Journal of Research in Dental

Abstract

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Background and Aim: Extraction of teeth in diabetic patients can be followed by some complications such as delay in recovery of hard and soft tissues, dry socket and increased risk of infection. This study investigate the impact of low-level laser therapy on tooth socket healing following extraction of maxillary molars in male rat.
Materials and Methods: In this experimental study 30 male rats divided into control and case groups. All rats made diabetic with streptozotocin (STZ). After one week, the second molar of all rats were extracted. Tooth sockets in the experimental group were immediately irradiated by He-Ne laser from a distance of 5 mm for 120s, which was continued until the third day. All of the rats were sacrificed on days 3, 7 and 14 after extraction in both groups for pathological evaluation the amount of granulation tissue, angiogenesis and the newly formed bone trabeculae, fibroblasts, macrophages, neutrophils and lymphocytes. The results were statistically analyzed by Kruskal Wallis and One-Way Analysis of Variance (One-way ANOVA) tests.
Results: Low-level laser therapy could increase angiogenesis until day three (P-value=0.009), reduce the number of lymphocytes after day fourteen (P-value=0.001), increase the number of fibroblasts and neutrophils (P-value<0.05), reduce the extent of granulation tissue during study (P-value<0.05) and elevate the extent of bone trabeculae after day fourteen (P-value=0.001).
Conclusion: The results also indicated low-level He-Ne laser therapy can reduce inflammation and accelerate the wound healing process in diabetic rats.
Key words: Diabetes Mellitus, Low-Level Laser Therapy, Rats, Tooth Socket
 

 Introduction

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Diabetes mellitus is a syndrome of impaired carbohydrate, fat and protein metabolism which is caused by lack of insulin secretion or tissue sensitivity to insulin. Vascular disease, kidney damage, blindness and atherosclerosis are more –prevalent in the diabetic patients than in the normal population.^{(1, 2)} Previous studies have reported that diabetes weakens the function of endothelial cells, which are in the vessel wall and take an important task in the normal functioning of blood vessel.⁽³⁾ Bone formation and amount of osteoids decrease in diabetics which shows low bone turnover in them;⁽⁴⁾ diabetics moreover show 30–50% decrease in bone synthesis and implant osseointegration.⁽⁵⁾ Tooth extraction in diabetic patients also can cause some complications, including more infection risk, dry socket and delayed wound healing.^{(6, 7)}
Laser therapy has had advantages to a lot of medical and dental issues such as articular, cartilage and bone problems since the past few decades.^{(8, 9)} In dental applications, lasers have Been applied for soft tissue operations such as biopsy and gingivectomy.⁽¹⁰⁾ -Level Laser Therapy (LLLT) systems increase the synthesis of cytokine growth factors and apoptosis induction and also has bio modulation effect on cells which improve wound healing, and has analgesic, disinfecting, and anti-inflammatory effects.^{(11, 12)} It also reduces pain and inflammation by increasing the activity of wound cells and enhances collagen synthesis.^{(13, 14)}
According to the literature LLLT seems to promote vascularization and new bone formation to a limited extent in diabetic rats.⁽¹⁵⁾ Some studies also indicated that LLLT enhanced osteoblast density but it was not able to enhance new bone formation in diabetic male rats as it does in nondiabetics.⁽¹⁶⁾
Very few studies have examined the efficiency of LLLT in eliminating the complications induced by teeth extraction in diabetic patients. On the other hand, different methods have been used in such studies many of which lack adequate information about the physical parameters of the laser which has been used. Therefore, this study conducted to experimentally evaluate the efficacy of LLLT on the tooth socket repair in male diabetic male rats.
 
Materials and methods

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Experimental protocol was approved by the Ethical Committee of Rafsanjan University of Medical Sciences (IR.RUMS.REC.1396.69). In this experimental study   male Wistar rats weighted 250-300g were selected from the animal house of Rafsanjan Medical School. Before the test, the rats were examined by a veterinarian and evaluated for the surgical site and general health. Before surgery, the rats were kept under standard conditions in the animal laboratory of Rafsanjan Medical School for one week. All rats were injected intraperitoneally by streptozotocin (STZ) (Sigma, MO, USA) dissolved in 50 mg/kg 0.1 citrate buffer to induce diabetes mellitus type 1. One week after STZ injection, the blood glucose of rats was measured by glucometers (Roche, Mannhei, Germany). Blood sugar >250 mg/dl showed the rats were diabetic. Blood glucose level was checked once a week during the test. Subjects were randomly divided into two groups of case and control (15 rats in each group), were subjected to general anesthesia, and injected with  a combination of 100mg/ml anesthetic/ Ketamine (Irandarou, Tehran, Iran) and 100 mg/ml Xylazine with a ratio of 1.9cc/0.6cc for each 10g weight of rats by intraperitoneal insulin syringes. Then, the upper right second molars of all rats were extracted. In the case group, after tooth extraction and bleeding control, the device laser He-Ne Plasmax IV, LHN 9709 (KLD Biossistemas®) was used.  socket was irradiated with He-Ne laser with an optical output power of 100 mW and wavelength of 808 nm to a spot size area of 0.028 cm². Energy density was 78.5 J/cm² and tooth socket was irradiated at a distance of 5 mm of the target location for 120 seconds.
Laser therapy was performed for 120 seconds every day until the third day. In the control group, the laser probe was located the same as the experimental group conditions and without laser activation immediately after tooth extraction. After putting animals in a hot enviroment and following their recovery, they were kept in separate cages with free access to food and water in the same conditions.
On day 3, the day of inflammation and proliferation phases, one-third of mice in each group (5 mice), on day 7, the day of restriction and closure phases, half of the remaining mice in each group (5 mice), and on day 14, the time of bone tissue formation, the remaining animals in each group (5 mice) were sacrificed in compliance with the protocols and tricks recommended by the National Committee for the Protection of Animals used for Scientific Purposes. After detaching their maxilla, they were fixed in 10% formalin solution for a week (Dr. Mojallali, Tehran, Iran). They were then placed in 4%EDTA  solution at 40°c for bone tissue decalcification (Merck, Berlin, Germany). After 4 weeks, softening of the bone tissue was checked by a scalpel. Samples were placed in a tissue processor (Sukura fine technical, Tokyo, Japan). They were soaked in paraffin 19 hours later and 5 micron sections were prepared from them using a microtome (SITE medical, Mainz, Germany). They were stained by hematoxylin and eosin staining and were pathologically examined, by a pathologist who was unaware of the sections of case and control groups on different days, under a light microscope at 400× magnification  (Olympus BX51 microscope). The slides were assessed in terms of the extent of granulation tissue, angiogenesis, number of fibroblasts, macrophages, neutrophils, lymphocytes, and the amount of newly formed bone trabeculae and granulation tissue. Results were recorded by the pathologist in a checklist. Data were statistically analyzed by Kruskal-Wallis tests were used to determine the effect of the groups and the evaluation days on the study variables and one-way ANOVA tests were used for multiple comparisons. P<0.05 was considered statistically significant.


Results

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LLLT on tooth socket all 30 diabetic rats could increase angiogenesis until day three (P-value=0.009), reduce the number of lymphocytes after day fourteen (P-value=0.001), increase the number of fibroblasts and neutrophils (P-value<0.05), reduce the extent of granulation tissue during study (P-value<0.05) and increase the extent of bone trabeculae after day fourteen (P-value=0.001).
As shown in Fig. 1, on the third day after extraction, the mean number of blood vessels was 8.2 ± 2.89 in the control group and 14.2 ± 5.24 in the laser treated group, indicating a significant difference between groups (P-value = 0.009).
Fig 1.  Mean number of newly formed vessels in both groups on days 3, 7, and 14
 
As indicated in Fig. 2, the number of neutrophil cells was significantly higher 3, 7 and 14 days after extraction in comparison with laser treated group (P-value<0.05).
Fig 2. Mean number of newly formed neutrophils in both groups on days 3, 4, and 14
Fig. 3 indicates no significant difference between the laser treated and control groups in the number of lymphocyte cells 3 and 7 days after surgery (P-value=0.09). However, on there was a significant reduction in the laser treated group on day 14 (P-value=0.001).
Fig 3.  Mean number of newly formed lymphocytes in both groups on days 3,7, and 14
Figures 4 and 5 also show a significant difference between the laser treated and control groups in the number of macrophages and fibroblasts on day 3.
Fig 4. Mean number of newly formed macrophages in both groups on days 3,7, and 14
Fig 5. Mean number of newly formed fibroblasts in both groups on days 3,7, and 14

Also shown in Fig. 6, there is no significant difference between the control and laser treated groups in the extent of granulation tissue 3 and 7 days after surgery (P-value=0.08).
Fig 6. Mean extent of newly formed granulation tissue in both groups on days 3, 7, and 14
Interestingly, however, the mean extent of granulation tissue was significantly lower in the laser treated group than in the control group on the day 14 (P-value<0.005).
Fig 7. Mean number of newly formed trabeculae in both groups on days 7 and 14
Due to lack of bone formation on day 3, the extent of trabecular bone was studied on the days 7 and 14. The extent of trabecular bone was significantly lower in the control group than in the laser treated group (p-value = 0.001).


Discussion

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  of diabetes as one of most common disease in the world has reported to increase especially in less developed countries.⁽¹⁷⁾]treatments should be modified to have the least Complications due to diabetes; LLLT can be a way to reduce them after tooth extraction. As clinical  trials cause limitations in method of study, male rats were used as subjects. STZ is an agent which necrosis β-cells in pancreas and cause type 1 diabetes⁽¹⁸⁾ and were used to induce diabetes in present study.⁽¹⁹⁾ study showed that the application of He-Ne laser (100 mW, 808 nm) on tooth socket could enhance bone formation and wound healing.
LLLT can affect cell proliferation and collagen synthesis, decrease the hypoxia caused by damage, and induce releasing growth factors.⁽²⁰⁾ On the other side, efficacy of LLLT relies upon a few elements, for example, wavelength, frequency, power, spot size and total treatment time.⁽²¹⁾ Results of present study showed that using LLLT until day 3 could significantly increase the amount of angiogenesis although the differences was not significant on days 7 and 14. Other studies have also reported an increase of angiogenesis in the laser-treated groups.^(22-24) Yildirimturk et.al. ⁽¹⁵⁾ also applied Ga-Al-As (820 nm, 0.5 W, three times a week) to diabetic animals and observed an increase in blood vessel count.
The present study shows that fibroblast formation in laser group was higher than the control, same results were obtained by G Ustaoglu et al.  who determined the effects of LLLT on wound healing at human free gingival graft (FGG) and concluded that LLLT enhanced FGG donor site wound healing and preserved tissue thickness at palatinal donor sites.⁽²⁵⁾ Also M Eissa and WHM Salih ⁽²⁴⁾ reported laser enhances diabetic rats wound healing and turn down the healing time to the half although the method and procedure they applied was different (He-Ne laser with a wavelength of 632.8 nm at power density of 4.0 mW/cm2, five times a week). This finding was consistent with the results of other studies.⁽¹⁴⁾ which can be due to different laser parameters and also soft tissue repair conditions.
Use of laser could decrease the number of neutrophils until day 14, indicating a significant reduction. Using laser increased the number of lymphocytes and macrophages on day 3 but mean number of these cells was lower in the laser group than in the control group in day 14, which can indicate the effect of laser on reducing the course of wound inflammation. Rabelo et al.⁽²⁶⁾ also reported that LLLT reduced wound inflammation. Rabelo's study showed use of He-Ne laser at 634.8 nm wavelength with an output power of 15 mW accelerate the wound healing by decreasing inflammatory cells in both diabetic and non-diabetic groups in comparison with the control groups. According to Alves et. al.⁽²⁷⁾ LLLT (arsenide and aluminum gallium-type diode laser) with output power of 50 mW was more efficient in modulating inflammatory cells (macrophages and neutrophils), that showed a reduction in the inflammatory process.
The mean extent of trabecular bone on days 7 and 14 was more in the laser group than in the control. Similar to these results, Oliveira FA et al. ⁽²⁸⁾ reported an increase of 31-58% in survival rate of osteoblasts which irradiated by He-Ne laser and concluded osteoblasts proliferation and maturation increased as the osteogenic markers elevated. Diker N et. al. ⁽¹⁶⁾  reported that application of Gallium-aluminium-arsenide (GaAlAs) diode laser at wavelength of 808 nm with an optical output of 100mW enhanced osteoblast density but was not able to enhance the new bone formation which shows that different irradiation parameters can affect treatment procedure and also result of the treatment. Yildirimturk et. al. ⁽¹⁵⁾ applied Gallium-Aluminum-Arsenide (GaAlAs) laser at wavelength of 820 nm on animals with STZ induced diabetes which promoted vascularization and new bone formation. Results of other studies and the present study indicate positive role of LLLT in tooth socket repair but in different gratitude because of different laser parameters.
 
 
Conclusion

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The results of this study support the idea that low-level laser therapy can reduce inflammation after tooth extraction and accelerate bone healing process in diabetic male rats.