Fetal mice dermal mesenchymal stem cells promote wound healing by inducing M2 type macrophage polarization

Abstract

BACKGROUND

Mesenchymal stem cells, found in various tissues, possess significant healing and immunomodulatory properties, influencing macrophage polarization, which is essential for wound repair. However, chronic wounds present significant therapeutic challenges, requiring novel strategies to improve healing outcomes.

AIM

To investigate the potential of fetal dermal mesenchymal stem cells (FDMSCs) in enhancing wound healing through modulation of macrophage polarization, specifically by promoting the M2 phenotype to address inflammatory responses in chronic wounds.

METHODS

FDMSCs were isolated from BalB/C mice and co-cultured with RAW264.7 macrophages to assess their effects on macrophage polarization. Flow cytometry, quantitative reverse transcriptase polymerase chain reaction, and histological analyses were employed to evaluate shifts in macrophage phenotype and wound healing in a mouse model. Statistical analysis was performed using GraphPad Prism.

RESULTS

FDMSCs induced macrophage polarization from the M1 to M2 phenotype, as demonstrated by a reduction in pro-inflammatory markers (inducible nitric oxide synthase, interleukin-6) and an increase in anti-inflammatory markers [mannose receptor (CD206), arginase-1] in co-cultured RAW264.7 macrophages. These shifts were confirmed by flow cytometry. In an acute skin wound model, FDMSC-treated mice exhibited faster wound healing, enhanced collagen deposition, and improved vascular regeneration compared to controls. Significantly higher expression of arginase-1 further indicated an enriched M2 macrophage environment.

CONCLUSION

FDMSCs effectively modulate macrophage polarization from M1 to M2, reduce inflammation, and enhance tissue repair, demonstrating their potential as an immunomodulatory strategy in wound healing. These findings highlight the promising therapeutic application of FDMSCs in managing chronic wounds.

Key Words: Fetal dermal mesenchymal stem cells; Macrophage polarization; Wound healing; Immunomodulation; M2 phenotype

Core Tip: This study investigates the role of fetal dermal mesenchymal stem cells in wound healing by modulating macrophage polarization towards a pro-healing M2 phenotype. Using flow cytometry, reverse transcriptase polymerase chain reaction, and histology, we demonstrate that fetal dermal mesenchymal stem cells promote a reparative immune environment, accelerating wound closure, enhancing collagen deposition, and supporting vascular regeneration in a mouse model.

INTRODUCTION

Mesenchymal stem cells (MSCs) are multipotent precursor cells with self-renewal capacity, found in various tissues such as bone marrow, adipose tissue, umbilical cord blood, dental pulp, dermis, and the endometrial lining of the uterus[1]. MSCs are known for their robust regenerative properties, including the ability to migrate to injury sites, differentiate into various cell types, and release cytokines, growth factors, and chemokines that promote tissue repair[2]. In addition to their direct regenerative effects, MSCs modulate the immune response during healing, particularly by influencing macrophage behavior, which plays a critical role in tissue repair and inflammation resolution[3]. This immunomodulatory function enhances the healing process, positioning MSCs as promising candidates for treating chronic wounds, autoimmune disorders, and inflammation-driven diseases[4,5].

Macrophages are critical immune cells involved in the inflammatory, proliferative, and remodeling phases of wound healing[6]. Initially, granulocytes and monocytes are recruited to the injury site, with monocytes differentiating into pro-inflammatory M1 macrophages characterized by high CD86 and programmed death-ligand 1 expression[7]. These M1 macrophages secrete inflammatory mediators such as inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha, interleukin-1 beta (IL-1β), and IL-6, and play a key role in phagocytosis and debris clearance[8]. As healing progresses, macrophages transition to an anti-inflammatory M2 phenotype, marked by high mannose receptor (CD206), arginase-1 (Arg-1), and FIZZ1 expression, along with the secretion of transforming growth factor-beta and IL-10[9]. M2 macrophages support tissue repair by promoting angiogenesis and collagen deposition[10]. They also produce cytokines like vascular endothelial growth factor and fibroblast growth factor to stimulate fibroblast and endothelial cell activity[11]. An overabundance of pro-inflammatory macrophages in chronic wounds can impair healing by hindering tissue remodeling[12]. Recent research has revealed that macrophage polarization is more complex than the classical M1/M2 dichotomy, with various intermediate phenotypes influencing the wound healing process. These phenotypes, shaped by factors such as the tissue microenvironment, activation timing, and the presence of other immune cells, demonstrate that macrophage polarization exists on a spectrum. This dynamic understanding is essential for developing more targeted immunomodulatory therapies for chronic wounds and inflammation[13].

Fetal dermal MSCs (FDMSCs) are a distinct category of MSCs extracted from the dermal tissue of BalB/C mice on the fifteenth day of gestation. In comparison to adult MSCs, including those sourced from bone marrow and adipose tissue, FDMSCs exhibit enhanced multidirectional differentiation potential, decreased immunogenicity, and a significant correlation with the scarless healing properties inherent to fetal skin. Previous studies have demonstrated that FDMSCs play a crucial role in promoting wound healing and angiogenesis, with evidence showing that they facilitate tissue repair by inducing macrophage polarization toward the M2 phenotype[14,15]. This study aims to investigate the role of FDMSCs in modulating macrophage polarization, specifically promoting the transition from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, and to evaluate their impact on wound healing.

MATERIALS AND METHODS

Animals

BalB/C mice, aged 6 to 8 weeks, were procured from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. and were maintained in groups at the Animal Facility of our hospital. All animal care and experimental techniques were conducted in accordance with the protocols sanctioned by the Institutional Animal Care and Use Committee at our hospital. Furthermore, all procedures were conducted in compliance with applicable rules and legislation to guarantee the ethical treatment of animals. This study adhered to ARRIVE principles, and all techniques are documented to guarantee transparency and reproducibility of the experimental processes. Upon the completion of the experiments, euthanasia was performed to alleviate suffering. Mice were executed via CO₂ inhalation, followed by cervical dislocation as a supplementary method to confirm death, in compliance with the American Veterinary Medical Association euthanasia recommendations. All treatments were performed under the oversight of veterinary personnel to guarantee humane care.

Cells isolation and culture

FDMSC was isolated from the dorsal dermis of BalB/C mice on gestational day 15. The epidermis and dermis were dissociated using neutral protease, followed by digestion of the dermis with type I collagenase. The FDMSCs were subjected to filtration using a 70-micrometer filter and subsequently cultivated in DMEM low glucose medium (HyClone, UT, United States) supplemented with 10% fetal bovine serum (Gibco, CA, United States) and 1% Penicillin-Streptomycin at a concentration of 100 U/mL (Gibco, CA, United States).

Flow cytometry

The surface markers of FDMSCs and M1/M2 type RAW264.7 were evaluated by flow cytometry. Macrophages were stimulated to the M1 phenotype using 100 ng/mL lipopolysaccharide (LPS) and 20 ng/mL interferon (IFN)-β, and to the M2 phenotype using 20 ng/mL IL-4 for intracellular cytokine detection. In summary, 5 × 10⁵ cells were harvested and resuspended in phosphate buffered saline buffer, followed by a 15-minute incubation with particular antibodies in the dark. The cells were subsequently rinsed with phosphate buffered saline solution and evaluated by flow cytometry. Results were evaluated using FlowJo Software (Ashland, OR, United States).

Co-culture of FDMSCs and RAW264.7

Co-culture investigations were conducted utilizing 0.4 μm pore transwell chambers (Corning, CA, United States). RAW264.7 cells were cultured in the lower compartment and polarized to the M1 phenotype using 100 ng/mL LPS and 20 ng/mL IFN-β; 20 ng/mL IL-4 was employed to produce the M2 macrophage phenotype. The FDMSCs were cultured in the upper chamber. Co-culture for a duration of 24 hours prior to evaluation.

Acute skin wound healing model and FDMSCs treatment

Mice were randomly assigned to control and FDMSCs-treated groups. A full-thickness skin splinting model in mice with a diameter of 1 cm was established. In the control group, 100 μL of normal saline was administered at the wound’s periphery. In the FDMSCs group, 100 μL of normal saline containing approximately 10⁶ FDMSCs was administered. The injury was dressed with sterile gauze.

Total RNA extraction and quantitative reverse transcriptase polymerase chain reaction

Total cellular RNA was extracted using a TRIzol reagent (Invitrogen, Carlsbad, CA, United States), according to the manufacturer’s instructions. Isolated total RNA was then subjected to reverse transcription using Oligo dT primer and PrimeScript^® RTase (Takara, Dalian, China), according to the manufacturer’s instructions. Quantitative reverse transcriptase polymerase chain reaction was performed with SYBR^® Premix Ex Taq™ II (Takara, Dalian, China) using the C1000™ Thermal Cycler (Bio-Rad, Hercules, CA, United States). The expression levels of the target genes were normalized to that of the housekeeping gene, β-actin.

Histological analysis

The excised tissues from wound sites were preserved in 4% paraformaldehyde for 24 hours and subsequently dehydrated using graded ethanol. Following the embedding of collected tissues in paraffin, the samples were sectioned into 4 μm thick slices. Hematoxylin and eosin (HE) staining was performed to examine the histological alterations in the wound healing process. The Masson’s trichrome staining assay was conducted to assess collagen maturity in accordance with the manufacturer’s guidelines. Microscopic images were obtained and analyzed using Image J software.

Statistical analysis

Statistical analysis was conducted on GraphPad Prism 8. Three or more separate experiments were conducted for each outcome, and the mean and standard deviation were computed. One-way ANOVA or Student’s t-test were employed to identify statistically significant differences. A P value of less than 0.05 was deemed statistically significant.

RESULTS

Characterization of FDMSC and M1/2 macrophages

In accordance with the MSC identification criterion, we discovered three biomarkers: CD29, CD44, and CD45. Among these, CD29 and CD44 were highly expressed, while CD45 was little expressed. RAW264.7 was stimulated with conditioned media for 24 hours, and the expression levels of CD86 and CD206 were assessed using flow cytometry. In comparison to uninduced RAW264.7 cells, M1-type macrophages exhibited elevated expression of CD86, while M2-type macrophages demonstrated increased expression of CD206. The induction with 100 ng/mL LPS, 20 ng/mL IFN-γ, and 20 ng/mL IL-4 effectively generated M1/2-type macrophages for further tests (Figure 1).

Figure 1 Characterization of fetal dermal mesenchymal stem cell M1/2 macrophages. A: Detection of CD29 (FITC), CD44 (APC), and CD45 (PE) expression, characteristic markers of mesenchymal stem cells by flow cytometry; B: Flow cytometric analysis of CD86 (PE) cell surface marker expression (red - RAW264.7, blue - M1-type macrophages induced); C: Flow cytometric analysis of CD206 (FITC) cell surface marker expression (red - RAW264.7, blue - M2-type macrophages induced).

FDMSCs facilitate the phenotype switch of RAW264.7 from M1 to M2 type

We investigated the influence of FDMSCs on macrophage polarization through a coculture method. Prior to co-culture, RAW264.7 was polarized to the M1 phenotype using LPS and IFN-γ, followed by co-culturing with FDMSCs. We identified the gene expression of iNOS and IL-6, which are hallmark pro-inflammatory components of M1 macrophages. In comparison to the control group, the expressions of iNOS and IL-6 in M1 macrophages co-cultured with FDMSC were decreased. Furthermore, we identified the expression of the anti-inflammatory cytokine IL-10. The expression of the IL-10 gene was elevated in the MSC group relative to the control group. Ultimately, we identified two cell markers, CD86 and CD206, by flow cytometry. The results indicated a decrease in CD86 expression, indicative of M1 polarization, and an increase in CD206 expression, indicative of M2 polarization. Consequently, we have demonstrated that FDMSC can diminish the polarization of M1 macrophages, decrease the expression levels of pro-inflammatory factors IL-6 and iNOS; concurrently, it can facilitate the transformation of M1 macrophages to the M2 phenotype, resulting in an elevated expression level of IL-10 (Figure 2).

Figure 2 Fetal dermal mesenchymal stem cells facilitate the phenotypic switch of RAW264.7 from M1 to M2 type. A: Quantitative reverse transcriptase polymerase chain reaction analysis of genes characterizing M1, including interleukin-6 and inducible nitric oxide synthase, and the anti-inflammatory factor interleukin-10; B and C: Cells were immunostained with PE-CD86 and FITC-CD206 antibodies and analyzed by flow cytometry. ^aP < 0.05. IL: Interleukin; iNOS: Inducible nitric oxide synthase; FDMSC: Fetal dermal mesenchymal stem cell.

FDMSC promotes macrophage polarization towards M2

We employed the identical methodology to investigate the influence of FDMSCs on M2-polarized macrophages, which were induced to the M2 phenotype using IL-4 prior to co-culture. We subsequently analyze the distinctive cellularity that indicates the degree of polarization of M2 macrophages.

Following FDMSC injection, the gene expression of IL-10 in M2 macrophages was elevated in comparison to the control group. The flow cytometry data indicated a reduction in CD86 expression and an elevation in CD206 expression relative to the control group. Furthermore, we observed that the expression of the Arg-1 gene in M2 macrophages increased following the administration of FDMSC, in comparison to the control group. It may be inferred that FDMCS can enhance macrophage polarization towards the M2 phenotype and elevate the gene expression of key anti-inflammatory proteins IL-10 and Arg-1 (Figure 3).

Figure 3 Fetal dermal mesenchymal stem cell promotes macrophage polarization towards M2. A: Quantitative reverse transcriptase polymerase chain reaction analysis of genes characterizing M2, including interleukin-10 and arginase-1; B and C: Cells were immunostained with PE-CD86 and FITC-CD206 antibodies and analyzed by flow cytometry. ^aP < 0.05; ^bP < 0.01. IL: Interleukin; Arg-1: Arginase-1; FDMSC: Fetal dermal mesenchymal stem cell.

FDMSCs promote acute wound healing in mice

We developed a model of acute total trauma in mice and treated the trauma with FDMSCs. The healing process demonstrated that trauma in the FDMSCs intervention group resolved more rapidly than in the non-intervention control group, achieving substantial healing by day 14 (Figure 4A). ImageJ software was employed to quantify the disparity in wound healing rates between the two groups, revealing that the wound healing rate was accelerated in the FDMSCs intervention group (Figure 4B). Ultimately, we excised the entire skin from the day 14 trauma for paraffin embedding, followed by HE and Masson staining to examine the healing process of the damage. In HE stains, the FDMSCs exhibited closely grouped fibroblasts, an increased number of neoplastic capillaries, and discernible neoplastic hair follicles in comparison to the control group. Masson staining revealed a greater abundance of neoplastic collagen fibers (Figure 4C and D). Subsequently, we assessed the area filled by blue-stained collagen fibers in Masson-stained sections utilizing ImageJ software, and the results indicated that the FDMSCs intervention group facilitated collagen deposition in comparison to the control group (Figure 4E). This indicated that FDMSCs facilitated the healing of acute full-thickness wounds in mice, encompassing the enhancement of collagen deposition, vascular regeneration, and re-epithelialization.

Figure 4 Evaluation of wound healing and tissue analysis in mice following thoracoscopic surgery. A: Mice were euthanized on postoperative days 0, 7, and 14; wound healing was photographed and documented; B: Analysis of wound healing rate; C and D: Hematoxylin and eosin and Masson’s trichrome staining of traumatized skin on day 14 (× 40 magnification); E: The area of blue-stained collagen in Masson’s trichrome-stained sections was quantified using ImageJ software and statistically analyzed; F: Mouse trauma tissue extracted on day 14 for quantitative reverse transcriptase polymerase chain reaction analysis to detect arginase-1 expression. ^bP < 0.01. HE: Hematoxylin and eosin; Arg-1: Arginase-1; FDMSC: Fetal dermal mesenchymal stem cell.

To further substantiate the role of FDMSCs in the wound healing process, we collected wound specimens on day 14 and assessed the expression of iNOS and Arg-1 via quantitative reverse transcriptase polymerase chain reaction. The results indicated that Arg-1 expression was elevated in the FDMSCs-intervention group relative to the control group (Figure 4F), whereas no statistically significant difference was noted for iNOS. This indicates a potentially elevated proportion of M2-type macrophages in the FDMSCs group during the wound healing process, hence facilitating wound repair.

DISCUSSION

Our study reveals the innovative potential of FDMSCs for wound healing, highlighting their capability to shift macrophage polarization from M1 to M2, thereby fostering an anti-inflammatory milieu conducive to tissue repair. This effect was validated through co-culture experiments showing decreased pro-inflammatory markers and increased anti-inflammatory markers. Further, FDMSC treatment markedly improved wound closure, collagen deposition, and vascular regeneration in a mouse model, supported by increased expression of the M2 marker Arg-1. These findings underscore the therapeutic promise of FDMSCs for chronic wound management and tissue regeneration, leveraging their low immunogenicity and high biocompatibility. This research advances the field of regenerative medicine, offering new strategies for treating non-healing wounds and enhancing tissue repair outcomes. Current therapies for wound healing primarily involve the use of growth factors, stem cell therapies, and skin grafts. However, these approaches often face limitations such as prolonged healing times, immune rejection, and risk of scarring. Our study introduces FDMSCs as a promising alternative. FDMSCs promote macrophage polarization toward the M2 phenotype, enhancing tissue regeneration, collagen deposition, and vascularization. Compared to existing methods, FDMSC-based treatment offers potential advantages in reducing inflammation, accelerating healing, and improving scar-free recovery, providing a more sustainable, less invasive option for wound care.

Numerous studies have documented the beneficial effects of MSCs on various diseases, particularly through their influence on macrophage polarization, which impacts the progression of certain inflammatory disorders, myocardial infarction, spinal cord injury, and wound healing[16,17]. MSCs operate by multiple mechanisms, including the secretion of diverse cytokines and exosomes, facilitation of angiogenesis, tissue healing, immunological modulation, and cell recruitment. Our prior research has established that FDMSCs contribute positively to angiogenesis, wound healing, and keloid management. We concentrated on the impact of FDMSC on macrophage polarization. During tissue regeneration, MSCs can facilitate the change of macrophages from the inflammatory M1 phenotype to the anti-inflammatory M2 phenotype[18]. Bone marrow MSCs and human placenta MSCs have been shown to suppress M1 marker expression and facilitate polarization towards the M2 phenotype, characterized by decreased levels of tumor necrosis factor-alpha and iNOS, alongside increased levels of IL-10, CD206, and Arg1.

Macrophages engage in tissue repair via phenotypic conversion, facilitate the phagocytosis of pathogenic microbes and cellular debris during the inflammatory phase, release various essential cytokines during this period, and aid in cell recruitment[19]. During the tissue repair phase, it demonstrates an anti-inflammatory activity, releases various cytokines, and activates fibroblasts and endothelial cells. Research has established that MSCs are pivotal in cardiac healing and the reduction of fibrosis following acute kidney damage via macrophage polarization[20].

FDMSC, a type of MSC isolated from fetal mouse dermis, has low immunogenicity and excellent biocompatibility. Its dermal origin suggests a superior capacity for in situ skin wound repair and it is considered a crucial cell in scarless healing. The interaction between FDMSCs and macrophages, essential immune cells in tissue regeneration, remains unexplored. This study demonstrates that coculturing FDMSC with macrophages facilitates the polarization of macrophages towards the M2 phenotype, hence reducing inflammation and enhancing tissue healing. Flow cytometry revealed that, relative to the control group, CD206 expression in M1 and M2 macrophages rose and CD86 expression reduced following FDMSC intervention, suggesting that FDMSC can promote macrophage polarization towards the M2 phenotype. Subsequent polymerase chain reaction results indicated that FDMSC could not only suppress the expression of pro-inflammatory factors IL-6 and iNOS in M1 macrophages but also enhance the expression of the anti-inflammatory factor IL-10, thereby mitigating the pro-inflammatory effects of M1 macrophages. Following FDMSC intervention in M2 macrophages, the expression of the anti-inflammatory factor IL-10 and the growth factor transforming growth factor-beta increased, enhancing their capacity to control inflammation and facilitate tissue repair. The results indicate that FDMSC can drive macrophage polarization towards the M2 phenotype, hence augmenting the macrophages’ capacity to suppress inflammation and facilitate tissue regeneration.

To further validate the characteristics of FDMSCs, we conducted a morphological examination and confirmed their typical fibroblast-like spindle-shaped appearance under a microscope, which is consistent with MSC properties. Additionally, the multipotent differentiation potential of FDMSCs was demonstrated through their ability to differentiate into adipocytes, osteocytes, and chondrocytes under specific culture conditions, as expected for MSCs. This differentiation capacity underscores the functional versatility of FDMSCs. Furthermore, to ensure the purity of the FDMSC population, we assessed the expression of negative markers, including CD45, CD34, and HLA-DR, all of which were absent, further confirming their mesenchymal identity. Prior to the injection of FDMSCs into the wound healing model, we ensured that the number and viability of the cells met stringent quality control standards. Cell counting was performed using a hemocytometer, and cell viability was assessed by the Trypan blue exclusion assay. Only FDMSCs with a viability greater than 95% were used, ensuring that only healthy and functional cells were administered. These rigorous procedures were carried out immediately prior to injection, ensuring the reproducibility and reliability of the experimental outcomes.

While our study demonstrates the promising therapeutic potential of FDMSCs in promoting wound healing and macrophage polarization, several limitations need to be addressed. First, the mouse model used in this study, though informative, may not fully replicate the complexity of human chronic wounds. In particular, human-specific factors such as age, comorbidities, and impaired immune responses are not adequately modeled. To improve clinical relevance, future studies should incorporate advanced models such as diabetic or aged mice, which more closely resemble the delayed and inflammatory healing seen in human patients. Additionally, while FDMSCs showed effective wound healing in the short term, long-term outcomes, including the potential for fibrosis, scarring, and immune responses post-treatment, remain unexplored. These aspects are crucial for assessing the translational potential of FDMSC therapy. Finally, while we assessed macrophage polarization, a more comprehensive evaluation of immune responses, including cytokine profiling and T-cell interactions, is needed to fully understand the immunomodulatory effects of FDMSCs.

To address these limitations, future studies should employ clinically relevant models, such as 3D bioprinted human skin constructs or diabetic wound models, to better simulate human wound healing. We also plan to investigate the potential differential effects of male and female hormones on FDMSC efficacy in wound healing, as hormonal variations may influence immune responses and healing dynamics. Additionally, we will explore the optimization of co-culture durations to determine whether extending or shortening the co-culture period enhances macrophage polarization and macrophage-driven healing outcomes. These directions will provide deeper insights into the mechanisms of FDMSC action and further validate their clinical applicability.

CONCLUSION

Our research demonstrates that FDMSC can induce a phenotypic transition of RAW264.7 from M1 to M2, hence inhibiting inflammation during the initial phase of wound healing and enhancing tissue regeneration in the subsequent phase. This establishes a basis for more research on FDMSCs in relation to wound healing via the immunomodulation of macrophages.