Tissue injury has emerged as one of the major global health challenges, with more than 14 million people suffering from it annually (Martinengo et al., 2019). The repair of tissue represents one of the most intricate biological processes in human life. To restore tissue integrity and homeostasis following an injury, various intracellular and intercellular pathways must be activated and coordinated instantaneously (Gurtner et al., 2008). Across all organ systems, the body’s response to injury can be categorized into four distinct phases, including clot formation, inflammation, new tissue formation, and tissue remodeling (Werner and Grose, 2003). Within this process, the immune microenvironment plays a crucial role in tissue repair. Numerous studies have demonstrated that modulating the immune microenvironment can promote the repair of tissues such as bone (Fu et al., 2025; Yang et al., 2023), nerve (Liu et al., 2024; Fan L. et al., 2022), and skin (Valvis et al., 2015; Yang et al., 2025).

Macrophages, as a crucial component of the immune microenvironment, possess a wide range of functions, including tissue repair and regulation of homeostasis and immunity (Zhang et al., 2024; Qi et al., 2024). Therefore, modulating macrophages serves as a significant means of controlling the immune microenvironment for tissue repair. This can be achieved by regulating their phagocytic function, migration and differentiation to influence their immune response behavior (Chen et al., 2023a; Groh et al., 2018). Furthermore, regulating the phenotypic transformation of macrophages has emerged as a research hotspot in the biomedical field in recent years. The phenotypic transformation of macrophages from pro-inflammatory (M1) to anti-inflammatory (M2) alters their cytokine secretion profile, promoting the secretion of anti-inflammatory factors such as IL-10 and Transforming Growth Factor (TGF)-β1, thus playing a profound role in immune regulation (Wang et al., 2024; Domínguez-Andrés and Netea, 2019).

Traditionally, it was believed that the functions of immune cells were entirely modulated by the numerous biochemical signals they receive from the environment (Koppes et al., 2016; Willand et al., 2016; Anand and Chou, 1992). In recent years, it has been discovered that immune cells possess the same electrical properties as other human cells. Physical signals present in the cellular microenvironment also play a crucial role in determining the activity of immune cells, among which electrical stimulation (ES) is a potential physical signal (Zhao et al., 2020; Roy and Jhunjhunwala, 2024). Studies have indicated that exogenous ES can facilitate the transformation of macrophages into an anti-inflammatory (M2) phenotype, demonstrating beneficial effects in tissue repair (Hoare et al., 2016; Li J. et al., 2021). Therefore, the combination of ES and wound dressings to promote immune modulation during the tissue repair represents a breakthrough in the design of novel wound dressings.

The design of scaffold materials with good electrical conductivity is required for ES combined with wound dressings. The main method for fabricating conductive materials involves hybridizing polymers with conductive nanomaterials (Radhakrishnan et al., 2015). Previous research has employed 3D printing technology to produce conductive scaffolds with complex structures, better simulating the tissue microenvironment for various types of tissue repair (Lee et al., 2024; Yu et al., 2021). Nowadays, electrospun aligned fibrous scaffolds have emerged as a significant scaffold preparation method due to their excellent mechanical properties and biocompatibility (Xue et al., 2019; Carvalho et al., 2019; Allison et al., 2017). Polyurethane (PU) is a thermoplastic polymer with a series of excellent properties, such as good mechanical properties, superior elastic performance, high durability, good biocompatibility and biodegradability (Zhang et al., 2023a; Ghorbani et al., 2020; Pedicini and Farris, 2003),making it a promising scaffold material in tissue repair when combined with the unique characteristics of nanofiber structures through electrospinning technology (Mi et al., 2017; Kucinska-Lipka et al., 2015).

The conductive nanomaterials used in conductive scaffolds, such as polypyrrole (PPY), polyaniline (PANI), poly(p-phenylene vinylene) (PPV), have shown great potential for biological stimulation through in-situ charge transfer (Dong et al., 2020). However, most of these conductive materials are non-biodegradable or have poor biodegradability, which may cause immune responses after long-term implantation. Additionally, their complex chemical structures make them difficult to process and thus limit their widespread use (Wang et al., 1999; Ghasemi-Mobarakeh et al., 2011; Chen et al., 2025). In contrast, carbon nanotubes (CNTs) stand out as widely used conductive nanomaterials due to their strong mechanical properties, conductivity, high biosafety, and relative ease of manufacture and functionalization. Currently, numerous studies have utilized CNTs to prepare conductive scaffolds for tissue repair, confirming their effectiveness and feasibility in scaffold fabrication (Zhang et al., 2023b; Parikh et al., 2023).

Therefore, in this study, we employed electrospinning technology to fabricate conductive PU/CNT fibrous scaffolds. By combining these scaffolds with exogenous ES, we cultured macrophages on the conductive scaffolds to evaluate the impact of both the conductive scaffolds and exogenous ES on macrophages. And a rat model of wound healing was established to evaluate their effects on wound repair. Our investigation aimed to explore their roles in macrophage phenotypic transformation and cytokine secretion, and to assess their functions in wound repair in vivo, providing valuable guidance for the future development of tissue repair biological scaffold.

Tissue repair is typically divided into four stages: clot formation, inflammation, new tissue formation (including re-epithelialization and granulation tissue formation), and tissue remodeling and resolution, with each stage involving multiple cell types (Peña and Martin, 2024). Macrophages, a heterogeneous population of myeloid cells, play numerous coordinating roles in the repair process. Currently, strategies for tissue repair primarily focus on the intrinsic properties of the materials themselves, including the natural mimicry of scaffolds, moisturizing properties, porosity, biodegradability, and biocompatibility (Hao et al., 2024), as well as the loading capabilities of the materials, including the ability to carry stem cells, drugs, and factors (Beavers et al., 2015; Yu et al., 2023). However, research on strategies involving additional physical stimuli remains inadequate. Nevertheless, it is worth noting that all cells in the body, not only excited nerve and muscle cells, can generate and receive steady-state bioelectric signals, which are key factors for regulating and controlling the quantity (cell proliferation and apoptosis), location (migration and orientation), and type (cell differentiation) of each cell (Liang et al., 2023). Therefore, exogenous ES may represent a new strategy to mimic the regenerative microenvironment in the body and promote tissue repair. In this study, we designed a tissue repair strategy combining a PU/CNT scaffold with excellent conductivity and exogenous ES and discovered its effective role in tissue repair.

This study successfully prepared PU/CNT nanofiber scaffolds using electrospinning technology. These scaffolds feature parallel-aligned fibers and well-organized surface structures, resembling the complex architecture of the extracellular matrix. The nanofiber scaffolds exhibit high porosity, providing ample space that is conducive to promoting cell proliferation and differentiation (Chen et al., 2016; Han et al., 2024). PU has excellent biomechanical properties, providing appropriate tensile strength, fatigue resistance and flexibility for tissue repair, and enabling better structural support compared to hydrogels (Nasab et al., 2022). Previous research has demonstrated that composite nanofiber scaffolds containing CNTs can spatially guide cell growth and enhance signal transmission, thereby facilitating tissue repair in vitro (Huang et al., 2020; Fabbro et al., 2013). In this study, CNTs were integrated to improve the conductivity and mechanical strength of the nanofiber scaffolds. The incorporation of CNTs significantly increased the electrical conductivity of the scaffolds from 10 to 6 S/m (PU group) to 100 S/m (PU/CNT group), providing an ideal platform for effective ES delivery. Additionally, SEM and TEM results revealed that both groups of fiber scaffolds exhibited good orientation, indicating that the addition of CNTs did not affect the orientation of the scaffolds. The orientation of nanofiber scaffolds serves as an important topographical guidance cue that plays a crucial role in processes such as cell directional growth and migration (Jin et al., 2023; Huang L. et al., 2018; Zhao et al., 2024). At the same time, the highly oriented structure can optimize the conduction and output performance of ES on the scaffolds (Zhang M. et al., 2023). Overall, PU/CNT scaffolds demonstrated the expected electrical conductivity and in vivo microenvironment simulation, creating a favorable microenvironment for tissue repair.

In the early stages, tissue macrophages produce mitochondrial ROS and drive several early repair processes, amplifying the inflammatory response. Later, the characteristics of macrophages are mitochondrial respiration and catabolic functions, suppressing inflammation, regulating ECM deposition, and promoting tissue repair (Willenborg et al., 2021; Sheppe and Edelmann, 2021). Excessive early inflammation can lead to excessive ROS production after injury, which disrupts calcium homeostasis, causes lipid peroxidation of cell membranes, and leads to damage to nucleic acids and proteins (Xia et al., 2021). In addition, ROS can promote M1 polarization of macrophages, which in turn activates inflammatory activation through multiple signaling pathways such as STAT 1, NFκB, p38 MAPK, and NLRP 3 inflammasome (Pawate et al., 2004; Padgett et al., 2014). Evidently, regulating the inflammatory response and promoting the anti-inflammatory transition of macrophages is crucial in tissue repair, and the dynamic transition in macrophage phenotype from pro-inflammatory to anti-inflammatory plays a key role (Peña and Martin, 2024). When macrophages polarize, they differentiate into functionally opposite M1 and M2 macrophages. M2 macrophages express increased levels of Arg-1, mannose receptor (CD206), IL-10, and chemokines CCL17 and CCL22, which play significant roles in tissue repair, angiogenesis, and metabolism (Yunna et al., 2020). Therefore, the combined strategy designed in this study aims to alleviate the harmful effects of excessive inflammation and promote macrophage M2 polarization to facilitate the tissue repair process.

In the selection of macrophage models for experimental design, various macrophage lines exhibit distinct advantages and disadvantages. Among them, RAW264.7 and THP-1 are widely utilized macrophage models (Chen et al., 2017; Liu X. et al., 2021; Fan Q. et al., 2022). THP-1 is a human myeloid leukemia monocytic cell line, and RAW264.7 is a mouse leukemia monocytic cell line derived from myeloid cells (Chen et al., 2023a). RAW264.7 cells, originating from mouse leukemic monocytes, demonstrate high sensitivity to physical/chemical stimuli such as ES and nanomaterials (Chen et al., 2023a). However, polarization macrophages still have significant differences between human and mouse cell lines (Li P. et al., 2021). The advantage of human THP-1 cells lies in being closer to the human pathological physiological environment, but they need to be induced to differentiate into macrophage-like states by PMA, which may introduce additional variables and have lower phenotypic stability (Li P. et al., 2021; Chanput et al., 2014). Therefore, if focusing on clinical translation, THP-1 cells should be used to verify key mechanisms to compensate for the differences in human and mouse immune responses. If the focus is on high-throughput screening or mechanism exploration, RAW264.7 is still the preferred choice due to its operational simplicity (Li P. et al., 2021). In this study, we utilized RAW264.7 cells as the cellular model for initial mechanistic exploration. In the future, a multi-model complementary strategy, such as initial screening with mouse-derived cells followed by validation with human-derived cells, will further enhance the reliability and generalizability of research.

Good biocompatibility is the foundation for the function of biomaterials. It refers to the appropriate host response during the expected clinical usage period in the body. In tissue engineering, scaffolds provide support for cell attachment, proliferation, and differentiation. Therefore, if cells adhere to and migrate across the surface and through the extracellular matrix of the scaffold, followed by cell proliferation and deposition of new matrix, the tissue engineering scaffolds and implant materials can be considered biocompatible (Munir et al., 2019). In this study, WMCNTs were utilized to impart conductive properties to PU nanofibrous scaffolds due to their unique mechanical, physical, and electronic characteristics. However, the safety and toxicity of WMCNTs remain controversial. Some studies have reported the formation of granulomas in the lungs of mice administered with different concentrations of CNTs, exhibiting lung-related cytotoxicity (Lam et al., 2004; Warheit et al., 2004). Conversely, other studies indicate that CNTs can purposefully interact with proteins, nucleic acids, and cells, demonstrating good cytocompatibility and playing a positive role in tissue repair (Munir et al., 2019). CCK-8 and Live/Dead staining are commonly used reagents to evaluate scaffold cell proliferation and cytotoxicity (Yi et al., 2024; Ja et al., 2022). The results of this study show that cells in all groups exhibited good proliferation and survival rates. The addition of CNTs did not affect the values of CCK-8 and even promoted cell proliferation. This could be attributed to various factors influencing the toxicity level of CNTs in the physiological environment, including metal catalysts and processing methods (Li et al., 2013). After integration into PU scaffold via electrostatic spinning technology, CNTs appeared non-toxic under physiological conditions. Additionally, their conductivity and ES exhibited a synergistic effect, thereby promoting cell proliferation. In the evaluation of in vivo biocompatibility, H&E staining of organs in each group of rats revealed no significant damage, further confirming the biosafety of the nanofiber membrane.

Numerous studies have demonstrated the beneficial effects of ES in tissue repair (Hoare et al., 2016; Li J. et al., 2021). iNOS and Arg1 serve as markers for M1 and M2 macrophages, respectively (Kieler et al., 2021). In this study, immunofluorescence experiments revealed that ES can upregulate Arg1 expression while downregulating iNOS expression, indicating that ES promotes M2 polarization of macrophages, which is consistent with previous findings that ES can modulate macrophage phenotype by inhibiting the MAPK/JNK signaling pathway and activating oxidative phosphorylation and ATP synthesis (Wu et al., 2023). Besides, observations of wound healing in animal experiments, along with results from HE and Masson staining, demonstrated that ES can reduce wound length, regenerate granulation tissue, and increase collagen ratio, thereby facilitating wound healing. The adjustment of ES parameters is crucial for exerting these effects. Therefore, setting the safest and most effective ES parameters, such as voltage, frequency, time, ES mode, is a key point for future research. Low voltage (<50 mV/mm) has limited effectiveness, while excessively high voltage (>200 mV/mm) may damage cells (Zhao et al., 2020). ES at different frequencies also exhibits varying effects. You et al. found that the cell survival rate was highest in the 500 Hz ES group (You et al., 2019). Additionally, different ES modes, including direct current (DC), alternating current (AC), pulsed current (PC), and pulsed electromagnetic fields (PEMF), have been proven to have beneficial effects in tissue repair (Thakral et al., 2013; Zhao et al., 2006; Zhao, 2009), but their impact on macrophage metabolism and phenotypic transformation remains unclear.

In mechanistic studies, PU/CNT nanofiber scaffolds upregulated the expression of genes associated with the M2 phenotype of macrophages (such as Arg1 and IL-10 (Opal and DePalo, 2000)) and suppressed the expression of genes linked to the M1 phenotype (such as TNF-α and IL-6 (Liu C. et al., 2021)) through the transmission of exogenous ES signals. ELISA is an immunoassay technique for the quantitative detection of antigens or antibodies in biological samples (Lequin, 2005), and its results also provides compelling evidence that exogenous ES signals can enhance the expression of the anti-inflammatory protein IL-10 and reduce the expression of the inflammatory proteins TNF-α and IL-6. In vivo immunohistochemical analysis further confirmed ES promoted the expression of IL-10 and suppressed the expression of TNF-α. These findings suggest that ES promotes M2 polarization of macrophages, further modulating their cytokine secretion profile and shifting macrophage function from pro-inflammatory to anti-inflammatory. On the other hand, IL-10 is commonly used to induce M2 polarization of macrophages in vitro (Huang X. et al., 2018). Numerous studies have also shown that the IL-10 factor is a key factor regulating the activation pathways of macrophages, and its high expression has been confirmed to promote M2 polarization of macrophages in vivo and in vitro (Chen et al., 2023b; Li et al., 2022; Sun et al., 2024). This indicates that macrophage polarization and cytokine secretion by macrophages have a mutually reinforcing effect, jointly reshaping the immune microenvironment during regeneration.

The coherent alignment between our in vitro and in vivo findings strongly suggests that the promoted wound healing phenotype observed in rats is mechanistically rooted in the ES-driven immunomodulation at the cellular level. While our data clearly demonstrate that the PU/CNT + ES group promotes an M2-like cytokine profile, an intriguing question remains regarding how the physical cue of ES is transduced into a sustained pro-regenerative macrophage phenotype. Beyond the potential signaling pathways like MAPK/JNK (Wu et al., 2023), recent insights into immunometabolism propose that the functional polarization of macrophages is underpinned by a fundamental metabolic reprogramming (Bianconi et al., 2024; Srirussamee et al., 2019). Typically, M1 macrophages exhibit enhanced glycolysis, while M2 cells show increased fatty acid oxidation and oxidative phosphorylation (Viola et al., 2019). It is plausible that ES not only provides topographical and electrical cues but may also orchestrate a shift in macrophage metabolic state. For instance, ES may promote a metabolic shift from glycolysis towards oxidative phosphorylation, a shift characterized by the M2 phenotype (Bianconi et al., 2024). This metabolic reprogramming could improve the stability of the anti-inflammatory state and the consequent secretion of pro-regenerative factors like IL-10, thereby directly linking the observed in vitro polarization to the enhanced tissue repair in vivo. Future studies focusing on the metabolic landscape of macrophages under ES will be crucial to unravel this intricate connection.

In summary, the PU/CNT electrospun conductive nanofiber scaffold combined with exogenous ES exerts an inhibitory effect on inflammatory responses and accelerates tissue repair by regulating macrophage phenotype polarization and cytokine secretion profiles. This provides a theoretical foundation and practical guidance for the development of novel immunomodulatory nanofiber scaffolds. In the future, it is necessary to further optimize the parameters and types of ES and explore more complex models, such as the efficacy of the scaffold in diabetic wounds, promoting the clinical translation of nanofiber scaffolds.

In this study, conductive PU/CNT nanofiber scaffolds were successfully fabricated by electrospinning technology. Combined with exogenous ES (150 mV/mm), it was confirmed that these scaffolds could significantly promote the polarization of macrophages to the M2 phenotype, manifested by the upregulation of Arg1 expression and the downregulation of iNOS, while the secretion of pro-inflammatory factors (TNF-α, IL-6) was reduced and the anti-inflammatory factor (IL-10) was increased. In vivo studies further demonstrated both the biological safety of the scaffolds and its efficacy in promoting immune regulation and wound healing. This finding provides experimental evidence for the development of novel immunomodulatory biological scaffolds. Future research can focus on optimizing ES parameters, such as frequency and mode, and incorporating growth factor loading strategies to further advance their clinical application in tissue repair.