Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes

The possibility of obtaining transplantable oral epithelia opens new perspectives for oral treatments. Most of them are surgical, resulting in mucosal failures. As reconstructive material this in vitro epithelia would be also useful for other parts of the human body. Many researchers still use controversial methods; therefore it was evaluated and compared the efficiency of the enzymatic and direct explant methods to obtain oral keratinocytes. To this project oral epithelia fragments were used. This work compared: time needed for cell obtainment, best cell amount, life-span and epithelia forming cell capacity. The results showed the possibility to obtain keratinocytes from a small oral fragment and we could verify the advantages and peculiar restrictions. We concluded that under our conditions the enzymatic method showed the best results: in the cells obtaining time needed, cell amount and life-span. Both methods showed the same capacity to form in vitro epithelia.

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.

Correlation between c-Jun and human papillomavirus in oral premalignant and malignant lesions

c-Jun, one of the components of the transcription factor activating protein-1 (AP-1), is suggested as a factor in malignant progression of oral lesions. c-Jun and other AP-1 components relationships with human papillomavirus (HPV) infection have been investigated, but not yet focusing on oral carcinogenesis. The aim of this study was to verify whether c-Jun immunohistochemical expression is related to HPV DNA detection in oral premalignant and malignant lesions. Fifty cases diagnosed as oral leukoplakias, with different degrees of epithelial dysplasia, and as oral squamous cell carcinomas (OSCC) were submitted to immunohistochemistry to detect c-Jun and to in situ hybridization with signal amplification to assess HPV DNA. It was verified that c-Jun nuclear expression increased according to the degree of dysplasia within the lesion, with the greatest expression in OSCC. The same did not happen concerning HPV infection - a discrete proportional relation was observed in indexes found in leukoplakia with no dysplasia, leukoplakia with dysplasia and OSCC, but statistically insignificant. When separating the group of leukoplakia by degrees of dysplasia, this relation of proportion was not observed. Nevertheless, the overall prevalence of HPV infection was 24% and the high-risk HPV types were the most frequently identified, which does not allow excluding HPV as a risk factor in oral carcinogenesis. When relating c-Jun expression and HPV infection, no statistically significant relationship is observed. Results suggest then that malignant progression mediated by c-Jun is independent of the presence of HPV in oral carcinogenesis. (C) 2007 Elsevier Ltd. All rights reserved.

Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: Immunolocalization according to the odontogenesis phases

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunobistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3 were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

Micro-Shear Bond Strength of Different Adhesives to Human Dental Enamel

The aim of this study was to evaluate the micro-shear bond strength of 5 adhesive systems to enamel, one single-bottle acid-etch adhesive (O), two self-etching primers (P) and two all-in-one self-etching adhesives (S). Method: Sixty premolar enamel surfaces (buccal or lingual) were ground flat with 400- and 600-grit SiC papers and randomly divided into 5 groups (n=12), according to the adhesive system.. SB2 - Single Bond 2 (O); CSE - Clearfil SE Bond (P); ADS - AdheSE (P); PLP - Adper Prompt L-Pop (S); XE3 - Xeno III (S). Tygon tubing (inner diameter of 0.8mm) restricted the bonding area to obtain the resin composite (Z250) cylinders. After storage in distilled water at 37 degrees C for 24h and thermocycling, micro-shear testing was performed (crosshead speed of 0.5mm/min). Data were submitted to one-way ANOVA and Tukey test (a=5%). Samples were also subjected to stereomicroscopic and SEM evaluations after micro-shear testing. Mean bond strength values (MPa +/- SD) and the results of Tukey test were: SB2: 36.36(+/- 3.34)a; ADS: 33.03(+/- 7.83)a; XE3: 32.76(+/- 5.61)a; CSE: 30.61(+/- 6.68)a; PLP: 22.17(+/- 6.05)b. Groups with the same letter were not statistically different. It can be concluded that no significant difference was there between SB2, ADS, XE3 and CSE, in spite of different etching patterns of these adhesives. Only PLP presented statistically lower bond strengths compared with others. J Clin Pediatr Dent 35(3): 301-304, 2011

Evaluation of two mineral trioxide aggregate compounds as pulp-capping agents in human teeth

The present randomized, controlled prospective study evaluated the histomorphological response of human dental pulps capped with two grey mineral trioxide aggregate (MTA) compounds. Pulp exposures were performed on the occlusal floor of 40 human permanent pre-molars. The pulp was capped either with ProRoot (Dentsply) or MTA-Angelus (Angelus) and restored with zinc oxide eugenol cement. After 30 and 60 days, teeth were extracted and processed for histological examination and the effects on the pulp were scored. The data were subjected to Kruskal-Wallis and Conover tests (alpha = 0.05). In five out of the 40 teeth bacteria were present in pulp tissue. No significant difference was observed between the two materials (P > 0.05) in terms of overall histological features (hard tissue bridge, inflammatory response, giant cells and particles of capping materials). Overall, 94% and 88% of the specimens capped with MTA-Angelus and ProRoot, respectively, showed either total or partial hard tissue bridge formation (P > 0.05). Both commercial materials ProRoot (Dentsply) and MTA-Angelus (Angelus) produced similar responses in the pulp when used for pulp capping in intact, caries-free teeth.

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.

Roughness of Human Enamel Surface Submitted to Different Prophylaxis Methods

The purpose of this in vitro study was to evaluate alterations in the surface roughness and micromorphology of human enamel submitted to three prophylaxis methods. Sixty-nine caries-free molars with exposed labial surfaces were divided into three groups. Group I was treated with a rotary instrument set at a low speed, rubber clip and a mixture of water and pumice; group II with a rotary instrument set at a low speed, rubber cup and prophylaxis paste Herjos-F (Vigodent S/A Industria e Comercio, Rio de Janeiro, Brazil); and group III with sodium bicarbonate spray Profi II Ceramic (Dabi A dante Indtistrias Medico Odontologicas Ltda, Ribeirao Preto, Brazil). All procedures were performed by the same operator for 10 s, and samples were rinsed and stored in distilled water. Pre and post-treatment surface evaluation was completed using a surface profilometer (Perthometer S8P Marh, Perthen, Germany) in 54 samples. In addition, the other samples were coated with gold and examined in a scanning electron microscope (SEM). The results of this study were statistically analyzed with the paired t-test (Student), the Kruskal-Wallis test and the Dunn (5%) test. The sodium bicarbonate spray led to significantly rougher surfaces than the pumice paste. The use of prophylaxis paste showed no statistically significant difference when compared with the other methods. Based on SEM analysis, the sodium bicarbonate spray presented an irregular surface with granular material and erosions. Based on this study, it can be concluded that there was an increased enamel stuface roughness when teeth were treated with sodium bicarbonate spray when compared with teeth treated with pumice paste.

Activity of Matrix Metalloproteinases in Bovine versus Human Dentine

Metalloproteinases (MMPs) have been implicated with metabolism of collagen in physiological and pathological processes in human dentine. As bovine teeth have been used as a substitute for human teeth in laboratory analysis, this study evaluated the activity of MMP-2 and -9 in bovine versus human dentine. Bovine and human dentine fragments, from crowns and roots, were powderized. Protein extraction was performed by two protocols: a neutral extraction with guanidine-HCl/EDTA (pH 7.4) and an acidic extraction with citric acid (pH 2.3). Gelatinolytic activities of extracts were revealed by zymography. MMP-2 and -9 were detected in crown and root dentine from bovine and human teeth. Total activities of MMP-2 were 11.4 +/- 2.2, 14.6 +/- 2.0, 9.7 +/- 1.2 and 12.4 +/- 0.9 ng/ml for bovine root, human root, bovine crown and human crown dentine, respectively. Corresponding activities for MMP-9 were 14.9 +/- 2.0, 15.3 +/- 1.3, 15.4 +/- 1.3 and 15.5 +/- 1.3 ng/ml, respectively. Bovine dentine was found to be a reliable substrate for studies involving the activity of MMP-2 and -9. Copyright (C) 2011 S. Karger AG, Basel

Cross-Sectional Microhardness of Human Enamel Subjected to Erosive, Cariogenic or Combined Erosive/Cariogenic Challenges

This in situ study evaluated the interaction between caries and erosion processes. In the first phase, enamel specimens were subjected to erosion without dental plaque (EO) or to erosion with plaque (EP); in the second phase, they were subjected to erosion plus cariogenic challenge (EC) or cariogenic challenge (CO), both with plaque accumulation. Cross-sectional hardness data (10-330 mu m depth) were tested using ANOVA (alpha = 0.05). EO and EP showed surface softening to 10 mu m depth. CO and EC produced subsurface lesions, of similar depth (up to 220 mu m), with CO showing higher integrated loss of hardness than EC, indicating that cariogenic and erosive challenges did not have an additive effect. Copyright (C) 2010 S. Karger AG, Basel

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.