激光扫描共焦显微镜活体组织诊断技术的应用进展

激光扫描共焦显微镜作为一种新的无创诊断技术,能对人体体表组织细胞在活体进行观察,相对于常规组织学检查,它不需要标本切取、固定、包埋、切片、染色等繁琐的过程,具有很大的优势。它能在活体组织观察细胞形态和组织结构,甚至在使用增强对照的试剂后,能在分子水平观察细胞的活动。口腔黏膜、体表皮肤由于其特殊的位置及结构,使激光扫描共焦显微镜在这些方面得到广泛的应用。作者对激光扫描共焦显微镜在口腔黏膜、皮肤病变的活体组织诊断以及外科手术中的应用进行综述。     

Effect of cariogenic challenge on the stability of dentin bonds

Objective

The oral environment is subject to biofilm accumulation and cariogenic challenge, and few studies exist on the effect of these factors on the bond strength of adhesive systems. The aim of this study was to test if the exposure of adhesive interfaces to cariogenic challenge under biofilm accumulation could promote higher degradation than the exposure to biofilm accumulation alone.

Material And Methods

Five molars were ground until exposure of medium dentin and then restored (Single Bond 2 and Z250 3M ESPE). The tooth/resin sets were cut to obtain beam-shaped specimens, which were distributed according to the aging conditions (n=20): water for 24 h (control); biofilm under cariogenic challenge for 3, 5 or 10 days; biofilm without cariogenic challenge for 10 days; and water for 3 months. Microcosm biofilms were formed from human saliva and grown in a saliva analogue medium, supplemented or not with sucrose to promote cariogenic challenge. Specimens were tested for microtensile bond strength, and failure modes were classified using light microscopy. Bond strength data were analyzed using ANOVA and failure modes were analyzed using ANOVA on ranks (α=0.05).

Results

No significant differences in bond strength were detected among the aging methods (P=0.248). The aging period was associated with an increase in the frequency of adhesive failures for the groups aged for 10 days or longer (P<0.001).

Conclusion

Aging leads to a higher prevalence of interfacial adhesive failures, although this effect is not associated with cariogenic challenge or reduction in bond strengths.     

变异链球菌荧光报告株在树脂、玻璃离子表面形成生物膜能力的初步研究

目的：本研究通过报告基因技术与激光共聚焦显微镜（Confocal Laser Scanning Microscopy,CLSM）研究变异链球菌荧光报告株（UA140-mrfp）在不同修复材料表面形成生物膜能力,比较树脂与玻璃离子的抗菌性能,为研究双菌、多菌生物膜在材料表面的黏附打下基础,探索CLSM技术在研究生物膜领域中的优势。方法：制作（直径15mm,厚度0.5mm）大小相同的树脂片和玻璃离子片,在其表面形成单菌生物膜,用CLSM分别观察4h、8h、12h、24h生物膜生长情况,在不同位置用CLSM沿Z轴进行扫描,测量其厚度和组织结构。结果：CLSM观察结果显示在不同时段玻璃离子表面形成的生物膜量及其厚度均少于同一时间段树脂表面。结论：变异链球菌荧光报告株在玻璃离子表面形成生物膜的能力低于树脂表面。     

Quantitative analysis of S. mutans and S. sobrinus cultivated independently and adhered to polished orthodontic composite resins

In Orthodontics, fixed appliances placed in the oral cavity are colonized by microorganisms.

Objective

The purpose of this study was to quantitatively determine the independent bacterial colonization of S. mutans and S. sobrinus in orthodontic composite resins.

Material and methods

Seven orthodontic composite adhesives for bonding brackets were selected and classified into 14 groups; (GIm, GIs) Enlight, (GIIm, GIIs) Grengloo, (GIIIm, GIIIs) Kurasper F, (GIVm, GIVs) BeautyOrtho Bond, (GVm, GVs) Transbond CC, (GVIm, GVIs) Turbo Bond II, (GVIIm, GVIIs) Blugloo. 60 blocks of 4x4x1 mm of each orthodontic composite resin were made (total 420 blocks), and gently polished with sand-paper and ultrasonically cleaned. S. mutans and S. sobrinus were independently cultivated. For the quantitative analysis, a radioactive marker was used to codify the bacteria ⁽³H) adhered to the surface of the materials. The blocks were submerged in a solution with microorganisms previously radiolabeled and separated (210 blocks for S. mutans and 210 blocks for S. sobrinus) for 2 hours at 37ºC. Next, the blocks were placed in a combustion system, to capture the residues and measure the radiation. The statistical analysis was calculated with the ANOVA test (Sheffè post-hoc).

Results

Significant differences of bacterial adhesion were found amongst the groups. In the GIm and GIs the significant lowest scores for both microorganisms were shown; in contrast, the values of GVII for both bacteria were significantly the highest.

Conclusions

This study showed that the orthodontic composite resin evaluated in the GIm and GIs, obtained the lowest adherence of S. mutans and S. sobrinus, which may reduce the enamel demineralization and the risk of white spot lesion formation.     

The influence of Brazilian plant extracts on Streptococcus mutans biofilm

Nineteen plant extracts obtained from plants from the Brazilian Amazon showed activity against planktonic Streptococcus mutans, an important bacterium involved in the first steps of biofilm formation and the subsequent initiation of several oral diseases.

Objective

Our goal was to verify whether plant extracts that showed activity against planktonic S. mutans could prevent the organization of or even disrupt a single-species biofilm made by the same bacteria.

Material and Methods

Plant extracts were tested on a single-bacteria biofilm prepared using the Zürich method. Each plant extract was tested at a concentration 5 times higher than its minimum inhibitory concentration (MIC). Discs of hydroxyapatite were submersed overnight in brain-heart infusion broth enriched with saccharose 5%, which provided sufficient time for biofilm formation. The discs were then submersed in extract solutions for one minute, three times per day, for two subsequent days. The discs were then washed with saline three times, at ten seconds each, after each treatment. Supports were allowed to remain in the enriched medium for one additional night. At the end of the process, the bacteria were removed from the discs by vortexing and were counted.

Results

Only two of 19 plant extracts showed activity in the present assay: EB1779, obtained from Dioscorea altissima, and EB1673, obtained from Annona hypoglauca. Although the antibacterial activity of the plant extracts was first observed against planktonic S. mutans, influence over biofilm formation was not necessarily observed in the biofilm model. The present results motivate us to find new natural products to be used in dentistry.     

扫描同聚焦显微镜观察血链菌和粘性放线菌生物膜的形成

目的：观察血链菌和粘放菌体外形成生物膜的动态过程。方法：采用同聚焦显微镜技术,原位、立体、动态观察血链菌和粘放菌体外形成的生物膜。结果：生物膜形成8h厚度达15．4μm,细菌已聚集成簇,菌细胞分布是表层和基底较少,中间层较厚并较密。生物膜形成16h后已基本成熟,细菌成簇厚度增加达34．3μm.48h后生物膜已成熟成谷穗状。结论：血链菌和粘放菌具有相互作用促进体外形成生物膜。CLSM观察生物膜有其独到优越性。     

细菌素免疫蛋白调控变形链球菌抗菌敏感性及生物膜形成的实验研究

目的 观察细菌素免疫蛋白相关基因对变形链球菌抗菌敏感性及生物膜形成的影响,探讨细菌素免疫蛋白与细菌抗菌剂耐受性的关系,为生物膜抗菌敏感性的研究提供基础数据.方法筛选培养细菌素免疫蛋白基因突变株,绘制生长曲线.酶标仪检测不同质量浓度氨苄青霉素(0.04、0.05、0.06、0.07及0.08 mg/L)、氟化钠(50、100、150、200及250 mg/L)及不同质量分数的次氯酸钠(0.078%、0.156%、0.313%、0.625%及1.250%)作用下变形链球菌标准株、△immA-和△immB-突变株菌液的吸光度值.应用最小生物膜清除浓度(minimal biofilm eradicatin concentration,MBEC)桩钉96孔板以连续稀释法检测醋酸氯己定对3种菌株生物膜的MBEC.应用激光共聚焦扫描显微镜(confocal laser scanning microscope,CLSM)定量分析标准株和突变株生物膜结构.结果 △immA-和△immB-突变株的迟缓期和稳定生长期均比标准株延时1 h.氨苄青霉素为0.06 mg/L时,标准株、△immA-突变株和△immB-突变株菌液吸光度值分别为0.334±0.016、0.027±0.016及0.047±0.018;氟化钠质量浓度为150 mg/L时,3种菌株菌液吸光度值分别为0.254±0.018、0.129±0.011及0.167±0.01;当次氯酸钠质量分数为0.313%时,3种菌株菌液吸光度值分别为0.467±0.008、0.017±0.006及0.050±0.006,以上各组抗菌剂中,标准株与突变株吸光度值差异均有统计学意义(P<0.01).醋酸氯己定对3种菌株的MBEC分别为6.25、1.57及3.13 mg/L.标准株生物膜厚度显著高于△immA-和△immB-突变株(P<0.01);标准株各层活菌比例均高于△immA-突变株(P<0.05);标准株中、外层活菌比例高于△immB-突变株(P<0.01),但内层活菌比例差异无统计学意义(P=0.191).结论细菌素免疫蛋白参与调控浮游细菌生长,尤其在生长初期;细菌素免疫蛋白相关基因缺陷使浮游态变形链球菌抗菌敏感性提高、抗菌剂MBEC降低及生物膜结构不成熟.     

改良恒化器中牙周致病菌和致龋菌的激光共聚焦显微镜动态观察

目的　模拟口腔条件下研究牙周致病菌和致龋菌的动态关系。方法　选用牙龈卟啉单胞菌、伴放线放线杆菌、具核梭杆菌、中间普氏菌、变形链球菌、血链球菌、黏性放线菌和嗜酸乳杆菌8种牙周致病菌和致龋菌。按分组接种于模拟口腔的改良恒化器中 ,连续培养 1、2 4、4 8和 96h ,活菌革兰荧光染色与激光共聚焦显微镜结合测量羟基磷灰石圆片表面生物膜厚度 ,连续断层扫描及三维重建。结果　随时间变化各组生物膜厚度均显著增加 (P <0 0 0 1) ;同一时间点血链球菌生物膜明显厚于伴放线放线杆菌 ,8种菌明显比血链球菌和伴放线放线杆菌形成的生物膜厚。三维重建显示 ,G-牙周致病菌主要分布于G 致龋菌菌团中或膜表层。结论　人工菌斑生物膜中G 致龋菌首先定植 ,G -牙周致病菌的量和比例随时间增加 ;牙周致病菌和致龋菌的生态平衡与疾病之间的关系值得深入研究     

Effects of recombinase A deficiency on biofilm formation by Streptococcus mutans

Background/aim: Recombinase A (RecA) is essential for the transformation of both plasmid and chromosomal DNA in Streptococcus pneumoniae and is considered to be related to the SOS‐response in Streptococcus mutans. Methods: In the present study, a RecA‐deficient mutant strain (RAD) was constructed by insertional inactivation of the recA gene encoding the RecA protein in strain MT8148 of S. mutans, after which the biological functions of acid tolerance and biofilm formation were investigated. Results: RAD showed reduced acid tolerance and produced lower density biofilm compared with the wild‐type strain. In addition, confocal microscopic observation indicated that the biofilm produced by RAD was composed of cells with significantly lower viability compared with that produced by strain MT8148. Conclusion: These results suggest that RecA has a relationship with biofilm formation.     

变形链球菌luxS基因突变对口腔混合细菌生物膜的影响

目的 研究致龋菌变形链球菌luxS基因在口腔细菌混合培养形成牙菌斑生物膜中的作用。方法 将变形链球菌野生株(UA159)及其2种luxS基因突变株（luxS基因高表达株和luxS基因缺陷株）分别与口腔细菌嗜酸乳杆菌（ATCC4356）按照1∶1比例接种于牛心脑浸液培养基,体外混合培养不同时间,包括生物膜形成过程中的初期（4 h）、中期（14 h）、晚期（24 h）,通过MTT法检测混合菌在生物膜形成的量。通过激光共聚焦显微镜观察混合细菌24 h形成的生物膜结构,实时定量PCR检测变形链球菌相关基因（ftf, smu630, brpA, gbpB, gtfB, vicR, comDE和relA）的表达。采用SPSS17.0软件包对数据进行统计学分析。结果 变形链球菌野生株及其2种luxS基因突变株与嗜酸乳杆菌混合培养14 h后,生物膜的量分别为0.481±0.024、0.591±0.023和0.279±0.019;24 h后,混合细菌形成生物膜的量趋势与该时间点一致,变形链球菌高表达株高于野生株,而缺陷株明显降低;但4 h后形成的生物膜组间无显著差异。激光共聚焦显微镜结果表明,高表达株和野生株的集聚程度更高,形成生物膜的结构更加紧密;而缺陷株生物膜菌间结构比较稀疏。以变形链球菌野生株和嗜酸乳杆菌混合形成的生物膜中相关基因的表达为标准,高表达株相关基因的表达均增加,缺陷株表达均降低,且各组间存在显著差异（P<0.05）。结论 变形链球菌luxS基因影响与具核梭杆菌混合培养形成的牙菌斑生物膜,为进一步研究该基因在生物膜中的作用及其调控机制提供了依据。     

变形链球菌luxS基因缺失对生物膜早期形成的影响

目的 利用变形链球菌luxS基因敲除突变株,研究这一种属间密度感应系统对生物膜早期形成的影响.方法通过在生物膜培养悬液中加入与菌细胞直径相近的磁性小珠,利用这些小珠在磁场中受到生物膜的位移约束力的原理,采用生物膜定量分析仪,定量比较、分析变形链球菌luxS基因knockout突变株与野生株在生物膜形成上的差异.结果变形链球菌luxS基因突变株与野生株在生物膜形成模式上有显著差别,突变株生物膜自第6小时起开始形成,生物膜形成指数(biofilm index,BFI)差值ABFI=2.015,约第10小时突变株形成的生物膜可完全限制磁珠在磁场中的位移(ABFI=7.025);而野生株生物膜约第10小时开始形成(ABFI=1.875),明显晚于突变株.12h后两菌株生物膜形成未见明显差异(P>0.05).结论变形链球菌luxS基因的缺失可影响生物膜的早期形成.     

十肽对变异链球菌生物膜生长和结构影响的实验研究

目的：评价人工合成抗菌肽（十肽）对口腔变异链球菌生物膜生长、结构及活性的影响能力。方法???人工合成十肽（氨基酸序列：KKVVFKVKFK-NH2）通过对细菌生物膜药物最低抑制生物膜形成浓度（MBIC）和药物最低清除已形成生物膜浓度（MBRC）2个指标的测定,研究和评估十肽对变异链球菌单菌生物膜的抑制和清除作用;在激光共聚焦扫描显微镜（CLSM）下观察十肽作用下变异链球菌生物膜的形态、结构及活性变化。结果???当十肽浓度为62.5~125μg?mL-1时,能够抑制变异链球菌生物膜的形成;当十肽浓度为250~500μg?mL-1时,可破坏已形成24?h后的生物膜,CLSM下观察到十肽作用后生物膜结构残存,死菌增多,生物膜厚度明显降低。结论??新型人工合成抗菌肽（十肽）不仅可抑制主要致龋菌变异链球菌单菌生物膜的生长形成,而且可破坏已经形成24?h后的生物膜结构。     

蔗糖环境对寡发酵链球菌与变异链球菌双菌种生物膜形成的影响

目的探讨蔗糖环境对寡发酵链球菌与变异链球菌双菌种生物膜形成的影响,并与血链球菌和变异链球菌的双菌种生物膜形成进行比较。方法运用菌落计数法观察蔗糖环境下唾液包被的玻璃生物模型中单/双菌株寡发酵链球菌,变异链球菌和血链球菌24 h生物膜形成情况;运用激光共聚焦显微镜观察蔗糖环境下寡发酵链球菌,变异链球菌和血链球菌单/双菌株生物模型中24h生物膜形成厚度。结果无糖环境下,单菌株模型中,菌落数:血链球菌(55.67±5.36)>变异链球菌(53.48±2.63)(P>0.05)>寡发酵链球菌(46.24±2.34)(P<0.05);生物膜厚度:血链球菌(17.23±3.82)>变异链球菌(15.16±4.21)(P>0.05)>寡发酵链球菌(10.54±4.37)(P<0.05)。双菌株模型中,变异链球菌菌落数降低幅度:血链球菌组>寡发酵链球菌组(P<0.05)。生物膜厚度:血链球菌组(8.12±2.82)<寡发酵链球菌组(11.27±3.55)(P<0.05)。蔗糖环境下,单菌株模型中,菌落数:变异链球菌(58.54±2.74)>血链球菌(51.87±5.35)>寡发酵链球菌(48.57±3.05)(P<0.05)生物膜厚度:变异链球菌(20.63±5.71)>血链球菌(13.37±4.93)>寡发酵链球菌(12.45±4.62)(P<0.05)双菌株模型中;变异链球菌菌落数降低幅度,寡发酵链球菌组>血链球菌组(P<0.05),生物膜厚度:寡发酵链球菌组(6.67±2.19)<血链球菌组(10.45±2.72)(P<0.05)。结论外界糖环境影响寡发酵链球菌和血链球菌对变异链球菌的抑制作用,蔗糖环境下,寡发酵链球菌的抑制作用强于血链球菌。     

变形链球菌与血链球菌的质子移位膜ATP酶的研究概况

口腔变形链球菌是龋病的主要致病菌,血链球菌为牙周有益菌。口腔致龋菌的致龋能力主要是其产酸性和耐酸性,而ATP酶是耐酸性的决定因素,是变形链球菌等致龋菌的固有毒力因子。本文以大肠杆菌为参照,对变形链球菌和血链球菌ATP酶的基因结构进行比较,探讨口腔致龋菌的质子移位膜ATP酶的结构特性。     

密度感应拮抗剂C-30对变异链球菌生物膜的影响

目的:研究密度感应拮抗剂呋喃C-30对变异链球菌生物膜形成的影响。方法:将已合成的密度感应拮抗剂呋喃C-30用BHI培养基配制至终浓度分别为2.0,4.0μg/mL,再加入新鲜培养的变异链球菌液,37℃微需氧培养24 h,在96孔板上形成体外生物膜,用MTT法检测生物膜的量。激光共聚焦显微镜观察不同浓度呋喃C-30处理后的生物膜结构。实验中以不含呋喃C-30的BHI培养基作为阴性对照。结果:实验发现呋喃C-30不会对变异链球菌的生长产生影响,但随着呋喃C-30的浓度增加,变异链球菌形成生物膜的量显著降低(P<0.05);激光共聚焦显微镜的结果也表明呋喃C-30浓度的增加使生物膜菌间结构变得稀疏,集聚程度明显减轻。结论:密度感应拮抗剂呋喃C-30能有效抑制变异链球菌生物膜的形成。     

几种制剂对口腔主要致龋菌抑制作用的比较

目的 :研究玉洁纯、硫酸锌、茶多酚以及它们相对配伍的制剂应用于龋病预防的可能性。方法 :采用液体稀释法体外抑菌实验比较玉洁纯、硫酸锌、茶多酚以及它们配伍使用时对口腔主要致龋菌S .mutansIngbritt、S .sobrinus　 6 715的抑制效果。结果 :以上三种制剂均能有效地抑制主要致龋菌的生长 ,其中玉洁纯的抑菌效果最为显著 ;硫酸锌和茶多酚都不能有效地促进玉洁纯的抑菌作用。结论 :玉洁纯作为一种高效低毒的抑菌剂可以考虑用于龋病预防 ,硫酸锌对玉洁纯抑菌效果的影响有待进一步研究。     

Analysis of Streptococcus mutans biofilm proteins recognized by salivary immunoglobulin A

Introduction:  The purpose of this study was to examine the Streptococcus mutans biofilm cellular proteins recognized by immunoglobulin A (IgA) in saliva from various caries-defined populations.
Methods:  Biofilm and planktonic S. mutans UA159 cells were prepared. The proteins were extracted, separated by two-dimensional gel electrophoresis, transferred to blotting membranes, and probed for IgA using individual saliva samples from three groups of subjects; those who developed 0 caries (no active caries), 5–9 caries (medium), or more than 10 caries (severe) over a 12-month interval.
Results:  Several proteins were recognized by salivary IgA in all groups of saliva but spot distribution and intensity varied greatly between the groups, and some proteins were recognized more strongly in biofilm cells than in planktonic culture, and vice versa. Furthermore, 15 proteins were only recognized by saliva from the 'no active caries' group, and four proteins were recognized by saliva samples from subjects in all three groups. Specifically, antigen I/II was recognized less in biofilm cells by caries-free saliva compared with planktonic cells. However, salivary IgA antibody to antigen I/II was absent in blots using saliva from the 'medium caries' and 'severe caries' groups.
Conclusion:  The bacterial molecules recognized by caries-free saliva are significant factors for S. mutans caries formation, and their inhibition could be a therapeutic target. In addition, saliva of caries-free subjects includes significant IgA antibody against antigen I/II of S. mutans , indicating a protective mechanism. However, microorganisms may protect themselves from host immune attack by forming biofilms and decreasing expression of antigen I/II.     

新型含氟正畸托槽对变形链球菌抑制性的实验研究

目的研究新型含氟正畸托槽对主要致龋菌变形链球菌生长繁殖的抑制性,探讨新型含氟正畸托槽对正畸固定矫治中牙釉质脱矿的预防作用。方法将新型含氟正畸托槽浸泡在去离子水中,每24h换液1次,留取2-5d的浸泡液,然后将浸泡液与变形链球菌菌悬液同时加入变形链球菌液体培养基中,厌氧培养后,观察菌悬液的光密度OD值变化。再将测过OD值的菌悬液经过固体培养基培养观察菌落数（CFU）。结果2、3、4d所得的含氟正畸托槽24h浸泡液经细菌培养后OD值与对照组相比,OD值减小且差异非常显著,5dOD值与对照组相比也减小,但差异无统计学意义。而菌落数的变化只是2、3d的与对照组相比,数目减少并且差异有统计学意义,4、5d的与对照组相比,数目也减少但差异无统计学意义。结论新型含氟正畸托槽在一定时间内对变形链球菌有抑制作用,其在正畸临床治疗中预防牙釉质脱矿的作用尚待进一步研究。     

A new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy

Objectives

To compare three methods of intratubular contamination that simulate endodontic infections using confocal laser scanning microscopy (CLSM).

Material and Methods

Two pre-existing models of dentinal contamination were used to induce intratubular infection (groups A and B). These methods were modified in an attempt to improve the model (group C). Among the modifications it may be included: specimen contamination for five days, ultrasonic bath with BHI broth after specimen sterilization, use of E. faecalis during the exponential growth phase, greater concentration of inoculum, and two cycles of centrifugation on alternate days with changes of culture media. All specimens were longitudinally sectioned and stained with of LIVE/DEAD^® for 20 min. Specimens were assessed using CLSM, which provided images of the depth of viable bacterial proliferation inside the dentinal tubules. Additionally, three examiners used scores to classify the CLSM images according to the following parameters: homogeneity, density, and depth of the bacterial contamination inside the dentinal tubules. Kruskal-Wallis and Dunn’s tests were used to evaluate the live and dead cells rates, and the scores obtained.

Results

The contamination scores revealed higher contamination levels in group C when compared with groups A and B (p<0.05). No differences were observed between group A and B (p>0.05). The volume of live cells in group C was higher than in groups A and B (p<0.05).

Conclusion

The new protocol for intratubular infection resulted in high and uniform patterns of bacterial contamination and higher cell viability in all specimens when compared with the current methods.     

Extracellular polysaccharides do not inhibit the reaction between Streptococcus mutans and its specific immunoglobulin G (IgG) or penetration of the IgG through S. mutans biofilm

The present study investigated whether extracellular polysaccharides inhibit reaction between Streptococcus mutans and its specific immunoglobulin G (IgG) and penetration of the IgG through S. mutans biofilm. The planktonic organisms with or without extracellular polysaccharides were prepared, incubated with rabbit IgG against whole cell of S. mutans and fluorescein isothiocyanate (FITC)-conjugated goat affinity purified antibody to rabbit IgG. Biofilms with or without extracellular polysaccharides were formed on cover glasses and incubated with rabbit IgG against S. mutans and FITC-conjugated goat antibody to rabbit IgG. Then, biofilms were stained with propidium iodide. The amount of specific IgG binding on S. mutans was determined by FITC intensity with a fluorescence microplate reader. The penetration of IgG through biofilms was determined by confocal laser scanning microscopy. The results showed that the fluorescence intensity of FITC in planktonic organisms with extracellular polysaccharides was similar to that in planktonic organisms without extracellular polysaccharides, indicating that extracellular polysaccharides did not inhibit the reaction between S. mutans and its specific IgG. Although biofilms of S. mutans with extracellular polysaccharides were much thicker and denser than those without extracellular polysaccharides, the speed with which IgG penetrated through both of the biofilms did not differ significantly, suggesting that penetration of IgG through S. mutans biofilm was not affected by extracellular polysaccharides.