Recent progress in immunomodulation-based strategies for bone repair

Abstract

Bone regeneration is a highly coordinated process shaped by the interplay between immune responses and osteogenic mechanisms. Immune cells such as neutrophils, macrophages, T cells, and B cells dynamically regulate the local microenvironment through cytokine secretion and signaling pathways, thereby influencing osteogenesis, angiogenesis and bone remodeling, while dysregulated or prolonged inflammation can disrupt healing. Growing evidence has highlighted the potential of leveraging immunomodulation to enhance bone repair. This review synthesizes recent progress in immunoregulatory strategies by comparing cellular therapies, molecular interventions and biomaterial-based approaches in terms of their mechanisms, their effects on osteogenesis and angiogenesis, and their translational potential. Particular emphasis is placed on immune cell specific signaling pathways, biomaterial design parameters including surface topography, porosity, ion release and stiffness, and emerging technologies such as immune responsive hydrogels, programmable scaffolds and exosome based delivery systems. Current findings indicate that mesenchymal stem cells and regulatory T cells not only provide progenitor sources but also reshape the immune milieu through paracrine factors and exosomes; cytokines, small molecules, microRNAs and pro resolving mediators effectively modulate inflammatory cascades to promote vascularized bone formation; and immunomodulatory biomaterials enable spatiotemporal regulation of macrophage polarization, particularly the transition from the pro inflammatory M1 phenotype to the reparative M2 phenotype. Collectively, these advances highlight that bone repair is fundamentally an immunologically driven process, and integrating temporal immune regulation with emerging therapeutic platforms offers a promising pathway toward precise and personalized bone regeneration.

Graphical abstract

1. Introduction

Bone tissue, as one of the most critical structural systems of the human body, not only maintains body shape, protects internal organs, and enables locomotion, but also serves as a central site for mineral metabolism and hematopoiesis [1,2]. Under physiological conditions, bone possesses a remarkable capacity for self-repair and regeneration. Following fractures or small-scale defects, natural healing can occur through the dynamic balance between osteoblasts and osteoclasts, regulated by the local microenvironment [3]. However, in cases of trauma, infection, tumor resection, or nonunion, large or complex bone defects often arise. Such defects exceed the intrinsic repair threshold of bone tissue, and without intervention, they can lead to severe functional impairment and a marked decline in quality of life [4,5]. Consequently, promoting effective repair and regeneration of large-scale bone defects has remained a key focus in both orthopedics and tissue engineering.

Clinically, bone grafting is the most widely used and established method for treating bone defects. Among these, autologous bone grafts are considered the gold standard due to their inherent osteogenic, angiogenic, and chondrogenic potential [6]. Nevertheless, autologous transplantation is limited by donor site morbidity, restricted tissue availability, and additional surgical trauma [7]. Allogeneic bone grafts, while alleviating donor shortages to some extent, are associated with risks of immune rejection, disease transmission, and poor integration [8]. Artificial bone substitutes, such as hydroxyapatite, bioactive glass, and polymer-based materials, can partially compensate for these shortcomings. Yet, challenges remain in achieving ideal bioactivity, degradation kinetics, and mechanical compatibility [9]. Therefore, traditional approaches alone often fail to deliver satisfactory outcomes in the repair of complex bone defects. In recent years, with the emergence of the concept of osteoimmunology, researchers have increasingly recognized the indispensable role of the immune system in bone repair and regeneration [10,11]. Following bone injury, a rapid local inflammatory response is initiated, in which macrophages act as key innate immune cells. Classically activated M1 macrophages secrete proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), contributing to the clearance of necrotic tissue and pathogens in the early stage, while simultaneously activating signaling pathways related to bone repair [12,13]. In contrast, alternatively activated M2 macrophages release anti-inflammatory mediators such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), which promote angiogenesis and osteogenic differentiation, thereby facilitating tissue regeneration during later stages of healing [14]. Beyond macrophages, other immune cells including T cells, B cells, and neutrophils also influence the balance between osteogenesis and osteoclastogenesis through complex cytokine networks and signaling pathways [15,16]. As a result, strategies that modulate immune cell polarization and function to create a pro-regenerative microenvironment are increasingly becoming a major research direction in bone regeneration.

Against this backdrop, immunomodulation-based strategies for bone repair have rapidly gained attention. First, stem cell therapy, owing to its immunoregulatory properties and osteogenic differentiation potential, has emerged as a research hotspot. Mesenchymal stem cells (MSCs), for example, not only differentiate into osteoblasts but also modulate immune cell functions via paracrine mechanisms, thereby facilitating the resolution of inflammation and promoting bone formation [[17], [18], [19], [20], [21]]. Second, growth factors such as bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) can regulate inflammatory responses, enhance angiogenesis, and stimulate osteogenesis. Their combined application with biomaterials has been widely investigated [[22], [23], [24]]. Third, decellularized extracellular matrix (dECM), which retains natural extracellular matrix components and bioactivity, effectively modulates immune cell behavior and reshapes the bone repair microenvironment [[25], [26], [27], [28]]. In addition, various novel biomaterials—including metallic scaffolds, biodegradable polymers, magnesium-releasing materials, and polycaprolactone (PCL)-based composites—have been engineered with surface modifications or compositional optimization to precisely regulate the immune microenvironment, thereby enhancing bone regeneration [29,30].

In summary, bone repair and regeneration rely not only on mechanical support and osteogenic mechanisms but also on the dynamic regulation of the immune system. A deeper understanding of the distinct roles of immune cells during bone healing, combined with the integration of diverse immunomodulatory strategies (including stem cells, growth factors, dECM, and functionalized biomaterials), holds promise for achieving more precise and efficient bone tissue regeneration. This review systematically summarizes the roles of various immune cells in bone repair and highlights recent advances in immunomodulation-based strategies for bone regeneration, with the aim of providing a theoretical foundation and translational reference for the development of effective immunoregulatory approaches in bone repair.

1.1. Mechanisms by which immune cells participate in bone repair

Bone regeneration after a defect is not accomplished solely by rebuilding bone mass through osteoblasts and osteoclasts; rather, it unfolds within a microenvironment profoundly shaped by immune responses. Injury immediately triggers coagulation and inflammation. Mediators released from platelets and the complement cascade rapidly recruit neutrophils and monocytes/macrophages to the defect site, followed by the stepwise engagement of adaptive immune cells. Through soluble mediators and cell–cell contact, these immune cells regulate the receptor activator of nuclear factor κB ligand (RANKL)–receptor activator of nuclear factor κB (RANK)–osteoprotegerin (OPG) axis, transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) and Wnt/β-catenin signaling, thereby setting the pace of osteoclast–osteoblast coupling, the quality of neovascularization, and the rate of matrix maturation [23,31,32]. Defining the temporal functions of distinct immune populations and their effects on osteoblasts and osteoclasts provides the foundation for immunoregulatory strategies in bone regeneration.

1.1.1. Neutrophils

Neutrophils are the earliest effector population to arrive at the fracture microenvironment. Driven by chemokines such as chemokine (C-X-C motif) ligand 1/2 (CXCL1/2), CXCL8 [interleukin-8 (IL-8)], and complement component 5a (C5a), they accumulate within hours of injury and clear necrotic tissue and potential pathogens via phagocytosis, degranulation, and the generation of reactive oxygen species (ROS) [33,34]. Although predominantly pro-inflammatory, this early response is prerequisite for subsequent regeneration. On one hand, neutrophil-derived matrix metalloproteinase-9 (MMP-9) and elastase degrade damaged matrix and liberate matrix-bound vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β), thereby promoting local angiogenesis and paving the way for MSC recruitment [35]. On the other hand, short-lived increases in interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) can transiently enhance chemotactic and proliferative signals related to osteogenesis; however, sustained elevation prolongs nuclear factor κB/mitogen-activated protein kinase (NF-κB/MAPK) activation, suppresses Runx2 and Osterix (Sp7), reduces alkaline phosphatase (ALP) activity, and impairs mineralization [36,37]. In addition, neutrophils can produce modest amounts of RANKL or amplify RANKL signals originating from stromal cells and T cells, thereby fostering pre-osteoclast fusion and potentially exacerbating bone resorption when inflammation persists [[38], [39], [40]]. Recent work on neutrophil extracellular traps (NETs) further shows that while transient NET formation aids pathogen control, their persistent accumulation and heightened protease activity can damage microvasculature and matrix, delaying both cartilaginous and bony callus formation [[41], [42], [43]]. Collectively, neutrophil effects on osteogenesis and osteoclastogenesis are highly time-dependent: rapid arrival followed by timely resolution is desirable, whereas overactivation or retention correlates with a higher risk of nonunion.

1.1.2. Macrophages

Macrophages play central roles in skeletal homeostasis and repair, with ontogeny and differentiation routes underpinning their functional diversity. Broadly, macrophages comprise monocyte-derived macrophages arising from circulating monocytes and tissue-resident macrophages (TRMs) that are seeded independently of adult hematopoiesis and persist long-term in tissues [44,45]. In bone, osteal macrophages are considered prototypical TRMs; they exhibit self-renewal and can maintain local populations without exogenous input [46]. Owing to their marked heterogeneity and plasticity, macrophages are abundant throughout all phases of repair, dynamically modulating healing via cytokine secretion and intercellular crosstalk. In particular, they rapidly sense local immune cues and adopt distinct functional phenotypes—most notably classically activated M1 and alternatively activated M2 states—which assume complementary regulatory roles during inflammatory initiation and tissue regeneration, respectively [45].

Among immune populations, macrophages function as a hub for phase transitions in repair and for osteoimmune coupling. Inflammatory macrophages recruited from the circulation initially assume an M1-like phenotype under stimuli such as interferon-γ (IFN-γ), TNF-α, and microbe-associated molecular patterns, expressing inducible nitric oxide synthase (iNOS) and releasing TNF-α, IL-1β, interleukin-6 (IL-6), and CXCL9/10 to enhance pathogen clearance and antigen presentation [47,48]. M1-skewed signals upregulate RANKL expression on stromal cells and early osteoprogenitors while reducing OPG availability, thereby lowering the threshold for osteoclastogenesis; concurrently, sustained TNF-α/IL-1β suppress Runx2, Osterix, and β-catenin, diminishing osteoid formation and delaying mineralization [49,50]. However, macrophages are highly plastic. As apoptotic neutrophils and debris are efferocytosed and as IL-4, interleukin-13 (IL-13), and TGF-β increase, macrophage metabolism shifts from glycolysis toward fatty acid oxidation and oxidative phosphorylation, and phenotypes transition to M2-like states [47]. M2 macrophages are themselves heterogeneous—operationally classified as M2a, M2b, M2c, and M2d based on inducing cues and functions [51]. M2a cells, driven by IL-4/IL-13, secrete interleukin-10 (IL-10) and TGF-β, activate fibroblasts, and support collagen deposition, thereby fostering early matrix remodeling and osteogenic differentiation; M2b cells, induced by immune complexes and Toll-like receptor signals, exhibit mixed pro-/anti-inflammatory features and may balance inflammatory and reparative cues; M2c cells, induced by IL-10 and glucocorticoids, are strongly immunosuppressive and pro-remodeling, able to suppress osteoclastogenesis by lowering the RANKL/OPG ratio; M2d cells, driven by adenosine and IL-6, are pro-angiogenic and release VEGF and platelet-derived growth factor (PDGF), indirectly promoting osteogenesis by improving vascularization [52,53] (Fig. 1A). Overall, M2 macrophages dampen inflammation through IL-10, TGF-β, CCL18, and arginase-1 (Arg-1), while producing regenerative mediators such as VEGF, PDGF, and oncostatin M [OSM], the latter enhancing osteoblast differentiation and type I collagen expression via the gp130/STAT3 pathway to directly boost mineralization [[54], [55], [56]]. Through coordinated control of adaptive immunity and skeletal metabolic signaling, macrophages orchestrate the transition from inflammatory osteolysis to regenerative osteogenesis.

Fig. 1.

Macrophage polarization and bone reconstruction. (A) Unpolarized M0 macrophages can differentiate into M1 or M2 subtypes under distinct signals, exerting pro-inflammatory or reparative functions, respectively.(B) M1 macrophages secrete pro-inflammatory cytokines that promote osteoclastogenesis, whereas M2 macrophages support osteogenesis and bone repair through anti-inflammatory and osteogenic mediators. Together, they coordinate the balance of bone reconstruction. Created with BioRender.com.

The identification of bone-surface osteal macrophages further expands the recognized role of macrophages in bone formation. These resident cells are broadly distributed along the periosteum and osteogenic fronts, where they clear apoptotic osteoblasts, maintain a “clean” bone surface, and stabilize osteoblast adhesion and alignment. Experimental depletion markedly reduces osteoid deposition and de novo bone formation in animal models [52,57]. At the cellular level, macrophage–MSC interactions are strongly bidirectional: M2-like macrophages promote MSC osteogenic commitment and mineralization via TGF-β, BMP-2, OSM, and exosomal microRNAs (miRNAs) [58,59], whereas MSCs and their extracellular vesicles drive macrophage polarization toward M2-like states through prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO), and miR-21/miR-146a signaling, in part by inhibiting NF-κB and activating STAT6 and peroxisome proliferator-activated receptor-γ (PPARγ) pathways [60]. This immuno-osteogenic positive feedback facilitates a smooth handover from inflammation to regeneration. With respect to osteoclasts, macrophages influence multiple checkpoints: during the M1 phase they lower the resorptive threshold by increasing RANKL availability and pro-inflammatory cytokine levels; during the M2 phase they raise this threshold through OPG upregulation and IFN-β/IL-10-mediated inhibitory cues, and by clearing surface debris optimize the spatial conditions for osteoclast–osteoblast coupling [46,55]. Notably, subsets of bone marrow macrophages and osteoclasts arise from closely related monocyte progenitors; macrophage colony-stimulating factor (M-CSF) and RANKL signals that drive osteoclastogenesis are amplified in inflammatory settings, suggesting that chronic low-grade inflammation can bias macrophage lineages toward osteoclast commitment, contributing to bone loss [[61], [62], [63]] (Fig. 1B). In sum, through phenotype polarization, metabolic reprogramming, and dense interactions with bone cells and stem cells, macrophages determine the timing and quality of the inflammation–regeneration–remodeling sequence.

1.1.3. T cells

The roles of T cells in bone repair are strikingly subset-dependent. Upon antigen and cytokine stimulation, naïve T cells differentiate into CD8⁺ cytotoxic T cells and multiple CD4⁺ T helper (Th) subsets—including Th1, Th2, Th17 cells—and regulatory T cells (Tregs), each exerting bidirectional control over skeletal metabolism [64,65]. On the pro-inflammatory, pro-resorptive side, Th17 cells secrete interleukin-17A/F (IL-17A/F), which induces and amplifies RANKL expression on stromal cells, osteoprogenitors, and T cells themselves, thereby promoting pre-osteoclast differentiation and fusion. This mechanism is well documented in chronic inflammatory bone loss and estrogen-deficiency osteoporosis models, indicating that heightened Th17 activity augments resorption and delays repair [66,67]. In contrast, Tregs suppress dendritic cell and effector T-cell activation via IL-10 and TGF-β, directly inhibit pre-osteoclast differentiation into mature osteoclasts, and—through cytotoxic T-lymphocyte–associated protein 4 [CTLA-4] engagement with CD80/86 on stromal cells—downregulate RANKL, indirectly reducing osteoclastogenesis. Importantly, Treg expansion often coincides with inflammation resolution and callus maturation; their indirect activation of Wnt/β-catenin and BMP/Smad signaling underscores a central role in promoting osteogenesis and stabilizing remodeling [68,69]. Among other adaptive subsets, CD8⁺ T cells and some natural killer T (NK-T) cells release IFN-γ, which interferes with TRAF6 [TNF receptor–associated factor 6] and NF-κB downstream of RANK, thereby interrupting osteoclastogenic signaling and helping preserve bone mass. By comparison, Th1/Th17 bias prolongs inflammation, lowering callus quality and delaying osteogenesis, whereas Th2 cells, via IL-4 and IL-13, generally aid the establishment of an anti-inflammatory, pro-osteogenic milieu [70,71] (Fig. 2A and 2B). Notably, bone-forming cells are not merely targets but also regulators of adaptive immunity: osteoblasts and osteocytes secrete TGF-β and OPG, which restrain excessive T-cell responses, while osteocyte-derived RANKL becomes a key driver of osteoclastogenesis when adaptive signals are amplified [72].

Fig. 2.

Schematic representation of immunoregulatory mechanisms in bone repair. (A) Dynamic roles of T cells during different phases of bone repair.(B) Regulation of osteogenesis and osteoclastogenesis by distinct T-cell subsets through cytokine signaling [65]. Copyright 2025 ELSEVIER (C) Estrogen influences bone homeostasis via modulation of immune cytokines and contributes to immunoregulation of bone repair through B cells.(D) Dendritic cells act as a bridge between innate and adaptive immunity, participating in the regulation of bone repair [82]. Copyright 2016 ELSEVIER.

1.1.4. B cells

Within skeletal homeostasis and controlled inflammation, B cells are pivotal modulators of osteoclast activity. By secreting OPG, which competes with RANKL for binding to RANK, B cells inhibit the differentiation of pre-osteoclasts into mature osteoclasts, slowing resorption and providing a temporal window favorable for osteogenesis [73,74]. Under normal conditions, B cells are considered a major source of OPG and thus are important for maintaining bone mass. In chronic inflammatory or autoimmune contexts, however, aberrant B-cell activation can upregulate RANKL, lowering the OPG/RANKL ratio, enhancing osteoclastogenesis, and culminating in bone loss and osteoporosis [[75], [76], [77]] (Fig. 2C). Beyond the RANKL/OPG axis, B-cell-derived cytokines exert dual effects on bone metabolism: anti-inflammatory mediators such as IL-10 mitigate osteogenesis-adverse inflammation, whereas pro-inflammatory cytokines such as IL-6 can indirectly impede repair by boosting resorptive activity or suppressing osteogenic signaling [78,79]. Moreover, B cell derived immune complexes can engage Fc-gamma receptors (FcγRs) on pre-osteoclasts to modulate their differentiation thresholds and functional states [80,81]. Overall, B cells participate in the dynamic regulation of bone resorption and formation via the RANKL/OPG balance and multifaceted cytokine outputs, with their effects substantially shaped by upstream T-cell and dendritic-cell networks.

1.1.5. Other inflammatory cells

Although not dominant players, dendritic cells (DCs), natural killer (NK) cells, and mast cells exert non-negligible effects at specific pathological or temporal nodes. DCs govern T-cell polarization through antigen presentation, indirectly affecting the Th17/Treg balance and RANKL availability; under combined macrophage colony-stimulating factor (M-CSF) and RANKL stimulation, subsets of DCs can transdifferentiate into osteoclast-like cells, suggesting a potential direct contribution to bone resorption [[82], [83], [84]] (Fig. 2D). NK cells secrete IFN-γ to inhibit osteoclastogenesis and, through crosstalk with DCs, calibrate the strength of local adaptive responses; in infection-related defects, NK cells help limit excessive inflammation and stabilize the microenvironment [85]. Mast cells release histamine, TNF-α, and proteases that regulate vascular permeability and matrix remodeling actions that can transiently favor cell infiltration and angiogenesis but, when prolonged, intensify chronic inflammation and matrix degradation, thereby impairing osteogenesis [86,87]. The effects of these cell types are largely mediated by alterations in the physical and chemical milieu across vasculature, matrix, and cytokine gradients, ultimately converging on the dynamic coupling between resorption and formation.

1.2. Temporal dynamics and chronobiology of the immune response

The immune response during bone repair exhibits a well-defined temporal sequence. Immediately after injury, an acute inflammatory phase ensues in which neutrophils constitute the predominant effector population for several hours to a few days [88]. Guided by chemokine (C-X-C motif) ligand 1/2 (CXCL1/2), CXCL8 [interleukin-8 (IL-8)], and complement fragment C5a, neutrophils rapidly accumulate within the fracture hematoma, peaking early and declining markedly by days 7–10 [89,90]. During this stage, neutrophils release matrix metalloproteinase-9 (MMP-9), elastase, and reactive oxygen species (ROS), thereby clearing necrotic tissue and degrading damaged matrix while liberating matrix-bound vascular endothelial growth factor (VEGF) to enable subsequent neovascularization. However, excessive ROS and protease activity can suppress osteogenic transcription factors—Runt-related transcription factor 2 (Runx2) and Osterix (Sp7)—via nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, delaying osteoid formation and mineralization [[91], [92], [93]]. Animal studies indicate that moderately enhancing tumor necrosis factor-α (TNF-α) signaling within the first 24 h post-injury promotes monocyte recruitment and accelerates callus formation, whereas prolonging this window or completely blocking TNF signaling compromises repair—underscoring that precise matching of inflammatory intensity and timing is decisive for optimal bone healing [94,95].

From approximately days 3–7 onward, monocytes/macrophages progressively become the core regulators of the immune response. As they efferocytose apoptotic neutrophils, macrophages undergo metabolic reprogramming from a glycolysis-dominant, M1-like state to an M2-like phenotype characterized by fatty acid oxidation and mitochondrial oxidative phosphorylation [[96], [97], [98]]. This transition coincides with the biosynthesis of specialized pro-resolving mediators (SPMs), such as resolvins and maresins, which downregulate pro-inflammatory pathways while upregulating signals linked to angiogenesis and osteogenesis. In animal models, the timing of SPM administration critically shapes outcomes: Maresin-1 initiated from day 3 post-injury rather than immediately yields greater bone mass and superior callus histology, mirroring the natural macrophage phenotypic shift [99,100]. During this period, the role of bone-surface resident macrophages (osteal macrophages, often termed osteomacs) also becomes evident. CD169⁺ osteomacs form a continuous layer along the periosteum and osteogenic surfaces, support osteoblast function, and maintain the integrity of the osteoblast monolayer by clearing apoptotic osteoblasts. Recent experimental work further shows that inducing osteoblast apoptosis enhances osteomac efferocytosis, stabilizing the continuity of osteoid deposition and directly linking resolution of inflammation to ongoing osteogenesis [101].

Upon entry into the bony callus phase (approximately weeks 2–4), the immune milieu shifts toward a low-inflammation, pro-osteogenic program. M2-like macrophages secrete interleukin-10 (IL-10), transforming growth factor-β (TGF-β), VEGF, platelet-derived growth factor (PDGF), and oncostatin M (OSM), collectively reducing osteoclastogenic activity by tuning the RANKL/OPG balance while enhancing osteoblast differentiation and mineralization via gp130/STAT3, BMP/Smad, and Wnt/β-catenin signaling. Concurrently, regulatory T cells increase and suppress the Th17/IL-17 axis, further limiting RANKL availability [67]. During the subsequent remodeling phase, osteocytes—through dominant control of RANKL and osteoprotegerin (OPG)—emerge as principal modulators of osteoclast–osteoblast coupling. By integrating mechanical cues with low-grade inflammatory signals, osteocytes sustain the long-term rhythm of skeletal remodeling [66,102].

1.3. Bone cells (osteoblasts, osteoclasts, and osteocytes) as immune targets

Osteoblasts are not only matrix-synthesizing effector cells during bone repair and remodeling but also direct responders to immune signals. They express receptors for multiple inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and IFN-γ, enabling them to dynamically adapt to the inflammatory microenvironment. Acute, transient inflammatory stimulation can activate stress-responsive repair pathways, promote transcription of osteogenic genes such as Runt-related transcription factor 2 (Runx2) and Osterix (Sp7), and enhance type I collagen deposition. In contrast, persistent high levels of inflammatory signals chronically activate nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, markedly suppressing osteogenic transcriptional programs and reducing mineralization capacity [103]. During the resolution phase, when M2-like macrophages predominate, specialized pro-resolving mediators and IL-6 family cytokines—particularly oncostatin M (OSM)—enhance osteoblast differentiation and matrix synthesis via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Specifically, OSM downregulates sclerostin expression through the leukemia inhibitory factor receptor (LIFR) pathway, thereby relieving inhibition of the Wnt/β-catenin pathway, while via the OSM receptor (OSMR) pathway it enhances receptor activator of nuclear factor κB ligand (RANKL) expression, suggesting a bidirectional regulatory role of osteoblasts in balancing bone formation and resorption [104,105]. Moreover, osteoblasts are major sources of macrophage colony-stimulating factor (M-CSF) and RANKL, and together with osteoprotegerin (OPG) secretion, they critically regulate the threshold and rhythmic activity of osteoclast differentiation.

Osteoclasts are the principal effectors of the RANKL/RANK/OPG signaling axis, with differentiation dependent on osteoblast- and osteocyte-derived RANKL and M-CSF. The immune system also directly regulates osteoclastogenesis. For example, IFN-γ produced by CD8⁺ T cells and natural killer (NK) cells inhibits TNF receptor-associated factor 6 (TRAF6)/NF-κB signaling, thereby suppressing osteoclast formation [70]. During the resolution phase, regulatory Tregs secrete interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), further suppressing pre-osteoclast differentiation and limiting RANKL activity in the local niche. Importantly, osteoclasts themselves are not merely effectors of bone resorption but also active regulators of the immune microenvironment, for instance, by secreting TGF-β, thus forming an osteoclast–immune feedback loop [106]. Recent studies have described an intermediate “osteomorph” state during osteoclast resorption, highlighting a reversible differentiation phenomenon that provides a novel perspective on cellular plasticity in bone remodeling [107].

Osteocytes, the most abundant cell type in adult bone, represent the dominant source of RANKL and play a decisive role in regulating osteoclast activity and the overall rhythm of bone remodeling. Genetic studies in murine models demonstrate that osteocyte-specific deletion of Tnfsf11 (encoding RANKL) significantly impairs osteoclastogenesis and increases bone mass, definitively establishing the central role of osteocytes in remodeling [108]. The contribution of osteocytes varies across physiological stages: chondrocytes and osteoblasts are the main sources of RANKL during growth, whereas in mature bone, osteocytes predominate [109]. Under mechanical unloading or chronic inflammatory conditions, osteocyte-derived RANKL expression is upregulated, enhancing osteoclast activity and resulting in bone loss [110,111]. Conversely, during resolution phases enriched with regulatory immune cells, osteocytes increase OPG while downregulating RANKL, thereby suppressing osteoclastogenesis and supporting an osteoblast-dominant remodeling state.

2. Advances in immunomodulatory strategies for bone repair

2.1. Cell therapy and immune reprogramming

The temporal dynamics of immune responses after bone injury are tightly interconnected with the activity of bone MSC, bone marrow stem cells (BMSCs), and vascular endothelial cells. The core of cell therapy and immune reprogramming lies not in simply replenishing osteoblasts but in delivering cell populations with immunoregulatory properties, or in using engineered materials and molecular cues to redirect innate and adaptive immune responses toward pathways that favor bone regeneration. Representative approaches include:

• (1).Direct chemotaxis or in situ polarization of regulatory Tregs and macrophages;
• (2).MSC-based therapy and their extracellular vesicles (EVs)/exosomes;
• (3).Immunoregulatory scaffolds or hydrogels delivering cytokines such as IL-4 or IL-10;
• (4).Engineering immune cells to establish closed-loop systems for inflammation sensing and repair execution.

2.1.1. Direct immune cell therapy

Regulatory T cells play a pivotal role in maintaining immune homeostasis during bone repair. They not only suppress excessive inflammation but also induce macrophage polarization from M1-to M2-like states, thereby facilitating tissue regeneration. Experimental studies demonstrate that local delivery of exogenous Tregs via fibrin hydrogels into murine calvarial critical-sized defects, skeletal muscle injuries, and skin wounds significantly increases defect coverage and new bone volume. Mechanistic investigations reveal that Tregs rapidly acquire injury-associated phenotypes within the lesion microenvironment and enhance IL-10 secretion, which drives monocytes and macrophages into an anti-inflammatory, reparative state. Genetic manipulation further confirms this pathway: when Il10 is deleted in Tregs, their reparative effect is abolished, underscoring the indispensable role of IL-10–dependent mechanisms [112] (Fig. 3D–E). Moreover, both allogeneic and human-derived Tregs exhibit comparable pro-repair effects in xenogeneic models, highlighting their translational potential.

Fig. 3.

Cell therapies promote bone repair through immune reprogramming. (A) Adoptive transfer of M1 or M2 macrophages in a femoral roughened titanium implant model. M1 macrophages increase pro-inflammatory cells and cytokines while reducing T cells and mesenchymal stem cells (MSCs), resulting in decreased bone–implant contact; conversely, M2 macrophages suppress inflammation and enhance osseointegration. (B) Macrophage depletion increases neutrophil infiltration and decreases T cells and MSCs. In immunocompetent mice, M1 adoptive transfer exacerbates inflammation, whereas M2 markedly alleviates it. In macrophage-depleted models, adoptive transfer fails to rescue the inflammatory microenvironment. (C) Delivery of differentially polarized macrophages (M1 vs. M2) around titanium alloy implants significantly alters bone–implant integration. M1 macrophages aggravate inflammation and inhibit bone contact, whereas M2 macrophages promote osseointegration by reducing neutrophil and T-cell infiltration, enhancing MSC recruitment, and increasing anti-inflammatory factor secretion [114]. Copyright 2023 Springer Nature (D) Regulatory T-cell (Treg) therapy. Local delivery of exogenous Tregs in bone, muscle, and skin injury models increases bone mass and defect coverage, reduces muscle fibrosis, and accelerates wound closure. Delivered Tregs acquire injury-specific transcriptional profiles and upregulate immunoregulatory factors such as interleukin-10 (IL-10), driving monocytes/macrophages toward anti-inflammatory reparative states. (E) Dependency validation: the pro-healing effect of Tregs disappears after clodronate liposome–mediated depletion of monocytes/macrophages, confirming that Treg-mediated repair is largely dependent on macrophage reprogramming [112]. Copyright 2024 ELSEVIER.

Beyond immunomodulation, Tregs can also directly promote osteogenesis. Recent work shows that CCR8⁺ injury-reactive Tregs accumulate in human fracture tissues and murine bone injury models under CCL1 signaling. These CCR8⁺ Tregs upregulate the transcription factor BATF, enhancing secretion of progranulin (PGRN), which directly recruits skeletal stem cells (SSCs) and promotes their osteogenic differentiation. Blocking the CCR8/CCL1 axis decreases SSC frequency and markedly impairs callus formation, demonstrating its key role in Treg-mediated osteogenic regulation [113].

Macrophages are equally crucial regulators in the immunomodulation of bone repair. Adoptive transfer studies show that delivery of M2-like macrophages around titanium implants reduces inflammatory infiltration and enhances bone implant contact, whereas M1-like macrophages exacerbate inflammation and impair integration [114]. In macrophage-depletion models, prolonged inflammation and impaired osseointegration become more severe, underscoring the indispensable role of macrophages in inflammation resolution and bone rebuilding. However, other reports note that transplantation of poorly characterized or unstable macrophages into critical-sized defects fails to improve repair, suggesting that macrophage phenotypic stability, delivery timing, and microenvironmental compatibility are key determinants of therapeutic efficacy [115] (Fig. 3A–C).

Among other T-cell subsets, γδ T cells and their secretion of IL-17A have also been implicated in bone repair. In murine fracture models, γδ T cell derived IL-17A promotes bone formation and healing, whereas IL-17A deficiency impairs repair. Yet excessive IL-17A may suppress periosteal osteogenic differentiation or inhibit bone formation through the Wnt pathway. This biphasic effect highlights that IL-17A influences bone repair in a dose-, time-, and context-dependent manner [116,117]. Thus, the design of immune cell–based therapies must carefully consider the dynamic balance between early immune mobilization and subsequent osteogenic differentiation.

2.1.2. Mesenchymal stem cells and their extracellular vesicles

The therapeutic effects of MSCs are now increasingly attributed to their immunomodulatory and paracrine functions rather than solely to their osteogenic differentiation capacity. MSCs suppress NF-κB/NLRP3 inflammatory signaling and promote M2 macrophage polarization and Treg expansion through pathways including TNF-stimulated gene 6 (TSG-6)–CD44, prostaglandin E2 (PGE2)–EP4, indoleamine 2,3-dioxygenase (IDO), and programmed death-ligand 1 (PD-L1). These mechanisms collectively restore the Th17/Treg balance and promote angiogenesis and osteogenesis. For instance, exosomes from lipopolysaccharide (LPS)-preconditioned BMSCs induce M2 polarization through the TSG-6/NF-κB/NLRP3 pathway and improve the inflammatory microenvironment, providing a programmable strategy for paracrine based therapy [118].

In animal models of bone defects, MSC-derived exosomes have demonstrated direct efficacy. For example, MSC exosomes combined with mesoporous bioactive glass significantly increase new bone volume and angiogenesis in rat calvarial defects, while raising the proportion of CD206⁺ (M2) macrophages and reducing iNOS⁺ (M1) macrophages, reflecting synergy among immunomodulation, angiogenesis, and osteogenesis [119]. Under pro-inflammatory conditions such as diabetes, BMP-2–induced BMSC exosomes display stronger osteogenic and reparative functions than unmodified exosomes, suggesting that preconditioning MSCs with specific signals can enhance exosome efficacy and overcome the adverse effects of metabolic inflammation [120] (Fig. 4D–E). Furthermore, BMSC exosomes enriched in tripartite motif-containing protein 25 (TRIM25) suppress triggering receptor expressed on myeloid cells 1 (TREM-1) signaling, thereby reducing inflammation and promoting bone repair, directly linking immune suppression with osteogenic promotion [121].

Fig. 4.

Exosome-based immunomodulatory strategies for enhancing bone repair and regeneration. (A) Schematic illustration of the mechanism of M2 macrophage–derived exosomes (M2-Exos) in a diabetic fracture model. M2-Exos deliver immunoregulatory factors that suppress M1-driven pro-inflammatory responses, establish an anti-inflammatory microenvironment, and activate the PI3K/AKT signaling pathway, thereby improving bone repair. (B) In vitro experiments demonstrate that M2-Exos promote macrophage polarization toward an anti-inflammatory phenotype (upregulation of CD206, Arg-1, interleukin-10 [IL-10], CD163; downregulation of CD86) while enhancing the osteogenic differentiation potential of bone marrow mesenchymal stem cells (BMSCs), as indicated by increased alkaline phosphatase (ALP) activity and Alizarin Red staining. These findings suggest dual regulation of immunity and osteogenesis through intercellular communication. (C) In diabetic fracture mice, M2-Exos significantly improve histological repair at the fracture site (hematoxylin and eosin [HE] and Safranin O/Fast Green [SOFG] staining show enhanced new bone formation and reduced fibrous tissue deposition). Inhibition of the PI3K/AKT pathway attenuates these repair effects, validating the underlying molecular mechanism [122]. Copyright 2023 ELSEVIER. (D) Bone morphogenetic protein-2 (BMP-2)–preconditioned BMSCs secrete exosomes (BMP2-Exos) with enhanced osteoinductive capacity. These exosomes deliver specific signaling molecules that promote MSC osteogenic differentiation and regulate immune cells (e.g., polarization of M2 macrophages), thereby accelerating angiogenesis and bone regeneration in defect models. (E) Histological and immunohistochemical analyses show that the BMP2-Exos group exhibits superior bone defect repair compared with controls, including faster bone bridge formation, increased bone matrix deposition, and upregulation of osteogenic proteins (RUNX2, osteocalcin [OCN], type I collagen [COL I]) and angiogenic markers (CD31, vascular endothelial growth factor [VEGF]) [120]. Copyright 2024 ELSEVIER.

Exosomes derived from immune cells, such as M2-like macrophages, also carry microRNA cargo that regulates BMSC osteogenic differentiation and accelerate healing in diabetic fracture models, further highlighting the importance of intercellular communication between immune and mesenchymal cells in bone regeneration [122] (Fig. 4A–C). As acellular therapies, exosomes hold advantages including low immunogenicity, scalability, and engineering flexibility. However, challenges such as dose optimization, in vivo targeting, and consistency of therapeutic efficacy remain to be addressed for clinical translation.

2.1.3. Immunomodulatory scaffolds and hydrogels

Engineered biomaterials can achieve spatially and temporally controlled release of immune mediators in situ, enabling precise, window-specific modulation of local immunity to promote bone repair. Studies have reported that hydrogels loaded with IL-4 not only induce macrophage polarization from an M1 to an M2 phenotype, but also promote bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation via the transforming growth factor-β1 (TGF-β1)/Smad pathway, thereby significantly increasing new bone formation in murine calvarial defect models. Likewise, alginate hydrogels delivering interleukin-10 (IL-10) display dual actions—suppressing inflammation and enhancing osteogenesis—in alveolar bone defect models, supporting the feasibility of integrating immunoregulatory factors as “built-in drugs” within hydrogels or scaffolds [123].

Further efforts have focused on programmable release systems that align immune and osteogenic signals with the temporal sequence of bone healing. For example, embedding IL-4 nanoparticles together with the osteoinductive small molecule icariin into a phase-change hydrogel enables early preferential release of IL-4 to drive M2 polarization, followed by subsequent release of the small molecule to activate osteogenic pathways—better recapitulating the natural progression from inflammatory resolution to tissue rebuilding [124]. In addition, next-generation responsive hydrogels can dynamically release epigenetic modulators in response to inflammatory cues, thereby reprogramming macrophage transcriptional states; such self-adaptive immunomaterials are emerging as a promising direction [125] (Fig. 6A and B). In parallel, micro/nanostructural surface modifications of materials tuning roughness, wettability, charge, and ion release—have been shown to bias macrophages toward M2 polarization even without exogenous factors, via integrin–focal adhesion kinase (FAK) signaling, Ca²⁺ channel activity, and metabolic reprogramming. These effects, together with enhanced MSC recruitment and angiogenesis, indirectly strengthen osteogenesis [126].

Fig. 6.

Immunoregulatory biomaterials promoting bone repair. (A) Schematic workflow of composite hydrogel design and a bone defect treatment strategy that promotes multi-stage bone regeneration through macrophage reprogramming. (B) In vitro effects of the composite hydrogel in regulating anti-inflammatory gene expression and enhancing osteogenesis- and angiogenesis-related protein levels [125]. Copyright 2024 BMC. (C) Critical role of macrophages in magnesium ion (Mg²⁺) induced promotion of new bone formation. (D) Mg²⁺ modulates mesenchymal stem cell (MSC) osteogenic differentiation and mineralization by regulating macrophage-secreted interleukin-8 (IL-8). Experimental results show that different concentrations of Mg²⁺ and IL-8 significantly affect MSC alkaline phosphatase (ALP) activity, osteogenic gene expression, and mineralized nodule formation, indicating that Mg²⁺ indirectly promotes bone healing by reshaping the inflammatory microenvironment [148]. Copyright 2021 Springer.

2.1.4. Immune engineering and programmable cells

Concepts from immune engineering are expanding from oncology into regenerative medicine. Chimeric antigen receptor macrophages (CAR-M) represent a new class of engineered immune cells. A recent design incorporated a receptor that senses tumor necrosis factor (TNF) and triggers intracellular IL-4 signaling; when TNF levels rise in inflamed tissues, these CAR-M cells convert to an anti-inflammatory phenotype, forming a closed loop of inflammation sensing and immune reprogramming [127]. Although presently demonstrated outside the skeletal context, this strategy highlights opportunities for intelligent immunomodulation in future bone repair. In parallel, exosome engineering is gaining traction: loading defined microRNAs or proteins, adding bone-targeting ligands, or compositing with bioactive glass can simultaneously optimize targeting, release kinetics, and the combined immuno-osteogenic effects [119].

Taken together, immune reprogramming and bone repair are tightly coupled processes. Initially, anti-inflammatory signaling suppresses nuclear factor κB (NF-κB)/NLRP3 inflammasome pathways and induces M2 polarization, reducing neutrophil and T helper 1 (Th1) infiltration and mitigating early inflammatory damage. Subsequently, progranulin (PGRN) secreted by regulatory Tregs increases skeletal stem cell (SSC) numbers and lowers the threshold for osteogenic differentiation, while macrophage–MSC interactions couple immunity, angiogenesis, and osteogenesis through chemokine (C-X-C motif) ligand (CXCL) cues and factors such as TGF-β, vascular endothelial growth factor (VEGF), and bone morphogenetic protein-2 (BMP-2) [114,120]. Ultimately, under the influence of mechanical cues, ion release, and matrix guidance, cells and materials together drive trabecular reconstruction and mineralization. From a translational viewpoint, both cell- and material-based therapies must resolve issues of temporal matching, phenotypic stability, dosing control, and host variability. Current animal data provide a foundation for these approaches; future work in large-animal models and early-phase clinical studies should define dose–response relationships, safety, and peripheral immunoprofiles to enable individualized treatment.

2.2. Molecular and pharmacologic modulation strategies

The post-injury immune response displays pronounced spatiotemporal heterogeneity. Moderate early inflammation helps clear necrotic tissue and initiate angiogenesis, whereas sustained M1 bias or excessive inflammasome activation suppresses recruitment and differentiation of osteoprogenitors, enhances osteoclast activity, lowers callus quality, and increases nonunion risk. Molecular and pharmacologic interventions aim to retune the microenvironment toward regeneration by targeting the intersections of inflammatory networks and osteogenic signaling. Current research centers on cytokines and chemokines, small molecules and pathway inhibitors, lipid-derived specialized pro-resolving mediators, nucleic acids and extracellular vesicles, and sequential delivery strategies.

Cytokines and chemokines. IL-4 induces macrophage M2 polarization via the STAT6–peroxisome proliferator-activated receptor-γ (PPARγ) axis and concurrently augments TGF-β/Smad and Runx2 signaling to promote BMSC osteogenesis. In murine calvarial critical-sized defects, IL-4–loaded gels significantly increase the proportion of M2 macrophages and elevate new bone volume and mineralization. In aged models, local IL-4 injections on postoperative days 3–7 reduce NLRP3–interleukin-1β (IL-1β) activity and downregulate osteoclast-related genes; although the effect wanes over time, these data suggest a specific dosing window under immunosenescent conditions. Interleukin-10 (IL-10) attenuates NF-κB signaling through STAT3–suppressor of cytokine signaling 3 (SOCS3), lowering tumor necrosis factor-α (TNF-α) and IL-1β. In alveolar and calvarial defect models, IL-10 injections or sustained-release hydrogels increase vascular density and new bone formation, evidencing combined anti-inflammatory, pro-angiogenic, and pro-osteogenic effects [128,129]. Within vascular–osteogenic coupling, VEGF and platelet-derived growth factor-BB (PDGF-BB) are particularly prominent. Adeno-associated virus–mediated delivery of VEGF165 increases microvessel density and callus volume in rat fracture models, while PDGF-BB enhances MSC recruitment, angiogenesis, and mechanical recovery in rabbit tibial osteotomy and calvarial defects. Notably, PDGF and BMP-2 exhibit sensitive temporal interplay: premature or excessive PDGF can dampen the osteoinductive actions of BMP-2, indicating that multi-factor combinations should prioritize angiogenesis before intensifying osteogenesis [130]. Chemokines also regulate cell homing and repair; for example, scaffolds delivering CXCL12/stromal cell–derived factor-1α (SDF-1α) significantly enhance MSC recruitment and the coordinated reconstruction of vasculature and bone. The complement system shows duality: overactivation of the C5a/C5a receptor (C5aR) pathway amplifies inflammation and impairs angiogenesis; C5aR antagonists improve long-term bone strength in severe trauma models but show limited benefit in simple fractures, implying pathology-dependent therapeutic potential [131].

Small molecules and pathway inhibitors.The NOD-, LRR- and pyrin domain–containing protein 3 (NLRP3) inflammasome is a critical node influencing bone repair. Under rheumatoid or high-inflammation conditions, the NLRP3 inhibitor MCC950 markedly reverses delayed fracture healing and improves callus mineralization; in diabetic alveolar defects, short hairpin RNA (shRNA)–mediated NLRP3 knockdown elevates Runx2 and osteocalcin (OCN) while reducing inflammation. Recently, responsive hydrogels that release natural compounds on demand have shown potential to inhibit NLRP3 and improve hard-to-heal fractures. Toll-like receptor 4 (TLR4)–NF-κB signaling constitutes another key axis: TLR4 deficiency or pharmacologic inhibition with TAK-242 (resatorvid) alleviates local inflammation and enhances angiogenesis and bone bridging in calvarial defect and bone-substitute implantation models. The prostaglandin–EP receptor pathway underscores the complexity of inflammatory control. While systemic cyclooxygenase-2 (COX-2) inhibitors provide analgesia, they are often associated with delayed healing in animal studies and clinical observations [132]. By contrast, selective prostaglandin E receptor 4 (EP4) agonists accelerate bone formation in drill-hole and segmental defect models and can restore normal healing in COX-2–deficient settings, suggesting that redirecting downstream signaling better balances inflammation and osteogenesis. Additionally, adenosine A2A receptor (A2AR) agonists promote repair in fracture models, in part by enhancing Tregs and suppressing Th17 responses [133]. Local sustained-release carriers can extend drug action and reduce systemic side effects, achieving dual goals of bone repair and analgesia.

Lipid-derived specialized pro-resolving mediators (SPMs). SPMs such as resolvin D1/E1 and maresin-1 orchestrate orderly inflammatory resolution and microenvironmental homeostasis. In models of bone injury and periodontal destruction, these mediators downregulate M1-associated genes, upregulate M2 markers, and promote angiogenesis. Administration of maresin-1 during the delayed phase of fracture healing improves callus quality, emphasizing the importance of dosing at times that avoid suppressing the essential early inflammatory response [16].

Nucleic acid and extracellular vesicle (EV) approaches offer new molecular-level interventions. MicroRNA-26a (miR-26a) has been shown to simultaneously upregulate osteogenic and angiogenic genes and to promote calvarial defect repair. Encapsulating miR-26a within mesenchymal stem cell (MSC)–derived exosomes and delivering them via hydrogel enables concurrent modulation of macrophage and osteoclast activity, yielding superior outcomes compared with single growth-factor delivery. Conversely, inhibiting inflammation-associated microRNAs—such as miR-155 and miR-182-5p—can also improve bone formation [134].

Hypoxic preconditioning upregulates microRNA-126 (miR-126) in small extracellular vesicles (sEVs) derived from MSCs via hypoxia-inducible factor-1α (HIF-1α) activation. This elevation improves fracture healing through the miR-126/SPRED1/Ras/Erk pathway [135] (Fig. 5C). In addition, sEVs from hypoxia-conditioned MSCs stimulate osteogenesis and promote neovascularization in murine bone defects. Under hypoxia, microRNA sequencing reveals increased levels of miR-210-3p in sEVs. Upregulation of miR-210-3p suppresses ephrin-A3 (EFNA3) and activates the phosphoinositide 3-kinase (PI3K)/AKT pathway, thereby enhancing vascularized bone regeneration. Moreover, MSC sEVs derived from hypoxia-preconditioned microenvironments induce macrophage polarization from M1 to M2 phenotypes by enriching microRNA-216a-5p (miR-216a-5p) and activating the Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB)/PI3K/AKT axis [136] (Fig. 5A–B). It is noteworthy that prolonged or excessively deep hypoxia can impair MSC function, even though hypoxic preconditioning is effective in enhancing MSC regenerative and therapeutic potential. Compared with protein therapeutics, exosomes exhibit intrinsic inflammation homing, and when combined with carriers such as hydrogels, they achieve longer local residence and on-demand release.

Fig. 5.

Hypoxic preconditioning enhances the osteogenic and immunoregulatory effects of mesenchymal stem cell–derived small extracellular vesicles (sEVs). (A) Schematic illustration of MSCs cultured under hypoxic conditions to produce hypoxia-conditioned sEVs (Hypo-sEVs), which are subsequently loaded into hydrogels for delivery. Hypoxic preconditioning enriches sEVs with hypoxia-responsive microRNAs, such as miR-126 and miR-210-3p, which promote osteogenesis and angiogenesis through the SPRED1/Ras/Erk and EFNA3/PI3K/AKT pathways, respectively.(B) In bone defect animal models, Hypo-sEVs markedly enhance new bone formation and angiogenesis compared with normoxic sEVs (Nor-sEVs) [136]. Copyright 2022 ELSEVIER. (C) In a mouse calvarial critical-size defect model, Hypo-sEV treatment significantly increases callus volume, vascular density, and angiogenic activity [135]. Copyright 2020 ELSEVIER.

Spatiotemporally controlled delivery and sequential immunomodulation emphasize the staged regularity linking immune responses and bone repair. Extensive experimental evidence indicates that the phased transition from M1 to M2 macrophages and the timing of angiogenesis versus osteogenesis are critical determinants of successful healing. Programmable scaffolds can effect short-term release of granulocyte–macrophage colony-stimulating factor (GM-CSF) or IFN-γ to promote early cell recruitment, followed by gradual release of IL-4 and IL-10 to induce M2 polarization and osteogenic differentiation. Another design releases stromal cell–derived factor-1α (SDF-1α/CXCL12) first to recruit BMSCs and endothelial progenitor cells, then delivers vascular endothelial growth factor (VEGF) or platelet-derived growth factor (PDGF) to strengthen angiogenesis and matrix remodeling [137]. Such strategies markedly enhance healing efficiency and vessel–bone coupling in animal models.

At the clinical translation level, the repurposing of common drugs is attracting attention. Nonsteroidal anti-inflammatory drugs (NSAIDs) are associated in animals and systematic reviews with increased risk of delayed union when used at high doses or for prolonged periods—especially cyclooxygenase-2 (COX-2)–selective agents. However, conclusions regarding low-dose, short-term, or context-specific use are inconsistent. Consequently, more targeted prostaglandin E receptor 4 (EP4) agonists or pathway retargeting may be better suited for bone repair. Metabolic modulators such as metformin improve inflammation and osteogenesis in diabetic models, but in normoglycemic settings or with suboptimal dosing may suppress early cell proliferation and angiogenesis—highlighting host metabolic status as a decisive determinant of efficacy [138]. Complement inhibitors or immune checkpoint interventions can be effective in complex injuries with high inflammatory burden but show limited benefit in simple fracture models, underscoring the importance of patient stratification and inflammation monitoring.

In summary, molecular and pharmacologic strategies promote bone repair by reshaping the crosstalk among immunity, osteogenesis, and angiogenesis. Despite diverse mechanisms, their common goal is fine control of the phases and amplitudes of immune–osteo–vascular interactions. Future studies should define optimal timing, dosing, and safety in large-animal models and early clinical trials, and establish quantifiable immune-phenotype stratification to enable precise, individualized therapy.

2.3. Immunoregulatory biomaterials

The success of bone repair is determined first by the inflammatory response within hours to days after implantation. In this critical window, contact between the material surface and body fluids rapidly forms a protein corona, triggers complement cascades, and drives neutrophil and monocyte/macrophage adhesion and phenotype transitions—thereby setting the immune tone for subsequent osteogenesis and remodeling. Traditionally, immunoregulatory materials were conceived as chemical platforms that deliver cytokines or drugs. However, extensive studies over the past decade show that intrinsic physical surface features—wettability and surface energy, nano-/microtopography and roughness, pore architecture and interconnectivity, surface charge, and stiffness—function as immunomodulatory cues in their own right. These cues can reprogram complement and macrophage pathways and, indirectly, the interactions among osteoblasts, osteoclasts, and MSCs.

In titanium-based orthopedic implants, differences such as smooth versus rough, hydrophobic versus hydrophilic, and nanotube versus nanopore modifications markedly alter surface protein adsorption patterns, thereby affecting generation of complement fragments C3a/C5a, Fc receptor crosslinking, and activation of the NF-κB pathway [139,140]. In rodent defect models, the balance between inhibitory and activating complement signals correlates bidirectionally with osseointegration quality, highlighting “complement compatibility” as a critical, underappreciated dimension in biomaterial design [141].

Following protein corona formation and complement activation, wettability and surface energy further determine the quantity and conformation of early-adhesion proteins (e.g., fibronectin, albumin, and IgG). These, in turn, selectively engage distinct integrin clusters on macrophages and rewire downstream signaling. In vitro, micro-sandblasted/acid-etched surfaces with subsequent hydrophilization (modSLA/SLActive) downregulate macrophage interleukin-1β (IL-1β), interleukin-6 (IL-6), and TNF-α within 24–72 h relative to hydrophobic SLA surfaces, while upregulating IL-4 and IL-10. This effect is reproduced on both commercially pure titanium and Ti–Zr alloys, indicating it is not substrate-specific. In vivo, hydrophilic surfaces induce macrophage phenotypes characterized by low inflammation and high pro-osteogenic signaling, thereby promoting early osseointegration [142,143]. Mechanistically, the benefit of hydrophilicity extends beyond wetting: preferential engagement of integrin β1 on fibronectin and suppression of integrin β2–driven NF-κB activation together bias macrophages toward an M2-like state and enhance secretion of osteogenesis-related cytokines.

Nanoscale topography further amplifies immunoregulation. Ordered titanium dioxide (TiO₂) nanotubes and nanopores modulate macrophage spreading and cytoskeletal tension, directly suppress MAPK/NF-κB signaling, reduce pro-inflammatory mediator release, and induce M2 polarization. In parallel, they foster endothelial–MSC crosstalk by promoting secretion of angiogenic and osteogenic factors [144]. Structural parameters—tube diameter, wall sharpness, and array order—produce distinct immunologic outcomes within defined ranges. Coordinated in vitro/in vivo studies show that nanotube arrays, compared with unmodified surfaces, lower pro-inflammatory signals, increase anti-inflammatory mediators, and enhance peri-implant microvascularization. Mechanistically, altered adhesion-site density and integrin clustering modulate focal adhesion kinase (FAK)/Src signaling and downstream metabolic reprogramming, sculpting a characteristic cytokine network [145].

Stiffness, a macroscopic physical parameter, also transduces into immune cues via mechanosensitive channels and nuclear transcription programs. When bone marrow–derived macrophages are cultured on polyacrylamide substrates of varying Young's moduli, stiffer substrates increase Piezo1 expression and promote nuclear translocation of Yes-associated protein (YAP), biasing macrophages toward an M1 phenotype. Softer or intermediate stiffness favors M2 polarization. Inhibiting YAP under high-stiffness conditions reverses this trend and improves implant osseointegration in animal models. The Piezo1–YAP axis provides direct evidence linking mechanics to immunoregulation and osteogenesis, and suggests that interfacial elastic layers, stiffness gradients, or deformable coatings could precisely regulate macrophage phenotypes during the critical transition from inflammation to repair [146].

Pore architecture and interconnectivity shape the immune microenvironment at the tissue scale. Studies using mineralized collagen scaffolds demonstrate that pore size influences macrophage phenotypic distribution, reactive oxygen species levels, and protease release, thereby tuning the coupling rhythm between scaffold degradation and new bone formation. Pore interconnectivity dictates oxygen tension and nutrient diffusion gradients, which reprogram macrophage M1/M2 metabolism and cytokine profiles. Angiogenesis-guided porosity allows maintenance of moderate early M1 activity for debridement and cell recruitment, then—via nano–micro features at pore walls and adjusted surface energy—drives a gradual shift to M2 dominance, ultimately promoting MSC osteogenic differentiation. This pattern has been validated in vitro and in vivo with porous mineralized collagen systems [147].

Ion Release as an Immunoregulatory Cue of Material Surfaces. Ion release is another critical immunoregulatory factor at the material interface. Magnesium ions (Mg²⁺), recognized modulators of bone metabolism, exert dose- and time-dependent effects on bone regeneration. In rat defect models, short-term early Mg²⁺ elevation promotes macrophage-dependent repair: nuclear translocation of transient receptor potential melastatin 7 (TRPM7) kinase fragments and phosphorylation of histone H3S10 induce a cytokine profile characterized by upregulation of CCL5 and interleukin-8 (IL-8) with concurrent downregulation of interleukin-1β (IL-1β). This cytokine milieu enhances mesenchymal stem cell (MSC) homing and angiogenesis. However, sustained Mg²⁺ release can excessively activate NF-κB and osteoclast-associated pathways, thereby inhibiting mineralization. Importantly, Mg²⁺-mediated bone repair effects vanish when macrophages are depleted, underscoring that its benefits arise primarily from immune modulation rather than direct action on osteoblasts (Fig. 6C and D) [148]. Similar mechanisms have been observed with zinc ions (Zn²⁺) and their metal–organic framework (MOF) carriers: surface-released Zn²⁺, in synergy with nano/microtopography, suppresses macrophage glycolysis, activates the AMP-activated protein kinase (AMPK) pathway, promotes M2 polarization, and enhances downstream angiogenic and osteogenic signaling.

The roles of surface wettability and surface energy in shaping protein corona formation and complement activation warrant special emphasis. Titanium and titanium oxide surfaces activate complement C3 cleavage through classical and alternative pathways, producing C3a and C5a that drive inflammatory cell infiltration and osteoclastogenic signaling. Heat or ultraviolet treatment can markedly reduce complement deposition, suggesting that tuning complement compatibility could be a strategy to enhance the immunological safety of implants. Similarly, nanoporous structures on ceramics and polymers affect C3 deposition and membrane attack complex formation, with major implications for fibrous encapsulation and bone–implant interface maturation. Together with systematic studies on hydrophilized surfaces and macrophage polarization, these findings suggest that enhancing surface energy, reducing complement adsorption, and optimizing integrin engagement can improve osseointegration quality [142,149].

Micro- and nanoscale topography not only directly modulates macrophage phenotypes but also amplifies effects through MSC paracrine signaling. For instance, on nanoporous titanium, MSCs release exosomes in response to topographic stimulation rather than soluble factors, and these exosomes play a pivotal role in driving macrophage polarization from M1 to M2. Inhibition of exosome production abrogates the immunoregulatory influence of nanoscale features, highlighting a coupled feedback loop among materials, MSCs, and macrophages. This suggests that rational material design should integrate MSC migration, exosomal cargo transfer, and osteoclast signaling modulation into a holistic optimization framework [150].

Fiber orientation and anisotropy likewise regulate immune responses through mechanical and nuclear signaling pathways. Aligned electrospun nanofibers reshape macrophage nuclear morphology and mechano-transduction, suppress Janus kinase/signal transducer and activator of transcription (JAK/STAT) and NF-κB activation, and downregulate M1 markers and inflammatory mediators, creating a local microenvironment more conducive to angiogenesis and bone regeneration [151]. This underscores intrinsic links among structural morphology, nuclear signaling, and immune regulation.

Taken together, these findings support a staged framework for immunomodulation and bone regeneration. During the first 0–24 h post-implantation, hydrophilic surfaces and nanostructures with low complement deposition dampen initial inflammation and neutrophil activity. Within 24–72 h, modulation of stiffness and topography influences integrin–focal adhesion kinase (FAK)/PI3K and Piezo1–Yes-associated protein (YAP) pathways, promoting M2 polarization and the release of angiogenic and osteogenic mediators. Over 1–2 weeks, controlled Mg²⁺/Zn²⁺ release and persistent nanostructural cues maintain immune homeostasis while suppressing excessive osteoclastogenesis. With tissue maturation, pore size and interconnectivity provide a low-shear, oxygen-rich niche, while MSC-derived exosomes further stabilize the bone–implant interface [146,152].

This framework highlights the need for coordinated coupling of physical, chemical, and biological signals across temporal stages. It also explains why seemingly similar surface treatments can yield opposing results in different models or time windows. From a translational perspective, immunoregulatory bone materials should not pursue single-parameter optimization (e.g., roughness, hydrophilicity, or ion release), but instead target quantifiable immune endpoints such as early C3a/C5a and IL-1β kinetics, the IL-10/IL-6 ratio at 48–72 h, macrophage metabolic states (glycolysis versus oxidative phosphorylation), and MSC exosome signatures. Guided by such immune readouts, material design can be iteratively refined to optimize nanoscale dimensions, surface energy, stiffness gradients, and ion release kinetics. As mechanistic insights deepen into Piezo1–YAP, TRPM7–chromatin modification, and AMPK–metabolic reprogramming pathways, the design of material surface topography and mechanics will evolve from passive compatibility toward active immune programming, establishing itself as a major driver of bone repair and regeneration.

2.4. Physical and environmental modulation

Bone repair is not determined solely by growth factors and cells. Local physical and environmental factors after implantation—including mechanical loading, acoustic and electromagnetic stimulation, electrical currents, light exposure, and oxygen tension—continuously influence immune pathways over hours to weeks, thereby shaping angiogenesis and osteogenesis. Growing evidence shows that these physical cues first act on mechanosensing and metabolic pathways in myeloid cells such as macrophages, steering inflammation toward resolution and repair. Subsequently, through paracrine interactions with endothelial cells and skeletal stem/progenitor cells, they orchestrate the coupling of angiogenesis and osteogenesis. This mechanistic cascade is increasingly being validated by experimental studies. The following sections will review these mechanisms with a focus on mechanical forces, electromagnetic fields, ultrasound, light stimulation, and oxygen tension, emphasizing how parameter selection and timing critically determine immunological outcomes.

The mechanical environment at the fracture site is a primary determinant of the immune and regenerative cascade. Moderate interfragmentary motion and strain recruit and shape macrophages into phenotypes more favorable for repair, whereas excessively strong or weak signals prolong inflammation or bias the response toward fibrosis. Recent animal and in vitro studies demonstrate that mechanical loading not only increases reparative macrophage populations but also synchronously enhances angiogenesis and osteogenesis. Importantly, deletion of the mechanosensitive ion channel Piezo1 in macrophages abolishes these pro-healing effects, identifying Piezo1 as a pivotal mediator of macrophage polarization and angio-osteogenic coupling under mechanical stimuli. Further work shows that mechanical stretch induces Piezo1-dependent Ca²⁺ influx, which promotes p53 deacetylation and upregulates transforming growth factor-β1 (TGF-β1), thereby driving M2 polarization and enhancing bone marrow mesenchymal stem cell (BMSC) osteogenesis [153] (Fig. 7A–C). In parallel, the transcriptional regulators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) also link macrophage phenotype to osteogenic outcomes, together forming a strain–Piezo1–YAP/TAZ–immune–bone signaling axis. These insights provide molecular underpinnings for optimizing clinical fixation strategies and early weight-bearing protocols [154].

Fig. 7.

Physical and environmental factors regulating immune responses to promote bone repair. (A) Schematic diagram of the Piezo1-mediated mechanism in macrophages during mechanically induced bone regeneration. (B) Key role of Piezo1 in angiogenesis–osteogenesis coupling. Piezo1-deficient mice exhibit impaired load-induced bone repair characterized by reduced type H vessel volume, decreased osteoprogenitor numbers, and weakened spatial association between osteoprogenitors and vessels. (C) Importance of Piezo1 in macrophage-mediated coupling of angiogenesis and cell proliferation. Piezo1-deficient mice show defective proliferative responses during load-induced repair, manifested by reduced Ki67+ proliferating cells, decreased F4/80+ macrophage volume, and diminished spatial association between proliferating cells and vessels [153]. Copyright 2025 OXFORD (D) Implantation of piezoelectric barium titanate coated porous Ti6Al4V scaffolds into a sheep cervical lamina defect model promotes macrophage polarization toward the M2 phenotype, suppresses MAPK/JNK signaling, and enhances oxidative phosphorylation in macrophages, thereby accelerating bone repair. (E) In vitro effects of electrical stimulation on MC-3T3 osteoblasts, showing that osteogenesis is enhanced via M2 macrophage polarization [157]. Copyright 2023 ELSEVIER.

Within this mechano-immunology framework, electrical and electromagnetic stimulation offer programmable external interventions. Pulsed electromagnetic fields (PEMFs) consistently promote osteogenesis in multiple bone repair models. One study demonstrated that PEMFs synergize with exosomes derived from M2 macrophages to suppress osteoclastogenesis and upregulate osteogenic genes, suggesting that electromagnetic cues amplify macrophage–osteoblast crosstalk and reprogram the immune–bone axis [155]. Direct current (DC) stimulation provides further evidence: THP-1–derived macrophages exposed to a 100 mV/mm DC field for 1 h per day over 3 days downregulated M1 markers (CD86, IL1B) while upregulating M2-related genes (IL10, CD163, PPARG), with concomitant decreases in interleukin-1β (IL-1β) and interleukin-6 (IL-6) secretion. These findings highlight that low-intensity DC fields can shift macrophage polarization toward repair without exogenous factors [156]. Such electro-immunomodulation aligns naturally with functional biomaterials. For instance, piezoelectric materials such as barium titanate (BaTiO₃) or polyvinylidene fluoride (PVDF) generate spontaneous surface potentials under micro-mechanical perturbations, and in large-animal jaw and spine models, these materials enhanced M2 polarization, bone mass, and mechanical integration [157] (Fig. 7D and E). In vivo analyses of piezoelectric coatings and composite scaffolds further revealed immunologic signatures including MAPK/JNK suppression and augmented oxidative metabolism. Ultimately, electric field strength, duration, waveform, and material-specific piezoelectric coefficients jointly determine the thresholds and directionality of immune and osteogenic effects.

Ultrasound, as a noninvasive modality capable of reaching deep tissues, also regulates bone repair through immunological pathways. In vitro, low-intensity pulsed ultrasound (LIPUS; 8–15 mW/cm², 1 MHz, duty cycle 1:4) upregulated Wnt2b in RAW264.7 macrophages, inhibited AXIN/β-catenin degradation, and consequently suppressed TNF-α, IL-1β, and inducible nitric oxide synthase (iNOS), yielding an anti-inflammatory phenotype [158,159]. Animal and clinical data support the potential of LIPUS in treating fresh fractures, delayed unions, and nonunions. In tendon–bone interface models, initiating LIPUS one week post-surgery improved histological and biomechanical repair by modulating macrophage polarization. However, the therapeutic window for LIPUS is narrow: variations in frequency, intensity, duty cycle, and duration strongly influence immune–bone coupling. High-intensity ultrasound can accelerate mineralization but may divert immune trajectories, emphasizing the need for individualized dosing based on injury type and timing [160].

Optical energy provides high spatiotemporal precision for immunomodulation. Photobiomodulation (PBM) with near-infrared or red light significantly increased new bone volume and improved histology in rat calvarial defects. In other tissue injury models, PBM suppressed NF-κB–related inflammation and altered macrophage polarization, with direct evidence of increased M2 and decreased M1 macrophages in the injury zone [161,162] (Fig. 8A and B). Overall, wavelengths between 650 and 810 nm and low-to-moderate energy densities most reliably balance anti-inflammatory and pro-osteogenic effects [163]. Nevertheless, different cell types exhibit varying sensitivities to irradiance and pulsing parameters, necessitating tissue- and stage-specific optimization.

Fig. 8.

Oxygen tension and metabolic environment regulate bone repair through immune responses. (A) Schematic illustration of the effects of visible light on angiogenesis. (B) Light irradiation (650 nm, 808 nm) promotes vascular endothelial growth factor (VEGF) secretion from endothelial cells and macrophages under hypoxic conditions. The upregulated VEGF further enhances the formation of capillary-like structures (CLS) by endothelial cells, highlighting the critical role of light-induced VEGF production in angiogenesis [162]. Copyright 2022 ELSEVIER. (C) In mice exposed to different inhaled oxygen concentrations (21 %–14 % pO₂), two-photon lifetime imaging microscopy (2PLIM) was used to measure phosphorescence lifetimes of CX3CR1⁺ cells and local oxygen tension. Results showed a strong correlation between cellular pO₂ and peripheral blood oxygen saturation (SpO₂) [164]. Copyright 2022 Springer. (D) Under physiological hypoxia (5 % and 2 % O₂), IL-4 induced bone marrow–derived macrophages (BMMs) exhibited significantly increased proportions and mean fluorescence intensity (MFI) of CD115⁺CD169⁺CD206⁺ double-positive cells, suggesting that hypoxia promotes monocyte differentiation toward immunoregulatory phenotypes. (E) Hyperbaric oxygen treatment significantly enhances extracellular matrix mineralization and calcium deposition of human adipose-derived stem cells (hADSCs) during osteogenic induction culture, with marked differences compared with controls between days 7 and 21 [169]. Copyright 2023 MDPI.

Oxygen tension and metabolic environment form a continuous backdrop from early inflammation to late remodeling [164] (Fig. 8C and D). Transient hypoxia supports angiogenesis and cell migration, whereas prolonged or excessive hypoxia entrenches inflammatory phenotypes. At the molecular level, hypoxia-inducible factor-1α (HIF-1α) aligns with glycolysis and M1 polarization, while HIF-2α enhances arginase-1 (Arg1) expression and alternative activation. Bone tissue engineering solutions are advancing along two lines: (1) oxygen-releasing biomaterials to alleviate deep hypoxia—for example, oxygen-releasing thermosensitive hydrogels improve bone volume and angiogenesis while reducing HIF-1α expression in long-bone defect models; and (2) strategies leveraging HIF stabilization under controlled hypoxia to promote angiogenesis and osteogenesis [[165], [166], [167]]. In contrast, hyperbaric oxygen therapy (HBOT) has produced inconsistent results in randomized controlled trials of fracture patients, suggesting that locally controlled, material-mediated oxygen regulation is a more feasible avenue for bone repair [168,169] (Fig. 8E).

These physical and environmental modalities do not act in isolation; they can be combined with surface engineering, drug delivery, and cell therapies to achieve concerted effects. Piezoelectric scaffolds transduce micro mechanical perturbations into local electric fields and can be superimposed with pulsed electromagnetic fields (PEMFs) or direct-current (DC) stimulation to further enhance immunoregulation. Low-intensity pulsed ultrasound (LIPUS) strengthens integrin and ion-channel signaling through matrix–cell interactions and, in tandem with porous, hydrophilic surfaces, attenuates the early inflammatory peak. Photobiomodulation (PBM) coupled with oxygen-releasing hydrogels simultaneously alleviates hypoxia and dampens pro-inflammatory macrophage metabolism via mitochondrial mechanisms.

Crucially, efficacy hinges on precise matching of phase, dose, and target cell: during the first 0–72 h post-injury, low-dose electrical/optical/acoustic stimuli combined with hydrophilic surfaces help restrain excessive inflammation; between 72 h and 2 weeks, emphasis should shift toward pro-angiogenic and pro-osteogenic inputs—e.g., controlled mechanical loading, piezoelectric scaffolds, or PEMFs; during the remodeling phase, the priority is microenvironmental homeostasis, favoring gentle mechanical stimuli or oxygen modulation [170]. Immunologic readouts such as the interleukin-10/interleukin-6 (IL-10/IL-6) ratio, M2/M1 macrophage ratio, nuclear factor κB (NF-κB) activity, and cellular metabolic state—should guide parameter optimization to mitigate interindividual variability.

From a translational standpoint, physical modalities are repeatable, programmable, and naturally compatible with material design and surgical workflows. Current evidence suggests three principles. First, each intervention has thresholds for intensity and timing; values too high or too low can provoke undesirable immune responses. Second, host factors—age, diabetes, osteoporosis—shape immunity and warrant stratified selection based on inflammatory and metabolic status. Third, localized and sequential strategies outperform prolonged systemic anti-inflammation, reducing complications stemming from impaired host defense. Future studies should implement a “physical prescription immune readout bone reconstruction” closed-loop design, validate interactions among loading/electric/sonic/light cues and materials in large-animal models, and apply multi-omics to delineate shared and specific pathways along the Piezo1–Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), NF-κB, and hypoxia-inducible factor (HIF) axes, ultimately enabling a generalizable, individualized parameter framework.

2.5. Combination and translational strategies

Immunoregulation of bone repair rarely succeeds through a single lever. Early post-trauma immune clearance, subsequent angiogenic remodeling, and osteogenic differentiation operate on different timescales and involve distinct effector cells. Consequently, assembling cellular or molecular cues, material platforms, and physical environments in a spatiotemporally ordered fashion is more reliable than single-line interventions for steering the defect microenvironment from inflammation toward repair and rebuilding. This concept supported across multiple animal models and select preclinical/clinical studies coalesces into two complementary approaches: (i) programmed release of agents within synthetic scaffolds to recapitulate the immune–vascular–osteogenic cascade, and (ii) integration of intrinsic material immune cues with ionic, electromagnetic, and mechanical regulation to keep host responses within favorable windows, iteratively tuned by measurable immune endpoints.

Programmed release. Spiller and colleagues demonstrated the decisive impact of sequential delivery on macrophage programming: early IFN-γ release induced an M1 phenotype to support debridement and angiogenic initiation, followed by sustained IL-4 release that drove M1 to M2 transition, markedly increasing intragraft vessel density and perfusion and yielding mature, vascularized bone-like tissue [171]. This logic has been extended to osteogenic timing. For example, early release of deferoxamine (DFO) stabilizes HIF-1α and promotes angiogenesis, followed by low-dose bone morphogenetic protein-2 (BMP-2) to support later osteogenic differentiation significantly improving bone volume fraction and lamellar maturity in rat and beagle defect models while reducing soft-tissue reactions and ectopic ossification associated with high-dose BMP-2 [172].

Exosome–scaffold integration. A dual-responsive (thermo- and photosensitive) F127/hyaluronic acid nanobrush (F127/HA-NB) hydrogel has been used to deliver M2 macrophage–derived exosomes (M2-Exos), achieving robust regeneration in rat calvarial defects by upregulating Wnt/β-catenin signaling, enhancing osteogenesis, and reshaping the immune niche [173]. Building further, combinations of ions, exosomes, and metal scaffolds have emerged: a biodegradable porous zinc scaffold paired with high-activity serum-derived exosomes (BF-Exo) greatly improved bone bridging and angiogenesis in rabbit radial defects compared with scaffold alone while mitigating short-term hepatic/renal burden from metal degradation. Mechanistically, p38/STAT1 modulation suppressed osteoclast activity and enhanced osteogenesis [174].

Materials as immunologic entry points. Magnesium-based systems exemplify this paradigm. Early Mg²⁺ release upregulates transient receptor potential melastatin 7 (TRPM7) in macrophages, enabling nuclear translocation of TRPM7 kinase fragments (M7CKs) and phosphorylation of histone H3 at Ser10 (H3S10), which together establish a pro-osteogenic immune milieu. Prolonged Mg²⁺ release, however, can overactivate NF-κB and impede maturation [148]. In early phases, Mg²⁺ promotes macrophage infiltration, polarization, and activation with interleukin-8 (IL-8) upregulation and interleukin-1β (IL-1β) downregulation, thereby enhancing MSC homing and angiogenesis; the pro-repair effect disappears upon macrophage depletion, underscoring immune reprogramming as the primary mechanism. In rabbits, high-purity magnesium (HP–Mg) screws elicited milder foreign-body responses and earlier mineralized contact at the bone–implant interface than poly(lactic acid) (PLA) screws [175]. Prospective clinical data indicate that HP-Mg screws for ankle fracture fixation yield reduction and union rates comparable to titanium plates/screws, with progressive degradation and no breakage or infection, suggesting degradable, immune-friendly metals are entering clinical pathways.

Physical modalities in combination. As a noninvasive adjunct, PEMF is supported by mechanistic and animal data. In rabbit femoral defects and porous-titanium models, prolonged PEMF enhanced expression of osteogenic genes such as Runt-related transcription factor 2 (Runx2) and Wnt/β-catenin components and accelerated intrabony growth. At the immune level, specific extremely low-frequency PEMF (ELF-PEMF) parameters can steer macrophage activity and indirectly enhance MSC function, thereby promoting regeneration [176,177]. Nevertheless, systematic reviews and randomized trials report inconsistent acceleration of acute fracture healing with PEMF, likely reflecting parameter and population heterogeneity. This points to combining PEMF with immune-programmable scaffolds or exosomes, guided by immunologic biomarkers to refine dosing and timing for more consistent clinical outcomes.

A clinically tractable testbed: oral implants. In vitro and animal studies show that, relative to conventional sandblasted, large-grit, acid-etched (SLA) titanium, hydrophilized rough titanium surfaces significantly reduce macrophage IL-1β, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) within 24–72 h while increasing IL-10 and IL-4, thus establishing an early cytokine milieu favorable for osseointegration [139,178]. Clinical randomized trials now incorporate inflammatory and anti-inflammatory markers and implant stability quotient (ISQ) as early endpoints—reflecting a translational logic from surface modification to clinical immune outcomes. Concurrently, reports of innate immune overactivation by shed titanium ions/particles are increasing, flagging the need to manage particulate effects and complement activation even as roughness and hydrophilicity are optimized to avoid chronic inflammation and bone loss [179].

Population stratification and immune monitoring. Early peripheral immune phenotypes after fracture (e.g., elevated myeloid-derived suppressor cells, higher IL-10) correlate with callus quality and healing trajectory in animal studies and can be translated clinically to guide therapeutic windows and personalization. Looking ahead, large-animal and multicenter clinical studies should integrate immune metrics with imaging and biomechanical endpoints to preserve reproducibility and comparability across the rodent-to-clinic gap [180].

In sum, the value of combination and translation strategies lies not in piling on factors but in synchronizing timing, intensity, and localization. By using material-encoded immune signals and programmed release of exosomes/cytokines to stabilize deleterious inflammation within 24–72 h, prioritizing angiogenesis-first, osteogenesis-next, maintaining immune homeostasis with controlled Mg²⁺/Zn²⁺ release and persistent nano-features during weeks 1–2, optionally amplifying osteogenic signaling with PEMF, and closing the loop with quantifiable immune and skeletal endpoints, these mechanism-to-clinic strategies have a realistic chance to cross the translational threshold from rodents to large animals to early clinical trials making immunomodulation a reproducible, actionable paradigm for bone regeneration.

3. Conclusion

Bone repair and regeneration are not merely a balance between osteoblasts and osteoclasts, but rather a complex, dynamic process co-orchestrated by the immune system, the vascular network, stem/progenitor cells, and the physical environment. Advances in osteoimmunology underscore that the polarization and functional states of immune cells determine the speed and quality of the transition from inflammation to repair; this transition is not spontaneous but shaped by a convergence of cell therapies, molecular/pharmacologic interventions, biomaterial interfaces, and exogenous physical cues. Cell therapy and immune reprogramming leveraging regulatory Tregs macrophages, or MSC derived exosomes have demonstrated the ability to reroute inflammatory trajectories, particularly under high-inflammation or immunosenescent conditions, thereby improving angio-osteogenic coupling. Molecular and drug strategies modulate inflammasomes, cytokine networks, or defined signaling pathways to balance inflammation control with osteogenesis within specific windows. Immunoregulatory biomaterials extend this logic: surface wettability, nano/micro-architecture, stiffness, and ion release not only shape immune responses directly but also amplify effects via MSC exosomes and paracrine circuits realizing materials-as-signals and active immune programming. Physical/environmental factors mechanical strain, PEMF, LIPUS, light, and oxygen tension offer spatiotemporally precise tools that act on macrophages and MSCs through Piezo1–YAP/TAZ, NF-κB, and HIF pathways to sequentially promote inflammatory resolution, angiogenesis, and bone rebuilding. Across these domains, three trends merit emphasis. First, temporal matching is paramount: curb excessive inflammation early, trigger angiogenesis at key nodes, and sustain osteogenic homeostasis mid-to long-term. Second, combination therapy outperforms monotherapy: coordinated cell–factor–material–physical interventions create a closed loop that reduces same prescription, different response. Third, translation requires quantitative immune and tissue endpoints IL-10/IL-6 ratio, dynamic M1/M2 profiling, vascular density, and bone volume fraction validated stepwise in large animals and early clinical studies for reproducibility and safety. Altogether, the future of immunomodulation-enabled bone regeneration will not rely on single cells, single factors, or single materials. It will be integration-centric, programmable, quantifiable, and translational driven by synergy among immunology, materials science, bioengineering, and clinical practice. This framework provides a mechanistic foundation for treating complex bone defects and a roadmap toward individualized, precision orthopaedic regeneration.

Author contributions

WYQ investigated and summarized the literature and wrote the original draft. ZZY and SXD conducted deep review and editing. HWD WSJ and conducted deep review. PCD helped revise the paper, supervised the paper. All authors have read and approved this manuscript for publication.

Ethical approval

This article does not contain any studies with human or animal subjects performed by any of the authors.

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.