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.

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.

Afin de résoudre la problématique des substituts osseux faiblement vascularisés, un des challenges majeurs en ingénierie tissulaire osseuse est de favoriser le développement précoce d’une microvascularisation. La reproduction du microenvironnement local et l’organisation cellulaire in situ sont des approches innovantes pour optimiser la formation osseuse. En Biofabrication, la Bioimpression Assistée par Laser (LAB) est une technologie émergente permettant l’impression de cellules et de biomatériaux avec une résolution micrométrique. L’objectif de ce travail était d’étudier l’effet de l’organisation de la pré-vascularisation par LAB sur la régénération osseuse. La station de bioimpression Novalase a été utilisée pour imprimer des motifs de cellules endothéliales sur un « biopaper » constitué de collagène et de cellules souches issues de la papille apicale. Les paramètres d’impression, densités cellulaires et conditions de recouvrement ont été optimisés afin de favoriser la formation d’un réseau microvasculaire avec une architecture définie in vitro. Ce modèle a ensuite été transposé in vivo, grâce à la bioimpression in situ de cellules endothéliales au niveau de défauts osseux critiques chez la souris, afin d’évaluer si la prévascularisation organisée par LAB permettait de promouvoir et contrôler spatialement le processus de régénération osseuse. Les résultats ont montré que la bioimpression permettait d’augmenter la densité de vaisseaux dans les défauts osseux et de favoriser la régénération osseuse. / In order to solve the issue of poorly vascularized bone substitutes, development of a microvasculature into tissue-engineered bone substitutes represents a current challenge. The reproduction of local microenvironment and in situ organization of cells are innovating approaches to optimize bone formation. In Biofabrication, Laser-Assisted Bioprinting (LAB) has emerged as a relevant method to print living cells and biomaterials with micrometric resolution. The aim of this work was to study the effect of prevascularization organized by LAB on bone regeneration. The laser workstation Novalase was used to print patterns of endothelial cells onto a « biopaper » of collagen hydrogel seeded with stem cells from the apical papilla. Printing parameters, cell densities and overlay conditions were optimized to enhance the formation of microvascular networks with a defined architecture in vitro. This model was then transposed in vivo, through in situ bioprinting of endothelial cells into mouse calvarial bone defects of critical size, to investigate if prevascularization organized by LAB can promote and spatially control bone regeneration. The results showed that bioprinting allowed to increase blood vessel density in bone defects and promote bone regeneration.