Culture bag systems for clinical applications of adult human neural crest-derived stem cells

Introduction Facing the challenging treatment of neurodegenerative diseases as well as complex craniofacial injuries such as those common after cancer therapy, the field of regenerative medicine increasingly relies on stem cell transplantation strategies. Here, neural crest-derived stem cells (NCSCs) offer many promising applications, although scale up of clinical-grade processes prior to potential transplantations is currently limiting. In this study, we aimed to establish a clinical-grade, cost-reducing cultivation system for NCSCs isolated from the adult human nose using cGMP-grade Afc-FEP bags. Methods We cultivated human neural crest-derived stem cells from inferior turbinate (ITSCs) in a cell culture bag system using Afc-FEP bags in human blood plasma-supplemented medium. Investigations of viability, proliferation and expression profile of bag-cultured ITSCs were followed by DNA-content and telomerase activity determination. Cultivated ITSCs were introduced to directed in vitro differentiation assays to assess their potential for mesodermal and ectodermal differentiation. Mesodermal differentiation was determined using an enzyme activity assay (alkaline phosphatase, ALP), respective stainings (Alizarin Red S, Von Kossa and Oil Red O), and RT-PCR, while immunocytochemistry and synaptic vesicle recycling were applied to assay neuroectodermal differentiation of ITSCs. Results When cultivated within Afc-FEP bags, ITSCs grew three-dimensionally in a human blood plasma-derived matrix, thereby showing unchanged morphology, proliferation capability, viability and expression profile in comparison to three dimensionally-cultured ITSCs growing in standard cell culture plastics. Genetic stability of bag-cultured ITSCs was further accompanied by unchanged telomerase activity. Importantly, ITSCs retained their potential to differentiate into mesodermal cell types, particularly including ALP-active, Alizarin Red S-, and Von Kossa-positive osteogenic cell types, as well as adipocytes positive in Oil Red O assays. Bag culture further did not affect the potential of ITSCs to undergo differentiation into neuroectodermal cell types coexpressing β-III-tubulin and MAP2 and exhibiting the capability for synaptic vesicle recycling. Conclusions Here, we report for the first time the successful cultivation of human NCSCs within cGMP-grade Afc-FEP bags using a human blood plasma-supplemented medium. Our findings particularly demonstrate the unchanged differentiation capability and genetic stability of the cultivated NCSCs, suggesting the great potential of this culture system for future medical applications in the field of regenerative medicine.

Anticipatory engineering: anticipation in sensory-motor systems of human

In visual tracking experiments, distributions of the relative phase be-tween target and tracer showed positive relative phase indicating that the tracer precedes the target position. We found a mode transition from the reactive to anticipatory mode. The proposed integrated model provides a framework to understand the antici-patory behaviour of human, focusing on the integration of visual and soma-tosensory information. The time delays in visual processing and somatosensory feedback are explicitly treated in the simultaneous differential equations. The anticipatory behaviour observed in the visual tracking experiments can be ex-plained by the feedforward term of target velocity, internal dynamics, and time delay in somatosensory feedback.

Effect of refrigeration systems upon frozen bull sperm viability assessed by computer-assisted sperm analysis and fluorescent probes

Sperm cryopreservation success depends upon the maintenance of spermatozoa fertility potential. Sperm cells must preserve both integrity and functionality of several cell structures. The stabilization phase must allow the exit of water from the sperm cells via osmosis. This study aimed to compare the effect of refrigeration in the commercial refrigerator (CR) and the transport/refrigeration box (TRB) upon the viability of frozen bull sperm diluted in three different extenders (A, B and C). Ten Nellore bulls, Bos taurus indicus maintained in Artificial Insemination Center were used and the spermatozoa samples was assessed for Plasma Membrane Integrity and CASA evaluation. The stabilization phase (5 degrees C/4 hours) was performed in the CR as well as in the TRB, and then samples were exposed to nitrogen vapor during 20 minutes and then plunged into nitrogen. The statistical analysis was done using the variance analysis and the significance level was set at 5%. In the CR the post-thawing parameters for PM and ALH were higher (p < 0.05) in the extender A (glicine egg-yolk) and extender B (glicine egg-free) when compared with extender C (TRIS egg-yolk). As for BCF, STR and LIN, the parameters were higher (p < 0.05) in extender B than in C. Samples that were stabilized in the TRB presented higher post-thawing parameters (p < 0.05) for PM and LIN in extender A and extender B when compared with C. BCF and STR parameters were higher (p < 0.05) in extemder B when compared with C. Extender B samples had higher (p < 0.05) PMI when stabilized in CR. The findings in this experiment enable us to say that both CR and TRB were effective in keeping the viability of post-thawing bull semen.

Influence of human dentine on the antibacterial activity of self-etching adhesive systems against cariogenic bacteria

Objectives: The incorporation of antibacterial agents into adhesive systems has been proposed to eliminate residual bacteria from dentine. This study used the agar diffusion method to evaluate the antibacterial activity of Clearfil Protect Bond (CPB), Clearfil SE Bond (CSEB), Clearfil Tri-S Bond (C3SB) and Xeno-III (XIII) self-etching adhesive systems, with or without light-activation, against cariogenic bacteria, and to assess the influence of human dentine on the antibacterial activity of these materials.Methods: An aliquot of 10 mu l per material (and individual components) were pipetted onto paper and dentine discs distributed in Petri dishes containing bacterial culture in BHI agar. Positive control was 0.2% chlorhexidine digluconate (CHX).Results: After incubation, the adhesive components of CPB and CSEB, liquid A of XIII and C3SB did not present antibacterial activity when applied to paper discs. The non-light-activated CPB primer + adhesive promoted the greatest inhibition of Streptococcus mutans (p < 0.05), whereas with light-activation, there was no significant difference between primer + adhesive and primer alone. For Lactobacillus acidophilus, CPB primer presented the greatest antibacterial activity in both light-activation conditions (p < 0.05). Regarding the dentine discs, only CHX promoted an inhibitory effect, though less intense than on paper discs (p < 0.05). CHX presented greater antibacterial activity against S. mutans than against L. acidophilus (p < 0.05).Conclusions: Light-activation significantly reduced the antibacterial activity of the self-etching adhesive systems; MDPB incorporation contributed to the effect of adhesive systems against cariogenic bacteria; the components eluted from the adhesive systems were not capable to diffuse through 400 mu m-thick dentine disc to exert their antibacterial activity against cariogenic bacteria. (C) 2008 Elsevier Ltd. All rights reserved.

Response of human pulps capped with different self-etch adhesive systems

The aim of this study was to evaluate the response of human pulps capped with a calcium hydroxide hard-setting cement or with two-step self-etch adhesive systems. Pulp exposures were performed on the occlusal floor, and the bleeding control was performed with saline solution. The exposed pulp tissue was capped with Clearfil LB 2V (2V) or Clearfil SE Bond (SE) and restored with a composite resin. In control group, the pulpal wound was capped with Ca(OH)(2) cement and restored with Clearfil LB 2V or Clearfil SE Bond + composite resin. After 30 and 90 days, the teeth were extracted, processed for hematoxylin and eosin, and categorized in a histological score system. The pulpal response was worse for groups capped with the self-etch adhesive systems (2V and SE) in both periods of evaluation, when compared to their respective control groups at 90 days (p < 0.05). For both self-etch systems evaluated, the pulp tissue exhibited moderate to severe inflammatory cell infiltrate involving the coronal pulp with chronic abscesses. Dentin bridging was observed in a few specimens. For the calcium hydroxide groups, almost all specimens showed dentin bridge formation, with few scattered inflammatory cells and normal tissue below the pulp exposure site. Calcium hydroxide should be used as the material of choice for pulp capping, and the use of two-step self-etch adhesives for human pulp capping is contraindicated.

A machine learning approach for genome-wide prediction of morbid and druggable human genes based on systems-level data

Background: The genome-wide identification of both morbid genes, i.e., those genes whose mutations cause hereditary human diseases, and druggable genes, i.e., genes coding for proteins whose modulation by small molecules elicits phenotypic effects, requires experimental approaches that are time-consuming and laborious. Thus, a computational approach which could accurately predict such genes on a genome-wide scale would be invaluable for accelerating the pace of discovery of causal relationships between genes and diseases as well as the determination of druggability of gene products.Results: In this paper we propose a machine learning-based computational approach to predict morbid and druggable genes on a genome-wide scale. For this purpose, we constructed a decision tree-based meta-classifier and trained it on datasets containing, for each morbid and druggable gene, network topological features, tissue expression profile and subcellular localization data as learning attributes. This meta-classifier correctly recovered 65% of known morbid genes with a precision of 66% and correctly recovered 78% of known druggable genes with a precision of 75%. It was than used to assign morbidity and druggability scores to genes not known to be morbid and druggable and we showed a good match between these scores and literature data. Finally, we generated decision trees by training the J48 algorithm on the morbidity and druggability datasets to discover cellular rules for morbidity and druggability and, among the rules, we found that the number of regulating transcription factors and plasma membrane localization are the most important factors to morbidity and druggability, respectively.Conclusions: We were able to demonstrate that network topological features along with tissue expression profile and subcellular localization can reliably predict human morbid and druggable genes on a genome-wide scale. Moreover, by constructing decision trees based on these data, we could discover cellular rules governing morbidity and druggability.

Response of human dental pulp to dentin adhesive systems

Many in vivo studies have stated that the response of the dentin/pulp complex does not depend on the dental material used as the liner or pulp-capping agent. However, several in vitro studies have reported the metabolic cytotoxic effects of resin components applied to fibroblast and odontoblast cell lines. The aim of this study was to evaluate the human pulp response following direct pulp capping with current bonding agents and calcium hydroxide (CH). Sound premolars scheduled for orthodontic extraction had their pulp tissue mechanically exposed. After hemorrhage control and total acid conditioning, the experimental bonding agents, including All Bond 2, Scotchbond MP-Plus, Clearfil Liner Bond 2, and Prime & Bond 2.1 were applied on the pulp exposure site. CH saline paste was used as the control pulp-capping agent. All cavities were restored with Z-100 resin composite according to the manufacturer's instructions. Following extractions, the teeth were processed for microscopic evaluation. In the short term, the bonding agents elicited a moderate inflammatory pulp response with associated dilated and congested blood vessels adjacent to the pulp exposure site. A mild inflammatory pulp response was observed when Clearfil Liner Bond 2 or CH was applied on the pulp exposures. With time, macrophages and giant cells engulfing globules and components of all experimental bonding agents displaced into the pulp space were seen. This chronic inflammatory response did not allow complete pulp repair, which interfered with the dentin bridge formation. Pulp exposures capped with CH exhibited an initial organization of elongated pulp cells underneath the coagulation necrosis. CH stimulated early pulp repair and dentin bridging that extended into the longest period. The bonding agents evaluated in the present study cannot be recommended for pulp therapy on sound human teeth.

Computer-assisted analysis of p53 and PCNA expression in oral lesions infected with human papillomavirus

OBJECTIVE: To carry out a retrospective study to determine whether human papillomavirus (HPV) infection and immunohistochemical expression of p53 and proliferating cell nuclear antigen (PCNA) are related to the risk of oral cancer. STUDY DESIGN: Fifty-seven oral biopsies, consisting of 30 oral squamous papillomas (OSPs) and 27 oral squamous cell carcinomas (OSCCs) were tested for the presence of HPV 6/11 and 16/18 by in situ hybridization using catalyzed signal amplification and in situ hybridization. p53 And PCNA expression was analyzed by immunohistochemistry and evaluated quantitatively by image analysis. RESULTS: Nineteen of the 57 oral lesions (33.3%) were positive for HPV. HPV 6/11 was found in 6 of 30 (20%) OSPs and 1 of 27 (3.7%) OSCCs. HPV 16/18 was found in 10 of 27 (37%) OSCCs and 2 of 30 (6.7%) OSPs. Sixteen of the 19 HPV-positive cases (84.2%) were p53 negative; 5 (9%) were HPV 6/11 and 11 (19%) HPV 16/18, with an inverse correlation between the presence of HPV DNA and p53 expression (P=.017, P < .05). PCNA expression appeared in 18 (94.7%) of HPV positive cases, showing that HPV 16/18 was associated with intensity of PCNA expression and with OSCCs (P=.037, P < .05). CONCLUSION: Quantitative evaluation of p53 by image analysis showed an inverse correlation between p53 expression and HPV presence, suggesting protein degradation. Image analysis also demonstrated that PCNA expression was more intense in HPV DNA 16/18 OSCCs. These findings suggest involvement of high-risk HPV types in oral carcinogenesis.

Adaptative techniques for the human faces detection

This paper presents results from an efficient approach to an automatic detection and extraction of human faces from images with any color, texture or objects in background, that consist in find isosceles triangles formed by the eyes and mouth.

Bond strength of adhesive systems to human tooth enamel

The purpose of this study was to evaluate in vitro three adhesive systems: a total etching single-component system (G1 Prime & Bond 2.1), a self-etching primer (G2 Clearfil SE Bond), and a self-etching adhesive (G3 One Up Bond F), through shear bond strength to enamel of human teeth, evaluating the type of fracture through stereomicroscopy, following the ISO guidance on adhesive testing. Thirty sound premolars were bisected mesiodistally and the buccal and lingual surfaces were embedded in acrylic resin, polished up to 600-grit sandpapers, and randomly assigned to three experimental groups (n = 20). Composite resin cylinders were added to the tested surfaces. The specimens were kept in distilled water (37°C/24 h), thermocycled for 500 cycles (5°C-55°C) and submitted to shear testing at a crosshead speed of 0.5 mm/min. The type of fracture was analyzed under stereomicroscopy and the data were submitted to Anova, Tukey and Chi-squared (5%) statistical analyses. The mean adhesive strengths were G1: 18.13 ± 6.49 MPa, (55% of resin cohesive fractures); G2: 17.12 ± 5.80 MPa (90% of adhesive fractures); and G3: 10.47 ± 3.14 MPa (85% of adhesive fractures). In terms of bond strength, there were no significant differences between G1 and G2, and G3 was significantly different from the other groups. G1 presented a different type of fracture from that of G2 and G3. In conclusion, although the total etching and self-etching systems presented similar shear bond strength values, the types of fracture presented by them were different, which can have clinical implications.

Computer tools to aid the learning and design steps for photovoltaic systems

This paper presents novel simulation tools to assist the lecturers about learning processes on renewable energy sources, considering photovoltaic (PV) systems. The PV behavior, functionality and its interaction with power electronic converters are investigated in the simulation tools. The main PV output characteristics, I (current) versus V (voltage) and P (power) versus V (voltage), were implemented in the tools, in order to aid the users for the design steps. In order to verify the effectiveness of the developed tools the simulation results were compared with Matlab. Finally, a prototype was implemented with the purpose to compare the experimental results with the results from the proposed tools, validating its operational feasibility. © 2011 IEEE.

Fluorescence Level of Composites Assessed by Computer Processing of Digital Images - ScanWhite

The human dentition is naturally translucent, opalescent and fluorescent. Differences between the level of fluorescence of tooth structure and restorative materials may result in distinct metameric properties and consequently perceptible disparate esthetic behavior, which impairs the esthetic result of the restorations, frustrating both patients and staff. In this study, we evaluated the level of fluorescence of different composites (Durafill in tones A2 (Du), Charisma in tones A2 (Ch), Venus in tone A2 (Ve), Opallis enamel and dentin in tones A2 (OPD and OPE), Point 4 in tones A2 (P4), Z100 in tones A2 ( Z1), Z250 in tones A2 (Z2), Te-Econom in tones A2 (TE), Tetric Ceram in tones A2 (TC), Tetric Ceram N in tones A1, A2, A4 (TN1, TN2, TN4), Four seasons enamel and dentin in tones A2 (and 4SD 4SE), Empress Direct enamel and dentin in tones A2 (EDE and EDD) and Brilliant in tones A2 (Br)). Cylindrical specimens were prepared, coded and photographed in a standardized manner with a Canon EOS digital camera (400 ISO, 2.8 aperture and 1/ 30 speed), in a dark environment under the action of UV light (25 W). The images were analyzed with the software ScanWhite©-DMC/Darwin systems. The results showed statistical differences between the groups (p < 0.05), and between these same groups and the average fluorescence of the dentition of young (18 to 25 years) and adults (40 to 45 years) taken as control. It can be concluded that: Composites Z100, Z250 (3M ESPE) and Point 4 (Kerr) do not match with the fluorescence of human dentition and the fluorescence of the materials was found to be affected by their own tone.

Computer graphics applied to anatomy: a study of two bio-cad modeling methods on finite element analysis of human edentulous hemi-mandible

Modeling is a step to perform a finite element analysis. Different methods of model construction are reported in literature, as the Bio-CAD modeling. The purpose of this study was to perform a model evaluation and application using two methods of Bio-CAD modeling from human edentulous hemi-mandible on the finite element analysis. From CT scans of dried human skull was reconstructed a stereolithographic model. Two methods of modeling were performed: STL conversion approach (Model 1) associated to STL simplification and reverse engineering approach (Model 2). For finite element analysis was used the action of lateral pterygoid muscle as loading condition to assess total displacement (D), equivalent von-Mises stress (VM) and maximum principal stress (MP). Two models presented differences on the geometry regarding surface number (1834 (model 1); 282 (model 2)). Were observed differences in finite element mesh regarding element number (30428 nodes/16683 elements (model 1); 15801 nodes/8410 elements (model 2). D, VM and MP stress areas presented similar distribution in two models. The values were different regarding maximum and minimum values of D (ranging 0-0.511 mm (model 1) and 0-0.544 mm (model 2), VM stress (6.36E-04-11.4 MPa (model 1) and 2.15E-04-14.7 MPa (model 2) and MP stress (-1.43-9.14 MPa (model 1) and -1.2-11.6 MPa (model 2). From two methods of Bio-CAD modeling, the reverse engineering presented better anatomical representation compared to the STL conversion approach. The models presented differences in the finite element mesh, total displacement and stress distribution.

Ergonomics and interfaces of traditional information systems: packaging

The contemporary world is characterized, among other factors, by the influence of the new computer information systems on the behavior of individuals. However, traditional information systems still have interaction problems with users. The aim of this study was to determine whether the interaction aspects between user versus traditional information systems (particularly the graphics) have been fully studied. To do so, the ergonomic aspects and usability of such systems were reviewed, with emphasis on the problems of visibility, legibility and readability. From that criteria, the evolution of ergonomic studies of information systems was reviewed (bibliometrics technique); and examples of ergonomic and usability problems in packaging were demonstrated (case study). The results confirm that traditional information systems still have problems of interaction between human X system, hindering the effective perception of information.