Journal of Oral Biology and Craniofacial Research, 01 Sep 2015, 6(1):49-53 DOI: 10.1016/j.jobcr.2015.08.005PMID: 26937370 PMCID: PMC4756064
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Abstract
BACKGROUND:Carbonated drinks are the second most consumed non-alcoholic beverages in the world after tea. The effects of these drinks on hard tissues and vital organs of the body have been proved beyond doubt. This study, however, explains the effect of these drinks on wound healing of oral epithelium. METHODS:Thirty-six male Wistar rats were considered for the study. A circular wound of 3.0 mm was created on the buccal mucosa of all animals and they were divided into two groups. Animals in group 1 were fed with chow pellet and water, while those in group 2 were fed with a commercially available carbonated drink instead of water. Six animals from each group were euthanized at 0, 7, and 21 days. Wound site was histologically assessed for differences in thickness and characteristics of the regenerating epithelium between two groups. RESULTS:There was a marked difference in the healing pattern between the two groups. Animals in group 1 showed a normal healing pattern at the end of day 21. In the group 2, the regenerated epithelium showed hyperplasia and hyperkeratosis along with acanthosis at the end of the experiment with a subsequent delayed inflammatory reaction at day 21. CONCLUSION:Consumption of carbonated drinks can disrupt oral wound healing. The contents in carbonated drinks have a proinflammatory action on the soft tissue. Results suggest that epithelial changes seen in experimental group 2 could be a result of constant irritation by the acidic and fizzy nature of carbonated drinks.
nbcnews.com / Today
By TODAY Health
Pop quiz! What's the single biggest source of calories for Americans? White bread? Big Macs? Actually, try soda. The average American drinks about two cans of the stuff every day. "But I drink diet soda," you say. "With no calories or sugar, it's the perfect alternative for weight watchers...Right?"
Not so fast. Before you pop the top off the caramel-colored bubbly, know this: guzzling diet soda comes with its own set of side effects that may harm your health--from kickstarting kidney problems to adding inches to your waistline.
Unfortunately, diet soda is more in vogue than ever. Kids consume the stuff at more than double the rate of last decade, according to research in the American Journal of Clinical Nutrition. Among adults, consumption has grown almost 25 percent.
But knowing these 7 side effects of drinking diet soda may help you kick the can for good.
Kidney Problems
Here's something you didn't know about your diet soda: It might be bad for your kidneys. In an 11-year-long Harvard Medical School study of more than 3,000 women, researchers found that diet cola is associated with a two-fold increased risk for kidney decline. Kidney function started declining when women drank more than two sodas a day. Even more interesting: Since kidney decline was not associated with sugar-sweetened sodas, researchers suspect that the diet sweeteners are responsible.
According to a 2008 University of Minnesota study of almost 10,000 adults, even just one diet soda a day is linked to a 34% higher risk of metabolic syndrome, the group of symptoms including belly fat and high cholesterol that puts you at risk for heart disease. Whether that link is attributed to an ingredient in diet soda or the drinkers' eating habits is unclear. But is that one can really worth it?
You read that right: Diet soda doesn't help you lose weight after all. A University of Texas Health Science Center study found that the more diet sodas a person drank, the greater their risk of becoming overweight. Downing just two or more cans a day increased waistlines by 500%. Why? Artificial sweeteners can disrupt the body's natural ability to regulate calorie intake based on the sweetness of foods, suggested an animal study from Purdue University. That means people who consume diet foods might be more likely to overeat, because your body is being tricked into thinking it's eating sugar, and you crave more.
Your first bad decision was ordering that whiskey-and-diet-cola -- and you may make the next one sooner than you thought. Cocktails made with diet soda get you drunker, faster, according to a study out of the Royal Adelaide Hospital in Australia. That's because sugar-free mixers allow liquor to enter your bloodstream much quicker than those with sugar, leaving you with a bigger buzz.
Cell Damage
Diet sodas contain something many regular sodas don't: mold inhibitors. They go by the names sodium benzoate or potassium benzoate, and they're in nearly all diet sodas. But many regular sodas, such as Coke and Pepsi, don't contain this preservative.
That's bad news for diet drinkers. "These chemicals have the ability to cause severe damage to DNA in the mitochondria to the point that they totally inactivate it - they knock it out altogether," Peter Piper, a professor of molecular biology and biotechnology at the University of Sheffield in the U.K., told a British newspaper in 1999. The preservative has also been linked to hives, asthma, and other allergic conditions, according to the Center for Science in the Public Interest.
Since then, some companies have phased out sodium benzoate. Diet Coke and Diet Pepsi have replaced it with another preservative, potassium benzoate. Both sodium and potassium benzoate were classified by the Food Commission in the UK as mild irritants to the skin, eyes, and mucous membranes.
Rotting Teeth
With a pH of 3.2, diet soda is very acidic. (As a point of reference, the pH of battery acid is 1. Water is 7.) The acid is what readily dissolves enamel, and just because a soda is diet doesn't make it acid-light. Adults who drink three or more sodas a day have worse dental health, says a University of Michigan analysis of dental checkup data. Soda drinkers had far greater decay, more missing teeth, and more fillings.
Reproductive Issues
Sometimes, the vessel for your beverage is just as harmful. Diet or not, soft drink cans are coated with the endocrine disruptor bisphenol A (BPA), which has been linked to everything from heart disease to obesity to reproductive problems. That's a lot of risktaking for one can of pop.
Effect of carbonated drinks on wound healing of oral epithelium
Drinks that contain phosphoric acid have been shown to have erosive effects and cola drinks are strongly acidic (pH 2.5). Gingivitis may be caused by dietary ...
A linear mixed model statistic did not show the caffeine effect on salivary flow rate. ... mucosa for 10 seconds and moisture content estimated with the Periotron.
Find patient medical information for Mouthwash W/Baking Soda Mucous Membrane on WebMD including its uses, side effects and safety, interactions, pictures, ...
Oct 19, 2012 — "But I drink diet soda," you say. ... But knowing these 7 side effects of drinking diet soda may help you kick the can for good. ... by the Food Commission in the UK as mild irritants to the skin, eyes, and mucous membranes.
The presence of these enzymes in the mouth is likely to short to achieve a positive effect. Carbonated water, carbonated drinks, or rinsing with dark beer (with a low alcohol content of 1.5%) may dissolve mucus somewhat. This advice is unsuitable for patients with an alcohol addiction.
May 10, 2019 — Should I be worried that my soda contains brominated vegetable oil? ... Bromine can irritate the skin and mucous membranes (the moist lining of the ... Long-term exposure can cause neurologic symptoms such as headache, ...
... W/Baking Soda mucous membrane on RxList including its uses, side effects, ... This information is not individual medical advice and does not substitute for the ...
Some patients have problems with thick mucus or phlegm, which can make them uncomfortable. If you are experiencing this side effect, here are a few tips: Drinking club soda or hot tea with lemon, or sucking on sugar-free sour lemon drops ...
Caffeine by itself failed to increase [Ca2+]i and affect membrane currents, while it dose-dependently inhibited agonist (acetylcholine (ACh) or histamine)-induced ...
Soft drinks containing caffeine have been associated with more aggressive forms of dental decay. Cariogenicity of caffeinated soft drinks may be attributed to the effect of caffeine on salivary flow. This study assessed whether caffeinated soft drinks produced short-term oral dryness in healthy adults.
Methods
The authors collected saliva on two separate days from 35 participants before and one hour after drinking a soft drink. On one of the days the soft drink was caffeinated and on the other day it was not. Saliva collection involved 15 minutes unstimulated whole saliva, 5 minutes paraffin-stimulated whole saliva, and 10 seconds labial minor salivary gland output.
Results
Unstimulated and stimulated flow rates slightly increased and minor gland output slightly decreased one hour after the soft drink consumption regardless of caffeine content. These changes were not statistically significant (two-period two-treatment crossover trial using two-stage Grizzle model, p>0.05). A linear mixed model statistic did not show the caffeine effect on salivary flow rate.
Conclusions
Caffeinated soft drink consumption had no significant effect on salivary flow rate after one hour by any of the three measures employed in this study. Caffeine's contribution to the cariogenicity of soft drinks is likely by centrally-mediated effects on consumption patterns.
Frequent exposure to fermentable carbohydrates has been recognized as the hallmark of a cariogenic diet.1 In particular, regular consumption of sweetened soft drinks has been associated with an increase rate of dental caries.2 Less described in the literature has been the association between the more aggressive forms of dental caries and regular consumption of caffeinated soft drinks. Caffeine-free soft drinks appear to be less frequently associated with aggressive forms of decay.3 One possible explanation for this association might be that the caffeine tends to promote patterns of consumption that are deleterious to dental health—more frequent, more prolonged, and perpetual. One mechanism by which caffeine could support these changes in diet is through its effect on salivary flow.
Sufficient salivary flow is necessary to maintain oral health and integrity of the dentition. Saliva acts as a lubricant, washes away residue, contains various host defense systems, and helps maintains dental mineral integrity. Hyposalivation is therefore associated with an increased risk of oral diseases, including dental caries.4 Additionally, a diminished salivary flow is associated with the adoption of deleterious dietary habits, such as sucking on hard candies or using sweetened beverages to combat the sensation of oral dryness.5
Caffeine is a central nervous system stimulant with diuretic properties. Caffeine may reduce salivary flow by direct effects upon the salivary glands, through effects on the autonomic nervous system, or through diuresis and dehydration.6 Studies reported an increase in urine production after ingestion of caffeine equivalent to 3–6 cups of coffee.7,8 However, others found no effect of caffeine in standard serving sizes on hydration status.9,10
The primary constituent of saliva is water. Degree of hydration is potentially the most important factor influencing salivary flow.11 Dehydration or even hypohydration can cause decreased salivary flow.12 Although negative fluid balance has not been evident when caffeinated beverages are consumed in moderation,13 whether or not oral dryness is a consequence of caffeine consumption has not yet been reported.
Children and adolescents who consume large amounts of carbonated soft drinks have high caries experience.2,14,15 The presence of fermentable carbohydrates in the beverage no doubt plays a major role in early initiation and rapid progression of dental caries. What is often overlooked is that over 60% of soft drinks sold in the United States contain caffeine as a flavor additive.16 The combination of sugar and caffeine may encourage frequent and perpetual patterns of consumption, leading to early initiation and rapid progression of dental caries.15 Patients with rampant caries are frequently seen at the University of Minnesota School of Dentistry dental clinics (See Fig. 1). A common finding with these patients is the regular consumption of soda pop containing fermentable carbohydrates and caffeine.
Rampant caries in a patient who regularly consumed Mountain Dew. Cervical areas of all the teeth had bands of demineralization wrapped around the gingival area, with cavitated lesions and recurrent caries lesions present.
The aim of the study was to examine the effect of caffeinated soft drinks on oral dryness. The hypothesis is that caffeinated soft drinks will lead to oral dryness by decreasing salivary flow, as compared to soft drinks without caffeine.
The University of Minnesota Institutional Review Board approved the conduct of this clinical study (IRB# 1005M81575).
Subject recruitment
Sample size was determined using the following mean and standard deviation flow rates: 0.3±0.2 mL/min for unstimulated whole saliva, 3.0±1.4 μL/cm2·min for labial minor salivary gland secretion, and 2.2±1.1 mL/min for stimulated whole saliva.17 Using α=0.05, 34 participants per group would give 80% power to detect a 25% difference between baseline and post-caffeine stimulated and labial minor salivary gland flow rates and a 33% difference between baseline and post-caffeine unstimulated salivary flow rates.
We recruited 38 healthy adults of both genders, ages 19–63. The participants did not take stimulant-containing medications and did not have oral removable appliances. We asked the participants to abstain from caffeinated food or drink starting the night before and the morning of their appointment. Written informed consent was secured from, and demographic data recorded on, each participant.
Study protocol
The saliva collection took place at the same time for each participant on two separate mornings. After baseline saliva collection, subjects consumed 355 mL (12 oz) of either caffeinated drink (Mountain Dew; PepsiCo) or caffeine-free drink (Caffeine-Free Mountain Dew; PepsiCo) on the first day of the study, followed by the alternate version on the second day. The order was randomly determined by a coin flip during the first appointment. The soft drinks were poured into unmarked plastic cups so that the subjects were blinded to the caffeine content. Subjects consumed the soft drinks within a 30-minute period, and were asked to refrain from eating, drinking, brushing, or chewing gum until after the second saliva collection was performed one hour later.
Saliva collection
Salivary flow rates were measured in this sequence: unstimulated whole saliva, minor salivary gland secretion, and stimulated whole saliva.
Unstimulated whole saliva
After swallowing to clear the mouth, participants sat quietly and expectorated any saliva that collected in the mouth into a preweighed paper cup for 15 minutes. To ensure natural salivary flow, participants were instructed not to think of food, talk, or chew. The weight (g) of the collected saliva was measured and the volume (mL) inferred, assuming salivary density of 1 g/mL.
Minor labial salivary secretion
Secretion from labial minor salivary glands was estimated using a Periotron (Model 8000; Oraflow, Inc.).18 The participant's lower lip was gently extended and dried with a gauze square, then a SialoPaper strip (Oraflow, Inc.) was placed on the midline of the labial mucosa for 10 seconds and moisture content estimated with the Periotron. Three consecutive measurements were recorded and averaged. The Periotron output was calibrated with known volumes of deionized water using a linear regression technique.
Stimulated whole saliva
After swallowing to clear the mouth, subjects chewed a 5-cm square of Parafilm (American National Can) and expectorated any saliva that developed into a preweighed paper cup for 5 minutes. The weight (g) of the collected saliva was measured and the volume (mL) inferred.
Statistical analysis
The effect of caffeinated soft drink on unstimulated, stimulated, and minor gland saliva production was analyzed using the two-stage Grizzle model for the two-period two-treatment crossover trial.19,20 First, the data were tested for the presence of a carry-over effect according to the sequence of consumption, that is, caffeine or caffeine-free on the first day. Then the caffeine effect was estimated using a linear mixed model. Caffeine effect refers to the difference between the average change in flow rate after caffeinated soft drink and the average change in flow rate after caffeine-free soft drink.
Of the 38 subjects enrolled in the study, 3 did not return for the second appointment due to scheduling conflicts. Only 35 completed the study. Table 1 reports demographic and soft drink sequences for the 35 participants.
Table 1.
Demographics and Sequence of Soft Drink Consumption of Study Participants (n=35)
Age (mean±SD)
27.7±10.6
Gender (N, percentage)
Male
18 (51%)
Female
17 (49%)
Ethnicity (N, percentage)
African American
8 (23%)
Asian
9 (26%)
Hispanic
1 (3%)
White
17 (48%)
Sequence of soft drink consumption (N, percentage)
Caffeine on first day
16 (46%)
Caffeine-free on first day
19 (54%)
Table 2 shows the salivary flow rates before and after each soft drink was consumed and the caffeine effects. Both unstimulated and stimulated flow rates slightly increased one hour after soft drink consumption, whereas labial minor salivary gland output slightly decreased regardless of the beverage types. The two-stage Grizzle model indicated no carry-over effect presented in the data. Since there was no carry-over effect, the caffeine effects were estimated using the linear mixed model applied to the salivary flow rates of both days. No significant difference was found between the changes in salivary flow rates after caffeinated or caffeine-free soft drinks (linear mixed model with significance level of 0.05).
Table 2.
Caffeine Effect and Salivary Flow Rates Before and After Caffeinated and Caffeine-Free Soft Drink Consumption (n=35)
Δ=difference in salivary flow rate before and after soft drink consumption. Positive values represent increased salivary flow rate after the consumption, negative values represent decreased salivary flow rate after the consumption.
Caffeine effect, 95% confidence interval (CI), and p-value were analyzed using linear mixed model. Caffeine effect is defined as the difference between the average change in flow rate after consuming caffeine-containing soft drink and the average change in flow rate after consuming caffeine-free soft drink.
Caffeine is one of the most widely consumed dietary ingredients in the world. Approximately 80% of the world's population and 90% of adults in North America consume caffeine daily. Main sources of caffeine consumption are coffee (71%), soft drinks (16%), and tea (12%).21 These beverages each contain different amounts of caffeine. A standard 8 oz (240 mL) cup of brewed coffee contains 100–200 mg of caffeine, while instant coffee and tea contain ∼90 and 50 mg of caffeine, respectively.21 Cola and many non-cola soft drinks contain about 40 mg in a 12 oz (355 mL) can. Mountain Dew contains relatively more caffeine (55 mg) than other soft drinks.22
The alarming increase in dental caries seen in some young adults has caught the interest of the news media and has been named ‘Mountain Dew Mouth’.23 A recent study by Keast et al. demonstrated that caffeine suppresses sweetness in soft drinks resulting in the need for extra sugar to achieve an equivalent level of perceived sweetness.24 However, commercial non-caffeinated sodas have a sugar concentration ranging from 3.1–3.6 g/oz, whereas caffeinated sodas range from 3.2–3.9 g/oz.25 This small difference in sugar concentration would not be enough to explain the differences in caries rates seen between regular users of the two types of beverages.
The rapid progress of dental caries among caffeinated soft drink consumers might be a result of habitual consumption due to a physical desire for the caffeine in the beverages.26 Based on the hypothesis that caffeine causes oral dryness leading to further beverage consumption, we investigated whether a caffeinated soft drink could be related to short-term oral dryness as a possible contributing factor in observed aggressive caries patterns.
The results of this study do not support the hypothesis that caffeine leads to oral dryness, as salivary flow rates did not decrease after consuming a caffeinated soft drink compared to a caffeine-free soft drink. Following consumption of a caffeine-containing beverage, peak serum levels of caffeine are attained in 15 minutes to 2 hours.27 It would be expected that one hour after caffeine ingestion caffeine effects, if any, on saliva flow would be detectible. Rather, the flow rates of unstimulated and stimulated whole saliva slightly increased one hour after soft drink consumption regardless of the caffeine content. Minor salivary gland secretion slightly decreased, also regardless of the caffeine content. Any potential diuretic effect of caffeine in the amount consumed in the present study is not reflected in salivary flow rates.
The baseline flow rates measured before caffeinated and caffeine-free soft drink consumption (Table 2) closely resemble the values reported by Rudney et al.28 and Eliasson et al.,17,18 and summarized by Dawes.11 Some variation in the results was natural. Collection of stimulated and unstimulated whole saliva depended to some degree on participant cooperation. However, the participants had no control over their minor gland secretion. Therefore, this method of saliva collection may have been the most objective.
Degree of hydration is a factor that may influence salivary flow. Salivary flow rate decreases during dehydration.11,29 However, we found slightly increased flow rates for whole saliva in both caffeinated and caffeine-free groups one hour after beverage consumption. Although it seems intuitive that caffeine would have a diuretic effect, this decrease may be balanced out by the fluid intake.11 In addition, the high-fructose corn syrup, citric acid, sodium citrate, and flavoring agents of Mountain Dew may have increased the saliva flow rates. Interestingly, we found a slight decrease in the flow rate of the labial minor salivary gland in both caffeinated and caffeine-free soft drinks. Reduced minor labial salivary gland secretions have been reported among individuals with subjective oral dryness.17 Although it is conceivable that the sensation of “dry mouth” arising from decreased minor salivary gland secretion could drive individuals to consume more soft drink, in the present study the magnitude of the reduction was very small (5%–10%).
A review of the literature identified associations between low saliva flow and dental disease and between low saliva flow and dehydration, but it was not able to find a direct link between dehydration and dental disease.30 Whether or not a state of dehydration can be caused by ingested caffeine has been questioned. A recent review concluded that the caffeine dose in standard servings of coffee or carbonated soft drinks does not have diuretic action.10 A short-term increase in urine volume was reported with large doses of caffeine intake (250–300 mg, equivalent to 2–3 cups of coffee), but the effect is confounded by higher tolerance in individuals who regularly consume caffeine-containing beverages. Caffeinated beverages consumed in moderation did not cause negative fluid balance, even for athletes and exercising adults.13,31 Our study result tends to support the concept that the amount of caffeine in a single carbonated beverage is not high enough to cause a diuretic effect. Recognizing that it is possible that caffeine could affect salivary flow by mechanisms other than diuresis and dehydration, such as by direct effects upon the salivary glands or through effects on the autonomic nervous system,6 we were still unable to demonstrate any significant effect of caffeine on saliva gland function.
The physiological effects of caffeine diminish with regular use as tolerance builds up.32 We did ask each study participant whether they considered themselves heavy “caffeine users.” Six study participants reported that they were heavy caffeine users, while 26 reported that they were not heavy users. Assuming the self-report is accurate, it would be considered unlikely that the lack of effect of the studied beverage on salivary flow was caused by tolerance to caffeine among the study participants.
Manufacturers justify the addition of caffeine to soft drinks as a flavoring agent. A recent study of the effects of caffeine added to novel-flavored drinks found that, with repeated exposure, the caffeine increased subject preference for the beverages.33 The results of our study do not support short-term oral dryness caused by caffeine. Therefore, caffeine is unlikely to contribute to cariogenesis via effects on salivary flow. How caffeine in soft drinks affects consumption patterns remains to be demonstrated. We did not evaluate the impact of caffeine consumed on a regular basis. A single dose of caffeine may affect the body differently than caffeine consumed daily, because the response may depend on tolerance level. We did not attempt to record subjective sensations of dry mouth after consumption of caffeinated soft drinks. The sensation of oral dryness, irrespective of actual salivary parameters, could compel one to consume more of the caffeinated beverages.
Both caffeinated and caffeine-free soft drinks were associated with a slight increase in unstimulated and stimulated salivary flow rates and a slight decrease in the flow rate from labial minor salivary glands, however, these trends were not statistically significant. Any potential diuretic effects of caffeine in the amount found in a single soft drink were not reflected in salivary flow.
The project described was supported by Award Number UL1RR033183 from the National Center For Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. Additional support was received from the University of Minnesota School of Dentistry Summer Fellowship program and a faculty start-up fund.
Yes, but not under ordinary conditions, as it would require decarboxylation of benzoic acid into benzene.
You could force it with extremely high temperatures and placing iron and copper catalysts, but it won't happen significantly in broad daylight.
Benzene could form within soft drinks by reaction of sodium benzoate with ascorbic acid (vitamin C), but sparingly... If you drink roughly 20 L of soda a day, if it contained the usual ~10μg of benzene, you would ingest the equivalent of the benzene you would breathe from city air (that's not a lot...).
So yes, but not to the extent that we should care. See here.
State and national drinking water standards and health guidelines
EWG Health Guideline0.15 ppb
The EWG Health Guideline of 0.15 ppb for benzene was defined by the California Office of Environmental Health Hazard Assessment as a public health goal, the level of a drinking water contaminant that does not pose a significant health risk. This health guideline protects against cancer.
EPA Maximum Contaminant Level (MCL)5 ppb
The legal limit for benzene, established in 1987, was based on analytical detection limits at the time that the standard was set. This limit may not fully protect against the risk of cancer due to benzene exposure.
Benzene
Roberts Creek Water District
Benzene is a known human carcinogen. It also damages blood cells and the nervous system. Emissions from petroleum processing, hazardous waste landfills and underground storage tanks contaminate drinking water with benzene.
To protect against cancer risks, California set a public health goal for benzene in drinking water at 0.15 parts per billion, 30 times lower than the federal legal limit of 5 parts per billion.
Cancer-causing Volatile Organic Compounds
October 2019
Summary
Many tap water contaminants can move from water into air and can enter the body through the skin after a shower or bath. The federal government classifies such substances as volatile organic compounds, or VOCs. These pollutants originate in multiple sources, including gasoline, solvents, paints, cars, carpets and shower curtains.
Between 1987 and 1992, the federal government set legal limits for 21 different VOCs in tap water. No new or updated federal standards for VOCs have been set since then. The legal limits allow much greater VOC exposure than the amounts many public health agencies consider pose minimal harm to human health.
The full range of potential VOC contaminants in everyday environments may include hundreds of chemicals. Most worrisome are those that harm the developing fetus or increase the risk of cancer. This report highlights 12 carcinogenic VOCs EWG believes should be regulated as a group of tap water contaminants.
Carcinogenic VOCs in tap water
In 2011 the Environmental Protection Agency recommended establishing a single regulatory standard for a group of carcinogenic VOCs, but it has not yet proposed a new standard. Of the 12 VOCs listed in the table below, the EPA recommended including nine in a group of potentially carcinogenic VOCs. EWG believes three additional chemicals should be added to the list: 1,4-dioxane, styrene and 1,1,2-trichloroethane.
Saliva has a pH normal range of 6.2-7.6 with 6.7 being the average pH. Resting pH of mouth does not fall below 6.3. In the oral cavity, the pH is maintained near neutrality (6.7-7.3) by saliva. The saliva contributes to maintenance of the pH by two mechanisms.
There are numerous small salivary glands within the tongue, lips cheeks and palate. Humans also have four large glands termed the parotid, submaxillary, submandibular and sublingualsalivary glands. Collectively, the primary function of these glands is the production and secretion of saliva. The components of saliva are produced by serous and mucous acinarcells within the glands, and saliva is then drained though a network of ducts into the oral cavity.
Aside from the production of saliva, the large salivary glands also help protect us from the numerous microbes that we are constantly exposed to through our mouth. Plasma B cells reside in the salivary glands and produce IgA antibody which is then secreted in the saliva. IgA binds the mucus layer that covers the epithelia lining of the oral cavity, thus providing a barrier against potentially dangerous pathogens.
As part of the oral mucosal system, the salivary gland is also exposed to a large number of harmless antigens in the form of foods. To ensure that our white blood cells do not react to these proteins, immune responses in the salivary glands are tightly regulated. One mechanism by which regulation occurs is through the production of a suppressive immune molecule (cytokine) called transforming growth factor–b (TGF–b). TGF–b limits the expansion of T and B cells and inhibits their ability to induce inflammation, therefore preventing accidental tissue damage that may otherwise be triggered by food.
Mechanisms employed by our immune system to stop reactivity to harmless antigens can also be exploited by unwanted pathogens. Viruses including human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) can spread via infected saliva. In the case of cytomegalovirus, virus infection of the salivary gland (Figure 1) triggers the production of another immune suppressive cytokine called interleukin-10 (IL-10). Like TGF-b , IL-10 can inhibit the accumulation and function of T cells in the salivary glands. Because T cells are required to kill cytomegalovirus, IL-10 mediated inhibition of these cells results in persistent viral replication in, and dissemination from, the salivary glands.
Salivary transmission by the 30 million human immunodeficiency virus (HIV) carriers is rare, despite kissing, aerosolization, and dental treatment. The main protective mechanism of saliva is reported to be inactivation of HIV-transmitting leukocytes by its unique hypotonicity; however, the successful oral transmission of HIV by seminal fluid and milk is unexplained. Whether seminal fluid and milk successfully transmit HIV orally by overcoming the recipient's salivary hypotonic inactivation of HIV-transmitting leukocytes was tested. Isotonic salt solution and normal donor samples of milk, colostrum, seminal fluid, and blood were studied for their ability to overcome the salivary hypotonic inactivation. All samples, in physiologic volumes, prevented the hypotonie saliva from inactivating HIV-transmitting leukocytes by providing solutes and retarding diffusion. This indicates that successful oral transmission of HIV by seminal fluid, milk, and colostrum may be due to their isotonicity, which overcomes hypotonie salivary inactivation of HIV-transmitting leukocytes.
Oral transmission of human immunodeficiency virus (HIV) by the 30 million HIV carriers is rare during kissing, biting, aerosolization, and dental treatment, even when infected blood or exudate is shed into the mouth [1–8]. This phenomenon is reportedly due to salivary inactivation of HIV-infected leukocytes that are shed into the mouth [9]. Saliva's disruption of leukocytes has been confirmed in recent studies [10]. The inactivation of leukocytes is attributable primarily to saliva's strong hypotonicity, although other, less active inhibitors of HIV also are present in saliva [9, 11–28]. Oral transmission of HIV, however, does occur epidemiologically if infected seminal fluid [29–34] or milk [35–39] is deposited orally, despite the presence of saliva. By use of patient samples, we studied the mechanisms by which the carrier's seminal fluid or milk may overcome protection by the recipient's saliva in vitr
Outbreak pneumonia announced in Wuhan, China, in December 2019, had its causative factor classified as a new coronavirus (SARS-CoV-2). Since saliva can host several viruses including SARS-CoV-2, the transmission chance of viruses through saliva, particularly those causing respiratory infections, is unavoidable. COVID-19 can be detected through salivary diagnostic testing which has lots of advantages for medical care professionals and patients. It should be noted that not only does saliva offer an ecological niche for the colonization and development of oral microorganisms, but it also prevents the overgrowth of particular pathogens such as viral factors. The aim of this study is to gather all the information about saliva and its association with COVID-19 for the whole health care professionals across the world.
Outbreak pneumonia announced in Wuhan, China, in December 2019, had its causative factor classified as a new coronavirus. On March 11, 2020, the World Health Organization (WHO) announced that the epidemic of the latest coronavirus, Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), is pandemic currently known COronaVIrus Disease (COVID-19). Until May 20, 2020, the number of confirmed cases is 4,789,205 with over 318,789 deaths in 213 countries or territories [1], [2].
The clinical symptoms of COVID-19 are cough, fever, shortness of breath, muscle pain, sore throat, confusion, chest pain, headache, rhinorrhea (4%), diarrhea, and nausea and vomiting. SARS-CoV-2 transmits human-to-human by either direct transmission such as cough, sneeze, and droplet inhalation, or contact transmission like ocular contact, saliva, mucous membranes of the nose and eyes [2], [3].
Since saliva can host several viruses including SARS-CoV-2, the transmission chance of viruses through saliva, particularly those causing respiratory infections, is unavoidable in a dental office. Based on experience in combating the COVID-19 outbreak, stopping disease transmission by saliva in the dental clinic is vital to the safety of doctors and patients.
The analysis of saliva in COVID-19 cases can help to explain the pathogenesis because epithelial oral cavity cells demonstrated ample expression of the Angiotensin-Converting Enzyme 2 (ACE2) receptor that plays a critical role in allowing SARS-CoV-2 to enter the cells [4].
A quick and efficient diagnosis of COVID-19 is essential in monitoring the pandemic. The suggested upper respiratory tract specimen types to diagnose COVID-19 are oropharyngeal and nasopharyngeal swabs. Nevertheless, gathering these specimen types involves direct interaction between health workers and patients, presenting a high risk of virus transmission. Moreover, collecting oropharyngeal or nasopharyngeal specimens comes with pain and can lead to bleeding, particularly in thrombocytopenia patients. Thus, oropharyngeal or nasopharyngeal swabs cannot be suitable for serial controlling of viral load. Specimens of saliva can be conveniently given by telling patients to spit into a sterile container [5].
The aim of this study is to gather all the information about saliva and its association with COVID-19 for the whole health care professionals across the world.
Human saliva is a distinctive body fluid that is produced by the salivary glands. Saliva mostly consists of water (94–99%) with organic molecules accounting for nearly 0.5% and inorganic ones for 0.2%. It plays an important role in digesting food, lubricating oral mucosa, cleaning and preserving the oral cavity, and influencing the homeostasis of the oral cavity. A normal adult usually generates about 600 ml of saliva every day. Besides salivary gland excreta, saliva also includes food particles, serum elements, oral microorganisms and their metabolites, white blood cells, and exfoliated epithelial cells.
By now, over 700 microbial species have been detected in saliva, many of which are linked to oral and systemic diseases. Not only does saliva offer an ecological niche for the colonization and development of oral microorganisms, but it also prevents the overgrowth of particular pathogens to preserve the homeostasis of the oral cavity. In addition, saliva may serve as a gatekeeper, and prevent pathogens from spreading to the gastrointestinal and respiratory tract [6].
SARS-CoV-2 has at least three separate routes to present in saliva. SARS-CoV-2 in the lower and upper respiratory tract reaches the oral cavity along with the liquid droplets; SARS-CoV-2 in the blood may enter the mouth through the gingival crevicular fluid; and major and minor infection of the salivary gland, with the ensuing release of particles into the saliva through salivary ducts [7].
Human saliva is a complicated fluid and plays a crucial role in preventing from a viral infection, especially through the innate immune system, which is a notable first-line defense [8]. Iwabuchi et al. proposed that hyposalivation could result in severe respiratory infection. Two possible explanations for enhancing the incidence rate of this infection are as follows:
• Lowered saliva secretion can disrupt the oral and airway mucosal surfaces as a physical barrier, thereby enhancing the viral colonization and adhesion.
• This decrease may also hinder the secretion of antimicrobial peptides and proteins [8], [9].
Considering the existence of various proteins with established antiviral characteristics in saliva such as lysozyme, mucins, cathelicidin (LL-37), lactoferrin, peroxidase, sIgA SLPI, salivary agglutinin (gp340, DMBT1), alpha-defensins, beta-defensins, and cystatins, some of which may potentially impede virus replication especially SARS-CoV-2. Besides, antiviral activity in saliva can be due to salivary microvesicles including at least 20 microRNA’s, which may restrict the replication of some types of viruses. This provides the idea that these salivary proteins can have the same defensive impact against SARS-CoV-2. A possible risk factor for severe respiratory infection could be hyposalivation. It can leave patients at a significant risk of getting COVID-19 [8].
Salivary glands as potential reservoirs for COVID-19
ACE-2 is a critical COVID-19 receptor. Liu et al. studied SARS-CoV and showed that epithelial cells of salivary gland having elevated ACE-2 expression were infected [10]. The ACE-2 expression in minor salivary glands was greater than that in the lungs, indicating that a target for COVID-19 may possibly be salivary glands. Furthermore, before lung lesions emerge, SARS-CoV RNA can be found in the saliva. This could account for asymptomatic infections. For SARS-CoV, the salivary gland is a significant reservoir of the virus in saliva. The positive rate of COVID-19 in the saliva of patients can exceed about 92%, and the live virus can also be cultivated through saliva samples. This proposes that COVID-19 spread through asymptomatic infection may come from the contaminated saliva. Consequently, the source of asymptomatic infection could be salivary glands [11].
The size of droplets can determine how far and long they can fly along with the airflow. Huge droplets within a short distance or touching infected surfaces spread majority of transmissible respiratory infections. Huge droplets with a diameter of greater than 60 μm appear to settle rapidly from the air, making the transmission risk minimal for individuals in close vicinity to the source of the saliva droplet. Small droplets with a diameter of less than or equal to 60 μm can cause short-range transmission for individuals with distance less than one meter. In a desirable environment, small droplets are likely to fade away into droplet nuclei with a diameter of less than 10 μm, and then become capable of long-range aerosol transmission.
When speaking, coughing, sneezing, or even breathing, saliva droplets are produced and shaped as particles in a combination of moisture and droplet nuclei of microorganisms. The quantity, distance, and size of saliva droplets vary among individuals, indicating that the infectious intensity and transmission route of saliva droplets differ when the same pathogen is contracted. Each cough can produce about 3000 saliva droplets nuclei, which is approximately equivalent to the quantity generated during a 5-min chat. Each sneeze can produce roughly 40,000 droplets of saliva covering several meters in the air. A regular exhalation may create saliva droplets that exceed one meter in the air. Huge saliva droplets with more mass typically fall to the ground ballistically and small saliva droplets fly by airflow like a cloud over longer distances.
For a susceptible host to develop infectious droplets of saliva, they can enter the mouth, eyes, or be inhaled directly into the lungs. The virus may lead to another individual’s respiratory infections by inducing ocular complications. Thus, the SARS-CoV contamination was minimized to a degree by wearing surgical masks and protective eyewear or face shield in vulnerable healthcare workers [2], [12].
SARS-CoV-2 may cause acute and chronic sialadenitis
Wang et al. proposed that SARS-CoV-2 might induce acute sialadenitis and associated symptoms, such as pain, discomfort, inflammation, and secretory dysfunction in salivary glands.
SARS-CoV-2 can attach to ACE-2 receptors on the epithelium of salivary glands, fuse with them, replicate, and lyse cells to trigger apparent signs and symptoms, such as discomfort, inflammation, and pain in major salivary glands. After the cytolytic activity of SARS-CoV-2 lyses the acinar cells, salivary amylase is unleashed into the peripheral blood. It can be inferred that the amylase rises in peripheral blood during the early contamination process. Secreted inflammatory cytokines facilitate the inflammatory reaction that destroys the tissue of the salivary glands as the immunopathological process continues. Granulation and fibrogenesis can restore the inflammatory damage by decreasing immunoreaction. After the severe stage, the function of salivary glands can be anomalous due to contamination with SARS-CoV-2, which may induce chronic sialadenitis [13].
The main strategy of identification for COVID-19 is Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR), which is commonly used to extract viral RNA from oropharyngeal and nasopharyngeal swabs or sputum samples. In addition, a chest X-ray may be an invaluable diagnostic method for identifying bilateral pneumonia, displaying as multi-lobar ground-glass opacities with an asymmetric, peripheral, and posterior distribution [14].
Saliva is now widely established as a reservoir for biological indicators that range from modifications in nucleic acids, proteins, and biochemicals to the microflora. As a diagnostic fluid, saliva has tremendous potential and advantage over other biological fluids because the sampling process of saliva does not entail an invasive intervention, and it is inexpensive and helpful for controlling the systemic health. In the near future, designing accurate and responsive salivary diagnostic instruments and the implementation of established guidelines following meticulous testing will enable the use of salivary diagnostic as chair-side tests for diversified oral and systemic diseases. The benefits of salivary diagnostic tests are economical, noninvasive, healthier to apply than serum sampling, diagnostic values in real-time, no requirement for specialized healthcare workers, numerous samples are simple to obtain, collecting and monitoring are doable at home, minimizing the possibility of cross-infection, better shipping and storage than serum sampling, lesser agitation during the diagnostic process, screening assays are commercially available, and saliva does not clot and can be handled more efficiently than blood. Thus, salivary diagnostic testing can offer a convenient and cost-effective mechanism for early-diagnosis of Covid-19 [15].
It has been documented that three methods capture saliva thus far: saliva swabs, coughing out, and directly from the salivary gland duct. For clinical applications needing a strong positive rate of virus identification, saliva from deep throat provides the strongest positive rate, which could account for early-diagnosis of COVID-19. Saliva extracted from saliva gland ducts is consistent with acute COVID-19 which may likely be a reliable and noninvasive test for acute patients [12].
There are several diagnostic methods that can be conducted before commencing a dental emergency treatment in dental clinics including loop-mediated isothermal amplification (LAMP) tests, antibody testing, and microfluidic RT-PCR devices [16], [7].
There are six studies evaluating the diagnosis efficiency of saliva and all the associated information is shown in Table 1 .
Table 1
The most recent clinical findings using salivary diagnostic tests for COVID-19.
Evaluation and treatment of patients with salivary gland disease in this pandemic situation.
A three-step guideline to survey patients with salivary gland disease is as follows [22]:
(1)
Primary telemedicine examination
1.1. Recognize patients having touchable or visible lesions, mass presence in the majority of salivary gland region, and neoplasm symptoms
1.2. Ask the history of skin cancer of the head and neck, lymphoma, etc.
1.3. Ask the clinical symptoms and history of inflammatory salivary gland disease
(2)
Diagnostic examination for patients suspected of having salivary gland neoplasm
2.1. COVID-19 screening
2.2. FNA biopsy
2.3. Ultrasound of salivary gland
2.4. Extra imaging such as MRI, if needed
2.5. Make sure of the existence of salivary gland disorder
2.6. Identify signs and symptoms of non-neoplastic illness
(3)
Patient counseling and recommendations for treatment
3.1. Survey FNA biopsy outcomes
3.2. Assess treatment options and prognosis
3.3. Prescribe COVID-19 test before any surgery
3.4. Pay attention to age comorbidities of the patient
3.5. Radiation oncology and hematology consultations, if required
3.6. Considering the outbreak situation
Patients can optimally receive the test for COVID-19 before any surgical procedures. Health professionals need to be conscious about COVID-19 false negatives, and the infeasibility of repeating COVID-19 testing for most patients. In some selected cases, chest CT can be recommended for further assessment and risk stratification to evaluate signs of COVID-19-associated pulmonary activity or metastases.
Surgery will be postponed for any patients with abnormal chest CT results, those either with symptoms of COVID-19 or confirmed cases of COVID-19. If dental surgery is an emergency, dentists must address the possibility of aerosolization. Sinonasal area and pharynx, suitable PPE of the whole operating room team are strongly advised for minor salivary gland tumors of the oral cavity. The number of staff in the operating room must be minimized.
The risk of aerosolized mucosal secretions is only found during intubation and extubation for parotid and submandibular neoplasm surgeries. During intubation and extubation, all the staff in the room must wear suitable PPE while all unneeded staff must leave the room.
Controlling the obstructive salivary gland disease can be remotely conducted in most cases. In rare situations, intraoperative drainage and biopsy or office-based assessment and treatment procedures may be needed for handling an abscess, detecting cancer, relieving acute pain which has not been treated with proper medical care. Due to irrigation and aerosolization, sialendoscopy should be stopped during the outbreak of COVID-19 [22].
In addition, all dentists should know that the use of a mouthrinse and/or local nasal products, which include beta-cyclodextrins in conjunction with flavonoid agents, might provide invaluable adjunctive care to minimize the viral load of saliva and nasopharyngeal microbiota, including SARS-CoV-2 [23]. Therefore, dental professionals can create a safer atmosphere for themselves as well as their patients.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Soft drinks containing caffeine have been associated with more aggressive forms of dental decay. Cariogenicity of caffeinated soft drinks may be attributed to the effect of caffeine on salivary flow. This study assessed whether caffeinated soft drinks produced short-term oral dryness in healthy adults.
Methods
The authors collected saliva on two separate days from 35 participants before and one hour after drinking a soft drink. On one of the days the soft drink was caffeinated and on the other day it was not. Saliva collection involved 15 minutes unstimulated whole saliva, 5 minutes paraffin-stimulated whole saliva, and 10 seconds labial minor salivary gland output.
Results
Unstimulated and stimulated flow rates slightly increased and minor gland output slightly decreased one hour after the soft drink consumption regardless of caffeine content. These changes were not statistically significant (two-period two-treatment crossover trial using two-stage Grizzle model, p>0.05). A linear mixed model statistic did not show the caffeine effect on salivary flow rate.
Conclusions
Caffeinated soft drink consumption had no significant effect on salivary flow rate after one hour by any of the three measures employed in this study. Caffeine's contribution to the cariogenicity of soft drinks is likely by centrally-mediated effects on consumption patterns.
Frequent exposure to fermentable carbohydrates has been recognized as the hallmark of a cariogenic diet.1 In particular, regular consumption of sweetened soft drinks has been associated with an increase rate of dental caries.2 Less described in the literature has been the association between the more aggressive forms of dental caries and regular consumption of caffeinated soft drinks. Caffeine-free soft drinks appear to be less frequently associated with aggressive forms of decay.3 One possible explanation for this association might be that the caffeine tends to promote patterns of consumption that are deleterious to dental health—more frequent, more prolonged, and perpetual. One mechanism by which caffeine could support these changes in diet is through its effect on salivary flow.
Sufficient salivary flow is necessary to maintain oral health and integrity of the dentition. Saliva acts as a lubricant, washes away residue, contains various host defense systems, and helps maintains dental mineral integrity. Hyposalivation is therefore associated with an increased risk of oral diseases, including dental caries.4 Additionally, a diminished salivary flow is associated with the adoption of deleterious dietary habits, such as sucking on hard candies or using sweetened beverages to combat the sensation of oral dryness.5
Caffeine is a central nervous system stimulant with diuretic properties. Caffeine may reduce salivary flow by direct effects upon the salivary glands, through effects on the autonomic nervous system, or through diuresis and dehydration.6 Studies reported an increase in urine production after ingestion of caffeine equivalent to 3–6 cups of coffee.7,8 However, others found no effect of caffeine in standard serving sizes on hydration status.9,10
The primary constituent of saliva is water. Degree of hydration is potentially the most important factor influencing salivary flow.11 Dehydration or even hypohydration can cause decreased salivary flow.12 Although negative fluid balance has not been evident when caffeinated beverages are consumed in moderation,13 whether or not oral dryness is a consequence of caffeine consumption has not yet been reported.
Children and adolescents who consume large amounts of carbonated soft drinks have high caries experience.2,14,15 The presence of fermentable carbohydrates in the beverage no doubt plays a major role in early initiation and rapid progression of dental caries. What is often overlooked is that over 60% of soft drinks sold in the United States contain caffeine as a flavor additive.16 The combination of sugar and caffeine may encourage frequent and perpetual patterns of consumption, leading to early initiation and rapid progression of dental caries.15 Patients with rampant caries are frequently seen at the University of Minnesota School of Dentistry dental clinics (See Fig. 1). A common finding with these patients is the regular consumption of soda pop containing fermentable carbohydrates and caffeine.
Rampant caries in a patient who regularly consumed Mountain Dew. Cervical areas of all the teeth had bands of demineralization wrapped around the gingival area, with cavitated lesions and recurrent caries lesions present.
The aim of the study was to examine the effect of caffeinated soft drinks on oral dryness. The hypothesis is that caffeinated soft drinks will lead to oral dryness by decreasing salivary flow, as compared to soft drinks without caffeine.
The University of Minnesota Institutional Review Board approved the conduct of this clinical study (IRB# 1005M81575).
Subject recruitment
Sample size was determined using the following mean and standard deviation flow rates: 0.3±0.2 mL/min for unstimulated whole saliva, 3.0±1.4 μL/cm2·min for labial minor salivary gland secretion, and 2.2±1.1 mL/min for stimulated whole saliva.17 Using α=0.05, 34 participants per group would give 80% power to detect a 25% difference between baseline and post-caffeine stimulated and labial minor salivary gland flow rates and a 33% difference between baseline and post-caffeine unstimulated salivary flow rates.
We recruited 38 healthy adults of both genders, ages 19–63. The participants did not take stimulant-containing medications and did not have oral removable appliances. We asked the participants to abstain from caffeinated food or drink starting the night before and the morning of their appointment. Written informed consent was secured from, and demographic data recorded on, each participant.
Study protocol
The saliva collection took place at the same time for each participant on two separate mornings. After baseline saliva collection, subjects consumed 355 mL (12 oz) of either caffeinated drink (Mountain Dew; PepsiCo) or caffeine-free drink (Caffeine-Free Mountain Dew; PepsiCo) on the first day of the study, followed by the alternate version on the second day. The order was randomly determined by a coin flip during the first appointment. The soft drinks were poured into unmarked plastic cups so that the subjects were blinded to the caffeine content. Subjects consumed the soft drinks within a 30-minute period, and were asked to refrain from eating, drinking, brushing, or chewing gum until after the second saliva collection was performed one hour later.
Saliva collection
Salivary flow rates were measured in this sequence: unstimulated whole saliva, minor salivary gland secretion, and stimulated whole saliva.
Unstimulated whole saliva
After swallowing to clear the mouth, participants sat quietly and expectorated any saliva that collected in the mouth into a preweighed paper cup for 15 minutes. To ensure natural salivary flow, participants were instructed not to think of food, talk, or chew. The weight (g) of the collected saliva was measured and the volume (mL) inferred, assuming salivary density of 1 g/mL.
Minor labial salivary secretion
Secretion from labial minor salivary glands was estimated using a Periotron (Model 8000; Oraflow, Inc.).18 The participant's lower lip was gently extended and dried with a gauze square, then a SialoPaper strip (Oraflow, Inc.) was placed on the midline of the labial mucosa for 10 seconds and moisture content estimated with the Periotron. Three consecutive measurements were recorded and averaged. The Periotron output was calibrated with known volumes of deionized water using a linear regression technique.
Stimulated whole saliva
After swallowing to clear the mouth, subjects chewed a 5-cm square of Parafilm (American National Can) and expectorated any saliva that developed into a preweighed paper cup for 5 minutes. The weight (g) of the collected saliva was measured and the volume (mL) inferred.
Statistical analysis
The effect of caffeinated soft drink on unstimulated, stimulated, and minor gland saliva production was analyzed using the two-stage Grizzle model for the two-period two-treatment crossover trial.19,20 First, the data were tested for the presence of a carry-over effect according to the sequence of consumption, that is, caffeine or caffeine-free on the first day. Then the caffeine effect was estimated using a linear mixed model. Caffeine effect refers to the difference between the average change in flow rate after caffeinated soft drink and the average change in flow rate after caffeine-free soft drink.
Of the 38 subjects enrolled in the study, 3 did not return for the second appointment due to scheduling conflicts. Only 35 completed the study. Table 1 reports demographic and soft drink sequences for the 35 participants.
Table 1.
Demographics and Sequence of Soft Drink Consumption of Study Participants (n=35)
Age (mean±SD)
27.7±10.6
Gender (N, percentage)
Male
18 (51%)
Female
17 (49%)
Ethnicity (N, percentage)
African American
8 (23%)
Asian
9 (26%)
Hispanic
1 (3%)
White
17 (48%)
Sequence of soft drink consumption (N, percentage)
Caffeine on first day
16 (46%)
Caffeine-free on first day
19 (54%)
Table 2 shows the salivary flow rates before and after each soft drink was consumed and the caffeine effects. Both unstimulated and stimulated flow rates slightly increased one hour after soft drink consumption, whereas labial minor salivary gland output slightly decreased regardless of the beverage types. The two-stage Grizzle model indicated no carry-over effect presented in the data. Since there was no carry-over effect, the caffeine effects were estimated using the linear mixed model applied to the salivary flow rates of both days. No significant difference was found between the changes in salivary flow rates after caffeinated or caffeine-free soft drinks (linear mixed model with significance level of 0.05).
Table 2.
Caffeine Effect and Salivary Flow Rates Before and After Caffeinated and Caffeine-Free Soft Drink Consumption (n=35)
Δ=difference in salivary flow rate before and after soft drink consumption. Positive values represent increased salivary flow rate after the consumption, negative values represent decreased salivary flow rate after the consumption.
Caffeine effect, 95% confidence interval (CI), and p-value were analyzed using linear mixed model. Caffeine effect is defined as the difference between the average change in flow rate after consuming caffeine-containing soft drink and the average change in flow rate after consuming caffeine-free soft drink.
Caffeine is one of the most widely consumed dietary ingredients in the world. Approximately 80% of the world's population and 90% of adults in North America consume caffeine daily. Main sources of caffeine consumption are coffee (71%), soft drinks (16%), and tea (12%).21 These beverages each contain different amounts of caffeine. A standard 8 oz (240 mL) cup of brewed coffee contains 100–200 mg of caffeine, while instant coffee and tea contain ∼90 and 50 mg of caffeine, respectively.21 Cola and many non-cola soft drinks contain about 40 mg in a 12 oz (355 mL) can. Mountain Dew contains relatively more caffeine (55 mg) than other soft drinks.22
The alarming increase in dental caries seen in some young adults has caught the interest of the news media and has been named ‘Mountain Dew Mouth’.23 A recent study by Keast et al. demonstrated that caffeine suppresses sweetness in soft drinks resulting in the need for extra sugar to achieve an equivalent level of perceived sweetness.24 However, commercial non-caffeinated sodas have a sugar concentration ranging from 3.1–3.6 g/oz, whereas caffeinated sodas range from 3.2–3.9 g/oz.25 This small difference in sugar concentration would not be enough to explain the differences in caries rates seen between regular users of the two types of beverages.
The rapid progress of dental caries among caffeinated soft drink consumers might be a result of habitual consumption due to a physical desire for the caffeine in the beverages.26 Based on the hypothesis that caffeine causes oral dryness leading to further beverage consumption, we investigated whether a caffeinated soft drink could be related to short-term oral dryness as a possible contributing factor in observed aggressive caries patterns.
The results of this study do not support the hypothesis that caffeine leads to oral dryness, as salivary flow rates did not decrease after consuming a caffeinated soft drink compared to a caffeine-free soft drink. Following consumption of a caffeine-containing beverage, peak serum levels of caffeine are attained in 15 minutes to 2 hours.27 It would be expected that one hour after caffeine ingestion caffeine effects, if any, on saliva flow would be detectible. Rather, the flow rates of unstimulated and stimulated whole saliva slightly increased one hour after soft drink consumption regardless of the caffeine content. Minor salivary gland secretion slightly decreased, also regardless of the caffeine content. Any potential diuretic effect of caffeine in the amount consumed in the present study is not reflected in salivary flow rates.
The baseline flow rates measured before caffeinated and caffeine-free soft drink consumption (Table 2) closely resemble the values reported by Rudney et al.28 and Eliasson et al.,17,18 and summarized by Dawes.11 Some variation in the results was natural. Collection of stimulated and unstimulated whole saliva depended to some degree on participant cooperation. However, the participants had no control over their minor gland secretion. Therefore, this method of saliva collection may have been the most objective.
Degree of hydration is a factor that may influence salivary flow. Salivary flow rate decreases during dehydration.11,29 However, we found slightly increased flow rates for whole saliva in both caffeinated and caffeine-free groups one hour after beverage consumption. Although it seems intuitive that caffeine would have a diuretic effect, this decrease may be balanced out by the fluid intake.11 In addition, the high-fructose corn syrup, citric acid, sodium citrate, and flavoring agents of Mountain Dew may have increased the saliva flow rates. Interestingly, we found a slight decrease in the flow rate of the labial minor salivary gland in both caffeinated and caffeine-free soft drinks. Reduced minor labial salivary gland secretions have been reported among individuals with subjective oral dryness.17 Although it is conceivable that the sensation of “dry mouth” arising from decreased minor salivary gland secretion could drive individuals to consume more soft drink, in the present study the magnitude of the reduction was very small (5%–10%).
A review of the literature identified associations between low saliva flow and dental disease and between low saliva flow and dehydration, but it was not able to find a direct link between dehydration and dental disease.30 Whether or not a state of dehydration can be caused by ingested caffeine has been questioned. A recent review concluded that the caffeine dose in standard servings of coffee or carbonated soft drinks does not have diuretic action.10 A short-term increase in urine volume was reported with large doses of caffeine intake (250–300 mg, equivalent to 2–3 cups of coffee), but the effect is confounded by higher tolerance in individuals who regularly consume caffeine-containing beverages. Caffeinated beverages consumed in moderation did not cause negative fluid balance, even for athletes and exercising adults.13,31 Our study result tends to support the concept that the amount of caffeine in a single carbonated beverage is not high enough to cause a diuretic effect. Recognizing that it is possible that caffeine could affect salivary flow by mechanisms other than diuresis and dehydration, such as by direct effects upon the salivary glands or through effects on the autonomic nervous system,6 we were still unable to demonstrate any significant effect of caffeine on saliva gland function.
The physiological effects of caffeine diminish with regular use as tolerance builds up.32 We did ask each study participant whether they considered themselves heavy “caffeine users.” Six study participants reported that they were heavy caffeine users, while 26 reported that they were not heavy users. Assuming the self-report is accurate, it would be considered unlikely that the lack of effect of the studied beverage on salivary flow was caused by tolerance to caffeine among the study participants.
Manufacturers justify the addition of caffeine to soft drinks as a flavoring agent. A recent study of the effects of caffeine added to novel-flavored drinks found that, with repeated exposure, the caffeine increased subject preference for the beverages.33 The results of our study do not support short-term oral dryness caused by caffeine. Therefore, caffeine is unlikely to contribute to cariogenesis via effects on salivary flow. How caffeine in soft drinks affects consumption patterns remains to be demonstrated. We did not evaluate the impact of caffeine consumed on a regular basis. A single dose of caffeine may affect the body differently than caffeine consumed daily, because the response may depend on tolerance level. We did not attempt to record subjective sensations of dry mouth after consumption of caffeinated soft drinks. The sensation of oral dryness, irrespective of actual salivary parameters, could compel one to consume more of the caffeinated beverages.
Both caffeinated and caffeine-free soft drinks were associated with a slight increase in unstimulated and stimulated salivary flow rates and a slight decrease in the flow rate from labial minor salivary glands, however, these trends were not statistically significant. Any potential diuretic effects of caffeine in the amount found in a single soft drink were not reflected in salivary flow.
The project described was supported by Award Number UL1RR033183 from the National Center For Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. Additional support was received from the University of Minnesota School of Dentistry Summer Fellowship program and a faculty start-up fund.
Diet soda is doing these 7 awful things to your body
nbcnews.com / Today
By TODAY Health
Pop quiz! What's the single biggest source of calories for Americans? White bread? Big Macs? Actually, try soda. The average American drinks about two cans of the stuff every day. "But I drink diet soda," you say. "With no calories or sugar, it's the perfect alternative for weight watchers...Right?"
Not so fast. Before you pop the top off the caramel-colored bubbly, know this: guzzling diet soda comes with its own set of side effects that may harm your health--from kickstarting kidney problems to adding inches to your waistline.
Unfortunately, diet soda is more in vogue than ever. Kids consume the stuff at more than double the rate of last decade, according to research in the American Journal of Clinical Nutrition. Among adults, consumption has grown almost 25 percent.
But knowing these 7 side effects of drinking diet soda may help you kick the can for good.
Kidney Problems
Here's something you didn't know about your diet soda: It might be bad for your kidneys. In an 11-year-long Harvard Medical School study of more than 3,000 women, researchers found that diet cola is associated with a two-fold increased risk for kidney decline. Kidney function started declining when women drank more than two sodas a day. Even more interesting: Since kidney decline was not associated with sugar-sweetened sodas, researchers suspect that the diet sweeteners are responsible.
According to a 2008 University of Minnesota study of almost 10,000 adults, even just one diet soda a day is linked to a 34% higher risk of metabolic syndrome, the group of symptoms including belly fat and high cholesterol that puts you at risk for heart disease. Whether that link is attributed to an ingredient in diet soda or the drinkers' eating habits is unclear. But is that one can really worth it?
You read that right: Diet soda doesn't help you lose weight after all. A University of Texas Health Science Center study found that the more diet sodas a person drank, the greater their risk of becoming overweight. Downing just two or more cans a day increased waistlines by 500%. Why? Artificial sweeteners can disrupt the body's natural ability to regulate calorie intake based on the sweetness of foods, suggested an animal study from Purdue University. That means people who consume diet foods might be more likely to overeat, because your body is being tricked into thinking it's eating sugar, and you crave more.
Your first bad decision was ordering that whiskey-and-diet-cola -- and you may make the next one sooner than you thought. Cocktails made with diet soda get you drunker, faster, according to a study out of the Royal Adelaide Hospital in Australia. That's because sugar-free mixers allow liquor to enter your bloodstream much quicker than those with sugar, leaving you with a bigger buzz.
Cell Damage
Diet sodas contain something many regular sodas don't: mold inhibitors. They go by the names sodium benzoate or potassium benzoate, and they're in nearly all diet sodas. But many regular sodas, such as Coke and Pepsi, don't contain this preservative.
That's bad news for diet drinkers. "These chemicals have the ability to cause severe damage to DNA in the mitochondria to the point that they totally inactivate it - they knock it out altogether," Peter Piper, a professor of molecular biology and biotechnology at the University of Sheffield in the U.K., told a British newspaper in 1999. The preservative has also been linked to hives, asthma, and other allergic conditions, according to the Center for Science in the Public Interest.
Since then, some companies have phased out sodium benzoate. Diet Coke and Diet Pepsi have replaced it with another preservative, potassium benzoate. Both sodium and potassium benzoate were classified by the Food Commission in the UK as mild irritants to the skin, eyes, and mucous membranes.
Rotting Teeth
With a pH of 3.2, diet soda is very acidic. (As a point of reference, the pH of battery acid is 1. Water is 7.) The acid is what readily dissolves enamel, and just because a soda is diet doesn't make it acid-light. Adults who drink three or more sodas a day have worse dental health, says a University of Michigan analysis of dental checkup data. Soda drinkers had far greater decay, more missing teeth, and more fillings.
Reproductive Issues
Sometimes, the vessel for your beverage is just as harmful. Diet or not, soft drink cans are coated with the endocrine disruptor bisphenol A (BPA), which has been linked to everything from heart disease to obesity to reproductive problems. That's a lot of risktaking for one can of pop.
C4 pH value of beverages – How acidic is it in the stomach?
The pH value is a measure of the concentration of protons in an aqueous solution. Protons can
significantly alter certain substances with which they come into contact (“etching agents”). Students will be familiar with this from the example of the effect of acetic acid for cleaning lime deposits in the kitchen or bathroom.
Carbonated water, which is thereby acidified, only produces a thirst quenching and refreshing effect because of the tingling sensation it gives. Carbon dioxide in water also has a preserving effect.
If, for example, mineral water was not absolutely sterile when it was bottled, it still remains “fresh”
thanks to the carbon dioxide. For the body or stomach, the carbon dioxide is superfluous and lowers the pH value in the stomach unnecessarily.
Humans can drink highly acidic beverages, at least in small quantities, without doing any harm.
That suggests that a very low pH value is also present in the stomach.
Nevertheless, excessive consumption of acidic drinks can stress the mucus membrane of the
stomach and stimulate it to activate its mucosal barrier “unnecessarily”. That is obvious to the students from their observations and previous knowledge. They will recognize that different beverages vary with regard to how stomach-friendly they are.
The presence of these enzymes in the mouth is likely to short to achieve a positive effect. Carbonated water, carbonated drinks, or rinsing with dark beer (with a low alcohol content of 1.5%) may dissolve mucus somewhat. This advice is unsuitable for patients with an alcohol addiction.
Prof. R. Fass, University of Arizona, Southern Arizona VA Health Care System, GI Section (1‐111‐GI), 3601 South 6th Avenue, Tucson, AZ 85723‐0001, USA. E‐mail: Ronnie.Fass@va.gov
Abstract
Aliment Pharmacol Ther31, 607–614
Summary
Background Carbonated beverages have unique properties that may potentially exacerbate gastro‐oesophageal reflux disease (GERD), such as high acidity and carbonation. Cessation of carbonated beverage consumption is commonly recommended as part of lifestyle modifications for patients with GERD.
Aims To evaluate the relationship of carbonated beverages with oesophageal pH, oesophageal motility, oesophageal damage, GERD symptoms and GERD complications.
Methods A systematic review.
Results Carbonated beverage consumption results in a very short decline in intra‐oesophageal pH. In addition, carbonated beverages may lead to a transient reduction in lower oesophageal sphincter basal pressure. There is no evidence that carbonated beverages directly cause oesophageal damage. Carbonated beverages have not been consistently shown to cause GERD‐related symptoms. Furthermore, there is no evidence that these popular drinks lead to GERD complications or oesophageal cancer.
Conclusions Based on the currently available literature, it appears that there is no direct evidence that carbonated beverages promote or exacerbate GERD.
Introduction
Man‐made sparkling water was ‘invented’ in the mid to late 1700s in England, where scientist and clergyman Joseph Priestley suspended water above a vat of fermenting beer, producing carbon dioxide, which was then passively dissolved in the water. The effervescence was found by people to be pleasing. Presently, carbonated beverages are ubiquitous, commonplace and day‐to‐day drinks. They are a staple of the modern culinary repertoire, ranging from sparkling water to beer, soda pop or cola. Carbonated beverages, which include sodas, sparkling waters and beers, have unique properties. Although not necessarily common to all types of carbonated beverages, the principal properties of note are carbonation, acidity and high levels of sugar or artificial sweeteners. Carbonation, which produces the characteristic effervescence and bubbling associated with these drinks, is the result of dissolving gaseous CO2 in a liquid under pressure. Temperature and pressure influence the rate at which dissolved carbon dioxide converts into gas and is released, thus producing bubbles when a beverage container is opened. Acidity is a common chemical property of many carbonated beverages. Colas, sodas and beers are known to be among the most acidic beverages consumed in modern society. In part, the acidic nature of sodas (pH ∼3) is derived from conversion of dissolved CO2 to HCO3− and H+ by interaction with H2O as well as additives such as citric acid and phosphoric acid. Sugars and artificial sweeteners are added to many carbonated beverages, such as sodas, to impact a sweet taste, while beverages like beer have other forms of carbohydrate used in the fermentation process. Sugars and other carbohydrates add calories and increase the osmolality of the beverage. Artificial sweeteners provide a sugary taste without increasing the caloric content of the beverage. Other additives such as caffeine and alcohol may also be present, depending on the type of beverage, and may have unique or synergistic effects on the gastrointestinal tract. These will not be discussed in this review.
The effects of carbonated beverages on the human body have been the centre of much attention in the last decade, specifically, the consequences of carbonation, acid load and high carbohydrate consumption. Several studies have suggested that carbonated beverages may exacerbate GERD, dyspepsia and bloating.1 In addition, high consumption of sweetened carbonated beverages has been linked to the increased body mass index (BMI) and obesity epidemic.2 Obesity and elevated BMI have in turn been associated with increased frequency and severity of GERD‐related symptoms and oesophageal mucosal injury.3-6 The rate of soft drink consumption is at an all time high in the United States for both youth and adults (Table 1 and Figure 1). Recent statistics also suggested that daily calories from fruit and soda drinks have tripled since the mid 1970s, significantly contributing to increased daily calorie consumption and weight gain.7, 8
Table 1. Daily beverage consumption among men and women aged 20–39 years according to data from the National Health and Nutrition Examination Survey (NHANES) 1999–20028 (used with permission)
Estimated average daily energy intake from beverages in the National Health and Nutrition Examination Survey 1999–2002 by males and females (kcal/day). The numbers above the bars represent the mean energy (kcal/day) from beverages for that age category. Bars within gender sharing a common superscript (a, b, c, d, e and x, y, z) are not statistically distinguishable from one another at the P = 0.05 level (used with permission).8
Cessation of carbonated beverage consumption is commonly recommended as part of lifestyle modifications for patients with GERD.9 Thus far, it has been assumed that carbonated beverages can exacerbate GERD. Consequently, the aim of this systematic review is to determine the impact of carbonated beverages on GERD. Overall, this systematic review will provide a summary of the recent published literature and thus elucidate the role of carbonated soft drinks on GERD and its associated symptoms as well as complications.
Methods
We conducted a systematic review of the medical literature to identify original studies evaluating a potential relationship between GERD and carbonated beverages. We searched Ovid Medline, Cochrane Library, Web of Science, BIOSIS Previews, and CINAHL (Cumulated Index to Nursing and Allied Health Literature) for English‐language articles that were published between 1965 and 2009. To be included in the systematic review, we required that studies be fully published articles including at least 1 search term related to oesophageal diseases and another search term related to carbonated beverages. We included studies that described a potential relationship between carbonated beverages and heartburn by any methodology including oesophageal pH monitoring, manometric analysis, symptom survey, or other methodology as reported in Table 2.
Table 2. Characteristics of the studies included in the systematic review
The terms included in the bibliographic search were oesophageal diseases, indigestion, GERD or GORD, esophagitis, reflux, heartburn, regurgitation, oesophageal cancer, Barrett’s oesophagus, oesophageal stricture and soda, soft drink, sparkling, cola, pop, pepsi, coke, mountain dew, carbonated beverages, beverages, carbonation, carbonated or carbon dioxide. Additional articles were identified through manual search and from other doctors and experts in the field.
Results
We identified 393 records through database searching including 87 from BIOSIS Previews, 24 in CINAHL, 34 from Cochrane Library, 132 in Ovid Medicine, and 101 in the Web of Science. Thirteen additional articles were obtained that did not appear in the search. We screened 346 records and identified 29 full‐text relevant articles. Twelve articles were excluded for the following reasons: (1) article did not cover the subject matter (N = 3); (2) article did not present data (N = 6); (3) article described treatment of GERD with cola (N = 1); (4) article described effect of soft drinks on dental erosions (N = 2). Overall, 17 published studies were included in the review (Figure 2).
Given the fact that many carbonated beverages are highly acidic, alterations in intra‐oesophageal pH that can result in GERD‐like symptoms have been of major concern (see Table 3). In addition, carbonated beverages may serve as an acid load and thus may raise gastric acid volume, leading to increased likelihood of gastro‐oesophageal reflux. Two studies have demonstrated that carbonated beverages can reduce the oesophageal pH < 4 and potentially cause GERD‐related symptoms. By using oesophageal impedance and pH in normal subjects, Agrawal et al. compared lemonade, strawberry juice, white wine, red wine, ketchup, apple juice, orange juice, coffee and tea with carbonated beverages.10 The authors demonstrated that consumption of carbonated beverages resulted in the lowest intra‐oesophageal pH (1.9). However, while the drop in intra‐oesophageal pH was immediate, it lasted only 90 s on an average, which was the shortest period as compared with the other consumed beverages. The authors also found, from 100 patient diaries, that carbonated beverages are the most commonly consumed acidic food (45%) as compared with the other products. Shoenut et al. demonstrated that while acidic beverages altered intra‐oesophageal pH, the effect was temporary and not long lasting.11 The total time pH < 4 during consumption of cola (7.7 ± 6.0 min) was significantly longer than for beer (3.3 ± 3.7 min), tea/coffee (1.4 ± 6.5 min) and water (1.1 ± 25 min). Although cola and juice in this study had the greatest impact on intra‐oesophageal pH, the effect did not exceed 0.5% of the entire 24‐h pH study. Thus, the authors of this study concluded that the impact of cola (as well as other acidic fluids) on intra‐oesophageal pH is so minimal that their effect could be disregarded in most patients.
Table 3. The pH values of the different carbonated beverages27
RC Cola
2.4
Coke
2.5
Pepsi
2.5
Cherry Coke
2.5
Dr. Pepper
2.9
Squirt
2.9
Diet Pepsi
3.0
Mountain Dew
3.2
Diet Dr Pepper
3.2
7‐Up
3.2
Diet Coke
3.3
Sprite
3.3
Diet Mountain Dew
3.4
Diet 7‐Up
3.7
Mug Root Beer
4.0
Carbonated beverages have been shown to increase postprandial oesophageal acid exposure.12 However, only alcoholic carbonated beverages were studied, and thus it remains unclear if it is the alcohol or the carbonation that causes an increase in oesophageal acid exposure.
The aforementioned studies have numerous limitations, as they did not assess intragastric volume or pH. In addition, the magnitude of carbonated beverage consumption is significantly different from one individual to another. Nevertheless, the impact of carbonated beverages on intra‐oesophageal pH appears to be very limited.
The effect of carbonated beverages on upper gut motility
The effect of carbonated beverages on lower oesophageal sphincter (LES) tone has been evaluated by one study.13 In this study, oesophageal manometry was performed in nine healthy volunteers after ingestion of tap water and different carbonated beverages (caffeinated and noncaffeinated Pepsi and carbonated water). All carbonated beverages produced at least a 20‐min reduction of approximately 30–50% in lower oesophageal sphincter (LES) resting pressure, overall length, and intra‐abdominal length. The authors postulated that the decrease in LES pressure is mediated by gaseous distention of the stomach because of the carbonation. The authors further proposed that the aforementioned effect of carbonated beverages on the LES is the underlying mechanism for heartburn in subjects consuming these drinks. However, the study was conducted in a small number of healthy subjects. In addition, the authors did not measure changes in transient LES relaxation rate or demonstrated any evidence of increase in oesophageal acid exposure as a result of carbonated beverage consumption.
Thus far, there are no studies that evaluated the effect of carbonated beverages on oesophageal body motor function. However, several studies evaluated the effect of carbonated beverages on gastric function. Overall, it does not appear that carbonated beverages alter gastric motility. In a study by Zachwieja et al., eight male cyclists consumed four different beverages (carbonated, carbonated and sugary, noncarbonated and noncarbonated and sugary) during 120‐min period of cycling.14 Following each exercise, the content of each cyclist’s stomach was aspirated. There was no difference in the retained volume after each of the beverages. The authors concluded that carbonated beverages have no impact on gastric emptying. These study results corroborated the results of an earlier study by the same authors in which there was no difference in gastric emptying characteristics of 20 exercising subjects who again were given different drinks including sugary carbonated and noncarbonated sports drinks as well as water and carbonated water.15 In another study by Cuomo et al., 13 healthy subjects consumed 300 mL of either water containing increased concentrations of carbon dioxide or sweetened commercial flavoured drinks with and without carbon dioxide.16 Thereafter, gastric emptying was assessed using octanoic acid breath test. The authors were not able to show any significant difference in gastric emptying among all beverages studied. Similar findings were documented by Cuomo and colleagues in an earlier study.17 In that study, 22 patients with dyspepsia and secondary constipation underwent gastric emptying assessment after consuming carbonated water or tap water for almost 15 days. Water without carbonation demonstrated a shorter gastric emptying duration, but it did not reach statistical significance.
Lastly, Pouderoux et al. compared the gastric emptying time of a radio‐labelled meal in eight healthy volunteers who also consumed either distilled or carbonated water.18 The authors found no significant difference in gastric emptying of both solids and liquids, including the duration of the lag phase between the two drinks. However, there was a greater retention of food (both solids and liquids) in the proximal stomach with carbonated water as compared with distilled water. There was retention of the meal within the proximal stomach, which ended with the lag phase and was likely related to proximal gastric distention. The authors concluded that gastric distention by liberated CO2 from carbonation was not associated with alteration in overall gastric emptying, but more with modification of intragastric distribution of a meal.
Overall, the aforementioned studies demonstrated that consumption of a carbonated beverage with a meal does not significantly alter gastric emptying.
Oesophageal damage
It has been hypothesized that chronic, repetitious ingestion of carbonated, highly acidic beverages might cause oesophageal mucosal damage that can potentially lead to chronic oesophageal inflammation and even cancer. Presently, there are very little data to support or contradict this hypothesis. Rats that were exposed to either cola or saline demonstrated no evidence of oesophageal histopathological abnormalities.19 However, the rats that were exposed to cola demonstrated a higher regenerative index by flow cytometry than those exposed to saline, suggesting some irritating effect.
Despite the high consumption of acidic carbonated beverages, there are no studies clearly demonstrating an increased risk for oesophageal mucosal injury in the form of oesophagitis, oesophageal ulceration or even oesophageal stricture. Part of the explanation is likely due to the unique capability of oesophageal mucosa to withstand acid even in very high concentrations.
Carbonated beverages and GERD symptoms
Overall, carbonated beverages have not been consistently shown to cause GERD‐related symptoms.1 Fass et al. have demonstrated that consumption of carbonated beverages increased the risk of having heartburn that awakens subjects from sleep during the night by 24% (OR 1.24 95% CI 1.07–1.45).20 In contrast, several population‐based studies were unable to demonstrate any relationship between GERD‐related symptoms and carbonated beverage consumption.21, 22 Furthermore, a systematic review of lifestyle modifications for GERD could not find even one study that evaluated the effect of cessation of carbonated beverage consumption on GERD‐related symptoms.9 Consequently, the authors were unable to make any evidence‐based recommendation related to carbonated beverage consumption.
The relationship between acidity and osmolality of various beverages and their propensity to cause GERD symptoms was evaluated by Feldman and Barnett.23 The authors studied 11 different carbonated beverages as well as citrus drinks and juices, alcoholic beverages and milk. The authors surveyed participants who rated frequency of heartburn symptoms associated with specific drinks. Of those who returned the questionnaire, between 10% and 19.8% reported frequent heartburn with consumption of the different carbonated beverages (see Table 4). Of the soft drinks studied, Diet Dr Pepper was the most commonly associated with heartburn (21.1%) and 7UP the least (10.0%). The frequency of reported heartburn symptoms was not different between diet and regular carbonated beverages. The authors also found that osmolality of the beverage was not an important factor in determining association with GERD‐related symptoms. Unfortunately, the authors of this study did not evaluate sparkling water alone. That would have allowed the determination if carbonation per se is responsible for subjects’ heartburn.
Table 4. Prevalence of frequent heartburn reported with carbonated beverages23 (used with permission)
Beverage
% with frequent heartburn
Diet Dr Pepper
21.1
Coca Cola
20.8
Pepsi Cola
19.8
Dr Pepper
19.3
Diet Coke
15.9
Diet Pepsi
15.3
Diet 7‐Up
13.7
Root beer
13.3
Mountain Dew
12.3
Sprite
12.3
7‐Up
10.1
Presently, there are very few studies supporting the role of carbonated beverages in causing heartburn or any other GERD‐related symptom. Despite the general impression that carbonated beverages are strongly associated with gastro‐oesophageal reflux disease, the literature lacks clear data to support this notion. As the consumption of carbonated beverages continues to be highly popular, a prospective trial should determine if carbonated beverages are a significant risk factor for GERD symptoms.
Carbonated beverages and GERD complications
Currently, there are no studies demonstrating any relationship between carbonated beverage consumption and complications of GERD such as oesophageal ulcers, peptic stricture, or Barrett’s oesophagus. However, three studies did evaluate carbonated beverage consumption and risk for oesophageal malignancy. In a study by Lagergren et al.24 that utilized Swedish nationwide population‐based data, the authors demonstrated that frequency of intake of carbonated soft drinks was not associated with increased risk of oesophageal or cardia adenocarcinoma. However, high consumers (>6 time a week) were at a statistically nonsignificant decreased risk compared with newer users. In a U.S multicentre, population‐based, case‐control study, the authors found that high carbonated soft drink consumption was not associated with increased risk of any oesophageal or gastric cancer subtypes in men or women.25 In fact, the authors found an inverse relationship between carbonated soft drink consumption (especially diet) and the risk of oesophageal adenocarcinoma. Ibiebele and colleagues also could not find any relationship between consumption of carbonated beverages and the risk of either adenocarcinoma or squamous cell carcinoma of the oesophagus.26 The authors did demonstrate an inverse relationship between soda consumption and squamous cell carcinoma of the oesophagus (OR = 0.4, 95% CI 0.20–0.78).
Overall, it does not appear that carbonated beverage consumption is more commonly associated with severe GERD. Surprisingly, soda consumption appears to be protective against both adenocarcinoma and squamous cell carcinoma of the oesophagus through an unknown mechanism. Further support for the protective effect of soda against oesophageal cancer is needed from well‐designed prospective trials.
Conclusions
This systematic review revealed that despite the increased consumption of carbonated beverages in the last few decades and their proposed relationship with GERD, studies that specifically evaluated their role in promoting acid reflux or reflux‐related symptoms are relatively scarce. The currently available literature does not support a strong relationship between carbonated beverages and GERD. Thus far, there are only conflicting data about their role in generating GERD‐related symptoms and apparently no evidence that they directly cause oesophageal inflammation or GERD‐related complications. Thus, it appears that there is no direct evidence that carbonated beverages promote or exacerbate GERD. Consequently, sweeping recommendations for cessation of all carbonated beverages as part of lifestyle modifications should be re‐evaluated. Regardless, sugary carbonated beverages have been blamed for their contribution to the obesity epidemic in westernized societies and may contribute to GERD through increase in BMI. Additional research is required to delineate further the role of these ubiquitous soft drinks in causing GERD.
Acknowledgement
Declaration of personal interests: Dr Ronnie Fass is a consultant for Takeda, Vecta, AstraZeneca, GSK, Eisai and XenoPort. He receives grant/research support from Takeda, Wyeth, Addex and AstraZeneca and is a member of the speaker’s bureau at Takeda and Eisai. Dr Lauren Gerson is a consultant for Takeda, Eisai and XenoPort and receives grant support from Santarus and XenoPort. Dr Timothy Johnson, Dr Tiberiu Hershcovici and Mr Christopher Stave have no disclosures or competing interests. Declaration of funding interests: This manuscript was supported in part by The Coca‐Cola Company in Portugal.
Wounds in the mouth heal faster and with less scarring than wounds in other locations on the body. To understand differences in healing, Iglesias-Bartolome et al. performed transcriptional analysis on sequential, paired oral and skin biopsies from healthy human subjects. Compared to baseline, skin samples showed a larger number of up-regulated genes on subsequent biopsies than oral samples, indicating that healing was unresolved. Oral wounds healed faster than skin wounds, and certain transcription factors were consistently up-regulated in the oral wounds but not in skin wounds. Overexpressing some of these transcription factors in a mouse model of skin wounding enhanced healing. The authors suggest that the molecular signature of the oral mucosa could be used to develop therapies for wound healing.
Abstract
Oral mucosal wound healing has long been regarded as an ideal system of wound resolution. However, the intrinsic characteristics that mediate optimal healing at mucosal surfaces are poorly understood, particularly in humans. We present a unique comparative analysis between human oral and cutaneous wound healing using paired and sequential biopsies during the repair process. Using molecular profiling, we determined that wound-activated transcriptional networks are present at basal state in the oral mucosa, priming the epithelium for wound repair. We show that oral mucosal wound–related networks control epithelial cell differentiation and regulate inflammatory responses, highlighting fundamental global mechanisms of repair and inflammatory responses in humans. The paired comparative analysis allowed for the identification of differentially expressed SOX2 (sex-determining region Y-box 2) and PITX1 (paired-like homeodomain 1) transcriptional regulators in oral versus skin keratinocytes, conferring a unique identity to oral keratinocytes. We show that SOX2 and PITX1 transcriptional function has the potential to reprogram skin keratinocytes to increase cell migration and improve wound resolution in vivo. Our data provide insights into therapeutic targeting of chronic and nonhealing wounds based on greater understanding of the biology of healing in human mucosal and cutaneous environments.
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INTRODUCTION
Improving wound healing resolution is a major medical and social priority due to the increase in incidence of traumatic injury, chronic wounds, and scarring (1). Although several studies characterize in detail the mechanisms and pathways altered in these deficient wounds, a different approach that defines factors involved in accelerated wound healing would allow for the identification of novel therapeutic targets to improve tissue repair. In this regard, oral wound healing and embryonic wound healing have long been considered models of optimal wound resolution characterized by rapid and scarless wound healing (1–4). Dissecting the different molecular events that drive wound healing resolution in oral mucosa compared with those of the skin will help us define why these oral lesions heal more efficiently and will provide a basis from which to translate those findings to treat deficient healing processes.
Cutaneous wound healing is well documented, with the overall classic interpretation for the repair pathway having four systematic phases: hemostasis, inflammation, proliferation, and remodeling (5). The molecular circuitries that drive these different phases of cutaneous repair have been characterized, whereas the unique environment of the oral cavity represents a different wound healing paradigm that remains poorly understood. Oral wounds heal at an accelerated rate compared to cutaneous wounds, and in vitro or animal model studies have attributed this to a variety of mechanisms including differential inflammatory response, distinct modulation of stem cell, proliferative and differentiation programs, and more efficient epithelial remodeling (1, 3). Despite this progress, the lack of detailed clinical studies in humans comparing oral and cutaneous wound healing in vivo has limited the advancement of our knowledge on the mechanisms mediating accelerated wound healing. To close this gap, we characterized the molecular and histological aspects of wound healing in paired samples of oral mucosa and the skin in healthy human subjects. Wounds were induced simultaneously in the oral buccal mucosa and the skin and sequentially biopsied for comparison at progressive time points. We show that the oral cavity is primed for wound repair and that oral keratinocyte limited differentiation and proinflammatory responses may contribute to accelerated wound healing in oral mucosa. We also present evidence that transcriptional networks established by transcription factors such as SOX2 (sex-determining region Y-box 2) and PITX1 (paired-like homeodomain 1) mediate this phenotype and can be exploited to reprogram cutaneous keratinocytes to present oral keratinocyte features, including accelerated wound closure.
RESULTS
Wound-activated transcriptional networks present at basal state prime the oral mucosa for wound repair
Using paired and sequential biopsy samples, we contrasted oral mucosal healing with cutaneous healing to determine the differential regulation of these processes in the human setting (Fig. 1A). After clinical screening (day 0), baseline biopsies of paired, identically sized wounds in the oral buccal mucosa and skin were obtained (day 1) (Fig. 1B). Day 1 biopsies allowed for evaluation of homeostatic transcriptional profiles in human mucosal versus cutaneous surfaces. Follow-up biopsies of the wound areas were collected at day 3 (48 hours after the first biopsy) and day 6 (120 hours after the first biopsy). This approach evaluated distinct phases during the physiologic process of human wound healing.
Fig. 1Comparison of paired oral and skin wounds in human subjects.
(A) Table and schematic of spatiotemporal human biopsy sample collection (ClinicalTrials.gov #NCT01078467). Baseline biopsies were performed to create paired identically sized wounds in the oral mucosa (blue) and skin (orange) on day 1. Follow-up biopsies of the wound areas were collected on days 3 and 6 of healing in two different groups. (B) Representative hematoxylin and eosin (H&E) pictures of longitudinal sections of biopsies taken at day 1. Thirty healthy subjects were randomized in three groups (with 10 subjects on each group). SM, smooth muscle. (C) Representative pictures and quantitation of healing time course of oral wounds and skin wounds in group 1 after a 3-mm primary biopsy and of group 2 after a 5-mm secondary biopsy. Markings on the dental periodontal probe are in millimeters. Biopsy sites were demarcated using blue polypropylene sutures. Data are mean values, and error bars represent SDs. Number of samples for each group are as follows: 3-mm wounds, day 1, n = 29; day 3, n = 30; day 6, n = 20; day 9, n = 9; day 13, n = 9; day 15, n = 9; 5-mm wounds, day 3, n = 11; day 6, n = 20; day 9, n = 21; day 13, n = 21; day 15, n = 21. Nonparametric two-sided t test. (D) Graphs show the healing rate of 3-mm wounds in group 1 and of 5-mm wounds in group 2 (top) and percentage of contraction immediately after the 3-mm biopsy in group 1 and after the 5-mm biopsy in group 2 (bottom). n.s., not significant. *P < 0.05 by unpaired t test. Error bars represent SDs. (E) Representative H&E pictures of oral and skin wounds at days 3 and 6. Magnification of dotted boxes is shown on the right. In magnification, epithelium is marked with a dotted line.
Analysis of the healing time course revealed that oral wounds resolved significantly faster than skin wounds (P < 0.001; Fig. 1C). This was observed after the first wound-inducing biopsy (3-mm wound; Fig. 1C, top) and after the secondary biopsy of the wound area (5-mm wound; Fig. 1C, bottom). There was no significant difference in healing rates between different sized wounds created in the same tissue bed; however, 5-mm oral wounds exhibited less contraction than 3-mm oral wounds (Fig. 1D). Histological analysis of the wound sections showed earlier re-epithelialization of the oral mucosa wounds compared to the skin (Fig. 1E). By day 3, oral wounds were almost completely covered by squamous epithelium, even in the absence of stromal healing.
RNA sequencing (RNA-seq) was used to characterize molecular mechanisms of the human wound repair process (Fig. 2A). Unsupervised clustering analysis of the gene expression data provided three major observations. First, oral and skin samples clustered separately, indicating distinct transcriptional identities consistent with unique tissue microenvironments. Although there is differential gene regulation between oral mucosa and skin during wound healing, most of these differences were already evident at starting basal conditions (day 1; Fig. 2, A and B, and fig. S1). Second, within the skin samples, there was separation between baseline biopsies and wound biopsies at days 3 and 6 with an overall up-regulation of gene activity in the skin evident during the healing process, indicating nonresolution of the skin wounds at the time points evaluated (Fig. 2A, skin). Third, and in contrast to the skin, the oral day 3 biopsies separated from days 1 and 6, indicating wound healing activity at day 3 that resolved by day 6 (return of gene expression to basal conditions; Fig. 2A, oral).
Fig. 2Wound-activated transcriptional networks are present in the unwounded oral mucosa.
(A) Schematic representation of biopsy site in the mucosa of the cheek and posterior axillary region of the arm (left) and unsupervised clustering analysis of RNA-seq gene expression data of the 24 paired samples at days 1, 3, and 6. O, oral; S, skin. Numbers indicate matching subjects. Paired oral and skin samples were chosen randomly from four subjects for each day (24 total samples from 12 individual subjects) and were a mix of males and females. (B) Circular ideogram plot (CIRCOS) of the differential gene expression during wound healing (ANOVA). No significant differences were found in oral biopsies taken at day 6 versus day 1 (day 6/day 1). Ribbon connectors indicate the same genes present in different data sets. Number of genes with differential expression in each comparison: oral day 3 versus day 1 (day 3/day 1), 410 genes; skin day 3 versus day 1 (day 3/day 1), 1473 genes; skin day 6 versus day 1 (day 6/day 1), 1836 genes. See fig. S1A for explanation on CIRCOS plot. RPKM, reads per kilobase million; |FC|, absolute fold change. (C) Volcano plot indicating differential gene expression between unwounded (day 1) oral mucosa and skin. Dotted line region is magnified on the right panel, highlighting some of the most significantly up-regulated genes in the oral mucosa compared to the skin. P < 0.05 by paired t test. (D) Ingenuity Pathway Analysis (IPA) showing diseases and functions terms in up-regulated genes in the oral mucosa compared to the skin. (E) Unsupervised hierarchical clustering using a psoriasis gene signature with the gene expression of oral mucosa and skin data at baseline (day 1) and during wound healing (days 3 and 6).
Consistent with these observations, analysis of variance (ANOVA) of the differential gene expression during wound healing in oral and skin revealed few significant gene expression changes during oral healing at day 3 (410) and none at day 6, whereas a large number of genes were differentially regulated during skin wound healing at both time points (skin day 3 versus day 1, 1473; skin day 6 versus day 1, 1836; Fig. 2B). These results demonstrate an enhanced transcriptional activity during skin wound healing but minimal differential transcriptional regulation in oral wounds, raising the possibility that the transcriptional regulatory networks responsible for the accelerated healing in oral mucosa are already present in the unwounded state.
To explore the intrinsic differences between oral mucosa and skin at baseline, we determined the significant differentially expressed genes between unwounded (day 1) oral mucosa and unwounded skin (q < 0.05 and fold change ≥ 2; table S1). Transcripts up-regulated in the oral environment were consistent with increased keratinocyte activation and with heightened antimicrobial defenses (day 1; Fig. 2C). Among the up-regulated transcripts in the oral samples, we found genes described in wound-activated keratinocytes (6), including keratins 6 (KRT6) and 16 (KRT16), small proline-rich (SPRR) and S100 proteins, defensins, serpins, and annexins among others (Fig. 2C). IPA showed that the top processes represented in the transcriptome of the oral mucosa were related to inflammatory skin disorders such as psoriasis, dermatitis, and skin hyperplasia (Fig. 2D), conditions in which transcriptional networks resemble those of the wound-activated skin (7–10). This indicated that gene networks related to increased proliferation, migration, and wound resolution were potentially already present in the oral mucosa at basal state.
Because psoriasis has been shown to present a particular expression signature of genes, inflammatory cytokines, and proteins related to wound healing, we used the gene list from the psoriasis gene signature in IPA to perform an unsupervised clustering of our samples (Fig. 2E). Results showed that skin wound samples (days 3 and 6) clustered more closely with oral samples at all days than unwounded skin (Fig. 2E). These results support our hypothesis that wound-activated networks are present in the oral epithelium at basal state. Psoriasis patients have accelerated wound healing with reduced scarring (10, 11), suggesting that the presence of gene networks related to wound healing at basal state might be the key for the accelerated wound healing observed in psoriatic skin and oral mucosa.
Analysis of the transcriptome of the oral mucosa also revealed that gene networks related to cell movement and migration were highly activated in the unwounded oral mucosa (fig. S2A), showing up-regulation of genes linked to epithelial and immune cell migration (fig. S2B). Together, these results indicate that wound-activated transcriptional networks are present at basal state in the oral mucosa, priming the epithelium for wound repair.
Oral mucosa shows reduced differentiation and inflammatory response during wound healing
Consistent with the possibility that the transcriptional regulatory networks responsible for the accelerated healing in oral mucosa are already present in the unwounded state, we identified 250 genes (q < 0.05 and fold change ≥ 2; table S2) that are highly expressed in the unwounded oral mucosa but are only up-regulated in the skin during wound healing (Fig. 3A, black line in CIRCOS plot). Although there is ample evidence of the importance of the extracellular matrix and underlying stroma during re-epithelialization (1), the topmost significant gene ontologies (GOs) represented in this gene list were processes related to keratinization, epidermal cell differentiation, and responses to biotic stimulus and bacterium (Fig. 3A and table S3). Terms related to epithelial and immune cell migration were also represented (table S3). These results highlight specific gene networks, both intrinsic to keratinocyte biology and related to immune responses, as critical elements mediating the priming of the oral mucosa to wound repair.
Fig. 3Oral keratinocytes show reduced differentiation during wound healing.
(A) CIRCOS plot summarizing cross-reference of transcripts up-regulated during skin wound healing (skin day 3/day 1 and skin day 6/day 1) with those up-regulated in the oral mucosa with respect to the skin at basal conditions (oral day 1/skin day 1). The black line indicates genes up-regulated in the unwounded oral mucosa that are up-regulated during skin wound healing (250 unique genes; see table S2). Ribbon connectors indicate that the same genes are present in different data sets. Right: Graph indicates GO biological process terms enriched in the oral day 1/skin day 1 data set. Oral day 1/skin day 1, 760 genes; skin day 3/ day 1, 971 genes; skin day 6/day 1, 1089 genes. B&H, Benjamini and Hochberg’s; FDR, false discovery rate. (B) Relative mRNA expression of keratinization and epidermal cell differentiation markers throughout the wound healing process. (C) Representative pictures of unwounded (day 1) and wounded (days 3 and 6) oral mucosa and skin stained to show expression of the basal cell marker keratin 5 (K5, red) and differentiation markers keratin 4 (K4) for the oral mucosa and keratin 10 (K10) for the skin (green). Magnification of the dotted box is shown on the right of each picture. (D) Representative pictures of unwounded (day 1) and wounded (day 3) oral mucosa and skin stained to show expression of the basal marker cytokeratin 5 (K5, red) and activated epithelium keratin 6 (K6, green). (E) Representative pictures of unwounded (day 1) and wounded (days 3 and 6) oral mucosa and skin stained to show expression of the basal marker keratin 5 (K5, green) and proliferation marker proliferating cell nuclear antigen (PCNA; red). Quantification of number of cells positive for the proliferation marker PCNA per area (mm2). **P < 0.01 by unpaired ttest, oral versus skin day 6. No asterisk means not statistically different between oral and skin. Data are mean values, and error bars represent SDs. n = 3 independent samples. For day 1, magnification shows the basal, unwounded expression of corresponding marker. For days 3 and 6, magnification shows the migratory tongue or wound area (I) or an adjacent epithelial area to the wound (II). D1, day 1; D3, day 3; D6, day 6.
Distinct keratin and structural protein expression profiles between oral mucosa and skin are indicative of inherent differences in the epithelial compartments of these tissues (Fig. 3B). Correspondingly, we found that characteristic signature keratins for each epithelium (12) were present: K4 in the oral mucosa and K10 in the cornified skin (Fig. 3C). We also examined reciprocal staining for K4 and K10 in skin and oral mucosa (fig. S3A) and confirmed the tissue-specific expression for each keratin.
Stress- and wound-activated keratins, including the keratin 6 family (KRT6A, KRT6B, and KRT6C) and KRT16, are uniquely active in unwounded oral epithelium and remain highly up-regulated during wound healing, whereas they are only expressed in the skin during the wound healing process (Fig. 3, B and D). These keratins are essential for keratinocyte migration and epithelial structure in vivo in murine oral epithelia at basal state and during wound healing (13, 14).
Analyzing these human data sets identified distinct expression profiles for genes clustered in the epidermal differentiation complex (EDC) (15). Most of the EDC genes (S100s, SPRRs, and cell envelope precursors) that were up-regulated in skin during wounding (Fig. 3B) presented heightened expression at baseline day 1 in the oral mucosa.
Differentiation markers such as involucrin (IVL), usually present in both epithelia, were down-regulated in the oral mucosa during healing but were present in the migratory tongue of the wounded skin (fig. S3B). Although the oral mucosa has a more extensive total area of proliferating cells during wound healing (Fig. 3E), there is no significant difference in the number of proliferating cells when this is corrected for surface area involved (PCNA+ cells per square millimeter; Fig. 3E). In aggregate, these results indicate that oral wounds do not engage differentiation pathways during wound healing but, instead, are primed for wound repair by maintaining a larger pool of regenerative epithelial keratinocytes that aid in accelerated wound closure, as corroborated by activated keratin expression.
The differential expression of structural keratins observed between oral and cutaneous samples reflects the unique characteristics of these distinct epithelia and highlights the fact that the oral buccal mucosa is noncornified and therefore is more exposed to environmental signals (16). Hence, the oral epithelium is increasingly exposed to the commensal microbiota that inhabit barrier surfaces. The oral environment is home to some of the most rich and diverse microbial communities harbored on human body surfaces (17) and is an environment of constant mechanical stimulation during mastication shown to induce heightened immune responsiveness (18). Oral epithelia had minimally up-regulated inflammatory pathways during the healing process (Fig. 4A and fig. S4A) and increased antimicrobial defenses (fig. S4B). In contrast, in the cutaneous microenvironment, inflammatory responses were less active at steady state but became up-regulated throughout the healing process and did not resolve by day 6, suggestive of a chronic inflammatory response when compared to the oral mucosa (Fig. 4, A to C, and fig. S4A). Chronic inflammation is a hallmark of nonhealing wounds, and overactivation of immune processes during healing has detrimental effects on wound resolution, delaying closure and increasing fibrosis and scarring (19). Several immune mediators including proinflammatory cytokines, chemokines, and cyclooxygenases showed higher expression in the skin at basal state and were up-regulated in skin continuously through day 6 (Fig. 4C). Markers of fibrosis, including transforming growth factor–β targets (20), were up-regulated in skin wounds compared to oral wounds (fig. S4, A and C). Analysis of the gene expression changes in skin wound healing from day 3 to day 6 revealed that, although gene networks related to keratinocyte differentiation (peptide cross-linking and keratinization) were up-regulated by day 6, additional networks related to immune response were still active at this time (fig. S4D). This further corroborates the nonresolution of the inflammatory response in the skin wounds.
Fig. 4Inflammatory pathways are more active and sustained in skin wounds than in oral wounds.
(A) IPA analysis showing diseases and functions terms found in differentially regulated genes during the wound healing process relative to day 1, including terms related to inflammatory processes. (B) CIRCOS plot showing the genes exclusively up-regulated during skin wound healing (black line) and GO biological process terms enriched in this data set. Ribbon connectors indicate that the same genes are present in different data sets. Oral day 3/day 1, 276 genes; skin day 3/day 1, 971 genes. (C) Relative mRNA expression of interleukins, chemokines, and other inflammatory regulators during wound healing. (D) Representative pictures of recruitment of immune cells during the wound healing process in the oral mucosa and the skin. Bottom: Quantification of recruitment of specific immune cell types during the wound healing process in the oral mucosa and the skin. *P < 0.05, **P < 0.01 by unpaired t test. No asterisk means not statistically different, comparisons between days 3 and 6 versus day 1 (D1) oral or skin, respectively. Data are mean values, and error bars represent SDs. n = 3. MPO, myeloperoxidase. (E) Representative images of unwounded (day 1) and wounded (day 3) oral mucosa and skin stained to detect expression of the basal marker keratin 5 (K5, red) and the immune modulators secretory leukocyte peptidase inhibitor (SLPI) (green) and ANXA1 (magenta). Magnification of dotted boxes is shown on the right of each picture.
Histological and quantitative evaluation of the cellular infiltrate during healing reflected an acute response in the oral environment with complete resolution by day 6, consistent with transcriptional responses that reverted to baseline by day 6 in the oral environment but continued to amplify in the skin (Fig. 4D). Associated with this rapid and controlled inflammatory response in the oral environment, we observed constitutive activation of several proresolution molecules [ANXA1, SLPI, ALOX12, and IL1RN (21)] in the oral mucosa (Fig. 4C). We confirmed that annexin A1 (ANXA1) and SLPI, two proteins that ameliorate wound healing by moderating chronic inflammation (22–24), showed increased expression in the oral mucosa and oral wounds compared to skin (Fig. 4E). These results show that oral mucosa is primed for wound repair by a series of wound-signature networks that help control epithelial cell differentiation and regulate proinflammatory responses.
Transcriptional networks in oral mucosa contribute to rapid wound resolution
Rapid oral versus skin wound healing has previously been identified in mouse and pig models (3, 25, 26) and replicated in vitro in oral keratinocyte models (4), suggesting a conserved mechanism in oral keratinocytes that allows for faster wound resolution. To identify conserved factors and signaling pathways in oral keratinocytes that may have physiological relevance for improved healing, we searched databases for overexpressed transcripts present in our human oral mucosa data set (Fig. 3A), which were also overexpressed in mouse oral mucosa in vivo (25) and in human (27) and mouse (3) oral keratinocytes (fig. S5). This analysis identified eight genes that were consistently up-regulated in oral mucosa and oral keratinocytes (Fig. 5A), four of which encode for transcriptional regulators (PAX9, PITX1, PITX2, and SOX2), one that encodes the LIM domain–only 7 emerin-binding factor (LMO7) (28), and three that encode factors involved in various oral mucosa biological processes (ALDH3A1, ATP1B1, and IGFBP2) (4, 29, 30).
Fig. 5Transcriptional networks in oral keratinocytes contribute to rapid wound resolution.
(A) Relative mRNA expression of genes consistently up-regulated in oral mucosa and oral keratinocytes, presented by fold change of the unwounded oral mucosa with respect to the unwounded skin (O1/S1) in the wound healing data set. (B) Western blot of PAX9, PITX2, PITX1, and SOX2 in primary cultures of human oral (NOK) and skin (NHEK) keratinocytes. n = 2. (C) Representative pictures of unwounded (day 1) and wounded (days 3 and 6) oral mucosa and skin stained to show expression of the basal marker keratin 5 (K5, red) and the indicated transcription factor (SOX2, PITX1, or PAX9; green). Magnification of the dotted boxes is shown on the right of each picture. For day 1, magnification shows the basal, unwounded expression of corresponding marker. For days 3 and 6, magnification shows the migratory tongue or wound area (I) or an adjacent epithelial area to the wound (II).
Transcription factors have the potential to reprogram cells to specific developmental states (31). Within the group of oral up-regulated transcription factors, we found the paired-like homeodomain PITX factors (PITX1 and PITX2), the paired box homeodomain factor PAX9, and the HMG-domain SOX2, which is part of the SOX family of transcriptional drivers of somatic cell reprogramming (32–34).We confirmed the differential expression of the PAX9, PITX1, PITX2, and SOX2 transcriptional regulators in primary human oral [NOK (normal oral keratinocytes)], and skin [NHEK (normal human epidermal keratinocytes)], keratinocytes in vitro and in human biopsies, at baseline and during healing (Fig. 5, B and C). PITX1 and SOX2 were expressed in NOK cells and unwounded oral mucosa, whereas their expression was almost undetectable in NHEK cells and skin. Expression of these factors in the epithelial layer of the oral mucosa increased upon wounding. PAX9 showed increased expression in epithelia and dermis after wounding in both oral mucosa and skin and was less expressed in NHEK cells than in NOK cells.
To functionally validate the role of these factors in controlling a transcription-regulated wound healing program, we analyzed the gene expression changes in NOK cells treated with small interfering RNA (siRNAs) for PAX9, PITX1, PITX2, and SOX2 (Fig. 6 and fig. S6A). Knockdown of these factors altered gene networks related to cell movement and migration (Fig. 6B and fig. S6, A to C). siRNA for PITX1 and SOX2 significantly reduced the migration capacity of NOK cells (**P < 0.01; Fig. 6C). siSOX2 affected gene networks related to the immune and defense responses, whereas siPITX1 altered epidermal developmental and differentiation and keratinization pathways (fig. S6, B and C).
Fig. 6Knockdown of oral signature genes reduces migratory capacity of primary oral keratinocytes.
(A) Western blot of SOX2, PITX1, and total protein (RPS14) after transfection with respective siRNAs in NOK cells. (B) IPA analysis of RNA-seq data from NOK cells treated with siRNAs for SOX2 and PITX1: Diseases and functions terms related to migration and cell movement found in differentially regulated genes. P < 0.05 by IPA. (C) Relative cell migration distance of NOK cells transduced with siRNA for SOX2, PITX1, and siControl (siCON). Values were determined from three microscopic fields in n = 3 per group. **P < 0.01 by ANOVA, siSOX2 and siPITX1 versus control. Data are mean values, and error bars represent SDs. Scale bar, 100 μm.
We then evaluated the functional effects of overexpressing PITX1 and SOX2 in NHEK cells (Fig. 7and fig. S7), which do not express these factors under normal conditions (Fig. 5, B and C). Transcriptomic analysis of NHEK cells transduced with PITX1 and SOX2 revealed a similar pattern of GO biological process as oral keratinocytes, with SOX2 affecting pathways related to the immune response including cytokines and antibacterial peptides and PITX1 expression resulting in the alteration of developmental and differentiation pathways (Fig. 7B). Both PITX1 and SOX2 expression in skin keratinocytes reduced expression of the skin differentiation marker KRT1 and overexpression of the oral epithelial marker cornulin (CRNN; Fig. 7C). adPITX1 also increased expression of several markers enriched in the oral epithelium, including keratins (KRT13 and KRT78), SPRRs (SPRR2A, SPRR2E, and SPRR3), LCE3D, IGFBP2, and ALDH3A1 (Fig. 7C and fig. S7B). Transduction with PITX1 and SOX2 significantly increased the migratory capacity of NHEK cells (**P < 0.01, *P < 0.05; Fig. 7D and fig. S7C). These results show that the differential expression of transcriptional regulators in oral versus skin keratinocytes confers a unique identity to oral keratinocytes and that skin keratinocytes can be reprogrammed to increase cell migration and improve wound resolution.
Fig. 7Overexpression of SOX2 and PITX1 increases migratory capacity of primary skin keratinocytes.
(A) Representative pictures of NHEK cells transduced with indicated adenoviruses and stained to show overexpression of corresponding proteins. Scale bars, 20 μm. (B) GO biological process terms enriched in data sets of genes differentially regulated by PITX1 and SOX2 overexpression in NHEK cells. (C) Fold change of the expression of genes related to differentiation and response to biotic stimulus in NHEK cells transduced with PITX1 and SOX2, presented as log2 fold change (log FC) over green fluorescent protein (GFP) expression. (D) Migrating NHEK cells transduced with SOX2, PITX1, and GFP (control) by adenoviral delivery. Images were taken at 0 and 24 hours after removal of silicone insert. Values were determined by counting the number of migrating cells at 24 hours in six microscopic fields in n = 3 per group. **P < 0.01, *P < 0.05 by ANOVA, adSOX2 and adPITX1 versus control. Data are mean values, and error bars represent SDs.
To investigate the effect of overexpressing SOX2 in epidermal keratinocyte on cutaneous wound healing in vivo, we generated epidermal-specific SOX2-overexpressing mice (35). Mice expressing a tamoxifen-inducible Cre driven by the keratin 14 promoter (K14CreERTM) were crossed with lox-stop-lox (LSL)–SOX2 mice to generate K14CreERTM/LSL-SOX2 mice. We compared the kinetics of cutaneous wound healing in K14CreERTM/LSL-SOX2 mice without and with tamoxifen-induced expression of SOX2 in basal keratinocytes (Fig. 8A and fig. S8). Immunohistochemical analysis showed that tamoxifen treatment induced SOX2 expression in epidermal keratinocytes in K14CreERTM/LSL-SOX2 mice (Fig. 8B), and short-term SOX2 expression in the skin amplified the K5 stem cell compartment (Fig. 8B).
Fig. 8Conditional overexpression of SOX2 contributes to cutaneous wound healing.
(A) Schematic representation of the experimental design used for evaluating cutaneous wound healing in K14CreERTM/LSL-SOX2 mice. Mice were treated with vehicle (ethanol) or tamoxifen for five consecutive days by topical application on dorsal skin (day −4 to day 0, red arrow). Wounds were created as a 6-mm full-thickness excisional dorsal skin wound by biopsy punch (day 1). (B) Representative images of unwounded skin stained to show expression of SOX2 (red) and the basal marker keratin 5 (K5, green) in K14CreERTM/LSL-SOX2 mice treated with vehicle or tamoxifen. DAPI (4′,6-diamidino-2-phenylindole) in blue. Epithelium is marked with dotted lines. (C) Photographs of the wound areas after topical treatment with vehicle or tamoxifen in K14CreERTM/SOX2 mice at 1, 3, 5, 7, and 9 days after wounding. Scale bar, 1 mm. (D) Percent wound area at each time point relative to the original wound area in K14CreERTM/LSL-SOX2 mice treated with vehicle or tamoxifen. Quantification of the wound areas in n = 7 wounds per group was performed using ImageJ software. *P < 0.05, tamoxifen versus vehicle at each day. Error bars represent SDs of seven wounds. (E) Representative H&E-stained sections of skin day 1 (unwounded) and day 5 (during wound healing, wound edges including epithelial tongue). Skin sections were from K14CreERTM/LSL-SOX2 mice after treatment with tamoxifen or vehicle. (F) Representative images of unwounded skin stained to show expression of PCNA+proliferating epithelial cells (red). DAPI in blue. *P < 0.05 by unpaired ttest.
Wound healing was significantly promoted in SOX2-overexpressing skin from 3 to 9 days after wounding compared to mice treated with vehicle (*P < 0.05; Fig. 8, C and D). SOX2 overexpression in epithelial keratinocytes led to skin acanthosis (Fig. 8E). Histological analysis in SOX2-overexpressing mice day 5 after wounding showed differences in the migratory tongue when compared to that of control mice (Fig. 8E). SOX2 overexpression significantly increased PCNA+proliferating epithelial cells (*P < 0.05; Fig. 8F). These findings demonstrate that genetic or pharmacological approaches to increase the expression or activity of the SOX2 transcription factor in the skin can improve cutaneous wound healing.
DISCUSSION
Wound healing is a major medical and social priority, and broadening our understanding of the mechanisms involved in wound repair is needed to improve wound care. Oral wounds are able to heal more rapidly and with fewer complications than cutaneous wounds; however, the lack of detailed comparative analysis in humans has limited the advancement of our knowledge in this area. Here, we identified the physiological and molecular determinants for this repair paradigm. Our findings could have widespread implications for the wound healing field. Pathways and molecules characterized in this study may facilitate rapid, scarless healing and could be considered for therapeutic application to non-oral mucosal sites.
To identify and explain the mechanisms that define accelerated oral wound healing, we analyzed the gene expression signature changes during oral mucosal and skin wound resolution in healthy human subjects. Oral mucosa samples obtained before wounding exhibited transcriptional networks that primed the epithelium for wound repair. Our data indicate that the major processes driving acute wound repair in healthy individuals were keratinocyte-driven. These networks were determined in part by the differential expression of a set of transcriptional regulators in oral versus skin keratinocytes, suggesting that pathways established during development are responsible for the differential wound resolution capacity of these cells.
Priming allows the oral mucosa to rapidly control and limit inflammatory responses, leading to fast inflammatory resolution. Oral keratinocyte activation and reduced differentiation allows for a rapid re-epithelialization of the wound area. It has been recently shown that re-epithelialization in mice is dependent on tissue-scale coordination of proliferation, differentiation, and migration (36), with acquisition of stem cell properties in de-differentiated epidermal cells (37). In human wound repair, the reduced expression of cytokines and fibrosis mediators such as annexin 1 and SLPI potentially contributes to the scarless wound healing observed in the oral mucosa.
Transcriptional regulators hold the key to the activation of the molecular events responsible for accelerated wound resolution in oral healing. Here, we identified factors that define oral keratinocytes and demonstrated that two of them, SOX2 and PITX1, regulate networks involved in wound closure. The SOX and PITX family of transcription factors have important roles in development, ranging from regulation of cell fate to axis and pattern formation (32, 34). Our results support that these transcription factors are responsible for establishing an oral mucosa–specific network that primes these epithelia for rapid and efficient wound healing. We further showed that SOX2 and PITX1 can be exploited to reprogram skin keratinocytes to present oral keratinocyte features, including accelerated wound resolution in vitro and in vivo. SOX2 is involved in adult stem cell maintenance for a myriad of epithelial tissues (38) and has been shown to regulate the functions of skin tumor–initiating cells (39, 40). We showed that SOX2 induced an expansion of the K5+ basal stem cell compartment of the skin in mice, indicating that the wound regenerative capacity of SOX2 might be linked to its stem cell regulatory functions. Our data also suggest that PITX1 regulates a separate set of processes related to the expression of structural proteins, including keratins, LCEs and SPRRs. PITX2 is an essential component of the genetic network activated by tissue damage during heart repair (41); however, more studies are needed to identify the specific pathways activated by these transcriptional regulators and their potential for wound repair and tissue regeneration.
Our analysis of wound resolution was limited by clinical concerns that restricted the location of the wound site and timing of the of biopsies. Although our study focused on buccal mucosa as a model for oral wound healing, the oral palate more closely resembles the histological features and differentiation profile of the skin. The location of the oral biopsy in the buccal mucosa was chosen on the basis of clinical parameters. Performing multiple wounds in the palate was not possible due to the extreme discomfort of unsutured or repeated wounds in that region and an increased risk of potential complications, including exposure of bone or tooth surfaces. Despite having different keratinization and terminal differentiation profiles, buccal mucosa, gingiva, and palate show similar accelerated wound healing when compared to cutaneous wounds (3, 20). This indicates that there are mechanisms inherent within the oral cavity that increase wound resolution, beyond local differences in epithelial structure. Choosing buccal mucosa provided the most effective way to minimize discomfort and risk of clinical complications while still being able to study oral mucosa wound healing in subjects. In regard to the timing of the biopsies, our study focused on pairing oral mucosa and skin wounds, and this presented the challenge of comparing two tissue sites with differences in rate of healing that made it unfeasible to compare wounds at the same stage of closure. The number of time points was determined by taking into consideration the paired wounds in both locations, comfort of the subjects, and number of subjects in the clinical protocol.
Overall, we present human clinical data and histological and gene expression analysis that provide a comprehensive comparative analysis of the molecular and cellular mechanisms underlying the different wound healing processes in oral and skin epithelia. Our data indicate that the unique environment of the oral cavity represents a wound healing program geared toward rapid wound resolution (fig. S9). Ultimately, this human transcriptomic data set highlights fundamental global mechanisms of inflammation and repair in humans that will serve as an invaluable resource, providing insights into therapeutic targeting of chronic and nonhealing wounds.
MATERIALS AND METHODS
Study design
We performed a clinical study to obtain paired oral and skin baseline and wound samples and to analyze the healing profile of both locations. The clinical study was approved by the Institutional Review Board at National Institute of Dental and Craniofacial Research (NIDCR; ClinicalTrials.gov #NCT01078467). Inclusion criteria for enrollment consisted of participants being nonsmokers and having only occasional (social) alcohol consumption. Pregnancy was also an exclusion criterion. A sterile 3-mm punch biopsy (McKesson) was used to create a uniform, full-thickness biopsy in the mucosa of the cheek and posterior axillary region of the arm and followed up for up to 15 days. All subjects received an oral and skin biopsy at their day 1 time point (1 day after initial exam). The 30 healthy subjects were randomized in three groups with 10 subjects in each group: Group 1 subjects received one set of biopsies at their day 1 time point, group 2 subjects received a second set of biopsies at their day 3 time point, and group 3 subjects received a second set of biopsies at their day 6 time point. Day 3 and 6 biopsies were performed with a sterile 5-mm punch biopsy. For RNA-seq analysis, paired oral and skin samples were chosen randomly from four subjects for each day (24 total samples from 12 individual subjects). Wound healing was also analyzed in mice according to the protocol approved by the Animal Use and Care Committee at the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS). Full-thickness wounds were created in K14CreERTM/LSL-SOX2 mice and examined as described previously (42, 43). Changes in wound area are expressed as percentages of the initial wound area in control mice. Supplementary Materials and Methods include full clinical study protocol and experimental details. Individual subject-level data are reported in table S4.
Statistical analysis
For RNA-seq data, statistical analysis was performed with the Partek Genomics Suite (www.partek.com): ANOVA was performed to compare the wound healing process in the oral and skin samples by comparing each wound day (days 3 and 6) with the nonwounded samples (day 1); paired t test was performed to compare oral with skin wound healing at each time point. q < 0.05 was considered statistically significant (q value is an adjusted P value taking into account the FDR). For all other data sets, data analysis was performed with GraphPad Prism version 5.01 for Windows (GraphPad Software); P < 0.05 was considered statistically significant. ANOVA followed by the Tukey’s t test were used to analyze the differences in the groups for migration assays. Two-tailed, unpaired t tests were used to analyze the healing time and size differences between the groups, differences in PCNA expression between the groups, and differences in immune markers.
, Evidence for an alternative pathway of keratinocyte maturation in psoriasis from an antigen found in psoriatic but not normal epidermis. J. Invest. Dermatol.83, 296–301 (1984).
, Psoriasis as a model of exaggerated, not dysregulated, wound healing presents potential therapeutic targets for enhanced tissue repair integrity and diminished scaling. Surgery156, 15–19 (2014).
, Introducing a null mutation in the mouse K6α and K6β genes reveals their essential structural role in the oral mucosa. J. Cell Biol. 150,921–928 (2000).
, Genes encoding structural proteins of epidermal cornification and S100 calcium-binding proteins form a gene complex (“epidermal differentiation complex“) on human chromosome 1q21. J. Invest. Dermatol. 106, 989–992 (1996).
, Wound healing in oral mucosa results in reduced scar formation as compared with skin: Evidence from the red Duroc pig model and humans. Wound Repair Regen. 17, 717–729 (2009).
, Lmo7 is an emerin-binding protein that regulates the transcription of emerin and many other muscle-relevant genes. Hum. Mol. Genet. 15, 3459–3472 (2006).
K. G. Leelahavanichkul, J. S. Gutkind, Oral and Pharyngeal Epithelial Keratinocyte Culture. Method in Molecular Biology: Epithelial Cell Culture Protocols (SpringerLink, 2012), vol. 945, pp. 67–79.
Acknowledgments: We thank members of the Laboratory of Skin Biology and of the Laboratory of Cancer Biology and Genetics for helpful suggestions and discussions. We also thank G. Gutierrez-Cruz and S. Dell’Orso of the NIAMS Genome Analysis Core Facility and the NIAMS Light Imaging Core Facility. This work used the computational resources of the NIH High-Performance Computing Biowulf Cluster. We would like to thank the participants of the clinical trial #NCT01078467 and research teams whose contributions made this work possible. Funding: This work was supported by the Intramural Research Programs of the NIAMS (ZIA-AR041124 to M.I.M.) and the NIDCR (Z01DE00558 to J.S.G.) of the NIH. Author contributions: A.A.M., D.E., and J.S.G. performed clinical trial design, execution, and specimen collection. R.I.-B., A.U., J.S.G., and M.I.M. designed experiments. R.I.-B., A.U., A.A.M., J.L.C.-V., C.D., M.-L.A.-L., and M.I.M. performed the experimental work and data analysis. L.A. and N.M.M. performed immune cell staining and analysis. M.-L.A.-L. and M.W.O. provided LSL-SOX2 mice. R.I.-B., A.U., S.R.B., and M.I.M. analyzed RNA-seq data. R.I.B., A.U., D.E., C.D., N.M.M., J.S.G., and M.I.M. wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data associated with this study are present in the paper or the Supplementary Materials. Raw and analyzed RNA-seq data have been deposited in the Gene Expression Omnibus site (GSE97615, GSE97616, and GSE97617).
The in vitro-engineered mucosa equivalent was developed using autologous mucosa and fibrin.
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The mucosal and skin cell sheets had similar histological structures to oral mucosa or skin.
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The mucosal and skin cell sheet promoted skin wound healing with early closure and less scarring.
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The oral mucosal cell sheet showed in vivo tissue plasticity in cutaneous wounds.
Abstract
Objectives
Wound healing is generally faster and associated with less scarring in the oral mucosa than in the skin. Although rarely studied, oral mucosa equivalents may contribute to rapid, scarless cutaneous wound healing. Therefore, we examined the potential utility of our newly developed oral mucosal cell sheet in skin wound healing.
Materials and methods
Oral mucosa and skin samples were obtained from surgical patients and Sprague-Dawley rats. Keratinocytes and fibroblasts were primarily cultured for in vitro cell expansion. Mucosa and skin equivalents were produced with a mixture of cultured fibroblasts and autologous fibrin from plasma and seeding keratinocytes. Mucosal and skin cell sheets were transplanted in full-thickness excisional wounds of rat skin with control wounds. Gross, histological, and molecular characteristics of wound healing according to different postsurgical days were compared in control and cell sheet-covered wounds.
Results
Keratinocytes and fibroblasts derived from the oral mucosa were cultured faster than those derived from the skin. The in vitro-engineered oral mucosa and skin equivalents were successfully produced using complete autologous mucosa or skin and plasma fibrin, showing similarity to the histological characteristics of the skin or mucosa. In the in vivo rat model, the oral mucosal and skin cell sheet promoted wound healing with early wound closure and less scarring. The cell sheet-treated wounds showed lower TGF-β1, α-smooth muscle actin, and fibronectin mRNA expression than the control wounds.
Conclusions
The oral mucosal cell sheet demonstrated in vivo tissue plasticity through good adaptation to skin wounds, contributing to accelerated and scarless healing.
Objectives: Dermal and mucosal healing are mechanistically similar. However, scarring and closure rates are dramatically improved in mucosal healing, possibly due to differences in apoptosis. Apoptosis, nature's preprogrammed form of cell death, occurs via two major pathways, extrinsic and intrinsic, which intersect at caspase3 (Casp3) cleavage and activation. The purpose of this experiment was to identify the predominant pathways of apoptosis in mucosal and dermal wound healing.
Approach: Wounds (1 mm biopsy punch) were made in the dorsal skin (n=3) or tongue (n=3) of female Balb/C mice aged 6 weeks. Wounds were harvested at 6 h, 24 h, day 3 (D3), D5, D7, and D10. RNA was isolated and analyzed using real time reverse transcriptase–polymerase chain reaction. Expression levels for genes in the intrinsic and extrinsic apoptotic pathways were compared in dermal and mucosal wounds.
Results: Compared to mucosal healing, dermal wounds exhibited significantly higher expression of Casp3 (at D5; p<0.05), Casp7 (at D5; p<0.05), Trp53 (at 24 h and D5; p<0.05), Tnfrsf1b (at 24 h; p<0.05), FasR (at 24 h, D5, and D7; p<0.05), and Casp8 (at 24 h; p<0.05) and significantly lower gene expression of Tradd (at 24 h; p<0.05).
Innovation: Our observations indicate differential execution of apoptosis in oral wound healing compared to skin.
Conclusion: Expression patterns of key regulators of apoptosis in wound healing indicate that apoptosis occurs predominantly through the intrinsic pathway in the healing mucosa, but predominantly through the extrinsic pathway in the healing skin. The identification of differences in the apoptotic pathways in skin and mucosal wounds may allow the development of therapeutics to improve skin healing.
wound healing is a complex process that requires succinct yet overlapping phases of hemostasis, inflammation, proliferation, and remodeling. Our lab and others have extensively examined the differences between mucosal and dermal healing. The differences range from macroscopic differences in wound closure rates and scarring outcomes to the microscopic differences in inflammatory cell infiltrates and rates of re-epithelialization, and differential prohealing and proangiogenesis protein production.1–4 Mucosal healing has several key features that mimic regeneration. Mucosal wounds are faster to re-epithelialize, have a decreased inflammatory response, and have a blunted angiogenic response with concomitant reduction in vascular endothelial growth factor (VEGF) gene and protein expression.4 Altogether, the phases of wound healing after mucosal injury are shortened in duration and generally have reduced gene and protein expression changes in comparison to skin wound healing.5
Apoptosis is an important mechanism for cellular elimination during wound healing and maintains tissue homeostasis in normal, uninjured tissue. In a recent study, the overall gene expression of mucosal and skin wounds was compared via microarray analysis.5 Among the multiple differences that were noted, the data suggested that wound healing in these two tissues might exhibit differential signatures of apoptosis related genes, such as tumor necrosis factor alpha (TNF-α) and several downstream signaling factors.5
Apoptosis can be induced via two main pathways, termed intrinsic and extrinsic. The intrinsic pathway is related to DNA damage from ultraviolet (UV) light, chemotherapy, ischemia, and oxidative stress. The extrinsic pathway requires extracellular input, specifically, activation of the intracellular portion of the death receptor (DR) by binding of a death ligand. Apoptosis in general is associated with an intracellular caspase cleavage cascade. Caspases (CASP) involved in apoptosis can be broken down into 3 broad categories: the initiators of apoptosis (Casp2, Casp8, and Casp9), executioners of apoptosis (Casp3 and Casp7), and inflammation-related (Casp1, Casp4, Casp5, and Casp12).6–8
In the intrinsic pathway Casp2, the balance of Bcl-2 and Bax, and the levels of p53 (Trp53) determine cytochrome-c (Cycs) release from mitochondria after mitochondrial membrane disruption. Cycs then forms the apoptosome with apoptotic peptidase activating factor 1 (Apaf1), which cleaves and activates Casp9. The resulting caspase cleavage cascade ends with Casp3 cleavage and activation. Casp3 cleavage and activation represents the point of convergence for the intrinsic and extrinsic pathways and is the final step in initiation of cell death through further DNA fragmentation, and cleavage of cytoskeletal proteins (Fig. 1).
Diagram of the intrinsic and extrinsic apoptosis signaling pathways. Intrinsic apoptosis (the left side of the figure) is usually the result of hypoxia, ischemia, or UV damage. These induce cell stress which is propagated through Casp2 and Trp53 disruption of the balance of the mitochondrial membrane. Cycs is released after mitochondrial membrane disruption and recruited to form the apoptosome with Apaf1 and pro-Casp9. Pro-Casp9 is cleaved and activates the caspase cascade resulting in cleavage and activation of Casp3, Casp6, and Casp7. The downstream effect of activation of the caspase cascade is DNA fragmentation, formation of apoptotic bodies, and cell death. The extrinsic apoptosis signaling pathway (the right side of the figure) requires binding of the death ligand (FasL or TNF-α) to its respective receptor (FasR or Tnfrsf1b). Binding of the death ligand to the death signals the recruitment of Tradd, Fadd, and pro-Casp8. Pro-Casp8 is cleaved by the complex and begins the caspase cleavage cascade resulting in the cleavage and activation of Casp3, Casp6, and Casp7. Similar to the intrinsic pathway, the result of the caspase cleavage cascade is DNA fragmentation, formation of apoptotic bodies, and cell death. ▲, significantly increased in oral mucosa versus skin; ▼, significantly decreased in oral mucosa versus skin; =, no significant difference in oral mucosa versus skin gene expression. Cycs, cytochrome C; Casp, caspase; TNF-α, tumor necrosis factor alpha; UV, ultraviolet
The extrinsic apoptotic signaling pathway involves transmembrane DRs of the TNF receptor gene superfamily, FasR, TnfR1, and DRs 3, 4, and 5. Upon binding of the DR ligand, Fas ligand (FasL) or TNF-α, the TnfR-associated death domain (Tradd) is activated and recruits21 Fas-associated death domain (Fadd) to the intracellular portion of the DR. This begins the intracellular signaling cascade of recruitment and cleavage of pro-Casp8 and ultimately cleavage and activation of Casp3 (Fig. 1).
The purpose of this study was to determine if differential apoptotic responses occur in oral and skin wound healing. Equally-sized wounds from the oral mucosa and the dorsal skin were compared at five different time points (6 h, 24 h, day 3 [D3], D5, and D7) over the course of wound healing for changes in gene expression of key factors in the apoptosis pathways. We hypothesized that apoptosis would be initiated through different pathways in the oral wounds compared to skin.
In the skin, fibrosis, or scarring can vary from normal to hypertrophic scars, keloids, or painful contractures. Effective antifibrotic or antiscarring treatments are currently limited. Oral wound healing, like fetal wound healing, closely resembles optimal healing with very rare occurrences of keloids or hypertrophic scars. Further examination of the mechanisms involved in cellular clearance may direct the development of therapeutic tools to improve the healing process and in turn, patient scarring outcomes.
All animal procedures were approved by the University of Illinois at Chicago Institutional Animal Care and Use Committee. The standard skin and tongue wounding protocols were described previously.5 Briefly, female 6-week-old Balb/c mice (Harlan, Inc. Indianapolis, IN) were anesthetized with intraperitoneal injection of 100 mg/kg ketamine and 0.05 mg/kg xylazine. For mice with dorsal wounds (n=3, per time point, six wounds per mouse) the dorsal skin was shaved and six excisional dermal wounds were placed using a 1 mm punch biopsy (Acu-Punch; Acuderm, Inc., Ft. Lauderdale, FL) on opposing sides of the midline starting at the scapula level and continuing caudally. For mice with mucosal wounds (n=3, per time point, 1 wound per mouse), a 1 mm biopsy punch (Acu-punch; Acuderm) was used to make wounds lateral to and equal distance from the midline of the tongue.
Tissue harvesting and fixation
All mice were euthanized via CO2 inhalation combined with cervical dislocation. Dorsal skin wounds were excised by first cutting a 2 cm×2 cm square encompassing all dorsal wounds (6 total) followed by 2 mm biopsy punch (Acu-punch; Acuderm) of the original wound sites. Oral wound tissue was harvested by excision of the tongue as close to the base as possible. The tongue was then bisected laterally, followed by 2 mm biopsy punch at the site of the original wound on each half of the bisected tongue. Uninjured tissue was harvested in a similar manner from 2 mm biopsy punches taken at the beginning of the experiment in euthanized mice. The wounds and surrounding tissues were collected, and placed in 0.5 mL RNAlater (Sigma, St. Louis, MO) for RNA isolation and stored at −20°C before analysis, or snap frozen in OCT compound (Sakura Finetechnical, Tokyo, Japan) for cryotome sectioning and immunofluorescence, and stored at−80°C before analysis. For RNA analysis and isolation, wounds were harvested at 6 h, 24 h, D3, D5, and D7 (n=3 mice per time point, per tissue type) postinjury for RNA isolation. The small size of the mouse oral cavity and tongue only allow for 1 mm wounds, making it the standard protocol for mucosal injury in mice. Although these small wounds heal quickly, significant site-specific patterns of healing have been identified in this model.1,4,5,9,10
Real time reverse-transcriptase polymerase chain reaction
Total RNA was isolated from 3 wounds per time point per group using TriZol (Invitrogen, Carlsbad, CA) according to the instructions of the manufacturer. The concentration of RNA was determined with Nanodrop 1000 (Thermo Scientific, Wilmington, DE), and 1 μg of RNA was used from each sample for the remainder of the reverse transcriptase–polymerase chain reaction (RT-PCR) protocol. RNA was treated with DNase I (Invitrogen), and reverse transcription performed with Retroscript kit (Ambion/Life Technologies, Grand Island, NY) according to the manufacturer's instructions. The cDNA was amplified on an ABI Step One Plus Real Time PCR System (Applied Biosystems, Life Technologies, Foster City, CA) in 96-well plate reactions with 3 reference gene wells and 3 target gene wells per sample. Primer sequences used for target genes analyzed are listed in Table 1. To quantify relative differences in mRNA expression, the comparative CT method (ΔΔCT) was used to determine relative quantity.11 All target genes were normalized to Gapdh expression in uninjured tongue tissue. Gene expression patterns of 18S RNA, β-actin, and ribosomal protein large, p0 (Rplp0) were also examined and Gapdh was determined to be the most stable reference gene. To compare expression over the time, and to assess differences between the uninjured tongue and skin, gene expression was normalized to the reference uninjured tongue as a single baseline. Results were analyzed with two-way analysis of variance to analyze the time and tissue effects comparing skin and tongue at each time point to each other and to uninjured tissue followed by a Bonferroni's post-test with α=0.05.
table 1.
reverse transcriptase–polymerase chain reaction primer sequences
For immunofluorescence, tissue (n=2 per group) was sectioned with a cryotome (Leica 3050CS; Buffalo Grove, IL) at a 8 μm thickness and placed on UltraStick glass slides (Gold Seal, Portsmouth, NH). Tissue sections were fixed with ice cold acetone for 5 min and then washed 2×5 min with Tris-buffered saline (TBS) 0.025% Triton X-100, followed by 1× TBS wash 3×5 min. The slides were then blocked with normal goat serum (10% normal goat serum in 0.1% BSA 1× PBS) for 2 h. The primary antibody, rabbit antiactive Casp3 (1:100; Abcam, Cambridge, MA) diluted with 1% BSA in PBS was applied in a humidified chamber overnight at 4°C. Slides were rinsed 3×5 min with TBS. Secondary antibody, Alexafluor 488 conjugated goat anti rabbit (1:1,000; Invitrogen Molecular Probes, Grand Island, NY) with 0.1 μg/mL Hoescht nuclear stain (Immunochemistry, Bloomington, MN) diluted with 1% BSA in PBS was applied in a humidified chamber at room temperature for 2 h in the dark. Slides were rinsed 3×5 min with 1× TBS and 0.5% Tween 20. Slides were then mounted in aqueous mounting media (VectaMount AQ; Vector Laboratories, Burlingame, CA) followed by a coverslip and sealed with nail polish. All slides were visualized on a Carl Zeiss fluorescence microscope using AxioVision LE (Thornwood, NY).
To identify the relative amounts of apoptosis occurring over the course of wound healing, the levels of gene expression of Casp3 and Casp7, the executioner caspases involved in the final steps of both intrinsic and extrinsic apoptosis pathways, were examined. Casp3 expression was higher in uninjured skin and over the time course of wound healing in skin, significantly at D5 (Fig. 2a) as compared to tongue wound healing. Casp3 expression in skin wounds showed a significant increase compared to uninjured skin at D3 and D5 (Fig. 2a). When compared to tongue, Casp7 expression was significantly higher in uninjured skin (Fig. 2b). Over the time course of wound healing, skin wounds exhibited significantly increased levels of Casp7 at D5 (Fig. 2b), when compared to tongue wounds.
Apoptosis markers in skin and tongue. Real time RT-PCR of (a)Casp3 and (b)Casp7 were performed on RNA isolated from uninjured tissue (U) and wound samples at 6 h, 24 h, D3, D5, and D7 postinjury. To determine RQ of mRNA levels during wound healing, all samples were normalized to Gapdh expression in uninjured tongue tissue. The results are shown as the mean±SEM; n=3. Data were analyzed by two-way ANOVA and Bonferroni's posttest (*p<0.05 for skin vs. tongue wounds, #p<0.05 for skin vs. uninjured skin). ANOVA, analysis of variance; RT-PCR, reverse transcriptase–polymerase chain reaction; RQ, relative quantity.
CASP3 protein expression was also qualitatively examined by immunofluorescence staining of active (cleaved) CASP3 in uninjured, D3, and D5 skin and tongue tissue (Fig. 3). These time points were identified as having significant differences in Casp3 gene expression. Qualitatively, active CASP3 protein was seen in both uninjured skin and skin wounds; minimal expression was seen in tongue. These results support the concept that, as compared to skin, both normal tongue tissue and tongue wounds exhibit significantly less active, cleaved CASP3.
Active CASP3 protein expression. Immunofluorescence for cleaved (active) CASP3 was performed on uninjured, D3, and D5 postinjury tissues (n=2). The images were not quantified, merely observed to detect the presence of active CASP3 protein. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound
Intrinsic apoptosis pathway
To determine if differential involvement of the intrinsic apoptosis pathway occurs in wound healing of the oral mucosa and the dermis, gene expression of Trp53, Casp2, Casp9, Cycs, and Apaf1 was examined in skin and oral wounds. Significantly lower gene expression of Trp53 was seen in oral wounds at 24 h and D5 (Fig. 4a). Compared to uninjured skin, levels of Trp53 showed a significant increase in skin wounds through D7 (Fig. 4a). Casp2 expression was initially higher in uninjured tongue compared to skin, followed by a peak at D3 in oral wound healing. In contrast, skin wounds demonstrated little change in Casp2 expression (Fig. 4b). Casp9 expression was significantly higher in uninjured skin compared to tongue (Fig. 4c). In tongue wounds, the expression of Casp9 significantly increased at 24 h (Fig. 4c) followed by a decrease to baseline levels by D7. However, skin wounds exhibited a significant decrease in Casp9 expression at 6 h postwounding (Fig. 4c) and then increased back to baseline levels by D7. Cycs expression was not significantly different for uninjured tissues. In skin wounds, Cycs levels increased at 6 h after injury (Fig. 4d), while a significant increase in tongue wound healing did not occur until 24 h (Fig. 4d) compared to uninjured tongue tissue. Apaf1 expression in uninjured tissues was not significantly different. Both skin and tongue wounds showed significantly increased Apaf1 at 24 h after injury (Fig. 4e); Apaf1 levels remained elevated through D7. Overall, the pattern of expression of the signaling factors of the apoptosis pathway suggested that intrinsic apoptosis may play a more significant role in oral wound healing compared to skin wound healing.
Intrinsic pathway markers. Real time RT-PCR of (a)Trp53, (b)Casp2, (c)Casp9, (d)Cycs, and (e)Apaf1 were performed on RNA isolated from uninjured tissue (U) and wound samples at 6 h, 24 h, D3, D5, and D7 postinjury. To determine relative changes in mRNA levels during wound healing, all samples were normalized to Gapdh expression in uninjured tongue tissue. The results are shown as the mean±SEM; n=3. Data were analyzed by two-way ANOVA and Bonferroni's posttest (*p<0.05 for skin vs. tongue wounds, #p<0.05 for skin vs. uninjured skin, &p<0.05 for tongue vs. uninjured tongue).
Extrinsic apoptosis pathway
To assess differences in the contribution of the extrinsic pathway to apoptosis in skin and oral wound healing, we examined the relative gene expression of Tnfrsf1b, FasR, Casp8, Tradd, and Fadd. Tnfrsf1b expression was similar in uninjured skin and tongue; however, a significant increase in expression was observed in skin wounds at 24 h (Fig. 5a) followed by a return to baseline levels. FasR expression was higher in uninjured skin, and significantly increased by 24 h postinjury in skin wounds (Fig. 5b). No corresponding increase in FasR was seen in oral wounds, and, in fact, FasR expression was significantly lower in oral wounds at 24 h, D3, and D5 (Fig. 5b) compared to skin wounds. Casp8 expression was higher in uninjured skin, and significantly increased by 24 h postinjury in both skin and tongue wound healing compared to uninjured tissues (Fig. 5c),with skin showing greater levels than tongue at 24 h (Fig. 5c). Casp8 expression also decreased significantly by D7 in skin wound healing compared to uninjured skin (Fig. 5c). Tradd expression was not significantly different in uninjured tissues; however, by 24 h a significant increase in expression was seen in oral but not skin wounds (Fig. 5d) compared to uninjured tongue and 24 h skin wounds. No corresponding peaks in skin wound healing were observed over the course of wound healing. Fadd expression was higher in uninjured skin (Fig. 5e), and increased over the time course of the experiment, trending to higher than baseline levels (Fig. 5e). When Fadd expression in healing wounds was examined, no significant differences were seen (Fig. 5e). Overall, the expression of genes involved in the extrinsic pathway tended to be significantly increased in skin wound healing as compared to oral wound healing, suggesting more involvement of the extrinsic apoptosis pathway in skin wound healing.
Extrinsic pathway markers. Real time RT-PCR of (a)Tnfrsf1b, (b)FasR, (c)Casp8, (d)Tradd, and (e)Fadd were performed on RNA isolated from uninjured tissue (U) and wound samples at 6 h, 24 h, D3, D5, and D7 postinjury. To determine relative changes in mRNA levels during wound healing, all samples were normalized to Gapdh expression in uninjured tongue tissue. The results are shown as the mean±SEM; n=3. Data were analyzed by two-way ANOVA and Bonferroni's post-test (*p<0.05 for skin vs. tongue wounds, #p<0.05 for skin vs. uninjured skin, &p<0.05 for tongue vs. uninjured tongue).
Oral mucosal wound healing has previously been shown to exhibit reduced scar formation, a faster rate of re-epithelialization, lower levels of inflammation, and lower levels of angiogenesis compared to wound repair in the skin.1,4 However, very little attention has been given to the mechanisms that regulate cell death in wounds of these two tissues. Cell death and the mechanism of cell elimination may play an important role in the scarring outcome via paracrine signaling or immune modulation. Recent studies have suggested that apoptotic cells secrete factors that can modulate immune cell phenotypes to affect myofibroblast differentiation, fibroblast and myofibroblast proliferation, and apoptosis resistance.12 Since increased levels of myofibroblasts, increased fibroblast and myofibroblast proliferation, and increased apoptosis resistance are known to influence scarring and fibrosis, apoptotic cells may play an important role in determining the final result of wound healing. The current study demonstrates that the dominant mechanisms of apoptosis differ for wounds of the oral mucosa and skin. Given the differential scar formation in these two anatomic sites, these results suggest possible connections for apoptotic mechanisms and scarring outcomes.
The mechanism of apoptosis is known to derive from the local environment of preapoptotic cells. The intrinsic apoptotic pathway is generally initiated by ischemia, DNA damage, and a reduction in the levels of growth factors, cytokines, or hormones. In oral wound healing the predominance of the intrinsic apoptosis pathway is early, generally peaking at 24 h. This early peak may have to do with lower levels of pro-survival growth factors in oral wounds, such as VEGF, EGF, and TGF-β1. Previous studies have shown lower levels of these key antiapoptotic growth factors in oral mucosal wounds.1,10 The lower levels of important antiapoptotic growth factors in oral wound healing may be responsible for triggering the intrinsic apoptosis pathway by reducing pro-survival signaling. In contrast, the extrinsic apoptosis pathway requires extracellular input to initiate cell death. Skin wound healing is characterized by robust growth factor production, and hyperproliferation, effectively preventing the initiation of the intrinsic apoptosis pathway. In this situation, activation of the extrinsic apoptosis signaling pathway may be required to induce cell death.
Several other characteristics of oral wound healing have been suggested to play a role in the increased healing rate: faster re-epithelialization, increase proliferation of oral keratinocytes, decreased immune response, increased oxygen availability in the oral cavity, the moist wound environment, temperature, saliva flow, and local microflora. Previous studies have determined that the saliva-based, moist-wound environment plays a role in oral healing;13,14 however, the presence of saliva seems to be more important in larger wounds. Smaller mucosal wounds heal at similar rates independent of salivary influence.15 Saliva contains growth factors, including EGF and VEGF, both of which have been suggested to important to oral wound healing.16,17 The tissue levels of these growth factors, however, is low when compared to skin wound healing. Correspondingly, skin that is transplanted into the oral cavity and shows a healing response that more closely resembled that of skin rather than that of oral mucosa.18,19 Together these studies suggest that environmental factors have a somewhat limited role in healing of oral mucosa. Intrinsic differences between oral mucosa and skin tissue seem likely to play an important part in defining the improved healing of oral wounds.
Our observations indicate that there are distinct and often significant differences in the gene expression of key mediators of both the intrinsic and extrinsic apoptosis pathways in oral wound healing compared to skin wound healing. Overall, our results show that the gene expression of the mediators of both intrinsic and extrinsic apoptosis pathways generally maintain low levels over the course of oral wound healing and return to baseline levels faster (Figs. 2–5) than skin wounds. This observation leads to the conclusion that apoptosis in oral wounds occurs via rapid and concise mechanisms. Although changes in gene expression levels do not necessarily translate to protein expression or function, our findings show that cleaved Casp3 protein levels follow similar trends to that of gene expression (Fig. 3). Our work does not address the protein levels and activation status of the remaining elements of the apoptotic cascade. Further studies will be necessary to quantitatively determine how translational regulation, post-translational modification, and release of intracellular stores influence the many other elements of the apoptotic pathways.
The data here suggest that overall, expression of genes related to the intrinsic pathway are generally higher in oral wound healing compared to skin wound healing (Fig. 1). The timing of the peak of the gene expression related to intrinsic apoptosis in oral wound healing was most commonly seen at 24 h (Fig. 4b–e). This peak may correspond with the particular events occurring at that time. Specifically, inflammatory cells in an oral wound peak at around 24 h and the peak in intrinsic apoptosis may be related to the resolution of inflammation and the elimination of inflammatory cells present in the wound bed.
In contrast to the intrinsic pathway, our studies suggest that mediators of the extrinsic pathway are significantly increased in skin versus oral wound healing. Here again the timing of the peak may be related to the other events occurring in the wound. For example, the peak gene expression of Tnfrsf1b and Casp 8 occurs at 24 h for both oral and skin wound healing (Fig. 5a, c, respectively), although the levels are significantly higher in skin wounds. Apoptosis occurring at 24 h may again be related to the elimination of inflammatory cells and the resolution of inflammation. Interestingly, FasR gene expression also peaks at 24 h in skin wound healing, but there is no corresponding peak in oral wound healing (Fig. 5b). This phenomenon may be due to a more significant role of Fas-mediated apoptosis in skin wound healing, both in general and at 24 h. Also of interest, Tradd gene expression peaks in oral wound healing at 24 h, but there is no corresponding peak in skin wound healing (Fig. 5d). Tradd may be the rate limiting mediator in extrinsic apoptosis in skin wound healing, while it may be in excess in oral wound healing. The significant differences later on in skin wound healing (FasR at D5 and D7, Fig. 5b) could correspond to apoptosis occurring during vessel regression. As opposed to oral mucosa, the robust angiogenesis seen in skin wounds requires a pruning of the overabundant new vessels. As normal skin wound healing progresses into the remodeling phase, large numbers of endothelial cells undergo apoptosis as unnecessary vessels regress.
Recent studies have suggested differences in the mechanism of apoptosis in fetal wound healing compared to adult wound healing. Cleavage of Casp7 and PARP were significantly increased in scarless fetal wound healing (embryonic day 15) compared to fetal wound that resulted in a scar (embryonic day 18).20 Similar to oral mucosal wound healing, regeneration or improved scarring outcomes have been identified in fetal wound healing; several observations suggest potential reasons for the similarities. First, similar to oral wound healing, the immune response in fetal wound healing is significantly lower than adult wound healing.21 Second, the extracellular matrix in fetal and oral wounds have lower collagen I to collagen III ratios compared to normal adult skin wound healing.22–30 Third, the presence and persistence of myofibroblasts during fetal and oral healing are lower compared to adult skin wound healing.31,32 On the same thread, the growth factors that stimulate myofibroblast differentiation have been identified as differentially regulated, with predominance of transforming growth factor (TGF)-β3 in fetal wound healing and TGF-β1 in adult wound healing.33 TGF-β1 protein levels are significantly lower in oral wound healing compared to tongue,10in vitro, oral fibroblasts exhibit a decreased fibrotic response to the same levels of TGF-β1 compared to dermal fibroblasts.34 Lastly, similar to oral wound healing, the angiogenic response in fetal wound healing is significantly lower than adult wound healing.1 Given the numerous similarities between fetal and oral wound healing, the finding of differences in the levels and mechanisms of apoptosis in both oral and fetal wounds20 suggests that apoptosis may play a significant role in the determination of scarring outcomes.
In summary, our results indicate that intrinsic apoptosis may be the predominant mechanism of induction of apoptosis in oral wound healing, while extrinsic apoptosis may play a more significant role in skin wound healing. The differences in the pathways for apoptosis induction may provide potential targets for modifying skin wound healing outcomes to resemble the regeneration seen in oral and fetal wound healing.
Apoptosis maintains normal tissue homeostasis, but in wound healing the process of apoptosis is not completely understood. Fetal and oral wound healing are examples of wound repair that result in regeneration. Further examination of the mechanisms involved in tissue repair in fetal and oral wound healing may generate therapeutic targets to improve skin wound healing. Our observations indicate differential execution of apoptosis in oral wound healing compared to skin. Oral wound healing is characterized by increased gene expression of mediators in the intrinsic apoptosis pathway, while skin wound healing has increased gene expression of mediators in the extrinsic pathway.
Key Findings
• Execution of apoptosis in oral wound healing occurs at lower levels over the entire course of wound healing in oral wounds compared to skin.
• Intrinsic apoptosis is the predominant mechanism of apoptosis in oral wound healing.
• Extrinsic apoptosis is the predominant mechanism of apoptosis in skin wound healing.
The authors wish to thank Ms. Angelica Lagunas, College of Dentistry, University of Illinois at Chicago, for her help in the preliminary studies that led to the experiments discussed in this manuscript. The work described in this article was funded in part by NIGMS R01 GM50875 (LAD) and NIDCR T32 DE018381(AJ LD).
No competing financial interests exist. The content of this article was expressly written by the author(s) listed. No ghostwriters were used to write this article.
Dr. Luisa A. DiPietro is a Professor and the Director of the Center for Wound Healing and Tissue Regeneration at the University of Illinois at Chicago (UIC) College of Dentistry. She currently directs the UIC Multidisciplinary Oral Sciences Training Program (MOST); this NIH-supported program provides research training for graduate students (PhD), postdoctoral fellows, and dual degree (DMD-PhD) students, including Ms. Ariel R. L. Johnson, who is currently a 4th year PhD candidate working on her dissertation research. Research in the DiPietro laboratory focuses on how wounds heal, with the ultimate goal of developing therapies that will allow humans to regenerate perfect tissue after an injury. Ms. Marybeth M. Francis, also a MOST trainee (DMD-PhD candidate), worked closely with Ms. Johnson on this research project.
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Articles from Advances in Wound Care are provided here courtesy of Mary Ann Liebert, Inc.
Wound healing in mucosal tissues differs in many aspects from cutaneous wound healing, a unique aspect being the lack of scar formation. Oral mucosa wound healing comprises a series of sequential responses that allow the closure of ruptures in this tissue. This process is of critical importance to prevent the invasion of microorganisms or other agents into tissues avoiding the establishment of chronic inflammation. Since the oral mucosa is continually exposed to traumatic and infectious challenges, this tissue has developed evolutionary strategies to circumvent this adverse environment. Therefore, the oral mucosa has several advantages in terms of the efficiency of the wound healing response. Wound healing may also play an important role during the cell and tissue reactions that occur during the development of chronic inflammatory diseases and cancer. Therefore, knowledge of the mechanisms that regulate wound healing is essential for the comprehension of pathological events in this tissue.
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Sodium bicarbonate is a salt that breaks down to form sodium and bicarbonate inwater. This breakdown makes a solution alkaline, meaning it is able to neutralize acid. Because of this, sodium bicarbonate is often used to treat conditions caused by high acidity in the body, such as heartburn.
People take sodium bicarbonate by mouth for bowel cleansing, poor kidney function, indigestion, exercise performance, high potassium in the blood, reviving newborns, stomach ulcers, and urinary stones.
Sodium bicarbonate is injected intravenously (by IV) for heartresuscitation, poor kidney function, cocainetoxicity, to prevent kidney damage caused by dyes used during some X-ray exams, poisoning from certain allergy medications, reviving newborns, pesticide poisoning, prevention of chemotherapyside effects, breakdown of muscles, and fluid build-up in the lungs caused by a certain chemical.
People also use sodium bicarbonate, or baking soda, as an ingredient in baking.
In Muc2−/− mice, transient de novo expression of Muc6 messenger RNA was observed in the distal colon. On day 2 of DSS treatment, the histologic damage was ...
Rabbit IgG polyclonal antibody for MUC2 detection. Tested with ... one of these 3 criteria: COVID-19 related;; rare species;; replacing discountinued antibodies.
We are open and processing orders during the COVID-19 global pandemic ... It has been selected for its ability to recognize MUC2 in immunohistochemical ...
Mucin 2 (MUC2) expression is detected in human tissues such as normal colon, breast, prostate, and salivary gland, as well as in gastrointestinal, colonic, breast ...
Mucin-2 (MUC2) is just one of several human mucin proteins but it is the major one lining the intestine and airways. Its role there is largely as a protective or ...
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