Rol de la osteocalcina más allá del hueso / Role of osteocalcin beyond bone

Los hallazgos osteológicos se intensi!caron en los últimos años. Se demostró que el esqueleto se comporta, además de sus funciones clásicas, como un órgano de secreción endocrina que sintetiza al menos dos hormonas: el factor de crecimiento de !broblastos 23 (FGF-23) y la osteocalcina (Ocn). La Ocn es un péptido pequeño que contiene 3 residuos de ácido glutámico. Estos residuos se carboxilan postraduccionalmente, quedando retenida en la matriz ósea. La forma decarboxilada en el primer residuo de ácido glutámico (GluOcn) fue reportada por poseer efectos biológicos; la resorción ósea es el mecanismo clave para su bioactivación. La presente revisión se centra en los conocimientos actuales sobre la función hormonal de la Ocn. A la fecha se reporta que la Ocn regularía el metabolismo energético aumentando la proliferación de células ` pancreáticas, y la secreción de insulina y de adiponectina. Sobre el músculo esquelético actuaría favoreciendo la absorción y el catabolismo de nutrientes. La función reproductiva masculina estaría regulada mediante el estímulo a las células de Leydig para sintetizar testosterona; en el desarrollo cerebral y la cognición, la Ocn aumentaría la síntesis de neurotransmisores monoaminados y disminuiría el neurotransmisor inhibidor GABA. Si bien son indispensables mayores evidencias para dilucidar los mecanismos reguladores por medio de los cuales actuaría la Ocn, los resultados enumerados en los distintos estudios experimentales establecen la importancia de este novedoso integrante molecular. Dilucidar su rol dentro de estos procesos interrelacionados en seres humanos abriría la posibilidad de utilizar a la Ocn en el tratamiento de enfermedades endocrino-metabólicas. (AU)

Osteological !ndings have intensi!ed in recent years. The skeleton behaves as an endocrine secretion organ that synthesizes at least two hormones: osteocalcin (Ocn) and !broblast growth factor 23 (FGF-23). Ocn is a small peptide that contains 3 glutamic acid residues. After translation, these residues are carboxylated to make possible its retention into the bone matrix. Decarboxylation on the !rst glutamic acid residue (GluOcn) has been reported to have biological effects. Bone resorption is the key mechanism for its bioactivation. This review focuses on current knowledge on Ocn hormonal function. It has been reported that Ocn regulates energy metabolism by increasing the proliferation of pancreatic ` cells, and the secretion of insulin and adiponectin. On the skeletal muscle, it may act by favoring the absorption and catabolism of nutrients. Male reproductive function might be regulated by stimulating Leydig cells to synthesize testosterone. Regarding brain development and cognition, Ocn would increase monoamine neurotransmitters synthesis and decrease inhibitory neurotransmitter GABA. Although more evidence is needed to elucidate the regulatory mechanisms of Ocn, different experimental studies establish the importance of this novel molecular mediator. Clarifying its role within interrelated processes in humans, might open the possibility of using Ocn in different treatments of endocrine-metabolic diseases. (AU)

In-office tooth bleaching with 38% hydrogen peroxide promotes moderate/severe pulp inflammation and production of ll-1ß, TNF-ß, GPX, FGF-2 and osteocalcin in rats

Abstract Objectives: To study the intensity of inflammatory infiltrate and production of interleukin-1β (ll-1β), tumor necrosis factor-β (TNF-β), fibroblast growth factor-2 (FGF-2), glutathione peroxidase (GPX), and osteocalcin in response to in-office tooth bleaching in rats. Material and Methods: Twenty male Wistar rats were randomized into four groups (n=5) according to the received treatment (tooth bleaching or no treatment - control) and the period of euthanasia after treatment (24 h or 10 days). We performed tooth bleaching using a 38% hydrogen peroxide gel on maxillary and mandibular incisors. After euthanasia, incisors (20 per group) were processed for histological analysis, immunohistochemistry staining of ll-1β, TNF-β, FGF-2 and GPX and osteocalcin by immunofluorescence. We analyzed data using the Mann-Whitney and Kruskal-Wallis/Dunn tests (p<0.05). Results: The bleached groups presented statistically significant differences regarding the pulp inflammation stage compared with the control groups. Bleached teeth showed moderate/severe inflammatory infiltrate and control groups presented absent inflammatory cells or a negligible number of mononuclear cells (p<0.001) at two times (24 h and 10 days). There was strong staining for ll-1β, TNF-β, and GPX in bleached groups at 24 h and strong staining for ll-1β, TNF-β, GPX and FGF-2 at 10 days. After 10 days of tooth bleaching, the bleached group showed a statistically superior amount of osteocalcin than the other groups (p<0.01). Conclusions: Tooth bleaching with 38% hydrogen peroxide causes severe pulp inflammation, but characteristics of tissue repair after 10 days.

El esqueleto como órgano endocrino: funciones metabólicas de la osteocalcina / The skeleton as an endocrine organ: metabolic functions of osteocalcin

El esqueleto es uno de los sistemas más grandes de un vertebrado y, como tal, es razonable especular que no puede funcionar aislado del resto del organismo. De hecho, sabemos que existen sistemas complejos de regulación cruzada entre el esqueleto y muchos otros órganos. Hoy poseemos herramientas que nos permiten realizar supresión genética en células o tejidos específicos. Esto nos ha permitido comprender cómo los órganos se comunican entre sí y ha revitalizado el concepto de fisiología del organismo como un todo. Efectivamente, los últimos años han sido testigos del descubrimiento de funciones inesperadas que ejerce el esqueleto y que afectan al organismo en su totalidad. Una de tales funciones reconocidas recientemente es el control del metabolismo energético, a través de la secreción de osteocalcina. La osteocalcina es una hormona producida por los osteoblastos que regula la secreción de insulina, la sensibilidad a esta hormona y el metabolismo energético. Los hallazgos iniciales suscitaron varias preguntas fundamentales sobre la naturaleza de la acción de la insulina sobre el hueso. Pero esto solo fue la punta del iceberg. Efectivamente, más adelante se descubrió, mediante el análisis de ratones que carecen del receptor de insulina (Ins R) solamente en osteoblastos, que la acción de la insulina sobre estas células favorecía la homeostasis de la glucosa en todo el cuerpo. Es importante destacar que esta función de la insulina en los osteoblastos opera mediante la regulación negativa de la carboxilación y la biodisponibilidad de la osteocalcina. Más aún, se observó que las vías de señalización de la insulina en los osteoblastos regulan positivamente no solo la formación sino también la resorción del hueso. Curiosamente, parece que las vías de señalización de la insulina en osteoblastos pueden inducir la activación de la osteocalcina mediante la estimulación de la actividad de los osteoclastos. De hecho, el bajo pH generado durante la resorción ósea es suficiente para desencadenar la descarboxilación (y subsiguiente activación) de la osteocalcina. En breve discutiremos dos nuevas proposiciones: 1) los osteoblastos son un blanco utilizado por la insulina para controlar la homeostasis de la glucosa en todo el organismo y 2) la resorción ósea desempeña un papel fundamental en la regulación de la activación de la osteocalcina. (AU)

The skeleton is one of the biggest systems in a vertebrate animal and, as such, it is reasonable to speculate that it cannot function isolated from the rest of the organism. In fact, we know that complex systems exist for the cross-regulation between the skeleton and several other organs. Today, we have the tools that allow us to perform genetic suppression in specific cells or tissues. This has allow us understand the mechanisms by which the organs communicate with each other and has revitalized the concept of organismal physiology as a whole. Studies conducted in recent years have uncovered unexpected functions performed by the skeleton. One of these is the control of global energy metabolism, through the secretion of osteocalcin, a protein produced by osteoblasts that acts as a hormone regulating insulin secretion, insulin sensitivity and energy expenditure. The evidence comes from the analysis of mice lacking insulin receptor (InsR) exclusively in osteoblasts. These mice have a global metabolic phenotype demonstrating that the action of insulin in osteoblasts promotes the homeostasis of glucose throughout the body. This action of insulin in osteoblasts is mediated by the negative regulation of the carboxylation (and bioavailability) of osteocalcin. The decarboxylation (and activation) of osteocalcin, in turn, occurs in the osteoclastic resorption pit. Briefly: the osteoblast is a target used by insulin to control the homeostasis of glucose throughout the body and bone resorption is the mechanism that regulates the activation of osteocalcin. (AU)

Effects of low-level laser therapy on human osteoblastic cells grown on titanium

The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.

Este estudo teve como objetivo investigar o efeito do laser diodo de gálio-alumínio-arsênio (GaAlAs) em células osteoblásticas humanas cultivadas sobre discos de Ti. Para tanto, células osteoblásticas foram obtidas por digestão enzimática de osso alveolar humano e cultivadas sobre discos de Ti por 17 dias. As células foram submetidas à irradiação no 3º e 7º dias na dose de 3 J/cm2 e comprimento de onda de 780 nm e células não irradiadas foram usadas como controle. A irradiação não alterou a proliferação celular, atividade de ALP e formação de matriz mineralizada. Microscopia por epifluorescência indicou que após 24 h da aplicação do laser, as culturas irradiadas apresentaram áreas sem células, que mais tarde foram repovoadas por células em fase de proliferação e menos diferenciadas. O laser aumentou a expressão gênica relativa da ALP, OC, BSP e BMP-7 e reduziu a de RUNX2, OPN e OPG. Os resultados indicam que a terapia com laser modula de forma complexa as respostas celulares, estimulando a diferenciação osteoblástica. Assim, é possível sugerir possíveis benefícios do laser na osseointegração de implantes de Ti apesar do efeito deletério às células imediatamente após a irradiação.

Effect of titanium surface roughness on human bone marrow cell proliferation and differentiation: an experimental study / Efeito da rugosidade de superfície de discos de titânio sobre a proliferação e diferenciação de células de medula óssea humana: estudo experimental

PURPOSE: To assess the proliferation and differentiation of human bone marrow-derived cells cultured on titanium surfaces with different roughness characteristics. METHODS: Cells obtained from the iliac crest of an adult human donor were routinely processed and cultured on titanium surfaces of varying roughness, according to their preparation method: polishing only (smooth surface) and polishing followed by etching with HF/HNO3 for 15 and 30 minutes (rough surfaces). Surfaces were assessed using scanning electronic microscopy and profilometry. RESULTS: Titanium disks etched with acid for 15 minutes allowed greater cell proliferation in all culture periods. The level of osteopontin and osteocalcin expression was increased in both acid-etched groups, which indicates an advanced stage of differentiation of cells into osteoblasts. CONCLUSIONS: Increased surface roughness accelerates the differentiation of undifferentiated mesenchymal cells into osteogenic lineage cells, but does not necessarily favor cell proliferation. An intermediate surface roughness of 0.5µm (acid etching for 15 minutes) favors both initial and final cell responses.

OBJETIVO: Avaliar a proliferação e diferenciação de células derivadas da medula óssea humana sobre superfícies de titânio com diferentes rugosidades de superfície. MÉTODOS: Células obtidas da crista ilíaca de um doador humano adulto foram rotineiramente processadas e cultivadas sobre superfícies de titânio preparadas através de polimento apenas, ou polimento seguido de condicionamento com HF/HNO3 por 15 e 30 minutos, para produzir superfícies com rugosidades variadas, conforme determinado por Microscopia Eletrônica de Varredura e perfilometria. RESULTADOS: Discos de titânio condicionados com ácido por 15 minutos permitiram maior proliferação celular em todos os períodos de cultura. O nível de expressão das proteínas osteopontina e osteocalcina estava aumentado em ambos os grupos condicionados com ácido, indicando que as células estavam comprometidas com o fenótipo osteogênico. CONCLUSÕES: O aumento na rugosidade de superfície acelera a diferenciação de células mesenquimais indiferenciadas em células de linhagem osteogênica, mas não necessariamente favorece a proliferação celular. Uma superfície com rugosidade intermediária de 0,5µm (condicionada com ácido por 15 minutos) favorece tanto as respostas celulares iniciais quanto as finais.

Influence of the microenvironment on gene and protein expression of odontogenic-like and osteogenic-like cells

Progenitor cells play an important biological role in tooth and bone formation, and previous analyses during bone and dentine induction have indicated that they may be a good alternative for tissue engineering. Thus, to clarify the influence of the microenvironment on protein and gene expression, MDPC-23 cells (mouse dental papilla cell line) and KUSA/A1 cells (bone marrow stromal cell line) were used, both in vitro cell culture and in intra-abdominal diffusion chambers implanted in 4-week-old male immunodefficient mice (SCID mice). Our results indicate that KUSA/A1 cells differentiated into osteoblast-like cells and induced bone tissue inside the chamber, whereas, MDPC-23 showed odontoblast-like characteristics but with a low ability to induce dentin formation. This study shows that MDPC-23 cells are especial cel ls, which possess morphological and functional characteristics of odontoblast-like cells expressing dentin sialophosphoprotein in vivo. In contrast, dentin sialophosphoprotein gene and protein expression was not detected in both cell lines in vitro. The intra-abdominal diffusion chamber appears as an interesting experimental model for studying phenotypic expression of dental pulp cells in vivo.

Induction of type I collagen and osteocalcin in human dental pulp cells by retinoic acid.

Retinoic acid has been known to play a key role in the regulation of bone cell differentiation and function. The effects of retinoic acid on human dental pulp cells, which contain several characteristics similar to those of bone cells, has yet to be elucidated extensively. The effects of retinoic acid on human dental pulp cells in terms of type I collagen and osteocalcin induction were investigated in vitro. Dental pulp cells obtained from the teeth of young patients (age between 18-22 years) were cultured and subsequently treated with various concentrations of retinoic acid (0, 10(-7), 10(-6), 10(-5) M) in serum-free DMEM. At different time intervals (8, 12 and 24 hours), the levels of type I collagen and osteocalcin secreted were determined using Type I Procollagen C-Peptide and Gla-type Osteocalcin EIA kits, respectively. Induction effects were evaluated using analysis of variance and the Duncan's multiple rank test. Retinoic acid at concentrations of 10(-5), 10(-6), 10(-7) M was able to induce type I collagen and osteocalcin production in human dental pulp cells within 12 hours of exposure. Dose-dependent induction was observed only after 24 hours. A two-fold increase in osteocalcin level was detected after exposed to 10(-5) M retinoic acid within 24 hours. Our data suggest that retinoic acid at concentrations of 10(-5), 10(-6), 10(-7) M has the ability to induce type I collagen and osteocalcin secretions in human dental pulp cells in vitro.