RESUMO
Several studies have developed efficient oral mucosa constructs using different types of scaffold. However, the changes in the morphology and gene and protein expression profile that could occur in these artificial constructs remain unknown. This study compared the histology and expression of several extracellular matrix molecules in human artificial oral mucosa developed using two different types of scaffolds: fibrin and fibrin-agarose. To that end, bioengineered oral mucosa stromas were constructed from biopsy samples of human oral mucosa and the substitute generated was analyzed at different periods of time in culture. Histological analysis was carried out by light and transmission electron microscopy and the expression of collagen types I, III, and VI, the proteoglycans decorin and biglycan, and the different chains of laminin, were assessed by immunoperoxidase technique. This study found that fibrin scaffolds accelerated fibroblast growth and remodeling of the scaffold, thus enhancing collagen fibrillogenesis. In the fibrin-agarose scaffold, the morphology and organization of the fibroblasts did not change during the culture period. All extracellular matrix proteins analyzed were expressed in both scaffolds. However, in fibrin scaffolds, these proteins were widely distributed and replaced the scaffold during the follow-up period. These results show that the substitutes generated showed histological and molecular similarities with native human oral mucosa stroma. In addition, it was observed that the nature of the biomaterial influenced the behaviour of the oral stromal fibroblasts, thereby modulating their growth, protein synthesis, and collagen fibrillogenesis.
Assuntos
Matriz Extracelular/metabolismo , Fibrina/fisiologia , Mucosa Bucal/fisiologia , Sefarose/química , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Engenharia Biomédica/métodos , Clostridium histolyticum/metabolismo , Fibrina/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Mucosa Bucal/metabolismo , Engenharia Tecidual/métodos , Alicerces TeciduaisRESUMO
In regenerative medicine, the generation of biocompatible substitutes of tissues by in vitro tissue engineering must fulfil certain requirements. In the case of human oral mucosa, the rheological properties of tissues deserve special attention because of their influence in the acoustics and biomechanics of voice production. This work is devoted to the rheological characterization of substitutes of the connective tissue of the human oral mucosa. Two substitutes, composed of fibrin and fibrin-agarose, were prepared in cell culture for periods in the range 1-21 days. The time evolution of the rheological properties of both substitutes was studied by two different experimental procedures: steady-state and oscillatory measurements. The former allows the plastic behaviour of the substitutes to be characterized by estimating their yield stress; the latter is employed to quantify their viscoelastic responses by obtaining the elastic (G') and viscous (G'') moduli. The results demonstrate that both substitutes are characterized by a predominant elastic response, in which G' (order 100 Pa) is roughly one order of magnitude larger than G'' (order 10 Pa). But the most relevant insight is the stability, throughout the 21 days of culture time, of the rheological quantities in the case of fibrin-agarose, whereas the fibrin substitute shows a significant hardening. This result provides evidence that the addition to fibrin of a small amount of agarose allows the rheological stability of the oral mucosa substitute to be maintained. This feature, together with its viscoelastic similitude with native tissues, makes this biomaterial appropriate for potential use as a scaffold in regenerative therapies of human oral mucosa.
Assuntos
Fibrina/química , Teste de Materiais , Mucosa Bucal/fisiologia , Reologia , Sefarose/química , Engenharia Tecidual/métodos , Separação Celular , Células Cultivadas , Módulo de Elasticidade , Humanos , Oscilometria , Estresse Mecânico , Fatores de Tempo , ViscosidadeRESUMO
Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues. This way, tissue ingineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we have developed a new model for artificial oral mucosa generated by tissue engineering using a fibrin-agarosa scaffold. For that purpose, we have generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal mucosa oral using enzymatic treatments. Then, we have determined the viability of cultured cells by electron probe quantitative X-ray microanalysis, and we have demonstrated that most of the cells in the primary cultures were alive and hd high K/Na ratios. Once cell viability was determined, we used cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarosa extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and de oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.
Assuntos
Mucosa Bucal/cirurgia , Engenharia Tecidual/métodos , Técnicas de Cultura de Células , Humanos , Procedimentos de Cirurgia Plástica/métodosRESUMO
Dentin responds to different alterations in the enamel with hypermineralization, and is a biomarker of fluoride exposure. We hypothesized that severe fluorosis would lead to hypermineralization of the dentin when the enamel was severely affected. We used scanning electron microscopy and quantitative electron-probe microanalysis to compare dentin and enamel from healthy and fluorotic teeth. The dentin in fluorotic teeth was characterized by a highly mineralized sclerotic pattern, in comparison with control teeth (p < 0.001) and fluorotic enamel lesions (p < 0.001). Enamel near the lesions showed hypercalcification in comparison with dentin (p < 0.001). In response to the effects of severe fluorosis in the enamel, the dentin showed hypermineralization, as found in other enamel disorders. The hypermineralization response of the dentin in our samples suggests that the mechanism of the response should be taken into account in dental caries and other dental disorders associated with severe fluorosis.
Assuntos
Dentina/patologia , Fluorose Dentária/patologia , Calcinose , Cálcio/análise , Esmalte Dentário/patologia , Esmalte Dentário/ultraestrutura , Dentina/ultraestrutura , Microanálise por Sonda Eletrônica , Humanos , Microscopia Eletrônica de Varredura , Fósforo/análiseRESUMO
Incontinentia pigmenti (IP) is a genodermatosis with an X-linked dominant mode of inheritance, characterized by ectodermal, mesodermal, neurological, ocular, and dental manifestations. The purpose of this case study was to report the oral and dental manifestations of an IP case in a Venezuelan pediatric patient. A 9 year-old Venezuelan girl was evaluated. She showed macular pigmented lesions in her face, trunk, back, legs, and fingers as well as abnormal hair distribution, alopecia on the vertex, and hypoplasia of eyebrows. During the dental examination, conical shaped-teeth and delayed dental eruption was evidenced. The microanalytical examination showed dentin without significant alterations in the mineralization except for hypermineralization in focal areas. In addition, a decrease in the enamel mineralization was observed.
Assuntos
Incontinência Pigmentar/complicações , Anormalidades Dentárias/etiologia , Criança , Esmalte Dentário/anormalidades , Esmalte Dentário/química , Dentina/anormalidades , Dentina/química , Microanálise por Sonda Eletrônica , Feminino , Humanos , Radiografia , Anormalidades Dentárias/diagnóstico por imagem , Calcificação de Dente , Coroa do Dente/anormalidades , Erupção DentáriaRESUMO
El presente trabajo se realizó con el objeto de corroborar y relacionar los aspectos estructurales de los molares primarios observados con MO y MEB y aplicar las técnicas de histoquímica microanalítica para determinar los patrones cuantitativos de mineralización del esmalte y dentina. Se utilizó un total de 30 molares (1º y 2º) clínicamente sanos, exfoliados. El estudio para microscopía óptica (MO) fue mediante la técnica por desgaste y cortes procesados y metalizados para la electrónica de barrido (MEB). En el esmalte se identificó la zona aprismática periférica, la líne
Assuntos
Pré-Escolar , Criança , Dente Decíduo/ultraestrutura , Dente Molar/ultraestrutura , Dentina/ultraestrutura , Esmalte Dentário/ultraestrutura , Microscopia de Polarização , Microscopia Eletrônica de Varredura , Microanálise por Sonda Eletrônica , Histocitoquímica/métodos , Estudos de Avaliação como Assunto , Interpretação Estatística de DadosRESUMO
El presente trabajo se realizó con el objeto de corroborar y relacionar los aspectos estructurales de los molares primarios observados con MO y MEB y aplicar las técnicas de histoquímica microanalítica para determinar los patrones cuantitativos de mineralización del esmalte y dentina. Se utilizó un total de 30 molares (1º y 2º) clínicamente sanos, exfoliados. El estudio para microscopía óptica (MO) fue mediante la técnica por desgaste y cortes procesados y metalizados para la electrónica de barrido (MEB). En el esmalte se identificó la zona aprismática periférica, la línea neonatal, abundantes husos, conductillos dentinarios remanentes y microdefectos (laminillas o cracks de morfología y longitud variables). La dentina se caracterizó por la amplitud de los túbulos y escasa o nula dentina intertubular a nivel circumpulpar. El microanálisis con energía dispersiva de rayos X puso de relieve que el índice calcio/fósforo, tanto en esmalte como en dentina, alcanza el valor de 1.7, lo que sugiere que en la configuración de los cristales de apatita existen aniones carbonato. Los hallazgos de frecuentes microdefectos y la presencia de numerosas estructuras sin esmalte (husos y conductillos penetrantes) en la conexión amelodentinaria, especialmente en la porción coronaria oclusal nos advierte de los cuidados o precauciones a tomar en el momento de usar ácidos grabadores. Asimismo, la existencia de patrones de mineralización similares entre elementos primarios y permanentes, permite inferir que la terapéutica odontológica basada en la naturaleza de lamineralización podría aplicarse con idénticas pautas, pero respetando las variaciones estructurales respectivas (AU)
Assuntos
Pré-Escolar , Criança , Dente Decíduo/ultraestrutura , Dente Molar/ultraestrutura , Dentina/ultraestrutura , Esmalte Dentário/ultraestrutura , Microscopia de Polarização , Microscopia Eletrônica de Varredura , Microanálise por Sonda Eletrônica , Histocitoquímica/métodos , Estudos de Avaliação como Assunto , Interpretação Estatística de DadosRESUMO
El presente trabajo se realizó con el objeto de corroborar y relacionar los aspectos estructurales de los molares primarios observados con MO y MEB y aplicar las técnicas de histoquímica microanalítica para determinar los patrones cuantitativos de mineralización del esmalte y dentina. Se utilizó un total de 30 molares (1º y 2º) clínicamente sanos, exfoliados. El estudio para microscopía óptica (MO) fue mediante la técnica por desgaste y cortes procesados y metalizados para la electrónica de barrido (MEB). En el esmalte se identificó la zona aprismática periférica, la línea neonatal, abundantes husos, conductillos dentinarios remanentes y microdefectos (laminillas o cracks de morfología y longitud variables). La dentina se caracterizó por la amplitud de los túbulos y escasa o nula dentina intertubular a nivel circumpulpar. El microanálisis con energía dispersiva de rayos X puso de relieve que el índice calcio/fósforo, tanto en esmalte como en dentina, alcanza el valor de 1.7, lo que sugiere que en la configuración de los cristales de apatita existen aniones carbonato. Los hallazgos de frecuentes microdefectos y la presencia de numerosas estructuras sin esmalte (husos y conductillos penetrantes) en la conexión amelodentinaria, especialmente en la porción coronaria oclusal nos advierte de los cuidados o precauciones a tomar en el momento de usar ácidos grabadores. Asimismo, la existencia de patrones de mineralización similares entre elementos primarios y permanentes, permite inferir que la terapéutica odontológica basada en la naturaleza de lamineralización podría aplicarse con idénticas pautas, pero respetando las variaciones estructurales respectivas