The usual appearance of the dorsal surface of the tongue is either pinkish or with a thin white coating. This surface is colonized by large amounts of bacteria, mostly in the presence of fissures, crypts and high mucosal papillae. These anatomical niches create an environmental condition where microorganisms are embedded and well-protected from the flushing action of the saliva. Also, oxygen levels in such environment are low, thus promoting the development of anaerobic microbiota (1). The coating of the tongue consists of a visible white-brownish layer adhering to the dorsum of the tongue and embedding desquamated epithelial cells, blood cells and metabolites, nutrients and bacteria. Indeed, more than 100 bacterial species were found attached to a single epithelial cell on the dorsum of the tongue, whereas only about 25 bacteria adhere to each cell in other areas of the oral cavity (2). Different indexes were developed for quantifying the degree of tongue coating: Miyazaki et al. (3) reported that tongue coating is present or absent in three areas, while no indication of thickness was recorded. Winkel at al. (4) divided the tongue in six areas, scoring each one independently from 0 (no coating) to 1 (light coating) and 2 (heavy coating). The final value of the Winkel Tongue Coating Index has been obtained by adding all six scores. Therefore, the tongue microflora offering a large surface area represents a unique ecological niche in the oral cavity. The dorso-posterior surface of the tongue was reported to harbor a high quantity of attached microbes (109 or 1010 CFU cm2) (5). Oral malodor is a common problem affecting a large percentage of the adult population (6, 7) and volatile sulfur compounds (VSC) are thought to be the most important volatile components.
The surface of the tongue has been described by Maeda (11) as a surface consisting of papillae oriented perpendicular to the tongue plane. Hesse modelled a three dimensional structure of the substratum and biofilm with the first layer consisting of the connective tissue core of the papillae, the second layer having a thin biofilm cover and the last one consisting of a thick biofilm cover. The uppermost layer of the biofilm is considered to be aerated and hence aerobic, whereas deeper layers of the biofilm are anaerobic (12). Hesse (13) reported that in case the forces are applied to the papillae, for example, when scraping the tongue, the papillae bend slightly and protect the biofilm, thus remaining in the interstitial volume. This demonstrates the significant influence of the substratum structure on the stability of the tongue biofilm under mechanical stress. Each papilla itself cannot be seen just as a simple stud sticking out from the surface of the tongue but a cluster of individual strands, as showed by Kobayashi et al. (14). The presence of deep fissures has been related to twice the total counts of bacteria and to significantly higher mouth and tongue odor scores (15), although other authors have failed to confirm the association of higher bacterial counts with increased surface roughness of the tongue (16, 17).
The aim of this research was to correlate the roughness of the tongue with the presence of the tongue coating biofilm in a halitosis patient. A preliminary protocol was designed for this purpose.
Material and Methods
One subject with halitosis was selected and included in the study. A picture of the lingual dorsum was taken to spot the areas where the coating was visible (Figure 1).
The first impression was taken by alginate obtaining an exact replica in plaster and an impression tray was modelled utilizing silicone putty impression material. Subsequently, the second impression was taken combining the tray in silicone putty with a silicone material, thus having a very low-light density (using the 2-step double-mix impression technique) (Figures 2-3).
The obtained impression was divided and cut with a blade in six parts, according to Winkel Tongue Coated Index (18), and their contours were observed with the stereo-microscope LEICA LED2000. The images were analyzed by the Image J software (19), and the depths among papillae were considered to be parameters.
The obtained data were processed by descriptive statistical analysis (Mean, SD, Shapiro-Wilk Normality, and T-Student) using the XLSTAT software (2015.4.01. version).
The stereomicroscope observation revealed some interesting outcomes. The parameter considered was the depth of the fissures on the long and on the short side of each zone (Figures 4-5), and such mean values were compared between the 6 zones. The 6 zones were divided in two groups, based on the visibility of the biofilm on the taken picture.
All of the six zones resulted by following a normal distribution (Figures 6).
The zones A, B and C mean values resulted respectively as 0.25 + 0.05 mm, 0.46 + 0.23 mm and 0.17 + 0.03 mm. These three zones produced those with high visible presence of biofilm on the taken picture.
The zones D, E and F mean values resulted respectively as 0.20 + 0.07 mm, 0.28 + 0.12 mm and 0.14 + 0.05 mm. These three zones produced those with a low visible presence of biofilm on the taken picture. The first group (A-B-C) showed higher values of depth fissures compared to the second group (D-E-F). The difference between the two groups was statistically significant with a p value < 0.05 (Tables 1-2).
|ZONE||Mean Value (mm)||SD||Shapiro-Wilk Normality test (P value)|
|Zone A||0.25||0.05||> 0 .05|
|Zone B||0.46||0.23||> 0 .05|
|Zone C||0.17||0.03||> 0 .05|
|Zone D||0.20||0.07||> 0 .05|
|Zone E||0.28||0.12||> 0 .05|
|Zone F||0.14||0.05||> 0 .05|
The papillary structure of the dorsum represents a unique ecological niche in the oral cavity, offering a large surface area that favors the accumulation of oral debris and microorganisms. In addition, its location as a crossroad between the oral cavity and the pharynges provides access to many different types of nutrients, products and bacteria (20).
It is a well-known fact that dorsal surface of the human tongue includes four distinct types of papillae: filiform, distributed over the dorsum; fungiform, located anteriorly; foliate found in the lateral posterior regions and circumvallate located along the sulcus (21).
The dorsal surface of the tongue is described as a set of various papillae and taste buds. Each papilla itself can be seen as a cluster embedded in a matrix, and the saliva, which in turn is exposed to the mouth gaseous air. This implies a particular oxygenation of the biofilm that is created on this surface: the oxygen level is very low in deep surfaces of the papilla and it is consumed by the metabolisms of the bacteria. Conversely, the level of oxygen is higher at the top of the surface (13).
This structure and its ecological layout favors the harbor of different species of bacteria, and its roughness allows the adhesion and the formation of a biofilm where the bacteria coexist (22).
The surface roughness degree can change due to several factors including age, sex salivary secretion, immunological defense, gastrointestinal disorders (21).
Murayama and Kobayashi reported that the protrusions of filiform papillae and taste pores of fungiform papillae were successfully reproduced by using a low viscous silicone impression material (25).
In 2012, Uemori et al. carried out a study on reliability of the tongue impression in order to determine the roughness of the tongue dorsum and to exploit it as diagnostic tool to quantify the degree of atrophy as well as morphology of the lingual papillae (26).
The results obtained in the present study reveal that the depths of papillae and fissures were expressed quantitatively. Also, the relationship between the coated biofilm found in a halitosis-affected subject and the abovementioned depths was analyzed. The method used in this study appeared to be correct, as confirmed by the cited authors. It allowed us to investigate morphologically the papillary structure and to associate it with a visible biofilm presence.
Our study confirmed the fact that the tongue dorsum morphology offers a unique environmental niche for biofilm coating and adhesion. Thus, the study of its roughness and its papillary structure is fundamental for diagnosis and personalized treatments of pathologies affecting this organ.