Materials

L. rhamnosus (Strain Number: IBRC-M 11409), L. acidophilus (Strain Number: IBRC-M 10815), L. casei (Strain Number: IBRC-M 10711), and L. fermentum (Strain Number: IBRC-M 10816) were purchased from the Iranian Biological Resource Center. All raw materials listed as follows were purchased from Sigma Chemical Co. (St. Louis, MO): streptozotocin (STZ), De Man, Rogosa, and Sharpe (MRS) medium, agar, glycerol (C3H8O3), paraffin, polyethylene glycol (PEG) 400 and 4000, formalin, and Mueller- Hinton broth. Tetracycline 3% ointment (TC) was purchased from Iran Darou Pharmaceutical Co, Iran. Xylazine hydrochloride and ketamine hydrochloride were purchased from Molteni Pharmaceutical Co., Italy. Thiopental was purchased from Loghman Pharmaceutical Co, Iran. Moreover, male Sprague-Dawley rats were purchased from the Laboratory Animals Research Center (Shiraz University of Medical Sciences, Iran).

Formulation development

The oleogel-based formulation was developed according to our prior study [21], containing a particular percentage of glycerol, PEG, and a particular precipitate concentration of each probiotic strain individually as an active pharmaceutical ingredient (API). Four Lactobacillus probiotic species of L. rhamnosus, L. casei, L. fermentum, and L. acidophilus included probiotic contents with a 1 × 109 colony forming unit (CFU) per ml of formulation.

Biofilm formation assay

Antibiofilm activity of L. rhamnosus was carried out against Escherichia coli ATCC 35150 (E. coli), Listeria monocytogenes ATCC 7644 (L. monocytogenes) and Salmonella typhimurium ATCC 14028 (S. typhimurium) biofilms, employing 12-well microtiter plates each containing 2 ml of MRS broth inoculated with 1% v/v of probiotic, were incubated at 30 °C for 48 h. The broth was discarded, and the biofilms formed on the plate wells were washed with 2 ml PBS (pH 7.1) to remove loosely adherent and planktonic cells. Pathogenic bacterial suspensions in TSB, with concentrations of 108 CFU/ml, were added to biofilms and incubated at 30 °C for 24, 48,72, and 96 h. Half of the broth in the plate wells (1 ml) was substituted with fresh broth every 24 h. After the incubation period, the removal of the planktonic cultures from the wells was executed carefully and the biofilms were suspended by shaking and scratching the well. The collected suspensions were employed to determine the adhering pathogen viable count in the biofilm. Saline solution 0.85% (w/v) was used to prepare the appropriate dilution of each suspension and plated on MacConkey sorbitol agar (SM) for E. coli, Modified Oxford agar (MOX) for L. monocytogenes and xylose lysine deoxycholate agar (XLD) for S. typhimurium. The bacterial count was determined after 48 h of incubation at 37 °C. Furthermore, the wells containing 108 CFU/ml of pathogenic bacterial suspension were considered as the control group of each pathogen. All assays were made in triplicate. \(}\) of L. rhamnosus against three pathogens was calculated using the following formula:

$$}=\frac}}_}}-}}}}_}}}\times 100$$

where AC and A represent the log CFUs of the control group individually and log CFUs of each pathogen group treated by L. rhamnosus, respectively.

Experimental animals

Forty-eight male Sprague-Dawley rats (weighing 200–300 g, 8–10 weeks) were purchased from the laboratory animals research center of Shiraz University of Medical Sciences, Iran. The animals were monitored in the animal laboratory in stainless steel cages for two weeks to adapt to the controlled environment at 22–25 °C, humidity 55%, and lighting 12 h light/dark cycles. Their diet consisted of rat chow (Pars Dam Co., Tehran, Iran). The protocol of animal experiments was approved locally by the ethics committee of Shiraz University of Medical Sciences (Code: ir.sums.aec.1400.027), and the guidelines for the care, handling, and use of laboratory animals were precisely followed.

Ethics committee approval

The protocol of this study was approved by the Ethics Committee of Shiraz University of Medical Sciences and performed according to the Ethical Standards laid down in the Declaration of Helsinki of 1964 and its later amendments. It follows the ARRIVE (Animal Research: Reporting of in vivo Experiments) guidelines about using and coring experimental animals.

Induction of DM

The overnight fasted rats received a single dose of intraperitoneal injection of 60 mg/kg body weight STZ that was freshly dissolved in a 0.1 mol/L citrate buffer (pH 4.5) to induce diabetes in rats. After one-week, fasting blood sugar was monitored using the glucometer (Accu-Chek Active, Roche, Germany). The rats with blood glucose levels of more than 300 mg/dl were considered diabetic models and entered the study. Rats weight and water intake were screened throughout the study [22].

Skin wound model

The animals were generally anesthetized by intraperitoneal injection of 5 mg/kg xylazine hydrochloride and 80 mg/kg ketamine hydrochloride. Following the removal of hair and preparation of the dorsal skin with povidone-iodine solution, a biopsy punch is used to outline a circle of 2 cm in diameter under aseptic conditions.

Experimental design

In this experimental study, rats were randomly divided into eight groups of 6 rats each as follows: “control” group, nondiabetic rats without any treatment; the “DM” group, diabetic rats without any treatment; the “DM + B.g” group, diabetic rats received gel formulation without any bacteria once daily for 14 days and the “DM + TC” group, diabetic rats topically received TC, once daily for 14 days, as well as “DM + LBF”, “DM + LBR”, “DM + LBC” and “DM + LBA” groups which defined as diabetic animals received gel formulation containing L. fermentum, L. rhamnosus, L. casei, and L. acidophilus respectively once daily for 14 days. After the intervention, a morphological assay of wound sites and then the biological and histopathological analysis of tissue samples, including hydroxyproline contents, re-epithelization, hair follicle formation, fibroblast population, collagen deposition, and neovascularization been carried out to assess and compare the efficacy of probiotic formulations in the wound healing process. Finally, the euthanasia of all experimental rats was performed with a single dose of thiopental, 100 mg/kg.

Morphological analysis

To quantitatively and qualitatively evaluate the wound healing process in diabetic rats, the wound area was photographed on 0th, 3th, 7th, and 14th post-wounding days (n = 3). The illustrations were analyzed using Image Pro Plus software® V.6 (Media Cybernetics, Inc., Silver Spring, USA). Then, the wound closure percentage was calculated using the initial wound sizes using the following formula:

$$}=\frac}-}}}}\times 100$$

The higher wound closure percentage assigned to the effective wound healing performance.

Biological analysisTissue sampling

Tissue sampling is required before biological assays from biochemical to histopathological analysis. For doing so, the tissue samples were excised from the wound areas on 7th and 14th post-wounding days using a biopsy punch (n = 3). Vertical Uniform Random (VUR) method was used to create random and uniform cuts from tissue samples. Then, tissue samples were applied for biochemical tests. Furthermore, to perform the histopathological and stereological assay, the separated skins were fixed in formalin buffer solution (10%) and embedded in paraffin wax, followed by tissue passage and serial sections of sample blocks [23].

Biochemical analysis

The hydroxyproline content of skin tissues was analyzed as an index of collagen deposition in the wound site for biochemical studies. For this purpose, 500 μl homogenized tissue samples (in 2 ml deionized water, pH = 7.4) were digested in 1 ml of hydrochloric acid (6 N) for 8 h at 120 °C. Then, the suspension was mixed with an equal proportion of citrate-acetate buffer (pH = 6, 25 μl), and 500 μl of chloramines-t-solution, 56 mM, was added and left for 20 min at 25 °C. Finally, 500 μl of Ehrlich’s reagent containing 15 g of p-Dimethyl amino benzaldehyde in n-propanol/perchloric acid (2:1 v/v) was added to each sample, and the resultant mixture was incubated at 65 °C for 15 min. After cooling, the intensity of the developed color of samples was measured at 550 nm using an Ultrospec 2000® UV spectrophotometer (Pharmacia Biotech, Uppsala, Sweden).

Histopathological analysis

Histopathological analysis was performed on 7th and 14th post-wounding days. After the intervention, samples of rats (N = 3) of each group were collected from the wound area by biopsy punch and stored in 10% formaldehyde solution for further histopathological studies with standard procedures. For this purpose, the separated skins were blocked in paraffin after tissue passage and serial section. The histopathological slides were prepared by sectioning, staining, and mounting the paraffin-embedded tissues [23]. The volume and numerical density of skin tissue structures were determined to measure the re-epithelization, hair follicle formation, fibroblast population, collagen deposition, and neovascularization as the histopathological factors studied in the wound healing process. Finally, all animals were euthanized with a single dose of thiopental (100 mg/kg) after each tissue sampling.

The volume density of various components of skin tissue, including epidermis, collagen fibrils, hair follicles, and neovascularization, were determined by using the Delesse formula as follows, which involves counting cross points:

$$\left(\right)=\mathop\limits_^p\frac\right)}\nolimits_^p\left(\right)}$$

∑P (structure) is equal to the number of points that collided with different parts of the skin tissue, and ∑P (Reference) is similar to the total number of points that collided with the entire skin tissue [23].

Furthermore, the numerical density of the fibroblast cell was determined by the optical dissector method using a micrometer using the following formula:

$$=\frac\nolimits_^Q}\nolimits_^P\times h\times \left(\frac\right)}\times \frac}$$

∑Q is the number of fibroblast cells counted at the height of the dissector, ∑p is the total number of fields in which the counting was done, a/f is equal to the area of the counting frame in all microscopic fields, h is the height of the dissector, t is the average slice thickness in different sections, and BA is the total thickness of the cut [23].

Statistical analysis

Statistical analysis was performed using IBM SPSS software. One-way ANOVA and Tukey’s post hoc test were used to evaluate group differences. All experiments were carried out in triplicate, and a P value ≤ 0.05 was considered statistically significant.