Association of polypropylene scaffolds and mesenchymal stem cells for use in tissue engineering / Associação de arcabouço de polipropileno e células tronco mesenquimais para uso em engenharia de tecido

Tissue engineering replaces injured tissues by manipulating cells, making scaffolds, and using molecules that stimulate the tissue. Mesenchymal stem cells (MSCs) are good candidates for tissue engineering, as this is one of the cell types which are recruited to repair injured tissues. Scaffolds are structural devices that allow cell fixation and migration, with polypropylene meshes being an example. This study aims to evaluate the culture of adipose tissue-derived mesenchymal stem cells (ADSCs), isolated from C57Bl/6 GFP + mice, in two types of polypropylene meshes (macroporous and microporous) in conventional culture plates and plates coated with methacrylate, over a period of fifteen days. The objective was to obtain the best interaction protocol between the mesh and the cells. The choice of the best method was based on adherence, maintenance of adherence and viability during culture. The amount of ADSCs adhering was checked daily by counting in a Neubauer Chamber and by using a growth curve performed with the MTT assay. The ADSCs adhering to the meshes were visualized with DAPI, panotic, hematoxylin and eosin, immunohistochemistry (integrin), and immunofluorescence (actin). ADSCs adhere to all forms of culture and to the two types of polypropylene mesh. ADSCs adhered more to the microporous mesh, within the seven day period of culture and in the plates without methacrylate. Thus, polypropylene meshes offer a good scaffold for ADSCs to adhere to.

A engenharia de tecidos substitui tecidos danificados com a manipulação de células, confecção de arcabouços e a utilização de moléculas que estimulem o tecido. As células-tronco mesenquimais (MSCs) são boas candidatas para engenharia de tecido, pois são um dos tipos celulares recrutadas para a reparação de tecidos lesionados. O arcabouço deve ser um dispositivo estrutural que forneça uma estrutura para o crescimento e a diferenciação celular no sítio, sendo a tela de polipropileno um exemplo. O objetivo deste estudo foi avaliar o cultivo de células-tronco mesenquimais de tecido de adiposo (ADSCs), isoladas de camundongos C57Bl/6 GFP+, em dois tipos de telas de polipropileno (macroporosa e microporosa) em placas de cultura convencionais e revestidas com metacrilato, durante quinze dias, para obter o melhor protocolo de interação entre a tela e as células. A escolha do melhor método foi baseada na adesão, manutenção da adesão e viabilidade durante cultivo. A quantidade de ADSCs aderidas foi verificada diariamente em contagem em Câmara de Neubauer e através de uma curva de crescimento realizada através de ensaio de MTT. As ADSCs aderidas nas telas foram visualizadas com a marcação de DAPI, panótico, hematoxilina e eosina, imumo-histoquímica (integrina) e imunofluorescência (actina). Nas duas formas de cultivo e nos dois tipos de telas de polipropileno houve aderência das ADSCs. Houve maior aderência na tela microporosa, no período de sete dias de cultivo e em placas sem metacrilato. Conclui-se que a tela de polipropileno oferece um bom arcabouço para as ADSCs se aderirem.

An optimized method for embedding undecalcified mouse tibias in plastic blocks / 南方医科大学学报

OBJECTIVE@#To optimize the method for embedding multiple undecalcified mouse tibias in plastic blocks, improve the efficiency and stability of plastic embedding and reduce the detachment rate of plastic slides.@*METHODS@#Thirty undecalcified tibias from 15 B6 mice were used for plastic embedding after calcein labeling, fixation, dehydration and infiltration. The tibias were embedded in cylindrical plastic blocks with a diameter of 4 mm. For each bone, the 1/4 proximal tibia was cut off, and the remaining 3/4 was used for re-embedding. Five bones were embedded in a single block with each bone standing closely on the surface of a flat plate. The samples were randomized into control and experimental groups in all the processes of embedding, sectioning and staining. In the 3 groups with modified embedment, flowing CO was added into the embedding solution, embedding solution was applied to the section surface, and the slides were heated at 95 ℃ for 15 min. The polymerization time, slide detachment rate, bone formation and osteoblast parameters were analyzed.@*RESULTS@#We prepared 6 plastic blocks, each containing 5 tibias, whose cross sections were on the same plane. The blocks were completely polymerized and suitable for sectioning. Flowing CO into the embedding solution reduced the polymerization time and increased the rate of complete polymerization. Application of the embedding solution on the section surface significantly reduced the detachment rate of the sections ( < 0.05) without affecting bone formation analysis ( > 0.05). Heating the slides significantly lowered the detachment rate of the sections ( < 0.05) without affecting osteoblast analysis ( > 0.05).@*CONCLUSIONS@#The optimized method allows effective embedding of multiple undecalcified mice tibias in the same block and can be an ideal method for histological analysis of undecalcified bones.

Validation of a new technique to freezing and embedding tissue in Mohs surgery, using an animal model

ABSTRACT PURPOSE: To validate the innovative Dry Ice method, comparing it with two standard methods currently used for tissue processing in Mohs surgery, the Heat Sink method and the Miami Special. METHODS: Forty eight samples of pigs kin with the standard beveled Mohs technique were used, and randomly allocated into six groups. Each group was processed with one of the 3 methods and evaluated for: The freezing time, the depth required to cut into the block to obtain a complete section, and the quality of histological slides analyzed with a image software. The statistical analysis was performed with the software SAS(r) System. The inferential analysis was made by one-way ANOVA. RESULTS: The Miami Special showed a processing time significantly shorter than Dry Ice method and Heat Sink method. There was no significant difference in the depth required to cut into the blocks, and area of surgical margins visualized. CONCLUSION: The Dry Ice method was as efficient as the other two methods currently used in Mohs surgery, considering the individual advantages and disadvantages of each method.

New and probable ultrastructural criteria for identification of cytoplasmic sol ­ gel states / Nuevo y probable criterio ultraestructural para la identificación de los estados citoplasmáticos sol ­ gel

In embedment-free transmission electron microscopy without employing epoxy embedding media, the cytoplasmic matrix, in which cell organelles and elements including the cytoskeletons are held in place, lattices of strands are clearly and constantly disclosed in every cell. Their compactness is variable in different kinds of cells and in different domains of one and the same cell, and it is changeable under hypo- or hyper-osmolarity. In addition, the appearance of strand-lattices is duplicable in artificial proteins at different sol/gel states and concentrations. All taken together, a new and probable ultrastructural criteria has been proposed for identification of cytoplasmic sol/gel states with a hope that the dynamic properties of the cell is understood not only by the cytoskeleton but also by the sol/gel states of cytosolic proteins and their concentration in distinct association with cellular ultrastructural entities.

En microscopía electrónica de transmisión, la inclusión-libre sin el uso de medios de inclusión epoxi, la matriz citoplasmática (los orgánulos celulares y elementos, incluyendo los citoesqueletos) se mantienen en su lugar y las redes de hebras aparecen claramente y constantemente en cada célula. Su tamaño compacto es variable en diferentes tipos de células y en diferentes dominios de una y la misma célula, y es modificable bajo hipo o hiper-osmolaridad. Además, la aparición de redes de hebras es duplicable en las proteínas artificiales en diferentes estados de concentraciones sol / gel. En este contexto se ha propuesto un criterio ultraestructural nuevo y probable para la identificación de los estados sol / gel citoplasmáticos, con el objetivo de que las propiedades dinámicas de la célula se comprendan no solo a partir del citoesqueleto, sino también a partir de los estados sol / gel de proteínas citosólicas y su concentración en relación con una asociación indistinta con las entidades celulares ultraestructurales.

Avaliação da diluição do DNA extraído de material parafinado para amplificação em PCR / Evaluation of DNA dilution extracted of paraffin-embedded material for PCR amplification

Introdução: Alguns fatores, como a presença de substâncias inibidoras e degradação do DNA, podem contribuir para a falha na detecção de genes, através da reação em cadeia da polimerase (PCR), a partir de DNA extraído de material parafinado. A diluição do DNA frequentemente pode reduzir o número de inibidores e contaminantes e ainda assim conter DNA suficiente para a amplificação em PCR. Objetivo: Avaliar a influência da diluição de soluções de DNA extraído de material parafinado na amplificação por PCR do gene ß-globina humana. Material e método: Foram utilizados 30 blocos parafinados de carcinomas epidermoides de orofaringe referentes a pacientes diagnosticados e tratados no Centro de Oncologia Bucal da FOA-UNESP. A extração do DNA foi realizada com o sistema QIAamp DNA minikit (Quiagen). O DNA obtido foi quantificado e avaliado quanto à pureza por espectrofotometria. Dois grupos foram formados com diferentes quantidades de DNA, sendo que o Grupo I foi constituído pelo DNA originalmente extraído e o Grupo II com o mesmo DNA , porém diluído com adição de água ultrapura.

Foi realizada a PCR utilizando-se oligonucleotídeos iniciadores para ß-globina. Resultados: No grupo I, 33,33% das amostras foram positivas para o gene ß-globina, enquanto no Grupo II, 23,33% foram positivas. Conclusão: Neste estudo, a diluição do DNA extraído de material parafinado não alterou estatisticamente a quantidade de amostras positivas por PCR para o gene ß-globina, embora os resultados obtidos sugiram que esta seja uma das formas de aumentar a eficácia do método de amplificação por PCR

Effect of histological processing and methacrylate sectioning on the area of gill tissue in teleost / Efeito do processamento histológico e seccionamento em metacrilato sobre a área de tecido branquial em teleósteo

Deformation of biological tissues may occur during histological processing and results in loss of accuracy when quantitative information about cells, tissues and organs is necessary. In this study, the gill tissue from armored catfish (Pterygoplichthys anisitsi) was quantified in each step of processing using the stereological principles. During processing for glycol methacrylate embedding, gill tissue from shrinks significantly but regains its original dimensions after sectioning.

Deformações nos tecidos podem ocorrer durante o processamento histológico e resultar em informações errôneas quando há necessidade de dados quantitativos sobre células, tecidos e órgãos. Neste estudo, o tecido branquial do cascudo (Pterygoplichthys anisitsi) foi quantificado em cada etapa do processamento utilizando os princípios de estereologia. O tecido branquial reduziu significativamente durante processamento histológico com metacrilato, mas retornou às suas dimensões iniciais depois de seccionadas, o que indica não ocorrer nenhuma perda na informação quantitativa do tecido.

Clinical and pathological correlation of the microdeletion of Y chromosome for the 30 patients with azoospermia and severe oligoasthenospermia / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To review the accumulated 30 patients with different area of Y chromosome microdeletions, focusing on their correlation with the clinical and pathological findings.</p><p><b>METHODS</b>A total of 334 consecutive infertile men with azoospermia (218 patients) and severe oligoasthenospermia (116 patients) were screened. Complete physical and endocrinological examinations, general chromosome study and multiplex polymerase chain reaction assay to evaluate the Y chromosome microdeletion were performed. Ten patients received testicular biopsy. Then the clinical and pathological findings were analyzed with reference to the areas of Y chromosome microdeletion.</p><p><b>RESULTS</b>There is a decline of the percentage of sperm appearing in semen in the group that the gene deletion region from AZFc to AZFb. The clinical evidence of the impairment (decreased testicular size and elevated serum FSH) is also relevantly aggravated in this group. However, the pathology of testicular biopsy specimen was poorly correlated with the different deletion areas of the Y chromosome, which may be due to the limited number of specimens.</p><p><b>CONCLUSION</b>The clinical correlation of spermatogenic impairment to the different AZF deletion regions may provide the information for the infertile couples in pre-treatment counseling.</p>

Demonstração de um protocolo para o estudo anatomopatológico dos vasos linfáticos no linfedema / Demonstration protocol for the anatomic-pathologic study of lymphatic vessels in lymphedema

Objetivo: sugerir um protocolo de estudo dos vasos linfáticos com o propósito de obter os dados necessários para uma melhor compreensão da morfologia e das alterações parafisiológicas e francamente patológicas do círculo linfático na patologia do linfedema, tanto de origem primária como secundária.Método: A primeira etapa do protocolo é a obteção do material, que pode ser um grupo de tecido fibroadiposo, em que estão envoltos os vasos linfáticos, ou um segmento isolado de coletor linfático. Após, segue-se uma série de etapas: fixação e inclusão do material, preparação de 11 lâminas com coloração histoquímica e imuno-histoquímica e, por último, leitura das lâminas em busca de alterações, desde os componentes dos vasos linfáticos até a matriz periadventicial.Resultados: Conforme o grupo de células predominante na parede do vaso e na matriz periadventicial, pode-se suspeitar de uma reação actínica, como, por exemplo, nos casos em que há muita fibrose e regressão das fibras contráteis ou, ainda, no caso de um linfedema crônico pós-cirúrgico, quando há predominância de um processo degenerativo da parede linfática.Conclusão: Para a realização de um estudo amplo dos vasos linfáticos em diversos laboratórios de anatomia patológica, faz-se necessária a criação de um protocolo para melhor compreensão desta patologia tão complexa.

Comparative analysis of duodenal biopsy tissue orientation supported on vegetable matrix versus filter paper.

BACKGROUND: Biopsy tissue may get disoriented if filter paper is used as a supporting medium. Use of vegetable matrix (cucumber) as a supporting medium may obviate the need of lifting the tissue while making blocks and thus avoid disorientation that occurs during this step. OBJECTIVE: To compare the orientation of duodenal biopsy tissue supported on vegetable matrix (cucumber) and on filter paper. METHODS: Over one year, 40 patients (20 with large-volume diarrhea, 20 with dyspepsia) were included in the study. Two pairs of duodenal biopsy tissues were obtained during gastroscopy; one pair was placed on filter paper, the other on vegetable matrix. Tissue and vegetable matrix were embedded together while making blocks, whereas the tissue had to be lifted off in case of filter paper. Sections were stained and assessed for crypt-villous alignment, parallel orientation of crypts and presence of visible muscularis mucosae with the help of a scoring system. RESULTS: Compared to biopsy tissue supported on filter paper, vegetable matrix-supported tissues were better oriented. Scores were rated as bad, good and very good in 8, 11 and 21 vegetable matrix-mounted tissues and in 21, 11 and 8 filter paper-mounted tissues, respectively. CONCLUSION: Duodenal biopsy tissue supported on vegetable matrix (cucumber) is better oriented than that on filter paper.

Silicone gel implants for calf augmentation: 14 years experience / Implantes de silicone gel em panturrilhas: 14 anos de experiência

Based on experience of 131 operated patients submitted to calf augmentation, the authors describe the indications, surgical technique and complications of the method. The procedure is recommended to correct calf volume of congenital origin, neurological or traumatic sequela.

Transparent tooth model system. An aid in the study of root canal anatomy.

Clearing techniques of tooth have long since been advocated in the demonstration of root canal anatomy. We have compared the efficacy of various clearing agents such as Xylene, Benzene, Methyl salicylate and Eugenol, using 40 maxillary second premolars as experimental subjects. We have further tried to select a suitable dye for the demonstration of root canal anatomy and have compared the efficacy of various mounting media such as D.P.X. medium, Canada balsam and Commercial plastic dissolved in benzene, in preserving the cleared state. Results of our study indicate that Eugenol is a superior clearing agent and that oil-based dyes are better suited for root canal demonstration. Canada balsam is the most effective mounting media and D.P.X. medium has been found to serve both as a clearing agent and as a mounting media. We have introduced a miniature museum jar technique, for preserving the transparency permanently. The Transparent Tooth Model System presented here, thus serves as a much simpler tool, in the study of root canal anatomy, three dimensionally.

Morfologia do eptélio intestinal de ratos adultos utilizando-se como meio de inclusäo o paraplast e o glicometacrilato / Intestinal epithelium morphology of adults rats using as inclusion media paraplast and glycometacrylatel

Através da adequaçäo ao processamento histológico rotineiro buscamos aperfeiçoar a descriçäo morfológica por meio de melhor visualizaçäo das células presentes no epitélio intestinal. Para isso utilizamos segmentos iniciais e finais do intestino delgado de ratos adultos. As células absortivas, caliciformes, células de Paneth e células indiferenciadas apresentaram melhor nitidez na forma e na distribuiçäo dos elementos intracelulares graças às nuances de colorações distintas obtidos pela inclusäo do material em glicometacrilato e cortes de 2 g.m feitos com navalha de aço. As células enteroendócrinas, puderam ter seus grânulos evidenciados após inclusäo em paraplast, cortes de 4 g.m em navalha de aço, sendo corados pela prata amoniacal. A aplicaçäo destas técnicas, ao nosso ver, contribuem com melhorias do material didático para aulas práticas sobre o epitélio intestinal.

Matrices de inclusión para obtención de cortes de tejidos en vibrátomo en estudios citoquímicos / Embedding matrixes for obtaining vibratome tissue slices for cytochemistry studies

Para estudios citoquímicos en diferentes muestras de tejidos, de tamaño pequeños, se hace necesaria la utilización de una matriz inerte que genere soporte y estabilidad al tejido en el momento de obtener cortes de 50µm en vibrátomo. Por esta razón se ensayaron cinco matrices: agar granulado, agar purificado, agar-agar, agarosa y gelatina, a diferentes concentraciones. Tanto con el agar-agar como con la agarosa se obtuvieron los mejores resultados, puesto que estos ofrecen, mayor estabilidad al tejido, fluyen fácilmente al servirse y su textura es muy homogénea, requisitos necesarios para la obtención de cortes precisos, utilizables en citoquímica, inmunocitoquímica y trazados histoquímicos

Alterations of CDKN2 (MTS1/p16INK4A) gene in paraffin-embedded tumor tissues of human stomach, lung, cervix and liver cancers

The CDKN2 (MTS1/p16INK4A) gene, encoding cyclin dependent kinase inhibitor, was found to be homozygously deleted at a high frequency in cell lines from many different types of cancer and some primary cancers. To determine the frequency of CDKN2 mutations in most common human cancers in Korea, PCR and PCR-SSCP analyses for the exon 2 of CDKN2 were performed on each set of 20 formalin-fixed and paraffin-embedded tumor tissues of stomach adenocarcinomas, lung cancers, cervix cancers and hepatocellular carcinomas. No mutations in exon 2 of CDKN2 were found in 20 stomach adenocarcinomas. In contrast to rare mutations in stomach adenocarcinomas, a high frequency of CDKN2 mutations was identified in other 3 cancers, 11 of 20 (55%) lung cancers (7 of 10 NSCLCs and 4 of 10 SCLCs), 14 of 20 (70%) cervix cancers and 11 of 20 (55%) hepatocellular carcinomas. These results suggest that mutations of the CDKN2 gene might be an important genetic change in NSCLCs, cervix cancers and hepatocellular carcinomas.

Standardization of fixation, processing and staining methods for the central nervous system of vertebrates

This paper reports the standardization of methods used for processing and embedding various vertebrate brains of different size in paraffin. Other technical details developed for avoiding frequent difficulties arising during laboratory routine are also reported. Some modifications of the Nissl and Klüver-Barrera staining methods are proposed. These modifications include: 1) a Nissl stain solution with a rapid and efficient action with easier differentiation; 2) the use of a cheap microwave oven for the Klüver-Barrera stain. These procedures have the advantage of permitting Nissl and Klüver-Barrera staining of nervous tissue in about five and fifteen minutes respectively. The proposed procedures have been tested in brains obtained from fish, amphibians, reptiles and mammals of different body sizes. They are the result of our long experience in preparing slides for comparative studies. Serial sections of excellent quality were regularly obtained in all the specimens studied. These standardized methods, being simple and quick, are recommended for routine use in neurobiological laboratories.

Los cianoacrilatos en cirugía / Cyano-acrylates in surgery

Desde su introducción en la cirugía hace más de 35 años, los cianoacrilatos se han usado en diversos campos de las especialidades quirúrgicas. A pesar de sus múltiples ventajas sobre la sutura convencional, muchos cirujanos dejaron de usarlos por su gran histotoxicidad y las dificultades de su aplicación. En los últimos años, nuevamente se han comenzado a utilizar los olvidadod adhesivos con excelentes resultados. En esta revisión se comentan aspectos históricos, características físicas, químicas y biológicas de estos adhesivos; Métodos de síntesis, metabolismo, respuesta inmunitaria y carcinogénesis después de aplicarse en el organismo y usos actuales y potenciales de los cianoacrilatos en la cirugía

Histology revisited: technical improvement promoted by the use of hydrophilic resin embedding

The use of isotonic 4 per cent paraformaldeyde fixation and glycolmethacrylate embedding permits to obtain undistorted light microscope images with an increased resolution to some extent similar to low power images of electron microscopy. This allows an improved analysis of cell and tissue biology with light microscopy.