Evaluation of Ilex guayusa and Piper marginatum Extract Cytotoxicity on Human Dental Pulp Mesenchymal Stem Cells.

BACKGROUND: Amelogenesis imperfecta is a hereditary disorder affecting dental enamel. Among its phenotypes, hypocalcified AI is characterized by mineral deficiency, leading to tissue wear and, consequently, dental sensitivity. Excessive fluoride intake (through drinking water, fluoride supplements, toothpaste, or by ingesting products such as pesticides or insecticides) can lead to a condition known as dental fluorosis, which manifests as stains and teeth discoloration affecting their structure. Our recent studies have shown that extracts from Colombian native plants, Ilex guayusa and Piper marginatum, deposit mineral ions such as phosphate and orthophosphate into the dental enamel structure; however, it is unknown whether these extracts produce toxic effects on the dental pulp. OBJECTIVE: To assess cytotoxicity effects on human dental pulp stem cells (hDPSCs) exposed to extracts isolated from I. guayusa and P. marginatum and, hence, their safety for clinical use. METHODS: Raman spectroscopy, fluorescence microscopy, and flow cytometry techniques were employed. For Raman spectroscopy, hDPSCs were seeded onto nanobiochips designed to provide surface-enhanced Raman spectroscopy (SERS effect), which enhances their Raman signal by several orders of magnitude. After eight days in culture, I. guayusa and P. marginatum extracts at different concentrations (10, 50, and 100 ppm) were added. Raman measurements were performed at 0, 12, and 24 h following extract application. Fluorescence microscopy was conducted using an OLIMPUS fv1000 microscope, a live-dead assay was performed using a kit employing a BD FACS Canto TM II flow cytometer, and data analysis was determined using a FlowJo program. RESULTS: The Raman spectroscopy results showed spectra consistent with viable cells. These findings were corroborated using fluorescence microscopy and flow cytometry techniques, confirming high cellular viability. CONCLUSIONS: The analyzed extracts exhibited low cytotoxicity, suggesting that they could be safely applied on enamel for remineralization purposes. The use of nanobiochips for SERS effect improved the cell viability assessment.

Cytotoxic effects on human dental pulp stem Cells after exposure to adhesive bonding agents.

Studies regarding cytotoxic effects attributed to the use of adhesive bonding agents on pulp tissue are not conclusive. To point out whether these materials are safe for clinical use, in vivo exposure of dental pulp to adhesive bonding agents was simulated using an experimental setup in which Human Dental Pulp Stem Cells (hDPSC) are exposed to the action of two kinds of adhesives: self-etching adhesives and two-step bonding agents through a dentine barrier. Cytotoxic effects on these cells were evaluated by MTT assay protocol and fluorescence microscopy, and their results were contrasted to those obtained through Raman spectra taken on single hDPSCs. Overall, no significant cytotoxic effects were observed by combining all the techniques, and cell viability close to 90% was achieved for a dentine barrier of at least 1 mm thick. Moreover, Raman spectroscopy was able to detect structural DNA damage in some dental pulp cells when exposed to two-step bonding agents, suggesting that this technique could be considered a complementary tool with the potential to evaluate cell toxicity beyond cell viability.

Manifestaciones de trastornos musculoesqueléticos en moldeadores manuales metalúrgicos

Introducción: Los trastornos musculoesqueléticos en el contexto laboral metalúrgico repercuten negativamente desde la perspectiva personal, económica y social de los trabajadores. Objetivo: Identificar las manifestaciones de trastornos musculoesqueléticos en los moldeadores manuales metalúrgicos del área de fundición de la empresa Holmeca de la provincia Holguín, Cuba, en el período comprendido enero-marzo de 2021. Método: Se realizó un estudio observacional, no experimental de corte transversal en 19 moldeadores manuales metalúrgicos, en el que se incluyeron variables sociodemográficas, ubicación de zonas dolorosas, intensidad del dolor, tiempo de aparición y duración del trastorno. Se emplearon métodos teóricos, empíricos. Fue utilizado el Cuestionario Nórdico estandarizado de Kuorinka y la Escala Visual Analógica. El procesamiento de la información se llevó a cabo mediante la estadística descriptiva. Resultados: Se identificaron las zonas dolorosas y la prevalencia más alta de dolor musculoesqueléticos en: muñeca/mano (94,74 %), hombro (89,47 %), dorsal/lumbar (89,47 %) y codo/antebrazo (84,21 %). La intensidad del dolor fue determinada como intenso en el 36,84 % y moderado en el 42,11 % de los trabajadores; mientras que en el 21,05 % fue leve. El tiempo de padecimiento delos trastornos fue de 3-38 años,que aparecieron luego de 5 a 7 años de haber comenzado las labores en el área de estudio. Conclusiones: La prevalencia de manifestaciones de trastornos musculoesqueléticos en los moldeadores manuales metalúrgicos del área de fundición de la empresa Holmeca de Holguín es elevada. Se identifican en mano/muñeca, hombro, lumbar y codo/antebrazo, como las zonas anatómicas más afectadas en este grupo poblacional.

Introduction: Musculoskeletal disorders in the metallurgical work context have a negative impact from the personal, economic and social perspective of workers. Objective: To identify the manifestations of musculoskeletal disorders in metallurgical manual molders in the foundry area of the Holmeca company in the province of Holguín, Cuba, in the period from January to March 2021. Method: An observational, non-experimental study was carried out. cross-sectional study in 19 metallurgical manual shapers, which included sociodemographic variables, location of painful areas, pain intensity, time of onset and duration of the disorder. Theoretical and empirical methods were used. The standardized Kuorinka Nordic Questionnaire and the Visual Analog Scale were used. The information processing was carried out using descriptive statistics. Results: The painful areas and the highest prevalence of musculoskeletal pain were identified in: wrist/hand (94.74%), shoulder (89.47%), dorsal/lumbar (89.47%) and elbow/forearm (84.21%). The intensity of the pain was determined as intense in 36.84% and moderate in 42.11% of the workers; while in 21.05% it was mild. The duration of the disorders was 3-38 years, which appeared after 5 to 7 years after beginning work in the study area. Conclusions: The prevalence of manifestations of musculoskeletal disorders in metallurgical manual molders in the foundry area of the Holmeca company in Holguín is high. They are identified in the hand/wrist, shoulder, lumbar and elbow/forearm, as the anatomical areas most affected in this population group.

Introdução: As lesões osteomusculares no contexto do trabalho metalúrgico têm um impacto negativo na perspectiva pessoal, económica e social dos trabalhadores. Objetivo: Identificar as manifestações de distúrbios osteomusculares em moldadores manuais metalúrgicos da área de fundição da empresa Holmeca, na província de Holguín, Cuba, no período de janeiro a março de 2021. Método: Foi realizado um estudo observacional, não experimental. estudo transversal em 19 modeladores manuais metalúrgicos, que incluiu variáveis sociodemográficas, localização das áreas dolorosas, intensidade da dor, tempo de início e duração do distúrbio. Foram utilizados métodos teóricos e empíricos. Foram utilizados o Questionário Nórdico Kuorinka padronizado e a Escala Visual Analógica. O processamento das informações foi realizado por meio de estatística descritiva. Resultados: As áreas dolorosas e as maiores prevalências de dores musculoesqueléticas foram identificadas em: punho/mão (94,74%), ombro (89,47%), dorsal/lombar (89,47%) e cotovelo/antebraço (84,21%). A intensidade da dor foi determinada como intensa em 36,84% e moderada em 42,11% dos trabalhadores; enquanto em 21,05% foi leve. A duração dos distúrbios foi de 3 a 38 anos, surgindo após 5 a 7 anos após o início do trabalho na área de estudo. Conclusões: É elevada a prevalência de manifestações de distúrbios musculoesqueléticos em moldadores manuais metalúrgicos na área de fundição da empresa Holmeca em Holguín. São identificadas na mão/punho, ombro, lombar e cotovelo/antebraço, como as áreas anatômicas mais acometidas neste grupo populacional.

Viability and Adhesion of Periodontal Ligament Fibroblasts on a Hydroxyapatite Scaffold Combined with Collagen, Polylactic Acid-Polyglycolic Acid Copolymer and Platelet-Rich Fibrin: A Preclinical Pilot Study.

Background: Conventional periodontal therapy relies on bone regeneration strategies utilizing scaffolds made of diverse materials, among which collagen, to promote cell adhesion and growth. Objective: To evaluate periodontal ligament fibroblast (HPdLF) cell adhesion and viability for periodontal regeneration purposes on hydroxyapatite scaffolds containing collagen (HAp-egg shell) combined with polylactic acid−polyglycolic acid copolymer (PLGA) and Platelet-Rich Fibrin (PRF). Methods: Four variations of the HAp-egg shell were used to seed HPdLF for 24 h and evaluate cell viability through a live/dead assay: (1) (HAp-egg shell/PLGA), (2) (HAp-egg shell/PLGA + collagen), (3) (HAp-egg shell/PLGA + PRF) and (4) (HAp-egg shell/PLGA + PRF + collagen). Cell adhesion and viability were determined using confocal microscopy and quantified using central tendency and dispersion measurements; significant differences were determined using ANOVA (p < 0.05). Results: Group 1 presented low cell viability and adhesion (3.70−10.17%); groups 2 and 3 presented high cell viability and low cell adhesion (group 2, 59.2−11.1%, group 3, 58−4.6%); group 4 presented the highest cell viability (82.8%) and moderate cell adhesion (45%) (p = 0.474). Conclusions: The effect of collagen on the HAp-egg shell/PLGA scaffold combined with PRF favored HPdLF cell adhesion and viability and could clinically have a positive effect on bone defect resolution and the regeneration of periodontal ligament tissue.

In vitro mineral apposition analysis of two Colombian plant extracts on Amelogenesis imperfecta teeth.

OBJECTIVE: To determine if native Colombian Piper marginatum Jacq. and Ilex guayusa Loes plant extracts have a remineralizing effect on teeth with Amelogenesis imperfecta in comparison with the commercial products Clinpro-3M and Recaldent™. MATERIAL AND METHODS: An in vitro study was carried out with 128 human teeth slices (64 healthy and 64 with Amelogenesis imperfecta) on which an initial Raman spectroscopy was performed followed by Raman spectroscopies at 0, 24, 48, and 72 h to determine possible remineralization by observing mineral increase or decrease as a result of P. marginatum Jacq. and I. guayusa Loes extract application in comparison to control substance (Clinpro and Recaldent™) application. Obtained data were analyzed using a bivariate method with a t unidirectional test. Significant differences among groups were determined by an ANOVA with Dunnett post hoc tests. RESULTS: Native I. guayusa Loes and P. marginatum Jacq. Colombian plants extracts exhibited phosphate and orthophosphate mineral apposition, where P. marginatum Jacq. presented better results. CONCLUSIONS: Native Colombian I. guayusa Loes and P. marginatum Jacq plant extract might in the future be useful for dental tissue remineralization, as they induced phosphate and orthophosphate mineral apposition, main components of tooth enamel. These types of natural compounds can become an alternative to fluorine, whose ingestion is harmful to the human body.

Clinical and Molecular Study of the NOG Gene in Families with Mandibular Micrognathism.

OBJECTIVES: Previous studies showed that noggin gene (NOG) sequence alterations, as well as epigenetic factors, could influence mandibular development. The aim of this study was to analyze clinical characteristics, NOG gene sequences, and promoter methylation sites in patients with mandibular micrognathism. MATERIALS AND METHODS: A total of 35 individuals of five Colombian families were subject to clinical and cephalometric analysis for mandibular micrognathism. One nonaffected individual of each family was included as a control. DNA was isolated from whole blood sample from all individuals by salting out method. Nine NOG gene fragments were amplified by polymerase chain reaction (PCR) and sequenced. Identification of CpG islands for methylation analysis at the NOG gene promoter was performed by MSP-PCR kit (Qiagen R). STATISTICAL ANALYSIS: A descriptive statistical analysis was carried out evaluating the presence or absence of genetics variants and the methylation sites in the NOG gene. RESULTS: NOG sequence results of affected individuals with mandibular micrognathism for one of the families studied demonstrated that they were heterozygous for 672 C/A (new mutation). For a second family, individuals were heterozygous for 567 G/C (single nucleotide polymorphism [SNP] RS116716909). For DNA analyzed from all patients studied, no methylations were observed at the NOG gene promoter region. CONCLUSION: Our results suggested that 672 C/A and 567 G/C variants could be involved in the presence of mandibular micrognathism. Moreover, lack of methylation sites at the NOG gene promoter region of all individuals studied suggests possibly other epigenetic factors could modulate mandibular growth. The search of genetic variants related with mandibular micrognathism will allow to predict in an integral way the development patterns of the patients and therefore establish a better clinical treatment.

A Light-Sheet-Based Imaaging Spectrometer to Characterize Acridine Orange Fluorescence within Leukocytes.

Low-cost imaging systems that utilize exogenous fluorescent dyes, such as acridine orange (AO), have recently been developed for use as point-of-care (POC) blood analyzers. AO-based fluorescence imaging exploits variations in emission wavelength within different cell types to enumerate and classify leukocyte subpopulations from whole blood specimens. This approach to leukocyte classification relies on accurate and reproducible colorimetric features, which have previously been demonstrated to be highly dependent on the cell staining protocols (such as specific AO concentration, timing, and pH). We have developed a light-sheet-based fluorescence imaging spectrometer, featuring a spectral resolution of 9 nm, with an automated spectral extraction algorithm as an investigative tool to study the spectral features from AO-stained leukocytes. Whole blood specimens were collected from human subjects, stained with AO using POC methods, and leukocyte spectra were acquired on a cell-by-cell basis. The post-processing method involves three steps: image segmentation to isolate individual cells in each spectral image; image quality control to exclude cells with low emission intensity, out-of-focus cells, and cellular debris; and the extraction of spectra for each cell. An increase in AO concentration was determined to contribute to the red-shift in AO-fluorescence, while varied pH values did not cause a change in fluorescence. In relation to the spectra of AO-stained leukocytes, there were corresponding red-shift trends associated with dye accumulation within acidic vesicles and at increasing incubation periods. The system presented here could guide future development of POC systems reliant on AO fluorescence and colorimetric features to identify leukocyte subpopulations in whole blood specimens.

Depicting the battle between nectarine and Monilinia laxa: the fruit developmental stage dictates the effectiveness of the host defenses and the pathogen's infection strategies.

Infections by the fungus Monilinia laxa, the main cause of brown rot in Europe, result in considerable losses of stone fruit. Herein, we present a comprehensive transcriptomic approach to unravel strategies deployed by nectarine fruit and M. laxa during their interaction. We used M. laxa-inoculated immature and mature fruit, which was resistant and susceptible to brown rot, respectively, to perform a dual RNA-Seq analysis. In immature fruit, host responses, pathogen biomass, and pathogen transcriptional activity peaked at 14-24 h post inoculation (hpi), at which point M. laxa appeared to switch its transcriptional response to either quiescence or death. Mature fruit experienced an exponential increase in host and pathogen activity beginning at 6 hpi. Functional analyses in both host and pathogen highlighted differences in stage-dependent strategies. For example, in immature fruit, M. laxa unsuccessfully employed carbohydrate-active enzymes (CAZymes) for penetration, which the fruit was able to combat with tightly regulated hormone responses and an oxidative burst that challenged the pathogen's survival at later time points. In contrast, in mature fruit, M. laxa was more dependent on proteolytic effectors than CAZymes, and was able to invest in filamentous growth early during the interaction. Hormone analyses of mature fruit infected with M. laxa indicated that, while jasmonic acid activity was likely useful for defense, high ethylene activity may have promoted susceptibility through the induction of ripening processes. Lastly, we identified M. laxa genes that were highly induced in both quiescent and active infections and may serve as targets for control of brown rot.

Elaboration and Biocompatibility of an Eggshell-Derived Hydroxyapatite Material Modified with Si/PLGA for Bone Regeneration in Dentistry.

Hydroxyapatite (HAp) is the most commonly used biomaterial in modern bone regeneration studies because of its chemical similarity to bone, biocompatibility with different polymers, osteoconductivity, low cost, and lack of immune response. However, to overcome the disadvantages of HAp, which include fragility and low mechanical strength, current studies typically focus on property modification through the addition of other materials. Objective. To develop and evaluate the biocompatibility of a HAp material extracted from eggshells and modified with silicon (Si) and poly(lactic-co-glycolic) acid (PLGA). Materials and Methods. An in vitro experimental study in which a HAp material prepared from eggshells was synthesized by wet chemical and conventional chemical precipitation. Subsequently, this material was reinforced with Si/PLGA using the freezing/lyophilization method, and then osteoblast cells were seeded on the experimental material (HAp/Si/PLGA). To analyse the biocompatibility of this composite material, scanning electron microscopy (SEM) and fluorescence confocal microscopy (FCM) techniques were used. PLGA, bovine bone/PLGA (BB/PLGA), and HAp/PLGA were used as controls. Results. A cellular viability of 96% was observed for the experimental HAp/Si/PLGA material as well as for the PLGA. The viability for the BB/PLGA material was 90%, and the viability for the HAp/PLGA was 86%. Cell adhesion was observed on the exterior surface of all materials. However, a continuous monolayer and the presence of filopodia were observed over both external and internal surface of the experimental materials. Conclusions. The HAp/Si/PLGA material is highly biocompatible with osteoblastic cells and can be considered promising for the construction of three-dimensional scaffolds for bone regeneration in dentistry.

Double-sided battle: The role of ethylene during Monilinia spp. infection in peach at different phenological stages.

Controversy exists on whether ethylene is involved in determining fruit resistance or susceptibility against biotic stress. In this work, the hypothesis that ethylene biosynthesis in peaches at different phenological stages may be modulated by Monilinia spp. was tested. To achieve this, at 49 and 126 d after full bloom (DAFB), ethylene biosynthesis of healthy and infected 'Merryl O'Henry' peaches with three strains of Monilinia spp. (M. fructicola (CPMC6) and M. laxa (CPML11 and ML8L) that differ in terms of aggressiveness) was analysed at the biochemical and molecular level along the course of infection in fruit stored at 20 °C. At 49 DAFB, results evidenced that infected fruit showed inhibition of ethylene production in comparison with non-inoculated fruit, suggesting that the three Monilinia strains were somehow suppressing ethylene biosynthesis to modify fruit defences to successfully infect the host. On the contrary, at 126 DAFB ethylene production increased concomitantly with brown rot spread, and values for non-inoculated fruit were almost undetectable throughout storage at 20 °C. The expression of several target genes involved in the ethylene biosynthetic pathway confirmed that they were differentially expressed upon Monilinia infection, pointing to a strain-dependent regulation. Notably, Prunus persica 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) (PpACS) family was the most over-expressed over time, demonstrating a positive ethylene regulation, especially at 126 DAFB. At this phenological stage it was demonstrated the ability of Monilinia spp. to alter ethylene biosynthesis through PpACS1 and benefit from the consequences of an ethylene burst likely on cell wall softening. Overall, our results put forward that infection not only among different strains but also at each stage is achieved by different mechanisms, with ethylene being a key factor in determining peach resistance or susceptibility to brown rot.

Evaluation of Acridine Orange Staining for a Semi-Automated Urinalysis Microscopic Examination at the Point-of-Care.

A urinary tract infection (UTI) can be diagnosed via urinalysis, consisting of a dipstick test and manual microscopic examination. Point-of-care (POC) image-based systems have been designed to automate the microscopic examination for low-volume laboratories or low-resource clinics. In this pilot study, acridine orange (AO) was evaluated as a fluorescence-based contrast agent to aid in detecting and enumerating urine sediment specific for diagnosing a UTI. Acridine orange staining of epithelial cells, leukocytes, and bacteria provided sufficient contrast to successfully implement image segmentation techniques, which enabled the extraction of classifiable morphologic features. Surface area bounded by each cell border was used to differentiate the sediment; epithelial cells were larger than 500µm2, bacteria were less than 30µm2, and leukocytes in between. This image-based semi-automated technique using AO resulted in similar cell counts to the clinical results, which demonstrates the feasibility of AO as an aid for POC urinalysis systems.

Differences in colonic crypt morphology of spontaneous and colitis-associated murine models via second harmonic generation imaging to quantify colon cancer development.

BACKGROUND: Colorectal cancer remains the second leading cause of cancer death in the United States, and increased risk in patients with ulcerative colitis (a subset of inflammatory bowel disease) has motivated studies into early markers of dysplasia. The development of clinically translatable multiphoton imaging systems has allowed for the potential of in vivo label-free imaging of epithelial crypt structures via autofluorescence and/or second harmonic generation (SHG). SHG has been used to investigate collagen structures in various types of cancer, though the changes that colorectal epithelial collagen structures undergo during tumor development, specifically colitis-associated tumors, have not been fully investigated. METHODS: This study used two murine models, using A/J mice, one for spontaneous carcinoma and one for colitis-associated carcinoma, to investigate and quantify SHG image features that could potentially inform future study designs of endoscopic multiphoton imaging systems. The spontaneous tumor model comprised a series of six weekly injections of azoxymethane (AOM model). The colitis-associated tumor model comprised a single injection of AOM, followed by cycles of drinking water with dissolved dextran sodium sulfate salt (AOM-DSS model). SHG images of freshly resected murine colon were acquired with a multiphoton imaging system, and image features, such as crypt size, shape and distribution, were quantified using an automated algorithm. RESULTS: The comparison of quantified features of crypt morphology demonstrated the ability of our quantitative image feature algorithms to detect differences between spontaneous (AOM model) and colitis-associated (AOM-DSS model) murine colorectal tissue specimens. There were statistically significant differences in the mean and standard deviation of nearest neighbor (distance between crypts) and circularity between the Control cohort, AOM and AOM-DSS cohorts. We also saw significance between AOM and AOM-DSS cohorts when calculating nearest neighbor in images acquired at fixed depths. CONCLUSION: The results provide insight into the ability of SHG imaging to yield relevant data about the crypt microstructure in colorectal epithelium, specifically the potential to distinguish between spontaneous and colitis-associated murine models using quantification of crypt shape and distribution, informing future design of translational multiphoton imaging systems and protocols.

Analysis of In Vitro Osteoblast Culture on Scaffolds for Future Bone Regeneration Purposes in Dentistry.

One of the main focuses of tissue engineering is to search for tridimensional scaffold materials, complying with nature's properties for tissue regeneration. Determining material biocompatibility is a fundamental step in considering its use. Therefore, the purpose of this study was to analyze osteoblast cell adhesion and viability on different materials to determine which was more compatible for future bone regeneration. Tridimensional structures were fabricated with hydroxyapatite, collagen, and porous silica. The bovine bone was used as material control. Biocompatibility was determined by seeding primary osteoblasts on each tridimensional structure. Cellular morphology was assessed by SEM and viability through confocal microscopy. Osteoblast colonization was observed on all evaluated materials' surface, revealing they did not elicit osteoblast cytotoxicity. Analyses of four different materials studied with diverse compositions and characteristics showed that adhesiveness was best seen for HA and viability for collagen. In general, the results of this investigation suggest these materials can be used in combination, as scaffolds intended for bone regeneration in dental and medical fields.

Static magnetic fields reduce epileptiform activity in anesthetized rat and monkey.

Increasing evidence indicates that static magnetic fields (SMF) reduce cortical activity in both human and animal models. The aim of this work was to investigate the effect of SMF on epileptiform cortical activity, a condition related to an abnormal increase in neuronal excitability. The first experimental block included a Pilocarpine rat model of epilepsy, in which a magnetic neodymium nickel-plated cylinder, a magnetic field of 0.5 T, or "sham" were placed over the skull. In the second experimental block, we recorded epileptic-like activity in the visual cortex of a monkey (Macaca mulatta) under control conditions and in the presence of the magnet. Between 15 and 30 minutes after the second dose of Pilocarpine, EEG changes compatible with seizure like events induced by Pilocarpine were clearly observed in the control animals (sham stimulation). Similar effects were visible in the animals exposed to the real magnet after 1-2 hours. In the monkey, SMF over the cortical focus clearly reduced abnormal activity: the intensity threshold for visual induction increased and the severity and duration decreased. These results reinforce the view that static magnets modulate cortical activity and open the door to the future therapeutic use of SMF in epilepsy as a complement to current pharmacological treatments.

Fiber-Optic Pyrometer with Optically Powered Switch for Temperature Measurements.

We report the experimental results on a new infrared fiber-optic pyrometer for very localized and high-speed temperature measurements ranging from 170 to 530 °C using low-noise photodetectors and high-gain transimpedance amplifiers with a single gain mode in the whole temperature range. We also report a shutter based on an optical fiber switch which is optically powered to provide a reference signal in an optical fiber pyrometer measuring from 200 to 550 °C. The tests show the potential of remotely powering via optical means a 300 mW power-hungry optical switch at a distance of 100 m, avoiding any electromagnetic interference close to the measuring point.

Fluorescein as a topical fluorescent contrast agent for quantitative microendoscopic inspection of colorectal epithelium.

Fiber bundle microendoscopic imaging of colorectal tissue has shown promising results, for both qualitative and quantitative analysis. A quantitative image quality control and image feature extraction algorithm was previously designed for quantitative image feature analysis of proflavine-stained ex vivo colorectal tissue. We investigated fluorescein as an alternative topical stain. Images of ex vivo porcine, caprine, and human colorectal tissue were used to compare microendoscopic images of tissue topically stained with fluorescein and proflavine solutions. Fluorescein was shown to be comparable for automated crypt detection, with an average crypt detection sensitivity exceeding 90% using a combination of three contrast limit pairs.

Evaluación del efecto del plasma rico en plaquetas en diferentes tiempos y conce ntraciones sobre la viabilidad de fibroblastos de ligamento periodontal y osteoblastos / Evaluation of platelet-rich plasma effect at different times and concentrations on periodontal ligament fibroblast and osteoblast viability

Antecedentes: Los factores de crecimiento utilizados en salud se pueden obtener de una fuente autóloga de primera generación llamada plasma rico en plaquetas (PRP). La diversidad de protocolos para prepararlos genera resultados variables en cuanto al tiempo entre la activación del PRP y sus efectos sobre la proliferación y viabilidad celular. Objetivo: Evaluar proliferación y viabilidad celular de fibroblastos de ligamento periodontal y osteoblastos tratados con PRP en diferentes concentraciones y tiempos de aplicación. Métodos: Se cultivaron líneas celulares de fibroblastos y osteoblastos y se preparó el PRP de sangre venosa de un adulto sano mediante centrifugación, seguido de activación con CaCl2 al 10 %. El efecto sobre la proliferación de las líneas celulares tras la aplicación de PRP y plasma pobre en plaquetas al 1 %, al 3 % y al 5 % se evaluó a las 0, 12, 24, 48 y 72 horas después de su activación mediante MTS. El grupo control consistió en cultivos sin tratamiento. Los datos se analizaron mediante las pruebas de Chi cuadrado, Fischer y McNemar. Resultados: Se observó un aumento de la viabilidad en células tratadas con PRP 24 horas después de su activación en una concentración del 5%. El ensayo de viabilidad celular mostró diferencias estadísticamente significativas entre el grupo experimental y el grupo control (p = 0,05). Conclusión: Los cultivos de fibroblastos y osteoblastos mostraron una tendencia a mayor viabilidad 24 horas después de a la activación con PRP al 5 %.

Background : Growth factors used in health treatments can be obtained from a first-generation source called platelet-rich plasma. The variety of protocols to prepare PRP produces variable results regarding PRP activation time and its effects on cell proliferation and viability. Purpose: To evaluate proliferation and cell viability of periodontal ligament fibroblasts and osteoblasts stimulated with PRP in several concentrations and times after PRP activation. Methods: An in vitro study was carried out using periodontal ligament fibroblast and osteoblast cell cultures. PRP from venous blood of a healthy adult was prepared through centrifugation and activated with 10% CaCl2. The effect on cell proliferation after application of 1%, 3%, and 5% PRP and platelet-poor plasma was evaluated at 0, 12, 24, 48, and 72 hours after activation through MTS. The control group consisted of culture that did not receive any treatment. Data were analyzed using Chi square, Fisher, and McNemar tests. Results: The cell viability assay showed statistically significant differences between the experimental and the control groups. Cell viability increased in cells treated with 5% PRP 24 hours after activation (p = 0.05). Conclusions: Fibroblast and osteoblast cell lines tended to be more viable 24 hours after activation with 5% PRP.

Quantitative analysis of ex vivo colorectal epithelium using an automated feature extraction algorithm for microendoscopy image data.

Qualitative screening for colorectal polyps via fiber bundle microendoscopy imaging has shown promising results, with studies reporting high rates of sensitivity and specificity, as well as low interobserver variability with trained clinicians. A quantitative image quality control and image feature extraction algorithm (QFEA) was designed to lessen the burden of training and provide objective data for improved clinical efficacy of this method. After a quantitative image quality control step, QFEA extracts field-of-view area, crypt area, crypt circularity, and crypt number per image. To develop and validate this QFEA, a training set of microendoscopy images was collected from freshly resected porcine colon epithelium. The algorithm was then further validated on ex vivo image data collected from eight human subjects, selected from clinically normal appearing regions distant from grossly visible tumor in surgically resected colorectal tissue. QFEA has proven flexible in application to both mosaics and individual images, and its automated crypt detection sensitivity ranges from 71 to 94% despite intensity and contrast variation within the field of view. It also demonstrates the ability to detect and quantify differences in grossly normal regions among different subjects, suggesting the potential efficacy of this approach in detecting occult regions of dysplasia.

Quantitative structural markers of colorectal dysplasia in a cross sectional study of ex vivo murine tissue using label-free multiphoton microscopy.

Two-photon excitation of label-free tissue is of increasing interest, as advances have been made in endoscopic clinical application of multiphoton microscopy, such as second harmonic generation (SHG) scanning endoscopy used to monitor cervical collagen in mice1. We used C57BL mice as a model to investigate the progression of gastrointestinal structures, specifically glandular area and circularity. We used multiphoton microscopy to image ex-vivo label-free murine colon, focusing on the collagen structure changes over time, in mice ranging from 10 to 20 weeks of age. Series of images were acquired within the colonic and intestinal tissue at depth intervals of 20 microns from muscularis to the epithelium, up to a maximum depth of 180 microns. The imaging system comprised a two-photon laser tuned to 800nm wavelength excitation, and the SHG emission was filtered with a 400/40 bandpass filter before reaching the photomultiplier tube. Images were acquired at 15 frames per second, for 200 to 300 cumulative frames, with a field of view of 261um by 261um, and 40mW at sample. Image series were compared to histopathology H&E slides taken from adjacent locations. Quantitative metrics for determining differences between murine glandular structures were applied, specifically glandular area and circularity.