The influence of LLLT applied on applied on calvarial defect in rats under effect of cigarette smoke

Abstract Objective Considering the global public health problem of smoking, which can negatively influence bone tissue repair, the aim of this study is to analyze the influence of photobiomodulation therapy (PBM) on calvaria defects created surgically in specimens under the effect of cigarette smoke and analyzed with use of histomorphometric and immunohistochemistry techniques. Methodology Calvaria defects 4.1 mm in diameter were surgically created in the calvaria of 90-day-old rats (n=60) that were randomly divided into 4 experimental groups containing 15 animals each: control group (C), smoking group (S), laser group (L), and smoke associated with laser group (S+L). The animals were subjected to surgery for calvaria defects and underwent PBM, being evaluated at 21, 45, and 60 days post-surgery. The specimens were then processed for histomorphometric and immunohistochemistry analyses. The area of bone neoformation (ABN), percentage of bone neoformation (PBNF), and the remaining distance between the edges of the defects (D) were analyzed histometrically. Quantitative analysis of the TRAP immunolabeled cells was also performed. The data were subjected to analysis of variance (ANOVA) in conjunction with Tukey’s test to verify the statistical differences between groups (p<0.05). Results The smoking group showed less ABN compared to the other experimental groups in all periods, and it also showed more D at 21 days compared to the remaining groups and at 45 days compared to the laser group. The smoking group showed a lower PNBF compared to the laser group in all experimental periods and compared to smoking combined with LLLT group at 21 days. Conclusions PBM acted on bone biomodulation, thus stimulating new bone formation and compensating for the negative factor of smoking, which can be used as a supportive therapy during bone repair processes.


Introduction
Bone healing is a complex process that involves multiple cellular and molecular events, and therefore requires a longer time for cells to completely regenerate the affected area in this tissue. 1 Furthermore, there are several factors that can negatively influence bone tissue repair. A vast amount of literature shows smoking as having a primarily negative effect on bone tissue repair. [2][3][4][5][6] Smoking is a public health problem worldwide. The World Health Organization (WHO) estimated that 10 million deaths will have occurred by 2020 as a result of tobacco-related diseases, given that 70% of these cases are to be reported in developed countries. 7 César-Neto, et al. 2 (2003) have shown that cigarette smoke inhalation is harmful to bone metabolism, suggesting that cigarette smoke constituents (toxic heavy metals, polychlorinated biphenyls, dioxins and polycyclic aromatic hydrocarbons) and nicotine can have a negative impact on bone wound healing.
Other major negative effects of smoking are high rates of failure in clinical dental procedures, such as periodontal surgery, implants, and different types of bone grafts. 2,[8][9][10] Bone healing can be stimulated by applying various molecules, such as bone morphogenetic proteins (BMPs), physical stimuli, (e.g. ultrasonography or electromagnetic field) and, more recently, photobiomodulation therapy (PBM). 11 PBM is a locally applied therapeutic modality that produces biostimulatory effects, and it is used as an adjuvant in tissue healing and repair processes. [12][13][14] Its mechanism of action on bone repair is not yet well understood, 15 although it has been regarded as promoting angiogenesis 16 and an increase in in situ blood flow, thus increasing the supply of circulating cells, nutrition, oxygen and inorganic salts to the bone defect. This process stimulates cellular growth, such as fibroblasts that are related to collagen production 17,18 , through an increase in osteoblasts proliferation, viability and differentiation 14,19,20 , and promoting mitochondrial respiration and ATP synthesis. 21 All these events can promote bone healing and mineralization. 21,22 Several pre-clinical and clinical studies have evaluated the effects of different PBM protocols. It has been established that PBM increases bone formation in calvaria defects models in rats. 23 rats (weighing 250 to 300 g and 3 months-old) were kept in cages at room temperature with a 12-hour daylight cycle, fed with Guabi Nutrilabor ® (Mogiana Alimentos; São Paulo, SP, Brazil) and water ad libitum supplied by a qualified staff, at the animal house of compartment for lit cigarettes and another one for the animals. The animals were subjected to an adaptation period in which they were exposed to cigarette smoke inhalation for 5 minutes on the first day, 6 minutes on the second day, and 7 minutes on the third day.
Afterwards, they were exposed at the same time to the smoke of 10 cigarettes for 8 minutes, three times a day for 5 days a week. The cigarette brand used (MINISTER King Size Unique -Souza Cruz; Rio de Janeiro, RJ, Brazil) contained different tobacco blends, sugar, cigarette paper, 10 mg tar, 0.8 mg nicotine, and 10 mg carbon monoxide, according to the product label information. Nonsmoking animals were also placed in acrylic boxes of the same size for the same period in order to simulate the same conditions. Calvarial defects creation and photobiomodulation therapy session

Statistical analysis
The data were previously subjected to Shapiro Wilk test and the results indicated that the residuals were normally distributed and, by plotting against predicted values, uniformity was verified and no ANOVA assumptions were violated. Analysis of variance (two-way ANOVA) was performed to evaluate the relationship between the subgroups subjected to the treatment and sacrifice time.
The data were analyzed statistically with use of the application software GraphPad Prism (version 7.00 for Windows, GraphPad Software; La Jolla, California, USA) and Tukey's multiple comparison test (α=5%).

Quantitative and qualitative histological analysis
The qualitative histological comparisons between groups are described below and resumed in the Figure   1, according to the analyzed periods.

days
In the L group, it is possible to observe the total covering of the wound by a narrow thickness of bone tissue with areas of immature bone tissue covered by a thin band of connective tissue that has been vascularized and cellularized, with thin collagen fibers parallel to the wound and throughout its length ( Figure   1A). In group C, a bone formation can be observed in the wound edges which extend towards the center, but   inflammatory response, which allows the healing proliferation stage to be reached sooner than it can be under smoke inhalation conditions. 32 In addition, in our study, there was only one application of PBM similar to the studies by Cunha, et al. 32 (2014), Almeida, et al. 33 (2014) and Moreira, et al. 34 (2018), who also observed greater initial bone formation compared to the control groups. In a protocol with higher number of PBM applications compared to our study, Oliveira, et al. 35  The use of laser on several tissues, including bone tissue in experimental models under other conditions, 13 has been investigated and the literature has proven its stimulatory effects. However, the exact regulation mechanism by which the laser acts on the tissues is not fully understood, 38  The influence of LLLT applied on applied on calvarial defect in rats under effect of cigarette smoke