Additional Insights Into the Role of Osteocalcin in Osteoblast Differentiation and in the Early Steps of Developing Alveolar Process of Rat Molars.

This study investigated whether osteocalcin (OCN) is present in osteoblast precursors and its relationship with initial phases of alveolar process formation. Samples of maxillae of 16-, 18-, and 20-day-old rat embryos (E16, E18, and E20, respectively), and 05-, 10-, and 15-day-old postnatal rats (P05, P10, and P15, respectively) were fixed and embedded in paraffin or araldite. Immunohistochemistry for osterix (Osx), alkaline phosphatase (ALP), and OCN detection was performed and the number of immunolabelled cells was computed. Non-decalcified sections were subjected to the von Kossa method combined with immunohistochemistry for Osx or OCN detection. For OCN immunolocalization, samples were fixed in 0.5% glutaraldehyde/2% formaldehyde and embedded in LR White resin. The highest number of ALP- and OCN-immunolabelled cells was observed in dental follicle of E16 specimens, mainly in basal portions of dental alveolus. In corresponding regions, osteoblasts in differentiation adjacent to von Kossa-positive bone matrix exhibited Osx and OCN immunoreactivity. Ultrastructural analysis revealed OCN immunoreactive particles inside osteoblast in differentiation, and in bone matrix associated with collagen fibrils and within matrix vesicles, at early stages of alveolar process formation. Our results indicate that OCN plays a role in osteoblast differentiation and may regulate calcium/phosphate precipitation during early mineralization of the alveolar process.

Histomorphometric analysis of the endometrium in an ectopic model of endometriosis in mice.

Objective: Evaluate histomorphometry of ectopic and eutopic endometrial tissues in receptor mice. Method: Eighteen female Balb/c were divided into 3 groups, 6 animals each: GI Control, no procedure; GII - Sham, animals that had the same procedures as GIII without receiving the ectopic endometrial implant. Instead, they received saline solution; GIII - endometriosis model, animals had surgical intervention with an ectopic endometrial implant. GI and GIII mice were treated with 17ß-estradiol, 100 µg/kg each. All animals were euthanized to collect uterine horns, which were fixed in 4% paraformaldehyde, embedded in paraffin, stained with Hematoxilin and Eosin and submitted to histomorphometric analyzes. Data underwent one-way ANOVA followed by Tukey's test. Results: Local tissue growth, showing important lesions and adhesions, as well as dark cysts were noticed. In GIII group, there was an increase in number of blood vessels and glands (GIII ≥ GI and GIII p > .001). Thickening of the GIII endometrial epithelial was also evident (GIII ≥ GI and GIII. p > .001). We also noticed an increase in the number of eosinophils (GIII (GIII ≥ GI and GIII. p > .001). Conclusion: Easy to perform model, capable of reproducing morphological endometriosis characteristics. From our findings, there was an increase of endometrial thickness as well as an increase in the eosinophils population.

Isoflavone Protects the Renal Tissue of Diabetic Ovariectomized Rats via PPARγ.

Diabetes associated with post-menopause is related to a worse condition of kidney disease. Taking into consideration that this disorder may be regulated by estrogenic mediators, we evaluated the renal protective effect of isoflavone. We investigated the role of the PPARγ in the pathogenesis of the disease. For this study, we used diabetic female rats in a postmenopausal model through ovariectomy. The animals were treated with isoflavone or 17ß-estradiol. A dosage was administered to bring on blood glycemia, and through immunohistochemistry, we evaluated the immunoreactivity of PPARγ in the endometrium and renal tissue. We analyzed the immunoreactivity of renal injury molecule KIM-1 and the collagen and glycogen densities in the kidney. Through bioinformatics analysis, we observed PPARγ and COL1A1 gene expression under the influence of different glucose doses. In particular, we observed that isoflavone and 17ß-estradiol regulate blood glycemia. Renal injury was inhibited by isoflavone, observed by a reduction in KIM-1, along with glycogen accumulation. These benefits of isoflavone may be associated with PPARγ overexpression in the kidneys and endometrium of diabetic ovariectomized rats.

Spatio-temporal immunolocalization of VEGF-A, Runx2, and osterix during the early steps of intramembranous ossification of the alveolar process in rat embryos.

Vascular endothelial growth factor A (VEGF-A) is expressed by several cell types and is a crucial factor for angiogenic-osteogenic coupling. However, the immunolocalization of VEGF-A during the early stages of the alveolar process formation remains underexplored. Thus, we analyzed the spatio-temporal immunolocalization of VEGF-A and its relationship with Runt-related transcription factor 2 (Runx2) and osterix (Osx) during the early steps of intramembranous ossification of the alveolar process in rat embryos. Embryo heads (E) of 16, 18 and 20-day-old rats were processed for paraffin embedding. Histomorphometry and immunohistochemistry to detect VEGF-A, Runx2, and Osx (osteoblast differentiation markers) were performed. The volume density of bone tissue including bone cells and blood vessels increased significantly in E18 and E20. Cells showing high VEGF-A immunoreactivity were initially observed within a perivascular niche in the ectomesenchyme; afterwards, these cells were diffusely located near bone formation sites. Runx2-and Osx-immunopositive cells were observed in corresponded regions of cells showing strong VEGF-A immunoreactivity. Although these immunostained cells were observed in all specimens, this immunolocalization pattern was more evident in E16 specimens and gradually decreased in E18 and E20 specimens. Double immunofluorescence labelling showed intracellular co-localization of Osx and VEGF-A in cells surrounding the developing alveolar process, indicating a crucial role of VEGF-A in osteoblast differentiation. Our results showed VEGF-A immunoexpression in osteoblasts and its precursors during the maxillary alveolar process formation of rat embryos. Moreover, the VEGF-A-positive cells located within a perivascular niche at the early stages of the alveolar process development suggest a crosstalk between endothelium and ectomesenchymal cells, reinforcing the angiogenic-osteogenic coupling in this process.

Analysis of hyaluronic acid in the endometrium of women with polycystic ovary syndrome.

Endometrium extracellular matrix provides a wide range of signals at different cellular levels, like cell death and proliferation, which can be important for regulating menses and reducing the proliferative processes. The objective of this study is to evaluate hyaluronic acid concentration, the enzymes of hyaluronic acid synthases in the endometrium of patients with polycystic ovary syndrome (PCOS) and eumenorrheic women. A total of 60 endometrial samples from 30 patients with PCOS and 30 women with regular menstrual cycles in the proliferative phase, attended at Gynecology Division of Clinical Hospital of the FMUSP (HC-USP). Profile determination and the concentration of hyaluronic acid was performed by the biochemical method of the fluorimetric assay (ELISA-like). Its location in the endometrial tissue as well as the dosage of enzymes synthases (HAS1, HAS2 and HAS3) was done by immunohistochemistry and western blotting. Statistical analyses were performed with one-way ANOVA, followed by the Bonferroni test. Regarding hyaluronic acid synthases, there was a higher HAS1 and HAS2 reactivity and lower HAS3 reactivity in the PCOS endometrium compared to women with regular menstrual cycles in the proliferative phase. We suggest that PCOS patients have different composition of hyaluronic acid in relation to a regular cycle in the proliferative phase.