Piezo1-mediated mechanotransduction in bone formation and fracture repair

Abstract

The mechanosensitive ion channel Piezo1 is a crucial transducer of mechanical forces into biological signals governing skeletal development and fracture repair. This review summarizes its central role in bone formation, primarily through endochondral ossification. We detail how Piezo1 regulates key processes including chondrocytes-to-osteoblasts transdifferentiation, osteogenic fate determination in skeletal stem/progenitor cells, and functional activation of mature osteoblasts. Evidence from genetic models demonstrates that Piezo1 deficiency in these cell lineages leads to profound skeletal defects and fragility. Furthermore, Piezo1 is essential for fracture healing, coordinating periosteal stem cell activity, chondrocyte fate and macrophages regulation within the repair callus to enable successful bone regeneration. Finally, we discuss emerging Piezo1-targeted therapeutic strategies, such as low-intensity pulsed ultrasound, biomaterial-based and pharmacological approaches, highlighting its potential as a novel target for treating bone disorders and enhancing fracture repair.

Highlights

• •Skeletal development and molecular basis of Piezo1.
• •The role of Piezo1 in physiological bone formation.
• •The role of Piezo1 in fracture repair.
• •Novel therapeutic strategy targeting Piezo1 for bone growth.

1. Introduction

In vertebrates, bone is a highly specialized and complex tissue with a well-structured architecture that protects internal organs, provides attachment sites for muscles, and facilitates locomotion. The formation of bone is an intricate process. During embryonic development, bone formation typically occurs through two pathways: intramembranous ossification or endochondral ossification [1]. Intramembranous ossification arises from the condensation of mesenchymal cells, while endochondral ossification begins with the formation of a cartilaginous framework, upon which bone tissue subsequently develops [1]. Concurrently, bone resorption accompanies bone formation. These two processes are temporally and spatially interconnected, collectively constituting the stages of bone remodeling—a dynamic process that persists throughout human life. Beyond physiological bone formation, bones exhibit significant regenerative capacity after injury. This process closely resembles the mechanism of endochondral ossification [2]. Bone formation is crucial for maintaining the skeletal system and works in tandem with bone resorption. Abnormalities in bone formation can lead to various skeletal disorders, making research into its mechanisms highly significant.

Bone formation is influenced by numerous factors, with mechanical forces being particularly significant. Humans exist within a constantly changing mechanical environment, so the sensing of mechanical cues is vital for physiological functions—including the osteogenic process. Numerous microscopic structures within the body have been identified that sense changes in mechanical force, such as TRPV4 and primary cilia. Among these, the large transmembrane mechanosensitive cation channel Piezo1, discovered in 2010 [3], plays a central role in sensing external mechanical force variations at the cellular and systemic levels and transducing these changes into intracellular biological signals. Piezo1 is widely expressed across multiple cell types within the skeletal system, including osteoblasts, osteoclasts, and chondrocytes [4]. Upon activation of Piezo1 channels, cells typically exhibit changes in intracellular calcium ion concentration and translocation of YAP, demonstrating close connections between Piezo1 and signaling pathways such as Wnt and Hippo. This review summarizes recent research on the interactions between Piezo1 and bone formation-related signaling pathways and factors. The roles of these Piezo1-associated pathways and factors in bone formation may provide novel insights for the clinical management of skeletal system disorders.

2. Skeletal development and molecular basis of Piezo1

2.1. Embryonic bone development: intramembranous ossification and endochondral ossification

Intramembranous ossification is a mode of bone development, primarily occurring in flat bones, such as those of the skull and mandible [5]. This process begins with the aggregation of mesenchymal cells, which directly differentiate into osteoblasts [1,5]. These osteoblasts then secrete osteoid that subsequently calcifies to form a network of trabeculae [1,5]. As the trabeculae thicken and fuse, the woven bone gradually forms, and periosteal osteoblasts promote thickening and subsequent remodeling into mature lamellar bone.

Endochondral ossification, in contrast, is the primary mode of bone formation for most long bones in the human skeleton. Moreover, the repair process following bone injury closely resembles the process in endochondral ossification. During endochondral ossification, the mesenchymal cells first condense and gradually differentiate into chondrocytes. The cells surrounding this newly formed cartilage constitute the perichondrium, which defines the boundary of the developing skeletal element (which defines the boundary of bone formation) [1,5]. Cells in the perichondrium progressively withdraw from the cell cycle and undergo hypertrophy, subsequently promoting osteoblast differentiation of perichondrial cells. Vascular invasion into the hypertrophic cartilage is a pivotal event, inducing osteogenic differentiation and the emergence of a primary bone marrow cavity. This process facilitates the recruitment of chondroclasts, which degrade the existing cartilage, and osteoprogenitor cells, which promote osteoblast differentiation, while also allowing perichondrial cells to enter the developing marrow cavity [6,7]. This vascular invasion marks the formation of the primary ossification center, which expands and is eventually succeeded by the formation of secondary ossification centers form at the epiphysis [1]. The metaphysis, located between the diaphysis and epiphysis, contains a growth plate where chondrocytes in the proliferative zone undergo rapid replication [1,7]. As chondrocytes continuously proliferate and migrate away from the epiphyseal plate, most of them undergo hypertrophy and promote osteoblast differentiation of the surrounding progenitor cells via paracrine signaling, while a subset may directly differentiate into osteoblasts to participate in bone formation [[6], [7], [8]]. Blood vessels invading the medullary cavity deliver osteoclasts that resorb the mineralized cartilage matrix, upon which osteoblasts deposit new mineralized bone [5]. Postnatally, bones continue growing through adolescence until skeletal maturity is achieved, marked by the eventual fusion of epiphyseal plates.

2.2. Molecular structure and mechanistic mechanism of Piezo1 channel

Piezo1 is a large and conserved transmembrane protein found in various organisms. It is one of the Piezo proteins—a family of mechanically activated ion channels discovered in 2010 [3]. In mammals, this family comprises two homologous Piezo proteins: PIEZO1 and PIEZO2. Human genome studies indicate that loss-of-function mutations in the PIEZO1 gene lead to multiple human defects, suggesting this gene plays extensive roles in human development and homeostasis [9,10]. Piezo1 is essential for vascular development and function [11], red blood cell volume regulation [12], epithelial homeostasis [13], neural stem cell lineage selection [14], axonal growth [15], and urinary osmolality [16].

Piezo1 is characterized as a mechanosensor. The molecular structure of the Piezo1 protein has been systematically characterized using cryo-electron microscopy. Its conformation on the cell membrane resembles a three-bladed, propeller-shaped trimer. The unique topological features and ion-conducting pathway of the Piezo1 protein enable a lever-like mechanical transduction mechanism. This mechanism, formed by curved blades and long beams, converts large conformational changes in the distal blades into a relatively slight opening of the central pore, facilitating selective cation permeation [17]. This massive propeller-like assembly of three lever-like devices endows Piezo1 with coordinated mechanosensitivity. Compared to other mechanosensitive ion channels, it employs distinct structural and functional mechanisms for mechanical force transduction [17].

At the molecular mechanism level, the lineage commitment, proliferation, and function of osteogenic lineage cells are jointly regulated by multiple signaling pathways. The Wnt, TGF-β, Hedgehog, and FGF pathways are well-known pathways influencing osteoblast differentiation, while the Notch, Hippo, and NF-κB signaling pathways also participate in this process [18]. Given the structural basis of Piezo1-mediated mechanotransduction, it is pertinent to explore how this channel regulates bone formation and repair. The following sections will address its roles in physiological bone formation—covering chondrocyte transdifferentiation, stem/progenitor cell fate, and osteoblast function—and then extend to fracture repair, where Piezo1 orchestrates callus formation and remodeling. These insights may ultimately guide Piezo1-targeted therapeutic strategies.

3. The role of Piezo1 in physiological bone formation

Physiological bone formation is a sophisticated process involving the coordinated action of multiple cell types, primarily participating in embryonic skeletal formation and postnatal bone remodeling. Piezo1, as a key mechanosensor, plays crucial roles in multiple stages of this process. This section will focus on elucidating the core functions of Piezo1 in the typical paradigm of endochondral ossification. Specifically, it will examine Piezo1's pivotal roles in chondrocyte transdifferentiation, fate determination of stem/progenitor cells, and functional regulation of mature osteoblasts.

3.1. Regulating chondrocyte hypertrophy and transdifferentiation into osteoblasts

During endochondral ossification, the replacement of the cartilage template into bone represents a critical step. At the cellular level, a critical question is the source of the osteoblasts responsible for secreting osteoid. In this process, chondrocytes undergo a strict differentiation sequence: resting chondrocytes, proliferating chondrocytes, prehypertrophic chondrocytes, and hypertrophic chondrocytes [6,19]. Current research suggests that, in addition to stem cells differentiating into osteoblasts via blood vessels invading the cartilage matrix, chondrocytes can also directly differentiate into osteoblasts—a process termed chondrocyte-to-osteoblast transdifferentiation [8,20]. There are three mainstream models of chondrocyte-to-osteoblast transdifferentiation, reflecting the diversity of this process, which may be influenced by the specific chondrocyte subtypes and their distinct locations during skeletal development [21]. During the physiological process of endochondral ossification, most hypertrophic chondrocytes undergo programmed apoptosis to make space for incoming osteoblasts, while a portion directly transforms into osteoblasts to acquire osteogenic functions, as demonstrated by cell lineage tracing studies. The current paradigm regarding the transdifferentiation of hypertrophic chondrocytes (HCs) into osteoblasts is primarily built upon genetic lineage-tracing studies using Col10a1-Cre and Agc1-CreERT2 tools. Using both constitutive and inducible Col10a1-Cre mice to permanently label HCs, detected the expression of osteoblast markers (Col1a1, Osterix/Sp7) and the osteocyte marker SOST in the progeny cells. These labeled cells persisted from embryonic stages through to adult bone tissue, providing direct in vivo evidence that HCs can survive and transdifferentiate into functional osteoblasts [8]. Zhou et al. employing the Agc1-CreERT2 system in a pulse-chase experiment, further quantified this contribution, showing that approximately 60% of trabecular osteoblasts in this region are derived from the chondrocyte lineage, and the same transdifferentiation phenomenon was also observed during fracture healing [20]. Thus, chondrocyte-to-osteoblast transdifferentiation is a well-established fact. However, the molecular signaling pathways that govern this process still require in-depth scientific investigation. Previous studies have implicated changes in calcium and its receptor, IHH signaling, and the Wnt/β-catenin pathway in chondrocyte-to-osteoblast transdifferentiation [21]. More recently, Piezo1 protein on the chondrocyte surface has been shown to play an important regulatory role in this process, thereby influencing osteogenesis and fracture healing.

In Piezo1^Runx2Cre and Piezo1^Col2a1Cre transgenic mice generated by Gretl Hendrickx et al., multiple primary fractures and impaired formation of secondary trabecular bone were observed. Their experiments revealed near-complete absence of trabecular bone formation beneath the growth plates of long bones in Piezo1^Col2a1Cre mice [22,23]. This defect stems from Piezo1 deficiency in Col2a1-positive chondrocytes. Gretl Hendrickx's team further observed that “osteoblasts” near the growth plate exhibited an atypical and flat morphology and were type X-collagen positive [23]. Sequencing analysis revealed elevated expression of chondrogenic genes in these cells [23]. Based on this evidence, it is suggested that trabecular bone formation under the growth plate is critically dependent on Piezo1 in chondrocytes. In other words, the osteoblasts forming these trabeculae under the growth plate originate predominantly from transdifferentiation of chondrocytes, a process that is very likely regulated by Piezo1. Upon Piezo1 deletion, the expression levels of chondrocyte-related marker genes such as Col10a1 and SOX9 increased.

It is noteworthy that, although Piezo1^Col2a1Cre mice exhibited severely impaired trabecular bone formation, their growth plate architecture showed only mild widening, and chondrocyte alignment was not obviously disturbed [22,23]. Moreover, TRAP staining did not reveal an increased number of osteoclasts, suggesting that this phenotype is not caused by excessive bone resorption but rather by insufficient bone formation [22,23]. These findings further support the hypothesis that Piezo1 deficiency may impair the transdifferentiation of hypertrophic chondrocytes into osteoblasts. Collectively, there is compelling evidence that Piezo1 in chondrocytes plays a critical role in physiological endochondral ossification. This is supported by the observation that mice with chondrocyte-specific Piezo1 deletion using Col2a1Cre exhibit defective secondary spongiosa formation and subsequent spontaneous fractures near the growth plate. However, direct demonstration of whether this transdifferentiation process is regulated by Piezo1 still requires definitive lineage-tracing experiments and more in-depth molecular mechanistic studies.

3.2. Influencing the osteogenic differentiation fate of skeletal stem/progenitor cells

Skeletal stem/progenitor cells (SSCs) broadly encompass all immature precursor cells within bone tissue and possess the capacity to differentiate into various cell types that constitute bone tissue [24]. Bone marrow stromal cells (BMSCs) serve as a crucial cellular model for studying skeletal stem cells. Knockout of Piezo1 in these cells leads to phosphorylation of Yap1 and Ctnnb1, causing their intracellular inactivation and degradation. This impairs the osteoblastic differentiation response of BMSCs to fluid shear stress [25]. Mechanistically, Piezo1-dependent activation of Yap1 and Ctnnb1 is mediated through the intracellular calcium sensor Ppp3ca activating NFAT. Notably, Nfat, YAP1, and Ctnnb1 exhibit reciprocal activation, forming a concerted signaling hub that amplifies the downstream response (Fig. 1) [25]. The functional significance of this pathway is further supported by small-molecule interventions: Yoda1 treatment enhances the Ppp3ca/Nfat/YAP1/Ctnnb1 signaling pathway, increasing the activity of all components, while this effect is blocked by calcium channel blockers and Ppp3ca inhibitors [25]. Consequently, the activity of Nfat, YAP1, and Ctnnb1 in BMSCs is robustly enhanced upon mechanical stimulation or Yoda1 treatment [25].

Fig. 1.

Piezo1 converges multiple pathways to govern the osteogenic fate of skeletal stem/progenitor cells.

Abbreviations: MSC, mesenchymal stem cells; SSCs, skeletal stem/progenitor cells; BMSCs, bone marrow stromal cells; BMMSC, bone marrow-derived mesenchymal stem cells; UE7T-13, UE7T-13 cell lines; HP, hydrostatic pressure. Evidence grading: Nfat/Yap/β-catenin is well-established via in vivo knockout and pharmacology. BMP2 is supported by in vitro pharmacological and siRNA studies but lacks in vivo validation. Oln⁺LEPR⁺ and Gli1⁺ pathways are emerging, each supported by a single study; their universality across distinct skeletal niches remains speculative.

Bone marrow-derived mesenchymal stem cells (BMMSCs) constitute a subpopulation of bone marrow stromal cells with enhanced osteogenic differentiation potential [26]. Their differentiation into osteoblasts is closely linked to the external mechanical force environment. In the absence of normal external mechanical forces, the osteogenic differentiation of BMMSCs is impaired due to reduced Piezo1 activity, ultimately compromising bone formation [27]. Furthermore, upon mechanical stimulation, the Piezo1 protein channel on Gli⁺ mesenchymal stem cells is activated, facilitating β-catenin nuclear translocation. This activates its target gene ATF4, thereby promoting mesenchymal stem cells’ proliferation and differentiation (Fig. 1) [27]. Regarding the identification of surface markers for bone marrow stromal cells and their subpopulations, LEPR⁺ cells are recognized as a stem-like cell population in adult bone marrow. Recent studies reveal that LEPR⁺Oln⁺ bone marrow stromal cells surrounding small arteries possess osteogenic differentiation capacity (Fig. 1) [28]. Their numbers significantly increase after fracture, representing a rapidly dividing, short-lived osteogenic progenitor cell population [28]. Crucially, both osteogenic and lymphoid differentiation potentials of these cells depend on Piezo1 and mechanical stress from the external environment. Under conditions of gravity deprivation or Piezo1 knockout, LEPR⁺Oln⁺ cell numbers decrease and osteogenic differentiation is impaired [28]. Sugimoto et al. experimentally demonstrated that under hydrostatic pressure (HP) stimulation, Piezo1 mediates mechanical force transduction and elevates BMP2 levels, thereby promoting mesenchymal stem cell differentiation toward osteoblasts and partially determining their differentiation fate (Fig. 1) [29].

Among the various bone stem cell subtypes labeled with the surface markers, Piezo1 is closely associated with their osteogenic differentiation. When studying skeletal stem/progenitor cells, research has primarily focused on simulating physiological bone formation to investigate the origin of osteoblasts. Current research increasingly focuses on the osteogenic potential of periosteal stem cells under the condition of fracture healing [30]. The relationship between other skeletal stem cell subtypes, mechanical forces, and Piezo1 remains to be further explored.

3.3. Activating mature osteoblast function and promote bone matrix formation

Recent studies have established a critical role for Piezo1 in osteoblast-lineage cells [31,32]. At the animal model level, For instance, Sun et al. generated conditional knockout mice Piezo1^OcnCre to specifically ablate Piezo1 in osteoblasts and observed severe skeletal defects, including a high incidence of spontaneous fractures [32]. The biomechanical data suggest that these fractures stem primarily from structural weakness, as evidenced by marked reductions in cortical thickness and cross-sectional area [32], without concomitant changes in Young's modulus or tissue mineral density [31]. The observed decrease in bone formation rate and osteoblast marker expression points to impaired osteogenesis [32], while the unaltered matrix material quality indicates that the remaining osteoblasts are functionally competent [31]. Therefore, the net bone loss likely reflects a reduction in osteoblast number. This could arise from several non-exclusive mechanisms, including compromised mesenchymal progenitor recruitment or osteogenic commitment, increased osteoblast apoptosis, or defective chondrocyte-mediated endochondral ossification—a process recently identified as Piezo1-dependent. Given that current studies have largely focused on mature osteoblasts, future lineage-specific tracing and conditional deletion studies are necessary to dissect the relative contributions of distinct skeletal cell populations to this phenotype.

In vitro studies of the relationship between osteoblasts and Piezo1 have relied heavily on the MC3T3-E1 osteoblast-like cell line. In MC3T3-E1 cells, Piezo1 is activated by fluid shear stress (FSS). This activation promotes PKB/AKT phosphorylation, which in turn leads to GSK-3β phosphorylation, β-catenin stabilization and nuclear translocation, and subsequently upregulates the expression of the key osteogenic transcription factor Runx2 (Fig. 2) [33]. Conversely, in MC3T3-E1 cells with Piezo1 expression knocked down via siRNA, the expression of osteogenesis-related marker genes Alp, Ocn, and Col1a1 was reduced [32]. In addition to Piezo1, TRPV4 is another crucial mechanosensor in osteoblasts, and these two channels may interact. Yoneda et al. demonstrated that while both the Piezo1 agonist Yoda1 and FSS elicit cation influx in MC3T3-E1 cells, the underlying mechanisms differ: the response to Yoda1 involves both Piezo1 and TRPV4, whereas the response to FSS is primarily mediated by TRPV4 (Fig. 2) [34]. Therefore, the relationship between TRPV4 and Piezo1 in sensing mechanical forces warrants further investigation. In addition, the effect of Piezo1 on osteoblast proliferation remains controversial. While some studies show that mechanical activation of Piezo1 and TRPV4 inhibits osteoblast proliferation, others have reported that acoustic radiation force promotes both proliferation and migration [35]. These discrepancies suggest that the proliferative response may be highly dependent on the type, intensity, and duration of the mechanical stimulus.

Fig. 2.

Piezo1-activated cascades in osteoblasts.

Abbreviations: FSS, fluid shear stress. Evidence grading: YAP-ECM regulation is validated by in vivo knockout and in vitro mechanical loading/promoter assays. AKT/Runx2 is only supported by in vitro siRNA and inhibitor studies. CaMKII/Creb is inferred from reduced phosphorylation in Piezo1-deficient osteoblasts, but lacks direct causal validation. TRPV4 interplay and proliferative effects are speculative, derived solely from MC3T3-E1 data with conflicting results.

The MC3T3-E1 cell line is a valuable tool for initial screening and mechanistic studies, whereas primary osteoblasts offer greater physiological relevance. Together, they provide a complementary approach that balances experimental throughput with biological fidelity. In primary osteoblasts, exposure to FSS for 2 h upregulated Piezo1 and osteogenic marker gene expression, whereas simulated microgravity exerted the opposite effect [32]. A hallmark of Piezo1 activation is robust calcium influx. In Piezo1-deficient primary osteoblasts, this influx is diminished, leading to reduced phosphorylation of CaMKII and Creb, and decreased expression of Runx2 and Atf4 [32]. Given that Ca²⁺/CaMKII signaling promotes osteoblast differentiation via the CREB pathway [36,37], these findings implicate a functional link between Piezo1 and the CaMKII/CREB axis in the regulation of osteoblast differentiation (Fig. 2). Wang et al. employed Piezo1^Prx1 mice to conditionally delete Piezo1 in limb mesenchymal progenitors [38]. They found that Piezo1-deficient osteoblasts derived from these mice exhibited significantly reduced expression of the extracellular matrix (ECM) genes Col2a1 and Col9a2. This regulation was shown to occur through the YAP signaling pathway. [38]. Given that both type II collagen [39] and type IX collagen [40] can inhibit osteoclast activation, these findings reveal a crucial role for Piezo1 in osteoblast-osteoclast crosstalk: by controlling the secretion of specific collagens, Piezo1 in osteoblasts can indirectly restrain bone resorption (Fig. 2).

4. The role of Piezo1 in fracture repair

The process of fracture healing closely parallels endochondral ossification and has consequently been conceptualized as a form of pathological endochondral ossification. Fracture healing proceeds through sequential stages: the inflammatory phase, the cartilaginous callus formation phase, the hard callus formation phase, and the bone remodeling phase. Multiple cell types participate in fracture healing, and Piezo1 expressed on some of these cells is directly linked to fracture repair. Based on the distinct cell types in which Piezo1 functions, its roles in fracture healing can be categorized as follows.

4.1. Piezo1 in skeletal progenitor cells: promoting early callus formation

Fracture healing, particularly during the early phases of callus formation and maturation, critically depends on the activation, migration, and proper differentiation of periosteal stem cells (PSCs), which serve as the principal source of chondrocytes and osteoblasts in the callus. Liu et al. discovered that Piezo1 is highly expressed in periosteal stem cells and plays a crucial role in fracture healing. As a key source of chondrocytes and osteoblast lineage cells, periosteal progenitor cells are essential for chondrogenesis, osteogenesis, and the transition from cartilaginous callus to hard callus [30]. In periosteal stem cells, mechanical force activates Piezo1, which in turn elevates cytoplasmic YAP expression and initiates its nuclear translocation. YAP forms a complex with β-catenin, translocates to the nucleus, and modulates the expression of osteogenic, chondrogenic, and angiogenesis-related factors, ultimately influencing fracture healing (Fig. 3) [30]. The impact of Piezo1 deficiency in periosteal progenitor cells on fracture healing primarily manifests in impaired osteogenic and chondrogenic differentiation, as well as indirectly affected angiogenesis [30]. These effects are intrinsically linked to the initiation of fracture healing and the supply of cell types involved in repair.

Fig. 3.

Roles of Piezo1 in fracture repair.

Abbreviations:sEVs, small extracellular vesicles. Evidence grading: All mechanistic pathways depicted in this figure have been phenotypically validated by both in vivo and in vitro experiments, as reported in the cited studies. However, the intracellular signaling cascade linking Piezo1 activation in macrophages to M2 polarization and subsequent release of pro-repair small extracellular vesicles (sEVs) remains largely undefined. Furthermore, Piezo1 has been shown to mediate both M1 and M2 polarization under different pathological or biomechanical contexts; the mechanistic basis for this functional dichotomy—namely, how Piezo1 engagement leads to opposing polarization outcomes—remains an open question that warrants in-depth investigation.

4.2. Piezo1 in chondrocytes: mediating cartilage-to-bone transition

As fracture healing progresses from the cartilaginous callus to the hard callus, a transitional stage occurs where the hard callus replaces the cartilaginous callus. From a microscopic perspective, this transitional phase is closely linked to chondrocytes' differentiation, senescence, and transdifferentiation into osteoblasts. Experimental evidence demonstrates that Piezo1's function in chondrocytes also impacts this critical conversion. Specific deletion of Piezo1 in chondrocytes accelerates their senescence, delaying the transition from cartilage to bone [41]. This occurs because Piezo1 deficiency leads to substantial accumulation of ApoE within cells, causing heterochromatin disruption in chondrocytes and thereby accelerating their senescence and apoptosis (Fig. 3) [42]. These alterations may indirectly influence chondrocyte-to-osteoblast transdifferentiation, ultimately affecting cartilage-to-bone conversion.

Beyond senescence, Piezo1 deficiency also inflicts multiple deleterious effects on chondrocyte mitochondria, the powerhouses essential for endochondral ossification. Subsequent studies provided deeper insights: while Piezo1 does not directly mediate chondrocyte-to-osteoblast transdifferentiation, its specific absence in chondrocytes impairs mitochondrial function. This manifests as reduced membrane potential, decreased ATP synthesis, lowered oxygen consumption rate, and increased mitochondrial superoxide production, thereby impairing the cartilage-to-bone transition during fracture healing (Fig. 3) [2]. Mechanistically, Piezo1 in chondrocytes connects to the downstream β-catenin signaling pathway, thereby influencing Lars2 expression (Fig. 3) [2]. Knockout of Piezo1 elevates Lars2 expression, impairing mitochondrial function and ultimately impairing endochondral ossification during fracture healing [2]. Intriguingly, mitochondrial dysfunction in this context is not solely a metabolic consequence. Recent findings reveal a parallel inflammatory axis where Piezo1 deletion drastically upregulates Fstl1, a pro-inflammatory protein, which in turn activates NF-κB signaling within chondrocytes [43]. This autonomous inflammatory cascade triggers a burst of reactive oxygen species (ROS), directly provoking mitochondrial oxidative stress, opening the mitochondrial permeability transition pore (mPTP), and disrupting mitochondrial dynamics (Fig. 3). Therefore, the impairment of cartilage-to-bone conversion arises from a synergy between metabolic reprogramming (via β-catenin/LARS2) and oxidative/inflammatory damage (via Fstl1/NF-κB), both converging to cripple the bioenergetic capacity required for chondrocyte-to-osteoblast transdifferentiation [2,43].

4.3. Piezo1 in macrophages: a mechano-immune hub across inflammatory and regenerative phases

Beyond its well-characterized roles in chondrocytes, osteoblasts, and skeletal progenitors, increasing evidence highlights Piezo1 as a critical mechanosensor in immune cells, especially macrophages, during bone repair and remodeling. Macrophages are key orchestrators of both the inflammatory and regenerative phases of fracture healing, and Piezo1 mediates their responses to mechanical stimuli in a context-dependent manner, thereby influencing multiple stages of the repair process.

First, mechanical force activates Piezo1 in macrophages, triggering Ca²⁺ influx and subsequent upregulation of Z-DNA binding protein 1 (ZBP1). This Piezo1--ZBP1 axis drives a pro-inflammatory response, releasing IL-1β and TNF-α, which promotes osteoclastogenesis and bone resorption. Interestingly, in a femoral fracture model, macrophage-specific deletion of Piezo1 accelerated healing by reducing osteoclast numbers and shifting the balance toward bone formation; however, the long-term consequences of pharmacological inhibition of this pathway remain to be fully elucidated (Fig. 3) [44]. Second, mechanical loading stimulates Piezo1 in macrophages to secrete small extracellular vesicles (sEVs) enriched with specific microRNAs, such as let-7g-5p. These sEVs target Adrb2 in osteoblasts, suppressing β2-adrenergic receptor signaling and thereby preserving osteogenic differentiation and activity, which favors fracture repair (Fig. 3) [45]. Third, in a tibial defect model, mechanical loading via Piezo1 significantly increased the proportion of CD206⁺ regenerative (M2-like) macrophages while reducing iNOS⁺ inflammatory (M1-like) macrophages within the defect, promoting angiogenesis--osteogenesis coupling and cell proliferation. Conversely, macrophage-specific Piezo1 knockout impaired these loading-induced effects, confirming that Piezo1 is an indispensable molecular switch for mechanical stimulation to drive a pro-repair macrophage phenotype (Fig. 3) [46].

Importantly, the functional outcome of Piezo1 activation in macrophages is highly dependent on the local microenvironment. In non-inflammatory regenerative settings, Piezo1 promotes M2 polarization and tissue repair; however, in inflammatory conditions Piezo1 upregulation can instead drive M1 polarization, exacerbate inflammation, and impair osteogenic differentiation of mesenchymal stem cells. Collectively, these findings establish macrophage Piezo1 as a central mechano-immune hub that integrates physical signals with inflammatory and osteogenic responses. Targeting this pathway holds therapeutic promise for modulating fracture healing, but requires careful consideration of the inflammatory context and timing to avoid unintended outcomes.

4.4. Integrative summary: Piezo1 functions across the sequential phases of fracture healing

Macrophages persist across multiple phases of fracture healing, with their M1/M2 polarization states dynamically shifting in response to the local microenvironment. This functional plasticity shapes both the early inflammatory response and subsequent callus formation (Fig. 3). Piezo1 in periosteal stem cells primarily operates during the cartilaginous callus phase, driving their migration and chondrogenic differentiation to establish the cartilaginous template (Fig. 3). Piezo1 in chondrocytes exerts its dominant effect during the hard callus formation and cartilage-to-bone transition, supporting mitochondrial homeostasis and energy metabolism to sustain the biosynthetic capacity required for chondrocyte-to-osteoblast transdifferentiation (Fig. 3). Importantly, these cell types are not strictly confined to a single healing phase; rather, they also exert critical functions at the transitional interfaces between phases, where their overlapping and synergistic actions ensure the continuous and orderly progression of fracture repair. Nevertheless, several key questions remain—whether Piezo1 directly regulates the initiation factors of chondrocyte transdifferentiation, how it precisely modulates macrophage polarization, and how the dynamic balance between M1 and M2 macrophages is achieved across different phases—all of which warrant further investigation.

5. Novel therapeutic strategy targeting Piezo1 for bone growth

Targeting Piezo1 for bone regeneration has evolved from purely mechanical concepts into a multidisciplinary field encompassing physical therapy, biomaterial engineering, and pharmacology. Although no Piezo1-targeted drug has yet entered clinical trials for bone repair, substantial preclinical evidence supports the therapeutic potential of modulating this mechanosensitive channel. This section summarizes current strategies according to their primary intervention modality—physical factors, biomaterials, and pharmacological agents—and concludes with a critical discussion of translational barriers that are common across all approaches.

5.1. Physical factor-based therapies

Physical modalities represent the most clinically advanced approaches, owing to their non-invasiveness, established safety profiles, and direct engagement of Piezo1 as a native mechanotransducer.

Ultrasound-based interventions. Ultrasound, as a mechanical acoustic radiation force, is intrinsically suited to activate Piezo1. Low-intensity pulsed ultrasound (LIPUS) has already received FDA approval for non-invasive bone repair. Mechanistically, LIPUS promotes calcium influx via Piezo1 in MC3T3-E1 osteoblasts, leading to ERK1/2 phosphorylation and perinuclear F-actin aggregation [35]. In zebrafish fin fracture models, osteocytes have been identified as primary targets of LIPUS [47]; however, whether this osteocyte-centric mechanism operates equivalently in mammals remains to be confirmed. Beyond LIPUS, higher-intensity pulsed ultrasound has also been demonstrated to accelerate tibial fracture healing in mice via Piezo1 [48], suggesting a broader therapeutic window than previously appreciated. In parallel, ultrasound can be combined with piezoelectric biomaterials to generate localized electrical cues—for instance, a BaTiO₃/multi-walled carbon nanotube/collagen (BMC) biomimetic periosteum [49] and a polydopamine-modified black phosphorus/decellularized adipose tissue (pDA-mBP@DAT) scaffold [50] both deliver multiple physical signals (mechanical, thermal, and electrical) upon LIPUS exposure, converging on Piezo1 activation. These studies illustrate that ultrasound is not merely a standalone therapy but also a versatile “remote control” for smart biomaterials.

Hyperbaric oxygen (HBO) therapy. HBO therapy has long been used for ischemic conditions, and recent evidence links its bone-regenerative effects to Piezo1. In a mouse tibial defect model, HBO treatment promoted osteogenic differentiation of progenitor cells through the Piezo1/Yap/Runx2 axis [51]. This finding expands the repertoire of physical stimuli engaging Piezo1 beyond direct mechanical forces, suggesting oxygen tension as an upstream regulator. Nevertheless, the molecular crosstalk between hyperoxia and Piezo1 gating remains poorly understood, and optimal pressure/duration protocols for bone repair have yet to be standardized.

5.2. Biomaterial-based microenvironmental regulation and delivery systems

Biomaterials offer an alternative to direct physical energy application by modulating Piezo1 signaling through alterations in the metabolic or mechanical microenvironment, or via smart delivery vehicles. These approaches offer spatiotemporal control and can be tailored to different healing phases. According to their mode of action, current strategies can be classified into three categories: implant surface modifications, mechano-active hydrogels, and targeted delivery platforms.

Implant coatings that remodel the metabolic microenvironment. Pei et al. engineered single-atom catalytic coatings (VN₄/RhN₄) on titanium and stainless-steel implants. These coatings sustain lactate dehydrogenase- and NADH oxidase-like activities for over two years, reshaping the local metabolic milieu to indirectly activate Piezo1 and promote osteogenic differentiation [52]. This strategy exploits the metabolic sensitivity of Piezo1 without pharmacological agonists, yet the long-term retention of metal single atoms and their potential systemic toxicity require careful evaluation [52].

Mechano-responsive hydrogels. Yang et al. fabricated a NIR-II-responsive NIPAM/Nb₂C hydrogel that undergoes reversible shrinkage/swelling upon photothermal stimulation, generating controlled micromotion (optimised at ∼1/300 Hz) that directly activates Piezo1 in mesenchymal stem cells and enhances bone formation in both calvarial and femoral defects [53]. While remote near-infrared control is attractive, tissue penetration depth and repeated irradiation safety are practical concerns for deep fractures.

Targeted delivery platforms – gene knockdown and small-molecule controlled release. Zhang et al. constructed a hyaluronic acid-phenylboronic acid/tannic acid (HA-PBA/TA) self-healing hydrogel loaded with chondrocyte-targeting lipid nanoparticles encapsulating Fstl1 shRNA. Local injection suppresses Fstl1 expression, attenuates inflammation, reduces mitochondrial oxidative stress, and facilitates endochondral ossification [43]. Gan et al. developed two smart delivery systems: an oral yeast microcapsule (YC) that packages SRT2104 or resveratrol and exploits M-cell transcytosis and macrophage-mediated inflammatory homing for fracture-site-specific targeting; and an injectable gelatin-dopamine hydrogel (RSV@GelDa) that provides sustained local release for up to 28 days [54]. These platforms bridge systemic administration and local action, yet batch-to-batch reproducibility of yeast microcapsules and the degradation profile of gelatin-dopamine matrices require further optimisation.

5.3. Pharmacological modulation of the Piezo1 signalling network

Pharmacological agents offer the advantage of defined molecular targets and systemic bioavailability, acting either directly on Piezo1 or on its upstream modifiers and downstream effectors.

Direct agonists, antagonists, and natural product binders. Beyond the widely used synthetic agonist Yoda1 and antagonist GsMTx4, Dong et al. identified Asperosaponin Ⅵ (ASP) as a direct Piezo1 binder via molecular docking, Cellular Thermal Shift Assay (CETSA) and Drug Affinity Responsive Target Stability (DARTS). ASP activates Piezo1 and its downstream ERK1/2–YAP–VEGF axis, promoting osteogenic–angiogenic coupling in LEPR⁺ bone marrow stromal cells and endothelial cells, thus accelerating osteoporotic fracture healing [55]. The natural origin of ASP may offer a favorable safety profile, but its selectivity for Piezo1 over other mechanosensitive channels has not been rigorously quantified.

Post-translational regulation via Sirt1-mediated deacetylation. Gan et al. discovered that Sirt1 directly binds to and deacetylates Piezo1 at lysine residue K2174, and that Sirt1 activators (SRT2104 and resveratrol) potently enhance Piezo1-mediated calcium influx [54]. This post-translational mechanism represents a previously unrecognised route for Piezo1 activation. Oral or local delivery of these agents significantly improves fracture repair in mice, an effect abolished by chondrocyte-specific Piezo1 deletion [54]. This finding opens new avenues using clinically tested Sirt1 activators, but their pleiotropic effects on other Sirt1 substrates complicate the interpretation of bone-specific outcomes.

Downstream effector blockade. Genetic or pharmacological silencing of downstream mediators has proven effective in rescuing Piezo1-deficiency phenotypes. Jia et al. designed a thermosensitive injectable gellan gum hydrogel that releases an ApoE antagonist, reversing Piezo1-deficiency-induced chondrocyte senescence and restoring cartilage-to-bone transition [41]. Similarly, the Fstl1-silencing system described above (Section 5.2) targets a downstream inflammatory mediator [43]. These indirect approaches may avoid the pitfalls of directly modulating the channel (e.g., off-target effects, desensitisation), but they require a deep understanding of tissue- and stage-specific downstream networks. Moreover, Zhu et al. identified JNJ-10311795 as a ZBP1 modulator through virtual screening targeting the Zα domain [44]. By inhibiting ZBP1, this compound blocks Piezo1-driven pro-inflammatory cytokine release and osteoclastogenesis, enabling bidirectional modulation of bone remodeling—slowing orthodontic tooth movement while accelerating fracture healing [44]. This dual functionality illustrates the complexity of targeting Piezo1-associated inflammatory cascades, as the desired outcome may vary according to the clinical context.

5.4. Common challenges and future directions for clinical translation

Despite the encouraging preclinical data, several universal hurdles impede the clinical adoption of Piezo1-targeted therapies across all three categories. First, the cell-type-dependent functional duality of Piezo1—pro-osteogenic in osteoblastic progenitors but pro-inflammatory in macrophages—demands precise cell-type-specific targeting, a goal that remains technically challenging to achieve. Second, the stage-specific requirements during endochondral ossification imply that Piezo1 activation may be beneficial at callus formation but detrimental during early stage of inflammation; temporal control of drug/material release is therefore critical but challenging. Third, interspecies differences between rodents and humans in Piezo1 expression levels, splice variants, and associated signalling partners cast doubt on direct translatability of current murine data. Fourth, long-term safety concerns—metal nanoparticle retention, multi-target drug effects (e.g., SRT2104 affecting multiple Sirt1 substrates), and repeated administration of biological delivery systems—have not been adequately addressed in large animal models. Finally, the manufacturing complexity of multi-component hydrogels, yeast microcapsules, and piezoelectric scaffolds poses significant hurdles in GMP compliance and batch consistency.

In summary, the current landscape offers a rich toolkit—from physical energy and smart materials to small molecules—for fine-tuning Piezo1 activity. However, no single strategy is universally superior; the optimal choice will likely depend on fracture type, patient age, and co-morbidities. Future research should prioritize head-to-head comparisons of different modalities in clinically relevant large-animal models, develop real-time monitoring techniques for Piezo1 activity in vivo, and design adaptive delivery systems that respond to the dynamic healing milieu. Only through such integrative efforts can Piezo1-targeted therapies move from bench to bedside.

6. Discussion

Bone formation is a complex and finely orchestrated process involving multiple cell types and intricate interactions among various factors, which underlies its remarkable regenerative capacity. Currently, bone biology research has entered a more refined and subdivided phase. Piezo1 plays a pivotal role in mediating the involvement of various cell types in osteogenesis, influencing both longitudinal and transverse skeletal growth, as well as the development of trabecular bone density and cortical bone thickness. Piezo1 is expressed in multiple skeletal cell types, collectively regulating bone development. Based on studies of physiological bone formation, using endochondral ossification as a reference template to investigate fracture healing mechanisms has become an important concept in current fracture research. Macrophages, periosteal progenitor cells, chondrocytes, and others have been found to influence fracture healing through Piezo1-mediated biological changes. Activation of Piezo1 in most cells favors the maintenance or enhancement of anabolic bone formation during fracture healing, whereas in a few cell types, such as macrophages, Piezo1 activation enhances osteoclastogenesis, thereby delaying healing. Against this backdrop of research on Piezo1-mediated mechanotransduction in both physiological osteogenesis and pathological fracture repair, the development of therapeutic strategies targeting Piezo1 is showing diversified trends. With substantial basic and translational research efforts, many mechanisms are gradually being elucidated, while new questions and challenges are also emerging.

6.1. Regarding experimental models

In current studies, whether in physiological osteogenesis or pathological fracture repair, mice with specific Piezo1 knockout are frequently used as key in vivo experimental subjects, employing genetic engineering tools such as Cre-driver systems for gene ablation. Data obtained from such mice are widely considered robust and provide critical mechanistic insights. However, the studies reviewed here also reveal several potential caveats of the Cre-loxP system that must be critically examined when interpreting these transgenic models. First, incomplete recombination efficiency is a common pitfall: as noted by Sun et al., Ocn-Cre achieves only approximately 88% excision efficiency, and residual Piezo1 function may mask true dose-dependent effects [32]. Second, the broad targeting range of Prrx1-Cre [25] complicates the interpretation, as it deletes Piezo1/2 not only in osteoblasts but also in cartilage, tendon, and connective tissue progenitors within the limb mesenchyme, making it difficult to attribute the bone defects solely to osteoblast dysfunction. Finally, the tamoxifen administration window in inducible systems (often occurring during the acute inflammatory phase of fracture healing) may independently interfere with healing dynamics. Consequently, future loss-of-function studies should prioritize spatially restricted dual-recombinase systems (e.g., Dre-rox combined with Cre-loxP) coupled with delayed induction strategies. Such a strategy ensures cell-type-specific ablation of Piezo1 and, crucially, preserves the fidelity of fracture healing readouts by segregating genuine mechanosensing responses from developmental or drug-induced artifacts.

In transgenic mice with cell-type-specific Piezo1 knockout, although many develop spontaneous fractures, the fracture locations vary considerably. A substantial body of experimental evidence demonstrates that Piezo1 deletion in different cell types results in divergent skeletal phenotypes. For example, in Col2a1-Cre transgenic mice, Piezo1 deficiency profoundly affects secondary trabecular bone formation, leading to spontaneous fractures near the rib growth plate; in contrast, Piezo1 deletion in osteoblasts primarily manifests as cortical bone thinning. Moreover, Piezo1 deficiency also affects bone length during osteogenesis, thereby disturbing axial skeletal development. This suggests that different cell types contribute variably to bone phenotypes at distinct stages of osteogenesis. Even within the same cell type, their roles may differ across regions of long bones due to variations in the local microenvironment. In future studies of physiological bone formation, when comparing skeletal phenotypes, both axial and transverse bone growth should be considered as important parameters, rather than focusing solely on bone mass or bone mineral density.

Secondly, the anatomic site-specificity of current fracture models may constrain in-depth mechanistic exploration. Vast majority of fracture healing studies employ rodent tibial or femoral mid-diaphyseal fracture models, and these anatomical sites possess abundant blood supply and exhibit relatively stable healing. However, physiological osteogenesis and pathological bone repair studies have unequivocally shown that bone formation mechanisms differ substantially across anatomical sites—diaphysis, metaphysis, and epiphysis. Such site-specific mechanistic differences imply that pro-healing conclusions drawn from diaphyseal models may not be directly extrapolated to metaphyseal or epiphyseal fractures. Bones at different anatomical sites differ in embryonic origin, blood supply patterns, loading regimes, and cellular composition, all of which may profoundly influence Piezo1-mediated mechanoresponses.

Therefore, the development of refined animal models represents an urgent technical bottleneck. To achieve deeper mechanistic insights into bone formation and repair, it is essential to establish standardized fractures at distinct sites—diaphysis, metaphysis, and epiphysis—with corresponding fixation methods. However, this poses a substantial technical challenge, especially in small animals like mice: the metaphyseal/epiphyseal regions are spatially confined and anatomically complex, making precise fracture creation and subsequent stable fixation extremely difficult. Different sites require different fixation strategies—intramedullary pins or external fixators for diaphyseal fractures, whereas metaphyseal/epiphyseal fractures may require more sophisticated screw or plate fixation, which is technically demanding at a miniature scale. Overcoming this bottleneck will rely on innovations in micro-surgical instruments, the development of 3D-printed patient-specific fixation devices, and multidisciplinary collaboration.

6.2. Regarding clinical translation

Clinical translation should target challenging orthopedic problems rather than merely validating known efficacy. Most current Piezo1-related studies focus on standard fracture models (e.g., murine mid-femoral fractures), yet mechanistic exploration of truly difficult clinical situations—such as delayed fracture healing, non-union, and fractures in areas with poor blood supply (e.g., talus, scaphoid, femoral head)—remains extremely limited. This is partly constrained by the challenges of establishing site-specific fracture models mentioned above. Hence, future studies should consciously orient animal model design toward these clinically difficult issues, rather than perpetuating validation of already known concepts. Moreover, even when evaluating potential therapeutics in standard fracture models, the field must confront a critical temporal and mechanistic caveat. In the study by Zhu et al., although the small-molecule compound successfully inhibited Piezo1 activation and accelerated early callus formation, the authors limited their observation window to early time points without extended longitudinal follow-up into the remodeling phase [44]. This highlights a fundamental concern: pharmacological perturbation of Piezo1-mediated mechanotransduction, whether through agonists or inhibitors, inevitably disrupts the dynamic equilibrium between osteoblast-mediated bone formation and osteoclast-mediated resorption. While such tilting may yield transiently high bone volume and rapid radiographic bridging, it does not guarantee superior mechanical properties or functional recovery in the long run. Given that the dynamic interplay between these two cell types and their microenvironment is highly complex, single-agent strategies that override this equilibrium demand rigorous long-term biomechanical and histomorphometric validation—especially before being extrapolated to delayed unions or poorly vascularized fractures, where the biological milieu is even more precarious.

Furthermore, based on studies of physiological osteogenesis and pathological bone repair, it is evident that Piezo1 activation in different cell surfaces elicits distinct intracellular and extracellular cascades. This heterogeneity has novel implications for therapeutic development. When investigating the microscopic mechanisms of therapeutic approaches, one should first avoid single-pathway interpretations, as the mechanisms are complex and the responsive cell types are not singular. Second, the timing of intervention is critical—for example, in fracture healing, excessive Piezo1 activation during the inflammatory-immune phase may be counterproductive, as it can trigger macrophage Piezo1-mediated osteoclastogenesis and delay healing. In contrast, Piezo1 activation during the transition from cartilaginous to hard callus may exert more pronounced pro-healing effects.

7. Conclusion

Currently, research directions on Piezo1 in osteogenesis and fracture healing are diverse, and mechanistic exploration has progressed substantially. A relatively comprehensive understanding of Piezo1's role in the skeletal system and related diseases has been achieved, with numerous studies advancing the field considerably. At the same time, new questions and challenges continue to emerge. To address current knowledge gaps and promote clinical translation, future research should focus on the following areas: (1) developing site-specific fracture models to systematically compare Piezo1 mechanisms in diaphyseal, metaphyseal, and epiphyseal fracture healing; (2) anchoring on challenging clinical problems by establishing animal models tailored to non-union and fractures in poorly vascularized sites, rather than relying solely on standard healing models; (3) extending observation windows to complete remodeling (e.g., 6–8 weeks post-surgery) and incorporating three-point bending or torsional mechanical testing to assess callus quality, rather than relying only on micro-CT-derived bone volume parameters; (4) integrating single-cell sequencing and spatial transcriptomics to dissect the real-time dynamics and signaling networks of Piezo1 across different sites and cell types; and (5) promoting multi-center, standardized sharing of animal experimental data to build a comprehensive Piezo1 phenotypic database covering diverse strains, ages, sexes, and skeletal sites, thereby laying the groundwork for a “mechanistic map” of bone biology. Only through such systematic model innovation, mechanistic exploration, and data integration can fundamental findings on Piezo1 be effectively translated into precision therapeutic strategies for challenging orthopedic problems, ultimately improving long-term outcomes for fracture patients.

CRediT authorship contribution statement

Rongrui Wu: Writing – review & editing, Writing – original draft, Data curation, Conceptualization. Yan Zhuang: Writing – review & editing, Writing – original draft, Conceptualization. Hui Yang: Writing – review & editing, Writing – original draft. Qi Dong: Writing – review & editing, Writing – original draft. Shilin Wang: Writing – review & editing, Writing – original draft. Jierong Zhang: Writing – review & editing, Writing – original draft, Conceptualization. Yunqiang Chen: Writing – review & editing, Writing – original draft, Methodology, Data curation, Conceptualization.

Declaration of generative AI and AI-assisted technologies in the manuscript preparation process

During the preparation of this work, the authors used DeepSeek(https://chat.deepseek.com/) solely for the purpose of language polishing and grammar correction. After using this tool, the authors reviewed and edited the content as necessary and take full responsibility for the final content of the publication.

Funding

This work was supported by the Sanya Science and Technology Fanxing Special Research Project (grant number 2024KJFX077, 2024); the Key Project of Scientific Research in Higher Education Institutions of Hainan Province (grant number Hnky2025ZD-8, 2025); Hainan Medical University 2025 Academic Advancement Initiative (grant number XSTS2025012), and the Hainan Province Clinical Medical Center (Sanya TCM Hospital).

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.

Data availability

The data used in this article are sourced from materials mentioned in the References section.