Efferocytosis in tissue engineering: A comprehensive review of emerging therapeutic strategies for enhanced tissue repair and regeneration

Abstract

This comprehensive review elucidates the critical role of efferocytosis in tissue repair and regeneration processes, While systematically exploring innovative approaches through which tissue engineering strategies can modulate efferocytosis to optimize these biological processes. The manuscript is structured to first establish a fundamental understanding of efferocytosis, encompassing its core concepts, molecular mechanisms, and physiological functions within tissue repair. Subsequently, it provides an in-depth analysis of the regulatory role of efferocytosis in inflammatory response modulation during tissue repair cascades. The review culminates in a detailed investigation of cutting-edge tissue engineering applications specifically designed to manipulate efferocytosis pathways. Substantial evidence from recent studies has unequivocally demonstrated that efferocytosis serves as a crucial biological process in maintaining tissue homeostasis and orchestrating injury repair mechanisms. In this context, tissue engineering has emerged as a transformative approach, offering precise control over efferocytosis enhancement, inflammation resolution, and tissue regeneration processes. Through sophisticated integration of bioactive factor regulation, advanced scaffolding materials, and targeted cellular interactions, tissue engineering platforms have established novel therapeutic paradigms, providing unprecedented insights and innovative strategies for the treatment of diverse pathological conditions.

Headnote

ARTICLE INFO

Keywords:

Efferocytosis

Tissue repair

Tissue engineering

Inflammatory response

ABSTRACTS

This comprehensive review elucidates the critical role of efferocytosis in tissue repair and regeneration processes, While systematically exploring innovative approaches through which tissue engineering strategies can modulate efferocytosis to optimize these biological processes. The manuscript is structured to first establish a fundamental understanding of efferocytosis, encompassing its core concepts, molecular mechanisms, and physiological functions within tissue repair. Subsequently, it provides an in-depth analysis of the regulatory role of efferocytosis in inflammatory response modulation during tissue repair cascades. The review culminates in a detailed investigation of cutting-edge tissue engineering applications specifically designed to manipulate efferocytosis pathways. Substantial evidence from recent studies has unequivocally demonstrated that efferocytosis serves as a crucial biological process in maintaining tissue homeostasis and orchestrating injury repair mechanisms. In this context, tissue engineering has emerged as a transformative approach, offering precise control over efferocytosis enhancement, inflammation resolution, and tissue regeneration processes. Through sophisticated integration of bioactive factor regulation, advanced scaffolding materials, and targeted cellular interactions, tissue engineering platforms have established novel therapeutic paradigms, providing unprecedented insights and innovative strategies for the treatment of diverse pathological conditions.

1. Introduction

In adult organisms, the daily turnover of billions of cells generates substantial apoptotic debris, which is efficiently eliminated by phagocytes through the evolutionarily conserved process known as efferocytosis. Efferocytosis refers to the process by which phagocytes clear apoptotic cells. This biological mechanism is essential for maintaining tissue homeostasis under physiological conditions [1]. However, efferocytosis dysregulation leads to apoptotic cell accumulation and secondary necrosis, forming necrotic cores that release pro-inflammatory mediators. These mediators amplify inflammatory cascades and compromise tissue repair processes, ultimately driving the progression of chronic inflammatory diseases [2-4]. Therefore, unraveling the molecular mechanisms governing efferocytosis is pivotal for the advancement of precision therapeutics that foster tissue repair and mitigate sustained inflammatory responses.

Efferocytosis, as a fundamental biological process, prevents secondary necrosis and the subsequent amplification of inflammation through the macrophage-mediated recognition and engulfment of apoptotic debris. This clearance mechanism not only inhibits the release of pro-inflammatory mediators but also promotes the production of antiinflammatory cytokines, thereby fostering a regenerative tissue microenvironment [5]. Furthermore, recent studies highlight the potential role of efferocytosis in promoting macrophage polarization toward the M2 phenotype, a pivotal process in the resolution of inflammation and the repair of damaged tissues [6-8].

Recent advances in deciphering the molecular mechanisms of efferocytosis have propelled tissue engineering strategies to the forefront of regenerative medicine. By precisely modulating efferocytic pathways through biomimetic approaches and cell-specific targeting, these innovative strategies demonstrate significant potential in enhancing therapeutic efficacy, minimizing adverse effects, and promoting tissue regeneration [9]. Specifically, tissue engineering interventions for efferocytosis regulation can be categorized into three mechanistically distinct approaches: (1) bioactive factor-mediated enhancement of efferocytic capacity and inflammation resolution, (2) scaffold-based strategies that either recapitulate extracellular matrix architecture or deliver specific molecular cues to optimize efferocytosis and tissue repair, and (3) cellular engineering approaches that directly participate in apoptotic cell clearance while simultaneously modulating inflammatory responses and promoting regenerative microenvironments (Fig. 1).

This review aims to systematically elucidate the fundamental principles of efferocytosis and its regulatory role in tissue repair processes. Through a comprehensive analysis of the molecular mechanisms and physiological functions underlying efferocytosis, we will investigate its pivotal role in modulating inflammatory responses during tissue regeneration. Furthermore, we will explore innovative tissue engineering strategies that harness efferocytosis regulation to enhance repair outcomes, thereby establishing both theoretical frameworks and practical paradigms for developing next-generation therapeutic interventions. As efferocytosis research advances, we anticipate the emergence of sophisticated biomaterial systems capable of precise spatiotemporal control over efferocytic processes, ultimately enabling comprehensive structural and functional restoration of damaged tissues.

2. Efferocytosis and tissue repair

2.1. Mechanisms of efferocytosis

The efferocytosis process comprises three mechanistically distinct phases (Fig. 2). In the initial recognition phase, apoptotic cells actively recruit phagocytes through the release of specific "find-me" signals, rather than relying on passive encounter mechanisms. Subsequently, during the engulfment phase, phagocytes identify characteristic "eatme" signals, particularly phosphatidylserine exposure, on apoptotic cell surfaces via specific receptor-ligand interactions. This enables selective recognition and initiation of phagocytic uptake. In the final degradation phase, intracellular signaling cascades are activated, triggering cytoskeletal remodeling for apoptotic cell internalization and phagosome formation. This process culminates in phagosome-lysosome fusion, generating phagolysosomes where specialized lysosomal enzymes systematically degrade cellular debris. Collectively, this sophisticated process integrates extracellular recognition, intracellular signaling, and metabolic degradation mechanisms to ensure efficient apoptotic cell clearance.

The recognition phase initiates efferocytosis through the release of diverse soluble mediators, collectively termed "find-me" signals. These signals encompass lipid-derived molecules (e.g., lysophosphatidylcholine [LPC] and sphingosine 1-phosphate [S1P]) [1,10], specific chemokines (e.g., Fractalkine) [11], and purinergic signals (e.g., ATP and UTP) [12,13]. "Find-me" signals serve dual physiological functions: they establish chemotactic gradients to recruit phagocytes to apoptotic sites, while simultaneously enhancing phagocytic clearance capacity and promoting anti-inflammatory phenotypic polarization [14,15].

Lauber et al. showed that apoptotic cells release lipid attraction signals to induce phagocyte migration through a caspase-3-mediated mechanism [1]. Caspase-3, a key enzyme in the apoptotic process,

(1) Recognition phase

facilitates the release of lipid signals such as LPC, which attracts phagocytes to remove apoptotic cells. In recent years, several studies have investigated the mechanisms underlying LPC-mediated phagocyte migration. For instance, Riederer et al. found that LPC exerts an acyl-chain-dependent effect on endothelial cell prostacyclin production, which may influence phagocyte migration and elucidate how lipid signals released by apoptotic cells affect subsequent phagocytosis [16]. Moreover, Peter et al. demonstrated that the G2A receptor and ATP-binding cassette transporter Al (ABCA1) also play crucial roles in phagocyte migration in response to the "find-me" signals released by apoptotic cells [17,18]. In addition to LPC signaling, apoptotic cells can release S1P via sphingosine kinase 1 (SphK1) expression, which acts as another "find-me" signal to promote phagocytosis [10,19]. As an important bioactive lipid, S1P is involved in regulating cell migration and survival and is associated with pathological processes such as atherosclerosis. Weigert et al. revealed that S1P release from apoptotic cells involves caspase-1-mediated cleavage of sphingosine kinase 2 (SphK2), which may affect the release and function of SIP [20]. CX3CL1/fractalkine, a unique transmembrane chemokine, also plays a significant role in the efferocytosis recognition phase. Apoptotic cells actively recruit phagocytes by releasing apoptotic vesicles containing CX3CL1 to facilitate their clearance own [21]. Both membrane-bound and soluble forms of CX3CL1 stimulate macrophage chemotaxis, participating in the clearance of apoptotic cells [11]. Furthermore, Rodriguez et al. noted that the cytoplasmic fragment of CX3CL1 exhibits transcriptional activation during apoptotic cell processing and immunomodulation, highlighting its complex role in apoptotic cell clearance and immune regulation [22].

Apoptotic cells actively facilitate their clearance through the release of purinergic "find-me" signals, particularly ATP and UTP. These nucleotides play crucial roles in maintaining immune homeostasis [12]. They are selectively released via Pannexin-1 channel activation, establishing chemotactic gradients that enhance phagocyte recruitment and phagocytic capacity [13]. Chiu et al. established a direct correlation between Pannexin channel activation and nucleotide release during apoptosis, while Qu et al. further demonstrated that Pannexin-1-mediated ATP release is essential for apoptotic clearance yet independent of inflammasome activation, providing critical insights into the distinct roles of Pannexin-1 in apoptosis and inflammation [23].

Beyond their chemotactic functions, extracellular nucleotides modulate inflammatory responses through specific receptor activation, thereby influencing both apoptotic cell clearance and the progression of chronic inflammatory diseases [24,25]. This dual regulatory capacity positions nucleotide signaling as a key mediator in the maintenance of tissue homeostasis.

Collectively, the recognition phase of efferocytosis is characterized by the coordinated release of diverse signaling molecules, including lipid mediators (LPC, S1P), chemokines (CX3CL1), and nucleotides (ATP, UTP). These molecules actively recruit and prime phagocytes for efficient apoptotic cell clearance. These mechanistic insights not only advance our understanding of efferocytosis regulation but also identify novel therapeutic targets for diseases associated with impaired apoptotic cell clearance.

(2) Phagocytosis phase

The phagocytosis phase represents a critical juncture in efferocytosis, wherein phagocytes precisely recognize and internalize apoptotic cells through sophisticated molecular interactions. This process initiates when phagocyte surface receptors engage with "eat-me" signals displayed on apoptotic cells, triggering actin cytoskeletal remodeling and subsequent phagosome formation for cellular engulfment.

Central to this phase is the surface exposure of phosphatidylserine (PS), an amphipathic phospholipid that translocates from the inner to outer leaflet of the plasma membrane during apoptosis. First identified by Fadok et al. [2], PS serves as a principal "eat-me" signal, marking cells for programmed removal while simultaneously activating phagocytic receptors [26]. Beyond its role in apoptotic cell clearance, PS exposure critically regulates inflammatory responses and tissue repair processes [27]. The molecular machinery governing PS externalization involves multiple protein families, including ATP11C (the major erythrocyte flippase whose deficiency causes congenital hemolytic anemia) [28], Xk-related proteins (XkR8 and CED-8), and TMEM16 family members (which exhibit calcium-dependent phospholipid scramblase activity) [29,30].

PS recognition occurs through both direct and indirect receptor mechanisms. Direct recognition is mediated by specialized receptors including brain-specific angiogenesis inhibitor 1 (ВАП), T-cell immunoglobulin mucin (TIM) family members (TIM-1, TIM-3, TIM-4), and Stabilin-2 [2,31,32]. These receptors employ unique structural domains for PS binding, with BAI1's thrombospondin type 1 repeats (TSR) domain additionally recognizing bacterial lipopolysaccharide (LPS), enabling dual pathogen and apoptotic cell clearance [33]. Indirect recognition involves bridging molecules such as growth arrest-specific factor 6 (Gas6) and protein S (PROS), which facilitate interactions between PS and TAM receptor tyrosine kinases (TYRO3, AXL, MerTK) [34]. Additional bridging molecules like milk fat globule-EGF factor 8 (МЕСЕ8) mediate PS recognition through integrin receptors (avp3, avß5) [31].

Beyond PS, apoptotic cells display additional "eat-me" signals including calreticulin, which complexes with PS or C1q to engage CD91/ LRP1 receptors, enhancing phagocytic recognition [35-37]. Conversely, healthy cells express "don't-eat-me" signals, such as CD47 and CD24, which engage SIRPa and Siglec-10 receptors, respectively, to inhibit phagocytosis [38,39]. The evolutionary conservation of these inhibitory interactions underscores their physiological importance, while their therapeutic blockade represents an emerging strategy in cancer immunotherapy.

The coordinated recognition of apoptotic signals serves dual physiological purposes: ensuring efficient cellular clearance while preventing inflammatory responses. PS exposure promotes anti-inflammatory cytokine production and prevents inflammasome activation by containing cellular debris [32,40]. This intricate balance between "eat-me" and "don't-eat-me" signaling represents a sophisticated evolutionary adaptation for maintaining tissue homeostasis and immune tolerance.

(3) Digestion phase

The digestive phase of efferocytosis encompasses the molecular processes governing apoptotic cell degradation following phagocytic internalization, comprising three distinct stages: phagosome formation, maturation, and lysosomal fusion. This intricate process is regulated by an ensemble of molecular regulators and signaling pathways.

Racl, a Rho family GTPase, orchestrates critical events through cytoskeletal remodeling and signal transduction [41]. Racl activation initiates phagocytic cup formation via two primary mechanisms: (1) WAVE complex activation through interaction with WASP family proteins, driving actin filament nucleation and polymerization [42]; and (2) formation of the ELMO/Dock180/Rac signaling module, which amplifies actin polymerization through the C-terminal polyproline region-mediated interaction between ELMO and Dock180 [43,44]. These processes are further regulated by PS receptors (TIM4 and MerTK), which transduce phagocytic signals upon PS recognition [45, 46]. Notably, the TLR4-PI3K-Rac1 signaling axis has been identified as a crucial regulator of macrophage cytoskeletal reorganization and phagocytic capacity [47].

Phagosome maturation is governed by the sequential action of Rab GTPases, particularly Rab5 and Rab7, through the classical maturation pathway [48]. Rab5 initiates early phagosome maturation by coordinating vesicle fusion and cargo sorting through interactions with effector proteins Rabaptin5 and Rabex-5 [49,50]. Subsequently, Rab7 assumes control, facilitating late phagosome-lysosome fusion via interactions with RILP and HOPS complexes while simultaneously regulating microtubule-dependent phagosome trafficking through FYCO1 binding [49]. The transition from Rab5 to Rab7 represents a critical molecular switch marking the progression from early to late phagosomal stages [51].

Complementing the classical pathway, LC3-associated phagocytosis (LAP) represents an alternative degradation mechanism. LAP utilizes LC3 lipidation (LC3-II formation) to enhance phagosome-lysosome fusion efficiency, thereby accelerating apoptotic cell degradation [52, 53]. LC3-II functions dually in both phagosome formation and lysosomal fusion, ensuring efficient degradation of apoptotic contents [53].

Additionally, LC3-associated phagocytosis (LC3-associated phagocytosis, LAP) is a non-canonical autophagy-related pathway characterized by the covalent conjugation of LC3 protein (microtubule-associated protein 1 light chain 3) to the inner membrane of phagosomes via an ATG5/ATG7-dependent lipidation process, forming the degradative LC3-II complex [52,53]. Unlike canonical autophagy, LAP is triggered not by starvation signals but by phagocytosis mediated via pattern recognition receptors (e.g., TLRs) or Fcy receptors. This process requires Rubicon protein involvement, which recruits the Beclin-1/VPS34 complex to promote PI3P lipid generation and guide phagosome maturation [53,54]. Mechanistically, LAP enhances phagocytic efficiency through two actions: (1) LC3-II anchors to the phagosome membrane via its hydrophobic domain, forming a "molecular zipper" to promote membrane extension and closure; and (2) LC3-II directly interacts with lysosomal membrane proteins LAMP1/2, accelerating phagosome-lysosome fusion via Rab7-mediated membrane trafficking. Notably, this process also depends on reactive oxygen species (ROS) generated by NADPH oxidase to regulate phagosome acidification, enabling LAP to achieve a degradation efficiency 3-5 times higher than classical phagocytosis when degrading pathogens or apoptotic cells [52, 54,55]. The critical role of LAP in efferocytosis may offer new insights for biomaterial design. By coordinating lysosomal fusion in phagocytes and anti-inflammatory signaling, LAP can optimize tissue repair mediated by biomaterials. For example, modifying LAP activators in wound dressings or biodegradable scaffolds may enhance macrophage phagocytic function, thereby reducing local inflammation and accelerating healing.

Collectively, the digestive phase of efferocytosis integrates the coordinated actions of Rac1, Rab GTPases, and LC3 to regulate phagosome dynamics, ultimately ensuring efficient apoptotic cell degradation and clearance. This molecular synergy not only maintains tissue homeostasis but also provides potential therapeutic targets for disorders of apoptotic cell clearance.

2.2. Efferocytosis regulates the inflammatory response during tissue repair

Tissue repair is a complex and highly coordinated biological process aimed at restoring the structural integrity and physiological function of damaged tissues. It can be broadly divided into several overlapping stages: hemostasis and inflammation, proliferation, and remodeling. Among these, the inflammatory response serves as the initial and critical phase, facilitating the removal of damaged tissue and the prevention of infection. This is achieved through mechanisms such as vascular dilation, increased vascular permeability, leukocyte recruitment, and the release of inflammatory mediators, all of which collectively establish the foundation for subsequent cell proliferation and tissue remodeling [56, 57]. In this process, macrophages, as key components of the innate immune system, become the core link connecting inflammatory response and tissue repair [58].

Macrophages play a central regulatory role in all stages of tissue repair. They can quickly respond to tissue damage signals, migrate to the damaged site, and release inflammatory mediators such as cytokines and chemokines, which further activate and recruit other immune cells, thereby initiating and maintaining the inflammatory response. This process not only helps to remove blood clots, necrotic tissue, and cell debris, creating a favorable environment for the proliferation and differentiation of new cells, but also directly promotes the proliferation and differentiation of key cells such as fibroblasts and endothelial cells through the release of growth factors and cytokines [59,60]. Macrophages can be polarized into two phenotypes: M1 phenotype (pro-inflammatory) and M2 phenotype (anti-inflammatory) [61]. In conventional wound repair, Mi-type macrophages produce pro-inflammatory cytokines such as TNF-a, IL-6, and IL-16, thereby activating the immune response during the inflammatory phase [60]. During the proliferation phase, anti-inflammatory or M2 phenotype macrophages gradually dominate over time, releasing anti-inflammatory cytokines such as IL-10 and TGF-p, triggering wound regeneration and recovery of wound integrity and function [62,63]. In addition, in the stage of tissue remodeling, macrophages release proteases and other enzymes, participate in the cross-linking and hydrolysis of collagen fibers, fine-regulate tissue remodeling, and ensure the functional recovery of damaged tissues.

The efferocytosis of macrophages is also of great significance in tissue repair. Efferocytosis not only prevents tissue damage and excessive inflammation but also promotes pro-resolving signaling in macrophages, which is essential for tissue repair and remodeling following injury or serious inflammation. This function is mediated through three principal mechanisms: (1) the clearance of dead and dying cells, thereby preventing secondary necrosis and further exacerbation of inflammation; (2) the suppression of pro-inflammatory cytokine production, alongside the upregulation of anti-inflammatory cytokines and specialized pro-resolving mediators (SPMs); and (3) the regulation of macrophage polarization toward an anti-inflammatory (M2) phenotype as well as improving [60-63] (Fig. 3).

The efferocytosis of macrophages is also of great significance in tissue repair. Efferocytosis of macrophages refers to the phagocytic process of apoptotic cells. This process can not only prevent tissue damage and inflammation caused by secondary necrosis of apoptotic cells, but also inhibit the production of pro-inflammatory cytokines by removing dead and dying cells, and increase the expression of anti-inflammatory cytokines and SPMs. In addition, efferocytosis can regulate the polarization of macrophages to anti-inflammatory phenotypes, such as M2, thereby promoting resolution of inflammation and tissue repair [64-66]. Through efferocytosis, macrophages realize the transition from inflammatory response to tissue regeneration in tissue repair, providing a key microenvironmental regulation for tissue repair.

Next, we will introduce the regulation of inflammatory response during tissue repair by efferocytosis from three aspects.

The accumulation of apoptotic cells (ACs) poses a significant immunological challenge, as secondary necrosis can trigger detrimental inflammatory responses. Efferocytosis serves as a critical homeostatic mechanism, facilitating the non-inflammatory clearance of ACs through a dual cytokine regulatory strategy: enhancing anti-inflammatory mediator production while suppressing pro-inflammatory cytokine release, thereby promoting efficient inflammation resolution.

Related studies have shown that efferocytosis promotes the clearance of apoptotic cells and reduces the accumulation of inflammatory mediators through specific phosphatidylserine receptors, such as kidney injury molecule-1 (KIM-1) and Stabilin-2 [67,68]. KIM-1-mediated efferocytosis promotes the resolution of inflammation by inhibiting the production of pro-inflammatory cytokines such as TNF-a, IL-6, and CC-chemokine ligand 5 (CCL5) [67]. Stabilin-2 reduces the accumulation of these pro-inflammatory cytokines by enhancing the rapid clearance of apoptotic cells by macrophages [68]. Efferocytosis involves the regulation of the NF-kB signaling pathway, a key transcription factor that controls the expression of a variety of pro-inflammatory cytokines. By inhibiting NF-kB activation, efferocytosis reduces the transcription and production of these pro-inflammatory cytokines, which in turn promotes resolution of inflammation [69]. The TAM receptor family (including Tyro3, Axl, and Mer) upregulates the E3 ubiquitin ligase inhibitors of cytokine signaling 1 (SOCS1) and SOCS3, thus blocking IFNa-mediated signal transducer and activator of transcription 1 (STAT1) signal transduction and pro-inflammatory gene expression [70], TAM receptors play a role in efferocytosis and promote the resolution of inflammation by inhibiting the production of pro-inflammatory cytokines. Studies have shown that efferocytosis inhibits the activation of NF-xB through Twist-mediated type I IFNs and Axl signaling pathways, thereby reducing the production of TNF-a, IL-6, and other pro-inflammatory cytokines and promoting the resolution of inflammation [71]. This regulatory effect not only helps to control the intensity of the inflammatory response but also can shorten the duration of inflammation and promote the resolution of inflammation.

Efferocytosis plays a pivotal role in immune regulation by stimulating anti-inflammatory cytokine production and maintaining tissue homeostasis [72,73]. This process specifically enhances the secretion of IL-4 and IL-13, which are crucial for macrophage functional polarization [74]. These cytokines bind to macrophage surface receptors, driving M2 phenotype polarization characterized by increased IL-10 production and decreased TNF-o and IL-1f release [75]. IL-10, as a master anti-inflammatory regulator, not only suppresses inflammatory responses and promotes tissue repair but also inhibits T-cell proliferation and activation, thereby reducing inflammatory mediator production [75]. Beyond cytokine regulation, efferocytosis modulates macrophage metabolic states to influence inflammatory responses [76]. The clearance of apoptotic cells enhances fatty acid oxidation and electron transport chain activity, metabolic reprogramming essential for anti-inflammatory macrophage polarization [77]. This metabolic shift provides both energy and biosynthetic precursors necessary for anti-inflammatory cytokine production and tissue repair functions [77]. Furthermore, efferocytosis activates Liver X receptors (LXRs), nuclear receptors that regulate inflammatory and metabolic pathways in macrophages [78]. The interaction between phosphatidylserine (PtdSer) on apoptotic cells and macrophage scavenger receptors not only initiates efferocytosis but also triggers LXRs signaling, promoting cholesterol metabolism and anti-inflammatory cytokine production while suppressing inflammatory mediators [79]. This mechanism ensures immune homeostasis and prevents excessive inflammation.

Beyond IL-4, IL-13, and IL-10, efferocytosis also stimulates the production of transforming growth factor-B (ТСЕ-В), а multifunctional cytokine with potent anti-inflammatory and immunomodulatory properties. The interaction between apoptotic cells and the receptor Stabilin2 2 phagocytes triggers TGF-f production [68]. TGF-B plays a critical role in immune regulation by promoting regulatory T cell differentiation. These differentiated T cells further enhance anti-inflammatory responses through IL-10 production, creating a positive regulatory loop for inflammation suppression. Moreover, TGF-B contributes to tissue homeostasis by orchestrating extracellular matrix remodeling and repair processes. Its dual role in immune modulation and tissue reconstruction underscores its importance in maintaining the structural integrity and functional recovery of damaged tissues [75].

(2) Driving the biosynthesis of pro-decomposition mediators.

SPMs constitute a class of bioactive lipid mediators synthesized from essential fatty acids, exhibiting potent anti-inflammatory and proresolving properties. A critical mechanism in inflammation resolution involves the enzymatic conversion of polyunsaturated fatty acids into diverse SPMs classes-including lipoxins, resolvins, protectins, and maresins-that collectively orchestrate inflammation suppression and tissue repair, ultimately restoring tissue homeostasis.

Efferocytosis activates SPMs biosynthesis through distinct molecular pathways. Following apoptotic neutrophil clearance, macrophages significantly upregulate the production of SPMs, including lipoxin A4 (LXA4), resolvin D1 (RvD1), RvD2, and RvE2. Notably, LXA4 and RvD1 enhance efferocytic capacity, establishing a positive feedback loop that amplifies inflammation resolution [64]. Dalli and Serhan found that the efferocytosis of macrophages can stimulate the production of RvD1, RvD2 and RvE2, and these SPMs can regulate the function of macrophages and promote the resolution of inflammation [64]. Saturated efferocytosis can produce macrophages with low CD11b expression, which can be regulated by the synthesis of pro-resolving lipid mediators such as RvEl and RvD1 by macrophages undergoing efferocytosis and by glucocorticoids [80]. The MerTK signaling pathway plays an important role in efferocytosis, promoting SPM synthesis by regulating macrophage metabolic and signaling pathways. Cai et al. found that MerTK cleavage limits the biosynthesis of pro-resolving mediators and exacerbates tissue inflammation [81]. However, activation of the MerTK signaling pathway promotes the synthesis of inflammation resolution mediators by inhibiting CaMKII activity [82]. This suggests that MerTK signaling plays a key role in regulating SPM synthesis and efferocytosis. Efferocytosis also involves SPM biosynthetic pathways, such as the 12/15-lipoxygenase (12/15-LOX) and 5-lipoxygenase (5-LOX) pathways. These pathways are activated in macrophages and promote the synthesis of lipoxins and resolvins, thereby regulating the inflammatory response [83].

The generation of SPMs creates a positive feedback loop that enhances efferocytosis efficiency. Uderhardt et al. demonstrated that 12/ 15-LOX drives the biosynthesis of pro-resolving lipid mediators, including LXA4, RvE1, and protectin D1 (PD1) [83]. These mediators not only initiate inflammation resolution but also promote apoptotic cell clearance by selectively activating macrophages. Specifically, LXA4 enhances the phagocytic capacity of macrophages, while RvE1 and PD1 regulate leukocyte infiltration and increase apoptotic neutrophil uptake both in vivo and in vitro [84]. Experimental evidence further supports the pro-resolving effects of SPMs. Grazda et al. reported that RvE1 significantly improves efferocytosis efficiency and accelerates inflammation resolution in murine models [85]. Similarly, RvD1 promotes efficient apoptotic cell clearance by modulating NADPH oxidase activity and controlling reactive oxygen species (ROS) levels in macrophages, thereby limiting inflammation and tissue damage, particularly in aging contexts [86,87]. Additionally, 15-epi-LXA5 and RvE4 enhance splenic macrophage efferocytosis, facilitating neutrophil clearance from tissue exudates and promoting local inflammation resolution [88]. These findings collectively underscore the critical role of SPMs in orchestrating a coordinated response that links enhanced efferocytosis with effective inflammation resolution.

(3) Regulation of macrophage phenotypic.

Macrophage polarization is a dynamic process that is influenced by a variety of cytokines and environmental factors. Compared to M1 macrophages, which are more effective at pathogen elimination through the high expression of phagocytosis-related receptors (e.g., Fcy receptors and complement receptors) and the production of reactive oxygen and nitrogen species (ROS/RNS), M2 macrophages exhibit enhanced apoptotic cells clearing capacity, secretion of anti-inflammatory cytokines such as IL-10 and TGF-B, and properties that promote tissue repair [65,66].

Several studies have shown that the efferocytosis of specific apoptotic cells can regulate the polarization of macrophages to an antiinflammatory phenotype. Following the clearance of apoptotic cells, bone marrow-derived macrophages from mice exhibit increased secretion of IL-10, i.e., characteristic of M2 phenotype. This response can be further enhanced by stimulation with lipopolysaccharide (LPS) and attenuated by the depletion of CD36 and platelet-activating factor receptor (PAFR) [8,89]. Similarly, efferocytosis of apoptotic mesenchymal stem cells (MSCs), anti-inflammatory neutrophils has been shown to elevate the levels of anti-inflammatory markers (e.g. IL-10, CD206, TGF-B, PPAR-y, Nur77, and KLF4) and efferocytosis receptors (e.g. MerTK, CD36, CX3CR1, and integrins a v/ß 5) in macrophages while concurrently reducing the levels of pro-inflammatory markers (e.g. TNF-a and nitric oxide (NO) [90-92]. In addition, efferocytosis also promotes macrophage polarization to tissue repair by affecting fatty acid oxidation and the demand for electron transport chains [77]. This process involves metabolic reprogramming of macrophages, providing energy and biosynthetic precursors to macrophages with an anti-inflammatory phenotype.

Additional evidence of cross-talk among signaling pathways further reinforces the concept that efferocytosis plays a pivotal role in modulating macrophage phenotype. Arginase-1, a key enzyme expressed in M2 macrophages, contributes to the suppression of inflammatory responses by metabolizing L-arginine into L-ornithine and urea. In GMCSF-differentiated human macrophages, efferocytosis has been shown to regulate the expression of Arginase-1 and the tyrosine kinase Mer, both of which are critical for the establishment of an anti-inflammatory phenotype [65]. Similarly, COX-2 is a key enzyme in the synthesis of prostaglandins, and its products, such as prostaglandin E2 (PGE2), can promote the anti-inflammatory phenotype of macrophages [93]. Macrophage COX-2 plays a key role in mediating efferocytosis function, macrophage reprogramming, and intestinal epithelial repair.

In the polarization process of anti-inflammatory macrophages, the efferocytosis related receptors, namely TAM receptor family members (including Mer, Axl and Tyro3) and their ligands Gas6 and protein S have important effects in regulating macrophage polarization and inflammatory response [94]. These receptors promote efferocytosis by binding to phosphatidylserines on the surface of apoptotic cells, which in turn affect the polarization state of macrophages. It has also been shown that apoptotic vesicles can restore the homeostasis of liver macrophages, enhance macrophage efferocytosis, combat the development of type 2 diabetes, and promote the transformation of macrophages to an anti-inflammatory phenotype through calreticulin located in T2 diabetic liver vesicles [95]. This suggests that apoptotic bodies may influence the progression of metabolic diseases by regulating the polarized state of macrophages. De Maeyer et al. found that blocking p38 MAPK restored and accelerated the clearance of apoptotic cells by macrophages, while modulation of these pathways contributed to the restoration of the reparative macrophage phenotype and promoted resolution of inflammation [96].

Taken together, these findings suggest that the inflammatory response can potentially be modulated through efferocytosis-mediated macrophage phenotype switching, thereby promoting effective tissue repair. In-depth understanding and precise intervention of this biological process may not only help to promote swift tissue repair but also effectively inhibit adverse outcomes of repair, such as pathological fibrosis. This, in turn, could offer novel therapeutic strategies aimed at improving patient prognosis and overall quality of life.

3. Specific application of efferocytosis in tissue repair

Efferocytosis is one of the key mechanisms for organisms to maintain tissue homeostasis and promote damage repair. This process involves the recognition, phagocytosis, and degradation of apoptotic cells by macrophages and other phagocytes, thereby preventing the development of harmful autoimmune responses and inflammatory reactions. Effective and continuous efferocytosis not only helps to remove dead cells and avoid their release of damage-associated molecular patterns (DAMPs) that trigger inflammation, but also directly contributes to the repair process of different tissues in the human body by releasing antiinflammatory cytokines and promoting the release of tissue repair signaling molecules. Thus, efferocytosis not only plays a role in preventing inflammation but is also a critical link in the tissue repair process (Fig. 4, Table 1).

3.1. Bone tissue repair

The regulatory role of efferocytosis in bone tissue repair has been extensively investigated. Studies have shown that bone tissue healing can be promoted by regulating the efferocytosis of macrophages. For example, it was mentioned in the study of Geng et al. that MFG-E8 promoted tendon-bone healing after anterior cruciate ligament reconstruction by regulating macrophage efferocytosis and M2 polarization [97]. This suggests that efferocytosis not only plays a role in eliminating apoptotic cells, but also plays a key role in regulating inflammatory response and promoting tissue repair.

Further studies have also shown that efferocytosis has a bidirectional regulatory effect on bone tissue repair. Wang et al. found that by promoting efferocytosis and bi-directional regulation of pyroptosis, it is possible to balance the elimination of bioresponsive biofilms and guide tissue repair during implantation surgery [98]. This suggests efferocytosis modulation as a promising strategy to enhance osseointegration and reduce implant-related complications. In addition, the role of efferocytosis in bone formation has been demonstrated. Batoon et al. experimentally demonstrated that induction of osteoblast apoptosis can stimulate macrophage efferocytosis and trigger paradoxical bone formation [99]. This finding reveals the complexity of efferocytosis in bone tissue repair and its potential role in promoting bone formation. In the process of bone repair, continuous macrophage efferocytosis is of great significance for reversing inflammation and promoting bone repair. The therapeutic potential of efferocytosis extends to inflammation modulation and cartilage repair. Wang et al. successfully reversed the inflammatory response in bone repair by activating the continuous efferocytosis of macrophages by short fibers that mimic the microenvironment [100]. This further emphasizes the importance of efferocytosis in regulating inflammation and promoting bone repair. Efferocytosis also has a significant effect on cartilage repair. Additionally, efferocytosis has a significant effect on cartilage repair. In the study of Xiong et al., cartilage senescence was reversed by local microspheres that regulate macrophage efferocytosis [7]. This study expands the understanding of efferocytosis in bone tissue repair and provides a new therapeutic strategy for cartilage repair.

Efferocytosis plays an important role in bone tissue repair. By regulating the efferocytosis of macrophages, it can not only promote bone healing, but also regulate inflammatory response, promote bone formation, and provide a new treatment strategy for cartilage repair. These studies provide new perspectives and potential therapeutic targets for future bone tissue repair therapy.

3.2. Skin tissue repair

Following skin injury, the timely clearance of apoptotic neutrophils by macrophages at the wound site represents a critical step in tissue repair [101]. This efferocytic process serves dual roles: preventing secondary necrosis-induced inflammation and promoting resolution through macrophage polarization to an M2 phenotype characterized by reduced pro-inflammatory cytokine expression and increased production of reparative mediators (TGF-B1, IL-10) [101]. Mechanistic studies by Jun et al. identified CCN1 as a pivotal molecular bridge in this process. Through its TSR domain, CCN1 binds phosphatidylserine (PS) on apoptotic neutrophils, while its von Willebrand factor type C (VWC) domain engages macrophage integrins (avp3/avp5), thereby activating Racl-mediated efferocytosis [101]. This molecular bridging mechanism establishes CCN1 as an essential opsonin in cutaneous wound healing, with potential therapeutic applications for chronic or non-healing wounds.

In diabetic wound healing models, impaired macrophage function significantly compromises inflammation resolution, highlighting the critical role of efferocytosis in diabetic tissue repair [102]. Maruyama et al. further demonstrated that reduced macrophage numbers and activation levels impair lymphangiogenesis, creating a pathological cycle that hinders diabetic wound closure [103]. This highlights the role of macrophages in promoting lymphangiogenesis and wound healing.

In addition, C1q, as part of the complement system, has a unique role in angiogenesis and wound healing. Bossi et al. identified C1q's capacity to induce angiogenesis in both in vitro and in vivo models, suggesting its therapeutic potential for enhancing wound repair [103]. At the cellular level, Justynski et al. employed single-cell RNA sequencing to reveal that apoptotic pathway regulation orchestrates the transition from inflammatory resolution to proliferative phases [104]. Their findings further demonstrated that inhibiting efferocytosis receptors (Axl and TIMD4) disrupts proper wound healing, emphasizing the functional importance of these receptors in skin repair.

Efferocytosis plays a multifaceted role in skin tissue repair, including promoting the resolution of inflammation, regulating the polarization of macrophages, participating in lymphangiogenesis, and coordinating the clearance of apoptotic cells. These studies provide a comprehensive molecular mechanistic understanding of the application of efferocytosis in skin wound healing and identify potential targets for the development of novel therapeutic strategies.

3.3. Liver tissue repair

Efferocytosis plays a multifaceted role in liver tissue repair, primarily through inflammation resolution and hepatocyte regeneration. Miao et al. demonstrated that GAS6-containing extracellular vesicles protect against ischemia-reperfusion injury by enhancing macrophage efferocytosis via the MerTK-ERK-COX2 signaling pathway [105]. This process involves macrophage recognition and clearance of apoptotic hepatocytes, triggering anti-inflammatory responses and tissue repair mechanisms [105]. This suggests that efferocytosis plays a key role in liver repair after ischemia-reperfusion injury. Liver macrophages, including resident Kupffer cells and infiltrating macrophages, exhibit remarkable phenotypic plasticity in response to injury. As reviewed by Li and Weinman [106], these cells regulate hepatic inflammation through efferocytosis-mediated clearance of apoptotic cells, reducing inflammatory mediator release while promoting anti-inflammatory cytokine production. This regulatory mechanism is essential for controlling liver inflammation and fibrosis progression.

The Gas6/TAM system has emerged as a key regulator in liver pathology. Bellan et al. identified Gas6 as a potential biomarker for chronic liver disease (CLD), with its levels correlating with fibrosis progression [107]. The Gas6/TAM axis modulates both hepatic stellate cell (HSC) activation and macrophage function, suggesting its potential as a therapeutic target for CLD treatment [107].

Collectively, these studies demonstrate that efferocytosis orchestrates liver repair through dual mechanisms: 1) inflammation resolution via apoptotic cell clearance and 2) promotion of hepatocyte regeneration. These insights provide a molecular framework for developing novel therapeutic strategies targeting efferocytosis in liver diseases.

3.4. Cardiac tissue repair

Efferocytosis serves as a pivotal mechanism in cardiac tissue repair, orchestrating the clearance of apoptotic cells by macrophages and other phagocytes. This process not only eliminates cellular debris and mitigates inflammation but also drives macrophage polarization from proinflammatory (M1) to anti-inflammatory (M2) phenotypes, creating a regenerative microenvironment essential for myocardial repair [108]. Through these mechanisms, efferocytosis significantly enhances post-injury cardiac recovery by reducing inflammation and promoting tissue regeneration [109]. Furthermore, it facilitates cardiac cell reprogramming and intercellular communication, critical processes for functional restoration [110].

The regulatory mechanisms of phagocyte-mediated efferocytosis in cardiac repair have been validated through multi-dimensional experiments at the molecular, cellular, and tissue levels. From a cellular mechanism perspective, macrophages significantly promote neovascularization and lymphatic remodeling in infarcted areas by upregulating vascular endothelial growth factor C (VEGFC), increasing local vascular density by 135 % and lymphatic density by 89 % simultaneously. This biphasic vascular system remodeling provides essential material transport channels and structural support for ischemic myocardium [111]. Concurrently, mesenchymal stem cells enhance the efferocytic function of neutrophils, drastically improving the clearance efficiency of apoptotic cardiomyocytes from 27.4 % to 68.9 %. This process is accompanied by a significant 54 % reduction in pro-inflammatory cytokine IL-6 levels, accelerating the repair and remodeling of the myocardial microenvironment by inhibiting excessive inflammation [112].

Based on this in-depth analysis of efferocytic regulatory mechanisms, innovative therapeutic strategies targeting the efferocytosis pathway have demonstrated clear efficacy in myocardial injury repair. In the field of nanodrug delivery, Song et al.'s biomimetic nanocarriers, through dual regulation of ferroptosis inhibition and efferocytosis activation, not only reduced the myocardial infarction area by 43.2 % but also significantly improved cardiac pump function by increasing LVEF from 28.4 % to 41.1 %, a key indicator of cardiac function [113]. Reverse validation studies by Wang et al. showed that Sectmla gene deletion reduced macrophage efferocytosis efficiency by 61 %, directly leading to a 2.3-fold expansion of myocardial injury area after ischemia-reperfusion, revealing the necessity of intact efferocytic function for myocardial protection at the pathological level [114]. In signaling pathway-targeted interventions, Wan et al. activated the MER tyrosine kinase-mediated efferocytic signaling axis, increasing the clearance rate of apoptotic cardiomyocytes by 3.1-fold and improving LVEF by 9.7 percentage points, confirming the critical role of this pathway in promoting cell clearance and cardiac function recovery [115]. Studies on immune checkpoint regulation demonstrated that Gao et al.'s nanodegradants, by blocking the CD47-SIRPa "don't eat me" signaling pathway, not only reduced myocardial fibrosis by 58 % but also restored capillary density to 82 % of normal myocardial tissue, effectively curbing the pathological process of post-infarction myocardial remodeling [116].

These findings collectively reveal the stage-dependent regulatory mechanisms of phagocytosis in cardiac repair, offering new therapeutic perspectives for cardiovascular diseases. Efferocytosis is a cascade process encompassing apoptotic cell recognition, internalization and transport, and lysosomal degradation, where the integrity of functional coupling among stages dictates the ultimate biological outcomes. By finely regulating macrophage polarization and targeting specific stages (e.g., recognition or degradation) to enhance apoptotic cell clearance efficiency while maintaining normal lysosomal function, phagocytosis can effectively alleviate inflammation and promote tissue regeneration, thereby significantly improving cardiac functional recovery and longterm prognosis. Notably, intervention strategies must account for the complete execution chain of efferocytosis: for instance, in models with lysosomal dysfunction, solely enhancing the recognition stage may increase pro-inflammatory factor release due to incomplete clearance, highlighting the need for therapeutic design based on precise evaluation of each stage of the phagocytic process.

3.5. Intestinal tissue repair

Efferocytosis plays a pivotal role in intestinal tissue repair, particularly in the context of inflammatory bowel disease (IBD) and other intestinal injuries. This process, which involves the clearance of apoptotic cells by phagocytes such as macrophages, is essential for maintaining intestinal mucosal integrity and facilitating wound healing [117-119]. Yurdagul et al. demonstrated that macrophages enhance efferocytosis and damage resolution by metabolizing arginine released from apoptotic cells [120]. This mechanism not only removes dead cells and mitigates inflammation but also drives the polarization of macrophages from pro-inflammatory to anti-inflammatory phenotypes, which is crucial for intestinal tissue repair and regeneration. Studies have shown that following efferocytosis, macrophages predominantly transition to the anti-inflammatory M2 phenotype, enabling them to promote inflammation resolution and tissue repair [101].

The pro-resolving factors released by macrophages post-efferocytosis are vital for mucosal wound healing, particularly in IBD [121]. These factors not only aid in resolving inflammation but also directly contribute to intestinal epithelial repair. For instance, arginase-1 (Arg-1) produced by M2 macrophages converts L-arginine into polyamines such as spermine and spermidine, which stimulate the proliferation and division of nearby intestinal stem cells through c-Myc and p21 signaling, thereby facilitating wound healing [101]. Beyond macrophages, other cell types, such as neutrophils, also participate in efferocytosis. While neutrophil efferocytosis is known to aid skin wound healing, a similar role is likely in intestinal barrier repair [101,109]. In intestinal repair surgery, the regulation of efferocytosis and the bidirectional regulation of pyroptosis balance the eradication of bioresponsive biofilms and guided tissue repair [98].

At the molecular level, Zhao et al. revealed that cystathionine y-lyase (Cth) induces macrophage efferocytosis via the ERK1/2 signaling pathway, thereby regulating intestinal barrier repair [122]. This finding provides critical molecular insights into the role of efferocytosis in maintaining intestinal barrier function. Additionally, Meriwether et al. highlighted the role of COX2 in macrophages during efferocytosis, inflammation resolution, and intestinal epithelial repair [93], further underscoring the importance of efferocytosis in these processes. Efferocytic function regulates the inflammatory microenvironment and promotes intestinal tissue regeneration through multi-dimensional molecular mechanisms. A series of studies by Wu MY and colleagues demonstrate that NRBF2, a core subunit of the PI3KC3 complex, enhances macrophage clearance of apoptotic cells by regulating autophagosome and phagosome maturation, thereby restricting intestinal inflammation-clinical research has found that NRBF2 expression in colonic tissues of ulcerative colitis patients is positively correlated with macrophage infiltration, and its deficiency leads to apoptotic cell accumulation and increased release of inflammatory factors, while exogenous supplementation with wild-type macrophages significantly alleviates intestinal injury. Targeted activation of the FPR2 receptor promotes the expression of efferocytosis-related genes such as MERTK and CD36 via the Gi protein-coupled pathway, while inhibiting the NF-kB pathway to reduce the release of pro-inflammatory factors like IL-1p and TNF-a. This receptor also drives actin rearrangement to form phagocytic cups by recognizing phosphatidylserine (PS) and lipoxin A4 on the surface of apoptotic cells, accelerating apoptotic cell clearance and promoting anti-inflammatory macrophage polarization. As a non-canonical autophagic pathway, LC3-associated efferocytosis (LAP) enhances lysosomal fusion efficiency and apoptotic cell degradation through LC3 protein modification of the phagosomal membrane. In a DSS-induced colitis model, enhancing LAP activity promotes the release of anti-inflammatory mediators such as IL-10 and RvD1 via NADPH oxidase (NOX2)-dependent reactive oxygen species (ROS) production and p38 MAPK pathway activation, significantly reducing intestinal mucosal damage [117,118,123].

In summary, efferocytosis contributes to intestinal tissue repair through multiple mechanisms, including macrophage polarization, the release of pro-resolving factors, regulation of intestinal barrier repair, and promotion of epithelial regeneration. These interconnected processes provide a robust biological foundation for understanding and enhancing intestinal repair following injury, offering potential therapeutic avenues for conditions such as IBD and other intestinal pathologies.

3.6. Lung tissue repair

Efferocytosis serves as a critical regulator in pulmonary tissue repair and homeostasis maintenance, orchestrating apoptotic cell clearance, inflammatory modulation, and tissue regeneration. Recent advances have elucidated its therapeutic potential in various lung injury models.

For example, Ji et al. [124] found that inhaled pro-efferocytosis nanoenzymes can promote the resolution of acute lung injury by enhancing efferocytosis, clearing apoptotic cells in the alveoli and reducing inflammatory responses. Kang et al. [125] further confirmed that innate immune training protects the lung from injury by initiating efferocytosis, which may involve modulating the pulmonary innate immune system to more efficiently recognize and eliminate apoptotic cells. Yan et al. [126] showed that PTX3 attenuates hard metal-induced acute lung injury by enhancing efferocytosis. This finding provides a new perspective on the molecular mechanisms underlying hard metal-induced lung injury and reveals PTX3 as a potential therapeutic approach. Similarly, Li et al. [127] found that dexmedetomidine reduces sepsis-associated acute lung injury by regulating macrophage efferocytosis via the ROS/ADAM10/AXL pathway, suggesting that drug interventions can modulate efferocytosis to affect lung injury repair.

These studies collectively position efferocytosis as a central therapeutic target for pulmonary diseases, offering novel opportunities for drug development, advanced delivery systems such as nanoenzymes, combination therapy strategies, and personalized treatment approaches. Future research directions should focus on developing efferocytosisspecific biomarkers, optimizing targeted delivery systems, exploring epigenetic regulation mechanisms, and investigating long-term tissue remodeling effects. These advancements promise to revolutionize pulmonary disease management, particularly for acute lung injuries and environmental toxin exposures.

3.7. Nervous system repair

Microglia, the resident immune cells of the central nervous system (CNS), play a pivotal role in maintaining neural homeostasis through immune surveillance, inflammatory regulation, and efferocytosis-the clearance of pathological cells and harmful substances. Comprising 5-20 % of the total glial cell population, microglia are essential for CNS integrity and function. Their ability to perform efferocytosis is particularly critical in neural tissue repair and disease modulation.

Following ischemic stroke, macrophages in the brain undergo reprogramming to enhance efferocytosis and resolve inflammation, a process essential for neural tissue recovery [128]. Neumann emphasized the significance of microglial and macrophage phagocytosis in brain injury repair, highlighting their dual roles in damage response and tissue regeneration [129].Further studies by Ulrich and Holtzman demonstrated that microglial phagocytosis is crucial for neuronal survival and the progression of neurodegenerative diseases [130]. Studies have demonstrated that macrophages in the brains of mice following ischemic stroke undergo functional remodeling via the TREM2/PI3K/Akt signaling pathway. Activation of this pathway not only promotes efferocytosis and accelerates the clearance of apoptotic neurons but also suppresses pro-inflammatory cytokine synthesis to create an immunological microenvironment conducive to neural repair [131]. Cantoni et al. found in demyelination studies that TREM2 receptors on microglial surfaces reduce the release of pro-inflammatory factors such as IL-18 and TNF-a by mediating NF-kB pathway inhibition, while simultaneously enhancing phagocytic activity through upregulation of phagocytosis-related gene expression [131,132]. The neuroprotective effects of TREM2 are multidimensional; its activation of the PI3K/Akt/mTOR pathway sustains microglial survival and proliferation under aging and pathological conditions, providing sustained effector cell support for long-term tissue repair [131]. In acute ischemic injury, the Syk kinase-Rho GTPases pathway activated by TREM2 binding to DAP12 is critical for phagocytic synapse formation. This pathway promotes phagocytic cup maturation and closure through actin cytoskeleton reorganization, while TREM2 deficiency impairs this process, reducing the clearance efficiency of apoptotic cells in the ischemic penumbra and worsening ischemic damage [133]. In terms of nutritional intervention, Belayev et al. confirmed that DHA (docosahexaenoic acid) enhances microglial phagocytic function and reduces infarct volume by upregulating TREM2 and MANF expression through PPARy pathway activation. Additionally, its metabolites inhibit the NF-kB inflammatory pathway via GPR32 receptors, suppressing inflammation while synergistically promoting neurogenesis and functional recovery [134]. Koizumi et al. found that UDP (uridine diphosphate) activation of the P2Y6 receptor drives actin reorganization to form phagocytic cups for clearing apoptotic cells via the Rho/ROCK pathway [135]. Wen et al. demonstrated that inhibiting the P2Y6 receptor reduces Syk kinase phosphorylation levels, impedes the recruitment of phagocytosis-related proteins such as Dock180 and CrkII, suppresses efferocytosis, and exacerbates brain injury after ischemia [136]. These findings suggest that targeting the P2Y6 receptor-Rho/ROCK/Syk axis may represent a novel strategy for regulating efferocytosis.

Efferocytosis in neural tissue repair encompasses multiple mechanisms, including the regulation of microglial and macrophage activity, inflammation resolution, and neuroprotection in neurodegenerative diseases and brain injuries. These insights provide a molecular foundation for developing novel therapeutic strategies aimed at enhancing efferocytosis to promote tissue repair.

The maintenance of efferocytosis is vital for post-injury tissue repair, and its dysfunction can lead to pathological processes that compromise health. Investigating the mechanisms of efferocytosis and developing strategies to enhance its efficacy hold significant clinical promise for advancing tissue repair and treating CNS disorders.

4. Application of tissue engineering in the regulation of efferocytosis

In the field of tissue engineering, enhancing efferocytosis is increasingly recognized as a promising strategy for the repair and regeneration of damaged tissues. By integrating bioactive factors, scaffold materials, and cellular components, tissue engineering approaches can effectively augment macrophage efferocytosis, modulate the inflammatory response, and facilitate tissue repair and regeneration [Fig. 5]. Bioactive factors directly influence macrophage behavior, while scaffold materials offer structural support and mimic the functional properties of the extracellular matrix. At the cellular level, targeted regulation can significantly impact the efficiency of efferocytosis. A carefully tailored combination of these strategies-adapted to the specific pathological context-offers a comprehensive approach to modulate efferocytosis, thereby playing a pivotal role in advancing tissue engineering. The following sections provide an in-depth examination of how these components synergistically promote efferocytosis.

4.1. The role of active factors in the regulation of efferocytosis

Active factors play multifaceted roles in the regulation of efferocytosis; they finely tune the process through multiple mechanisms and pathways to maintain tissue homeostasis, promote damage repair, modulate immune responses, and play a role in the treatment of related diseases (Fig. 6).

For example, isoflurane promotes macrophage phagocytosis of apoptotic neutrophils through the AMP-mediated ADAM17/Mer signaling pathway [140]. Targeted delivery of MerTK protein by cell membrane-engineered nanoparticles enhances efferocytosis and alleviates atherosclerosis in diabetic ApoE-/- mice [141]. Resolvin E1 rescues severe aplastic anemia in mice by improving efferocytosis [138]. Additionally, resveratrol enhances macrophage efferocytosis through the AMPK/STAT3/S1PR1 pathway [142]. These factors also affect efferocytosis by regulating metabolic pathways. For example, 3,3'-diindolomethane enhances macrophage efferocytosis through the AhR/Nrf2/Arg-1-mediated arginine metabolic pathway [143], while CRBN controls calcium influx by regulating Orail [144].

In immunoregulation and inflammatory response, active factors such as LPS alleviate multi-tissue injury by promoting macrophage efferocytosis [139], while MRP8/14 complex mediates macrophage efferocytosis through RAGE and Gas6/MFG-E8 and induces polarization through TLR4-dependent pathway [145]. Active factors also affect efferocytosis by regulating the polarization state of macrophages. For example, MFG-E8 regulates macrophage efferocytosis and M2 polarization, promoting tendon-bone healing [97], while baicalin enhances macrophage efferocytosis by inhibiting the RhoA/ROCK signaling pathway and regulating macrophage polarization [146].

Active factors can regulate the surface receptors of macrophages, affecting the recognition and phagocytosis of apoptotic cells. For example, dipotassium glycyrrhizic acid and hinokitiol enhance efferocytosis by regulating the recognition, uptake, and metabolism of apoptotic cells [147]. They also enhance efferocytosis by regulating macrophage mobility. For instance, PLAG enhances macrophage mobility through membrane P2Y2 redistribution, promoting the efferocytosis of apoptotic neutrophils [148].

Active factors enhance efferocytosis by promoting the differentiation of macrophages. For example, retinoids promote the differentiation and efferocytosis of mouse bone marrow-derived macrophages by upregulating BMP-2 and Smad3 [149]. Active factors also improve the phagocytic function of macrophages. For instance, alpha-1 antitrypsin supplementation improves efferocytosis and phagocytosis in alveolar macrophages after cigarette smoke exposure [149], while procysteine treatment improves the phenotypic and functional changes in alveolar macrophages caused by cigarette smoke [150].

Active factors restore efferocytosis by reversing changes in macrophage programming. In chronic granulomatous disease (CGD), impaired clearance of apoptotic cells due to changes in macrophage programming is reversed by phosphatidylserine-dependent IL-4 production [151]. Active factors also contribute to spleen macrophage clearance by promoting the expulsion of neutrophils from the exudate. For example, 15-epi-lipoxin A5 promotes neutrophil exit from the exudate, facilitating their clearance by spleen macrophages [152].

Therefore, through these multifaceted regulatory mechanisms, active factors not only enhance the ability of macrophages to clear apoptotic cells, but also affect the inflammatory response and tissue repair process, providing new strategies and targets for the treatment of a variety of diseases.

4.2. The role of scaffold materials in the regulation of efferocytosis

Scaffold materials offer significant advantages in the targeted delivery of therapeutic molecules, enhancing the precision of drug delivery, flexibly regulating efferocytosis, and providing innovative strategies for treating a wide range of diseases. Their unique biological and physicochemical properties make them highly promising candidates as drug carriers, enabling advanced therapeutic applications (Fig. 7).

(1) Microspheres and microfiber-based scaffold materials

Microsphere and microfiber-based scaffold materials can regulate efferocytosis by releasing apoptotic signals to induce cell apoptosis or carrying apoptotic signals to promote the phagocytic function of cells, thereby promoting tissue repair. For instance, Xiong et al. [7]. developed aldehyde-modified microsphere scaffolds that release pro-apoptotic liposomes (A-Lipo) to induce apoptosis in senescent chondrocytes. Subsequently, the apoptotic cells are recognized and phagocytosed by macrophages, initiating the efferocytosis process and establishing a favorable microenvironment for repair (Fig. 7a). Additionally, the microsphere scaffolds deliver factors such as PDGF-BB to recruit stem cells to the injury site, promoting tissue repair and regeneration. Moreover, coating "apoptotic signals" on the surface of microspheres or microfibers is also beneficial for regulating efferocytosis to promote tissue repair. For example, Wang et al. [100]. coated "biomimetic apoptotic signals (АМ/СеО2)" on the surface of polydopamine short fibers and implanted them in situ into bone defect areas. By mimicking a healthy microenvironment with abundant apoptotic signals, it activates the continuous efferocytosis of macrophages, reverses inflammation, and accelerates the bone repair process (Fig. 7b). Besides, modifying or carrying ligands that can bind to the surface receptors of phagocytes (such as PS, Annexin Al, and Gas6) on the surface of microspheres or microfibers to activate the phagocytic signaling pathways of phagocytes can also achieve the goal of regulating efferocytosis to promote tissue repair.

(2) Nanoparticle-based scaffolds materials

Nanoparticle-based scaffolds materials have emerged as versatile tools for targeted drug delivery and efferocytosis modulation. Qiu et al. [141] employed cell membrane-engineered nanoparticles to deliver MerTK protein specifically to target sites, enhancing efferocytosis and demonstrating therapeutic efficacy in treating atherosclerosis in diabetic ApoE mice. Patel et al. [154] developed pro-resolving and pro-efferocytosis nanoparticles capable of carrying multiple drugs or active molecules. These nanoparticle-based scaffolds promote macrophage efferocytosis and repolarization, offering significant potential for treating cardiovascular diseases, particularly atherosclerosis. Tang et al. designed CpG-conjugated silver nanoparticles as multifunctional nanomedicine scaffolds [155], which not only enhance macrophage efferocytosis but also regulate macrophage phenotypes, providing a robust strategy for atherosclerosis treatment. Liu et al. utilized licorice protein nanoparticles to mediate in situ neutrophil apoptosis and macrophage efferocytosis, offering a novel approach for treating acute inflammation [156]. These nanoparticles exhibit excellent biocompatibility and interact with cells through their protein components, balancing apoptosis and efferocytosis to accelerate inflammation resolution. Ji et al. developed inhaled nanoenzyme scaffolds that carry active ingredients to enhance alveolar macrophage efferocytosis, effectively treating acute lung injury [124]. The catalytic activity of nanoenzymes, combined with the scaffold's functionality, enhances the regulation of the lung microenvironment, demonstrating the therapeutic potential of nanoparticle-based scaffolds in pulmonary diseases.

(3) Biomimetic scaffold materials

Biomimetic scaffold materials have demonstrated remarkable potential in enhancing efferocytosis and targeted drug delivery. Han et al. [157] synthesized biomimetic liposome scaffolds that leverage the efferocytosis mechanism to achieve efficient macrophage-targeted drug delivery. The biomimetic structure of these liposomes facilitates better integration with the host's up-regulating miR-136-5p and inhibiting the GNAS/STAT3 pathway. This demonstrates a strategy for optimizing EVs function [169].

Overall, EVs significantly influence the efferocytosis process through multiple mechanisms, such as regulating macrophage polarization, providing phagocytic signals, modulating inflammatory responses, and promoting intercellular communication. These mechanisms provide a powerful strategy for designing biomaterials targeting efferocytosis to promote tissue repair.

(7) Other special scaffold materials

Innovative scaffold materials continue to expand the possibilities for efferocytosis regulation and tissue repair. Zou et al. [170] designed an exosome-loaded vascular stent with a composite structure that prevents in-stent restenosis through an Lp-PLA2-triggered release mechanism. This scaffold combines the bioactive transport capabilities of exosomes with the physical support of vascular stents, enabling localized release of efferocytosis-related molecules and regulating vascular tissue repair. Lyu et al. [171] developed in situ hydrogel scaffolds that enhance non-efferocytic phagocytic activity for postoperative tumor treatment. The unique physical properties of hydrogels create a localized environment for sustained drug release, interacting with surrounding tissues and cells to modulate phagocytic behavior in the tumor microenvironment. This approach offers a novel strategy for cancer therapy.

Van der Meeren et al. [172] introduced a novel "eat-me" signal-based scaffold material, leveraging the mechanobiology of ferroptotic cancer cells. This innovative coating structure serves as a potential drug-loading and release platform, offering new insights into efferocytosis regulation and demonstrating significant potential for future research.

Scaffold materials show unique potential as targeted drug delivery carriers for regulating efferocytosis-related molecules. By mimicking biological processes and precisely targeting specific cells, these materials enhance therapeutic efficacy, reduce side effects, and promote tissue repair. From biomimetic liposomes and nanovesicles to genetically engineered macrophages and specialized hydrogels, scaffold materials are revolutionizing treatments for various diseases and offering innovative strategies for regenerative medicine [173]. However, current technologies face several challenges.

Drug release kinetics contradictions: Burst release-induced local concentration overload and temporal mismatches in multi-molecule co-delivery limit intervention efficacy in complex pathological processes, exerting significant negative impacts on the recognition, phagocytosis, and digestion phases of efferocytosis. These effects hinder normal apoptotic cell clearance, potentially leading to secondary inflammation and tissue damage [174-176]. Excessive local drug concentrations from burst release disrupt phagocyte recognition of apoptotic cells. For example, high concentrations of certain chemotherapeutic drugs alter the expression patterns or conformations of "eat me" signals (e.g., phosphatidylserine [PS], calreticulin) on apoptotic cells while interfering with phagocyte surface receptors (e.g., TIM-4, CD36, CD91). These receptors must bind to specific ligands on apoptotic cells to initiate efferocytosis; studies show the binding affinity of these receptors to ligands decreases at high drug concentrations, rendering phagocytes unable to effectively recognize apoptotic cells [177]. During phagocytosis, precise temporal control of molecular release and coordination (e.g., cytokines, chemokines) is required to guide phagocyte migration and phagocytic cup formation. Temporal mismatches in drug release disrupt this molecular synergy, delaying or preventing efficient phagocytosis. Additionally, high local drug concentrations impair phagocyte cytoskeletal reorganization-a critical step in pseudopod formation and apoptotic cell engulfment [178,179]. Drug release kinetics also negatively impact the digestion phase of efferocytosis. Burst release-induced concentration overload alters the acid-base balance within phagosomes. Lysosomal enzyme activity is highly pH-dependent; excessive drug concentrations can disrupt phagosomal pH, reducing lysosomal enzyme activity and impairing digestion of apoptotic cell contents. Moreover, temporal mismatches in multi-molecule co-delivery disrupt the synergistic action of digestive enzymes, which must be activated at specific time points to sequentially degrade biomolecules within apoptotic cells. Such mismatches interfere with enzymatic coordination, blocking efficient digestion [180-182].

Biocompatibility bottlenecks: Scaffold materials may induce immune responses and inflammation, inhibiting efferocytosis and disrupting tissue integration/repair. Degradation products of some scaffolds can trigger inflammatory reactions, impair efferocytic function, and complicate healing. Immune/inflammatory responses to scaffolds significantly disrupt efferocytosis: excessively rapid degradation may lose tissue support before repair is complete, while slow degradation can lead to material retention and persistent inflammation. The immune system recognizes implanted materials and releases inflammatory mediators (e.g., cytokines, chemokines), which interfere with phagocyte recognition/engulfment of apoptotic cells. Inflammation can also activate the complement system, producing components (e.g., C3b, C4b) that bind to apoptotic cell receptors and disrupt efferocytosis-though these components normally aid pathogen clearance [183,184]. The chemical properties of degradation products are equally critical; some may be immunogenic, further complicating healing. For example, acidic degradation products (e.g., from polyester scaffolds) alter local tissue pH, inhibiting lysosomal enzyme activity and reducing phagocyte digestion efficiency of apoptotic cell contents. Additionally, degradation products may bind to phagocyte surface receptors, interfering with signal transduction and disrupting efferocytosis initiation/progression [185,186].

Material intrinsic property limitations: The physicochemical properties of scaffolds, such as surface charge and pore size distribution, significantly influence efferocytosis efficiency [173]. Surface charge affects interactions with biomolecules and cells, impacting targeting precision and specific efferocytic processes. Mismatched surface charge can hinder binding between efferocytosis-related molecules and their targets, impairing efferocytosis. Pore size distribution is also critical: appropriate pore sizes facilitate biomolecule transport and normal cell migration/interaction, which are essential for phagocytes to approach and recognize apoptotic cells [187,188]. Excessively small or large pores can disrupt ligand-receptor binding required for efferocytosis. Additionally, low pH and high ROS levels in inflamed microenvironments can alter the conformation of material surface ligands, directly impairing molecular recognition and binding during efferocytosis [187-189]. This reduces the binding efficiency of efferocytosis-regulating molecules and the precision of targeted delivery, compromising multiple stages of efferocytosis (recognition, phagocytosis, digestion) and failing to promote normal efferocytic function [187-189].

Lack of specificity: The effect of scaffold materials on efferocytosis receptors, such as MerTK, Axl, etc., usually lacks specificity. For example, some scaffold materials may indirectly affect the expression of the efferocytosis receptor by releasing ions or growth factors, but they cannot target the specific receptor as precisely as drug molecules. Depending on the cell source difference: different cells (such as macrophages, smooth muscle cells, etc.) have different responses to the efferocytosis receptor, and scaffold materials may have a significant effect on some cells, but a limited effect on others. In addition, the complexity of signaling pathways: efferocytosis involves a variety of signaling pathways (such as PI3K/Akt, MAPK, etc.). The mechanism of action of scaffolds is relatively simple, and it is difficult to regulate multiple pathways at the same time, and it cannot specifically activate or inhibit a certain signaling pathway like gene therapy or small molecule drugs. Differences in cellular microenvironment: The microenvironment of scaffolds in different locations or tissues in vivo may be different, leading to differences in their regulatory effects on the efferocytosis signaling.

4.3. The role of cells in the regulation of efferocytosis

Cell regulation of efferocytosis is a key mechanism to maintain tissue homeostasis and promote wound repair. During this process, multiple cell types enhance macrophage efferocytosis by secreting specific molecules, regulating metabolic pathways, and direct cell-to-cell interactions.

Extracellular vesicles (EVs) play a crucial role in regulating efferocytosis. They not only enhance MerTK-dependent efferocytosis in macrophages, thereby promoting tissue repair and inflammation resolution [191], but also augment the efferocytosis capacity of macrophages through specific signaling pathways. For example, EVs containing GAS6 enhance macrophage efferocytosis and protect the liver from ischemia-reperfusion injury through the MerTK-ERK-COX2 signaling pathway [105] (Fig. 8a). Additionally, exosome-loaded pro-efferocytosis vascular scaffolds have been used to prevent in-stent restenosis via Lp-PLA2-triggered release [170], demonstrating the potential application of EVs in promoting efferocytosis and treating cardiovascular diseases.

(2) Regulation of macrophage metabolites

Metabolic regulation of macrophages during efferocytosis is crucial. Macrophages promote ongoing efferocytosis and resolution of damage by metabolizing arginine released by apoptotic cells [120]. This metabolic process not only provides energy for macrophages but also regulates efferocytosis efficiency through the metabolites produced (Fig. 8b). The critical role of macrophages in efferocytosis and inflammation resolution is further emphasized by the fact that EVs released from efferocytosis cells resolve inflammation and tissue damage through prosaposin-GPR37 signaling [192], whereas macrophage-derived protein S promotes the clearance of apoptotic neutrophils and supports metabolic reprogramming [193]. These findings highlight the important role of metabolites in the regulation of efferocytosis.

(3) Efferocytosis of mesenchymal stem cells (MSCs)

Mesenchymal stem cells (MSCs) demonstrate remarkable potential in regulating efferocytosis through multifaceted molecular and cellular interactions. Studies reveal that MSCs modulate phagocyte function directly or indirectly via mitochondrial remodeling. For example, in the bone marrow microenvironment, MSCs inhibit osteoclast precursor differentiation and enhance apoptotic cell clearance by transferring mitochondria to disrupt metabolic reprogramming, a process regulated by mitochondrial reactive oxygen species (ROS) homeostasis [194]. In cardiac ischemia-reperfusion injury models, MSCs secrete anti-inflammatory factors (e.g., TGF-B, IL-10) and lipid mediators (e.g., resolvin D1) via paracrine signaling, promoting neutrophil polarization toward a pro-resolving phenotype and enhancing their efferocytic capacity, thereby accelerating inflammation resolution and reducing myocardial fibrosis [112]. Furthermore, in endometrial injury repair, MSCs regulate macrophage lipid metabolism (e.g., the GPX4/ACSL4 axis) to suppress ferroptosis while upregulating efferocytosis-related receptors (e.g., MerTK) on large peritoneal macrophages, facilitating the clearance of ferroptotic monocytes/macrophages and improving tissue regeneration [190] (Fig. 8c). Collectively, these mechanisms highlight how MSCs systemically enhance efferocytosis through mitochondrial regulation, paracrine signaling, and phenotypic reprogramming of target cells, offering novel therapeutic strategies for inflammatory diseases and tissue repair.

Efferocytosis, a critical process for apoptotic cell clearance, plays a pivotal role in tumor progression and immune evasion through its regulation in the tumor microenvironment (TME). Emerging evidence indicates that tumor cells manipulate efferocytosis via multiple mechanisms to reshape the TME and immune responses. For instance, enhancing macrophage-mediated efferocytosis of tumor cells could promote tumor cell clearance, whereas inhibiting efferocytosis might amplify anti-tumor immunity by altering tumor-associated macrophage (TAM) functionality [195]. Tumor cells also exploit dysregulated phosphatidylserine (PS) externalization-a key "eat-me" signal for efferocytosis-to evade immune detection. This aberrant PS exposure disrupts immune recognition and fosters a pro-tumorigenic microenvironment [196]. Additionally, efferocytosis in the TME releases pro-tumor factors (e.g., IL-6, IL-10) that drive tumor proliferation, invasion, and immunosuppression, thereby sustaining tumor progression [197]. These findings underscore the dual role of efferocytosis in cancer biology and its potential as a therapeutic target. Future research should prioritize elucidating the molecular intricacies of efferocytosis to develop strategies that harness or disrupt this process for improved cancer therapy.

In summary, the regulation of efferocytosis in tissue engineering relies on the synergistic interplay of bioactive factors, scaffold materials, and cells, forming a "signaling regulation-structural support-functional execution" tripartite system.

Bioactive factors: Serve as core regulators of efferocytosis by targeting critical pathways (e.g., the MerTK/AXL-Tyro3 receptor tyrosine kinase axis) to directly modulate phagocytic functions of macrophages and other phagocytes, reducing inflammation and promoting tissue repair.

Scaffold materials: Provide the physical foundation for synergy through three mechanisms: 1) Guiding macrophage polarization via biomimetic topological structures and mechanical cues; 2) Acting as intelligent carriers for sustained factor release to maintain localized therapeutic concentrations; 3) Promoting seed cell adhesion through surface modifications to establish functional cell-material interfaces.

Cellular participation: Cells act as dynamic regulatory hubs: Directly executing efferocytosis (e.g., a single macrophage can clear 3-5 apoptotic cells); Amplifying factor effects via paracrine networks; Engaging in bidirectional interactions with scaffolds-scaffolds guide cell migration, while cells remodel scaffold structures via bioactive factors, dynamically shaping adaptive repair microenvironments.

Bioactive factors directly modulate phagocyte function by targeting key signaling pathways, thereby enhancing efferocytosis. Scaffold materials, through their biomimetic structures and factor delivery systems, promote cell adhesion and provide a supportive microenvironment for cells. Cells, in turn, execute efferocytosis, amplify the effects of bioactive factors, and engage in bidirectional interactions with scaffolds. The synergistic effects of these elements manifest as scaffolds providing sustained-release platforms for factors, seed cells supplementing exogenous factors, and factors enhancing cell-material interactions. This tripartite "scaffold support-factor regulation-cell execution" system enhances efferocytosis efficiency and optimizes the spatiotemporal coordination of repair microenvironments, offering innovative solutions for tissue regeneration. The synergy is exemplified by: scaffolds providing sustained factor release synchronized with phagocytic cycles (e.g., degradation rates matching macrophage activity peaks); seed cells supplementing exogenous factors via autocrine/paracrine loops; and factors enhancing cell-material interactions.

However, when designing regenerative materials that target efferocytosis regulation, it is important to consider the context-dependent plasticity of macrophages. For example, STAT3 has been shown to mediate both pro- and anti-inflammatory responses, depending on the specific cellular and tissue context. Therefore, it is essential to account for the particular disease environment and individual patient conditions in order to achieve more effective and tailored therapeutic outcomes.

5. Summary and Prospect

With the rapid development of biomaterials science and tissue engineering technology, biomaterials targeting efferocytosis have shown significant therapeutic potential. Future research will focus on understanding the molecular mechanism of efferocytosis and exploring how to achieve precise regulation of this process through biomaterials. Based on the further elucidation of the mechanism of efferocytosis, more new therapeutic strategies targeting efferocytosis are expected to be developed, so as to provide more accurate and efficient treatment options for the repair and functional recovery of damaged tissues (Fig. 9).

5.1. Specifically, the in-depth study of the molecular mechanism of efferocytosis can be carried out from the following aspects

(1) Analyze the key signaling pathways and their target points

A comprehensive exploration of the signaling pathways involved in the process of efferocytosis is a core task for a profound understanding of this complex physiological phenomenon [198]. It is currently known that after apoptosis, cells release "eat-me" signals such as the externalization of phosphatidylserine. Phagocytes, such as macrophages and dendritic cells, possess recognition receptors like Mer TK and Axl on their surfaces [199]. Subsequent research should not only further deeply analyze the interaction mechanisms among them but also utilize advanced technologies to identify the key nodes in the signaling pathways [200,201]. For instance, the CRISPR-Cas9 gene editing technology can achieve precise knockout or overexpression operations of specific genes in the cell genome [27,202]. We can edit the genes encoding Mer TK and Axl receptors or related signal transduction proteins. By constructing gene knockout or overexpression cell lines, we can observe the behavioral changes of cells during the efferocytosis process, including the ability to recognize apoptotic cells, the phagocytic rate, and the differences in the activation of subsequent signaling pathways, among others [203]. At the same time, deeply analyze the activation mechanisms of downstream signaling pathways within phagocytes, such as the PI3K-Akt and Rho GTPase pathways [181]. The PI3K-Akt pathway plays a crucial role in aspects such as cell survival, metabolism, and cytoskeleton rearrangement, while the Rho GTPase family is essential for cell shape change and motility [201]. Through proteomics techniques, such as the mass spectrometry-based quantitative proteomics method, it is possible to comprehensively analyze the expression changes, modification states, and protein-protein interaction networks of proteins within phagocytes during the efferocytosis process. Taking core signaling molecules such as MerTK, Gas6, and Annexin Al as entry points, and with the aid of bioinformatics analysis methods, construct signaling pathway network models to reveal the dynamic regulatory mechanisms of signaling pathways under different microenvironments (such as inflammation, hypoxia, etc.) [204].

(2) Conduct in-depth research on the influence of efferocytosis on other stages of tissue repair

Tissue repair is an orderly and complex process, which can usually be divided into four key stages: hemostasis, inflammation, proliferation, and remodeling. These stages are closely linked and influence each other, jointly promoting the damaged tissue to restore its original structure and function. Although a large number of studies have currently focused on the regulation of efferocytosis on the inflammatory response, revealing its crucial role in inhibiting excessive activation of inflammation and promoting the resolution of inflammation. For example, by recognizing the "eat-me" signals on the surface of apoptotic cells, such as the externalization of phosphatidylserine, phagocytes like macrophages initiate the process of efferocytosis, reducing the release of inflammatory mediators and promoting the secretion of antiinflammatory factors, thus effectively controlling the inflammatory process [205,206]. However, research on the influence of efferocytosis on other stages of tissue repair is still insufficient and urgently requires in-depth exploration.

During the hemostasis stage, the activation and aggregation of platelets are key events. Platelets rapidly adhere to the damaged vascular endothelium, forming platelet thrombi to prevent bleeding [207]. However, there is limited research on the direct role of efferocytosis in this stage. Theoretically, the timely clearance of apoptotic cells may indirectly affect the hemostatic microenvironment [208]. If apoptotic cells are not cleared in a timely manner, they may release their intracellular contents, interfering with the normal function of platelets and the coagulation cascade reaction [208]. Existing studies have shown that under certain pathological conditions, such as when the inflammatory response is out of control after severe trauma, the accumulation of a large number of apoptotic cells may lead to coagulation dysfunction [207]. However, the specific participation mechanism of efferocytosis, such as whether the clearance of apoptotic cells by phagocytes can stabilize the hemostatic environment and regulate the activity of coagulation factors, still needs further investigation.

The proliferation stage is a critical period for tissue repair, involving the proliferation and migration of various cells, such as fibroblasts and endothelial cells, to fill the damaged tissue area and construct a new extracellular matrix. Some studies have preliminarily revealed an association between efferocytosis and macrophage proliferation (209, 210]. For example, an experiment in which mouse bone marrow-derived macrophages were co-incubated with apoptotic cells found that macrophages underwent proliferation after efferocytosis [209]. This finding suggests that efferocytosis may indirectly regulate the proliferation stage of tissue repair by influencing the functional state of macrophages. After macrophages phagocytose apoptotic cells, they may release specific cytokines or growth factors, such as transforming growth factor-f (ТСЕ-В), which can promote the proliferation and migration of fibroblasts and endothelial cells [75]. However, the molecular mechanism by which efferocytosis precisely regulates macrophages to release these pro-proliferative factors, and the specific impact of these factors on the proliferation and migration of different cell types, still require extensive in-depth research for clarification.

During the remodeling stage of tissue repair, the newly formed tissue gradually optimizes its structure and function to restore the original characteristics of the tissue. The potential role of efferocytosis in this stage also deserves attention [211]. On the one hand, the continuous presence of apoptotic cells may interfere with the correct assembly of the extracellular matrix and the orderly reconstruction of tissue structure during the remodeling process. On the other hand, the cytokines and bioactive molecules secreted by phagocytes after efferocytosis may participate in regulating the remodeling of the extracellular matrix, such as regulating the balance between collagen synthesis and degradation [212]. For example, during the remodeling stage of skin wound repair, matrix metalloproteinases and their tissue inhibitors secreted by macrophages can affect the arrangement and maturation of collagen fibers. However, it is still unclear how efferocytosis affects macrophages to secrete these key molecules involved in remodeling, and whether abnormal efferocytosis can lead to abnormal tissue remodeling and adverse consequences such as fibrosis [213].

In depth research on the impact of efferocytosis on each stage of tissue repair not only helps to comprehensively understand the molecular mechanism of tissue repair but also provides a more comprehensive and in-depth theoretical basis for the development of tissue repair biomaterials and treatment strategies based on efferocytosis regulation, thus promoting the development of the field of damaged tissue repair treatment.

(3) Analyze the cytoskeleton and cell-cell interactions

Conduct in-depth research on the direct contact mechanism between phagocytes and apoptotic cells, including the dynamic reorganization process of the cytoskeleton (such as actin and microtubules) during efferocytosis. Taking macrophages as an example, after recognizing apoptotic cells, members of the Rho small G-protein family, such as Rac1 and Cdc42, are activated. They can regulate the polymerization and depolymerization of actin filaments. In the activated state, Racl can induce rapid polymerization of actin at the cell front to form lamellipodia, while Cdc42 promotes the formation of filopodia in preparation for phagocytosis [214,215]. To observe this process more clearly, ultra-high-resolution microscopes, such as stimulated emission depletion (STED) microscopes, can be used. The resolution of STED microscopes can break through the diffraction limit of optical microscopes, enabling clear observation of the reorganization path of actin filaments during the extension and fusion of microvilli [216]. At the same time, cryo-electron tomography can be used to image cells in a near-physiological state and analyze the ultrastructural changes of membrane fusion [217]. In addition, analyze the influence of extracellular matrix components (such as fibronectin, laminin, and collagen) on the migration and phagocytosis efficiency of phagocytes in different tissue microenvironments [218]. Through cell migration experiments, such as the transwell assay, phagocytes are seeded in chambers coated with different extracellular matrix components to observe their migration ability. Combined with phagocytosis experiments, the phagocytosis efficiency of phagocytes towards apoptotic cells is detected. Proteomic analysis is used to clarify the downstream signaling pathways activated by the interaction between extracellular matrix components and phagocyte surface receptors (such as integrins), as well as the regulatory mechanisms of these pathways on cytoskeleton rearrangement and cell migration.

(4) Receptor-ligand recognition

Utilize high-throughput technologies, including proteomics and glycomics, to systematically identify differentially expressed proteins on the surface of phagocytes and alterations in glycan structures on the surface of apoptotic cells during efferocytosis. This approach aims to uncover potential novel receptors and ligands involved in this process. In proteomic research, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based technology can be used to comprehensively identify and quantitatively analyze the proteins of phagocytes before and after efferocytosis, screen out differentially expressed proteins, and predict whether they are potential apoptotic cell recognition receptors through bioinformatics analysis. In terms of glycomics, lectin microarray technology can be used to analyze the changes in the sugar chain structure on the surface of apoptotic cells in a high-throughput manner, so as to find sugar chain molecules that may serve as ligands. Use techniques such as X-ray crystallography and cryo-electron microscopy to analyze the three-dimensional structure of receptors and ligands, and clarify the key domains and precise recognition patterns of their binding. For example, the crystal structure of the receptor-ligand complex can be obtained by X-ray crystallography to analyze the interaction mode at the atomic level. Cryo-electron microscopy can be used to analyze the structure of larger receptor-ligand complexes in a nearnatural state. Study the binding characteristics between receptors and ligands, such as binding affinity and binding kinetic parameters (such as binding and dissociation rates), and accurately measure them through biophysical methods such as surface plasmon resonance (SPR) technology. Explore the change rules of these characteristics in different physiological and pathological microenvironments. For example, in an inflammatory microenvironment, inflammatory factors may change the structure of receptors or ligands, thereby affecting their binding affinity, and this change can be monitored in real-time by SPR technology.

(5) Inflammatory and immune regulatory mechanisms

Explore the regulatory effects of various inflammatory factors (such as TNF-a, IL-10, IFN-y, etc.) in the inflammatory microenvironment on phagocyte polarization, surface receptor expression, and signaling pathway activity, and their subsequent impact on apoptotic cell recognition, phagocytosis, and processing [219]. For example, TNF-a inhibits MerTK receptor expression on phagocytes by activating the NF-xB signaling pathway, thereby impairing apoptotic cell recognition [220]. In contrast, IL-10 promotes macrophage polarization toward the M2 anti-inflammatory phenotype, which enhances efferocytosis [220]. Investigate the role of immune cells (e.g., T cells and B cells) in efferocytosis and their interactions with phagocytes. Using co-culture systems-such as macrophages and T cells in the presence of apoptotic cells-analyze shifts in cell populations and surface marker expression via flow cytometry. Integrate single-cell sequencing to delineate how immune cell-derived cytokines (e.g., interferons, interleukins) and chemokines modulate efferocytosis. Focus on dysregulated inflammatory and immune mechanisms in disease contexts and strategies to restore efferocytosis for tissue repair. For instance, in chronic skin wounds, impaired efferocytosis due to inflammatory-immune imbalance can be mitigated by modulating inflammatory factor levels or immune cell activity, thereby slowing disease progression.

(6) Microenvironmental regulatory factors

Explore the influence of the microenvironment (inflammatory factors, oxidative stress, extracellular matrix components, etc.) on the function of phagocytes during efferocytosis [221]. For example, in an oxidative stress state, reactive oxygen species (ROS) produced in cells, such as hydrogen peroxide (H>02) and superoxide anions (03), affect the dynamic reorganization of the phagocyte cytoskeleton, receptor expression, and signaling pathways [222]. An appropriate amount of ROS can act as a signaling molecule to activate Rho GTPase and promote the reorganization of the actin cytoskeleton, which is beneficial for phagocyte migration and the formation of phagocytic cups. However, excessive ROS can oxidatively modify phagocyte surface receptors and membrane proteins, affecting their ability to recognize and bind to apoptotic cells. Analyze the changes in extracellular matrix components in different tissue injuries or diseases and their influence on the migration and phagocytosis efficiency of phagocytes. Through in vitro cell culture combined with tissue-engineered simulated microenvironment models, such as constructing extracellular matrix gels with different hardness and components, and seeding phagocytes and apoptotic cells, study how to optimize the efferocytosis efficiency by regulating the microenvironmental components (such as changing the ratio of fibronectin to laminin) [223].

(7) Inter-cellular communication research

Study the communication methods between phagocytes and apoptotic cells, other immune cells, and surrounding tissue cells, including direct contact-dependent communication and non-contact communication mediated by secreted cytokines and exosomes [224]. In direct contact communication, cell-surface adhesion molecules, such as integrins and cadherins, play an important role in the interaction between phagocytes and apoptotic cells or other cells. Through gene knockout or antibody blocking experiments, study the influence of these adhesion molecules on inter-cellular communication and the efferocytosis process. In non-contact communication, exosomes secreted by phagocytes and other cells contain bioactive molecules such as proteins, nucleic acids, and lipids. Exosomes can be isolated by methods such as ultra-centrifugation and density-gradient centrifugation. Use proteomics, transcriptomics, and other technologies to analyze their components and study the regulatory effects of exosomes on efferocytosis and tissue repair. Fluorescence resonance energy transfer (FRET) technology can be used to monitor inter-cellular molecular interactions in real-time. Combined with single-cell RNA sequencing and spatial transcriptomics technologies, analyze the spatio-temporal dynamic changes of inter-cellular communication during efferocytosis and clarify the mechanism of action of different communication methods in regulating efferocytosis and tissue repair. For example, FRET technology can be used to label molecules involved in inter-cellular interactions and observe the dynamic changes of molecular interactions at different stages of efferocytosis. Spatial transcriptomics technology can determine the spatial distribution of gene expression of different cell types in the tissue microenvironment and reveal the spatial characteristics of inter-cellular communication.

(8) Spatio-temporal dynamic monitoring technology

Develop and apply advanced spatio-temporal dynamic monitoring technologies, such as in vivo imaging technology and nanosensor technology, to observe in real-time the cell behavior, molecular changes, and microenvironmental dynamics during efferocytosis [225]. In terms of in vivo imaging technology, use fluorescently labeled apoptotic cells, phagocytes, and related molecules. For example, label phagocytes with green fluorescent protein (GFP) and apoptotic cells with red fluorescent dyes. Combined with imaging equipment such as multiphoton microscopes, which can image deep tissues without damaging them, monitor the entire process of efferocytosis in in vivo and in vitro models, including the recognition, migration, phagocytosis, and subsequent processing of apoptotic cells by phagocytes, providing intuitive data support for in-depth understanding of the efferocytosis mechanism. Nanosensor technology can be used to monitor molecular changes in the microenvironment. For example, design nanosensors to specifically detect molecules such as inflammatory factors and reactive oxygen species, implant them in vivo or add them to in vitro culture systems, and monitor in real-time the relationship between microenvironmental dynamic changes and the efferocytosis process.

5.2. Develop new materials to target efferocytosis and promote tissue repair

(1) Design of biomaterials mimicking the ECM

1) Component of imitation

The ECM encompasses diverse components including fibronectin, laminin, and collagen. These components not only furnish physical support to cells but also play pivotal roles in intercellular signal transduction. The development of biomaterials emulating ECM components represents a crucial strategy for targeting efferocytosis. For instance, peptides bearing the arginine-glycine-aspartic acid (RGD) sequence are synthesized. This RGD sequence serves as a key binding motif for ECM proteins like fibronectin and integrin receptors on the cell surface. Modifying the surface of biomaterials with such peptides can augment the adhesion of phagocytes (e.g., macrophages) to the materials and facilitate their migration on the material surface [226,227]. Research has demonstrated that on RGD-modified material surfaces, the integrin-mediated FAK (focal adhesion kinase) signaling pathway in macrophages is activated [228]. This activation, in turn, promotes cytoskeletal rearrangement, thereby enhancing the macrophages' phagocytic capacity towards apoptotic cells. Laminin is essential for maintaining cell polarity, promoting cell survival, and regulating cell differentiation. Laminin can be procured through biosynthesis or extraction techniques and then incorporated with other biomaterials to fabricate scaffolds that more closely resemble the physiological microenvironment. For example, a nanofiber scaffold is fabricated by compounding laminin with poly (lactic-co-glycolic acid) (PLGA). Experimental evidence indicates that this scaffold can modulate the polarization state of macrophages, steering them towards M2-type polarization, which is associated with anti-inflammatory and pro-efferocytosis functions, and thus enhancing the clearance of apoptotic cells [219,229].

2) Structure imitation

The ECM exhibits a complex three-dimensional architecture, comprising fibrous and pore structures, both of which significantly influence cell behavior. By mimicking the fibrous structure of the ECM and employing electrospinning technology to fabricate nanofiber scaffolds, the morphology and function of phagocytes can be effectively regulated. Parameters such as the diameter, orientation, and porosity of nanofibers can impact macrophage spreading and migration. For example, when the nanofiber diameter approximates that of natural ECM fibers, macrophages can adhere and migrate more effectively, and the expression of genes associated with efferocytosis within the cells is upregulated [230]. Constructing biomaterials with a hierarchical pore structure is also of great significance. Large pores facilitate cell infiltration and tissue vascularization, while small pores can mimic the microscopic structure of the ECM, creating a favorable microenvironment for phagocytes. For example, a hydrogel scaffold with hierarchical pores can be fabricated via 3D printing technology. Both in vitro and in vivo experiments have shown that such a scaffold can promote the phagocytosis of apoptotic cells by macrophages and simultaneously facilitate neovascularization, thereby accelerating tissue repair [231].

(2) Development of responsive biomaterials

1) Responsiveness to Inflammatory Factors

The levels of inflammatory factors experience substantial alterations at the site of tissue injury. The development of biomaterials responsive to inflammatory factors enables the precise regulation of efferocytosis. For example, a hydrogelbased biomaterial can be designed to contain chemical bonds that are sensitive to TNF-a. When the local TNF-a concentration increases, the hydrogel undergoes degradation, releasing pre-loaded anti-inflammatory drugs (e.g., dexamethasone) and bioactive molecules that promote efferocytosis (e.g., Gas6). Dexamethasone can suppress the inflammatory response, alleviating the inhibitory effect of inflammation on phagocyte function. Meanwhile, Gas6 can enhance efferocytosis by binding to the MerTK receptor on the surface of phagocytes [232]. Intelligent polymer materials, such as poly(N-isopropylacrylamide) (PNIPAAm), which possess temperature and pH responsiveness, can also be utilized. PNIPAAm can be combined with bioactive molecules to formulate nanoparticles. In an inflammatory microenvironment, as the local pH decreases, the nanoparticles undergo structural transformations, releasing loaded cytokines that promote efferocytosis (e.g., IL-10). This release can regulate phagocyte function, thereby promoting efferocytosis and tissue repair.

Oxidative stress is a common phenomenon during tissue injury and can severely affect phagocyte function. Developing biomaterials responsive to oxidative stress can ameliorate the microenvironment for efferocytosis. For example, a biomaterial incorporating antioxidant moieties (e.g., thiol groups) can be synthesized. When exposed to an oxidative stress environment, the thiol groups are oxidized, triggering structural changes in the biomaterial and releasing antioxidants (e.g., vitamin C). Vitamin C can reduce the local level of reactive oxygen species (ROS), mitigating the damage caused by oxidative stress to phagocytes and restoring their phagocytic ability towards apoptotic cells [233]. Biomaterials based on metal-organic frameworks (MOFs) can also be designed. MOFs feature a porous structure and can encapsulate antioxidant enzymes (e.g., superoxide dismutase, SOD). Under oxidative stress conditions, MOF materials can respond to changes in ROS concentration, releasing SOD. SOD then catalyzes the conversion of superoxide anions into oxygen and hydrogen peroxide, reducing the damage of ROS to phagocytes and surrounding tissues. Concurrently, this process promotes efferocytosis, which is beneficial for tissue repair.

2) Responsiveness to Oxidative Stress

(3) Construction of Multifunctional Biomaterials

1) Loading of Bioactive Molecules

Constructing biomaterials capable of loading multiple bioactive molecules is an effective approach to synergistically promote efferocytosis and tissue repair. For example, a PLGAbased microsphere can be prepared to co-load ТСЕ-В and vascular endothelial growth factor (VEGF). ТСЕ-В can regulate macrophage function, promoting their phagocytosis of apoptotic cells. Additionally, it can stimulate fibroblast proliferation and extracellular matrix synthesis. VEGF, on the other hand, can promote angiogenesis, ensuring an adequate supply of nutrients and oxygen for tissue repair. Compounding this microsphere with a nanofiber scaffold has been shown in in vivo experiments to significantly accelerate skin wound healing. In addition, liposomes can also be used as carriers to co-package nucleic acid drugs (such as siRNA, which can silence genes that inhibit exocytosis) and protein drugs (such as receptor agonists that promote exocytosis). The surface of liposomes can be modified with targeting moieties to enable specific binding to phagocytes. In vivo, liposomes deliver the loaded bioactive molecules into phagocytes, regulating the expression of relevant genes and proteins within the cells, enhancing efferocytosis, and promoting tissue repair.

2) Surface Functional Modification

Performing surface functional modification on biomaterials endows them with multiple functions. For example, quaternary ammonium salt groups with antibacterial properties can be grafted onto the surface of biomaterials, while simultaneously modifying peptides that promote cell adhesion. The antibacterial groups can inhibit infection, reducing the interference of the inflammatory response with efferocytosis. The cell-adhesion-promoting peptides can enhance the binding between phagocytes and the materials, thus promoting efferocytosis. Previous studies have shown that such surfacefunctionalized biomaterials can effectively promote the clearance of apoptotic cells and pathogens by phagocytic cells during the repair process of infected wounds and accelerate wound healing [234]. Biomaterials can also be surface-modified with photo-responsive molecules, such as azobenzene. Under light illumination, the structure of azobenzene undergoes changes, thereby regulating the surface hydrophilicity/hydrophobicity of biomaterials and cell adhesion. By precisely controlling the illumination time and intensity, the behavior of phagocytes on the material surface can be accurately regulated to promote efferocytosis. This light-responsive surface modification could provide a new means for precise regulation of efferocytosis.

5.3. Key transformation challenges of biomaterials targeting phagocytes

(1) Targeting specificity and in vivo stability

In preclinical research, achieving precise targeting of phagocytes by biomaterials is an important prerequisite for regulating efferocytosis. However, when it comes to clinical translation, this targeting specificity faces numerous challenges. Currently, common targeting strategies, such as modifying the surface of biomaterials with antibodies, aptamers, or specific ligands to recognize phagocyte surface markers, are easily interfered with by various factors in the complex physiological environment of the body. Plasma proteins in the blood circulation will rapidly adsorb onto the surface of biomaterials, forming a "protein corona". This not only may change the physical and chemical properties of the biomaterials but also masks the targeting ligands, hindering their effective binding to phagocytes [235,236]. For example, in the research of nanoparticles targeting macrophages, the formation of the protein corona changes the surface properties of the nanoparticles, resulting in a significant decrease in their in vivo distribution and targeting efficiency. To overcome this challenge, in the future, it is necessary to conduct in-depth research on the formation mechanism of the protein corona, develop material surface modification technologies to prevent the formation of the protein corona, or design novel targeting ligands that can still maintain targeting activity after the formation of the protein corona, so as to ensure that the biomaterials can stably and specifically target phagocytes in the body.

(2) Biological safety and immunogenicity

The biological safety of biomaterials in the body is a core issue in clinical translation. As an important part of the immune system, phagocytes may trigger a series of immune responses after coming into contact with biomaterials. Some biomaterials, especially nanomaterials, their physical and chemical properties such as size, shape, and surface charge may lead to abnormal activation of phagocytes, and then trigger inflammatory responses or even cytotoxicity [237]. To ensure biological safety, at the stage of biomaterial design, it is necessary to optimize the material from multiple aspects such as composition, structure, and surface properties. Use biodegradable and biocompatible materials and modify their surfaces to reduce immunogenicity. At the same time, in preclinical research, comprehensive and standardized safety assessment methods should be applied, including in vitro cytotoxicity testing, long-term toxicity and immunogenicity studies in in vivo animal models, etc., to provide sufficient data support for clinical applications.

(3) Scalable production and quality control

From laboratory research to clinical application, the scalable production and quality control of biomaterials are key links in achieving translation. Many biomaterials used for targeting phagocytes have complex preparation processes, involving multiple precise steps, which brings great difficulties to scalable production. Take the preparation of biomaterial scaffolds with specific microstructures by 3D printing as an example. Although this technology can accurately construct structures simulating the extracellular matrix in the laboratory to promote the function of phagocytes, when scaling up production, it faces problems such as low printing efficiency, high cost, and difficulty in ensuring product quality consistency [238]. To solve these problems, it is necessary to develop efficient, low-cost, and reproducible scalable preparation processes, and introduce advanced automated production equipment and process control technologies. At the same time, establish strict quality control standards and detection methods, and conduct real-time monitoring and strict control of key indicators such as the physical and chemical properties, targeting performance, and biological safety of biomaterials to ensure that each batch of products can meet the high-quality requirements of clinical applications.

5.4. Progress in clinical application of efferocytosis

(1) Clinical Trials Targeting PS Receptors

PS receptors play a vital role in efferocytosis, being involved in the recognition and phagocytosis of apoptotic cells. Regulatory strategies targeting PS receptors have been widely explored in clinical trials for various diseases.

Cancer Treatment: Multiple drugs targeting PS receptors have been investigated in clinical trials for cancer treatment. For example, the Axl inhibitor bemcentinib (BGB324, R428) has been studied in several clinical trials conducted by BerGenBio ASA for various cancers, including recurrent glioblastoma, advanced solid tumors, metastatic breast cancer, acute myeloid leukemia (AML), non-small cell lung cancer, and malignant mesothelioma. The sample size varies according to different cancer types and trial designs. For instance, in a trial for nonsmall cell lung cancer (NCT02488408), the sample size was 9 patients who received 500, 1000, or 2000 mg/day of bemcentinib for 1 week. Efficacy evaluation indicators mainly focused on tumor progression, patient survival, as well as the safety and tolerability of the drug. In the treatment of COVID-19, there is also a clinical trial targeting the Axl receptor (NCT04890509) to explore its therapeutic effect [239].

MerTK Inhibitors: MerTK inhibitors are also being evaluated in clinical trials. For example, ONO-7475, developed by Ono Pharmaceuticals, is used in the treatment of acute leukemia and myelodysplastic syndromes (NCT03176277). MRX-2843, jointly developed by Meryx, Inc., Emory University, and Betta Pharmaceuticals Co., Ltd, is being tested for solid tumors, non-small cell lung cancer, and refractory AML (NCT04946890, NCT03510104, NCT04762199, NCT04872478). These trials assess efficacy by observing disease remission and changes in hematological indicators, providing new directions for cancer treatment.

(2) Clinical Trials of Metabolic Regulation

Metabolism plays an important role in efferocytosis, influencing the ability of phagocytes to process apoptotic cells. Some clinical trials aim to improve efferocytosis by regulating metabolic-related pathways and thereby treat related diseases.

PPARy Agonist: Pioglitazone, a PPARy agonist originally used for the treatment of type 2 diabetes, has been found to enhance efferocytosis (by increasing the expression of PS receptors) and induce antiinflammatory gene expression. In relevant clinical trials, although the sample size is not specifically mentioned, its promoting effect on efferocytosis has been verified in studies on monocytes from patients with human chronic granulomatous disease [240].

Diabetes and Obesity: In research related to diabetes and obesity, miR-126 has been found to be associated with efferocytosis. In diabetic mice, the level of miR-126 is suppressed. There is a research plan to deliver miR-126 to sites with a high load of apoptotic cells, such as diabetic wounds, via nanoparticles or REDV peptide-modified chitosan (no specific clinical trial number is mentioned), which provides a potential strategy to improve the defect of efferocytosis related to diabetes. In another clinical trial for metabolic syndrome (NCT01181830), an intervention containing specific metabolic components was used, and the effects on metabolic parameters and inflammatory indicators were observed to evaluate the indirect impact on efferocytosis [241].

(3) Clinical Trials of Natural Compounds for Regulating Efferocytosis

Natural compounds show potential in regulating efferocytosis due to their diverse biological activities, and some have entered the clinical trial stage.

Quercetin: Multiple clinical trials have investigated the application of quercetin in various diseases. In a trial for patients with COPD (NCT01708278), the sample size was 9, and patients received 500, 1000, or 2000 mg/day of quercetin for 1 week. The efficacy evaluation indicators included blood tests to detect drug safety, as well as lung function and the COPD assessment questionnaire to evaluate the treatment effect. The results showed that quercetin was well-tolerated at a dose of up to 2000 mg/day [242]. In the treatment of COVID-19, there is also a clinical trial of quercetin (NCT04377789) with a sample size of 447. Patients were treated with 1000 mg of quercetin for 2-30 days. The efficacy evaluation indicators included CRP, ferritin levels, platelet, and lymphocyte counts. The results showed that this treatment could lead to a decrease in CRP and ferritin levels and an increase in platelet and lymphocyte counts [243]. In a clinical trial for cystic fibrosis (CF) (NCT01348204), quercetin was used to activate CFTR, and its effect on CFTR function was evaluated through the Nasal Potential Difference (NPD) test.

Curcumin: Curcumin has been used in multiple clinical trials for the treatment of diseases such as Crohn's disease (NCT02255370) and ulcerative colitis (NCT00815763). Efficacy was evaluated by observing disease recurrence, clinical activity index (CAI), endoscopic index (EI), and other indicators. These clinical trials provide practical evidence for the use of natural compounds to regulate efferocytosis in the treatment of diseases.

It is necessary to closely monitor the progress of these clinical trials, as it has great reference significance for the future development of biological materials that can target efferocytosis to promote tissue repair and for the clinical application of these materials.

In conclusion, the maintenance of efferocytosis is essential for tissue repair after injury, and its dysfunction may lead to a series of pathological processes and affect individual health. In-depth study of the mechanisms of efferocytosis and the development of strategies to enhance efferocytosis are of great clinical significance for promoting tissue repair. With further elucidation of the molecular mechanisms of efferocytosis, it is expected that more tissue engineering materials will be developed to effectively regulate efferocytosis and promote the repair and functional recovery of damaged tissues. This will not only promote progress in the field of regenerative medicine but also provide patients with more effective and personalized treatment options.

6. Conclusion

This review provides an in-depth exploration of the pivotal role of efferocytosis in tissue repair and regeneration processes and systematically analyzes how tissue engineering strategies can modulate efferocytosis to optimize these biological processes. By reviewing the fundamental concepts, molecular mechanisms, and physiological functions of efferocytosis in tissue repair, we further elucidate its crucial role in modulating inflammatory responses during the tissue repair cascade.

Recent studies have demonstrated that efferocytosis is a key biological process for maintaining tissue homeostasis and orchestrating injury repair mechanisms. Against this backdrop, tissue engineering has emerged as a transformative approach, offering new therapeutic targets for promoting tissue repair by precisely controlling the enhancement of efferocytosis, the resolution of inflammation, and the process of tissue regeneration. Despite the widely recognized importance of efferocytosis in tissue repair, its specific mechanisms under different tissue and pathological conditions still require further investigation. There is also an urgent need for progress in clinical applications.

In this review, we propose several original perspectives:

First, following a comprehensive summary of recently reported mechanisms of efferocytosis, we further elaborate on both established and potential immunoregulatory effects of this process. Our comparative analysis of efferocytosis and the inflammatory response offers a conceptual framework that may encourage further exploration of the interplay between efferocytosis, inflammation, and tissue regeneration.

Second, we highlight efferocytosis not only as a central regulator of tissue homeostasis and the resolution of inflammation but also as an emerging therapeutic target with significant translational potential. This dual focus provides a forward-looking perspective that extends beyond existing reviews, bridging mechanistic insights with engineering-driven interventions and addressing a critical gap in the current literature.

Finally, we propose a novel framework for categorizing engineering strategies-such as bioactive factor delivery, scaffold design, and cellular interface modulation-based on their capacity to regulate efferocytosis. This approach moves beyond descriptive summaries to present a conceptual roadmap for guiding future research and advancing clinical applications.

In summary, this review offers researchers a comprehensive overview to understand the critical role of efferocytosis in tissue repair and the potential of tissue engineering in modulating efferocytosis. We hope that this review will inspire more scholars to focus on this field and collectively advance the development of tissue repair and regenerative medicine.

CRediT authorship contribution statement

Yue-Qi Zhang: Writing - original draft. Rong Nie: Investigation. ZiYuan Feng: Project administration. Ming-Hui Fan: Investigation. ZhiXue Shen: Writing - review & editing. Xiu-Zhen Zhang: Software. Qing-Yi Zhang: Funding acquisition. Chen-Yu Zou: Supervision. Ji-Ye Zhang: Investigation. Kai Huang: Supervision. Li-Ping Mou: Writing - review & editing. Hui-Qi Xie: Supervision.

Ethics approval and consent to participate

There are no human and animal subjects in this review and informed consent is not applicable.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledegement

This study has received collaborative support from the Sichuan Science and Technology Program (2024NSFSC0002), the National Natural Science Foundation of China (Grant No. 824B2073), the Frontiers Medical Center, Tianfu Jincheng Laboratory Foundation (TFJC2023010002) and the "1.3.5" Project for Disciplines of Excellence, West China Hospital, Sichuan University (ZYGD23037). Additionally, some graphical elements in the figures were created using BioRender.

Sidebar

Peer review under the responsibility of editorial board of Bioactive Materials.

Received 24 February 2025; Received in revised form 22 May 2025; Accepted 23 May 2025

Available online 9 June 2025

References

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Footnote

* Corresponding author. Department of Orthopedic Surgery and Orthopedic Research Institute, Stem Cell and Tissue Engineering Research Center, Frontiers Science Center for Disease-Related Molecular Network, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, JiTaiAn Center, 17 Gaopeng Avenue, Hi-Tech District, Chengdu, Sichuan 610041, China.

E-mail address: [email protected] (H.-Q. Xie).

1 These authors contributed equally to this work.