Open research questions in Bone Metabolism and Diseases

What the literature leaves open

• In addition, although we investigated the effects of DKK1 on neutrophils, osteoclast-like cells, and macrophages in bone TME, the precise downstream signaling pathways involved in DKK1–CKAP4-mediated immune modulation remain to be explored. Finally, the potential contribution of canonical DKK1 receptors (LRP5/6) to immune regulation within the bone TME was not addressed in this study and whether these classical pathways cooperate with or compensate for CKAP4- dependent signaling warrants further investigation.

  Myeloid cell reprogramming combined with zoledronic acid effectively suppresses bone metastasis · 2026 · DOI

• Osteoblast senescence has recently been recognized as an important contributor to skeletal aging; however, its upstream regulatory mechanisms remain unclear [35]. Previous studies have mainly focused on osteoclast activation and BMSC dysfunction [18–20], whereas its role in osteoblast senescence remains insufficiently explored.

  Gut Microbiota-Derived TMAO Drives MC3T3-E1 Senescence and Osteogenic Dysfunction via cGAS–STING–NF-κB Signaling: Implications for Age-Related Bone Loss · 2026 · DOI

• Nevertheless, considering the multifaceted regulatory potential inherent in natural compounds and the central position of NF-κB in RANKL-induced signaling, the potential involvement of this canonical pathway in SG-mediated effects cannot be definitively ruled out and warrants further investigation.

  Stigmasterol glucoside attenuates RANKL-induced osteoclastogenesis and bone resorption by suppressing MAPK and STAT3 signaling pathways: an integrative pharmacological study · 2026 · DOI

• However, beyond the canonical role of gasdermin D (GSDMD) in mediating pyroptosis in immune cells, its function in osteocytes remains poorly understood.

  Osteocyte-specific gasdermin D deletion accelerates osteoarthritis via promoting subchondral inflammation and remodeling · 2026 · DOI

• Based on the molecular mechanisms summarized in this review, future research in osteoporosis should advance along four strategic directions to achieve a shift from “symptom management” to “etiological intervention.” Each direction must be pursued with concurrent attention to its specific translational barriers and clear entry points for precision medicine. 6.1 Precise classification and biomarker development Path-directed actions: A multi-omics-integrated molecular subtyping system should be established. Specifically, m6A-seq, single-cell sequencing, and other technologies should be employed for patient stratification, and combined diagnostic panels based on circulating miRNAs, SASP factors, or m6A modification profiles should be developed. Translational barriers include the high cost and lack of standardization in multi-omics data; most biomarker studies remain cross-sectional, lacking prospective cohort valida- tion; and the scarcity of bone tissue-specific markers limits the ability of circulating biomarkers to accurately reflect local skeletal changes. Precision medicine opportunities: Large-scale longitudinal cohorts should be established, integrating multi-modal data through machine learning to systematically evaluate the clinical utility of candidate biomarkers in fracture risk prediction and therapeutic response monitoring. 6.2 Drug development targeting novel mechanisms Three most promising intervention strategies: First, senolytics (e.g., dasatinib + quercetin) require clinical validation in senile osteoporosis, though challenges remain in bone tissue targeting, long-term safety, and drug resistance. Second, epigenetic modula- tors (targeting METTL3, KDM7A, etc.) can precisely regulate osteogenic or osteoclastic differentiation, but genome-wide off- target effects and the lack of mature bone cell-specific delivery strategies pose major safety and feasibility concerns. Third, mechanosensitive channel agonists (e.g., Yoda1 derivatives) could mimic mechanical stimulation to promote bone formation, yet current agonists lack subtype selectivity and have poor bioavaila- bility, placing them at a considerable distance from clini- cal application. 6.3 Smart delivery and regenerative medicine Path-directed actions: Bone-targeted nanocarriers should be developed for the precise delivery of siRNA or epigenetic drugs; smart biomaterial scaffolds responsive to the local microenviron- ment should be designed for controlled release of growth factors, or for loading mechanically preconditioned MSC-derived exosomes for bone defect repair. Core barriers include: suboptimal bone- targeting efficiency and poor in vivo stability of nanocarriers; insufficient systematic evaluation of long-term degradation behav- ior and biocompatibility of biomaterial scaffolds; and unresolved challenges in large-scale standardized exosome production and quality control. Without overcoming these bottlenecks, regenerative strategies will struggle to transition from bench to bedside. 6.4 Artificial intelligence and systems integration Path-directed actions: artificial intelligence (AI) should be used to integrate clinical imaging, biomarkers, and multi-omics data to con- struct individualized fracture risk prediction models; machine learning should be employed to screen drug libraries for novel multi-target compounds capable of synergistically regulating the RANKL-Wnt- epigenetic network. Major challenges include: heterogeneity and an- notation inconsistencies across multi-modal data modalities, which severely compromise model transferability; the “black box” nature of AI models, which limits their interpretability and acceptability in clinical decision-making; and the predominance of predictions without functional validation, creating a “more prediction, less validation” dilemma. Future efforts should establish standardized data-sharing platforms and strengthen iterative closed-loop cycles between AI prediction and experimental validation. 7.Conclusion Future prevention and treatment strategies should shift from “symptom management” to “etiological intervention.” Through interdisciplinary collaboration, mechanistic insights should be translated into individualized diagnostic tools, novel therapeutic agents, and regenerative strategies, ultimately achieving the goal of restoring skeletal health.

  Molecular mechanisms and translational prospects in osteoporosis · 2026 · DOI

• These findings suggest that the metabolic phenotype in OI mice is complex, driven both by OCN, likely elevated due to high bone remodeling, and by OCN-independent mechanisms that warrant further investigation.

  Osteocalcin-dependent and -independent metabolic dysregulation in a mouse model of Osteogenesis imperfecta · 2026 · DOI

• Superoxide dismutase (SOD) is a key antioxidant enzyme, yet its sex-specific link with osteoporosis in type 2 diabetes mellitus (T2DM) remains unclear.

  Sex-Specific Association Between Serum SOD Activity and Osteoporosis in Type 2 Diabetes: A Cross-Sectional Study with Functional Validation · 2026 · DOI

• miRNAs are emerging as powerful regulators of osteo- blast diferentiation and bone formation, exerting their efects through post-transcriptional control of key signalling pathways. Numerous miRNAs have been shown to modu- late critical phases of osteogenesis, osteoclastogenesis, and macrophage polarization, thereby linking bone regeneration with immune modulation.169–171 Despite increasing evidence from in vitro and in vivo studies on bone regeneration and osteoimmunology, there is limited direct evidence connecting specifc miRNAs to improved osseointegration. Thus, while the application of miRNA-based therapies in the context of implant integration is biologically plausible, it remains largely hypothetical and requires further validation.125,172 Osseointe- gration is a complex process that is regulated by a wide range of miRNAs. These miRNAs, many of which remain unidenti- fed or incompletely characterized, control cell proliferation, osteoblast diferentiation, ECM synthesis, and matrix miner- alization.171 Several miRNAs, such as those of the miR-21, miR-155, miR-451, and miR-181 families, have been presented in this review, which both modulate osteoblast and osteo- clast activity as well as immune cell behaviour, suggesting a coordinated regulatory network. However, their efects are not generally defned and depend on the specifc conditions of the experiment. This alignment suggests that miRNAs act as shared molecular regulators at the bone-immune interface, thereby synchronizing infammatory cues with osteogenic responses. These fndings propose the potential of miRNA- based strategies to improve bone regeneration by infuencing both immune responses and bone formation.

  Functionalizing biomaterials for osseointegration – microRNAs at the crossroads of bone formation and immune modulation · 2026 · DOI

• Despite their considerable potential for skeletal regeneration, the early molecular and cellular events underlying BM-MSCs commitment to EO remain elusive.

  Multi-omics profiling links epigenetic and lncRNA changes to early human endochondral ossification priming · 2026 · DOI

• Differentiation programs remain incompletely understood across stem cell types, including for human bone marrow mesenchymal stromal/stem (BM-MSCs) cells, a heterogenous population orchestrating bone formation and establishing a functional hematopoietic niche in the bone marrow.

  Multi-omics profiling links epigenetic and lncRNA changes to early human endochondral ossification priming · 2026 · DOI

• The bHLH family is recognized for its role in regulating cell fate decisions; however, the precise function of Bhlhe22 in bone metabolism had not been fully investigated [78-80].

  Aptamer-functionalized tetrahedral framework nucleic acid delivery of siBhlhe22 for repairing osteoporotic bone defects via dual modulation of PI3K-Akt signaling and purine metabolism · 2026 · DOI

• Evans WJ, Guralnik J, cawthon P, Appleby J, Landi F, clarke L, Vellas B, Ferrucci L and Roubenoff R: Sarcopenia: No consensus, no diagnostic criteria, and no approved indication‑How did we get here? Geroscience 46: 183‑190, 2024.

  Myokines in exercise‑mediated bone homeostasis: Molecular signaling mechanisms and therapeutic implications for bone disorders (Review) · 2026 · DOI

• While further investigations are warranted to fully elucidate these intricate modes of action, the present findings nonetheless underscore a clear advantage: the combined administration of BMP-2 and DNB leads to more abundant reparative tissue, superior tissue quality, and ultimately, more effective skeletal healing.

  Synergistic effects of denosumab and BMP-2 on bone regeneration in critical-sized femoral defects in ovariectomized rats · 2026 · DOI

• Since the levels of Cxcl12 and CCcl5 mRNA in human bone samples had not been assessed when we started the study, we were unable to use a power analysis to determine the number of samples that would be required. No data were excluded from the analyses.

  A RANKL+/CXCR4+ B cell population accumulates in bone marrow and causes age-related osteoporosis in mice · 2026 · DOI

• Although the OVX rat model is the gold standard for simulating postmenopausal estrogen deficiency, bilateral ovariectomy is a systemic intervention that affects overall metabolic states, such as weight gain and lipid metabolism, which may independently affect the composition of the gut microbiota. While a sham-operated group was utilized to control for the confounding effects of surgical trauma, the multiorgan effect of estrogen withdrawal implies that the observed microbial changes may reflect broader metabolic alterations rather than bone metabolism alone. Second, the sample size is relatively small (n = 6 per group), and the duration of 3 months only captures short-term effects. Future research needs larger cohorts and extended longitudinal designs. Third, this study relies on 16 S rRNA sequencing. The integration of metagenomics and metabolomics will better elucidate the metabolic pathways and downstream metabolites involved in the antiosteoporosis effect of JTG.

  Effect of Jintiange capsule on the gut microbiota of rats with ovariectomized osteoporosis · 2026 · DOI

• Dento-maxillofacial bones, which develop from both mesoderm and neural crest cells through endochondral and intramembranous ossification, present unique developmental characteristics that warrant focused investigation of JAK-STAT signaling functions in this region. Notably, mutations in JAK1, JAK2, STAT1, STAT3, STAT5, and STAT6 have been linked to skeletal abnormalities, with patients carrying STAT3 GOF, LOF, and STAT6 GOF mutations displaying specific dento-maxillofacial anomalies. Supplementary Table 1 provides a summary of gene-edited mouse models associated with the JAK-STAT signaling, reinforcing their relevance in disease modeling. The majority of existing research has mainly examined molecular changes at a systemic level, which limits the ability to elucidate the specific roles of particular cell types. Moreover, reliance on classical markers from long bone studies, such as Osx, Col1, and Prx1, has impeded the specificity needed for targeted investigations within craniofacial contexts. Nevertheless, the recent identification of distinct stem cell markers in the maxillofacial future exploration, particularly regarding the influence of the JAK-STAT signaling on these cells and its potential implications for stem cell therapies addressing craniofacial abnormalities. region presents a promising avenue for The development of dento-maxillofacial complex originates from complex sources, and the function of JAK-STAT in long bones may not entirely align with its role in the complex. STAT1 is a prominent regulator, exhibiting different mechanisms in regulating endochondral ossification between cranial and long bones. JAK1, JAK2, STAT3, STAT5, and STAT6 demonstrate dual functions in bone formation and resorption during the development of both long bones and dento-maxillofacial bones. Notably, STAT1 and STAT3 also promote the function of tooth-forming cell populations which is a unique cellular cohort specific to the dentalperiodontal complex. This specialization necessitates careful consideration when administering systemic inhibitors targeting STAT1 or STAT3 activity, as such treatments may potentially compromise dental development.

  JAK-STAT signaling: molecular mechanism and targeted treatment in dento-maxillofacial abnormalities · 2026 · DOI

• the methods of intervention may Despite the promising results demonstrated in this metaanalysis, several limitations should be considered. First, while the inclusion of studies across different animal models provides a comprehensive assessment, the variations in animal species, models, and introduce heterogeneity that could affect the generalizability of the results. For example, the OVX model is widely used for postmenopausal OP, but other models, such as glucocorticoid-induced and diabetesinduced OP, were also included, which might respond differently to treatment. Moreover, the intervention duration and dosage varied significantly across studies, which may impact the overall efficacy of S. miltiorrhiza-derived interventions. Moreover, the broad dose range reported across studies should not be interpreted as directly clinically translatable, because crude extracts and purified constituents differ substantially in composition, potency, and likely pharmacokinetic behavior. In addition, several secondary outcomes were based on a very limited number of studies, such as ultimate stress (n = 2) and PINP (n = 3), which increase the uncertainty of these pooled estimates and raise the possibility of false-positive findings. Therefore, these results should be considered exploratory and require confirmation in future studies. In addition, the included interventions encompassed multiple phytochemical classes and both extracts and isolated constituents. Therefore, the overall pooled effect should not be interpreted as evidence of pharmacological interchangeability among all S. miltiorrhiza-derived interventions. Because the number of studies within many individual compound categories was limited, fully compound-specific meta-analyses were not feasible and should be addressed in future studies. Second, the quality of the included studies could be a concern. Despite following rigorous inclusion criteria, most studies did not report detailed information about random sequence generation, allocation concealment, or blinding of outcome assessors. This may lead to potential bias, affecting the reliability of the findings. Many studies were rated as having unclear rather than confirmed high risk in some domains because key methodological details were insufficiently reported. Therefore, the available evidence should be interpreted cautiously as low-certainty preclinical evidence. In addition, the relatively large pooled effect size for BMD may have been influenced, at least in part, by methodological bias or small-study effects. Although sensitivity analysis excluding high-risk-of-bias studies and trim-and-fill analysis did not materially change the overall direction or statistical significance of the main finding, the adjusted effect size was attenuated, suggesting that the magnitude of the pooled effect should be interpreted cautiously. Furthermore, the studies predominantly used animal models, and the findings may not fully translate to clinical human applications, as animal models do not always replicate human responses accurately. In particular, commonly used rodent models such as ovariectomized (OVX) rats reproduce only selected aspects of human osteoporosis and cannot fully capture the complexity of human disease progression, treatment response, or interspecies pharmacokinetic differences associated with botanical drug interventions. Finally, although S.

  Pharmacological effects of Salvia miltiorrhiza-derived interventions on osteoporosis in animal models: a systematic review and meta-analysis · 2026 · DOI

• PI3K/AKT signaling and ROS regulation are involved in the action of MR, the causal relationship between these two processes remains to be fully clarified. , glucocorticoid-induced or age-related osteoporosis) and in humans remains to be evaluated.

  5-methoxyresorcinol mitigates postmenopausal osteoporosis through regulation of the PI3K–AKT–GSK3β signaling pathway and ROS homeostasis · 2026 · DOI

• In conclusion, emerging biomaterials targeting the gut micro- biota have emerged as a transformative therapeutic strategy for TABLE 2 Critical comparison of administration routes for gut microbiota-targeted biomaterials in osteoporosis therapy.

  The role of gut microbiota in osteoporosis: underlying mechanisms, clinical associations, and emerging biomaterials · 2026 · DOI

• Limited, mainly associative studies linking fecal/serum SCFA levels with BMD and fracture risk.

  The role of gut microbiota in osteoporosis: underlying mechanisms, clinical associations, and emerging biomaterials · 2026 · DOI

• Estrogen deficiency triggers complex alterations in the bone remodeling process, which typically involves multiple signaling Frontiers in Cell and Developmental Biology 10 frontiersin.org Chen et al. 10.3389/fcell.2026.1788812 FIGURE 3 Mechanistic regulatory network of bone metabolism in postmenopausal osteoporosis (PMOP) by E3 ligases and deubiquitinating enzymes (DUBs). Mesenchymal stem cells differentiate into osteoblasts to regulate bone formation, while hematopoietic stem cells differentiate into osteoclasts to regulate bone resorption. In the estrogen-deficient PMOP model, an imbalance in the reversible ubiquitin degradation system of E3 ligases and DUBs modulates bone metabolic pathways through multiple mechanisms, thereby affecting the course of PMOP. Straight arrows indicate activation or positive regulation. T-shaped arrows indicate inhibition or negative regulation; blue icons represent E3 ligases; blue “Ub” labels indicate ubiquitination; purple icons represent DUBs; purple “Ub” labels indicate deubiquitination; green icons denote inhibitors, and orange icons indicate regulated cytokines. and leading osteoclast differentiation, pathways. The evidence summarized in this review suggests that ubiquitin-mediated protein regulation disrupts bone metabolism through synergistic (and sometimes even antagonistic) effects on to osteoblast postmenopausal bone loss. Ubiquitin E3 ligases and DUBs do not function independently but form a reversible regulatory network that modulates bone cell function in a contextdependent manner. Furthermore, most ubiquitin proteases reported in the review do not act directly on mature bone cells but rather affect the differentiation of precursor cell populations such as MSCs or osteoclast progenitor cells, thereby indirectly regulating the differentiation of osteoblasts and osteoclasts. We cannot simply define ubiquitin-related pathways as entirely beneficial or entirely detrimental. PMOP is more likely the turnover result of a selective dysregulation of protein mechanisms, disrupting the delicate balance between bone formation and resorption.

  Ubiquitin-proteasome system regulation of bone remodeling in postmenopausal osteoporosis · 2026 · DOI

• While the cumulative knowledge of osteoclast regulatory molecules, such as receptor activator of nuclear factor-kB ligand (RANKL) and nuclear factor of activated T cells 1 (NFATc1), contributes to the understanding of the developmental progression of osteoclasts, little is known about how the discrete steps of osteoclastogenesis modify osteoclast status but not the absolute number of osteoclasts.

  Unraveling the intricacies of osteoclast differentiation and maturation: insight into novel therapeutic strategies for bone-destructive diseases · 2024 · DOI

• PTH-related peptide (PTHrP) improves the bone marrow micro-environment to activate the bone-remodelling, but the coordinated regulation of PTHrP and transforming growth factor-β (TGFβ) signalling in TMJ-OA remains incompletely understood.

  PTHrP promotes subchondral bone formation in TMJ-OA · 2022 · DOI

• Inflammatory Bowel Disease encompasses Crohns Disease, which is capable of affecting the entire GI tract, although usually favors the ileocolonic and perianal areas, and Ulcerative Colitis, which is limited to the colon.

  Bone Metabolism in Inflammatory Bowel Diseases 2 · 2020 · DOI

• PURPOSE: Interferon (IFN)-γ is a major cytokine produced by immune cells that plays diverse roles in modulating both the immune system and bone metabolism, but its role in autogenous bone grafting remains unknown.

  Interferon-γ enhances the efficacy of autogenous bone grafts by inhibiting postoperative bone resorption in rat calvarial defects · 2016 · DOI

Most-cited papers in Bone Metabolism and Diseases

• Cell signaling and transcriptional regulation of osteoblast lineage commitment, differentiation, bone formation, and homeostasis · Cell Discovery · 2024 · 354 citations
• Osteoporosis and periodontal diseases – An update on their association and mechanistic links · Periodontology 2000 · 2022 · 347 citations
• Cellular and molecular toxicity of lead in bone. · Environmental Health Perspectives · 1991 · 273 citations
• Bone Strength: Current Concepts · Annals of the New York Academy of Sciences · 2006 · 272 citations
• Dynamics of the transition from osteoblast to osteocyte · Annals of the New York Academy of Sciences · 2010 · 264 citations
• Mechanism of alveolar bone destruction in periodontitis — Periodontal bacteria and inflammation · Japanese Dental Science Review · 2021 · 206 citations
• Alcohol-Induced Bone Loss and Deficient Bone Repair · Alcoholism Clinical and Experimental Research · 2005 · 201 citations
• Estrogen Receptor Gene Polymorphisms and the Genetics of Osteoporosis: A HuGE Review · American Journal of Epidemiology · 2005 · 173 citations
• Is All Bone the Same? Distinctive Distributions and Properties of Non-Collagenous Matrix Proteins in Lamellar Vs. Woven Bone Imply the Existence of Different Underlying Osteogenic Mechanisms · Critical Reviews in Oral Biology & Medicine · 1998 · 123 citations
• Unraveling the intricacies of osteoclast differentiation and maturation: insight into novel therapeutic strategies for bone-destructive diseases · Experimental & Molecular Medicine · 2024 · 122 citations

Most recent work

• Cuproptosis promotes inflammatory osteolysis via GYS1-mediated glycogen metabolism · International Journal of Oral Science · 2026
• KDM6B/Pdk1 glycolytic pathway-driven ZEB2 lactylation promotes cellular cementum formation · International Journal of Oral Science · 2026
• COMPARATIVE STUDIES OF CISSUS QUADRANGULARIS AND PISONIA GRANDIS ON ANTI OSTEOPOROTIC ACTIVITY – REVIEW · Zenodo (CERN European Organization for Nuclear Research) · 2026
• LincRNA-EPS alleviates osteoclastogenesis under inflammatory microenvironment through preventing excessive iron metabolism · Cell Death & Disease · 2026
• Osteoimmunology and stem cells: two forefronts in bone biology · Journal of Bone and Mineral Metabolism · 2026
• Angelic Acid Prevents RANKL-Induced Osteoclastogenesis Through Pathway-Biased Inhibition of MAPK–NFATc1 Signaling · Current Issues in Molecular Biology · 2026
• Ubiquitin-proteasome system regulation of bone remodeling in postmenopausal osteoporosis · Frontiers in Cell and Developmental Biology · 2026
• Integrative Machine Learning Screening and Functional Validation of Autophagy-Related Genes in Osteoporosis · Russian Journal of Bioorganic Chemistry · 2026
• Immunomodulatory effects of TAK-242 on osteogenesis of mesenchymal stem cells in acute inflammatory conditions · BMC Molecular and Cell Biology · 2026
• Branch site mutated mice revealed distinct roles of two Runx2 isoforms in bone development · Frontiers in Cell and Developmental Biology · 2026

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