Coordinated regulation of follicle development by germ and somatic cells

The continuum of folliculogenesis begins in the fetal ovary with the differentiation of the oogonia and their isolation within the primordial follicles. Primordial follicle activation is an enigmatic process, whereby some follicles enter the growing pool to become primary follicles, thereby embarking on an irreversible progression towards ovulation or atresia. This process is under the coordinated regulation of factors from the oocyte itself, as well as from the somatic cells of the ovary, in particular the theca and granulosa cells, which are structural components of the follicle. These two influences provide the principal stimuli for the growth of the follicle to the late preantral or early antral stage of development. The endocrine effects of the gonadotrophins FSH and LH are essential to the continued progression of the follicle and most atresia can be attributed to the failure to receive or process the gonadotrophin signals. The peri-ovulatory state has received intensive investigation recently, demonstrating a coordinated role for gonadotrophins, steroids, epidermal growth factor family proteins and prostaglandins. Thus, a complex programme of coordinated interaction of governing elements from both germ and somatic cell sources is required for successful follicle development.

Oestrogen and Progesterone Receptor Gene Expression in Canine Oocytes and Cumulus Cells Throughout the Oestrous Cycle

The aim of this research was to analyze oestrogen receptor-alpha (ER alpha), ER beta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells` total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ER beta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ER beta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ER alpha and ER beta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ER alpha was expressed throughout the oestrous cycle.

Bovine sperm cells viability during incubation with or without exogenous DNA

The aim of this study was to assess the effect of exogenous DNA and incubation time on the viability of bovine sperm. Sperm were incubated at a concentration of 5 x 10(6)/ml with or without plasmid pEYFP-NUC. Fluorescent probes, propidium iodide/Hoechst 33342, FITC-PSA and JC-1, were used to assess plasma membrane integrity (PMI), acrosome membrane integrity (AMI) and mitochondrial membrane potential (MMP) respectively at 0, 1, 2, 3 and 4 h of incubation. Exogenous DNA addition did not affect sperm viability; however, incubation time was related to sperm deterioration. Simultaneous assessment of PMI, AMI and MMP showed a reduction in the number of sperm with higher viability (integrity of plasma and acrosome membranes and high mitochondrial membrane potential) from 58.7% at 0 h to 7.5% after 4 h of incubation. Lower viability sperm (damaged plasma and acrosome membranes and low mitochondrial membrane potential) increased from 4.6% at 0 h to 25.99% after 4 h of incubation. When PMI, AMI and MMP were assessed separately we noticed a reduction in plasma and acrosome membrane integrity and mitochondrial membrane potential throughout the incubation period. Therefore, exogenous DNA addition does not affect sperm viability, but the viability is reduced by incubation time.

Pulp Vitality in Patients with Intraoral and Oropharyngeal Malignant Tumors Undergoing Radiation Therapy Assessed by Pulse Oximetry

Introduction: The aim of this study was to evaluate pulp oxygenation levels (%SpO(2)) in patients with malignant intraoral and oropharyngeal tumors treated by radiotherapy (RT). Methods: Pulp oxygenation levels were measured by pulse oximetry. Twenty patients were selected, and two teeth of each participant (n = 40) were analyzed, regardless of the quadrant and the area irradiated, at four different time points: TP1, before RI; TP2, at the beginning of RI with radiation doses between 30 and 35 Gy; TP3, at the end of RI with radiation dose! between 60 and 70 Gy; and TP4, 4 to 5 months after the beginning of cancer treatment. Results: Mean %SpO(2) at the different time points were 93% (TP1), 83% (TP2), 77% (TP3), and 85% (TP4). The Student`s t test showed statistically significant differences between TP1 and TP2 (P < .01), TP3 (P <.01), and TP4 (P <.01). TP3 was also statistically significantly different when compared with TP2 (P <.01) and TP4 (P <.01). No statistically significant difference could be observed between TP2 and TP4. Conclusion`s: Because the mean %SpO(2) before RI was greater than during and after therapy and values obtained 4 to 5 months after the beginning of RI were close to the initiation of RI, pulp tissue may be able to regain normal blood flow after RT. If the changes in the microcirculation of the dental pulp were indeed transitory, preventive endodontic treatment or extraction in patients who are currently undergoing or recently received RI and who show negative signs of pulp sensitivity may rot be necessary for pulpal reasons. (J Endod 2011;37:1197-1200)

Determination of pulp vitality in vivo with pulse oximetry

Aim To evaluate the use of pulse oximetry as a test for pulp vitality, by comparing in the same patient, the levels of oxygen saturation of the index finger and of the maxillary central incisor and canine teeth without clinically detectable pulp inflammation. Methodology Seventeen male and female patients aged between 26 and 38 years participated and a total of 32 maxillary central incisor and 32 canine teeth were analysed. Selection criteria required the teeth to have healthy crowns, or with restorations no more than 2 mm in diameter and no clinical and radiographical signs or symptoms of pulp or periapical inflammatory changes. The negative control group consisted of 10 root filled teeth. Measurements were first taken from the index finger of patients. Their teeth were then subjected to a thermal test with refrigerant gas and then to a vitality test with pulse oximetry. Data were analysed by Pearson`s and paired t-tests. Results There were no significant statistical correlations between blood oxygen levels in the index finger and in the teeth of the patient (P > 0.05). There was a statistically significant difference in the oxygen levels between the two tooth groups studied and the index finger (P <= 0.002). Mean oxygen values in the index finger of patients were 95% (SD = 1.6), oxygen values in the maxillary central incisor were 91.29% (SD = 2.61) and mean oxygen values in maxillary canine were 90.69% (SD = 2.71). Conclusion The method determined consistently the level of blood oxygen saturation of the pulp in maxillary central incisor and canine teeth and can therefore be used for pulp vitality testing. Further studies are required to assess the effectiveness and validity of pulse oximetry in determining pulp vitality in traumatized teeth.

Stem cell proliferation under low intensity laser irradiation: A preliminary study

Background and Objectives: Phototherapy with low intensity laser irradiation has shown to be effective in promoting the proliferation of different cells. The aim of this in vitro study was to evaluate the potential effect of laser phototherapy (660 nm) on human dental pulp stem cell (hDPSC) proliferation. Study Design/Materials and Methods: The hDPSC cell strain was used. Cells cultured under nutritional deficit (10% FBS) were either irradiated or not (control) using two different power settings (20 mW/6 seconds to 40 mW/3 seconds), with an InGaAIP diode laser. The cell growth was indirectly assessed by measuring the cell mitochondrial activity through the MTT reduction-based cytotoxicity assay. Results: The group irradiated with the 20 mW setting presented significantly higher MTT activity at 72 hours than the other two groups (negative control-10% FBSand lased 40 mW with 3 seconds exposure time). After 24 hours of the first irradiation, cultures grown under nutritional deficit (10% FBS) and irradiated presented significantly higher viable cells than the non-irradiated cultures grown under the same nutritional conditions. Conclusions: Under the conditions of this study it was possible to conclude that the cell strain hDPSC responds positively to laser phototherapy by improving the cell growth when cultured under nutritional deficit conditions. Thus, the association of laser phototherapy and hDPSC cells could be of importance for future tissue engineering and regenerative medicine. Moreover, it opens the possibility of using laser phototherapy for improving the cell growth of other types of stem cells.

Tooth Tissue Engineering: Optimal Dental Stem Cell Harvest Based on Tooth Development

Our long-term objective is to devise reliable methods to generate biological replacement teeth exhibiting the physical properties and functions of naturally formed human teeth. Previously, we demonstrated the successful use of tissue engineering approaches to generate small, bioengineered tooth crowns from harvested pig and rat postnatal dental stem cells (DSCs). To facilitate characterizations of human DSCs, we have developed a novel radiographic staging system to accurately correlate human third molar tooth developmental stage with anticipated harvested DSC yield. Our results demonstrated that DSC yields were higher in less developed teeth (Stages 1 and 2), and lower in more developed teeth (Stages 3, 4, and 5). The greatest cell yields and colony-forming units (CFUs) capability was obtained from Stages 1 and 2 tooth dental pulp. We conclude that radiographic developmental staging can be used to accurately assess the utility of harvested human teeth for future dental tissue engineering applications.

Release of cytokines by stimulated peripheral blood mononuclear cells in chronic periodontitis

Objective: The emergence of periodontal medicine increased interest in defining the behaviour of peripheral blood cells in periodontitis subjects in comparison with healthy group. The aim of this study was to evaluate the levels of interleukin (IL)-8, tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-10 released by Escherichia coli lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC) obtained from the peripheral blood of chronic periodontitis subjects. Design: PBMC samples were isolated from 19 systemically healthy donors, divided into generalized chronic periodontitis (n = 10) and healthy (n = 9) subjects. Cells were incubated for 24-48 h in 500 mu L wells containing RPM! 1640 and stimulated with 1.0 ng/mL of E. coli LPS. Supernatants were used to quantify the amounts of IL-8, TNF-alpha, IL-6 and IL-10 released using enzyme-linked immunosorbent assay (ELISA). Results: PBMC cells from periodontitis subjects released higher levels of TNF-alpha and IL-6 than those from healthy subjects (P < 0.05). Conversely, the supernatants of the stimulated PBMC cells obtained from healthy subjects presented higher amounts of IL-8 than those from periodontitis (P < 0.05). No differences were observed in the levels of IL-10 (P > 0.05) between groups. Conclusion: In conclusion, the results of the present study showed that E. coli LPS-stimulated PBMC from subjects with periodontitis present a different pattern of cytokine release when compared to PBMC from healthy subjects. This phenomenon could have implications locally, in periodontitis, as well as in systemic diseases. (C) 2010 Elsevier Ltd. All rights reserved.

Cysteine Cathepsins in Human Dentin-Pulp Complex

Introduction: Collagen-degrading matrix metalloproteinases (MMPs) are expressed by odontoblasts and present in dentin. We hypothesized that odontoblasts express other collagen-degrading enzymes such as cysteine cathepsins, and their activity would be present in dentin, because odontoblasts are known to express at least cathepsin D. Effect of transforming growth factor beta (TGF-beta) on cathepsin expression was also analyzed. Methods: Human odontoblasts and pulp tissue were cultured with and without TGF-beta, and cathepsin gene expression was analyzed with DNA microarrays. Dentin cathepsin and MMP activities were analyzed by degradation of respective specific fluorogenic substrates. Results: Both odontoblasts and pulp tissue demonstrated a wide range of cysteine cathepsin expression that gave minor responses to TGF-beta. Cathepsin and MMP activities were observed in all dentin samples, with significant negative correlations in their activities with tooth age. Conclusions: These results demonstrate for the first time the presence of cysteine cathepsins in dentin and suggest their role, along with MMPs, in dentin modification with aging. (J Endod 2010;36:475-481)

Expression of proteins in the extracellular matrix of pulp tissue in human primary teeth during physiologic root resorption

Objectives: To analyze the expression of tenascin, fibronectin, collagens I and III, osteonectin, and bone morphogenetic protein 4 (BMP4) in the extracellular matrix of pulp tissue in primary teeth during physiologic root resorption. Method and Materials: Eighteen teeth were decalcified and equally distributed into 3 groups (group I, teeth with two-thirds root length; group II, teeth with one-third root length; and group III, teeth lacking the root). Results: Immunohistochemical analysis showed that all the proteins were expressed. Tenascin, collagen I, and osteonectin showed strong and broad reactivity in group I, with weaker and rare reactivity in groups II and III. The expression of fibronectin, collagen III, and BMP4 did not vary with root resorption phase. Conclusion: The expression of tenascin, collagen I, and osteonectin was reduced in the extracellular matrix and odontoblasts during root resorption. This fact may be related to the decreasing pulp response to damage and treatment during the progression of root resorption. (Quintessence Int 2009; 40: 553-558)

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.

Clastic cells: Mineralized tissue resorption in health and disease

Clastic cells are responsible for mineralized tissue resorption. Bone resorbing cells are called osteo-clasts; however, they are able to resorb mineralized dental tissues or calcified cartilage and then they are called odontoclasts and chondroclasts, respectively. They derive from mononuclear precursors of the monocyte-macrophage lineage from hemopoietic tissue, reach target mineralized tissues and degrade them under many different physiologic or pathologic stimuli. Clastic cells play a key role in calcium homeostasis, and participate in skeletal growth, tooth movement, and other physiological and pathological events. They interact tightly with forming cells in bone and dental hard tissues; their unbalance may result in disturbed resorptive activity thus, causing local or systemic diseases. (C) 2008 Elsevier Ltd. All rights reserved.

Response of human dental pulp capped with MTA and calcium hydroxide powder

Objectives: To compare the response of human dental pulp capped with a mineral trioxide aggregate (MTA) and Ca(OH)(2) powder. Methods and Material: Pulp exposures were performed on the occlusal floor of 40 permanent premolars. The pulp was then capped with either Ca(OH)(2) powder (CH) or MTA and restored with resin composite. After 30 days (groups CH30 and MTA30) and 60 days (groups CH60 and MTA60), the teeth were extracted and processed for HE and categorized in a histological score system. The data were subjected to Kruskal-Wallis and Conover tests (alpha=0.05). Results: In regard to dentin bridge formation, CH30 showed a tendency towards superior performance compared to MTA30 (p>0.05), although the products showed comparable results at day 60. In the item ""Inflammation"" and ""General State of the Pulp"" (p>0.05), CH showed a tendency towards presenting a higher inflammatory response. In the item ""Other Pulpal Findings,"" MTA and Ca(OH)(2) showed equal and excellent performance after 30 and 60 days (p>0.05). Conclusion: After 30 days, Ca(OH)(2) powder covered with calcium hydroxide cement showed faster hard tissue bridge formation compared to MTA. After 60 days, Ca(OH)(2) powder or NITA materials showed a similar and excellent histological response with the formation of a hard tissue bridge in almost all cases with low inflammatory infiltrate.

Specific infiltration of langerin-positive dendritic cells in EBV-infected tonsil, Hodgkin lymphoma and nasopharyngeal carcinoma

We report here the existence of a novel subset of langerin (CD207)-positive, immature dendritic cells (DCs) (CD83(neg)) abundantly infiltrating Epstein Barr virus (EBV)-infected areas in tonsil, Hodgkin lymphoma and nasopharyngeal carcinoma. These CD207(+) DCs differ from conventional epidermal Langerhans cells in their lack of CD1a and CCR6 and their unusual tissue localization. CD207(+) DC infiltration strongly correlates with EBV infection because it was neither detected in EBV negative specimens nor in tissues infected with other human viruses. These immature DCs might represent good candidates for induction of the EBV-specific immune response.

Patients with oral squamous cell carcinoma are characterized by increased frequency of suppressive regulatory T cells in the blood and tumor microenvironment

Oral squamous cell carcinoma (OSCC) is a cancerous lesion with high incidence worldwide. The immunoregulatory events leading to OSCC persistence remain to be elucidated. Our hypothesis is that regulatory T cells (Tregs) are important to obstruct antitumor immune responses in patients with OSCC. In the present study, we investigated the frequency, phenotype, and activity of Tregs from blood and lesions of patients with OSCC. Our data showed that > 80% of CD4(+)CD25(+) T cells isolated from PBMC and tumor sites express FoxP3. Also, these cells express surface Treg markers, such as GITR, CD45RO, CD69, LAP, CTLA-4, CCR4, and IL-10. Purified CD4(+)CD25(+) T cells exhibited stronger suppressive activity inhibiting allogeneic T-cell proliferation and IFN-gamma production when compared with CD4(+)CD25(+) T cells isolated from healthy individuals. Interestingly, approximately 25% of CD4(+)CD25(-) T cells of PBMC from patients also expressed FoxP3 and, although these cells weakly suppress allogeneic T cells proliferative response, they inhibited IFN-gamma and induced IL-10 and TGF-beta secretion in these co-cultures. Thus, our data show that Treg cells are present in OSCC lesions and PBMC, and these cells appear to suppress immune responses both systemically and in the tumor microenvironment.