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Tooth extraction causes an open wound in the soft and hard tissues. During the inflammatory phase of the healing process, a large amount of free radicals are produced and cause oxidative stress, which leads to tissue damage and delayed wound healing. Thus, in this study, we evaluated the effect of tert-butylhydroquinone (TBHQ), as an antioxidant, on the healing process of tooth sockets in rats.
To conduct this experimental study, male Wistar rats (n = 42) were divided into two groups. In each case, one upper second molar was extracted under general anesthesia. After the extraction, the tooth sockets of the experimental group were treated with a 0.02% TBHQ solution (0.1 ml) while the same volume of distilled water placed in the sockets of the control group. On days 3, 7, and 21 postoperatively, 7 rats from each group were euthanized, and histological slides were prepared from their tooth sockets. The prepared slides were examined histopathologically using a light microscope and compared using an independent two-sample t-test. The significance level was set at 0.05.
In the experimental group, a statistically significant (P = 0.003) increase in granulation tissue was observed on day 3, in comparison to the control group. The extent of bony trabeculation was also significantly higher in the TBHQ-treated group than in the control group on day 21 (P < 0.001).
Considering the limitations of an experimental study, it can be concluded that TBHQ may enhance the healing of the hard tissue in the tooth sockets.
Tooth extraction causes an open wound in the soft tissue and bone. The wound healing process is divided into four phases such as coagulation, inflammation, proliferation, and reconstruction.
Tert-Butylhydroquinone (TBHQ) is an antioxidant with a documented effect on reducing oxidative stress. It has been considered the gold standard for antioxidants and is used in food products.
This double-blinded experimental study was performed on male Wistar rats (n = 42) of the same size, weight (weighing 120–180 g), and age (3–4 months). All procedures were approved by the Institutional Animal Care Committee and were in accordance with guidelines approved by the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, Revised 1978).
The rats were kept in individual cages under standard conditions in the Department of Physiology of Rafsanjan University of Medical Sciences, Iran. A veterinarian assessed the animals regarding general health and the required conditions for surgery before the beginning of the study. Then, the rats were placed under general anesthesia using an intraperitoneal injection of 0.1 ml/100 g with a combination of anesthetic ketamine 100 mg/ml (Iran Darou, Tehran, Iran) and xylazine 100 mg/ml (Iran Darou) in a ratio of 1:1.
One maxillary second molar of each animal was extracted. In the experimental group (21 rats), approximately 0.1 ml of the 0.02% TBHQ solution (Sigma, MO, USA) was placed in the tooth socket (20 mg TBHQ powder was dissolved in 100 ml of distilled water). This concentration was the maximum acceptable concentration of TBHQ used in the food industry. To control the procedure influence, the same volume of distilled water was placed in the sockets of the control group (21 rats). For retaining the compound in the sockets, the animals were kept under anesthesia for approximately 4 h in the supine position.
The extent of granulation tissue (mm 2), extent of bone trabeculae (mm 2), inflammatory cell count, number of fibroblasts, and angiogenesis were the variables that were evaluated to assess restoration under a CXR2 microscope (Labomed, Los Angeles, USA). For the inflammatory cell count of neutrophils, lymphocytes, macrophages, and fibroblasts, the number of cells was counted under ×400 magnification in at least five different fields. Next, the mean cell count in these five fields was calculated for statistical analysis. The number of blood vessels was counted in ten microscopic fields under ×400 magnification. Subsequently, their mean was calculated and recorded for statistical analysis.
Statistical analysis methods
Data were then analyzed using SPSS software (version 18, SPSS Inc., Chicago, IL, USA). The quantitative data were expressed as mean ± standard deviation, and the change range was reported as the largest observation–smallest observation. The independent two-sample t-test was used to compare the mean value of histopathological variables (lymphocytes, macrophages, neutrophils, fibroblasts, the number of newly formed vessels, extent of granulation tissue, and newly formed bone trabeculae) on days 3, 7, and 21 in both of the groups. P < 0.05 was considered statistically significant.
The independent t-test showed that the mean size of the granulation tissue (mm
2) was significantly greater in the TBHQ group than in the control group (P = 0.003) on day 3
Healing of extracted tooth socket on day 21. (a) Experimental group (b) control group.
In the process of wound healing, the production of reactive oxygen species (ROS) is necessary to defend body against pathogens.
Increased granulation tissue on day 3 was statistcally significant in the experimental group compared to the control group. This could indicate the acceleration of wound healing in the presence of TBHQ. The study by Jelenko et al. also showed acceleration in the healing of skin burns in the presence of TBHQ.
In this study, the extent of bone trabeculation on day 21 was significantly greater in the TBHQ group than in the control group. This finding indicates that the recovery of hard tissue is faster in the presence of TBHQ. Yoneda et al. concluded that the antioxidant coenzyme Q
10is more effective on soft tissue repair than on hard tissue repair.
In this study, the neutrophils count on day 3 in the experimental group was lower than the control group. Although this difference was not statistically significant, the reduction in the number of these cells on day 3 showed a reduction of the inflammatory phase and progression of the natural process of wound healing. In addition, in the study by Yoneda et al., the neutrophils count decreased in the presence of antioxidants on day 3.
In this study, the macrophages were the largest number of cells present on day 3. These cells stimulate the proliferation of fibroblasts through lactate synthesis and are gradually reduced during the process of restoration through the replacement of fibroblasts with these cells.
On day 7 of the study, a decrease in neutrophils and an increase in fibroblasts counts were observed in the TBHQ group as compared to the control group, but this difference was not statistically significant. The low volume and concentration of TBHQ in the tooth sockets were perhaps the reasons for the insignificant results of this study. Due to the limitation of budget, it was impossible to determine the maximum TBHQ concentration without toxic effects on the mouth tissues before the study. Because no study was conducted on the effect of TBHQ on tooth socket healing, the concentration used in this study was the maximum acceptable concentration of TBHQ used in the food industry. In this concentration, the TBHQ was in solution form and not paste and this was one of the limitations of this study. This limitation may increase the chance of washing-out the material from socket by blood and saliva. Hence, we decided to decrease the chance of washing-out by 4 h supine position after extraction that has been recommended in Mendes study.
It appears that the use of antioxidants in oral and periodontal surgery are the way forward for improving and accelerating healing procedures.
Considering the limitations of an experimental study, it can be concluded that TBHQ may enhance the healing of the hard tissue in teeth sockets.
Acknowledgment
Our sincere appreciation goes to the Research Deputy of Rafsanjan University of Medical Sciences for the financial support of this study.
Financial support and sponsorship
Rafsanjan University of Medical Sciences.
Conflicts of interest
The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.