Glucosamine and chondroitin sulfate association increases tibial epiphyseal growth plate proliferation and bone formation in ovariectomized rats

OBJECTIVE: The growth plate consists of organized hyaline cartilage and serves as a scaffold for endochondral ossification, a process that mediates longitudinal bone growth. Based on evidence showing that the oral administration of glucosamine sulfate (GS) and/or chondroitin sulfate (CS) is clinically valuable for the treatment of compromised articular cartilage, the current study evaluated the effects of these molecules on the tibial epiphyseal growth plate in female rats. METHOD: The animals were divided into two control groups, including vehicle treatment for 45 days (GC45) and 60 days (GC60) and six ovariectomized (OVX) groups, including vehicle treatment for 45 days (GV45), GS for 45 days (GE45GS), GS+CS for 45 days (GE45GS+CS), vehicle for 60 days (GV60), GS for 60 days (GE60GS) and GS+CS for 60 days (GE60GS+CS). At the end of treatment, the tibias were dissected, decalcified and processed for paraffin embedding. Morphological and morphometric methods were employed for analyzing the distal tibial growth plates using picrosirius red staining and the samples were processed for histochemical hyaluronan detection. Morphometric analyses were performed using the 6.0ProPlus¯ Image system. RESULTS: Notably, after 60 days of treatment, the number of proliferative chondrocytes increased two-fold, the percentage of remaining cartilage increased four-fold and the percentage of trabecular bone increased three-fold in comparison to the control animals. CONCLUSION: GS and CS treatment drugs led to marked cellular proliferation of the growth plate and bone formation, showing that drug targeting of the tibial epiphyseal growth plate promoted longitudinal bone growth

Glucosamine and chondroitin sulfate association increases tibial epiphyseal growth plate proliferation and bone formation in ovariectomized rats

OBJECTIVE: the growth plate consists of organized hyaline cartilage and serves as a scaffold for endochondral ossification, a process that mediates longitudinal bone growth. Based on evidence showing that the oral administration of glucosamine sulfate (GS) and/or chondroitin sulfate (CS) is clinically valuable for the treatment of compromised articular cartilage, the current study evaluated the effects of these molecules on the tibial epiphyseal growth plate in female rats.METHOD: the animals were divided into two control groups, including vehicle treatment for 45 days (GC45) and 60 days (GC60) and six ovariectomized (OVX) groups, including vehicle treatment for 45 days (GV45), GS for 45 days (GE45GS), GS+CS for 45 days (GE45GS+CS), vehicle for 60 days (GV60), GS for 60 days (GE60GS) and GS+CS for 60 days (GE60GS+CS). At the end of treatment, the tibias were dissected, decalcified and processed for paraffin embedding. Morphological and morphometric methods were employed for analyzing the distal tibial growth plates using picrosirius red staining and the samples were processed for histochemical hyaluronan detection. Morphometric analyses were performed using the 6.0ProPlus (R) Image system.RESULTS: Notably, after 60 days of treatment, the number of proliferative chondrocytes increased two-fold, the percentage of remaining cartilage increased four-fold and the percentage of trabecular bone increased threefold in comparison to the control animals.CONCLUSION: GS and CS treatment drugs led to marked cellular proliferation of the growth plate and bone formation, showing that drug targeting of the tibial epiphyseal growth plate promoted longitudinal bone growth.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Dept Gynecol & Climaterium, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Gynecol & Climaterium, São Paulo, BrazilFAPESP: 05/57914-8Web of Scienc

Regenerative Eigenschaften von Laufbandtraining auf die Struktur des peripheren Skelettmuskes der orchiektomierten Ratte

Der Alterungsprozess des Menschen steht im Zusammenhang mit Funktionseinschränkungen des Bewegungsapparates und geht mit Veränderungen der strukturellen und mechanischen Eigenschaften des peripheren Skelettmuskels einher. In der Arbeit wurde der regenerative Einfluss von körperlicher Aktivität auf den sarkopenen Muskel im andropausalen Organismus der orchiektomierten Ratte untersucht. Die Ergebnisse zeigen, dass Laufbandtraining in unterschiedlichen Trainingsmodalitäten geeignet ist, den Muskelschwund bei Sarkopenie aufzuhalten

Extraction et analyse comparative des propriétés mécaniques intrinsèques des plaques de croissance porcines à quatre stades développementaux

RÉSUMÉ La croissance longitudinale du tissu osseux est régulée par les hormones, la nutrition, l‘environnement génétique et les forces mécaniques. L‘environnement mécanique constitue un élément clé dans ce processus, car des forces excessives peuvent être impliquées dans la progression des maladies musculosquelettiques infantiles et juvéniles. L‘accentuation de leurs progressions coïncide souvent avec des périodes de croissance accélérées, notamment, les poussées de croissance juvéniles et adolescentes. La croissance longitudinale des os longs et des vertèbres s‘opère au droit des plaques de croissance qui sont divisées en trois zones histologiques distinctes (réserve, proliférative, hypertrophique). Des études antérieures ont investigué le comportement biomécanique des plaques de croissance, ainsi que leurs zones histologiques, mais aucune étude n‘a, à ce jour, caractérisé la variation de ce comportement avec le stade de développement. Cette étude vise à extraire les propriétés mécaniques intrinsèques des plaques de croissance porcines à quatre stades de développement afin de caractériser la sensibilité biomécanique des plaques de croissance en fonction du stade développemental. Dans des expériences antérieures, des explants de plaques de croissance porcines à quatre stades de développement (nouveau-né, quatre, huit et 18 semaines) ont été testés sous compression non confinée en relaxation de contraintes. Ces explants ont été soumis à une déformation de 1.5x10-3 s-1 jusqu‘à l‘obtention de l‘équilibre, suivant un critère de relaxation de 0.05 g/min. Les données expérimentales ont été récupérées et modélisées selon une loi de comportement biphasique isotrope transverse décrit par les perméabilités transverse (k1) et hors plan (k3), les modules d‘Young transverse (E1) et hors plan (E3), et les coefficients de Poisson transverse (ν21) et hors plan (ν31). Le module d‘Young hors plan (E3) a été déterminé expérimentalement et la perméabilité hors plan (k3) n‘a pas été étudiée car elle est seulement déterminée de la compression confinée. L‘extraction des quatre autres propriétés mécaniques (k1, E1, ν21, ν31) a été réalisée par un recalage des courbes expérimentales avec ce modèle mathématique en utilisant un algorithme d‘optimisation. Afin d‘améliorer la corrélation graphique avec les courbes expérimentales, une étude de sensibilité a été effectuée pour déterminer les propriétés mécaniques les plus influentes sur le comportement de ce modèle avec le but de potentiellement modifier la définition de la fonction d‘erreur. Une procédure d‘extraction des propriétés mécaniques intrinsèques a été développée pour ce recalage en utilisant----------ABSTRACT Bone growth is a process regulated by hormones, nutrition, genetics and mechanical forces. When excessive, mechanical forces have key implications in the progression of infantile and juvenile musculoskeletal deformities, where marked progression is clinically observed during periods of rapid growth, notably, the juvenile and adolescent growth spurts. Longitudinal growth of long bones and vertebrae occurs in growth plates, which are divided into three distinct histological zones (reserve, proliferative and hypertrophic). Previous studies have investigated the biomechanical behaviour of growth plates and its histological zones, but no study has characterized the variation of this behaviour with developmental stage. The objective of this study is to extract the intrinsic mechanical properties of the porcine growth plate at four developmental stages in order to characterize the biomechanical sensitivity of growth plates as a function of developmental stage. In previous experiments, porcine growth plate explants at four developmental stages (newborn, four, eight and 18 weeks) were tested in unconfined compression under stress relaxation. These explants were subjected to a deformation of 1.5x10-3 s-1 until equilibrium; corresponding to relaxation criteria of 0.05 g/min. Experimental data obtained from these experiments were modeled with a transversely isotropic biphasic mathematical model which is described by the transverse and out-of-plane permeabilities (k1, k3), Young‘s moduli (E1, E3), and Poisson‘s ratios (ν21, ν31). The out-of-plane Young‘s modulus (E3) was determined experimentally and the out-of-plane permeability (k3) was not studied as it is only determined from confined compression. Extraction of the four remaining mechanical properties (k1, E1, ν21, ν31) was done by curve fitting experimental curves with this mathematical model using an optimization algorithm. In order to improve graphical correlation with experimental curves, a sensitivity study was done to determine the mechanical properties that have the greatest effect on the behaviour of this model, with the goal of potentially modifying the error function. A procedure to extract intrinsic mechanical properties was developed for curve fitting using a global optimization algorithm and a new error function based the relative error and the maximal stress. The criteria necessary for the use of this algorithm were determined though a rigorous process that evaluated their performance with analytical and experimental curves. Using this curve fitting procedure, mechanical properties were extracted for all samples, at all developmental age

Variação genética em mecanismos de neurotransmissão na osteoporose e em parâmetros metabólicos associados

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017A osteoporose é uma doença multifatorial cuja prevalência aumenta drasticamente com a idade. É caracterizada pela diminuição da densidade mineral óssea com aumento significativo do risco de fraturas e subsequentes complicações. Esta diminuição pode ser resultado de um aumento da reabsorção óssea por células osteoclastogénicas ou de uma diminuição da formação de nova matriz óssea por células osteoblásticas. Vários estudos têm demonstrado um papel relevante de diversas vias metabólicas no controlo da função destas células e têm sido identificados fatores genéticos do hospedeiro, envolvidos nestas vias metabólicas, que alteram a suscetibilidade para o desenvolvimento desta patologia. Diferentes estudos têm demonstrado a relevância do metabolismo do neurotransmissor serotonina no desenvolvimento de osteoporose e no risco de fraturas. A serotonina produzida no Sistema Nervoso Central (SNC) e no Sistema Nervoso Autónomo do intestino (células enterocromafins) tem recetores localizados nos osteoblastos e nos osteoclastos que estão envolvidos na ativação do turnover ósseo mediado por influência de mediadores parácrinos (RANKL ou ligando do recetor ativador do NFκB e osteocalcina) nos osteoclastos. O transportador de recaptação da serotonina (SERT) é um gene polimórfico tendo sido identificados 2 polimorfismos genéticos. Um deles localiza-se no intrão 4 (5HTTVNTR) e está associado a variações na estabilidade do mRNA e, consequentemente, a diferenças na taxa de recaptação. Também os recetores específicos da serotonina (5-HTR) têm polimorfismos genéticos que afetam a capacidade de ligação a este neurotransmissor. Estudos demonstraram que o recetor β2-adrenérgico (ADRB2), identificado em osteoblastos e osteoclastos, está envolvido na inibição da formação óssea pelos osteoblastos e que promove a osteoclastogénese através do aumento do RANKL. O gene que o codifica é polimórfico estando estas alterações genéticas associadas a variações na função deste recetor. Objetivo: estudar a associação dos polimorfismos genéticos em mecanismos de neurotransmissores na osteoporose e em parâmetros metabólicos. Métodos: foram estudados 272 indivíduos seguidos na consulta de endocrinologia da Clínica de Endocrinologia Diabetes e Metabolismo de Lisboa. A massa óssea foi avaliada por DXA em diferentes partes do esqueleto e os doentes divididos em osteoporose (n=120) e densidade mineral óssea normal (n=152). Os indivíduos foram genotipados para os polimorfismos 5HTTVNTR por PCR e 5HT2A_T102C, Arg16Gly e Glu276Gln por PCR-RFLP. A visualização dos produtos de amplificação bem como de restrição foi efetuada num transiluminador de ultravioleta após eletroforese dos produtos em gel de agarose. Foi feita a análise estatística dos dados por SPSS 23.0 e considerada significância estatística para p<0,05. Resultados: obtiveram-se resultados para a condição de menopausa tendo sido, por isso, feita a análise separada para mulheres pre-menopáusicas, pós-menopáusicas e homens. Numa análise geral, verificaram-se diferenças nos níveis de glicémia e de serotonina nas plaquetas nas mulheres pré-menopáusicas com e sem osteoporose e diferenças nos níveis de insulina, colesterol total, osteocalcina e serotonina plasmática nas mulheres pós-menopáusicas com e sem osteoporose; verificou-se também relações diretas e inversas tanto na análise da população em geral (densidade mineral óssea normal + osteoporose), como na análise das duas populações em separado; a nível genético, observou-se uma diferença entre a população controlo e estudo no polimorfismo Arg16Gly em mulheres pós-menopáusicas e no polimorfismo 5HTR2A em homens; na relação dos polimorfismos com os parâmetros metabólicos, para o polimorfismo 5HTTVNTR verificaram-se diferenças nos níveis de serotonina plasmática, HDL e osteocalcina; para o polimorfismo 5HTR2A houve diferenças nos níveis de glicémia e HDL; para o polimorfismo Arg16Gly verificaram-se diferenças nos níveis de glicémia e colesterol total e por fim, no polimorfismo Glu27Gln observaram-se diferenças nos níveis de serotonina plaquetária e do HOMAIR. É de salientar que estes polimorfismos, também foram estudados para a população em geral e para as duas populações (densidade mineral óssea normal e osteoporose) em separado. A única condição constante foi a separação entre mulheres pré-menopáusicas, mulheres pós-menopáusicas e homens. Conclusões: com este trabalho conseguiu-se concluir que o osso não é um órgão isolado e que está em constante atividade e interação com outros sistemas do organismo humano. De facto, a suscetibilidade à osteoporose está relacionada tanto com os parâmetros ósseos como com parâmetros do metabolismo lipídico e hormonas do sistema endócrino, havendo um aumento de possibilidades na sua prevenção e tratamento. Sendo assim, este estudo pode ser um ponto de partida para outras equipas de investigação para a pesquisa ou adaptação de novas terapêuticas existentes, a grupos com um background genético específico.Osteoporosis is a multifactor disease which drastically increases with age. The source is the low bone density created by an increase of bone resorption by osteoclasts and / or decreased bone formation by osteoblasts in bone remodeling. Several studies show that these two types of cells are controlled by several metabolic pathways, whereby this pathology are associated with many others. Because of this, the study of these pathways is the best form of prevention. Objective: To study an association of genetic polymorphisms in neurotransmitter mechanisms in osteoporosis and metabolic diseases. Methods: 272 subjects were followed up at the endocrinology clinic of the Diabetes and Metabolism Clinic of Lisbon. Bone mass was assessed by DXA in different parts of the skeleton and patients divided in osteoporosis (n = 120) and normal bone mineral density (n = 152). Subjects were genotyped for 5HTTVNTR polymorphisms by PCR and 5HT2A_T102C, Arg16Gly and Glu276Gln by PCR-RFLP. The visualization of the amplification products as well as restriction was performed in an ultraviolet transilluminator after electrophoresis of the agarose gel products. Statistical analysis of the data by SPSS 23.0 was performed and statistical significance was considered for p <0.05. Results: Results were obtained for menopausal status, and separate analysis was performed for premenopausal, postmenopausal and men. In general analysis, there were differences in glycemia and platelet serotonin levels in premenopausal women with and without osteoporosis and differences in insulin, total cholesterol, osteocalcin and plasma serotonin levels in postmenopausal women with and without osteoporosis; there were also direct and inverse relationships both in the analysis of the population in general (control and with osteoporosis) and in the analysis of the two populations separately; At the genetic level, a difference was observed between the control and study population in the Arg16Gly polymorphism in postmenopausal women and in the 5HTR2A polymorphism in men; in the relation of the polymorphisms with the metabolic parameters, for the 5HTTVNTR polymorphism there were differences in the levels of plasma serotonin, HDL and osteocalin, for the 5HTR2A polymorphism there were differences in glycemia and HDL levels, for Arg16Gly polymorphism there were differences in levels of glycemia and total cholesterol and, finally, in the Glu27Gln polymorphism, differences in platelet serotonin and HOMAIR levels were observed. It should be noted that for these polymorphisms, they were also studied for the general population and for the two populations (control + osteoporosis) separately. The only constant condition was the separation between premenopausal women, postmenopausal women and men. Conclusions: with this work it was possible to conclude that the bone is not an isolated organ and that it is in constant activity and interaction with other systems of the human organism. In fact, susceptibility to osteoporosis is related to bone parameters, parameters of the lipid metabolism and hormones of the endocrine system. That increase the possibilities in its prevention and treatment. This study can be a starting point for other research teams towards the search for new and/or adapting existing therapeutics, to groups with a specific genetic "background"

Normal bone growth requires optimal estrogen levels: negative effects of both high and low dose estrogen on the number of growth plate chondrocytes

Endochondral bone formation at epiphyseal growth plate consists of the synchronized processes of chondrogenesis and cartilage ossification. Estrogen, the major female sex hormone, plays an important role in this process, particularly during the pubertal growth spurt. However, its effects on the growth plate are not completely understood. The aims of this study were to clarify the effects of estrogen on the kinetics of chondrocytes in the growth plates of 10- to 25-week-old female rabbits by studying the effects of ovariectomy or high-dose administration of estrogen on the balance between cell proliferation and death. Forty-eight Japanese white rabbits were divided into three groups: sham operated, ovariectomized, or ovariectomized with subsequent weekly injection of high dose estrogen from 10 weeks. The chondrocyte kinetics was investigated by histomorphometry and immunohistochemistry, using antibodies for caspase-3, a marker of apoptosis, and for proliferating cell nuclear antigen. Both ovariectomized and estrogen-injected rabbits showed a declination of the chondrocyte number although the latter animals indicated a more dramatic effect. Estrogen-injected rabbits showed a decrease in the cell proliferating ability together with an increase in chondrocytes undergoing apoptosis while ovariectomy mainly reduced the cell proliferating ability. Given the known importance of estrogen for bone growth, one would expect that ovariectomy and high-dose administration of estrogen would have opposite effects. However, the present study indicated that both low and high concentration had a similar effect: a decrease in the chondrocyte number compared with control, suggesting that estrogen has to be maintained within a narrow range for optimal bone growth