction. Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
LASER FOR BONE FORMATION IN EXTRACTION SOCKET HISTOMORPHOMETRIC ANALYSIS
Histomorphometric evaluation of the newly formed bone was performed using ImageJ 1.46r (National Institutes of Health, Bethesda, MD). Three buccolingual sections from each specimen were obtained at different standardized depths. For each section, a photograph was taken at the apical third of the socket18,19 using the same magnification (100). On each photograph, the total surface area was measured. Then, the bone marrow spaces were outlined using the brush tool from the tool bar. Afterward, all bone marrow spaces were selected using the wand tracing tool and their surface area was measured. To obtain the surface area occupied only by bone, the bone marrow surface area was subtracted from the total area. The same procedure was repeated for the other 2 sections and the mean was obtained. The percentage of newly formed bone was calculated as follows20: % newly formed bone = (bone surface area/total surface area) 100 The same procedure was repeated for each of the 6 samples in each group. The results obtained were expressed as mean and standard deviation. Analysis of variance was used to compare all groups. The significance level was set at .05, and P values less than .05 were statistically significant. SPSS 20.0 (IBM Corp, Armonk, NY) was used for statistical analysis.
Results HISTOLOGIC RESULTS
After 3 Weeks Histologic examination of specimens obtained from the C group showed limited new bone formation radiating from the socket wall. The bone consisted of spicules containing large, numerous, and haphazardly arranged osteocytic lacunae covered by osteoblasts. Some osteoclasts also were seen lining some parts of the bone trabeculae. The bone marrow spaces appeared well vascularized (Fig 3). The SL group exhibited greater bone formation. The bone appeared more mature, with relatively thick cancellous bone trabeculae, which contained numerous parallel resting lines indicating successive bone formation (Fig 4). Similar to the SL group, the ML group exhibited more numerous and mature bony trabeculae compared with the C group. Wellvascularized bone marrow spaces were visible (Fig 5). After 6 Weeks Light microscopic examination of the 6-week specimens showed the formation of more bone compared with the 3-week specimens of the C, SL, and ML groups. In the C group, the socket
1763
KHALIL AND NOURELDIN
FIGURE 3. Light micrographs of socket healing in the control group after 3 weeks. A, Newly formed bone spicules (arrows) can be seen extending from the socket wall for a limited distance toward the center of the socket and surrounding vascularized bone marrow spaces (stars). The central part of the defect contains dense fibrous tissue (arrowheads). B, Higher magnification of the inset in A showing the structure of the bony spicules, which contain numerous osteocytes (short arrows) and are lined by osteoblasts (long arrows). Areas of active bone formation are noted by the high density of bone-forming cells (arrowheads). Upper inset, Plump osteoblasts (arrows). Lower inset, Osteoclast cells on the endosteal surface of bone trabeculae (arrows) (hematoxylin and eosin stain; magnification, 40 in A, 100 in B, 400 in insets). Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
was filled with cancellous bone trabeculae, which appeared relatively thin and disconnected, that surrounded well-vascularized bone marrow spaces. Moreover, the osteocytic lacunae were haphazardly
arranged (Fig 6). In contrast, in the SL and ML groups, more bone formation was observed, which appeared more organized than in the C group. In the SL group, compact and cancellous bone types
FIGURE 4. Light micrographs of the healing socket at 3 weeks in the single laser group. A, A larger amount of newly formed bone is seen extending from the socket wall toward its center. The socket exhibits thin newly formed bony spicules (solid stars), and the deeper portions contain denser and more mature bone trabeculae (hollow stars), which contain numerous resting lines (arrows). Blood vessels containing red blood corpuscles are seen in the bone marrow spaces (arrowheads). B, Higher magnification of the inset in A showing the structure of the woven bone. A line of demarcation (arrows) between the old and newly formed bone is visible. In the deeper areas of the socket, dense fibrous tissue is visible (arrowheads). Inset, Osteoblasts (short arrows) lining the bone surface, osteoclasts (long arrows), and large, irregularly arranged osteocytic lacunae (arrowheads) (hematoxylin and eosin stain; magnification, 40 in A, 100 in B, 400 in insets). Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
1764
LASER FOR BONE FORMATION IN EXTRACTION SOCKET
FIGURE 5. Light micrographs of the healing socket at 3 weeks in the multiple laser group. A, Newly formed bone with different trabecular thicknesses. Some trabeculae appear relatively thin (solid stars), whereas others appear relatively thick (hollow stars). B, Higher magnification of the inset in A showing the structure of the cancellous bone trabeculae surrounding well-vascularized bone marrow spaces (arrows). A line of demarcation is visible between the old and newly formed bone (arrowheads) (hematoxylin and eosin stain; magnification, 40 in A, 100 in B). Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
were seen. The compact bone consisted of small osteons composed of a centrally located Haversian canal and concentric bone lamellae. Relatively plump osteoblasts were seen lining the bone trabeculae (Fig 7). Moreover, in the ML group, thick cancellous bone trabeculae were seen traversing the socket area and contained regularly distributed osteocytes. Bone marrow spaces were well vascularized. Remodeling of bone was evident by the presence of some osteoclasts in their lacunae (Fig 8).
HISTOMORPHOMETRIC ANALYSIS
The percentage of bone surface area in the different groups is presented in Table 1. At 3 weeks, the SL and ML groups showed an increase in the percentage of bone surface area compared with the C group; however, the difference between the SL and C groups was not significant (P = .108). At 6 weeks, there was a statistically significant increase in the percentage of bone surface area in the SL and ML groups compared with the C group (P = .008 and .019, respectively). In addition, no statistically significant difference between the SL and
FIGURE 6. Light micrographs of the socket healing at 6 weeks in the control group. A, Cancellous bone trabeculae fill the defect area especially at the central part of the socket (arrows), whereas other parts appear with relatively wide marrow spaces (arrowheads). B, Higher magnification of the inset in A showing the organization of the bone trabeculae, which appear relatively irregular and discontinuous. Wellvascularized bone marrow spaces (arrows) are seen. Inset, Flattened osteoblasts lining bone surface (arrows) and a relatively irregular arrangement of osteocytes (arrowheads) (hematoxylin and eosin stain; magnification, 40 in A, 100 in B, 400 in inset). Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
1765
KHALIL AND NOURELDIN
FIGURE 7. Light micrographs of the healing socket at 6 weeks in the single laser group. A, Formation of cancellous (solid stars) and compact bone (arrows) filling the socket. B, Higher magnification of the inset in A showing the structure of the compact bone, which consists of many small osteons (arrows). Inset, Continuous layer of osteoblasts (arrows) can be seen lining the bone trabeculae, which appear more voluminous than those seen in control group (hematoxylin and eosin stain; magnification, 40 in A, 100 in B, 400 in inset). Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
ML groups was noted at 3 and 6 weeks postoperatively (P = .873 and .896, respectively).
Discussion LLLT can involve multiple laser applications, which require more time and higher costs compared with a single laser application. Most previous studies investigated the effect of multiple applications of low-level laser on bone healing.21-25 Few studies investigated the effect of a single laser application on bone
healing.26,27 Therefore, the present study was conducted to assess and compare the effect of a single dose versus multiple doses of diode laser on bone repair after tooth extraction in rabbits and whether a single laser application could be sufficient to provide similar effects as multiple laser applications. This was determined histologically and histomorphometrically. The rabbit model was chosen because it is simple to handle compared with rat and mouse models. Moreover, the Haversian systems of rabbits are similar to
FIGURE 8. Light micrographs of the healing socket at 6 weeks in the multiple laser group. A, Formation of relatively thick and interconnected cancellous bone trabeculae (solid stars) occupying the socket area and surrounding marrow spaces of different sizes and obvious vascularity (arrows). B, Higher magnification of the inset in A showing the organization of the bony trabeculae with relatively regularly distributed osteocytes (short arrows) and osteoblasts (long arrows) lining the outer surface of the trabeculae. Numerous blood vessels are visible (arrowheads). Inset, Osteoclasts (arrows) in Howship lacunae (hematoxylin and eosin stain; magnification, 40 in A, 100 in B, 400 in inset). Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
1766
LASER FOR BONE FORMATION IN EXTRACTION SOCKET
Table 1. COMPARISON AMONG THE 3 STUDIED GROUPS ACCORDING TO PERCENTAGE OF BONE SURFACE AREA
Control Group (n = 6) 3 wk Significance among groups 6 wk Significance among groups P0
57.9 8.1 P1 = .108, P2 = .043*, P3 = .873 68.7 4.8 P1 = .008*, P2 = .019*, P3 = .896 .018*
Single Laser Group (n = 6)
Multiple Laser Group (n = 6)
F Value
P Value
66.0 5.6
67.8 5.3
4.045*
.039*
80.0 4.4
78.6 7.1
7.483*
.006*
.001*
.014*
Note: Analysis of variance was used to compare F values, and pairwise comparison between 2 groups was performed using the Tukey post hoc test. The P value compared the 3 study groups. Abbreviations: P0, P value by Student t test for 3 versus 6 weeks; P1, P value for control versus single-laser group; P2, P value for control versus multiple-laser group; P3, P value for single-laser versus multiple-laser group. * Statistically significant at P # .05. Khalil and Noureldin. Laser for Bone Formation in Extraction Socket. J Oral Maxillofac Surg 2019.
those of humans, which is a vital advantage in terms of using the outcomes obtained with such animals for understanding human bone healing.28 LLLT involves the use of low levels of red and near infrared light for therapeutic purposes. It also is called ‘‘cold laser’’ because it does not produce heat in the penetrated tissues. The wavelength range of LLLT is 600 to 1,070 nm.29 Shorter wavelengths (<700 nm) are used for superficial tissues, and longer wavelengths have deeper penetration and a greater biological stimulatory effect.29-33 Therefore, in this study the wavelength of the diode laser used was 980 nm to ensure better penetration of the socket after extraction. In the present work, results of the single and multiple diode laser applications showed enhanced new bone formation and rapid maturation after tooth extraction. In the C group after 3 weeks, the bone consisted mainly of woven bone. However, in the SL and ML groups, mature bone was seen, which showed more thick cancellous bone trabeculae. In addition, at 6 weeks the osteoblasts lining the bone trabeculae in the SL group appeared more voluminous compared with the C group. This was in agreement with the report by Mirdan27 who used a single 980-nm dose of diode laser on rabbit dental sockets and concluded that there was stimulation and facilitation of the bone healing process in addition to its effect on coagulation of the blood, which provided a dressing for the wound site. This enhancement of new bone formation can be explained by the stimulatory effect of laser on the function of osteoblasts. Several studies found that LLLT was effective in the stimulation of osteoblastic
differentiation.34-36 Furthermore, laser can increase the expression of osteocalcin and several growth factors, including transforming growth factor-b1 and bone morphogenetic protein-2 from osteoblasts.37 In addition, histologic observations of the present work showed that LLLT at 6 weeks led to enhanced bone formation, which exhibited regularly distributed osteocytic lacunae, compared with the C group, which exhibited haphazardly arranged osteocytes. This is in accordance with the work of Tas Deynek and Ramoglu25 who found that laser application to expanded sutures in rats led to marked increases in osteoblasts and osteocytes compared with the control group. The effect of LLLT on cells is known as biostimulation. When the light reaches the cell, it is absorbed by a certain chromophore inside the mitochondria.29 This leads to excitation of electrons,38 controls reactive oxygen species production, increases the production of adenosine triphosphate,39 and upregulates the production of transcription factors.40 These transcription factors are key molecules for the regulation of other cellular activities, including the proliferation, differentiation, and secretion of growth factors and cytokines.30 Contrary to the present results, Bouvet-Gerbettaz et al41 and Coombe et al42 found that laser had no beneficial effect on the proliferation and differentiation of osteoblasts. Moreover, Atasoy et al23 studied the effect of multiple LLLT irradiation on tibia bone defects in rats and found no relevant difference in the number of osteoblasts, osteocytes, and osteoclasts and new blood vessel formation between the control and laser-irradiated groups. These conflicting
1767
KHALIL AND NOURELDIN
outcomes of LLLT application on bone repair could have been caused by the variation of the physical parameters used by different researchers. The present histologic observations showed the presence of some osteoclasts on the endostea surface of bone trabeculae in the SL group at 3 weeks and in the ML group at 6 weeks. Schell et al43 reported that osteoclasts play an important role in all phases of bone healing. These cells not only resorb bone but also maintain the structural integrity of bone and its physiologic remodeling throughout life. Furthermore, Cackowski et al44 proved that osteoclasts can stimulate angiogenesis through the secretion of matrix metalloproteinase-9 (MMP-9). In adults, MMP-9 is mainly produced by osteoclasts and to a lesser extent by cells such as osteoblasts, macrophages, and neutrophils.45-47 One of the suggested mechanisms for the role of MMP-9 in enhancing angiogenesis is that it can release growth factors such as vascular endothelial growth factor from the bone matrix.48-50 Light microscopic observations of the ML group in the present study showed well-vascularized bone marrow spaces at 3 and 6 weeks and in the SL group at 3 weeks. Good vascularity is important during bone healing. This finding corresponds to the results of Tim et al51 who investigated the effect of LLLT (830 nm) on angiogenic genes in rat tibia bone defects. They concluded that laser increased the expression of angiogenic genes, such as angiopoietin-2 and -4, platelet-derived growth factor, and fibroblast growth factors-2 and -14, especially during the early stages of bone healing. Moreover, Garavello et al52 reported on the dual action of low-level laser on angiogenesis in tibia bone defect healing in rats. They found an increase in the number of blood vessels in the irradiated group after 1 week; however, after 2 weeks there was a notable decrease in the blood vessel count. In the authors’ opinion, this could be attributed to the shorter wavelength used in that study (633 nm), resulting in insufficient laser penetration. In the present study, histologic qualitative analysis showed the formation of compact bone in the SL group at 6 weeks. This indicates the rapid and enhanced bone remodeling and structural organization of the formed bone inside the socket after laser irradiation. Histomorphometric analysis of the present work was accomplished using ImageJ 1.46r software. Histomorphometry is used to evaluate bone healing and provide a numerical assessment to facilitate comparison among different groups.18 ImageJ has many advantages and is considered a reliable and inexpensive software for the evaluation of changes in different tissue structures.53 The present histomorphometric analysis confirmed the light microscopic observations, in which an increase in the percentage of bone surface
area was noted in the SL and ML groups compared with the C group at the 2 observational periods. However, the increase was not relevant in the SL group at 3 weeks. In addition, no relevant difference was detected between the SL and ML groups at the 2 observational periods. Furthermore, at 6 weeks, the SL group showed a larger percentage of bone surface area compared with the ML group (80 and 78.6%, respectively), but the difference was not significant (P = .896). These results suggest that the single laser application can be effective in enhancing bone formation compared with the multiple laser application. These findings corroborate those of Pretel et al26 who concluded that the application of diode laser at a single dose directly to surgically created bone defects in rats abbreviated the bone healing process and was effective in accelerating bone repair compared with non-lasered surgical wounds. However, Ng et al13 compared single versus multiple laser applications on the repair of the medial collateral ligament and stated that multiple laser treatments produced better outcomes compared with a single treatment using similar parameters. In conclusion, based on the present results, laser irradiation after tooth extraction can be effective in enhancing bone formation in the rabbit model. This can be attributed to its stimulatory effect on boneforming cells and enhanced angiogenesis. Furthermore, the single laser application can be as effective as multiple laser applications. This could be an important advantage in clinical practice to save time and lower the cost of several laser irradiations.
References 1. Blum IR: Contemporary views on dry socket (alveolar osteitis): A clinical appraisal of standardization, aetiopathogenesis and management: A critical review. Int J Oral Maxillofac Surg 31:309, 2002 2. Okamoto T, Okamoto R, Alves Rezende MC, Gabrielli MF: Interference of the blood clot on granulation tissue formation after tooth extraction. Histomorphological study in rats. Braz Dent J 5:85, 1994 3. Rubin CT, Lanyon LE: Regulation of bone mass by mechanical strain magnitude. Calcif Tissue Int 37:411, 1985 4. Turner CH, Forwood MR, Rho JY, Yoshikawa T: Mechanical loading thresholds for lamellar and woven bone formation. J Bone Miner Res 9:87, 1994 5. Hasuike A, Sato S, Udagawa A, et al: In vivo bone regenerative effect of low-intensity pulsed ultrasound in rat calvarial defects. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 111:e12, 2011 6. Fındık Y, Baykul T: Effects of low-intensity pulsed ultrasound on autogenous bone graft healing. Oral Surg Oral Med Oral Pathol Oral Radiol 117:e255, 2014 7. Kooistra BW, Jain A, Hanson BP: Electrical stimulation: Nonunions. Indian J Orthop 43:149, 2009 8. Adie S, Harris IA, Naylor JM, et al: Pulsed electromagnetic field stimulation for acute tibial shaft fractures: A multicenter, double-blind, randomized trial. J Bone Joint Surg Am 93:1569, 2011 9. McCarthy TL, Ji C, Centrella M: Links among growth factors, hormones, and nuclear factors with essential roles in bone formation. Crit Rev Oral Biol 11:409, 2000
1768
LASER FOR BONE FORMATION IN EXTRACTION SOCKET
10. Kazancioglu HO, Ezirganli S, Aydin MS: Effects of laser and ozone therapies on bone healing in the calvarial defects. J Craniofac Surg 24:2141, 2013 11. Nagata MJH, Santinoni CS, Pola NM, et al: Bone marrow aspirate combined with low-level laser therapy: A new therapeutic approach to enhance bone healing. J Photochem Photobiol B 121:6, 2013 12. Kawasaki K, Shimizu N: Effects of low-energy laser irradiation on bone remodeling during experimental tooth movement in rats. Lasers Surg Med 26:282, 2000 13. Ng GYF, Fung DTC, Leung MCP, Guo X: Ultrastructural comparison of medial collateral ligament repair after single or multiple applications of GaAlAs laser in rats. Lasers Surg Med 35: 317, 2004 14. Woodruff LD, Bounkeo JM, Brannon WM, et al: The efficacy of laser therapy in wound repair: A meta-analysis of the literature. Photomed Laser Surg 22:241, 2004 15. MacNeill SR, Cobb CM, Rapley JW, et al: In vivo comparison of synthetic osseous graft materials. A preliminary study. J Clin Periodontol 26:239, 1999 16. Hamad SA, Naif JS, Abdullah MA: Effect of diode laser on healing of tooth extraction socket: An experimental study in rabbits. J Maxillofac Oral Surg 15:308, 2016 17. Feldman AT, Wolfe D: Tissue processing and hematoxylin and eosin staining. Methods Mol Biol 1180:31, 2014 18. Gorustovich AA, Steimetz T, Nielsen FH, Guglielmotti MB: Histomorphometric study of alveolar bone healing in rats fed a borondeficient diet. Anat Rec 291:441, 2008 19. Steimetz T, Gorustovich AA, Collet AM, et al: Histologic and histomorphometric study of bone repair under acute Trypanosoma cruzi infection in rats. Acta Odontol Latinoam 25:193, 2012 20. Chen J, He Y, Shan C, et al: Topical combined application of dexamethasone, vitamin C, and b-sodium glycerophosphate for healing the extraction socket in rabbits. Int J Oral Maxillofac Surg 44:1317, 2015 21. Son J, Kim Y-B, Ge Z, et al: Bone healing effects of diode laser (808 nm) on a rat tibial fracture model. Vivo 26:703, 2012 22. do Nascimento MF, de Almeida BM, Cunha JLS, et al: Improvement of bone repair in diabetic rats subjected to ƛ780 nm lowlevel laser therapy. Acta Cir Bras 30:660, 2015 23. Atasoy KT, Korkmaz YT, Odaci E, Hanci H: The efficacy of lowlevel 940 nm laser therapy with different energy intensities on bone healing. Braz Oral Res 31:e7, 2017 24. Gurler G, Gursoy B: Investigation of effects of low level laser therapy in distraction osteogenesis. J Stomatol Oral Maxillofac Surg 119:469, 2018 25. Tas Deynek G, Ramoglu SI: Effects of different settings for 940 nm diode laser on expanded suture in rats. Angle Orthod; https://doi.org/10.2319/052318-392.1, January 2, 2019 [Epub ahead of print] 26. Pretel H, Lizarelli RFZ, Ramalho LTO: Effect of low-level laser therapy on bone repair: Histological study in rats. Lasers Surg Med 39:788, 2007 27. Mirdan BM: A 980nm diode laser clot formation of the rabbit’s dental sockets after teeth extraction. Iraqi J Laser 11: 37, 2018 28. Nunamaker DM: Experimental models of fracture repair. Clin Orthop 355(suppl):S56, 1998 29. Chung H, Dai T, Sharma SK, et al: The nuts and bolts of low-level laser (light) therapy. Ann Biomed Eng 40:516, 2012 30. Karu TI, Kolyakov SF: Exact action spectra for cellular responses relevant to phototherapy. Photomed Laser Surg 23:355, 2005 31. Medina-Huertas R, Manzano-Moreno FJ, De Luna-Bertos E, et al: The effects of low-level diode laser irradiation on differentiation, antigenic profile, and phagocytic capacity of osteoblast-like cells (MG-63). Lasers Med Sci 29:1479, 2014 32. Park JJ, Kang KL: Effect of 980-nm GaAlAs diode laser irradiation on healing of extraction sockets in streptozotocin-induced diabetic rats: A pilot study. Lasers Med Sci 27:223, 2012
33. Pires-Oliveira DA, Oliveira RF, Amadei SU, et al: Laser 904 nm action on bone repair in rats with osteoporosis. Osteoporos Int 21: 2109, 2010 34. Petri AD, Teixeira LN, Crippa GE, et al: Effects of low-level laser therapy on human osteoblastic cells grown on titanium. Braz Dent J 21:491, 2010 35. Soleimani M, Abbasnia E, Fathi M, et al: The effects of low-level laser irradiation on differentiation and proliferation of human bone marrow mesenchymal stem cells into neurons and osteoblasts—An in vitro study. Lasers Med Sci 27:423, 2012 36. Saygun I, Nizam N, Ural AU, et al: Low-level laser irradiation affects the release of basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), and receptor of IGF-I (IGFBP3) from osteoblasts. Photomed Laser Surg 30:149, 2012 37. Pyo S-J, Song W-W, Kim I-R, et al: Low-level laser therapy induces the expressions of BMP-2, osteocalcin, and TGF-b1 in hypoxiccultured human osteoblasts. Lasers Med Sci 28:543, 2013 38. Sutherland JC: Biological effects of polychromatic light. Photochem Photobiol 76:164, 2002 39. Karu T: Primary and secondary mechanisms of action of visible to near-IR radiation on cells. J Photochem Photobiol B 49:1, 1999 40. Chen AC-H, Arany PR, Huang Y-Y, et al: Low-level laser therapy activates NF-kB via generation of reactive oxygen species in mouse embryonic fibroblasts. PLoS One 6:e22453, 2011 41. Bouvet-Gerbettaz S, Merigo E, Rocca J-P, et al: Effects of low-level laser therapy on proliferation and differentiation of murine bone marrow cells into osteoblasts and osteoclasts. Lasers Surg Med 41:291, 2009 42. Coombe AR, Ho CT, Darendeliler MA, et al: The effects of low level laser irradiation on osteoblastic cells. Clin Orthod Res 4: 3, 2001 43. Schell H, Lienau J, Epari DR, et al: Osteoclastic activity begins early and increases over the course of bone healing. Bone 38: 547, 2006 44. Cackowski FC, Anderson JL, Patrene KD, et al: Osteoclasts are important for bone angiogenesis. Blood 115:140, 2010 45. Colnot C, Thompson Z, Miclau T, et al: Altered fracture repair in the absence of MMP9. Development 130:4123, 2003 46. Haeusler G, Walter I, Helmreich M, Egerbacher M: Localization of matrix metalloproteinases (MMPs), their tissue inhibitors, and vascular endothelial growth factor (VEGF) in growth plates of children and adolescents indicates a role for MMPs in human postnatal growth and skeletal maturation. Calcif Tissue Int 76: 326, 2005 47. Vu TH, Shipley JM, Bergers G, et al: MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93:411, 1998 48. Bergers G, Brekken R, McMahon G, et al: Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol 2:737, 2000 49. Engsig MT, Chen QJ, Vu TH, et al: Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J Cell Biol 151:879, 2000 50. Ishibashi O, Niwa S, Kadoyama K, Inui T: MMP-9 antisense oligodeoxynucleotide exerts an inhibitory effect on osteoclastic bone resorption by suppressing cell migration. Life Sci 79: 1657, 2006 51. Tim CR, Bossini PS, Kido HW, et al: Effects of low level laser therapy on inflammatory and angiogenic gene expression during the process of bone healing: A microarray analysis. J Photochem Photobiol B 154:8, 2016 52. Garavello I, Baranauskas V, da Cruz-H€ ofling MA: The effects of low laser irradiation on angiogenesis in injured rat tibiae. Histol Histopathol 19:43, 2004 53. Egan KP, Brennan TA, Pignolo RJ: Bone histomorphometry using free and commonly available software. Histopathology 61: 1168, 2012