Tissue transglutaminase (TG2) activity regulates osteoblast differentiation and mineralization in the SAOS-2 cell line

Tissue transglutaminase (type II, TG2) has long been postulated to directly promote skeletal matrix calcification and play an important role in ossification. However, limited information is available on the expression, function and modulating mechanism of TG2 during osteoblast differentiation and mineralization. To address these issues, we cultured the well-established human osteosarcoma cell line SAOS-2 with osteo-inductive conditioned medium and set up three time points (culture days 4, 7, and 14) to represent different stages of SAOS-2 differentiation. Osteoblast markers, mineralization, as well as TG2 expression and activity, were then assayed in each stage. Furthermore, we inhibited TG activity with cystamine and then checked SAOS-2 differentiation and mineralization in each stage. The results showed that during the progression of osteoblast differentiation SAOS-2 cells presented significantly high levels of osteocalcin (OC) mRNA, bone morphogenetic protein-2 (BMP-2) and collagen I, significantly high alkaline phosphatase (ALP) activity, and the increased formation of calcified matrix. With the same tendency, TG2 expression and activity were up-regulated. Furthermore, inhibition of TG activity resulted in a significant decrease of OC, collagen I, and BMP-2 mRNA and of ALP activity and mineralization. This study demonstrated that TG2 is involved in osteoblast differentiation and may play a role in the initiation and regulation of the mineralization processes. Moreover, the modulating effects of TG2 on osteoblasts may be related to BMP-2.

Effect of JJYMD-C, a novel synthetic derivative of gallic acid, on proliferation and phenotype maintenance in rabbit articular chondrocytes in vitro

Tissue engineering encapsulated cells such as chondrocytes in the carrier matrix have been widely used to repair cartilage defects. However, chondrocyte phenotype is easily lost when chondrocytes are expanded in vitro by a process defined as “dedifferentiation”. To ensure successful therapy, an effective pro-chondrogenic agent is necessary to overcome the obstacle of limited cell numbers in the restoration process, and dedifferentiation is a prerequisite. Gallic acid (GA) has been used in the treatment of arthritis, but its biocompatibility is inferior to that of other compounds. In this study, we modified GA by incorporating sulfamonomethoxine sodium and synthesized a sulfonamido-based gallate, JJYMD-C, and evaluated its effect on chondrocyte metabolism. Our results showed that JJYMD-C could effectively increase the levels of the collagen II, Sox9, and aggrecan genes, promote chondrocyte growth, and enhance secretion and synthesis of cartilage extracellular matrix. On the other hand, expression of the collagen I gene was effectively down-regulated, demonstrating inhibition of chondrocyte dedifferentiation by JJYMD-C. Hypertrophy, as a characteristic of chondrocyte ossification, was undetectable in the JJYMD-C groups. We used JJYMD-C at doses of 0.125, 0.25, and 0.5 µg/mL, and the strongest response was observed with 0.25 µg/mL. This study provides a basis for further studies on a novel agent in the treatment of articular cartilage defects.

Human fetal malformations associated with the use of an angiotensin II receptor antagonist: Case Report

Introduction: The potential risks related to drug exposure during pregnancy represent a vast chapter in modern obstetrics and data regarding the safety of antihypertensive drugs during pregnancy are relatively scarce. Case report: A 37-year-old patient discovered her fifth pregnancy at our hospital after 26 weeks and 4 days of gestation. She reported a history of hypertension and was currently being treated with Losartan. Hospitalization was recommended for the patient and further evaluation of fetal vitality was performed. On the fourth day an ultrasound was performed, resulting in a severe oligohydramnios, fetal centralization and abnormal ductus venosus. After 36 hours, the newborn died. Pathologic evaluation: At autopsy, the skullcap had large fontanels and deficient ossification. The kidneys were slightly enlarged. A microscopic examination detected underdevelopment of the tubules and the presence of some dilated lumens. Immunohistochemical detection of epithelial membrane antigen was positive. Immunoreactivity of CD 15 was also assayed to characterize the proximal tubules, and lumen collapse was observed in some regions. Discussion: Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor antagonists (ARAs) are among the most widely prescribed drugs for hypertension. They are often used by hypertensive women who are considering become pregnant. While their fetal toxicity in the second or third trimesters has been documented, their teratogenic effect during the first trimester has only recently been demonstrated. Conclusion: Constant awareness by physicians and patients should be encouraged, particularly in regard to the prescription of antihypertensive drugs in women of childbearing age who are or intend to become pregnant.

Prédiction de l'évolution de la scoliose idiopathique de l'adolescent à l'aide des paramètres tridimensionnels du rachis

La scoliose idiopathique de l’adolescent est une déformation 3D du rachis. La littérature comporte une multitude d’études sur la prédiction de l’évolution et l’identification de facteurs de risque de progression. Pour l’instant les facteurs de risque établis sont l’amplitude de la déformation, la maturité squelettique et le type de courbure. Plusieurs autres champs ont été explorés comme les aspects génétiques, biochimiques, mécaniques, posturaux et topographiques, sans vraiment apporter beaucoup de précision à la prédiction de l’évolution. L’avancement de la technologie permet maintenant de générer des reconstructions 3D du rachis à l’aide des radiographies standard et d’obtenir des mesures de paramètres 3D. L’intégration de ces paramètres 3D dans un modèle prédictif représente une avenue encore inexplorée qui est tout à fait logique dans le contexte de cette déformation 3D du rachis. L’objectif général de cette thèse est de développer un modèle de prédiction de l’angle de Cobb à maturité squelettique à partir de l’information disponible au moment de la première visite, soit l’angle de Cobb initial, le type de courbure, l’âge osseux et des paramètres 3D du rachis. Dans une première étude, un indice d’âge osseux a été développé basé sur l’ossification de l’apophyse iliaque et sur le statut du cartilage triradié. Cet indice comporte 3 stades et le second stade, qui est défini par un cartilage triradié fermé avec maximum 1/3 d’ossification de l’apophyse iliaque, représente le moment pendant lequel la progression de la scoliose idiopathique de l’adolescent est la plus rapide. Une seconde étude rétrospective a permis de mettre en évidence le potentiel des paramètres 3D pour améliorer la prédiction de l’évolution. Il a été démontré qu’à la première visite il existe des différences pour 5 paramètres 3D du rachis entre un groupe de patients qui sera éventuellement opéré et un groupe qui ne progressera pas. Ces paramètres sont : la moyenne da la cunéiformisation 3D des disques apicaux, la rotation intervertébrale à la jonction inférieure de la courbure, la torsion, le ratio hauteur/largeur du corps vertébral de T6 et de la colonne complète. Les deux dernières études sont basées sur une cohorte prospective de 133 patients avec une scoliose idiopathique de l’adolescent suivi dès leur première visite à l’hôpital jusqu’à maturité squelettique. Une première étude a permis de mettre en évidence les différences morphologiques à la première visite entre les patients ayant progresser de plus ou moins de 6°. Des différences ont été mise en évidence pour la cyphose, l’angle de plan de déformation maximal, la rotation ntervertébrale l’apex, la torsion et plusieurs paramètres de «slenderness». Ensuite une seconde étude a permis de développer un modèle prédictif basé sur un modèle linéaire général en incluant l’indice d’âge osseux développé dans la première étude, le type de courbure, l’amplitude de l’angle de Cobb à la première visite, l’angle de déformation du plan maximale, la cunéiformisation 3D des disques T3-T4, T8-­T9, T11-­T12 et la somme des cunéiformisation 3D de tous les disques thoraciques et lombaires. Le coefficient de détermination multiple pour cette modélisation est de 0.715. Le modèle prédictif développé renforce l’importance de considérer la scoliose idiopathique dans les trois dimensions et il permettra d’optimiser la prédiction de l’évolution au moment de la première visite.

Impacto funcional con el uso de clavo telescopico tipo Fassier Duval en pacientes con osteogénesis imperfecta

Introducción: la osteogénesis es una patología de origen genético caracterizada por fragilidad ósea, en su curso natural los pacientes que la padecen se enfrentan a múltiples fracturas y múltiples intervenciones quirúrgicas, este tipo de pacientes por ser de alto riesgo necesitan técnicas quirúrgicas que aumenten el tiempo entre cada intervención y que demuestren un mayor impacto en el estado funcional. Objetivo: Determinar el impacto en el estado funcional de los pacientes con osteogénesis imperfecta llevados a tratamiento quirúrgico con clavos telescopados tipo Fassier Duval. Diseño: Estudio descriptivo prospectivo en el que se incluyeron 8 pacientes con diagnóstico de osteogénesis imperfecta, llevados a tratamiento quirúrgico con clavos telescopados tipo Fassier Duval desde el 2009 al 2013 a los cuales se les realizó seguimiento menor de 1 año del post operatorio. Resultados: La respuesta encontrada fue satisfactoria en la mayoría de los pacientes analizados 6 de 8, con cercanía a un estado funcional normal; un riesgo de caída bajo, incorporación y deambulación adecuada y una valoración funcional motora gruesa con valores cercanos al 100% identificando un buen nivel de independencia funcional. Se pudo demostrar que existieron cambios en los valores de la escala y que estos fueron estadísticamente significativos con p=0,028 indicando que el aumento dichos valores en el posoperatorio están relacionados con el procedimiento quirúrgico al utilizado en este grupo de pacientes. Conclusión: El tratamiento quirúrgico con el clavo telescopado de Fassier Duval en nuestra experiencia demostró tener una mejoría en el estado funcional de los pacientes del presente estudio, por lo tanto se sugiere la posibilidad de implementar su uso según este indicado con el fin de obtener un mejor resultado quirúrgico y funcional. Palabras clave: Osteogénesis Imperfecta, Clavo de Fassier Duval, Valoración Funcional Motora

Desmite dos Ligamentos Colaterais da Articulação Interfalângica Distal em Equinos

Os objectivos deste estudo basearam-se na análise dos aspectos clínicos de cavalos de desporto, aos quais foi diagnosticado através de ecografia, desmite dos Ligamentos Colaterais (LCs) da articulação Interfalângica Distal (IFD). Os cavalos incluídos na amostra foram examinados entre Setembro de 2011 e Junho de 2012 tendo sido selecionados aqueles que apresentaram os critérios de inclusão requeridos, nomeadamente o diagnóstico de desmite em pelo menos um dos LCs da articulação IFD. Foram incluídos 8 animais no estudo, sendo que o diagnóstico foi feito através de um cuidadoso exame ortopédico e de ecografia, tendo-se verificado as lesões nos membros anteriores. O LC Lateral foi o mais afectado (5 cavalos) seguido do Medial (2 cavalos). Apenas em 1 dos casos as lesões eram bilaterais. A maioria dos animais apresentavam distensão da articulação IFD confirmada ecograficamente. A claudicação foi invariavelmente exacerbada em círculo no piso duro, sendo que 7 dos 8 (87.5%) animais aumentaram o grau de claudicação quando o membro afectado estava no interior do círculo. Após o bloqueio digital palmar a claudicação foi atenuada em 7 dos 8 animais (87.5%) sendo totalmente abolida em 3 (37.5%) deles. O bloqueio da articulação IFD foi positivo em todos os animais, e o bloqueio da bursa podotroclear negativa em 7 dos 8 cavalos. Metade dos animais (50%) apresentaram alterações radiográficas como osteoartrite da articulação IFD, remodelação óssea da origem/inserção do LC afectado e um deles apresentava ossificação de uma cartilagem ungular. Todos os animais apresentaram sinais evidentes de lesão na ecografia, sendo que alguns demostraram sinais de desmite crónica e outros aguda. Os tratamentos instituídos variaram de acordo com a história, sinais clínicos, sinais radiográficos e severidade das lesões ecográficas. A desmite dos LC da articulação IFD deve ser considerada como uma causa de claudicação que afecta a performance dos cavalos de desporto. Mais estudos são necessários, de modo a caraterizar melhor estas lesões e avaliar as melhorias após a terapia por forma a determinar os factores que mais influenciam o prognóstico. Palavras-Chave: Equinos, Ligamentos colaterais, Desmite, articulação Interfalângica distal

On the threshold of adulthood: a new approach for the use of maturation indicators to assess puberty in adolescents from medieval England

Objectives: This study provides the first large scale analysis of the age at which adolescents in medieval England entered and completed the pubertal growth spurt. This new method has implications for expanding our knowledge of adolescent maturation across different time periods and regions. Methods: In total, 994 adolescent skeletons (10-25 years) from four urban sites in medieval England (AD 900-1550) were analysed for evidence of pubertal stage using new osteological techniques developed from the clinical literature (i.e. hamate hook development, CVM, canine mineralisation, iliac crest ossification, radial fusion). Results: Adolescents began puberty at a similar age to modern children at around 10-12 years, but the onset of menarche in girls was delayed by up to 3 years, occurring around 15 for most in the study sample and 17 years for females living in London. Modern European males usually complete their maturation by 16-18 years; medieval males took longer with the deceleration stage of the growth spurt extending as late as 21 years. Conclusions: This research provides the first attempt to directly assess the age of pubertal development in adolescents during the tenth to seventeenth centuries. Poor diet, infections, and physical exertion may have contributed to delayed development in the medieval adolescents, particularly for those living in the city of London. This study sheds new light on the nature of adolescence in the medieval period, highlighting an extended period of physical and social transition.

Immunohistochemical, tomographic and histological study on onlay bone graft remodeling. Part II: calvarial bone

Objectives Little information is available on the molecular events that occur during graft incorporation over time. The calvarial bone (Cb) grafts have been reported to produce greater responses compared with other donor regions in maxillofacial reconstructions, but the scientific evidences for this are still lacking. The objectives of this study are (1) to study the morphological pattern of Cb onlay bone grafts and compare them with the biological events through immunohistochemical responses and (2) to establish the effects of perforations in maintaining the volume and bone density of the receptor bed. Material and methods Sixty New Zealand White rabbits were submitted to Cb onlay bone grafts on the mandible. In 30 rabbits, the receptor bed was perforated (perforated group), while for the remaining animals the bed was kept intact (non-perforated group). Six animals from each group were sacrificed at 5, 7, 10, 20 and 60 days after surgery. Histological sections from the grafted area were prepared for immunohistochemical and histological analyses. Immuno-labeling was found for proteins Osteoprotegerin (OPG), receptor activator of nuclear factor-kappa beta ligand (RANKL), alkaline phosphatase (ALP), osteopontin (OPN), vascular endothelial growth factor (VEGF), tartrate-resistant acid phosphatase (TRAP), Type I collagen (COL I) and osteocalcin (OC). The tomography examination [computerized tomography (CT) scan] was conducted just after surgery and at the sacrifice. Results The histological findings revealed that the perforations contributed to higher bone deposition during the initial stages at the graft-receptor bed interface, accelerating the graft incorporation process. The results of the CT scan showed lower resorption for the perforated group (P < 0.05), and both groups showed high bone density rates at 60 days. This set of evidences is corroborated by the immunohistochemical outcomes indicating that proteins associated with revascularization and osteogenesis (VEGF, OPN, TRAP and ALP) were found in higher levels in the perforated group. Conclusions These findings indicate that the bone volume of calvarial grafts is better maintained when the receptor bed is perforated, probably resulting from more effective graft revascularization and greater bone deposition. The process of bone resorption peaked between 20 and 60 days post-operatively in both groups although significantly less in the perforated group. To cite this article:Pedrosa Jr WF, Okamoto R, Faria PEP, Arnez MFM, Xavier SP, Salata LA. Immunohistochemical, tomographic and histological study on onlay bone grafts remodeling. Part II: calvarial bone.Clin. Oral Impl. Res. 20, 2009; 1254-1264.doi: 10.1111/j.1600-0501.2009.01747.x.

Characteristics of the equine embryo and fetus from days 15 to 107 of pregnancy

In spite of numerous, substantial advances in equine reproduction, many stages of embryonic and fetal morphological development are poorly understood, with no apparent single source of comprehensive information. Hence, the objective of the present study was to provide a complete macroscopic and microscopic description of the equine embryo/fetus at various gestational ages. Thirty-four embryos/fetuses were aged based on their crown rump length (CRL), and submitted to macroscopic description, biometry, light and scanning microscopy, as well as the alizarin technique. All observed developmental changes were chronologically ordered and described. As examples of the main observed features, an accentuated cervical curvature was observed upon macroscopic examination in all specimens. In the nervous system, the encephalic fourth ventricle and the encephalic vesicles forebrain, midbrain, and hindbrain, were visualized from Day 19 (ovulation = Day 0). The thoracic and pelvic limbs were also visualized; their extremities gave rise to the hoof during development from Day 27. Development of other structures such as pigmented optical vesicle, liver, tail, cardiac area, lungs, and dermal vascularization started on Days 25, 25, 19, 19, 34, and 35, respectively. Light and scanning microscopy facilitated detailed examinations of several organs, e.g., heart, kidneys, lungs, and intestine, whereas the alizarin technique enabled visualization of ossification. Observations in this study contributed to the knowledge regarding equine embryogenesis, and included much detailed data from many specimens collected over a long developmental interval. (C) 2011 Elsevier Inc. All rights reserved.

Placas maxilo-dentárias em Rincossauros Hyperodapedon Huxley, 1859. Histologia e morfogênese maxilo-dental: nova abordagem.

O estudo das estruturas da placas maxilo-dentárias dos rincossauros Hyperodapedon Huxley, 1859, do Triássico da Formação Santa Maria, em nova abordagem histológica e ontogênica resultou na identificação da natureza do esmalte aprismático verdadeiro, de variados elementos histológicos dentinários e osteológicos, e de um centro de ossificação periosteal primário. Também foram encontradas evidências histológicas dos mecanismos de fusionamento maxilo-dentinário e amelo-maxilar. Com estes elementos, inferimos os modelos de organogênese dental, da ontogênese maxilar e dos mecanismos de fusionamento maxilodental. Encontrou-se uma singular e raríssima coroa dental, imatura e ainda não erupcionada, na região posterior da placa dental e assim evidenciou-se a correta posição da margem odontogenicamente ativa. Adicionalmente inferiu-se a localização da posição da lâmina dentária embrionária. Constatou-se a não formação de alvéolos dentários, de cemento radicular e do espaço necessário à formação do ligamento periodontal e, assim, se deduziu a não formação do folículo dental embriônico. As presenças de especiais elementos anatômicos e histológicos nos tecidos ósseos periapicais evidenciam o crescimento radicular contínuo, enquanto a forma e o fusionamento radicular imediato depõe a favor de uma função dentária fisiológica diferenciada para as baterias dentárias maxilares dos Rincossauros do gênero Hyperodapedon. Os mecanismos que possibilitaram o controle embriônico para a deposição das lamelas de tecido ósseo coronal e seu preciso fusionamento sobre o esmalte dentário, declinam por modificações nas funções tardias do órgão reduzido do esmalte e pela presença de uma membrana oral com funções osteogênicas e também protetivas, situada nas porções posteriores da placa maxilo-dentária em desenvolvimento. Mudanças heterocrônicas no tempo de diferenciação das células da crista neural embriônica e em seus derivados, como a lâmina dentária e órgãos dentários embrionários ou correlacionadas com a organogênese das placas maxilo-dentárias e seus anexos periodontais, todos como condições plesiomórficas para Diápsidas Triássicos, poderiam ser as causas responsáveis pela origem e evolução deste estranho aparelho estomatognático nos clados de Hyperodapedon sp..

Kinetic Analysis of Substrate Utilization by Native and TNAP-, NPP1-, or PHOSPHO1-Deficient Matrix Vesicles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação histológica da regeneração óssea do rádio e ulna em cães submetidos ao alongamento com o fixador de Ilizarov

O objetivo do estudo foi analisar histologicamente a qualidade, tipo e orientação dos tecidos formados na área de regeneração óssea produzida durante alongamento simultâneo do rádio e da ulna, com fixador externo de Ilizarov, a partir de osteotomia subperiosteal diafisária distal, com dois incrementos diários de 0,5 mm. A montagem foi composta de dois anéis e quatro hastes telescópicas, sendo a distração óssea iniciada no sexto dia de pós-operatório. Utilizaram-se 15 cães, sem raça definida, adultos, com peso corpóreo entre 17 e 30 kg, divididos por sorteio em subgrupos (A, B, C, D e E) compostos por três animais, que foram submetidos a eutanásia após os seguintes procedimentos: A- oito dias de alongamento, B- 15 dias de alongamento, C- 22 dias de alongamento, D- 28 dias de alongamento e oito dias de fase neutra com o fixador, E- 28 dias de alongamento, 60 dias de fase neutra com fixador e 45 dias sem fixador. Os resultados obtidos mostram que o tipo e localização da osteotomia, bem como o ritmo de distração utilizado, não interferiram sobremaneira na regeneração óssea. A reparação óssea foi eficiente, notadamente intramembranosa, contudo nos subgrupos B e C foi observada a presença de cartilagem.

Repercussions of mild diabetes on pregnancy in Wistar rats and on the fetal development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of exercise on the reproductive outcome and fetal development of diabetic rats

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Evaluation of neonatally-induced mild diabetes in rats: Maternal and fetal repercussions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)