Histochemical assessment on osteoclasts in long bones of toll-like receptor 2 (TLR2) deficient mice

ObjectiveToll-like receptor 2 (TLR2), recognizes a wide variety of pathogen-associated molecular patterns such as lipopolysaccharides, peptidoglycans, and lipopeptides, and is generally believed to be present in monocytes, macrophages, dendritic cells, and vascular endothelial cells. However, no histological examination of osteoclasts, which differentiate from precursors common to macrophages/monocytes, has been performed in a non-infected state of TLR2 deficiency. The objective of this study was to examine the histological properties and function of osteoclasts in the long bones of 8-week-old male TLR2 deficient (TLR2^−/−) mice to gain insight into TLR2 function in biological circumstances without microbial infection.MethodsEight-week-old male wild-type and TLR2^−/− mice were fixed with paraformaldehyde solution, and their tibiae and femora were used for micro-CT analysis, immunohistochemistry, transmission electron microscopy, and real-time PCR analysis.ResultsTLR2^−/− tibiae and femora exhibited increased bone volume of metaphyseal trabeculae and elevated numbers of TRAP-positive osteoclasts. However, the number of multinucleated TRAP-positive osteoclasts was reduced, whereas mononuclear TRAP-positive cells increased, despite the high expression levels of Dc-Stamp and Oc-Stamp. Although TRAP-positive multinucleated and mononuclear osteoclasts showed the immunoreactivity and elevated expression of RANK and siglec-15, they revealed weak cathepsin K-positivity and less incorporation of the mineralized bone matrix, and often missing ruffled borders. It seemed likely that, despite the increased numbers, TLR2^−/− osteoclasts reduced cell fusion and bone resorption activity.ConclusionIt seems likely that even without bacterial infection, TLR2 might participate in cell fusion and subsequent bone resorption of osteoclasts.     

Proteolytic activities in the supragingival plaque of monkeys (Macaca fascicularis)

Thirteen peptidase substrates were hydrolysed by this plaque. After fasting the monkeys for 24 h, the rates of hydrolysis of N-leucyl-, N-alanyl-, N-isoleucyl- and N-tyrosyl-2-naphthylamines, glycyl-L-proline 4-nitroanilide, N alpha-benzoyl-DL-arginine-2-naphthylamine and L-leucyglycine were significantly increased. Changes in these peptidase activities may serve to enhance the mobilization of amino acids and peptides, and they may explain why the growth rates of dental-plaque bacteria in vivo are generally unaffected by the availability of the host's diet.     

Relation between the presence of supragingival calculus and protease activity in dental plaque

Protease activity was measured in dental plaque collected from patients with or without supragingival calculus. Plaque specimens from the calculus group showed significantly greater protease activity in the presence of 0.05% sodium thioglycollate than did those from the non-calculus group. No significant difference between the two groups was observed in the protease activity without sodium thioglycollate. There was no significant correlation between the age of the patients and the protease activity.     

Expression of toll-like receptor 2 and 4 in dental pulp

Toll-like receptors (TLRs) are important factors in innate immune responses because they mediate signals from bacterial cell wall components during inflammatory reactions. However, the role of TLR in dental pulp, which is bounded by hard tissues, is little understood. The present study investigated the expression of TLR-2 and TLR-4 in experimentally inflamed pulp by quantitative real-time polymerase chain reaction and immunohistochemistry. Total RNA isolated from pulp tissue from 0 to 72 hours after bacterial dentinal infection. The TLR-2 messenger RNA (mRNA) level was 30-fold higher than the TLR-4 mRNA level at 9 hours. The TLR-2 mRNA level in pulp began to increase by 3 hours after bacterial infection, reaching a maximum level after 9 hours and gradually decreasing from 9 to 72 hours. Numerous TLR-2- and CD64-positive cells detected on macrophage and dendritic-like cells, TLR-4-positive cells detected a little in the pulp at 9 hours. These results suggest that TLR-2 may be mainly regulated during the early stage of pulp inflammation triggered by bacterial infection.     

炎症信号受体TLR4在大鼠正常牙髓和牙周组织中的分布及意义

目的:研究大鼠正常牙髓和牙周组织中炎症信号受体TLR4表达特点,探讨其在健康牙髓及牙周组织天然免疫防御中的可能作用.方法:采用石蜡及冰冻切片和免疫组化技术检测大鼠健康牙髓及牙周组织中TLR4的表达情况.结果:正常牙髓组织中成牙本质细胞、血管内皮细胞及参与先天免疫的巨噬细胞、树突状细胞、中性粒细胞等细胞TLR4免疫反应阳性,牙髓成纤维细胞不表达TLR4.TLR4表达于牙周结缔组织,可见与上皮层临近的固有层结缔组织强阳性染色.结论:TLR4可能参与了牙髓牙周组织的先天免疫反应,在牙髓牙周组织抵御外来病原微生物入侵中可能起重要作用.     

Evaluation of gingival suppuration and supragingival plaque following 4 modalities of periodontal therapy

This study evaluated the effect of coronal scaling (CS), root planing (RP), modified Widman surgery (MW) and flap with osseous resectional surgery (FO) upon the presence of gingival suppuration and supragingival plaque. 75 patients completed split-mouth therapy and 2 years of maintenance care. Data were collected prior to the initiation of therapy, following initial therapy, following surgical therapy and yearly during 2 years of maintenance care. All 4 types of therapy reduced the prevalence of suppuration with RP, MW and FO producing a greater reduction than CS in sites greater than or equal to 5 mm. Sites were grouped according to presence of suppuration at 2 consecutive examinations. The mean changes in probing depth and probing attachment level for each time period were compared. Sites that began to suppurate between 2 exams or were suppurating at both exams had a less favorable response in mean probing depth reduction and mean probing attachment gain when compared to sites that stopped suppurating between exams or did not suppurate at either exam. The prevalence of supragingival plaque decreased during active therapy and 2 years of maintenance. There was no difference in the prevalence between the therapy groups except for FO-treated sites showing more plaque accumulation after surgical therapy. The presence or absence of supragingival plaque at specific sites was dynamic, frequently converting to a new status between 2 examinations.     

甲苯胺蓝为光敏剂的光动力疗法对龈上菌斑灭菌效果的研究

目的:比较不同浓度甲苯胺蓝对龈上菌斑的灭菌效果.方法:从门诊20例牙周病病人下前牙区取龈上菌斑一份,放入10mL生理盐水中振荡均匀,在2 h内送到微生物室,用微量移液器取25 ul放在琼脂培养皿中,在37℃恒温箱中培养24 h后,分别滴人0.01,0.1,0.5,1.0,2.5,5.0 ms/mL甲苯胺蓝,用12 J/cm2,159mW/em2波长为630衄的二极管激光照射57 s后,观察比较各种浓度的灭菌效果.结果:在相同的光波长、光剂量、光强度和光照时间下,1.0mg/mL的甲苯胺蓝的灭菌效果最好,灭菌率为79%.结论:以甲苯胺蓝为光敏剂的光动力疗法对龈上菌斑有灭菌作用.     

Comparative effects of 2 mouthrinses on the development of supragingival dental plaque and gingivitis

A 6-month double-blind, controlled clinical study was completed with 124 healthy adult subjects to determine the efficacy of 2 mouthrinses, Listerine (LA) and Peridex (PX), used as supplements to regular oral hygiene measures in reducing supragingival dental plaque and gingivitis. Following screening examinations for entry levels of existing gingivitis and plaque, baseline gingival and plaque area indices, extrinsic tooth stain, supragingival calculus, bleeding and soft tissue condition were recorded. All subjects then received a complete dental prophylaxis to remove plaque, calculus and extrinsic stain. Subjects were randomly assigned to 1 of 3 groups and performed supervised rinses twice daily for 30 s in addition to their normal oral hygiene, for 6 months. All indices were again evaluated at 3 and 6 months. After 6 months, LA and PX significantly (p less than 0.001) inhibited development of plaque by 36.1% and 50.3%, respectively, and the development of gingivitis by 35.9% and 30.5%, respectively, compared to a hydroalcohol control. PX was more effective in inhibiting plaque and both mouthrinses appeared to be equally effective in inhibiting gingivitis. LA patients did not develop significant levels of stain or supragingival calculus at 6 months, compared to baseline or control. PX patients developed significant levels of extrinsic stain and supragingival calculus compared to baseline and control. Though PX was more effective than LA in the control of plaque, this study indicates that both LA and PX were effective agents in a regimen for the control of plaque and gingivitis.     

Microsatellite GT polymorphism in intron 2 of human Toll-like receptor (TLR) 2 gene and susceptibility to periodontitis

As pattern recognition receptor, Toll-like receptor (TLR) 2 is a signal molecule essential for the cellular response to Porphyromonas gingivalis. A recently described guanine-thymine (GT) repeat microsatellite polymorphism of the human TLR2 gene leads to impaired promotor activity. A total of 380 patients with chronic periodontitis and 590 unrelated healthy control individuals of Caucasian descent were genotyped for the GT repeat microsatellite polymorphism of the TLR2 gene. The mutations were identified with polymerase chain reaction followed by sizing of GT repeat length. The number of GT repeats ranged from 13 to 29 in both study groups. There was no significant difference between periodontitis patients and healthy controls regarding the frequency of GT repeats (p = 0.365). Subclassification of alleles into three subclasses (S allele, M allele, and L allele) revealed no significant association (p = 0.810). Moreover, also the comparison of the summarized prevalence of S and L alleles (high promotor activity) with that of the M allele (low promotor activity) was not significantly different among study groups (p = 0.377). The susceptibility to chronic peridontitis was not associated with the functional effective GT repeat microsatellite polymorphism in the human TLR2 gene.     

Effectiveness of a new toothbrush with irrigation and suctioning capabilities (ty e-Brush) for removing supragingival plaque

Patients with dysphagia typically have poor oral health. Because of improper swallowing, they cannot easily and safely clean their mouths. As a solution for such a problem, a manual toothbrush with both irrigation and suctioning functions has been developed, called the "e-Brush". The purpose of this study was to evaluate the cleaning effectiveness of the new e-Brush (9 mm and 11 mm in bristle length) for removing supragingival plaque, compared with a conventional toothbrush, GUM # 211 by Butler. In this study, 12 subjects (12 female of average age 20.6) were selected, and plaque control record (PCR) and scrubbing method were used. The following results were obtained: 1. Significant differences (p < 0.05) were recognized between e-Brush/9 mm (55.54 +/- 18.27%) and the others (e-Brush/11 mm: 30.88 +/- 8.14%, GUM # 211: 35.42 +/- 9.32%). 2. Bristles 9 mm in length were more effective than 11 mm bristles (p < 0.05). 3. Irrigation/suctioning function is more effective than the conventional tooth-brushing method. 4. The irrigation function of e-Brush was meritorious in making almost all users comfortable. These results suggest that this new oral hygiene device, "e-Brush/9 mm", is effective for improving oral care management for patients with dysphagia.     

Relationship of gingival bleeding, gingival suppuration, and supragingival plaque to attachment loss

This study evaluated the relationship between the presence of gingival bleeding, gingival suppuration, and supragingival plaque at 3 month appointments to the incidence of probing attachment loss during a 2-year period of maintenance therapy. The data included in this report were taken during the second and third year of maintenance from 75 periodontal patients who had previously received active therapy in an ongoing longitudinal study. The diagnostic sensitivity, specificity, and positive and negative predictive values were calculated for different frequencies of positive responses for each clinical parameter in relation to sites demonstrating greater than or equal to 2 mm probing attachment loss. Gingival bleeding and plaque were not prognosticators and gingival suppuration was a weak prognosticator of attachment loss during a 2 year maintenance period.     

Streptococci and activities of sucrases and alpha-amylases in supragingival dental plaque and saliva in three caries activity groups

Thirty-eight young adults participated in the study. They were divided in a caries-inactive group, a low caries activity group and a moderate to high caries activity group. Total cultivable bacteria, Streptococcus salivarius, and S. mutans in plaque and saliva were quantitated on TSA, MS, and MSB plates, respectively. Sucrase activity was determined by measuring reducing sugars in plaque and saliva after incubation with sucrose. alpha-Amylase activity was determined by Pharmacia Phadebas Amylase test. The data were analyzed with the non-parametric Mann-Whitney U test. The only significant difference was observed for plaque alpha-amylase activity between the caries-inactive group and the moderate-high caries activity group (P less than 0.05). The lack of differences concerning the other variables is discussed mainly on the basis of the multifactorial character of dental caries and the possible insufficiency of the applied methods.     

Comparison of 2 toothbrushing methods for efficacy in supragingival plaque removal The Toothpick method and the Bass method

Abstract. Plaque removing efficacy was compared between a new toothbrushing method named the Toothpick method and the Bass method, 20 male university dental students participated in the study, which consisted of 2 experiments. Prior to each experiment, all subjects were asked to abstain from all oral hygiene procedures for 48 h. In Exp. I, plaque-removing efficacy was assessed after each participant's mouth had been professionally brushed by a dentist. In this experiment, 2 quadrants were brushed professionally using the Toothpick method, and the remaining 2 quadrants were brushed using the Bass method. The brushing time amounted to a total of 2 min per mouth. In Exp. II, the subjects brushed their own teeth after receiving toothbrushing instruction. A blind examiner assessed dental plaque using a modification of the Quigley and Hein index at 6 sites around each tooth, Toothbrushing using the Toothpick method was found to remove significantly more plaque from proximal tooth surfaces than brushing using the Bass method.     

低氧促进口腔鳞癌细胞TLR3及TLR4表达的相关研究

目的　　检测常氧培养条件下口腔鳞癌细胞HSC3中Toll样受体3、4（Toll like receptor 3,4,TLR3、TLR4）的表达水平,并进一步探讨低氧微环境对其表达可能的影响。方法　常氧条件下,将口腔鳞癌细胞HSC3培养至指数增长期,收集细胞,采用Real-time PCR方法在mRNA水平检测TLR3、4的表达,继之,采用Western blotting在蛋白水平检测其表达。模拟体内低氧微环境,在体外采用1% O2浓度分别刺激细胞0、3、6、12及24 h,采用Real-time PCR方法在mRNA水平检测TLR3、4的表达变化,进一步通过Western blotting在蛋白水平检测其表达改变。结果　在常氧培养条件下, HSC3细胞中可检测到TLR3和TLR4的表达。采用低氧刺激可以显著促进TLR3、TLR4的表达水平,其中低氧刺激24 h后,与对照组相比,实验组TLR3的蛋白表达水平增高至(6.2±0.1)倍,而TLR4在低氧刺激6 h后蛋白表达水平可增高至(5.6±0.1)倍,其结果在统计学上具有显著性差异（P＜0.05）。结论　口腔鳞癌细胞HSC3在常氧培养下可检测到TLR3、TLR4的表达,肿瘤低氧微环境可进一步促进其表达水平。     

Hand and ultrasonic instrumentation in the treatment of chronic periodontitis after supragingival plaque control

This study compared the clinical effects of hand or ultrasonic scaling and root planing on the treatment of chronic periodontitis. After supragingival plaque control, twenty patients were examined by a blinded and calibrated examiner for probing pocket depth (PPD), clinical attachment level (CAL) and bleeding on probing (BOP). Experimental teeth were allocated to the following subgingival treatment groups according to PPD: 1) hand instrumentation; 2) hand instrumentation with irrigation; 3) ultrasonic instrumentation; 4) ultrasonic followed by hand instrumentation. Time used in the procedures was recorded. Follow-up examinations were performed at 30 and 90 days after treatment. Each patient's individual BOP, PPD and CAL means were analyzed with repeated-measures ANOVA. Differences in the instrumentation time were analyzed with 1-way ANOVA. Significance level was established at 5%. All treatments produced significant changes in the clinical parameters. BOP reduced, in proximal surfaces, from 67.21-79.17% at baseline to 45.75-51.54% at 90 days. Significant reductions were also found for PPD and CAL in all groups, both in proximal and free surfaces. Reductions in mean PPD at 90 days ranged from 0.92 to 1.14 mm for the free surfaces and from 1.01 to 1.35 mm for proximal surfaces, whereas reductions in CAL ranged from 0.43 to 0.82 mm and from 0.60 to 0.73 mm for free and proximal surfaces, respectively. Mean instrumentation time ranged from 4.77 to 5.30 minutes. No statistically significant differences were found among the four study groups. It can be concluded that the four methods of subgingival instrumentation were equally efficacious in the improvement of the studied clinical parameters.     

不同牙龋病状况患者龈上菌斑及龋损组织微生物多样性分析

目的： 探讨不同龋病状况患者龈上菌斑及龋损组织微生物的组成特点及多样性差异。方法： 随机双盲法选择2019年1月—12月于西安医学院第一附属医院口腔科确诊的龋病患者33例（轻度、中度、重度各11例）,无龋病健康者10例。收集龈上牙菌斑和龋损组织,采用细菌DNA测序方法对细菌16S rRNA-cDNA高可变区进行测序,比较各组微生态种属和相对丰度。采用SPSS 23.0软件包对数据进行统计学分析。结果： 与无龋组相比,龋病组龈上菌斑、龋损组织中微生物含量和丰富度均显著降低（P<0.05）。轻、中、重度组患者龋损组织微生物主要为拟杆菌门、螺旋体门、变形菌门、梭杆菌门、厚壁菌门、放线菌门,各组菌属比例有显著差异（P<0.05）。轻、中、重度组患者龈上菌斑微生物主要有21种,其中,梭杆菌、科里亚细菌、奈瑟菌目、放线菌目、乳杆菌目占比高,其他均小于1%,各组5种菌属比例无显著差异（P>0.05）。结论： 龋病的发生由多菌种所致,且为多种细菌的共同作用。不同龋病状况患者龈上菌斑及龋损组织微生物丰度有显著差异,随着龋病的发生,微生物的多样性下降。     

Relative proportions of pathogen-related oral spirochetes (PROS) and Treponema denticola in supragingival and subgingival plaque from patients with periodontitis.

The purpose of this study was to use monoclonal antibodies to enumerate spirochetes in dental plaque, including the newly recognized pathogen-related oral spirochete (PROS) and specific serovars of Treponema denticola. Plaque was collected from control subjects with no apparent periodontal disease and from sites of moderate to severe chronic periodontitis in patients with inflammatory periodontal disease. Individual monoclonal antibodies were used to determine whether spirochetes were present and then a double-staining protocol was employed to count total spirochetes and specific treponemes in individual microscopic fields. Results indicate that spirochetes are more common at diseased sites and in subgingival plaque than at healthy sites or in supragingival plaque. Together PROS and T. denticola comprised the majority of all spirochetes in all samples and PROS and T. denticola serovars "B" and D were most numerous in plaque from patients with periodontitis. PROS were the majority of all spirochetes in supragingival plaque (76.2% +/- 23.8%) and subgingival plaque (60.9% +/- 19.1%) from periodontitis patients, significantly larger than the percentage of T. denticola serovar "B" (P less than .001 for both supragingival and subgingival plaque) and serovar D (P less than .01 for supragingival and P less than .001 for subgingival plaque). These observations indicate that PROS are the predominant spirochete in plaque from sites of patients with periodontitis, but other analytical approaches are necessary to determine if PROS or T. denticola are pathogenic.     

Toll样受体4对牙周膜细胞增殖的影响

目的：了解Toll样受体4对人牙周膜细胞增殖的影响。方法：以改良组织块法体外获得人牙周膜细胞,取生长良好的第3代细胞随机分为3组：空白对照组、NC对照组（NC－shRNA转染组）和TLR4－shRNA转染组,并于转染48 h 后用倒置荧光显微镜观察转染效果、用荧光实时定量 PCR检测各组细胞中TLR4mRNA的表达水平;最后将3组细胞分别接种于96孔细胞培养板进行培养,分别于培养24、48、72 h用MTT法检测各组细胞的增殖能力。结果：TLR4－shRNA可明显降低人牙周膜细胞的TLR4mRNA表达水平（P＜0．05）;MTT检测结果显示,TLR4－shRNA转染组在常规培养48、72 h的OD值均明显低于空白对照组和NC对照组（P＜0．05）,而NC对照组的OD值在72 h时明显低于空白对照组（P＜0．05）。结论：TLR4对牙周膜细胞的增殖有一定调控作用,转染慢病毒后该作用增强。