2.1 Osteoblastogenesis

Osteoblasts, which are derived from bone marrow mesenchymal stem cells (BMSCs), are responsible for bone formation¹⁸ (Figure 1). The development and differentiation of osteoblasts are regulated by a series of signaling pathways and the activation of transcription factors.⁷ Given the multilineage differentiation potential of BMSCs, their initial differentiation step involves committing to a common osteochondroprogenitor cell, triggered by the activation of key osteogenic transcription factors, including drosophila distal-less 5 (DLX5), Osterix (OSX) and RUNX2. This commitment leads to the formation of an osteoprogenitor cell, which then progresses to a preosteoblast through the expression of initial osteogenic genes, including alkaline phosphatase (ALP) and COL1A1, and the expression of these genes is maintained throughout the lifespan of mature osteoblasts. Moreover, mature osteoblasts secrete several extracellular proteins essential for bone formation, including COL1, osteocalcin (OCN), bone sialoprotein II (BSP II), and osteopontin (OPN). The extracellular matrix (ECM), which is initially produced as a nonmineralized osteoid, eventually undergoes mineralization through the accumulation of calcium phosphate to form hydroxyapatite. After osteoblasts deposit bone where necessary, they can end up in one of three states: 60–80% of osteoblasts undergo apoptosis; transform into bone lining cells; or become osteocytes.¹⁹

2.2 Osteoclastogenesis

Osteoclasts, which originate from hematopoietic progenitors within the monocyte lineage, exhibit a distinctive capacity for bone resorption, rendering them essential for the maintenance of bone homeostasis²⁰ (Figure 1). Osteoclast precursors (pOCs) are guided to the bone resorption surface from the bone marrow and circulation through sphingosine 1-phosphate signaling. Subsequently, pOCs fuse into multinucleated osteoclasts (mOCs), which are associated with the availability of RANKL and macrophage colony-stimulating factor (M-CSF).²¹ Upon these stimuli, a wide array of signaling cascades, including both canonical and noncanonical NF-κB pathways, MAPK pathways, calcium signaling pathways, and PI3K/AKT pathways, are activated.²² These signaling cascades mobilize a complex transcriptional network that upregulates osteoclast-promoting factors such as NF-κB, activator protein 1 (AP-1), cAMP-response element binding protein (CREB), and B-lymphocyte-induced maturation protein 1, and downregulating osteoclast differentiation suppressors such as interferon regulatory factor 8, MAF BZIP transcription factor B, and B-cell lymphoma 6 (BCL-6), which coordinately induces the expression of nuclear factors that are the “master regulators” of activated T-cell c1 (NFATc1). As a crucial transcription factor, NFATc1 enhances osteoclast formation by increasing the transcription of genes associated with bone resorption, including matrix metalloproteinase 9 (MMP9), cathepsin K (CTSK), acid phosphatase 5, and tartrate-resistant acid phosphatase (TRAP).²¹ In addition, mOCs are specialized cells that exhibit polarity and adhere to the bone surface, creating resorption lacuna by secreting lysosomal proteases, including MMPs, CTSK, and TRAP, in which hydrochloric acid is actively secreted, leading to the dissolution of bone mineral hydroxyapatite.²³ In addition to RANK/RANKL and M-CSF, other factors, such as the Wnt pathway, FGF signaling pathway, and transforming growth factor beta (TGF-β) pathway, have been shown to participate in osteoclastogenesis.²⁴

2.3 Osteocytes

Osteocytes, matured from OBs, are the main component of the skeleton that maintains the bone tissue environment and contributes to the osteoblasts and osteoclasts regulation during bone remodeling.²⁵ Osteocytes release RANKL to induce osteoclast differentiation and OPG to inhibit osteoclast formation.²⁶ In addition, osteocytes also release FGF-23, BMP, and sclerostin (SOST) to regulate osteoblast activity. These functions largely depend on endocrine, paracrine, and autocrine mechanisms to regulate bone homeostasis.²⁷ In addition, as mechanosensors of bone, osteocytes sense mechanical signals and transduce them into specific ion channel signaling.²⁸ Osteocytes embed in bone tissues, forming a complex network within the bone structure and maintaining close communication with the bloodstream and other bone cells. The recently identified signaling pathway of osteocytes presents numerous possibilities for treating different bone disorders.

2.4 Equilibrium and communication of bone homeostasis

The equilibrium between bone anabolism and catabolism is crucial to the maintenance of bone homeostasis.²⁹ Bone resorption and formation normally follow each other rather than mutually independent processes. The switch between the osteoblastic and osteoclastic phases is regulated by direct cell-to-cell contact.³⁰ EphrinB2–EphB4 signaling involves communication between osteoblasts and osteoclasts.³¹ EphrinB2, a transmembrane protein on osteoclasts, which can bind with the receptor EphB4 on osteoblast membranes, activating signaling pathways in both osteoblasts and osteoclasts.²¹ In osteoblasts, EphB4 promotes osteogenic differentiation and suppresses osteoblast apoptosis. In osteoclasts, reversing EphrinB2 signaling can inhibit osteoclastogenesis by downregulating AP-1 and NFATc1 expression.³²

Furthermore, FAS (CD95) and FAS ligand (FAS–FASL) signaling has recently been shown to be involved in postmenopausal osteoporosis.³³ FAS is a death domain-containing member of the TNFR superfamily, and FASL mediate apoptosis upon binding to FAS. Estrogen deficiency decreases FASL expression in osteoblasts, leading to decreased osteoclast apoptosis and increased bone resorption. Estrogen deficiency decreases FASL expression in osteoblasts, resulting in reduced osteoclast apoptosis and increased bone resorption. In addition, cell vesicle communication has been shown to contribute to this transition.²

In addition, extracellular vesicles (EVs) are also involved in communication between osteoblasts and osteoclasts. Small osteoclast vesicles are osteoblast-derived EVs that inhibit osteoblast differentiation by inhibiting RUNX2 and enhancing RANKL expression, which induces osteoclast differentiation.³⁴ Although direct modes of conversation, such as cell-to-cell direct contact or cell vesicle communication between osteoblasts and osteoclasts, already exist locally to regulate bone homeostatic balance, several signaling pathways are involved in more complex bone homeostatic homeostasis, as we will discuss in the next section.

3.1 RANKL/RANK/OPG signaling system

The RANKL/RANK/OPG signaling system function as pivotal molecules in bone homeostasis.²¹ Produced by osteocytes, osteoblasts, and BMSCs, RANKL is a member of the tumor necrosis factor (TNF) family and is characterized as a trimeric transmembrane protein.³⁶ RANK, a type I transmembrane protein, is found on osteoclast progenitors, mature osteoclasts, and some immune cells. The interaction between RANKL in osteoblasts and RANK on osteoclast progenitors triggers a series of intracellular signals via TNF receptor associated factor 6 (TRAF6), which engages pathways such as the MAPK, NF-κB, and PI3K pathways. This cascade amplifies NFATc1 expression, fostering osteoclastogenesis and bone resorption. Interestingly, while RANK was traditionally viewed as the exclusive receptor for RANKL, research by Luo et al.³⁷ revealed that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) is another receptor for RANKL. This receptor competes with RANK for RANKL, hindering the typical RANK signaling pathway involved in osteoclast differentiation. The interaction of RANKL with LGR4 stimulates the G protein subunit alpha Q (Gαq) and glycogen synthase kinase 3 beta (GSK-3β) pathways, consequently diminishing NFATc1 expression and function in osteoclastogenesis.³⁷

Furthermore, RANKL is also a receptor in osteoblasts and activates an intracellular “reverse” signaling pathway upon binding with RANK-RANKL, which further promotes osteoblast differentiation and mineralization. It was suggested that the activation of the PI3K/AKT/mechanistic target of rapamycin kinase (mTOR) pathway through the interplay between RANKL and Src family kinases leads to the entry of RUNX2 into the nucleus and increases the expression of markers of early differentiation. By acting as a decoy receptor for RANKL, OPG is produced by osteoblasts and blocks the interaction between RANKL and RANK, thus preventing the formation of osteoclasts and the subsequent breakdown of bone. OPG is regulated by many factors. For example, in vitro studies have demonstrated that estrogen increases OPG production by osteoblasts,³⁸ suggesting a link between estrogen withdrawal postmenopause and the development of osteoporosis due to increased OPG levels. In addition to estrogen, several signaling pathways and molecules, including cytokines such as IL-1β, IL-11, TNF-α, 1α,25-dihydroxyvitamin D3, PTH, and prostaglandin (PG), play indirect roles in bone metabolism by influencing the RANK/RANKL/OPG signaling cascade, primarily by promoting the expression of RANKL on the cell membrane, which in turn stimulates bone resorption³⁹ (Figure 2).

In addition, osteocytes, the major source of RANKL, produce RANKL in a membrane-bound and possibly soluble form and transmit RANKL signals in the bone matrix through their dendritic projections,⁴⁰ thereby communicating with pOCs in the bone marrow and periosteal regions to further promote osteoclast differentiation and activity. Mechanical stress enhances RANKL expression in osteoblasts through the NF-κB pathway.⁴¹ PTH also upregulates RANKL expression in osteoblasts. In addition, osteocytes secrete OPG, which inhibits osteoclastogenesis and bone resorption. Thus, osteocytes regulate osteoclast activity and bone resorption directly through RANKL and indirectly through OPG.⁴² In addition, osteocytes detect microdamage in bone and initiate bone remodeling through signaling. Apoptotic osteocytes in the damaged area release ATP signals that activate the release of RANKL from nearby healthy osteocytes, a process that involves the activation of pannexin-1 (Panx1) and P2X7 receptors, which transmit signals to attract remodeling units.⁴³

3.2 Hormone and growth factor signaling pathways

3.2.1 PTH signaling pathway

PTH, the main secretory hormone, regulating calcium and phosphorus metabolism, and it is crucial for regulating bone homeostasis by influencing the activity of osteoblasts and osteoclasts.⁴⁴ Binding of PTH to PTH-receptor 1 (PTH1R) activates the G protein-coupled receptor (GPCR) signaling pathway, which promotes the production of cAMP, which acts as a second messenger and activates protein kinase A (PKA) in osteoblast lineage cells. The cAMP/PKA signaling pathway increases RUNX2 and OSX activity and expression, thereby promoting the differentiation and maturation of osteoblasts. In addition, it coordinates the activity of BMP, thereby promoting bone formation.⁴⁵ Specifically, the interaction between PTH and its receptor PTH1R triggers endocytosis of the PTH1R-low-density lipoprotein receptor-related protein 6 (LRP6) complex, resulting in enhanced downstream BMP/SMAD family member 1 (SMAD1) signaling. This cascade ultimately promotes the differentiation of BMSCs into osteoblasts.⁴⁶

PTH also indirectly activates the Wnt signaling pathway, increasing β-catenin stability and intranuclear aggregation and promoting osteoblast differentiation. This is achieved by inhibiting the SOST, a protein that inhibits Wnt signaling, expression. Furthermore, PTH increases RANKL expression in osteoblasts and BMSCs and decreases the expression of OPG, which promotes osteoclasts survival and differentiation, enhances osteoclast activity, and indirectly results in bone resorption.⁴⁷ Furthermore, PTH has also been shown to be associated with insulin-like growth factor 1 (IGF-1),⁴⁸ which is induced by PTH in mature osteoblasts and enables PTH to induce RANKL and M-CSF, promoting osteoclastogenesis. In addition, it has been shown that PTH exerts dual effects on bone formation and bone resorption, which depend on its mode of action.⁴⁹ Under sustained high doses of PTH, osteoclast activity exceeds that of osteoblasts, ultimately leading to greater bone loss than bone formation.⁵⁰

In addition to osteoblasts and osteoclasts, PTH significantly influences osteocytes. PTH targets osteocytes to regulate the expression of key genes such as SOST and RANKL. The expression of SOST, which encodes SOST, an inhibitor of bone formation, is downregulated by PTH, leading to increased bone formation. Moreover, PTH upregulates RANKL expression in osteocytes, which is essential for bone resorption by promoting osteoclast activity.⁵¹ Additionally, PTH affects osteocyte morphology and function, including perilacunar remodeling, which helps mobilize calcium from the bone matrix, particularly during physiological states like lactation. Osteocytes also exhibit rapid changes in response to PTH, indicating their role in immediate calcium regulation and bone remodeling dynamics⁵² (Figure 3).

3.3 Wnt signaling pathway

The Wnt signaling pathway is evolutionarily preserved and controls the maintenance of tissue homeostasis. The Wnt signaling pathway can be categorized into two main pathways: the canonical pathway (also termed the Wnt/β-catenin pathway), which relies on the activity of β-catenin, and the noncanonical pathway (consisting of the Wnt/PCP pathway and Wnt/Ca²⁺ pathway), which operates independently of the activity of β-catenin.¹²⁰ In the Wnt/β-catenin signaling pathway, upon binding of Wnt ligands to frizzled class receptor (FZD) and LRP5/6, the multiprotein complex known as the β-catenin “destruction complex” is mobilized to the cellular membrane through its interaction with FZD. Consequently, the complex undergoes a loss of function in degrading β-catenin. Subsequently, β-catenin is translocated to the nucleus where it facilitates the activation of target genes by engaging with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF).¹²¹ The canonical Wnt signaling pathway is crucial for osteoblast differentiation, bone development, bone homeostasis, and bone remodeling in living organisms.¹²² When different types of Wnt ligands (such as Wnt-1, Wnt-3a, Wnt-10a, and Wnt-10b¹²³) interact with receptors on the cell membrane, they initiate downstream gene expression and determine the specificity of the Wnt pathway.¹¹⁶ Traditionally, Wnt-10a, Wnt-10b, Wnt-1, and Wnt-6 suppress the differentiation of BMSCs to adipocytes and facilitate the differentiation of BMSCs to osteoblasts through the canonical Wnt pathway.¹²⁴ Wnt-3a, Wnt-4, and Wnt-7b activate osteoblast differentiation and mineralization through the Gαq/phospholipase C-β/protein kinase c delta (Gαq/11/PLCβ/PKCδ) pathway.⁷ Wnt-7a modulates osteoblast proliferation by influencing RUNX2 expression.¹²⁵ These Wnt ligands collectively establish a regulatory framework indispensable for the proper formation and functionality of osteoblasts.

According to traditional views, the Wnt signaling pathway also exerts a significant influence on energy metabolism within osteoblasts. Wnt/β-catenin regulates fatty acid β-oxidation through the activation of β-catenin, which is dependent on the activation of mTORC2 and mTORC1. These factors in turn regulate the use of glucose and glutamine, respectively.¹²⁶

In addition, extensive evidence supports the interplay between the Wnt signaling pathway and other signaling pathways, such as the BMP, TGF-β, FGF, Hh, Notch, and PDGF pathways, in regulating the gene network responsible for controlling osteoblast differentiation and bone formation.¹¹⁶ Activation of the canonical pathway leads to the transcription of BMP-2.¹²⁷ BMP-2 serves as a significant autocrine and paracrine growth factor, and its TCF/LEF-responsive component governs signals that initiate specific transcriptional programs necessary for bone formation. This mechanism facilitates the differentiation of mesenchymal precursor cells into mature osteoblasts.¹²⁸ Furthermore, stabilized β-catenin can enhance the activation of BMP-9-induced ALP and the expression of OCN and OPN in osteoblasts.¹²⁹ Similarly, the noncanonical pathways also play crucial roles in osteocyte migration and osteoblast differentiation.⁷ Wnt-1 accelerates bone fracture healing and enhances bone formation by activating the yes-associated protein (YAP)/BMP signaling pathway.¹³⁰ Wnt5a was shown to promote BMP-2-mediated osteoblast differentiation via a SMAD-independent pathway by activating nonclassical Wnt signaling through ROR2.¹³¹ Furthermore, the Wnt/β-catenin signaling pathway in osteoblasts and osteocytes indirectly inhibits the differentiation of osteoclasts and the resorption of bone by promoting the secretion of OPG.¹³²

In osteoclasts, the Wnt pathway has been shown to indirectly influence osteoclast formation by controlling the production of RANKL and OPG under normal physiological conditions.¹³³ Primarily, the classical pathway plays a pivotal role in osteoclastogenesis via β-catenin, which has varying impacts at different developmental stages. The activation of β-catenin in early precursors promotes their proliferation, yet at advanced stages, it impedes osteoclast formation.¹³⁴ In contrast to the spatial and temporal regulation of β-catenin by the nonclassical pathway, the nonclassical pathway has been shown to promote the differentiation of pOCs.¹³⁵ It also plays a crucial role in blocking NFATc1 transcription factor activation by RANKL. The administration of Wnt-3a triggers a rapid increase in cAMP levels, which leads to the phosphorylation and subsequent activation of PKA, culminating in the increased phosphorylation (and thus inactivation) of NFATc1¹³⁶ (Figure 4).

3.4 NF-κB signaling pathway

The NF-κB family, encompassing transcription factors crucial for skeletal cell differentiation, is pivotal for skeletal health.^{137, 138} The NF-κB signaling pathway can be categorized as the canonical and noncanonical pathways. In the canonical pathway, upon exposure to RANKL, TNF-α, IGF signaling, IκB kinase β (IKKβ) and IκB kinase γ (IKKγ, also known as NEMO) phosphorylate and degrade IκB kinase α (IKKα), resulting in the nuclear translocation of the NF-κB1 (p50) and RelA (p65) complex.¹³⁹ Conversely, in the noncanonical route, cytokine stimulation leads to TNF receptor associated factor 3 (TRAF3) degradation after binding to the cytokine receptor, stabilizing NF-κB-inducing kinase (NIK), continuously degraded through the TRAF3 interaction. This stabilization activates IKKα, prompting the conversion of NF-κB2 (p100) into its active RelB (p52) form and enabling RelB/p52 complexes to enter the nucleus.¹⁴⁰ However, not all TNF family cytokines can initiate this pathway; RANKL does, but TNF-α does not.¹⁴¹

In osteoclasts, activation of both pathways is instrumental for differentiation, longevity, and functionality.¹⁴² Mice lacking NF-κB p50 and p52 exhibit notable dwarfism, thickened hypertrophic chondrocyte layers, severe osteopetrosis, and a lack of osteoclasts.¹⁴³ Whereas NF-κB activation in osteoblasts can either stimulate or inhibit osteogenesis. For example, TNF-induced NF-κB activation suppresses bone formation by obstructing RUNX2, thus interfering with BMP signaling.¹⁴⁴ The activity of the IKK complex also hampers osteoblast functionality. A study involving IKKalpha knockdown in OSX⁺ cells reported no impact on osteolineage cells.¹⁴⁵ Conversely, inhibiting NIK increases p100 levels, enhancing bone creation and osteoblast count, while disrupting RelB bolsters bone formation.¹⁴⁶ Early osteogenesis is promoted by NF-κB activation, and p65/RelA is crucial for osteoblast functionality and survival. At low doses, TNF-α can promote osteogenesis through NF-κB activation.¹⁴⁷ These findings highlight that the role of NF-κB in osteoblast differentiation is complex and dependent on context.

Crosstalk between the noncanonical NF-κB pathway and other signaling pathways also plays an important regulatory role in osteoblasts and osteoclasts. TRAF3 serves to restrict the activation of GSK-3β induced by TGF-β1 (via phosphorylation at Tyr216) and the degradation of β-catenin in mesenchymal progenitor cells. This mechanism enables β-catenin to sustain osteoblast differentiation and promote the expression of OPG, thereby mitigating bone degradation. RelA and RelB enhance osteoblastic RANKL expression, promoting osteoclastogenesis and a self-sustaining cycle of bone destruction, TGF-β release, TRAF3 degradation, and NF-κB activation.¹⁴⁸

In osteocytes, NF-κB responds to mechanical stress by mediating the cellular response to bone loading and unloading.¹⁴⁹ Mechanical stress induces the activation of NF-κB in osteocytes, leading to the expression of genes such as RANKL, which is crucial for osteoclastogenesis. This process facilitates communication between osteocytes and other bone cells, coordinating bone remodeling. NF-κB activation in osteocytes also plays a role in regulating bone resorption and formation and maintaining bone homeostasis.⁴¹ Additionally, the interaction of NF-κB with other signaling molecules in osteocytes is critical for adapting bone structure to mechanical demands¹⁵⁰ (Figure 5).

3.5 PI3K/AKT signaling pathway

The PI3K/AKT signaling pathway regulates cellular biological processes, such as cell proliferation and metabolism, by activating PI3K and causing AKT phosphorylation.¹⁵¹ The major components of the PI3K/AKT signaling pathway include PI3K family and the AKT family (also known as protein kinase B family; PKB).¹⁵² PI3Ks are an important class of kinases that include three classes of molecules (I, II, III).¹⁵³ PI3K Class I includes two isoforms (IA and IB), which use phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2) as substrates, phosphorylating inositol ring position 3 of the substrate. PI3K Class I can be activated by both receptor tyrosine kinases (RTKs) and non-RTKs on the cell surface.¹⁵⁴ PI3K Class II has a C2 structural domain and mainly phosphorylates PI and PIP,¹⁵⁵ while Class III involves vacuolar protein sorting 34 homolog (Vps34) and p150, targeting PI and primarily concerned with cell growth and survival regulation.¹⁵⁶ AKT, a serine/threonine kinase, consists of approximately 480 amino acid residues, which is one of the major downstream effector molecules of PI3K and can be involved in the regulation of a variety of life activity processes by directly phosphorylating various transcription factors, such as NF-κB and mTOR.¹⁵⁷ The AKT molecule consists of a short carboxy-terminal regulatory domain, a PH domain and a central catalytic domain.¹⁵⁸ Among them, the PH structural domain mediates the process of membrane translocation after AKT activation¹⁵⁹; the catalytic structural domain contains an ATP-binding site¹⁶⁰; and the C-terminal regulatory domain is proline-rich and contains another phosphorylation site, Ser473.¹⁶¹ AKT family members include AKT1/PKBα, AKT2/PKBβ, and AKT3/PKBγ.¹⁶²

The PI3K/AKT signaling pathway is regulated by various factors, including RTK and PI3K for upstream regulation¹⁶³; phosphatase and tensin homolog and PKB for internal regulation¹⁶⁴; and mTORC1, GSK3, and BCL-2 for downstream regulation.¹⁶⁵ In recent years, studies have supported the involvement of the PI3K/AKT pathway in regulating bone homeostasis through various cells.¹⁶⁶ In osteoblasts, PI3K inhibits osteoblast apoptosis through the activation of AKT and further activates PI3K/AKT through Wnt-3a.¹⁶⁷ The PI3K/AKT pathway also plays an important role in osteoclast formation. Recently, several researchers have reported that Akt activation can limit osteoclast differentiation by activating the GSK-3β/NFATc1 signaling cascade.¹⁶⁸ Moreover, Karkache et al.¹⁶⁹ reported that AKT, a downstream target of PI3K, can mediate the proliferation and survival of RANKL- and/or M-CSF-stimulated osteoclast lineage cells. Similarly, Wang et al.¹⁷⁰ demonstrated that the PI3K/AKT signaling pathway is closely related to bone homeostasis through a network pharmacological study of the antiosteoporotic effect of dulcolax cyclen ether terpene glycosides, which is also in line with previously reported results. In conclusion, the PI3K/AKT signaling pathway can promote osteoclast differentiation and facilitate osteoblast apoptosis.

In addition, the PI3K signaling pathway in osteocytes is essential for their survival and function, particularly in response to mechanical loading. Mechanical stress activates the PI3K/Akt pathway in osteocytes, promoting their survival by inhibiting apoptosis.¹⁷¹ This activation also enhances the release of signaling molecules such as BMP-7, which protects osteocytes from glucocorticoid-induced apoptosis¹⁷² (Figure 5).

3.6 JAK/STAT signaling pathway

The JAK/STAT signaling pathway is involved in cell proliferation, differentiation, survival, and apoptosis and mediates immune dysregulation and tumorigenesis in organisms.¹⁷³ It consists of ligand‒receptor complexes, the JAK family and the STAT family.¹⁷⁴ Canonical JAK/STAT signaling is activated when cytokines (e.g., interferon, IL, etc.) bind to the plasma membrane receptor. It triggers a conformational change in the receptor and leads to dimerization, which further activates the activity of JAK.¹⁷⁵ Activated JAK further phosphorylates the receptor to create a docking site for STAT. JAK-phosphorylated STAT detaches, forming homodimers or heterodimers through SH2 domain-phosphotyrosine interactions, which then regulate target gene transcription.¹⁷⁶ In addition, previous studies have demonstrated that the JAK/STAT signaling pathway is also involved in complex nonclassical signaling pathways, interconjugating with other signaling pathways, such as the MAPK/ERK and PI3K/AKT/mTOR pathways, to mediate a series of biological effects.¹⁷⁷ Increasing evidence indicates that JAKs and STATs are responsible for signaling via multiple hormones, growth factors, and cytokines.¹⁷⁸ Therefore, they are associated with the proliferation and differentiation of osteoblasts and osteoclasts, which is crucial for skeletal development and bone homeostasis.¹⁷⁹

First, osteoclast formation is usually stimulated indirectly through multiple pathways via the actions of JAK1 and STAT3 in inflammatory and subsidiary cells.¹⁸⁰ Additionally, osteoblast differentiation is secondary to stimulated osteoclast formation through IL-6 family cytokines.¹⁸¹ IL-6 can activate STAT3 signaling within osteoblasts, leading to the upregulation of various factors, including RANKL and C-X-C motif chemokine ligand 1. These factors play a crucial role in initiating pOC cells to differentiate into osteoclasts and initiate the process of bone resorption.¹⁸² In addition, STAT3 signaling also promotes bone formation through direct signaling in osteocytes, mainly by stimulating the expression of transcription factors (e.g., C/EBPδ and C/EBPβ) to induce osteoblast differentiation.¹⁸³ In conclusion, normal bone remodeling and bone homeostasis depend on the JAK1/STAT3/suppressor of cytokine signaling 3 (SOCS3) signaling pathway.¹⁸⁰ Moreover, the JAK2/STAT5B pathway is also fundamental for osteoblast formation,¹⁸⁴ as it acts on important transcription factors, such as T box transcription factor 3, RUNX2, and BMP-7,¹⁸⁵ which further illustrates the role of JAKs and STATs in bone conversion under inflammatory conditions. JAK inhibition increases the expression of OCN and the Wnt signaling pathway (by stabilizing β-catenin).¹⁸⁶

Furthermore, osteocytes are regulated by factors such as LIF and subsequent activation of LIFR, which are modulated by the JAK/STAT pathway. This pathway supports the expression of SOST and other osteocyte-specific genes that control bone mineral density and structural integrity. Through miR-30 and its effect on RUNX2, the pathway indirectly influences osteocyte maturation and function, highlighting its critical role in skeletal biology and disease processes such as osteoporosis^{187, 188} (Figure 5).

3.7 MAPK signaling pathway

MAPK, a member of the Ser/Thr kinase family,¹⁸⁹ plays a crucial role in regulating cell proliferation, differentiation, and migration.¹⁹⁰ The MAPK pathway consists of at least three cascade reactions involving three enzymes MAPK, MAPK kinase (MAPKK), and MAPK kinase kinase (MAPKKK).¹⁹¹ Each cascade reaction is initiated by specific extracellular signals and leads to the activation of a specific MAPK upon sequential activation of MAPKKK and MAPKK.¹⁹² The MAPK family is composed of three kinases: ERK, JNK, and p38 MAPK (MAPK14).¹⁹³ The RAF–MEK–ERK pathway is the most representative MAPK signaling pathway, and its important upstream regulators include cell surface receptors such as RTKs, GPCRs, and integrins, as well as GTPases, Ras, and Rap.¹⁹⁴ ERKs can translocate to the nucleus and phosphorylate different transcription factors, including Elk-1, Sap-1a, and c-Myc, altering gene expression to promote growth, differentiation, or mitosis.¹⁹⁵

According to several studies, MAPKs have been demonstrated to have significant implications in regulating bone density and bone homeostasis through their influence on the differentiation processes of osteoblasts and osteoclasts.¹⁹⁶ First, mechanical stimuli can increase the expression of Focal adhesion kinase (Fak), which is an important member of integrin-mediated signal transduction, thereby activating the Fak–MAPK pathway by activating Erk, Jnk, and p38–MAPK during distraction osteogenesis.¹⁹⁷ In addition, the activation of the ERK, p38, and JNK signaling pathways can expedite osteoclast differentiation by modulating the activity of AP-1, which serves as a critical regulator of osteoclast formation.¹⁹⁸ In osteoblasts, TNF stimulates the activation of SHN3 through ERK MAPK-mediated phosphorylation. Subsequently, phosphorylated SHN3 suppresses Wnt/β-catenin signaling while increasing the expression of RANKL. Consequently, introducing a mutation in Shn3 that hinders its binding with ERK MAPK enhances bone formation in mice that overexpress human TNF-α by boosting Wnt/β-catenin signaling.¹⁹⁹ In addition, the ERK signaling pathway plays a crucial role in regulating various phases of osteoblast differentiation through the phosphorylation of essential transcription factors, including RUNX2, and activation of transcription factor 4.²⁰⁰ Similarly, activation of the p38 signaling pathway facilitates the differentiation of osteoblasts through the phosphorylation of DLX5, OSX, and RUNX2.²⁰¹ In addition, in a study by Xu et al.,²⁰² endothelial progenitor cells were found to have a significant effect on BMSC differentiation and osteogenesis through the MAPK pathway. In accordance with prior research findings, the p38 signaling pathway has been shown to play a role in the regulation of ALP activity during the process of osteoblast differentiation.²⁰³ Moreover, this pathway is essential for the expression of BSP and OPN through the activation of p38MAPK–RUNX2 signaling.²⁰⁴ The absence of p38α in pre-osteoblasts has been shown to lead to impaired osteoblast differentiation, as indicated by decreased levels of collagen 1, ALP, BSP, and OCN.²⁰⁵ Furthermore, Luo et al.¹⁹⁸ discovered that the increased expression of disintegrin-like and metalloproteinase with thrombospondin and MMPs through the activation of the ERK signaling pathway is significantly involved in the initial stages of OA progression.

In osteocytes, MAPK signaling is essential for regulating autophagy and protecting osteocytes from oxidative stress and other cytotoxic insults. It also responses to mechanical stimuli and interacts with other signaling pathways, such as the ERK, JNK, and p38–MAPK pathways, to maintain bone homeostasis²⁰⁶ (Figure 5).

3.8 AMPK signaling pathway

AMPK, a serine–threonine kinase,²⁰⁷ is a heterotrimeric complex consisting of an α-subunit, a β-subunit, and a γ-subunit,²⁰⁸ of which the α-subunit plays a catalytic role, while the β- and γ-subunits play important roles in maintaining trimeric stability and substrate specificity.²⁰⁹ The AMPK signaling pathway functions as a detector of cellular energy levels and is triggered by elevated cellular AMP/ATP ratios resulting from metabolic stress, including interference with ATP production (e.g., glucose or oxygen deprivation) or accelerated ATP consumption (e.g., muscle contraction).²¹⁰ Upon activation, AMPK simultaneously suppresses energy-expending biosynthetic processes, including glycogen, protein and fatty acid synthesis, while stimulating ATP-generating catabolic pathways such as glycolysis and fatty acid oxidation.²¹¹ In essence, AMPK regulates cellular energy allocation by suppressing protein synthesis and cell growth, as well as modulating cell cycle arrest by reducing the activity of mTOR, a protein that is overactive in many cancer cells.²¹² Consequently, AMPK has emerged as a promising target for the treatment of metabolic disorders and cancer.²¹³ In addition, according to many studies, the activation of AMPK has the potential to influence the promotion of bone formation and bone density, indicating the significance of AMPK signaling as a crucial pathway in skeletal physiology.²⁰⁹

Initially, the regulatory impact of AMPK on osteoblasts primarily occurs via the activation of the osteoblast-specific transcription factor RUNX2.²¹⁴ Furthermore, AMPK can influence bone formation by promoting osteoblastogenesis through the down-regulation of AGEs and the activation of IR deformation.²¹⁵ Conversely, the AMPK signaling pathway suppresses RANKL by reducing the expression of peroxisome proliferator-activated receptor gamma 1 (PPARγ1), NFATc1, PTH-related protein (PTHrP), and mevalonate, consequently impeding the process of osteoclast formation.²¹⁶ Notably, adipocytes and osteoblasts share a lineage with BMSCs as common progenitors.²¹⁷ PPARγ2 inhibits osteoblast differentiation, promoting the differentiation of BMSCs into adipocytes,²¹⁸ whereas the differentiation of BMSCs into osteoblasts is regulated by AMPK through the modulation of RUNX2 and a newly identified pathway, the Wnt/β-catenin pathway.²¹⁹ In addition, the mevalonate pathway, which is involved in the prenylation of regulatory proteins such as Ras and Rho GTPase, has been found to have a detrimental impact on bone tissue.²¹⁶ AMPK has been shown to modulate the mevalonate pathway by inhibiting 3-hydroxy-3-methylglutaryl-coa reductase.²²⁰

Furthermore, AMPK signaling in osteocytes is vital for protecting against oxidative stress, regulating bone remodeling, and interacting with glucose metabolism. By modulating the expression of RANKL and SOST, AMPK helps maintain a balance between bone formation and resorption.²²¹ Additionally, its role in enhancing OCN expression links bone metabolism with systemic glucose homeostasis, highlighting its importance in both skeletal and metabolic health²²² (Figure 5).

3.9 Hh signaling pathway

The Hh signaling pathway consists of the Hh ligand, patched receptor (Ptch), smoothened receptor (Smo), suppressor of fused (Sufu), and the transcription factor glioma-associated oncogene (Gli).²²³ There are three subtypes of Hh genes in mammals: Indian Hh, sonic Hh (Shh) and desert Hh, of which Ihh plays a major role in skeletal development. Ptch represents a transmembrane receptor consisting of 12 passes that interact with Hh ligands, encompassing two similar genes known as Ptch1 and Ptch2.²²⁷ Smo is a seven-transmembrane protein that functions as a signal sensor and is inhibited by Ptch. Generally, the Hh protein relieves the inhibitory effect of Ptch on Smo by binding to Ptch on target cells, leading to Smo activation. Sufu is a negative regulator of the Hh pathway. However, activated Glis with a zinc finger structure dissociates from the Sufu-containing repressor complex and contributes to the regulation of certain downstream target genes of cell-active Hh signaling. Typically, Gli1 and Gli2 function primarily as transcriptional activators, whereas Gli3 acts as a repressor of Hh signaling.²²⁴

According to many studies, Hh signaling inhibits the differentiation of BMSCs into adipocytes while stimulating their differentiation into chondrocytes and osteoblasts, further maintaining bone homeostasis and promoting endochondral bone formation.²²⁵ In the process of embryonic development, the Hh signaling pathway triggers the upregulation of PTHrP in chondrocytes, thereby controlling the rate of chondrocyte hypertrophy within the developing growth plates.⁸ Apart from that, it is essential for the differentiation of osteoblasts through the regulation of RUNX2 expression. However, in postnatal bone remodeling, it has been shown that Hh signaling serves dual roles in regulating bone mass. A partial enhancement of Hh signaling due to Ptch1 haploinsufficiency leads to an increase in bone mass, while a widespread elevation of Hh signaling particularly in mature osteoblasts stimulates both bone formation and bone resorption. The non-cell-autonomous function of Hh signaling in the process of osteoclast differentiation is facilitated through the release of PTHrP from fully developed osteoblasts, leading to the stimulation of RANKL expression.²²⁶ The information presented indicates that Hh signaling in bones utilizes both intrinsic and extrinsic mechanisms to intricately oversee bone remodeling. In addition, the Hh signaling pathway also has key functions in synergistic cascades with other signaling pathways, such as the Wnt/β-catenin, BMP, and PThrP pathways.^{227, 228} In osteocytes, the Hh pathway is integral for maintaining bone homeostasis and responding to mechanical stimuli. It plays a non-cell-autonomous role by affecting the differentiation and activity of other bone cells through the modulation of signaling molecules and pathways.²²⁹

In summary, once activated, the Hh pathway promotes the differentiation of stem cells to osteoblasts and the deposition of bone matrix and inhibits the apoptosis and destruction of osteoblasts through a complex of direct actions and interactions with other pathways. This is corroborated by current studies on osteoporosis, where inhibition of the Hh signaling pathway leads to inhibition of osteoblast proliferation and differentiation, thereby affecting bone formation and reducing bone density (Figure 5).

3.10 Notch signaling pathway

The Notch signaling pathway is a crucial intercellular communication mechanism playing a pivotal role in cell differentiation, proliferation and fate determination.²³⁰ The pathway includes four receptors, Notch1 to Notch4, which are transmembrane proteins capable of receiving external signals on the cell surface. The activation of Notch receptors is dependent on direct binding with ligands from two main families: the Jagged family (Jagged1 and Jagged2) and the Delta-like family (including Delta-like 1, Delta-like 3, etc.). These ligands are also cell surface proteins that are expressed on neighboring cells and trigger Notch signal transduction. The binding of ligands to Notch receptors triggers a series of cleavage events, resulting in the release of the Notch intracellular domain (NICD) from the membrane. The NICD is a key executor of Notch signaling. The released NICD subsequently translocates to the cell nucleus, where it acts as a transcription factor to activate the transcription of specific genes. The primary target genes include the Hes families (hairy and Enhancer of split E(spl) homologs) and Hey families (Hes-related proteins containing the YRPW motif).²³¹ The expression of these genes affects cell proliferation, differentiation, and survival. Recent studies have emphasized the role of Notch signaling in regulating bone homeostasis, particularly its inhibitory effect on osteoblasts.²³²

The Notch pathway's activation hinders the differentiation of osteoblastic cells and results in impaired bone formation. Notch inhibits early osteoblast differentiation, preventing the maturation of matrix-synthesizing cells, while in mature cells, it blocks further differentiation, causing an accumulation of dysfunctional osteoblasts. NICD binds to RUNX2, obstructing Bglap transactivation, and Hes and Hey proteins inhibit osteoblastogenesis by suppressing RUNX2 function.^233-235 Additionally, in cells overexpressing Notch, cytosolic β-catenin levels and Wnt-3-induced ALP activity are reduced. Hes1 complexes with Groucho and T-cell-specific factors, inhibiting the interaction between T-cell factors and β-catenin, which is essential for the transcription of Wnt-dependent genes.²³⁶ Thus, osteoblastic differentiation was inhibited. Notch also regulates osteoblast differentiation and bone homeostasis via antiapoptotic actions. Jagged1 (JAG1), a notch activator, enhances the activation of antiapoptotic factor BCL-2 and reduces the proapoptotic factor Caspase3 in osteoblasts.²³⁷ In osteocytes, the presence of Notch1 leads to specific outcomes, including the suppression of bone resorption through the upregulation of OPG and downregulation of SOST, ultimately resulting in increased activation of Wnt signaling.²³¹ Moreover, Notch1 increases Wnt/β-catenin activity in osteocytes by downregulating SOST and Dkk1 (Dickkopf Wnt signaling pathway inhibitor 1).²³⁸ There is a degree of overlap between Notch and Wnt activation in osteocytes. Notch receptors are induced in mice expressing a constitutively active β-catenin mutant, and Notch further activates the Wnt/β-catenin pathway in osteocytes, which creates a positive feedback loop.²³³

The roles of Notch1 and Notch2 in osteoclastogenesis and bone resorption are distinctly different. Stimulation of Notch1 receptors in pOCs leads to direct suppression of osteoclast formation.²³⁹ Concurrently, Notch1 activation in mature osteoblasts and osteoclasts stimulates OPG production, which hinders bone resorption.²⁴⁰ Conversely, Notch2 NICD interacts with NF-κB, promoting the transcription of NFATc1, a key gene for osteoclastogenesis, thus enhancing osteoclastogenesis through both direct and indirect pathways. Furthermore, Notch2 activation in osteoblasts indirectly triggers RANKL, boosting osteoclastogenesis²⁴¹ (Figure 5).

3.11 Ion channel signaling pathways

Cellular mechanotransduction plays a crucial role in the development and maintenance of bone tissue, with mechanical stimuli playing a key role in promoting bone formation and preventing bone loss.²⁴² BMSCs, osteoblasts, osteocytes, and osteoclasts, which are the primary cell types found in bone tissue, respond to various mechanical cues such as matrix stiffness, fluid shear stress, tension, and compression across the lifespan of an individual.²⁴³ Physiological mechanotransduction activates relevant signaling pathways and maintains bone homeostasis. However, aberrant cellular mechanotransduction may lead to a variety of bone diseases, including osteoporosis and OA.²⁴⁴ Recent studies have revealed that certain ion channels, such as Piezo1, TMEM16A, P2X7, TRPV4, and other ion channels, which are closely related to skeletal homeostasis, play key roles in cellular mechanotransduction.²⁴⁵

3.12 Noncoding RNA signaling pathways

Noncoding RNA refers to RNA that does not encode proteins, including microRNAs (miRNAs), long noncoding RNAs (lncRNAs), circular RNAs, and other RNAs with known functions.²⁶¹ It is well recognized that miRNAs are small noncoding RNAs that can recognize cognate sequences and interfere with transcriptional, translational, or epigenetic processes.²⁶² LncRNAs are defined as RNA transcripts >200 nucleotides that do not encode proteins.²⁶³ There is accumulating evidence that these RNAs are transcribed from the genome, and although they cannot encode proteins, they can exert their biological functions at the RNA level as regulatory factors, affecting various biological processes such as bone metabolism.²⁶⁴

In general, miRNAs can induce gene silencing by binding to mRNAs, while competing endogenous RNAs may modulate gene expression through competitive combining to miRNA response elements.^{265, 266} In contrast, both circRNAs and lncRNAs have miRNA-binding sites, which bind to miRNAs in cells, thus counteracting the inhibitory effect of miRNAs on their target genes and thus increasing the level of target gene expression.^{267, 268}

Some noncoding RNAs, such as lncRNAs ORLNC1,²⁶⁹ lncRNA-POIR,²⁷⁰ miRNA-433-3p,²⁷¹ circRNA-0016624,²⁷² and circRNA-AFF4,²⁷³ have been shown to promote osteoblast differentiation by regulating substances associated with their differentiation. Moreover, some noncoding RNAs can also affect the differentiation of other cells, such as osteoclasts, thus promoting bone destruction.^274-276 Notably, the corresponding biological functions of these noncoding RNAs need to be targeted by genes or pathways to produce indirect effects.

For instance, miRNA-214, an important regulator of musculoskeletal metabolism and disease,²⁷⁷ can be expressed in human osteoporotic bone tissue to prevent osteoporosis by effectively regulating bone metabolism bidirectionally, promoting osteoblasts but inhibiting osteoclast activity, reducing bone loss, and effectively delaying osteoporosis pathology.²⁷⁸ Moreover, lncRNA KCNQ1OT1 can silence this gene to promote BMP2 expression to regulate osteogenic differentiation.²⁷⁹ Therefore, these RNAs play important regulatory roles in various signaling pathways through their interactions with each other, thus affecting the differentiation of osteoblasts and osteoclasts. Finally, they can regulate the balance of bone homeostasis.²⁸⁰

And noncoding RNAs have been implicated in many orthopedic diseases in which they play important roles.^281-284 For instance, lncRNA AC006064.4-201 disrupts the stability of CDKN1B mRNA through interaction with PTBP1, thereby alleviating cartilage aging and protecting against OA.²⁸⁵ It has also been reported that the lncRNAs MALAT1,²⁸⁶ TUG1,²⁸⁷ and DANCR²⁸⁸ play different roles in OA by targeting different miRNAs. For instance, miR-214-3p can downregulate IKK-β expression and lead to dysfunction of the NF-κB signaling pathway, thus relieving the damage caused by OA.²⁸⁹

In addition, noncoding RNAs can also reach other cells via EVs and act as significant messengers in cellular crosstalk and biological activities.³⁰ For example, EVs derived from BMSCs can deliver the lncRNA NEAT1 to relieve arthritis.²⁹⁰ EVs can be encapsulated by miR-206,²⁹¹ miRNA-208a,²⁹² the lncRNA PVT1,²⁹¹ and other noncoding RNAs, and transported to osteosarcoma cells, leading to the proliferation, migration and invasion of cancer cells. However, EVs can regulate Wnt/β-catenin signaling by decreasing TCF/LEF activity to further inhibit the proliferation and induce the apoptosis of osteosarcoma cells.²⁹³

4.1 Osteoporosis

Osteoporosis is a chronic disease characterized by reduced bone mineral density and structural deterioration, leading to an increased risk of fracture, functional limitations, and mortality.⁷ It has emerged as a major global health challenge, affecting people of all ages, sexes, and races, with the greatest impact on postmenopausal women and the elderly.²⁹⁴ Primary osteoporosis can generally be divided into postmenopausal and age-related osteoporosis.

Menopause disrupts bone homeostasis due to estrogen withdrawal, which affects the activity of osteoblasts and osteoclasts. Physiologically, estrogen protects osteoblasts by preventing apoptosis and promoting their growth, maturation, and mineralization through various signaling pathways. Chang et al.²⁹⁵ reported that estrogen deficiency triggers NF-κB activation in osteoblasts, inhibiting osteoblast function. For osteoclasts, estrogen depletion encourages osteoclastogenesis and bone resorption while deterring osteoclast apoptosis through various pathways. The absence of estrogen increases RANKL expression by the osteoblast lineage, including BMSCs, osteocytes, and bone lining cells, and diminishes OPG production, thereby intensifying osteoclast formation via the RANK–RANKL–OPG signaling system. Estrogen scarcity also curtails osteoclast apoptosis by inhibiting Fas/FasL signaling.²⁹⁶ Moreover, estrogen scarcity also boosts T-cell activity and elevates the levels of inflammatory cytokines such as IL-1, IL-6, IL-17, and TNF-α, which indirectly stimulate osteoclast proliferation and differentiation by promoting RANKL and M-CSF production.²⁹⁷ In conclusion, studies have highlighted the role of estrogen in protecting osteoblasts and its absence in promoting osteoclastogenesis.

In age-related osteoporosis, there is a notable shift from osteogenesis to adipogenesis within BMSCs, guided by signaling pathways such as the ERK 1/2, TGF-β, and BMP2/4 pathways.²⁹⁸ An increase in bone marrow adipocytes further exacerbates the development of osteoporosis, which not only impedes osteoblastogenesis but also favors osteoclastogenesis through the activation of PPARγ by free fatty acids (FFAs).²⁹⁹ Palmitic acid, a specific FFA, notably hinders osteoblast development and mineralization by suppressing the Wnt/β-catenin and BMP2/RUNX2/SMAD pathways and induces apoptosis and autophagy dysfunction in osteoblasts.³⁰⁰ In addition, the interaction of PPARγ with specific promoter elements in hematopoietic stem cells promotes osteoclastogenesis,³⁰¹ highlighting the complex interplay between factors that drive the progression of osteoporosis.

4.2 Osteogenesis imperfecta

OI is a hereditary skeletal disorder characterized by mutations in the COL1A1/COL1A2 genes, leading to increased bone fragility and skeletal deformities.³⁰² Recent research has identified several signaling pathways, such as the Wnt, BMP, and TGF pathways, as significant contributors to the development of OI.³⁰³ For instance, mutations in the Wnt1 gene can disrupt the Wnt/β-catenin pathway, impacting osteoblast growth, differentiation, and function.³⁰⁴ Studies by Grafe et al.³⁰⁵ have shown that modulating TGF-β signaling in an OI mouse model can correct bone abnormalities and improve lung function. Gene ontology analysis of human samples has highlighted the upregulation of SMAD phosphorylation in OI patients, with TGF-β identified as a critical activation signal through genomic enrichment and pathway analyses.³⁰⁶ FAM46A, a member of the nucleotidyltransferase folding protein superfamily, interacts with SMAD and promotes the transcription of BMP target genes, with mutations in FAM46A linked to autosomal recessive OI inheritance.⁷ Additionally, BMP1 plays a crucial role in cleaving the C-terminal propeptide of procollagen types I, II, and III, and defects in this process have been implicated in OI pathogenesis.⁴

4.3 Osteoarthritis

OA is a degenerative condition of the joints marked by the gradual breakdown of articular cartilage, subchondral sclerosis, osteophyte development, varying levels of synovial inflammation, and meniscus degeneration. Persistent pain, swelling, deformity, limited joint function, and reduced mobility impose significant financial and emotional burdens on OA patients.³⁰⁷ In OA, the subchondral bone experiences remodeling, with simultaneous increases in bone resorption and formation.²⁰³ Abnormal mechanical strain disrupts osteoblast metabolism, leading to higher levels of IL-6, PGE2, MMP, and RANKL, and lower levels of OPG. IL-6 and PGE2 promote osteoclast formation by reducing OPG secretion and increasing RANKL production in osteoblasts or by elevating RANK expression in osteoclasts. PGE2 also triggers IL-6 release, which in turn increases PGE2 secretion by osteoblasts.³⁰⁸ This creates a positive feedback loop between PGE2 and IL-6 signaling, enhancing osteoclast differentiation by modulating the RANK/RANKL/OPG pathway.³⁰⁹ Additionally, heightened mechanical stress causes osteocytes to influence osteoblast mineralization by increasing Wnt protein production and decreasing SOST secretion. In vitro studies indicate that osteoclastic bone resorption significantly raises active TGF-β1 levels in OA subchondral bone, promoting osteoblast-driven bone formation through the SMAD2/3 pathway in advanced OA.⁷ Consequently, increased osteoblast activity results in spatial remineralization and osteosclerosis in late-stage OA. Furthermore, TGF-β can elevate neurofactors in OA via the ALK5–SMAD2/3 pathway and may contribute to joint pain in OA patients.³¹⁰

4.4 Rheumatoid arthritis

RA is a systemic autoimmune disease characterized by a complex interplay of cellular activities and signaling pathways that leads to the deterioration of joints.³¹¹ Synovial inflammation and irreversible bone destruction are prominent features of RA that lead to an imbalance in bone homeostasis.³¹² Overactivated fibroblast-like synoviocytes (FLSs) increase the expression of proangiogenic factors such as IL-6 and chemokines and cause an imbalance between γδTreg and γδT17 cells.³¹³ This environment causes acidic and hypoxic conditions in arthritic joints, which fosters the activation of osteoclasts and the apoptosis of osteoblasts, contributing to bone degradation.³¹⁴ In detail, acidic and hypoxic conditions diminish the osteoblast production of ALP and impede mineralization. Moreover, hypoxia interferes with Wnt signaling by blocking β-catenin transduction, and inflammatory factors such as TNF-α disrupt this pathway further by inducing the overexpression of DKK-1 in synovial fibroblasts.³¹⁵ TNF-α also negatively affects the synthesis of collagen type I, ALP activity, and the expression of OCN in vitro.⁷ In addition, peripheral blood monocytes (PBMCs) are thought to be the major source of osteoclasts in inflammatory environments, and osteoclasts derived from PBMCs show an enhanced bone resorption capacity.³¹⁶ Notably, the synovial fluid of RA patients contains mononuclear cells that express osteoclast-related genes and can form osteoclasts spontaneously, indicating a heightened osteoclastogenic potential in RA.⁷ These developments are further amplified by inflammatory cytokines such as TNF-α and IL-6, which activate the JAK–STAT signaling pathway and promote the differentiation of PBMCs into osteoclasts, leading to increased bone resorption and bone destruction.³¹⁷

4.5 Paget's disease of bone

PDB is a chronic focal disorder characterized by disturbed bone homeostasis, leading to asymmetric skeletal deformities. These deformities result from excessive bone resorption by abnormally large osteoclasts and disorganized bone formation by osteoblasts.^{318, 319} Pagetic osteoclasts, characterized by an increased number of nuclei, exhibit heightened responsiveness to osteoclastogenic factors, including RANKL and 1,25-dihydroxyvitamin D3. This heightened responsiveness not only boosts their bone resorption capacity but also increases their resistance to apoptosis.^{7, 320} In response to the activity of these osteoclasts, coupling factors are released, increasing the number of immature osteoblasts at resorption sites. Notably, these immature osteoblasts produce more RANKL than their mature counterparts, exacerbating bone formation.³²¹ Additionally, individuals with PDB exhibit elevated levels of IL-6, which promotes RANKL expression on osteoblasts and stromal cells, potentially leading to osteoclast hyperactivation.³²² This localized increase in RANKL is critical for disease progression. Although less is known about osteoblasts in the PDB, emerging research suggests that osteoclast-osteoblast communication plays a vital role. For instance, the measles virus nucleocapsid protein can stimulate osteoclasts to upregulate IL-6 and IGF-1, enhancing EphrinB2–EphB4 coupling and thereby stimulating bone formation by osteoblasts.^{323, 324} Furthermore, pagetic phenotypes may boost osteoclast-derived IGF-1 expression, which, in turn, triggers additional RANKL production in osteocytes. This increase in RANKL production is a key factor in the development of pagetic lesions.³²⁵ In conclusion, disordered osteoclast and osteoblast activity together contribute to the development of PDB.

4.6 Bone tumors

Bone is a frequent site of metastasis for many cancers, particularly breast and prostate cancers, aside from primary bone tumors like osteosarcoma. Cancer cells facilitate bone degradation by inducing osteoclast-mediated bone resorption, leading to substantial structural damage and severe morbidity. This process results in significant pain, fractures, and reduced quality of life for patients.³²⁶ Bone is a frequent site of metastasis for many cancers, particularly breast and prostate cancers, aside from primary bone tumors like osteosarcoma. Cancer cells facilitate bone degradation by inducing osteoclast-mediated bone resorption, leading to substantial structural damage and severe morbidity. This process results in significant pain, fractures, and reduced quality of life for patients.³²⁷ Tumor-derived M-CSF and RANKL on osteoblasts, BMSCs, and immune cells bind to c-fms and RANK on pOCs, initiating their differentiation and activation. Consequently, cancer cells stimulate osteoclasts to break down bone tissue, releasing growth factors (TGF-β, IGF-1, PDGF, and FGF) stored within the bone matrix and enhancing tumor growth.³²⁸ The interaction between cancer cells and the bone microenvironment creates a vicious cycle and induces bone tumor growth and bone metastasis.³²⁹ RANKL secreted by osteoblasts attracts cancer cells expressing RANK and promotes their migration within the bone microenvironment.¹⁰⁷ Furthermore, osteoclasts contribute to this cycle by releasing factors that promote tumor growth and metastasis. Zuo and Wan³³⁰ found that PDL1 on tumor cells can bind to PD-1 on osteoclasts, affecting the function of osteoclasts and further promoting tumor growth. Anti-PDL1 treatment inhibited bone metastasis in breast cancer and melanoma and did so by inhibiting osteoclast formation and enhancing the immune response within the bone. In addition, another study showed that PD-1 blockade inhibited osteoclast formation and bone cancer pain in mice.³³¹ These findings suggest that although PD-L1 is expressed on tumor cells primarily to inhibit T-cell activity and help tumor cells evade immune surveillance, it also plays a significant role in bone metastasis and associated pain.

4.7 Osteonecrosis of the femoral head

ONFH is a prevalent orthopedic clinical condition marked by the demise of bone and marrow cells. This condition can be attributed to a range of factors including hip trauma, corticosteroid administration, alcohol misuse, hemoglobin disorders, bone marrow transplants, chemotherapy, and radiation exposure.³³² ONFH results in pain, restricted mobility, joint collapse, and subsequent development of secondary OA.³³³ Glucocorticoids have a multifaceted impact, directly causing apoptosis of osteoblasts and osteocytes, extending the lifespan of osteoclasts, and inducing the apoptosis of endothelial cells.⁷ These actions contribute collectively to femoral head ischemia and bone marrow changes, illustrating the detrimental effects of corticosteroids on bone integrity.³³⁴ The induction of osteonecrosis by glucocorticoids in animal models has been attributed to GSK3β-mediated osteoblast apoptosis, highlighting a specific pathway through which corticosteroids may exert their harmful effects.³³⁵ Necrotic osteocytes release macrophage C-type lectin (Mincle) that induces osteoclast formation through ITAM-based calcium signaling, leading to OxPhos and increased bone loss.⁷ In addition, osteoclasts and osteoblasts may undergo apoptosis after prolonged treatment with glucocorticoids through the FASL/FAS pathway. In corticosteroid-associated osteonecrosis, corticosteroids can stimulate bone loss through their action on the RANK/RANKL/OPG signaling system. Several potential signaling pathways are involved in osteocyte apoptosis mitigation, such as the mitochondrial, MAPK, PI3K/Ak, Wnt/β-catenin, and HIF-1 signaling networks.⁷

4.8 Periprosthetic osteolysis

PPO, a critical complication after artificial joint replacement, jeopardizes prosthesis longevity and outcomes and increases treatment costs.³³⁶ Titanium implants generate wear particles over time due to friction, disrupting bone homeostasis by engaging immune cells and altering osteoblast and osteoclast activity. These particles provoke macrophage activation and inflammatory cytokine release (IL-6, PGE2, IL-1β, TNF-α), driving osteoclastogenesis and jeopardizing BMSC osteogenic differentiation, which contributes to osteolysis and aseptic prosthesis loosening.³³⁷ Wear particles affect osteogenic differentiation through activating the NF-κB signaling pathway and reducing the involvement of the Wnt/β-catenin signaling pathway in BMSC activation of the ERK signaling pathway.³³⁸ Moreover, Ti particles inhibit the osteogenic differentiation of osteoblasts via the BMP/SMAD signaling pathway.³³⁹ Furthermore, we found that titanium particles lead to an aberrant increase in GSK-3β activity, which inhibits the Hh/Gli1 signaling pathway, ultimately leading to an inhibition of the osteogenic process.⁷ Furthermore, we demonstrated that up-regulation of SIRT3 expression enhanced the osteogenic differentiation of PPO289 by decreasing NLRP3 inflammasome expression via the GSK-3β/β-catenin signaling pathway.⁷ Wang et al.³⁴⁰ found that Netrin-1 promotes autophagy induced by Ti particles and enhances osteoblast formation via the ERK1/2 signaling pathway.

5.1 Anabolic agents

5.2 Antiresorptive agents

5.3 Anabolic–antiresorptive agents

5.4 Anti-inflammatory agents