The mucosa of the oral cavity is composed of a stratified squamous epithelium and a lamina propria, separated by a basal membrane. The epithelium consists of a proliferative basal layer, a partially differentiated intermediate layer, and a terminally differentiated superficial layer. Epithelial differentiation may also vary according to the region of the mouth. The epithelium can be keratinized, para-keratinized or non-keratinized (1). The surfaces of the mucous membranes are kept hydrated, lubricated and protected by the mucus layer. In addition to capturing microorganisms and particles, the mucus acts as a physical and chemical barrier against external agents and acid secretions from the stomach.
The homeostasis of the oral cavity is preserved by the integrity of mucous membranes lined with mucus, by the constant presence of saliva, by the action of enzymes, and by the production of mucosal immunoglobulins (SIgA). It can often be altered by the action of both internal and external agents, leading to the onset of oral diseases such as mucositis, pharyngitis, and xerostomia (i.e., dry mouth).
Hyaluronic acid (sodium hyaluronate) is a high molecular weight polysaccharide that is widely distributed in body tissues and fluids. It is made of a repeated sequence of D-glucuronic acid and N-acetyl-D-glucosamine, thus belonging to the group of glycosaminoglycans. The molecule is flexible, extremely polar and has a high solubility in water, which is important for the hydration of mucous membranes, and thus for its protective and supportive function. Thanks to its molecular characteristics, this molecule looks like an ideal mucoadhesive polymer (2–5).
Hyaluronic acid also plays a major role in tissue repair and healing, promoting fibrin formation and attracting inflammation mediators to the damaged area. Another important feature of hyaluronate is its viscoelastic property, due to this property it can affect cellular functions leading to changes in the surrounding cells and the extracellular environment. Viscoelastic properties can also slow down the penetration of viruses and bacteria, which is of particular interest in oral diseases (6).
Carbomer is a synthetic polymer that is widely used in controlled drug delivery systems. (7). The molecule contains a high percentage of carboxylic acid groups, allowing it to swell in water and produce a certain viscosity; this characteristic is the basis of its mucoadhesive capacity. The oral hydrogels prepared with carbomer are suitable for dispersing and delivery of minitablets or pellets and then they can be used also in paediatric patients (8).
Xanthan gum is a natural polysaccharide whose use is common in the food industry as a stabilizer and thickener. Recent clinical studies (9) on the preparation of a mucoadhesive gel based on dimenhydrinate (DMH) have shown that the addition of xanthan gum to this compound provides better physicochemical and mechanical properties compared to a previous association with hydroxyethyl cellulose.
The residence time of formulations for the treatment of disease of the oral cavity is largely influenced by the combined effects of salivation, the swallowing reflex, speech, mastication and the passage of food bolus. Consequently, in the last years there has been an increasing interest in specific mucoadhesion, i.e. the adherence of a material to a mucosal surface for a certain period of time, a complex phenomenon which involves wetting, adsorption and interpenetration of polymer chains (10).
Advances in hydrogel, mucoadhesive polymers made from macromolecular organic substances with a high water content (80–90%), have a rapid rate. Hydrogels are highly biocompatible and their mucoadhesiveness allows adhesion to the oral and pharyngeal mucosa.
Aim, Materials and Methods
The aim of the study was to evaluate the in vivo adhesiveness of a product based on hyaluronic acid combined with xanthan gum and carbomer to the oral cavity mucosa in human healthy subjects.
Ten healthy volunteers (6 females, mean age 28.5 y.), referred to the Ear, Nose and Throat (ENT) Unit of Sant’Andrea University Hospital, Rome, Italy, were enrolled from December 2017 to January 2018. Written informed consent was obtained from all study participants, and all procedures were conducted in accordance with the Helsinki declaration.
The inclusion criteria in the study included enrollment of individuals without distinction of age or sex, able to comply with planned procedures (methods and timings of tests), and with no diseases of the oral cavity. Individuals with previous and/or current oral diseases, subjects with a history of known hypersensitivity to any components of the study product, and those under treatment with other products for the oral cavity were excluded from the study.
The study protocol included two tests, carried out one week apart.
For each test, participants were asked to intake the study product (Karos® tablet, sodium hyaluronate) and then underwent sample collection by brushing the oral mucosa. The samples were taken immediately after the administration of the product (T0), and then at 1 hour (T1), 2 hours (T2), 3 hours (T3), and 4 hours (T4) after intake. Samples of the oral mucosa were collected, at the different timepoints, from different areas within the mouth in order not to affect subsequent samples.
In the first test (Test 1), participants were asked to abstain from drinking and eating for the whole duration of the test; in the second test (Test 2), participants were instead allowed to follow their daily routine, so they were free to eat and drink at will.
After each collection, the sample was transferred to a slide and then fixed. Slides were sent to the respective laboratory for cytological evaluation.
The qualitative and quantitative analysis of the compound was carried out by observation of samples under microscope after cytochemical staining with Alcian Blue at pH 2.5.
Densitometric analysis of Alcian blue staining
Oral brush samples were stained with Alcian blue at pH 2.5 (Bio-Optica, Milan, Italy), according to the manufacturer's instructions. For the quantification of the signal, digital photomicrographs of all samples were obtained with an Axiovert inverted optical microscope (Zeiss, Oberhocken, Germany) equipped with Axiocam CCD camera (Zeiss) and Axiovision image analysis software (Zeiss), using a random sampling method. All slides were digitally scanned and densitometrically analyzed with the dedicated Axiovision software (Zeiss).
The sample area with positive staining for Alcian blue was expressed in µm² ± Standard Deviation (SD); the color intensity was expressed in arbitrary units (AU, mean ± standard error [SE]), corresponding to the brightness values of the dye on a gray scale from 0 to 256 (max value).
Statistical Analysis and Results
Analysis of statistical significance for quantitative variables was performed by Student’s t-test for paired data; the statistical significance threshold was defined as p < 0.05; the results of the study are shown in Table 1, and in Figs. 1–2.
Table 1
Densitometric analysis of Alcian blue.
| | T0 | T1 | T2 | T3 | T4 |
| Test 1 (fasting) | 44.2 ± 5.6 | 33.2 ± 4.1 | 36.1 ± 4.6 | 34.8 ± 4.3 | 35.3 ± 4.5 |
| Test 2 (normal routine) | 45.4 ± 4.3 | 38.1 ± 2.6 | 36.6 ± 1.6 | 31.9 ± 2.8 | 31.4 ± 3.8 |
| Values are expressed as Arbitrary Units staining intensity (mean ± Standard Error; reference intensity of untreated sample: 34.2 AU) |
The results of this preliminary study demonstrate that the combination of hyaluronic acid, xanthan gum and carbomer has adhesive properties on the oral mucosa, satisfying the adhesion criteria of the classic mucoadhesive hydrogels. The effective adhesive duration of this product, based on hyaluronic acid, xanthan gum and carbomer, is at least 4 hours. Based on a quantitative analysis of time-intensity data, both in the fasted state and in the normal fed state, product adhesiveness does not appear to be significantly affected by the intake of solid foods and/or drinks.
The combination of hyaluronic acid, xanthan gum and carbomer is more effective, in terms of duration, than the individual mucoadhesive polymers; accordingly, this product may have positive effects on the mucosa of the oral cavity as a result of the long-lasting effect of hyaluronic acid, including better hydration, improved repair capacity of the mucosa, and a protective effect against internal or external insults.