Beta‐Glucan: A Macrophage Differentiation Modulator That Accelerates the Wound Healing Response

ABSTRACT

Beta Glucans (β‐glucans) are naturally occurring polysaccharides that have positive effects on healing in acute and chronic wounds. This study aimed to identify how β‐glucans modulate macrophage polarisation and inflammation to aid the healing response. Flow cytometry was used to assess the effect of β‐glucan on human monocytes during differentiation into M0, M1 and M2 macrophages. Subsequently, a murine full‐thickness excisional wound healing model was conducted where wounds were treated with either β‐glucan hydrogel or vehicle, at the time of wounding. The wounds were analysed to determine the rate of wound closure, the effect on inflammation, and matrix deposition. β‐glucan promoted differentiation of monocytes to M0 macrophages but inhibited differentiation of M0 macrophages to pro‐inflammatory M1 macrophages with no effect on M2 macrophage formation. In vivo, treatment of excisional wounds with β‐glucan hydrogel accelerated healing with an earlier, more resolved inflammatory phase containing greater numbers of M2 macrophages and fewer neutrophils within the wound. No statistically significant effect on matrix deposition was observed. β‐glucans modulate macrophage differentiation and accelerate healing in excisional wounds with no adverse effect on matrix formation. β‐glucans are a potential therapeutic approach for treatment of hard‐to‐heal wounds in humans.

Highlights

• The key points of the work showed that treatment with beta‐glucan was able to inhibit monocyte to macrophage3 differentiation in vitro and in vivo.

• In vivo this led to a reduced inflammatory response resulting in an accelerated rate of healing.

Abbreviations and Acronyms

ATCC

American Type Culture Collection

CD

cluster of differentiation

CR3

complement receptor

DAMPS

damage associated molecular pathogens

DAPI

4′,6‐diamidino‐2‐phenylindole

DRFU

diabetic related foot ulcer

FMO

fluorescence minus one

IFN‐γ

interferon gamma

IL

interleukin

LPS

lipopolysaccharide

Ltd

limited

NF‐κB

nuclear factor kappa B

NOS

nitric oxide synthase

PAMPs

pathogen associated molecular pathogens

PBS

phosphate buffered saline

PMA

phorbol 12‐myristate 13 acetate

ROS

reactive oxygen species

TLR

toll‐like receptors

TNF

tumour necrosis factor

β‐glucan

beta‐glucan

1. Introduction

Wound healing is a highly complex process that involves coordinating the action of numerous skin and immune cell types, with the aim of reforming the skin barrier as quickly as possible. Due to its complex nature, this process can often be disrupted due to factors such as ageing, infection, peripheral vascular disease and diabetes [1]. This often results in hard‐to‐heal wounds termed chronic wounds. The cost of healing these wounds is estimated to be upwards of US$96.7 billion in the United States [2], which emphasises the need for new and more effective therapeutics.

Beta‐glucans (β‐glucans) are a group of β‐d‐glucose polysaccharides that occur naturally in cereals, bacteria, yeast, and fungi. Their study has expanded significantly in recent years due to the wide‐ranging effects they can have on the body, including the modulation of the gut microbiome, the immune response, and lipid and glucose metabolism [3]. This has led to their use as therapies in diet regulation, diabetes, cardiovascular disease, infection, cancer, and wound healing [3, 4]. They can vary in structure, which can result in numerous different conformations, which in turn can affect their solubility and action in biological systems [3, 4, 5, 6]. The solubility and conformation of β‐glucans determine what they can interact with and affect their mechanism of action. The two main areas that β‐glucans can affect the body are the metabolism and immune response. The glucans from cereals, consisting mainly of β1,3/1,4 glucans, affect metabolism and glucans from fungi, consisting of β1,3/1,6 glucans, have an immunomodulatory effect [3].

The immunomodulatory effects of β‐glucans have been shown to work through Complement Receptor 3 (CR3), Dectin‐1 receptor and toll‐like receptors (TLR), all of which are involved with wound healing [7, 8]. TLRs and Dectin‐1 receptors are present on most skin and immune cells such as keratinocytes, fibroblasts, endothelial cells, macrophages and neutrophils [4, 7, 9, 10, 11]. Both signalling pathways are activated by damage‐associated molecular patterns (DAMPs) and pathogen‐associated molecular patterns (PAMPs), such as cellular and extracellular matrix debris and invading pathogens, which results in the release of pro‐inflammatory cytokines that promote an innate immune response [3, 12].

The innate response plays a key role in the inflammatory phase of wound healing, through its action on macrophage polarisation [13]. Macrophages have dual and opposing functions within the wound [14]. Initially, monocytes migrate to the wound site and polarise into M1 macrophages, which are pro‐inflammatory in nature, releasing factors such as TNF, and act to phagocytose any cellular debris and pathogens. Once this action is achieved, macrophages polarise to more of an M2 phenotype, releasing factors including interleukin‐10 (IL‐10), which acts to resolve the inflammatory response and to promote the proliferative phase of wound healing [12, 13]. Early activation of the innate response, through receptors like TLRs, can lead to an earlier resolution of the inflammatory phase, resulting in faster healing times [13].

β‐glucan activation of TLR2 and Dectin‐1, in murine and human macrophages, has been reported to increase the expression of NF‐κB, TNF, IL‐1β, nitric oxide synthase (NOS), and reactive oxygen species (ROS) [7]. This activation of the innate immune response would recruit immune cells to the wound site in vivo. This was shown to be the case in a study by LeBlanc et al. [15], where infected wounds created on Fisher‐344 rats were treated with β‐glucan, which led to an increase in the neutrophil influx into the wound site. This helped to clear the wound of infection and increase the rate of healing [15]. β‐glucan treatment can also reduce the expression of pro‐inflammatory cytokines IL‐1α/β and IL‐6 in infected rat wounds; however, the effect on inflammatory cells within the wounds was not investigated [16]. The ability of β‐glucans to inhibit the inflammatory response has been observed in a study of corneal alkali burns in Sprague–Dawley rats and an excisional wound model in mice [17, 18]. In both studies, β‐glucan treatment suppressed acute inflammatory reactions with fewer polymorphonuclear lymphocytes at the wound site [17].

β‐glucans have been shown to affect the proliferative phase of wound healing. In a study by Kang et al., β‐glucan derived from barley halted proliferation but increased migration of human dermal fibroblasts after 24 h [19]. In addition to this, Yamamoto and Kimura [20] showed that in vitro treatment of adult dermal human fibroblasts with β‐glucan could increase collagen production. This effect was further seen in vivo where infected rat wounds treated with β‐glucan showed increased fibroblast migration into the wound with more organised collagen formation compared to controls [16]. Increased collagen expression was also seen in β‐glucan treatment of mouse excisional wounds [18]. The overall effects of β‐glucan treatment of wounds reported in the literature appeared to be beneficial with an accelerated healing response in acute [16, 18, 21, 22, 23], infected [24, 25, 26] and diabetic wound models [20, 27, 28].

These in vivo studies have led to β‐glucans being used as a wound treatment in eight human clinical trials. Four had been completed at the time of writing (April 2026), two were terminated, one was active but not recruiting, and one had an unknown status. They have been used to treat a variety of wounds including venous ulcers [29], diabetic foot ulcers [30], non‐healing ulcers associated with diabetes [31] and ‘hard to heal’ wounds [32]. All studies reported beneficial effects of β‐glucan treatment on these wounds, though the data was not always significant. Comparisons between the studies were difficult due to the differences in the wounds treated, the varying β‐glucan formulations used and the treatment regimes.

The above studies have given us some data on how β‐glucan treatment of wounds can help to improve the wound healing response, even in hard‐to‐heal wounds. Whilst β‐glucans have been shown to influence the immune response overall, few studies have assessed their effect on specific immune cells present within a wound. Here the effect of β‐glucan, derived from Saccharomyces cerevisiae , on macrophage polarisation was investigated in vitro, and its effect on inflammation and healing determined when applied as a wound treatment in a hydrogel formulation.

2. Materials and Methods

2.1. In Vitro Assays to Determine the Effect of β‐Glucan on Macrophage Polarisation

A human THP‐1 monocyte cell line (ATCC, VA, USA; Table S2) was used to determine the effects of β‐glucan on the activation of monocytes and the polarisation of macrophages, which was adapted from [33]. Cells were maintained in RPMI (ATCC, VA, USA), supplemented with 10% foetal bovine serum and penicillin/streptomycin (Thermo Fisher, VIC, Australia) termed growth media. For assays, cells were seeded at 300,000 cells/mL. Control cells were left untreated. To differentiate THP‐1 cells to macrophages, cells were treated with 5 ng/mL of phorbol 12‐myristate 13 acetate (PMA; Merck, VIC, Australia) for 72 h, after which the media was replaced with growth media and the cells left to culture for a further 4 days to create M0 macrophages. Cells were then further differentiated to M1 macrophages by treating with 20 ng/mL of lipopolysaccharide (LPS; Merck, VIC, Australia) and 20 ng/mL interferon gamma (Merck, VIC, Australia) for 24 h. M0 macrophages were also differentiated into M2 macrophages using 20 ng/mL of IL‐10 (R&D Systems, MN, USA). Cells were treated, then left for 3 days before a further treatment with IL‐10, after which time the cells were left for a further 3 days. At this point the media was replaced without the addition of IL‐10 and the cells were then cultured for a further 3 days. When differentiating to M0, M1, and M2 macrophages, cells were treated with or without β‐glucan (20 μg/mL) to determine its effects on the formation of these three cell states. For treatments refer to Table S1.

2.2. Antibodies and General Reagents

Primary antibodies used for immunohistochemistry are as follows: NIMP‐R14 (rat anti‐mouse) and TNF (goat anti‐mouse) were sourced from Santa Cruz (TX, USA), F4/80 (rabbit anti‐mouse; Cell Signalling Technology, VIC, Australia), IL‐10 (rabbit anti‐mouse; Abcam, VIC, Australia), Ym‐1 (rabbit anti‐mouse; StemCell, VIC, Australia) and collagen I and collagen III (rabbit anti‐mouse) were from Rockland Immunochemicals (PA, USA). Secondary antibodies were Alexa‐Fluor 488, 633 and 635 from Thermo Fisher Scientific (VIC, Australia).

2.3. β‐Glucan

The β‐glucan (Figure 1A) was kindly donated by Tissue Repair Ltd. (Tissue Repair, NSW, Australia). It had a molecular weight of 0.8–1.5 MDa and was greater than 85% pure. It was solubility in water was 0.64–0.66 mg/mL and > 16 mg/mL in DMSO. Wounds were treated with a hydrogel that contained 0.1% of the active β‐glucan ingredient. This dosage was determined via a dose response study investigating doses of 0.1%, 1% and 10% in an in vivo murine wound healing model. The 0.1% treatment had the fastest rate of wound closure and was the same dosage which has previously been used in human clinical trials. Wounds were treated with a hydrogel that contained 0.1% of the active β‐glucan ingredient. Control wounds were treated with the hydrogel alone. For in vitro assays, the active β‐glucan ingredient was resuspended in PBS.

FIGURE 1.

(A) 3D structure of the β‐glucan. Graphs of the population percentages of CD11b⁺, CD14⁺, CD80⁺ and CD206⁺ cells from (B) β‐glucan treated THP‐1s s, (C) β‐glucan treatment of PMA differentiated THP‐1 cells, (D) β‐glucan treatment of PMA, IFN‐γ and LPS differentiated THP‐1s (M1 macrophages) and (E) β‐glucan treatment of PMA and IL‐10 differentiated THP‐1 cells (M2 macrophages). * p > 0.05 and **p > 0.01.

2.4. Immunostaining to Determine Cell Differentiation State via Flow Cytometry

Cells were collected, centrifuged and washed using PBS (Merck, VIC, Australia) before being stained with Fixable Viability Stain 780 diluted 1/1000 and blocked with Human FC Block for 10 mins. Cells were then washed in FACS buffer (PBS, 1% BSA, 0.04% azide) and stained for 30 min with directly conjugated antibodies in Brilliant Stain Buffer (see Table S2 for staining reagents). Cells were stained for CD11b, CD14, CD80 and CD206 (for antibody details see Table S3). Control staining and FMO were determined before analysis was carried out (Figure S1).

2.5. Murine Models Used to Determine the Effect of β‐Glucan on the Rate of Wound Closure

All experiments were approved by University of South Australia Animal Ethics Committee (#U09‐23) and followed the Australian Code for the Care and Use of Animals for Scientific Purposes. Mice were ordered from Animal Resource Centre (WA, Australia) and were group housed upon arrival. They acclimatised for 7 days and kept in a 12‐h day/night cycle with free access to food and water. On the day of surgery mice were administered analgesic 30 min prior to surgery and anaesthetised using isoflurane inhalation. Anaesthetic induction was carried out with 2 L/min of oxygen and isoflurane, and maintenance was carried out at 2 L/min of oxygen and 0.2 L/min of isoflurane. One full thickness 10 mm circular excision was made on the dorsum of each mouse (C57BL/6, female, 8–12 weeks). The excisions were 1 cm from the occipital protuberance and either side of the midline.

The wounds were then treated with either 0.1 g of 0.1% β‐glucan hydrogel (β‐glucan) or vehicle (hydrogel alone) immediately after wounding. A Tegaderm dressing was then placed over the wound to keep the treatment in the wound area. If the dressing was removed by the mouse, it was replaced under inhaled anaesthetic.

Once the mice were awake and mobile, they were individually caged, and the wounds left to heal by secondary intention. At Day 3, the Tegaderm dressings were removed. The endpoints investigated were either Day 3 or 7. Wounds were photographed daily for macroscopic measurements. At the endpoint, the wounds were removed and bisected. Half the wound was snap frozen in liquid nitrogen and then stored at −80°C for molecular analysis. The other half was fixed in 10% neutral buffered formalin for 24 h, then processed into paraffin for immunohistochemistry.

2.6. Immunohistochemistry Assays to Determine the Wound Cell Infiltrate and Collagen Production

Mouse sections were cut from paraffin embedded tissue at 4 μm using a microtome. Antigen retrieval was performed by heating the sections, using a pressure cooker, in 0.1 M citric acid buffer (pH 7.4) at 90°C for 10 min, then allowed to cool to room temperature. All antibodies were used at 1:100 dilution and appropriate secondary antibodies were used at 1:200 dilution. Five drops of DAPI (concentration of 1:10000) were added to each section and incubated for 5 min prior to the end of the staining procedure. Secondary antibodies that were used included: goat anti‐rabbit Alexa Fluor 635, donkey anti‐rabbit Alexa Fluor 568, donkey anti‐goat Alexa Fluor 633 and donkey anti‐rat Alexa Fluor 488 (Thermo Fisher Scientific, VIC, Australia). The staining protocol was adapted from Mills et al. [13].

2.7. Statistical Analysis

Analysis of wound measurements and cell counts was carried out using CellSens software (Olympus, SA, Australia). Statistical differences were determined using a student's t‐test. For non‐parametric data, a Mann–Whitney U test was used. A p value of < 0.05 was considered significant. Electronic laboratory notebook platform was not used.

3. Results

β‐glucan treatment of the THP‐1 cells altered the surface expression markers CD11b, CD14, CD80 and CD206. The effect of β‐glucan treatment alone on THP‐1 cells had no significant change in any of the markers measured by flow cytometry (Figures 1B and S2B). THP‐1 monocytes, treated with PMA to induce their differentiation into macrophages, showed a significant increase in the expression of CD14 when also treated with β‐glucan compared to those treated with PMA alone, suggesting that β‐glucan may promote the differentiation of the monocytes to macrophages when compared to PMA treatment alone (58%–62% respectively; p = 0.048, Figures 1C and S2C). There were no significant differences seen in the other markers after PMA treatment.

3.1. β‐Glucan Treatment Inhibited the Differentiation of Macrophages to M1 Macrophages

A highly significant effect was seen when the cells were treated with IFN‐γ and LPS to polarise the cells into M1 macrophages and treated with and without β‐glucan (Figures 1D and S2D). The expression of all cell markers was reduced in the β‐glucan treated groups (CD11b; 75% and 53%; p = 0.006, CD14; 64% and 51%; p = 0.014, CD80; 65% and 44%; p = 0.003 and CD206; 5% and 3%; p = 0.014, respectively). This suggested that the addition of β‐glucan into the media was inhibiting the polarisation of the cells to an M1 macrophage phenotype. No statistically significant differences were observed in cells treated with IL‐10 and with and without β‐glucan. There was a trend to a greater percentage of CD206 positive cells (an M2 macrophage marker) in the β‐glucan treated group vs. non‐β‐glucan treated group (28% and 33% respectively), but this was not statistically significant (Figures 1E and S2E).

3.2. β‐Glucan Treatment Accelerated Healing of Murine Excisional Wounds

To determine the effect of β‐glucan treatment on the rate of wound closure, murine excisional wounds were treated with a β‐glucan hydrogel or hydrogel alone (vehicle). The macroscopic wound gape measurements showed no significant differences at Day 0 or Day 3. β‐glucan treated wounds were 10% smaller at Day 7 (p = 0.005), which was significant when compared to vehicle treated wounds (Figure 2A,B). The microscopic wounds measurements (Figure 3C) showed no significant differences at Day 3. At Day 7, there was a trend towards improved epithelisation that did not achieve statistical significance for the percentage of the wound that had been re‐epithelialised (Figure 2D). More compelling, however, was the wound widths (p = 0.01) and the wound areas (p = 0.0031), both of which were significantly smaller in β‐glucan treated wounds (Figure 2E,F).

FIGURE 2.

Representative (A) photographs and (C) microscopic images of H&E‐stained Day 7 mouse wounds treated with β‐glucan or vehicle. Graphs of (B) macroscopic wound gape and microscopic (D) percentage re‐epithelialisation, (E) wound width and (F) wound area. Black arrows denote wound width. Scale bars in (A) = 5 mm and (C) = 2 mm. * p > 0.05 and **p > 0.01.

FIGURE 3.

Representative images of Day 3 and Day 7 (A) NIMP‐R14 and (C) F4/80 and TNF‐stained wounds treated with β‐glucan or vehicle. Graphs showing (B) neutrophil (NIMP‐R14⁺), (D) TNF⁺ macrophages and (E) total macrophages numbers in the Day 3 and Day 7 wounds, treated with β‐glucan or vehicle. White arrows denote positive cell staining. Images in (A) and (C) taken at ×20 magnification ((A) scale bar = 20 μm and (C) scale bar = 50 μm), panels with the white asterisk are a magnified image taken at ×40 magnification (scale bar = 10 μm). **p > 0.01.

3.3. β‐Glucan Treatment Reduced the Number of TNF+ Macrophages in Murine Excisional Wounds

To determine the effect of β‐glucan on inflammation, wounds treated with the β‐glucan hydrogel or vehicle were stained for the presence of neutrophils and TNF⁺ macrophages. At Day 3 no significant difference was observed in the number of neutrophils but by Day 7, β‐glucan treated wounds had 1.6 times (p = 0.006) fewer neutrophils in the wounds than vehicle treated wounds (Figure 3A,B).

To further investigate the effects of β‐glucan treatment on the inflammatory cell influx into the wound, sections were stained for the macrophage marker F4/80 and the pro‐inflammatory cytokine TNF, a marker of M1 macrophages. When investigating the staining of TNF⁺ macrophages, there were no significant differences seen at Day 3. In contrast, at Day 7, there were 50% fewer TNF positive macrophages in β‐glucan treated wounds when compared to vehicle (p = 0.0002) treated wounds (Figure 3C,D). No differences in the number of overall macrophages or the number of M1 macrophages were observed between the two treatment groups at Day 3 and Day 7 (Figure 3E).

3.4. β‐Glucan Treatment Increased the Number of M2 Macrophages in Murine Excisional Wounds

The wounds from both treatment groups were also stained to determine the presence of M2 macrophages (Ym1⁺) and IL‐10⁺ macrophages, to determine how advanced the resolution of the inflammatory phase was progressing. Cells were also co‐stained with F4/80 and IL‐10, a cytokine expressed by M2 macrophages (Figure 4A,B) and F4/80 and Ym1, an M2 macrophage marker (Figure 4C,D). β‐glucan treated wounds had less than half the number of IL‐10⁺ macrophages (p = 0.00036) compared to vehicle treated wounds at Day 3 (Figure 4B). Additionally, there were significant differences seen at Day 7, with the β‐glucan treated wounds having 48.9% less IL‐10⁺ macrophages in the wounds when compared to the vehicle treated wounds. There were also differences in the number of M2 macrophages with 2.5 times greater numbers of M2 macrophages seen in the wounds treated with β‐glucan (Figure 4E), when compared to vehicle treated wounds at Day 7 (p = 0.0097).

FIGURE 4.

Representative images of Day 3 and Day 7 (A) F4/80 and IL‐10 and (C) F4/80 and Ym1 stained wounds treated with β‐glucan or vehicle. White arrows denote positive cell staining. Graphs showing (B) IL‐10⁺ macrophages, (D) M2 macrophages and (E) the ratio of M1:M2 macrophages in the Day 3 and Day 7 wounds, treated with β‐glucan or vehicle. Images in (A) and (C) taken at ×20 magnification (scale bar = 50 μm), panels with the white asterisk are a magnified image taken at ×40 magnification (scale bar = 10 μm). **p > 0.0.1 and ***p > 0.001.

When comparing the ratio of M1 and M2 macrophages there were no differences at Day 3. On Day 7, however, there was a significantly higher ratio of M2 to M1 macrophages, with twice as many M2 to M1 macrophages in the β‐glucan treated wounds compared to vehicle treated wounds (Figure 4E).

3.5. β‐Glucan Treatment Had no Effect on Collagen Deposition in Murine Excisional Wounds

No significant differences were seen in the Masson's trichrome staining between the two treatment groups but there was a trend to suggest that the β‐glucan treated wounds had a collagen content closer to that of the surrounding unwounded skin (Figure S3A,B). The effect of β‐glucan treatment on the matrix deposition phase of wound healing was also investigated. No significant differences were seen in the collagen I and III staining between the two groups and no significant differences were seen in the collagen I to III ratios (Figure S3C–F).

4. Discussion

β‐glucans have been investigated as therapies, in clinical trials, for the treatment of a variety of skin conditions including atopic dermatitis, irradiated skin, skin barrier dysfunction, photoaging and UV damage, as well as metabolic disorders and cancer (clinicaltrials.gov search parameter ‘treatment—glucan’ in April 2026). In the study investigating the effects on atopic dermatitis, the yeast‐derived β 1,3/1,6 glucan was administered orally and was able to modulate mRNA expression of pro‐inflammatory transcription factors, which led to a reduction in vasodilation and pruritus oedema [34]. Ceyhan et al. [35] investigated the effects of β‐glucan on oxidative injury caused by electromagnetic radiation. In this study, the oral administration of β‐glucan was able to reduce superoxide dismutase and levels of malondialdehyde, which resulted in a reduction in skin damage from the oxidative injury.

When considering human clinical trials investigating the effect of β‐glucan on the wound healing process, numerous aetiologies have been investigated, including burns, venous leg ulcers, infection, uveitis, chronic diabetic foot ulcers, anal fissures and haemorrhoids [3]. In these studies, treatments appear to promote healing [29, 31] and to potentially restart the healing process in wounds where it appeared to have stalled [32]. The potential mechanism promoting the healing response was linked to the increased recruitment of inflammatory cells, proliferation of fibroblasts, increased rates of re‐epithelialisation and angiogenesis [29].

β‐glucans have been found to have numerous biological functions within the body, many of which are immunomodulatory, signalling through receptors including CR3, TLR2, 4 and 6 and the Dectin‐1 receptors [7, 8]. This has been investigated more thoroughly using in vitro and in vivo models, as these are less restrictive the human clinical trials. β‐glucans have also shown to dampen the inflammatory response in human monocytes to reduce pro‐inflammatory cytokine expression (TNF) and increase anti‐inflammatory cytokines (IL‐10) [36]. This mechanism has previously been shown to increase rate of healing [13]. β‐glucan activation of dectin‐1 has shown to promote human keratinocyte proliferation and migration potentially providing a mechanism to explain the increased rate of re‐epithelialisation seen in human clinical trials, using β‐glucan for the treatment of wounds [37].

This study used a β‐glucan, derived from Saccharomyces cerevisiae , for in vitro studies and a hydrogel containing 0.1% β‐glucan for the in vivo wound studies. This β‐glucan formulation used in this study was derived from yeast, but it is possible that similar effects could be seen in other fungal‐derived β‐glucans containing β 1,3/1,6 glucans. Whilst murine skin is inherently different to human skin, it does offer a model where the effects of β‐glucan treatment on the immune and wound healing process can be investigated. The dosage was determined after testing concentrations of 0.1%, 1%, and 10% in a murine wound healing model. The 0.1% dosage was chosen as it had the fastest wound closure rate and was also the dosage which had previously been used in human clinical studies using this β‐glucan formulation [38]. β‐glucan treatment was applied once at the time of wounding and was shown to accelerate healing after 7 days when compared to vehicle controls (Figure 2). This was in accordance with other studies that have shown β‐glucan treatment to accelerate healing in acute [16, 18, 21, 22, 23], infected [24, 25, 26] and diabetic wound models [20, 27, 28]. To identify the mechanism responsible for the accelerated healing observed, in vitro and in vivo analyses were carried out.

Studies using β‐glucan treatment have shown it to have varying effects on the inflammatory phase of wound healing, with some studies showing an increase in immune cell influx [15] and others a reduction [16, 17, 18]. For this current study, we built upon previous work using the same β‐glucan formulation, which was shown to increase the expression of pro‐inflammatory mediators, TNF and ROS, in human macrophages, through TLR2 and Dectin‐1 signalling [7]. This study investigated how this may affect the polarisation of macrophages (Figures 1 and S1) and showed that β‐glucan treatment promoted monocyte differentiation into macrophages (Figure 1C). Interestingly, it additionally showed that β‐glucan treatment significantly inhibited macrophage polarisation to an M1 phenotype (Figure 1D) but had no significant effect on M2 macrophage polarisation (Figure 1E). This finding was significant as the initial study by Roy et al. [7] suggested that β‐glucan treatment would promote a pro‐inflammatory response, while this study suggested that the mechanism may be more nuanced and may control the degree of inflammation by regulating macrophage polarisation.

β‐glucan and vehicle‐treated wounds were assessed to identify the inflammatory cells within the wounds. At Day 7, there was a significantly reduced inflammatory response with fewer neutrophils and TNF⁺ macrophages, within the β‐glucan treated wounds (Figure 3). This mirrored the in vitro mechanism which showed that β‐glucan treatment resulted in an inhibition in the formation of M1 macrophages, which are known to produce TNF [12, 13]. This was further confirmed when the numbers of M2 macrophages were shown to be increased in the β‐glucan treated wounds at Day 7 (Figure 4D), which resulted in a greater ratio of M2 macrophages when compared to M1 macrophages (Figure 4F). This suggested that the β‐glucan treated wounds were in a more advanced stage of healing, with a more resolved inflammatory phase, compared to vehicle control wounds that showed higher numbers of immune cells present within the wounds. These findings appeared to support the in vitro mechanism that β‐glucan treatment could control macrophage polarisation to regulate inflammation.

IL‐10 has previously been shown to be induced by β‐glucan treatment in human monocyte‐derived macrophages that could potentially promote a polarisation to an M2c macrophage phenotype, which is specifically formed in response to IL‐10 [7]. This was investigated in this study and whilst there was a trend to suggest that β‐glucan treatment could increase the percentage of M2c polarisation, this data was not significant (Figure 1E). Interestingly, in vivo vehicle control treated wounds contained higher numbers of IL‐10⁺ macrophages. The reason for this could have been differences in the in vitro and in vivo environments or that a different subset of M2 macrophages was present and expressing IL‐10. It has also been reported that alternate sources of IL‐10, such as neutrophils, can also be present after treatment with β‐glucan [39]. A reduction in IL‐10 expression has been reported in another in vivo excisional wound healing model treated with a β‐glucan hydrogel [16]. It could be hypothesised that in these two wound healing studies, where there were a reduced number of pro‐inflammatory cytokines and macrophages, in the case of the β‐glucan treated wounds, only a subsequent and reduced IL‐10 induced resolution of the inflammatory process was necessary. Overall, the inflammatory data from this study suggested that treating the wounds with β‐glucan significantly reduced the immune cell influx into the wounds, shortening the inflammatory response and increasing the rate of wound closure (Figure 5). This mechanism has been observed in other work, which has shown that an early initiation of the immune response after wounding, through receptors like TLRs, can result in a diminished and shortened inflammatory phase that results in faster healing times [13].

FIGURE 5.

Illustration showing the mechanism of beta‐glucan action on macrophages, which inhibits M1 formation and promotes a higher M2/M1 ratio, resulting in accelerated healing.

β‐glucan treatment has also been shown to increase fibroblast migration and collagen deposition in vitro [20, 40] and to increase fibroblast migration into a wound site resulting in a more organised deposition of collagen [16, 18]. The effect of β‐glucan treatment in this study showed limited effects on the collagen I and III deposition. The Masson's trichrome staining suggested that β‐glucan treated wounds had a more mature collagen content, which reflected that these wounds had a more progressed healing response when compared to the vehicle‐control treated wounds, though this was not statistically significant (Figure S3). The non‐significant effects on collagen in this model may be due to multiple factors. It should be noted that mouse wounding models are not optimal for investigating scarring and collagen deposition due to the inherent and significant differences in the skin architecture compared to humans. An investigation into later time points, in this study, may have revealed a more significant effect of the β‐glucan treatment on collagen formation.

5. Conclusion

β‐glucans are an area of research interest for their use in various conditions due to their safety profile and beneficial effects on various biological activities. This study showed that treatment with a β‐glucan hydrogel accelerated the closure of murine excisional wounds. This was achieved by modulating the differentiation of macrophages, inhibiting the formation of M1 pro‐inflammatory macrophages, resulting in an earlier increase in M2 macrophage numbers. This promoted an earlier resolution of inflammation and faster healing after 7 days (Figure 5). This mechanism has previously been shown to be initiated by an activation of TLRs, of which β‐glucan is a known activator [13].

This accelerated healing has been observed in clinical trials where diabetic and ‘hard to heal’ wounds have been treated with β‐glucans [31, 32] One trial treating venous ulcers showed that the accelerated healing was through immunomodulator effects and increased rates of re‐epithelialisation, which mirrored the findings from this study [29]. Together these data suggest β‐glucan treatment appears to offer a safe effective therapeutic for the treatment of various wound types including hard to heal wounds.

Funding

This work was supported by Tissue Repair Ltd.

Disclosure

Tissue Repair Ltd. supplied the β‐glucan used in this study. A powdered form was supplied for the in vitro study, which has previously been termed Glucoprime/Glyc101 and a hydrogel containing 0.1% of the active β‐glucan, previously termed TR Pro⁺, was supplied for the in vivo studies. The author(s) declare that no Generative AI was used in the creation of this manuscript.

Ethics Statement

All experiments were approved by the Adelaide University Animal Ethics Committee (#U09‐23) and followed the Australian Code for the Care and Use of Animals for Scientific Purposes.

Conflicts of Interest

S.J.M., C.S. and A.J.C. are employed by Adelaide University. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflicts of interest. D.R. and T.C. are employees of Tissue Repair Ltd. but had no input or influence on the study design, the carrying out of the experiments or the analysis and reporting of the results.

Supporting information

Figure S1: (A) FMO Staining using unstained, isotype and antibody staining and (B) the gating strategy used for the flow.

Figure S2: Flow data showing, (A) isotype control staining. Flow staining showing the population percentages of CD11b⁺, CD14⁺, CD80⁺ and CD206⁺ cells of (B) THP‐01 cells treated with and without β‐glucan, (C) β‐glucan treatment of PMA differentiated THP‐1 cells, (D) β‐glucan treatment of PMA, IFN‐γ and LPS differentiated THP‐1 s (M1 macrophages) and (E) β‐glucan treatment of PMA and IL‐10 differentiated THP‐1 cells (M2 macrophages).

Figure S3: Representative images of Day 7 (A) Masson's Trichrome staining and Day 3 and Day 7 (C) collagen I and collagen III‐stained wounds treated with β‐glucan or vehicle. Graphs showing (B) Masson's staining intensities, (D) collagen I and (E) collagen III intensities and (F) the ratio of collagen I:collagen III staining intensities, in the Day 3 and Day 7 wounds, treated with β‐glucan or vehicle.

Table S1: Treatment groups for the macrophage differentiation studies

Table 2. List of materials and reagents for flow cytometry.

Table 3. Antibodies used for flow cytometry analysis.

Data Availability Statement

The original contributions presented in the study are included in the article/Supporting Information. Further inquiries can be directed to the corresponding author.