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The 3-D configuration of excisional skin wound healing after topical probiotic application

Open AccessPublished:February 05, 2022DOI:https://doi.org/10.1016/j.injury.2022.02.006

      Abstract

      Nowadays, there is an increasing knowledge that probiotic bacteria, topically applied, affects skin pathology. The objective of this study is to evaluate the effect on wound healing of locally applied probiotics by calculating the 3-D configuration of a standardized excisional wound. Fifty-two male Wistar rats were randomly allocated into groups: control, PRO1 [L. plantarum] and PRO2 [L. rhamnosus, B. longum]. Six excisional full-thickness wounds were created on each dorsum by an 8-mm circular biopsy punch; probiotics or saline were applied on days 0, 2, 4, 8, 16, photos of the wounds taken and specimens excised for histology [4 rats/group/time-point]. Both probiotic-groups exhibited accelerated healing significantly faster than the control, throughout, PRO2 exhibiting finally the best results [day 16]. However, only on day 2, did PRO1 exhibit the best results [wounded area, borders distance and epitheliazation line]. The results clearly demonstrate that the topical application of probiotics significantly improves the healing process, each strain working differently and more effectively in different healing phases. Thus, a combined formula containing different probiotics to modulate various healing phases is desirable. To this end our research continous.

      Keywords

      Introduction

      Wound healing is an evolutionary, complex, curative response to tissue injury, aiming to restore tissue continuity and resistance to externally applied forces. In the case of skin injury, quick self-repair is vital to prevent the entrance of harmful micro-organisms, to avoid blood loss and body dehydration and to restore skin barrier function; even more to achieve tissue functionality and cosmetic outcome [
      • Burger B
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      Oral administration of EPA-rich oil impairs collagen reorganization due to elevated production of IL-10 during skin wound healing in mice.
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      Skin wound healing: an update on the current knowledge and concepts.
      ].
      After injury, the skin, besides the obvious loss of integrity, exhibits changes in the beta-diversity of resident microbiota [
      • Johnson TR
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      • Dubick MA
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      The cutaneous microbiome and wounds: new molecular targets to promote wound healing.
      ], which normally act as a first line of defense by producing anti-microbial peptides and by boosting the immune arsenal of toll-like receptors, Langerhans cells and T cells [
      • Tavaria FK.
      Topical use of probiotics: the natural balance.
      ]; these perturbations in the local microbiota might accelerate a chain of serious processes, including the dysregulation of the immune and inflammatory response and the delay in re-epitheliazation and fibrin deposition, in cellular/molecular basis [
      • Johnson TR
      • Gómez BI
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      The cutaneous microbiome and wounds: new molecular targets to promote wound healing.
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      Skin wound healing is accelerated and scarless in the absence of commensal microbiota.
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      ] as well as, in clinical conditions, those of bacterial colonization, local wound infection, and even clinical sepsis.
      Both in-vivo and in-vitro studies, as well as clinical applications which target the microbiome and its restoration, have supported a general consensus that the topical appliance of probiotics on skin wounds, of any kind, positively stimulate the wound healing process [
      • Poutahidis T
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      Microbial symbionts accelerate wound healing via the neuropeptide hormone oxytocin.
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      Probiotics and prebiotics in dermatology.
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      ,
      • Knackstedt R
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      The role of topical probiotics on wound healing: a review of animal and human studies.
      ]. However, there still remain many unanswered questions; there is a marked heterogeneity among studies regarding the insult investigated, the type and dosing regimen of the probiotic utilized, and a lack of standardized outcome measures [
      • Knackstedt R
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      • Tsiouris CG
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      The efficacy of probiotics as pharmacological treatment of cutaneous wounds: Meta-analysis of animal studies.
      ], thus further research is clearly required.
      Since it has been variously documented that different probiotic strains exert profound anti-inflammatory properties, as in thermal injuries [
      • Oryan A
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      Kefir accelerates burn wound healing through inducing fibroblast cell migration in vitro and modulating the expression of IL-1ß, TGF-ß1, and bFGF genes in vivo.
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      • Argenta A
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      Local application of probiotic bacteria prophylaxes against sepsis and death resulting from burn wound infection.
      ], in reactive skin [
      • Guéniche A
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      ] or after CO2 laser therapy [
      • Zoccali G
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      • Romano L
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      Improving the outcome of fractional CO2 laser resurfacing using a probiotic skin cream: preliminary clinical evaluation.
      ], it is supposed that they work mainly in the first, i.e., the inflammatory, phase of healing; as well as on fibroblast differentiation and collagen production. Hence, the object of this study is to evaluate and compare the phase-by-phase wound healing properties of different probiotics, by means of calculating and describing the 3-D configuration of a standardized excisional wound-model in male Wistar rats, throughout the wound healing process.

      Material and methods

      Probiotic treatment

      The probiotics used in the present study, the decision based on previous literature, were: Lactobacillus plantarum UBLP-40, and a combined formula of Lactobacillus rhamnosus UBLR-58 and Bifidobacterium longum UBBL-64. They were delivered as a fresh purified stock culture in the form of a dry powder, containing 1011cfu/gr; donated by the Pharmaceutical Company UniPharma, SA, Greece. For its use in the present experiment, a volume of 0.3 mL of normal saline 0.9% was freshly added to each pre-weighed gram of dry probiotic culture, making it a type of “ointment”, ready for application to the wound.

      Probiotics viability, identity and purity testing

      In order to verify the viability of the probiotic strains to be used, a trypan-blue staining was performed, on the basis of its ability to penetrate dead cells, staining them blue [
      • Strober W.
      Trypan blue exclusion test of cell viability.
      ]. The probiotic strains were reconstituted in phosphate-buffered saline [PBS; Sigma-Aldrich, St. Louis, Missouri, United States] at a final concentration of 109cfu/mL and were incubated at 37 ⁰C for 15 min. Probiotics were then stained with trypan blue [Gibco, Waltham, Massachusetts, United States] for 3 min and then observed under a brightfield microscope [Leica DM2000, Leica Microsystems GmbH, Germany].
      In order to verify the identity and purity of the probiotic strains, Gram staining was then performed [
      • Tarapatzi G
      • Filidou E
      • Kandilogiannakis L
      • Arvanitidis K
      • Vradelis K
      • Kotzampassi K
      • Kolios G.
      The effect of a probiotic mix on mucosal healing and fibrotic responses in healthy colonic subepithelial myofibroblasts.
      ], since it is already well known that all these strains are Gram-positive [
      • Goldstein E.J.C.
      • Tyrrell K.L.
      • Citron D.M.
      Lactobacillus species: taxonomic complexity and controversial susceptibilities.
      ,
      • Lee J.H.
      • O'Sullivan D.J.
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      ]. Briefly, probiotics were first heat-fixed on a microscope slide, stained with Crystal violet for 1 min and then washed with tap water. Iodine solution was added for another 1 min and then washed off with tap water. Decolorizer was added for a few seconds and then washed off, again with tap water. Finally, safranin was added for 30 s, washed off with tap water and the slides were then observed under a brightfield microscope [Leica DM2000, Leica Microsystems GmbH, Germany]. All reagents were purchased from Sigma-Aldrich, St. Louis, Missouri, United States.

      Animals

      Fifty-two healthy male Wistar rats weighing 200–250 g were housed individually in polypropylene cages under controlled light, temperature and humidity conditions; a 12-h light-dark cycle, at 22±2 °C and 50±2% humidity. Water and food [standard laboratory chow diet] were provided ad libitum and the rats allowed to acclimatize for a week. Housing, anesthesia, wound induction and post-operative care complied with the European Guidelines for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Local Governmental Committee for the Control and Supervision of Experiments on Animals [EU Directive 2010/63/EU, Protocol registration number 227933(934)/06.05.2021].

      Wound induction

      On experimentation day, all the rats were anaesthetized intraperitoneally with a mixture of ketamine [100 mg/kg] and xylazine [10 mg/kg]; the dorsal skin was shaved with an electrical clipper, rinsed with an alcohol swab and sterilely prepped with betadine, and finally draped with sterile sheets.
      Six full-thickness open excision wounds extending through the panniculus carnosus were surgically performed in the dorsal skin, using a sterile 8 mm diameter biopsy punch. Excisional wounds were created on each side of the midline, starting 2 cm from the ears, the next two pairs being 1 cm apart and 1 cm below the previous. A blinded operator filled the excisional wound area with either 0.3 mL of normal saline 0.9% or freshly prepared probiotic ointment by means of a metallic dentist spatula according to the allocation group [1011cfu/gr per rat]. Treatment was re-applied every two days, the rats being under ether anesthesia (Fig. 1A, 1B).
      Fig. 1
      Fig. 1A. Full-thickness open excision wounds just filled [right side] with the freshly prepared probiotic “ointment”, while the left side are empty yet.
      B. Freshly prepared probiotic “ointment”.
      The total wounded area, after the appropriate treatment application, was allowed to heal by secondary intension. Wounds were immediately covered with sterile gauze dressing reinforced with a self-adhesive bandage and the rats were returned to their own individual cages to recover anesthesia.

      Study design

      Rats, by means of random numbers, were allocated as follows: four rats, serving as baseline; 16 rats as placebo– control group [CNTR]; 16 rats as single probiotic-treatment [L. plantarum] group [PRO1]; and 16 rats as duo-probiotics [L. rhamnosus and B. longum] treatment group [PRO2].
      The baseline-group rats, upon wound induction, were photographed and immediately sacrificed with an anesthesia-formula overdosing, being thus used as day 0 for all groups. The wounded tissue was en-bloc — full-thickness skin — excised with 1 cm normal skin surrounding margins; then specimens pinned flat on a cork-board and immersed in buffered formalin for further processing. For the remaining rats, photographs were similarly taken on days 2, 4, 8 and 16. On these time-points, a total of four rats of each group were sacrificed and tissue specimens were similarly excised and treated (Table 1).
      Table 1Study Design: Number of rats in each group, divided in the time points for photography, scarification and tissue specimen obtained.
      daysgroups024816
      baseline4----
      CNTR-4444
      PRO1-4444
      PRO2-4444

      Wound healing studies

      Photography

      Digital photographs were taken on days 0, 2, 4, 8 and 16, by using a high-resolution camera and lens [Cannon EOS-50D, EF-100mmf/2.8 L Macro IS USM lens], with a standardized focus and consistent room lighting. Photographs were captured perpendicular to the wound surface and included a paper-ruler tape in the same plane as the wounds as a comparison of their actual sizes [
      • Galiano RD
      • Michaels 5th, J
      • Dobryansky M
      • Levine JP
      • Gurtner GC
      Quantitative and reproducible murine model of excisional wound healing.
      ,
      • Kim BS
      • Breuer B
      • Arnke K
      • Ruhl T
      • Hofer T
      • Simons D
      • Knobe M
      • Ganse B
      • Guidi M
      • Beier JP
      • Fuchs PC
      • Pallua N
      • Bernhagen J
      • Grieb G.
      The effect of the macrophage migration inhibitory factor (MIF) on excisional wound healing in vivo.
      ]. Each rat study number, being a combination of the group, the study time-point and the number of the rat, was written on the paper tape.
      A blinded single observer performed the analysis of digital photographs by tracing each wound margin by means of a fine-resolution computer mouse and thus calculating the pixel area, which was then converted to square mm, using the free image analysis software Image J [Image J, Bethesda, MD, http://imagej.nih.gov/ij/]. The wound area at each time-point was additionally expressed as a percentage closure of the original wound: wound size at baseline in mm2 minus wound area on a specific day in mm2, divided by wound size at baseline in mm2 X 100 [
      • Lombardi F
      • Palumbo P
      • Mattei A
      • Augello FR
      • Cifone MG
      • Giuliani M
      • Cinque B.
      Soluble fraction from lysates of selected probiotic strains differently influences Re-epithelialization of HaCaT scratched monolayer through a mechanism involving nitric oxide synthase 2.
      ].

      Histology

      Four wounds per rat were sectioned longitudinally from one edge to the other to well past the center of the wound, at its deepest point, keeping a 3 mm margins of peripheral [surrounding] unwounded skin. All specimens underwent routine histological processing with hematoxylin and eosin staining. Digital images at X2 magnification were obtained from four [two consecutive slices from each half of the wound], 3 μm sections, judged to be at the actual center of each wound and used for measurements of various healing related lengths; each one then averaged to one measurement over sections to provide a representative value for each wound [
      • Weinheimer-Haus EM
      • Mirza RE
      • Koh TJ.
      Nod-like receptor protein-3 inflammasome plays an important role during early stages of wound healing.
      ]. We thus have a total of 16 measurements per group, per time-point. A Nikon ECLIPSE E600 microscope and a Nikon CFI Plan Achromat UW 2x/0.06 objective lens providing a very large field of view was used. A Nikon DS-Fi1 digital color camera mounted to the microscope was also used and the measurements performed by means of the Image J image analysis software, as previously.
      Based on the mathematical model for the healing and re-modeling described by Lemo et al in rabbits [
      • Lemo N
      • Marignac G
      • Reyes-Gomez E
      • Lilin T
      • Crosaz O
      • David M
      • Ehrenfest D
      Cutaneous reepithelialization and wound contraction after skin biopsies in rabbits: a mathematical model for healing and remodelling index.
      ] we decided to measure the following healing related lengths: [i], the distance between the borders of the wound, following the straight line of the epidermis, assigned S; [ii], the length of the re-epitheliazation zone, i.e. the length of tissue between the borders of the wound, assigned L; [iii], the depth of the wound, from the epidermis line [S] to the first connective tissue layer at the deepest point of the wound, assigned D; and [iv], the thickness of the natural dermis, from the epidermis to the muscle in the healthy area surrounding the wound, assigned N.
      From these measurements, the Superficial Contraction Index [SCI = (L – S)/L] and the Deep Contraction Index [DCI = (N – D)/N were then calculated for each wound and their sum expressed in the Wound Contraction Index WCI = SCI + DCI. Theoretically, during the healthy healing process, the SCI decreases due to reduction of L and S and the DCI increases due to decrease of D [
      • Lemo N
      • Marignac G
      • Reyes-Gomez E
      • Lilin T
      • Crosaz O
      • David M
      • Ehrenfest D
      Cutaneous reepithelialization and wound contraction after skin biopsies in rabbits: a mathematical model for healing and remodelling index.
      ].

      Statistical analysis

      The normality of data was verified by the Shapiro-Wilk test. One-way analysis of variance [ANOVA] was used for between and within group comparisons, followed by Tukey–Kramer and LSD [post Hoc tests]. Data are presented as mean ± SD. For statistical analysis purposes GraphPad Prism software version 9.2.0 for Windows, (GraphPad Software, San Diego, California USA, www.graphpad.com) was used, while a P value of less than 0.05 was considered statistically significant.

      Results

      Probiotics viability, identity and purity testing

      As seen in Fig. 2 (A, B and C), trypan blue staining revealed that the probiotics were alive after their reconstitution, as no positive trypan blue-stained bacteria were found. As also expected, all probiotic strains were stained Gram-positive, suggesting that the probiotics used in this study were pure and uncontaminated by other Gram-negative bacteria (Fig. 3 A, B and C).
      Fig. 2
      Fig. 2Viability examination of probiotic strains L. plantarum, L. rhamnosus and B. longum using trypan blue. Trypan blue staining revealed no positive-stained bacteria, suggesting a high viability rate among the different strains. Representative 100x brightfield snapshots of L. plantarum (A), L. rhamnosus (B) and B. longum (C) are shown.
      Fig. 3
      Fig. 3Gram identification of probiotic strains L. plantarum, L. rhamnosus and B. longum. Gram staining revealed only positive-stained bacteria, verifying their identity and purity, as no Gram-negative bacteria were found. Representative 100x brightfield snapshots of L. plantarum (A), L. rhamnosus (B) and B. longum (C) are shown.

      Wound healing studies

      Photography

      The wound area revealed a significant reduction in rats treated with probiotics in relation to control treatment [p<0.0001 on day 16]. By comparing the two probiotics groups, PRO1 and PRO2, we observed that healing began on day 2 in the PRO2 in relation to control [p=0.006], but afterward there was less significant progress up to day 4 in relation to the progress of PRO1 [PRO1 vs PRO2, p=0.001]; thereafter both PRO1 and PRO2 groups exhibited equal reduction [p<0.0001] in the wounded area, culminating on day 16 with a small, non-significant difference over the PRO2. Detailed results are shown in Fig. 4, Fig. 5A.
      Fig. 4
      Fig. 4Photos of the excisional wound in the 3 study-groups: Controls, PRO 1 and PRO 2 groups. The step-by-step progress of healing throughout the days 0 to 16, as well as the difference in healing rate among groups in the same time-point are prominent.
      Fig. 5
      Fig. 5A. AREA: Comparative presentation of wound area [in mm2] of all study groups, throughout the experimentation study-periods [days 0, 2, 4, 8 and 16]. B. S-DISTANCE: Comparative presentation of the distance S of epidermis gap of all study groups [in mm], throughout the experimentation study-periods [days 0, 2, 4, 8 and 16]. C. L-DISTANCE: Comparative presentation of the length L of the re-epithelization zone [in mm] of all study groups, throughout the experimentation study-periods [days 0, 2, 4, 8 and 16]. D. SCI: Comparative presentation of the Superficial Contraction Index SCI [in mm] of all study groups, throughout the experimentation study-periods [days 0, 2, 4, 8 and 16]. E. D-DISTANCE: Comparative presentation of the wound depth D [in mm] of all study groups, throughout the experimentation study-periods [days 0, 2, 4, 8 and 16]. F. DCI: Comparative presentation of the Deep Contraction Index DCI [in mm] of all study groups, throughout the experimentation study-periods [days 0, 2, 4, 8 and 16]. G. SCI + DCI: Comparative presentation of the Wound Contraction Index WCI [in mm] of all study groups, throughout the experimentation study-periods [days 0, 2, 4, 8 and 16].
      By considering the initial wound area, baseline, as 100%, and transforming the results into percentage of healing area in relation to the initial area, it becomes more clear that over the days of healing from day 0 to day 2, 4, 8 and 16 we observed a progress in healing, [i.e. the percentage of shrinkage] of 11.5%, 12.2%, 51.8% and 75.7% in control group. In the PRO1 group the progress of healing begins dynamically: 20% on day 2 and continues to 41.2%, 58.8% and 93.8%; surprisingly, PRO2 treatment begins more slowly by healing 22% and 29.5% on days 2 and 4, respectively, and then speeds up immediately after to 77.2% and finally to 96.1% at days 8 and 16.

      Histology

      The distance S of epidermis gap — L. plantarum more effective in the early phase of healing

      Measurements of the distance S between the borders of the wound revealed a significant tendency to decrease, in both control and the probiotics-treated groups, which finally resulted in the lowest overall value, on day 16, achieved in the PRO2 group. However, what is worth mentioning is that on day 2 a significant decrease [p<0.0001] was found compared to the control in the L. plantarum treated rats. After day 2 the PRO2 group achieved a greater reduction at each time-point compared to PRO1, but the difference became statistically significant [p=0.048] only on day 16. [Fig. 5B]

      The length L of the re-epithelization zone — the double probiotic better than one

      In the fresh specimen, upon creation of the skin wound, the L line represents the sum of 2.5+8+2.5 mm [wound depth plus diameter]; however, in the processed specimen this distance is equal to 12.8±0.82 mm. After two days, on day 2, there was a significant reduction [p=0.04] in only the PRO1 group in relation to the control, the finding relating to the corresponding reduction of S distance in the same group on day 2. Thereafter, a highly significant decrease [p<0.0001] was observed on days 4, 8 and 16 in PRO2 group in relation to the control, and a similar downward trend, but less marked, in the PRO1 group. [Fig. 5C].

      Superficial contraction index — the double probiotic work more slowly, but steadily

      The Superficial Contraction Index revealed a rather weak contraction, since there is no statistical difference either among consecutive time-points or between groups at the same time-point, a fact that can be interpreted as evidence that probiotics do not interfere in the healing process through the tissue contraction mechanism. However, assuming this to be reliable since there is no statistically significant difference but only a trend, the two probiotics seem not to share the same mechanism of action: the L. plantarum values increased up to day 4 — self-explained by the decrease of S value on day 2 — and then began declining to day 16 and achieving the lowest value ever. On the other hand, the combo regimen L. rhamnosus plus B. longum seems to make a slight steady increase, however, until day 16, when it has statistically significantly exceeded the value of PRO1 [Fig. 5D].

      The wound depth D — the double probiotic works towards deep healing

      A significant reduction [p=0.046] of D was prominent as early as day 4 in relation to day 2, followed by further decrease between days 4 and 8 [p<0.0001], as well as days 8 and 16 [p<0.0001] in the PRO2 group. It is of interest that from day 4 and thereafter D similarly reduced in the PRO1 group; however there was a highly statistically significant difference [p<0.0001] in the D values between PRO1 and PRO2 on days 8 and 16; the L. plantarum alone never succeed in reaching the low values achieved by the combined regimen of L. rhamnosus plus B. longum. [Fig. 5E].

      The thickness N of the natural dermis

      The thickness of the healthy dermis, measured more than 5 mm from the wound border was found to range from 1.72±0.5 to 1.74±0.5 mm, with no statistically significance between values in all groups. This is mainly due to the homogeneity of the rats used, i.e., same age and gender. This parameter was measured only to be used as a factor in the equation for calculating deep contraction index.

      Deep contraction index DCI — the double probiotic positively affects deep healing

      The Deep Contraction Index revealed a highly significant increase [p<0.0001] on day 8 and thereafter, for the PRO1 and PRO2 groups only in relation to the control group; furthermore, PRO2 exhibited a further increase [p<0.001] in relation to PRO1, both on day 8 and day 16. However, the DCI of the control was found to be increased [p<0.0001] only on day 16 in relation to its value on day 8, while at the same time-point PRO1 and PRO2 DCI indexes were highly statistically significantly increased [p<0.0001], in relation the control [Fig. 5F].

      Wound contraction index WCI— PRO1 more effective in the early phase of healing, PRO2 more effective in deep healing

      The information derived from this formula is evidenced of the way PRO1 and PRO2 act. From day 4 to 8, significant healing was observed, in more or less all groups [control p=0.043, PRO1 and PRO2 p<0.0001], but from day 8 to 16 the RPO1 group remained rather stable in contrast to the PRO2 group, which was again found significantly increased [p<0.0001] [Fig. 5G].

      Discussion

      Skin wounds, defined as a disruption of cellular, anatomical and functional continuity of a living tissue [
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      ], are inherently associated with perturbations in the local microbiome, because of both the injury itself and the activation of the immune responses as a consequence of trauma [
      • Johnson TR
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      ]. Such wounds, and notably, excisional wounds, independently of a small or extensive skin loss, remain a major problem in surgical procedures, since skin barrier function must be quickly and promptly restored in order to prevent further damage or infection and to achieve an aesthetically acceptable scar.
      Nowadays, there is an increasing knowledge that beneficial bacteria residing on the skin ‒ or in the gut, if we accept Arck's concept [
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      ] of “gut-brain-skin axis” ‒ are involved in the complex process of defense against pathogens and in tissue healing. This operates in various ways, especially through the production of antimicrobial molecules and the regulation of immune and inflammatory responses [
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      ]; thus, an intuitive means to prevent infection, regulate inflammation, restore skin homeostasis and potentially augment healing should be the modulation of the microbiome, by means of the topical application of probiotics.
      According to the current definition endorsed by both the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO), as well as the International Scientific Association for Probiotics and Prebiotics (ISAPP), “probiotics are live microorganisms that, when administered in adequate amounts, confer a health effect on the host” [
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      ]. In vitro and in vivo experiments have already indicated that probiotics exert potential suppressive effects on different infectious, immune-related as well as inflammatory conditions. They have the ability to control inflammation and induce local and systemic immune responses, while various positive stimulatory, and even mutually reversible effects on wound healing have been reported in a number of studies [
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      • Satish L
      • Gallo PH
      • Johnson S
      • Yates CC
      • Kathju S
      Local probiotic therapy with lactobacillus plantarum mitigates scar formation in rabbits after burn injury and infection.
      ]. However, there is a marked heterogeneity among studies: the probiotic strain or the combination of strains, the concentration of probiotic utilized, the dosing, the treatment duration, also the study purpose, the model under investigation, the time-plan for repeated measurements, the desired outcome, etc, make it difficult to move toward a consensus on how exactly probiotics work, and, much more, what is the most appropriate strain for particular treatment. Thus, further research is clearly mandatory [
      • Lee GR
      • Maarouf M
      • Hendricks AJ
      • Lee DE
      • Shi VY.
      Topical probiotics: the unknowns behind their rising popularity.
      ].
      In the present study we aimed to assess the wound healing properties and mainly the way that probiotics interfere in the wound healing process of an acute wound, by using a totally different methodological approach. Since the acute wound healing is a complex, highly dynamic, multi-factorial process involving three precisely and highly programmed, overlapping stages, namely hemostasis/inflammation, proliferation, and tissue remodeling [2, 25, 27, driven by a complex sequence of cellular and hormonal/biochemical signaling [
      • Rodrigues M
      • Kosaric N
      • Bonham CA
      • Gurtner GC.
      Wound healing: a cellular perspective.
      ], it is very difficult to assess the precise involvement of probiotics on the healing process, knowing well that each strain has separate, distinctive, even multi-factorial features of action [
      • Shavandi A
      • Saeedi P
      • Gérard P
      • Jalalvandi E
      • Cannella D
      • Bekhit AE.
      The role of microbiota in tissue repair and regeneration.
      ]. Thus, we decided to simply compare the three-dimensional transforming morphology of the excisional wound, throughout the healing process, namely on days 2, 4, 8 and 16, which cover the healing phases. In order to obtain a complete view of the continually changing area of the excisional wound we not only assessed it through consecutive digital photographs, as most studies already do [
      • Galiano RD
      • Michaels 5th, J
      • Dobryansky M
      • Levine JP
      • Gurtner GC
      Quantitative and reproducible murine model of excisional wound healing.
      ,
      • Masson-Meyers DS
      • Andrade TAM
      • Caetano GF
      • Guimaraes FR
      • Leite MN
      • Leite SN
      • Frade MAC.
      Experimental models and methods for cutaneous wound healing assessment.
      ], but we also measured the wound dimensions [
      • Lemo N
      • Marignac G
      • Reyes-Gomez E
      • Lilin T
      • Crosaz O
      • David M
      • Ehrenfest D
      Cutaneous reepithelialization and wound contraction after skin biopsies in rabbits: a mathematical model for healing and remodelling index.
      ], that is [i] the distance between the borders of the wound, [ii] the length of the re-epithelization zone, and [iii] the depth of the wound, in paraffin embedded specimens cut longitudinally at the actual center of each wound.
      The excisional wounds are one of the most frequently used wound healing models, considered to reliably resemble acute clinical wounds, requiring healing by second intention. Additionally, just from the practical point of view, an excisional wound is a cavity able to contain the topically applied treatment, which should ideally improve one or more phases of healing, without producing deleterious side effects. For this experiment our animals were chosen to be entirely male in gender, since wounds in males exhibit a greater tendency to close by epithelialization [
      • WA D.-M.
      Rat models of skin wound healing: a review.
      ], probably because of estrogen absence and the thicker and firmer skin, in comparison to females [
      • Zomer HD
      • Trentin AG.
      Skin wound healing in humans and mice: challenges in translational research.
      ,
      • Mohtashami M
      • Mohamadi M
      • Azimi-Nezhad M
      • Saeidi J
      • Nia FF
      • Ghasemi A.
      Lactobacillus bulgaricus and Lactobacillus plantarum improve diabetic wound healing through modulating inflammatory factors.
      ,
      • Simoes RM
      • Teixeira D
      • de Maldonado EPRW
      • Zezell DM.
      Effects of 1047-nm neodymium laser radiation on skin wound healing.
      ].
      In the present study we decided to use probiotics, which generally seem to exert beneficial effects for skin healing in human clinical studies, even though their — strain-specific — potential benefits on wound healing are still unknown [
      • Lee GR
      • Maarouf M
      • Hendricks AJ
      • Lee DE
      • Shi VY.
      Topical probiotics: the unknowns behind their rising popularity.
      ]. The probiotics tested in the present study were: The Lactobacillus plantarum [1011CFU/gr dry powder] as monotherapy and the laboratory mixture of Lactobacillus rhamnosus and Bifidobacterium longum [1011CFU/gr dry powder for each]; the decision for their selection and dosing arising from previous in vitro and in vivo studies [
      • Zahedi F
      • Heydari Nasrabadi M
      • Tajabadi E
      • Aboutalebi H
      Comparison of the effects of Lactobacillus brevis and Lactobacillus plantarum on cutaneous wound healing in rats.
      ,
      • Heydari Nasrabadi M
      • Tajabadi E
      • Dehghan Banadaki Sh Torabi
      • Kajousangi M
      • Zahedi F.
      Study of cutaneous wound healing in rats treated with Lactobacillus plantarum on days 1, 3, 7, 14 and 21.
      ].
      Lactobacillus plantarum was found to significantly accelerate re-epithelialization by the NOS2/NO pathway [
      • Lombardi F
      • Palumbo P
      • Mattei A
      • Augello FR
      • Cifone MG
      • Giuliani M
      • Cinque B.
      Soluble fraction from lysates of selected probiotic strains differently influences Re-epithelialization of HaCaT scratched monolayer through a mechanism involving nitric oxide synthase 2.
      ] and by Plantaricin A production, which both stimulate keratinocyte migration and proliferation [
      • Mohammedsaeed W
      • Cruickshank S
      • McBain AJ
      • O'Neill CA.
      Lactobacillus rhamnosus GG lysate increases Re-epithelialization of keratinocyte scratch assays by promoting migration.
      ]. It is of interest that the scratch area in human keratinocytes monolayer was re-epithelialized at 16 h and 24 h by 74% and 96% compared to 47% and 72% (p < 0.0001) in a control [
      • Brandi J
      • Cheri S
      • Manfredi M
      • Di Carlo C
      • Vita Vanella V
      • Federici F
      • Bombiero E
      • Bazaj A
      • Rizzi E
      • Manna L
      • Cornaglia G
      • Marini U
      • Valenti MT
      • Marengo E
      • Cecconi D
      Exploring the wound healing, anti-inflammatory, anti-pathogenic and proteomic effects of lactic acid bacteria on keratinocytes.
      ], while an increase of tight barrier function of keratinocytes was also prominent [
      • Lukic J
      • Chen V
      • Strahinic I
      • Begovic J
      • Lev-Tov H
      • Davis SC
      • Tomic-Canic M
      • Pastar I.
      Probiotics or pro-healers: the role of beneficial bacteria in tissue repair.
      ]. Similarly, in in-vivo experiments, L. plantarum seems to play a role in shortening the early/inflammatory phase of the wound healing process [
      • Heydari Nasrabadi M
      • Tajabadi E
      • Dehghan Banadaki Sh Torabi
      • Kajousangi M
      • Zahedi F.
      Study of cutaneous wound healing in rats treated with Lactobacillus plantarum on days 1, 3, 7, 14 and 21.
      ]; and, in H&E staining keratinocyte migration over the wound edge towards the center was prominent over days 4 to 16, the better wound closure and complete re-epithelization achieved by day 12 [
      • Ong JS
      • Taylor TD
      • Yong CC
      • Khoo BY
      • Sasidharan S
      • Choi SB
      • Ohno H
      • Liong MT.
      Lactobacillus plantarum USM8613 aids in wound healing and suppresses staphylococcus aureus infection at wound sites.
      ].
      With regard to L. rhamnosus GG, given orally in a Swiss mouse model, it accelerates skin wound healing, as assessed by the epithelial tongue length and the epithelial gap extent, and reduces scar formation through reduction of inflammation and collagen deposition and increase of angiogenesis [
      • Moreira CF
      • Cassini-Vieira P
      • Canesso MCC
      • Felipetto M
      • Ranfley H
      • Teixeira MM
      • Nicoli JR
      • Martins FS
      • Barcelos LS.
      Lactobacillus rhamnosus CGMCC 1.3724 (LPR) improves skin wound healing and reduces scar formation in mice.
      ]. When applied topically, it increases re-epithelization, through induction of the chemokine CXCL2 and its receptor CXCR2, which stimulate keratinocyte proliferation and migration, the cell migration considered as the dominant mechanism of action [
      • Lombardi F
      • Palumbo P
      • Mattei A
      • Augello FR
      • Cifone MG
      • Giuliani M
      • Cinque B.
      Soluble fraction from lysates of selected probiotic strains differently influences Re-epithelialization of HaCaT scratched monolayer through a mechanism involving nitric oxide synthase 2.
      ,
      • Mohammedsaeed W
      • Cruickshank S
      • McBain AJ
      • O'Neill CA.
      Lactobacillus rhamnosus GG lysate increases Re-epithelialization of keratinocyte scratch assays by promoting migration.
      ]. It also increases the tight barrier function of keratinocytes via expression of claudin 1, zonula occludens-1, and occluding [
      • Sultana R
      • McBain AJ
      • O'Neill CA.
      Strain-dependent augmentation of tight-junction barrier function in human primary epidermal keratinocytes by Lactobacillus and Bifidobacterium lysates.
      ], as occurs on gut epithelium, too; the process enhanced via the mitogen activated protein kinases (MAP) pathway, modulating the ERK and p38 [
      • Dai C
      • Zhao DH
      • Jiang M
      VSL#3 probiotics regulate the intestinal epithelial barrier in vivo and in vitro via the p38 and ERK signaling pathways.
      ].
      Similarly, the B. longum exerts protective abilities towards keratinocytes through enhancing tight junction functionality, by increasing the expression of tight junction proteins [
      • Sultana R
      • McBain AJ
      • O'Neill CA.
      Strain-dependent augmentation of tight-junction barrier function in human primary epidermal keratinocytes by Lactobacillus and Bifidobacterium lysates.
      ]; however, B. longum enhanced the expression of claudin 4, and not claudin 1 expressed by L. rhamnosus GG, suggesting that B. longum can influence tight junction function via an alternative, TLR2-dependent, mechanism by decreasing paracellular permeability and thus preventing pathogen invasion [
      • Yuki T
      • Yoshida H
      • Akazawa Y
      • Komiya A
      • Sugiyama Y
      • Inoue S.
      Activation of TLR2 enhances tight junction barrier in epidermal keratinocytes.
      ]. Moreover, evidence exists that B. longum decreases skin sensitivity and increases skin resistance to physicochemical aggregation, since its lysate suppresses the release of the calcitonin gene-related peptide from neurons stimulated by capsaicin [
      • Guéniche A
      • Bastien P
      • Ovigne JM
      • Kermici M
      • Courchay G
      • Chevalier V
      • Breton L
      • Castiel-Higounenc I.
      Bifidobacterium longum lysate, a new ingredient for reactive skin.
      ]. In the same manner, in an ex-vivo human skin model, B. longum was found to lower inflammation by decreasing vasodilation, edema, TNF-alpha release, and mast cell degeneration, suggesting that the bacterial extract may be used for therapies targeting sensitive skin [
      • Guéniche A
      • Bastien P
      • Ovigne JM
      • Kermici M
      • Courchay G
      • Chevalier V
      • Breton L
      • Castiel-Higounenc I.
      Bifidobacterium longum lysate, a new ingredient for reactive skin.
      ].
      The results of the present study clearly demonstrate the superiority of the double-probiotic combined-formula, which is L. rhamnosus plus B. longum, as opposed to the single probiotic treatment of L. plantarum, as an alternative, topically applied regimen, for the healing of a standardized acute skin wound defect in rats. However, the L. plantarum group was found to start the healing process much earlier than the L. rhamnosus plus B. longum, the wounded area, on day 4, being reduced by 41.2% against 12,2% [p<0.0001] in controls and 29.5% [p=0.0011] in the PRO2 group. Similarly, the distance S between the borders of the wound, having initially a diameter of 8 mm, and measured in paraffin embedded sections as 7.04±0.8 mm, was found to be significantly reduced on day 2 in the PRO1 group in relation to the control group [p<0.0001], but not to the PRO2 group. After two days, on day 4, the S distance was found further reduced in relation to the control group [p=0.002]; however, at the same time-point, PRO2 exhibited a huge reduction in relation to control [p<0.0001]. This finding is compatible with those in a similar study in mice [
      • Strober W.
      Trypan blue exclusion test of cell viability.
      ], to which L. rhamnosus was given orally. They found a significant reduction in the epithelial gap, along with reduced macrophages, mast cell infiltration and collagen deposition, as well as improved angiogenesis and blood flow.
      Our results are compatible with those of others [
      • Lombardi F
      • Palumbo P
      • Mattei A
      • Augello FR
      • Cifone MG
      • Giuliani M
      • Cinque B.
      Soluble fraction from lysates of selected probiotic strains differently influences Re-epithelialization of HaCaT scratched monolayer through a mechanism involving nitric oxide synthase 2.
      ,
      • Weinheimer-Haus EM
      • Mirza RE
      • Koh TJ.
      Nod-like receptor protein-3 inflammasome plays an important role during early stages of wound healing.
      ,
      • Lemo N
      • Marignac G
      • Reyes-Gomez E
      • Lilin T
      • Crosaz O
      • David M
      • Ehrenfest D
      Cutaneous reepithelialization and wound contraction after skin biopsies in rabbits: a mathematical model for healing and remodelling index.
      ,
      • Sultana R
      • McBain AJ
      • O'Neill CA.
      Strain-dependent augmentation of tight-junction barrier function in human primary epidermal keratinocytes by Lactobacillus and Bifidobacterium lysates.
      ,
      • Gottrup F
      • Agren MS
      • Karlsmark T.
      Models for use in wound healing research: a survey focusing on in vitro and in vivo adult soft tissue.
      ], supporting the knowledge that L. plantarum promotes the healing process, by shortening the inflammatory phase; although, Zadedi et al reports that L. Brevis starts healing faster than L. plantarum in rats, but on day 7 and afterwards the wounds treated with L. plantarum become smaller [
      • Zahedi F
      • Heydari Nasrabadi M
      • Tajabadi E
      • Aboutalebi H
      Comparison of the effects of Lactobacillus brevis and Lactobacillus plantarum on cutaneous wound healing in rats.
      ]. This reference is in agreement with our findings that L. plantarum begins working on healing process very early, probably as scavenger of inflammatory cells, but other bacteria work step-by-step by increasing their effectiveness and finally exhibiting superior healing effects.
      The same findings were observed with the length of the epithelization zone [L]. The L. plantarum significantly reduced the L parameter on day 2, in relation to the control group [p=0.04], while the PRO2-induced reduction was practically unaffected. However, on day 4, the control to PRO1 group reduction was smaller, but still significant in relation to their difference in day 2 [p=0.0006], while PRO2 exhibited a highly significant decrease, both in relation to control [p<0.0001], as well as in relation to PRO1 [p<0.0001]. In other words, the combo-regimen seems to work through a mechanism promoting rather deep tissue contraction, although, in parallel, the superficial contraction index proves the healing capacity at superficial level from day 8 onward.
      Furthermore, the D line, representing the wound depth, was progressively reduced, beginning from day 4 in PRO2, but not in PRO1. On day 8, although a statistically significant reduction [p<0.0001] was observed in both PRO1 and PRO2 groups in relation to day 4 measurements, the PRO2 group exhibited a significant decrease in relation to the PRO1 group [p<0.0001] the same reduction also being observed on day 16 [p<0.0001]; this difference is also reflected in the DCI values.
      The superiority of the combined-probiotic regimen over the L. plantarum as a single treatment is also clear from the value of the Deep Contraction Index. Here, it is prominent that the PRO2 rats exhibited higher DCI on day 16, the difference from the value of the PRO1 group being significantly smaller [p<0.0001], the same difference also observable on day 8 [p<0.0001]. However, we must emphasize that both probiotic treatments significantly promote deep healing, if one compares their CDI values with those of the control group on day 16 [p<0.0001].
      However, analyzing the findings in detail, we observed that on day 2 the L. plantarum treated group exhibits a significantly greater healing response in relation to the double-regimen group, as can be verified by the data obtained from the wound area reduction, the S length and the D length, thus confirming the previous knowledge that L. plantarum shorten the inflammatory phase [
      • Satish L
      • Gallo PH
      • Johnson S
      • Yates CC
      • Kathju S
      Local probiotic therapy with lactobacillus plantarum mitigates scar formation in rabbits after burn injury and infection.
      ]. However, two days later, at the beginning of the proliferation phase, the findings are reversed, with the PRO2 group gradually becoming more effective up to day 16; this finding fits well with the knowledge that L. rhamnosus also supports keratinocytes proliferation and migration through the CXCL2 chemokine expression [
      • Heydari Nasrabadi M
      • Tajabadi E
      • Dehghan Banadaki Sh Torabi
      • Kajousangi M
      • Zahedi F.
      Study of cutaneous wound healing in rats treated with Lactobacillus plantarum on days 1, 3, 7, 14 and 21.
      ].
      Our experiment has some limitations:
      • 1.
        The findings of the present study in acute wounds in rats cannot be directly transferred to humans. It is widely recognized that rat skin does not perfectly mimic human skin because rats are described as loose-skinned animals allowing contraction to play a significant role in the approximation of the rat skin margins [
        • WA D.-M.
        Rat models of skin wound healing: a review.
        ,
        • Mogford JE
        • Mustoe TA.
        Experimental models of wound healing.
        ], while the presence of panniculus carnosus muscle, not existing in humans, contributes to the final wound healing by both contraction and collagen formation [
        • Zomer HD
        • Trentin AG.
        Skin wound healing in humans and mice: challenges in translational research.
        ,
        • Gottrup F
        • Agren MS
        • Karlsmark T.
        Models for use in wound healing research: a survey focusing on in vitro and in vivo adult soft tissue.
        ]. On the other hand, the round-shaped excisional wound model used exhibited a decreased contraction due to the lack of extensibility [
        • WA D.-M.
        Rat models of skin wound healing: a review.
        ], allowing the epithelization to be more extensively studied. Furthermore, in no case we can directly transfer the present results to chronic persistent wounds due to tissue ischemia, or to infected wounds, to recumbent or to diabetic ulcers; and finally we have to underline once again that the probiotics’ beneficial effects are absolutely strain and dose specific.
      • 2.
        According to Kullander and Olsson [
        • Kullander S
        • Olsson A.
        On the tensile strength of healing cutaneous wounds in pregnant rats.
        ] it is important to compare cutaneous wounds in exactly corresponding positions, because, based on their research, tensile strength of dorsal incisions diminished the more caudally they were performed, the finding probable relating to the lack of a strong adherence to the underlying structures. In our material, we try to analyze healing in relation to the exact location of the wound, but we noted only a slight tendency, not evaluable between the 1st and the 3rd line of wounds, measured from the head towards the tail, thus we finally assessed all as it is in the same place.
      • 3.
        The PRO2 group was treated with a probiotic formula, being a combination of two strains, L. rhamnosus and B. longum. Ideally, we should also have tested each one of these strains separately. However, our purpose was to compare a single strain with a combination of strains; unfortunately, the L. rhamnosus plus B. longum were available from our donors only as a mixture.
      • 4.
        According to our knowledge, there is no other study systematically comparing probiotic strains by these parameters, so there is no data for judgment and discussion.

      Conclusion

      The conclusion drawn from this experimental study supports the general thought that the modulation of the environment of the wounded area by means of beneficial bacteria, namely probiotics, positively affects the healing process. Moreover, from our results it became clear once again that probiotics work by means of different, perhaps individually unique ways, which are strain-specific, although the final effect is similar: the wound healing occurs faster. In this study, their mechanism of action was typically not investigated, but an indirect method was used to perceive their contribution on the healing process, in each time-phase. Thus, initially, it is clear that L. plantarum plays a significant role in the early, inflammatory stage of healing, allowing the re-epithelization to begin earlier. From the clinical point of view, the early shrinkage of wounded area offers the desired effect of protection from potential pathogen ingression. At this early time-point the combined-probiotics regimen, L. rhamnosus plus B. longum seems to be slower compared to L. plantarum. Nevertheless, their roles became reversed, the former speeding up the cell migration and differentiation process for tissue reconstruction and, eventually, on day 16, achieves the greatest healing rate, in comparison to the L. plantarum.
      From this assessment, which was essentially like a 16-day road race, it would be wrong to come up with a winner: the combination formula is better than the single one, just because it achieved the better final result. Our conclusion — which leads to new studies — is to finally find the most appropriate combination to bring together all the advantages, based on what each probiotic strain is really able to contribute in every phase of healing. Or, in other words, to compound a regimen capable modulating all the various stages of healing. Therefore, this new regimen must definitely contain a L. plantarum strain, or similar, to regulate the local inflammatory response; and two or more other strains, like the L. rhamnosus and B. longum, to induce keratinocyte and fibroblast migration and differentiation and collagen formation, to finally achieve the perfect healing effect. To this end we already working.

      Acknowledgments

      We would like to thank the Pharmaceutical Company UniPharma, SA, Greece for the kind donation of probiotics strains. We express our gratitude to Ms. Filidou E, MSc, PhD, Postdoc, and Ms. Tarapatzi G, MSc, of Pharmacology Lab, Department of Medicine, Democritus University of Thrace, Greece for their kind support for the viability testing and identification of probiotics used. We would also like to thank Ms. Anne Shrewsbury BA, MA for editing and language clarification. A part of this publication will be included in the doctorial thesis of the first author.

      Institutional Review Board Statement

      All procedures performed complied with the European Guidelines for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Local Governmental Committee for the Control and Supervision of Experiments on Animals [EU Directive 2010/63/EU, Protocol registration number 227933(934)/06.05.2021].

      Author Contributions

      Conceptualization: KK; Methodology: KK, MM, and AI; Formal Analysis: GT, GS; Investigation: KK, MM, AC and IAD; Data curation: GS, GT and DK; Writing – Original Draft Preparation: GS, AC, IAD, MM, and VB; Writing – Review and Editing: GS, GT and KK; Supervision: KK; Project Administration: JT. All authors are in agreement with the final form of the manuscript.

      Conflicts of Interest

      All authors have no conflict of interest or financial ties to disclose.

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