Pfaffia paniculata (Brazilian ginseng) roots decrease proliferation and increase apoptosis but do not affect cell communication in murine hepatocarcinogenesis

Pfaffia paniculata (Brazilian ginseng) roots and/or its extracts have shown anti-neoplastic, chemopreventive, and anti-angiogenic properties. The aim of this work was to investigate the chemopreventive mechanisms of this root in Mice Submitted to the infant model of hepatocarcinogenesis, evaluating the effects oil cellular proliferation, apoptosis. and intercellular communication. Fifteen-day-old BALB/c male mice were given, i.p., 10 mu g/g of the carcinogen N-nitrosodiethylamine (DEN). Animals were separated into three groups at weaning and were given different concentrations of powdered P. paniculata root (0%, 2%, or 10%) added to commercial food for 27 weeks. Control group (CT) was not exposed to the carcinogen and was given ration without the root. After euthanasia, the animals` liver and body weight were measured. Liver fragments were sampled to Study intercellular communication, molecular biology, and histopathological analysis. Cellular proliferation was evaluated by immunohistochemistry for PCNA, apoptosis was evaluated by apoptotic bodies count and alkaline cornet technique, and inter-cellular communication by diffusion of lucifer yellow dye, immunofluorescence, western blot and real-time PCR for connexins 26 and 32. Chronic treatment with powdered P. paniculata root reduced cellular proliferation and increased apoptosis in the 2%, group. Animals in the 10% group had an increase in apoptosis with chronic inflammatory process. Intercellular communication showed no alterations in any of the groups analyzed. These results Indicate that chemopreventive effects of P. paniculata are related to the control of cellular proliferation and apoptosis, but not to cell communication and/or connexin expression, and are directly Influenced by the root concentration. (C) 2009 Elsevier GmbH. All rights reserved.

Hepatic function evolution in dogs anesthetized with Zolazepam/Tiletamine

The hepatic effects of the anesthetic association zolazepam/tiletamine were investigated in dogs by analyzing the serum concentration of hepatic enzymes. Ten healthy dogs were divided into two groups of five, group I (GI) and group II (GII). The animals of GI received a single dose of 6,6 mg/kg of zolazepam/tiletamine, by intramuscular (IM) injection. GII dogs received 6,6 mg/kg of zolazepam/tiletamine by the IM route; after a period of 50 - 80 minutes the animals received two additional doses (3,3 mg/kg) by intravenous administration[SAH1]. The hepatic function were analyzed by monitoring the serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl-transferase (GGT). Four blood samples were collected in different moments during the analyses: M0, before the first application of the drug; and M1 to M4. M1 through M3 was collected with intervals of 20 minutes before M0, while M4 was obtained 24 hours after M1. The normality of the obtained results was analyzed by Kolmogorov-Smirnov Test; while the Tukey`s test compared the means, using a level of significance of 5% for both statistical analyses. The mean values of all enzymes evaluated were within normal limits for both experimental groups, without any significant statistical alteration being observed between and within these groups. These results demonstrated that the association of zolazepam/tiletamine at the dosage of 6.6 mg/kg, followed by two applications additional of 3.3 mg/kg resulted in elevation of the evaluated hepatic enzymes without exceeding the physiologic values. Additionally, a single application of 6.6 mg/kg of zolazepam/tiletamine by the intramuscular route resulted in lower values when compared to three applications.

Prognostic impact of adenomatous polyposis coil gene expression in osteosarcoma of the extremities

Purpose: To evaluate the impact of adjuvant chemotherapy on the outcome of osteosarcoma of the extremities, and to identify prognostic factors using the expression of adenomatous polyposis coli (APC), cadherin and beta-catenin Wnt-signalling markers. Methods: The clinical, demographic, anatomic and pathological factors including a detailed analysis of the immunohistochemical expression of cadherin, B-catenin and APC were retrospectively examined in 97 patients with osteosarcoma of the extremities (metastatic and non-metastatic at diagnosis), treated with surgery and/or chemotherapy from 1985 to 2000. Results: APC immunoreactivity showed a statistically significant association with age and serum alkaline phosphatase levels (p = 0.025 and p = 0.038). When survival was the end-point of multivariate analysis, race segregated patients with poor survival as did lack of cadherin expression. For overall survival, cadherin immunoreactivity and the interaction between APC expression and response to adjuvant chemotherapy were significant (p = 0.012 and p < 0.001). No significant clinical association was evident with immunohistochemical expression of cadherin, beta-catenin. Conclusion: Lack of expression of cadherin was a significant variable to overall and disease-free survival. Significantly, positive APC immunoreactivity and adjuvant chemotherapy were associated with a favourable treatment response. Studies using newer immunohistochemical markers within the Wnt-signalling pathway may guide the development of more appropriate therapeutic targets for future individualised treatment. (C) 2010 Elsevier Ltd. All rights reserved.

Cigarette smoke inhalation modulates gene expression in sites of bone healing: a study in rats

Objective. The aim of this study was to assess the effect of cigarette smoke inhalation (CSI) on gene expression in alveolar bone healing sites. Study design. Wistar rats were randomly assigned to the groups: control [animals not exposed to CSI (n = 20)] and test [animals exposed to CSI, starting 3 days before teeth extraction and maintained until killing them (n = 20)]. First mandibular molars were bilaterally extracted, and the expression of alkaline phosphatase, bone morphogenetic protein (BMP) 2 and 7, receptor activator of nuclear factor kappa B ligand, osteoprotegerin, and d2 isoform of vacuolar adenosine triphosphatase V(o) domain were assessed by quantitative polymerase chain reaction in the newly formed tissue in the sockets. Results. Overall, data analysis demonstrated that CSI significantly affected the expression pattern of all of the studied genes except BMP-7. Conclusion. The expression of key genes for bone healing may be affected by CSI in tooth extraction sites. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;110:447-452)

Effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) on the viability, proliferation and differentiation of osteoblast-like cells cultured on a chemically modified titanium surface

Aim: The aim of the present study was to assess the influence of the chemical characteristics and roughness of titanium surfaces on the viability, proliferation and differentiation of osteoblast-like cells cultured in a medium supplemented with recombinant human bone morphogenetic protein-7 (rhBMP-7). Material and methods: Osteo-1 cells were grown on titanium disks presenting with the following surfaces: (1) machined, (2) coarse grit-blasted and acid-attacked (SLA) and (3) chemically modified SLA (SLAmod) in the absence or presence of 20 ng/ml rhBMP-7 in culture medium. The viability and number of osteo-1 cells were evaluated after 24 h. Analyses of total protein content (TP) and alkaline phosphatase (AP) activity at 7, 14 and 21 days, collagen content at 7 and 21 days and mineralized matrix formation at 21 days were performed. Results: Cell viability (P=0.5516), cell number (P=0.3485), collagen content (P=0.1165) and mineralized matrix formation (P=0.5319) were not affected by the different surface configurations or by the addition of rhBMP-7 to the medium. Osteo-1 cells cultured on SLA surfaces showed a significant increase in TP at 21 days. The ALPase/TP ratio (P=0.00001) was affected by treatment and time. Conclusion: The results suggest that the addition of rhBMP-7 to the culture medium did not exert any effect on the viability, proliferation or differentiation of osteoblast-like cells grown on the different surfaces tested. All titanium surfaces analyzed allowed the complete expression of the osteoblast phenotype such as matrix mineralization by osteo-1 cells.

A possible mechanism of low molecular weight protein tyrosine phosphatase (LMW-PTP) activity modulation by glutathione action during human osteoblast differentiation

Objective: Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are a family of enzymes strongly involved in the regulation of cell growth and differentiation. Since there is no information concerning the relationship between osteoblastic differentiation and LMW-PTP expression/activity, we investigated its involvement during human osteoblast-like cells (hFOB 1.19) differentiation. It is known that LMW-PTP is regulated by an elegant redox mechanism, so we also observed how the osteoblastic differentiation affected the reduced glutathione levels. Design: hFOB 1.19 cells were cultured in DMEM/F12 up to 35 days. The osteoblast phenotype acquisition was monitored by measuring alkaline phosphatase activity and mineralized nodule formation by Von Kossa staining. LMW-PTP activity and expression were measured using the p-nitrophenylphosphate as substrate and Western blotting respectively. Crystal violet assay determined the cell number in each experimental point. Glutathione level was determined by both HPLC and DNTB assays. Results: LMW-PTP modulation was coincident with the osteoblastic differentiation biomarkers, such as alkaline phosphatase activity and presence of nodules of mineralization in Vitro. Likewise LMW-PTP, the reduced glutathione-dependent microenvironment was modulated during osteoblastic differentiation. During this process, LMW-PTP expression/activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day (p < 0.001) of culturing, decreasing thereafter. Conclusions: Our results clearly suggest that LMW-PTP expression/activity was rigorously modulated during osteoblastic differentiation, possibly in response to the redox status of the cells, since it seems to depend on suitable levels of reduced glutathione. in this way, we pointed out LMW-PTP as an important signaling molecule in osteoblast biology and bone formation. (C) 2009 Elsevier Ltd. All rights reserved.

Tartrate-resistant acid phosphatase activity and glutathione levels are modulated during hFOB 1.19 osteoblastic differentiation

Tartrate-resistant acid phosphatase (TRAP) is a well-known marker of osteoclasts and bone resorption. Here we have investigated whether osteoblast-like cells (hFOB 1.19) present TRAP activity and how would be its pattern of expression during osteoblastic differentiation. We also observed how the osteoblastic differentiation affected the reduced glutathione levels. TRAP activity was measured using the p-nitrophenylphosphate substrate. The osteogenic potential of hFOB 1.19 cells was studied by measuring alkaline phosphatase activity and mineralized nodule formation. Oxidative stress was determined by HPLC and DNTB assays. TRAP activity and the reduced glutathione-dependent microenvironment were modulated during osteoblastic differentiation. During this phase, TRAP activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day, decreasing thereafter. We demonstrate that TRAP activity is modulated during osteoblastic differentiation, possibly in response to the redox state of the cell, since it seemed to depend on suitable levels of reduced glutathione.

Evaluation of the physical and chemical properties of two commercial and three experimental root-end filling materials

Objective. The aim of this study was to evaluate the pH, calcium release, setting time, and solubility of two commercially available mineral trioxide aggregate (MTA) cements (white MTA Angelus and MTA Bio), and of three experimental cements (light-cured MTA, Portland cement with 20% bismuth oxide and 5% calcium sulfate, and an epoxy resin-based cement). Study design. For evaluation of pH and calcium ion release, polyethylene tubes with 1.0 mm internal diameter and 10.0 mm length were filled with the cements and immediately immersed in flasks containing 10 mL deionized water. After 3, 24, 72, and 168 hours, the tubes were removed and the water from the previous container was measured for its pH and calcium content with a pH meter and an atomic absorption spectrophotometer. For analysis of the setting time, Gilmore needles weighing 100 g and 456.5 g were used, in accordance with the American Society for Testing and Materials specification no. C266-03. Solubility of each cement was also tested. Results. All the cements were alkaline and released calcium ions, with a declining trend over time. After 3 hours, Portland cement + bismuth oxide and MTA Bio had the highest pH and light-cured MTA the lowest. After 1 week, MTA Bio had the highest pH and light-cured MTA and epoxy resin-based cement the lowest. Regarding calcium ion release, after 3 hours, Portland cement + bismuth oxide showed the highest release. After 1 week, MTA Bio had the highest. Epoxy resin-based cement and light-cured MTA had the lowest calcium release in all evaluation periods. Regarding setting times, white MTA Angelus and MTA Bio had the shortest, Portland cement + bismuth oxide had an intermediate setting time, and the epoxy resin-based cement had the longest. The materials that showed the lowest solubility values were the epoxy resin-based cement, Portland cement + bismuth oxide, and light-cured MTA. The highest solubility values were presented in white MTA Angelus and MTA Bio. Conclusions. The white MTA Angelus and MTA Bio had the shortest setting times, higher pH and calcium ion release, and the highest solubility. In contrast, the epoxy resin-based cement and light-cured MTA showed lower values of solubility, pH, and calcium ion release. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 110: 250-256)

Evaluation of pH and calcium ion release of new root-end filling materials

Objective. The purpose of this study was to evaluate the pH and calcium ion release of 6 materials used for root-end filling and perforation repair. Study design. Gray ProRoot MTA, gray MTA-Angelus, white MTA-Angelus, and CPM were compared to 2 experimental ones: MTA-exp, also based in Portland cement with a modified mixing liquid, and MBPc, an epoxy-resin based cement containing calcium hydroxide. After 3, 24, 72, and 168 hours the water in which each sample had been immersed was tested to determine the ph and calcium ion release. Results. All the analyzed materials showed alkaline pH and capacity to release calcium ions; however, a tendency of reduction of these characteristics was noted for all the analyzed materials, except for the MBPc, which showed a slight increase of pH among the 3 initial periods. Conclusion. The results suggest that all materials investigated presented alkaline pH and ability of release of calcium ions. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: 135-139)

Growth hormone induces bone morphogenetic proteins and bone-related proteins in the developing rat periodontium

The hypothesis that growth hormone (GH) up-regulates the expression of enzymes, matrix proteins, and differentiation markers involved in mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with GH over 5 days, The molar teeth and associated alveolar bone were processed for immunohistochemical demonstration of bone morphogenetic proteins 2 and 4 (BMP-2 and -4), bone morphogenetic protein type IA receptor (BMPR-IA), bone alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OPN), bone sialoprotein (BSP), and E11 protein (E11), The cementoblasts, osteoblasts, and periodontal ligament (PDL) cells responded to GH by expressing BMP-2 and -4, BMPR-IA, ALP, OC, and OPN and increasing the numbers of these cells. No changes were found in patterns of expression of the late differentiation markers BSP and E11 in response to GH, Thus, GH evokes expression of bone markers of early differentiation in cementoblasts, PDL cells, and osteoblasts of the periodontium. We propose that the induction of BMP-2 and -4 and their receptor by GH compliments the role of GH-induced insulin-like growth factor 1 (IGF-1) in promoting bone and tooth root formation.

A fluorometric microassay for histamine release from human gingival mast cells

Mast cells are important effector cells of the immune system. We describe a rapid and inexpensive microassay to determine histamine release from human gingival mast cells. The assay is based on the coupling of histamine with o-phthalaldehyde (OPT) at a highly alkaline pH to form a fluorescent product. Using this assay with a sample volume of 10 mul/well in a 384 black well microplate, the histamine detection limit was 0.031 mug/ml. The human mast cell line (HMC-1) and fresh mast cells isolated from human gingival tissue (n = 10) were stimulated with substance P, anti-IgE or calcium ionophore A23187, Calcium ionophore significantly increased histamine release from HMC-1 cells and gingival mast cells (p < 0.05). This microassay will facilitate the study of mast cell histamine release in diseased oral mucosa.

Chronic ethanol intake inhibits in vitro osteogenesis induced by osteoblasts differentiated from stem cells

The study investigated whether chronic ethanol (ETH) intake and subsequent ETH exposure of cell cultures affects osteoblast differentiation by evaluating key parameters of in vitro osteogenesis. Rats were treated with 5-20% (0.85-3.43 mM) ETH, increasing by 5% per week for a period of 4 weeks (habituation), after which the 20% level was maintained for 15 days (chronic intake). Bone-marrow stem cells from control (CONT) or ETH-treated rats were cultured in osteogenic medium which was either supplemented (ETH) or not supplemented (CONT) with 1.3 mm ethanol. Thus, four groups relating to rat treatment/culture supplementation were evaluated: (1) CONT/CONT, (2) ETH/CONT, (3) CONT/ETH and (4) ETH/ETH Cell morphology, proliferation and viability, total protein content, alkaline phosphatase (ALP) activity and bone-like nodule formation were evaluated. Chronic ethanol intake significantly reduced both food and liquid consumption and body weight gain. No difference was seen in cell morphology among treatments. Cell number was affected at 7 and 10 days as follows: CONT/CONT = CONT/ETH < ETH/CONT = ETH/ETH. Doubling time between 3 and 10 days was greater in groups of CONT animals: ETH/ETH = ETH/CONT < CONT/ETH = CONT/CONT. Cell viability and ALP activity were not affected by either animal treatment or culture exposure to ethanol. At day 21, the total protein content was affected as follows: ETH/ETH = CONT/ETH < ETH/CONT = CONT/CONT. Bone-like nodule formation was affected as follows: ETH/ETH < CONT/ETH < ETH/CONT < CONT/CONT. These results show that chronic ethanol intake, followed by the exposure of osteoblasts to ethanol, inhibited the differentiation of osteoblasts, as indicated by an increased proliferation rate and reduced bone-like nodule formation. Copyright (C) 2007 John Wiley & Sons, Ltd.

In vitro osteogenesis induced by cells derived from sites submitted to sinus grafting with anorganic bovine bone

Objectives: This study evaluated key parameters of the in vitro osteogenesis induced by osteoblastic cells obtained from sites submitted to sinus grafting with anorganic bovine bone (ABB) in comparison with cells derived from bone sites of the same patients. Materials and methods: In three patients, the augmentation of maxillary sinus was carried out using ABB (Bio-Oss (R)). After at least 6 months, during the surgical intervention for titanium implants placement, biopsies were taken from these areas using trephine burs (grafted group). Bone fragments, of the same patients, from sites that had not received graft were also obtained with trephine burs and used as a control group. Osteoblastic cells were obtained from grafted and control groups by enzymatic digestion and cultured under standard osteogenic condition until subconfluence. First passaged cells were cultured in 24-well culture plates. Cell adhesion was evaluated at 24 h. For proliferation and viability assay, cells were cultured for 1, 3, 7, and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 3, 7, 10, 14, 17, and 21 days. Cultures were stained with Alizarin red S at 21 days, for detection of mineralized matrix. Data were compared by Student`s t-test. Results: Cell adhesion and viability were not affected by cell source (P>0.05). Total protein content was greater (P<0.05) for grafted group. Cell proliferation, ALP activity, and bone-like nodule formation were all greater (P<0.05) for the control group. Conclusions: Taken together, these results indicate that the in vivo long-term contact of cells with ABB downregulates the expression of osteoblast phenotype and consequently the in vitro osteogenesis.

Response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate membrane

This study investigated the response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane. Osteoblastic cells were cultured in osteogenic conditions either on P(VDF-TrFE)/BT or polytetrafluoroethylene (PTFE) for up to 14 days. At 7 and 14 days, the mRNA expression of Runt-related transcription factor 2 (RUNX2), Type I collagen (COL I), Osteopontin (OPN), Alkaline phosphatase (ALP), Bone sialoprotein (BSP), and Osteocalcin (OC), key markers of the osteoblastic phenotype, and of Bcl2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and Survivin (SUR), associated with the control of the apoptotic cell death, was assayed by real-time PCR. In situ ALP activity was qualitatively evaluated by means of Fast red staining. Surface characterization was also qualitatively and quantitatively assayed in terms of topography, roughness, and wettability. Cells grown on P(VDF-TrFE)/BT exhibited a significantly higher mRNA expression for all markers compared to the ones on PTFE, except for Bcl-2, which was not detected for both groups. Additionally, Fast red staining was noticeably stronger in cultures on P(VDF-TrFE)/BT at 7 and 14 days. At micron-and submicron scale, SEM images and roughness analysis revealed that PTFE and P(VDF-TrFE)/BT exhibited a smooth topography and a similar roughness, respectively. PTFE membrane displayed higher contact angles compared with P(VDF-TrFE)/BT, as indicated by wettability assay. The novel P(VDF-TrFE)/BT membrane supports the acquisition of the osteoblastic phenotype in vitro, while up-regulating the expression of apoptotic markers. Further in vivo experiments should be carried out to confirm the capacity of P(VDF-TrFE)/BT membrane in promoting bone formation in guided bone regeneration.

In vitro biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate composite using cultures of human periodontal ligament fibroblasts and keratinocytes

The mm of this work was to evaluate the biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane to be used in guided tissue regeneration (GTR) Fibroblasts from human periodontal ligament (hPDLF) and keratinocytes (SCC9) were plated on P(VDF-TrFE)/BT and polytetrafluorethylene membranes at a cell density of 20.000 cells well(-1) and Cultured for up to 21 days Cell morphology, adhesion and proliferation were evaluated in hPDLF and keratinocytes, while total protein content and alkaline phosphatase (ALP) activity were assayed only for hPDLF Using a higher cell density. real-time polymerase chain reaction (PCR) was performed to assess the expression of typical genes of hPDLF, such as periostin, PDLs17, S100A4 and fibromodulin, and key phenotypic markers of keratinocytes, including involucrin, keratins 1. 10 and 14 Expression of the apoptotic genes bax, bcl-2 and Survivin was evaluated for both cultures hPDLF adhered and spread more oil P(VDF-TrFE)/BT, whereas keratinocytes showed a round shape on both membranes. hPDLF adhesion was greater oil P(VDF-TrFE)/BT at 2 and 4 h, while keratinocyte adhesion was similar for both membranes. Whereas proliferation was significantly higher for hPDLF on P(VDF-TrFE)/BT at days 1 and 7. no signs of keratinocyte proliferation could be noticed for both membranes Total protein content was greater on P(VDF-TrFE)/BT at 7, 14 and 21 days, and higher levels of ALP activity were observed oil P(VDF-TrFE)/BT at 21 days. Real-time PCR revealed higher expression of phenotypic markers of hPDLF and keratinocytes as well as greater expression of apoptotic genes in cultures grown on P(VDF-TrFE)/BT. These results indicate that, by favoring hPDLF adhesion. spreading. proliferation and typical mRNA expression, P(VDF-TrFE)/BT membrane should be considered an advantageous alternative for GTR (C) 2009 Acta Materialia Inc Published by Elsevier Ltd All rights reserved