Document Type: Original Article

Authors

1 Department of Clinical Studies, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.

2 Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.

3 Department of Biotechnology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.

4 Department of Biochemistry, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.

5 Department of Clinical Science, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.

Abstract

Objective- This study was designed to investigate the effect of Lactobacillus plantarum gel on cutaneous burn wound healing in diabetic rats.
Design- Randomized experimental study
Animals- Forty adult male rats
Procedures- Two circular 1 cm cutaneous wounds were created in the dorsum back of each rat. 48 h post-burning, debridement with a 1 cm biopsy punch was performed. The wounds were divided into the following four treatment groups (n= 10, each): 1. Untreated or negative control (NC), 2. silver sulfadiazine (positive control-SSD), 3. base gel (BG) 4. Lactobacillus plantarum Gel (LP gel). The wound surface area and epithelialization were monitored. The animals were euthanized at 10 (n = 5), and 20 (n = 5) days post-injury (DPI) and the skin samples were used for histopathological, biochemical, TGF-β gene expression and biomechanical investigations.
Results- It was indicated that the L. plantarum and SSD treated lesions had the lowest percentage of wound size and collagen content and also the L. plantarum treated group showed shortest inflammatory period and highest amount of TGF-β at 10 days post injury. The L. plantarum gel treated lesions also demonstrated greater ultimate load compared to the untreated and based gel treated wounds.
Conclusions and Clinical Relevance- In conclusion, L. plantarum gel therapy improved wound healing and resulted in better outcomes after severe burn injury in diabetic rats compared with the silver sulfadiazine treatment.

Keywords

Main Subjects

1. Introduction

Wound healing is a process in skin or other organs that occurs after injury.1,2 It is a body’s natural process of regenerating dermal and epidermal tissue in skin and is a complex process involving inflammation, cellular proliferation, and tissue remodeling which is regulated by a cascade of inflammatory mediators, including cytokines and growth factors. Scientists are still interested in wound healing, especially with regard to factors that delay or hinder this process.3 A fibrous scar with collagen is the end product of this process. Collagen provides strength and integrity to dermis.4 Although numerous growth factors are involved in the process of wound healing, transforming growth factor-beta (TGF-β) exerts the greatest fibrogenic effect during repair.3 Complications may happen in the wound healing process in patients with underlying metabolic disorders. As an instant, in both experimental and clinical diabetes disease, complications such as compromises in cellular migration, vascular proliferation, and extracellular matrix remodeling make negative impact on tensile strength and wound healing process.5

Diabetes mellitus is a chronic metabolic disease. In diabetes hyperglycemia due to impaired secretion and/or action of insulin, and disturbances in carbohydrate, fat and protein metabolism may occur.6 In diabetic patients poor wound healing is a common complication, but the mechanisms underlying impaired healing is not completely understood.7,8 One of the reasons of delayed healing is connective tissue abnormalities. In diabetes, as a result of reduced synthesis and enhanced degradation of collagen the collagen content is reduced and this may be one of the abnormalities contributed to poor wound healing seen in diabetes.9 Treatment of chronic wounds in diabetes has remained a challenging clinical problem.10

Burn is characterized by a hypermetabolic state which compromises the immune system leading to chronic wound healing. Thermal exposure to the body surface causes damage to the skin by membrane destabilization, protein coagulation, associated energy depletion and hypoxia at the cellular level which leads to extensive tissue necrosis.6 In burn patients, diabetes may have implications for length of hospitalization, hospital course, number of surgical procedures, and burn outcome.11 Silver sulfadiazine (SSD) is the gold standard therapy in topical burn treatment,12 but due to its adverse effects, bacterial resistance and its ineffectiveness on the healing process, scientists are still searching for alternative compounds that can enhance the wound healing process.13-15

Probiotics are either a single strain or a mixture of different organisms and have the ability to strengthen the immune system, produce anti-inflammatory action and enhance the wound healing process following accumulation of inflammatory cells like polymorphonuclear cells, macrophages, lymphocytes and plasma cells in the wound bed.16 In new ways, for treating wounds, probiotics can eliminate pathogenic microbes and improve wound healing. Probiotics have also anti-infective properties and can also promote wound healing, hence they have potency to inhibit infection.17 L. plantarum is a probiotic organism producing lactic acid, this product has antibacterial properties that can inhibit the proliferation of pathogenic microorganisms. L. plantarum is a commensal micro-organism that does not produce virulence factors and succumbs easily to the antimicrobial battery of host defenses, particularly the polymorphonuclear cell activity, and for these properties, the effects of L. plantarum gel was tested on cutaneous wound healing in the burn induced injury in diabetic rats. 18

2. Materials and Methods

All the procedures were conducted in accordance with the Iranian community guidelines for laboratory animals and the principles of laboratory animal care (NIH publication NO. 86-23, revised 1985) was followed. This experiment was approved by the local Ethics Committee of “Regulations for using animals in scientific procedures” in School of Veterinary Medicine of our University.

L. Plantarum and gel preparation

Lactobacillus plantarum was grown in MRS broth at 37 °C for 24 h in an incubator. The gel was formulated by adding 2% carboxy methyl cellulose (CMC) in medium with Lactobacillus plantarum (1×109 CFU/ml). The solution was stirred at room temperature for 2 h.19

Experimental animals

Forty mature male Sprague-Dawley rats, weighing 200-250 g were housed in individual cages with controlled light, temperature and humidity. The rats were fed commercial rat food and water ad libitum.

Induction of diabetes mellitus in rats

Diabetes was induced after an overnight fasting, using one single shot intraperitoneal injection of 65 mg/kg streptozotocin (STZ) (Sigma, St. Louis, MO), a toxin specific for insulin-producing cells, in saline-sodium citrate buffer. The blood glucose level was measured, using an acute glucometer (Johnson & Johnson Co., USA). 21 days after STZ injection, the animals with blood glucose level above 300 mg/dl were defined as diabetic and entered in the study.20

Skin burn wound model

The animals were positioned in sternal recumbency and generally anesthetized by intramuscular (IM) injection of ketamine (Ketamin hydrochloride, Alfasan, Woerden Holland, Netherlands, 40 mg/kg) and xylazine (Xylazine hydrochloride, Alfasan, Woerden Holland, Netherlands, 5 mg/kg). The dorsum was then shaved and sterilized with povidone-iodine solution. A template was used to standardize the location and distance of the burn wounds. A standard iron bar, 1 cm in diameter, was maintained at a temperature of 100 °C and applied for 15 seconds to create second degree burns wounds. The rats were placed in an isolated cage to inhibit transmission of infection. The wounds were examined after 24 h and for debridement of necrotic tissue two full-thickness excisional wounds (10 mm in diameter, Figure 1A) were created under aseptic conditions in the back of each rat, using a biopsy punch.

Treatment design

The animals were randomly divided into the following groups (n=10, each):

  • Group 1: Diabetic animals received no treatment (NC)
  • Group 2: Diabetic animals received silver sulfadiazine (SSD) twice a day for 14 days
  • Group 3: Diabetic animals received base gel (BG) twice a day for 14 days
  • Group 4: Diabetic animals received L. plantarum gel (LP) twice a day for 14 days

Tissue sampling

The rats were euthanized at 10 (n=5) and 20 (n=5) days post-injury (DPI). The right side wounds were collected for histological investigation and hydroxyproline content estimation and mRNA TGF-β expression. Tissue sections were collected from the center of the lesions to a rim of normal skin surrounding the wound. Moreover, the left wounds were carefully dissected for biomechanical testing (Figure 1 A).

Gross Morphology of the Wounds

The wound area was observed and photographed at 10 and 20 DPI. The images were transferred to Image Pro Plus software® V.6 (Media Cybernetics, Inc., Silver Spring, USA) for morphometric analysis including measurement of the wound surface area at these time points (Figure 1A).

Histopathologic and histomorphometric analysis

Five animals from each group were euthanized at 10 and five other at 20 days post-treatment and the skin tissues were harvested and immediately fixed in 10% neutral buffered formalin (pH=7.26) for 48 h. The fixed tissue samples were then processed, embedded in paraffin, and sectioned to 5 µm thickness. Finally, the sections were stained with hematoxylin and eosin (H&E). The histological sections were evaluated by two independent pathologists, using light microscopy (Olympus BX51; Olympus, Tokyo, Japan) in a double-blind fashion. Epithelialization, inflammatory cell infiltration, fibroplasia and granulation tissue formation were assessed in different groups, comparatively. Magnification ×400 was employed to count different cells and calculation was repeated in five fields for each tissue section. Finally, the average number of each criterion for these fields was then recorded. Epithelialization was assessed, on 10 and 20 DPI, semi-quantitatively on 5-point scores: 0 (without new epithelialization), 1 (25%), 2 (50%), 3 (75%), and 4 (100%) epithelialization.21 For these parameters, the results were validated by comparative analysis of two independent observers blinded to the treatment groups.

Tensile testing

After shaving, the skin samples containing the burned area were excised in a rectangular shape (10 × 2 cm). The samples were kept frozen (-20 °C) until the time of testing. Prior to testing the samples were then thawed at room temperature. A Universal Instron testing machine was used for tensile testing of the specimens (TT-CM-L, United Kingdom). The skin samples were mounted on tensiometer holders, where both sides of the skin strip were clamped into a pair of holders so that a constant length of 4 cm, with the wound in the middle of the strip, was used for tensile testing. The skin strip was stretched, using a constant strain rate of 10 mm/min and the ultimate tensile strength was calculated based on the created load-deformation curve.22

Hydroxyproline content

After thawing, the samples were dried in a hot air oven at 60-70˚ C until a constant weight was achieved. The samples were then hydrolyzed with 6 N HCL for 2 hours at 120˚ C. The hydrolyzed samples were adjusted to pH=7 and subjected to chloramines T oxidation, and finally, the colored adduct formed with the aldehyde perchloric acid reagent at 60˚ C, was read at 550 nm after cooling for 5 min. A modified assay to determine hydroxyproline content in a tissue hydrolizate was used.23

Real-time RT-PCR

The mRNA expression of TGF-β were determined by real-time RT-PCR. RNA was isolated from wound tissues using High Pure RNA Isolation Kit (Roche, Germany) and cDNA was synthesized using Transcriptor First Strand cDNA Synthesis kit (Roche, Germany) according to the manufacturer’s instruction. CDNA was used as a template for the subsequent real time RT-PCR. The real time PCR assay was performed, using Light Cycler 480 SYBR Green Ι Master (Roche, Germany) in the LightCycler 480 real time PCR system (Roche, USA). The real time RT-PCR experiment was carried out according to the manufacturer’s instruction and after an initial denaturation of 95 for 10 min, the following thermal cycling profile was used (50 cycles): 95 C for 10 s, 55 C for 20 s and 72 C for 30 s. Finally melting curve analysis from 65 to 95 was performed and the data was processed using the LightCycler 480 software with basic relative quantification program (ΔΔCT method). Primer sequences (Supplementary Table 1) were designed by Beacon Designer software (Version 7.9).

Statistical analysis

The quantitative data were presented as the mean ± standard deviation (SD) and One-way ANOVA with subsequent tukey post-hoc tests was used to compare the data between the groups. Kruskal-Wallis H and non-parametric ANOVA were used for statistical analysis of the qualitative data obtained from the scored values, and if the differences were significant (P<0.05), then analyzed by Mann-Whitney U test. The results with P values less than 0.05 were considered statistically significant. Statistical analyses were performed using the SPSS software (IBM SPSS Statistics for Windows, Version 20.0, Armonk, NY: IBM Corp).

3. Results

Gross morphology

Gross morphology of the lesions is illustrated in Figure 1, A. At the end of day 10 PI the wound sizes were significantly lower in the L. plantarum gel (p<0.05) treated group as compared to the base gel and untreated groups. The wounds size was lower in the L. plantarum and SSD, in 20 DPI, as compared to other two treatment groups, but there were no significant differences between the L. plantarum gel treated and the other groups. The treated wounds with L. plantarum and silver sulfadiazine had better cosmetic appearance at 20 DPI than the other experimental groups. No wounds became infected and normal healing response occurred in all the wounds (Figure 1A).

 

Biomechanical performance

Treatment with L. plantarum and SSD increased the ultimate tensile strength compared to the negative control and base gel lesions, at 10 and 20 DPI. However, the differences were not statistically significant at 10 DPI, whereas the differences between the L. plantarum and other groups were statistically significant at 20 DPI and the L. plantarum treatedgroup showed higher ultimate tensile strength (p<0.05) at this time point (Figure 1B).

Histopathologic and histomorphometric findings

The wounds in the negative control group (diabetic rats without any treatment) displayed evident inflammatory cell infiltration and granulation tissue formation, however, re-epithelialization was not completed, even at the 20 DPI. Histopathological evaluation of the SSD group showed moderate infiltration of leukocytes into the wound area, at 10 days. The epithelialization process was completed at 20-day post-treatment. The inflammatory cells were significantly fewer when compared to the negative control group, at 20 DPI.

The histological findings of the base gel treated group showed a close similarity to the negative control group, with hemorrhages, hyperemia and infiltration of inflammatory cells in the tissue sections, at 10-DPI. Re-epithelialization was not completed at 20 DPI, and the inflammatory response significantly decreased in comparison to day 10 post-injury in this group. The photomicrographs of the wounds treated by L. plantarum showed a considerable reduction in inflammatory cells, at 10 DPI and 20 DPI in comparison to other groups (p<0.05). A complete epithelial layer with presence of rete ridges was formed in the L. plantarum treated wounds, at 20 DPI. This group showed more resemblance to normal skin and demonstrated a thin epidermis with presence of normal rete ridges, rejuvenation  of  hair follicles and other skin appendages.24

Figure 1. A, Template of burn wounds on the dorsum of rat (four full thickness burn wounds at day 0), gross morpho-logy of the burn wounds and the percentage of wounds size after inducing burn wounds at the 10th and 20th days of treatment in different ex-perimental groups (Mean ±SD). The mean surface area of the wounds (mm2) was significantly lower in the L. plantarum and SSD treated wound when compared to the base gel and untreated group at 10 DPI. However, there was no statistically sig-nificant differences between all groups in terms of wounds’ mean surface area. B, Ultimate tensile strength in different experimental groups at the 10th and 20th DPI. Picture of the Universal Instron tensile testing machine while loading a skin specimen. The L. plantarum treated group showed a considerable improve-ment in biomechanical properties at 20 DPI when compared to other groups. *p<0.05, *p<0.01.  SSD: Silver sulfadiazine, NC: negative control (untreated), BG: Base Gel, L. plantarum Gel.


Figure 2. Histopathological sections of the healing incisional wounds at 10 and 20 DPI. The re-epithelialization process was completed in the SSD and L. plantarum treated groups at 20 DPI (stained by H&E). Moreover, rejuvenation of the skin appendages such as hair follicles was seen in the L. plantarum treated group at 20 DPI. Thick arrows: crusty scab, thin arrows, infiltration of inflammatory cells, arrowheads: Re-epithelialization, white arrows: rejuvenation of the skin appendages.

Table 1. Histomorphometric analysis of wounds at the 10th and 20th day post-treatment

Groups

Epithelialization Scores

(N=5)

Fibrocytes and fibroblasts

174.2±15.2

Inflammatory cells

86.2±4.1

Blood vessels

5.7±1.2

Negative control

0,0,0,0,0 (10 d)

0,1,0,1,0 (20 d)

 

174.2±15.2 (10 d)

275.2±14.5 (20 d)

86.2±4.1(10 d)

54.4±7.5(20 d)

5.7±1.2 (10 d)

14.5± 1.3(20 d)

SSD

1,0,2,1,2 (10 d)*

4,2,4,3,3 (20 d)***

 

193.5±9.6(10 d)

104.5±28.2 (20 d) ***

42.8±4.6 (10 d) *

17.3±2.8 (20 d) **

12.0±2.5 (10 d) *

7.2± 1.6 (20 d) *

Gel

1,0,0,1,0 (10 d)

3,2,3,2,1 (20 d)**

 

221.3±11.1*(10 d)

217.8±11.3 (20 d) *

75.5±7.4(10 d)

29.7±2.5 (20 d) **

10.3±1.8 (10 d)

15.1± 3.4 (20 d)

Lactobacillus spp.

2,2,1,3,3 (10 d)**

4,2,3,4,2 (20 d)***

325.7±19.8 (10 d) ***

135.9±7.4 (20 d) **

26.2±4.5 (10 d) **

7.8±1.1 (20 d) ***

8.1±1.3 (10 d)

10.5± 2.8 (20 d)

 

 

 

 

 

 

 

 

 

 

 

 

*, **, ***: values indicates treatment group versus un-treatment group (negative control); * p<0.05, ** p<0.01, ***p<0.001

 

skin appendages.24 Therefore, the L. plantarum treated wounds showed the best results when compared to the negative control and other experimental groups.

The histomorphometric analysis was done at 10 and 20 days after burn injuries and the results have been presented in Table 1. Amongst all groups, re-epithelialization was minimum in the negative control group and the wounds in the animals of this group were mostly filled with immature granulation tissue (p<0.05). The best re-epithelialization was seen in the L. plantarum treated group. Moreover, the total inflammatory cell counts significantly reduced in the L. plantarum treated group in comparison to other groups, at 10 and 20 DPI (p<0.05). Overall, the healing condition of the L. plantarum treated diabetic wounds was more similar to those of the normal skin, and the wounds had the best cosmetic appearance with normal thickness of the epidermal layer and rejuvenation of the skin appendages (Figure 2).

Hydroxyproline content

Since collagen formation is a critical step in wound healing, so we evaluated the hydroxyproline content as a marker of collagen content.25 Hydroxyproline content in the L. plantarum and SSD treated wounds were higher than the negative control and base gel groups in 10 and 20 PDI. Although the hydroxyproline contents in the L. plantarum and SSD treated groups were more than the other groups but the differences between the groups were not statistically significant (Figure 3A).

TGF-β mRNA expression

Relative mRNA expression of TGF- β significantly increased in the L. plantarum treated group on day 10 post-wounding as compared to the SSD, NC and untreated groups (p<0.05). Although the expression level of TGF- β in the SSD group was higher among the other groups, no significant differences were seen, at day 20 PI (Figure 3B). The result of gene expression level in Day 10 post-treatment are shown in the supplementary Table 2.

4. Discussion

The results of the present study indicate that local application of L. plantarum can successfully suppress the inflammatory response and promote burn wound healing in diabetic rats. Development of new and effective interventions in wound care remains an area of intense research. Several studies have been done to find new products for treating wounds.26-29 Some diseases like diabetes may cause hinder in wound healing.30 Diabetes even in its early stages impairs the normal course of wound healing, thus leading to chronic wounds. It has been speculated that diabetes interferes with various phases of repair by decreasing growth-factor levels in the wound environment.5 Delay in cellular infiltration and formation of granulation tissue are possible reasons for prolonged epithelialization in diabetes.31 Diabetes causes reductions in collagen organization; diminished blood supply; impaired leukocyte function; unbalanced production of growth factors, cytokines and proteases; increased blood viscosity and delayed conversion of acute to chronic inflammation.4,9,32,33 In the present study, therefore, the STZ-induced diabetic rats were used as the model of diabetes to study diabetic wound healing. Our results showed that L. plantarum can accelerate the wound healing process by decreasing duration of inflammatory response.

A probiotic is a single strain or a mixture of different organisms and it is proposed that probiotics can enhance wellbeing through immunomodulatory, metabolic and barrier activities against pathological processes.3 Probiotics can provide local immunity and protection to the gastric ulcers and it has been stated that the probiotic microbes such as Lactobacillus improved healing of the gastric ulcers in rats.34 It has also been showed that probiotics have the ability to enhance healing of the gastric ulcers specifically by enhanced angiogenesis and expression of growth factors, such as VEGF, TGF-β and EGF.35 Probiotics can enhance the overall body immunity by immunomodulation.36 New data based on human fibroblast cultures and DNA microarrays suggest that probiotics possess the distinct ability to naturally stimulate the skin’s immune response. In essence, probiotics boost skin repair and healing.16 L. plantarum is a probiotic organism that produces lactic acid.37 It is a micro-organism that does not produce virulent factors and succumbs easily to the antimicrobial battery of host defenses, particularly activation of the polymorphonuclear cell activity.18

L. plantarum has been shown to have beneficial effects in the wound healing process in our study and our results were in harmony with other studies. The antimicrobial activity of kefir 96 h gel has been shown to be similar to silver sulfadiazine 1% ointment but the wound healing time has been shown to be lower in kefir 96 h gel when compared to silver sulfadiazine ointment.17 Rodrigues et al found that probiotics can both decrease inflammation and restore the wounded area.38 The anti-inflammatory properties of polysaccharides present in the Kefir extracts also influence the wound healing process and stimulate innate immune responses in defense against pathogens.17

Figure 3. A, Hydroxyproline content in the wound tissue during wound healing. Values are mean ± SD, n = 5 for each group. There were no significant differences between all groups in terms of mean hydroxyproline content (µg/mg tissue) on the 10th or 20th DPI. Bb mRNA expression of TGF-β in different experimental groups on days 10 and 20 PI. The TGF-β mRNA expressions significantly increased on day 10 in the L. plantarum treated wounds. P value indicates the L. plantarum treated group in comparison to all other treated and un-treated groups: *p < 0.05.

Our  investigation  was  in  harmony with other studies and the total number of inflammatory cells in the L. plantarum treated group was significantly lower in comparison to others at 10 and 20 days post-treatment.

The wound status in the L. plantarum and SSD treated groups was obviously improved on day 10 compared to the gel based treated and untreated groups while the wound area in the gel treated and untreated groups showed little recovery on day 10. The wound surface area was significantly smaller in the L. plantarum treated group in both 10 and 20 days post injury in our study and this emphasizes better wound contraction compared to the other groups. L. plantarum treatment, in this study, decreased inflammatory cell infiltration by reducing the number of inflammatory cells at day 10 and 20 post injury and increased the number of fibroblasts and fibrocytes at this stage.

Collagen formation is a critical step in wound healing and collagen is a major extracellular matrix protein which confer strength and integrity to tissue and also plays a role in hemostasis by interacting with thrombocytes.39 Collagen provides tensile strength, organization and integrity to connective tissues, and in the present study we evaluated the hydroxyproline content as a marker of collagen content.40 It has been shown that diabetes can cause reductions in collagen content.9 Treatment by L. plantarum gel, in the present study, resulted in enhanced level of hydroxyproline content on day 10 after injury in comparison to the negative control and untreated group and the hydroxyproline content was comparable to the silver sulfadiazine treated group at this time point. Increased level of hydroxylproline in the L. plantarum and SSD treated groups provided more strength to the wound and this data was supported by enhanced biomechanical performance of the L. plantarum and SSD treated groups that showed higher ultimate tensile strength compared to the other groups although the difference in biomechanical evaluation were not significant.

It has been speculated that the effect of L. plantarum culture on diabetic burn wounds could be because of the fact that the cytokine and growth factor pattern induced by L. plantarum in inflammatory cells is different from those induced by pathogens like P. aeruginosa.41 P. aeruginosa induced higher levels of PGE2 (prostagladin E2) compared with the low level induced by L. plantarum, and it also should be highlighted that L. plantarum demonstrated a great anti-inflammatory activity.42-44 Our study also showed that treatment by L. plantarum enhances the expression level of TGF-β in wound at early stages of wound healing. TGF-β was significantly higher in the L. plantarum treated lesions than the base gel and untreated wounds at day 10 post injury. TGF-β is a critical peptide which is responsible to control repair, chemotactically attract inflammatory cells to a wound, and promote deposition of ground substance and collagen; therefore, it has been aptly called a “wound hormone”.45 TGF-β has been shown to be deficient in models of impaired healing.

Topical application of TGF-β accelerated biomechanical performance in incisional wounds in rats.45 In addition, increase in TGF-β expressions could promote fibroblasts to synthesize collagen proteins, fiber connexin and integrin, and enhance migration of epithelial cells.45 Increased TGF-β in the L. plantarum treated group could be the reason for earlier wound closure, greater degree of collagen deposition, better biomechanical performance and also having the best cosmetic appearance compared to other groups. It should be mentioned that Becaplermin, a drug for treatment of diabetic wounds, promotes healing in diabetic wounds by increasing the expression level of TGF-β.45,46 In addition, it has been stated that L. plantarum showed great anti-inflammatory activity and this feature could be another reason for its potent wound healing capacity.18 It has been reported that the positive effects of hyaluronic acid on burn injuries are due to its anti-inflammatory effects.47 Therefore, the anti-inflammatory properties present in L. plantarum may have a potential role in enhanced wound healing.

Amongst all groups, re-epithelialization was minimum in the negative control group and it was mostly filled with immature granulation tissue. The best re-epithelialization was seen in the L. plantarum treated group specially, at day 20 after injury, and this treatment regimen showed better results than silver sulfadiazine which is a standard burn wound treatment regimen. Therefore, quicker collagen deposition, earliest and highest TGF-β expression and the anti-inflammatory effect of L. plantarum may be act as the main mechanisms in improving wound healing in diabetic rats. Overall, the healing criteria of the L. plantarum treated diabetic wounds were more similar to those of the normal skin and demonstrated the best cosmetic appearance, with normal thickness of epidermal layer and rejuvenation of the hair follicles and skin appendages.

Topical application of L. plantarum gel not only promoted wound healing by enhancing collagen synthesis, it also increased the number of fibroblasts and fibrocytes and increased TGF-β level and decreased the risk of infection. Thereby, L. plantarum may provide a safe, effective, and less expensive alternative in managing diabetic and also burn wound treatment. The results of our investigation demonstrated that L. plantarum can be used as a topical treatment regimen in treatment of delayed wound healing and can be introduced as a promising alternative to conventional methods in treating delayed wound healing.

 

Conflict of interest

 

The authors declare that they have no conflict of interests.

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