**1. Introduction**

Periodontal disease (PD) is one of the most prevalent inflammatory diseases in dogs [1]. Affecting over 80% of animals over two years old, PD is initiated by the formation of a polymicrobial biofilm on the tooth surface; this is also known as dental plaque and is responsible for a subsequent local host inflammatory reaction [1,2]. PD can evolve from a reversible stage of gingivitis to an irreversible stage of periodontitis [3]. The periodontal damage can be assessed by evaluating gingivitis, furcation, and mobility indices or stages and by measuring the periodontal pocket depth and the clinical attachment level [4]. According to the severity of the periodontium damage, four stages can be considered in PD classification. Stage 1 includes animals with only gingivitis; stage 2 includes animals with early periodontitis with less than 25% of attachment loss and/or stage 1 furcation; stage 3 comprises animals with moderate periodontitis, revealing an attachment loss of 25 to 50% and/or stage 2 furcation involvement; and the final stage, stage 4, includes dogs with advanced periodontitis, more than 50% of attachment loss and/or stage 3 furcation [4,5]. Besides damaging the periodontium, the persistent contact of dental plaque bacteria with periodontal structures facilitates their migration to the bloodstream and, consequently, the appearance of PD-related systemic consequences in distant organs [6–9].

**Citation:** Cunha, E.; Carreira, L.M.; Nunes, T.; Videira, M.; Tavares, L.; Veiga, A.S.; Oliveira, M. In Vivo Evaluation of the Efficacy of a Nisin–Biogel as a New Approach for Canine Periodontal Disease Control. *Pharmaceutics* **2022**, *14*, 2716. https://doi.org/10.3390/ pharmaceutics14122716

Academic Editors: Scavello Francesco, Jean-Eric Ghia and Amiche Mohamed

Received: 28 October 2022 Accepted: 1 December 2022 Published: 4 December 2022

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Several strategies can be used for PD control. Professional removal of the dental plaque along with the application of measures that inhibit dental plaque formation, such as daily toothbrushing (the gold standard method), application of chemical or natural anti-plaque compounds, administration of dental treats and biscuits, or a specific dental diet, are useful for PD control [4]. However, when periodontitis is present, apart from the previously mentioned measures and according to the case severity, PD treatment may include specific surgical approaches, the use of host modulation drugs, and even antimicrobial therapy [5].

Recently, an innovative approach based on the application of the antimicrobial peptide nisin incorporated in a delivery system formed by guar gum gel (nisin–biogel) has shown inhibitory and eradication abilities against pre-formed biofilms composed by PD enterococci, as well as towards canine dental plaque polymicrobial biofilms, in vitro [10,11]. Previous studies have also revealed that nisin–biogel activity is maintained in the presence of canine saliva and over a long-term storage period at distinct temperatures. Furthermore, nisin–biogel has shown an absence of cytotoxicity up to 200 μg/mL towards several cell lines, reinforcing the potential of nisin–biogel as a promising compound for canine PD control [12,13].

The main goal of this in vivo study was to evaluate the influence of the long-term dental application of the nisin–biogel on the dental plaque and gingivitis indices, the periodontal pocket depth, and total oral bacterial counts in dogs through a randomized controlled clinical trial. In addition, hematological and biochemical parameters were monitored during the trial, as well as general side effects.

## **2. Materials and Methods**
