Osteoprotegerin (OPG) and OPG-ligand (OPGL) potently inhibit and stimulate, respectively, osteoclast differentiation (Simonet, W.S., D.L. Lacey, C.R. Dunstan, M. Kelley, M.-S. Chang, R. Luethy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al. 1997. Cell. 89:309-319; Lacey, D.L., E. Timms, H.-L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Cell. 93: 165-176), but their effects on mature osteoclasts are not well understood. Using primary cultures of rat osteoclasts on bone slices, we find that OPGL causes approximately sevenfold increase in total bone surface erosion. By scanning electron microscopy, OPGL-treated osteoclasts generate more clusters of lacunae on bone suggesting that multiple, spatially associated cycles of resorption have occurred. However, the size of individual resorption events are unchanged by OPGL treatment. Mechanistically, OPGL binds specifically to mature OCs and rapidly (within 30 min) induces actin ring formation; a marked cytoskeletal rearrangement that necessarily precedes bone resorption. Furthermore, we show that antibodies raised against the OPGL receptor, RANK, also induce actin ring formation. OPGL-treated mice exhibit increases in blood ionized Ca++ within 1 h after injections, consistent with immediate OC activation in vivo. Finally, we find that OPG blocks OPGL's effects on both actin ring formation and bone resorption. Together, these findings indicate that, in addition to their effects on OC precursors, OPGL and OPG have profound and direct effects on mature OCs and indicate that the OC receptor, RANK, mediates OPGL's effects.

Bone tissues have trabecular bone with a high bone turnover and cortical bone with a low turnover. The mechanisms by which the turnover rate of these bone tissues is determined remain unclear. Osteocytes secrete sclerostin, a Wnt/β-catenin signaling antagonist, and inhibit bone formation. We found that sclerostin expression in cortical bone is more marked than in trabecular bone in Sost reporter mice. Leukemia inhibitory factor (LIF) secreted from osteoclasts reportedly suppressed sclerostin expression and promoted bone formation. Here, we report that osteoclasts downregulate sclerostin expression in trabecular bone and promote bone turnover. Treatment of C57BL/6 mice with an anti-RANKL antibody eliminated the number of osteoclasts and LIF-positive cells in trabecular bone. The number of sclerostin-positive cells was increased in trabecular bone, while the number of β-catenin-positive cells and bone formation were decreased in trabecular bone. Besides, Tnfsf11 heterozygous (Rankl+/-) mice exhibited a decreased number of LIF-positive cells and increased number of sclerostin-positive cells in trabecular bone. Rankl+/- mice exhibited a decreased number of β-catenin-positive cells and reduced bone formation in trabecular bone. Furthermore, in cultured osteoclasts, RANKL stimulation increased Lif mRNA expression, suggesting that RANKL signal increased LIF expression. In conclusion, osteoclasts downregulate sclerostin expression and promote trabecular bone turnover.

Cellular senescence, that is, the withdrawal from the cell cycle, combined with the acquirement of the senescence associated secretory phenotype has important roles during health and disease and is essential for tissue remodeling during embryonic development. Osteoclasts are multinucleated cells, responsible for bone resorption, and cell cycle arrest during osteoclastogenesis is well recognized. Therefore, the aim of this study was to investigate whether these cells should be considered senescent and to assess the influence of hypoxia on their potential senescence status. Osteoclastogenesis and bone resorption capacity of osteoclasts, cultured from CD14+ monocytes, were evaluated in two oxygen concentrations, normoxia (21% O2 ) and hypoxia (5% O2 ). Osteoclasts were profiled by using specific staining for proliferation and senescence markers, qPCR of a number of osteoclast and senescence-related genes and a bone resorption assay. Results show that during in vitro osteoclastogenesis, osteoclasts heterogeneously obtain a senescent phenotype. Furthermore, osteoclastogenesis was delayed at hypoxic compared to normoxic conditions, without negatively affecting the bone resorption capacity. It is concluded that osteoclasts can be considered senescent, although senescence is not uniformly present in the osteoclast population. Hypoxia negatively affects the expression of some senescence markers. Based on the direct relationship between senescence and osteoclastogenesis, it is tempting to hypothesize that contents of the so-called senescence associated secretory phenotype (SASP) not only play a functional role in matrix resorption, but also may regulate osteoclastogenesis.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are Ca2+ channels that localize to intracellular Ca2+ stores such as the endoplasmic reticulum (ER). Recently, IP3Rs were found to participate in the formation of the cytoskeleton and cellular adhesions. In this study, we examined the cellular localization of type I, II, and III IP3Rs to assess their role in cellular adhesion in rat osteoclasts. Rat bone marrow cells were cultured in alpha-MEM with 10% fetal bovine serum, M-CSF, RANKL, and 1,25(OH)2D3 for 1 week to promote osteoclast formation. Type I, II, and III IP3R expression in the osteoclasts was then examined by RT-PCR. Double-staining was performed using antibodies against type I, II, and III IP3Rs and DiOC6, an ER marker, or TRITC-phalloidin, an actin filament marker. Expression of all three IP3Rs was detected in the newly formed osteoclasts; however, the localization of the type I and II IP3Rs was predominantly close to nuclear, and possibly colocalized with the ER, while the type III IP3Rs were localized to the ER and podosomes, actin-rich adhesion structures in osteoclasts. These findings suggest that type III IP3Rs are associated with osteoclast adhesion.

As the most important bone-resorbing cells, osteoclasts play fundamental roles in bone remodeling and skeletal health. Much effort has been focused on identifying the regulators of osteoclast metabolism. Noncoding RNAs (ncRNAs) reportedly regulate osteoclast formation, differentiation, survival, and bone-resorbing activity to participate in bone physiology and pathology. The present review intends to provide a general framework for how ncRNAs and their targets regulate osteoclast differentiation and the important events of osteoclastogenesis they are involved in, including osteoclast precursor generation, early differentiation, mononuclear osteoclast fusion, and multinucleated osteoclast function and survival. This framework is beneficial for understanding bone biology and for identifying the potential biomarkers or therapeutic targets of bone diseases. The review also summarizes the results of in vivo experiments and classic experiment methods for osteoclast-related researches.

The coupling of bone resorption and bone formation is well-recognized in the bone remodeling process, in which osteoblasts and osteoclasts are key players. However, the anabolic effect of human primary osteoclasts has rarely been reported as mouse and cell line derived osteoclasts were mostly used in previous reports. Therefore, a comprehensive comparison of mouse and human osteoclasts and their corresponding functions is needed to study cell-cell interactions between osteoclasts and osteoblasts. Osteoclasts from mouse and human origin were generated, characterized and compared, after which their anabolic effects on the osteogenic differentiation of mouse and human MSCs were assessed. Both murine RAW264.7 derived osteoclasts (mOCs) and primary human osteoclasts (hOCs) derived from buffy coats characteristically displayed multinuclearity, marked integrin β3 expression and enhanced TRAP activity. Despite comparable cell size, mOCs showed higher osteoclast density (number of osteoclasts per cm2 culture dish) and osteoclast nuclearity (average number of nuclei per osteoclast), but lower TRAP activity compared to hOCs. Culturing primary rat and human bone marrow MSCs with the conditioned medium of mOCs or hOCs showed anabolic effects regarding the osteogenic differentiation of MSCs with superiority of hOCs over mOCs. We conclude that despite morphological and functional differences between mouse and human osteoclasts, their secretory factors evoke similar anabolic effects on MSC osteogenic differentiation.

Calcium signaling controls multiple cellular functions and is regulated by the release from internal stores and entry from extracellular fluid. In bone, osteoclast differentiation is induced by RANKL (receptor activator of NF-kappaB ligand)-evoked intracellular Ca(2+) oscillations, which trigger nuclear factor-activated T cells (NFAT) c1-responsive gene transcription. However, the Ca(2+) channels involved remain largely unidentified. Here we show that genetic ablation in mice of Trpv4, a Ca(2+)-permeable channel of the transient receptor potential (TRP) family, increases bone mass by impairing bone resorption. TRPV4 mediates basolateral Ca(2+) influx specifically in large osteoclasts when Ca(2+) oscillations decline. TRPV4-mediated Ca(2+) influx hereby secures intracellular Ca(2+) concentrations, ensures NFATc1-regulated gene transcription, and regulates the terminal differentiation and activity of osteoclasts. In conclusion, our data indicate that Ca(2+) oscillations and TRPV4-mediated Ca(2+) influx are sequentially required to sustain NFATc1-dependent gene expression throughout osteoclast differentiation, and we propose TRPV4 as a therapeutic target for bone diseases.

Autosomal recessive osteopetrosis (ARO) is a heterogeneous disorder, characterized by defective osteoclastic resorption of bone that results in increased bone density. We have studied nine individuals with an intermediate form of ARO, from the county of Västerbotten in Northern Sweden. All afflicted individuals had an onset in early infancy with optic atrophy, and in four patients anemia was present at diagnosis. Tonsillar herniation, foramen magnum stenosis, and severe osteomyelitis of the jaw were common clinical features. Whole exome sequencing, verified by Sanger sequencing, identified a splice site mutation c.212 + 1 G > T in the SNX10 gene encoding sorting nexin 10. Sequence analysis of the SNX10 transcript in patients revealed activation of a cryptic splice site in intron 4 resulting in a frame shift and a premature stop (p.S66Nfs * 15). Haplotype analysis showed that all cases originated from a single mutational event, and the age of the mutation was estimated to be approximately 950 years. Functional analysis of osteoclast progenitors isolated from peripheral blood of patients revealed that stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL) resulted in a robust formation of large, multinucleated osteoclasts which generated sealing zones; however these osteoclasts exhibited defective ruffled borders and were unable to resorb bone in vitro.

In vitro osteoclastogenesis is a central assay in bone biology to study the effect of genetic and pharmacologic cues on the differentiation of bone resorbing osteoclasts. To date, identification of TRAP+ multinucleated cells and measurements of osteoclast number and surface rely on a manual tracing requiring specially trained lab personnel. This task is tedious, time-consuming, and prone to operator bias. Here, we propose to replace this laborious manual task with a completely automatic process using algorithms developed for computer vision. To this end, we manually annotated full cultures by contouring each cell, and trained a machine learning algorithm to detect and classify cells into preosteoclast (TRAP+ cells with 1-2 nuclei), osteoclast type I (cells with more than 3 nuclei and less than 15 nuclei), and osteoclast type II (cells with more than 15 nuclei). The training usually requires thousands of annotated samples and we developed an approach to minimize this requirement. Our novel strategy was to train the algorithm by working at "patch-level" instead of on the full culture, thus amplifying by >20-fold the number of patches to train on. To assess the accuracy of our algorithm, we asked whether our model measures osteoclast number and area at least as well as any two trained human annotators. The results indicated that for osteoclast type I cells, our new model achieves a Pearson correlation (r) of 0.916 to 0.951 with human annotators in the estimation of osteoclast number, and 0.773 to 0.879 for estimating the osteoclast area. Because the correlation between 3 different trained annotators ranged between 0.948 and 0.958 for the cell count and between 0.915 and 0.936 for the area, we can conclude that our trained model is in good agreement with trained lab personnel, with a correlation that is similar to inter-annotator correlation. Automation of osteoclast culture quantification is a useful labor-saving and unbiased technique, and we suggest that a similar machine-learning approach may prove beneficial for other morphometrical analyses.

Osteoclasts are a driver of a vicious bone-destructive cycle with breast cancer cells. Here, we examined whether this vicious cycle can be altered into a beneficial one by activating Wnt signaling with its activating agent, BML284. The conditioned medium, derived from Wnt-activated RAW264.7 pre-osteoclast cells (BM CM), reduced the proliferation, migration, and invasion of EO771 mammary tumor cells. The same inhibitory effect was obtained with BML284-treated primary human macrophages. In a mouse model, BM CM reduced the progression of mammary tumors and tumor-induced osteolysis and suppressed the tumor invasion to the lung. It also inhibited the differentiation of RANKL-stimulated osteoclasts and enhanced osteoblast differentiation. BM CM was enriched with atypical tumor-suppressing proteins such as Hsp90ab1 and enolase 1 (Eno1). Immunoprecipitation revealed that extracellular Hsp90ab1 interacted with latent TGFβ (LAP-TGFβ) as an inhibitor of TGFβ activation, while Hsp90ab1 and Eno1 interacted and suppressed tumor progression via CD44, a cell-adhesion receptor and a cancer stem cell marker. This study demonstrated that osteoclast-derived CM can be converted into a bone-protective, tumor-suppressing agent by activating Wnt signaling. The results shed a novel insight on the unexplored function of osteoclasts as a potential bone protector that may develop an unconventional strategy to combat bone metastasis.

Osteopontin (OPN) is a non-collagenous extracellular sialylated glycoprotein located in bone. It is believed to be one of the key components in osteoclast attachment to bone during resorption. In this study, we characterized OPN and other glycoproteins found in the resorption lacunae to confirm the role of osteoclasts in OPN secretion using electron microscopy and mass spectrometry. Additionally, we examined the glycan epitopes of resorption pits and the effects of different glycan epitopes on the differentiation and function of osteoclasts. Osteoarthritic femoral heads were examined by immunohistochemistry to reveal the presence of OPN in areas of increased bone metabolism in vivo. Our results demonstrate that human osteoclasts secrete OPN into resorption lacunae on native human bone and on carbonated hydroxyapatite devoid of natural OPN. OPN is associated with an elevated bone turnover in osteoarthritic bone under experimental conditions. Our data further confirm that osteoclasts secrete OPN into the resorption pit where it may function as a chemokine for subsequent bone formation. We show that α2,3- and α2,6-linked sialic acids have a role in the process of osteoclast differentiation. OPN is one of the proteins that has both of the above sialic residues, hence we propose that de-sialylation can effect osteoclast differentiation in bone.

Osteoclasts (OCs) seeded on bone slices either drill round pits or dig long trenches. Whereas pits correspond to intermittent resorption, trenches correspond to continuous and faster resorption and require a distinct assembly of the resorption apparatus. It is unknown whether the distinction between pits and trenches has any biological relevance. Using OCs prepared from different blood donors, we found that female OCs achieved increased resorption mainly through pit formation, whereas male OCs did so through trench formation. Trench formation went along with high collagenolytic activity and high cathepsin K (CatK) expression, thereby allowing deeper demineralization. A specific CatK inhibitor abrogated the generation of trenches, while still allowing the generation of pits. OCs obtained from bone marrow were more prone to generate trenches than those obtained from blood. Scanning electron microscopy of bone surfaces eroded in vivo showed trenches and pits of similar size as those made by OCs in culture. We conclude that the distinction between trench- and pit-forming OCs is relevant to the differences among OCs from different skeletal sites, different individuals, including gender, and results from differences in collagenolytic power. This indicates a biological relevance and highlights the importance of discriminating between pits and trenches when assessing resorption.

Homozygosity for the R51Q mutation in sorting nexin 10 (SNX10) inactivates osteoclasts (OCLs) and induces autosomal recessive osteopetrosis in humans and in mice. We show here that the fusion of wild-type murine monocytes to form OCLs is highly regulated, and that its extent is limited by blocking fusion between mature OCLs. In contrast, monocytes from homozygous R51Q SNX10 mice fuse uncontrollably, forming giant dysfunctional OCLs that can become 10- to 100-fold larger than their wild-type counterparts. Furthermore, mutant OCLs display reduced endocytotic activity, suggesting that their deregulated fusion is due to alterations in membrane homeostasis caused by loss of SNX10 function. This is supported by the finding that the R51Q SNX10 protein is unstable and exhibits altered lipid-binding properties, and is consistent with a key role for SNX10 in vesicular trafficking. We propose that OCL size and functionality are regulated by a cell-autonomous SNX10-dependent mechanism that downregulates fusion between mature OCLs. The R51Q mutation abolishes this regulatory activity, leading to excessive fusion, loss of bone resorption capacity and, consequently, to an osteopetrotic phenotype in vivo. This article has an associated First Person interview with the joint first authors of the paper.

Bone fractures are common traumatic injuries of the musculoskeletal system. However, delayed union and non‑union fractures are a major clinical problem that present significant socioeconomic burden to patients and the public health sector. The bone‑resorbing osteoclasts and bone‑forming osteoblasts serve important roles in the fracture repair/healing process. Osteoclast deficiency or decreased osteoblast activity negatively impacts fracture healing. We previously demonstrated that the specific deletion of the serine/threonine kinase 3‑phosphoinositide‑dependent protein kinase 1 (PDK1) in osteoclasts leads to abrogated osteoclast formation and bone resorption in response to receptor activator of nuclear factor‑κB in vitro and protected mice against ovariectomized‑induced bone loss and lipopolysaccharide‑induced osteolysis in vivo. Given the importance of osteoclasts in fracture repair, we hypothesized that the specific loss of PDK1 in osteoclasts will alter the fracture healing process. Mice of tibial fracture were constructed, and tibial specimens were sampled at 7‑, 14‑, 21‑ and 28‑days post‑fracture to observe the effect of PDK1 gene regulated osteoclasts on fracture healing process by X‑ray radiography, microcomputed tomography scanning, histomorphological staining and biomechanical testing. The present study revealed, using the tibial fracture model, that the specific deletion of the PDK1 gene in osteoclasts impeded the fracture healing process by delaying the resorption of the cartilaginous callus and subsequent remodeling of immature woven bone to structurally and mechanically ensure lamellar bone is stronger. No effect on osteoblast bone formation and osteogenesis was observed, thus indicating that delayed fracture healing is primarily due to defective osteoclast activity. These results provide important clinical implications for the use of anti‑resorptive agents, such as bisphosphonates, for the treatment of osteolytic conditions. Such anti‑resorptive therapies may detrimentally delay fracture healing and repair.

Osteoclasts (OCs), the only cells capable of remodeling bone, can demineralize calcium minerals biologically. Naive OCs have limitations for the removal of ectopic calcification, such as in heterotopic ossification (HO), due to their restricted activity, migration and poor adhesion to sites of ectopic calcification. HO is the formation of pathological mature bone within extraskeletal soft tissues, and there are currently no reliable methods for removing these unexpected calcified plaques. In the present study, we develop a chemical approach to modify OCs with tetracycline (TC) to produce engineered OCs (TC-OCs) with an enhanced capacity for targeting and adhering to ectopic calcified tissue due to a broad affinity for calcium minerals. Unlike naive OCs, TC-OCs are able to effectively remove HO both in vitro and in vivo. This achievement indicates that HO can be reversed using modified OCs and holds promise for engineering cells as "living treatment agents" for cell therapy.

Osteoporosis is a bone metabolic disease, characterized by loss of bone density leading to fractures. Its incidence increases with age and affects patient quality of life. Although osteoclasts play a significant role in osteoporosis, their underlying regulatory mechanisms remain unclear. In this study, we found that microRNA (miR)-25-3p negatively regulates osteoclast function through nuclear factor I X (NFIX). Overexpression of NFIX promoted osteoclast proliferation and increased the expression of the osteoclast differentiation and activity markers tartrate-resistant acid phosphatase and cathepsin K. MiR-25-3p transfection inhibited NFIX expression, which in turn inhibited osteoclast proliferation. Collectively, our results suggest that miR-25-3p promotes osteoclast activity by regulating the expression of NFIX. Therefore, targeting miR-25-3p in osteoclasts could be a promising strategy for treating skeletal disorders involving reduced bone formation.

Bone homeostasis is regulated by a balance of bone formation and bone resorption; dysregulation of bone homeostasis may cause bone-related diseases (e.g., osteoporosis, osteopetrosis, bone fracture). Members of the nuclear factor of activated T cells (NFAT) family of transcription factors play crucial roles in the regulation of immune system, inflammatory responses, cardiac formation, skeletal muscle development, and bone homeostasis. Of these, NFATc1 is a key transcription factor mediating osteoclast differentiation, which is regulated by phosphorylation by distinct NFAT kinases including casein kinase 1 (CK1), glycogen synthase kinase 3 (GSK3), and dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs). In this study, we report that cell division control protein 2 homolog (cdc2) is a novel NFAT protein kinase that inhibits NFATc1 activation by direct phosphorylation of the NFATc1 S263 residue. Cdc2 inhibitors such as Roscovitine and BMI-1026 induce reduction of phosphorylation of NFATc1, and this process leads to the inhibition of NFATc1 translocation from the nucleus to the cytoplasm, consequently increasing the nuclear pool of NFATc1. Additionally, the inhibition of cdc2-mediated NFATc1 phosphorylation causes an elevation of osteoclast differentiation or TRAP-positive staining in zebrafish scales. Our results suggest that cdc2 is a novel NFAT protein kinase that negatively regulates osteoclast differentiation.

Elevated activity of bone-degrading osteoclasts (OC) contributes to pathological bone degradation in diseases such as multiple myeloma. Several proinflammatory cytokines, including TNF, contribute to osteoclastogenesis. The receptor-interacting protein kinase 1 (RIPK1) regulates inflammation and cell death. It is recruited to the TNF-receptor complex, where it is ubiquitinated, and activates transcription factor NF-κB and mitogen-activated protein kinases (MAPK). Smac-mimetics (SM) is a group of drugs that block RIPK1 ubiquitination and shifts RIPK1 to activation of apoptosis or necroptosis. In this manuscript, we show that the two SM birinapant and LCL-161 reduced the number and viability of primary human OC, and induced TNF-dependent cell death in OC precursors (pre-OC). Birinapant was more cytotoxic than LCL-161 and induced predominantly apoptosis and to some degree necroptosis. Both inhibitors restrained osteoclastogenesis induced by myeloma patient bone-marrow aspirates. SM has gained attention as novel treatment strategies both for cancer and chronic inflammatory pathologies, but limited information has been available on interactions with primary human immune cells. As LCL-161 is in phase 2 clinical studies for multiple myeloma, we propose that SM might possess additional benefits in reducing bone degradation in myeloma patients. Taken together, we show that SM reduces human osteoclastogenesis, and that these compounds may represent promising drug candidates for pathological bone degradation.

Prostaglandin E2 (PGE2), a major cyclooxygenase-2 (COX-2) product, is highly secreted by the osteoblast lineage in the subchondral bone tissue of osteoarthritis (OA) patients. However, NSAIDs, including COX-2 inhibitors, have severe side effects during OA treatment. Therefore, the identification of novel drug targets of PGE2 signaling in OA progression is urgently needed. Osteoclasts play a critical role in subchondral bone homeostasis and OA-related pain. However, the mechanisms by which PGE2 regulates osteoclast function and subsequently subchondral bone homeostasis are largely unknown. Here, we show that PGE2 acts via EP4 receptors on osteoclasts during the progression of OA and OA-related pain. Our data show that while PGE2 mediates migration and osteoclastogenesis via its EP2 and EP4 receptors, tissue-specific knockout of only the EP4 receptor in osteoclasts (EP4LysM) reduced disease progression and osteophyte formation in a murine model of OA. Furthermore, OA-related pain was alleviated in the EP4LysM mice, with reduced Netrin-1 secretion and CGRP-positive sensory innervation of the subchondral bone. The expression of platelet-derived growth factor-BB (PDGF-BB) was also lower in the EP4LysM mice, which resulted in reduced type H blood vessel formation in subchondral bone. Importantly, we identified a novel potent EP4 antagonist, HL-43, which showed in vitro and in vivo effects consistent with those observed in the EP4LysM mice. Finally, we showed that the Gαs/PI3K/AKT/MAPK signaling pathway is downstream of EP4 activation via PGE2 in osteoclasts. Together, our data demonstrate that PGE2/EP4 signaling in osteoclasts mediates angiogenesis and sensory neuron innervation in subchondral bone, promoting OA progression and pain, and that inhibition of EP4 with HL-43 has therapeutic potential in OA.

Spinal pain affects individuals of all ages and is the most common musculoskeletal problem globally. Its clinical management remains a challenge as the underlying mechanisms leading to it are still unclear. Here, we report that greater numbers of senescent osteoclasts (SnOCs) are observed in mouse models of spinal hypersensitivity, like lumbar spine instability (LSI) or aging, compared to controls. The larger population of SnOCs is associated with induced sensory nerve innervation, as well as the growth of H-type vessels, in the porous endplate. We show that deletion of senescent cells by administration of the senolytic drug Navitoclax (ABT263) results in significantly less spinal hypersensitivity, spinal degeneration, porosity of the endplate, sensory nerve innervation and H-type vessel growth in the endplate. We also show that there is greater SnOC-mediated secretion of Netrin-1 and NGF, two well-established sensory nerve growth factors, compared to non-senescent OCs. These findings suggest that pharmacological elimination of SnOCs may be a potent therapy to treat spinal pain.