Collection and culture of human connective tissue cells from gingival explant technique for oral tissue bioengineering / Colección de células de tejido conectivo humano a partir de la técnica de explante gingival para bioingeniería de tejidos orales

SUMMARY: Cell culture is an important tool in medical, odontological and biological research laboratories, supporting cell therapies and tissue bioengineering strategies. Gingival fibroblasts present structural function, being able to modulate their metabolic capacity, which is reflected in the tissue morphology. The possibility of culturing fibroblasts in vitro, in monolayer or on three-dimensional scaffolds, for subsequent transplants in vivo opens important perspectives for the periodontal surgical clinic. The objective of the present article is to present a method of obtaining and cultivating viable human gingival fibroblasts for in vitro research. Explants derived from periodontal surgical discards were used, grown in 25 cm2 bottles to obtain a primary cell culture. After observing the proliferation and growth of the fibroblasts that interconnected and formed a monolayer network, involving the periphery of the explants, it was possible to remove the explants, to make the passage and the new subcultures were obtained in a ratio of 1:1. After 7 days, the amount of viable cells was analyzed in triplicate, using the Neubauer chamber technique, in cell culture bottles of 25 mm2 (T25) and 75 mm2 (T75). Fibroblasts were described and subclassified morphologically. The results showed a growth pattern in both bottles, but with a larger number in bottles of 75 cm2. Cells with fibroblastic morphology were subclassified into reticular and fusiform, being predominant those with fusiform morphology. In conclusion, culture of explant of human gingival connective tissue is a viable method for obtaining gingival connective tissue cells suitable for laboratory tests in cell culture, aiming at obtaining constructs for gingival tissue engineering.

RESUMEN: El cultivo celular es una herramienta importante en los laboratorios de investigación médica, odontológica y biológica, que apoyan las terapias celulares y las estrategias de bioingeniería de tejidos. Los fibroblastos gingivales presentan una función estructural, pudiendo modular su capacidad metabólica, que se refleja en la morfología tisular. La posibilidad de cultivar fibroblastos in vitro, en monocapa o en andamios tridimensionales, para trasplantes posteriores in vivo abre perspectivas importantes para la clínica de cirugía periodontal. El objetivo del presente artículo es presentar un método para obtener y cultivar fibroblastos gingivales humanos viables para investigación in vitro. Se utilizaron explantes derivados de los descartes quirúrgicos periodontales, crecidos en frascos de 25 cm2 para obtener un cultivo de células primarias. Después de observar la proliferación y el crecimiento de los fibroblastos que se interconectaron y formaron una red de monocapa, que involucraba la periferia de los explantes, fue posible eliminar los explantes, hacer el pasaje y los nuevos subcultivos se obtuvieron en una proporción de 1:1. Después de 7 días, la cantidad de células viables se analizó por triplicado, utilizando la técnica de cámara de Neubauer, en botellas de cultivo celular de 25 mm2 (T25) y 75 mm2 (T75). Los fibroblastos fueron descritos y sub-clasificados morfológicamente. Los resultados mostraron un patrón de crecimiento en ambas botellas, pero con un número mayor en botellas de 75 cm2. Las células con morfología fibroblástica se subclasificaron en reticulares y fusiformes, predominando aquellas con morfología fusiforme. En conclusión, el cultivo de explante de tejido conectivo gingival humano es un método viable para obtener células de tejido conectivo gingival adecuadas para pruebas de laboratorio en cultivos celulares, con el objetivo de obtener construcciones para la ingeniería del tejido gingival.

Perfil de prescrição de antimicrobianos nas unidades básicas de saúde conveniadas com a universidade de Itaúna/MG / Antimicrobial prescription profile in the basic health units agreed with the university of Itaúna/MG

INTRODUÇÃO: O uso abusivo de antibacterianos está intimamente relacionado ao desenvolvimento de resistência bacteriana, considerada, atualmente, um problema de saúde pública mundial. Segundo a Organização Mundial de Saúde, em 2001, mais da metade das prescrições de antimicrobianos foram inapropriadas e dois terços de sua utilização foram feitas sem prescrição médica. Assim, o uso racional desse medicamento requer uma seleção criteriosa e bom senso clínico do prescritor. OBJETIVOS: Analisar o perfil de prescrição de antimicrobianos nas Unidades Básicas de Saúde da Família (UBSF) do município de Itaúna-MG, conveniados com a Universidade de Itaúna (UIT) e contribuir para que futuras intervenções possam ser conduzidas promovendo do uso racional dos antimicrobianos na atenção primária. MÉTODOS: Estudo transversal de prontuários médicos de pacientes atendidos nas UBSF de Itaúna/MG conveniadas com a UIT, realizado entre março de 2013 e março de 2014, para os quais foram prescritos antibióticos. RESULTADOS: A classe de antimicrobianos mais prescrita foi a das penicilinas seguido pelas quinolonas e macrolídeos. Quanto à duração do tratamento, o período de cinco a dez dias foi observado na maioria das prescrições. As principais indicações clínicas foram infecção das vias aéreas superiores não especificadas, amigdalite, otite, sinusite, infecção do trato urinário entre outros. A solicitação de culturas foi realizada em apenas 5,5% dos casos. CONCLUSÃO: A análise do perfil das prescrições revelou a necessidade de reciclagem da equipe e adoção de protocolos clínicos. Tais medidas permitirão a uniformização das condutas, otimizando as prescrições e reduzindo o risco do uso inapropriado de antimicrobianos. (AU)

Introduction: The overuse of antibacterials is closely related to the development of bacterial resistance, currently considered a problem of public health worldwide. According to the World Health Organization, in 2001 over half of the antimicrobial prescriptions were inappropriate and two thirds of its use were made without prescription. Thus, the rational use of this drug requires careful selection and clinical judgment of the prescriber. Objectives: To analyze the antimicrobial prescription profile in the Basic Health Units of family of Itaúna-MG, that have agreements with the University of Itaúna (UIT) and contribute to future interventions that can be conducted to promote the rational use of antimicrobials in primary care. Methods: Cross-sectional study of medical records of patients treated in Basic Health Units of family Itaúna / MG with agreement with the UIT, held between March 2013 and March 2014, for which antibiotics were prescribed. Results: The most prescribed class of antimicrobials was the penicillins followed by quinolones and macrolides. About the duration of treatment, five to ten days was observed in the majority of prescriptions. The main clinical indications were infection of the upper airways unspecified, tonsillitis, otitis, sinusitis, urinary tract infection, among others. The request of cultures was performed in only 5.5% of cases. Conclusion: The analysis of the profile of prescriptions revealed the need for retraining of staff and adoption of clinical protocols. These measures will enable to uniform the procedures, optimizing the regulations and reducing the risk of inappropriate use of antimicrobials. (AU)

Rat dental pulp stem cells: isolation and phenotypic characterization method aiming bone tissue bioengineering

ABSTRACT: Dental pulp stem cells (DPSC) have been showing a considerable potential for regenerative medicine. Pulps were collected from lower incisors (n=2) through direct access of the tooth pulp chamber. The isolated cells were cultured in alfa-MEM 10% FBS, in standard culture conditions. At the third passage, DPSC were characterized by flow cytometry (MHCI, CD54, CD73, CD90, CD45, CD11 and CD34); RT-PCR for Nanog gene; and their differentiation capacity in osteogenic, adipogenic and chondrogenic cell lines. Isolated cells exhibited adhesion capacity to plastic; fusiform morphology, and 80% confluence reached in approximately 3 days. These cells have also revealed positive expression for CD54, CD73 and CD90 markers; and negative expression for CD11, CD34 and CD45. Nanog expression was detected by RT-PCR, expected for a mesenchymal stem cell profile. DPSC chondrogenic differentiation was confirmed by positive staining in Alcian Blue; lipidic droplets stained with oil red confirmed their capacity to differentiate in adipogenic fate; while mineralized beads, stained with alizarin red, confirmed their differentiation in osteogenic phenotype. These results indicate the viability of the isolation and expansion of rat DPSC following this method, and osteogenic differentiation potential opens new perspectives for in vivo studies and the use of these cells in cellular therapies and tissue bioengineering, aiming bone repair.

Effect of PDGF-BB, IGF-I Growth Factors and their Combination Carried by Liposomes in Tooth Socket Healing

This work evaluated the bone-forming potential of the platelet-derived growth factor isoform BB (PDGF-BB), insulin-like growth factor I (IGF-I), and mixed PDGF-BB/IGF-I delivered in liposomes compared with phosphate buffered saline (PBS), in the healing process of rat tooth sockets. One hundred and twelve Wistar rats were randomized into 7 groups of 16 animals each and were evaluated at 3, 7, 14 and 21 days after extraction of the maxillary second molars. The left sockets were treated with PBS (P), empty liposome (L), IGF-I in PBS (IP), IGF-I in liposome (IL), PDGF-BB in PBS (PDP), PDGF-BB in liposome (PDL) and both growth factors (GFs) together within liposomes (PDIL). The right sockets were filled with blood clot (BC). Histological and histomorphometric analyses were used to evaluate the formation of new bone and blood vessels. Immunohistochemistry was performed to evaluate the expression of osteocalcin and vascular endothelial growth factor (VEGF) during bone repair. Data were tested statistically using a Tukey's test according to a Dunn's analysis and Mann-Whitney U test followed by Kruskal-Wallis one-way analysis. Results were considered significant when p<0.05. A significantly higher percentage of bone trabeculae and a higher number of blood vessels were observed in the IL, PDL and PDIL groups (p<0.05). However, these GF-liposome groups had statistically similar results. Immunohistochemical assays first detected osteocalcin and VEGF expression at 3 days followed by a peak at 7 days. Lower immunoreactivity levels were observed in the BC, L, P, IP and PDP groups compared with the IL, PDL and PDIL groups (p<0.05). The results suggest that GFs carried by liposomes, either in isolated or mixed forms, enhanced the healing process in rat tooth sockets. The differential expression of the osteogenic markers VEGF and osteocalcin in the early phases of bone healing support these findings.

Este trabalho avaliou o potencial de formação óssea do fator de crescimento derivado de plaquetas na isoforma BB (PDGF-BB), fator de crescimento semelhante à insulina I (IGF-I), e a mistura PDGF-BB/IGF-I administrada em lipossomas comparando com tampão fosfato salino (PBS), no processo de cicatrização de alvéolos dentários de ratos. Cento e doze ratos Wistar foram distribuídos aleatoriamente em 7 grupos de 16 animais cada e foram avaliados aos 3, 7, 14 e 21 dias após a extração dos segundos molares maxilares. Os alvéolos esquerdos foram tratados com PBS (P), lipossomas vazios (L), IGF-I em PBS (IP), IGF-I em lipossomas (IL), PDGF-BB em PBS (PDP), PDGF-BB em lipossomas (PDL) e ambos os fatores de crescimento (GFs) em associação dentro de lipossomas (PDIL). Os alvéolos direitos foram preenchidos com coágulo sanguíneo (BC). As análises histomorfométrica e histológica foram utilizadas para avaliar a formação de novo osso e vasos sanguíneos. Imunohistoquímica foi realizada para avaliar a expressão de osteocalcina e o fator de crescimento endotelial vascular (VEGF) durante o reparo ósseo. Os dados foram testados estatisticamente utilizando o teste de Tukey em acordo com análise de Dunn e o teste Mann-Whitney U seguido pela análise de um passo de Kruskal-Wallis. Os resultados foram considerados significantes quando p<0,05. Uma percentagem altamente significativa de osso trabecular e alto número de vasos sanguíneos foram observados nos grupos IL, PDL e PDIL (p<0,05). Todavia, esses grupos lipossoma-GF tiveram resultados similares estatisticamente. Ensaios de imunohistoquímica inicialmente detectaram a expressão de osteocalcina e VEGF aos 3 dias, seguida por um pico aos 7 dias. Niveis mais baixos de imunorreatividade foram observados em BC, L, P, PI e PDP quando comparados com os grupos IL, PDL e PDIL (p<0,05). Os resultados sugerem que GFs carreados por lipossomas, na forma isolada ou em combinação, aceleram o processo de cicatrização em alvéolos dentários de rato. A expressão diferencial dos marcadores osteogênicos VEGF e osteocalcina, nas fases iniciais de cicatrização óssea, confirma esses achados.

Ultrastructural evaluation of the hybrid layer after cementation of fiber posts using adhesive systems with different curing modes

This in vitro study evaluated the adhesive interface of intraradicular fiber glass posts and root dentin using scanning electron microscopy (SEM). Forty-eight single-rooted premolars were randomly divided into 6 groups consisting of chemical, dual, or light cured adhesive systems combined with either chemical or dual cure resin cements. Scanning electron microscopic analysis showed the best results for continuity, density and morphology of the hybrid layer and resin tags for the combination of a self-cure adhesive with self-cure cement resin, followed by a dual-cure adhesive with self-cure cement resin, and finally a light-cure adhesive with self-cure cement. For the dual-cure resin cement, the same relation may be observed. The apical third was the most critical region for evaluated the criteria for all combinations of materials (Kruskal-Wallis and Friedman tests; p<0.001). Generally, the simplification of steps in the adhesive system and the polymerization reaction of resin adhesives and cements produced a direct effect on the quality of the adhesive post/dentin substrate interface.

Este estudo in vitro avaliou as interfaces adesivas de pinos intra-radiculares de fibra de vidro e a dentina radicular utilizando microscópio eletrônico de varredura (MEV). Quarenta e oito pré-molares unirradiculares foram divididos ao acaso em seis grupos, compostos por sistemas adesivos de cura química, dual ou fotopolimerizável, associado com cimentos resinosos de polimerização química ou dual. As análises microscópicas mostraram a maior continuidade, densidade e morfologia da camada híbrida e prolongamentos resinosos para a associação entre adesivos e cimentos auto-polimerizáveis seguido pelo grupo do adesivo de dupla polimerização e cimento de resina auto-polimerizável, e finalmente pelo adesivo fotopolimerizável e cimento de resina auto-polimerizável . Para os cimentos resinosos de dupla polimerização a mesma relação pode ser observada. O terço apical mostrou ser o substrato mais crítico em relação aos critérios avaliados para todas as associações entre os materiais usados(testes de Kruskal-Wallis e Friedman p<0,001). De maneira geral, a simplificação dos passos do sistema adesivo e a reação de polimerização dos adesivos e cimentos resinosos produzem efeitos diretos na qualidade da interface adesivo pino/dentina.