Periarticular calcifications containing giant pseudo-crystals of francolite in skeletal fluorosis from 1,1-difluoroethane "huffing".

Inhalant use disorder is a psychiatric condition characterized by repeated deliberate inhalation from among a broad range of household and industrial chemical products with the intention of producing psychoactive effects. In addition to acute intoxication, prolonged inhalation of fluorinated compounds can cause skeletal fluorosis (SF). We report a young woman referred for hypophosphatasemia and carrying a heterozygous ALPL gene variant (c.457T>C, p.Trp153Arg) associated with hypophosphatasia, the heritable metabolic bone disease featuring impaired skeletal mineralization, who instead suffered from SF. Manifestations of her SF included recurrent articular pain, axial osteosclerosis, elevated bone mineral density, maxillary exostoses, and multifocal periarticular calcifications. SF was suspected when a long history was discovered of 'huffing' a computer cleaner containing 1,1-difluoroethane. Investigation revealed markedly elevated serum and urine levels of F-. Histopathology and imaging techniques including backscattered electron mode scanning electron microscopy, X-ray microtomography, energy dispersive and wavelength dispersive X-ray emission microanalysis, and polarized light microscopy revealed that her periarticular calcifications were dystrophic deposition of giant pseudo-crystals of francolite, a carbonate-rich fluorapatite. Identifying unusual circumstances of F- exposure is key for diagnosing non-endemic SF. Increased awareness of the disorder can be lifesaving.

Gene expression analysis of subchondral bone, cartilage, and synovium in naturally occurring equine palmar/plantar osteochondral disease.

Osteoarthritis (OA) is a disease of the entire joint but the relationship between pathological events in various joint tissues is poorly understood. We examined concurrent changes in bone, cartilage, and synovium in a naturally occurring equine model of joint degeneration. Joints (n = 64) were grossly assessed for palmar/plantar osteochondral disease (POD) in racehorses that required euthanasia for unrelated reasons and assigned a grade of 0 (n = 34), 1 (n = 17), 2 or 3 (n = 13) using a recognized grading scheme. Synovium, cartilage, and subchondral bone were collected for histological and gene expression analysis. Relations between POD grade, cartilage histological score, and gene expression levels were examined using one-way analysis of variance or Kruskal-Wallis test and Spearman's correlation coefficient with corrections for multiple comparisons. Cartilage histological score increased in joints with POD grade 1 (p = 0.002) and 2 or 3 (p < 0.001) compared to 0. At grade 1, expression of COL1A1, COL2A1, and MMP1 increased and BGN decreased in subchondral bone while expression of BGN and ACAN decreased in cartilage. These changes further progressed at grades 2 and 3. POD grades 2 and 3 were associated with decreased expression of osteoclast inhibitor OPG and increased markers of cartilage degeneration (MMP13, COL1A1). Expression of the vascular endothelial growth factor decreased with POD grade and negatively correlated with cartilage histological score. Synovium showed no histological or transcriptomic changes related to pathology grade. Cartilage degeneration in POD is likely to be secondary to remodeling of the subchondral bone. Limited activation of proinflammatory and catabolic genes and moderate synovial pathology suggests distinct molecular phenotype of POD compared with OA.

Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition.

Collagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF; also known as VEGFA) in bone-forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we used murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Label-free and non-destructive polarisation-resolved second-harmonic generation (p-SHG) microscopy revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed that the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-strand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure, including Adamts2, Spp1, Mmp9 and Lama1. Our results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders that clinically manifest in pathological nano- and macro-level disorganisation. This article has an associated First Person interview with the first author of the paper.

Accelerating functional gene discovery in osteoarthritis.

Osteoarthritis causes debilitating pain and disability, resulting in a considerable socioeconomic burden, yet no drugs are available that prevent disease onset or progression. Here, we develop, validate and use rapid-throughput imaging techniques to identify abnormal joint phenotypes in randomly selected mutant mice generated by the International Knockout Mouse Consortium. We identify 14 genes with functional involvement in osteoarthritis pathogenesis, including the homeobox gene Pitx1, and functionally characterize 6 candidate human osteoarthritis genes in mouse models. We demonstrate sensitivity of the methods by identifying age-related degenerative joint damage in wild-type mice. Finally, we phenotype previously generated mutant mice with an osteoarthritis-associated polymorphism in the Dio2 gene by CRISPR/Cas9 genome editing and demonstrate a protective role in disease onset with public health implications. We hope this expanding resource of mutant mice will accelerate functional gene discovery in osteoarthritis and offer drug discovery opportunities for this common, incapacitating chronic disease.

RANKL Inhibition in Fibrous Dysplasia of Bone: A Preclinical Study in a Mouse Model of the Human Disease.

Fibrous dysplasia of bone/McCune-Albright syndrome (Polyostotic FD/MAS; OMIM#174800) is a crippling skeletal disease caused by gain-of-function mutations of Gs α. Enhanced bone resorption is a recurrent histological feature of FD and a major cause of fragility of affected bones. Previous work suggests that increased bone resorption in FD is driven by RANKL and some studies have shown that the anti-RANKL monoclonal antibody, denosumab, reduces bone turnover and bone pain in FD patients. However, the effect of RANKL inhibition on the histopathology of FD and its impact on the natural history of the disease remain to be assessed. In this study, we treated the EF1α-Gs αR201C mice, which develop an FD-like phenotype, with an anti-mouse RANKL monoclonal antibody. We found that the treatment induced marked radiographic and microscopic changes at affected skeletal sites in 2-month-old mice. The involved skeletal segments became sclerotic due to the deposition of new, highly mineralized bone within developing FD lesions and showed a higher mechanical resistance compared to affected segments from untreated transgenic mice. Similar changes were also detected in older mice with a full-blown skeletal phenotype. The administration of anti-mouse RANKL antibody arrested the growth of established lesions and, in young mice, prevented the appearance of new ones. However, after drug withdrawal, the newly formed bone was remodelled into FD tissue and the disease progression resumed in young mice. Taken together, our results show that the anti-RANKL antibody significantly affected the bone pathology and natural history of FD in the mouse. Pending further work on the prevention and management of relapse after treatment discontinuation, our preclinical study suggests that RANKL inhibition may be an effective therapeutic option for FD patients. © 2019 American Society for Bone and Mineral Research.

PYY is a negative regulator of bone mass and strength.

OBJECTIVE: Bone loss in anorexia nervosa and following bariatric surgery is associated with an elevated circulating concentration of the gastrointestinal, anorexigenic hormone, peptide YY (PYY). Selective deletion of the PYY receptor Y1R in osteoblasts or Y2R in the hypothalamus results in high bone mass, but deletion of PYY in mice has resulted in conflicting skeletal phenotypes leading to uncertainty regarding its role in the regulation of bone mass. As PYY analogs are under development for treatment of obesity, we aimed to clarify the relationship between PYY and bone mass. METHODS: The skeletal phenotype of Pyy knockout (KO) mice was investigated during growth (postnatal day P14) and adulthood (P70 and P186) using X-ray microradiography, micro-CT, back-scattered electron scanning electron microscopy (BSE-SEM), histomorphometry and biomechanical testing. RESULTS: Bones from juvenile and Pyy KO mice were longer (P < 0.001), with decreased bone mineral content (P < 0.001). Whereas, bones from adult Pyy KO mice had increased bone mineral content (P < 0.05) with increased mineralisation of both cortical (P < 0.001) and trabecular (P < 0.001) compartments. Long bones from adult Pyy KO mice were stronger (maximum load P < 0.001), with increased stiffness (P < 0.01) and toughness (P < 0.05) compared to wild-type (WT) control mice despite increased cortical vascularity and porosity (P < 0.001). The increased bone mass and strength in Pyy KO mice resulted from increases in trabecular (P < 0.01) and cortical bone formation (P < 0.05). CONCLUSIONS: These findings demonstrate that PYY acts as a negative regulator of osteoblastic bone formation, implicating increased PYY levels in the pathogenesis of bone loss during anorexia or following bariatric surgery.

Regulation of the Bone Vascular Network is Sexually Dimorphic.

Osteoblast (OB) lineage cells are an important source of vascular endothelial growth factor (VEGF), which is critical for bone growth and repair. During bone development, pubertal differences in males and females exist, but little is known about whether VEGF signaling contributes to skeletal sexual dimorphism. We have found that in mice, conditional disruption of VEGF in osteocalcin-expressing cells (OcnVEGFKO) exerts a divergent influence on morphological, cellular, and whole bone properties between sexes. Furthermore, we describe an underlying sexual divergence in VEGF signaling in OB cultures in vitro independent of circulating sex hormones. High-resolution synchrotron computed tomography and backscattered scanning electron microscopy revealed, in males, extensive unmineralized osteoid encasing enlarged blood vessel canals and osteocyte lacunae in cortical bone after VEGF deletion, which contributed to increased porosity. VEGF was deleted in male and female long bone-derived OBs (OBVEGKO) in vitro and Raman spectroscopic analyses of mineral and matrix repertoires highlighted differences between male and female OBVEGFKO cells, with increased immature phosphate species prevalent in male OBVEGFKO cultures versus wild type (WT). Further sexual dimorphism was observed in bone marrow endothelial cell gene expression in vitro after VEGF deletion and in sclerostin protein expression, which was increased in male OcnVEGFKO bones versus WT. The impact of altered OB matrix composition after VEGF deletion on whole bone geometry was assessed between sexes, although significant differences between OcnVEGFKO and WT were identified only in females. Our results suggest that bone-derived VEGF regulates matrix mineralization and vascularization distinctly in males and females, which results in divergent physical bone traits.

Bi-allelic CSF1R Mutations Cause Skeletal Dysplasia of Dysosteosclerosis-Pyle Disease Spectrum and Degenerative Encephalopathy with Brain Malformation.

Colony stimulating factor 1 receptor (CSF1R) plays key roles in regulating development and function of the monocyte/macrophage lineage, including microglia and osteoclasts. Mono-allelic mutations of CSF1R are known to cause hereditary diffuse leukoencephalopathy with spheroids (HDLS), an adult-onset progressive neurodegenerative disorder. Here, we report seven affected individuals from three unrelated families who had bi-allelic CSF1R mutations. In addition to early-onset HDLS-like neurological disorders, they had brain malformations and skeletal dysplasia compatible to dysosteosclerosis (DOS) or Pyle disease. We identified five CSF1R mutations that were homozygous or compound heterozygous in these affected individuals. Two of them were deep intronic mutations resulting in abnormal inclusion of intron sequences in the mRNA. Compared with Csf1r-null mice, the skeletal and neural phenotypes of the affected individuals appeared milder and variable, suggesting that at least one of the mutations in each affected individual is hypomorphic. Our results characterized a unique human skeletal phenotype caused by CSF1R deficiency and implied that bi-allelic CSF1R mutations cause a spectrum of neurological and skeletal disorders, probably depending on the residual CSF1R function.

Activating the unfolded protein response in osteocytes causes hyperostosis consistent with craniodiaphyseal dysplasia.

Bone remodeling is a balanced process between bone synthesis and degradation, maintaining homeostasis and a constant bone mass in adult life. Imbalance will lead to conditions such as osteoporosis or hyperostosis. Osteoblasts build bone, becoming embedded in bone matrix as mature osteocytes. Osteocytes have a role in sensing and translating mechanical loads into biochemical signals, regulating the differentiation and activity of osteoblasts residing at the bone surface through the secretion of Sclerostin (SOST), an inhibitor of WNT signaling. Excessive mechanical load can lead to activation of cellular stress responses altering cell behavior and differentiation. The unfolded protein response (UPR) is a shared pathway utilized by cells to cope with stress stimuli. We showed that in a transgenic mouse model, activation of the UPR in early differentiating osteocytes delays maturation, maintaining active bone synthesis. In addition, expression of SOST is delayed or suppressed; resulting in active WNT signaling and enhanced periosteal bone formation, and the combined outcome is generalized hyperostosis. A clear relationship between the activation of the unfolded protein response was established and the onset of hyperostosis that can be suppressed with a chemical chaperone, sodium 4-phenobutyrate (4-PBA). As the phenotype is highly consistent with craniodiaphyseal dysplasia (CDD; OMIM 122860), we propose activation of the UPR could be part of the disease mechanism for CDD patients as these patients are heterozygous for SOST mutations that impair protein folding and secretion. Thus, therapeutic agents ameliorating protein folding or the UPR can be considered as a potential therapeutic treatment.

A distinctive patchy osteomalacia characterises Phospho1-deficient mice.

The phosphatase PHOSPHO1 is involved in the initiation of biomineralisation. Bones in Phospho1 knockout (KO) mice show histological osteomalacia with frequent bowing of long bones and spontaneous fractures: they contain less mineral, with smaller mineral crystals. However, the consequences of Phospho1 ablation on the microscale structure of bone are not yet fully elucidated. Tibias and femurs obtained from wild-type and Phospho1 null (KO) mice (25-32 weeks old) were embedded in PMMA, cut and polished to produce near longitudinal sections. Block surfaces were studied using 20 kV backscattered-electron (BSE) imaging, and again after iodine staining to reveal non-mineralised matrix and cellular components. For 3D characterisation, we used X-ray micro-tomography. Bones opened with carbide milling tools to expose endosteal surfaces were macerated using an alkaline bacterial pronase enzyme detergent, 5% hydrogen peroxide and 7% sodium hypochlorite solutions to produce 3D surfaces for study with 3D BSE scanning electron microscopy (SEM). Extensive regions of both compact cortical and trabecular bone matrix in Phospho1 KO mice contained no significant mineral and/or showed arrested mineralisation fronts, characterised by a failure in the fusion of the calcospherite-like, separately mineralising, individual micro-volumes within bone. Osteoclastic resorption of the uncalcified matrix in Phospho1 KO mice was attenuated compared with surrounding normally mineralised bone. The extent and position of this aberrant biomineralisation varied considerably between animals, contralateral limbs and anatomical sites. The most frequent manifestation lay, however, in the nearly complete failure of mineralisation in the bone surrounding the numerous transverse blood vessel canals in the cortices. In conclusion, SEM disclosed defective mineralising fronts and extensive patchy osteomalacia, which has previously not been recognised. These data further confirm the role of this phosphatase in physiological skeletal mineralisation.

Bisphosphonate-induced zebra lines in fibrous dysplasia of bone: histo-radiographic correlation in a case of McCune-Albright syndrome.

Bisphosphonates (BPs) are currently used in the treatment of diverse bone diseases including fibrous dysplasia of bone (FD). In pediatric patients, a radiographic consequence of cyclical administration of BPs is the development of apo-, epi-, and meta-physeal sclerotic bands, otherwise known as zebra lines, which result from the temporary inhibition of osteoclastic activity at the time of drug treatment. We report here on a child with McCune-Albright syndrome (FD in addition to hyperfunctioning endocrinopathies and skin hyperpigmentation) treated with cyclical intravenous infusions of pamidronate in which conventional radiography, contact microradiography, histology, and backscattered electron image analysis demonstrated that zebra lines formed only where bone was normal, were arrested at the boundary between FD-unaffected and FD-affected bone where bone is sclerotic, and were absent within the undermineralized FD bone. Moreover, in spite of the treatment, the FD lesions continued to expand. This case report is unique because no previously published studies correlated the radiographic and the histologic features of BP-induced zebra lines in the metaphysis of an FD-affected long bone of the limbs.

Lessons from rare diseases of cartilage and bone.

Studying severe phenotypes of rare syndromes can elucidate disease mechanisms of more common disorders and identify potential therapeutic targets. Lessons from rare bone diseases contributed to the development of the most successful class of bone active agents, the bisphosphonates. More recent research on rare bone diseases has helped elucidate key pathways and identify new targets in bone resorption and bone formation including cathepsin K and sclerostin, for which drugs are now in clinical trials. By contrast, there has been much less focus on rare cartilage diseases and osteoarthritis (OA) remains a common disease with no effective therapy. Investigation of rare cartilage syndromes is identifying new potential targets in OA including GDF5 and lubricin. Research on the arthropathy of the ultra-rare disease alkaptonuria has identified several new features of the OA phenotype, including high density mineralized protrusions (HDMPs) which constitute a newly identified mechanism of joint destruction.

High density infill in cracks and protrusions from the articular calcified cartilage in osteoarthritis in standardbred horse carpal bones.

We studied changes in articular calcified cartilage (ACC) and subchondral bone (SCB) in the third carpal bones (C3) of Standardbred racehorses with naturally-occurring repetitive loading-induced osteoarthritis (OA). Two osteochondral cores were harvested from dorsal sites from each of 15 post-mortem C3 and classified as control or as showing early or advanced OA changes from visual inspection. We re-examined X-ray micro-computed tomography (µCT) image sets for the presence of high-density mineral infill (HDMI) in ACC cracks and possible high-density mineralized protrusions (HDMP) from the ACC mineralizing (tidemark) front (MF) into hyaline articular cartilage (HAC). We hypothesized and we show that 20-µm µCT resolution in 10-mm diameter samples is sufficient to detect HDMI and HDMP: these are lost upon tissue decalcification for routine paraffin wax histology owing to their predominant mineral content. The findings show that µCT is sufficient to discover HDMI and HDMP, which were seen in 2/10 controls, 6/9 early OA and 8/10 advanced OA cases. This is the first report of HDMI and HDMP in the equine carpus and in the Standardbred breed and the first to rely solely on µCT. HDMP are a candidate cause for mechanical tissue destruction in OA.

Osteoblast-specific expression of the fibrous dysplasia (FD)-causing mutation Gsα(R201C) produces a high bone mass phenotype but does not reproduce FD in the mouse.

We recently reported the generation and initial characterization of the first direct model of human fibrous dysplasia (FD; OMIM #174800), obtained through the constitutive systemic expression of one of the disease-causing mutations, Gsα(R201C) , in the mouse. To define the specific pathogenetic role(s) of individual cell types within the stromal/osteogenic system in FD, we generated mice expressing Gsα(R201C) selectively in mature osteoblasts using the 2.3kb Col1a1 promoter. We show here that this results in a striking high bone mass phenotype but not in a mimicry of human FD. The high bone mass phenotype involves specifically a deforming excess of cortical bone and prolonged and ectopic cortical bone remodeling. Expression of genes characteristic of late stages of bone cell differentiation/maturation is profoundly altered as a result of expression of Gsα(R201C) in osteoblasts, and expression of the Wnt inhibitor Sost is reduced. Although high bone mass is, in fact, a feature of some types/stages of FD lesions in humans, it is marrow fibrosis, localized loss of adipocytes and hematopoietic tissue, osteomalacia, and osteolytic changes that together represent the characteristic pathological profile of FD, as well as the sources of specific morbidity. None of these features are reproduced in mice with osteoblast-specific expression of Gsα(R201C) . We further show that hematopoietic progenitor/stem cells, as well as more mature cell compartments, and adipocyte development are normal in these mice. These data demonstrate that effects of Gsα mutations underpinning FD-defining tissue changes and morbidity do not reflect the effects of the mutations on osteoblasts proper.

Thyroid hormone receptor α mutation causes a severe and thyroxine-resistant skeletal dysplasia in female mice.

A new genetic disorder has been identified that results from mutation of THRA, encoding thyroid hormone receptor α1 (TRα1). Affected children have a high serum T3:T4 ratio and variable degrees of intellectual deficit and constipation but exhibit a consistently severe skeletal dysplasia. In an attempt to improve developmental delay and alleviate symptoms of hypothyroidism, patients are receiving varying doses and durations of T4 treatment, but responses have been inconsistent so far. Thra1(PV/+) mice express a similar potent dominant-negative mutant TRα1 to affected individuals, and thus represent an excellent disease model. We hypothesized that Thra1(PV/+) mice could be used to predict the skeletal outcome of human THRA mutations and determine whether prolonged treatment with a supraphysiological dose of T4 ameliorates the skeletal abnormalities. Adult female Thra1(PV/+) mice had short stature, grossly abnormal bone morphology but normal bone strength despite high bone mass. Although T4 treatment suppressed TSH secretion, it had no effect on skeletal maturation, linear growth, or bone mineralization, thus demonstrating profound tissue resistance to thyroid hormone. Despite this, prolonged T4 treatment abnormally increased bone stiffness and strength, suggesting the potential for detrimental consequences in the long term. Our studies establish that TRα1 has an essential role in the developing and adult skeleton and predict that patients with different THRA mutations will display variable responses to T4 treatment, which depend on the severity of the causative mutation.

Constitutive expression of Gsα(R201C) in mice produces a heritable, direct replica of human fibrous dysplasia bone pathology and demonstrates its natural history.

Fibrous dysplasia of bone (FD) is a crippling skeletal disease associated with postzygotic mutations (R201C, R201H) of the gene encoding the α subunit of the stimulatory G protein, Gs. By causing a characteristic structural subversion of bone and bone marrow, the disease results in deformity, hypomineralization, and fracture of the affected bones, with severe morbidity arising in childhood or adolescence. Lack of inheritance of the disease in humans is thought to reflect embryonic lethality of germline-transmitted activating Gsα mutations, which would only survive through somatic mosaicism. We have generated multiple lines of mice that express Gsα(R201C) constitutively and develop an inherited, histopathologically exact replica of human FD. Robust transgene expression in neonatal and embryonic tissues and embryonic stem (ES) cells were associated with normal development of skeletal tissues and differentiation of skeletal cells. As in humans, FD lesions in mice developed only in the postnatal life; a defined spatial and temporal pattern characterized the onset and progression of lesions across the skeleton. In individual bones, lesions developed through a sequence of three distinct histopathological stages: a primary modeling phase defined by endosteal/medullary excess bone formation and normal resorption; a secondary phase, with excess, inappropriate remodeling; and a tertiary fibrous dysplastic phase, which reproduced a full-blown replica of the human bone pathology in mice of age ≥1 year. Gsα mutations are sufficient to cause FD, and are per se compatible with germline transmission and normal embryonic development in mice. Our novel murine lines constitute the first model of FD.

Mice lacking the calcineurin inhibitor Rcan2 have an isolated defect of osteoblast function.

Calcineurin-nuclear factor of activated T cells signaling controls the differentiation and function of osteoclasts and osteoblasts, and regulator of calcineurin-2 (Rcan2) is a physiological inhibitor of this pathway. Rcan2 expression is regulated by T(3), which also has a central role in skeletal development and bone turnover. To investigate the role of Rcan2 in bone development and maintenance, we characterized Rcan2(-/-) mice and determined its skeletal expression in T(3) receptor (TR) knockout and thyroid-manipulated mice. Rcan2(-/-) mice had normal linear growth but displayed delayed intramembranous ossification, impaired cortical bone formation, and reduced bone mineral accrual during development as well as increased mineralization of adult bone. These abnormalities resulted from an isolated defect in osteoblast function and are similar to skeletal phenotypes of mice lacking the type 2 deiodinase thyroid hormone activating enzyme or with dominant-negative mutations of TRα, the predominant TR isoform in bone. Rcan2 mRNA was expressed in primary osteoclasts and osteoblasts, and its expression in bone was differentially regulated in TRα and TRß knockout and thyroid-manipulated mice. However, in primary osteoblast cultures, T(3) treatment did not affect Rcan2 mRNA expression or nuclear factor of activated T cells c1 expression and phosphorylation. Overall, these studies establish that Rcan2 regulates osteoblast function and its expression in bone is regulated by thyroid status in vivo.

A mouse model for spondyloepiphyseal dysplasia congenita with secondary osteoarthritis due to a Col2a1 mutation.

Progeny of mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) revealed a mouse, designated Longpockets (Lpk), with short humeri, abnormal vertebrae, and disorganized growth plates, features consistent with spondyloepiphyseal dysplasia congenita (SEDC). The Lpk phenotype was inherited as an autosomal dominant trait. Lpk/+ mice were viable and fertile and Lpk/Lpk mice died perinatally. Lpk was mapped to chromosome 15 and mutational analysis of likely candidates from the interval revealed a Col2a1 missense Ser1386Pro mutation. Transient transfection of wild-type and Ser1386Pro mutant Col2a1 c-Myc constructs in COS-7 cells and CH8 chondrocytes demonstrated abnormal processing and endoplasmic reticulum retention of the mutant protein. Histology revealed growth plate disorganization in 14-day-old Lpk/+ mice and embryonic cartilage from Lpk/+ and Lpk/Lpk mice had reduced safranin-O and type-II collagen staining in the extracellular matrix. The wild-type and Lpk/+ embryos had vertical columns of proliferating chondrocytes, whereas those in Lpk/Lpk mice were perpendicular to the direction of bone growth. Electron microscopy of cartilage from 18.5 dpc wild-type, Lpk/+, and Lpk/Lpk embryos revealed fewer and less elaborate collagen fibrils in the mutants, with enlarged vacuoles in the endoplasmic reticulum that contained amorphous inclusions. Micro-computed tomography (CT) scans of 12-week-old Lpk/+ mice revealed them to have decreased bone mineral density, and total bone volume, with erosions and osteophytes at the joints. Thus, an ENU mouse model with a Ser1386Pro mutation of the Col2a1 C-propeptide domain that results in abnormal collagen processing and phenotypic features consistent with SEDC and secondary osteoarthritis has been established.

Activated invariant NKT cells regulate osteoclast development and function.

Invariant NKT (iNKT) cells modulate innate and adaptive immune responses through activation of myeloid dendritic cells and macrophages and via enhanced clonogenicity, differentiation, and egress of their shared myeloid progenitors. Because these same progenitors give rise to osteoclasts (OCs), which also mediate the egress of hematopoietic progenitors and orchestrate bone remodeling, we hypothesized that iNKT cells would extend their myeloid cell regulatory role to the development and function of OCs. In this study, we report that selective activation of iNKT cells by α-galactosylceramide causes myeloid cell egress, enhances OC progenitor and precursor development, modifies the intramedullary kinetics of mature OCs, and enhances their resorptive activity. OC progenitor activity is positively regulated by TNF-α and negatively regulated by IFN-γ, but is IL-4 and IL-17 independent. These data demonstrate a novel role of iNKT cells that couples osteoclastogenesis with myeloid cell egress in conditions of immune activation.