Tau-Marin Mucoadhesive Gel for Prevention and Treatment of Gum Diseases

An innovative and stable probiotic-containing mucoadhesive gel (AL0020), integrated with botanical extracts, has been developed to rebalance the dysbiosis associated with periodontal diseases. Tau-Marin gel, prepared with anhydrous ingredients to prevent the replication of bacteria and ensure good stability over time, was tested against some pathogenic bacteria, belonging to the so-called “red complex”, recognized as the most important pathogens in plaque specimens, adherent to the epithelial lining of periodontal pockets. This lipogel was tested in vitro, in a physiological solution (PS) and in a simulated saliva (SS), for up to 8 h, to monitor its ability to release probiotics over time. Probiotics were enumerated through two different techniques, Lacto-Counter Assay (LCA) and Colony Forming Unit (CFU). A detailed physico-chemical profile of AL0020 and its in vitro efficacy in protecting activity against pathogenic bacteria as well as soothing or irritative effect on gingival epithelium were reported. Moreover, a clinical-dermatological trial on 20 volunteers using the product once a day for 30 days was also performed, where the efficacy of the gel in the control of gum disorders was observed.


Introduction
Oral diseases have remained the most dominant morbid condition globally since 1990, and among these, untreated caries of permanent teeth is the most prevalent with about 2 billion cases, followed by severe periodontal disease affecting about 1 billion cases and then untreated caries of deciduous teeth with about 510 million cases and edentulism with 350 million cases (only in 2019). The estimated combined number of oral disease cases globally is about 1 billion higher than the cases of all five major noncommunicable diseases combined (mental disorders, cardiovascular disease, diabetes mellitus, chronic respiratory diseases, and cancers). These are the data from the WHO Report on the State of World Oral Health: towards universal coverage for oral health by 2030 [1].
In such a scenario, a greater effort to find efficient and inexpensive solutions can no longer be derogated from. An alteration of the oral microbial flora (dysbiosis) plays an important role in generating health problems, both locally (caries, gingivitis, and periodontitis) and at a systemic level. Gingivitis is a condition often characterized by bleeding gums swollen and painful. If left untreated, gingivitis progresses to periodontitis which involves the loss of the periodontal ligament attachment and of the bone surrounding and supporting the teeth. Periodontal diseases are one of the most common diseases in humans, Gels 2023, 9, 607 3 of 22 prolonged probiotics dispersion was much more efficient than the dispersion lasting few minutes.
The results presented here are the continuation of a previous exploratory study, where it was demonstrated that probiotics were stable over time (more than one year) and were slowly released into the oral cavity after application, if formulated in an anhydrous mucoadhesive gel. We had also demonstrated the effectiveness of this formula in contrasting bone reduction following an inflammatory stress and in counteracting-in vitro-some pathogenic bacteria belonging to the "red complex" [19].
Here, we report the results obtained with a new formula (lab code: AL0020) containing probiotics and botanical extracts, where the mucoadhesive gel has been loaded with three probiotics at 1%, Lacticaseibacillus rhamnosus SP1 (L. rhamnosus SP1), Lactobacillus helveticus SP27 (L. helveticus SP27), and Lacticaseibacillus paracasei CBA-L87 (L. paracasei CBA-L87), so defined according to the latest nomenclature [20], and with three botanical extracts, Aloe Barbadensis leaf, Vaccinium Myrtillus Fruit, and Malva Sylvestris Leaf. The final formula (AL0020) was evaluated both in in vitro tests (competition, mucosa, and epithelia) and in a survey of 20 volunteers who used Tau-Marin gel (AL0020) once a day for 30 days, with a positive response based on the protective effect, a reduction in gingival inflammation and bleeding, associated with a better general condition of gum health. Also, the gel was appreciated without any revulsion.

Mucoadhesive Gel (AL0038) and Ingredients
The mucoadhesive gel (AL0038) does not contain any active ingredients but consists of an oily basic gel inside of which mucoadhesive polymers are dispersed. The resulting gel is viscous and beige/ivory in color and menthol flavor. The composition of mucoadhesive gel (AL0038) is reported in Table 1.

Tau-Marin Mucoadhesive Gel (AL0019, AL0039, and AL0020) Preparation
To the previous basic formulation (AL0038) were added three botanical extracts in powder (Aloe vera leaf extract, blueberry extract, and mallow leaf extract). The resulting lipogel is AL0019, viscous and beige/ivory in color, with green and violet dots and menthol flavor.
At the same time, the basic formulation (AL0038) was added with three probiotics (L. rhamnosus SP1, L. helveticus SP27, and L. paracasei CBA-L87). The resulting product is AL0039, viscous and beige/ivory in color and menthol flavor (Tables 2 and 3).
At the end, to the basic formulation (AL0038) were added both three botanical extracts and three probiotics. The resulting final formulation of lipogel is AL0020, viscous and beige/ivory in color, with green and violet dots and menthol flavor. Table 2. Bacterial strain, percentage and theorical title used in the preparation, and minimum titer of the bacterial consortium in the final preparation.

Antimicrobial Activity: Antagonist Effects
In Table 4, a scheme of each combination evaluated in the competition test was reported. Chlorhexidine digluconate 0.2% was included as positive inhibition control.  Results obtained from the competition tests (inhibition zone) were consistent with those obtained in the previous project [19], confirming the anti-bacterial activity of the tested ingredients of the product, although evaluated by means of in vitro assays. In the second assessment, the antimicrobial action of the single herbal extracts, of the probiotic mixture, and of the different matrices was measured.
The results of the agar diffusion well test are reported in Table 5, showing the different inhibitory effect of the gels compared to the positive control, chlorhexidine (CHX). As shown in Table 5, a neutral gel (AL0038) and the two gels containing the separate probiotic mixture (AL0039) or botanical extracts (AL0019) showed a reduced inhibiting Gels 2023, 9, 607 6 of 22 power compared to the complete formulation AL0020 against the single components of the "red complex" as well as against the pathogen blend.
An inhibition test provided interesting results in order to understand the contribution given by the individual components in contrasting the pathogenic bacteria of the oral cavity. The probiotic strains, L. rhamnosus, L. helveticus, and L. paracasei, individually tested showed minimal inhibition on some representatives of the "red complex". The efficacy was increased towards single pathogenic strains examined when the same three probiotics were added into the mucoadhesive gel (AL0039), also including an efficacy on the pathogenic bacteria mixture (blend). Similar results were observed by the single botanical extracts, and again the efficacy was extended to all pathogens when single extracts were mixed in the same matrix (AL0019).
In detail, Aloe vera and mallow leaves powders weakly inhibited all pathogens and their blend, with the only exception of T. denticola. Blueberry extract weakly inhibited the pathogens' blend and P. melaninogenica, without any influence on the other members of the "red complex".
The pathogens' blend clearly showed that the so-called "red complex" was actually able to create a very efficient biofilm, in which the different strains were integrated and able to optimize growth even in less favorable conditions, as the presence of potentially inhibiting agents.
However, the pathogens' blend was demonstrated to be more prone to inhibition when all the ingredients (probiotics and botanicals) were added into the mucoadhesive gel: in fact, the final formulation (AL0020) turned out to be the best performing, and its action/efficacy was extended to the A. actinomycetemcomitans.
These results support the hypothesis that Tau-Marin gel (AL0020) could be a very effective device in counteracting the growth of pathogenic bacteria in the oral cavity. Tau-Marin (AL0020) Mucoadhesive Gel by LCA and CFU As described above, probiotics were enumerated in Tau-Marin gel (AL0020; batch L1538 K) by means of two different techniques, CFU and LCA, as shown in Tables 6 and 7.  5.1 × 10 9 3.9 × 10 9 8.6 × 10 6 6.7 × 10 6 2.5 × 10 9 2.0 × 10  Time 0 min refers to the number of lactobacilli in the lipogels, whereas timepoints 30 min and 2, 5, and 8 h indicate the number of bacteria remaining in the lipogel (1 g) or released (1 g/mL) in PS after different incubation times. The number of bacteria loaded and released from lipogels to PS was measured at the moment of the preparation (T0) and after 1 (T1), 3 (T3), 6 (T6), 9 (T9), and 12 (T12) months. The standard deviation (SD), ranging between 0.1 and 0.3 for all the means, was not reported.

Inserts of Gingival Epithelium Reconstructed In Vitro
To evaluate the irritating/protective effect of gel on an in vitro gingival model, an in vitro reconstructed epithelium of human gingiva with a surface area of 0.50 cm 2 was used, placed on a support consisting of a cellular multilayer. All were placed on top of an inert polycarbonate filter. This cellular multilayer histologically simulates the superficial part of the human gingiva.
In the present study, technical data of the Skinethic ® HGE gingival epithelium were used. The inserts of gingival epithelium reconstructed in vitro were treated topically with the sample under analysis for 4 h. At the same time, analogous inserts were treated with physiological solution for 1 h, as negative control, and with Sodium dodecyl sulphate solution (SDS 0.5%) for 1 h, as positive irritative control. At the end of each kind of exposure, the sample or the control solutions were removed from the surface of the inserts with a wash in saline, followed by gentle drying.
At the end of the 24 h rest period after treatment, cellular viability has been checked in all inserts analyzed using MTT titration, and the cellular medium was collected and analyzed for IL-1α expression.
Tau-Marin gel (AL0020) did not lead to a reduction in cell viability below 50%, which is placed as a criterion of acceptability for positive control. It can therefore be considered that the treatment with sample AL0020 did not affect the cell viability (Table 8). In addition, the treatment of gingival epithelium inserts with the irritative agent, consisting of a 0.5% aqueous solution of SDS, led to a drastic reduction in cellular viability of the multilayer. The pre-treatment of the inserts with Tau-Marin gel, followed by the application of the irritant, always consisting of the aqueous solution of 0.5% SDS, did not lead instead to a reduction in cell viability, compared to the negative control used as a reference. Therefore, the pre-treatment with the test sample determines an increase compared to the treatment performed only with the irritant, using the same time of application of the latter (Table 9). Table 9. Protective effect against an irritant agent (0.5% SDS) by pre-treating inserts with Tau-Marin gel.

Human IL-1 Alpha ELISA Test
The gel AL0020 was applied on the surface of the gingival epithelial layer reconstructed in vitro. The objective of the test was to evaluate the ability of gel to penetrate the cellular multilayer, affecting the living cells of the deeper layers. By applying the gel after having subjected the cell layer to an irritation treatment, it is possible to evaluate whether it has a soothing action. If, on the other hand, the gel is applied before the irritant treatment, it can be assessed whether it can form a protective barrier.
Evaluation of irritative potential of the sample: The inserts of gingival epithelium reconstructed in vitro were treated topically with the sample under analysis for 4 h. At the same time, analogous inserts were treated with physiological solution for 1 h, as a negative control, and with sodium dodecyl sulphate solution (SDS 0.5%) for 1 h, as a positive irritative control. At the end of each kind of exposure, the sample or the control solutions were removed from the surface of the inserts with a wash in saline, followed by gentle drying.
At the end of the 24 h rest period after treatment, cellular viability was checked in all inserts analyzed using MTT titration.
Evaluation of protective efficacy of the sample: The inserts of gingival epithelium reconstructed in vitro were treated topically with the sample under analysis, in sufficient quantity to cover the surface, followed by sodium dodecyl sulphate solution (SDS 0.5%) for 1 h. At the same time, analogous inserts were treated with physiological solution for 1 h, as a negative control, and with SDS 0.5% solution for 1 h, as a positive irritative control. At the end of each kind of exposure, the sample or the control solutions were removed from the surface of the inserts with a wash in saline, followed by gentle drying.
At the end of the 24 h rest period after treatment, cellular viability was checked in all inserts analyzed using MTT titration, and the cellular medium was collected and analyzed for IL-1α expression ( Figure 1).
The gingival epithelium, that undergoes treatment with the irritative agent (0.5% SDS), after treatment with Tau-Marin gel maintains a cellular viability and an IL-1αexpression similar to that evidenced by the inserts used as negative control, whereas the inserts treated only with the irritative agent show a significant decrease of cell viability and increase of IL-1α levels.
Evaluation of soothing effect of the sample: Gingival epithelium inserts underwent a pre-treatment with an irritant factor (lactic acid 1.0% for two hours) and, subsequently, were washed with saline and dried gently. At the end of each kind of exposure, the sample or the control solutions were removed from the surface of the inserts with a wash in saline, followed by gentle drying.
At the end of the 24 h rest period after treatment, cellular viability was checked in all inserts analyzed using MTT titration, and the cellular medium was collected and analyzed for IL-1α expression ( Figure 1). The gingival epithelium, that undergoes treatment with the irritative agent (0.5% SDS), after treatment with Tau-Marin gel maintains a cellular viability and an IL-1αexpression similar to that evidenced by the inserts used as negative control, whereas the inserts treated only with the irritative agent show a significant decrease of cell viability and increase of IL-1α levels.
Evaluation of soothing effect of the sample: Gingival epithelium inserts underwent a pre-treatment with an irritant factor (lactic acid 1.0% for two hours) and, subsequently, were washed with saline and dried gently.
To verify the soothing properties of the sample, two tissue inserts were treated with the sample analyzed, in sufficient quantity to cover the surface. The sample was applied to the epithelial inserts, and the exposure was maintained for four hours and was repeated for two days. At the end of each period of exposure to the sample, it was removed from the surface of the insert with a wash in saline, followed by gentle drying.
At the same time, two inserts were exposed to a treatment with acetylsalicylic acid (0.03% solution) for four hours as reference sample for a soothing effect.
At the end of the 48 h rest period after the initial irritative treatment, cellular viability was checked in all inserts using MTT titration, whereas the cellular medium was collected and analyzed for IL-1 expression at 24 and 48 h after the initial treatment ( Figure 2). To verify the soothing properties of the sample, two tissue inserts were treated with the sample analyzed, in sufficient quantity to cover the surface. The sample was applied to the epithelial inserts, and the exposure was maintained for four hours and was repeated for two days. At the end of each period of exposure to the sample, it was removed from the surface of the insert with a wash in saline, followed by gentle drying.
At the same time, two inserts were exposed to a treatment with acetylsalicylic acid (0.03% solution) for four hours as reference sample for a soothing effect.
At the end of the 48 h rest period after the initial irritative treatment, cellular viability was checked in all inserts using MTT titration, whereas the cellular medium was collected and analyzed for IL-1 expression at 24 and 48 h after the initial treatment ( Figure 2). The gingival epithelium, that undergoes treatment with the irritative agent (0.5% SDS), after treatment with Tau-Marin gel maintains a cellular viability and an IL-1αexpression similar to that evidenced by the inserts used as negative control, whereas the inserts treated only with the irritative agent show a significant decrease of cell viability and increase of IL-1α levels.
Evaluation of soothing effect of the sample: Gingival epithelium inserts underwent a pre-treatment with an irritant factor (lactic acid 1.0% for two hours) and, subsequently, were washed with saline and dried gently.
To verify the soothing properties of the sample, two tissue inserts were treated with the sample analyzed, in sufficient quantity to cover the surface. The sample was applied to the epithelial inserts, and the exposure was maintained for four hours and was repeated for two days. At the end of each period of exposure to the sample, it was removed from the surface of the insert with a wash in saline, followed by gentle drying.
At the same time, two inserts were exposed to a treatment with acetylsalicylic acid (0.03% solution) for four hours as reference sample for a soothing effect.
At the end of the 48 h rest period after the initial irritative treatment, cellular viability was checked in all inserts using MTT titration, whereas the cellular medium was collected and analyzed for IL-1 expression at 24 and 48 h after the initial treatment ( Figure 2). The gingival epithelium that undergoes treatment with the irritative agent (1.0% Lactic acid) demonstrates reduced cell viability and increased IL-1α expression. The treatment with Tau-Marin Gel, after the irritative agent, shows a rise in cellular viability and the lowering of IL-1α expression. The percentage viability achieved and the low expression of IL-1α allows for the attribution of an effective barrier action in the sample.

In Vitro Evaluation of Irritation on Human Oral Mucosa and TEWL Measurement
This test is based on in vitro reconstructed oral mucosa inserts. These are inserts with a surface area equal to 0.50 cm 2 of mucosa reconstituted in vitro from transformed human keratinocytes from a squamous cell carcinoma of the buccal mucosa.
This support consists of a cellular multilayer developed on top of an inert polycarbonate filter. The cellular multilayer histologically resembles the mucous membrane present in the oral cavity and, like this one, is devoid of a stratum corneum. It is therefore possible to reproduce in vitro the effect that the application of a product could have in vivo on the oral mucosa.
The MTT test measures the amount of formazan formed in cell culture. And this provides a measure of cell viability. For the MTT test to be valid, the following acceptance criteria for controls must be met:

•
Negative control: The average optical density of the extracted solution at the end of the MTT test must be greater than 0.8 and less than 3.0. The standard deviation should not exceed 18%.

•
Positive control: The percentage viability, obtained by comparing the optical density of the extracted solution at the end of the MTT test with that relating to the negative control, must be less than 50%, and the standard deviation of the percentage viability must not exceed 18%.
The sample under analysis (Tau-Marin gel) is considered not irritating on oral mucosa with viability results of 87.6% after an application lasting four hours, followed by the posttreatment rest period as it shows, compared to the negative reference control (Figure 3). and the lowering of IL-1α expression. The percentage viability achieved and the low expression of IL-1α allows for the attribution of an effective barrier action in the sample.

In Vitro Evaluation of Irritation on Human Oral Mucosa and TEWL Measurement
This test is based on in vitro reconstructed oral mucosa inserts. These are inserts with a surface area equal to 0.50 cm 2 of mucosa reconstituted in vitro from transformed human keratinocytes from a squamous cell carcinoma of the buccal mucosa.
This support consists of a cellular multilayer developed on top of an inert polycarbonate filter. The cellular multilayer histologically resembles the mucous membrane present in the oral cavity and, like this one, is devoid of a stratum corneum. It is therefore possible to reproduce in vitro the effect that the application of a product could have in vivo on the oral mucosa.
The MTT test measures the amount of formazan formed in cell culture. And this provides a measure of cell viability. For the MTT test to be valid, the following acceptance criteria for controls must be met: • Negative control: The average optical density of the extracted solution at the end of the MTT test must be greater than 0.8 and less than 3.0. The standard deviation should not exceed 18%.

•
Positive control: The percentage viability, obtained by comparing the optical density of the extracted solution at the end of the MTT test with that relating to the negative control, must be less than 50%, and the standard deviation of the percentage viability must not exceed 18%.
The sample under analysis (Tau-Marin gel) is considered not irritating on oral mucosa with viability results of 87.6% after an application lasting four hours, followed by the post-treatment rest period as it shows, compared to the negative reference control ( Figure 3).  TEWL (Transepidermal water loss), measured by the Vapometer-Delfin, is a key indicator of skin barrier function, and the ability to measure it accurately is essential in a wide range of clinical and personal care applications. In this test, the measurement of TEWL was performed on the mucosal inserts to verify their greater or lesser surface permeability.
For the TEWL test to be valid, its value for the positive control inserts must be higher than the value for the negative control inserts after treatment.
The trend of TEWL in the inserts treated with the sample shows a substantial maintenance of the barrier function of the treated mucosa throughout the study time (Table 10 and Figure S3 in S.I.).
The TEWL values remain optimal and comparable to those of the negative control, indicating a non-alteration of the barrier function of the mucous membrane, both at the end of the four hours of application and after the post-treatment rest period. (c) Drop out-reasons considered sufficient to terminate the participation of the subjects in the study: • Free choice of subject; • Medical reasons unrelated to treatment (e.g., onset of disease or surgery); • Reasons related to treatment (e.g., irritation or allergic reactions).
Restrictions: During the study, the subjects are instructed not to use hygiene products of the teeth and of the entire oral cavity (e.g., mouthwashes, pastes, chewing gum with xylitol, etc.) other than those delivered and not to apply the products under examination in parts other than those prescribed. The effectiveness of the product in improving the well-being of the oral cavity is evidenced by the decrease in the values of the parameters at the end of the treatment for erythema and gum bleeding, whereas it is supported by the maintenance of absence of erosions and leukoplastic lesions.
b. COPAN Swabs sampling and transport system. The COPAN Swabs sampling and transport system is intended for the collection, transport and storage of clinical samples that must be analyzed with nucleic acid amplification techniques. Sampling was performed on the upper and lower gums.
c. Subjective evaluation. At the end of the test, the volunteers expressed their subjective opinion on the effectiveness and pleasantness of the treatment by filling out a self-assessment questionnaire. For each question, the volunteers expressed their opinion on a four-point rating scale corresponding to four different intensities perceived according to the following values: 1 = insufficient; 2 = sufficient; 3 = good; 4 = excellent.  (Table 11). Table 11. Relative frequencies of the answers given to the questions in the final questionnaire.

Question
Opinion Expressed

Results
Regarding clinical evaluation, assessment of the microbiome, and self-assessment questionnaire of a cosmetic product for oral use, in order to simplify the interpretation of the c. Mathematical elaboration. c.1 Clinical-dermatological evaluation. The data obtained were statistically processed using the statistical program R-studio. For significance analyses, the Wilcoxon signed rank test was applied for paired data with a significance level of p-value < 0.05.
All the parameters considered were assigned a rating score according to the following four-point scale: 0 = absent; 1 = mild; 2 = moderate; 3 = severe.
After 30 days of treatment with the product, since p-value < 0.0001, it can be concluded that there is a significant change in the average evaluation rating of the presence of erythema, and therefore the hypothesis is accepted that data relating to this value after 30 days are different from those before use.
The average decrease in the assessment of the presence of erythema between T0 (initial) and TF (after 30 days of treatment) corresponds to −0.8 (Table 12).  A total of 100% of the volunteer subjects showed a significant improvement in the erythema evaluation (decrease) after 30 days of treatment.
After 30 days of treatment with the product, since p-value = 0.0003, it can be concluded that there is a significant change in the average evaluation rating of the presence of gum bleeding, and therefore the hypothesis is accepted that data relating to this value after 30 days are different from those before use.
The average decrease in the assessment of the presence of gum bleeding between T0 (initial) and TF (after 30 days of treatment) corresponds to −1.2 (Table 13).  A total of 90% of the volunteer subjects showed a significant improvement in the gum bleeding evaluation (decrease) after 30 days of treatment.
After 30 days of treatment with the products, since p-value = 0.35, it can be concluded that there is no significant change in the average value of the presence of erosions, and therefore the hypothesis is accepted that data relating to this value after 30 days are equal to those before use.
Treatment is proven not to cause erosion during its use (Table 14).  After 30 days of treatment with the products, since p-value = 0.37, it can be concluded that there is no significant change in the average value of the presence of leukoplastic lesions, and therefore the hypothesis is accepted that data relating to this value after 30 days are equal to those before use. Treatment is proven not to cause leukoplastic lesions during its Gels 2023, 9, 607 15 of 22 use. A total of 100% of the volunteer subjects showed that they maintain or improve their rating in the presence of leukoplastic lesion evaluation after 30 days of treatment (Table 15).  The whole data analysis workflow was performed using QIIME2 v2021.4. Raw reads were processed with cutadapt in order to remove primer sequences and were subsequently filtered, denoised, merged, and cleaned by chimera with DADA2, run with default parameters (--p-trunc-len-f 270, --p-trunc-len-r 215). The obtained amplicon sequence variants (ASVs) were then filtered by frequency applying a 0.01% threshold to remove singletons and poorly represented sequences. The feature-classifier plugin was applied to assign the proper taxonomy to ASVs, using trained OTUs at 99% from Silva (v 138) and Green Genes (v 13-8) databases. After the selection of the most feasible value, the feature table (sample in columns and ASV in rows) was rarefied, and alpha diversity indices (observed features, evenness, Faith PD, and Shannon) and beta diversity metrics (weighted and unweighted unifrac, Bray-Curtis, and Jaccard) were calculated using the diversity plugin. Moreover, statistical comparisons among the two time points were assessed with Kruskall-Wallis and Permanova tests, for alpha and beta diversity, respectively, while pairwise differences were calculated using the Wilcoxon signedrank test, integrated in the longitudinal plugin. Beta diversity PCoAs were calculated using the upgma clustering method with 200 iterations and drawn with the EMPeror visualization tool. Finally, differential abundance analysis was performed with ANCOM, collapsing the feature table at various levels (features, species, genus, family, and phylum).
d. Drop out cases. One subject stopped the treatment for personal reasons that arose after the start of the test but not related to the products used. Therefore, it has not been possible to collect the subjective evaluation in the self-assessment questionnaire from this subject. Moreover, the data relating to the measurements of this subject, initially collected, were included in microbiome evaluations at initial time (T0) but were not included in the statistical evaluation comparing date of initial time (T0) with those at the end of the testing time (TF). For the same reason, data relating to this subject were not included in clinical-dermatological evaluations, as they are expressed as comparative data between T0 and TF.

Microbiome Analysis
The study was performed to evaluate microbiota changes between two time points (T0 and TF). Overall, the investigations showed a stability of the microbial composition along the time, as alpha (S.I.; Figure S1A,B) and beta diversity (S.I.; Figure S1C) analyses and taxa differential abundances evaluation did not highlight significant differences.
Compositional data analysis of microbiota specimens was made by using ANCOM. In this way, we assessed if some taxa, at various levels (i.e., phylum and genus) (S.I.; Figure  S2), were significantly different between the two time points. Confirming previous results, no taxon was found to change its abundance significantly, even if some differences are visible analyzing grouped bar plots at family and genus levels.
In conclusion, we can affirm that the sample microbial composition has not undergone significant modifications between the two time points.
The non-variability of the microbiota following treatment can be seen as a positive result because, if the gum discomfort were not of microbiological origin, the treatment would eliminate/minimize the cause of the problem, without significantly changing the physiological composition of the microbiota. Otherwise (problem of microbiological origin), the lack of significance is not total, and therefore the reduction of gum problems in patients may partly derive from the improvement of alpha diversity indices that are significant by analyzing the samples in a coupled way.

Discussion
The results presented here are in continuation of what has already been published by our group, in a previous project aimed at the development of a mucoadhesive gel (AL0005), where we observed that probiotics are effective and counteract the action of bacterial pathogens, which had to be loaded into the lipogel at a concentration of not less than 1%. The formulation loaded with probiotics (blend) at a concentration equal to 2% and integrated with botanical extracts was stable over time. This lipogel, after application on the gingival mucosa, was able to release the probiotics slowly and constantly. This gel was found to have an antibacterial action, evaluated on some bacteria components of the plaque, the so-called "red complex", which starts on the gingival collar, threatening the health of the mouth and the stability of the teeth. Now, here we present the results of an improved formulation (AL0019) where the overall bacterial load has increased to 3% (three bacteria at a concentration of 1% each), and the composition of the botanical extracts has also been slightly modified with the aim of improving its plasticity (spreadability) and color. In fact, for this purpose, some natural ingredients have been replaced with respect to the previous formulation.
This new formulation (AL0020) has been tested both in different in vitro tests and in a clinical study on 20 volunteers.
The ability of probiotics, added to the gel, to compete with some of the bacteria belonging to the "red complex", was evaluated in vitro, and, interestingly, the individual components were less efficient than the final formulation in counteracting the development of pathogens.
The stability of the formulation over time, detected by CFU and LCA, was evaluated. This formulation remains stable at room temperature at least up to twelve months. We are confident that this new formulation could be stable over 18 months similarly to that already observed with the first formulation.
The gel was also subjected to a series of in vitro tests on safety, calming effect, and irritability (TEWL). In all tests, the formulation was found to be non-aggressive but, on the contrary, endowed with a protective effect.
Finally, the AL0020 gel was tested on a group of 20 volunteers, under medical supervision, who applied the gel to their gums every evening for 30 days and, at the end of the treatment, the dentist expressed an opinion on the health of their mouth. The result, evaluated with a self-assessment questionnaire, was positive. Both the dentist and the volunteers observed that after a month there was less bleeding, as well as an apparent improvement in the health of their gums, such that they considered the gel pleasant and not unpleasant.
Furthermore, saliva samples were analyzed microbiologically, at the beginning and after 30 days, at the end of the study. The results showed a stability of the buccal microbial composition along the time, as alpha and beta diversity analyses and taxa differential abundances evaluation did not highlight significant differences. This non-variability of the microbiota following treatment can be seen as a positive result because, if the gum discomfort were not of microbiological origin, the treatment would eliminate/minimize the cause of the problem, without significantly changing the physiological composition of the microbiota. Otherwise, the lack of significance is not total, and therefore the reduction of gum problems in patients may partly derive from the improvement of alpha diversity indices that are significant by analyzing the samples in a coupled way.

Conclusions
One of the most recent approaches to the treatment of oral dysbiosis, responsible for a series of pathologies of the oral cavity, starting from gingivitis up to periodontitis, is the local administration of probiotics or botanical extracts.
Some bacteria have been identified as the main responsible for oral dysbiosis and components of the so-called "red complex", consisting mainly of anaerobic facultative intracellular pathogens such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Tannerella forsythia, and Prevotella melaninogenica constituting bacterial plaque.
These are some of the bacteria described in the literature for their ability to colonize subgingival sites, penetrate inside the host cells, and thus elude the host's defense system, causing chronic inflammation with tissue damage. Thus, periodontitis may be associated with multifactorial chronic inflammation caused by oral dysbiosis.
Local administration of exogenous bacteria (probiotics) can promote oral eubiosis. For this purpose, botanical extracts have also been described as effective.
Previously, we had already described the results of a project aimed at developing a mucoadhesive gel that would keep the bacteria loaded in it viable and that would be able to release them regularly once the gel was applied to a mucosa, obviously in addition to having identified the most effective probiotics.
The new formulation (AL0020), described in this manuscript, has a higher bacterial load (3%) in addition to botanical extracts, Aloe, blueberry, and mallow.
The formulation was found to be stable, if kept at room temperature, non-aggressive on the gingiva models where it was tested, endowed with a protective effect, and capable of reducing inflammation (IL-1).
Furthermore, it was evaluated on a group of twenty volunteers who applied the gel to the gums for 30 days, every night after brushing their teeth and before going to sleep. The result was a reduction in gingival bleeding and a better condition of gum health. Also, the gel was appreciated without any revulsion, after evaluation with a self-questionnaire.

Mucoadhesive Gel (AL0038) Preparation
For the preparation of the mucoadhesive gels, botanical extracts were used together with cometic-grade ingredients compatible with the oral cavity. These ingredients are normally used in marketed products, such as toothpastes. To improve the stability of the lipogel, modified silicas (MPs) such as silica dimethyl silylate (AEROSIL ® R972) and hydroxypropyl methylcellulose (HPMC) (BENECEL K100M) were also used, as described in Table 1.

Tau-Marin Mucoadhesive Gel (AL0020) Preparation
We have previously reported [19] the preparation of a basic mucoadhesive gel, (AL0005). Now, the formula has been slightly modified, and mint has been chosen as a flavor (Lab code, AL0038).
The selection criteria of the three botanical extracts were different. Aloe vera and blueberry extract are supported by the literature data for their effectiveness in promoting well-being of the oral cavity through a high effect in protecting the teeth against bacteria responsible for accelerating tooth decay, [22][23][24] while the Malva Sylvestris leaf extract was selected for its emollient, [25] anti-inflammatory, antioxidant, and osteoblast differentiation properties [26].
The final formulation was added with "menta flavour" to give a pleasant taste.

In Vitro Evaluation of Irritative Potential, Protective Efficacy, and Soothing Effect on Gingival Epithelium
Another test used to evaluate the mucoadhesive gel was a three-dimensional model of in vitro reconstructed gingival epithelium (HE). This model was created by culturing normal human gingival cells on an inert polycarbonate filter, at the air-liquid interface, in a chemically defined medium. In culture, these cells form a highly differentiated multi-layered epithelium pattern histologically similar to the outer cell layers of human gum.
Such a support is treated topically with the tested product in order to study its irritative potential, its protective efficacy, and its soothing properties.
For the irritative potential study, the product is applied directly on the inserts and the treatment is maintained for four hours. This test evaluates whether the application of the sample under analysis can decrease cell viability in the inserts, proving in this way an irritative activity.
For the protective study, inserts are treated topically with the sample under test with a concomitant aggressive irritant product at the same time (sodium dodecyl sulphate 0.5%). In this case, the possible decrease of cell viability and the possible increase of Interleukin-1 alfa (IL-1α) expression can be informative about a product's protection properties.
The soothing test is performed exposing the inserts to a mild irritant treatment with lactic acid 1% for two hours, followed by the application of the product under analysis for four hours during the first day and for a further four hours during the next day. The presence of the sample in analysis, if it acts as a soothing factor, can facilitate the recovery of the damage caused by the initial irritant treatment and thus increase the cellular viability and decrease IL-1α expression of the inserts at the end of the study.
Cell viability is measured by considering the conversion by cellular enzymes of the yellow salt of MTT into a water-insoluble blue compound called "formazan". The amount of formazan formed during a defined contact period is proportional to the number of living cells present in the analyzed culture. IL-1α expression is measured by ELISA test.

Clinical
Evaluation, Assessment of the Microbiome, and Self-Assessment Questionnaire of a Cosmetic Product for Oral Use For a clinical evaluation of the activity of Tau-Marin gel, 20 volunteers applied AL0020 to their gums every evening, once a day for one month. The purpose of the study was to evaluate the ability of the product to rebalance the gingival mucosa altered by erythema, erosions, leukoplastic lesions, and bleeding, before and after treatment. The treatment activity was evaluated by a dentist at the end of the 30 days.
Microbiome rebalancing activity of the product is supported by the following: No alteration of the microbiota.
Rebalancing of the bacterial species present.
A clinical-dermatological evaluation of the condition of gums was performed to assess the presence of erythema, gum bleeding, erosions, and leukoplastic lesions before and after treatment.
All clinical assessments were scored according to the following 4-point scale: 0 = absent; 1 = mild; 2 = moderate; 3 = severe. The effectiveness of the product is evidenced by the decrease at the end of the treatment for erythema and gum bleeding parameters, whereas it is supported by the maintenance of absence of erosions and leukoplastic lesions.
At the end of the test, the volunteers expressed their subjective opinion on the effectiveness and pleasantness of the treatment by filling out a self-evaluation questionnaire.