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Chondrocyte differentiation controls skeleton development and stature. Here we provide a comprehensive map of chondrocyte-specific enhancers and show that they provide a mechanistic framework through which non-coding genetic variants can influence skeletal development and human stature. Working with fetal chondrocytes isolated from mice bearing a Col2a1 fluorescent regulatory sensor, we identify 780 genes and 2'704 putative enhancers specifically active in chondrocytes using a combination of RNA-seq, ATAC-seq and H3K27ac ChIP-seq. Most of these enhancers (74%) show pan-chondrogenic activity, with smaller populations being restricted to limb (18%) or trunk (8%) chondrocytes only. Notably, genetic variations overlapping these enhancers better explain height differences than those overlapping non-chondrogenic enhancers. Finally, targeted deletions of identified enhancers at the Fgfr3, Col2a1, Hhip and, Nkx3-2 loci confirm their role in regulating cognate genes. This enhancer map provides a framework for understanding how genes and non-coding variations influence bone development and diseases.

ObjectivesTo study total growth, rate of bone maturation, and menarche after discontinuation of Gonadotropin releasing hormone agonist (GnRHa) treatment for central precocious puberty (CPP).MethodsTwenty girls with CPP on treatment with GnRHa were followed from discontinuation of treatment to final height (FH). Height, height velocity (HV), and bone age were measured every 6 months. Age at menarche was collected.ResultsOnce treatment is discontinued, rate of bone maturation (bone age [BA]/chronological [CA]) accelerated from 0.7 ± 0.3 at end of treatment to 1.2 ± 0.8 post treatment, similar to BA/CA prior to treatment. BA at treatment discontinuation ranged from 11-14 years. On average, treatment was stopped when CA was within 9 months of BA. All girls continued to grow from end of treatment to menarche averaging an increase of 4.7 ± 3.7 cm, with HV 3.2 ± 2.0 cm/year. Post-menarche they grew an additional 4.6 ± 2.1 cm, with HV 2.4 ± 1.9 cm/year. Acceleration of HV was not seen post treatment. The younger the BA at initiation or completion of treatment, the longer time to menarche. No one had menarche prior to a BA of 12.5 year.ConclusionsA pubertal growth spurt does not usually occur after treatment with GnRHa in girls with CPP. Rate of bone maturation accelerates post treatment. These factors are important in assessing optimal height outcome and decisions regarding cessation of treatment. This study will help clinicians give patients and families better estimates of growth and onset of menarche post treatment.

ObjectivesGonadotropin-releasing hormone agonist treatment is important for optimal growth in girls with central precocious puberty (CPP). Data are lacking regarding benefit to height outcome when treatment is started after chronological age (CA) of 7 years, and if continued beyond CA of 10 years or bone age (BA) of 12 years.MethodsForty-eight girls with CPP were treated with monthly leuprolide depot. Change in predicted adult height (PAH) during treatment was assessed. Changes in PAH and growth velocity were compared between girls initiating treatment at CA 5 cm PAH change during treatment was similar, and PAH increased throughout treatment in most girls, regardless of age at treatment initiation. PAH continued to increase in 16/19 girls who continued treatment after BA of 12 years, and also in 16/22 girls who continued treatment after CA of 10 years.ConclusionsPAH improved in most girls who initiated treatment after CA of 7 years. It continued to improve in most girls with longer treatment, even past BA of 12 years or CA of 10 years, which suggests that no absolute CA or BA limit should define initiation or end of treatment. Treatment plans need to be individualized, and neither treatment initiation nor cessation should be based on BA or CA alone.

Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research.

Nonsteroidal anti-inflammatory drugs (NSAIDs) affect bone metabolism in vitro and in vivo. They delay but do not alter the outcome of healing processes in bone. In some bone loss models, they block bone resorption and slow the rate of loss. We studied the effect of naproxen, a potent NSAID, on cancellous bone of the proximal tibial metaphysis of 6-month-old adult female ovariectomized rats. Animals were ovariectomized, divided into groups, and fed standard diets differing only in naproxen content for 42 days. The rats of the groups ate 2.0, 5.5, 12.7, and 32 mg naproxen per kg body weight per day, respectively. Serum levels of naproxen were determined. Bone volume, mineralizing surface, osteoblast activity, osteoclast surface, and bone resorption rate were determined by bone histomorphometric techniques. The rats' dose-related serum naproxen levels ranged from 4 to 28 micrograms/ml. Naproxen inhibited up to 70% of the bone loss occurring after ovariectomy at a serum level of 4 micrograms/ml. We deduced that naproxen blocked bone resorption in ovariectomized rats by slowing osteoclast activity at all doses. In contrast, naproxen slowed bone formation only at serum levels greater than 20 micrograms/ml in ovariectomized rats. These findings may have clinical relevance in helping to prevent postmenopausal bone loss in women.

Human pluripotent stem cells (hPSCs) can provide a platform to model bone organogenesis and disease. To reflect the developmental process of the human skeleton, hPSC differentiation methods should include osteogenic progenitors (OPs) arising from three distinct embryonic lineages: the paraxial mesoderm, lateral plate mesoderm, and neural crest. Although OP differentiation protocols have been developed, the lineage from which they are derived, as well as characterization of their genetic and molecular differences, has not been well reported. Therefore, to generate lineage-specific OPs from human embryonic stem cells and human induced pluripotent stem cells, we employed stepwise differentiation of paraxial mesoderm-like cells, lateral plate mesoderm-like cells, and neural crest-like cells toward their respective OP subpopulation. Successful differentiation, confirmed through gene expression and in vivo assays, permitted the identification of transcriptomic signatures of all three cell populations. We also report, for the first time, high FGF1 levels in neural crest-derived OPs-a notable finding given the critical role of fibroblast growth factors (FGFs) in osteogenesis and mineral homeostasis. Our results indicate that FGF1 influences RUNX2 levels, with concomitant changes in ERK1/2 signaling. Overall, our study further validates hPSCs power to model bone development and disease and reveals new, potentially important pathways influencing these processes.

Human pluripotent stem cells (hPSCs) can provide a platform to model bone organogenesis and disease. To reflect the developmental process of the human skeleton, hPSC differentiation methods should include osteogenic progenitors (OPs) arising from three distinct embryonic lineages: the paraxial mesoderm, lateral plate mesoderm, and neural crest. Although OP differentiation protocols have been developed, the lineage from which they are derived, as well as characterization of their genetic and molecular differences, has not been well reported. Therefore, to generate lineage-specific OPs from human embryonic stem cells and human induced pluripotent stem cells, we employed stepwise differentiation of paraxial mesoderm-like cells, lateral plate mesoderm-like cells, and neural crest-like cells toward their respective OP subpopulation. Successful differentiation, confirmed through gene expression and in vivo assays, permitted the identification of transcriptomic signatures of all three cell populations. We also report, for the first time, high FGF1 levels in neural crest-derived OPs-a notable finding given the critical role of fibroblast growth factors (FGFs) in osteogenesis and mineral homeostasis. Our results indicate that FGF1 influences RUNX2 levels, with concomitant changes in ERK1/2 signaling. Overall, our study further validates hPSCs' power to model bone development and disease and reveals new, potentially important pathways influencing these processes.

Human pluripotent stem cells (hPSCs) can provide a platform to model bone organogenesis and disease. To reflect the developmental process of the human skeleton, hPSC differentiation methods should include osteogenic progenitors (OPs) arising from three distinct embryonic lineages: the paraxial mesoderm, lateral plate mesoderm, and neural crest. Although OP differentiation protocols have been developed, the lineage from which they are derived, as well as characterization of their genetic and molecular differences, has not been well reported. Therefore, to generate lineage-specific OPs from human embryonic stem cells and human induced pluripotent stem cells, we employed stepwise differentiation of paraxial mesoderm-like cells, lateral plate mesoderm-like cells, and neural crest-like cells toward their respective OP subpopulation. Successful differentiation, confirmed through gene expression and in vivo assays, permitted the identification of transcriptomic signatures of all three cell populations. We also report, for the first time, high FGF1 levels in neural crest-derived OPs-a notable finding given the critical role of fibroblast growth factors (FGFs) in osteogenesis and mineral homeostasis. Our results indicate that FGF1 influences RUNX2 levels, with concomitant changes in ERK1/2 signaling. Overall, our study further validates hPSCs' power to model bone development and disease and reveals new, potentially important pathways influencing these processes.

The skeleton is well-innervated, but only recently have the functions of this complex network in bone started to become known. Although our knowledge of skeletal sensory and sympathetic innervation is incomplete, including the specific locations and subtypes of nerves in bone, we are now able to reconcile early studies utilizing denervation models with recent work dissecting the molecular signaling between bone and nerve. In total, sensory innervation functions in bone much as it does elsewhere in the body-to sense and respond to stimuli, including mechanical loading. Similarly, sympathetic nerves regulate autonomic functions related to bone, including homeostatic remodeling and vascular tone. However, more study is required to translate our current knowledge of bone-nerve crosstalk to novel therapeutic strategies that can be effectively utilized to combat skeletal diseases, disorders of low bone mass, and age-related decreases in bone quality.

Bone fracture risk is influenced by bone quality, which encompasses bone's composition as well as its multiscale organization and architecture. Aging and disease deteriorate bone quality, leading to reduced mechanical properties and higher fracture incidence. Largely unexplored is how bone quality and mechanical competence progress during longitudinal bone growth. Human femoral cortical bone was acquired from fetal (n = 1), infantile (n = 3), and 2- to 14-year-old cases (n = 4) at the mid-diaphysis. Bone quality was assessed in terms of bone structure, osteocyte characteristics, mineralization, and collagen orientation. The mechanical properties were investigated by measuring tensile deformation at multiple length scales via synchrotron X-ray diffraction. We find dramatic differences in mechanical resistance with age. Specifically, cortical bone in 2- to 14-year-old cases exhibits a 160% greater stiffness and 83% higher strength than fetal/infantile cases. The higher mechanical resistance of the 2- to 14-year-old cases is associated with advantageous bone quality, specifically higher bone volume fraction, better micronscale organization (woven versus lamellar), and higher mean mineralization compared with fetal/infantile cases. Our study reveals that bone quality is superior after remodeling/modeling processes convert the primary woven bone structure to lamellar bone. In this cohort of female children, the microstructural differences at the femoral diaphysis were apparent between the 1- to 2-year-old cases. Indeed, the lamellar bone in 2- to 14-year-old cases had a superior structural organization (collagen and osteocyte characteristics) and composition for resisting deformation and fracture than fetal/infantile bone. Mechanistically, the changes in bone quality during longitudinal bone growth lead to higher fracture resistance because collagen fibrils are better aligned to resist tensile forces, while elevated mean mineralization reinforces the collagen scaffold. Thus, our results reveal inherent weaknesses of the fetal/infantile skeleton signifying its inferior bone quality. These results have implications for pediatric fracture risk, as bone produced at ossification centers during children's longitudinal bone growth could display similarly weak points. © 2019 American Society for Bone and Mineral Research.

Bone fracture risk is influenced by bone quality, which encompasses bone's composition as well as its multiscale organization and architecture. Aging and disease deteriorate bone quality, leading to reduced mechanical properties and higher fracture incidence. Largely unexplored is how bone quality and mechanical competence progress during longitudinal bone growth. Human femoral cortical bone was acquired from fetal (n = 1), infantile (n = 3), and 2- to 14-year-old cases (n = 4) at the mid-diaphysis. Bone quality was assessed in terms of bone structure, osteocyte characteristics, mineralization, and collagen orientation. The mechanical properties were investigated by measuring tensile deformation at multiple length scales via synchrotron X-ray diffraction. We find dramatic differences in mechanical resistance with age. Specifically, cortical bone in 2- to 14-year-old cases exhibits a 160% greater stiffness and 83% higher strength than fetal/infantile cases. The higher mechanical resistance of the 2- to 14-year-old cases is associated with advantageous bone quality, specifically higher bone volume fraction, better micronscale organization (woven versus lamellar), and higher mean mineralization compared with fetal/infantile cases. Our study reveals that bone quality is superior after remodeling/modeling processes convert the primary woven bone structure to lamellar bone. In this cohort of female children, the microstructural differences at the femoral diaphysis were apparent between the 1- to 2-year-old cases. Indeed, the lamellar bone in 2- to 14-year-old cases had a superior structural organization (collagen and osteocyte characteristics) and composition for resisting deformation and fracture than fetal/infantile bone. Mechanistically, the changes in bone quality during longitudinal bone growth lead to higher fracture resistance because collagen fibrils are better aligned to resist tensile forces, while elevated mean mineralization reinforces the collagen scaffold. Thus, our results reveal inherent weaknesses of the fetal/infantile skeleton signifying its inferior bone quality. These results have implications for pediatric fracture risk, as bone produced at ossification centers during children's longitudinal bone growth could display similarly weak points. © 2019 American Society for Bone and Mineral Research.

Since its initial discovery in 2002, the neuropeptide Kisspeptin (Kiss1) has been anointed as the master regulator controlling the onset of puberty in males and females. Over the last several years, multiple groups found that Kiss1 signaling is mediated by the 7TM surface receptor GPCR54. Kiss1 mRNA is highly enriched in the basal medial and lateral subregions of the arcuate nucleus (ARC) in the medial basal hypothalamus. Thus, Kiss1ARC neurons reside in a unique anatomical location ideal for sensing and responding to circulating steroid hormones as well as nutrients. Kiss1 expression is highly responsive to fluctuations of the gonadal hormone, estrogen, with nearly 90% of Kiss1ARC neurons expressing the nuclear hormone estrogen receptor alpha (ERa). Here we review recent research that extends the function of Kiss1ARC neurons beyond the regulation of puberty and highlight their emerging, novel roles in controlling energy allocation, behavioral outputs, and sex-dependent bone remodeling in females. Indeed, some of these previously unknown functions for Kiss1 neurons are quite striking as exemplified by the remarkable increase in bone mass after manipulating estrogen signaling in Kiss1ARC neurons. Taken together, we suggest that Kiss1ARC neurons are highly sensitive to nutritional and hormonal cues that dictate energy utilization and reproduction.

Since its initial discovery in 2002, the neuropeptide Kisspeptin (Kiss1) has been anointed as the master regulator controlling the onset of puberty in males and females. Over the last several years, multiple groups found that Kiss1 signaling is mediated by the 7TM surface receptor GPCR54. Kiss1 mRNA is highly enriched in the basal medial and lateral subregions of the arcuate nucleus (ARC) in the medial basal hypothalamus. Thus, Kiss1ARC neurons reside in a unique anatomical location ideal for sensing and responding to circulating steroid hormones as well as nutrients. Kiss1 expression is highly responsive to fluctuations of the gonadal hormone, estrogen, with nearly 90% of Kiss1ARC neurons expressing the nuclear hormone estrogen receptor alpha (ERa). Here we review recent research that extends the function of Kiss1ARC neurons beyond the regulation of puberty and highlight their emerging, novel roles in controlling energy allocation, behavioral outputs, and sex-dependent bone remodeling in females. Indeed, some of these previously unknown functions for Kiss1 neurons are quite striking as exemplified by the remarkable increase in bone mass after manipulating estrogen signaling in Kiss1ARC neurons. Taken together, we suggest that Kiss1ARC neurons are highly sensitive to nutritional and hormonal cues that dictate energy utilization and reproduction.

Since its initial discovery in 2002, the neuropeptide Kisspeptin (Kiss1) has been anointed as the master regulator controlling the onset of puberty in males and females. Over the last several years, multiple groups found that Kiss1 signaling is mediated by the 7TM surface receptor GPCR54. Kiss1 mRNA is highly enriched in the basal medial and lateral subregions of the arcuate nucleus (ARC) in the medial basal hypothalamus. Thus, Kiss1ARC neurons reside in a unique anatomical location ideal for sensing and responding to circulating steroid hormones as well as nutrients. Kiss1 expression is highly responsive to fluctuations of the gonadal hormone, estrogen, with nearly 90% of Kiss1ARC neurons expressing the nuclear hormone estrogen receptor alpha (ERa). Here we review recent research that extends the function of Kiss1ARC neurons beyond the regulation of puberty and highlight their emerging, novel roles in controlling energy allocation, behavioral outputs, and sex-dependent bone remodeling in females. Indeed, some of these previously unknown functions for Kiss1 neurons are quite striking as exemplified by the remarkable increase in bone mass after manipulating estrogen signaling in Kiss1ARC neurons. Taken together, we suggest that Kiss1ARC neurons are highly sensitive to nutritional and hormonal cues that dictate energy utilization and reproduction.

Stem cell regulation and hierarchical organization of human skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. VIDEO ABSTRACT.

AimTo determine the distribution of anthropometric parameter (AP)-z-scores and characterize associations between medications/serum biomarkers and AP-z-scores in pediatric Crohn's disease (CD).MethodsCD patients [< chronological age (CA) 21 years] were enrolled in a cross-sectional study. Descriptive statistics were generated for participants' demographic characteristics and key variables of interest. Paired t-tests were used to compare AP-z-scores calculated based on CA (CA z-scores) and bone age (BA) (BA z-scores) for interpretation of AP's. Linear regression was utilized to examine associations between medications and serum biomarkers with AP-z-scores calculated based on CA (n = 82) and BA (n = 49). We reported regression coefficients as well as their corresponding p-values and 95% confidence intervals.ResultsMean CA at the time of the study visit was 15.3 ± 3.5 (SD; range = 4.8-20.7) years. Mean triceps skinfold (P = 0.039), subscapular skinfold (P = 0.002) and mid-arm circumference (MAC) (P = 0.001) BA z-scores were higher than corresponding CA z-scores. Medications were positively associated with subscapular skinfold [adalimumab (P = 0.018) and methotrexate (P = 0.027)] and BMI CA z-scores [adalimumab (P = 0.029)]. Azathioprine/6-mercaptopurine were negatively associated with MAC (P = 0.045), subscapular skinfold (P = 0.014), weight (P = 0.002) and BMI (P = 0.013) CA z-scores. ESR, CRP, and WBC count were negatively associated, while albumin and IGF-1 BA z-scores were positively associated, with specific AP z-scores (P < 0.05). Mean height CA z-scores were higher in females, not males, treated with infliximab (P = 0.038). Hemoglobin (P = 0.018) was positively associated, while platelets (P = 0.005), ESR (P = 0.003) and CRP (P = 0.039) were negatively associated with height CA z-scores in males, not females.ConclusionOur results suggest poor efficacy of thiopurines and a possible sex difference in statural growth response to infliximab in pediatric CD. Prospective longitudinal studies are required.

AimTo determine the distribution of anthropometric parameter (AP)-z-scores and characterize associations between medications/serum biomarkers and AP-z-scores in pediatric Crohn's disease (CD).MethodsCD patients [< chronological age (CA) 21 years] were enrolled in a cross-sectional study. Descriptive statistics were generated for participants' demographic characteristics and key variables of interest. Paired t-tests were used to compare AP-z-scores calculated based on CA (CA z-scores) and bone age (BA) (BA z-scores) for interpretation of AP's. Linear regression was utilized to examine associations between medications and serum biomarkers with AP-z-scores calculated based on CA (n = 82) and BA (n = 49). We reported regression coefficients as well as their corresponding p-values and 95% confidence intervals.ResultsMean CA at the time of the study visit was 15.3 ± 3.5 (SD; range = 4.8-20.7) years. Mean triceps skinfold (P = 0.039), subscapular skinfold (P = 0.002) and mid-arm circumference (MAC) (P = 0.001) BA z-scores were higher than corresponding CA z-scores. Medications were positively associated with subscapular skinfold [adalimumab (P = 0.018) and methotrexate (P = 0.027)] and BMI CA z-scores [adalimumab (P = 0.029)]. Azathioprine/6-mercaptopurine were negatively associated with MAC (P = 0.045), subscapular skinfold (P = 0.014), weight (P = 0.002) and BMI (P = 0.013) CA z-scores. ESR, CRP, and WBC count were negatively associated, while albumin and IGF-1 BA z-scores were positively associated, with specific AP z-scores (P < 0.05). Mean height CA z-scores were higher in females, not males, treated with infliximab (P = 0.038). Hemoglobin (P = 0.018) was positively associated, while platelets (P = 0.005), ESR (P = 0.003) and CRP (P = 0.039) were negatively associated with height CA z-scores in males, not females.ConclusionOur results suggest poor efficacy of thiopurines and a possible sex difference in statural growth response to infliximab in pediatric CD. Prospective longitudinal studies are required.

Fibrous dysplasia (FD) is a disease caused by postzygotic activating mutations of GNAS (R201C and R201H) that encode the α-subunit of the Gs stimulatory protein. FD is characterized by the development of areas of abnormal fibroosseous tissue in the bones, resulting in skeletal deformities, fractures, and pain. Despite the well-defined genetic alterations underlying FD, whether GNAS activation is sufficient for FD initiation and the molecular and cellular consequences of GNAS mutations remains largely unresolved, and there are no currently available targeted therapeutic options for FD. Here, we have developed a conditional tetracycline (Tet)-inducible animal model expressing the GαsR201C in the skeletal stem cell (SSC) lineage (Tet-GαsR201C/Prrx1-Cre/LSL-rtTA-IRES-GFP mice), which develops typical FD bone lesions in both embryos and adult mice in less than 2 weeks following doxycycline (Dox) administration. Conditional GαsR201C expression promoted PKA activation and proliferation of SSCs along the osteogenic lineage but halted their differentiation to mature osteoblasts. Rather, as is seen clinically, areas of woven bone admixed with fibrous tissue were formed. GαsR201C caused the concomitant expression of receptor activator of nuclear factor kappa-B ligand (Rankl) that led to marked osteoclastogenesis and bone resorption. GαsR201C expression ablation by Dox withdrawal resulted in FD-like lesion regression, supporting the rationale for Gαs-targeted drugs to attempt FD cure. This model, which develops FD-like lesions that can form rapidly and revert on cessation of mutant Gαs expression, provides an opportunity to identify the molecular mechanism underlying FD initiation and progression and accelerate the development of new treatment options.

Fibrous dysplasia (FD) is a disease caused by postzygotic activating mutations of GNAS (R201C and R201H) that encode the α-subunit of the Gs stimulatory protein. FD is characterized by the development of areas of abnormal fibroosseous tissue in the bones, resulting in skeletal deformities, fractures, and pain. Despite the well-defined genetic alterations underlying FD, whether GNAS activation is sufficient for FD initiation and the molecular and cellular consequences of GNAS mutations remains largely unresolved, and there are no currently available targeted therapeutic options for FD. Here, we have developed a conditional tetracycline (Tet)-inducible animal model expressing the GαsR201C in the skeletal stem cell (SSC) lineage (Tet-GαsR201C/Prrx1-Cre/LSL-rtTA-IRES-GFP mice), which develops typical FD bone lesions in both embryos and adult mice in less than 2 weeks following doxycycline (Dox) administration. Conditional GαsR201C expression promoted PKA activation and proliferation of SSCs along the osteogenic lineage but halted their differentiation to mature osteoblasts. Rather, as is seen clinically, areas of woven bone admixed with fibrous tissue were formed. GαsR201C caused the concomitant expression of receptor activator of nuclear factor kappa-B ligand (Rankl) that led to marked osteoclastogenesis and bone resorption. GαsR201C expression ablation by Dox withdrawal resulted in FD-like lesion regression, supporting the rationale for Gαs-targeted drugs to attempt FD cure. This model, which develops FD-like lesions that can form rapidly and revert on cessation of mutant Gαs expression, provides an opportunity to identify the molecular mechanism underlying FD initiation and progression and accelerate the development of new treatment options.

Fibrous dysplasia (FD) is a disease caused by postzygotic activating mutations of GNAS (R201C and R201H) that encode the α-subunit of the Gs stimulatory protein. FD is characterized by the development of areas of abnormal fibroosseous tissue in the bones, resulting in skeletal deformities, fractures, and pain. Despite the well-defined genetic alterations underlying FD, whether GNAS activation is sufficient for FD initiation and the molecular and cellular consequences of GNAS mutations remains largely unresolved, and there are no currently available targeted therapeutic options for FD. Here, we have developed a conditional tetracycline (Tet)-inducible animal model expressing the GαsR201C in the skeletal stem cell (SSC) lineage (Tet-GαsR201C/Prrx1-Cre/LSL-rtTA-IRES-GFP mice), which develops typical FD bone lesions in both embryos and adult mice in less than 2 weeks following doxycycline (Dox) administration. Conditional GαsR201C expression promoted PKA activation and proliferation of SSCs along the osteogenic lineage but halted their differentiation to mature osteoblasts. Rather, as is seen clinically, areas of woven bone admixed with fibrous tissue were formed. GαsR201C caused the concomitant expression of receptor activator of nuclear factor kappa-B ligand (Rankl) that led to marked osteoclastogenesis and bone resorption. GαsR201C expression ablation by Dox withdrawal resulted in FD-like lesion regression, supporting the rationale for Gαs-targeted drugs to attempt FD cure. This model, which develops FD-like lesions that can form rapidly and revert on cessation of mutant Gαs expression, provides an opportunity to identify the molecular mechanism underlying FD initiation and progression and accelerate the development of new treatment options.

BackgroundPerinatally HIV-infected (PHIV) children have, on average, lower bone mineral density (BMD) than perinatally HIV-exposed uninfected (PHEU) and healthy children. Low 25-hydroxy vitamin D [25(OH)D] and elevated parathyroid hormone (PTH) concentrations may lead to suboptimal bone accrual.MethodsPHIV and PHEU children in the Pediatric HIV/AIDS Cohort Study had total body (TB) and lumbar spine (LS) BMD and bone mineral content (BMC) measured by dual-energy x-ray absorptiometry; BMD z-scores (BMDz) were calculated for age and sex. Low 25(OH)D was defined as ≤20 ng/mL and high PTH as >65 pg/mL. We fit linear regression models to estimate the average adjusted differences in BMD/BMC by 25(OH)D and PTH status and log binomial models to determine adjusted prevalence ratios of low 25(OH)D and high PTH in PHIV relative to PHEU children.ResultsPHIV children (n = 412) were older (13.0 vs. 10.8 years) and more often black (76% vs. 64%) than PHEU (n = 207). Among PHIV, children with low 25(OH)D had lower TB-BMDz [SD, -0.38; 95% confidence interval (CI), -0.60 to -0.16] and TB-BMC (SD, -59.1 g; 95% CI, -108.3 to -9.8); high PTH accompanied by low 25(OH)D was associated with lower TB-BMDz. Among PHEU, children with low 25(OH)D had lower TB-BMDz (SD, -0.34; 95% CI, -0.64 to -0.03). Prevalence of low 25(OH)D was similar by HIV status (adjusted prevalence ratio, 1.00; 95% CI, 0.81 to 1.24). High PTH was 3.17 (95% CI, 1.25 to 8.06) times more likely in PHIV children.ConclusionsPHIV and PHEU children with low 25(OH)D may have lower BMD. Vitamin D supplementation trials during critical periods of bone accrual are needed.

Purpose of reviewOsteogenesis is a complex process involving the specification of multiple progenitor cells and their maturation and differentiation into matrix-secreting osteoblasts. Osteogenesis occurs not only during embryogenesis but also during growth, after an injury, and in normal homeostatic maintenance. While much is known about osteogenesis-associated regulatory genes, the role of microRNAs (miRNAs), which are epigenetic regulators of protein expression, is just beginning to be explored. While miRNAs do not abrogate all protein expression, their purpose is to finely tune it, allowing for a timely and temporary protein down-regulation.Recent findingsThe last decade has unveiled a multitude of miRNAs that regulate key proteins within the osteogenic lineage, thus qualifying them as "ostemiRs." These miRNAs may endogenously target an activator or inhibitor of differentiation, and depending on the target, may either lead to the prolongation of a progenitor maintenance state or to early differentiation. Interestingly, cellular identity seems intimately coupled to the expression of miRNAs, which participate in the suppression of previous and subsequent differentiation steps. In such cases where key osteogenic proteins were identified as direct targets of miRNAs in non-bone cell types, or through bioinformatic prediction, future research illuminating the activity of these miRNAs during osteogenesis will be extremely valuable. Many bone-related diseases involve the dysregulation of transcription factors or other proteins found within osteoblasts and their progenitors, and the dysregulation of miRNAs, which target such factors, may play a pivotal role in disease etiology, or even as a possible therapy.

ATP6V1H is a component of a large protein complex with vacuolar ATPase (V-ATPase) activity. We identified two generations of individuals in which short stature and osteoporosis co-segregated with a mutation in ATP6V1H. Since V-ATPases are highly conserved between human and zebrafish, we generated loss-of-function mutants in atp6v1h in zebrafish through CRISPR/Cas9-mediated gene knockout. Homozygous mutant atp6v1h zebrafish exhibited a severe reduction in the number of mature calcified bone cells and a dramatic increase in the expression of mmp9 and mmp13. Heterozygous adults showed curved vertebra that lack calcified centrum structure and reduced bone mass and density. Treatment of mutant embryos with small molecule inhibitors of MMP9 and MMP13 significantly restored bone mass in the atp6v1h mutants. These studies have uncovered a new, ATP6V1H-mediated pathway that regulates bone formation, and defines a new mechanism of disease that leads to bone loss. We propose that MMP9/MMP13 could be therapeutic targets for patients with this rare genetic disease.

ATP6V1H is a component of a large protein complex with vacuolar ATPase (V-ATPase) activity. We identified two generations of individuals in which short stature and osteoporosis co-segregated with a mutation in ATP6V1H. Since V-ATPases are highly conserved between human and zebrafish, we generated loss-of-function mutants in atp6v1h in zebrafish through CRISPR/Cas9-mediated gene knockout. Homozygous mutant atp6v1h zebrafish exhibited a severe reduction in the number of mature calcified bone cells and a dramatic increase in the expression of mmp9 and mmp13. Heterozygous adults showed curved vertebra that lack calcified centrum structure and reduced bone mass and density. Treatment of mutant embryos with small molecule inhibitors of MMP9 and MMP13 significantly restored bone mass in the atp6v1h mutants. These studies have uncovered a new, ATP6V1H-mediated pathway that regulates bone formation, and defines a new mechanism of disease that leads to bone loss. We propose that MMP9/MMP13 could be therapeutic targets for patients with this rare genetic disease.

Vitamin D deficiency is associated with adverse health outcomes, including impaired bone growth, gingival inflammation and increased risk for autoimmune disease, but the relationship between vitamin D deficiency rickets in childhood and long-term health has not been studied. In this study, we assessed the effect of early vitamin D deficiency on growth, bone density, dental health and immune function in later childhood to determine if children previously diagnosed with rickets were at greater risk of adverse health outcomes compared with healthy children. We measured serum 25-hydroxyvitamin D, calcium, parathyroid hormone, bone mineral density, anthropometric measures, dietary habits, dental health, general health history, and markers of inflammation in 14 previously diagnosed rickets case children at Children's Hospital Oakland Research Center. We compared the findings in the rickets cases with 11 healthy children selected from the population of CHO staff families. Fourteen mothers of the rickets cases, five siblings of the rickets cases, and seven mothers of healthy children also participated. Children diagnosed with vitamin D deficiency rickets had a greater risk of fracture, greater prevalence of asthma, and more dental enamel defects compared with healthy children. Given the widespread actions of vitamin D, it is likely that early-life vitamin D deficiency may increase the risk of disease later in childhood. Further assessment of the long-term health effects of early deficiency is necessary to make appropriate dietary recommendations for infants at risk of deficiency.

Nutrition is an important determinant of bone health and attainment of peak bone mass. Diets containing dried plum (DP) have been shown to increase bone volume and strength. These effects may be linked to the immune system and DP-specific polyphenols. To better understand these relationships, we studied DP in skeletally mature (6-month-old) and growing (1- and 2-month-old) C57Bl/6 male mice. In adult mice, DP rapidly (

Undernourished children exhibit impaired development of their gut microbiota. Transplanting microbiota from 6- and 18-month-old healthy or undernourished Malawian donors into young germ-free mice that were fed a Malawian diet revealed that immature microbiota from undernourished infants and children transmit impaired growth phenotypes. The representation of several age-discriminatory taxa in recipient animals correlated with lean body mass gain; liver, muscle, and brain metabolism; and bone morphology. Mice were cohoused shortly after receiving microbiota from healthy or severely stunted and underweight infants; age- and growth-discriminatory taxa from the microbiota of the former were able to invade that of the latter, which prevented growth impairments in recipient animals. Adding two invasive species, Ruminococcus gnavus and Clostridium symbiosum, to the microbiota from undernourished donors also ameliorated growth and metabolic abnormalities in recipient animals. These results provide evidence that microbiota immaturity is causally related to undernutrition and reveal potential therapeutic targets and agents.

Prior to transplantation, mesenchymal stem/stromal cells (MSCs) can be induced toward the osteoblastic phenotype using a cocktail of soluble supplements. However, there is little evidence of differentiated MSCs directly participating in bone formation, suggesting that MSCs may either die or revert in phenotype upon transplantation. Cell-secreted decellularized extracellular matrices (DMs) are a promising platform to confer bioactivity and direct cell fate through the presentation of a complex and physiologically relevant milieu. Therefore, we examined the capacity of biomimetic DMs to preserve the mineral-producing phenotype upon withdrawal of the induction stimulus. Regardless of induction duration, ranging up to 6 weeks, MSCs exhibited up to a 5-fold reduction in osteogenic markers within 24 h following stimulus withdrawal. We show that seeding osteogenically induced MSCs on DMs yields up to 2-fold more calcium deposition than tissue culture plastic, and this improvement is at least partially mediated by increasing actin cytoskeletal tension via the ROCK II pathway. MSCs on DMs also secreted 25% more vascular endothelial growth factor (VEGF), a crucial endogenous proangiogenic factor that is abrogated during MSC osteogenic differentiation. The deployment of DMs into a subcutaneous ectopic site enhanced the persistence of MSCs 5-fold, vessel density 3-fold, and bone formation 2-fold more than MSCs delivered without DMs. These results underscore the need for deploying MSCs using biomaterial platforms such as DMs to preserve the in vitro-acquired mineral-producing phenotype and accelerate the process of bone repair.

To investigate the role of IGF-I signaling in osterix (OSX)-expressing cells in the skeleton, we generated IGF-I receptor (IGF-IR) knockout mice ((OSX)IGF-IRKO) (floxed-IGF-IR mice × OSX promoter-driven GFP-labeled cre-recombinase [(OSX)GFPcre]), and monitored postnatal bone development. At day 2 after birth (P2), (OSX)GFP-cre was highly expressed in the osteoblasts in the bone surface of the metaphysis and in the prehypertrophic chondrocytes (PHCs) and inner layer of perichondral cells (IPCs). From P7, (OSX)GFP-cre was highly expressed in PHCs, IPCs, cartilage canals (CCs), and osteoblasts (OBs) in the epiphyseal secondary ossification center (SOC), but was only slightly expressed in the OBs in the metaphysis. Compared with the control mice, the IPC proliferation was decreased in the (OSX)IGF-IRKOs. In these mice, fewer IPCs invaded into the cartilage, resulting in delayed formation of the CC and SOC. Immunohistochemistry indicated a reduction of vessel number and lower expression of VEGF and ephrin B2 in the IPCs and SOC of (OSX)IGF-IRKOs. Quantitative real-time PCR revealed that the mRNA levels of the matrix degradation markers, MMP-9, 13 and 14, were decreased in the (OSX)IGF-IRKOs compared with the controls. The (OSX)IGF-IRKO also showed irregular morphology of the growth plate and less trabecular bone in the tibia and femur from P7 to 7 weeks, accompanied by decreased chondrocyte proliferation, altered chondrocyte differentiation, and decreased osteoblast differentiation. Our data indicate that during postnatal bone development, IGF-I signaling in OSX-expressing IPCs promotes IPC proliferation and cartilage matrix degradation and increases ephrin B2 production to stimulate vascular endothelial growth factor (VEGF) expression and vascularization. These processes are required for normal CC formation in the establishment of the SOC. Moreover, IGF-I signaling in the OSX-expressing PHC is required for growth plate maturation and osteoblast differentiation in the development of the metaphysis.

The field of miRNA biology is relatively young, but its impact on our understanding of the regulation of a wide array of cell functions is far-reaching. The importance of miRNAs in development has become nearly ubiquitous, with miRNAs contributing to development of most cells and organs. Although miRNAs are clearly interwoven into known regulatory networks that control cell development, the specific modalities by which they intersect are often quite distinct and cleverly achieved. The frequently emerging theme of feed-back and feed-forward loops to either counterbalance or reinforce the gene programs that they influence is a common thread. Many of these examples of miRNAs as developmental regulators are presently found in organs with different miRNAs and targets, whereas novel, unexpected themes emerge in the context of mouse development as we learn more about this rapidly developing area of biology.

Advances in tissue engineering have offered new opportunities to restore anatomically and functionally compromised tissues. Although traditional tissue engineering approaches that utilize biomaterials and cells to create tissue constructs for implantation or biomaterials as a scaffold to deliver cells are promising, strategies that can activate endogenous cells to promote tissue repair are more clinically attractive. Here, we demonstrate that an engineered injectable matrix mimicking a calcium phosphate (CaP)-rich bone-specific microenvironment can recruit endogenous cells to form bone tissues in vivo. Comparison of matrix alone with that of bone marrow-soaked or bFGF-soaked matrix demonstrates similar extent of neo-bone formation and bridging of decorticated transverse processes in a posterolateral lumbar fusion rat model. Synthetic biomaterials that stimulate endogenous cells without the need for biologics to assist tissue repair could circumvent limitations associated with conventional tissue engineering approaches, including ex vivo cell processing and laborious efforts, thereby accelerating the translational aspects of regenerative medicine.

The primary goal of this study was to determine whether the IGF1R in mature osteoblasts and osteocytes was required for the catabolic actions of continuous parathyroid hormone (cPTH). Igf1r was deleted from male and female FVN/B mice by breeding with mice expressing cre recombinase under control of the osteocalcin promoter ((0CN) Igfr1(-/-) ). Littermates lacking the cre recombinase served as controls. PTH, 60 μg/kg/d, was administered continuously by Alzet minipumps for 4 weeks. Blood was obtained for indices of calcium metabolism. The femurs were examined by micro-computed tomography for structure, immunohistochemistry for IGF1R expression, histomorphometry for bone formation rates (BFR), mRNA levels by qPCR, and bone marrow stromal cell cultures (BMSC) for alkaline phosphatase activity (ALP(+) ), mineralization, and osteoblast-induced osteoclastogenesis. Whereas cPTH led to a reduction in trabecular bone volume/tissue volume (BV/TV) and cortical thickness in the control females, no change was found in the control males. Although trabecular BV/TV and cortical thickness were reduced in the (0CN) Igfr1(-/-) mice of both sexes, no further reduction after cPTH was found in the females, unlike the reduction in males. BFR was stimulated by cPTH in the controls but blocked by Igf1r deletion in the females. The (0CN) Igfr1(-/-) male mice showed a partial response. ALP(+) and mineralized colony formation were higher in BMSC from control males than from control females. These markers were increased by cPTH in both sexes, but BMSC from male (0CN) Igfr1(-/-) also were increased by cPTH, unlike those from female (0CN) Igfr1(-/-) . cPTH stimulated receptor activator of NF-κB ligand (RANKL) and decreased osteoprotegerin and alkaline phosphatase expression more in control female bone than in control male bone. Deletion of Igf1r blocked these effects of cPTH in the female but not in the male. However, PTH stimulation of osteoblast-driven osteoclastogenesis was blocked by deleting Igfr1 in both sexes. We conclude that cPTH is catabolic in female but not male mice. Moreover, IGF1 signaling plays a greater role in the skeletal actions of cPTH in the female mouse than in the male mouse, which may underlie the sex differences in the response to cPTH.

Craniosynostosis is the premature fusion of cranial sutures, which can result in progressive cranial deformations, increased intracranial pressure, and restricted brain growth. Most cases of craniosynostosis require surgical reconstruction of the cranial vault with the goal of increasing the intracranial volume and correcting the craniofacial deformities. However, patients often experience rapid post-operative bone regrowth, known as re-synostosis, which necessitates additional surgical intervention. Bone morphogenetic protein (BMP) inhibitors have tremendous potential to treat re-synostosis, but the realization of a clinically viable inhibitor-based therapeutic requires the development of a delivery vehicle that can localize the release to the site of administration. Here, we present an in situ rapidly crosslinking injectable hydrogel that has the properties necessary to encapsulate co-administered proteins and demonstrate that the delivery of rmGremlin1 via our hydrogel system delays bone regrowth in a weanling mouse model of re-synostosis. Our hydrogel is composed of two mutually reactive poly(ethylene glycol) macromolecules, which when mixed crosslink via a bio-orthogonal Cu free click reaction. Hydrogels containing Gremlin caused a dose dependent inhibition of bone regrowth. In addition to craniofacial applications, our injectable click hydrogel has the potential to provide customizable protein, small molecule, and cell delivery to any site accessible via needle or catheter.

In convergent evolution, similar phenotypes evolve repeatedly in independent populations, often reflecting adaptation to similar environments. Understanding whether convergent evolution proceeds via similar or different genetic and developmental mechanisms offers insight towards the repeatability and predictability of evolution. Oceanic populations of threespine stickleback fish, Gasterosteus aculeatus, have repeatedly colonized countless freshwater lakes and streams, where new diets lead to morphological adaptations related to feeding. Here, we show that heritable increases in branchial bone length have convergently evolved in two independently derived freshwater stickleback populations. In both populations, an increased bone growth rate in juveniles underlies the convergent adult phenotype, and one population also has a longer cartilage template. Using F2 crosses from these two freshwater populations, we show that two quantitative trait loci (QTL) control branchial bone length at distinct points in development. In both populations, a QTL on chromosome 21 controls bone length throughout juvenile development, and a QTL on chromosome 4 controls bone length only in adults. In addition to these similar developmental profiles, these QTL show similar chromosomal locations in both populations. Our results suggest that sticklebacks have convergently evolved longer branchial bones using similar genetic and developmental programmes in two independently derived populations.

ObjectiveTo assess skeletal maturity by measuring bone age (BA) in children with Cushing syndrome (CS) before and 1-year after transsphenoidal surgery or adrenalectomy, and to correlate BA with hormone levels and other measurements.Study designThis case series conducted at the National Institutes of Health Clinical Center included 93 children with Cushing disease (CD) (43 females; mean age, 12.3 ± 2.9 years) and 31 children with adrenocorticotropic hormone-independent CS (AICS) (22 females, mean age 10.3 ± 4.5 years). BA was obtained before surgery and at follow-up. Outcome measures were comparison of BA in CD vs AICS and analysis of the effects of hypercortisolism, insulin excess, body mass index, and androgen excess on BA.ResultsTwenty-six of the 124 children (21.0%) had advanced BA, compared with the expected general population prevalence of 2.5% (P < .0001). Only 4 of 124 (3.2%) had delayed BA. The majority of children (76%) had normal BA. The average BA z-score was similar in the children with CD and those with AICS (0.6 ± 1.4 vs 0.5 ± 1.8; P = .8865). Body mass index SDS and normalized values of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androsteonedione, estradiol, and testosterone were all significantly higher in the children with advanced BA vs those with normal or delayed BA. Fifty-nine children who remained in remission from CD had follow-up BA 1.2 ± 0.3 years after transsphenoidal surgery, demonstrating decreased BA z-score (1.0 ± 1.6 vs 0.3 ± 1.4; P < .0001).ConclusionContrary to common belief, endogenous CS in children appears to be associated with normal or even advanced skeletal maturation. When present, BA advancement in CS is related to obesity, insulin resistance, and elevated adrenal androgen levels and aromatization. This finding may have significant implications for treatment decisions and final height predictions in these children.

ObjectivesThe present study sought to determine the effects of decreased peripheral sensory nerve function on skeletal development and bone metabolism in mice.MethodsC57BL/6 neonatal mice were treated with capsaicin to induce peripheral sensory nerve degeneration, and compared to vehicle-treated controls at 4, 8 and 12 weeks of age. Changes in bone structure were assessed using micro-computed tomography, mechanical properties and fracture resistance were assessed using three-point bending of radii, and bone turnover was assessed using dynamic histomorphometry and serum biomarkers.ResultsCapsaicin treatment resulted in small but significant decreases in bone structure, particularly affecting trabecular bone. Capsaicin-treated mice exhibited lower trabecular thickness at the femoral metaphysis and L5 vertebral body compared with vehicle-treated mice. However, capsaicin- and vehicle-treated mice had similar mechanical properties and bone turnover rates.ConclusionNeonatal capsaicin treatment affected trabecular bone during development; however these small changes may not be meaningful with respect to bone strength under normal loading conditions. It is possible that capsaicin-sensitive neurons may be more important for bone under stress conditions such as increased mechanical loading or injury. Future studies will investigate this potential role of peripheral sensory nerves in bone adaptation.

BackgroundTo determine the prevalence and severity of bone deficits in a cohort of childhood cancer survivors (CCS) compared to a healthy sibling control group, and the modifiable factors associated with bone deficits in CCS.MethodsCross-sectional study of bone health in 319 CCS and 208 healthy sibling controls. Bone mineral density (BMD) was measured by dual-energy x-ray absorptiometry (DXA). Generalized estimating equations were used to compare measures between CCS and controls. Among CCS, multivariable logistic regression was used to evaluate odds ratios for BMD Z-score ≤ -1.ResultsAll subjects were younger than 18 years of age. Average time since treatment was 10.1 years (range 4.3 - 17.8 years). CCS were 3.3 times more likely to have whole body BMD Z-score ≤ -1 than controls (95% CI: 1.4-7.8; p = 0.007) and 1.7 times more likely to have lumbar spine BMD Z-score ≤ -1 than controls (95% CI: 1.0-2.7; p = 0.03). Among CCS, hypogonadism, lower lean body mass, higher daily television/computer screen time, lower physical activity, and higher inflammatory marker IL-6, increased the odds of having a BMD Z-score ≤ -1.ConclusionsCCS, less than 18 years of age, have bone deficits compared to a healthy control group. Sedentary lifestyle and inflammation may play a role in bone deficits in CCS. Counseling CCS and their caretakers on decreasing television/computer screen time and increasing activity may improve bone health.

BackgroundTo determine the prevalence and severity of bone deficits in a cohort of childhood cancer survivors (CCS) compared to a healthy sibling control group, and the modifiable factors associated with bone deficits in CCS.MethodsCross-sectional study of bone health in 319 CCS and 208 healthy sibling controls. Bone mineral density (BMD) was measured by dual-energy x-ray absorptiometry (DXA). Generalized estimating equations were used to compare measures between CCS and controls. Among CCS, multivariable logistic regression was used to evaluate odds ratios for BMD Z-score ≤ -1.ResultsAll subjects were younger than 18 years of age. Average time since treatment was 10.1 years (range 4.3 - 17.8 years). CCS were 3.3 times more likely to have whole body BMD Z-score ≤ -1 than controls (95% CI: 1.4-7.8; p = 0.007) and 1.7 times more likely to have lumbar spine BMD Z-score ≤ -1 than controls (95% CI: 1.0-2.7; p = 0.03). Among CCS, hypogonadism, lower lean body mass, higher daily television/computer screen time, lower physical activity, and higher inflammatory marker IL-6, increased the odds of having a BMD Z-score ≤ -1.ConclusionsCCS, less than 18 years of age, have bone deficits compared to a healthy control group. Sedentary lifestyle and inflammation may play a role in bone deficits in CCS. Counseling CCS and their caretakers on decreasing television/computer screen time and increasing activity may improve bone health.

Augmentation of the peak bone mass (PBM) may be one of the most effective interventions to reduce the risk of developing osteoporosis later in life; however treatments to augment PBM are currently limited. Our study evaluated whether a greater PBM could be achieved either in the progesterone nuclear receptor knockout mice (PRKO) or by using a nuclear progesterone receptor (nPR) antagonist, RU486 in mice. Compared to their wild type (WT) littermates the female PRKO mice developed significantly higher cancellous and cortical mass in the distal femurs, and this was associated with increased bone formation. The high bone mass phenotype was partially reproduced by administering RU486 in female WT mice from 1-3 months of age. Our results suggest that the inhibition of the nPR during the rapid bone growth period (1-3 months) increases osteogenesis, which results in acquisition of higher bone mass. Our findings suggest a crucial role for progesterone signaling in bone acquisition and inhibition of the nPR as a novel approach to augment bone mass, which may have the potential to reduce the burden of osteoporosis.

Augmentation of the peak bone mass (PBM) may be one of the most effective interventions to reduce the risk of developing osteoporosis later in life; however treatments to augment PBM are currently limited. Our study evaluated whether a greater PBM could be achieved either in the progesterone nuclear receptor knockout mice (PRKO) or by using a nuclear progesterone receptor (nPR) antagonist, RU486 in mice. Compared to their wild type (WT) littermates the female PRKO mice developed significantly higher cancellous and cortical mass in the distal femurs, and this was associated with increased bone formation. The high bone mass phenotype was partially reproduced by administering RU486 in female WT mice from 1-3 months of age. Our results suggest that the inhibition of the nPR during the rapid bone growth period (1-3 months) increases osteogenesis, which results in acquisition of higher bone mass. Our findings suggest a crucial role for progesterone signaling in bone acquisition and inhibition of the nPR as a novel approach to augment bone mass, which may have the potential to reduce the burden of osteoporosis.

Cornelia de Lange Syndrome (CdLS) is a multi-organ system birth defects disorder linked, in at least half of cases, to heterozygous mutations in the NIPBL gene. In animals and fungi, orthologs of NIPBL regulate cohesin, a complex of proteins that is essential for chromosome cohesion and is also implicated in DNA repair and transcriptional regulation. Mice heterozygous for a gene-trap mutation in Nipbl were produced and exhibited defects characteristic of CdLS, including small size, craniofacial anomalies, microbrachycephaly, heart defects, hearing abnormalities, delayed bone maturation, reduced body fat, behavioral disturbances, and high mortality (75-80%) during the first weeks of life. These phenotypes arose despite a decrease in Nipbl transcript levels of only approximately 30%, implying extreme sensitivity of development to small changes in Nipbl activity. Gene expression profiling demonstrated that Nipbl deficiency leads to modest but significant transcriptional dysregulation of many genes. Expression changes at the protocadherin beta (Pcdhb) locus, as well as at other loci, support the view that NIPBL influences long-range chromosomal regulatory interactions. In addition, evidence is presented that reduced expression of genes involved in adipogenic differentiation may underlie the low amounts of body fat observed both in Nipbl+/- mice and in individuals with CdLS.

Cornelia de Lange Syndrome (CdLS) is a multi-organ system birth defects disorder linked, in at least half of cases, to heterozygous mutations in the NIPBL gene. In animals and fungi, orthologs of NIPBL regulate cohesin, a complex of proteins that is essential for chromosome cohesion and is also implicated in DNA repair and transcriptional regulation. Mice heterozygous for a gene-trap mutation in Nipbl were produced and exhibited defects characteristic of CdLS, including small size, craniofacial anomalies, microbrachycephaly, heart defects, hearing abnormalities, delayed bone maturation, reduced body fat, behavioral disturbances, and high mortality (75-80%) during the first weeks of life. These phenotypes arose despite a decrease in Nipbl transcript levels of only approximately 30%, implying extreme sensitivity of development to small changes in Nipbl activity. Gene expression profiling demonstrated that Nipbl deficiency leads to modest but significant transcriptional dysregulation of many genes. Expression changes at the protocadherin beta (Pcdhb) locus, as well as at other loci, support the view that NIPBL influences long-range chromosomal regulatory interactions. In addition, evidence is presented that reduced expression of genes involved in adipogenic differentiation may underlie the low amounts of body fat observed both in Nipbl+/- mice and in individuals with CdLS.

During development, growth factors and hormones cooperate to establish the unique sizes, shapes and material properties of individual bones. Among these, TGF-beta has been shown to developmentally regulate bone mass and bone matrix properties. However, the mechanisms that control postnatal skeletal integrity in a dynamic biological and mechanical environment are distinct from those that regulate bone development. In addition, despite advances in understanding the roles of TGF-beta signaling in osteoblasts and osteoclasts, the net effects of altered postnatal TGF-beta signaling on bone remain unclear. To examine the role of TGF-beta in the maintenance of the postnatal skeleton, we evaluated the effects of pharmacological inhibition of the TGF-beta type I receptor (TbetaRI) kinase on bone mass, architecture and material properties. Inhibition of TbetaRI function increased bone mass and multiple aspects of bone quality, including trabecular bone architecture and macro-mechanical behavior of vertebral bone. TbetaRI inhibitors achieved these effects by increasing osteoblast differentiation and bone formation, while reducing osteoclast differentiation and bone resorption. Furthermore, they induced the expression of Runx2 and EphB4, which promote osteoblast differentiation, and ephrinB2, which antagonizes osteoclast differentiation. Through these anabolic and anti-catabolic effects, TbetaRI inhibitors coordinate changes in multiple bone parameters, including bone mass, architecture, matrix mineral concentration and material properties, that collectively increase bone fracture resistance. Therefore, TbetaRI inhibitors may be effective in treating conditions of skeletal fragility.

During development, growth factors and hormones cooperate to establish the unique sizes, shapes and material properties of individual bones. Among these, TGF-beta has been shown to developmentally regulate bone mass and bone matrix properties. However, the mechanisms that control postnatal skeletal integrity in a dynamic biological and mechanical environment are distinct from those that regulate bone development. In addition, despite advances in understanding the roles of TGF-beta signaling in osteoblasts and osteoclasts, the net effects of altered postnatal TGF-beta signaling on bone remain unclear. To examine the role of TGF-beta in the maintenance of the postnatal skeleton, we evaluated the effects of pharmacological inhibition of the TGF-beta type I receptor (TbetaRI) kinase on bone mass, architecture and material properties. Inhibition of TbetaRI function increased bone mass and multiple aspects of bone quality, including trabecular bone architecture and macro-mechanical behavior of vertebral bone. TbetaRI inhibitors achieved these effects by increasing osteoblast differentiation and bone formation, while reducing osteoclast differentiation and bone resorption. Furthermore, they induced the expression of Runx2 and EphB4, which promote osteoblast differentiation, and ephrinB2, which antagonizes osteoclast differentiation. Through these anabolic and anti-catabolic effects, TbetaRI inhibitors coordinate changes in multiple bone parameters, including bone mass, architecture, matrix mineral concentration and material properties, that collectively increase bone fracture resistance. Therefore, TbetaRI inhibitors may be effective in treating conditions of skeletal fragility.

This commentary is a concise discussion of the interactions between bone morphogenetic proteins (BMPs) and their binding proteins in bone and cartilage morphogenesis. BMPs are a family of growth and differentiation factors, and they act on mesenchymal cells to induce cartilage and bone differentiation in concentration-dependent thresholds. The BMP-BMP receptor binding leads to a cascade of signaling and transcription of BMP response genes. BMP binding proteins, noggin, chordin and DAN, act as antagonists and determine the bioavailability of BMPs for binding to cognate receptors to elicit the biological response. Noggin null mice with unrestricted action of BMPs exhibit defects in joint morphogenesis. BMPs and their binding proteins may reciprocally regulate the dynamic topography of joints, muscle, tendons and ligaments during morphogenesis of the skeleton. In addition, BMP actions may be potentiated by twisted gastrulation. BMPs and their binding proteins may play a critical role in regeneration of cartilage in osteoarthritis.

This commentary is a concise discussion of the interactions between bone morphogenetic proteins (BMPs) and their binding proteins in bone and cartilage morphogenesis. BMPs are a family of growth and differentiation factors, and they act on mesenchymal cells to induce cartilage and bone differentiation in concentration-dependent thresholds. The BMP-BMP receptor binding leads to a cascade of signaling and transcription of BMP response genes. BMP binding proteins, noggin, chordin and DAN, act as antagonists and determine the bioavailability of BMPs for binding to cognate receptors to elicit the biological response. Noggin null mice with unrestricted action of BMPs exhibit defects in joint morphogenesis. BMPs and their binding proteins may reciprocally regulate the dynamic topography of joints, muscle, tendons and ligaments during morphogenesis of the skeleton. In addition, BMP actions may be potentiated by twisted gastrulation. BMPs and their binding proteins may play a critical role in regeneration of cartilage in osteoarthritis.

Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737-744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.

Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737-744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.