**3. Results**

A total of 20 dogs were selected to participate in this clinical trial. The animals' gender distribution and mean age and weight are presented in Table 4. The results of the hemogram and biochemical parameters of all animals included in the trial agreed with the reference values established for healthy dogs at both timepoints (0 and 90) (Supplementary File S1).

**Table 4.** Gender, age, and weight distribution by group of animals in this study.


F—Female; M—Male; SD—Standard deviation.

### *3.1. Periodontal Evaluation*

Complete periodontal evaluations of every tooth of each dog were performed at timepoints 0 and 90. The mean results for each dog are presented in Table 5. At timepoint 0, after periodontal assessment, all dogs were submitted to scaling and dental polishing before proceeding to the clinical trial.

Statistical analysis with mixed models showed a significant reduction in mean PPD (estimate = −0.371, *p*-value < 0.001) for all tooth types evaluated (incisive, canine, molar, and pre-molar) in the animals in the treatment group at T90. For the DPI, a statistically significant reduction (estimate = −0.145, *p*-value < 0.05) was observed in the animals in the treatment group at T90, with a marked reduction (*p*-value < 0.001) in incisive teeth. In addition, a reduction in the GI was observed in the animals in the treatment group at T90, but it was not statistically significant (estimate = −0.056, *p*-value > 0.05). For the GI, it was possible to observe a significant reduction (*p*-value < 0.05) in the mandibular incisive teeth and the maxillary molars and pre-molars.



Intermediate evaluation of dental plaque coverage performed on days 30 and 60 using a disclosure solution revealed that the animals in the treatment group presented a mean dental plaque coverage of 24.58% and 33.08%, respectively. On the other hand, the dogs in the control group presented a mean dental plaque coverage of 42.37% on day 30 and 51.61% on day 60.

The results of the intermediate dental plaque coverage obtained by IMAGEJ are presented in Table 6.


**Table 6.** Mean dental plaque coverage (%) of each dog, determined after application of a disclosure solution and IMAGEJ analysis, on days 30 and 60.

ID—identification; SD—standard deviation; TG—treatment group; CG—control group.

#### *3.2. Dental Plaque Sample Processing*

Total oral aerobic bacteria counts were performed on days 0 and 90 using the dental plaque swab samples obtained from each animal. At T0, the samples revealed a mean total count of 8.6 × 107 CFU/mL, with those from the treatment group presenting 6.9 × <sup>10</sup><sup>7</sup> CFU/mL and those from the control group presenting 1 × <sup>10</sup><sup>8</sup> CFU/mL. At T90, the mean total counts were 7.4 × 107 CFU/mL, with the samples from the treatment group presenting 8.3 × 107 CFU/mL and those from the control group presenting 6.6 × <sup>10</sup><sup>7</sup> CFU/mL.

#### **4. Discussion**

Periodontal disease (PD) is well established as one of the most common oral inflammatory diseases in dogs, with 80% of these animals presenting some degree of PD by two years of age [2,19]. Its high prevalence, along with its potential local and systemic consequences, reinforces the need to improve PD control measures [6–9]. Nisin–biogel has been shown to be a promising compound against canine oral biofilms in vitro [10,11], with the ability to act on periodontopathogens present in the canine oral microbiome [20]. In this study, an in vivo clinical trial was performed to assess the efficacy of nisin–biogel dental topical application in dogs. A total of twenty animals were included in the trial after health parameter assessment through blood analysis to confirm the animals' health. During the study, all animals were observed daily to identify any adverse clinical signs or behavior deviations. Previous reports have described an absence of cytotoxicity of nisin at 200 μg/mL [12,13,21,22], which was confirmed by our study, in which none of the animals showed side effects during the 3-month trial. Moreover, a blood analysis was performed in all dogs at the end of the study, and it was observed that none presented deviations in the evaluated parameters.

In addition, oral samples were collected from all animals at T0 and T90 to evaluate the influence of the nisin–biogel on oral aerobic bacteria through total bacterial counts. No reduction in total bacteria counts was observed in the samples collected after the application of the nisin–biogel for 3 months, which is in agreement with previous reports that have suggested resilient behavior of dental plaque and oral microbiota after professional dental cleaning [15,23]. In addition, Cunha and collaborators (2021), who studied the influence of the dental application of the nisin–biogel in the dynamics of the oral microbiome of dogs, detected an increase in bacterial diversity after one week of application of this compound [20]. Considering these studies, the oral bacteria population seems to suffer a rearrangement after antimicrobial compound administration or physical aggression, maintaining microbiota dynamics and relative concentrations [20,23].

One of the main goals of this study was to evaluate the in vivo efficacy of nisin–biogel for PD control using periodontal measures, such as DPI, GI, and PPD, and it was observed that the nisin–biogel had the ability to reduce dental plaque accumulation. This reduction was particularly evident after 1 month of dental topical application, which resulted in 24.58% dental plaque coverage in the treatment group and 42.37% in the control group. Similar results were obtained by Howell et al. (1993), who applied a nisin mouth rinse in dogs for 88 days. In that study, researchers observed a 34–38% reduction in the dental plaque index in the animals submitted to nisin application [24]. Dental plaque is one of the key factors in PD onset, being responsible for the initial aggression to the periodontium [1,4]. Considering this, dental plaque reduction is an essential step for PD control [5]. At the end of our trial, a significant reduction in DPI was observed in the animals in the treatment group in comparison with those in the control group, reinforcing the potential of the nisin– biogel for PD management. This reduction may be caused by the direct action of nisin– biogel on the inner layers of the biofilm. Previous reports have shown that this compound can not only inhibit biofilm formation but also eradicate mono and polymicrobial oral biofilms [10,11]. It has been suggested that nisin's biochemical structure and mechanism of action contribute to its high activity against biofilms [25]. Specifically, nisin can penetrate these structures without being neutralized or bound by the biofilm cells or matrix and is able to target the extracellular polymeric substances of the surrounding matrix [26,27].

The other periodontal indicator evaluated was the gingivitis index. PD can be classified into four different stages, the first of which is characterized by gingivitis [4]. This is a reversible stage in which most periodontal structures are undamaged, with animals showing inflammation and swelling of the gingiva with or without bleeding upon probing [1,4]. At the end of our clinical trial, it was possible to observe that the application of the nisin–biogel induced a slight reduction in GI. Howell and collaborators (1993) also observed a reduction in GI after nisin oral application for 88 days, which may be related to the potential immunomodulatory effect of this antimicrobial peptide [24]. In fact, besides having antimicrobial activity against several periodontopathogens, nisin presents immunomodulatory and hound healing abilities [12,28]. It acts by binding to Lipid II, present in the bacterial cell membrane, and by interfering with cell wall biosynthesis, leading to bacterial death [10]. In addition, it has been shown that nisin promotes a reduction in pro-inflammatory cytokines, which contributes to PD progression, and presents some effects on immune cells [28–30]. In our trial, a significant PPD decrease was observed in the animals from the treatment group at T90, which may be directly related to dental plaque and gingivitis reduction. It is known that gingiva swelling can increase pocket depth, so the reduction of gingivitis after nisin–biogel therapy may have decreased local inflammation, leading to gingival shrinkage and reducing PPD [4].

It is important to note that the animals in the control group also exhibited a slight reduction in GI and PPD at T90, with no statistical significance, which was potentially caused by the scaling and dental polishing performed at the beginning of the study. In fact, dental scaling, polishing, irrigation, and home dental care are the standard procedures for the treatment of PD stage one cases (gingivitis), so a reduction in this periodontal parameter was expected. Nevertheless, the use of nisin–biogel promoted a higher and statistically significant decrease of the DPI and PPD and a reduction in the GI compared with the results from the control group. Moreover, nisin–biogel was well accepted by the animals, showing no adverse effects over a 3-month period of application.

This study has some limitations, such as the number of animals included, and a larger in vivo study would help to validate the nisin–biogel as a potential future compound to be applied by clinicians. Yet, the fact that it was necessary to use dogs with similar housing conditions, which needed to be maintained over a long period of time, prevented the inclusion of more animals in the trial. In addition, although the oral cavity is a complex and diverse environment [11,20], in our study we focused on supragingival dental plaque and, consequently, on the aerobic microbiota. However, anaerobic bacteria are also present in dental plaque and should be evaluated in a future trial [11,20]. Finally, as PD establishment is a multifactorial process [31,32] influenced by the oral microbiome, the host immune system and the local oral environment [1,4,33], in upcoming studies the influence of the nisin–biogel in the oral immune-inflammatory response could be a relevant issue to explore [33].

Considering the high prevalence and impact of canine PD, several other natural compounds are being studied to evaluate their potential for the control of this disease. Promising strategies include essential oils, alcoholic herbal products, and algae [34,35]. Most of these compounds have already been evaluated in vitro regarding their antimicrobial abilities. Moreover, in vivo studies have already shown the efficacy of oral products, including *Calendula officinalis* or the brown alga *Ascophyllum nodosum*, for PD control [34,35].

The nisin–biogel seems to be a promising compound for veterinary dentistry and human medicine [31,32]. Reports have described the potential application of this compound in human odontology and other areas, including for the treatment of diabetic foot infections [36], reinforcing the versatility of the nisin–biogel use in the biomedical field.

#### **5. Conclusions**

Periodontal disease is an inflammatory disease that is highly prevalent in dogs. The development of effective measures to control this disease is essential. Previous in vitro studies have revealed that the nisin–biogel is a safe antibiofilm agent with the potential to be used in canine PD control. Our study aimed to evaluate the in vivo efficacy of the nisin–biogel in dogs. Nisin–biogel showed the ability to reduce the DPI, GI, and PPD without any adverse effects on the animals in the study. These results suggest that the topical dental application of nisin–biogel may be used as an adjuvant measure for the prevention and control of canine PD. In the future, a large in vivo trial would be useful to fully validate this compound for commercial use. In addition, considering the high similarity between dog and human PD [31,32], nisin–biogel could also be a valuable compound to use in human dentistry.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/pharmaceutics14122716/s1, Supplementary File S1: Data blood analysis.

**Author Contributions:** Conceptualization, E.C, L.M.C. and M.O.; methodology, E.C., L.M.C. and M.V.; software, T.N. and E.C.; validation, E.C. and M.O.; formal analysis, E.C.; investigation, E.C., L.M.C., M.V. and A.S.V.; resources, L.T. and M.O.; data curation, M.O.; writing—original draft preparation E.C.; writing—review and editing, L.T. and M.O.; visualization, L.M.C. and M.O.; supervision, A.S.V. and M.O.; project administration, M.O.; funding acquisition, L.T. and M.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors would like to acknowledge the Foundation for Science and Technology (Eva Cunha Ph.D. fellowship SFRH/BD/131384/2017); CIISA—Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Project UIDB/ 00276/2020 (funded by FCT); and Laboratório Associado para Ciência Animal e Veterinária (LA/P/0059/ 2020—AL4AnimalS) for the financial support of this work. The funding bodies did not participate in the design, analysis, or report of this study.

**Institutional Review Board Statement:** All experimental procedures were approved by the Ethical Committee for Research and Teaching (CEIE) of the Faculty of Veterinary Medicine, University of Lisbon, Portugal (N/Ref 014/2020). This study complied with relevant institutional, national, and ARRIVE guidelines for the care and use of animals. All experiments were performed in accordance with relevant institutional, national, and ARRIVE guidelines for the care and use of animals. Written informed consent was obtained from the institution responsible for the animals.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request.

**Acknowledgments:** The authors would like to acknowledge all technical workers at the Casa dos Animais de Lisboa and the Centro de Medicina Veterinária Anjos de Assis for their support in this study.

**Conflicts of Interest:** The authors declare no conflict of interest.
