Aloinjertos análogos de mucosa bucal en ratas no consanguíneas / Oral mucosa analog allografts in non-consanguineous rats

Resumen Introducción: A pesar de que existen opciones terapéuticas para el tratamiento de defectos de la mucosa bucal, persiste la necesidad de encontrar sustitutos funcionales, anatómicos y estéticamente similares al tejido que se va a reemplazar, así como soluciones que reduzcan la morbilidad de los injertos autólogos. Objetivo: Determinar la compatibilidad clínica e histológica de aloinjertos equivalentes de mucosa bucal elaborados mediante ingeniería tisular en ratas no consanguíneas. Materiales y métodos: Se utilizó una muestra de mucosa bucal de ratas Sprague Dawley para la obtención de un cultivo de fibroblastos y otro de queratinocitos y fibroblastos. En ambos casos, se usó una membrana de colágeno comercial como soporte. Después de diez semanas de cultivo, las membranas resultantes se injertaron en cuatro ratas Wistar. La primera fase del estudio consistió en la elaboración de los tejidos análogos de mucosa bucal mediante ingeniería tisular, los cuales se implantaron en ratas Wistar inmunocompetentes; posteriormente, se evaluaron las características clínicas e histológicas del aloinjerto. Resultados: La evaluación in vivo de los tejidos análogos demostró que se habían integrado correctamente en los huéspedes inmunocompetentes, y se había logrado el aumento del biotipo periodontal y la creación de una zona con mayor queratinización. Desde el punto de vista histológico, el tejido adquirió características similares a las de la muestra de mucosa bucal de control, sin ningún tipo de reacción inflamatoria ni signos clínicos o histológicos de rechazo. Conclusión: Hubo compatibilidad clínica e histológica de los aloinjertos equivalentes de mucosa bucal obtenidos mediante ingeniería tisular.

Abstract Introduction: Although there are therapeutic options for the treatment of oral mucosa defects, the need for functional, anatomical and aesthetically similar substitutes persists, as well as for solutions to reduce autologous grafts morbidity. Objective: To determine clinical and histological compatibility of equivalent oral mucosa allografts generated through tissue engineering in non-consanguineous rats. Materials and methods: We used a sample of oral mucosa from Sprague Dawley rats to obtain a fibroblast culture and a keratinocytes and fibroblasts co-culture. In both cases, we used a commercial collagen membrane as "scaffold". After ten weeks of culture, we grafted the resulting membranes into four Wistar rats. The first phase of the study was the development of the oral mucosa equivalents generated by tissue engineering. Then, we implanted them in immunocompetent Wistar rats, and finally we evaluated the clinical and histological features of the allografts. Results: In vivo evaluation of mucosal substitutes showed a correct integration of artificial oral mucosa in immunocompetent hosts, with an increase in periodontal biotype and the creation of a zone with increased keratinization. Histologically, the tissue was similar to the control oral mucosa sample with no inflammatory reaction nor clinical or histological rejection signs. Conclusion: The equivalent oral mucosa allografts generated by tissue engineering showed clinical and histological compatibility.

Oral rapid test: an alternative to traditional HIV screening in Chile / Prueba oral rápida: una alternativa al tamizaje tradicional del VIH en Chile

OBJECTIVE: To compare the sensitivity and specificity of an Oral Rapid Test (ORT) to that of the Enzyme-Linked Immunosorbent Assay (ELISA) for HIV testing in Santiago, Chile; to track the number of study participants returning for ELISA testing results; and to analyze the participants' perceptions of the ORT compared to the ELISA. METHODS: A total of 497 people were recruited in Santiago, Chile: 153 had previously tested positive for HIV, and 344 were of unknown status. Participants were tested for HIV using both the ELISA and the ORT to examine and compare specificity and sensitivity. Qualitative data were collected from 22 participants to compare perceptions of the testing experience with ORT versus ELISA. RESULTS: The ELISA reported 184 (37%) of the 497 participants as being "positive" for HIV antibodies; the ORT showed 181 (36.4%) as being "reactive" for HIV. The ORT showed a sensitivity of 98.4% (95.7%-99.9%, 95% Confidence Interval) and specificity of 100%. The Kappa test produced K = 0.983 (P < 0.0001). Of the 344 participants whose HIV status was unknown at the start of the study, 55 failed to return for their ELISA results. Participants positively perceived ORT as having reduced both waiting time and anxiety over obtaining their test results. ORT oral swabbing appeared more practical and less invasive than drawing blood for the ELISA. CONCLUSIONS: The ORT and ELISA were statistically equal in specificity and sensitivity. ORT provides quicker results, potentially ensuring that more people receive them, and does not require handling of or exposure to potentially hazardous blood products. Trial number: ClinicalTrials.gov identifier: NCT01733927.

OBJETIVO: Comparar la sensibilidad y la especificidad de una prueba oral rápida con las del análisis de inmunoadsorción enzimática (ELISA) para la detección del VIH en Santiago de Chile, Chile; hacer un seguimiento del número de participantes en el estudio que regresan para saber los resultados del ELISA; y analizar las percepciones de los participantes con relación a la prueba oral rápida en comparación con el ELISA. MÉTODOS: Se incluyeron 497 personas en Santiago de Chile: 153 tenían resultados positivos para el VIH, y la situación de las restantes 344 era desconocida. Se sometió a los participantes a pruebas de detección del VIH tanto mediante el ELISA como mediante la prueba oral rápida, con objeto de analizar y comparar la especificidad y la sensibilidad. Se recopilaron datos cualitativos de 22 participantes para comparar sus impresiones con relación a la experiencia de someterse a la prueba oral rápida en comparación con el ELISA. RESULTADOS: Mediante el ELISA se notificó que 184 de los 497 participantes (37%) obtuvieron un resultado "positivo" en las pruebas de detección de anticuerpos contra el VIH; mediante la prueba oral rápida 181 participantes (36,4%) fueron "reactivos" para el VIH. Esta prueba demostró una sensibilidad de 98,4% (intervalo de confianza de 95%: 95,7-99,9%) y una especificidad de 100%. El coeficiente kappa (K) fue de 0,983 (P < 0,0001). De los 344 participantes cuyo estado con respecto a la infección por el VIH era desconocido al comienzo del estudio, 55 no regresaron para conocer los resultados del ELISA. Los participantes percibieron positivamente la prueba oral rápida debido al período de espera más breve y la reducción de la ansiedad por conocer los resultados de la prueba. La obtención de una muestra oral mediante hisopo resultó más práctica y menos invasora que la extracción de sangre necesaria para llevar a cabo un ELISA. CONCLUSIONES: La prueba oral rápida y el ELISA se mostraron estadísticamente equivalentes en cuanto a especificidad y sensibilidad. La primera proporciona resultados más rápidos, garantiza que más personas puedan conocerlos, y no requiere el manejo o la exposición a hemoderivados potencialmente peligrosos. Número de ensayo: Identificador de ClinicalTrials.gov, NCT01733927.

Argyrophilic nucleolar organizer regions in inflammatory, premalignant, and malignant oral lesions: a quantitative and qualitative assessment.

BACKGROUND AND OBJECTIVE: Argyrophilic nucleolar organizer regions (AgNORs) have found widespread application in the past, especially in tumor histopathology. This study was undertaken to evaluate the significance of various AgNOR parameters and to assess their role in differentiating hyperplastic, premalignant, and malignant lesions. MATERIALS AND METHODS: The study sample consisted of archival biopsy specimens of ten squamous cell carcinomas, ten premalignant lesions, and five inflammatory lesions. Two biopsies from normal mucosa acted as control. AgNORs were assessed both quantitatively and qualitatively. The data were analyzed using Student's independent t-test, one-way analysis of variance (ANOVA), and multiple range test (Tukey-HSD). RESULTS: Quantitatively significant difference existed in the number of AgNORs between the normal mucosa, inflammatory lesions, and carcinomas, but the premalignant lesions failed to differ significantly from the normal mucosa. The number of AgNORs was found to be related to epithelial proliferation. Qualitatively, in terms of size, shape, and pattern of distribution, the normal mucosa and inflammatory lesion were alike, but the premalignant and malignant lesions differed significantly from the normal, with a marked degree of AgNOR pleomorphism being observed in carcinomas. CONCLUSIONS: AgNOR quantity is strictly proportional to the proliferative activity of the cell and does not necessarily indicate malignancy. It is the qualitative characteristics of AgNOR that help to differentiate hyperplastic, premalignant, and malignant lesions.

A simple and cost-effective protocol for DNA isolation from buccal epithelial cells

Buccal cells provide a convenient source of DNA for epidemiological studies. The goal of this study was to develop a convenient method to obtain buccal cells from mouthwash samples to be used as a source of DNA, and to evaluate the stability of the DNA in mouthwash solution over time. The procedures used in the method described in this paper avoid the use of any organic solvents. This is achieved by salting out the cellular proteins by dehydration and precipitation with a saturated ammonium acetate solution. The protocol described here is fast, simple to perform, sensitive, economical and several samples can be processed at the same time. The analyses provide consistent evidence that DNA extracted by this methodology is sufficient for several PCR amplifications. The total DNA yield ranged from 5 to 93 µg (median 15 µg, mean 20.71 µg). DNA can be extracted and PCR amplified after storage of mouthwash solution at room temperature for periods of up to 30 days.

Células bucais são fontes convenientes de DNA para diagnóstico e estudos epidemiológicos. O objetivo deste trabalho foi desenvolver um método simples e prático para obter células epiteliais, através de bochechos, a fim de serem usadas como fonte de DNA e avaliar a estabilidade do DNA na solução de bochecho no decorrer do tempo. Os procedimentos usados neste estudo evitam o uso de solventes orgânicos permitindo uma pratica laboratorial mais segura. Isto é alcançado pela remoção das proteínas celulares por desidratação e precipitação com uma solução saturada de acetato de amônio. Este protocolo permite a extração de maneira rápida, simples, econômica e garante o processamento de várias amostras ao mesmo tempo, agilizando assim os procedimentos laboratoriais. Nossas análises forneceram evidências consistentes de que o DNA extraído por esta metodologia é suficiente para diversas amplificações por PCR (polymerase chain reaction - reação em cadeia pela polimerase). O produto total de DNA variou de 5 a 93 µg (mediana 15 µg; média 20,71 µg). Além disso, o DNA mostrou-se eficientemente preservado na solução de bochecho, a qual pode ser estocada em temperatura ambiente por até trinta dias.

Efeitos da laserterapia de גּ 660 nm na cicatrização de feridas cirúrgicas: estudo histológico e imunocitoquímico / Effects of lasertherapy of גּ 660 nm in wound healing: Histologic and immunocitochemical study

Os laseres têm sido usados em diversas especialidades médicas por mais de uma década. Um dos laseres mais usados na Odontologia é o laser do CO2, o qual tem uma excelente absorção em tecidos bucais, por isso é largamente utilizado em cirurgia de tecidos moles. Além dos laseres cirúrgicos, a laserterapia tem sido usada com sucesso em diversas circunstâncias como no combate a dores e em reparo de feridas. Neste estudo foram utilizados 54 ratos machos (Ratus norvegicus albinus, linhagem Wistar) com cerca de 8 semanas e pesando em média 250g, mantidos em ciclos de dia/noite de iluminação e temperatura ambiente. Os animais foram mantidos em caixas plásticas em grupos de três e alimentados com dieta padronizada, com água "ad libitum". Sob GA (hidrato de Cloral de 10 por cento) foi realizada ferida padronizada no dorso dos animais previamente tricotomizados, utilizando bisturi convencional (grupos 1, 2 e 3) ou com o laser do CO2 Sharplan, 20C, 5W, RSP (grupos 4, 5, e 6). Os grupos 1 e 4 foram os controles, os grupos 2 e 5 foram tratados com a terapia a laser de λ660nm (20J/cm2, 30mW, diâmetro de 3mm) e os grupos 3 e 6 foram tratados com laserterapia de λ830nm (20J/cm2, 40mW, diâmetro de 3mm). A terapia do laser foi aplicada transcutanamente em quatro pontos em torno da ferida e começada imediatamente depois da cirurgia, repetida no dia seguinte e depois a cada 48h durante o tempo experimental (3, 8 ou 14 dias). Os animais foram sacrificados dentro dos princípios da bioética, com overdose do anestésico geral e os espécimes foram processados para exame histológico, corados em H&E, Picrosirius e imunomarcados com α-actina e Vimentina. A análise histológica foi baseada em diversos aspectos do processo de reparo como: reepitelização, formação de colágeno, edema, neovascularização, infiltrado inflamatório, presença do miofibroblastos e fibroblastos. Os resultados evidenciaram reparo mais precoce no grupo 2, bem como maior maturação da matriz colagênica e.

Screening for fragile X syndrome among Brazilian mentally retarded male patients using PCR from buccal cell DNA

Fragile X syndrome is one of the most frequent causes of mental retardation. Since the phenotype in this syndrome is quite variable, clinical diagnosis is not easy and molecular laboratory diagnosis is necessary. Usually DNA from blood cells is used in molecular tests to detect the fragile X mutation which is characterized by an unstable expansion of a CGG repeat in the fragile X mental retardation gene (FMR1). In the present study, blood and buccal cells of 53 mentally retarded patients were molecularly analyzed for FMR1 mutation by PCR. Our data revealed that DNA extraction from buccal cells is a useful noninvasive alternative in the screening of the FMR1 mutation among mentally retarded males.

Occupational genotoxicity risk evaluation through the comet assay and the micronucleus test

The micronucleus (MN) test and the alkaline single cell gel or comet assay were applied to exfoliated cells of the buccal mucous in order to evaluate the genotoxic risk associated with occupational exposure of 10 storage battery renovation workers, and 10 car painters, with age matched controls, in Pelotas, RS, in southern Brazil. In the MN test, 2000 exfoliated buccal cells were analyzed for each individual, while 100 cells were examined in the comet assay. In the comet test, both comet tail length and a damage index were calculated. Highly significant effects of occupational exposure were found with both the MN test and the comet assay (P<0.001). The comet assay was found to be rapid, of simple visualization, and it is a sensitive technique for measuring and analyzing DNA damage in human cells

Expression of bcl-2 oncoprotien in oral squamous cell carcinoma--an immunohistochemical study.

Squamous cell carcinoma is the most common malignant neoplasm of the oral cavity. Number of mechanisms plays a role at the molecular level to transform normal cell into a neoplastic cell. There are a gamut of genes, which are expressed among which bcl-2, have gained a unique importance as inhibitor of apoptosis. In normal epithelial cells Bcl-2 is restricted to stem cells and cells which undergo mitosis. Bcl-2 blocks the post-mitotic phase from apoptosis. Reports of Bcl-2 protein expression in carcinomas are conflicting such as down regulation to elevated expression. In the present study 67 cases of squamous cell carcinomas of varying grades were studied and uniform cytoplasmic positivity were noted in 12 cases for Bcl-2 protein. Bcl-2 prolongs cell survival in epithelial cells and there by giving way to other external stimulus like action of carcinogens and viral agents and interaction with other genes and aids in progression to neoplasia. The possible roles of bcl-2 in the pathogenesis of oral squamous cell carcinoma are discussed.

Identification of volatile compounds from cheek glands of lesser bandicoot rats and assessment of behavioural response for identified compounds.

Check gland secretions were collected from sexually mature male and female rats to identify the volatile compounds using gas chromatography linked mass spectroscopy with a modified head space technique. Twenty one volatile fractions including alkanes, aliphatic acids, esters and alcohols were found. Three compounds viz. 3-octen-1-ol (E) (I), cyclopentane undecanoic acid (II) and 2,4,6,8 tetramethyl-l-undecane (III) were present in very high concentrations in both male and female rats. In addition, the estrus female contained another compound, L-alanine, 1,1-dimethyl ethyl ester. On the basis of odour preference tests the compounds I, II and III were found to attract both male and female of the homotypic species. In contrast, the fourth compound present in the female attracts the opposite sex. The results suggest that the volatile fractions have unique function and the level of attraction varies from compound to compound.

Screening for Turner syndrome: how useful is the buccal smear test?

The buccal smear test was used to screen for Turner syndrome in a sample of girls with severe short stature who did not have any other clinical features of that condition. The majority of the girls did not show X chromatin bodies in the buccal mucosal cells. None of those who qualified for chromosomal analysis showed an XO chromosomal pattern either. We conclude that reliability of the buccal smear test as a screening method for Turner syndrome remains doubtful.