During skeletal growth, it is thought that the lactate secreted by the glycolytic nucleus pulposus (NP) cells exits the intervertebral disc into circulation via endplates. Our current studies challenge this long-held notion. Mice with early postnatal, endplate, and annulus fibrosus-specific deletion of lactate importer, MCT1, exhibited disc degeneration characterized by NP cell loss and pronounced endplate structural changes. Using metabolic and transcriptomic approaches, we demonstrate that MCT1 loss inhibits endplate chondrocyte differentiation and that lactate serves both as a crucial TCA metabolite and promotes protein and histone lactylation and gene expression. These findings suggest that during skeletal growth, NP-derived lactate in part supports endplate cartilage differentiation into the vascularized subchondral bone, which, when absent, limits nutrient exchange and availability to the other disc compartments, affecting their homeostasis. This study provides the first in vivo evidence that loss of MCT1 mediated lactate uptake in endplate cells causes delayed maturation and intervertebral disc degeneration.

Abstract Aging is one of the most important risk factors for Intervertebral disc degeneration, a major contributor to chronic low back and neck pain. Recently, we demonstrated a critical role for SIRT6, a nuclear NAD⁺- dependent deacetylase and defatty acylase, in maintaining intervertebral disc health with aging. We therefore investigated whether pharmacological activation of SIRT6 improves disc health by examining the spinal phenotype of 24-month-old mice treated with the well-studied agonist MDL-800 for 6 months. Histological studies revealed healthy disc tissue morphology, enhanced cell viability, and lower degeneration scores in mice treated with MDL-800. Further mechanistic insights revealed that SIRT6 activation decreased H3K9ac levels, improved cell phenotype and matrix quality, and reduced the SASP burden in the disc, characterized by decreased abundance of p21, IL-6, and TGF-β. Tissue RNA-Seq, in vitro measurements of histone 3 modifications, and multi-omics ATAC-seq/RNA-seq analyses revealed that SIRT6 activation altered the epigenetic status (decreased H3K9ac, H3K36me3, and H3K79me2) and transcriptomic landscape of disc cells. Notably, MDL-800 treatment increased LC3II levels in disc cells, indicating enhanced autophagic flux. Furthermore, plasma LC-MS and nuclear magnetic resonance (NMR) analyses revealed minimal systemic metabolomic changes. ScRNA-sequencing of splenocytes and bone marrow cells and systemic cytokine profiling indicated good tolerance and the absence of systemic inflammation following MDL-800 treatment. Our study demonstrates that SIRT6 activation modulates autophagy, cell senescence, and matrix homeostasis in the disc, underscoring the feasibility of targeting SIRT6 activation as a promising pharmacological strategy to maintain disc health in the aging spine.

This protocol describes a standardized and reproducible minimally invasive approach for establishing mouse models of bisphosphonate-related osteonecrosis of the jaw (BRONJ) and osteoradionecrosis of the jaw (ORNJ). The method combines a unified low-trauma oral surgical procedure with disease-specific injury induction strategies to generate robust and clinically relevant models of jaw osteonecrosis. For BRONJ, systemic zoledronic acid administration is coupled with mandibular first molar extraction using tape-assisted mouth opening and customized bent micro-forceps, minimizing soft tissue damage and reducing procedural variability. For ORNJ, a customized lead-shielding platform enables precise, noninvasive mandible-targeted irradiation, producing reproducible bone injury while limiting off-target radiation exposure. Together, these complementary models provide a consistent and minimally invasive framework for investigating jaw osteonecrosis arising from distinct etiologies. The protocol supports comprehensive downstream analyses, including micro-computed tomography, histology, and immunofluorescence, and facilitates mechanistic studies of disease pathogenesis, bone regeneration, and therapeutic intervention.

Chondroadherin-like (CHADL), a small leucine-rich proteoglycan, plays a role in ECM assembly and chondrocyte differentiation. CHADL has been implicated in osteoarthritis (OA) due to the discovery of a rare frameshift variant (rs532464664) that confers one of the highest genetic risks for hip OA reported to date. In this study, we characterized the skeletal phenotypes of two mouse models of Chadl loss-of-function, a global null and an 8bp insertion that mimics the human frameshift variant. Surprisingly, while there was a slight increase in knee OA at 12 months, there were no genotype-based differences in knee or hip OA in aged mice. Interestingly, histological assessment revealed phenotypic changes in the intervertebral discs of mutant mice, along with increased disc height index, suggesting altered motion-segment biomechanics. Polarized imaging showed dysregulated collagen turnover in the annulus fibrosus (AF), evidenced by changes in the proportions of thin and intermediate fibers. There was decreased abundance of the nucleus pulposus (NP) marker CA3 and decreased staining for ECM proteins in the AF of mutants. CHADL loss affected vertebral trabecular architecture, cortical thickness, and mineral density. This study highlights an unrecognized role of CHADL in fine-tuning ECM homeostasis and health of the intervertebral disc and vertebral bone.

The intervertebral disc is a prime example of tissue adaptation to metabolic and mechanical extremes. As the largest avascular organ in the human body, the disc and the central nucleus pulposus (NP) endure constant compressive loading while sustaining a hypoxic, relatively acidic, and hyperosmotic microenvironment that would be inhospitable to most cell types. Over the past two decades, work from my laboratory has revealed extraordinary details of this type of adaptation, that hypoxia-inducible factors (HIFs), particularly HIF-1α, serve as master regulators of NP cell survival, metabolism, and matrix homeostasis. This review chronicles the scientific and personal journey that began with a single unexpected observation: the normoxic stabilization of HIF-1α in NP cells. That finding grew into a substantial understanding of oxygen-independent HIF regulation and biology in the disc. Through sustained collaborations with Irving Shapiro and other colleagues and a succession of talented trainees, we demonstrated that HIF-1α is indispensable for disc development and homeostasis. In contrast, HIF-2α plays a distinct, largely opposing role, promoting matrix catabolism and fibrosis. Our work has revealed the molecular architecture underlying the disc's adaptation to avascularity, encompassing HIF-dependent regulation of glycolytic enzymes, glucose transporters, carbonic anhydrases CA9 and CA12, monocarboxylate transporter 4 (MCT4), and BNIP3-mediated mitophagy, and has opened promising therapeutic avenues for disc regeneration. Recently, we discovered that NP-derived lactate exported via MCT4 is actively imported primarily by vertebral endplate cells, and, to a lesser extent, by annulus fibrosus cells via MCT1, where it serves dual roles as a TCA metabolite and an epigenetic regulator through lactylation. This review celebrates the intellectual journey, the collaborative spirit that sustained it, and the honor of the Alexander von Humboldt Research Award, while providing a synthesis of HIF biology in the intervertebral disc spanning the discovery of oxygen by Priestley 1774 to the 2019 Nobel Prize and beyond.

Bone regeneration relies on specialized vascular niches, yet the contribution of lymphatic vessels across distinct skeletal sites remains poorly understood. Here, we identify bone lymphatics as an essential component of the regenerative microenvironment in the mandible and long bones. We demonstrate that bone lymphatic endothelial cells (LECs) constitute a specialized endothelial population that is transcriptionally and spatially distinct from periosteal LECs. During skeletal repair, bone LECs reactivate a regenerative transcriptional programme, and promote mandibular and fracture healing. In osteonecrosis of the jaw and periodontitis, bone lymphatic-associated signalling is disrupted, identifying impaired lymphatic function as a shared feature of mandibular disease. Therapeutic activation of VEGFC-FLT4 signalling during injury or mandibular disease restores lymphangiogenesis, enhances osteogenesis, and markedly improves bone regeneration. Together, our findings advance the paradigm-shifting role of bone lymphatics positive regulators of bone regeneration and identify lymphatic activation as a promising therapeutic strategy to enhance bone regeneration in mandibular diseases.

Genetic background is a major determinant of disc degeneration, a leading cause of chronic back pain and disability. Herein, we demonstrate that premature disc cell senescence contributes to early-onset degeneration in SM/J mice and test two systemic senotherapeutic strategies to mitigate it: Navitoclax (Nav.) and a cocktail of Dasatinib and Quercetin (DQ). While Nav. treatment did not improve severe degeneration in SM/J mice, DQ-treated mice showed lower grades of degeneration and decreased abundance of senescence markers p19ARF and p21. DQ improved disc cell viability and phenotype retention and retarded fibrosis of the nucleus pulposus tissue. Transcriptomic analysis showed disc compartment-specific effects of the treatment, with cell cycle regulation and JNK signaling being commonly affected across tissue types. A comparison with DQ-mediated aging-dependent amelioration of disc degeneration in C57BL/6N mice identified Junb and Zfp36l1 signaling as shared DQ targets in the mouse disc. This study reinforces the efficacy of senolytic treatments in ameliorating local senescence and intervertebral disc fibrosis.

The seventh biennial ORS-PSRS International Spine Research Symposium was held from November 10-14, 2024, at Skytop Lodge in Pennsylvania, USA. Jointly organized by the PSRS and ORS, the meeting brought together over 195 participants from 13 countries. Selected contributors were invited to submit full-length manuscripts for this JOR Spine Special Issue.

Chondrocyte biology is being revolutionized by single-cell multi-omics technologies, revealing cellular heterogeneity within cartilaginous tissues. Although past research has implicated cellular heterogeneity in chondrocyte populations, advances over the past decade in single-cell transcriptomics now enable a more granular, functionally annotated classification of chondrocyte subtypes. These analyses provide crucial insights into the role of these subtypes in cartilage formation, maintenance and disease progression. Chondrocyte populations are implicated in tissue homeostasis, pathogenesis and responses to external stimuli, including pro-inflammatory mediators and novel therapeutic agents. This knowledge opens pathways for developing targeted treatments for diseases such as osteoarthritis and intervertebral disc disease. Insights into the molecular signatures of disease-critical chondrocyte populations provide a foundation for biomarker discovery and therapeutic targeting, and there are exciting opportunities for leveraging these findings to progress regenerative therapies. Spatial and temporal profiling of cellular markers, behaviour and metabolic activity will enhance understanding of disease pathogenesis and chondrosenescence and could possibly enable early intervention for osteoarthritis, thereby preventing irreversible joint damage. Future research must integrate advanced single-cell techniques with computational modelling to unravel the dynamic interplay of chondrocyte populations. These efforts could transform precision medicine in rheumatology, addressing the unmet clinical needs in cartilage-related diseases. This Review provides an update on chondrocyte heterogeneity in cartilaginous tissues in health, disease and senescence focusing on insights gained from single-cell analyses. The authors highlight how single-cell multi-omics techniques could reveal new biomarkers and therapeutic targets for conditions such as osteoarthritis and intervertebral disc degeneration.

Alterations in physiological loading of the spine are deleterious to intervertebral disc health. The base of the mouse caudal spine region Ca3-6 that naturally experiences increased flexion, showed adaptive tissue remodeling, reminiscent of disc degeneration in young adult mice. Given the role of Syndecan 4 (SDC4), a cell surface heparan sulfate proteoglycan in disc matrix turnover and mechanosensing, we investigated if deletion could mitigate this loading-dependent phenotype. Notably, at spinal levels Ca3-6, Sdc4-knockout (KO) mice did not exhibit increased collagen fibril and fibronectin deposition in the nucleus pulposus (NP) compartment or showed the alterations in collagen crosslinks observed in wild-type mice. Similarly, unlike wild-type mice, NP cells in Sdc4-KO mice retained transgelin (TAGLN) expression and showed absence of collagen type X (COL10) deposition, pointing to the preservation of their notochordal characteristics. Proteomic analysis revealed that NP tissues responded to the altered loading by increasing the abundance of proteins associated with extracellular matrix remodeling, chondrocyte development, and contractility. Similarly, downregulated proteins suggested decreased vesicle transport, autophagy-related pathway, and RNA quality control regulation. Notably, NP proteome from Sdc4-KO suggested that increased dynamin-mediated endocytosis, autophagy-related pathway, and RNA and DNA quality control may underscore the protection from adaptive tissue remodeling caused by this naturally observed altered loading. Our study highlights the important role of SDC4 in fine-tuning cellular homeostasis and extracellular matrix production in disc environment subjected to altered loading.

During skeletal growth, there is an increased secretion of lactate by glycolytic nucleus pulposus (NP) cells of the intervertebral disc. To investigate the role of this anion, we generated annulus fibrosus (AF) and endplate (EP) specific Mct1 cKO ( Slc16a1 Col2CreERT2 ) mice. Histological and spatial transcriptomic studies indicated significant disc degeneration in Mct1 cKO , characterized by NP cell loss and delayed EP maturation. Metabolic assays showed that while AF and EP cells were glycolytic, EP chondrocytes readily metabolized lactate. In EP cells, lactate promoted protein, and H3K18 lactylation, implying epigenetic programming. These findings suggest that NP-derived lactate promotes EP cartilage transdifferentiation into the subchondral bone, and in its absence, continued glucose consumption by the persistent EP cartilage reduces glucose availability to the NP and AF likely contributing to tissue degeneration. This study provides the first in vivo evidence that metabolic coupling between NP and EP cells is essential for disc growth and health.

Despite the high prevalence of age-dependent intervertebral disc calcification, there is a glaring lack of treatment options for this debilitating pathology. We investigated the efficacy of long-term oral K3Citrate supplementation in ameliorating disc calcification in LG/J mice, a model of spontaneous age-associated disc calcification. K3Citrate reduced the incidence of disc calcification without affecting the vertebral bone structure, knee calcification, plasma chemistry, or locomotion in LG/J mice. Notably, a positive effect on grip strength was evident in treated mice. FTIR spectroscopy of the persisting calcified nodules indicated K3Citrate did not alter the mineral composition. Mechanistically, activation of an endochondral differentiation in the cartilaginous endplates and nucleus pulposus (NP) compartment contributed to LG/J disc calcification. Importantly, K3Citrate reduced calcification incidence by Ca2+ chelation throughout the disc while exhibiting a differential effect on NP and endplate cell differentiation. In the NP compartment, K3Citrate reduced the NP cell acquisition of a hypertrophic chondrocytic fate, but the pathologic endochondral program was unimpacted in the endplates. Overall, this study for the first time shows the therapeutic potential of oral K3Citrate as a systemic intervention strategy to ameliorate disc calcification.

Nucleus pulposus (NP) cells of the intervertebral disc reside in a physiologically hypoxic niche and bioenergetically rely on anaerobic glycolysis generating lactate. Consequently, disc cells reside in an environment characterized by acidic pH. Pathologically acidic pH in NP cells has been shown to lead to matrix catabolism and decreased glucose uptake; therefore NP cells rely on robust HIF-1-dependent pH-regulating systems to maintain the optimal pH for cell survival. However, how disc cells handle high levels of lactate is not entirely understood. To address this long-standing question, we generated annulus fibrosus (AF) and endplate (EP) specific knockout mice for MCT1, a ubiquitously expressed channel involved in lactate influx. Our studies showed significant disc degeneration in knockout mice, characterized by NP cell loss and EP changes. Our studies provide the first in vivo evidence that metabolic coupling between disc compartments is essential for the growth and maintenance of health.

There are no appropriate mouse models to study the pathophysiology of spontaneous disc herniations in a wild-type setting. SM/J mice, a poor healer inbred strain, presented a high incidence of age-associated lumbar disc herniations with neurovascular innervations. Transcriptomic comparisons of the SM/J annulus fibrosus with human tissues showed shared pathways related to immune cell activation and inflammation. Notably, aged SM/J mice showed increased pain sensitization and neuroinflammation with altered extracellular matrix regulation in the dorsal root ganglia and spinal cord. There were increased T cells in the vertebral marrow, and cytometry by time-of-flight analysis showed increased splenic CD8+ T cells, nonspecific activation of CD8+ memory T cells, and enhanced interferon-γ production in the myeloid compartment. Single-cell RNA sequencing of peripheral blood mononuclear cells showed more B cells, with lower proportions of T cells, monocytes, and granulocytes. This study highlights the contribution of genetic background and aging to increased susceptibility of spontaneous intervertebral disc herniations in a clinically relevant murine model.

Intervertebral disc degeneration is a major risk factor contributing to chronic low back and neck pain. While the etiological factors for disc degeneration vary, age is still one of the most important risk factors. Recent studies have shown the promising role of SIRT6 in mammalian aging and skeletal tissue health, however its role in the intervertebral disc health remains unexplored. We investigated the contribution of SIRT6 to disc health by studying the age-dependent spinal phenotype of mice with conditional deletion of Sirt6 in the disc (AcanCreERT2; Sirt6fl/fl). Histological studies showed a degenerative phenotype in knockout mice compared to Sirt6fl/fl control mice at 12 months, which became pronounced at 24 months. RNA-Seq analysis of NP and AF tissues, in vitro quantitative histone analysis, and RNA-seq with ATAC-seq multiomic studies revealed that SIRT6-loss resulted in changes in acetylation and methylation status of specific Histone 3 lysine residues and affected DNA accessibility and transcriptomic landscape. A decrease in autophagy and an increase in DNA damage were also noted in Sirt6-deficient cells. Further mechanistic insights revealed that loss of SIRT6 increased senescence and SASP burden in the disc characterized by increased p21, p19, γH2AX, IL-6, IL-1β, and TGF-β abundance. Taken together, our study highlights the contribution of SIRT6 in modulating DNA damage, autophagy, and cell senescence and its importance in maintaining disc health during aging, thereby underscoring it as a potential therapeutic target to treat intervertebral disc degeneration.

Recent studies have highlighted the importance of mitochondria in NP cells and articular chondrocyte health. Since the understanding of mechanisms governing mitochondrial dynamics in these tissues is lacking, we investigated the role of OPA1, a mitochondrial fusion protein, in their homeostasis. OPA1 knockdown in NP cells altered mitochondrial size and cristae shape and increased the oxygen consumption rate. OPA1 governed the morphology of multiple organelles, including peroxisomes, early endosomes and cis-Golgi and loss resulted in the dysregulation of autophagy. Metabolic profiling and 13C-flux analyses revealed TCA cycle anaplerosis and altered metabolism in OPA1-deficient NP cells. Noteworthy, Opa1AcanCreERT2 mice showed age-dependent disc degeneration, osteoarthritis, and vertebral osteopenia. RNA-Sequencing of Opa1cKO NP tissue revealed dysregulation of metabolism, autophagy, cytoskeletal reorganization, and extracellular matrix and shared strong thematic similarities with a subset of human degenerative NP samples. Our findings underscore that maintenance of mitochondrial dynamics and multi-organelle cross-talk is critical in preserving metabolic homeostasis of disc and cartilage.

This paper presents a review of the potential role of the endoplasmic reticulum/Golgi complex and intracellular vesicles in mediating events leading to or associated with vertebrate tissue mineralization. The possible importance of these organelles in this process is suggested by observations that calcium ions accumulate in the tubules and lacunae of the endoplasmic reticulum and Golgi. Similar levels of calcium ions (approaching millimolar) are present in vesicles derived from endosomes, lysosomes and autophagosomes. The cellular level of phosphate ions in these organelles is also high (millimolar). While the source of these ions for mineral formation has not been identified, there are sound reasons for considering that they may be liberated from mitochondria during the utilization of ATP for anabolic purposes, perhaps linked to matrix synthesis. Published studies indicate that calcium and phosphate ions or their clusters contained as cargo within the intracellular organelles noted above lead to formation of extracellular mineral. The mineral sequestered in mitochondria has been documented as an amorphous calcium phosphate. The ion-, ion cluster- or mineral- containing vesicles exit the cell in plasma membrane blebs, secretory lysosomes or possibly intraluminal vesicles. Such a cell-regulated process provides a means for the rapid transport of ions or mineral particles to the mineralization front of skeletal and dental tissues. Within the extracellular matrix, the ions or mineral may associate to form larger aggregates and potential mineral nuclei, and they may bind to collagen and other proteins. How cells of hard tissues perform their housekeeping and other biosynthetic functions while transporting the very large volumes of ions required for mineralization of the extracellular matrix is far from clear. Addressing this and related questions raised in this review suggests guidelines for further investigations of the intracellular processes promoting the mineralization of the skeletal and dental tissues.

Syndecan 4 (SDC4), a cell surface heparan sulfate proteoglycan, is known to regulate matrix catabolism by nucleus pulposus cells in an inflammatory milieu. However, the role of SDC4 in the aging spine has never been explored. Here we analyzed the spinal phenotype of Sdc4 global knockout (KO) mice as a function of age. Micro-computed tomography showed that Sdc4 deletion severely reduced vertebral trabecular and cortical bone mass, and biomechanical properties of vertebrae were significantly altered in Sdc4 KO mice. These changes in vertebral bone were likely due to elevated osteoclastic activity. The histological assessment showed subtle phenotypic changes in the intervertebral disc. Imaging-Fourier transform-infrared analyses showed a reduced relative ratio of mature collagen crosslinks in young adult nucleus pulposus (NP) and annulus fibrosus (AF) of KO compared to wildtype discs. Additionally, relative chondroitin sulfate levels increased in the NP compartment of the KO mice. Transcriptomic analysis of NP tissue using CompBio, an AI-based tool showed biological themes associated with prominent dysregulation of heparan sulfate GAG degradation, mitochondria metabolism, autophagy, endoplasmic reticulum (ER)-associated misfolded protein processes and ER to Golgi protein processing. Overall, this study highlights the important role of SDC4 in fine-tuning vertebral bone homeostasis and extracellular matrix homeostasis in the mouse intervertebral disc.

This review examines the possible role of mitochondria in maintaining calcium and phosphate ion homeostasis and participating in the mineralization of bone, cartilage and other vertebrate hard tissues. The paper builds on the known structural features of mitochondria and the documented observations in these tissues that the organelles contain calcium phosphate granules. Such deposits in mitochondria putatively form to buffer excessively high cytosolic calcium ion concentrations and prevent metabolic deficits and even cell death. While mitochondria protect cytosolic enzyme systems through this buffering capacity, the accumulation of calcium ions by mitochondria promotes the activity of enzymes of the tricarboxylic acid (TCA/Krebs) cycle, increases oxidative phosphorylation and ATP synthesis, and leads to changes in intramitochondrial pH. These pH alterations influence ion solubility and possibly the transitions and composition in the mineral phase structure of the granules. Based on these considerations, mitochondria are proposed to support the mineralization process by providing a mobile store of calcium and phosphate ions, in smaller cluster or larger granule form, while maintaining critical cellular activities. The rise in the mitochondrial calcium level also increases the generation of citrate and other TCA cycle intermediates that contribute to cell function and the development of extracellular mineral. This paper suggests that another key role of the mitochondrion, along with the effects just noted, is to supply phosphate ions, derived from the breakdown of ATP, to endolysosomes and autophagic vesicles originating in the endoplasmic reticulum and Golgi and at the plasma membrane. These many separate but interdependent mitochondrial functions emphasize the critical importance of this organelle in the cellular control of vertebrate mineralization.