Abstract:

The use of probiotics has gained increasing attention as a strategy for wound healing to decrease microbial resistance to disinfectants and antibiotics. This study aimed to investigate the potential of a non-medicinal topical cocktail of probiotic bacteria (CPB) in promoting wound healing in dogs using in vitro scratch assay. Canine Progenitors Epidermal Keratinocytes (CPEK) were exposed to a prototype product formulated with CPB (PPP), non-formulated CPB, and the vehicle. The viability of CPB and CPEK cells was first evaluated in the co-culture model. Then, wound closure was analyzed over time. The CPB required a minimum concentration of 75 CFU/mL for better viability with CPEK. While the CPEK preserved 100% of their viability when PPP was diluted to up to 75,000 CFU/mL. At higher concentrations, the viability of CPEK was reduced by the concomitant effect of the non-formulated CPB and the vehicle. The formulated and non-formulated CPB and the vehicle seem to lead to a dose-dependent increase in cell migration compared to the control. Importantly, at the concentration of 750,000 CFU/mL, the PPP showed a 20% increase in wound closure. Taken together, our findings suggest the potential beneficial effects of the probiotic-based topical cocktail (PPP) on wound healing. However, to confirm and validate these effects, further experiments are necessary to provide more robust evidence and allow us to confidently establish the potential beneficial effects of the probiotic bacteria (CPB) in promoting wound healing.

Introduction

Dogs are a favorite household pet. Their number was estimated to be 90 million in 88 million pet-owning households in Europe (European Pet Food Federation, 2021). They are present in 65.1 million households in the U.S. (© Statista 2023). Regardless of their lifestyle, dogs are prone to accidents and injuries which disrupt the skin tissue continuity giving place to wounds. Independently of their severity, wounds require the immediate attention of the owner and the veterinarian. In all vertebrates, the skin is a barrier that plays a crucial role in maintaining homeostasis and preventing the invasion of pathogens and chemical and physical insults. The damaged skin is susceptible to microbial invasion, leading to infected wounds. Instantly, the skin activates the wound healing as a self-regeneration after damage to restore the protective barrier. The process of healing is complex and involves inter-cellular interactions, growth factors, and cytokines. It encompasses overlapping phases starting with hemostasis, then inflammation, reparation, and maturation [1–3]. In veterinary practices, wound management is carried out with conventional antiseptic solutions (e.g., sterile saline, diluted chlorhexidine, or diluted betadine) and topical antimicrobial agents [1,2]. These solutions eliminate beneficial bacteria and pathogens, disrupting the balance of the skin microbiota [4]. In addition, the activation of immune protection leads to the perturbance of the microbiota balance on the skin. To address this challenge, the use of probiotics has emerged as a promising approach to regulate inflammation and microbiota balance to potentially enhance the healing process [5–8]. Probiotics
have shown promising results in promoting wound healing in vivo animal models using for example L. plantarum, kefir, L. fermentum, and S. cerevisiae to treat burn or surgical wounds [9]. In a meta-analysis by Tsiouris et al. in 2017 [10], the sterile kefir extract and bacteria probiotic therapies (70% kefir gel, L. brevis, L. fermentum, L. plantarum, L. reuteri) were found to be accelerators of wound contraction. For improved efficacy, probiotics could be combined with other techniques, e.g., nanotechnology-based techniques [11].

In this study, our primary objective was to assess the potential of a non-medicinal topical cocktail of probiotic bacteria (CPB) in promoting wound healing in dog keratinocytes to provide support for the development of a probiotic-based topical cocktail intended for canine care purposes. Specifically, we focused on evaluating the influence of probiotics on cell migration in vitro using dog keratinocytes and scratch wound healing assay. The investigation involved multiple aspects. Firstly, we conducted viability tests on both probiotics and keratinocytes in co-culture to ensure their compatibility and assess their overall health. Subsequently, we investigated the wound healing process using the scratch test, allowing us to observe and analyze the effects of the CPB on cell migration and wound closure. Additionally, we examined the immune stimulation of keratinocytes through the quantification of cytokine expression levels. Through these comprehensive evaluations, we aimed to gain insights into the potential wound healing properties of the CPB and provide a scientific basis for the development of the PPP for use in canine care.

Results

Viability of Probiotic Bacteria in Culture Conditions

The CPEK were exposed to non-formulated CPB at three different concentrations: 75, 750, and 7500 CFU/mL for a duration of 24 h. When exposed to the concentration of 7500 CFU/mL, there were very few instances of dead bacteria, accounting for only 2% (Figure 1). However, at concentrations of 750 CFU/mL and 75 CFU/mL, a significantly higher number of dead bacteria were observed, representing 81% and 84%, respectively. Moreover, the concentration of 7500 CFU/mL exhibited a significantly greater number of viable bacteria compared to the lowest concentration of 75 CFU/mL (279 versus 5). This finding supports the inverse relationship between the number of viable bacteria and the number of dead bacteria, as higher concentrations result in fewer instances of dead bacteria.

Figure 1. Live / dead baterial viability assay of non-formulated cocktail probiotic bacteria. Canine Progenitors Epidermal Keratinocytes (CPEK) cultured in a 96-well plate were treated with nonformulatedprobiotic bacteria at 75, 750, or 7500 CFU/mL for 24 h. The bacteria were then stained with the BacLight™ double staining kit. The live and dead bacteria exhibited green and red fluorescence, respectively. The scale bar represents 100 μm.

Effect of the Cocktail of Probiotic Bacteria on the Viability of CPEK Cells

The CPEK were exposed to various concentrations of both formulated and non-formulated cocktail of probiotic bacteria, ranging from 7.5 to 7.500.000 CFU/mL, while untreated cells were used as a control. Following a 24 h period of co-culture with nonformulated CPB, a decrease in cell viability was observed (e.g., viability of 63.6% in the co-culture with 750,000 CFU/mL) only at concentrations above 75 CFU/mL of CPB (Figure 2). However, at lower concentrations of 75 CFU/mL and 750 CFU/mL of CPB, minimal to no cytotoxicity was observed. However, 100% of the viability of cells was maintained in the co-culture with up to 75,000 CFU/mL of formulated CPB. The highest concentration of the latter induced a higher decrease in the viability of cells than the non-formulated CPB, 44.8% and 77.2%, respectively. Taken together, these results indicated that the vehicle of formulated probiotic bacteria protected the CPEK from CPB toxicity when diluted to up to 75,000 CFU/mL This protection decreased with higher concentrations and could induce 32.4% of toxicity to CPEK in addition to the 44.8% of non-formulated CPB.

Figure 2. MTT cell viability assay. Canine Progenitors Epidermal Keratinocytes (CPEK) wereuntreated or treated with formulated or non-formulated probiotic bacteria at various concentrationsfor 24 h, and cell viability was determined using a colorimetric MTT assay. The results revealed that both the formulated and non-formulated probiotic bacteria were well tolerated by CPEK cells, except at very high concentrations. Here, the control (untreated) group is referred to as 100% viable cells. Data are presented as the mean  standard deviation of one experiment conducted in duplicate (N = 2). * indicates (p < 0.05).

 Effect of the Cocktail of Probiotic Bacteria on Cytokine Expression in CPEK Cells

The CPEK were exposed to non-formulated CPB at a concentration of 7500 CFU/mL for a duration of 24 h. Following the treatment, the mRNA expression levels of the inflammatory cytokine markers, namely IL-8, IL-6, and TNF-, were assessed using quantitative real-time RT-PCR. The findings depicted in Figure 3 demonstrate that the exposure to CPB at 7500 CFU/mL resulted in a substantial upregulation of IL-6, IL-8, and TNF-a mRNA expression, with fold increases of 3, 17, and 136, respectively. Moreover, an increase in IL-8 mRNA expression was observed even at the lowest concentration of 75 CFU/mL.

Figure 3. Effect of the cocktail of probiotic bacteria on mRNA expression of cytokine markers in
CPEK cells. Canine Progenitors Epidermal Keratinocytes (CPEK) were exposed to non-formulated
probiotic bacteria at 7500 CFU/mL for 24 h. mRNA expression of IL-6, IL-8, and TNF-α was
measured using quantitative RT-PCR.

Effect of the Cocktail of Probiotic Bacteria on Wound Healing

The CPEK were exposed to different treatments, including formulated and nonformulated CPB, as well as the vehicle used in the formulation of probiotics. The
experimental setup was validated by comparing the wound closure in the positive control
treated with taxol and the untreated control (Figure 4). In fact, the control of untreated
cells had an AUC (0 h–7 h) of 395 AU·h, whereas cells treated with 100 nM of taxol
exhibited an AUC (0 h–7 h) of 88 AU·h.

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