Mechanical Vibration Associated With Intermittent PTH Improves Bone Microarchitecture in Ovariectomized Rats.

INTRODUCTION: Intermittent 1-34 parathyroid hormone (iPTH) administration, a bone-forming treatment, is widely used as a therapy for severe osteoporosis. It can only be used for a maximum of 24 mo and must be followed by an antiresorptive drug to retain the new formed tissue. Mechanical load, in the form of low-intensity and high-frequency vibration, has received considerable attention due to its ability to prevent bone loss. AIM: To investigate the ability of whole body mechanical vibration (MV) to potentiate the anabolic effects of iPTH and to inhibit bone resorption following discontinuation of iPTH treatment in estrogen-deficient rats. METHODOLOGY: Fifty-four 6-month-old female Wistar rats were ovariectomized (OVX) or sham-operated. After 5 mo, they were divided into 7 groups: Sham - non-OVX; Control - OVX, vehicle for 60 d; MV - OVX, submitted to MV for 60 d; PTH60d - OVX, injected with iPTH for 60 d; PTH+MV - OVX, injected with iPTH combined with MV for 60 d; PTH30d - OVX, injected with iPTH for 30 d, and untreated for 30 d; PTH30d/MV30d - OVX, injected with iPTH for 30 d, followed by MV for 30 d. Bone mineral density (BMD) and body composition (lean mass and fat) were evaluated at OVX (T0), the beginning (T1), and at the end (T2) of treatments by dual X-ray absorptiometry (DXA). Femurs were processed for histomorphometry (bone volume - BV/TV and cortical thickness - Ct.Th) and tibias for biomechanical test. RESULTS: Body composition and BMD were similar among the groups at T0. In T2, MV presented higher fat than other groups (except PTH60d) and PTH30d/MV30d showed greater lean mass than Control. At T1, Sham presented the highest BMD, but between T1 vs T2 there was an increase in all iPTH-treated groups. At T2, BMD was higher in PTH60d and PTH+MV than in the Control and MV groups. The highest BV/TV was observed in the PTH+MV group, followed by PTH60d. Cortical thickness was increased in PTH60d and PTH+MV compared to Sham. Vibration applied post-iPTH (PTH30d/MV30d) improved the force at failure in tibias when compared to Sham and Control groups. CONCLUSION: MV potentiated iPTH anabolic effects in cancellous bone; however, MV was unable to maintain bone mass after stopping iPTH in ovariectomized rats.

Changes in human intervertebral disc biochemical composition and bony end plates between middle and old age.

OBJECTIVE: This study evaluates molecular, nutritional and biochemical alterations in human intervertebral discs between middle and old age. METHODS: Twenty-eight human lumbar intervertebral discs from donors were evaluated and separated into two groups: Middle-aged (35-50 years old, relatively non-degenerate discs of Pfirrmann grades 1-3, n = 15) and Old-aged (≥80 years old, all degenerate Pfirrmann grade 4 or 5, n = 13). Parameters which might be expected to to be related to nutrient supply and so the health of disc cells (eg the porosity of the vertebral endplate, cell viability and cell density) and to disc extracellular composition (ie quantification of glycosaminoglycan disaccharides and hyaluronic acid molecular weight) and collagen organization, were analyzed. Three regions of the intervertebral disc (anterior annulus fibrosus, nucleus pulposus, and posterior annulus fibrosus) were examined. RESULTS: The old-aged group showed a decrease in content of sulphated and non-sulphated glycosaminoglycans relative to middle-aged and there were also alterations in the proportion of GAG disaccharides and a decrease of collagen fiber size. Hyaluronic acid molecular weight was around 200 kDa in all regions and ages studied. The anterior annulus differed from the posterior annulus particularly in relation to cell density and GAG content. Additionally, there were changes in the bony endplate, with fewer openings observed in the caudal than cranial endplates of all discs in both groups. CONCLUSIONS: Results show the cranial vertebral endplate is the main vascular source for the intervertebral discs. Hylauronic acid molecular weight is the same through the intervertebral disc after age of 50 years.

Ionic and biochemical characterization of bovine intervertebral disk.

INTRODUCTION: Intervertebral disks have been associated with low back pain, and many therapies have been proposed for its treatment. The cellular and molecular knowledge of intervertebral disks composition and precise methods to quantify disk components are important for any type of proposed therapy. Thus, the aim of this study was to correlate glycosaminoglycans presence with the quantitation of cells, ions and collagen fiber distributions in different intervertebral disk sections. METHODS: In total, 14 intervertebral disks were used from cattle. All of the disks were dehydrated, separated in seven sections and digested in sodium-free papain buffer. Glycosaminoglycan measurements were performed in the samples according to agarose electrophoresis method; total cells were measured using the PicoGreen® technique, ions were quantified, and collagen fiber birefringence was analyzed with polarized light. RESULTS: Cations Na+ and K+ are more concentrate in the nucleus (Na(+) = 1688.50 ± 110 mmol/L; K(+) = 111.9 ± 28 mmol/L) of intervertebral disks than the annulus (Na(+) = 652.80 ± 75 mmol/L; K(+) = 55.6 ± 8 mmol/L). A negative correlation between cells number and sodium/potassium was observed (p < 0.001) Additionally, thin collagen fibers were largest in the nucleus, similar to hyaluronate distribution. CONCLUSIONS: The results suggest that annulus fibrosus cells are also sensitive to changes in ionic concentrations such as nucleus pulposus cells. Additionally, hyaluronate is related to thin collagen fibers type II.

Effects of early and late treatments of low-intensity, high-frequency mechanical vibration on bone parameters in rats.

Low-intensity, high-frequency mechanical vibration (LHMV) has shown to increase bone formation. However, studies comparing the effectiveness of early- and late-treatments of LHMV to counteract bone loss have not been documented. This study was designed to compare the effects of early- and late-treatments of LHMV (at 30 Hz/0.6 g, 20 min per day/five days per week, for 12 weeks) on bone parameters in ovariectomized (Ovx) rats. Thirty days after ovariectomy, 40 adult rats were randomly divided into four groups: GI (early control group); GII treated with LHMV 3 weeks after Ovx (early treatment); GIII (late control group) and GIV treated with LHMV twelve weeks after Ovx (late treatment). Bone mineral density (BMD) was analyzed before Ovx and after treatments. Then, animals were killed, and the femurs were collected and their length and diaphysis diameter were measured; the distal femurs were taken and processed for histomorphometry and polarized light microscopy for collagen fibers analysis or subjected to immunohistochemistry of cleaved caspase-3 in osteocytes. Statistical analysis was done by ANOVA followed by the Bonferroni post hoc test (p < 0.05). BMD was similar among the groups before Ovx, but after treatments, it was significantly higher in GII and GIV compared with their control groups (p < 0.05). Femur length and cortical bone thickness were similar among the groups, but the diaphysis diameter of GII was higher compared with GI. Trabecular bone area was higher in the vibrated groups, but it was greater in GII (p < 0.05). Also, the vibrated groups showed the higher content collagen fibers and lower presence apoptotic osteocytes (positive caspase-3 immunoreactivity) when compared with the other groups (p < 0.05). These results suggest that both early- and late-treatments with LHMV counteract bone loss, being the early treatment more effective than the late treatment.

Modifications in bone matrix of estrogen-deficient rats treated with intermittent PTH.

Bone matrix dictates strength, elasticity, and stiffness to the bone. Intermittent parathyroid hormone (iPTH), a bone-forming treatment, is widely used as a therapy for osteoporosis. We investigate whether low doses of intermittent PTH (1-34) change the profile of organic components in the bone matrix after 30 days of treatment. Forty 6-month-old female Wistar rats underwent ovariectomy and after 3 months received low doses of iPTH administered for 30 days: daily at 0.3 µg/kg/day (PTH03) or 5 µg/kg/day (PTH5); or 3 times per week at 0.25 µg/kg/day (PTH025). After euthanasia, distal femora were processed for bone histomorphometry, histochemistry for collagen and glycosaminoglycans, biochemical quantification of sulfated glycosaminoglycans, and hyaluronan by ELISA and TUNEL staining. Whole tibiae were used to estimate the bone mineral density (BMD). Histomorphometric analysis showed that PTH5 increased cancellous bone volume by 6% over vehicle-treated rats. In addition, PTH5 and PTH03 increased cortical thickness by 21% and 20%, respectively. Tibial BMD increased in PTH5-treated rats and this group exhibited lower levels of chondroitin sulfate; on the other hand, hyaluronan expression was increased. Hormonal administration in the PTH5 group led to decreased collagen maturity. Further, TUNEL-positive osteocytes were decreased in the cortical compartment of PTH5 whereas administration of PTH025 increased the osteocyte death. Our findings suggest that daily injections of PTH at low doses alter the pattern of organic components from the bone matrix, favoring the increase of bone mass.

Ação da melatonina no tecido cartilaginoso / The role of melatonin on cartilaginous tissue

Os autores fazem uma revisão que mostra a ação da melatonina sobre o tecido cartilaginoso. Referem sua estrutura química, seu local de síntese, seus receptores e sua ação. Relatam que os níveis baixos da mela to nina na menopausa poderiam ser um importante fator no desenvolvimento e na manutenção da osteoporose, visto que em ratas a sua reposição leva a um aumento da densidade mineral óssea e da espessura da cartilagem articular. Sugerem uma possível ação benéfica da melatonina na proteção das lesões da cartilagem articular, o que poderia estar relacionado ao bloqueio do estresse oxidativo, uma vez que produtos desse estresse, com resíduos de tiro sina, são observados no tecido cartilaginoso com doenças de degradação articular. Sugerem que a melatonina aumenta a síntese de matriz cartilaginosa. Esses fatos indicam que a mela to nina pode ser benéfica para o tecido cartilaginoso, uma vez que há uma redução da secreção do hormônio da melatonina, com o avançar da idade, o qual está relacionado ao aumento da incidência de osteoartrite.

The authors write a review showing the action of melatonin on the cartilaginous tissue and relate its chemical structure, site of synthesis, and receptors. They report that low levels of melatonin in menopause may be an important factor in the pathogenesis of osteoporosis, since its replacement in rats leads to an increase in bone mineral density and the thickness of articular cartilage. It is also suggested a possible beneficial effect of melatonin in the prevention of articular cartilage lesions, which could be related to the blockade of oxidative stress, since products of this stress, in addition to tyrosine residues, are observed in the cartilage tissue degradation in joint diseases. Furthermore, it is related that melatonin enhances cartilage matrix synthesis. These facts indicate that melatonin may be beneficial to the integrity of cartilaginous tissue, since there is a reduced secretion of melatonin with advancing age, which is related to increased incidence of osteoarthritis.

The low level laser therapy effect on the remodeling of bone extracellular matrix.

The low level laser therapy (LLLT) has been used as an option to accelerate the regeneration of bone tissue. In this study, both femurs of male Wistar rats (30 animals) were injured with a drill and the effect of LLLT using a laser diode (100 mW at 660 nm) in the bone matrix on the left paw measured. LLLT effect on the healing bone tissue matrix was evaluated by a combination of immunohistochemical histomorphometry, confocal immunofluorescence microscopy and isolation and characterization of glycosaminoglycans. Histomorphometric analysis showed that LLLT increased bone matrix and showing more organized. Alcian Blue and PAS staining seems to suggest differential glycosaminoglycans and glycoproteins. The data showed increased expression of chondroitin sulfate and hyaluronic acid, after reduction as the LLLT and mature bone, resembling the expression of osteonectin and biglycan. The difference in expression of siblings (DMP-1, OPN and BSP) is in accordance with the repair accelerated bone formation after the application of LLLT as compared with control. The expression of osteonectin and osteocalcin supports their role in bone mineralization protein, indicating that LLLT accelerates this process. The overall data show that LLLT bone changes dynamic array, shortening the time period involved in the bone repair.

Terapia gênica para osteoporose / Gene therapy for osteoporosis

A osteoporose é considerada um dos problemas de saúde mais comuns e sérios da população idosa mundial. É uma doença crônica e progressiva, caracterizada pela diminuição da massa óssea e deterioração da microarquitetura do tecido ósseo. A terapia gênica representa uma nova abordagem para o tratamento da osteoporose e tem como princípio devolver a função comprometida pelo metabolismo. Esta revisão visa focar os trabalhos relevantes desenvolvidos nos últimos anos, disponibilizados nas bases de dados médicas, e que utilizaram a terapia gênica para o tratamento da osteoporose em modelos animais, bem como, as perspectivas futuras desta terapia. A maioria dos estudos utiliza os genes BMPs, PTH e OPG na tentativa de restabelecer a massa óssea. Apesar da carência de novas moléculas, todos os genes empregados nos estudos se mostraram eficientes no tratamento da doença. Os benefícios que a terapia gênica proporcionará aos pacientes no futuro devem contribuir substancialmente para o aumento na qualidade de vida dos idosos. Em breve, protocolos clínicos envolvendo humanos irão beneficiar os indivíduos com osteoporose.

Osteoporosis is considered one of the most common and serious problems affecting the elderly population worldwide. It is a chronic and progressive disease, characterized by decreased bone mass and degeneration of the microarchitecture of the bone tissue. Gene therapy represents a new approach in osteoporosis treatment, and its main function is to restore the compromised function in the metabolism. This review aims to elucidate the main studies on gene therapy in recent years, in the medical databases, that use gene therapy for the treatment of osteoporosis in animal models, as well as the future prospects of this therapy. The majority of the studies use the BMP, PTH and OPG genes, in an attempt to reestablish bone mass. Despite the lack of new molecules, all genes employed in these studies have proven to be efficient in the treatment of the disease. The benefits that gene therapy will provide for patients in the future should contribute substantially to increasing the quality of life for the elderly. Soon, clinical trials involving humans will benefit individuals with osteoporosis.

Efeitos das isoflavonas da soja sobre o tecido ósseo de ratas / Effects of soy isoflavones on bone tissue of rats

Introdução: É sabido que as isoflavonas da soja exercem efeitos positivos contra a perda de massa óssea após a menopausa. No entanto, os efeitos de diferentes doses de isoflavonas e sua associação com o 17 beta-estradiol no tecido ósseo não são bem conhecidos. Objetivo: Nesse estudo, foram investigados os efeitos de diferentes doses de isoflavonas da soja e sua associação com o 17 beta-estradiol no tecido ósseo de ratas osteopênicas. Métodos: 50 ratas Wistar adultas foram ooforectomizadas e, após 90 dias, divididas em 5 grupos (10 ratas em cada): GI – grupo controle; GII, GIII e GIV – tratados diariamente com isoflavonas da soja por gavage nas doses de 80 mg, 200 mg e 350 mg/kg, respectivamente; GV – tratado com 350 mg/kg de isofl avonas da soja associadas ao 17 beta-estradiol (10 miug/kg/via subcutânea, diariamente). Após 90 dias, os fêmures e tíbias foram coletados, e seus comprimentos e espessuras foram medidos. Os fêmures distais foram processados para histomorfometria e as tíbias foram congeladas e,posteriormente, submetidas a testes físicos e biomecânicos. Resultados: Durante o período de tratamento, foi observada perda de peso no grupo submetido ao tratamento combinado. O comprimento dos fêmures e a espessura das tíbias foram maiores no grupo submetido ao tratamento combinado. O tratamento combinado promoveu maior área óssea trabecular e espessura do osso cortical. A densidade óssea e a capacidade das tíbias de receber carga foram maiores no grupo submetido ao tratamento combinado. Conclusão: Dessa forma, este estudo demonstrou que as isoflavonas da soja têm efeito positivo no tecido ósseo de ratas osteopênicas e sua associação com 17 beta-estradiol potencializa esse efeito.

Introduction: It has been shown that soy isoflavones counteract postmenopausal bone loss. However, the effects of different doses and its treatment combined with 17 beta-estradiol on bone tissue are not well-known. Objective: In this study we investigated the effects of different doses of soy isoflavones and their association with 17 beta-estradiol on bone tissue of osteopenic rats. Methods: 50 adult Wistar rats were ovariectomized and after90 days divided in 5 groups (10 rats in each): GI – control; GII, GIII and GIV – treated orally with soy isoflavones at the doses of 80 mg, 200 mg and 350 mg/kg/bodyweight/daily respectively; GV – soy isofl avones (350 mg/orally) + 17 beta-estradiol (10 miug/kg/body weight/subcutaneously, daily). After 90 days, the femurs and tibias were collected and its length and thickness were measured the distal femurs were processed for histomorphometry; the tibias were frozen and submitted to biomechanical tests. Results: During the period of treatment, loss of body weight in the group subjected to the combined treatment was observed. The femur length and the thickness of the tibias were higher in the combined treated group as well as.

Isoflavonas na pós-menopausa: uma revisão / Isoflavones in post-menopause: a review

Este artigo de revisão abordou os efeitos das isoflavonas na prevenção dos sintomas decorrentes da pós-menopausa na mulher. Isoflavonas são fenóis heterocíclicos com estrutura semelhante à do 17-beta-estradiol, atuando como modulador seletivo dos receptores de estrogênio (SERM). As ações na célula dependem do tecido-alvo, do status do receptor tecidual e dos níveis de estrogênios endógenos. As isoflavonas são produtos naturais que podem ser utilizados como uma alternativa à terapia hormonal na menopausa. Estudos in vitro e em modelos animais mostraram que agem de várias maneiras para exercer seus efeitos, podem atuar nas células através de vias genômicas e não-genômicas. Estudos epidemiológicos sugerem um efeito protetor das isoflavonas sobre o tecido mamário como é evidenciado pelas menores taxas de câncer de mama nos países do Leste Asiático, onde a soja é uma parte predominante da dieta. Os produtos que contêm isoflavonas também aliviam os sintomas da menopausa, reduzindo fogachos. No entanto ainda há necessidade de novos estudos relacionando a segurança em longo prazo de suplementos de isoflavonasna mulher na pós-menopausa.

This article review was designed to address the effects of isoflavones in postmenopausal women and their place in the prevention and treatment of postmenopausal symptoms. Isoflavones are heterocyclic phenols with structural similarity to estradiol-17 beta and selective estrogen receptor modulators (SERM). Actions at the cellular level depend on the target tissue, receptor status of the tissue, and the level of endogenous estrogen. Isoflavones are natural products that could be used as an alternative to menopausal hormone therapy. In vitro and animal studies have shown that they act in multiple ways to exert their postmenopausal effects. They act on both cells of through genomic and nongenomic pathways. Epidemiological studies suggest a protective effect of isoflavone on breast tissue as evidenced by the lower rates of breast cancer in East Asian countries where soy is a predominant part of the diet. Soy products also alleviate menopausal symptoms by reducing hot flashes. However there is still need for further studies relating to long-term safety of isoflavone supplements in postmenopausal women.

Morphological characterization of cell death during the ovary differentiation in worker honey bee.

Cell death that occurs during ovary differentiation in the honeybee worker's larval development accounts for ovariole reabsorption. From a morphological standpoint, three modes of death were detected. Germinative cells in the ovarioles die by an apoptotic-like process, whereas the somatic cells die by an autophagic process, type II cell death; and during pupation, stromatic and ovarian capsular cells die through cytoplasmic disintegration, releasing their components into the hemolymph. These modes of cell death are in part determined by the pattern of tissue organization within which the cell occurs.

Cell nucleus activity during post-embryonic development of Apis mellifera L. (Hymenoptera: Apidae). Intranuclear acid phosphatase.

We report nuclear acid phosphatase activity in the somatic (intra-ovariolar and stromatic) and germ cells of differentiating honey bee worker ovaries, as well as in the midgut cells of metamorphosing bees. There was heterogeneity in the intensity and distribution of electron dense deposits of lead phosphate, indicative of acid phosphatase activity in the nuclei of these tissues, during different phases of post-embryonic bee development. This heterogeneity was interpreted as a variation of the nuclear functional state, related to the cell functions in these tissues.