Association between recurrent aphthous stomatitis and inheritance of a single‐nucleotide polymorphism of the NOS2 gene encoding inducible nitric oxide synthase

J Oral Pathol Med (2011) 40 : 715–720 Background: Recurrent aphthous stomatitis is a common ulcerative disease of the oral mucosa. Recurrent oral aphthous ulceration is also a feature of the more serious and systemic Behçet’s disease. Nitric oxide is a free radical synthesized by one of a family of nitric oxide synthase (NOS) enzymes and is an important regulator of inflammation and immunity. Association of NOS3 gene polymorphisms encoding endothelial nitric oxide synthase has been reported in Behçet’s disease but not recurrent aphthous stomatitis. The aim of this study was to investigate any association between NOS2 gene polymorphisms that encode inducible nitric oxide synthase and recurrent aphthous stomatitis. Methods: This is a case control association study. Eighty‐three Jordanian recurrent aphthous stomatitis patients and 83 age, gender and ethnically matched controls were genotyped for three NOS2 single‐nucleotide polymorphisms, rs10459953, rs1060822 and rs2297518. Chi‐squared analysis was used to compare the allele frequencies and genotypes. Results: There was a significant association between recurrent aphthous stomatitis and inheritance of single‐nucleotide polymorphism rs2297518 (P = 0.006). Although no direct association was demonstrated between rs10459953 or rs1060822 and recurrent aphthous stomatitis, a strong linkage disequilibrium was identified between rs1060822 and rs2297518. Conclusion: Inheritence of a NOS2 single‐nucleotide polymorphism rs2297518 is associated with increased risk of recurrent aphthous stomatitis in a Jordanian population. Confirmatory studies in other populations and investigation of other NOS2 gene polymorphisms will enhance our understanding of the functional basis of this association and help elucidate the role of inducible nitric oxide synthase in recurrent aphthous stomatitis.     

Topical minocycline for managing symptoms of recurrent aphthous stomatitis

Recurrent aphthous stomatitis (RAS) is a common ulcerative condition of the oral mucosa. In this study, minocycline oral rinses were compared to a placebo in patients suffering from frequent episodes of RAS. Thirty‐three patients with RAS were randomly allocated to topical therapy with 0.2% minocycline or a placebo aqueous solution mouthwash. Seven patients also participated in a blind crossover study. The intensity of pain was recorded daily using a visual analogue scale. Minocycline mouthwashes resulted in significant reduction in the severity and duration of pain due to RAS. The findings in the subgroup that participated in the crossover were consistent with outcomes in the randomized study. The findings of this study show that minocycline oral rinses reduce pain in patients with RAS and may have implications for the use of minocycline in other non‐infectious inflammatory ulcerative oral mucosal diseases.     

Preliminary evidence for an association of Epstein-Barr virus with pre-ulcerative oral lesions in patients with recurrent aphthous ulcers or Behiçet's disease

In this study we used the polymerase chain reaction (PCR), slot blot and Southern blot hybridization, direct sequencing and in situ hybridization (ISH) to show the possible presence of EBV-DNA in pre-ulcerative oral aphthous lesions of patients with recurrent aphthous ulcers (RAU) or Behet's disease (BD). For this purpose, formalin-fixed biopsy specimens were obtained from 13 pre-ulcerative oral aphthous lesions of nine RAU and four BD patients. Five specimens of normal oral mucosa (NOM) from five normal control subjects and 10 specimens of oral erosive or ulcerative lesions from 10 patients with erosive lichen planus (ELP) were also included. EBV-DNA was detected by PCR in 5 of the 13 (38.5%) pre-ulcerative oral aphthous lesions, two from RAU patients and three from BD patients. However, no EBV-DNA was demonstrated in five NOM specimens from normal control subjects and in 10 specimens of oral lesions from ELP patients. EBV-DNA was also demonstrated in patients'peripheral blood lymphocytes and/ or plasma, suggesting that the lymphocytes may be the reservoir of latent EBV infection and there is EBV shedding in the plasma. EBV-DNA was detected by ISH in only one PCR-positive case; the reaction product was found to deposit on the nuclei of some of the epithelial cells and lymphocytes. By immunohistochemistry, expression of Epstein-Barr nuclear antigen and EBV/C3d receptors was also noted in some of the epithelial cells and lymphocytes in this ISH-positive case. Therefore, we suggest that the epithelial cells of pre-ulcerative oral aphthous lesions may be infected by EBV through EBV-infected lymphocytes; also, the cytotoxic T lymphocyte-induced lysis of the EBV-infected epithelial cells, but not the virus-induced cytolysis, may be the main mechanism causing oral ulcer formation. Our data provide preliminary evidence for an association of EBV with pre-ulcerative oral aphthous lesions in RAU and BD patients.     

Helicobacter pylori in oral ulcerations

Helicobacter pylori is an important pathogen involved in the development of gastrointestinal ulcers, but its involvement in oral ulcerous lesions is unclear. As culture is generally recognized as the gold standard for diagnosis of H. pylori infection, we employed this approach to assess the association of H. pylori with oral mucosal ulcerations. Samples were collected from patients with oral mucosal ulcerative disorders: 12 cases of recurrent aphthous stomatitis (RAS), 7 cases of herpes simplex virus (HSV) stomatitis, and 3 cases of erosive lichen planus (LP). Serum IgG antibodies against H. pylori were examined in all cases. All of the RAS and erosive LP cases were culture-negative for H. pylori, while two cases of HSV stomatitis were positive. The two culture-positive cases were also seropositve for the H. pylori antigen. It is suggested that H. pylori might not have a direct association with oral ulcerations. However, H. pylori in the oral cavity might exist in a non-culturable coccoid state without productive infection, and might form colonies only under special conditions such as HSV infection.     

Smokeless tobacco use prevents aphthous stomatitis.

Aphthous stomatitis is a common, recurrent, painful ulcerative condition of the oral mucosa. Cigarette smoking has been reported to protect against aphthous ulcers. To determine whether smokeless tobacco use also protects against aphthous ulcers, we examined the oral mucosa in 1456 professional baseball players, about half of whom were smokeless tobacco (ST) users. After controlling for the confounding effects of age, race, cigarette smoking, alcohol consumption, and dental hygiene practices, ST use was found to significantly reduce the risk of aphthous ulcers among these healthy young men (odds ratio = 0.4; p = 0.04). It has been suggested that cigarette smoking prevents aphthous ulcers by causing increased keratinization of the oral mucosa, and ST may protect by the same mechanism. Alternatively, a component of tobacco that is systemically absorbed might be responsible for protecting against aphthous ulcers. If the mechanism that protects ST users against aphthous ulcers is systemic, then nicotine is the likely protective factor.     

Serum IgG antibodies to Helicobacter pylori in patients with recurrent aphthous stomatitis and other oral disorders

Objectives. To compare the frequency of serum anti-Helicobacter pylori IgG antibodies in patients with recurrent aphthous stomatitis with patients with other oral ulcerative and nonulcerative disorders.Study design. Prospective study of serum IgG antibodies to H. pylori in 75 patients with recurrent aphthous ulcers, 15 patients with other oral ulcerative disorders, 41 patients with other oral mucosal lesions, 27 patients with oral dysaesthesia, and 25 healthy control patients without oral lesions.Results. The frequency of anti-H. pylori seropositivity was not significantly greater in patients with recurrent aphthous stomatitis (30.6%) compared with patients with other ulcerated oral mucosal lesions (33.0%) and controls (24%).Conclusions.Helicobacter pylori does not appear to be of etiologic significance in the development of recurrent aphthous stomatitis.     

Urban legends series: lichen planus

Oral Diseases (2012) Lichen planus (LP) is a common disorder affecting the oral cavity (OLP) and skin. Despite intensive research, LP/OLP etiology and treatment remain controversial. We investigated four controversial topics: (i) Is hepatitis C virus (HCV) infection associated with LP and involved in its pathogenesis? (ii) Should all patients with LP be screened for HCV? (iii) Should patients with OLP have all their amalgam restorations removed? (iv) Are there any new treatments for OLP? Results from extensive literature searches suggested that: (i) Robust evidence from three meta‐analyses indicate that HCV is associated with LP and might be involved in OLP pathogenesis (ii) It would be prudent to screen patients with LP/OLP at significant risk with an ELISA for HCV antibodies using country‐specific screening strategies (iii) There is no evidence that either OLP or oral lichenoid lesions patients would routinely benefit from having all their amalgam restorations replaced. Weak evidence from potentially very biased, small, non‐randomized, unblinded studies suggests that a small fraction of patients may benefit from targeted amalgam replacement. (iv) There is weak evidence that, among new OLP treatments, topical pimecrolimus, aloe vera, and oral curcuminoids may be useful. The development of specific formulations for oral delivery of topical medications is a promising field.     

Human cytomegalovirus as a potential etiologic agent in recurrent aphthous ulcers and Behçet's disease

In a preliminary study on anti-human cytomegalovirus (HCMV) antibody (Ab) by ELISA. the serum anti-HCMV/IgG Ab concentrations in 22 patients with recurrent aphthous ulcers (RAU) in the remission stage were found to be significantly higher than in 22 control subjects (121±42 vs 100±27, P<0.05) and in 39 patients with RAU in the active stage (121±42 vs 88±45, P<0.01). Therefore, the potential of HCMV as an etiologic agent in RAU was proposed and studies using the polymerase chain reaction (PCR) and in situ hybridization (ISH) have been performed to investigate the possible presence of HCMV DNA in pre-ulcerative oral aphthous lesions in patients with RAU or Behçet's disease (BD) of the mucocutaneous type. For this purpose, formalin-fixed biopsy specimens were obtained from 13 pre-ulcerative oral aphthous lesions, 2 samples of normal oral mucosa and 1 ileal mucosal lesion from 9 RAU patients and 4 BD patients. Five specimens of normal oral mucosa from 5 normal control subjects and 12 specimens of oral erosive or ulcerative lesions from 12 patients with erosive lichen planus (ELP) were also included. By PCR, HCMV DNA was detected in 5 of the 13 (38.5%) pre-ulcerative oral aphthous lesions. 3 from RAU patients and 2 from BD patients. The ileal mucosa specimen was also HCMV DNA-positive, whereas HCMV DNA was not demonstrated in any of the 7 specimens of normal oral mucosa from RAU patients and normal control subjects; 12 specimens of oral lesions from ELP patients were similarly negative. ISH did not detect HCMV DNA in any of the biopsy specimens from RAU patients and control subjects. Our findings suggest that HCMV may be an etiologic agent in some cases of RAU and BD.     

Effects of interleukin‐1β and tumor necrosis factor‐α on macrophage inflammatory protein‐3α production in synovial fibroblast‐like cells from human temporomandibular joints

Background

Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are key mediators of the intracapsular pathological conditions of the temporomandibular joint (TMJ). Therefore, the gene expression profiles in synovial fibroblast‐like cells (SFCs) from patients with internal derangement of the TMJ were examined after they were stimulated with IL‐1β or TNF‐α to determine which genes were altered.

Methods

Ribonucleic acid was isolated from SFCs after IL‐1β or TNF‐α treatment. Gene expression profiling was performed using oligonucleotide microarray analysis. On the basis of the results of this assay, we investigated the kinetics of macrophage inflammatory protein‐3α (MIP‐3α) gene expression using PCR, and protein production in TMJ SFCs stimulated by IL‐1β or TNF‐α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL‐1β or TNF‐α after treatment with inhibitors. The MIP‐3α levels were measured using an ELISA.

Results

Macrophage inflammatory protein‐3α was the gene most upregulated by IL‐1β‐ or TNF‐α stimulation. The mRNA and protein levels of MIP‐3α increased in response to IL‐1β in a time‐dependent manner. In contrast, during TNF‐α stimulation, the MIP‐3α mRNA levels peaked at 4 h, and the protein levels peaked at 8 h. In addition, the IL‐1β‐ and TNF‐α‐stimulated MIP‐3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors.

Conclusion

Interleukin‐1β and TNF‐α increased the MIP‐3α production in SFCs via the MAPK and NFκB pathways. These results suggest that the production of MIP‐3α from stimulation with IL‐1β or TNF‐α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.     

Changes in miRNA expression in sera and correlation to duration of disease in patients with multifocal mucosal lichen planus

J Oral Pathol Med (2012) 41 : 86–89 Background: Mucosal lichen planus is a severe variant of lichen planus, Lichen planus (LP), which in many ways affect patients’ lives. The aetiology is not fully understood, and there is no treatment clearing the disease once and for all. Oral LP has by the WHO been classified as a precancerous lesion. Micro‐RNAs, miRNAs, are non‐coding, small single‐stranded RNAs involved in biological processes like apoptosis, proliferation, differentiation, metastasis, angiogenesis and immune response. Methods and Results: In sera from 30 patients with multifocal mucosal LP, 15 miRNAs were identified as significantly differentially expressed compared with controls. The three most up‐regulated miRNAs are all connected to oral squamous cell carcinoma or epithelial carcinoma in general. Discussion: Even if no specific LP‐associated miRNA profile was found, data clearly indicate that miRNAs could play a role in the earlier phases of lichen planus.     

Increased production of tumour necrosis factor by peripheral blood leukocytes in patients with recurrent oral aphthous ulceration

Much evidence suggests that recurrent oral aphthous ulceration (RAU) is an immunologically mediated disease. Tumour necrosis factor has multiple biologic properties, some of which may be relevant to the pathogenesis of RAU. This study has assessed its production by peripheral blood leukocytes from aphthous patients in active and remission phases of disease and from patients with nonaphthous ulceration (diseased controls). Each ulcer patient was studied in parallel with a matched healthy control volunteer. A bioassay against the standard mouse fibrosarcoma line, L929, was used to assess the levels of tumour necrosis factor-alpha (TNF). Significantly greater amounts of TNF were released from unstimulated monocyte-enriched and monocyte-depleted leukocyte fractions in active RAU compared with those from healthy control donors, suggesting that this cytokine may be associated with RAU.     

Potential oral manifestations of cardiovascular drugs

Oral Diseases (2010) 16 , 769–773 Objective: The aim of this work was to determine the frequency and nature of oral manifestations secondary to use of cardiovascular drugs. Methods: Five hundred and thirty one patients attending an adult cardiology clinic in Saudi Arabia were questioned about the occurrence of oral dryness, dysgeusia, or burning sensation and were clinically evaluated for the presence of oral mucosal or gingival disease. Data were statistically analyzed with chi‐squared tests, odds ratios and Student’s t‐test. Results: Oral symptoms and/or signs were recorded in 75 (14.1%) patients with xerostomia being the most common (7.5%), followed by lichenoid (lichen planus‐like) lesions (3.6%) and dysgeusia (1.9%). Xerostomia was significantly more frequent in patients with a history of diabetes mellitus and in female patients (P < 0.05). There were no statistically significant differences (P > 0.05) between patients with or without oral manifestations when age, gender, cardiovascular risk factor, cardiac disease, type of cardiac drug used or the number of medications were assessed. There was a trend for xerostomia to be less frequent in patients receiving therapy with angiotensin converting enzyme inhibitors and a slight trend of xerostomia to be more likely with increased number of non‐cardiac and total number of agents per subject. The number of non‐cardiac and total medications taken by patients with potential oral manifestations tended to be greater than that of patients without oral manifestations. Conclusions: The frequency of potential oral manifestations in patients receiving cardiovascular agents was 14.1%. The occurrence and character of the oral manifestations had no significant relation with individual cardiac drugs, although there was a trend for oral manifestations to be likely with increasing number of drugs.     

Urban legends: recurrent aphthous stomatitis

Oral Diseases (2011) 17 , 755–770 Recurrent aphthous stomatitis (RAS) is the most common idiopathic intraoral ulcerative disease in the USA. Aphthae typically occur in apparently healthy individuals, although an association with certain systemic diseases has been reported. Despite the unclear etiopathogenesis, new drug trials are continuously conducted in an attempt to reduce pain and dysfunction. We investigated four controversial topics: (1) Is complex aphthosis a mild form of Behçet’s disease (BD)? (2) Is periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome a distinct medical entity? (3) Is RAS associated with other systemic diseases [e.g., celiac disease (CD) and B12 deficiency]? (4) Are there any new RAS treatments? Results from extensive literature searches, including a systematic review of RAS trials, suggested the following: (1) Complex aphthosis is not a mild form of BD in North America or Western Europe; (2) Diagnostic criteria for PFAPA have low specificity and the characteristics of the oral ulcers warrant further studies; (3) Oral ulcers may be associated with CD; however, these ulcers may not be RAS; RAS is rarely associated with B12 deficiency; nevertheless, B12 treatment may be beneficial, via mechanisms that warrant further study; (4) Thirty‐three controlled trials published in the past 6 years reported some effectiveness, although potential for bias was high.     

Salivary interleukin-6 and tumor necrosis factor alpha in patients with drug-induced xerostomia

It is well known that cytokines are involved in the homeostasis of oral cavity and that altered levels of either serum and/or salivary cytokines have been found in certain oral/systemic diseases. So far, cytokines in connection with xerostomia have been investigated in patients with Sjögren's syndrome. We wanted to find out whether drugs themselves influence salivary glands, which would result in altered cytokine level or whether xerostomia itself of different causes leads to the changes in salivary cytokine levels. Therefore, the aim of this study was to evaluate levels of salivary interleukin‐6 (IL‐6) and tumor necrosis factor (TNF)‐α in 30 patients with drug‐induced xerostomia, age range 29–84 and mean 63.9 years. Control group consisted of 30 healthy participants, age range 30–82 years and mean age 65.2 years. Enzyme‐linked immunoassay was performed on commercially available kits. Statistical analysis was performed by use of Student's test. No significant differences in salivary IL‐6 and TNF‐α between patients with drug‐induced xerostomia when compared with the healthy controls were found (P < 0.05). We might conclude that drugs do not induce damage to the salivary glands which could be seen in altered salivary IL‐6 and TNF‐α levels and that xerostomia itself, induced by drugs does not alter levels of the investigated salivary cytokines.     

Increased odds ratio for COVID‐19 in patients with recurrent aphthous stomatitis

The ACE2 receptor, the binding sites for the COVID‐19, is expressed abundantly in the oral cavity, raising the question of whether the mouth is a target for the virus in addition to organs such as kidneys and lungs. Recently, a flurry of individual case reports on oral manifestation of COVID‐19 including ulceration, blistering lesions, and stomatitis were published. However, it is not clear whether the oral presentations that are not unique to the virus are indeed related to the virus and appear at a higher prevalence than in the general population. We used the i2b2 platform of hospital patient's registry to determine the odds ratio for COVID‐19 in patients that were diagnosed with recurrent aphthous stomatitis, an entity restricted to the oral cavity. The overall odds ratio for COVID‐19 in patients with recurrent aphthous stomatitis before adjustments was 14 and after adjustment for gender, race, and age was 13.9, 6.5, and 2.93, respectively. The odds ratio remained increased after adjustments of the comorbidities such as respiratory disease, endocrine disease, obesity, diabetes, circulatory disease, and smoking and was 3.66, 7.46, 4.6, 10.54, 7.37, and 7.52, respectively. When adjusted for recurrent aphthous stomatitis, the respiratory disease had an odd ratio of 8.56 to be associated with COVID‐19. African American race and age‐group 18‐34 were additional significant risk factors. The present study has demonstrated a significant association between COVID‐19 and RAS; however, additional longitudinal and laboratory studies are necessary to establish a cause and effect relationship between these 2 conditions.     

Aqueous hydrocortisone mouthwash solution: clinical evaluation

Patients often experience difficulties in applying topical steroids in orabase to the oral mucosa, particularly when large areas need to be covered. An aqueous hydrocortisone mouthwash solution has been developed, one that was anticipated to be more acceptable to patients. The solution contains hydrocortisone (0.3% w/v) in a 4.5% (w/v) 2-hydroxypropyl-β-cyclodextrin solution. Hydroxypropylmethylcellulose (0.5% w/v) was used to increase the viscosity of the solution and to promote the hydrocortisone±cyclodextrin complex. One hundred and two patients with aphthous ulceration, lichen planus, and other mucosal conditions used the mouthwash in an open clinical efficacy study. Most patients reported some or considerable improvement following a 2-week course of treatment with the mouthwash: 26 of 33 (78.8%) patients with aphthous ulceration were `much better', as were 26 of 54 (48.1%) patients with lichen planus and 5 of 16 (31.3%) patients with other mucosal lesions. No serious side effects were reported. Aqueous mouthwash solutions offer a potential vehicle for topical steroid therapy of oral mucosal lesions.     

Serum ferritin in recurrent oral ulceration

A sensitive radio-assay for ferritin was developed and used to examine serum ferritin levels in 105 patients with recurrent oral ulceration (ROU), 41 patients with Behçet's syndrome (BS), 42 with other ulcerative oral lesions, 35 patients with non-ulcerative oral lesions and in 78 controls. Ferritin levels increased with age and were significantly higher in males than females. The mean ferritin concentrations in male patients with ROU, BS or with other oral ulcers were significantly reduced in comparison with controls, and in female patients were significantly reduced in those with major aphthous ulcers. The prevalence of low serum ferritin levels was about 8% in patients with ROU, 15% in BS and 9.5% in patients with other ulcerative oral lesions, compared with less than 3% in patients with non-ulcerative oral disorders and in controls. Most of the iron-deficient patients were female. Serum ferritin levels did not directly correlate with serum iron levels and may be a more accurate indicator of iron deficiency. Furthermore, serum ferritin can distinguish between patients with true iron deficiency and those with secondary sideropenia. It is suggested that in a small number of patients, oral ulceration may be a presenting sign of iron deficiency, and that in a further small proportion of patients, ROU already present will be exacerbated by concurrent iron deficiency. Both groups will show a therapeutic response to correction of the iron deficiency. The results suggest that serum ferritin levels are a useful part of the haematological investigations in patients with ROU.     

US1HIV – changing patterns in HAART era,patients’ quality of life and occupational risks

Oral manifestations are early and important indicators of HIV‐infection. Several lesions with strong association to HIV infection have been described: oral candidiasis (OC), oral hairy leukoplakia (OHL), Kaposi's sarcoma (KS), Non‐Hodgkin‐Lymphoma (NHL), necrotising ulcerative gingivitis and periodontitis. These lesions may be present in up to 50% of patients with HIV‐infection and up to 80% of those with AIDS. Changing patterns in HAART era: With the advent of highly active antiretroviral therapy (HAART) the prevalence of OC, OHL and HIV – associated periodontal disease has decreased in adults. The prevalence of KS has not changed. However, there has been an increase in HPV‐associated oral lesions (papillomas, condylomas and focal epithelial hyperplasia) and HIV‐related salivary gland disease. In children receiving HAART no change in the prevalence of HIV‐related oral lesions has been found. Quality of life: The presence of oral lesions has a marked impact on health related quality of life. HIV‐associated orofacial lesions may lead to facial disfigurement (KS, NHL) or may impair speech and swallowing. Consequently, weight loss and pain may be result. Studies have shown that patients with OC, angular cheilitis and OHL have a high score of decayed teeth (DMFT). Xerostomia and taste disturbances may also be factors with impact on quality of life. Occupational risks: Occupational exposure to HIV has resulted in 57 documented cases of HIV sero‐conversion among healthcare workers in the US (December 2001). Exposure to HBV and HCV carries a much higher risk of occupational infection than that for HIV‐exposure.     

Oral health related quality of life is affected by disease activity in Behçet's disease

Objectives: This study aimed to investigate oral and general health related quality of life (QoL) in patients with Behçet's Disease (BD) and to assess the performance of Turkish versions of oral health related quality questionnaires. Subjects and methods: Ninety‐four BD patients, 24 patients with recurrent aphthous stomatitis (RAS), 113 healthy controls (HC) and 44 dental patients were investigated. QoL was assessed by oral health impact profile‐14 (OHIP‐14), oral health related quality of life (OHQoL) and short form‐36 (SF‐36) questionnaires. Results: OHQoL, OHIP‐14 and SF‐36 subscale scores were significantly worse in patients with BD compared with those in HC (P < 0.05). Both OHIP‐14 and OHQoL scores were significantly worse in active patients compared with inactives in BD and RAS (P < 0.05). Scores of SF‐36 Role physical, Role emotional and Vitality were also lower in active patients than in inactives in BD (P < 0.05). Scores of OHIP‐14 and OHQoL were significantly worse in patients treated with colchicine compared with those treated with immunosuppressives (P < 0.05). Conclusions: Both oral and general QoL was impaired in BD and associated with disease activity and treatment modalities. Translated Turkish versions of OHIP‐14 and OHQoL were also observed to be valid and reliable questionnaires for further studies.