The abnormal differentiation of mesenchymal stem cell (MSC) in specific anatomical locations is a critical mechanism driving new bone formation in DISH (21). MSCs are primarily located in the outer fibrocartilage of entheseal sites, bony eminences at vertebral margins, and sesamoid bones, with the potential to proliferate and differentiate into osteoblasts or chondrocytes (21–23). Under the influence of metabolism-related growth factors, MSCs are activated. Insulin-like growth factors (IGF-1/2), through signaling with the IGF-1 receptor, induce osteogenic differentiation of these stem cells and enhance their osteogenic potential (24, 25). Bone morphogenetic proteins (BMPs) induce the differentiation of MSCs into chondrocytes, form new bone through endochondral ossification, and participate in the mineralization of fibrocartilage at the tendon attachment site and the growth of new bone (26, 27). Transforming growth factor-beta (TGF-β), in coordination with mechanical stress, influences the differentiation of mesenchymal cells into fibrocartilage or bone tissue (26, 27). Additionally, the function of MSCs derived from spinal ligaments is influenced by epigenetic processes; for example, demethylation of the WNT5A and GDNF genes enhances their osteogenic activity, suggesting that epigenetic modifications promote abnormal ossification by releasing transcriptional inhibition (28). This suggests that DNA methyltransferase inhibitors may be a potential treatment method, although their specificity for spinal entheses has not been confirmed.

The Wnt/β-catenin signaling pathway is a highly conserved and crucial pathway in skeletal development and homeostasis. Its aberrant activation is a significant mechanism driving pathological heterotopic bone formation in DISH (29, 30).

Clinical studies have found that serum levels of the Wnt pathway antagonist Dickkopf-1 (DKK1) are significantly lower in patients with DISH, suggesting systemic activation of Wnt signaling in vivo (31). Interestingly, another potent Wnt antagonist, sclerostin (SOST), has been reported to be elevated in the serum of DISH patients (32). This may represent a compensatory mechanism in response to the pathological ossification in DISH, although the specific underlying process remains unclear.

Transgenic mouse models powerfully demonstrate the pathogenic role of Wnt/β-catenin overactivation in DISH. Conditional activation of β-catenin in chondrocytes and intervertebral disc cells of β-catenin(ex3)^Col2CreER and β-catenin(ex3)^Agc1CreER mice successfully recapitulates the characteristic spinal phenotypes of human DISH, including extensive osteophyte formation and vertebral fusion (30).

Furthermore, recent studies have identified the lectin Galectin-3 as a key upstream factor activating the Wnt/β-catenin pathway in DISH (33). Bone marrow-derived mesenchymal stem cells (BMSCs) from DISH patients exhibit a high-secretory phenotype for Galectin-3, and its expression level directly correlates with the abnormally enhanced osteogenic differentiation capacity of these cells. Galectin-3 activates downstream signaling by directly binding to β-catenin and promoting its nuclear translocation, whereas inhibition of the Wnt/β-catenin pathway blocks the pro-osteogenic effects of Galectin-3. Upon pathway activation, the canonical β-catenin/TCF transcriptional program drives the expression of osteogenic genes, promoting BMSC osteogenic differentiation (33). Concurrently, it upregulates key degradative enzymes such as matrix metalloproteinase 13 (MMP13) and aggrecanases (ADAMTS4/5), which disrupt the cartilage matrix and create a permissive environment for heterotopic osteophyte deposition. Experimentally, knocking out the Mmp13 or Adamts5 gene in mice with activated β-catenin significantly alleviates the phenotypes of intervertebral disc destruction and osteophyte formation (30).

In summary, overactivation of the Wnt/β-catenin pathway constitutes a crucial signaling axis driving pathological ossification in DISH. This understanding provides potential theoretical targets for future development of disease-modifying therapies aimed at various components of this pathway.

Nuclear factor κB (NFκB) plays a regulatory role in the expression of multiple genes involved in cell growth and division as well as in the differentiation of multipotent cells (20). In ligament mesenchymal cells, NFκB activation is linked to osteoblastic differentiation. Kosaka et al. obtained tissue samples from patients with ossification of the spinal ligaments (OSL)/DISH and from individuals with non-ossified spinal ligaments during surgery, and found that the number of NFκB-positive samples was significantly higher in the OSL/DISH group, along with elevated levels of PDGF-BB and TGF-β1 in ligament tissues (34). The authors proposed that these growth factors may activate the NFκB signaling pathway, promoting the nuclear translocation of NFκB, which in turn regulates the expression of related genes (such as Sonic Hedgehog, Twist, and BMP-4) and thereby influences the differentiation of multipotent cells. Furthermore, increased alkaline phosphatase (ALP) activity was observed in ligament cells from the OSL/DISH group, indicating their characteristics as osteoprogenitor cells. The study suggests that NFκB activation may play an important role in the pathogenesis of DISH. Notably, this pathway was more significantly activated in patients complicated with non-insulin-dependent diabetes mellitus (NIDDM), highlighting a potential mechanism linking metabolic disturbances to enhanced local ossification signaling (34).

Bone morphogenetic protein (BMP) 2 signaling is one of the key drivers in the ossification process of DISH (20). It activates the Smad1/5/8 signaling pathway, enhancing the differentiation of MSCs into osteoblasts (35). Histological investigations have revealed a distinct spatial distribution of BMP−2, TGF−β, and decorin within ossified lesions from patients with DISH (36). Specifically, BMP−2 expression is markedly elevated in woven bone and in muscle tissue adjacent to the ossification sites, suggesting its pivotal role in the initiation of ectopic bone formation and the osteogenic transformation of muscle−derived cells. Concurrently, the enrichment of TGF−β and decorin in the bone matrix points to their cooperative involvement in regulating extracellular matrix assembly and mineralization. These findings provide direct evidence for the molecular pathological basis underlying the heightened susceptibility to severe ectopic ossification in individuals with DISH (36–38). Critically, functional characterization of the heterozygous ALK2 p.K400E mutation identified in DISH patients demonstrates enhanced signaling to osteogenic BMPs, providing a direct genetic-mechanistic link (39). The convergence of histological and genetic evidence strongly positions hyperactive BMP signaling as a key driver in DISH, although its precise interaction with other pathways in disease initiation needs clarification.

Obesity, particularly visceral adiposity, is one of the most robust clinical associations with DISH. The prevalence of DISH reaches 18% in patients aged 50 years or younger who suffer from severe obesity, a figure substantially higher than that in the general population (40). This means that the metabolic environment of severe obesity can strongly accelerate the ossification process much earlier than the traditional onset age of DISH. In obesity, visceral adipose tissue (VAT) secretes various adipokines, such as leptin and adiponectin (41). Especially the increase in leptin level has been confirmed in female patients with ossification of spinal ligament (42). Leptin promotes osteogenesis through the JAK/STAT signaling pathway, stimulates stromal cells to differentiate into osteoblasts, increases osteoblast proliferation, and inhibits osteoclast production (43), thereby promoting local ossification progression. Moreover, the visceral adiposity index (VAI), a substitute marker of visceral fat dysfunction, is significantly elevated in patients with the “fast ossifier” phenotype of DISH (44). This association is particularly pronounced in women, indicating that visceral obesity serves as a critical driver of accelerated ossification and concomitant early trabecular deterioration in this subgroup. These findings underscore that weight management and metabolic control may represent potential strategies to mitigate disease progression.

There is a close connection between DISH and type 2 diabetes mellitus (T2DM), with the prevalence of DISH in T2DM patients reaching 13% (4, 45–47). Even in cases where obesity is controlled, patients with impaired glucose metabolism show a higher incidence of DISH (48). Patients with type 2 diabetes often experience hyperinsulinemia, which promotes osteoblast activity and bone formation. Insulin may produce similar anabolic effects as IGF-1 through homology in its molecular structure and stimulate osteoblast differentiation (49). In addition, in the state of diabetes, the level of SOST in DISH patients is reduced (46), which weakens the inhibition of Wnt signaling pathway, and then promotes the expression of osteogenesis related genes. It is worth noting that DISH has not been recorded in patients with T1DM (50).

Growth hormone (GH) represents another significant metabolic factor influencing the development of DISH. GH can directly act on osteoblasts to promote their proliferation and differentiation, while also stimulating the production of IGF-1, which enhances the proliferation of bone and cartilage cells (51, 52). A case-control study observed that serum GH levels were significantly lower in asymptomatic DISH patients compared to symptomatic ones, thereby proposing serum GH as a potential biomarker for monitoring clinical inflammatory activity (53). However, this study had notable limitations: a small sample size, a cross-sectional comparison design, and a failure to fully account for confounding effects from symptomatic treatments on GH levels. Consequently, the precise role of the GH/IGF-I axis in osteophyte formation in DISH, remains to be elucidated through more rigorous, large-scale longitudinal studies.

Both COL6A6 and COL6A1 belong to the collagen gene family, which forms a critical component of the extracellular matrix (ECM) and is involved in membrane or intracondral osteogenesis (78, 79). Genetic studies have linked single nucleotide polymorphisms (SNPs) in COL6A1 and COL6A6 to DISH (80, 81). Specifically, the association of COL6A1 with DISH was significant in a Japanese cohort but not observed in a Czech population, suggesting the existence of population-specific genetic effects (80). These variants may contribute to the pathogenesis of DISH by altering the structure of the ECM, thereby creating a scaffold that facilitates HO, although the precise underlying mechanisms require further clarification.

Fibroblast growth factor 2 (FGF2) plays a multifaceted role in bone metabolism, promoting the recruitment of BMSCs and stimulating bone formation (82, 83). Jun et al. found through sequencing that two specific SNPs within the FGF2 gene (rs1476217 and rs3747676) that are significantly associated with DISH (84). This finding suggests that dysregulated FGF2 signaling may disrupt normal bone remodeling processes, thereby contributing to the pathological HO seen in DISH.

The Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene encodes a transmembrane enzyme that generates inorganic pyrophosphate (PPi), a potent inhibitor of ectopic mineralization, by hydrolyzing high-energy phosphate bonds (85, 86). An identified heterozygous missense mutation (c.1352A>G, p.Y451C) within the catalytic domain of ENPP1 has been linked to DISH (87). This mutation leads to decreased PPi levels in patients, consequently weakening the inhibition of hydroxyapatite crystal growth. This manifests clinically as a spectrum of ectopic calcification symptoms in patients, including ossification of paraspinal ligaments and calcification of the Achilles tendon (87, 88).

However, a critical paradox has emerged from recent animal studies. A knock-in mouse model carrying the corresponding ENPP1 Y433C mutation (corresponding to human Y451C mutation) showed no significant bone microstructural abnormalities or ectopic calcification at various time points (89).

This discrepancy may arise from compensatory effects of proteins like ENPP3 in mice, while DISH pathogenesis involves polygenic interactions and environmental factors (89). Species differences may also contribute to the discrepancy between the model and clinical phenotypes. Resolving this paradox is crucial for fully understanding the role of ENPP1 in DISH and requires further collection of human cases and the development of more precise animal models.

The Activin A receptor type I (ACVR1) gene encodes the bone morphogenetic protein (BMP) type I receptor ALK2, a pivotal component of the BMP signaling pathway essential for bone formation (90, 91). In DISH, a specific heterozygous missense mutation (p.K400E) within the kinase domain of ACVR1/ALK2 has been identified (92). This mutant receptor exhibits enhanced signaling in response to osteogenic BMPs but remains unresponsive to non-osteogenic ligands such as Activin A (39). This aberrant signaling leads to constitutive activation of the BMP-Smad pathway, resulting in the upregulation of osteogenic target genes like ID1 and Msx2 (90, 92). Furthermore, BMP type II receptors can further enhance the BMP signaling activity mediated by p.K400E through phosphorylating it (39). Collectively, the p.K400E mutation provides direct genetic evidence that hyperactive BMP signaling is an important driver of pathological ossification in DISH.

TGF-β1 is a pivotal cytokine in bone remodeling, which can promote the osteogenic differentiation of BMSCs while concurrently suppressing bone resorption by downregulating factors such as the osteoclast differentiation factor (93, 94). Evidence from both human studies and mouse models of HO has demonstrated activated TGF-β signaling at ectopic sites, and neutralization of TGF-β can alleviate HO progression (95). Genetically, an allelic analysis by Thomas M et al. revealed a significantly higher frequency of the SNP rs2241716 in the TGF-β1 gene among DISH patients compared to global reference frequencies (81). This finding strongly supports TGF-β1 as a susceptibility gene for DISH, implicating its enhanced signaling activity in the disease’s pathophysiology.

R-spondin 4 (RSPO4), a member of the R-spondin family of secreted proteins, is a potent activator of the Wnt/β-catenin signaling pathway, which plays a critical role in cell proliferation and osteogenesis (96). A whole-genome-wide linkage and identity-by-descent (IBD)/identity-by-state (IBS) study conducted on families with coexisting DISH and chondrocalcinosis (CC) from the Azores islands identified the RSPO4 gene as a potential candidate. Within this gene, nine genetic variants were discovered (73, 97). Notably, the rs146447064 variant was significantly more frequent in the control population, suggesting that it may confer a protective effect against the development of DISH/CC. This finding positions RSPO4-mediated Wnt signaling as a potentially important and modifiable pathway in the pathogenesis of DISH.

PPP2R2D encodes a regulatory subunit of protein phosphatase 2A (PP2A), a serine/threonine phosphatase involved in diverse cellular processes (98). Whole exome sequencing of DISH patients from the Azores identified a specific variant (rs34473884) in PPP2R2D that showed a significant association with the DISH phenotype (99). While the precise molecular mechanism by which this variant contributes to HO remains unclear, its discovery supports the polygenic nature of DISH and suggests that dysregulation of protein phosphorylation may play a role in its pathogenesis.