Growth defect in Grg5 null mice is associated with reduced Ihh signaling in growth plates

Gene-targeted disruption of Grg5, a mouse homologue of Drosophila groucho (gro), results in postnatal growth retardation in mice. The growth defect, most striking in approximately half of the Grg5 null mice, occurs during the first 4-5 weeks of age, but most mice recover retarded growth later. We used the nonlinear mixed-effects model to fit the growth data of wild-type, heterozygous, and Grg5 null mice. On the basis of preliminary evidence suggesting an interaction between Grg5 and the transcription factor Cbfa1/Runx2, critical for skeletal development, we further investigated the skeleton in the mice. A long bone growth plate defect was identified, which included shorter zones of proliferative and hypertrophic chondrocytes and decreased trabecular bone formation. This decreased trabecular bone formation is likely caused by a reduced recruitment of osteoblasts into the growth plate region of Grg5 null mice. Like the growth defect, the growth plate and trabecular bone abnormality improved as the mice grew older. The growth plate defect was associated with reduced Indian hedgehog expression and signaling. We suggest that Grg5, a transcriptional coregulator, modulates the activities of transcription factors, such as Cbfa1/Runx2 in vivo to affect Ihh expression and the function of long bone growth plates.

Magnetic field assisted stem cell differentiation - role of substrate magnetization in osteogenesis

Among the multiple modulatory physical cues explored to regulate cellular processes, the potential of magneto-responsive substrates in magnetic field stimulated stem cell differentiation is still unperceived. In this regard, the present work demonstrates how an external magnetic field can be applied to direct stem cell differentiation towards osteogenic commitment. A new culture methodology involving periodic delivery of 100 mT static magnetic field (SMF) in combination with HA-Fe3O4 magnetic substrates possessing a varying degree of substrate magnetization was designed for the study. The results demonstrate that an appropriate combination of weakly ferromagnetic substrates and SMF exposure enhanced cell viability, DNA synthesis and caused an early switchover to osteogenic lineage as supported by Runx2 immunocytochemistry and ALP expression. However, the mRNA expression profile of early osteogenic markers (Runx2, ALP, Col IA) was comparable despite varying substrate magnetic properties (diamagnetic to ferromagnetic). On the contrary, a remarkable upregulation of late bone development markers (OCN and OPN) was explicitly detected on weak and strongly ferromagnetic substrates. Furthermore, SMF induced matrix mineralization with elevated calcium deposition on similar substrates, even in the absence of osteogenic supplements. More specifically, the role of SMF in increasing intracellular calcium levels and in inducing cell cycle arrest at G0/G1 phase was elucidated as the major molecular event triggering osteogenic differentiation. Taken together, the above results demonstrate the competence of magnetic stimuli in combination with magneto-responsive biomaterials as a potential strategy for stem cell based bone tissue engineering.

Review of the crevalle jacks, Caranx hippos complex (Teleostei: Carangidae), with a description of a new species from West Africa

The Caranx hippos species complex comprises three extant species: crevalle jack (Caranx hippos) (Linnaeus, 1766) from both the western and eastern Atlantic oceans; Pacific crevalle jack (Caranx caninus) Günther, 1868 from the eastern Pacific Ocean; and longfin crevalle jack (Caranx fischeri) new species, from the eastern Atlantic, including the Mediterranean Sea and Ascension Island. Adults of all three species are superficially similar with a black blotch on the lower half of the pectoral fin, a black spot on the upper margin of opercle, one or two pairs of enlarged symphyseal canines on the lower jaw, and a similar pattern of breast squamation. Each species has a different pattern of hyperostotic bone development and anal-fin color. The two sympatric eastern Atlantic species also differ from each other in number of dorsal-and anal-fin rays, and in large adults of C. fischeri the lobes of these fins are longer and the body is deeper. Caranx hippos from opposite sides of the Atlantic are virtually indistinguishable externally but differ consistently in the expression of hyperostosis of the first dorsalfin pterygiophore. The fossil species Caranx carangopsis Steindachner 1859 appears to have been based on composite material of Trachurus sp. and a fourth species of the Caranx hippos complex. Patterns of hyperostotic bone development are compared in the nine (of 15 total) species of Caranx sensu stricto that exhibit hyperostosis.

Aspects of phosphorus pollution from aquaculture

Phosphorus is an essential element for living organisms and exists in waterbodies as dissolved and particulate forms. Phosphorus is required for optimum growth, feed efficiency, bone development and maintenance of acid-base regulation in fish. The presence of high concentration of phosphates in water may indicate presence of pollution as it may accelerate plant growth and disrupt the aquatic ecosystem thereby benefiting certain species and altering species diversity in affected areas. Eutrophication of waterbodies is often correlated with the phosphorus loading into the environment and aquaculture has been identified as one of the sources of phosphorus pollution. Details of the impacts of eutrophication is given in Bernhardt (1981). Phosphorus must be provided in fish feed because of its low concentration in water. Studies made in Europe and Northern America have revealed a phosphorus surplus in most commercial feeds which is above actual requirements; or is supplied in a form which is unavailable to the fish. Surplus phosphorus is excreted, while unavailable phosphorus is passed out in the feces. Discharge of phosphorus from fish farms and hatchery effluents have caused phosphorus pollution in Nordic countries, North America and Europe. This article examines the path of phosphorus pollution, quantification/prediction of phosphorus load from aquaculture and remedial measures.

Evidence for Positive Selection on the Osteogenin (BMP3) Gene in Human Populations

Background: Human skeletal system has evolved rapidly since the dispersal of modern humans from Africa, potentially driven by selection and adaptation. Osteogenin (BMP3) plays an important role in skeletal development and bone osteogenesis as an antagonist of the osteogenic bone morphogenetic proteins, and negatively regulates bone mineral density. Methodology/Principal Findings: Here, we resequenced the BMP3 gene from individuals in four geographically separated modern human populations. Features supportive of positive selection in the BMP3 gene were found including the presence of an excess of nonsynonymous mutations in modern humans, and a significantly lower genetic diversity that deviates from neutrality. The prevalent haplotypes of the first exon region in Europeans demonstrated features of long-range haplotype homogeneity. In contrast with findings in European, the derived allele SNP Arg192Gln shows higher extended haplotype homozygosity in East Asian. The worldwide allele frequency distribution of SNP shows not only a high-derived allele frequency in Asians, but also in Americans, which is suggestive of functional adaptation. Conclusions/Significance: In conclusion, we provide evidence for recent positive selection operating upon a crucial gene in skeletal development, which may provide new insight into the evolution of the skeletal system and bone development.

Caractérisation de nouveaux mécanismes transcriptionnels impliqués dans la biologie osseuse

Le développement et l'homéostasie des os requièrent l'orchestration spatio-temporelle d'un grand nombre de signaux moléculaires. Ces signaux entraînent l'activation ou l'inhibition de différents facteurs de transcription, lesquels sont en mesure de contrôler la prolifération et la différenciation des ostéoblastes et des chondrocytes. L'intégrité de ces différents mécanismes se doit d'être maintenu tout au long de la vie. Ainsi, une anomalie dans l'un de ces mécanismes conduit à l'apparition de pathologies osseuses et métaboliques telles qu’une hypophosphatémie, l'ostéoporose ou l'ostéoarthrite (OA). Afin d'en apprendre davantage sur la biologie osseuse, le projet décrit dans cette thèse a pour objectif de caractériser de nouveaux mécanismes de régulation transcriptionnelle pour deux gènes importants dans le développement des os et le maintien de leur intégrité. Il s’agit du Paired-like Homeodomain Transcription Factor 1 (PITX1) et du Phosphate-regulating gene with homology to endopeptidase on the X chromosome (PHEX). Le premier mécanisme présenté dans cette thèse concerne la régulation transcriptionnelle du gène PITX1, un facteur de transcription à homéodomaine nécessaire, notamment, au développement des os des membres inférieurs et au maintien de l'intégrité du cartilage articulaire chez l'adulte. Ainsi, dans les chondrocytes articulaires, on note que l'expression de PITX1 est assurée par le recrutement du facteur de transcription E2F1 à deux éléments de réponse présents dans la région proximale du promoteur de PITX1. Aussi, dans les chondrocytes articulaires de patients souffrant d'OA, dans lesquels l'expression de PITX1 est fortement diminuée, un mécanisme de répression transcriptionnelle, lequel implique la protéine multifonctionnelle Prohibitin (PHB1), semble être activé. En effet, dans ces chondroytes, on note une forte accumulation nucléaire de PHB1 comparativement aux chondrocytes articulaires de sujets sains. Le second mécanisme présenté dans cette thèse concerne la répression transcriptionnelle de PHEX, la peptidase mutée dans le syndrome d'hypophosphatémie lié au chromosome X (X-Linked Hypophosphatemia, XLH), lequel se caractérise par une hypophosphatémie et une ostéomalacie. Le traitement d'ostéoblastes à la Parathyroid hormone-related protein (PTHrP) permet d’observer la répression de PHEX. Afin de caractériser le mécanisme responsable de cette répression, des expériences de gènes rapporteurs ont révélé la présence de deux éléments de réponse pour le répresseur transcriptionnel E4BP4 dans le promoteur de PHEX. La suppression de l'expression d'E4BP4 par l'utilisation d'ARN d'interférence a permis de valider que ce facteur de transcription est responsable de la répression de PHEX suite au traitement d'ostéoblastes à la PTHrP. En somme ces nouveaux mécanismes de régulation transcriptionnelle permettent de mieux comprendre la régulation de l'expression de PITX1 et de PHEX. Aussi, cette nouvelle implication de PHB1 dans la pathogenèse de l'OA offre de nouvelles possibilités de traitement et pourrait servir pour le diagnostic précoce de cette pathologie. Enfin, la caractérisation d'E4BP4 en tant que médiateur pour la répression de PHEX par la PTHrP suggère que ce répresseur transcriptionnel pourrait être impliqué dans le contrôle de la minéralisation des os et des niveaux de phosphate sanguin.

Suivi longitudinal de la densité osseuse et du statut en vitamine D chez des enfants prépubères avec une allergie au lait de vache non résolue.

Problématique : L'allergie au lait de vache (ALV) est reconnue comme une condition transitoire qui disparaît chez la majorité des enfants avant l’âge de 3-5 ans, mais des données récentes révèlent une persistance de l’ALV. Les enfants souffrant d’une ALV sont à risque d’apports insuffisants en calcium et en vitamine D, deux nutriments impliqués dans la santé osseuse. Une première étude transversale portant sur la santé osseuse d’enfants prépubères ALV a observé que la densité osseuse (DMO) lombaire était significativement inférieure à celle d’enfants sans allergie au lait de vache (SALV). Objectifs : Sur la base de ces résultats, nous désirons documenter l’évolution longitudinale de la santé osseuse, du statut en vitamine D, des apports en calcium et en vitamine D et de l’adhérence à la supplémentation des enfants ALV (n=36) et de comparer ces données aux enfants SALV (n=19). Résultats : Le gain annualisé de la DMO lombaire est similaire entre les enfants ALV et SALV. Bien qu’il n’y ait pas de différence significative entre les deux groupes, la DMO lombaire des enfants ALV demeure cependant inférieure à celle des témoins. Qui plus est, le score-Z de la DMO du corps entier tend à être inférieur chez les enfants-cas comparé aux témoins. Au suivi, la concentration de 25OHD et le taux d’insuffisance en vitamine D sont similaires entre les deux groupes tout comme les apports en calcium et en vitamine D. Davantage d’enfants ALV prennent un supplément de calcium au suivi comparativement au temps initial (42% vs. 49%, p<0,05), mais le taux d’adhérence à la supplémentation a diminué à 4 jours/semaine. Conclusion : Une évaluation plus précoce ainsi qu’une prise en charge de la santé osseuse des enfants ALV pourraient être indiquées afin de modifier l’évolution naturelle de leur santé osseuse. Les résultats justifient aussi le suivi étroit des apports en calcium et vitamine D par une nutritionniste et la nécessité d'intégrer la supplémentation dans le plan de traitement de ces enfants et d’assurer une surveillance de l’adhérence à la supplémentation.

Efeitos do ultra-som terapêutico sobre o crescimento longitudinal do fêmur e da tíbia em ratos

Objectives: To determine the effects of ultrasound therapy on the femur and tibia growth in young rats. Method: Four-week-old male Ratus Norvegicus totaling 115 animals, divided into four groups, were submitted to ultrasound therapy (0.8 MHz, fixed tube head, continuous pulse, for 10 minutes, once a day, ten times) on the medial face of the right knee, with powers of 0.0 W/cm2 (group 31), 0.5 W/cm2 (group G2), 1.0 W/cm2 (group G3), and 1.5 W/cm2 (group G4). Histological slides of the epiphysis, growth plate and metaphysis and the femoral and tibial length measurements were studied in the sixth, thirteenth and twenty-sixth weeks of life. The data were submitted to factorial analysis of variance according to a one-way layout. Results: No statistically significant bone growth alteration was established between any of the three treated groups and the control group. However, alterations in femoral and tibial growth suggesting a decrease in G4 in relation to 02 and G3 were noted. In G4, histopathological alterations, such as cellular necrosis and post-necrosis bone neoformation were found. Conclusion: According to this study, no statistical evidence of bone growth stimulus or inhibition resulting from the application of ultrasound therapy was found when comparing the treated groups with the control group. Histological alterations regarded as pathological were only observed in G4. Also, smaller significant bone growth was found in G4 compared to G2 and G3. Level of Evidence: Level II, cross-sectional study.

Low-level laser on femoral growth plate in rats

OBJETIVO: Determinar a influência do Laser Terapêutico de Baixa Potência sobre a placa de crescimento de ratos. MÉTODOS: Trinta ratos Wistar machos com 40 dias de idade foram divididos em dois grupos, G1 e G2. O grupo G1 foi submetido à irradiação com laser GaAlAs 830 nm, potência de saída de 40 mW, e densidade de energia de 10 J/cm2. A irradiação foi aplicada diariamente por um período máximo de 21 dias. O mesmo procedimento foi realizado no grupo G2, com a probe desativada. Cinco animais em cada grupo foram sacrificados nos dias 7, 14 e 21 e submetidas à análise histomorfométrica. RESULTADOS: em ambos os grupos, o disco fisário esteve radiograficamente visível em todos os momentos nas incidências craniocaudal e médio-lateral. No 21º dia a porcentagem de comprimento longitudinal do fêmur foi maior em G1 que em G2 em relação ao valor basal, e o número de condrócitos da zona hipertrófica foi maior em G1 que em G2. A zona de cartilagem calcificada estava maior em G1 em relação a G2 em todos os momentos de avaliação. A angiogênese foi maior em G1 que em G2 nos 14º e 21º dias. CONCLUSÃO: A terapia com laser terapêutico de baixa potência influenciou negativamente o disco fisário distal do fêmur de ratos.

Natural killer activity in the experimental privational rickets

To study the 'in vivo' importance of vitamin D on the natural killer (NK) activity, rats were submitted to privational rickets induced by a diet deficient in vitamin D and phosphorus (D-P-). Thirty days after the beginning of treatment the animals showed low body weight, changes in the bone development, and decreased levels of 25-hydroxyvitamin D-3 (25-OH D-3). NK activity, evaluated using a cytotoxicity assay against Cr-51-labeled Yac.1 target cells, was not modified by the rickets-inducing treatment during the first 30 days. Following a long-term treatment (60 days) the rachitic rats (D-P-) exhibited higher NK activity than control animals (D + P +) (P < 0.05). on the other hand, D - P + animals showed higher cytotoxic activity than D - P - and D + P + groups. Feed replacement to the rachitic rats by a complete diet (D - P - /D + P +) led to a partial recuperation of growth, bone development, and 25-OH D-3 scrum. levels. The NK activity was also influenced by vitamin D intake, decreasing after treatment. (C) 2002 Elsevier B.V. B.V. All rights reserved.

KINETIC-PROPERTIES OF OSSEOUS PLATE ALKALINE-PHOSPHATASE FROM DIABETIC RATS

1. Increased levels of bone alkaline phosphatase activity were observed in diabetic rats. These animals exhibited impaired bone development without concomitant alterations of the sequence of cellular transformations.2. Alkaline phosphatase activity was delayed in diabetic rats but the kinetic parameters for the hydrolysis of p-Nitrophenylphosphate (PNPP) were virtually the same observed for controls (N = 1.2 and K0.5 = 43 muM).3. Alkaline phosphatase from diabetic rats had a better affinity (K0.5 = 38 muM) for magnesium ions than controls (K0.5 = 9 1 muM).4. Zinc ions affected alkaline phosphatase activity from control and diabetic rats in the same way (K0.5 = 10 muM).

Autogenous transplantation of rib cartilage preserved in glycerol, after removal of the perichondrium, to the malar process of rats--a histological study (Part I).

Seventy-two male albino rats received autogenous transplants of glycerol-preserved rib cartilage into the malar process. The animals were divided into two groups which received preserved cartilage with or without perichondrium. The implants were well tolerated and removal of the perichondrium enhanced the rate of resorption and bone replacement of the material.

Estudio radiográfico do desenvolvimento da dentição permanente de crianças brasileiras com idade cronológica variando entre 84 e 131 meses.

With the help of orthopantomograms we analyse the tooth development beside body weight and height from Brazilian healthy boys and girls. So, their dental age were compared to their chronological age. The results of this investigation, according to the methodology employed, indicated that: 1. girls showed accelerated formation of permanent teeth and mean values of dental age higher than boys; 2. the mean values of dental age in girls were higher than their chronological age in all groups; 3. the teeth development stages were appropriate in assessment of the degree of physiological maturity of a growing child.

Evolução do peso corporal e desenvolvimento da mandíbula de ratos em função da administração de flúor.

The body weights evolution and the development of mandible were analyzed in rats that received 0.25 mg F contained into a commercial product, during 30 and 60 days consecutively, beginning at birth. The study presented the following conclusions: 1. The treated animals showed body weights always inferior to the controls, with more evident difference at 60 days period; 2. Analysis showed a reduction in the horizontal linear measurements, but not in the length of the mandibular diastem. No influence was observed in relation to the verticals.

Free autogenous cartilage grafts to the mandible of rats-histological study.

The present study was designed to histologically evaluate the behavior of free autogenous cartilage grafts to the mandible of rats. A 3-mm segment was removed from the last rib of male adult rats and transplanted fresh to a receptor bed prepared on the mandibular ramus. The results showed that the grafts maintained their vitality up to 120 days and the perichondrium was biologically integrated to the osseous bed. Appositional growth of the grafts was found. New bone formation was observed in close proximity to the grafts, but newly formed trabeculae did not arise from perichondrium.