Long Bone Mineralization Research Articles

Mineral Metabolism in Children: Interrelation between Vitamin D and FGF23

Fibroblast growth factor 23 (FGF23) was identified at the turn of the century as the long-sought circulating phosphatonin in human pathology. Since then, several clinical and experimental studies have investigated the metabolism of FGF23 and revealed its relevant pathogenic role in various diseases. Most of these studies have been performed in adult individuals. However, the mineral metabolism of the child is, to a large extent, different from that of the adult because, in addition to bone remodeling, the child undergoes a specific process of endochondral ossification responsible for adequate mineralization of long bones' metaphysis and growth in height. Vitamin D metabolism is known to be deeply involved in these processes. FGF23 might have an influence on bones' growth as well as on the high and age-dependent serum phosphate concentrations found in infancy and childhood. However, the interaction between FGF23 and vitamin D in children is largely unknown. Thus, this review focuses on the following aspects of FGF23 metabolism in the pediatric age: circulating concentrations' reference values, as well as those of other major variables involved in mineral homeostasis, and the relationship with vitamin D metabolism in the neonatal period, in vitamin D deficiency, in chronic kidney disease (CKD) and in hypophosphatemic disorders.

Sustained release of recombinant human bone morphogenetic protein-2 combined with stromal vascular fraction cells in promoting posterolateral spinal fusion in rat model

To observe the effect of stromal vascular fraction cells (SVFs) from rat fat tissue combined with sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2) in promoting the lumbar fusion in rat model. SVFs were harvested from subcutaneous fat of bilateral inguinal region of 4-month-old rat through the collagenase I digestion. The sustained release carrier was prepared via covalent bond of the rhBMP-2 and β-tricalcium phosphate (β-TCP) by the biominetic apatite coating process. The sustained release effect was measured by BCA method. Thirty-two rats were selected to establish the posterolateral lumbar fusion model and were divided into 4 groups, 8 rats each group. The decalcified bone matrix (DBX) scaffold+PBS, DBX scaffold+rhBMP-2/β-TCP sustained release carrier, DBX scaffold+SVFs, and DBX scaffold+rhBMP-2/β-TCP sustained release carrier+SVFs were implanted in groups A, B, C, and D respectively. X-ray films, manual spine palpation, and high-resolution micro-CT were used to evaluate spinal fusion at 8 weeks after operation; bone mineral density (BMD) and bone volume fraction were analyzed; the new bone formation was evaluated by HE staining and Masson's trichrome staining, osteocalcin (OCN) was detected by immunohistochemical staining. The cumulative release amount of rhBMP-2 was about 40% at 2 weeks, indicating sustained release effect of rhBMP-2; while the control group was almost released within 2 weeks. At 8 weeks, the combination of manual spine palpation, X-ray, and micro-CT evaluation showed that group D had the strongest bone formation (100%, 8/8), followed by group B (75%, 6/8), group C (37.5%, 3/8), and group A (12.5%, 1/8). Micro-CT analysis showed BMD and bone volume fraction were significantly higher in group D than groups A, B, and C ( P<0.05), and in group B than groups A and C ( P<0.05). HE staining, Masson's trichrome staining, and immunohistochemistry staining for OCN staining exhibited a large number of cartilage cells with bone matrix deposition, and an active osteogenic process similar to the mineralization of long bones in group D. The bone formation of group B was weaker than that of group D, and there was no effective new bone formation in groups A and C. The combination of sustained release of rhBMP-2 and freshly SVFs can significantly promote spinal fusion in rat model, providing a theoretical basis for further clinical applications.

Correction: Altered Bone Development and an Increase in FGF-23 Expression in Enpp1−/−Mice

Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is required for the conversion of extracellular ATP into inorganic pyrophosphate (PPi), a recognised inhibitor of hydroxyapatite (HA) crystal formation. A detailed phenotypic assessment of a mouse model lacking NPP1 (Enpp1) was completed to determine the role of NPP1 in skeletal and soft tissue mineralization in juvenile and adult mice. Histopathological assessment of Enpp1 mice at 22 weeks of age revealed calcification in the aorta and kidney and ectopic cartilage formation in the joints and spine. Radiographic assessment of the hind-limb showed hyper-mineralization in the talocrural joint and hypo-mineralization in the femur and tibia. MicroCT analysis of the tibia and femur disclosed altered trabecular architecture and bone geometry at 6 and 22 weeks of age in Enpp1 mice. Trabecular number, trabecular bone volume, structure model index, trabecular and cortical thickness were all significantly reduced in tibiae and femurs from Enpp1 mice (P,0.05). Bone stiffness as determined by 3-point bending was significantly reduced in Enpp1 tibiae and femurs from 22-week-old mice (P,0.05). Circulating phosphate and calcium levels were reduced (P,0.05) in the Enpp1 null mice. Plasma levels of osteocalcin were significantly decreased at 6 weeks of age (P,0.05) in Enpp1 mice, with no differences noted at 22 weeks of age. Plasma levels of CTx (Ratlaps) and the phosphaturic hormone FGF-23 were significantly increased in the Enpp1 mice at 22 weeks of age (P,0.05). Fgf23 messenger RNA expression in cavarial osteoblasts was increased 12-fold in Enpp1 mice compared to controls. These results indicate that Enpp1 mice are characterized by severe disruption to the architecture and mineralization of longbones, dysregulation of calcium/phosphate homeostasis and changes in Fgf-23 expression. We conclude that NPP1 is essential for normal bone development and control of physiological bone mineralization. Citation: Mackenzie NCW, Zhu D, Milne EM, van ’t Hof R, Martin A, et al. (2012) Altered Bone Development and an Increase in FGF-23 Expression in Enpp1 Mice. PLoS ONE 7(2): e32177. doi:10.1371/journal.pone.0032177 Editor: Marlon R. Schneider, University of Munich, Germany Received October 11, 2011; Accepted January 22, 2012; Published February 16, 2012 Copyright: 2012 Mackenzie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an Institute Strategic Programme Grant and Institute Career Path Fellowship funding from the Biotechnology and Biological Sciences Research Council (BBSRC; www.bbsrc.ac.uk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: vicky.macrae@roslin.ed.ac.uk

Altered bone development and an increase in FGF-23 expression in Enpp1(-/-) mice.

Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is required for the conversion of extracellular ATP into inorganic pyrophosphate (PPi), a recognised inhibitor of hydroxyapatite (HA) crystal formation. A detailed phenotypic assessment of a mouse model lacking NPP1 (Enpp1−/−) was completed to determine the role of NPP1 in skeletal and soft tissue mineralization in juvenile and adult mice. Histopathological assessment of Enpp1−/− mice at 22 weeks of age revealed calcification in the aorta and kidney and ectopic cartilage formation in the joints and spine. Radiographic assessment of the hind-limb showed hyper-mineralization in the talocrural joint and hypo-mineralization in the femur and tibia. MicroCT analysis of the tibia and femur disclosed altered trabecular architecture and bone geometry at 6 and 22 weeks of age in Enpp1−/− mice. Trabecular number, trabecular bone volume, structure model index, trabecular and cortical thickness were all significantly reduced in tibiae and femurs from Enpp1−/− mice (P<0.05). Bone stiffness as determined by 3-point bending was significantly reduced in Enpp1−/− tibiae and femurs from 22-week-old mice (P<0.05). Circulating phosphate and calcium levels were reduced (P<0.05) in the Enpp1−/− null mice. Plasma levels of osteocalcin were significantly decreased at 6 weeks of age (P<0.05) in Enpp1−/− mice, with no differences noted at 22 weeks of age. Plasma levels of CTx (Ratlaps™) and the phosphaturic hormone FGF-23 were significantly increased in the Enpp1−/− mice at 22 weeks of age (P<0.05). Fgf-23 messenger RNA expression in cavarial osteoblasts was increased 12-fold in Enpp1−/− mice compared to controls. These results indicate that Enpp1−/− mice are characterized by severe disruption to the architecture and mineralization of long-bones, dysregulation of calcium/phosphate homeostasis and changes in Fgf-23 expression. We conclude that NPP1 is essential for normal bone development and control of physiological bone mineralization.

Efeito da inclusão de óleo de linhaça nas rações sobre o desempenho e os parâmetros ósseos de frangos de corte

O objetivo neste trabalho foi avaliar o desempenho e os parâmetros ósseos de frangos de corte alimentados com rações contendo diferentes níveis de óleo de linhaça no período de 1 a 42 dias de idade. Nas fases inicial e de crescimento (1 a 21 dias e 22 a 42 dias, respectivamente), foi adotado delineamento inteiramente casualizado com 4 níveis de óleo de linhaça (2,0; 3,5; 5,0 ou 6,5%), 6 repetições e 50 aves/unidade experimental. Os níveis de óleo de linhaça tiveram efeito linear crescente sobre o diâmetro e o peso absoluto da tíbia, mas não influenciaram o peso do fêmur. Os dados revelaram interação idade e nível de óleo de linhaça para o índice de Seedor, comprovando que os efeitos positivos e negativos dos maiores níveis de óleo de linhaça observados para o fêmur e a tíbia, respectivamente, aumentaram no final da fase de crescimento. Os parâmetros de crescimento e qualidade do fêmur e da tíbia apresentaram comportamento cúbico, decorrente da idade. Os níveis de óleo de linhaça e a idade tiveram efeito quadrático sobre o peso das cinzas do fêmur. A concentração de cinzas foi positivamente influenciada pelos níveis de óleo de linhaça nas rações, contudo, os melhores resultados foram evidenciados a partir dos 28 dias. A resistência óssea não foi influenciada pela ingestão de óleo de linhaça, mas sofreu efeito cúbico da idade. Verificou-se interação significativa idade × nível de óleo de linhaça para a densidade mineral óssea, e que os melhores efeitos do óleo ocorrem na fase inicial de criação das aves. Rações contendo 6,5% de óleo de linhaça são benéficas para o crescimento e a mineralização dos ossos longos de frangos de corte.

The Rate of Bone Mineralization in Birds Is Directly Related to Alkaline Phosphatase Activity

Recent studies have suggested that a biochemical marker, plasma alkaline phosphatase (ALP), can be used as a general indicator of skeletal development in vertebrate animals. In birds, age-related variation in ALP activity, presumably due to bone formation processes, has been demonstrated, but to date, a direct connection between bone mineralization and enzyme activity has been elusive. In this study, we show that the activity of a bone isoform of ALP (bone ALP) is closely related to the overall rate of skeletal mineralization in nestlings of a small passerine bird, the great tit (Parus major L). Moreover, bone ALP activity predicted the rate of mineralization of leg and wing bones but not that of the skull. Liver isoform of ALP was only marginally related to the overall rate of skeletal mineralization, while no association with the mineralization of long bones was found. We conclude that bone ALP activity in the blood plasma is a reliable biomarker for skeletal mineralization in birds. This marker enables detection of subtle developmental differences between chicks of similar structural size, potentially facilitating the prediction of offspring mid- and long-term survival.

Conditional deletion of the TGF-β type II receptor in Col2a expressing cells results in defects in the axial skeleton without alterations in chondrocyte differentiation or embryonic development of long bones

Members of the TGF-β superfamily are secreted signaling proteins that regulate many aspects of development including growth and differentiation in skeletal tissue. There are three isoforms of TGF-β that act through the same heteromeric receptor complex. To address the question of the role of TGF-β signaling in skeletal development, we generated mice with a conditional deletion of the TGF-β type II receptor gene ( Tgfbr2) specifically in Col2a expressing cells using the Cre/lox recombinase system. Alizarin red-/Alcian blue-stained skeletons were prepared from embryos at 17.5, 15.5, and 13.5 days of gestation. Col2acre +/−; Tgfbr loxP/loxP and Col2acre −/−; Tgfbr2 +/loxP skeletons were compared. Multiple defects were observed in the base of the skull and in the vertebrae. Specifically, the size and spacing of the vertebrae were altered, and defects were detected in the closure of the neural arches. In addition, alterations in transverse processes, costal joints, and zygapophyses were detected. While the vertebral bodies were only moderately affected, the intervertebral discs (IVDs) were either missing or incomplete. Alterations in the vertebrae could be detected as early as E13.5 days. Surprisingly, alterations in length and mineralization of long bones were not detected at E17.5 days. In addition, the expression patterns of markers for chondrocyte differentiation were not altered in vertebrae or long bones suggesting that loss of responsiveness to TGF-β in chondrocytes does not affect embryonic endochondral bone formation. In contrast, mice that survived postnatally demonstrated alterations in the length of specific bones. Skeletons from Col2acre +/−; Tgfbr2 loxP/loxP mice were compared to those from mice null for the TGF-β2 ligand. The differences observed between these models allow distinctions to be made between the roles of the various isoforms of TGF-β and the signaling in specific cell types. The data provide information regarding mechanisms of skeletal development and suggest that TGF-β signaling is a critical component.

Age- and genotype-dependence of bone material properties in the osteogenesis imperfecta murine model (oim)

Cortical mineralization of long bones was studied in collagen α2(I)-deficient mice (oim) used as a model for human osteogenesis imperfecta. Aspects of the age development of the mice were characterized by combining nanometer- to micrometer-scale structural analysis with microhardness measurements. Bone structure was determined from homozygous (oim/oim) and heterozygous (oim/+) mice and their normal (+/+) littermates as a function of animal age by small-angle X-ray scattering (SAXS) and quantitative backscattered electron imaging (qBEI) measurements. SAXS studies found anomalies in the size and arrangement of bone mineral crystals in both homozygous and heterozygous mice aged 1–14 months. Generally, the crystals were smaller in thickness and less well aligned in these mice compared with control animals. An increase in the mean crystal thickness of the bone was found within all three genotypes up to an age of 3 months. Vicker’s hardness measurements were significantly enhanced for oim bone (homozygotes and heterozygotes) compared with controls. The microhardness values were correlated directly with increased mineral content of homozygous and heterozygous compared with control bone, as determined by qBEI analysis. There was also a significant increase of mineral content with age. Two possibilities for collagen-mineral association are discussed for explaining the increased hardness and mineral content of oim/oim bone, together with its decreased toughness and thinner mineral crystals. As a consequence of the present measurements, one model for oim bone could incorporate small and densely packed mineral crystals. A second model for possible collagen-mineral association in oim material would consist of two families of mineral crystals, one being smaller and the other being much larger than the crystals found in normal mouse long bones.

Ultrastructural studies of initial stages of mineralization of long bones and vertebrae in human fetuses.

We studied 27 embryos of 5-12 weeks gestational age where pregnancy was interrupted due to paramedical reasons, in order to find the developmental stages at which matrix vesicles appear in cartilage, and whether they are involved in the mineralization process. Specimens of long bones, lumbar and thoracic vertebral column were prepared for light, transmission and scanning electron microscopic studies. In the cartilaginous models of long bones, matrix vesicles were found amongst maturing and hypertrophic chondrocytes already by the 6th week after fertilization. By that stage, bone rudiments consisted of only cartilage that was not yet mineralized. In the vertebral column matrix, vesicles were found in the vertebral bodies amongst maturing and hypertrophic chondrocytes at the beginning of the 8th week. At that stage, although hypertrophy of chondrocytes was observed, mineralization was still absent. No matrix vesicles were found in the perichondrium, investing mesenchyme and intervertebral discs. Mineralization of cartilage in long bone rudiments started in the form of hydroxyapatite crystals within or around the matrix vesicles at 7 weeks of age and in the vertebral column at 11 weeks. As mineralization progressed, more hydroxyapatite crystals were observed around the matrix vesicles, forming typical calcospherites . Mineralization then progressed in the form described in other animals.