Gisela Rodrigues da Silva Sasso

Biology of Bone Tissue: Structure, Function, and Factors That Influence Bone Cells

Bone tissue is continuously remodeled through the concerted actions of bone cells, which include bone resorption by osteoclasts and bone formation by osteoblasts, whereas osteocytes act as mechanosensors and orchestrators of the bone remodeling process. This process is under the control of local (e.g., growth factors and cytokines) and systemic (e.g., calcitonin and estrogens) factors that all together contribute for bone homeostasis. An imbalance between bone resorption and formation can result in bone diseases including osteoporosis. Recently, it has been recognized that, during bone remodeling, there are an intricate communication among bone cells. For instance, the coupling from bone resorption to bone formation is achieved by interaction between osteoclasts and osteoblasts. Moreover, osteocytes produce factors that influence osteoblast and osteoclast activities, whereas osteocyte apoptosis is followed by osteoclastic bone resorption. The increasing knowledge about the structure and functions of bone cells contributed to a better understanding of bone biology. It has been suggested that there is a complex communication between bone cells and other organs, indicating the dynamic nature of bone tissue. In this review, we discuss the current data about the structure and functions of bone cells and the factors that influence bone remodeling.

Elevated serum osteoprotegerin levels in women: friend or foe?

INTRODUCTION osteoprotegerin has emerged as a new candidate for the treatment of osteoporosis. However, high levels of osteoprotegerin have been linked to vascular calcification, an independent and well-defined risk factor for cardiovascular disease (CVD) and mortality. Thus, the action of osteoprotegerin in these situations has been questioned. OBJECTIVE to evaluate the effect of osteoprotegerin (OPG) on the human body, especially in bone tissue and in vascular diseases. METHODS the scientific databases consulted were PubMed-Medline and Cochrane, using keywords (MeSH terms) grouped into the following syntaxes: (Osteoprotegerin OR Osteoclastogenesis Inhibitory Factor OR Receptors, Tumor Necrosis Factor, Member 11b OR Tumor Necrosis Factor Receptor Superfamily, Member 11b OR FDCR-1 Protein OR FDCR 1 Protein OR OCIF Protein OR Follicular Dendritic Cell-Derived Receptor-1) AND (Bones AND Bone OR Bones AND Bone Tissue OR Bones OR Bone Tissue OR Cardiovascular Diseases). RESULTS Osteoprotegerin is present in various organs and binds to two ligands: nuclear factor kB (RANKL) related to the differentiation of osteoclasts, and tumor necrosis factor related to the apoptosis-inducing ligand (TRAIL). OPG inhibits the regulation effects of nuclear factor kB on inflammation and on the skeletal and vascular systems, preventing the apoptosis induced by TRAIL, being related to the preservation of bone tissue. CONCLUSION a deeper knowledge of the mechanisms involved in the association between OPG serum levels, bone integrity and cardiovascular disease can provide important data for future therapeutic interventions.

DNA and bone structure preservation in medieval human skeletons

Morphological and ultrastructural data from archaeological human bones are scarce, particularly data that have been correlated with information on the preservation of molecules such as DNA. Here we examine the bone structure of macroscopically well-preserved medieval human skeletons by transmission electron microscopy and immunohistochemistry, and the quantity and quality of DNA extracted from these skeletons. DNA technology has been increasingly used for analyzing physical evidence in archaeological forensics; however, the isolation of ancient DNA is difficult since it is highly degraded, extraction yields are low and the co-extraction of PCR inhibitors is a problem. We adapted and optimised a method that is frequently used for isolating DNA from modern samples, Chelex(®) 100 (Bio-Rad) extraction, for isolating DNA from archaeological human bones and teeth. The isolated DNA was analysed by real-time PCR using primers targeting the sex determining region on the Y chromosome (SRY) and STR typing using the AmpFlSTR(®) Identifiler PCR Amplification kit. Our results clearly show the preservation of bone matrix in medieval bones and the presence of intact osteocytes with well preserved encapsulated nuclei. In addition, we show how effective Chelex(®) 100 is for isolating ancient DNA from archaeological bones and teeth. This optimised method is suitable for STR typing using kits aimed specifically at degraded and difficult DNA templates since amplicons of up to 250bp were successfully amplified.

Osteoporosis and autophagy: What is the relationship?

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.

Steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation

OBJECTIVE: To analyze steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation. METHODS: Thirty-two virgin adult female rats were randomized to two groups as follows: the control group GI received vehicle and the experimental group GII received melatonin supplementation (10 µg/night per animal) for 60 consecutive days. After the treatment, animals were anesthetized and the collected ovaries were immediately placed in liquid nitrogen for complementary deoxyribonucleic acid microarray analyses. A GeneChip® Kit Rat Genome 230 2.0 Affymetrix Array was used for gene analysis and the experiment was repeated three times for each group. The results were normalized with the GeneChip® Operating Software program and confirmed through analysis with the secondary deoxyribonucleic acid-Chip Analyzer (dChip) software. The data were confirmed by real-time reverse transcription polymerase chain reaction analysis. Genes related to ovarian function were further confirmed by immunohistochemistry. RESULTS: We found the upregulation of the type 9 adenylate cyclase and inhibin beta B genes and the downregulation of the cyclic adenosine monophosphate response element modulator and cytochrome P450 family 17a1 genes in the ovarian tissue of GII compared to those of the control group. CONCLUSION: Our data suggest that melatonin supplementation decreases gene expression of cyclic adenosine monophosphate, which changes ovarian steroidogenesis.

Special dyeing, histochemistry, immunohistochemistry and ultrastructure: A study of mast cells/eosinophilic granules cells (MCs/EGC) from Centropomus parallelus intestine

ABSTRACT Intestine mast cells/eosinophilic granule cells (MCs/EGC) of the marine species Centropomus parallelus (fat snook) were first studied using light and electron microscopy techniques. Mast cells are cells from the connective tissue found in almost all organs and tissues of vertebrates. In fish, they appear in greater numbers in parts of their bodies that are exposed to their environment, such as skin, gills and intestine. The granules in fat snooks mast cell contain a variety of substances, such as histamine, heparin, chondroitin sulfate, serotonin, proteases and cytokines. The present study of intestine MCs/EGC was carried out in 20 specimens of fat snook. Samples of tissue were fixed in Bouin solution and in buffered formalin. Ferric hematoxylin ‐ Congo red, pH6 acridine orange, pH2.5 and pH0,5 Alcian Blue (AB), toluidine blue, PAS, AB + PAS and immunohistochemistry protocols were used. In the mucosa and submucosa layers, MCs/EGCs granules with basic contents were evidenced by Congo red staining, and with acid contents granules were identified through pH 2.5 and 0,5 AB, and acridine orange. Basic and acid contents were simultaneously evidenced using ferric hematoxylin ‐ Congo red stain. Metachromasia was observed in both mucosal and submucosal mast cells. Neutral glycoproteins were evidenced by using PAS protocol, glycosaminoglycan through AB and both simultaneously through AB + PAS. In immunohistochemistry assays, MCs/EGC were positive for tryptase, chymase and serotonin. As in mammals, the study of samples fixed in modified Karnovsky for transmission electron microscopy evidenced that most of the MCs granules were spherical and showed varying electron density, as described in previous reports on other teleost fish species. The metachromasia observed and the identification of tryptase, chymase and serotonin suggest a great similarity between fat snooks MCs/EGC and those described in the mucosa of mammals. HIGHLIGHTSMCs/EGC have granules with content exhibiting basic, acid and neutral character.Metachromasia was also a feature observed in fat snooks MCs/EGC.Serotonin, tryptase and chymase were found in plasmatic granules of MCs/EGC.Fat snooks MCs/EGC are very similar to mammals mast cells.

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

Abstract 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.

Collagen concentration on the facial skin of postmenopausal women after topical treatment with estradiol and genistein: a randomized double-blind controlled trial

Abstract Objective: The objective of this study is to compare the effects of topical estrogen and genistein (a soy isoflavone) on the facial skin collagen of postmenopausal women not undergoing systemic hormonal therapy. Methods: This is a prospective, double blind, randomized, controlled clinical trial. Volunteer women (N = 30) 45–55 year old from the Endocrine Gynecology sector of the Gynecology Department of the Federal University of São Paulo (UNIFESP). The Ethical Committee of the Federal University of São Paulo approved the study (report no. 386/2004; registration on ClinicalTrials.gov NCT01553773), were assigned to topical treatment with either estrogen or genistein for 24 weeks. We quantified and compared facial collagen concentration before and after each treatment by performing pre-auricular skin biopsies. Results: Our data showed an increase in the amount of both type I and type III facial collagen by the end of both treatments. However, the outcomes of the estrogen GI (ER) group were superior to the genistein GII (GEN) group, with statistical significance p < 000.1 Conclusion: Treatment with topical estrogen is superior to genistein, but both have positive impacts on facial skin collagen. Nevertheless, it is still unclear whether prolonged use of genistein and other topical phytoestrogens could produce systemic effects and further research is needed to clarify this question.

Effects of early and late treatment with soy isoflavones in the mammary gland of ovariectomized rats

ABSTRACT Introduction Soy isoflavones have been shown to be an alternative to hormone therapy at menopause, without causing side-effects such as breast cancer. However, the effects of early and late treatment with isoflavones on the mammary gland remain controversial. Objective To investigate the effects of early and late treatment with soy isoflavones on the mammary gland of ovariectomized rats. Methods Thirty 3-month-old rats were ovariectomized and divided equally into groups: Control, treated with vehicle solution; or with 150 mg/kg/body weight of isoflavones by gavage; or subcutaneously treated with 10 μg/kg/body weight with 17β-estradiol. Treatments started 3 days (early treatment) or 30 days (late treatment) after ovariectomy and lasted for 30 consecutive days. Thereafter, the animals were euthanized and the mammary glands were removed and processed for paraffin embedding. Sections were stained with hematoxylin and eosin for histomorphometry or subjected to immunohistochemical detection of Ki-67 and VEGF-A. Results The ductal, lobular and total epithelial fractions were similar between controls and the early/late isoflavone groups, but they were significantly higher in the groups treated with estradiol. In both epithelial and stromal regions, the immunoreactivity of VEGF-A and the percentage of Ki-67-positive cells were significantly higher in the groups treated with estradiol, while they were similar in the early/late isoflavone groups and control groups. Conclusion Our results indicate that early and late treatment with soy isoflavones at the dose of 150 mg/kg/body weight does not show proliferative and angiogenic effects on the mammary gland of ovariectomized rats.

The effects of soybean isoflavones and 17β-estradiol in uterus and mammary glands of diabetic rat models

Abstract The objective of this study was to evaluate the action of soy isoflavones and 17 beta estradiol on the extracellular matrix in the uterus and mammary gland of diabetic rats. Sixty adult female rats underwent ovariectomy, then randomized into seven groups of ten animals each: Non-diabetic: GI Sham control animals ovariectomized; and GII control ovariectomized that received propylene glycol vehicle. Diabetic: GIII Sham control diabetic animals ovariectomized; GIV ovariectomized diabetic animals receiving propylene glycol vehicle; GV diabetic ovariectomized animals treated with soy isoflavones (150 mg/kg by gavage); GVI ovariectomized diabetic rats treated with estrogen (17b-estradiol, 10 mg/kg, subcutaneously); GVII diabetic ovariectomized animals treated with soy isoflavones (150 mg/kg by gavage), and with estrogen (17b-estradiol, 10 mg/kg combination therapy). Treatments occurred during 30 consecutive days. After animals euthanasia, a portion of the uterus was immersed in liquid nitrogen for molecular biology analysis, the other portion of uterus and mammary glands were removed and processed for paraffin embedding. Soy isoflavones (GV) and 17b estradiol improved the production of compounds of extracellular matrix, such as small leucine-rich proteoglycans (SLRPs). The combination of both therapies had an additive effect in SLRPs expression. Soy isoflavones contribute to the uterine integrity of SLRPs of diabetic rats.