Expression Changes of Hypoxia-Inducible Factor-1α in G-CSF Induced Hematopoietic Stem Cell Mobilization / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression and its relative mechanism of hypoxia-inducible factor-1α(HIF-1α) in bone marrow(BM) of mice during G-CSF mobilization of hematopoietic stem cells (HSC) .@*METHODS@#Flow cytometry was used to detect the proportion of Lin-Sca-1+ c-kit+ (LSK) cells in peripheral blood of C57BL/6J mice before and after G-CSF mobilization. And the expression of HIF-1α and osteocalcin (OCN) mRNA and protein were detected by RQ-PCR and immunohistochemistry. The number of osteoblasts in bone marrow specimens of mice was counted under the microscope.@*RESULTS@#The proportion of LSK cells in peripheral blood began to increase at day 4 of G-CSF mobilization, and reached the peak at day 5, which was significantly higher than that of control group (P<0.05). There was no distinct difference in the expression of HIF-1α mRNA between bone marrow nucleated cells and osteoblasts of steady-state mice (P=0.073), while OCN mRNA was mainly expressed in osteoblasts, which was higher than that in bone marrow nucleated cells (P=0.034). After mobilization, the expression level of HIF-1α increased, but OCN decreased, and the number of endosteum osteoblasts decreased. The change of HIF-1α expression was later than that of OCN and was consistent with the proportion of LSK cells in peripheral blood.@*CONCLUSION@#The expression of HIF-1α in bone marrow was increased during the mobilization of HSC mediated by G-CSF, and one of the mechanisms may be related to the peripheral migration of HSC induced by osteoblasts inhibition.

Mechanism of miR-26a-5p/cAMP response element binding protein 1 molecular axis regulating osteogenic differentiation of adipose-derived mesenchymal stem cells / 中国修复重建外科杂志

OBJECTIVE@#To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1).@*METHODS@#The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture.@*RESULTS@#The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05).@*CONCLUSION@#Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.

Effects of 275 nm and 310 nm ultraviolet irradiation on bone metabolism in ovariectomized osteoporotic rats / 北京大学学报(医学版)

OBJECTIVE@#To investigate the effect of 275 nm and 310 nm ultraviolet irradiation on ovariectomized rats' bone metabolism.@*METHODS@#Twenty four 3-month-old female Sprague-Dawley (SD) rat were randomly divided into control group, sham operated group, 275 nm ultraviolet (UV) irradiation group and 310 nm UV irradiation group. Each group contained 6 rats. The rats in the two irradiation groups were treated with bilateral ovariectomy. The rats in sham operated group received sham operation (They were given the same back incision and a bit of par-ovarian fat were removed). Control group received no disposition. About 24 weeks after operation, all the rats received detailed bone mineral density (BMD) detection again. Detection regions include cervical vertebra, lumbar vertebra, proximal femur, mid femur and distal femur. Next, osteopenia rats in 275 nm irradiation group were UV irradiated 275 nm with fixed illumination intensity (15 μW/cm2) everyday for 16 weeks. The osteopenia rats in 310 nm irradiation group were UV irradiated 310 nm with fixed illumination intensity (15 μW/cm2) everyday for 16 weeks. The backs of the rats were shaved regularly as irradiation area (6 cm×8 cm). After 16-week irradiation, all the rats' BMD of cervical vertebra, lumbar vertebra, proximal femur, mid femur and distal femur were measured. At the end of the trial, all the rats' blood specimens were obtained and serum 25(OH)D, procollagen type Ⅰ N-peptide (PINP) and osteocalcin (OC) were measured.@*RESULTS@#Compared with control group [(238.78±26.74) mg/cm3], the BMD of the whole body were significantly lower in 275 nm [(193.34±13.28) mg/cm3] and 310 nm [(191.19±18.48) mg/cm3] irradiation groups (P=0.002, P=0.001). There were no significant difference between sham operated group [(227.20±14.32) mg/cm3] and control group. After 16-week ultraviolet irradiation, the BMD of the whole body were significantly increased in 275 nm [(193.34±13.28) mg/cm3 vs. (221.68±25.52) mg/cm3, P=0.005] and 310 nm groups [(191.19±18.48) mg/cm3 vs. (267.48±20.54) mg/cm3, P < 0.001] after corresponding irradiation. The BMD of the four body regions (lumbar vertebra, proximal femur, mid femur and distal femur) had significantly increased after irradiation in 275 nm irradiation group. For 310 nm irradiation group, the BMD in cervical vertebra, lumbar vertebra, proximal femur, mid femur and distal femur also had increased significantly after 310 nm ultraviolet irradiation. The concentration of serum 25(OH)D and OC was higher in 275 nm irradiation group than in control group [(46.78±5.59) μg/L vs. (21.32±6.65) μg/L, P=0.002;(2.05±0.53) U/L vs. (1.32±0.07) U/L, P=0.022]. Compared with the control, the concentration of serum 25(OH)D [(58.05±12.74) μg/L], OC [(2.04±0.53) U/L] and PINP [(176.16±24.18) U/L] was significantly higher (P < 0.001, P=0.015, P=0.005) in 310 nm irradiation group. However, there were no significantly difference between sham operated group and the control.@*CONCLUSION@#Both 275 nm and 310 nm ultraviolet could improve rats' vitamin D synthesis. Both 275 nm and 310 nm ultraviolet could improve osteopenia rats' bone condition. The irradiation of 310 nm might be more effective on bone condition improvement.

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)

Influence of low-level laser irradiation on osteocalcin protein and gene expression in bone tissue1

Abstract Purpose: To evaluate osteocalcin gene and protein expression in vitro and in an in vivo model of ostectomy. Methods: Twenty Wistar rats were assigned into two groups A (n=10, laser) and B (n=10, control). Ostectomy was performed in the femur diaphysis; the twenty fragments removed, composed in vitro groups named as in vivo (A and B) and cultivated in CO2 atmosphere for thirteen days. Low-level laser irradiation was performed in groups A (in vivo and in vitro) by an GaAlAs device (λ=808 nm, dose of 2J/cm2, power of 200mW, power density of 0.2W/cm2, total energy of 1.25J, spot diameter of 0.02mm) for 5 seconds, at one point, daily. It was performed immunocytochemistry assays in vivo and in vitro groups. In vitro groups were also submitted to RNA extraction, cDNA synthesis and gene expression by quantitative PCR. Statistical analysis was realized with p<0.05. Results: Immunocytochemistry scores showed no significant differences between control and laser groups either in vivo and in vitro. Gene expression also showed no statistical differences. Conclusion: Low-level laser irradiation did not alter osteocalcin protein and gene expression in vivo and in vitro in the studied period but it may have been expressed them in an earlier period.

Association between undercarboxylated osteocalcin, bone mineral density, and metabolic parameters in postmenopausal women

ABSTRACT Objective: Osteocalcin has been associated with several effects on energy and glucose metabolism. However, the physiological role of undercarboxylated osteocalcin (U-osc; the hormonally active isoform of osteocalcin) is still controversial. To correlate the serum levels of U-osc with bone mineral density (BMD) values and metabolic parameters in postmenopausal women. Subjects and methods: Cross-sectional study including 105 postmenopausal women (age 56.5 ± 6.1 years, body mass index [BMI] 28.2 ± 4.9 kg/m2) grouped based on the presence of three or less, four, or five criteria of metabolic syndrome according to the International Diabetes Federation (IDF). The subjects underwent dualenergy x-ray absorptiometry (DXA) for the assessment of body composition and BMD and blood tests for the measurement of U-osc and bone-specific alkaline phosphatase (BSAP) levels. Results: The mean U-osc level was 3.1 ± 3.4 ng/mL (median 2.3 ng/mL, range 0.0-18.4 ng/mL) and the mean BSAP level was 12.9 ± 4.0 ng/mL (median 12.1 ng/mL, range 73-24.4 ng/mL). There were no associations between U-osc and BSAP levels with serum metabolic parameters. Lower fasting glucose levels were observed in participants with increased values of U-osc/femoral BMD ratio (3.61 ± 4 ng/mL versus 10.2 ± 1.6 ng/mL, p = 0.036). When the participants were stratified into tertiles according to the U-osc/ femoral BMD and U-osc/lumbar BMD ratios, lower fasting glucose levels correlated with increased ratios (p = 0.029 and p = 0.042, respectively). Conclusion: Based on the ratio of U-osc to BMD, our study demonstrated an association between U-osc and glucose metabolism. However, no association was observed between U-osc and metabolic parameters.The U-osc/BMD ratio is an innovative way to correct the U-osc value for bone mass.

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)

Stimulating Effect of a Novel Synthesized Sulfonamido-Based Gallate ZXHA-TC on Primary Osteoblasts

PURPOSE: This study is intended to investigate the effects of plants or plant-derived antioxidants on prevention of osteoporosis through the maintenance of reactive oxygen species (ROS) at a favorable level. MATERIALS AND METHODS: In this study, a novel antioxidant, namely 3,4,5-Trihydroxy-N-[4-(5-hydroxy-6-methoxy-pyrimidin-4-ylsulfamoyl)-phenyl]-benzamide (ZXHA-TC) was synthesized from gallic acid and sulfadimoxine. Its effect on osteoblast metabolism was investigated via the detection of cell proliferation, cell viability, production of ROS, and expression of osteogenic-specific genes including runt-related transcription factor 2 (RUNX2), bone sialoprotein (BSP), osteocalcin (OCN), alpha-1 type I collagen (COL1A1), and osteogenic-related proteins after treatment for 2, 4, and 6 days respectively. RESULTS: The results showed that ZXHA-TC has a stimulating effect on the proliferation and osteogenic differentiation of primary osteoblasts by promoting cell proliferation, cell viability, and the expression of genes BSP and OCN. Productions of bone matrix and mineralization were also increased by ZXHA-TC treatment as a result of up-regulation of COL1A1 and alkaline phosphatase (ALP) at the early stage and down-regulation of both genes subsequently. A range of 6.25x10(-3) microg/mL to 6.25x10(-1) microg/mL is the recommended dose for ZXHA-TC, within which 6.25x10(-2) microg/mL showed the best performance. CONCLUSION: This study may hold promise for the development of a novel agent for the treatment of osteoporosis.

Immunoexpression of PPAR-? and Osteocalcin Proteins for Bone Repair of Critical-Size Defects Treated with Fragmented Autogenous Abdominal Adipose Tissue Graft

Immunoexpression of PPAR-γ and osteocalcin proteins was evaluated for bone repair of critical-size defects (CSDs), created in rat calvaria (n=42) and treated with fragmented abdominal autogenous adipose tissue graft. Three groups (n=14) were formed: C (control - blood clot), AB (autogenous bone) and AT (fragmented adipose tissue). The groups were divided into subgroups (n=7) for euthanasia at 30 and 90 days. Histological and immunohistochemical analyses were performed. Data were subjected to descriptive statistics (mode). A complete bone closure was observed in Group AB 90 days after surgery. In Group C, repair was achieved by the formation of collagen fiber bundles oriented parallel to the wound surface at both post-surgery periods. In Group AT the type of healing was characterized by dense connective tissue containing collagen fiber bundles arranged amidst the remaining adipose tissue, with rare heterotopic bone formation associated with fibrosis and different types of tissue necrosis. Immunostaining of PPAR-γ was not observed in any specimen from Groups C and AB. In Group AT, the immunostaining of PPAR-γ was more evident 30 days after surgery. Immunostaining of osteocalcin was present in all groups and at both postoperative periods. The fragmented autogenous abdominal adipose tissue graft did not favor the repair of critical-size bone defects created surgically in rat calvaria as evidenced by the positive immunostaining of PPAR-γ protein and the negative immunostaining of osteocalcin in the osteoblast-like cells and bone matrix.

A imunoexpressão das proteínas PPAR-γ e osteocalcina foi avaliada para o reparo ósseo de defeitos de tamanho crítico (DTC) criados em calvária de ratos (n = 42) e tratados com enxerto de tecido adiposo autógeno abdominal macerado. Foram formados três grupos (n=14): C (controlo - coágulo de sangue), AB (osso autógeno) e AT (tecido adiposo fragmentado). Estes grupos foram divididos em subgrupos (n = 7) para a eutanásia em 30 e 90 dias. Foram realizadas análises histológicas e imuno-histoquímico. Os dados foram submetidos à análise estatística descritiva (moda). Um fechamento ósseo completo foi observada no grupo AB 90 dias após a cirurgia. No grupo C a reparação foi alcançada através da formação de feixes de fibras de colágeno orientadas paralelamente à superfície da ferida nos dois períodos pós-operatórios. No Grupo AT, o tipo de cicatrização predominante foi caracterizada pela presença de tecido conjuntivo denso contendo feixes de colágeno de fibras dispostas em meio ao tecido adiposo restante, com raras formações ósseas heterotópicas associada à fibrose e diferentes tipos de necrose tecidual. A imunomarcação do PPAR- γ não foi observada em nenhum espécime dos grupos C e AB. No grupo AT a imunomarcação do PPAR-γ foi mais evidente aos 30 dias de pós-operátorio. A imunomarcação da osteocalcina estava presente em todos os grupos e nos dois períodos de pós-operatório O enxerto autógeno abdominal fragmentado tecido adiposo não favoreceu a reparação de defeitos ósseos de tamanho crítico criados cirurgicamente em calvária de ratos como evidenciado pela imunomarcação positiva da proteína PPAR-γ e a imunomarcação negativa de osteocalcina nos osteoblastos e matriz óssea.

Bone Mineral Density, Biochemical Bone Turnover Markers and Factors associated with Bone Health in Young Korean Women

PURPOSE: This study was done to assess the bone mineral density (BMD), biochemical bone turnover markers (BTMs), and factors associated with bone health in young Korean women. METHODS: Participants were 1,298 women, ages 18-29, recruited in Korea. Measurements were BMD by calcaneus quantitative ultrasound, BTMs for Calcium, Phosphorus, Osteocalcin, and C-telopeptide cross-links (CTX), body composition by physical measurements, nutrients by food frequency questionnaire and psychosocial factors associated with bone health by self-report. RESULTS: The mean BMD (Z-score) was -0.94. 8.7% women had lower BMD (Z-score or =0) and low-BMD (Z-score<0) women. However, Osteocalcin and CTX were higher in women preferring caffeine intake, sedentary lifestyle and alcoholic drinks. Body composition and Calcium intake were significantly higher in high-BMD. Low-BMD women reported significantly higher susceptibility and barriers to exercise in health beliefs, lower bone health self-efficacy and promoting behaviors. CONCLUSION: Results of this study indicate that bone health of young Korean women is not good. Development of diverse strategies to intervene in factors such as exercise, nutrients, self-efficacy, health beliefs and behaviors, shown to be important, are needed to improve bone health.

Osteocalcin Immunohistochemical Expression During Repair of Critical-Sized Bone Defects Treated with Subcutaneous Adipose Tissue in Rat and Rabbit Animal Model

The aim of this study was to evaluate the osteocalcin immunoexpression during the bone repair treated with subcutaneous adipose tissue in rats and rabbits. Fourteen rats and 14 rabbits were used in the study. A critical-sized calvarial defect was created in each animal. The animals were divided into 4 experimental groups: RC (rat control), RbC (rabbit control), RAT (rat adipose tissue), RbAT (rabbit adipose tissue). In the groups RC and RbC the defect was filled with a blood clot. In groups RAT and RbAT, the defect was filled with macerated subcutaneous adipose tissue. The euthanasia was performed at 30 days (RC and RAT) and 40 days (RbC and RbAT). Histological analysis and immunohistochemical analysis of osteocalcin protein expression were performed. Data were submitted to descriptive statistical analysis (mode). Osteocalcin immunoexpression was detected in the experimental models. Notwithstanding, RbAT showed higher positivity, especially in the adipocytes, than the group RAT group. In groups RC and RbC, the surgical wound was filled by collagen fibers. In Group RAT, the defect was composed by collagen fibers and adipocytes in the reparative sites. Similarly, in RbAT, the defect also was partially filled by collagen fibers and presence of adipocytes dispersed among the fibers. Additionally, chronic inflammatory process and areas of bone matrix deposition were observed. It may be concluded that in both animal models the adipose tissue demonstrated low osteogenic capacity. However, the rabbit animal model exhibited a more evident osteocalcin immunoexpression and a greater amount of newly formed bone matrix.

Este estudo avaliou a imunoexpressão da osteocalcina no reparo ósseo de defeitos críticos tratados com tecido adiposo subcutâneo em rato e coelho. Foram utilizados 14 ratos e 14 coelhos. Um defeito crítico foi realizado na calvária de cada animal. Os animais foram divididos em 4 grupos experimentais: CR (controle rato), CC (controle coelho), TAR (tecido adiposo do rato), TAC (tecido adiposo do coelho). Nos grupos CR e CC, o defeito foi preenchido com coágulo. Nos grupos TAR e TAC, o defeito foi preenchido com tecido adiposo subcutâneo macerado. A eutanásia dos grupos CR e TAR ocorreu com 30 dias e dos grupos CC e TAC com 40 dias. Foram realizadas análise histológica e imunoistoquímica com a proteína osteocalcina. Os dados foram submetidos à análise estatística descritiva (moda). Nos modelos experimentais foi observada a imunoexpressão de osteocalcina. Contudo, o TAC apresentou maior imunoexpressão entre os adipócitos quando comparado ao TAR. Nos grupos CR e CC a ferida cirúrgica foi preenchida por fibras colágenas. No grupo TAR foi observado o preenchimento por fibras colágenas e presença de adipócitos no interior deste tecido. No grupo TAC o defeito foi preenchido parcialmente por fibras colágenas, com presença de adipócitos dispersos entre as fibras. Além disso, foi observado processo inflamatório crônico e sinais de neoformação óssea. Conclui-se que em ambos os modelos animais o enxerto de tecido adiposo apresentou baixa capacidade osteogênica. Contudo, o modelo animal coelho apresentou uma imunoexpressão da osteocalcina mais evidente e maior quantidade de matriz óssea neoformada.

Alterações de parâmetros relacionados ao metabolismo ósseo em mulheres submetidas à derivação gástrica em Y de Roux / Changes in bone metabolism markers in women after Roux-en-Y gastric bypass

OBJETIVO: Avaliar o metabolismo ósseo e a densidade mineral óssea (DMO) em mulheres adultas pós-derivação gástrica em Y de Roux (DGYR). SUJEITOS E MÉTODOS: Estudo transversal com 48 mulheres submetidas a DGYR há três anos e 41 saudáveis. Dados obtidos: índice de massa corporal (IMC), atividade física, consumo alimentar e DMO da coluna lombar, colo e fêmur total. Dosagem de cálcio, fósforo, magnésio, albumina, fosfatase alcalina, telopeptídeo-C (CTX), paratormônio (PTH), 25-hidroxivitamina D (25OHD), osteocalcina e cálcio urinário. RESULTADOS: Maiores alterações no grupo DGYR observadas nos níveis de osteocalcina (p < 0,001), CTX (p < 0,001) e PTH (p < 0,001). Deficiência de 25OHD foi a mais frequente no grupo DGYR (p = 0,010). Deficiência/insuficiência de 25OHD associou-se com hiperparatiroidismo secundário (p = 0,025). Não houve diferença entre os grupos em relação à DMO. A ingestão de energia (p = 0,036) e proteína (p = 0,004) foi maior no grupo controle. CONCLUSÃO: Em mulheres pós-DGYR, encontraram-se alta frequência de deficiência de vitamina D, hiperparatireoidismo secundário e elevação nos marcadores de remodelação óssea, sem alteração na DMO quando comparado com o grupo controle não obeso.

OBJECTIVE: To evaluate bone turnover markers and bone mineral density (BMD) in women after Roux-en-Y (RYGB) gastric bypass. SUBJECTS AND METHODS: In a cross-sectional study, 48 women post-RYGB after three years, and 41 healthy women were evaluated. Evaluations: body mass index (BMI); physical activity; food intake; serum levels of calcium, phosphorus, magnesium, alkaline phosphatase, C-terminal telopeptide (CTX), intact parathyroid hormone (PTH), 25-hydroxyvitamin D (25OHD), osteocalcin, urinary calcium and BMD. RESULTS: Significantly higher levels were observed for osteocalcin (p < 0.001), CTX (p < 0.001), and PTH (p < 0.001) in the RYGB group when compared with the control group; 25OHD deficiency/insufficiency was more frequent in the RYGB group (p = 0.010), even after adjusted for nutritional status, and it was associated with secondary hyperparathyroidism (p = 0.025); there was no difference in BMD between the groups. Energy (p = 0.036) and protein intake (p = 0.004) were lower in the RYGB group. CONCLUSION: Patients submitted to RYGB showed a significantly higher frequency of vitamin D deficiency, secondary hyperparathyroidism, and increase in bone remodeling markers, with no difference in BMD status.

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.

Efeito da triiodotironina na expressão das proteínas ósseas durante a diferenciação osteogênica de células-tronco mesenquimais / Effect of triiodothyronine on the bone proteins expression during osteogenic differentiation of mesenchymal stem cells

OBJETIVO: O objetivo deste estudo foi avaliar o efeito da T3 na expressão da osteocalcina, osteo­pontina e colágeno I durante a diferenciação osteogênica das células-tronco mesenquimais (CTM). MATERIAIS E MÉTODOS: As células da medula óssea de ratas Wistar jovens foram extraídas, cultivadas e separadas em cinco grupos: controle (indiferenciado), diferenciado (estímulo osteogênico) e diferenciado com T3 (10-3 nM, 10-2 nM e 100 nM). Para cada grupo, foram cultivadas quatro amostras que foram analisadas por RT-PCR tempo real aos 7, 14 e 21 dias, para quantificação dos transcritos gênicos para osteocalcina, osteopontina e colágeno I. RESULTADOS: Todos os grupos diferenciados sem T3 ou com T3 independentemente da concentração apresentaram expressão de colágeno I significativamente menor e expressão de osteocalcina e osteopontina significativamente maior em comparação a das CTM indiferenciadas. Mas o grupo T3 100 nM apresentou concentração de osteocalcina mais elevada e semelhante à da cultura de osteoblastos. CONCLUSÃO: Conclui-se que a triiodotironina não altera a expressão de osteopontina e de colágeno pelas CTM, mas aumenta a expressão da osteocalcina durante a diferenciação osteogênica in vitro, sendo esse efeito dose-dependente.

OBJECTIVE: The aim of this study was to evaluate the effect of T3 on the expression of osteocalcin, osteopontin and collagen I during osteogenic differentiation of mesenchymal stem cells (MSC). MATERIALS AND METHODS: The bone marrow cells of Wistar rats with 30 days of age were extracted, cultured and separated into five groups: control (undifferentiated), differentiated (osteogenic stimulus) and differentiated with T3 (10-3 nM, 10-2 nM and 100 nM). For each group, four samples were cultured and were analyzed by real time RT-PCR at 7, 14 and 21 days for quantification of gene transcripts for osteocalcin, osteopontin and collagen I. RESULTS: All the different groups without T3 or with T3 regardless of the concentration, showed the collagen I expression significantly lower expression, and osteocalcin and osteopontin expression significantly greater than that of undifferentiated MSC. Nevertheless, the group T3 100 nM showed higher expression of osteocalcin and a similar expression of the osteoblast culture. CONCLUSION: In conclusion, the triiodothyronine does not affect the expression of osteopontin and collagen I, but increases ostecalcin expression during osteogenic differentiation in vitro of the MSC, and this effect is dose-dependent.

Use of calcium phosphate cement scaffolds for bone tissue engineering: in vitro study / A utilização do cimento de fosfato de cálcio como arcabouço para engenharia de tecido ósseo: estudo in vitro

Purpose: To evaluate the ability of macroporous tricalcium phosphate cement (CPC) scaffolds to enable the adhesion, proliferation, and differentiation of mesenchymal stem cells derived from human bone marrow. Methods: Cells from the iliac crest of an adult human donor were processed and cultured on macroporous CPC discs. Paraffin spheres sized between 100 and 250µm were used as porogens. Cells were cultured for 5, 10, and 15 days. Next, we assessed cells' behavior and morphology on the biomaterial by scanning electron microscopy. The expression levels of the BGLA and SSP1 genes and the alkaline phosphatase (ALP) activity were quantified by the quantitative real-time polymerase chain reaction technique (QT-PCR) using the fluorophore SYBR GREEN®. Results: QT-PCR detected the expression of the BGLA and SSP1 genes and the ALP activity in the periods of 10 and 15 days of culture. Thus, we found out that there was cell proliferation and differentiation in osteogenic cells. Conclusion: Macroporous CPC, with pore sized between 100 and 250µm and developed using paraffin spheres, enables adhesion, proliferation, and differentiation of mesenchymal stem cells in osteogenic cells and can be used as a scaffold for bone tissue engineering.

Objetivo: Avaliar a capacidade de suportes tridimensionais macroporosos de cimento de fosfato de cálcio (CFC), de permitir a adesão, proliferação e diferenciação de células-tronco mesenquimais derivadas da medula óssea humana. Métodos: células obtidas da crista ilíaca de um doador humano adulto foram processadas e cultivadas sobre suportes de CFC, macroporosos, que tiveram como corpo gerador de poros, microesferas de parafina, com tamanho entre 100 e 250µm. Os períodos de cultura estabelecidos foram de cinco, 10 e 15 dias. Após estes períodos, o comportamento e a morfologia das células junto ao biomaterial foram avaliados por meio de Microscopia Eletrônica de Varredura. Os níveis de expressão dos genes BGLA e SSP1 bem como a atividade da Fosfatase Alcalina (ALP) foram quantificados pela técnica de PCR em Tempo Real (QT-PCR) utilizando o fluoróforo SYBR Green®. Resultados: O QT-PCR detectou a expressão dos genes BGLA e SSP1 e a atividade da fosfatase alcalina nos períodos de 10 e 15 dias de cultura. No período de cinco dias, não foi observada a expressão de nenhum dos genes investigados. Conclusão: O CFC, macroporoso, com tamanho de poros entre 100 e 250µm, criados por meio da utilização de microesferas de parafina, permite a adesão, proliferação e diferenciação de células-tronco mesenquimais em células osteogênicas, podendo ser utilizado como arcabouço para engenharia de tecido ósseo.

Pulmonary Adenocarcinoma with Heterotopic Ossification

Pulmonary adenocarcinoma is a common malignancy that often involves calcification; however, bone formation in primary lung adenocarcinoma is extremely rare. In ten cases of primary pulmonary adenocarcinoma with heterotopic ossification, we detected immunoreactivity against TGF-beta1, osteopontin, osteocalcin and Runx2 in the fibroblastic stroma and tumor cells within the area of ossification. Our results suggest that in primary pulmonary adenocarcinoma, heterotopic ossification occurs via intramembranous bone formation. To our knowledge, only 11 other cases of pulmonary adenocarcinoma with heterotopic ossification have been reported. Here, we present ten cases of pulmonary adenocarcinoma showing heterotopic ossification with a description of previously published results and the histogenesis of heterotopic bone formation.

Pulmonary Adenocarcinoma with Heterotopic Ossification

Pulmonary adenocarcinoma is a common malignancy that often involves calcification; however, bone formation in primary lung adenocarcinoma is extremely rare. In ten cases of primary pulmonary adenocarcinoma with heterotopic ossification, we detected immunoreactivity against TGF-beta1, osteopontin, osteocalcin and Runx2 in the fibroblastic stroma and tumor cells within the area of ossification. Our results suggest that in primary pulmonary adenocarcinoma, heterotopic ossification occurs via intramembranous bone formation. To our knowledge, only 11 other cases of pulmonary adenocarcinoma with heterotopic ossification have been reported. Here, we present ten cases of pulmonary adenocarcinoma showing heterotopic ossification with a description of previously published results and the histogenesis of heterotopic bone formation.

Biochemical markers of bone turnover in angiographically-demonstrated coronary artery disease patients and healthy Thais.

The patients with coronary artery disease (CAD) were suffering from dyspnea. Physical activity of these patients was limited. Their lifestyle may be contributory factors for osteoporosis. Recent research has shown that biochemical markers may be used to predict future bone loss and identify individuals at risk for osteoporosis. Our objectives were to estimate reference ranges of bone markers in healthy Thais and to compare bone turnover between 105 healthy people and 118 CAD patients by using biochemical markers of bone formation and resorption. Mean values of bone markers in controls and patients were 22.9 +/- 12.9, 21.6 +/- 16.2 respectively for N-Mid osteocalcin and 0.45 +/- 0.30, 0.47 +/- 0.37 respectively for beta-Crosslaps. There was no statistical difference of N-Mid osteocalcin (p=0.50) and beta-Crosslaps (p=0.64) values between groups. Our data from this study suggested that that CAD patients have no higher risk for osteoporosis than healthy people.