Increased Osteoblastic and Osteocytic in Vitro Cell Viability by Yerba Mate (Ilex paraguariensis).

BACKGROUND: Yerba mate (YM, Ilex paraguariensis) consumption beneficially affects the bones. However, whether YM components exert their effect on bone cells directly remains elusive. METHODS: We evaluated how main YM components affect osteoblastic (MC3T3-E1) and osteocytic (MLO-Y4) cells in vitro when administered separately or in an aqueous extract. MC3T3-E1 and MLO-Y4 cells were exposed to three different experimental conditions: (1) Caffeine, chlorogenic acid, and their combinations; (2) Caffeine, rutin, and their combinations; (3) Aqueous YM extract. RESULTS: All polyphenol and caffeine concentrations as well as that of their tested combinations significantly increased MC3T3-E1 cell viability from 16.6% to 34.8% compared to the control. In MLO-Y4 cells, the lowest rutin and the two highest caffeine concentrations significantly increased cell viability by 11.9, 14.9, and 13.7%, respectively. While rutin and caffeine combinations tended to increase MLO-Y4 cell viability, different chlorogenic acid and caffeine combinations did not affect it. Finally, the aqueous YM extract significantly increased MLO-Y4, MC3T3-E1, and differentiated MC3T3-E1 cell viability compared to the control without treatment. CONCLUSIONS: YM components (rutin, chlorogenic acid, and caffeine) positively affected bone cells, mainly pre-osteoblast cells. Moreover, the aqueous YM extract significantly increased MLO-Y4, MC3T3-E1, and differentiated MC3T3-E1 cell viabilities indicating an additional relevant nutritional property of YM infusion. Further studies would be required to elucidate the underlying effector mechanism of YM on the bones and its relationship with previously described in vivo positive effects.

Cellular and Molecular Mechanisms Associating Obesity to Bone Loss.

Obesity is an alarming disease that favors the upset of other illnesses and enhances mortality. It is spreading fast worldwide may affect more than 1 billion people by 2030. The imbalance between excessive food ingestion and less energy expenditure leads to pathological adipose tissue expansion, characterized by increased production of proinflammatory mediators with harmful interferences in the whole organism. Bone tissue is one of those target tissues in obesity. Bone is a mineralized connective tissue that is constantly renewed to maintain its mechanical properties. Osteoblasts are responsible for extracellular matrix synthesis, while osteoclasts resorb damaged bone, and the osteocytes have a regulatory role in this process, releasing growth factors and other proteins. A balanced activity among these actors is necessary for healthy bone remodeling. In obesity, several mechanisms may trigger incorrect remodeling, increasing bone resorption to the detriment of bone formation rates. Thus, excessive weight gain may represent higher bone fragility and fracture risk. This review highlights recent insights on the central mechanisms related to obesity-associated abnormal bone. Publications from the last ten years have shown that the main molecular mechanisms associated with obesity and bone loss involve: proinflammatory adipokines and osteokines production, oxidative stress, non-coding RNA interference, insulin resistance, and changes in gut microbiota. The data collection unveils new targets for prevention and putative therapeutic tools against unbalancing bone metabolism during obesity.

Bone cells and their role in physiological remodeling.

PURPOSE: This work compiles the characteristics of bone cells involved in the physiological bone remodeling. METHODS: A narrative review of the literature was performed. RESULTS: Remodeling is a different process from modeling. Remodeling allows old or damaged bone tissue to be renewed, ensuring the maintenance of bone fracture resistance, as well as maintaining calcium and phosphorus homeostasis. We present the role of osteoclasts, a multinucleated cell with hematopoietic origin responsible for resorbing bone. The formation of osteoclasts depends on the cytokines macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL) and can be blocked by osteoprotegerin. Furthermore, this review highlights the features of osteoblasts, polarized cubic cells of mesenchymal origin that deposit bone and also covers osteocytes and bone lining cells. This review presents the five fundamental phases of bone remodeling and addresses aspects of its regulation through hormones and growth factors. CONCLUSIONS: Knowledge of the current concepts of physiological bone remodeling is necessary for the study of the different pathologies that affect the bone tissue and thus helps in the search for new therapies.

Bone fracture healing: perspectives according to molecular basis.

Fractures have a great impact on health all around the world and with fracture healing optimization; this problem could be resolved partially. To make a practical contribution to this issue, the knowledge of bone tissue, cellularity, and metabolism is essential, especially cytoskeletal architecture and its transformations according to external pressures. Special physical and chemical characteristics of the extracellular matrix (ECM) allow the transmission of mechanical stimuli from outside the cell to the plasmatic membrane. The osteocyte cytoskeleton is conformed by a complex network of actin and microtubules combined with crosslinker proteins like vinculin and fimbrin, connecting and transmitting outside stimuli through EMC to cytoplasm. Herein, critical signaling pathways like Cx43-depending ones, MAPK/ERK, Wnt, YAP/TAZ, Rho-ROCK, and others are activated due to mechanical stimuli, resulting in osteocyte cytoskeletal changes and ECM remodeling, altering the tissue and, therefore, the bone. In recent years, the osteocyte has gained more interest and value in relation to bone homeostasis as a great coordinator of other cell populations, thanks to its unique functions. By integrating the latest advances in relation to intracellular signaling pathways, mechanotransmission system of the osteocyte and bone tissue engineering, there are promising experimental strategies, while some are ready for clinical trials. This work aims to show clearly and precisely the integration between cytoskeleton and main molecular pathways in relation to mechanotransmission mechanism in osteocytes, and the use of this theoretical knowledge in therapeutic tools for bone fracture healing.

Bovine Milk Extracellular Vesicles Are Osteoprotective by Increasing Osteocyte Numbers and Targeting RANKL/OPG System in Experimental Models of Bone Loss.

Studying effects of milk components on bone may have a clinical impact as milk is highly associated with bone maintenance, and clinical studies provided controversial associations with dairy consumption. We aimed to evaluate the impact of milk extracellular vesicles (mEVs) on the dynamics of bone loss in mice. MEVs are nanoparticles containing proteins, mRNA and microRNA, and were supplemented into the drinking water of mice, either receiving diet-induced obesity or ovariectomy (OVX). Mice receiving mEVs were protected from the bone loss caused by diet-induced obesity. In a more severe model of bone loss, OVX, higher osteoclast numbers in the femur were found, which were lowered by mEV treatment. Additionally, the osteoclastogenic potential of bone marrow-derived precursor cells was lowered in mEV-treated mice. The reduced stiffness in the femur of OVX mice was consequently reversed by mEV treatment, accompanied by improvement in the bone microarchitecture. In general, the RANKL/OPG ratio increased systemically and locally in both models and was rescued by mEV treatment. The number of osteocytes, as primary regulators of the RANKL/OPG system, raised in the femur of the OVX mEVs-treated group compared to OVX non-treated mice. Also, the osteocyte cell line treated with mEVs demonstrated a lowered RANKL/OPG ratio. Thus, mEVs showed systemic and local osteoprotective properties in two mouse models of bone loss reflected in reduced osteoclast presence. Data reveal mEV potential in bone modulation, acting via osteocyte enhancement and RANKL/OPG regulation. We suggest that mEVs could be a therapeutic candidate for the treatment of bone loss.

Effects of ionizing radiation on woven bone: influence on the osteocyte lacunar network, collagen maturation, and microarchitecture.

OBJECTIVES: Evaluate the effects of ionizing radiation on microarchitecture, the osteocyte lacunar network, and collagen maturity in a bone repair site. MATERIALS AND METHODS: Bone defects were created on tibias of 20 New Zealand rabbits. After 2 weeks, the animals were randomly divided into (n = 10) NoIr (nonirradiated group) and Ir (irradiated group). In the Ir, the animals received single-dose irradiation of 30 Gy on the tibia and were euthanized after 2 weeks. Bone microarchitecture parameters were analyzed by using micro-CT, and the osteocyte lacunar network, bone matrix, and collagen maturation by histomorphometric analysis. The data were analyzed using unpaired Student's t test (α = 0.05). RESULTS: Trabecular thickness in Ir was lower than that in NoIr (P = 0.028). No difference was found for bone volume fraction and bone area. Lacunae filled with osteocytes were more numerous (P < 0.0001) in NoIr (2.6 ± 0.6) than in Ir (1.97 ± 0.53). Empty lacunae were more prevalent (P < 0.003) in Ir (0.14 ± 0.10) than in NoIr (0.1 ± 0.1). The mean osteocyte lacunae size was higher (P < 0.01) in Ir (15.4 ± 4.41) than in NoIr (12.7 ± 3.7). Picrosirius red analysis showed more (P < 0.05) mature collagen in NoIr (29.0 ± 5.3) than in Ir (23.4 ± 4.5). Immature collagen quantification revealed no difference between groups. CONCLUSIONS: Ionizing radiation compromised bone formation and an impairment in bone repair in irradiated woven bone was observed. CLINICAL RELEVANCE: Before radiotherapy, patients usually need surgical intervention, which may be better performed, if clinicians understand the repair process in irradiated bone, using novel approaches for treating these individuals.

Fibroblast-secreted trophic factors contribute with ECM remodeling stimulus and upmodulate osteocyte gene markers in osteoblasts.

As osteogenesis is a multifactorial mechanism, we wonder whether osteoblast-induced extracellular matrix (ECM) remodeling might be modulated by trophic factors released by fibroblasts in a paracrine signaling manner. To address this issue, fibroblasts were cultured for 72 h under conventional conditions when their conditioned medium was harvested and used to challenge pre-osteoblasts (MC3T3-E1 cells) for 14 days. Preliminarily, we validated the potential effect of fibroblasts in contributing to osteocyte phenotype, which specifically requires significant expression of Dentin Matrix Protein 1 (DMP1; about 10-fold changes) and Sclerostin (SOST; about 7-fold changes), both biomarkers of osteocyte. Fibroblasts also seem contributing to ECM remodeling in osteoblasts, because we detected a high level of both mRNA and enzyme activities of matrix metalloproteinase -9 (MMP-9) as well as a high level of reversion inducing cysteine rich protein with kazal motifs (RECK) transcripts (about 13-fold changes), a membrane-anchored MMP inhibitor, which seems to be a constitutive pathway in osteoblasts. Considering inflammatory panorama and using RTqPCR technology, both IL-13 (about 13-fold changes) and IL-33 (about 5-fold changes) genes were up-expressed in response to the fibroblast-secreted trophic factors, as were the receptor activator of NF-κB ligand (RANKL; about 8-fold changes) and osteoprotegerin (OPG; about 3-fold changes). Although preliminary, these data suggest a stimulus to finely control osteoclastogenesis, and this mechanism reinforces the role of fibroblasts in bone remodeling and homeostasis. Moreover, these results suggest an important crosstalk between fibroblast and osteoblast, when fibroblast-secreted trophic factors upmodulate osteocyte gene markers and contribute to ECM remodeling stimulus in osteoblast.

Alendronate inhibits osteocyte apoptosis and inflammation via IL-6, inhibiting bone resorption in periapical lesions of ovariectomized rats.

AIM: To evaluate the effect of alendronate (ALN) on the development of periapical lesions induced in ovariectomized rats. METHODOLOGY: Twenty-five rats were divided into three groups: sham (control), ovariectomy (OVX) and OVX + ALN. One day after OVX, animals from the OVX + ALN group received the medication via gavage. After 9 weeks, the first molars of all animals were submitted to periapical lesion induction. After 21 days, the animals were euthanized. Femurs were analysed for bone mineral density. The blocks of bone tissue containing the mandibular first molars were submitted to histotechnical processing and staining with haematoxylin and eosin (HE) for periapical lesion analysis under conventional microscopy. At the same time, the morphometric analysis of the periapical lesion area was performed in the fluorescence mode, as well as the histoenzimology for the quantification of osteoclasts and 4'-6-diamidino-2-phenylindole staining for the quantification of apoptotic osteocytes. In addition, the first maxillary molars were used for analysis of the gene expression of proinflammatory cytokines (IL-1ß, IL-6 and TNF-α) and osteoclastogenesis markers (RANKL/OPG). The results were submitted to ANOVA and Kruskal-Wallis tests and Tukey and Dunn post-tests (significance level of 5%). RESULTS: Ovariectomy reduced bone mineral density of the femur, and treatment with ALN was able to prevent bone loss (P < 0.001). Regarding the microscopic analysis of the periapical region, the sham and OVX + ALN groups had moderately increased periodontal ligament and inflammatory infiltrate, while the OVX group had these parameters increased intensely. The periapical lesions of the OVX group were significantly larger in area in comparison to the other groups (P < 0.001). The OVX group had the largest amount of apoptotic osteocytes, and ALN was able to prevent the apoptosis of these cells, in addition to significantly reducing IL-6 expression (P < 0.05). OVX and ALN had no effect on RANKL/OPG expression and did not influence the number of osteoclasts around the periapical lesion (P > 0.05). CONCLUSION: The hypoestrogenic condition induced by OVX aggravated bone resorption, inducing the death of osteocytes and provoking larger periapical lesions. ALN treatment inhibited osteocyte apoptosis and inflammation via IL-6, inhibiting bone resorption in periapical lesions of ovariectomized rats.

Effects of Nutritional Deficiency of Boron on the Bones of the Appendicular Skeleton of Mice.

Scientific evidence has shown the nutritional importance of boron (B) in the remodeling and repair of cancellous bone tissue. However, the effects of the nutritional deficiency of B on the cortical bone tissue of the appendicular skeleton have not yet been described. Thus, a study was performed to histomorphometrically evaluate the density of osteocyte lacunae of cortical bone of mouse femora under conditions of nutritional deficiency of B and to analyze the effects of the deficiency on the biomechanical properties of mouse tibiae. Weaning, 21-day-old male Swiss mice were assigned to the following two groups: controls (B+; n = 10) and experimental (B-; n = 10). Control mice were fed a basal diet containing 3 mg B/kg, whereas experimental mice were fed a B-deficient diet containing 0.07 mg B/kg for 9 weeks. The histological and histomorphometric evaluations of the mice fed a B-deficient diet showed a decrease in the density of osteocyte lacunae in the femoral cortical bone tissue and the evaluation of biomechanical properties showed lower bone rigidity in the tibia.

CKD-MBD: from the Pathogenesis to the Identification and Development of Potential Novel Therapeutic Targets.

PURPOSE OF REVIEW: Although we have seen tremendous advances in the comprehension of CKD-MBD pathophysiology during the last few years, this was not accompanied by a significant change in mortality rate and quality of life. This review will address the traditional and updated pathophysiology of CKD-MBD along with the therapeutic limitations that affect CKD-MBD and proposed alternative treatment targets. RECENT FINDINGS: An innovative concept brings the osteocyte to the center of CKD-MBD pathophysiology, in contrast to the traditional view of the skeleton as a target organ for disturbances in calcium, phosphate, parathyroid hormone, and vitamin D. Osteocytes, through the synthesis of FGF-23, sclerostin, among others, are able to interact with other organs, making bone an endocrine organ. Thus, osteocyte dysregulation might be an early event during the course of CKD. This review will revisit general concepts on the pathophysiology of CKD-MBD and discuss new perspectives for its treatment.

The p38 MAPK and NF-κB Pathways are Involved in Cyclic Compressive Force-induced IL-6 Secretion in MLO-Y4 Cells

ABSTRACT We previously revealed the involvement of extracellular regulated protein kinases 1/2 (ERK1/2) in interleukin-6 (IL-6) secretion induced by cyclic compressive force (CCF) in MLO-Y4 cells. In this study, we investigated the contributions of the p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways to IL-6 secretion by stimulating MLO-Y4 cells with CCF. At 80% confluence, different magnitudes (1000μstrain, 2000 μstrain and 4000 μstrain), frequencies (0.5 Hz, 1.0 Hz and 2.0 Hz) and durations (10 min, 30 min, 1 h, 3 h and 6 h) of CCF were loaded onto cells using a four-point bending system. Flow Cytometry (FCM) analysis was used to analyze cell mortality rates after CCF loading. p38 and p65 phosphorylation as well as IκBα degradation in MLO-Y4 cells were detected by Western blotting (WB). Changes in IL-6 secretion after inhibitor treatment were assessed by enzyme-linked immunosorbent assays (ELISAs). Cellular viability was over 90 percent after CCF. p38 and p65 phosphorylation increased under all conditions, whereas IκBα protein levels decreased. However, phosphorylation and degradation were not completely dependent on the loading magnitude, frequency or duration. Furthermore, p38 inhibition using the specific inhibitor SB203580 reduced both p38 phosphorylation and IL-6 secretion. Similarly, NF-κB inhibition using BAY 11-7082 decreased p65 phosphorylation and IL-6 secretion but increased the concentration of IκBα. These findings reveal significant roles for the p38 and NF-κB signaling pathways in IL-6 secretion induced by CCF in MLO-Y4 cells.

Osteoporosis and autophagy: What is the relationship? / Osteoporose e autofagia: qual é a relação?

Summary Autophagy is a survival pathway wherein non-functional proteins and organelles are degraded in lysosomes for recycling and energy production. Therefore, autophagy is fundamental for the maintenance of cell viability, acting as a quality control process that prevents the accumulation of unnecessary structures and oxidative stress. Increasing evidence has shown that autophagy dysfunction is related to several pathologies including neurodegenerative diseases and cancer. Moreover, recent studies have shown that autophagy plays an important role for the maintenance of bone homeostasis. For instance, in vitro and animal and human studies indicate that autophagy dysfunction in bone cells is associated with the onset of bone diseases such as osteoporosis. This review had the purpose of discussing the issue to confirm whether a relationship between autophagy dysfunction and osteoporosis exits.

Resumo A autofagia é uma via de sobrevivência celular pela qual proteínas e organelas não funcionais são degradadas nos lisossomos, para reciclagem e geração de energia. Assim, a autofagia é fundamental para a manutenção da homeostase e viabilidade da célula, agindo como um controle de qualidade que evita o acúmulo de estruturas desnecessárias e o estresse oxidativo. Um número crescente de estudos tem demonstrado que disfunções na via autofágica estão relacionadas ao surgimento de diversas doenças, como as neurodegenerativas e o câncer. Estudos também têm indicado que a autofagia exerce um importante papel para a manutenção da homeostase óssea; por exemplo, estudos in vitro e em animais e humanos mostram que disfunções da autofagia nas células ósseas estão associadas ao surgimento de doenças ósseas, como a osteoporose. Nesta revisão, foram abordados esses estudos, a fim de melhor esclarecer se há uma relação entre disfunção autofágica e osteoporose.

Osteocyte Alterations Induce Osteoclastogenesis in an In Vitro Model of Gaucher Disease.

Gaucher disease (GD) is caused by mutations in the glucosylceramidase ß (GBA 1) gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to the accumulation of the glycolipid glucocerebroside in the lysosomes of cells, mainly in the monocyte/macrophage lineage. Its mildest form is Type I GD, characterized by non-neuronopathic involvement. Bone compromise is the most disabling aspect of the Gaucher disease. However, the pathophysiological aspects of skeletal alterations are not yet fully understood. The bone tissue homeostasis is maintained by a balance between resorption of old bone by osteoclasts and new bone formation by osteoblasts. A central player in this balance is the osteocyte as it controls both processes. We studied the involvement of osteocytes in an in vitro chemical model of Gaucher disease. The osteocyte cell line MLO-Y4 was exposed to conduritol-ß-epoxide (CBE), an inhibitor of GCase, for a period of 7, 14 and 21 days. Conditioned media from CBE-treated osteocytes was found to induce osteoclast differentiation. GCase inhibition caused alterations in Cx43 expression and distribution pattern and an increase in osteocyte apoptosis. Osteoclast differentiation involved osteocyte apoptotic bodies, receptor activator of nuclear factor κ-B ligand (RANKL) and soluble factors. Thus, our results indicate that osteocytes may have a role to play in the bone pathophysiology of GD.

Osteocitos y la regulación de la formación ósea / Osteocytes and regulation of bone formation / Osteócitos e a regulação da formação óssea

Por muchos años los osteocitos han sido las células óseas "olvidadas" y consideradas espectadores inactivos enterrados en la matriz ósea. Hoy en día se sabe que los osteocitos detectan y responden a estímulos mecánicos y hormonales para coordinar tanto la resorción como la formación ósea. Actualmente se considera que los osteocitos proveen la mayoría de las moléculas que regulan la actividad de los osteoclastos y de los osteoblastos, como RANKL y esclerostina, ya que manipulaciones genéticas y famacológicas de cualquiera de estas dos moléculas afectan marcadamente la homeostasis ósea. Este artículo resume hallazgos recientes que delinean los mecanismos por los cuales los osteocitos regulan el número y actividad de los osteoblastos afectando de esta manera la formación ósea.

For many years, osteocytes have been the forgotten bone cells and considered as inactive spectators buried in the bone matrix. We now know that osteocytes detect and respond to mechanical and hormonal stimuli to coordinate bone resorption and bone formation. Osteocytes are currently considered a major source of molecules that regulate the activity of osteoclasts and osteoblasts, such as RANKL and sclerostin; and genetic and pharmacological manipulations of either molecule markedly affect bone homeostasis. This article summarizes recent findings demonstrating the mechanisms by which osteocytes regulate the number and activity of osteoblasts and thus affect bone formation.

Durante muitos anos, os osteócitos têm sido células ósseas "esquecidas" e consideradas como espectadores inativos enterrados na matriz óssea. Hoje sabemos que os osteócitos são capazes de detectar e responder a estímulos mecânicos e hormonais para coordenar tanto a reabsorção quanto a formação óssea. Os osteócitos são considerados atualmente como aquelesque fornecema maioria das moléculas que regulam a atividade dos osteoclastos e dos osteoblastos, tais como RANKL e a esclerostina,visto que manipulações genéticas e farmacológicas de qualquer uma destas moléculas afetam consideravelmente a homeostase óssea. Este artigo resume as recentes descobertas que demarcam os mecanismos pelos quais os osteócitos regulam o número e atividade dos osteoblastos, afetando assim a formação óssea.

Influence of osteoporosis on the osteocyte density of human mandibular bone samples: a controlled histological human study.

OBJECTIVES: Osteocytes have emerged as key regulators of skeletal and mineral homeostasis. The number of these cells could be influenced by the presence of osteoporosis and osteopenia. Hence, the aim this study was to evaluate the osteocyte density in patients with osteopenia, and in patients with osteoporosis treated with bisphosphonates. MATERIALS AND METHODS: Thirty-nine patients were selected for this study and divided into three groups: (A) nine healthy patients (control), (B) 15 patients with osteopenia, and (C) 15 patients with osteoporosis. During the surgical insertion of dental implants in the lower jaw, bone samples were retrieved and processed for histological analysis of osteocyte density, measured as number of osteocytes/bone tissue area (µm(2) ). RESULTS: Patients with osteopenia showed statistically higher values of osteocyte density than patients with osteoporosis (P < 0.05) No significant differences were detected between osteopenia and osteoporosis subjects vs. healthy patients (P > 0.05). CONCLUSIONS: Bone metabolism diseases (osteoporosis and osteopenia) do not seem to influence the osteocyte density; this could be due to the administration of bisphosphonates in patients with osteoporosis. This information could play a fundamental role in the diagnosis and treatment of patients in a postmenopausal stage.

ß-ARRESTIN/CONNEXIN 43 (Cx43) complex anchors ERKS outside the nucleus: a prerequisite for bisphosphonate antiapoptotic effect mediated by Cx43/ERK in osteocytes / El complejo ß-ARRESTINA/CONEXINA 43 (Cx43) retiene ERKS fuera del núcleo: un paso necesario para el efecto anti-apoptótico de los bisfosfonatos mediado por Cx34/ERK en osteocitos

Bisphosphonates (BPs) anti-fracture efficacy may be due in part to inhibition of osteocyte apoptosis. This effect requires opening of connexin (Cx) 43 hemichannels and phosphorylation of the extracellular signal regulated kinases (ERKs). However, unlike ERK activation by other stimuli, the Cx43/ERK pathway activated by BPs does not result in nuclear ERK accumulation. Instead, the anti-apoptotic effect of BPs depends on phosphorylation of cytoplasmic ERK targets and is abolished by forced nuclear retention of ERKs. We now report that ERKs and the scaffolding protein ß-arrestin co-immuno-precipitate with Cx43 in MLO-Y4 osteocytic cells and that the BP alendronate increases this association. Moreover, ERK2 fused to red fluorescent protein (ERK2-RFP) co-localizes with Cx43 fused to green fluorescent protein outside the nucleus in cells untreated or treated with alendronate. Alendronate does not induce ERK nuclear accumulation in cells transfected with wild type ß-arrestin (wtARR) or vector control, whereas it does in cells expressing a dominant negative ß-arrestin mutant (dnARR) consisting of the ß-arrestin-clathrin binding domain that competes with endogenous ß-arrestin for binding to clathrin. Alendronate activates ERKs in dnARRtransfected cells as effectively as in cells transfected with wtARR, demonstrating that dnARR only interferes with subcellular localization but not with activation of ERKs by BPs. Further, whereas alendronate inhibits apoptosis in cells expressing wtARR or vector control, it is ineffective in cells expressing dnARR. Thus, BPs induce the formation of a complex comprising Cx43, ß-arrestin, and clathrin, which directs ERKs outside the nucleus and is indispensable for osteocyte survival induced by BPs. (AU)

La efectividad de los bisfosfonatos (BPs) en la prevención de fracturas puede deberse en parte a la inhibición de la apoptosis de osteocitos. Este efecto depende de la apertura de hemicanales de conexina (Cx) 43 y la fosforilación de quinasas reguladas por señales extracelulares (ERKs). Sin embargo, a diferencia de la activación de ERKs debida a otros estímulos, la vía de señalización Cx43/ERK activada por BPs no conlleva la acumulación de ERKs en el núcleo. El efecto anti-apoptótico de los BPs depende de la fosforilación de blancos citoplasmáticos de ERKs y es inhibido cuando las quinasas son retenidas en el núcleo. En este estudio hemos demostrado que ERKs y la proteína "scaffolding" ß-arrestina co-inmunoprecipitan con Cx43 en células osteocíticas MLO-Y4 y que alendronato aumenta esta asociación. Más aún, ERK2 fusionada a la proteína roja fluorescente (ERK2-RFP) co-localiza con Cx43 fusionada con la proteína verde fluorescente fuera del núcleo en células tratadas con vehículo o alendronato. Alendronato no indujo la acumulación nuclear de ERK en células transfectadas con ß-arrestina nativa (wtARR) o con un vector control, pero si lo hizo en células que expresan una forma dominante negativa de ß-arrestina (dnARR), consistente en el dominio de interacción entre ß-arrestina y clatrina, y que compite con ß-arrestina endógena por la unión a clatrina. Alendronato activa ERKs con la misma eficiencia en células transfectadas con dnARR o wtARR, demostrando que dnARR sólo interfiere con la localización subcelular de ERKs, pero no con su activación inducida por los BPs. Más aún, mientras alendronato inhibe apoptosis en células que expresan wtARR o vector control, es inefectivo en células que expresan dnARR. En conclusión, los BPs inducen la formación de un complejo que incluye Cx43, ß-arrestina y clatrina, el cual retiene ERKs fuera del núcleo y es indispensable para la sobrevida de los osteocitos inducida por estas drogas. (AU)

Changes in cortical bone channels network and osteocyte organization after the use of zoledronic acid

Objective The aim of this study was to evaluate the effects of zoledronic acid (ZA) on the cortical bone channels network (CBCN) and osteocyte organization in relation to the bone channels. Materials and methods Eighteen male Wistar rats were divided into control (CG) and test groups (TG). Twelve animals from TG received 3 ZA doses (7.5 µg/kg), and 6 animals from CG did not receive any medication. TG animals were euthanized at 14 (n = 6) and 75 (n = 6) dadys after drug injection. CBCN was analyzed in mandibles and tibias using computational routines. The osteocyte organization was qualitatively evaluated in tibias using a three-dimensional reconstruction of images from serial histological sections. Results Significant differences in CBCN of tibia were found between the treated and untreated rats, with a wider range of sizes and shapes of the channels after the use of ZA (channels area p = 0.0063, channels area SD p = 0.0276) and less bone matrix (bone volume p = 0.0388). The alterations in the channels’ morphology were more evident at 75 days after the drug injection (channels perimeter p = 0.0286). No differences were found in mandibles CBCN. The osteocyte distribution revealed more variable patterns of cell distribution in ZA groups, with non-homogeneous distribution of cells in relation to the bone channels. Conclusion Zoledronic acid induces structural changes in CBCN and modifies the osteocyte arrangement in cortical bone in the tibia; also, the variability in the morphology of bone channels became more evident after a certain time of the use of the drug.

Oscillatory fluid flow elicits changes in morphology, cytoskeleton and integrin-associated molecules in MLO-Y4 cells, but not in MC3T3-E1 cells

Interstitial fluid flow stress is one of the most important mechanical stimulations of bone cells under physiological conditions. Osteocytes and osteoblasts act as primary mechanosensors within bones, and in vitro are able to respond to fluid shear stress, both morphologically and functionally. However, there is little information about the response of integrin-associated molecules using both osteoblasts and osteocytes. In this study, we investigated the changes in response to 2 hours of oscillatory fluid flow stress in the MLO-Y4 osteocyte-like cell line and the MC3T3-E1 osteoblast-like cell line. MLO-Y4 cells exhibited a significant increase in the expression of integrin-associated molecules, including OPN, CD44, vinculin and integrin avp3. However, there was no or limited increase observed in MC3T3-E1 osteoblast-like cells. Cell area and fiber stress formation were also markedly promoted by fluid flow only in MLO-Y4 cells. But the numbers of processes per cell remain unaffected in both cell lines.

Regulacin de la mineralizacin sea por factores inorgnicos y peptdicos / Regulation of Bone Mineralization by inorganic and peptide factors

La mineralizacin ortotpica comienza con la produccin de las vesculas de matriz, por brotacin polarizada de la superficie de condrocitos, osteoblastos y odontoblastos. Esta transcurre en dos etapas. La primera es la formacin de cristales de hidroxiapatita dentro de las vesculas de matriz, seguido por la propagacin de la hidroxiapatita a travs de la membrana de la vescula dentro de la matriz extracelular. En la regulacin de la mineralizacin ortotpica, aparte de las clulas tejido especficas, intervienen un gran nmero de enzimas, factores inorgnicos y peptdicos, que tienen complejas interacciones. Para que la mineralizacin normal contine se necesita un ajustado balance entre los niveles de fosfato inorgnico (Pi) y de pirofosfato inorgnico (PPi) extracelular. El PPi antagoniza la habilidad del Pi para cristalizar con el calcio y formar hidroxiapatita y por lo tanto suprime su propagacin. Se han identificado tres molculas reguladoras centrales de los niveles extracelulares de PPi: la fosfatasa alcalina tejido-no especfica (TNAP), que hidroliza el PPi, la nucletido pirofosfato fosfodiesterasa 1 (NPP1), que genera PPi de nuclesidos trifosfato y la protena transmembrana de mltiples-pasos ANK, que media la transferencia intracelular al extracelular de PPi. A su vez existen dos protenas SIBLING llamadas DMP1 y MEPE reguladoras de la mineralizacin. La expresin de DMP1 por el osteocito se induce en forma marcada en respuesta a la carga mecnica incrementando la mineralizacin sea. La protena MEPE contiene un motivo peptdico proteasa resistente llamado ASARM, que se cree es un candidato a ser un inhibidor de la mineralizacin (minhibina). La osteropontina es otro inhibidor de la mineralizacin en su forma fosforilada y su secrecin est marcadamente reducida en los ratones "knockout" para NPP1. Los datos actuales parecen sostener la hiptesis que estas molculas podran ser las transductoras del "strain" seo y participar en la regulacin de la mineralizacin del espacio osteoctico perilacunar.

Orthotopic mineralization begins with the production of matrix vesicles that are produced by polarized budding of the surface of condrocytes, osteoblasts and odontoblasts. It occurs in two steps: The first one is the formation of hydroxiapatite crystals within the matrix vesicles, followed by the propagation of the hydroxiapatite crystals through the membrane vesicle into the extra cellular matrix. In the regulation of orthotopic mineralization, apart from tissue-specific cells, a great number of enzymes, inorganic and peptide factors participate, that have complex interactions among them. Inorganic pyrophosphate (PPi) antagonizes the ability of phosphate (Pi) to crystallize with calcium and to form hydroxiapatite, thus suppressing its propagation. For the normal mineralization to continue, an adjusted balance of the extra cellular Pi and PPi levels is needed. Three molecules have been identified that have a central role in the regulation of extra cellular PPi levels: tissue non-specific alkaline phosphatase (TNAP), which hydrolyzes PPi, the nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), which generates PPi from triphosphate nucleosides, and the multiple-steps transmembrane protein ANK which transfers PPi from the intracellular to the extracellular compartment. There are, in turn, two SIBLING proteins called DMP1 and MEPE that regulate mineralization. The expression of DMP1 by the osteocyte is dramatically induced in response to mechanical loading increasing bone mineralization. MEPE protein contains a protease resistant motif called ASARM, which is believed to be the candidate for the mineralization inhibitor (minhibin). Osteopontin is another mineralization inhibitor in its phosphorilated form and its secretion is markedly reduced in knockout mice for NPP1. Present data seem to support the hypothesis that these molecules could be the translators of bone strain and participate in the regulation of mineralization of the perilacunar osteocytic space.

Tetraploidía en Ceratophrys (Anura, Leptodactylidae), análisis del registro fósil

It has been confirmed the existence of the tetraploid counterpart of the genus Ceratophrys Wied, 1824 (extint at the present time) in Argentina and Brazil since the Pliocene, and the apparition of the octaploidy in the Upper Pleistocene - Holocene. Fossil material of the diploid form, distinctive of the Chacoan District, has been reported from the horizons of the Montehermosense Formation in south-east of the province of Buenos Aires, Argentina. The coexistence of diploid-tetraploid and tetraploid-octaploid forms in the same province was documented.