Exploring effect of M2 macrophages on experimental full‐thickness wound healing in streptozotocin‐induced diabetic rats

Abstract

Diabetes mellitus is one of the most prevalent medical conditions, in both humans and animals. People with diabetes mellitus often experience slower than normal wound healing, making it a serious health concern. This study investigates the effect of M2 differentiated macrophages on full‐thickness wound healing in white Westar rats exposed to streptozocin 70 mg/kg. A full‐thickness skin defect with dimensions of 2 × 2 cm was created on the back of all the animals, and their blood sugar was simultaneously assessed. The monocytes were isolated from blood samples using the plastic adherence method and were exposed to dexamethasone (5–10 μ) for 24 h. Subsequently, they were washed with PBS and incubated in fresh cell culture medium for 5 days. The differentiated M2 cells were injected into four points of the experimental ulcers of the treatment group. Macroscopic and microscopic changes were evaluated and compared over a period of two weeks between the test and control groups. The infusion of these cells a few days after wounding enhances wound healing parameters significantly, as evidenced by an increase in germinating tissue formation, wound contraction, inflammation reduction, and collagen increase in the treated group.

1. INTRODUCTION

Wound healing is an intricate and ever‐evolving process that is influenced by a range of variables which can be divided into local and systemic categories. ¹ Identifying ways to expedite the wound healing process and forestall scarring, as well as a mechanism for tracking the healing process, is essential. Following a skin wound, dermal fibroblasts and keratinocytes move to the wound site and proliferate, thereby facilitating prompt healing. ² Diabetes is a widespread health condition that has a major impact on a large proportion of the global population. Among those affected by diabetes mellitus, a common problem is the delayed healing of wounds; 15% of individuals with diabetes experience difficulties with wound healing as a complication of their condition. ³ , ⁴

Research suggests that diabetic rats experience a slower recovery time after sustaining full‐thickness skin wounds than non‐diabetic rats. ⁵ , ⁶ Macrophages are pivotal in the healing of wounds, as they help to reduce inflammation, dispose of cellular remnants and manage tissue restoration. Inappropriately triggered macrophages, however, can prompt fibrosis in persistent (persisting) injuries, leading to unsatisfactory outcomes. ⁷ Excessive inflammation results in an upregulation of matrix metalloproteinase 9, which causes destruction of collagen, fibronectin, and elastin, thereby hindering the healing process of diabetic wounds. ⁸ Phenotypic adaptation enables macrophages to adjust their activities according to the organism's needs.

M2 macrophages, which possess abundant receptors involved in the clearance process, are implicated in wound healing. This type of macrophage displays heterogeneity, comprising M2c, M2b and M2a subsets. Specific drugs and cytokines, such as IL‐4, IL‐10, IL‐13, TGF‐β and CSF‐M, can be used to induce differentiation towards the M2 lineage, thereby enhancing and improving its effects. ⁹ Glucocorticoid drugs are known to induce differentiation of macrophages into M2 macrophages, which are characterized by the secretion of anti‐inflammatory cytokines and the suppression of proinflammatory cytokines. Moreover, MHCII, co‐stimulatory molecules and T lymphocyte activation are also reduced, while phagocytosis is increased. ¹⁰ , ¹¹

M2 Macrophages expressing Dectin‐1 (a C‐type lectin domain family 7 member A), CD206 (a Mannose Receptor) and CD204 (a Scavenger Receptor A) have a significant role in antigen presentation, aiding the activation of the adaptive immune system, as well as lipid metabolism, atherogenesis and various metabolic processes. Macrophages of the M2 type are weak antigen‐presenting cells, serving to balance tissue homeostasis, destroy residual material, and control inflammation. Furthermore, they are involved in the production of polyamines and ornithine, and stimulate collagen production in the wound area. ⁹

Jetten et al., ¹² showed that injection of M2 macrophages 1 and 3 days after the infliction of a wound did not have a substantial impact on the healing process. It is likely that the ineffectiveness of the macrophage infusion was a result of the absence of intrinsic inflammation in the wound bed. This study seeks to determine the efficacy of M2 differentiated macrophages, in combination with dexamethasone, in facilitating full‐thickness wound healing in diabetic rats. To this end, the injection should be administered 10 days after the wound induction, when the inflammation has subsided.

2. MATERIALS AND METHODS

2.1. Animals

Twenty‐four adult male Westar rats, with an average weight of 250–300 g, were randomly chosen from the Animal Breeding Centre of Shahid Chamran University of Ahvaz. The experimental procedures were approved by the Ethics Committee of Faculty of Veterinary Medicine at Shahid Chamran University of Ahvaz. The animals used in the study were kept under animal centre conditions at 22°C and 50% humidity with free access to food and water for 1 week prior to the experiments. An intraperitoneal injection of a ketamine‐xylazine mixture was used to anaesthetize the animals for surgical operations and for taking of blood samples.

2.2. Diabetic model

All animals were administered streptozotocin (70 mg/kg intraperitoneally). Five days post‐injection, rats with fasting blood glucose levels exceeding 180 mg/dL (via tail blood sampling and glucometer reading) were regarded as diabetic and used in further experiments. ¹³

2.3. Wound model

All animals were anaesthetized through intraperitoneal injection of a 2% xylazine solution (10 mg/kg) and a 10% ketamine solution (100 mg/kg). ¹⁴ Following this, their chest and lumbar areas were clipped and prepped for an aseptic procedure. Subsequently, a full‐thickness skin defect of 2 × 2 cm was created in the lumbar region.

2.4. Blood sampling and preparation of M2 macrophages

Simultaneously, a sterile syringe was used to collect blood samples, which were then placed in a sterile Falcon tube containing 10% EDTA. Subsequently, the samples were centrifuged for 12 min at 3000 rpm. The Buffy coat was removed and remaining red blood cells were lysed with a RBC lysis buffer containing 8% ammonium chloride. Subsequently, the leucocytes were placed in a T12 flask containing DMEM supplemented with 10% foetal bovine serum (FBS), penicillin (100 units/mL), streptomycin (100 μg/mL) and amphotericin‐B (2 μg/mL). Following 4 h of incubation at 37°C and 5% CO₂, the flasks were washed twice with phosphate‐buffered saline (PBS) and were incubated under the same environmental conditions for 48 h. After this, the cells were exposed to a concentration of 10⁻⁵ mM dexamethasone (Dex) (Decadron, OBS pharma) for 48 h. The treated cells were washed with PBS and incubated for 5 days under the specified conditions in fresh DMEM medium in the absence of Dex. After the aforementioned culturing period of 9 days, the treated macrophages were removed by trypsinization, followed by two washes with PBS.

2.5. Cell injection to wound

On the tenth day after incision, 250 μL of DMEM medium without FBS was injected intradermally into the animals of the treatment group. It is worth noting that the M2 cells were applied as autografts. The rats of the control group were administered DMEM medium without cells in the four corners of the wound on the 10th day post‐injury in order to subject them to injection stress.

2.6. Macroscopic and microscopic evaluation

The extent of wound contraction and percentage of wound healing were determined by images taken 10, 17 and 24 days post‐surgery. Subsequently, the rats were administered a combination of 10% ketamine and 2% xylazine for anaesthesia. The remaining area of the wound was excised and the skin sample was affixed to a foil plate and placed in 10% formalin for 24 h. The samples were placed in fresh conrainers and sent to the pathology laboratory. After preparing the tissue sections, H&E staining and trichrome staining techniques were employed to visualize the tissue, cells and collagen fibres.

2.7. Data analysis

spss version 26 (IBM Corporation) was used to analyse the data. One‐way analysis of variance (ANOVA) with Tukey post‐test was used to compare the data between the study groups, while repeated measures ANOVA coupled with LSD post‐test was employed to assess differences within groups. Non‐parametric data were assessed employing the Kruskal–Wallis test. The findings were displayed as mean ± standard deviation or median (minimum – maximum) and p‐values < .05 were deemed to be statistically significant.

3. RESULTS

3.1. Wound healing percentage

Ten days after surgery, the healing rate was reported as 66.78 ± 1.90. After 14 days of wound formation, the treatment group had the highest healing percentage, which was statistically significant compared to the control group (C) (p = .001). At the end of the study on the 24th day post‐wounding, the treatment group demonstrated the highest healing percentage, which was significantly higher than that of the control group (C) (p = .001) (Table 1, Figure 1).

TABLE 1.

Mean ± standard error of wound healing rate (percentage) in full‐thickness experimental skin wound in both control and treatment groups in rats (n = 5).

Group/Time	Day 10	Day 17	Day 24
Control (C)	66.78 ± 1.90	83.80 ± 2.96	85.67 ± 5.15
Treatment (T)		93.04 ± 2.22*	97.49 ± 0.34*

^*Asterisk mark indicates significant difference between both groups in each time (p ≤ .05).

FIGURE 1.

Macroscopic assessment of the experimental wound was conducted in both the control and treatment groups containing different macrophages on days 10, 17 and 24 post‐wounding.

3.2. Histopathology results

3.2.1. Qualitative review

Tissue evaluation after 10 days

On the 10th day, sections prepared from rats of different groups were microscopically examined. After the injection of cells, the microscopic characteristics of the control and treatment groups were similar, displaying the formation of scabs on the surface of the wound, as well as numerous inflammatory cells and fibrin strands. Immature flesh bud tissue was seen in the wound area, together with a considerable number of inflammatory cells beneath the scab. Moreover, the epidermis around the wound edges grew in thickness due to the advancement of keratinocytes, and some of these proliferated keratinocytes migrated to the wound exterior (Figure 2).

FIGURE 2.

The skin sections of the control group, which had been prepared prior to wound induction, were observed 10 days after the induction of M2 macrophages. (A: 4×, B: 20×, C: 40×; H&E and trichrome staining).

Tissue evaluation after 17 days

On the 17th day, the skin sections of both diabetic rat groups were microscopically examined. In the control group, immature fleshy bud tissue (high vascular and pericellular) was identified at the wound site, accompanied by a plethora of blood capillaries bordering the collagen fibres. Furthermore, a few inflammatory cells were observed in the same area. The proliferation of keratinocytes covered a substantial portion of the wound, yet a part of it remained uncovered. This was likewise observed in the treatment group (Figures 3 and 4).

FIGURE 3.

The skin sections obtained from the control and treatment groups that had been exposed to M2 macrophages 17 days after wound induction demonstrated a notable increase in collagen fibres at the wound site in the treatment group. (D–F) compared to the control group (A–C). (A, D: 4×) and (B, E: 20×) and (C, F: 40×); H&E).

FIGURE 4.

The prepared skin sections of the control and treatment group with M2 macrophage cells on day 17; note the presence of a large amount of blue collagen fibres in the wound site in the treatment group (C, D) compared to the control group (A, B). (A, C: 4×) and (B, D: 40×); Trichrome staining.

Tissue evaluation after 24 days

In a microscopic analysis of the skin samples from the control group on the 24th day, there were still numerous inflammatory cells present. The mature bud tissue had developed, with a decrease in the number of fibroblasts and blood vessels, and an increase in the amount of collagen fibres. In the treatment group, there was a fully mature fleshy tissue, along with a considerable number of collagen fibres and a large number of fibrocytes (Figures 5 and 6).

FIGURE 5.

The prepared skin sections of the control and treatment group with M2 macrophage cells on day 24; note the presence of a large amount of collagen fibres in the wound site in the treatment group (D–F) compared to the control group (A–C). (A, D: 4×) and (B, E: 20×) and (C, F: 40×); (H&E).

FIGURE 6.

The prepared skin sections of the control and treatment group with M2 macrophage cells on day 24; note the presence of a large number of blue collagen fibres in the wound site in the treatment group (C, D) compared to the control group (A, B). (A, C: 20×) and (B, D: 40×); Trichrome staining.

3.2.2. Quantitative review

Number of blood vessels

Examination of the number of blood vessels present in the wound revealed that, on days 17 and 24 post‐skin injury, the lowest quantity of vessels was observed in the treatment group; however, no statistically significant difference was observed (Table 2).

TABLE 2.

Mean ± standard error of number of blood vessels in experimental full‐thickness skin wound in both control and treatment groups in rats (n = 5).

Group/Time	Day 10	Day 17	Day 24
Control (C)	22.98 ± 0.92	21.33 ± 1.25	15.66 ± 1.62
Treatment (T)		20.12 ± 2.33	12.01 ± 1.51

Number of fibroblasts

The analysis of the amount of fibroblasts present in the wound indicated that the treatment group with M2 macrophages had the smallest number of fibroblasts. On days 17 (p = .002) and 24 (p = .002) post‐wounding, this numerical disparity was statistically significant (Table 3).

TABLE 3.

Mean ± standard error of number of fibroblasts in experimental full‐thickness skin wound in both control and treatment groups in rats (n = 5).

Group/Time	Day 10	Day 17	Day 24
Control (C)	33.33 ± 0.49	30.33 ± 1.40	29.31 ± 2.96
Treatment (T)		24.71 ± 1.99*	13.75 ± 1.37*

^*Asterisk mark indicates significant difference between both groups in each time (p ≤ .05).

Number of inflammatory cells

It was observed that the treatment group had the lowest number of inflammatory cells (neutrophils). This statistical difference was noteworthy on the 17th (p = .002) and 24th (p = .002) days following wound induction (Table 4).

TABLE 4.

Mean ± standard error of number of inflammatory cells in experimental full‐thickness skin wound in both control and treatment groups in rats (n = 5).

Group/Time	Day 10	Day 17	Day 24
Control (C)	30.58 ± 1.22	32.18 ± 3.59	11.90 ± 2.46
Treatment (T)		19.52 ± 2.61*	4.14 ± 0.74*

^*Asterisk mark indicates significant difference between both groups in each time (p ≤ .05).

4. DISCUSSION

Diabetes has been linked to a decrease in macrophage ability to phagocytose. ¹⁵ , ¹⁶ , ¹⁷ This study has provided the first evidence that the number of apoptotic cells in cutaneous wounds of diabetic mice and humans is associated with a decrease in macrophage activity in the clearance of dead cells from the wound site. Recent studies have employed macrophage‐depleted mouse models to demonstrate the critical role of these cells in wound healing. Macrophages recruited during the inflammatory phase of repair induce the formation of scar tissue that leads to scar formation. As shown by macroscopic analysis of wound closure, depletion of macrophages during the inflammatory phase resulted in a significant delay in the initial repair response compared to control mice. ¹⁸ Additionally, it has been observed that macrophages play a pivotal role during the inflammatory phase of repair by stimulating macrophage alternative activation, which subsequently promotes angiogenesis and myofibroblast differentiation. ¹⁹

A further study demonstrated that glucocorticoids stimulate the differentiation of monocytes into macrophages, which demonstrate a similar phenotype to Erythroblastic island macrophages, and that this differentiation process is contingent upon GC receptor activation. ²⁰ Macrophage deficiency has been found to be detrimental to wound healing, ²¹ while uncontrolled activation of macrophages may impede healing and result in chronic wounds. Moreover, studies have demonstrated that M2 macrophages can increase vessel number and improve blood supply to the wound site. ²² Therefore, it is believed that some level of early pro‐inflammatory reactions are not detrimental, but instead, are necessary to trigger later stages of healing. It is the indiscriminate, prolonged or excessive activation of inflammation that is detrimental. Administration of M2 macrophages during the early inflammatory stages of previous experiments could have disturbed the equilibrium; however, in this study, this was avoided by administering the injection on day 10. Jetten et al. ¹² indicated that, in the context of healing responses in mice, employing exogenous M2 macrophages to manipulate the wound environment is not a viable therapeutic approach.

The persistence of neutrophils and macrophages in the late stages of repair has been implicated in the delayed wound closure observed in diabetic mice. ²³ However, Jetten et al. ¹² surprisingly found that the administration of M2 macrophages did not enhance the rate of wound closure in mice, even when the animals were genetically diabetic. An analysis of gene expression on day 15 wounds revealed an uptick in certain markers indicative of an alternative activation state, including arginase‐1 and YM‐1, in the M2 wounds, suggesting that the M2 phenotype was still present after 15 days of macrophage injection.

At the given time point, there was no variance in cytokine expression between the groups, yet M2‐injected wounds demonstrated a notable decrease in MMP2 expression compared to the controls. ¹² It is believed that neutrophils retard wound repair, as these cells have the potential to damage healthy tissue. Research has revealed that their depletion hastens wound healing in both diabetic and wild‐type mice. ²⁴ Previous studies were inconclusive as to whether the raised number of neutrophils in M2‐injected wounds was due to their late infiltration or the unfinished resolution of inflammation and neutrophil clearance. However, it has been hypothesized that the persistence of neutrophils is a part of the healing process which, in turn, delays scarring. ¹² Upon the 10th day of the experiment, the injection of macrophages had a divergent effect, leading to a decrease in the number of neutrophil cells and not maintaining the same levels. It is uncertain whether the amplified presence of neutrophils in wounds injected with M2 in Dreymueller et al. ²⁵ research is due to late infiltration or to the unfulfilled resolution of inflammation and neutrophils clearance. Thus, it can be postulated that the lingering of neutrophils retards the healing process of these cells.

At the outset of the healing process, the depletion of macrophages can lead to a delay in re‐epithelialization and closure of mouse skin wounds. Macrophages that are recruited during the inflammatory part of repair are responsible for stimulating the creation of scar tissue, as evidenced by macroscopic examination of the wound healing process. ²⁶ Macrophages derived from diabetic mice demonstrate greater capacity as helper cells in the propagation of antigen‐dependent T cells than their non‐diabetic counterparts. This is attributed to the alteration of the “pseudo‐lectin” receptor, which is affected by exposure to elevated glucose levels.

Even a brief delay in activating the defence mechanisms through macrophage activity could have a profoundly detrimental outcome. It may partially account for the heightened vulnerability to infection that is characteristic of diabetic patients. ²⁷ Injecting activated macrophages into wounds was demonstrated to stimulate wound healing. ²⁸ At the onset of repair, macrophage ablation has been observed to occur in sequence ⁶ or loss of TNF ²⁹ was shown to delay re‐epithelialization and wound closure of murine skin wounds. Consequently, it can be postulated that a limited degree of initial pro‐inflammatory activity in the early days following injury is not detrimental. On the contrary, it is essential for activating the succeeding healing processes, which has been validated by our findings. It is an unrestrained, protracted or excessive inflammation that is damaging. Administration of M2 macrophages during early stages of inflammation in prior studies may have interfered with this delicate equilibrium.

5. CONCLUSION

Five days after treatment of the monocytes with dexamethasone, under conditions including 48 h incubation with 10⁻⁵ M concentration, resulted in the differentiation of macrophages, which had a beneficial effect on wound healing. Results from clinical and histopathological evidence suggest that the injection of the differentiated M2 cells enhances the quality of wound healing in diabetic rats. Furthermore, treating the wound with M2 cells a few days after the wound was created has a noteworthy effect on the wound healing process in comparison to the initial days.

FUNDING INFORMATION

This research did not receive any specific grant from funding agencies in the public, commercial or not‐for‐profit sectors.

CONFLICT OF INTEREST STATEMENT

None.

Khoshnoudi P, Sabiza S, Khosravi M, Mohamadian B. Exploring effect of M2 macrophages on experimental full‐thickness wound healing in streptozotocin‐induced diabetic rats. Int J Exp Path. 2024;105:13‐20. doi: 10.1111/iep.12496