Chlorhexidine induces DNA damage in rat peripheral leukocytes and oral mucosal cells

OBJECTIVE: Chlorhexidine digluconate is widely used in dental practice for decreasing plaque control, controlling gingivitis and disinfecting root canals. However, the undesirable effects of chlorhexidine digluconate regarding its genotoxicity are conflicting in the literature. Thus, the aim of this study was to investigate the genotoxicity of chlorhexidine digluconate in rat peripheral blood and oral mucosal cells by the single cell gel (comet) assay and micronucleus assay. METHODS: Thirty male Wistar rats were distributed into three groups: negative control; experimental group orally treated with 0.5 ml of 0.12% chlorhexidine digluconate, twice daily, during 8 days; and positive control, which received 4-nitroquinoline 1-oxide at 0.5 g/l by drinking water. RESULTS: A statistically significant increase of DNA damage was observed in leukocytes and oral mucosal cells of the chlorhexidine digluconate treated group, as assessed by the comet assay. However, no increase of micronucleated cells was detected in reticulocytes from peripheral blood cells. CONCLUSIONS: Taken together, the data indicate that chlorhexidine digluconate is able to induce primary DNA damage in leukocytes and in oral mucosal cells, but no chromosome breakage or loss in erythrocytes.     

The effect of toothpaste containing triclosan on oral mucosal desquamation

Abstract SLS-containing toothpaste has previously been shown to cause oral mucosal desquamations when used in an experimental cap splint model. The aim of the present study was to examine the effect of toothpastes containing SLS in combination with triclosan on oral mucosal desquamation in a similar cap splint model system. It has previously been shown that the antibacterial agent triclosan also may have anti-inflammatory properties. The concentration of triclosan in the experimental toothpastes was 0.3%, while SLS varied from 1.5% to 3%. No oral mucosal desquamations were observed after use of a 1.5% SLS – 0.3% triclosan containing toothpaste, contrary to the positive control toothpaste that contained 1.5% SLS without triclosan. Furthermore, a statistically significant reduction in severe desquamations was observed after use of a toothpaste containing 3% SLS – 0.3% triclosan compared with the positive control. It may thus be suggested that triclosan exerts a moderating effect on desquamative reactions caused by SLS and that the effect is dependent on the relative amount of triclosan and SLS in the toothpastes.     

口腔黏膜上皮细胞与猪小肠黏膜下层体外复合培养的实验研究

目的探讨体外快速培养犬口腔黏膜上皮细胞（OMECs）及其与猪小肠黏膜下层（SIS）体外复合培养的方法,为组织工程化黏膜研究提供实验依据。方法采用混合酶消化法,于6%胎牛血清的上皮细胞无血清培养基（DKSFM）中培养OMECs,观察OMECs的形态特征、绘制生长曲线并进行细胞表面标志物检测。将第2代犬OMECs接种在SIS上,行苏木精-伊红染色、免疫组织化学染色、扫描电镜等观察OMECs在SIS上的生长状况。结果OMECs在DKSFM中生长良好,CK19细胞角蛋白免疫组化染色阳性。OMECs接种在SIS上呈单层生长,多角形,铺路石样排列,复合培养8 d呈多层生长。结论采用混合酶消化法,用含6%胎牛血清的DKSFM培养犬OMECs,方法简便,适合推广应用。SIS与OMECs有良好的相容性,可作为构建组织工程化黏膜的理想支架材料。     

Effect of mobile phones on micronucleus frequency in human exfoliated oral mucosal cells

Oral Diseases (2012) 18, 786-792 Objective: In the last two decades, the use of mobile phones has increased enormously all over the world. The controversy regarding whether radiofrequency (RF) fields exert effects upon biological systems is a concern for the general population. An evaluation is made of DNA damage and cytokinetic defects, proliferative potential, and cell death because of RF radiation emitted by mobile phones in healthy young users. Study design: This cohort study was carried out in 50 Caucasian mobile phone users. We collected two cell samples from each subject (a total of 100 cell samples), corresponding to the right and left cheek mucosa, respectively. Case histories and personal information were assessed, including age, gender, body height and weight, history of cancer, smoking and alcohol consumption, exposure to chemical carcinogens or radiation, and dietary habits. Sampling comprised cell collection from both cheeks with a cytobrush, centrifugation, slide preparation, fixation, and staining, followed by fluorescent microscopic analysis. A total of 2000 exfoliated cells were screened for nuclear abnormalities, especially micronucleus. Results: No statistically significant changes were recorded in relation to age, gender, body mass index, or smoking status. A comparison of the results vs the control area according to the side of the face on which the mobile phone was placed, and in relation to the duration of exposure (years) to mobile phone radiation in the total 100 samples, yielded no significant differences. Conclusions: No genotoxic effects because of RF exposure were observed in relation to any of the study parameters.     

In vivo effects of zoledronic acid on oral mucosal epithelial cells

Oral Diseases (2011) 17 , 291–297 Objective: Osteonecrosis of the jaw is a serious complication of bisphosphonate treatment for which the pathophysiology is unknown. The purpose of this study was to investigate whether in vivo zoledronic acid (ZA) induces alterations in cell proliferation, apoptosis, and matrix metalloproteinases (MMPs) expression in oral mucosal epithelial cells. Methods: One‐year‐old dogs were either untreated (control group) or given high doses of intravenous ZA (ZA group) for 3 months. The doses of ZA were equivalent to those given to cancer patients, yet were administered two times more frequently (every 2 weeks). Mucosal tissues were assessed immunohistochemically for cell proliferation (proliferating cell nuclear antigen, PCNA), matrix metalloproteinase (MMP) expression, and apoptosis (caspase 3 and TUNEL). Results: There were no significant differences between the groups with respect to PCNA, MMP‐2, MMP‐14, and TUNEL positive cells. However, the expression of MMP‐9 was significantly higher in the control group than in the ZA group (P < 0.05), whereas the expression of caspase 3 was significantly lower in the control group than in the ZA group (P < 0.05). Conclusion: These results suggest that high doses of ZA resulted in higher levels of apoptosis and lower levels of MMP‐9 in the oral epithelial cells supporting the idea of bisphosphonate treatment affects the oral mucosa.     

In vivo evaluation of fluoride and sodium lauryl sulphate in toothpaste on buccal epithelial cells toxicity

Objectives: The present study addresses the effect of fluoride and sodium lauryl sulphate content of toothpaste on oral epithelial cells in vivo conditions.

Subjects and method: Forty volunteers were assigned into two experimental groups, each of them applying the different brand of toothpaste. Every group has been using three different types of toothpaste (non-fluoride and non-SLS, fluoride and non-SLS, and the fluoride and SLS) of the same brand for 6 months, each for 2 months. The buccal epithelial cells were sampled at baseline and 30, 60, 90, 120, 150 and 180 days after the beginning of the research. Effect on DNA damage was analyzed by micronucleus assay

Results: After 60 days of use, for both tested kinds of toothpaste with fluoride and without SLS, all studied parameters were not significantly different from the results obtained at the time when the participants used a non-fluoride toothpaste. While, after 60 days of use, for one kind of toothpaste with SLS and fluoride, was observed significantly higher incidence of pyknotic cells (2.20?±?0.95, 0.00?±?0.00 vs. 0.05?±?0.22, respectively; p?=?.001), cells with karyorrhexis (2.35?±?1.14, 0.85?±?0.93 vs. 0.40?±?0.68, respectively; p?=?.001), and nuclear buds (1.35?±?0.68, 0.45?±?0.51 vs. 0.45?±?0.60, respectively; p?=?.001), compared to toothpastes of the same brand with fluoride and without SLS, and without fluoride and without SLS, for the same period.

Conclusions: Based on the results, can be concluded that there is no fluorine-dependent cytotoxic or genotoxic effect, while SLS dentifrice increases the number of nuclear morphological changes in buccal epithelial cells.     

口腔黏膜上皮细胞和成纤维细胞的复合培养

目的 为癌前病变临床病理研究建立一种简便、迅速和有效的口腔黏膜上皮细胞和成纤维细胞复合培养的方法,实现体外模拟组织工程化口腔黏膜的发生发展。方法 用DispaseⅡ分离上皮和皮下组织,用KGM培养口腔黏膜上皮细胞。用细胞培养法和组织块培养法获取验用的口腔黏膜上皮细胞和成纤维细胞,并采用复合培养法对两种细胞进行共同培养。结果 用DispaseⅡ可成分地分离上皮和皮下组织。KGM可明显促进口腔黏膜上皮细胞的分裂繁殖。复合培养的HE染色切片显示薄层的结缔组织之上有方形的基底细胞、颗粒细胞和角化层。结论 KGM可明显促进口腔黏膜上皮细胞的分裂和成熟。采用组织培养法获取原代成纤维细胞是适合口腔黏膜取材等特点的有效方法。气液相培养的口腔黏膜下结缔组织可促进上皮细胞的分层和分化。     

Review of the extrinsic stain removal and enamel/dentine abrasion by a calcium carbonate and perlite containing whitening toothpaste

There has been an increase in the demand from consumers and patients for products that whiten teeth. To meet this demand, a whitening toothpaste containing calcium carbonate and perlite as the abrasive system and an efficacious fluoride source has recently been launched. The aim of the current paper is to review the toothpaste's stain removal efficacy and its effects on enamel and dentine wear. It has been shown to be effective at removing model extrinsic stain in vitro. Further, it has been shown to be more effective in removing naturally occurring extrinsic tooth stain than a silica non-whitening control toothpaste after two weeks of twice daily brushing in a parallel group, double-blind clinical study using 152 adult volunteers. In addition, the enhanced whitening effect did not give a clinically relevant level of wear to enamel or a significant increase in dentine wear compared to marketed non-whitening toothpaste formulations, as shown by using an in situ type model with ex vivo brushing.     

Cellular effects of genotoxic stress and gene silencing of the checkpoint kinases in human oral cells

Backround:  The human oral cells have different physiological properties and different origins in developmental stages. Mechanical, physiological, and chemical stress can cause damage and irritation during clinical treatment in various oral tissues.
Purpose:  The effects of DNA damage response and gene silencing of checkpoint kinases (Chk1/2) is not unclear in oral primary and cancer cells.
Method:  Treatment with doxorubicin involving DNA damage and gene silencing of Chk1/2 by shRNA constructs was performed in pulp, periodontal ligament, gingival tissues (HGF), and mouth epithelial carcinoma cells (KB).
Results:  The KB cells were more sensitive to genotoxic stress response than oral primary cells. Endogenous levels of Chk1/2 in KB cell were higher than in pulp cells. When doxorubicin was administered, Chk2 activation was induced in KB cell, but not in pulp cells. However, viability in KB cells did not decrease by the suppression of the checkpoint proteins, whereas primary cells were defective in gene silencing. When doxorubicin treatment and gene silencing were combined, both primary cells and KB cell were defective. Moreover, in case of KB cell, cell death was increased and activation of Chk2 was increased in doxorubicin dose-dependent.
Conclusion:  These data indicate that not only stress response mechanism may be different in oral primary and cancer cells but also Chk1/2 proteins may have essential roles in oral primary cells. Based on these data, checkpoint proteins may be crucial drug targets for oral cancer therapy.     

美白牙膏去除人离体牙咖啡及茶渍效果的比较研究

目的研究一种含过氧化物的美白牙膏对咖啡及茶渍色素去除的效果及差异。 方法选择2018年9月至2019年5月北京大学口腔医院综合科因正畸需要新鲜拔除的完整人双尖牙20颗,采用随机数字表法分为2组（咖啡溶液浸泡组与红茶溶液浸泡组）,每组10颗。美白牙膏配制为牙膏溶液,使用200 g压力,100次/分钟频率,140次作为1个刷牙周期（相当于刷牙1周）,连续刷牙8周。应用Crystaleye分光光度比色仪进行颜色测定。表面颜色测定选取的时间点为：基线、染色后及每个刷牙周期后。计算染色后及每个刷牙周期后与基线值之间,国际照明委员会（CIE）颜色的明度值之差ΔL_0-8*。各个样本染色后及每个刷牙周期后颜色与基线颜色之间的色差用ΔE_0-8表示,各组不同测量点组内差异应用配对t检验进行统计分析。各组组间差异应用ANOVA方差分析进行统计,P<0.05为差异有统计学意义。 结果咖啡溶液浸泡组及红茶溶液浸泡组,随着美白牙膏刷牙时间的延长,ΔE值较染色后逐渐降低。但直至美白牙膏刷牙第8周,仍无法恢复到基线水平。咖啡溶液浸泡组美白牙膏刷牙第1周起,牙齿颜色变化ΔE₁（5.2 ± 1.4）较染色后ΔE₀（6.1 ± 1.3）差异有统计学意义（t= 9.1,P<0.001）。红茶溶液浸泡组美白牙膏刷牙第4周起,牙齿颜色变化ΔE₄（5.6 ± 1.5）较染色后ΔE₀（6.4 ± 1.1）差异有统计学意义（t= 2.7,P= 0.025）。咖啡溶液浸泡组及红茶溶液浸泡组,离体牙样本明度值较基线值明显降低,从美白牙膏刷牙第1周起,ΔL₁*（-4.3 ± 0.9;-4.2 ± 1.0）较染色后ΔL₀*（-5.3 ± 0.9;-6.1 ± 1.2）差异有统计学意义（t_ΔL1*=-9.4,P_ΔL1*<0.001;t_ΔL0*=-12.1,P_ΔL0*<0.001）。 结论美白牙膏能够在一定程度上去除咖啡及红茶造成的牙面着色。其改善效果与使用时间成正相关。对于不同着色类型,美白牙膏去除外源性色素的效果不尽相同。     

Clinical and microbiological evaluation of the use of toothpaste containing 1% chlorhexidine and the influence of motivation on oral hygiene in patients with motor deficiency

Patients with motor deficiency have variable difficulties with mechanical plaque control, and as a consequence, the incidence of dental caries and periodontal disease can be higher in these patients. The objective of this study was to evaluate the clinical and microbiological efficacy of a toothpaste containing 1% chlorhexidine, which was used by patients with motor deficiency for 14 days. The reduction in plaque and gingival index and the impact on salivary microorganisms was evaluated. We conclude that the motivation of caregivers to carry out oral hygiene for patients with mental and motor deficiency is of great importance and is effective in reducing the formation of plaque as long as it is continuously reinforced. The use of chlorhexidine‐containing toothpaste significantly reduced the plaque index and microorganism count between days 0 and 14. A reduction was also observed in the group that used a dentifrice without the chlorhexidine, but this difference was not significant.     

KGF对口腔黏膜上皮细胞中增殖相关基因PCNAmRNA的作用

目的：检测不同浓度角质细胞生长因子（KGF）对体外培养的口腔黏膜上皮细胞形态学及对上皮细胞增殖相关基因PCNAmRNA表达的影响。方法：体外培养的口腔黏膜上皮细胞加入含不同浓度KGF（0ng／mL,5ng／rnL,25ng／mL,50ng／mL）的D—KFSM,分别培养12h、24h、48h后观察细胞形态改变并用荧光实时定量检测各组细胞内增殖相关基因PCNAmRNA的表达。结果：①相同时间段实验组较对照组上皮细胞贴壁明显,培养48h实验3组（50ng／mL）较其他组细胞核仁明显;②12h时,实验组较对照组上皮细胞内PCNAmRNA表达增加,但各实验组间PCNAmRNA表达逐渐降低（P〈0．05）;③24h时实验组较对照组PCNAmRNA表达增加,但各实验组间无统计学差异护〉0．05）：④48h时,实验组较对照组PCNAmRNA表达增加,且呈剂量依赖性（P〈0．05）。结论：外源性KGF可上调口腔黏膜上皮细胞增殖相关基因PCNAmRNA的表达,且在不同时间段、不同浓度调控作用存在差异。     

Comparison of the effect of toothpastes containing enzymes or antimicrobial compounds with a conventional fluoride toothpaste on the development of plaque and gingivitis

Many toothpastes have been formulated over recent years to contain antimicrobial compounds with the aim of preventing or reducing plaque, calculus, gingival inflammation or dental caries. For many, if not all of these toothpastes, it has yet to be proven whether they are significantly better at reducing plaque and gingivitis than conventional toothpastes, for which no such therapeutic effects have been claimed. This 12-day, incomplete block designed, cross-over study compared the development of plaque and gingivitis following rinsing with toothpaste slurries containing the following active ingredients: (1) hexetidine/zinc citrate, (2) 0.2% triclosan, (3) amyloglucosidase/glucose oxidase, (4) sodium fluoride/sodium monofluorophosphate (NaF, MFP). By the 8th day of the study, a significant difference in gingival crevicular fluid (GCF) and GI was found between the groups. By day 12, however, no significant difference in plaque index and gingival inflammation was found between the 4 toothpastes, although plaque area was significantly reduced with the hexetidine/zinc citrate paste when compared to the conventional fluoride paste. It was concluded that the active ingredients added to the toothpastes evaluated in this study provided little or no more additional benefit to oral hygiene and gingival health than could be achieved with a conventional fluoride toothpaste.     

角质细胞生长因子对口腔黏膜上皮细胞凋亡的作用研究

目的 研究不同浓度角质细胞生长因子（KGF）对口腔黏膜上皮细胞凋亡的作用,为探讨KGF在口腔黏膜病发生发展中的作用提供依据。方法 将不同浓度的KGF（对照组0 ng·mL-1,实验1组5 ng·mL-1,实验2组25 ng·mL-1,实验3组50 ng·mL-1）分别加入体外培养的口腔黏膜上皮细胞,培养12、24、48 h后,倒置显微镜下观察其对细胞形态的影响,并用流式细胞仪检测细胞凋亡情况,荧光实时定量检测细胞凋亡相关基因Bcl-2、Bax mRNA的表达水平。结果 1）实验组较对照组细胞贴壁明显,且48 h时实验3组细胞核仁明显。2）培养48 h时,4组之间的细胞凋亡率、Bcl-2 mRNA、Bax mRNA表达均有统计学差异,随着KGF浓度的增加,细胞凋亡率和Bax mRNA表达逐渐降低,Bcl-2 mRNA表达逐渐升高（P<0.05）。结论 KGF可通过上调Bcl-2 mRNA和下调Bax mRNA的表达抑制上皮细胞的凋亡。     

Histopathologic and ultrastructural studies of oral mucosa with Candida infection

Eighteen oral mucosal biopsies with Candida infection were studied with light and electron microscopy. Under light microscopy, candidal infected oral mucosa was classified with epithelial hyperplasia, 15 cases and epithelial dysplasia, three cases. Four of 15 epithelial hyperplasias showed marked parakeratosis, and high grade acanthosis with many eosinophilic cells in the spinous cell layers. Epithelial dysplasia was characterized by atrophy of the spinous cell layers and increased nucleocytoplasmic ratio in the basal cell layers. Ultrastructurally, candidal infected oral mucosa showed numerous small desmosomes and the interdigitation of cytoplasmic membranes between spinous cells in both epithelial hyperplasia and epithelial dysplasia. Moreover, eosinophilic spinous cells, observed predominantly in epithelial hyperplasia showed intricate arrangement of dense tonofibrils. These ultrastructural findings seemed to give rise to mechanical strength between spinous cells in oral mucous epithelium with Candida infection. Results in this study suggest that excessive hyperplasia of candidal infected oral mucosa might be a protective reaction to the invasion of candidal pseudohyphae, but not associated with precancerous conditions.     

Dentine hypersensitivity: the effects of brushing toothpaste on etched and unetched dentine in vitro

Although many people have exposed dentine, only a percentage exhibit symptoms of dentine hypersensitivity. This has been ascribed to opening and closing of the dentinal tubules by for example, smear layer changes or tubular occlusion. The aims of this study were to examine the surface morphological changes of etched and unetched dentine in vitro, attributed to the effects of toothbrushing with and without toothpastes designed for the alleviation of dentine hypersensitivity. A total of 96 etched and 96 unetched human dentine specimens were brushed with various toothpastes and water for 1, 2, 5 or 10-min periods in a toothbrushing machine and subsequently examined under scanning electron microscopy for surface changes. Analyses of brushed etched specimens demonstrated that time and treatment were significant variables (P < 0.05) for tubule occlusion. Further, the interaction between time and treatment was significant (P < 0.05). The artificial silica based paste was significantly better for all time intervals at occluding the dentine tubules. All toothpastes investigated caused dynamic changes to the smear layer of the unetched dentine, opening tubules. However, the artificial silica based paste resulted in occluded rather than patent tubules. All of the pastes evaluated had the capacity to remove the smear layer but some could then occlude tubules through the contained abrasives.     

Isolation of oral epithelial progenitors using collagen IV

Objective:  Although oral mucosal epithelial stem cells are thought to reside in the basal layer, such cells have not yet been isolated. We isolated a population of rabbit oral epithelial progenitor cells containing putative stem cells.
Materials and methods:  Epithelial cells harvested from rabbit buccal mucosa were allowed to adhere to dishes coated with collagen IV for periods ranging from 10 min to 16 h. The properties of individual cell populations were evaluated using BrdU, Ki-67, integrin β 1, integrin α 6 and keratin 13 using colony forming efficiency (CFE).
Results:  Cells that adhered to collagen IV-coated dishes within 10 min were enriched about sixfold in terms of BrdU incorporation, Ki-67, integrin α 6 and integrin β 1 were strongly expressed. Interestingly, keratin 13 was faintly expressed. The CFE of rapidly adherent cells among oral epithelial cells was significant compared with other cell populations.
Conclusions:  These results suggested that rabbit oral epithelial cells could be isolated by depending on adhesiveness to collagen IV, especially when segregated according to progenitor cell properties. Putative progenitor cells with stem cell properties were most effectively harvested within 10 min. Our separation procedure should be a useful tool with which to isolate epithelial stem cells for regenerative medicine.     

体外培养鼠口腔黏膜干细胞的超微结构改变

目的：观察体外培养鼠151腔黏膜干细胞超微结构的变化。方法：双重酶消化法分离、培养昆明小鼠口腔黏膜上皮干细胞，光镜下观察干细胞生长及分化特征，透射电镜鉴定细胞的超微结构。结果：鼠口腔黏膜干细胞初始保持未分化的克隆样生长，14d后分化成3种类型细胞，分别代表细胞不同的分化状态。光镜下细胞主要分为两种：一种细胞小而折光性强，另一种细胞大而折光性弱。电镜下，单个细胞的胞质中和满张力原纤维，细胞间形成桥粒结构，符合上皮细胞超微结构特征，可见3种细胞形态：一种细胞胞核大胞质少，一种细胞胞核小胞质多，另有少量细胞的核浓染、固缩。结论：光镜下观察到的小而折光性强的细胞和电镜下观察到的胞核大胞质少的细胞为口腔黏膜干细胞，通过对细胞超微结构的分析将有助于口腔黏膜干细胞的鉴定。