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DOI: 10.1039/D5MH01062D (Communication) Mater. Horiz., 2025, Advance Article

Human breast milk as a bioactive material platform enabling scarless skin regeneration via macrophage reprogramming and myofibroblast heterogeneity

Caihong Xianab, Zhipeng Gud and Jun Wu*ac
aBioscience and Biomedical Engineering Thrust, The Hong Kong University of Science and Technology (Guangzhou), Nansha, Guangzhou, 511400, Guangdong, China. E-mail: junwuhkust@ust.hk
bDepartment of Orthopedics, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, China
cDivision of Life Science, The Hong Kong University of Science and Technology, Hong Kong SAR, China
dSchool of Biomedical Engineering, Sun Yat-sen University, Shenzhen 518057, China

Received 5th June 2025 , Accepted 11th August 2025

First published on 19th August 2025

Abstract

The regulation of the immune system and promotion of myofibroblast heterogeneity in wounds are crucial for developing scar-reducing treatments. Currently, no approved multifaceted drug exists that can fully inhibit fibrosis. In this study, we explore the beneficial effects of human breast milk (HBM), focusing on its ability to regulate the macrophage phenotype and function, and promote myofibroblast heterogeneity to inhibit skin scar formation. Drawing inspiration from the scarless healing observed in fetuses, we utilize HBM to treat full-thickness cutaneous wounds. Our in vitro and in vivo experiments demonstrate that HBM, applied as a spray or in combination with a thermosensitive hydrogel, significantly increases M2 macrophage recruitment by at least 33.5% and enhanced angiogenic responses by up to 98.1%, while also improving skin stiffness and elasticity. Furthermore, we observe that HBM facilitates the formation of more neogenic hair follicles by activating the insulin-like growth factor 1 (IGF1) and platelet-derived growth factor C (PDGFC) signaling pathways in macrophages. This activation subsequently triggers BMP signaling, which stimulates adipocytes and promotes myofibroblast heterogeneity, resulting in normalized wound healing. These findings suggest that HBM holds significant potential for various medical applications, including the development of vascularized tissues, the reduction of stretch marks, and the promotion of wound regeneration without scarring. This study highlights the promising role of HBM in advancing regenerative medicine and tissue engineering.


New concepts

Our manuscript introduces a groundbreaking concept in scar-inhibiting wound therapy by demonstrating, for the first time, that human breast milk (HBM), applied as a spray and synergized with a thermosensitive hydrogel, serves as a multifaceted biomaterial to promote scarless healing. This breakthrough development leverages the HBM's innate ability to recruit M2 macrophages by over 33.5%, enhances angiogenic responses by up to 98.1%, and promotes myofibroblast heterogeneity, thereby normalizing skin repair without fibrosis. Diverging from existing scar interventions, which often target isolated pathways like TGF-β signaling through pharmaceuticals or single-target biomaterials, our approach uniquely addresses concurrent profibrotic mechanisms by activating IGF1 and PDGFC signaling in macrophages. This cascade stimulates BMP-mediated adipocyte differentiation, offering a holistic, natural solution that overcomes the limitations of current monotherapies. To materials science, our work delivers critical insights by revealing how thermosensitive hydrogels as dynamic and responsive delivery platforms, can synergize with biological agents like HBM to modulate immune responses and cellular heterogeneity spatially and temporally. This concept advances biomaterial design for regenerative applications, such as vascularized tissue engineering and scar-free wound dressings, by emphasizing multifunctionality and bio-inspired strategies to reduce the global healthcare burden.

1. Introduction

Fibrotic scar tissue typically forms in adult skin during the wound healing process, particularly following thermal and chemical burns. This can result in disfigurement, disability, and a diminished quality of life for the affected individuals.1–5 Treatment often requires multiple surgical revisions, leading to significant financial burden. By 2027, global spending on scar treatments is projected to reach approximately $32 billion.6 Given the prevalence of wounds and the associated costs of scarring, various approaches have been developed, including pharmaceutical products, cell therapies, tissue-engineered substitutes, and biomaterial-based dressings, all aimed at preventing or minimizing scar formation.7–10 Despite decades of research and clinical application, wound healing without scarring has not been achieved.

Tissue repair is a complex process traditionally categorized into three stages: inflammation, proliferation, and remodeling. Inflammatory cells, particularly macrophages, play crucial roles throughout all phases of wound healing by facilitating debridement and producing chemokines, metabolites, and growth factors that are often linked to scarring.11–13 Reprogramming macrophages toward a pro-regenerative phenotype directly enhances proliferative processes in tissue repair through augmented growth factor secretion and metabolic remodeling. Research studies on polyphenols, stem cells, and exosomes have demonstrated that the regulation of macrophage polarization is critical to the wound healing process.14–17 However, given that wound healing is a dynamic interplay of multiple cell types and stages, targeting only a single component may yield limited efficacy.

Additionally, fibroblasts are essential for synthesizing and organizing the extracellular matrix (ECM), making them key mediators in the scarring process.18 Research studies by Mascharak and colleagues demonstrated that disrupting the conversion of En1-lineage-negative fibroblasts to En1-lineage-positive fibroblasts, along with inhibiting mechanotransduction, reduced skin fibrosis in both mice and pigs.18,19 Notably, Cotsarelis and colleagues discovered that fat cells could regenerate from myofibroblasts during wound healing, leading to formation of new hair follicles and promoting normal wound healing.20 The heterogeneity of myofibroblasts is advantageous for achieving scarless wound healing. Fibromodulin, heparan sulfate, and engineered extracellular vesicle delivery systems show promise for clinical translation by modulating the fate of myofibroblasts.21–23 Despite significant advances in understanding the mechanisms and key players involved in both physiological and pathological tissue repair, no drug has yet been approved as a complete inhibitor of fibrosis.24 Given that regulating the macrophage phenotype and function and promoting myofibroblast heterogeneity in wounds are essential for the development of effective scar-reduction treatments, a multifaceted therapeutic approach may be essential for success.

Edible materials, similar to natural tissue both in biological and chemical properties, play crucial parts in regenerative medicine,25–28 yet their effect in healing without scarring is expected to be further improved. Therefore, it is imperative to identify suitable edible materials for wound with functional healing. Inspired by the scarless healing observed in fetuses, human breast milk (HBM) may offer significant potential for accelerated reconstructive healing and reduced scar formation. HBM consisting of abundant components like lipids, carbohydrates, and complex proteins, may modulate the microenvironment of the wound by delivering different mechanisms. Furthermore, in contrast to biological therapeutic agents such as exosomes, stem cells and growth factors, HBM is cost-effective, widely accessible, and inherently biocompatible. Herein, we explored the effect of HBM in promoting wound healing and normalizing the wounded skin. In this study, it was demonstrated that HBM exhibited good biocompatibility, low immunogenicity, angiogenesis, and antioxidant and anti-inflammatory activities. In vivo results showed that HBM could inhibit excessive inflammation, modulate reparative M2 macrophage phenotype, and enhance cell migration and proliferation during the inflammation phage. Furthermore, during the proliferation and remodeling phages, it could promote angiogenesis, collagen normalization, granulation tissue formation and hair follicle remodeling, which normalized the skin wound. Additionally, we found that HBM promoted formation of more hair follicles via IGF1 and PDGFC signaling activated by macrophages. Then, BMP signaling was triggered to activate adipocytes to promote myofibroblast heterogeneity (Scheme 1). These studies highlight that HBM has a profound impact on scar-free tissue reconstruction.


image file: d5mh01062d-s1.tif
Scheme 1 The schematic illustrates the preparation of HBM and its application as a spray or wound dressing to enhance wound healing and reduce scar formation.

2. Results and discussion

2.1 HBM with good biocompatibility and no immune response

Biocompatibility is a critical consideration in the selection of suitable biomaterials for tissue engineering applications. To assess the biomedical potential of HBM, we first investigated its biocompatibility, using the preparation method illustrated in Fig. 1(a). For this evaluation, we utilized fibroblast cells (NIH 3T3), endothelial cells (HUVECs), and macrophages (RAW264.7) to examine the cytocompatibility of HBM through MTT assays and live/dead staining after 1 and 2 days of incubation. Subsequently, we assessed hemocompatibility through hemolytic activity. The results indicated that cell viability of above 90% was retained across HBM concentrations of 0.01, 0.05, 0.1, 0.3, and 0.5 mg mL−1, compared to the control group (Fig. 1(b) and Fig. S1). Fluorescence imaging via live/dead staining demonstrated that all three cell types maintained good morphology and exhibited adequate proliferation, further confirming the cytocompatibility of HBM across the various concentrations tested (Fig. 1(c) and Fig. S2). Hemolysis tests revealed a notable difference in coloration between the five HBM concentration, physiological saline, and the Triton X-100 control groups (Fig. 1(d)). The supernatants of the HBM and physiological saline groups exhibited similar light-yellow hues, while the Triton X-100 group displayed a bright red coloration. The hemolysis ratios for the HBM groups were consistently below 1%, significantly lower than the 5% threshold indicated by the black line (Fig. 1(e)), thus demonstrating the excellent hemocompatibility of HBM. Collectively, these findings underscore the favorable biocompatibility of HBM. In addition to biocompatibility, it is imperative that biomaterials implanted or administered in vivo do not elicit an immune response. Numerous studies have established that excessive immune responses can hinder tissue repair processes. Upon activation, macrophages may transition to a pro-inflammatory M1 phenotype, characterized by elevated expression of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, IL-12, and IL-23. To investigate whether HBM triggers an immune response, we measured the expression levels of TNF-α and IL-1β using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis (Fig. 1(f) and (g)) and enzyme-linked immunosorbent assay (ELISA). The results indicated that treatment with HBM concentrations of 0.5 mg mL−1 or lower did not provoke an immune response in macrophages. This finding was corroborated by ELISA results (Fig. 1(h)). Thus, HBM demonstrates promise as a biocompatible biomaterial or drug for in vivo medical applications.
image file: d5mh01062d-f1.tif
Fig. 1 Evaluation of the biocompatibility and immunogenicity of HBM. (a) Schematic illustrates the preparation and cell culture of HBM. (b) Evaluation of the proliferation activity of HUVECs in 96-well culture plates under different concentrations of HBM in the cell culture medium after incubation for 1 and 2 days using MTT assay (n = 3). (c) Live/dead staining assess the viability of HUVECs treated with varying concentrations of HBM solution and seeded in 24-well plates for 1 and 2 days. Live cells emit green fluorescence, while dead cells emit red fluorescence. (d) Observation of RBCs after treatment with different samples, including Triton X-100, PBS, and various concentrations of HBM. (e) Analysis of the hemolysis ratio of RBCs following incubation with different samples (n = 3). qRT-PCR analysis for (f) TNF-α and (g) IL-1β mRNA expression in macrophages harvested 1 d after treatment with different concentrations of HBM. Values are represented relative to expression of the untreated group. Error bars, mean ± SEM of samples (n ≥ 3). (h) Measurement of TNF-α concentration in the supernatant using an Elisa assay, with macrophages treated with varying concentrations of HBM for 2 days (n = 3).

2.2 Immune modulation, cytoprotection against ROS damage, and promotion of cell

Migration and angiogenesis of HBM in vitro. Dysregulated inflammation and altered macrophage phenotypes significantly impede the closure of chronic wounds and promote scar formation. Notably, modulating the immune response and attenuating inflammation are beneficial for effective wound repair. Lipopolysaccharides (LPS), components of the outer membrane of Gram-negative bacteria, elicit robust immune responses and are commonly used to activate pro-inflammatory M1 phenotype macrophages, which release pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β. In this study, we measured TNF-α and IL-6 concentrations using ELISA following LPS-induced activation of macrophages. Treatment with HBM resulted in a notable downregulation of TNF-α and IL-6 expression (Fig. 2(a) and (b)). Importantly, the anti-inflammatory effect of HBM was observed even at a low concentration of 0.01 mg mL−1. Additionally, we measured cytokine IL-10, secreted by M2 macrophages via ELISA. While IL-10 secretion did not significantly increase on the first day compared to the untreated group, it was upregulated after macrophages were treated with HBM for 2 days, with the highest secretion being observed in the 0.1 mg mL−1 HBM group (Fig. 2(c)). To further elucidate the mechanisms through which HBM promotes wound healing, we conducted assays to assess cell migration, proliferation, and angiogenesis. Cell migration and proliferation during the inflammatory stage are crucial for facilitating angiogenesis and collagen deposition, which are essential for enhancing tissue reconstruction and closure rates in the remodeling phase of wound healing. In the context of traumatic inflammation, neutrophils and macrophages play a protective role against invading microbes, predominantly through the release of reactive oxygen species (ROS) that can inflict significant damage on normal cells. To simulate pathological conditions, NIH 3T3 cells were initially exposed to varying concentrations of H2O2 to determine the appropriate level of oxidative stress (Fig. S3). We observed dose-dependent effects of oxidative stress on the proliferation of NIH 3T3 cells, ultimately selecting a H2O2 concentration of 150 μM to mimic the increased ROS levels characteristic of the wound microenvironment. The results demonstrated that HBM effectively mitigated H2O2-induced oxidative stress and subsequent cell mortality (Fig. 2(d)). On the 1st day, exposure to 150 μM H2O2 resulted in mild oxidative stress, as indicated by AO/EB staining, with minimal cell mortality compared to the negative control group (without H2O2 or HBM treatment) (Fig. S4). However, on the 2nd day, H2O2 treatment reduced cell viability by approximately 50%, while HBM treatment significantly protected cells from oxidative damage in a concentration-dependent manner. Specifically, HBM enhanced cell viability to approximately 71.1%, 73.3%, 77.4%, 80%, and 97% at concentrations of 0.01, 0.05, 0.1, 0.3, and 0.5 mg mL−1, respectively, with consistent results observed on the 4th day. Notably, cells treated with HBM concentrations exceeding 0.3 mg mL−1 demonstrated continued proliferation despite oxidative stress. To investigate the mechanisms underlying HBM's protective effects against oxidative stress, we assessed DPPH and hydroxyl radical scavenging activities (Fig. 2(e) and (f)). HBM effectively scavenged radicals in a concentration-dependent manner, correlating with its protective effects on cell viability under oxidative conditions. Furthermore, HBM inhibited oxidative damage in NIH 3T3 cells induced by H2O2, as evidenced by fluorescent dye DCFH-DA assays. Cells without any stimulation exhibited low intracellular ROS levels (Fig. S5); however, ROS levels significantly increased following pre-treatment with 150 μM H2O2, while pretreatment with 0.01 and 0.05 mg mL−1 HBM markedly reduced ROS levels (Fig. S5). Remarkably, pretreatment with HBM concentrations of 0.1 mg mL−1 or higher resulted in negligible fluorescence in the cells, indicating robust intracellular antioxidant activity. These findings suggest that HBM promotes the growth of NIH 3T3 cells and preserves cell quality and function by scavenging radicals generated by oxidative stress. Subsequently, we investigated the impact of HBM on cell migration through a scratch assay. Co-culturing with HBM significantly enhanced the migration of HUVECs into the scratched area (Fig. 2(g) and (h)), with a markedly accelerated migration rate observed at 24 hours. Notably, after 48 hours, HBM concentrations above 0.3 mg mL−1 exhibited a migration rate surpassing that of the control group. Similar trends were observed in NIH 3T3 cell migration (Fig. S6), confirming that HBM treatment accelerated the migration of both HUVECs and fibroblasts compared to untreated controls. Cell migration is a critical component of angiogenesis. To further evaluate the capacity of HBM to promote angiogenesis, we conducted an endothelial tube formation assay in vitro. The number of branch points formed by HUVECs was significantly enhanced in the presence of HBM compared to the control group at both 2 and 4 hours (Fig. 2(i) and Fig. S7). While no significant differences were noted among the 0.01 to 0.1 mg mL−1 HBM groups, the 0.3 mg mL−1 group exhibited a notable increase in branch points compared to the lower concentration groups (Fig. 2(j) and (k)). Although statistical significance was not reached across the 0.01 to 0.5 mg mL−1 HBM groups, the capillary length of the formed tubes was significantly enhanced compared to the control group. Collectively, these results indicate that HBM not only modulates macrophage polarization from the M1 to the M2 phenotype but also promotes the migration and proliferation of HUVECs and NIH 3T3 cells, thereby facilitating angiogenesis and accelerating wound healing.
image file: d5mh01062d-f2.tif
Fig. 2 Impact of HBM on cellular responses and wound healing processes. (a) Measurement of TNF-α and (b) IL-6 concentrations in the supernatant using an Elisa assay, with macrophages treated with different concentrations of HBM after LPS induction (n = 5). (c) Evaluation of IL-10 concentration in the supernatant using an Elisa assay, with macrophages treated with varying HBM concentrations for 1 and 2 days (n = 5). (d) Assessment of the protective effect of HBM against H2O2-induced oxidative stress in NIH 3T3 cells using the MTT assay (n = 3). (e) Determination of the anti-radical efficiency of different HBM concentrations via a DPPH radical assay (n = 3). (f) Evaluation of anti-radical efficiency of various HBM concentrations using the hydroxyl radical assay (n = 3). (g) Analysis of HUVEC wound closure in response to different HBM concentrations over 24 and 48 hours. (h) Percentage of wound closure at 24 and 48 h (n = 5). (i) HUVECs were seeded on Matrigel and incubated in a medium with different concentrations of HBM for 4 h. Quantification of (j) capillary length and (k) tube numbers (n = 3).

2.3 HBM accelerates cutaneous wound closure and reduces scar formation

Building on the multifaceted functions of HBM and inspired by the scarless healing observed in fetuses, we applied an undecorated HBM spray for wound healing in normal SD rats in vivo. A full-thickness cutaneous wound model was established via excision of circular skin sections (1.5 cm in diameter) from the backs of the rats (Fig. 3(a)). Wound gross morphology and closure were assessed at various time points. The effects of HBM on wound healing were compared with an untreated control group, with a 0.3 mg mL−1 HBM solution sprayed onto the wounds once daily. Rats treated with HBM exhibited significantly smaller wounds by day 4 post-wound infliction compared to the control group (Fig. S8a and b). Moreover, observations of the regenerating skin tissues indicated that HBM-treated wounds had a smaller scar area (Fig. S8c). Histological evaluations, including hematoxylin and eosin (H&E) staining and Masson's trichrome staining, along with immunohistochemical analysis of VEGF-A, confirmed an increased presence of skin appendages, such as hair follicles, and elevated VEGF-A expression in the skin of HBM-treated rats (Fig. S9). These results collectively demonstrate that HBM promotes wound healing while reducing scar formation when directly applied to the wound. However, the volatility of HBM raised concerns about the limited duration of its action on the wound, potentially hindering its therapeutic efficacy. To address this issue, we incorporated HBM into Pluronic F-127 (HBM/PF), which forms a gel at a 20% concentration at room temperature (Fig. 3(b)). The wound healing efficacy of the HBM/PF hydrogel dressing was further evaluated in rats with full-thickness skin wounds, using Pluronic F-127 (PF) and a commercial dressing (Tegaderm, CD) as control groups. The HBM/PF and control groups were administered every 24 hours for 15 days (Fig. 3(c)). During the first three days, there were no significant differences in skin wound repair between the three groups (Fig. 3(d) and (e)). However, by day 5, wounds treated with HBM/PF exhibited accelerated closure compared to both the CD and PF groups. The early stages of healing involve macrophages scavenging cellular debris and pathogens in preparation for the proliferative phase. The HBM/PF dressing may protect fibroblasts and endothelial cells from ROS-induced damage, thereby promoting fibroblast differentiation, endothelial cell migration, and angiogenesis. Significant differences in closure area became evident after day 8, with the HBM/PF group achieving 73.7% closure compared to 46.9% in the PF group and 35.9% in the CD group. By day 11, wound contraction in the HBM/PF group reached 90.1%, while the PF and CD groups demonstrated contractions of 72% and 62%, respectively. After 16 days, wounds in the HBM/PF group were completely closed, whereas incomplete closure was observed in the other groups. By day 21, wounds in the PF and CD groups were also closed. Evaluation of the regenerated skin tissues revealed that the HBM/PF dressing resulted in a smaller scar area and improved the quality of the regenerated skin (Fig. 3(f) and (g)). To further assess the restoration of skin elasticity, we performed an excisional wound model at day 21 post-wound infliction (Fig. 3(h)). Stress at the healing site was measured using the mid-specimen cross-sectional area. Samples from the HBM/PF group exhibited skin stiffness and elasticity comparable to uninjured normal tissue, while the CD and PF groups showed significantly reduced mechanical properties (Fig. 3(i)). Additionally, Sirius Red staining was used to observe the distribution of type I and type III collagen in the closed wounds. Type I collagen is thicker, while type III collagen is thinner. The results indicated the highest ratio of type I/III collagen in the CD group, followed by the PF group. The HBM/PF group exhibited a lower ratio of type I/III, indicating restored skin elasticity and reduced keloid formation (Fig. 3(j) and (k)). These findings were consistent with the results of the tensile strength tests. In conclusion, these outcomes illustrate that HBM accelerates skin repair and reduces scar formation.
image file: d5mh01062d-f3.tif
Fig. 3 The effect of HBM's on cutaneous wound closure and scar formation in vivo. (a) Illustration of a full-thickness cutaneous wound model. (b) Gel formation of Pluronic F-127 at a 20% concentration at room temperature. (c) Application of Pluronic F-127 with or without HBM in the wound. (d) Percentage closure of the wound area at each time point (n = 3). (e) Photographs depicting the progression of wound healing and scar formation on days 0, 3, 5, 8, 11, and 16 post-treatment with CD, PF, and HBM/PF groups (n = 3). (f) Scar size on day 21 post-treatment with different groups. (g) Analysis of the scar area using Image J (n = 3). (h) Punched specimen from rat skin (n = 3). (i) Stress–strain curves of CD, PF, and HBM/PF groups obtained from tensile tests. (j) Sirius red staining on day 21 post-wound infliction. (k) Quantification of the ratio of type I/type III collagen. Green fibers correspond to type III collagen, while orange fibers represent type I collagen (magnification was 200 ×) (n = 6).

2.4 HBM inhibits inflammation and modulates reparative M2 phenotype in vivo

To elucidate the mechanisms through which HBM accelerates skin repair and reduces scar formation, we investigated macrophage phenotypes and re-epithelialization within the wound microenvironment following HBM treatment. The transition of macrophages from the pro-inflammatory “M1” phenotype to the reparative “M2” phenotype is critical for effective wound healing.29–31 M1 macrophages are predominantly present in the early stages of healing (up to day 5), whereas M2 macrophages become dominant in the later proliferative phase. We analyzed the expression of M1 and M2 phenotype markers, specifically inducible nitric oxide synthase (iNOS) for M1 and CD206 for M2. The expression of iNOS was found to be higher on day 3 compared to day 5 (Fig. 4(a) and (b)), while CD206 expression was lower on day 3 than on day 5 (Fig. 4(c) and (d)). Notably, wounds treated with HBM/PF exhibited the lowest expression of iNOS and the highest expression of CD206 compared to the CD and PF control groups. Furthermore, in vivo analyses confirmed that HBM treatment with Pluronic F127 (HBM/PF) significantly upregulated the mRNA expression of interleukin-10 (IL-10) in the healed tissue by day 5 post-wound infliction (Fig. 4(e)). These findings suggest that HBM reprograms macrophages toward a more pro-healing phenotype. Histomorphological evaluations demonstrated a reduced inflammatory response characterized by pro-inflammatory M1 macrophages in the HBM/PF group compared to both control groups on days 3 and 5 (Fig. 4(f) and (g)). Macrophages play a crucial role in regulating the proliferative phase by stimulating keratinocyte proliferation necessary for re-epithelialization. On day 5, the HBM/PF group exhibited a thicker epidermis compared to both the CD and PF groups (Fig. 4(h)). Collectively, these results indicate that HBM influences the activation of pro-regenerative macrophage phenotypes, thereby enhancing wound healing by modulating the wound microenvironment.
image file: d5mh01062d-f4.tif
Fig. 4 HBM inhibited excessive inflammation and transformed pro-inflammatory M1 phenotype to the pro-regenerative M2 phenotype. (a) Representative images of immunofluorescence staining for iNOS in wound sites treated with CD, PF and HBM/PF. Red: iNOS; blue: nucleus. (b) Percentage of iNOS+ macrophages shown in (a) (n = 6). (c) Representative images of immunofluorescence staining for CD206 in wound sites treated with CD, PF and HBM/PF. Red, CD206; blue: nucleus. (d) Percentage of CD206+ macrophages shown in (c) (n = 6). (e) qRT-PCR analysis of Il-10 mRNA expression in wound tissue after post-treatment in 5th day (n = 6). (f) Histological images depicting wound regeneration in CD, PF, and HBM/PF groups on the 3rd and (g) 5th day (boundary of epithelium: yellow lines). (h) Re-epithelialization of the wound at the specified time points shown in (g) (n = 3). Error bars, mean ± SEM of samples.

2.5 HBM promotes blood vessel formation, granulation tissue formation, hair follicle remodeling and normalization of collagen

To further investigate the activation of the angiogenic response in wounds in vivo, immunofluorescence staining of VEGF-A, CD31 and PDGF-BB in wound samples was conducted on postoperative days 3, 5 and 21. As illustrated in Fig. 5(a), minimal positive staining was observed in the CD and PF groups. In contrast, the HBM/PF group exhibited the strongest positive expression of VEGF-A, indicating enhanced cell migration for blood vessel formation. The strongest positive expression of CD31, which evaluates vascular formation, was also observed in the HBM/PF group, confirming these findings. By day 21, more mature blood vessels had formed in the HBM/PF group compared to the CD and PF groups (p < 0.001), as reflected by the highest expression of PDGF-BB in HBM/PF-treated wounds (Fig. 5(a)). Quantification of the areas positive for VEGF-A, CD31, and PDGF-BB further supported these results (Fig. 5(b)–(d)). Parenchymal cells alone are insufficient for wound repair,32 as granulation tissue also plays a pivotal role in this process. Thicker granulation tissue serves as a critical indicator for assessing repair efficacy. After 21 days of healing, the granulation tissue in the HBM/PF group was significantly thicker than that in the other groups (P < 0.05) (Fig. 5(e) and (f)), indicating superior wound healing efficacy. Notably, hair follicles were observed in the HBM/PF group (Fig. 5(e)), suggesting the involvement of HBM in extracellular matrix (ECM) remodeling and tissue regeneration, which contributes to reduced scar formation.
image file: d5mh01062d-f5.tif
Fig. 5 The role of HBM in wound regeneration and angiogenesis. (a) Blood vessels were visualized using VEGF-A (at day 3), CD31 (at day 5), and PDGF-BB (at day 21) immunofluorescence staining (blue, nucleus; green, VEGF-A, CD31 and PDGF-BB). Quantification of (b) VEGF-A+, (c) CD31+ and (d) PDGF-BB+ areas in over 5 random fields per group using imageJ software (n = 6). (e) Histological images illustrating wound regeneration in CD, PF, and HBM/PF groups on the 21st day (granulation tissue thickness: yellow arrows). (f) Quantitation of granulation tissue thickness (n = 3). (g) Masson's trichrome staining images for wound regeneration in CD, PF, and HBM/PF groups on the 21st day.

Throughout the healing process, collagen metabolism undergoes dynamic changes. Collagen content increased across all groups (Fig. S10 and Fig. 5(g)), with the HBM/PF group demonstrating higher collagen levels on day 11 of repair compared to the CD and PF groups, indicating that HBM enhances collagen deposition (Fig. S10). In healthy skin, collagen fibers typically form a lattice structure; however, in healing tissue, they are parallel to each other, leading to stiffness and weakness.33 On day 21, collagen fiber alignment in healed wounds displayed a lattice-like pattern in the HBM/PF group, contrasting sharply with the parallel orientation observed in the CD and PF groups (Fig. 5(e)). Collectively, these results demonstrate that HBM promotes blood vessel formation, granulation tissue formation, hair follicle remodeling, and normalization of collagen in wounds.

2.6 HBM activates IGF1 and PDGFC signaling for myofibroblast heterogeneity to reduce scar formation

Reducing scar formation is closely associated with the regeneration of hair follicles in healed skin. Neogenic hair follicles are known to activate adipocytes derived from myofibroblasts, thereby supporting myofibroblast heterogeneity and ultimately contributing to scar reduction.20,34 Previous studies have highlighted that signaling pathways involving insulin-like growth factor 1 (IGF1) and platelet-derived growth factor C (PDGFC), which are derived from macrophages, can stimulate the proliferation of adipocyte precursors (APs), promoting myofibroblast heterogeneity.34 Furthermore, research indicates that M2-polarized macrophages secrete growth factors such as hepatocyte growth factor (HGF) and IGF1 to stimulate hair regeneration.35 To investigate how HBM promotes hair follicle formation, we analyzed the gene expression of Igf1, Pdgfc, and Hgf in macrophages and wound tissue on day 5 using qRT-PCR. The mRNA expression of Hgf in both macrophages and wound tissue on day 5 showed no significant differences between the untreated and HBM-treated groups (Fig. 6(b) and (c)). In contrast, the mRNA expression of Igf1 in macrophages was significantly upregulated with HBM treatment at concentrations of 0.05 mg mL−1 and above (Fig. 6(d)). Moreover, Igf1 mRNA expression in the wound tissue of the HBM/PF group was also significantly elevated (Fig. 6(e)). The mRNA expression of Pdgfc in both macrophages and wound tissue on day 5 was similarly upregulated, exhibiting significant differences when treated with HBM concentrations of 0.1 mg mL−1 or higher (Fig. 6(f) and (g)). These results indicate that appropriate concentrations of HBM promote hair follicle formation by synergistically activating the Igf1 and Pdgfc signaling pathways. Myofibroblasts, which differentiate from fibroblasts, can transform into adipocytes to enhance myofibroblast heterogeneity, thereby reducing scar formation through the activation of bone morphogenetic protein (BMP) pathways. Analysis via qRT-PCR and Western blotting revealed that treatment with HBM at concentrations of 0.1 mg mL−1 or higher upregulated Bmp-2 mRNA expression and promoted BMP-2 protein secretion, with the highest levels observed in the 0.3 mg mL−1 group (Fig. 6(h) and (i)). Similarly, on day 5 post-wound infliction, the HBM/PF group exhibited significantly elevated Bmp-2 mRNA expression and BMP-2 protein secretion compared to both the CD and PF groups (Fig. 6(j) and (k)). Immunofluorescence staining of BMP-2 in wound samples on day 11 demonstrated the strongest positive expression in the HBM/PF group (Fig. 6(l) and (m)), indicating that HBM promotes myofibroblast heterogeneity. Collectively, these results suggest that appropriate concentrations of HBM may facilitate hair follicle formation by synergistically activating the IGF1 and PDGFC signaling pathways, subsequently triggering the BMP pathway to enhance adipocyte-induced myofibroblast heterogeneity, thereby reducing scarring during the healing process (Fig. 6(a)).
image file: d5mh01062d-f6.tif
Fig. 6 Mechanism of hair follicles neogenesis and BMP signaling. (a) Schematic illustrates the proposed mechanism by which HBM promote Igf1 and Pdgfc mRNA expression of macrophages to enhance hair follicles formation which secreted BMP to activate AP proliferation contributing to myofibroblast heterogeneity resulting in less scar formation. qRT-PCR analysis of (b) Hgf, (d) Igf1, (f) Pdgfc and (h) Bmp-2 mRNA expression in macrophages or NIH 3T3 cells treated with different concentrations of HBM for 5 days (untreated cells as the control group) (n = 5). qRT-PCR analysis for (c) Hgf, (e) Igf1, (g) Pdgfc and (k) Bmp-2 mRNA expression in wound tissue after post-treatment in 5th day (n = 6). (i) Immunoblotting analysis of BMP-2 protein levels in cells and (j) tissue. (l) Immunofluorescence staining of BMP-2 in healed skin wound tissue on day 11 post-wound infliction (blue: nucleus; red: BMP-2). (m) Quantification of the BMP-2+ area (n = 6). Error bars, mean ± SEM of samples.

3. Conclusions

In summary, our work reveals that HBM as a bioactive material platform significantly accelerates wound healing and minimizes scar formation in a full-thickness cutaneous wound model. The observed enhancement in closure rates is primarily attributed to the modulation of macrophage phenotype and function, increased cell migration and proliferation, and promotion of angiogenesis. Moreover, HBM facilitates hair follicle formation by synergistically activating the IGF1 and PDGFC signaling pathways, which subsequently trigger the BMP pathway to enhance adipocyte-induced myofibroblast heterogeneity, thereby reducing scar tissue formation. We propose that HBM holds significant potential for promoting tissue and organ regeneration beyond the skin, indicating a promising direction for future advancements in regenerative medicine. Future efforts will focus on isolating key components of HBM to create standardized therapeutics and mitigate donor variability for clinical translation.

Ethics statement

Human breast milk (HBM) was collected from five breastfeeding women who, after fulfilling their infants’ feeding needs, had surplus breast milk and were willing to donate the excess for our research. Approval from the Seventh Affiliated Hospital of Sun Yat-sen University ethics committee was obtained prior to the research (KY-2025-114-01), and the study complied with the principles and guidelines of the Declaration of Helsinki. Informed consent was obtained for any experimentation with human subjects.

Materials and methods

Full materials, experimental details, and the data supporting this article can be found in the SI.

Author contributions

Conceptualization: J. W.; methodology: C. H. X.; investigation: C. H. X., Z. P. G.; visualization: C. H. X., Z. P. G.; supervision: Z. P. G., J. W.; writing – original draft: C. H. X.; writing – review & editing: J. W.

Conflicts of interest

There are no conflicts of interest to declare.

Data availability

The data supporting this article have been included as part of the SI. See DOI: https://doi.org/10.1039/d5mh01062d

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 52173150 and U22A20315), the Guangzhou Science and Technology Program City-University Joint Funding Project (No. 2023A03J0001 and 2024A03J0604), and the Open Research Funds from the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital (No. 202301-211).

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