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.

Um infusor autônomo para um expansor de pele subcutâneo / An autonomous infuser for a subcutaneous skin expander

Introdução: A expansão da pele é um processo fisiológico definido como a capacidade de aumentar sua área superficial em resposta a uma tensão ou a uma dada deformação. Para realizar a cirurgia reconstrutiva, os expansores de pele são implantados sob a pele e periodicamente infiltrados com uma solução salina para fornecer um retalho extra de pele. Quando o volume interno prescrito do expansor é alcançado, a cirurgia reconstrutiva é realizada. Métodos: Foi desenvolvido um dispositivo semiautomático e portátil para facilitar um procedimento de expansão da pele. O dispositivo tem como objetivo simplificar o processo de infiltração, proporcionando mobilidade e independência para o paciente, e assegurando ao médico a qualidade e a precisão das infiltrações realizadas. O dispositivo também permite expansão contínua em pacientes hospitalizados. Resultados: Usando um código, o médico tem acesso ao menu do dispositivo e define a pressão máxima e/ou o valor máximo para cada expansor do paciente. O paciente pode realizar a infiltração e ter acesso ao controle da velocidade de infiltração, reverter ou parar a operação. Todos os dados são gravados em um SIM Card e incluem data, hora, volumes inicial e final, e pressão inicial e final de cada procedimento para cada expansor. Conclusões: O dispositivo automatiza e otimiza a expansão, de modo que o que o médico possa prescrever um limite para cada expansão, seja uma pressão máxima ou voluma infiltrado. Todos os dados são gravados, fornecendo um importante banco de dados sobre o comportamento de pele relacionado a gênero, raça, idade e local da expansão.

Introduction: Skin expansion is a physiological process defined as the ability of human skin to increase its superficial area in response to a stress or given deformation. In reconstructive surgery, skin expanders are implanted beneath the skin and periodically infiltrated with a saline solution to provide an extra flap of skin. When the prescribed internal volume of the expander is reached, reconstructive surgery is performed. Methods: A semiautomatic and portable device was developed and built to facilitate a skin expansion procedure. The device aims to simplify the infiltration process, providing mobility and independence to the patient and assuring the physician of the infiltration quality and precision. The device also enables continuous expansion in hospitalized patients. Results: Using a code, the doctor accesses the menu of the device and sets the maximum pressure and/or value for each expander of the patient. The patient can control the infiltration velocity and reverse or stop the operation. All data are recorded on a simcard and include date, time, initial and final volumes, and initial and final pressures of each procedure for each expander. Conclusions: The device motorizes and optimizes the expansion, allowing the doctor to prescribe a maximum infiltration pressure or volume. All data are recorded to provide an important database of skin behavior related to sex, race, age, and expansion site.

Elaboración de un biodiente: enfoque actual y desafíos / Making a Bio-tooth: Current Approaches and Challenges

Antecedentes: El edentulismo es uno de los mayores problemas de salud oral que cause alteraciones fisiológicas, sociales, estéticas, fonéticas y nutricionales. Las terapias actuales para el remplazo dental son artificiales y no satisfacen los requisitos básicos de un diente natural. La bioingeniería de tejidos constituye una alternativa para la sustitución de dientes perdidos. Objetivo: Identificar los enfoques/técnicas disponibles actualmente para obtener un diente completo por bioingeniería (biodiente), así como puntualizar sus desafíos y perspectivas futuras. Métodos: Se realizó una revisión integrativa de la literatura, por medio de las siguientes palabras clave: biodiente, bioingeniería de tejidos, diente entero y células madre. Los años de la búsqueda fueron 2000-2018, en las bases de datos: PubMed, Scopus, EBSCO, Science Direct, Wiley Online Library, Lilacs y Google Académico/Scholar, en inglés y español. Se seleccionaron únicamente artículos y libros de mayor relevancia y pertinencia. Resultados: Se obtuvieron 53 artículos y 10 libros. Para la elaboración de un biodiente se emplean los siguientes métodos: andamios, sin andamios, células madre pluripotentes inducidas, germen de órganos, diente quimérico y estimulación de la formación de la tercera dentición. El tamaño y forma normales del diente, así como la obtención de células epiteliales, son los principales desafíos. Conclusiones: La posibilidad de crear y desarrollar un biodiente en un ambiente oral adulto es cada vez más real gracias a los avances biotecnológicos que ocurren diariamente. Es posible que estos conceptos sean la base de la odontología restauradora en un futuro próximo.

Background: Edentulism is one of the major oral health problems that cause physiological, social, aesthetic, phonetic, and nutritional issues. Current therapies for dental replacement are artificial and do not satisfy the basic requirements of a natural tooth. Tissue bioengineering could be a viable alternative to substitute lost teeth. Objective: To identify current available approaches/techniques to obtain a complete bioengineered tooth (bio-tooth) and to point out future challenges and perspectives. Methods: This was an integrative literature review. Search keywords used were: bio-tooth, tissue bioengineering, whole tooth, stem cells. The search included the years 2000 through 2018, using the databases PubMed, Scopus, EBSCO, Science Direct, Wiley Online Library, Lilacs and Google Scholar, both in English and Spanish. Only relevant and pertinent articles and books were selected. Results: 53 articles and 10 books were obtained. Methods for bio-tooth generation found were: scaffolds, scaffold-free, induced pluripotent stem cells, tooth organ germ, chimeric tooth, and stimulation of third dentition formation. Achieving normal tooth size and shape and obtaining epithelial cells are the main challenges. Conclusions: The possibility of creating and developing a whole bioengineered tooth (bio-tooth) in an adult oral environment is becoming more realistic, considering the daily biotechnological advances. It is possible that these concepts will be the basis of restorative dentistry in a near future.

Aplicaciones del cultivo celular en odontología / Applications of cell culture in dentistry

Las ciencias básicas, la medicina oral y los nuevos avanzces en biotecnología y bioinformática constituyen un gran campo de investigación dentro de la odontología actual. En este sentido, dichos avances están proporcionandoun nuevo conjunto de estrategias terapéuticas para el manejo clínico de los pacientes con dolencias dentales y craneofaciales. Es importante destacar que las disciplinas relacionadas con las ciencias básicas, la medicina oral, la biotecnología y la bioinformática, han contribuido de manera trascendental al entendimiento de la fisiología y lasdiversas patologías que afectan las condiciones de normalidad del sistema bucal. La ingeniería tisular se considera como un enfoque prometedor para la odontología regenerativa, con el objetivofinal de reemplazar morfológica y funcionalmente los tejidos periodontales y/o los dientes perdidos a través dela síntesis in vitro de sustitutos análogos tisulares, considerando que el diente y las estructuras periodontales son importantes órganos del complejo craneofacial, los tratamientos utilizados para las enfermedades que los afectan no lo restauran completamente. La odontología clínica está incursionando en una nueva era en donde el enfoque terapéutico es el uso de terapia génica, terapia celular, ingeniería tisular y lamedicina regenerativa, ampliando el arsenal de posibilidades para nuestros pacientes. Una línea de investigaciónfundamental en ingeniería tisular y medicina regenerativa son las células madres. Como parte de los nuevos avances de la odontología a nivel mundial, científicos e investigadores del mundo aplican la bioingeniería para lograr reconstrucciones maxilofaciales,regeneraciones óseas y reconstrucciones de piezas dentales a partir de células madre como parte de tratamientos inovadores...

Estudo da interação entre células-tronco transplantadas e células do hospedeiro na bioengenharia pulpar / Study of the interaction between transplanted stem cells and host cells in pulp bioengineering

O uso de células tronco já é uma realidade em algumas áreas da Medicina, porém o mesmo não se aplica para a Odontologia, que segue utilizando materiais artificiais para substituir tecidos dentais perdidos. Desde 2000, quando Gronthos e colaboradores identificaram células-tronco na polpa de dentes permanentes, os estudos avançaram para que, num futuro breve, essas células possam ser de fato aplicadas para regenerar tecidos dentais, com destaque para a endodontia, onde o uso dessas células parece ser mais eminente. Dessa forma, esse trabalho procurou analisar a co-participação de células humanas transplantadas e células do hospedeiro em um modelo de engenharia pulpar. Para tanto, células de papila apical, enriquecidas ou não para o marcador CD146, foram transplantadas em câmaras pulpares despulpadas preenchidas com colágeno. Dois modelos animais foram utilizados, sendo um modelo transgênico para o gene GFP e outro imunocomprometido pela aplasia do timo (nude). Os resultados foram analisados nos dias 14 e 21 pós-transplante das amostras em cápsula renal. Nas amostras GFP realizou-se imunofluorescência para o marcador anti-GFP, com o objetivo de identificar as células do hospedeiro, enquanto nas amostras nude utilizou-se o marcador lâmina A para identificar as células humanas transplantadas.

Nas análises morfológicas de todas as coroas transplantadas houve a formação de um tecido conjuntivo frouxo de celularidade variável, com a presença de vasos bem formados com eritrócitos em seu interior, inclusive nas coroas que receberam somente colágeno. Somente as amostras que receberam células houve a formação de matriz mineralizada no espaço pulpar, mas nos tempos experimentais analisados não foi possível visualizar as células humanas. Nas amostras nude, o marcador lâmina A foi negativo para todos os grupos que receberam transplante de células. Nas amostras GFPs, o marcador anti-GFP foi positivo na totalidade das células em todas as amostras estudadas. A partir disso concluiu-se que as células-tronco humanas de papila apical transplantadas apesar de terem desempenhado alguma função fisiológica, não foram identificadas após 14 e 21 dias e o tecido neoformado no interior da câmara pulpar era proveniente do hospedeiro. Adicionalmente, concluímos que não é necessário o transplante de células para a formação de um tecido conjuntivo frouxo no interior da câmara pulpar.

The use of stem cells is already a reality in some areas of Medicine, but the same does not apply for Dentistry, which keeps on using artificial materials to replace lost dental tissues. Since 2000, when Gronthos and coleagues identified stem cells in the pulp of permanent teeth, studies advanced so that, in the near future, these cells may actually be applied to regenerate dental tissues, especially in endodontics, in which the use of these cells seems to be more imminent. Thereby, this study sought to examine the co-participation of transplanted human cells and host cells in a model of dental pulp engineering. For this, apical papilla cells enriched or not with CD146 marker, were transplanted into decelluarized pulp chambers (empty crowns) filled with collagen. Two animal models were used, a transgenic model for the GFP gene and an immunocompromised by thymus aplasia (nude). The results were analyzed on days 14 and 21 after transplantation of samples into renal capsule. In the GFP samples immunofluorescence was performed for the anti-GFP marker in order to identify host cells, while in the nude samples the lamina

A marker was used to identify the transplanted human cells. In the morphological analysis of all transplanted crowns there was formation of a loose connective tissue of variable cellularity, with the presence of well-formed vessels with erythrocytes inside, including in the crowns that received only collagen. Osteodentine was formed in the pulp chamber only in the samples that received cell, but after the wait time was not possible to visualize the human cells. In the nude samples the Lamina A marker was negative for all groups transplanted with cells. In the GFPs samples, the anti-GFP marker was positive for all cells in this group. We concluded that it was not necessary to use transplanted cells to form a connective tissue inside the pulp chamber and although transplanted human stem cells from apical papilla played some physiological function, after 14 and 21 days of transplantation, they were no longer present in the tissue newly formed by the host.

Células-tronco mesenquimais e sua aplicação na Odontologia / Mesenchymal stem cells and their application in Dentistry

Este estudo teve como objetivo realizar uma revisão de literatura sobre as células-tronco pulpares, células indiferenciadas que possuem uma elevada capacidade de proliferação, autorrenovação e diferenciação em variados tipos celulares e seu uso na engenharia de tecidos aplicada à Odontologia. Bases de dados: As bases de dados utilizadas foram Pubmed e Bireme. Seleção dos trabalhos: Foi realizada por meio das palavras-chave: “Células-tronco mesenquimais”; “Polpa dentária”; “Bioengenharia”. Conclusões: A capacidade de multidiferenciação, as altas taxas de proliferação e o acesso relativamente fácil fazem da polpa de dentes decíduos e permanentes uma atraente fonte de células-tronco mesenquimais, as quais poderiam ser empregadas na regeneração tecidual. Algumas pesquisas evidenciaram que células-tronco pulpares foram capazes de produzir tecidos dentários como dentina, polpa e até estruturas similares a dentes in vivo. Os avanços dos estudos utilizando células-tronco abrem oportunidades para o desenvolvimento novas terapias que procuram regenerar e/ou substituir dentes perdidos ou danificados por diferentes motivos...

This study aimed to conduct a review of the literature on pulp stem cells, undifferentiated cells that have a high capacity for proliferation, self-renewal and differentiation into various cell types and their use in tissue engineering applied to Dentistry. Data sources: The databases used were Pubmed and Bireme. Selection of works: Was performed using the keywords: “Células-tronco mesenquimais”; “Polpa dentária”; “Bioengenharia”. Conclusions: The ability to multipotent differentiation, high rates of proliferation and relatively easy access make the pulp tissue from deciduous and permanent teeth an attractive source of mesenchymal stem cells, which could be used for tissue regeneration. Some studies reported that dental pulp stem cells were capable of producing dental tissues like dentin, pulp and even teeth-like structures in vivo. The progress of stem cell biology research provides opportunities for developing new therapies that seek to regenerate and/ or replace lost or damaged teeth for different reasons...

Characterization of mesenchymal stem cells from human dental pulp, preapical follicle and periodontal ligament

Human dental stem cells have high proliferative potential for self-renewal that is important to the regenerative capacity of the tissue. The aim was to isolate human dental pulp stem cells [DPSC], periodontal ligament stem cells [PDLSC] and periapical follicle stem cells [PAFSC] for their potential role in tissue regeneration. In this experimental study, the postnatal stem cells were isolated from dental pulp, preapical follicle and periodontal ligament .The cells were stained for different stem cell markers by immunocytochemistry. To investigate the mesenchymal nature of cells, differentiation potential along osteoblastic and adipogenic lineages and gene expression profile were performed. For proliferation potential assay, Brdu staining and growth curve tests were performed. Finally, all three cell types were compared together regarding their proliferation, differentiation and displaying phenotype. The isolated cell populations have similar fibroblastic like morphology and expressed all examined cell surface molecule markers. These cells were capable of differentiating into osteocyte with different capability and adipocyte with the same rate. PAFSCs showed more significant proliferation rate than others. Reverse transcriptase PCR [RT-PCR] for nanog, oct4, Alkaline phosphatase [ALP] and glyceraldehydes-3-phosphate dehydrogenease [GADPH] as control gene showed strong positive expression of these genes in all three isolated cell types. PDLSCs, DPSCs and PAFSCs exist in various tissues of the teeth and can use as a source of mesenchymal stem cells for developing bioengineered organs and also in craniomaxillofacial reconstruction with varying efficiency in differentiation and proliferation

Future developments in brain-machine interface research

Neuroprosthetic devices based on brain-machine interface technology hold promise for the restoration of body mobility in patients suffering from devastating motor deficits caused by brain injury, neurologic diseases and limb loss. During the last decade, considerable progress has been achieved in this multidisciplinary research, mainly in the brain-machine interface that enacts upper-limb functionality. However, a considerable number of problems need to be resolved before fully functional limb neuroprostheses can be built. To move towards developing neuroprosthetic devices for humans, brain-machine interface research has to address a number of issues related to improving the quality of neuronal recordings, achieving stable, long-term performance, and extending the brain-machine interface approach to a broad range of motor and sensory functions. Here, we review the future steps that are part of the strategic plan of the Duke University Center for Neuroengineering, and its partners, the Brazilian National Institute of Brain-Machine Interfaces and the École Polytechnique Fédérale de Lausanne (EPFL) Center for Neuroprosthetics, to bring this new technology to clinical fruition.