Early life stress exacerbates bone resorption and inhibits anxiety-like behaviour induced by apical periodontitis in rats.

AIM: To evaluate the influence of the early life stress (ELS) on the severity of the apical periodontitis (AP) in Wistar rats. METHODOLOGY: Forty male Wistar rats were divided into four groups (n = 10): Control rats; AP-rats with AP; ELS-rats subject to ELS; AP + ELS-rats exposed to ELS and subject to AP. ELS was induced by maternal separation (MS) for a period of 3 h for 21 consecutive days. AP was induced via pulp exposure of the first and second right maxillary molars to the oral environment for 40 days. Three days before euthanasia, all rats underwent behavioural analysis to measure anxiety levels by elevated zero maze. Then, the rats were euthanized and the maxillas were removed to assess the occurrence and severity of AP. The periapical region was evaluated for the intensity of the inflammatory infiltrate and the extent of bone loss. The Mann-Whitney test was performed for nonparametric data, and the Tukey's or Student's t-test was performed for parametric data (p < .05). RESULTS: The intensity of the inflammatory infiltrate was significantly larger in the AP + ELS group when compared with AP group (p < .05). The AP + ELS group exhibited significantly greater alveolar bone loss, with a periapical lesion size of 103.5 ± 29.88, compared with 72.3 ± 22.28 in the AP group (p < .05). Rats with AP displayed higher anxiety-like behaviour in relation to the control group (p < .05). However, exposure to ELS abolished the AP-induced increased anxiety-like 'behaviour' throughout, since that rats from AP + ELS group attended more the open arms than non-stressed rats with AP (p < .05). CONCLUSION: Early life stress is predictive of the severity of AP exacerbating the inflammatory process and increasing periapical bone resorption.

Telmisartan impairs the in vitro osteogenic differentiation of mesenchymal stromal cells from spontaneously hypertensive male rats.

Telmisartan (TELM) is an angiotensin II (Ang II) type 1 receptor (Agtr1) antagonist, with partial agonism for Pparg, and has been shown to affect bone metabolism. Therefore, the aim of this study was to investigate the effects of TELM in the in vitro osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSC) from spontaneously hypertensive rats (SHRs). BMSC were obtained from male SHR, and the osteogenic medium (OM) was added to the cells concomitantly with TELM (0.005, 0.05, and 0.5 µM). Undifferentiated BMSC, in control medium (CM), showed an increased viability, while the addition of OM reduced this parameter, and TELM did not show cytotoxicity in the concentrations used. BMSC in OM had an alkaline phosphatase (ALP) activity peak at d10, which decreased at d14 and d21, and TELM reduced ALP at d10 in a dose-dependent manner. Mineralization was observed in the OM at d14, which intensified at d21, but was inhibited by TELM. Agtr1b was increased in the OM, and TELM inhibited its expression. TELM reduced Opn, Ocn, and Bsp and increased Pparg expression, and at the higher concentration TELM also increased the expression of adipogenic markers, Fabp4 and Adipoq. In addition, TELM 0.5 µM increased Irs1 and Glut4, insulin and glucose metabolism markers, known to be regulated by Pparg and to be related to adipogenic phenotype. Our data shows that TELM inhibited the osteogenic differentiation and mineralization of SHR BMSC, by favoring an adipogenic prone phenotype due to Pparg upregulation.

ß1-adrenergic receptor but not ß2 mediates osteogenic differentiation of bone marrow mesenchymal stem cells in normotensive and hypertensive rats.

The sympathetic nervous system regulates bone remodeling via adrenergic receptors on the surface of bone cells. Herein, we evaluated the role of beta-adrenergic receptors (ADRBs) in osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs) derived from normotensive (Wistar) and spontaneously hypertensive rats (SHRs). BMSCs were cultured in a proliferation medium or osteogenic medium (OM). Cells cultured in OM were treated with carvedilol (Cv) or nebivolol (Nb).In OM, cell proliferation was decreased in both strains. In Wistar rats, Cv increased BMSC proliferation and increased alkaline phosphatase (ALP) activity in OM. Both Cv and Nb decreased ALP activity. In addition, Cv and Nb reduced mineral deposition in Wistar rats. Moreover, NB decreased mineralization in SHRs, exhibiting superior efficacy. In OM, cells from Wistar rats and SHRs showed Adrb1 and Adrb2 expression. On day 7, Nb, but not Cv, reduced Adrb1 levels in BMSCs from Wistar rats. Nb inhibited Adrb2 in both strains, and Cv demonstrated superior efficacy. In BMSCs from Wistar rats, both antagonists inhibited Runx2, osterix, and ß-catenin; in SHRs, Cv and Nb inhibited only osterix. Cv decreased osteopontin (Opn), osteocalcin (Ocn), and bone morphogenetic protein (Bmp2) in BMSCs from Wistar rats, inhibiting only Opn in SHRs. Nb effectively inhibited Ocn, bone sialoprotein, and Bmp2, but not Ocn, in BMSCs from Wistar rats, while suppressing Opn in BMSCs from SHRs. In addition, Nb inhibited p-p38 in BMSCs from Wistar rats; Cv inhibited p-p38 in BMSCs from SHRs. In Wistar rats, both antagonists inhibited p-ERK and reduced p-JNK; Cv reduced these expressions only in SHRs. In conclusion, ADRB1, but not ADRB2, could be involved in the osteogenic differentiation of BMSCs from Wistar rats and SHRs. The high ADRB1 expression might suppress the effect of ADRB2 on BMSCs.

Soluble yerba mate (Ilex Paraguariensis) extract enhances in vitro osteoblastic differentiation of bone marrow-derived mesenchymal stromal cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Yerba mate (Ilex paraguariensis) consumption has been associated with beneficial effects on bone health. AIM OF THE STUDY: The purpose of this study was to evaluate the mechanism by which soluble yerba mate (SYM) stimulates osteoblast differentiation of bone marrow-derived mesenchymal stromal cells (BM-MSCs). MATERIALS AND METHODS: BM-MSCs from male Wistar rats were induced towards osteoblastic differentiation with different concentrations of SYM (10, 20, and 50 µg/mL). Osteoblastic differentiation was evaluated by measuring proliferation rates, alkaline phosphatase activity, MMP-2 activity, mineralization, and gene expression of Runx2, Osterix, ß-catenin (Catnb), collagen type I (Col1a1), osteopontin (Opn), osteocalcin (Ocn), bone sialoprotein (Bsp), bone morphogenetic protein-2 (Bmp2), osteoprotegerin (Opg), and Rankl. We also analyzed cytokine production and MAP kinase pathways. RESULTS: SYM (10 µg/mL) did not show a cytotoxic effect and induced a slight increase in ALP activity; however, a great increase in mineralization was observed. SYM was also able to reduce TNF-α and IL-10 production; increase the expression of transcription factors Runx2, Osterix, and Catnb; and increase matrix proteins Opn, Bsp, Ocn, and Bmp2. We also observed a decrease in intracellular signaling of ERK, JNK, and p38 MAPK, which seemed to be related to the SYM response. CONCLUSIONS: Together, these results help to explain the promoting effect on osteoblast differentiation produced by a low SYM concentration. However, a higher SYM concentration presented deleterious effects, including cytotoxicity, decreased ALP activity, increased cytokine production, decreased bone marker gene expression, increased MAPK signaling, and significant mineralization reduction. In conclusion, our results suggest a concentration-specific direct stimulatory effect of SYM on osteoblastic differentiation in vitro.

Bone marrow mesenchymal stem cells stimulated by bFGF up-regulated protein expression in comparison with periodontal fibroblasts in vitro.

OBJECTIVE: The aim of this study was to evaluate, in vitro, the role of bFGF in the proliferation and expression of collagen type I and fibronectin of dog bone marrow mesenchymal stem cells (dBMMSCs) in comparison with the expression of the same proteins in dog periodontal fibroblasts (dPLFs). DESIGN: dBMMSCs from the iliac crest were cultivated in Dulbecco's Modified Eagle's Medium (DMEM). Flow cytometry analysis (FCA) was used to characterize dBMMSC. Cells were stimulated with bFGF (1, 5 and 10 ng/mL) after 24 and 48 h. Real time RT-PCR was performed to verify collagen type I and fibronectin expressions. MTT assay was used to confirm cellular proliferation. Statistical analyses were performed (ANOVA and Kruskal-Wallis tests; p<0.05). RESULTS: FCA showed 55.98% of CD34+ and 32.67% of CD90+ after bone marrow aspiration; 3.33% of CD34+ and 33.0% of CD90+ before P1. After P2, 10.54% of dBMMSCs expressed CD90, whereas after P3, this number decreased to 1.58%. dPLFs presented 4.04% of CD90+ and 1.05% of CD34+ after P3. MTT evaluation showed increase in dBMSC proliferation with 5 ng/mL bFGF-stimulus after 24-h. Both collagen I and fibronectin expression were very similar between the two cells groups after 24-h stimulation with 1 ng/mL bFGF concentration. Fibronectin and collagen I expressions were higher after 24-h stimulation with 5 ng/mL bFGF. CONCLUSION: dBMMSCs (1 ng/mL-bFGF stimulus after 24 h) are very similar to dPLFs as regards morphological and immunostaining characteristics, and collagen and/or fibronectin production. The dBMMSCs presented the highest protein expression rates with 5 ng/mL-bFGF stimulus after 24-h.

Evaluation of the effects of endodontic materials on fibroblast viability and cytokine production.

INTRODUCTION: Recently, a new sealer composed of Portland cement named Endo-CPM-Sealer was developed. The aim of this study was to investigate the effects of Endo-CPM-Sealer (EGEO SRL, Buenos Aires, Argentina), Sealapex (Sybron Endo, Glendora, CA), and Angelus MTA (Angelus, Londrina, Brazil) on cell viability and cytokine (interleukin [IL]-1beta and IL-6) production by mouse fibroblasts. METHODS: Millipore culture plate inserts with polyethylene tubes filled with materials were placed into 24-well cell culture plates with mouse fibroblasts. Cells cultured with only empty polyethylene tubes were used as the control. After 24 hours, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to evaluate the cell viability. For cytokine assay, mouse fibroblasts were incubated in 24-well flat-bottom plates with set material disks at the bottom. Cells cultured without the material disks served as the negative control. After 24 hours of incubation, culture media were collected for cytokine evaluation by using an enzyme-linked immunosorbent assay. The data were statistically analyzed by analysis of variance and Bonferroni correction. RESULTS: Endo-CPM-Sealer, Sealapex, and Angelus MTA did not inhibit the cell viability. All materials induced IL-6 releasing, but the amount was not statistically significant compared with the control group. Angelus MTA induced IL-1beta releasing significantly more than the control. CONCLUSIONS: All materials were not considered cytotoxic in fibroblast culture.