Transcription analyses of differentially expressed mRNAs, lncRNAs, circRNAs, and miRNAs in the growth plate of rats with glucocorticoid-induced growth retardation.

Background: Glucocorticoids (GCs) are commonly used to treat autoimmune diseases and malignancies in children and adolescents. Growth retardation is a common adverse effect of GC treatment in pediatric patients. Accumulating evidence indicates that non-coding RNAs (ncRNAs) are involved in the pathogenesis of glucocorticoid-induced growth retardation (GIGR), but the roles of specific ncRNAs in growth remain largely unknown. Methods: In this study, 2-week-old male Sprague-Dawley rats had been treated with 2 mg/kg/d of dexamethasone for 7 or 14 days, after which the growth plate tissues were collected for high-throughput RNA sequencing to identify differentially expressed mRNAs, lncRNAs, circRNAs, and miRNAs in GIGR rats. Results: Transcriptomic analysis identified 1,718 mRNAs, 896 lncRNAs, 60 circRNAs, and 72 miRNAs with different expression levels in the 7d group. In the 14d group, 1,515 mRNAs, 880 lncRNAs, 46 circRNAs, and 55 miRNAs with differential expression were identified. Four mRNAs and four miRNAs that may be closely associated with the development of GIGR were further validated by real-time quantitative fluorescence PCR. Function enrichment analysis indicated that the PI3K-Akt signaling pathway, NF-kappa B signaling pathway, and TGF-ß signaling pathway participated in the development of the GIGR. Moreover, the constructed ceRNA networks suggested that several miRNAs (including miR-140-3p and miR-127-3p) might play an important role in the pathogenesis of GIGR. Conclusions: These results provide new insights and important clues for exploring the molecular mechanisms underlying GIGR.

High prevalence of variants in skeletal dysplasia associated genes in individuals with short stature and minor skeletal anomalies.

OBJECTIVE: Next generation sequencing (NGS) has expanded the diagnostic paradigm turning the focus to the growth plate. The aim of the study was to determine the prevalence of variants in genes implicated in skeletal dysplasias in probands with short stature and mild skeletal anomalies. DESIGN: Clinical and radiological data were collected from 108 probands with short stature and mild skeletal anomalies. METHODS: A customized skeletal dysplasia NGS panel was performed. Variants were classified using ACMG recommendations and Sherloc. Anthropometric measurements and skeletal anomalies were subsequently compared in those with or without an identified genetic defect. RESULTS: Heterozygous variants were identified in 21/108 probands (19.4%). Variants were most frequently identified in ACAN (n = 10) and IHH (n = 7) whilst one variant was detected in COL2A1, CREBBP, EXT1, and PTPN11. Statistically significant differences (P < 0.05) were observed for sitting height/height (SH/H) ratio, SH/H ratio standard deviation score (SDS), and the SH/H ratio SDS >1 in those with an identified variant compared to those without. CONCLUSIONS: A molecular defect was elucidated in a fifth of patients. Thus, the prevalence of mild forms of skeletal dysplasias is relatively high in individuals with short stature and mild skeletal anomalies, with variants in ACAN and IHH accounting for 81% of the cases. An elevated SH/H ratio appears to be associated with a greater probability in detecting a variant, but no other clinical or radiological feature has been found determinant to finding a genetic cause. Currently, we cannot perform extensive molecular studies in all short stature individuals so detailed clinical and radiological phenotyping may orientate which are the candidate patients to obtain worthwhile results. In addition, detailed phenotyping of probands and family members will often aid variant classification.

Kirner's deformity of the fifth finger: A case report.

RATIONALE: Kirner's deformity is an uncommon deformity of finger, characterized by palmo-radial curvature of distal phalanx of the fifth finger. The specific mechanism remains unknown yet. This study aims to present a case report to add the knowledge on this type of deformity. PATIENT CONCERNS: A 9-year-old girl presenting with deformity of her fifth finger since she was born was admitted to our hand surgery clinic. MRI findings showed widened epiphyseal plate, L-shaped physis, but normal flexor digitorum profundus tendon insertion, without any significantly enhanced soft issues. DIAGNOSIS: Kirner's deformity of the fifth finger. INTERVENTIONS: We presented 2 surgical choices for the patient: one was wedge osteotomy of the distal phalanx to correct the mechanical line of the distal phalanx and fixation with Kirschner wire and the other one was cut-off of deep flexor tendon insertion with brace immobilization, but her guardians refused either of them. OUTCOMES: Consecutive follow-up was performed for 19 months after the first visit, showing no any change in finger shape and function. LESSONS: The L-shaped epiphyses may be the cause of Kirner's deformity and further attention should be paid on in the clinic. This case report provided a basis for the etiological diagnosis and future treatment of Kirner's deformity.

Rabbit growth plate morphology in temporary bilateral blocking / Morfología de la placa de crecimiento de conejos durante bloqueo bilateral temporario

Blocking of the growth plate (GP) using plates with screws (tension band plating) is a modern method used to correct deformities and moderate leg length discrepancy in growing children. Determining the duration of temporary bilateral blocking without the occurrence of irreversible changes of GP is of paramount importance important. Methods: Two-month-old Californian breed male rabbits (n=30) were exposed to bilateral blocking of the distal GP of the right femur locking plates with screws for 3, 5, and 7 weeks. The fixators were removed after 5 and 7 weeks in 18 rabbits and 3 weeks after that, animals were sacri!ced. The contralateral limb was used as a control. Histological, histomorphometric, and X-ray analyses were performed. Results: During GP blocking, its height gradually decreased. This decreased was more pronounced after 7 weeks. Destructive changes progressed with an increase in the blocking duration. Three weeks after discontinuation of the bilateral blocking that lasted 5 weeks, the height of the GP signi!cantly increased 1.2 times on the lateral side and 1.9 times on the medial side (p<0.001) compared to the control. When blocking was discontinued after 7 weeks, the structure of the GP was partially restored after 3 weeks, the height of GP signi!cantly increased 1.2 times on the lateral side, and 1.07 times on the medial side (p<0.01) compared to the control. Conclusion: Restoration of the structuralfunctional features of the GP after the removal of the plates depends on the duration of temporary bilateral blocking, which must be taken into account in the clinical setting. (AU)

El bloqueo de la placa de crecimiento (PC) utilizando placas con tornillos (banda de tensión) es un método moderno utilizado para corregir deformidades y alteraciones moderadas en la longitud de las piernas en niños en crecimiento. Es de suma importancia determinar cuál debe ser la duración del bloqueo bilateral temporal sin que ocurran cambios irreversibles en la PC. Métodos: Conejos machos de raza californiana de dos meses de edad (n = 30) fueron expuestos al bloqueo bilateral de la PC distal colocando placas del fémur derecho con tornillos durante 3, 5 y 7 semanas. Los fijadores fueron retirados después de 5 y 7 semanas en 18 de los conejos, y 3 semanas después los animales fueron sacrificados. La extremidad contralateral se utilizó como control. Se realizaron análisis histológicos, histomorfométricos y de rayos X. Resultados: Durante el bloqueo de la PC, su altura disminuyó gradualmente. Esta disminución fue más pronunciada después de 7 semanas. Los cambios destructivos se incrementaron a medida aumentaba la duración del bloqueo. Tres semanas después de la interrupción del bloqueo bilateral que duró 5 semanas, la altura de la PC aumentó significativamente 1.2 veces en el lado lateral y 1.9 veces en el lado medial (p <0.001) en comparación con el control. Conclusión: La restauración de las características funcionales estructurales de la PC después de la extracción de las placas depende de la duración del bloqueo bilateral temporal, lo que debería tenerse en cuenta en el tratamiento clínico de estas alteraciones. (AU)

Mouse polycomb group gene Cbx2 promotes osteoblastic but suppresses adipogenic differentiation in postnatal long bones.

A set of key developmental genes is essential for skeletal growth from multipotent progenitor cells at weaning. Polycomb group proteins, which regulate such genes contributes to the cell lineage commitment and subsequent differentiation via epigenetic chromatin modification and remodeling. However, it is unclear which cell lineage and gene sets are targeted by polycomb proteins during skeletal growth. We now report that mice deficient in a polycomb group gene Cbx2cterm/cterm exhibited skeletal hypoplasia in the tibia, femur, and cranium. Long bone cavities in these mice contained fewer multipotent mesenchymal stromal cells. RNA-sequencing of bone marrow cells showed downregulation and upregulation of osteoblastic and adipogenic genes, respectively. Furthermore, the expression levels of genes specifically expressed in B-cell precursors were decreased. Forced expression of Cbx2 in Cbx2cterm/cterm bone marrow stromal cell recovered fibroblastic colony formation and suppressed adipogenic differentiation. Collectively, our results suggest that Cbx2 controls the maintenance and adipogenic differentiation of mesenchymal stromal cells in the bone marrow.

Inhibiting the integrated stress response pathway prevents aberrant chondrocyte differentiation thereby alleviating chondrodysplasia.

The integrated stress response (ISR) is activated by diverse forms of cellular stress, including endoplasmic reticulum (ER) stress, and is associated with diseases. However, the molecular mechanism(s) whereby the ISR impacts on differentiation is incompletely understood. Here, we exploited a mouse model of Metaphyseal Chondrodysplasia type Schmid (MCDS) to provide insight into the impact of the ISR on cell fate. We show the protein kinase RNA-like ER kinase (PERK) pathway that mediates preferential synthesis of ATF4 and CHOP, dominates in causing dysplasia by reverting chondrocyte differentiation via ATF4-directed transactivation of Sox9. Chondrocyte survival is enabled, cell autonomously, by CHOP and dual CHOP-ATF4 transactivation of Fgf21. Treatment of mutant mice with a chemical inhibitor of PERK signaling prevents the differentiation defects and ameliorates chondrodysplasia. By preventing aberrant differentiation, titrated inhibition of the ISR emerges as a rationale therapeutic strategy for stress-induced skeletal disorders.

Growth impairment in mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders that affect regulation of glycosaminoglycan (GAG) processing. In MPS, the lysosomes cannot efficiently break down GAGs, and the specific GAGs accumulated depend on the type of MPS. The level of impairment of breakdown varies between patients, making this one of the many factors that lead to a range of clinical presentations even in the same type of MPS. These clinical presentations usually involve skeletal dysplasia, in which the most common feature is bone growth impairment and successive short stature. Growth impairment occurs due to the deposition and retention of GAGs in bone and cartilage. The accumulation of GAGs in these tissues leads to progressive damage in cartilage that in turn reduces bone growth by destruction of the growth plate, incomplete ossification, and imbalance of growth. Imbalance of growth leads to various skeletal abnormalities including disproportionate dwarfism with short neck and trunk, prominent forehead, rigidity of joints, tracheal obstruction, kyphoscoliosis, pectus carinatum, platyspondyly, round-shaped vertebral bodies or beaking sign, underdeveloped acetabula, wide flared iliac, coxa valgus, flattered capital femoral epiphyses, and genu valgum. If left untreated, skeletal abnormalities including growth impairment result in a significant impact on these patients' quality of life and activity of daily living, leading to high morbidity and severe handicap. This review focuses on growth impairment in untreated patients with MPS. We comprehensively describe the growth abnormalities through height, weight, growth velocity, and BMI in each type of MPS and compare the status of growth with healthy age-matched controls. The timing, the degree, and the difference in growth impairment of each MPS are highlighted to understand the natural course of growth and to evaluate future therapeutic efficacy.

Post-weaning epiphysiolysis causes distal femur dysplasia and foreshortened hindlimbs in fetuin-A-deficient mice.

Fetuin-A / α2-Heremans-Schmid-glycoprotein (gene name Ahsg) is a systemic inhibitor of ectopic calcification. Due to its high affinity for calcium phosphate, fetuin-A is highly abundant in mineralized bone matrix. Foreshortened femora in fetuin-A-deficient Ahsg-/- mice indicated a role for fetuin-A in bone formation. We studied early postnatal bone development in fetuin-A-deficient mice and discovered that femora from Ahsg-/- mice exhibited severely displaced distal epiphyses and deformed growth plates, similar to the human disease slipped capital femoral epiphysis (SCFE). The growth plate slippage occurred in 70% of Ahsg-/- mice of both sexes around three weeks postnatal. At this time point, mice weaned and rapidly gained weight and mobility. Epiphysis slippage never occurred in wildtype and heterozygous Ahsg+/- mice. Homozygous fetuin-A-deficient Ahsg-/- mice and, to a lesser degree, heterozygous Ahsg+/- mice showed lesions separating the proliferative zone from the hypertrophic zone of the growth plate. The hypertrophic growth plate cartilage in long bones from Ahsg-/- mice was significantly elongated and V-shaped until three weeks of age and thus prior to the slippage. Genome-wide transcriptome analysis of laser-dissected distal femoral growth plates from 13-day-old Ahsg-/- mice revealed a JAK-STAT-mediated inflammatory response including a 550-fold induction of the chemokine Cxcl9. At this stage, vascularization of the elongated growth plates was impaired, which was visualized by immunofluorescence staining. Thus, fetuin-A-deficient mice may serve as a rodent model of growth plate pathologies including SCFE and inflammatory cartilage degradation.

Excesso de tiroxina materna associado ao hipertireoidismo pós-natal reduz o crescimento ósseo e o perfil proliferativo e angiogênico das cartilagens de crescimento de ratos / Excess maternal thyroxine associated with postnatal hyperthyroidism reduces bone growth and proliferative and angiogenic profile of rats growth cartilage

Foram estudados os efeitos do excesso da tiroxina materna associado ao hipertireoidismo pós-natal sobre o crescimento ósseo e o perfil proliferativo e angiogênico das cartilagens. Dezesseis ratas Wistar adultas foram distribuídas nos grupos tratados com L-tiroxina e controle. A prole do grupo tratado recebeu L-tiroxina do desmame até 40 dias de idade. Ao desmame, foi realizada dosagem plasmática de T4 livre nas mães. Na prole, foram realizados: dosagem plasmática de T3 total e T4 livre, morfometria das tireoides, mensuração do comprimento e largura do fêmur. Nas cartilagens, foi avaliada a expressão imuno-histoquímica e gênica de CDC-47, VEGF, Flk-1, Ang1, Ang2 e Tie2. As médias entre grupos foram comparadas pelo teste T de Student. As concentrações de T4 livre das mães tratadas e de T3 total e T4 livre da prole foram significativamente mais elevadas. A largura do fêmur foi menor nos animais tratados. Houve também redução da imunoexpressão de CDC-47 e de VEGF e dos transcritos gênicos para VEGF e Ang1 nas cartilagens. Conclui-se que o excesso de tiroxina materna associado ao hipertireoidismo pós-natal reduz a largura da diáfise femoral, a proliferação celular e a expressão de VEGF e de Ang1 nas cartilagens de crescimento de ratos.(AU)

The effects of excess of maternal thyroxine associated with postnatal hyperthyroidism at bone growth and proliferative and angiogenic profile of cartilage were studied. Sixteen adult Wistar rats were divided into treated and control groups. The offspring of the treated group received L-thyroxine from weaning to 40 days-old. At weaning, plasma assay of free T4 was measurement on female rats. In the offspring, the following assessments were performed: measurement of total T3 and free T4, histomorphometry analysis of the thyroid, measurement of body weight and length and width of the femur. In femoral growth cartilage, immunostaining of CDC-47, gene or protein expression of VEGF, Flk-1, Ang1, Ang2 and Tie2 were evaluated. Data were analyzed using Student's t-test. Free T4 was significantly higher in treated rats and total T3 and free T4 were significantly higher in offspring. The width of the femur was significantly lower in treated animals. There was lower immunoreactivity of CDC-47, VEGF and lower expression of gene transcripts for VEGF and Ang1. We concluded that the excess maternal thyroxine associated with postnatal hyperthyroidism reduces the width of the femoral shaft, the cell proliferation and gene and protein expression of VEGF and gene expression of Ang1 on the growth cartilage in rats.(AU)

Skull Base and Cervical Spine Involvement in Jansen Syndrome: Case Report.

INTRODUCTION: Metaphyseal chondrodysplasia, Jansen type (JMD), is a rare form of endochondral ossification resulting in short limbs and dwarfism. CASE REPORT: A child presented with JMD and was found to have involvement of the cervical spine. Conservative treatment was given to the patient who at the long-term follow-up continues to have no neurological findings or cervical spine instability. CONCLUSIONS: To our knowledge, this case represents the first report of involvement of the superior cervical spine in a patient with JMD. Clinicians should be aware of this potential albeit rare finding.

p38α MAPK Regulates Lineage Commitment and OPG Synthesis of Bone Marrow Stromal Cells to Prevent Bone Loss under Physiological and Pathological Conditions.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are capable of differentiating into osteoblasts, chondrocytes, and adipocytes. Skewed differentiation of BM-MSCs contributes to the pathogenesis of osteoporosis. Yet how BM-MSC lineage commitment is regulated remains unclear. We show that ablation of p38α in Prx1+ BM-MSCs produced osteoporotic phenotypes, growth plate defects, and increased bone marrow fat, secondary to biased BM-MSC differentiation from osteoblast/chondrocyte to adipocyte and increased osteoclastogenesis and bone resorption. p38α regulates BM-MSC osteogenic commitment through TAK1-NF-κB signaling and osteoclastogenesis through osteoprotegerin (OPG) production by BM-MSCs. Estrogen activates p38α to maintain OPG expression in BM-MSCs to preserve the bone. Ablation of p38α in BM-MSCs positive for Dermo1, a later BM-MSC marker, only affected osteogenic differentiation. Thus, p38α mitogen-activated protein kinase (MAPK) in Prx1+ BM-MSCs acts to preserve the bone by promoting osteogenic lineage commitment and sustaining OPG production. This study thus unravels previously unidentified roles for p38α MAPK in skeletal development and bone remodeling.

[Epiphyseal Bar Resection for Correction of Clinodactyly]. / Resektion der atypischen Wachstumsfuge zur Korrektur der Klinodaktylie.

BACKGROUND: Clinodactyly is a digital angulation in the radio-ulnar plane. Mostly it is seen on the little finger. The middle phalanx typically has a triangular or trapezoid shape (delta phalanx), resulting in radial deviation of the distal phalanx. Resection of the longitudinal epiphyseal bracket (physiolysis) with fat graft interposition is a possible surgical technique, which uses children's growth potential to correct the axial deviation. AIM: The purpose of our study was to review the degree of correction at least 2 years postoperatively and after an average of 5 years postoperatively, and to find out if children´s age influences the results. PATIENTS AND METHODS: 23 children (43 little fingers) underwent physiolysis and were retrospectively analyzed after a median follow-up of 5 years (2.1-7.9 years). The active range of motion of the little finger's MP, PIP and DIP joints and finger-palm-distance were measured. Lateral deviation was determined by using standardized radiographs and subsequently compared with preoperative values. Patients were divided into 2 subgroups: younger than 3 years (16 fingers, group A), older than 3 years (27 fingers, group B). The achieved correction of the lateral deviation was compared between both groups. RESULTS: All patients showed full active range of motion in all joints of treated fingers. Finger-palm-distance was 0 cm. No complications occurred. The mean preoperative deviation of all patients was 37°±11, which improved after surgery by 17°±11 (i. e. 44.0%±23.1 of initial findings). Group A demonstrated a mean preoperative lateral deviation of 40°±9, and group B a mean deviation of 36°±12. In both groups we saw a similar improvement (group A mean: 17°±10, group B mean: 17°±11). In group A there was a wider dispersion of postoperative results. In the age group 7 to 10, the results of individual cases show the large variability of the corrective potential. X-rays revealed the following incidental findings after surgery: a premature fusion of the proximal radial epiphyseal plates in 2 fingers and a sinuous-shaped proximal radial epiphyseal plate in 12 other fingers. CONCLUSION: Resection of the longitudinal epiphyseal bracket with fat graft interposition is a technically simple and effective treatment option for clinodactyly, particularly in children of 3 to 6 years of age.

Endoplasmic reticulum stress-mediated apoptosis contributes to a skeletal dysplasia resembling platyspondylic lethal skeletal dysplasia, Torrance type, in a novel Col2a1 mutant mouse line.

In humans, mutations in the COL2A1 gene encoding the α1(II) chain of type II collagen, create many clinical phenotypes collectively termed type II collagenopathies. However, the mechanisms generating this diversity remain to be determined. Here we identified a novel Col2a1 mutant mouse line by screening a large-scale N-ethyl-N-nitrosourea mutant mouse library. This mutant possessed a p.Tyr1391Ser missense mutation in the C-propeptide coding region, and this mutation was located in positions corresponding to the human COL2A1 mutation responsible for platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T). As expected, p.Tyr1391Ser homozygotes exhibited lethal skeletal dysplasias resembling PLSD-T, including extremely short limbs and severe dysplasia of the spine and pelvis. The secretion of the mutant proteins into the extracellular space was disrupted, accompanied by an abnormally expanded endoplasmic reticulum (ER) and the up-regulation of ER stress-related genes in chondrocytes. Chondrocyte apoptosis was severely induced in the growth plate of the homozygotes. These findings strongly suggest that ER stress-mediated apoptosis caused by the accumulated mutant proteins in ER contributes to skeletal dysplasia in Co12a1 mutant mice and PLSD-T patients.

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2.

Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.

Mechanisms of aberrant organization of growth plates in conditional transgenic mouse model of spondyloepiphyseal dysplasia associated with the R992C substitution in collagen II.

Mutations in collagen II, a main structural protein of cartilage, are associated with various forms of spondyloepiphyseal dysplasia (SED), whose main features include aberrations of linear growth. Here, we analyzed the pathomechanisms responsible for growth alterations in transgenic mice with conditional expression of the R992C collagen II mutation. Specifically, we studied the alterations of the growth plates of mutant mice in which chondrocytes lacked their typical columnar arrangement. Our studies demonstrated that chondrocytes expressing the thermolabile R992C mutant collagen II molecules endured endoplasmic reticulum stress, had atypical polarization, and had reduced proliferation. Moreover, we demonstrated aberrant organization and morphology of primary cilia. Analyses of the extracellular collagenous deposits in mice expressing the R992C mutant collagen II molecules indicated their poor formation and distribution. By contrast, transgenic mice expressing wild-type collagen II and mice in which the expression of the transgene encoding the R992C collagen II was switched off were characterized by normal growth, and the morphology of their growth plates was correct. Our study with the use of a conditional mouse SED model not only indicates a direct relation between the observed aberration of skeletal tissues and the presence of mutant collagen II, but also identifies cellular and matrix elements of the pathomechanism of SED.

Core binding factor beta (Cbfß) controls the balance of chondrocyte proliferation and differentiation by upregulating Indian hedgehog (Ihh) expression and inhibiting parathyroid hormone-related protein receptor (PPR) expression in postnatal cartilage and bone formation.

Core binding factor beta (Cbfß) is essential for embryonic bone morphogenesis. Yet the mechanisms by which Cbfß regulates chondrocyte proliferation and differentiation as well as postnatal cartilage and bone formation remain unclear. Hence, using paired-related homeobox transcription factor 1-Cre (Prx1-Cre) mice, mesenchymal stem cell-specific Cbfß-deficient (Cbfß(f/f) Prx1-Cre) mice were generated to study the role of Cbfß in postnatal cartilage and bone development. These mutant mice survived to adulthood but exhibited severe sternum and limb malformations. Sternum ossification was largely delayed in the Cbfß(f/f) Prx1-Cre mice and the xiphoid process was noncalcified and enlarged. In newborn and 7-day-old Cbfß(f/f) Prx1-Cre mice, the resting zone was dramatically elongated, the proliferation zone and hypertrophic zone of the growth plates were drastically shortened and disorganized, and trabecular bone formation was reduced. Moreover, in 1-month-old Cbfß(f/f) Prx1-Cre mice, the growth plates were severely deformed and trabecular bone was almost absent. In addition, Cbfß deficiency impaired intramembranous bone formation both in vivo and in vitro. Interestingly, although the expression of Indian hedgehog (Ihh) was largely reduced, the expression of parathyroid hormone-related protein (PTHrP) receptor (PPR) was dramatically increased in the Cbfß(f/f) Prx1-Cre growth plate, indicating that that Cbfß deficiency disrupted the Ihh-PTHrP negative regulatory loop. Chromatin immunoprecipitation (ChIP) analysis and promoter luciferase assay demonstrated that the Runx/Cbfß complex binds putative Runx-binding sites of the Ihh promoter regions, and also the Runx/Cbfß complex directly upregulates Ihh expression at the transcriptional level. Consistently, the expressions of Ihh target genes, including CyclinD1, Ptc, and Pthlh, were downregulated in Cbfß-deficient chondrocytes. Taken together, our study reveals not only that Cbfß is essential for chondrocyte proliferation and differentiation for the growth and maintenance of the skeleton in postnatal mice, but also that it functions in upregulating Ihh expression to promoter chondrocyte proliferation and osteoblast differentiation, and inhibiting PPR expression to enhance chondrocyte differentiation.

Long-term outcomes of lumbar posterior apophyseal end-plate lesions in children and adolescents.

BACKGROUND: A lesion of the lumbar posterior apophyseal end plate in children and adolescents causes symptoms similar to those associated with a herniated disc. However, the end-plate lesion and the herniated disc differ in terms of pathology. The purpose of this study was to clarify the long-term clinical and radiographic outcomes in children and adolescents who were treated either surgically or conservatively for a lumbar posterior apophyseal end-plate lesion. METHODS: We identified twenty-four consecutive patients who had been treated in the sports clinic of our hospital. The mean age at the first medical examination was 14.5 years. The mean follow-up time was 13.8 years. The mean age at the time of final follow-up was 28.4 years. All twenty-four patients had symptomatic low back pain with sciatica. All but two were active in sports. Sixteen patients were treated conservatively, and eight patients underwent surgical intervention. Skeletal maturity was evaluated on the basis of the appearance of the secondary ossification center of L3. RESULTS: The apophyseal stage ("A" stage), which was assigned when the secondary ossification center of the vertebral body was visible on radiographs, was seen most frequently. Both the surgically treated group and the conservatively treated group demonstrated progressive disc degeneration at the involved level. The average Roland-Morris Disability Questionnaire (RDQ) score was 1.3 for the patients treated conservatively and 1.8 for those treated surgically, a nonsignificant difference. One patient developed spinal stenosis after twelve years of conservative treatment. One patient treated surgically demonstrated severe lumbar instability. There were no significant associations between the magnetic resonance imaging (MRI) findings and RDQ scores. Histological examination of surgical specimens showed irregular alignment of the anulus fibrosus, with degenerative matrix and chondrocytes without a nucleus. CONCLUSIONS: The long-term outcome for patients with a posterior end-plate lesion is favorable, regardless of whether it is treated surgically or nonsurgically.

Constitutively activated NLRP3 inflammasome causes inflammation and abnormal skeletal development in mice.

The NLRP3 inflammasome complex is responsible for maturation of the pro-inflammatory cytokine, IL-1ß. Mutations in NLRP3 are responsible for the cryopyrinopathies, a spectrum of conditions including neonatal-onset multisystem inflammatory disease (NOMID). While excessive production of IL-1ß and systemic inflammation are common to all cryopyrinopathy disorders, skeletal abnormalities, prominently in the knees, and low bone mass are unique features of patients with NOMID. To gain insights into the mechanisms underlying skeletal abnormalities in NOMID, we generated knock-in mice globally expressing the D301N NLRP3 mutation (ortholog of D303N in human NLRP3). NOMID mice exhibit neutrophilia in blood and many tissues, including knee joints, and high levels of serum inflammatory mediators. They also exhibit growth retardation and severe postnatal osteopenia stemming at least in part from abnormally accelerated bone resorption, attended by increased osteoclastogenesis. Histologic analysis of knee joints revealed abnormal growth plates, with loss of chondrocytes and growth arrest in the central region of the epiphyses. Most strikingly, a tissue "spike" was observed in the mid-region of the growth plate in the long bones of all NOMID mice that may be the precursor to more severe deformations analogous to those observed in NOMID patients. These findings provide direct evidence linking a NOMID-associated NLRP3-activating mutation to abnormalities of postnatal skeletal growth and bone remodeling.

Inactivation of a novel FGF23 regulator, FAM20C, leads to hypophosphatemic rickets in mice.

Family with sequence similarity 20,-member C (FAM20C) is highly expressed in the mineralized tissues of mammals. Genetic studies showed that the loss-of-function mutations in FAM20C were associated with human lethal osteosclerotic bone dysplasia (Raine Syndrome), implying an inhibitory role of this molecule in bone formation. However, in vitro gain- and loss-of-function studies suggested that FAM20C promotes the differentiation and mineralization of mouse mesenchymal cells and odontoblasts. Recently, we generated Fam20c conditional knockout (cKO) mice in which Fam20c was globally inactivated (by crossbreeding with Sox2-Cre mice) or inactivated specifically in the mineralized tissues (by crossbreeding with 3.6 kb Col 1a1-Cre mice). Fam20c transgenic mice were also generated and crossbred with Fam20c cKO mice to introduce the transgene in the knockout background. In vitro gain- and loss-of-function were examined by adding recombinant FAM20C to MC3T3-E1 cells and by lentiviral shRNA-mediated knockdown of FAM20C in human and mouse osteogenic cell lines. Surprisingly, both the global and mineralized tissue-specific cKO mice developed hypophosphatemic rickets (but not osteosclerosis), along with a significant downregulation of osteoblast differentiation markers and a dramatic elevation of fibroblast growth factor 23 (FGF23) in the serum and bone. The mice expressing the Fam20c transgene in the wild-type background showed no abnormalities, while the expression of the Fam20c transgene fully rescued the skeletal defects in the cKO mice. Recombinant FAM20C promoted the differentiation and mineralization of MC3T3-E1 cells. Knockdown of FAM20C led to a remarkable downregulation of DMP1, along with a significant upregulation of FGF23 in both human and mouse osteogenic cell lines. These results indicate that FAM20C is a bone formation "promoter" but not an "inhibitor" in mouse osteogenesis. We conclude that FAM20C may regulate osteogenesis through its direct role in facilitating osteoblast differentiation and its systemic regulation of phosphate homeostasis via the mediation of FGF23.

Skeletal analysis of the long bone abnormality (lbab/lbab) mouse, a novel chondrodysplastic C-type natriuretic peptide mutant.

Long bone abnormality (lbab/lbab) is a strain of dwarf mice. Recent studies revealed that the phenotype is caused by a spontaneous mutation in the Nppc gene, which encodes mouse C-type natriuretic peptide (CNP). In this study, we analyzed the chondrodysplastic skeletal phenotype of lbab/lbab mice. At birth, lbab/lbab mice are only slightly shorter than their wild-type littermates. Nevertheless, lbab/lbab mice do not undergo a growth spurt, and their final body and bone lengths are only ~60% of those of wild-type mice. Histological analysis revealed that the growth plate in lbab/lbab mice, especially the hypertrophic chondrocyte layer, was significantly thinner than in wild-type mice. Overexpression of CNP in the cartilage of lbab/lbab mice restored their thinned growth plate, followed by the complete rescue of their impaired endochondral bone growth. Furthermore, the bone volume in lbab/lbab mouse was severely decreased and was recovered by CNP overexpression. On the other hand, the thickness of the growth plate of lbab/+ mice was not different from that of wild-type mice; accordingly, impaired endochondral bone growth was not observed in lbab/+ mice. In organ culture experiments, tibial explants from fetal lbab/lbab mice were significantly shorter than those from lbab/+ mice and elongated by addition of 10(-7) M CNP to the same extent as lbab/+ tibiae treated with the same dose of CNP. These results demonstrate that lbab/lbab is a novel mouse model of chondrodysplasia caused by insufficient CNP action on endochondral ossification.