Bone Mineral Homeostasis Research Articles

Michael Kleerekoper (August 18, 1944 to May 10, 2018): In Relentless Pursuit of a Better Way.

Michael Kleerekoper (August 18, 1944 to May 10, 2018): In Relentless Pursuit of a Better Way.

Aortic vascular calcification is inversely associated with the trabecular bone score in patients receiving dialysis.

Progressive chronic kidney disease (CKD) confers a marked increase in risk for vascular calcification, cardiovascular disease, fracture and mortality, with likely contributing factors including dysregulated bone metabolism and mineral homeostasis. In general population studies, increased vascular calcification is directly related to mortality and inversely related to bone mineral density (BMD) measured by dual-energy X-ray absorptiometry (DXA). In patients with CKD, abnormalities in turnover, mineralization and bone volume reduce the ability of DXA to predict fracture. The trabecular bone score (TBS) obtained from lumbar spine DXA images, provides a surrogate measure of microarchitectural integrity not captured by BMD. This study aimed to examine the association of the TBS to prevalent abdominal aortic calcification (AAC) in patients with CKD receiving dialysis. We performed a cross-sectional study of dialysis patients awaiting transplantation. All patients underwent laboratory testing, lateral spinal radiographs including the abdominal aorta, DXA imaging and TBS assessment. AAC scores were determined using the Kauppila method. Correlations and linear regression models were used to determine predictors of AAC scores. 146 patients (60% male, mean age 48 ± 13 years) were included, of whom 49% had prevalent calcification with an AAC score ≥ 1. Of those with calcification, the mean AAC score was 7 ± 5.5 and 42 patients had scores ≥ 6, considered to indicate severe AAC. TBS values corresponding to intermediate or high risk for fracture (<1.31) were present in 35% of patients. TBS values correlated inversely to AAC scores (β = -0.206, p = 0.013) and remained significant in multivariable linear regression, adjusting for age, BMI and time on dialysis (-0.160, p = 0.031). There was no significant correlation of AAC scores to any BMD parameter. There is a high prevalence of AAC in relatively young dialysis patients awaiting transplantation and their AAC scores are inversely related to the TBS but not to DXA-derived BMD parameters. In patients with CKD on dialysis, TBS assessment reflects microarchitectural abnormalities of bone not captured by DXA. The inverse relationship of TBS to vascular calcification may provide insights into bone-vascular interactions in CKD.

The vitamin D hormone and its nuclear receptor: molecular actions and disease states.

Vitamin D plays a major role in bone mineral homeostasis by promoting the transport of calcium and phosphate to ensure that the blood levels of these ions are sufficient for the normal mineralization of type I collagen matrix in the skeleton. In contrast to classic vitamin D-deficiency rickets, a number of vitamin D-resistant rachitic syndromes are caused by acquired and hereditary defects in the metabolic activation of the vitamin to its hormonal form, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), or in the subsequent functions of the hormone in target cells. The actions of 1,25(OH)2D3 are mediated by the nuclear vitamin D receptor (VDR), a phosphoprotein which binds the hormone with-high affinity and regulates the expression of genes via zinc finger-mediated DNA binding and protein-protein interactions. In hereditary hypocalcemic vitamin D-resistant rickets (HVDRR), natural mutations in human VDR that confer patients with tissue insensitivity to 1,25(OH)2D3 are particularly instructive in revealing VDR structure function relationships. These mutations fall into three categories: (i) DNA binding/nuclear localization, (ii) hormone binding and (iii) heterodimerization with retinoid X receptors (RXRs). That all three classes of VDR mutations generate the HVDRR phenotype is consistent with a basic model of the active receptor as a DNA-bound, 1,25(OH)2D3-liganded heterodimer of VDR and RXR. Vitamin D responsive elements (VDREs) consisting of direct hexanucleotide repeats with a spacer of three nucleotides have been identified in the promoter regions of positively controlled genes expressed in bone, such as osteocalcin, osteopontin, beta 3-integrin and vitamin D 24-OHase. The 1,25(OH)2D3 ligand promotes VDR-RXR heterodimerization and specific, high affinity VDRE binding, whereas the ligand for RXR, 9-cis retinoic acid (9-cis RA), is capable of suppressing 1,25(OH)2D3-stimulated transcription by diverting RXR to form homodimers. However, initial 1,25(OH)2D3 liganding of a VDR monomer renders it competent not only to recruit RXR into a heterodimer but also to conformationally silence the ability of its RXR partner to bind 9-cis RA and dissociate the heterodimer. Additional probing of protein-protein interactions has revealed that VDR also binds to basal transcription factor IIB (TFIIB) and, in the presence of 1,25(OH)2D3, an RXR-VDR-TFIIB ternary complex can be created in solution. Moreover, for transcriptional activation by 1,25(OH)2D3, both VDR and RXR require an intact short amphipathic alpha-helix, known as AF-2, positioned at their extreme C-termini. Because the AF-2 domains participate neither in VDR-RXR heterodimerization nor in TFIIB association, it is hypothesized that they contact, in a ligand-dependent fashion, transcriptional coactivators such as those of the steroid receptor coactivator family, constituting yet a third protein-protein interaction for VDR. Therefore, in VDR-mediated transcriptional activation, 1,25(OH)2D3 binding to VDR alters the conformation of the ligand binding domain such that it: (i) engages in strong heterodimerization with RXR to facilitate VDRE binding, (ii) influences the RXR ligand binding domain such that it is resistant to the binding of 9-cis RA but active in recruiting coactivator to its AF-2 and (iii) presents the AF-2 region in VDR for coactivator association. The above events, including bridging by coactivators to the TATA binding protein and associated factors, may position VDR such that it is able to attract TFIIB and the balance of the RNA polymerase II transcription machinery, culminating in repeated transcriptional initiation of VDRE-containing, vitamin D target genes. Such a model would explain the action of 1,25(OH)2D3 to elicit bone remodeling by stimulating osteoblast and osteoclast precursor gene expression, while concomitantly triggering the termination of its hormonal signal by inducing the 24-OHase catabolizing enzyme.

Assessment of Novel Vitamin D Receptor Antagonists that Mediate Suppression of Vitamin D Signaling

Antagonists are chemical compounds that bind to receptors without eliciting activation of signaling, therefore the biochemical response to the receptor ligand can be blocked or significantly decreased. Receptor antagonists display varying levels of binding affinity but do not produce the agonist‐mediated response upon binding. The vitamin D receptor (VDR) binds with high affinity to its endocrine agonist, 1,25‐dihydroxyvitamin D3 (1,25D) to regulate the expression of a suite of genes in target tissues including kidney, intestine, and bone to control biological processes such as calcium and phosphate bone mineral homeostasis. In conditions of VDR hyperactivity, excess vitamin D production, or significantly increased intake via diet or supplementation, there are risks associated with potential resultant hypercalcemia including kidney stones, GI dysfunction, behavioral changes such as depression, and cardiac problems. In the present study, we synthesized novel VDR antagonists to create synthetic ligands that have high affinity for VDR, and consequently inhibit the activation of 1,25D‐VDR regulated genes that lead to hypercalcemia. It is well established that agonist‐bound VDR forms a functional DNA‐binding heterodimer with the retinoid X receptor (RXR) that associates with vitamin D responsive elements (VDREs) to either induce or repress transcription. Therefore, we exploited this agonist‐dependent VDR‐RXR heterodimerization pathway as an initial screening assay to evaluate the efficacy of our putative VDR antagonists. The initial test employed the mammalian two‐hybrid assay (M2H), which can assess if the novel antagonists compete with 1,25D and bind to VDR to prevent the formation of a heterodimer with RXR, a prerequisite step in VDR signaling. A second assay utilized VDRE‐linked luciferase reporter plasmids to measure antagonist activity in the more natural environment of the VDRE DNA platform. This assay was followed by a third screen to determine the specificity of antagonist binding to VDR versus other closely related receptors in the nuclear receptor superfamily. Results from these assays revealed that two novel analogs bind as bona fide antagonists to the VDR, albeit with different affinity. Our findings also suggest that additional chemical modification of antagonists may result in analogs with enhanced ability to suppress 1,25D‐VDR activation. Thus, this study further broadens our understanding of VDR‐ligand binding interactions, and may facilitate the development of novel therapeutic compounds with the potential for clinical applications in treating VDR‐directed hypercalcemia.Support or Funding InformationSchool of Mathematical and Natural Sciences at Arizona State UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Accuracy of MRI-based finite element assessment of distal tibia compared to mechanical testing.

High-resolution MRI-derived finite element analysis (FEA) has been used in translational research to estimate the mechanical competence of human bone. However, this method has yet to be validated adequately under in vivo imaging spatial resolution or signal-to-noise conditions. We therefore compared MRI-based metrics of bone strength to those obtained from direct, mechanical testing. The study was conducted on tibiae from 17 human donors (12 males and five females, aged 33 to 88years) with no medical history of conditions affecting bone mineral homeostasis. A 25mm segment from each distal tibia underwent MR imaging in a clinical 3-Tesla scanner using a fast large-angle spin-echo (FLASE) sequence at 0.137mm×0.137mm×0.410mm voxel size, in accordance with in vivo scanning protocol. The resulting high-resolution MR images were processed and used to generate bone volume fraction maps, which served as input for the micro-level FEA model. Simulated compression was applied to compute stiffness, yield strength, ultimate strength, modulus of resilience, and toughness, which were then compared to metrics obtained from mechanical testing. Moderate to strong positive correlations were found between computationally and experimentally derived values of stiffness (R2=0.77, p<0.0001), yield strength (R2=0.38, p=0.0082), ultimate strength (R2=0.40, p=0.0067), and resilience (R2=0.46, p=0.0026), but only a weak, albeit significant, correlation was found for toughness (R2=0.26, p=0.036). Furthermore, experimentally derived yield strength and ultimate strength were moderately correlated with MRI-derived stiffness (R2=0.48, p=0.0022 and R2=0.58, p=0.0004, respectively). These results suggest that high-resolution MRI-based finite element (FE) models are effective in assessing mechanical parameters of distal skeletal extremities.

Reduced kidney function is associated with BMD, bone loss and markers of mineral homeostasis in older women: a 10-year longitudinal study

SummaryKidney function decreases with age; however, the long-term influence on bone density (BMD) in older women already at risk of osteoporosis is unknown. We followed kidney function and bone loss for 10 years. Declining kidney function was adversely associated with bone loss and mineral homeostasis in old women, though it attenuated with advanced aging.IntroductionExisting studies do not fully address the relationship between kidney function and bone metabolism with advanced aging in Caucasian women. This study describes the association between kidney function, BMD, bone loss and bone metabolism in older women and provides a review of the available literature for context.MethodsWe studied participants from the OPRA cohort with follow-up after 5 and 10 years. Using plasma cystatin C (cysC), estimated glomerular function rate (eGFR) was evaluated at age 75 (n = 981), 80 (n = 685) and 85 (n = 365). Women were stratified into “normal” function (CKD stages 1–2), “intermediate” (stage 3a) and “poor” (stages 3b–5), and outcome measures—BMD, bone loss and markers of mineral homeostasis—were compared.ResultsFemoral neck (FN) BMD positively associated with kidney function at 75 years old ( widehat{beta} = 0.001, p = 0.028) and 80 years old ( widehat{beta} = 0.001, p = 0.001), although with small effect size. Prevalence of osteoporosis (FN T-score ≤ − 2.5) did not differ with kidney function. Measured at age 75, women with poor kidney function had higher annual percentage bone loss over 5 years compared to those with normal function (2.3%, 95% CI 1.8–2.8 versus 1.3%, 95% CI 1.1–1.5, p = 0.007), although not when measured from age 80 or 85. Additionally, markers of mineral homeostasis (PTH, phosphate, vitamin D, calcium), CRP and osteocalcin differed by kidney function.ConclusionsIn old women, kidney function is associated with BMD, bone loss and altered mineral homeostasis; probably, a relationship attenuated in the very elderly.

PTH Reloaded: A New Evolutionary Perspective.

The parathyroid hormone (PTH) family is a group of structurally-related secreted peptides involved in bone mineral homeostasis and multitude of developmental processes in vertebrates. These peptides mediate actions through PTH receptors (PTHRs), which belong to the transmembrane G protein-coupled receptor group. To date, genes encoding for PTH and PTHR have only been identified in chordates, suggesting that this signaling pathway may be an evolutionary innovation of our phylum. In vertebrates, we found up to six PTH and three PTHR different paralogs, varying in number between mammals and teleost fishes due to the different rounds of whole-genome duplication and specific gene losses suffered between the two groups of animals. The diversification of the PTH gene family has been accompanied by both functional divergence and convergence, making sometimes difficult the comparison between PTH peptides of teleosts and mammals. Here, we review the roles of all Pth peptides in fishes, and based on the evolutionary history of PTH paralogs, we propose a new and simple nomenclature from PTH1 to PTH4. Moreover, the recent characterization of the Pth4 in zebrafish allows us to consider the prominent role of the brain-to-bone signaling pathway in the regulation of bone development and homeostasis. Finally, comparison between PTH peptides of fish and mammals allows us to discuss an evolutionary model for PTH functions related to bone mineral balance during the vertebrate transition from an aquatic to a terrestrial environment.

Effects of the different levels of dietary vitamin D on boar performance and semen quality

Vitamin D plays a major role primarily in bone mineral homeostasis; however, the action of vitamin D on male reproduction of boars remains unknown. This study was conducted to evaluate the effects of dietary vitamin D3 supplementation on reproductive performance of boars. Twenty-four Yorkshire boars with eighteen months were randomly allocated to one of three vitamin D3 levels diets: 200 IU/kg (C), 2000 IU/kg (T1) and 4000 IU/kg (T2) of vitamin D3 for a 16-week period. Results showed that sperm motility, effective sperm number per ejaculate, acrosome intactness ratio, and membrane intactness ratio in T1 group were significantly higher than those in C group. The sperm membrane intactness ratio was higher in T1 group than in T2 group, whereas the deformity ratio was significantly lower in T1 than in C and T2 groups. Dietary supplementation with vitamin D3 significantly increased 25-OHD3 concentration in the blood and seminal plasma, but there was no significant difference in its concentration in the seminal plasma between T1 and T2 groups. The concentrations of blood testosterone and aromatase activity increased following vitamin D supplementation. Seminal plasma Ca2+, fructose concentration, and acid phosphatase activity were higher in T1 group than in C group, but not significantly different from the corresponding values in T2 group. Furthermore, expression of CYP19, CYP24A1, and VDR mRNAs in sperm was up-regulated in T1 group compared to that in C group. In conclusion, supplementation of boar diets with 2000 IU/kg vitamin D3 increased the sperm motility and effective sperm number, which was in line with the elevated concentrations of vitamin D, hormone secretion and gene expression.

Denosumab: an Emerging Therapy in Pediatric Bone Disorders.

Denosumab is an inhibitor of receptor activator of nuclear factor kappa-B ligand (RANKL), and has emerged as an important novel therapy for skeletal disorders. This article examines the use of denosumab in children. Considerable safety and efficacy data exists for denosumab treatment of adults with osteoporosis, bone metastases, and giant cell tumors. Pediatric data is limited; however, evidence suggests denosumab may be beneficial in decreasing bone turnover, increasing bone density, and preventing growth of certain skeletal neoplasms in children. Denosumab's effect on bone turnover is rapidly reversible after drug discontinuation, representing a key difference from bisphosphonates. Rebound increased bone turnover has led to severe hypercalcemia in several pediatric patients. Denosumab is a promising therapy for pediatric skeletal disorders. At present, safety concerns related to rebounding bone turnover and mineral homeostasis impact use of denosumab in children. Research is needed to determine if and how these effects can be mitigated.

Class 3 semaphorins are transcriptionally regulated by 1,25(OH)2D3 in osteoblasts

The vitamin D endocrine system is essential for calcium metabolism and skeletal integrity. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] regulates bone mineral homeostasis and acts directly on osteoblasts. In the present study we characterized the transcriptional regulation of the class 3 semaphorin (Sema3) gene family by 1,25(OH)2D3 in osteoblastic cells. Class 3 semaphorins are secreted proteins that regulate cell growth, morphology and migration, and were recently shown to be involved in bone homeostasis. In ST2, MC3T3-E1 and primary calvarial osteoblast cell cultures we found that all members of the Sema3 gene family were expressed, and that Sema3e and Sema3f were the most strongly induced 1,25(OH)2D3 target genes among the studied cell types. In addition, transcription of Sema3b and Sema3c was upregulated, whereas Sema3d and Sema3g was downregulated by 1,25(OH)2D3 in different osteoblastic cells. Chromatin immunoprecipitation analysis linked to DNA sequencing (ChIP-seq analysis) revealed the presence of the vitamin D receptor at multiple genomic loci in the proximity of Sema3 genes, demonstrating that the genes are primary 1,25(OH)2D3 targets. Furthermore, we showed that recombinant SEMA3E and SEMA3F protein were able to inhibit osteoblast proliferation. However, recombinant SEMA3s did not affect ST2 cell migration. The expression of class 3 semaphorins in osteoblasts together with their regulation by 1,25(OH)2D3 suggests that these genes, involved in the regulation of bone homeostasis, are additional mediators for 1,25(OH)2D3 signaling in osteoblasts.

Vitamin D status and bone turnover marker levels in Greek women with fragility hip fracture

There is little data regarding Vitamin D deficiency in Greece. Also conflicting are the data regarding the bone turnover during the acute hip fracture interval and the situation is even more complex considering the recently developed bone turnover markers (BTMs). The aim of this study was to evaluate the parameters of bone mineral homeostasis of women with fragility hip fracture using the recently developed BTMs of carboxy-terminal collagen crosslinks (CTx) and the procollagen type 1 amino-terminal propeptide (PINP) and to determinate vitamin D (25-OHD) levels of elderly Greek women with acute hip fracture. 38 self-sufficient, community-living Greek women with acute hip fracture were included. A similar number of age- and sex-matched controls with no clinically evident fractures were included in the study meeting the same inclusion and exclusion criteria. To exclude the effect of trauma blood samples were drawn within 24 hours from the fracture. Hip fracture group had significantly lower serum 25-OHD levels and significantly higher intact parathormone (PTH) levels compared to the control group but no correlation between PTH and 25-OHD was found in both groups. CTx was significantly correlated with PINP in the total group and in both groups separately but there was no statistical difference of their levels between the two groups. There was high prevalence of severe Vitamin D deficiency within the postmenopausal Greek women with acute hip fracture and the fracture did not influence the above mentioned BTMs. The processes of bone resorption and bone production are in balance during the first 24 hours after the fracture.

Systematic Review on Vitamin D Level in Apparently Healthy Indian Population and Analysis of Its Associated Factors.

Background:Vitamin D which is involved in the maintenance of bone mineral homeostasis has been found to portray various pleiotropic effects. Although it has been widely accepted that serum 25-hydroxy Vitamin D level above 30 ng/ml is considered optimal for the biological actions of Vitamin D, there is a need to explore the levels of Vitamin D reported among Indians from various regions of the country. Hence, this systematic review aims to appraise the status of Vitamin D levels reported from apparently healthy Indians across various parts of India.Methodology:A comprehensive literature search was carried out to identify the range of Vitamin D levels among apparently healthy individuals from various parts of India, with the search term “Vitamin D and India” in the search portals of PubMed, Google Scholar, Indmed, and ScienceDirect. A total of 2998 articles were retrieved by the above search strategy, of which only forty studies fulfilled the criteria to be included in the systematic review. Studies done in various states were compiled under the respective zones based on the classification of Indian zones as specified in Zonal maps of India.Results:The level of Vitamin D from all the forty included studies ranged from 3.15 ± 1.4 to 52.9 ± 33.7 ng/ml. The effect size of Vitamin D level was higher in the South Zone compared to other zones.Conclusion:The present study shows that Vitamin D deficiency is prevalent among apparently healthy Indians living in different regions of India, irrespective of their exposure to sunlight.

Osteoimmunology and Beyond.

: ObjectiveOsteoimmunology investigates interactions between skeleton and immune system. In the light of recent discoveries in this field, a new reading register of osteoporosis is actually emerging, in which bone and immune cells are strictly interconnected. Osteoporosis could therefore be considered a chronic immune mediated disease which shares with other age related disorders a common inflammatory background. Here, we highlight these recent discoveries and the new landscape that is emerging.MethodExtensive literature search in PubMed central.ResultsWhile the inflammatory nature of osteoporosis has been clearly recognized, other interesting aspects of osteoimmunology are currently emerging. In addition, mounting evidence indicates that the immunoskeletal interface is involved in the regulation of important body functions beyond bone remodeling. Bone cells take part with cells of the immune system in various immunological functions, configuring a real expanded immune system, and are therefore variously involved not only as target but also as main actors in various pathological conditions affecting primarily the immune system, such as autoimmunity and immune deficiencies, as well as in aging, menopause and other diseases sharing an inflammatory background.ConclusionThe review highlights the complexity of interwoven pathways and shared mechanisms of the crosstalk between the immune and bone systems. More interestingly, the interdisciplinary field of osteoimmunology is now expanding beyond bone and immune cells, defining new homeostatic networks in which other organs and systems are functionally interconnected. Therefore, the correct skeletal integrity maintenance may be also relevant to other functions outside its involvement in bone mineral homeostasis, hemopoiesis and immunity.

Pth4, an ancient parathyroid hormone lost in eutherian mammals, reveals a new brain-to-bone signaling pathway.

Regulation of bone development, growth, and remodeling traditionally has been thought to depend on endocrine and autocrine/paracrine modulators. Recently, however, brain-derived signals have emerged as key regulators of bone metabolism, although their mechanisms of action have been poorly understood. We reveal the existence of an ancient parathyroid hormone (Pth)4 in zebrafish that was secondarily lost in the eutherian mammals' lineage, including humans, and that is specifically expressed in neurons of the hypothalamus and appears to be a central neural regulator of bone development and mineral homeostasis. Transgenic fish lines enabled mapping of axonal projections leading from the hypothalamus to the brainstem and spinal cord. Targeted laser ablation demonstrated an essential role for of pth4-expressing neurons in larval bone mineralization. Moreover, we show that Runx2 is a direct regulator of pth4 expression and that Pth4 can activate cAMP signaling mediated by Pth receptors. Finally, gain-of-function experiments show that Pth4 can alter calcium/phosphorus levels and affect expression of genes involved in phosphate homeostasis. Based on our discovery and characterization of Pth4, we propose a model for evolution of bone homeostasis in the context of the vertebrate transition from an aquatic to a terrestrial lifestyle.-Suarez-Bregua, P., Torres-Nuñez, E., Saxena, A., Guerreiro, P., Braasch, I., Prober, D. A., Moran, P., Cerda-Reverter, J. M., Du, S. J., Adrio, F., Power, D. M., Canario, A. V. M., Postlethwait, J. H., Bronner, M E., Cañestro, C., Rotllant, J. Pth4, an ancient parathyroid hormone lost in eutherian mammals, reveals a new brain-to-bone signaling pathway.

Changes in body composition of Indian lactating women: a longitudinal study.

Lactation places enormous demands on maternal bone mineral homeostasis. Indian middle class women (MSC) consume energy dense food supplements to meet these demands post-partum (PP) along with restricted physical activity (PA). Effects of these changes on body composition (BC) of PP women have not been studied. To examine longitudinal changes in: a) bone mineral density (BMD) at total body (TB), AP-spine (APS) and dual femur neck regions (DF) b) BC by body weight, lean mass, fat mass using dual energy X-ray absorptiometry (DXA) at baseline, 6-months and 1-year in urban MSC women. 76-primi-parous (28±3.2 yrs) randomly selected PP women (<7-days) were studied; 70 reassessed at 6- months and 42 1-yr PP. Data on anthropometry, BC, BMD at TB, APS and DF by DXA collected (baseline, 6- months, 1-yr PP). Weight, waist and body mass index (BMI) decreased both at 6-month and 1-yr PP with respect to baseline (p<0.05). BC changes showed increase in android fat % at 1-yr by 10% over baseline (p<0.05). BMD with initial decline at 6-months (-2.8%, -2.3% and -2.3% respectively) recovered partially by 1-yr (+2.5% +1.2% and +4.8% respectively) at DF and TB with complete recovery at APS (p<0.05). These urban relatively sedentary MSC women consumed fat rich food PP with higher android fat retention and partial recovery of BMD at DF and TB at 1-year. Modifications in activity and dietary nutrient intakes may be necessary to prevent cardiovascular and bone health related risks.

Minerals and extremely low birth weight infants

The 80% of bone mineralisation occurs in the third trimester of pregnancy. The daily fetal requirement is 310 mg of calcium and 170 mg of phosphorus[1]. The bone mineral density (BMD) correlates positively with gestational age, weight and length[2]. At birth, extremely premature infants (ELBWI)canpresent hypocalcaemiabecause of interruption of the maternal calcium supply, high level of calcitoninand immature response to PTH. Early parenteral nutrition solutionisnecessary to maintain normal serum calcium and phosphorus levels. Indeed, the exclusive use of dextrose and calcium gluconate leads to thehypophosphatemia, deleterious to intermediary metabolism, intracellular energy transfer and bone mineral homeostasis[3]. The ELBWI are at risk of Osteopenia of prematurity, also known as neonatal metabolic bone disease (MBD). The pathophysiology includesreduction in organic protein matrix (osteopenia) and/or a reduction in mineral component (osteomalacia). MBD causes rickets, fractures and may affect growth in infancy and childhood. The structural basis of osteopenia is decreased thickness or number of trabeculae and/or decreased thickness of the bone cortex. Osteomalaciais a disorder of the physiological process of mineralisation, when the incorporation of mineral into the organic bone matrix is disturbed, whereas “rickets” describes defective mineralisation of growth plate cartilage and its morphological consequences[4]. The ethiology is multifactorial: placental dysfunction, prolonged (>4 weeks) parenteral nutrition, diureticsand steroids treatment, immobilization and inadequate nutrient intake of calcium, phosphorus, and vitamin D[5]. The main ethiology factor is deficit of mineral substrates, whereas the critical factor is lack of phosphorus[4]. It should be needed an adequate mineral supplementation with parenteral and enteral nutrition, although the difficulties to fortify appropriately the human milk. The dosage recommended of vitamin D is between, 400 IU/d (AAP) and 1000 IU/d (ESPGHAN)[6]. Moreover hypotonia and immobilization are an additional risk factors [7], daily passive exercise canimproves the bone mineral content [8]. MBD could be detected by X-Ray, dual-energy x-ray absorptiometry (DEXA) or quantitative ultrasound. The serumdiagnostic markers are alkaline phosphatases, calcium and phosphateserum levels. Preterm infants with low serum phosphate (<2 mmol/l) are at risk of MBD and levels less than 1.8 mmol/l have been strongly associated with the presence of radiographically evident rickets. Because of preterm has a much lower renal phosphate threshold and this is not bound in the plasma like calcium, the tubular reabsorption is the best guide to calculate itssupplementation[8].

The role of the sphingosine-1-phosphate signaling pathway in osteocyte mechanotransduction

Osteocytes are proposed to be the mechanosensory cells that translate mechanical loading into biochemical signals during the process of bone adaptation. The lipid mediator sphingosine-1-phosphate (S1P) has been reported to play a role in the mechanotransduction process of blood vessels and also in the dynamic control of bone mineral homeostasis. Nevertheless, the potential role of S1P in bone mechanotransduction has yet to be elucidated. In this study, we hypothesized that a S1P cascade is involved in the activation of osteocytes in response to loading-induced oscillatory fluid flow (OFF) in bone. MLO-Y4 osteocyte-like cells express the necessary components of a functional S1P cascade. To examine the involvement of S1P signaling in osteocyte mechanotransduction, we applied OFF (1Pa, 1Hz) to osteocyte-like MLO-Y4 cells under conditions where the S1P signaling pathway was modulated. We found that decreased endogenous S1P levels significantly suppressed the OFF-induced intracellular calcium response. Addition of extracellular S1P to MLO-Y4 cells enhanced the synthesis and release of prostaglandin E2 (PGE2) under static cells and amplified OFF-induced PGE2 release. The stimulatory effect of OFF on the gene expression levels of osteoprotegerin (OPG) and receptor activator for nuclear factor κB ligand (RANKL) was S1P dependent. Furthermore, the S1P2 receptor subtype was shown to be involved in OFF-induced PGE2 synthesis and release, as well as down-regulation of RANKL/OPG gene expression ratio. In summary, our data suggest that S1P cascade is involved in OFF-induced mechanotransduction in MLO-Y4 cells and that extracellular S1P exerts its effect partly through S1P2 receptors.

UV-activated 7-dehydrocholesterol-coated titanium implants promote differentiation of human umbilical cord mesenchymal stem cells into osteoblasts.

Vitamin D metabolites are essential for bone regeneration and mineral homeostasis. The vitamin D precursor 7-dehydrocholesterol can be used after UV irradiation to locally produce active vitamin D by osteoblastic cells. Furthermore, UV-irradiated 7-dehydrocholesterol is a biocompatible coating for titanium implants with positive effects on osteoblast differentiation. In this study, we examined the impact of titanium implants surfaces coated with UV-irradiated 7-dehydrocholesterol on the osteogenic differentiation of human umbilical cord mesenchymal stem cells. First, the synthesis of cholecalciferol (D3) was achieved through the incubation of the UV-activated 7-dehydrocholesterol coating for 48 h at 23℃. Further, we investigated invitro the biocompatibility of this coating in human umbilical cord mesenchymal stem cells and its potential to enhance their differentiation towards the osteogenic lineage. Human umbilical cord mesenchymal stem cells cultured onto UV-irradiated 7-dehydrocholesterol-coated titanium implants surfaces, combined with osteogenic supplements, upregulated the gene expression of several osteogenic markers and showed higher alkaline phosphatase activity and calcein blue staining, suggesting increased mineralization. Thus, our results show that the use of UV irradiation on 7-dehydrocholesterol -treated titanium implants surfaces generates a bioactive coating that promotes the osteogenic differentiation of human umbilical cord mesenchymal stem cells, with regenerative potential for improving osseointegration in titanium-based bone anchored implants.

Selective Distal Enhancer Control of the Mmp13 Gene Identified through Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Genomic Deletions

Matrix metalloproteinase 13 (Mmp13, collagenase-3) plays an essential role in bone metabolism and mineral homeostasis. It is regulated by numerous factors, including BMP-2, parathyroid hormone, and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), through transcription factors such as Runt-related transcription factor 2 (RUNX2), CCAAT/enhancer-binding protein β (C/EBPβ), OSX, and vitamin D receptor (VDR). During osteoblast maturation, the basal expression of Mmp13 and its sensitivity to 1,25(OH)2D3 are strikingly increased. In this report, ChIP-sequencing analysis in mouse preosteoblasts revealed that the Mmp13 gene was probably regulated by three major enhancers located -10, -20, and -30 kb upstream of the gene promoter, occupied by activated VDR and prebound C/EBPβ and RUNX2, respectively. Initially, bacterial artificial chromosome clone recombineering and traditional mutagenesis defined binding sites for VDR and RUNX2. We then employed a CRISPR/Cas9 gene editing approach to delete the -10 and -30 kb Mmp13 enhancers, a region proximal to the promoter, and VDR or RUNX2. VDR-mediated up-regulation of Mmp13 transcription was completely abrogated upon removal of the -10 kb enhancer, resulting in a 1,25(OH)2D3-directed repression of Mmp13. Deletion of either the -30 kb enhancer or RUNX2 resulted in a complete loss of basal transcript activity and a ChIP-identified destabilization of the chromatin enhancer environment and factor binding. Whereas enhancer deletions only affected Mmp13 expression, the RUNX2 deletion led to changes in gene expression, a reduction in cellular proliferation, and an inability to differentiate. We conclude that the Mmp13 gene is regulated via at least three specific distal enhancers that display independent activities yet are able to integrate response from multiple signaling pathways in a model of activation and suppression.

Vitamin D deficiency and its predictors in a country with thirteen months of sunshine: the case of school children in central Ethiopia.

Studies examining vitamin D status among children living in sunny climates indicated that children did not receive adequate vitamin D, however, this has not been looked at among children living in Ethiopia. In this study, we determined vitamin D deficiency and its predictors among school children aged 11–18 years, examining circulating 25-hydroxy vitamin D [25(OH)D]. The school-based cross-sectional study was conducted in schools in Adama Town (n = 89) and in rural Adama (n = 85) for a total sample of 174. Students were randomly selected using multi-stage stratified sampling method from both settings. Socioeconomic status of parents and demographic, anthropometric, sun exposure status and blood 25(OH)D levels were obtained. Vitamin D deficiency, defined as circulating levels of 25(OH)D <50 nmol/L, was found in 42% of the entire study participants. Prevalence of deficiency was significantly higher among students in urban setting compared to rural (61.8% vs 21.2%, respectively, p<0.001). After controlling for potential confounders using multivariable logistic regression model, duration of exposure to sunlight, amount of body part exposed to sunlight, place of residence, maternal education, body fatness, having TV/computer at home and socioeconomic status were significant predictors of vitamin D deficiency. The findings suggest that Vitamin D deficiency was prevalent in healthy school children living both in urban and rural areas of a country with abundant year round sunshine providing UVB, with the prevalence of deficiency being significantly higher among urban school children who were less exposed to sunlight. Behaviour change communication to enhance exposure to ultraviolet light is critical to prevent vitamin D deficiency in tropical country like Ethiopia. Further study is required to assess the deleterious effect of its deficiency on bone mineral homeostasis of growing children in Ethiopia during their most critical period of bone development.