Human linkage studies suggest that craniofacial deformities result from either genetic mutations related to cholesterol metabolism or high-cholesterol maternal diets. However, little is known about the precise roles of intracellular cholesterol metabolism in the development of craniofacial bones, the majority of which are formed through intramembranous ossification. Here, we show that an altered cholesterol metabolic status results in abnormal osteogenesis through dysregulation of primary cilium formation during bone formation. We found that cholesterol metabolic aberrations, induced through disruption of either Dhcr7 (which encodes an enzyme involved in cholesterol synthesis) or Insig1 and Insig2 (which provide a negative feedback mechanism for cholesterol biosynthesis), result in osteoblast differentiation abnormalities. Notably, the primary cilia responsible for sensing extracellular cues were altered in number and length through dysregulated ciliary vesicle fusion in Dhcr7 and Insig1/2 mutant osteoblasts. As a consequence, WNT/β-catenin and hedgehog signaling activities were altered through dysregulated primary cilium formation. Strikingly, the normalization of defective cholesterol metabolism by simvastatin, a drug used in the treatment of cholesterol metabolic aberrations, rescued the abnormalities in both ciliogenesis and osteogenesis in vitro and in vivo. Thus, our results indicate that proper intracellular cholesterol status is crucial for primary cilium formation during skull formation and homeostasis.

Our recent studies demonstrate that the focal adhesion protein Kindlin-2 is critical for chondrogenesis and early skeletal development. Here, we show that deleting Kindlin-2 from osteoblasts using the 2.3-kb mouse Col1a1-Cre transgene minimally impacts bone mass in mice, but deleting Kindlin-2 using the 10-kb mouse Dmp1-Cre transgene, which targets osteocytes and mature osteoblasts, results in striking osteopenia in mice. Kindlin-2 loss reduces the osteoblastic population but increases the osteoclastic and adipocytic populations in the bone microenvironment. Kindlin-2 loss upregulates sclerostin in osteocytes, downregulates β-catenin in osteoblasts, and inhibits osteoblast formation and differentiation in vitro and in vivo. Upregulation of β-catenin in the mutant cells reverses the osteopenia induced by Kindlin-2 deficiency. Kindlin-2 loss additionally increases the expression of RANKL in osteocytes and increases osteoclast formation and bone resorption. Kindlin-2 deletion in osteocytes promotes osteoclast formation in osteocyte/bone marrow monocyte cocultures, which is significantly blocked by an anti-RANKL-neutralizing antibody. Finally, Kindlin-2 loss increases osteocyte apoptosis and impairs osteocyte spreading and dendrite formation. Thus, we demonstrate an important role of Kindlin-2 in the regulation of bone homeostasis and provide a potential target for the treatment of metabolic bone diseases.

Bone resorption requires the formation of complex, actin-rich cytoskeletal structures. During the early phase of sealing ring formation by osteoclasts, L-plastin regulates actin-bundling to form the nascent sealing zones (NSZ). Here, we show that L-plastin knockout mice produce osteoclasts that are deficient in the formation of NSZs, are hyporesorptive, and make superficial resorption pits in vitro. Transduction of TAT-fused full-length L-plastin peptide into osteoclasts from L-plastin knockout mice rescued the formation of nascent sealing zones and sealing rings in a time-dependent manner. This response was not observed with mutated full-length L-plastin (Ser-5 and -7 to Ala-5 and -7) peptide. In contrast to the observed defect in the NSZ, L-plastin deficiency did not affect podosome formation or adhesion of osteoclasts in vitro or in vivo. Histomorphometry analyses in 8- and 12-week-old female L-plastin knockout mice demonstrated a decrease in eroded perimeters and an increase in trabecular bone density, without a change in bone formation by osteoblasts. This decrease in eroded perimeters supports that osteoclast function is attenuated in L-plastin knockouts. Micro-CT analyses confirmed a marked increase in trabecular bone mass. In conclusion, female L-plastin knockout mice had increased trabecular bone density due to impaired bone resorption by osteoclasts. L-plastin could be a potential target for therapeutic interventions to treat trabecular bone loss.

There remain unmet clinical needs for safe and effective bone anabolic therapies to treat aging-related osteoporosis and to improve fracture healing in cases of nonunion or delayed union. Wnt signaling has emerged as a promising target pathway for developing novel bone anabolic drugs. Although neutralizing antibodies against the Wnt antagonist sclerostin have been tested, Wnt ligands themselves have not been fully explored as a potential therapy. Previous work has demonstrated Wnt7b as an endogenous ligand upregulated during osteoblast differentiation, and that Wnt7b overexpression potently stimulates bone accrual in the mouse. The earlier studies however did not address whether Wnt7b could promote bone formation when specifically applied to aged or fractured bones. Here we have developed a doxycycline-inducible strategy where Wnt7b is temporally induced in the bones of aged mice or during fracture healing. We report that forced expression of Wnt7b for 1 month starting at 15 months of age greatly stimulated trabecular and endosteal bone formation, resulting in a marked increase in bone mass. We further tested the effect of Wnt7b on bone healing in a murine closed femur fracture model. Induced expression of Wnt7b at the onset of fracture did not affect the initial cartilage formation but promoted mineralization of the subsequent bone callus. Thus, targeted delivery of Wnt7b to aged bones or fracture sites may be explored as a potential therapy.

Osteoclasts are multinucleated, giant cells derived from myeloid progenitors. While receptor activator of NF-κB ligand (RANKL) stimulation is the primary driver of osteoclast differentiation, additional signaling further contributes to osteoclast maturation. Here, we demonstrate that immunoglobulin superfamily member 11 (IgSF11), whose expression increases during osteoclast differentiation, regulates osteoclast differentiation through interaction with postsynaptic density protein 95 (PSD-95), a scaffold protein with multiple protein interaction domains. IgSF11 deficiency in vivo results in impaired osteoclast differentiation and bone resorption but no observed defect in bone formation. Consequently, IgSF11-deficient mice exhibit increased bone mass. Using in vitro osteoclast culture systems, we show that IgSF11 functions through homophilic interactions. Additionally, we demonstrate that impaired osteoclast differentiation in IgSF11-deficient cells is rescued by full-length IgSF11 and that the IgSF11-PSD-95 interaction requires the 75 C-terminal amino acids of IgSF11. Our findings reveal a critical role for IgSF11 during osteoclast differentiation and suggest a role for IgSF11 in a receptor- and signal transduction molecule-containing protein complex.

Osteoarthritis, a disorder characterized by articular cartilage deterioration, varying degrees of inflammation, and chondrocyte apoptosis, is the most common chronic joint disease. To slow or reverse its progression, inflammation should be inhibited, and chondrocyte proliferation should be promoted. Tetrahedral framework nucleic acids can be internalized by chondrocytes (even inflammatory chondrocytes) and can enhance their proliferation and migration. Wogonin, a naturally occurring flavonoid, suppresses oxidative stress and inhibits inflammation. In this study, tetrahedral framework nucleic acids were successfully self-assembled and used to load wogonin. We confirmed the effective formation of tetrahedral framework nucleic acid/wogonin complexes by dynamic light scattering, zeta potential analysis, transmission electron microscopy, and fluorescence spectrophotometry. Tetrahedral framework nucleic acids, wogonin, and especially tetrahedral framework nucleic acid/wogonin complexes effectively alleviated inflammation in vitro and in vivo and prevented cartilage destruction. In addition, these materials remarkably downregulated the expression of inflammatory mediators and matrix metalloproteinases, upregulated chondrogenic markers, and promoted tissue inhibitor of metalloproteinase 1 and B-cell lymphoma 2 expression. In vivo, after treatment with tetrahedral framework nucleic acid/wogonin complexes, the bone mineral density in regenerated tissues was much higher than that found in the untreated groups. Histologically, the complexes enhanced new tissue regeneration, significantly suppressed chondrocyte apoptosis, and promoted chondrogenic marker expression. They also inhibited cell apoptosis, increased chondrogenic marker expression, and suppressed the expression of inflammatory mediators in osteoarthritis. Therefore, we believe that tetrahedral framework nucleic acid/wogonin complexes can be used as an injectable form of therapy for osteoarthritis.

Lumbar degenerative disc diseases cause low back pain (LBP). The maintenance of the height and stability of the intervertebral disc (IVD) space is an effective treatment for LBP. The following study evaluated the effects of fibroblast injection on intervertebral disc degeneration (IDD) in a preclinical setting. Compared with the IDD group, the fibroblast treatment group demonstrated effective maintenance of IVD height, reduced endplate degeneration, and improved nuclear magnetic resonance signals and overall histological structure. In doing so, fibrotic IVDs maintained the stability and biomechanics of the vertebra. This finding is in agreement with clinical findings that human nucleus pulposus (NP) fibrosis is essential for the maintenance of IVD height and mechanical properties in patients following percutaneous endoscopic lumbar discectomy (PELD). Mechanistically, we demonstrated that injected fibroblasts not only proliferated but also induced NP cells to adopt a fibrotic phenotype via the secretion of TGF-β. Finally, to better mimic human conditions, the efficacy of autologous fibroblast injection in the treatment of IDD was further examined in a nonhuman primate cynomolgus monkey model due to their capacity for upright posture. We showed that the injection of fibroblasts could maintain the IVD height and rescue IVD signals in cynomolgus monkeys. Taken together, the results of our study reveal that autologous fibroblast injection can enhance the natural process of fibrosis during acute and subacute stages of stress-induced IDD. Fibrotic IVDs can maintain the stability, biological activity, and mechanical properties of the intervertebral space, thus providing a new direction for the treatment of intervertebral space-derived lumbar degenerative diseases.

The most severe sequelae after rehabilitation from SARS are femoral head necrosis and pulmonary fibrosis. We performed a 15-year follow-up on the lung and bone conditions of SARS patients. We evaluated the recovery from lung damage and femoral head necrosis in an observational cohort study of SARS patients using pulmonary CT scans, hip joint MRI examinations, pulmonary function tests and hip joint function questionnaires. Eighty medical staff contracted SARS in 2003. Two patients died of SARS, and 78 were enrolled in this study from August 2003 to March 2018. Seventy-one patients completed the 15-year follow-up. The percentage of pulmonary lesions on CT scans diminished from 2003 (9.40 ± 7.83)% to 2004 (3.20 ± 4.78)% (P < 0.001) and remained stable thereafter until 2018 (4.60 ± 6.37)%. Between 2006 and 2018, the proportion of patients with interstitial changes who had improved pulmonary function was lower than that of patients without lesions, as demonstrated by the one-second ratio (FEV₁/FVC%, t = 2.21, P = 0.04) and mid-flow of maximum expiration (FEF_25%–75%, t = 2.76, P = 0.01). The volume of femoral head necrosis decreased significantly from 2003 (38.83 ± 21.01)% to 2005 (30.38 ± 20.23)% (P = 0.000 2), then declined slowly from 2005 to 2013 (28.99 ± 20.59)% and plateaued until 2018 (25.52 ± 15.51)%. Pulmonary interstitial damage and functional decline caused by SARS mostly recovered, with a greater extent of recovery within 2 years after rehabilitation. Femoral head necrosis induced by large doses of steroid pulse therapy in SARS patients was not progressive and was partially reversible.

Osteocytes are mechanosensitive bone cells, but little is known about their effects on tumor cells in response to mechanical stimulation. We treated breast cancer cells with osteocyte-derived conditioned medium (CM) and fluid flow-treated conditioned medium (FFCM) with 0.25 Pa and 1 Pa shear stress. Notably, CM and FFCM at 0.25 Pa induced the mesenchymal-to-epithelial transition (MET), but FFCM at 1 Pa induced the epithelial-to-mesenchymal transition (EMT). This suggested that the effects of fluid flow on conditioned media depend on flow intensity. Fluorescence resonance energy transfer (FRET)-based evaluation of Src activity and vinculin molecular force showed that osteopontin was involved in EMT and MET switching. A mouse model of tumor-induced osteolysis was tested using dynamic tibia loadings of 1, 2, and 5 N. The low 1 N loading suppressed tumor-induced osteolysis, but this beneficial effect was lost and reversed with loads at 2 and 5 N, respectively. Changing the loading intensities in vivo also led to changes in serum TGFβ levels and the composition of tumor-associated volatile organic compounds in the urine. Collectively, this study demonstrated the critical role of intensity-dependent mechanotransduction and osteopontin in tumor-osteocyte communication, indicating that a biophysical factor can tangibly alter the behaviors of tumor cells in the bone microenvironment.

The inflammatory response is induced by the overexpression of inflammatory cytokines, mainly interleukin (IL)-1β, and is one of the main causes of intervertebral disc degeneration (IVDD). NLR pyrin domain containing 3 (NLRP3) inflammasome activation is an important source of IL-1β. As an anti-inflammatory neuroendocrine hormone, melatonin plays various roles in different pathophysiological conditions. However, its roles in IVDD are still not well understood and require more examination. First, we demonstrated that melatonin delayed the progression of IVDD and relieved IVDD-related low back pain in a rat needle puncture IVDD model; moreover, NLRP3 inflammasome activation (NLRP3, p20, and IL-1β levels) was significantly upregulated in severely degenerated human discs and a rat IVDD model. Subsequently, an IL-1β/NF-κB-NLRP3 inflammasome activation positive feedback loop was found in nucleus pulposus (NP) cells that were treated with IL-1β. In these cells, expression of NLRP3 and p20 was significantly increased, NF-κB signaling was involved in this regulation, and mitochondrial reactive oxygen species (mtROS) production increased. Furthermore, we found that melatonin disrupted the IL-1β/NF-κB-NLRP3 inflammasome activation positive feedback loop in vitro and in vivo. Melatonin treatment decreased NLRP3, p20, and IL-1β levels by inhibiting NF-κB signaling and downregulating mtROS production. Finally, we showed that melatonin mediated the disruption of the positive feedback loop of IL-1β in vivo. In this study, we showed for the first time that IL-1β promotes its own expression by upregulating NLRP3 inflammasome activation. Furthermore, melatonin disrupts the IL-1β positive feedback loop and may be a potential therapeutic agent for IVDD.

The axon guidance cue SLIT3 was identified as an osteoanabolic agent in two recent reports. However, these reports conflict in their nomination of osteoblasts versus osteoclasts as the key producers of skeletal SLIT3 and additionally offer conflicting data on the effects of SLIT3 on osteoclastogenesis. Here, aiming to address this discrepancy, we found no observable SLIT3 expression during human or mouse osteoclastogenesis and the only modest SLIT3-mediated effects on osteoclast differentiation. Conditional deletion of SLIT3 in cathepsin K (CTSK)-positive cells, including osteoclasts, had no effect on the number of osteoclast progenitors, in vitro osteoclast differentiation, overall bone mass, or bone resorption/formation parameters. Similar results were observed with the deletion of SLIT3 in LysM-positive cells, including osteoclast lineage cells. Consistent with this finding, bone marrow chimeras made from Slit3 ^−/− donors that lacked SLIT3 expression at all stages of osteoclast development displayed normal bone mass relative to controls. Taken in context, multiple lines of evidence were unable to identify the physiologic function of osteoclast-derived SLIT3, indicating that osteoblasts are the major source of skeletal SLIT3.

The lineage specification of mesenchymal stem/stromal cells (MSCs) is tightly regulated by a wide range of factors. Recently, the versatile functions of ZBP1 (also known as DAI or DLM-1) have been reported in the blood circulation and immune systems. However, the biological function of ZBP1 during the lineage specification of MSCs is still unknown. In the present study, we found that ZBP1 was upregulated during osteogenesis but downregulated during adipogenesis in mouse bone marrow-derived MSCs (mBMSCs). ZBP1 was highly expressed in osteoblasts but expressed at a relatively low level in marrow adipocytes. Knockdown of ZBP1 inhibited alkaline phosphataseactivity, extracellular matrix mineralization, and osteogenesis-related gene expression in vitro and reduced ectopic bone formation in vivo. Knockdown of ZBP1 also promoted adipogenesis in MSCs in vitro. Conversely, the overexpression of ZBP1 increased the osteogenesis but suppressed the adipogenesis of MSCs. When the expression of ZBP1 was rescued, the osteogenic capacity of ZBP1-depleted mBMSCs was restored at both the molecular and phenotypic levels. Furthermore, we demonstrated that ZBP1, a newly identified target of Wnt/β-catenin signaling, was required for β-catenin translocation into nuclei. Collectively, our results indicate that ZBP1 is a novel regulator of bone and fat transdifferentiation via Wnt/β-catenin signaling.

The human spinal column is a dynamic, segmented, bony, and cartilaginous structure that protects the neurologic system and simultaneously provides balance and flexibility. Children with developmental disorders that affect the patterning or shape of the spine can be at risk of neurologic and other physiologic dysfunctions. The most common developmental disorder of the spine is scoliosis, a lateral deformity in the shape of the spinal column. Scoliosis may be part of the clinical spectrum that is observed in many developmental disorders, but typically presents as an isolated symptom in otherwise healthy adolescent children. Adolescent idiopathic scoliosis (AIS) has defied understanding in part due to its genetic complexity. Breakthroughs have come from recent genome-wide association studies (GWAS) and next generation sequencing (NGS) of human AIS cohorts, as well as investigations of animal models. These studies have identified genetic associations with determinants of cartilage biogenesis and development of the intervertebral disc (IVD). Current evidence suggests that a fraction of AIS cases may arise from variation in factors involved in the structural integrity and homeostasis of the cartilaginous extracellular matrix (ECM). Here, we review the development of the spine and spinal cartilages, the composition of the cartilage ECM, the so-called “matrisome” and its functions, and the players involved in the genetic architecture of AIS. We also propose a molecular model by which the cartilage matrisome of the IVD contributes to AIS susceptibility.

It is well established that alterations in phosphate metabolism have a profound effect on hard and soft tissues of the oral cavity. The present-day clinical form of osteonecrosis of the jaw (ONJ) was preceded by phosphorus necrosis of the jaw, ca. 1860. The subsequent removal of yellow phosphorus from matches in the early 20th century saw a parallel decline in “phossy jaw” until the early 2000s, when similar reports of unusual jaw bone necrosis began to appear in the literature describing jaw necrosis in patients undergoing chemotherapy and concomitant steroid and bisphosphonate treatment. Today, the potential side effect of ONJ associated with medications that block osteoclast activity (antiresorptive) is well known, though the mechanism remains unclear and the management and outcomes are often unsatisfactory. Much of the existing literature has focused on the continuing concerns of appropriate use of bisphosphonates and other antiresorptive medications, the incomplete or underdeveloped research on ONJ, and the use of drugs with anabolic potential for treatment of osteoporosis. While recognizing that ONJ is a rare occurrence and ONJ-associated medications play an important role in fracture risk reduction in osteoporotic patients, evidence to date suggests that health care providers can lower the risk further by dental evaluations and care prior to initiating antiresorptive therapies and by monitoring dental health during and after treatment. This review describes the current clinical management guidelines for ONJ, the critical role of dental-medical management in mitigating risks, and the current understanding of the effects of predominantly osteoclast-modulating drugs on bone homeostasis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Diffuse idiopathic skeletal hyperostosis (DISH) is a prevalent noninflammatory spondyloarthropathy characterized by ectopic mineral formation along the anterolateral aspect of the vertebral column, yet little is known about its underlying pathogenesis. Our objective was to evaluate the histopathological features and composition of ectopic mineral within spinal tissues affected by DISH in humans. Thoracic spine segments from six embalmed cadaveric donors (one female and five males; median age 82 years) meeting the radiographic diagnostic criteria for DISH were evaluated using radiological, histological, and physical analyses. Overall, the histological features of ectopic mineralization at individual motion segments were heterogeneous, including regions of heterotopic ossification and dystrophic calcification. Heterotopic ossifications were characterized by woven and lamellar bone, multifocal areas of metaplastic cartilage, and bony bridges along the anterior aspect of the intervertebral disc space. Dystrophic calcifications were characterized by an amorphous appearance, a high content of calcium and phosphorus, an X-ray diffraction pattern matching that of hydroxyapatite, and radiodensities exceeding that of cortical bone. Dystrophic calcifications were found within the anterior longitudinal ligament and annulus fibrosus in motion segments both meeting and not meeting the radiographic criteria for DISH. In summary, our findings indicate that in DISH, ectopic mineral forms along the anterior aspect of the spine by both heterotopic ossification and dystrophic calcification of fibrocartilaginous tissues. Although both types of ectopic mineralization are captured by current radiographic criteria for DISH, dystrophic calcification may reflect a distinct disease process or an early stage in the pathogenesis of DISH.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Urokinase plasminogen activator receptor (uPAR) is implicated in tumor growth and metastasis due to its ability to activate latent growth factors, proteases, and different oncogenic signaling pathways upon binding to different ligands. Elevated uPAR expression is correlated with the increased aggressiveness of cancer cells, which led to its credentialing as an attractive diagnostic and therapeutic target in advanced solid cancer. Here, we examine the antitumor effects of a humanized anti-uPAR antibody (huATN-658) alone and in combination with the approved bisphosphonate Zometa (Zoledronic acid) on skeletal lesion through a series of studies in vitro and in vivo. Treatment with huATN-658 or Zometa alone significantly decreased human MDA-MB-231 cell proliferation and invasion in vitro, effects which were more pronounced when huATN-658 was combined with Zometa. In vivo studies demonstrated that huATN-658 treatment significantly reduced MDA-MB-231 primary tumor growth compared with controls. In a model of breast tumor-induced bone disease, huATN-658 and Zometa were equally effective in reducing skeletal lesions. The skeletal lesions were significantly reduced in animals receiving the combination of huATN-658 + Zometa compared with monotherapy treatment. These effects were due to a significant decrease in osteoclastic activity and tumor cell proliferation in the combination treatment group. Transcriptome analysis revealed that combination treatment significantly changes the expression of genes from signaling pathways implicated in tumor progression and bone remodeling. Results from these studies provide a rationale for the continued development of huATN-658 as a monotherapy and in combination with currently approved agents such as Zometa in patients with metastatic breast cancer.

The loss-of-function mutations in the ALPL result in hypophosphatasia (HPP), an inborn metabolic disorder that causes skeletal mineralization defects. In adults, the main clinical features are early loss of primary or secondary teeth, osteoporosis, bone pain, chondrocalcinosis, and fractures. However, guidelines for the treatment of adults with HPP are not available. Here, we show that ALPL deficiency caused a reduction in intracellular Ca²⁺ influx, resulting in an osteoporotic phenotype due to downregulated osteogenic differentiation and upregulated adipogenic differentiation in both human and mouse bone marrow mesenchymal stem cells (BMSCs). Increasing the intracellular level of calcium in BMSCs by ionomycin treatment rescued the osteoporotic phenotype in alpl ^+/− mice and BMSC-specific (Prrx1-alpl ^−/−) conditional alpl knockout mice. Mechanistically, ALPL was found to be required for the maintenance of intracellular Ca²⁺ influx, which it achieves by regulating L-type Ca²⁺ channel trafficking via binding to the α2δ subunits to regulate the internalization of the L-type Ca²⁺ channel. Decreased Ca²⁺ flux inactivates the Akt/GSK3β/β-catenin signaling pathway, which regulates lineage differentiation of BMSCs. This study identifies a previously unknown role of the ectoenzyme ALPL in the maintenance of calcium channel trafficking to regulate stem cell lineage differentiation and bone homeostasis. Accelerating Ca²⁺ flux through L-type Ca²⁺ channels by ionomycin treatment may be a promising therapeutic approach for adult patients with HPP.

Tissue innervation is a complex process controlled by the expression profile of signaling molecules secreted by tissue-resident cells that dictate the growth and guidance of axons. Sensory innervation is part of the neuronal network of the bone tissue with a defined spatiotemporal occurrence during bone development. Yet, the current understanding of the mechanisms regulating the map of sensory innervation in the bone tissue is still limited. Here, we demonstrated that differentiation of human mesenchymal stem cells to osteoblasts leads to a marked impairment of their ability to promote axonal growth, evidenced under sensory neurons and osteoblastic-lineage cells crosstalk. The mechanisms by which osteoblast lineage cells provide this nonpermissive environment for axons include paracrine-induced repulsion and loss of neurotrophic factors expression. We identified a drastic reduction of NGF and BDNF production and stimulation of Sema3A, Wnt4, and Shh expression culminating at late stage of OB differentiation. We noted a correlation between Shh expression profile, OB differentiation stages, and OB-mediated axonal repulsion. Blockade of Shh activity and signaling reversed the repulsive action of osteoblasts on sensory axons. Finally, to strengthen our model, we localized the expression of Shh by osteoblasts in bone tissue. Overall, our findings provide evidence that the signaling profile associated with osteoblast phenotype differentiating program can regulate the patterning of sensory innervation, and highlight osteoblast-derived Shh as an essential player in this cue-induced regulation.

Mutations in actin-bundling protein plastin 3 (PLS3) emerged as a cause of congenital osteoporosis, but neither the role of PLS3 in bone development nor the mechanisms underlying PLS3-dependent osteoporosis are understood. Of the over 20 identified osteoporosis-linked PLS3 mutations, we investigated all five that are expected to produce full-length protein. One of the mutations distorted an actin-binding loop in the second actin-binding domain of PLS3 and abolished F-actin bundling as revealed by cryo-EM reconstruction and protein interaction assays. Surprisingly, the remaining four mutants fully retained F-actin bundling ability. However, they displayed defects in Ca²⁺ sensitivity: two of the mutants lost the ability to be inhibited by Ca²⁺, while the other two became hypersensitive to Ca²⁺. Each group of the mutants with similar biochemical properties showed highly characteristic cellular behavior. Wild-type PLS3 was distributed between lamellipodia and focal adhesions. In striking contrast, the Ca²⁺-hyposensitive mutants were not found at the leading edge but localized exclusively at focal adhesions/stress fibers, which displayed reinforced morphology. Consistently, the Ca²⁺-hypersensitive PLS3 mutants were restricted to lamellipodia, while chelation of Ca²⁺ caused their redistribution to focal adhesions. Finally, the bundling-deficient mutant failed to co-localize with any F-actin structures in cells despite a preserved F-actin binding through a non-mutation-bearing actin-binding domain. Our findings revealed that severe osteoporosis can be caused by a mutational disruption of the Ca²⁺-controlled PLS3’s cycling between adhesion complexes and the leading edge. Integration of the structural, biochemical, and cell biology insights enabled us to propose a molecular mechanism of plastin activity regulation by Ca²⁺.

Human osteogenic progenitors are not precisely defined, being primarily studied as heterogeneous multipotent cell populations and termed mesenchymal stem cells (MSCs). Notably, select human pericytes can develop into bone-forming osteoblasts. Here, we sought to define the differentiation potential of CD146⁺ human pericytes from skeletal and soft tissue sources, with the underlying goal of defining cell surface markers that typify an osteoblastogenic pericyte. CD146⁺CD31⁻CD45⁻ pericytes were derived by fluorescence-activated cell sorting from human periosteum, adipose, or dermal tissue. Periosteal CD146⁺CD31⁻CD45⁻ cells retained canonical features of pericytes/MSC. Periosteal pericytes demonstrated a striking tendency to undergo osteoblastogenesis in vitro and skeletogenesis in vivo, while soft tissue pericytes did not readily. Transcriptome analysis revealed higher CXCR4 signaling among periosteal pericytes in comparison to their soft tissue counterparts, and CXCR4 chemical inhibition abrogated ectopic ossification by periosteal pericytes. Conversely, enrichment of CXCR4⁺ pericytes or stromal cells identified an osteoblastic/non-adipocytic precursor cell. In sum, human skeletal and soft tissue pericytes differ in their basal abilities to form bone. Diversity exists in soft tissue pericytes, however, and CXCR4⁺ pericytes represent an osteoblastogenic, non-adipocytic cell precursor. Indeed, enrichment for CXCR4-expressing stromal cells is a potential new tactic for skeletal tissue engineering.

Osteocytes, the most abundant and long-lived cells in bone, are the master regulators of bone remodeling. In addition to their functions in endocrine regulation and calcium and phosphate metabolism, osteocytes are the major responsive cells in force adaptation due to mechanical stimulation. Mechanically induced bone formation and adaptation, disuse-induced bone loss and skeletal fragility are mediated by osteocytes, which sense local mechanical cues and respond to these cues in both direct and indirect ways. The mechanotransduction process in osteocytes is a complex but exquisite regulatory process between cells and their environment, between neighboring cells, and between different functional mechanosensors in individual cells. Over the past two decades, great efforts have focused on finding various mechanosensors in osteocytes that transmit extracellular mechanical signals into osteocytes and regulate responsive gene expression. The osteocyte cytoskeleton, dendritic processes, Integrin-based focal adhesions, connexin-based intercellular junctions, primary cilium, ion channels, and extracellular matrix are the major mechanosensors in osteocytes reported so far with evidence from both in vitro and in vitro studies. This review aims to give a systematic introduction to osteocyte mechanobiology, provide details of osteocyte mechanosensors, and discuss the roles of osteocyte mechanosensitive signaling pathways in the regulation of bone homeostasis.

The GP130 cytokine receptor subunit encoded by IL6ST is the shared receptor for ten cytokines of the IL-6 family. We describe a homozygous non-synonymous variant in IL6ST (p.R281Q) in a patient with craniosynostosis and retained deciduous teeth. We characterize the impact of the variant on cytokine signaling in vitro using transfected cell lines as well as primary patient-derived cells and support these findings using a mouse model with the corresponding genome-edited variant Il6st p.R279Q. We show that human GP130 p.R281Q is associated with selective loss of IL-11 signaling without affecting IL-6, IL-27, OSM, LIF, CT1, CLC, and CNTF signaling. In mice Il6st p.R279Q lowers litter size and causes facial synostosis and teeth abnormalities. The effect on IL-11 signaling caused by the GP130 variant shows incomplete penetrance but phenocopies aspects of IL11RA deficiency in humans and mice. Our data show that a genetic variant in a pleiotropic cytokine receptor can have remarkably selective defects.

Exosomes participate in many physiological and pathological processes by regulating cell–cell communication, which are involved in numerous diseases, including osteoarthritis (OA). Exosomes are detectable in the human articular cavity and were observed to change with OA progression. Several joint cells, including chondrocytes, synovial fibroblasts, osteoblasts, and tenocytes, can produce and secrete exosomes that influence the biological effects of targeted cells. In addition, exosomes from stem cells can protect the OA joint from damage by promoting cartilage repair, inhibiting synovitis, and mediating subchondral bone remodeling. This review summarizes the roles and therapeutic potential of exosomes in OA and discusses the perspectives and challenges related to exosome-based treatment for OA patients in the future.

Osteoporosis is the most common metabolic bone disorder globally and is characterized by skeletal fragility and microarchitectural deterioration. Genetic pleiotropy occurs when a single genetic element is associated with more than one phenotype. We aimed to identify pleiotropic loci associated with bone mineral density (BMD) and nonbone phenotypes in genome-wide association studies. In the discovery stage, the NHGRI-EBI Catalog was searched for genome-wide significant associations (P value < 5 × 10⁻⁸), excluding bone-related phenotypes. SNiPA was used to identify proxies of the significantly associated single nucleotide polymorphisms (SNPs) (r ² = 1). We then assessed putative genetic associations of this set of SNPs with femoral neck (FN) and lumbar spine (LS) BMD data from the GEFOS Consortium. Pleiotropic variants were claimed at a false discovery rate < 1.4 × 10⁻³ for FN-BMD and < 1.5 × 10⁻³ for LS-BMD. Replication of these genetic markers was performed among more than 400 000 UK Biobank participants of European ancestry with available genetic and heel bone ultrasound data. In the discovery stage, 72 BMD-related pleiotropic SNPs were identified, and 12 SNPs located in 11 loci on 8 chromosomes were replicated in the UK Biobank. These SNPs were associated, in addition to BMD, with 14 different phenotypes. Most pleiotropic associations were exhibited by rs479844 (AP5B1, OVOL1 genes), which was associated with dermatological and allergic diseases, and rs4072037 (MUC1 gene), which was associated with magnesium levels and gastroenterological cancer. In conclusion, 12 BMD-related genome-wide significant SNPs showed pleiotropy with nonbone phenotypes. Pleiotropic associations can deepen the genetic understanding of bone-related diseases by identifying shared biological mechanisms with other diseases or traits.

Women gradually lose bone from the age of ~35 years, but around menopause, the rate of bone loss escalates due to increasing bone resorption and decreasing bone formation levels, rendering these individuals more prone to developing osteoporosis. The increased osteoclast activity has been linked to a reduced estrogen level and other hormonal changes. However, it is unclear whether intrinsic changes in osteoclast precursors around menopause can also explain the increased osteoclast activity. Therefore, we set up a protocol in which CD14⁺ blood monocytes were isolated from 49 female donors (40–66 years old). Cells were differentiated into osteoclasts, and data on differentiation and resorption activity were collected. Using multiple linear regression analyses combining in vitro and in vivo data, we found the following: (1) age and menopausal status correlate with aggressive osteoclastic bone resorption in vitro; (2) the type I procollagen N-terminal propeptide level in vivo inversely correlates with osteoclast resorption activity in vitro; (3) the protein level of mature cathepsin K in osteoclasts in vitro increases with age and menopause; and (4) the promoter of the gene encoding the dendritic cell-specific transmembrane protein is less methylated with age. We conclude that monocytes are “reprogrammed” in vivo, allowing them to “remember” age, the menopausal status, and the bone formation status in vitro, resulting in more aggressive osteoclasts. Our discovery suggests that this may be mediated through DNA methylation. We suggest that this may have clinical implications and could contribute to understanding individual differences in age- and menopause-induced bone loss.

Therapeutic targeting of metastatic breast cancer still remains a challenge as the tumor cells are highly heterogenous and exploit multiple pathways for their growth and metastatic spread that cannot always be targeted by a single-agent monotherapy regimen. Therefore, a rational approach through simultaneous targeting of several pathways may provide a better anti-cancer therapeutic effect. We tested this hypothesis using a combination of two nutraceutical agents S-adenosylmethionine (SAM) and Vitamin D (Vit. D) prohormone [25-hydroxyvitamin D; ‘25(OH)D’] that are individually known to exert distinct changes in the expression of genes involved in tumor growth and metastasis. Our results show that both SAM and 25(OH)D monotherapy significantly reduced proliferation and clonogenic survival of a panel of breast cancer cell lines in vitro and inhibited tumor growth, lung metastasis, and breast tumor cell colonization to the skeleton in vivo. However, these effects were significantly more pronounced in the combination setting. RNA-Sequencing revealed that the transcriptomic footprint on key cancer-related signaling pathways is broader in the combination setting than any of the monotherapies. Furthermore, comparison of the differentially expressed genes from our transcriptome analyses with publicly available cancer-related dataset demonstrated that the combination treatment upregulates genes from immune-related pathways that are otherwise downregulated in bone metastasis in vivo. Since SAM and Vit. D are both approved nutraceuticals with known safety profiles, this combination treatment may serve as a novel strategy to reduce breast cancer-associated morbidity and mortality.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

As one of the most common metastatic sites of malignancies, bone has a unique microenvironment that allows metastatic tumor cells to grow and flourish. The fenestrated capillaries in the bone, bone matrix, and bone cells, including osteoblasts and osteoclasts, together maintain the homeostasis of the bone microenvironment. In contrast, tumor-derived factors act on bone components, leading to subsequent bone resorption or excessive bone formation. The various pathways involved also provide multiple targets for therapeutic strategies against bone metastases. In this review, we summarize the current understanding of the mechanism of bone metastases. Based on the general process of bone metastases, we specifically highlight the complex crosstalk between tumor cells and the bone microenvironment and the current management of cancer bone metastases.

Axin1 is a negative regulator of β-catenin signaling and its role in osteoblast precursor cells remains undefined. In the present studies, we determined changes in postnatal bone growth by deletion of Axin1 in osteoblast precursor cells and analyzed bone growth in newborn and postnatal Axin1 ^Osx mice and found that hypertrophic cartilage area was largely expanded in Axin1 ^Osx KO mice. A larger number of chondrocytes and unabsorbed cartilage matrix were found in the bone marrow cavity of Axin1 ^Osx KO mice. Osteoclast formation in metaphyseal and subchondral bone areas was significantly decreased, demonstrated by decreased TRAP-positive cell numbers, associated with reduction of MMP9- and cathepsin K-positive cell numbers in Axin1 ^Osx KO mice. OPG expression and the ratio of Opg to Rankl were significantly increased in osteoblasts of Axin1 ^Osx KO mice. Osteoclast formation in primary bone marrow derived microphage (BMM) cells was significantly decreased when BMM cells were cultured with conditioned media (CM) collected from osteoblasts derived from Axin1 ^Osx mice compared with BMM cells cultured with CM derived from WT mice. Thus, the loss of Axin1 in osteoblast precursor cells caused increased OPG and the decrease in osteoclast formation, leading to delayed bone growth in postnatal Axin1 ^Osx KO mice.

Adult bone structural integrity is maintained by remodeling via the coupling of osteoclastic bone resorption and osteoblastic bone formation. Osteocytes or osteoblasts express receptor activator of nuclear factor κ-B ligand (Rankl) or osteoprotegerin (Opg) to promote or inhibit osteoclastogenesis, respectively. Bone morphogenetic protein (BMP) is a potent bone inducer, but its major role in adult bone is to induce osteocytes to upregulate sclerostin (Sost) and increase the Rankl/Opg expression ratio, resulting in promotion of osteoclastogenesis. However, the precise effect of BMP-target gene(s) in osteoblasts on the Rankl/Opg expression ratio remains unclear. In the present study, we identified atonal homolog 8 (Atoh8), which is directly upregulated by the BMP-Smad1 axis in osteoblasts. In vivo, Atoh8 was detected in osteoblasts but not osteocytes in adult mice. Although global Atoh8-knockout mice showed only a mild phenotype in the neonate skeleton, the bone volume was decreased and osteoclasts were increased in the adult phase. Atoh8-null marrow stroma cells were more potent than wild-type cells in inducing osteoclastogenesis in marrow cells. Atoh8 loss in osteoblasts increased Runx2 expression and the Rankl/Opg expression ratio, while Runx2 knockdown normalized the Rankl/Opg expression ratio. Moreover, Atoh8 formed a protein complex with Runx2 to inhibit Runx2 transcriptional activity and decrease the Rankl/Opg expression ratio. These results suggest that bone remodeling is regulated elaborately by BMP signaling; while BMP primarily promotes bone resorption, it simultaneously induces Atoh8 to inhibit Runx2 and reduce the Rankl/Opg expression ratio in osteoblasts, suppressing osteoclastogenesis and preventing excessive BMP-mediated bone resorption.

Rheumatoid arthritis and osteoarthritis, the most common forms of arthritis, are chronic, painful, and disabling conditions. Although both diseases differ in etiology, they manifest in progressive joint destruction characterized by pathological changes in the articular cartilage, bone, and synovium. While the potent anti-inflammatory properties of therapeutic (i.e., exogenous) glucocorticoids have been heavily researched and are widely used in clinical practice, the role of endogenous glucocorticoids in arthritis susceptibility and disease progression remains poorly understood. Current evidence from mouse models suggests that local endogenous glucocorticoid signaling is upregulated by the pro-inflammatory microenvironment in rheumatoid arthritis and by aging-related mechanisms in osteoarthritis. Furthermore, these models indicate that endogenous glucocorticoid signaling in macrophages, mast cells, and chondrocytes has anti-inflammatory effects, while signaling in fibroblast-like synoviocytes, myocytes, osteoblasts, and osteocytes has pro-inflammatory actions in rheumatoid arthritis. Conversely, in osteoarthritis, endogenous glucocorticoid signaling in both osteoblasts and chondrocytes has destructive actions. Together these studies provide insights into the role of endogenous glucocorticoids in the pathogenesis of both inflammatory and degenerative joint disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

We previously reported ¹⁸FPRGD₂ uptake by the coxofemoral lining, intervertebral discs and facet joint osteophytes in OA using PET/SCAN imaging. However, the molecular mechanism by which the PRGD₂ tracer interacts with joint tissues and osteophytes in OA remains unclear. As PRGD₂ ligands are expected to belong to the RGD-specific integrin family, the purpose of this study was (i) to determine which integrin complexes display the highest affinity for PRGD2-based ligands, (ii) to analyze integrin expression in relevant tissues, and (iii) to test integrin regulation in chondrocytes using OA-related stimuli to increase the levels of fibrosis and ossification markers. To this end, the affinity of PRGD₂-based ligands for five heterodimeric integrins was measured by competition with ¹²⁵I-echistatin. In situ analyses were performed in human normal vs. OA cartilage and spinal osteophytes. Osteophytes were characterized by (immuno-)histological staining. Integrin subunit expression was tested in chondrocytes undergoing dedifferentiation, osteogenic differentiation, and inflammatory stimulation. The integrins α_Vβ₅, α_Vβ₃, and α_Vβ₆ presented the highest affinity for PRGD₂-based ligands. In situ, the expression of these integrins was significantly increased in OA compared to normal cartilage. Within osteophytes, the mean integrin expression score was significantly higher in blood vessels, fibrous areas, and cells from the bone lining than in osteocytes and cartilaginous zones. In vitro, the levels of integrin subunits were significantly increased during chondrocyte dedifferentiation (except for β₆), fibrosis, and osteogenic differentiation as well as under inflammatory stimuli. In conclusion, anatomical zones (such as OA cartilage, intervertebral discs, and facet joint osteophytes) previously reported to show PRGD₂ ligand uptake in vivo expressed increased levels of α_Vβ₅, α_Vβ₃, and β₆ integrins, whose subunits are modulated in vitro by OA-associated conditions that increase fibrosis, inflammation, and osteogenic differentiation. These results suggest that the increased levels of integrins in OA compared to normal tissues favor PRGD2 uptake and might explain the molecular mechanism of OA imaging using the PRGD₂-based ligand PET/CT.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The LIM domain-containing proteins Pinch1/2 regulate integrin activation and cell–extracellular matrix interaction and adhesion. Here, we report that deleting Pinch1 in limb mesenchymal stem cells (MSCs) and Pinch2 globally (double knockout; dKO) in mice causes severe chondrodysplasia, while single mutant mice do not display marked defects. Pinch deletion decreases chondrocyte proliferation, accelerates cell differentiation and disrupts column formation. Pinch loss drastically reduces Smad2/3 protein expression in proliferative zone (PZ) chondrocytes and increases Runx2 and Col10a1 expression in both PZ and hypertrophic zone (HZ) chondrocytes. Pinch loss increases sclerostin and Rankl expression in HZ chondrocytes, reduces bone formation, and increases bone resorption, leading to low bone mass. In vitro studies revealed that Pinch1 and Smad2/3 colocalize in the nuclei of chondrocytes. Through its C-terminal region, Pinch1 interacts with Smad2/3 proteins. Pinch loss increases Smad2/3 ubiquitination and degradation in primary bone marrow stromal cells (BMSCs). Pinch loss reduces TGF-β-induced Smad2/3 phosphorylation and nuclear localization in primary BMSCs. Interestingly, compared to those from single mutant mice, BMSCs from dKO mice express dramatically lower protein levels of β-catenin and Yap1/Taz and display reduced osteogenic but increased adipogenic differentiation capacity. Finally, ablating Pinch1 in chondrocytes and Pinch2 globally causes severe osteopenia with subtle limb shortening. Collectively, our findings demonstrate critical roles for Pinch1/2 and a functional redundancy of both factors in the control of chondrogenesis and bone mass through distinct mechanisms.

The limited molecular classifications and disease signatures of osteoarthritis (OA) impede the development of prediagnosis and targeted therapeutics for OA patients. To classify and understand the subtypes of OA, we collected three types of tissue including cartilage, subchondral bone, and synovium from multiple clinical centers and constructed an extensive transcriptome atlas of OA patients. By applying unsupervised clustering analysis to the cartilage transcriptome, OA patients were classified into four subtypes with distinct molecular signatures: a glycosaminoglycan metabolic disorder subtype (C1), a collagen metabolic disorder subtype (C2), an activated sensory neuron subtype (C3), and an inflammation subtype (C4). Through ligand-receptor crosstalk analysis of the three knee tissue types, we linked molecular functions with the clinical symptoms of different OA subtypes. For example, the Gene Ontology functional term of vasculature development was enriched in the subchondral bone-cartilage crosstalk of C2 and the cartilage-subchondral bone crosstalk of C4, which might lead to severe osteophytes in C2 patients and apparent joint space narrowing in C4 patients. Based on the marker genes of the four OA subtypes identified in this study, we modeled OA subtypes with two independent published RNA-seq datasets through random forest classification. The findings of this work contradicted traditional OA diagnosis by medical imaging and revealed distinct molecular subtypes in knee OA patients, which may allow for precise diagnosis and treatment of OA.

Mesenchymal stem cells (MSCs) derived from human embryonic stem cells (hESCs) have significant potential for cell-mediated bone regeneration. Our recent study revealed that inhibiting the epigenetic regulator EZH2 plays a key role in promoting the mesodermal differentiation of hESCs. In this study, an epigenome-wide analysis of hESCs and MSCs revealed that growth differentiation factor 6 (GDF6), which is involved in bone formation, was the most upregulated gene associated with MSCs compared to hESCs. Furthermore, we identified GDF6 as a repressive target of EZH2 and found that ectopic GDF6 selectively promoted hESC differentiation towards the mesodermal lineage and enriched the MSC population. Our results provide molecular insights governing the mesenchymal commitment of hESCs and identify an inducing factor that offers strong promise for the future of regenerative medicine.

Exercise benefits the musculoskeletal system and reduces the effects of cancer. The effects of exercise are multifactorial, where metabolic changes and tissue adaptation influence outcomes. Mechanical signals, a principal component of exercise, are anabolic to the musculoskeletal system and restrict cancer progression. We examined the mechanisms through which cancer cells sense and respond to low-magnitude mechanical signals introduced in the form of vibration. Low-magnitude, high-frequency vibration was applied to human breast cancer cells in the form of low-intensity vibration (LIV). LIV decreased matrix invasion and impaired secretion of osteolytic factors PTHLH, IL-11, and RANKL. Furthermore, paracrine signals from mechanically stimulated cancer cells, reduced osteoclast differentiation and resorptive capacity. Disconnecting the nucleus by knockdown of SUN1 and SUN2 impaired LIV-mediated suppression of invasion and osteolytic factor secretion. LIV increased cell stiffness; an effect dependent on the LINC complex. These data show that mechanical vibration reduces the metastatic potential of human breast cancer cells, where the nucleus serves as a mechanosensory apparatus to alter cell structure and intercellular signaling.

Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) play important roles in bone metabolism. Smad ubiquitination regulatory factors (Smurfs) regulate TGF-β/BMP signaling via ubiquitination, resulting in degradation of signaling molecules to prevent excessive activation of TGF-β/BMP signaling. Though Smurf2 has been shown to negatively regulate TGF-β/Smad signaling, its involvement in BMP/Smad signaling in bone metabolism has not been thoroughly investigated. In the present study, we sought to evaluate the role of Smurf2 in BMP/Smad signaling in bone metabolism. Absorbable collagen sponges containing 3 μg of recombinant human BMP2 (rhBMP2) were implanted in the dorsal muscle pouches of wild type (WT) and Smurf2 ^−/− mice. The rhBMP2-induced ectopic bone in Smurf2 ^{− /−} mice showed greater bone mass, higher mineral apposition and bone formation rates, and greater osteoblast numbers than the ectopic bone in WT mice. In WT mice, the ectopic bone consisted of a thin discontinuous outer cortical shell and scant inner trabecular bone. In contrast, in Smurf2 ^−/− mice, the induced bone consisted of a thick, continuous outer cortical shell and abundant inner trabecular bone. Additionally, rhBMP2-stimulated bone marrow stromal cells (BMSCs) from Smurf2 ^−/− mice showed increased osteogenic differentiation. Smurf2 induced the ubiquitination of Smad1/5. BMP/Smad signaling was enhanced in Smurf2 ^−/− BMSCs stimulated with rhBMP2, and the inhibition of BMP/Smad signaling suppressed osteogenic differentiation of these BMSCs. These findings demonstrate that Smurf2 negatively regulates BMP/Smad signaling, thereby identifying a new regulatory mechanism in bone metabolism.

Neurological heterotopic ossification (NHO) is a debilitating condition where bone forms in soft tissue, such as muscle surrounding the hip and knee, following an injury to the brain or spinal cord. This abnormal formation of bone can result in nerve impingement, pain, contractures and impaired movement. Patients are often diagnosed with NHO after the bone tissue has completely mineralised, leaving invasive surgical resection the only remaining treatment option. Surgical resection of NHO creates potential for added complications, particularly in patients with concomitant injury to the central nervous system (CNS). Although recent work has begun to shed light on the physiological mechanisms involved in NHO, there remains a significant knowledge gap related to the prognostic biomarkers and prophylactic treatments which are necessary to prevent NHO and optimise patient outcomes. This article reviews the current understanding pertaining to NHO epidemiology, pathobiology, biomarkers and treatment options. In particular, we focus on how concomitant CNS injury may drive ectopic bone formation and discuss considerations for treating polytrauma patients with NHO. We conclude that understanding of the pathogenesis of NHO is rapidly advancing, and as such, there is the strong potential for future research to unearth methods capable of identifying patients likely to develop NHO, and targeted treatments to prevent its manifestation.

Extremity reconstruction surgery is increasingly performed rather than amputation for patients with large-segment pathologic bone loss. Debate persists as to the optimal void filler for this “limb salvage” surgery, whether metal or allograft bone. Clinicians focus on optimizing important functional gains for patients, and the risk of devastating implant infection has been thought to be similar regardless of implant material. Recent insights into infection pathophysiology are challenging this equipoise, however, with both basic science data suggesting a novel mechanism of infection of Staphylococcus aureus (the most common infecting agent) into the host lacunar–canaliculi network, and also clinical data revealing a higher rate of infection of allograft over metal. The current translational study was therefore developed to bridge the gap between these insights in a longitudinal murine model of infection of allograft bone and metal. Real-time Staphylococci infection characteristics were quantified in cortical bone vs metal, and both microarchitecture of host implant and presence of host immune response were assessed. An orders-of-magnitude higher bacterial burden was established in cortical allograft bone over both metal and cancellous bone. The establishment of immune-evading microabscesses was confirmed in both cortical allograft haversian canal and the submicron canaliculi network in an additional model of mouse femur bone infection. These study results reveal a mechanism by which Staphylococci evasion of host immunity is possible, contributing to elevated risks of infection in cortical bone. The presence of this local infection reservoir imparts massive clinical implications that may alter the current paradigm of osteomyelitis and bulk allograft infection treatment.

Mesenchymal stem cells (MSCs) closely interact with the immune system, and they are known to secrete inflammatory cytokines in response to stress stimuli. The biological function of MSC-derived inflammatory cytokines remains elusive. Here, we reveal that even under physiological conditions, MSCs produce and release a low level of tumor necrosis factor alpha (TNFα), which is unexpectedly required for preserving the self-renewal and differentiation of MSCs via autocrine/paracrine signaling. Furthermore, TNFα critically maintains MSC function in vivo during bone homeostasis. Mechanistically, we unexpectedly discovered that physiological levels of TNFα safeguard MSC homeostasis in a receptor-independent manner through mechanical force-driven endocytosis and that endocytosed TNFα binds to mammalian target of rapamycin (mTOR) complex 2 and restricts mTOR signaling. Importantly, inhibition of mTOR signaling by rapamycin serves as an effective osteoanabolic therapeutic strategy to protect against TNFα deficiency and mechanical unloading. Collectively, these findings unravel the physiological framework of the dynamic TNFα shuttle-based mTOR equilibrium that governs MSC and bone homeostasis.