| Abstract|| |
Background: Millions of people worldwide consume carbonated drinks every day. The effects of these drinks on hard tissues in the mouth have been proved beyond doubt. Only a little has been done so far to assess the effects of carbonated drinks on oral soft tissues. This study was an attempt to assess the effect of carbonated drinks on oral wound healing.
Materials and Methods: Twenty female Wistar rats were considered for the study. A circular wound was created on the palate and the animals were divided into two groups (experimental and control group). The experimental group animals were fed with a commercially available carbonated drink instead of water, and two animals from each group were euthanized at 3, 7, 14 and 21 days. Wound site was assessed morphometrically and histologically. Results: There was a marked difference in the healing pattern between the experimental group and control group animals. Control group animals showed a normal healing pattern with formation of a fibrous connective tissue at the end of 21 days. In the experimental group, healing was delayed and disrupted. The wound site showed a definite palatal perforation in experimental group animals after 14 days, but osteoclasts were not noticed in the histological sections.
Conclusion: Consumption of carbonated drinks can disrupt oral wound healing. Results suggest that the bone changes seen in experimental group samples are not mediated by osteoclasts, and acidity of the carbonated drinks could be one of the reasons for these changes.
Keywords: Carbonated drinks, Wistar rats, wound healing
|How to cite this article:|
Suragimath G, Krishnaprasad K R, Moogla S, Sridhara SU, Raju S. Effect of carbonated drink on excisional palatal wound healing: A study on Wistar rats. Indian J Dent Res 2010;21:330-3
Wound healing involves a complex, well-orchestrated series of events like hemostasis, inflammatory response, collagen synthesis etc. Wound healing in the oral cavity can be affected by a variety of factors including diet, which can influence intra oral wound healing directly or indirectly. Directly, it causes local irritation, due to the physical and chemical characteristics; indirectly it acts as a substrate for the oral microbial flora, and thus in turn, affects wound healing.
|How to cite this URL:|
Suragimath G, Krishnaprasad K R, Moogla S, Sridhara SU, Raju S. Effect of carbonated drink on excisional palatal wound healing: A study on Wistar rats. Indian J Dent Res [serial online] 2010 [cited 2015 Mar 27];21:330-3. Available from: http://www.ijdr.in/text.asp?2010/21/3/330/70789
Some of the most commonly consumed beverages all over the world, are carbonated drinks. It has been estimated that in the year 2007 per capita consumption of carbonated drinks in the United States was 187 liters.  All carbonated drinks are fizzy in nature as they are aerated; the neurological stimulation and sensory attributes of carbonated drinks have been implicated as reasons for addictive properties of the drinks. , In spite of all the controversies about the contents and their ill effects on health, carbonated drinks have become a part of our lives, especially the urban life style.
There has been an abundance of thorough and extensive research to explain the effect of carbonated drinks on the tooth surface. Carbonated drinks are known to cause erosion of the tooth surface.  But there is a scarcity of the data regarding the effect of carbonated drinks on soft tissues and oral wound healing. This study is an effort to assess the effect of carbonated drinks on oral wound healing, using an animal model. The null hypothesis "carbonated drinks have no effect on oral wound healing" was put to test in this study. Animal models were considered for the study as there was a need to create a wound, and to take samples for histological assessment.
| Materials and Methods|| |
Institutional animal ethical committee of Coorg Institute of Dental Sciences (CIDS), India, cleared the study protocol after a thorough review. The guidelines, put forth by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) were strictly followed during the entire course of the study. Twenty, three-month-old female Wistar rats, weighing 350 to 400 grams, were considered for the study. Animals were housed five per cage, in a well lit and well ventilated room. They were fed with standard chow pellets and water, ad libitum. All the procedures involving handling of the animals were carried out under the supervision of a veterinary officer.
After an acclimatization period of one week, animals were anesthetized with Ketamine (50 mg/ml) and Xylazine (23.32mg /ml). The anesthetic solution was prepared by mixing 2ml of Ketamine and 0.5ml of Xylazine with 2.5ml of distilled water. About 0.2ml of anesthetic solution per 100gm of body weight was administered intra peritoneally [Figure 1]a as the initial dose. If needed, 1/3 to 1/2 of the initial dose was used as maintenance dose.
|Figure 1 :(a) Intra peritoneal administration of anesthetic solution(b) Circular wound was created on the palate using a punch biopsy tool (c) Fresh wound with blood clot over the bone (d) Electrolyte pH meter|
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A punch biopsy tool with 4.5mm diameter was used to create a standardized circular wound outline on the anterior palate [Figure 1]b. The wound was created 2-3mm anterior to the imaginary line joining the mesial surfaces of the maxillary first molars. The soft tissue was removed by sharp dissection to expose the underlying bone [Figure 1]c. Cotton gauze was placed over the wound until hemostasis was achieved.
Four animals were euthanized immediately to get the baseline values. The remaining 16 animals were randomly divided into experimental and control groups. The animals in the control group were fed with chow pellets and water. The animals in the experimental group were fed with a randomly selected commercially available carbonated drink, instead of water. The pH of water and the carbonated drinks were tested on four instances during the study using an electrolyte pH meter [Figure 1]d
Two animals from each group were euthanized at 3, 7, 14 and 21 days post operatively, with a high dose of the anesthetic agent. Animals were decapitated, and maxillae were dissected out. Samples were photographically assessed and compared with the histological findings.
Photographs of the specimen were taken from standardized distance (40 cm) and magnification (2.4X optical zoom) using a digital camera. The surface area of the wound was morphometrically measured using the software, 'Biowizard - D Winter, Version 3'.
Specimens were fixed in 10% formalin for 48 hours. The samples were then decalcified in formic acid (10%) and nitric acid (10%) for 72 hours. Then, the samples were embedded in paraffin. Serial sections were prepared perpendicular to the palatal midline at the greatest diameter of the wound. The sections were stained with eosin and hematoxylin. Slides were viewed under light microscopy.
The control and experimental group data were compared with each other and to the base line values. Statistical analysis was done using the SPSS software. Repeated measure ANOVA and Students t test were applied.
| Results|| |
Photo morphometric assessment
The mean surface area of the wounds in both the groups at different time intervals was given in [Table 1]. The surface area of the wound at the base line was 14.640±0.25 sq mm [Figure 2]. Both the groups showed progressive decrease in the residual wound surface area, with time [Figure 2]. On day 3, there was no significant difference between the experimental and control group samples in terms of mean surface area of the residual wound [Table 1], but a small area of necrosis was noticed in the experimental group samples [Figure 3]. The difference between the experimental and the control group was significant (P<0.05) from day 7 onwards [Table 2]. On day 14, the experimental group samples showed a definite perforation of the palatal bone [Figure 3]. The surface area of the wound in the control group was just 0.05±0.012 Sq mm on day 14, whereas, in experimental group, surface area of the wound was least on day 21 (2.37±0.84 Sq mm). On day 21, there was no residual wound visible in the control group samples [Figure 3].
|Table 1 :Mean surface area of the residual wound (in mm2) as measured photomorphometrically |
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|Figure 2 :Mean surface area of the wound (y-axis) plotted against time (x- axis), Blue line - Control group values, Green line - Experimental group values|
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|Figure 3 :(a) Photograph of the wound site on day 0, showing fresh wound with blood clot over the bone, (b) Control group sample on day 3, (c) Experimental group sample on day 3, showing a small area of necrosis (encircled), (d) Control group sample on day 21, showing healed wound site, (e) Experimental group sample on day 14, showing palatal perforation on either side of the nasal septum, (f) Experimental group sample on day 21, showing oral epithelium extending into perforation in the palate|
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Microscopic findings correlated to the clinical and photographic appearance of the samples on all days. On day 0, the epithelium was sharply demarcated and extravasated RBCs were found over the bone with acute inflammatory cell infiltrate in the adjacent connective tissue [Figure 4].
|Figure 4 :(a) Section of the wound site on day 0, showing sharply cut epithelial margins(arrows) and blood clot over the bone(b) Experimental group sample on day 3, showing necrotic material in the encircled area (c) Control group sample on day 3, showing proliferating epithelium(arrows) (d) Osteoclasts (arrows) seen in control group sample on day 7 (e) Experimental group sample on day 21, showing irregularly formed epithelium extending beyond the underlying bone (f) Control group sample on day 21, showing a well formed epithelium over the connective tissue|
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On day 3, epithelial proliferation which was very much evident in the control group [Figure 4], but, was not appreciable in experimental group samples. The experimental group samples showed areas of superficial necrosis, with chronic inflammatory cell infiltrate in the connective tissue [Figure 4]. On day 14 and 21 the control group samples showed wound site completely covered with epithelium and well formed connective tissue over the bone [Figure 4].
The experimental group samples showed areas denuded of the soft tissues from day 7 onwards and there was a definite discontinuity in the bone. On day 21, the experimental group samples showed irregularly formed epithelium overlying a very thin plate of bone [Figure 4]. In spite of this, no osteoclastic activity could be found in the areas of bone loss. Osteoclasts were seen in control group samples from day 7 onwards indicating that the bone remodeling was taking place in those areas [Figure 4].
| Discussion|| |
The oral wounds are constantly subjected to test by our dietary habits. Carbonated drinks being the most commonly consumed beverage, cannot be neglected for its action on oral wound healing. During the present study, consumption of carbonated drink and water was not quantified, but it was noticed that the experimental group animals consumed more liquids than the control group animals. However, there was no significant difference in the body weight of control and the experimental group animals before and after the intervention. This craving for carbonated drinks noticed in experimental group animals could be due to the neurological sensations produced by the carbonated drinks in the oral cavity. 
The healing pattern [Figure 3] seen in case of control group animals was normal and in accordance with healing process reported by numerous studies.  Clinically, the wound site on day 3 appeared to be more clean and healthy in the experimental group samples, as compared to the control group [Figure 3]. The reason for this could be the cleansing action of the carbonated drink due to its acidity and fizziness. A low pH can have detrimental effects on the oral micro biota. The fizziness of the drink can dislodge the debris and thus help in keeping the oral cavity clean. On day 3, there was one finding that could not be neglected. A small area of necrosis was noticed in experimental group samples [Figure 3]. This finding was confirmed by microscopic examination [Figure 4]. It has been reported that, acidic environment inhibits the action of proteases and there by promotes wound healing.  The optimal range of pH which promotes wound healing and avoids degradation of the newly formed matrix is reported to be 4 - 7.  The acidity of the carbonated drink and the water that was provided to the animals were tested on four instances during the study. The pH of the carbonated drink was measured to be 2.3 to 2.45, average being 2.38. The pH of water provided to the control group animals was, 6.6 to 6.7. Such low pH levels of the carbonated drink may have lead to the soft tissue degradation, which was noticed in experimental group animals. Peter Lorenz and Michael T reported that presence of necrotic material delays wound healing.  From day 7 onwards, the healing was delayed in experimental group samples [Figure 2], with signs of tissue destruction observed both clinically and microscopically [Figure 3] and [Figure 4]. Optimal pH for viability and activity of the fibroblasts has been reported to be 7.2 to 7.5.  Changes in the fibroblast activity due to a low pH might have contributed for delayed healing in case of experimental group animals. On day 14, the experimental group samples showed a definite perforation of the palatal bone [Figure 3]. In fact, on day 21, the oral epithelium was found to extend into the palatal defect [Figure 3] and [Figure 4]. The perforation in the palate must have prevented the epithelium and the connective tissue from covering the whole wound site. Microscopically, areas of bone loss were seen, but osteoclasts could not be found in these sites. On day 21, the bone appeared to be very thin towards the end, with smooth borders [Figure 4], indicating that the bone demineralization was not mediated by osteoclasts.
As mentioned earlier, there was degradation of newly formed matrix at the healing site. Carbonated drinks being fizzy in nature might dislodge the new granulation tissue/ degraded tissues that are formed at the healing site. This exposes the under lying bone to the oral environment. Carbonated drinks being acidic, not only decrease the pH of the oral cavity directly, but also decrease the salivary pH.  It has been reported that, the consumption of carbonated drinks can decrease the bone density.  With the current knowledge and limitations of the present study, it can be hypothesized that when the palatal bone with decreased density is exposed to the highly acidic oral environment it gets demineralized easily to eventually cause palatal perforation.
Many dental procedures leave open wounds in the oral cavity (e.g., tooth extraction, gingivectomy, free gingival graft, etc.). If consumption of carbonated drinks during the healing period can have such complex outcomes as noticed in this study, it has the potential to affect the dentition in a periodontal perspective. It can be concluded from the results of this study that carbonated drinks have deleterious effects on open oral wounds. However, further studies are required to explain the underlying patho-physiology and its implications on humans.
| Acknowledgments|| |
The authors would like to thank Dr. Basavaraj (Veterinary Officer), Dr. Jagadish, Dr. Deepu Krishna, Dr. Bobby Wilson, and Dr. Soumyajit (Post Graduate students, CIDS) for their help and suggestions. We are greatly indebted to Dr. Sunil Muddaiah, Managing Trustee, Coorg Institute of Dental Sciences (CIDS), India, for constant support and encouragement.
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K R Krishnaprasad
Department of Periodontics and Implantology, Coorg Institute of Dental Sciences
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]