Angiogenic cell therapy is currently being developed as a treatment for coronary artery disease (CAD); however, endothelial dysfunction (ED), commonly found in patients with CAD, impairs the ability for revascularization to occur. We hypothesized that culture on a collagen matrix will improve survival and function of circulating progenitor cells (CPCs) isolated from a mouse model of ED. Overall, ED decreased the expression of endothelial markers in CPCs and impaired their function, compared to normal mice. Culture of CPCs from ED mice on collagen was able to increase cell marker expression, and improve migration and adhesion potential, compared to CPCs on fibronectin. Nitric oxide production was reduced for CPCs on collagen for the ED group; however, CPCs on collagen had better viability under conditions of serum deprivation and hypoxia, compared to fibronectin. This study suggests that a collagen matrix may improve the function of therapeutic CPCs that have been exposed to ED.

Angiogenic cell therapy is currently being developed as a treatment for coronary artery disease (CAD); however, endothelial dysfunction (ED), commonly found in patients with CAD, impairs the ability for revascularization to occur. We hypothesized that culture on a collagen matrix will improve survival and function of circulating progenitor cells (CPCs) isolated from a mouse model of ED. Overall, ED decreased the expression of endothelial markers in CPCs and impaired their function, compared to normal mice. Culture of CPCs from ED mice on collagen was able to increase cell marker expression, and improve migration and adhesion potential, compared to CPCs on fibronectin. Nitric oxide production was reduced for CPCs on collagen for the ED group; however, CPCs on collagen had better viability under conditions of serum deprivation and hypoxia, compared to fibronectin. This study suggests that a collagen matrix may improve the function of therapeutic CPCs that have been exposed to ED.

Angiogenic cell therapy is currently being developed as a treatment for coronary artery disease (CAD); however, endothelial dysfunction (ED), commonly found in patients with CAD, impairs the ability for revascularization to occur. We hypothesized that culture on a collagen matrix will improve survival and function of circulating progenitor cells (CPCs) isolated from a mouse model of ED. Overall, ED decreased the expression of endothelial markers in CPCs and impaired their function, compared to normal mice. Culture of CPCs from ED mice on collagen was able to increase cell marker expression, and improve migration and adhesion potential, compared to CPCs on fibronectin. Nitric oxide production was reduced for CPCs on collagen for the ED group; however, CPCs on collagen had better viability under conditions of serum deprivation and hypoxia, compared to fibronectin. This study suggests that a collagen matrix may improve the function of therapeutic CPCs that have been exposed to ED.

Injectable biomaterials have emerged as a treatment for myocardial infarction (MI). They can be applied either as an enhancement for cell therapy or as a stand-alone treatment for MI. The main focus of this study was to apply circulating angiogenic cells (CACs) with or without an injectable collagen matrix for MI treatment in a mouse model. Furthermore, a collagen-chitosan matrix was tested for modulating the myocardial maladaptive remodeling post-MI. First, the in vivo thermo-gelling and retention properties of the collagen matrix were validated using positron emission tomography (PET) tracer and quantum dot (Qdot) labelled matrix in MI mouse hearts. The therapeutic potential of the matrix ± CACs was then tested in a mouse MI model. The results showed that CACs-only and matrix-only treatments were associated with cardiac function preservation. However, in combination, CAC + matrix therapy had a synergistic effect and significantly improved cardiac function (echocardiography), perfusion and viability (PET scan), increased cell engraftment and arteriole density, and reduced the infarct size. CAC-matrix interaction through the integrin alpha2 receptor was essential for the observed therapeutic effect. In a third study, the addition of chitosan (a polysaccharide) to the collagen matrix was shown to reduce maladaptive remodeling post-MI by limiting cardiac fibroblast-to-myofibroblast differentiation and scar formation. In conclusion, these collagen-based hydrogels hold promise to enhance cardiac repair as a delivery scaffold for therapeutic cells, and/or as a stand-alone treatment, which can actively modulate the environment including the fibrotic process after MI.

Introdução: Fibroblastos gengivais desempenham um importante papel na regeneração de tecidos moles de proteção. Mucograft® (MCS-3D) é um substituto xenógeno novo que vem sendo utilizado na periodontia em técnicas de recobrimento radicular e aumento de tecido queratinizado. O objetivo do presente estudo foi avaliar morfológica e imunohistoquimicamente, se a MCS-3D é uma matriz tridimensional adequada, por meio de sua resposta à cultura de fibroblastos gengivais. Material e Métodos: Fibroblastos gengivais humanos (FGH) foram obtidos pela técnica do explante a partir de tecido conjuntivo gengival de três indivíduos. Os FGH foram cultivados sobre colágeno bovino bidimensional (ColB-2D), colágeno bovino tridimensional (ColB-3D) e matriz colágena suína tridimensional (MCS-3D) por até 10 dias. Em 3, 7 e 10 dias, os seguintes parâmetros foram avaliados: número, morfologia e distribuição de FGH por fluorescência direta; viabilidade celular por MTT; proliferação celular e expressão de proteínas citoesqueléticas: actina e tubulina e proteínas da matriz extracelular não colágena: fibronectina imunofluorescência indireta. Os dados quantitativos foram submetidos aos testes estatísticos de Friedman para comparação intra e intergrupos para as variáveis da análise de expressão das proteínas: fibronectina, actina e tubulina. O teste de Kruskal-Wallis foi utilizado para comparações intra e intergrupos, seguido do teste de Tukey para as variáveis das análises de viabidlidade, proliferação e número total de células. Resultados: A epifluorescência revelou que em 3 dias os FGH apresentavam-se aderidos em todos os grupos experimentais. FGH cultivados sobre ColB-2D exibiram forma menos alongada, ampla e achatada com maior quantidade de projeções e numerosas fibras de estresse em 3, 7 e 10 dias. Por outro lado, os FGH cultivados sobre ColB-3D e MCS-3D demonstraram, em todos os períodos experimentais, fenótipo fusiforme, alongado ou cilíndrico com menor quantidade de projeções do que observado no substrato 2D, com algumas das células cultivadas sobre MCS-3D demonstrando aspecto estrelado. O ensaio de MTT revelou viabilidade celular significantemente superior no ColB-2D e MCS-3D do que no ColB-3D (p<0,001), em todos os períodos experimentais. Em 3 dias, foi observado maior número de FGH cultivados sobre ColB-2D seguido por MCS-3D e ColB-3D respectivamente, havendo diferença estatística entre ColB-2D e MCS-3D (p<0,001) e entre ColB-2D e ColB-3D (p<0,001). No sétimo dia houve redução no número de células no ColB-2D e aumento na MCS-3D e ColB-3D, em que o número de células foi significantemente superior no ColB-2d com relação a MCS-3D (p=0,002). Ao final do período experimental, foi observado aumento no número de células em todos os grupos experimentais, sendo o número de células, mais uma vez, significantemente superior no ColB-2D do que na MCS-3D (p=0,002). Maior porcentagem de FGH no ciclo celular pode ser observado em ColB-2D seguido por ColB-3D e MCS-3D, respectivamente, em todos os períodos experimentais. Na MCS-3D praticamente não houve marcação por Ki-67 e o ColB-2D apresentou redução significativa de FGH ciclando entre o período de 3 e 10 dias (p=0,036). A expressão de proteínas não colágenas e citoesqueléticas foi similar em ambas as matrizes experimentais durante todo o estudo. Conclusão: Podemos concluir que a MCS-3D constitui uma matriz adequada para o cultivo de fibroblastos gengivais humanos, uma vez que, no interior da matriz, importantes propriedades foram verificadas, dentre as quais, morfologia, proliferação, e expressão de proteínas, semelhantes a uma matriz colágena tridimensional já consagrada na literatura. / Introduction: Gingival fibroblasts play a central role in oral soft tissue regeneration. Mucograft® (PCM-3D) is a new xenogeneic substitute that has been used in periodontics in techniques such as root coverage and increasing keratinized tissue. The aim of this investigation was to verify if PCM-3D is a suitable three-dimensional matrix though its in vitro response to the culture of HGF. Methods: Human gingival fibroblasts (HGF) culture was established by the explant technique of gingival connective tissues of three healthy patients. HGF were seeded on bidimensional bovine collagen matrix (BCol-2D), three-dimensional bovine collagen matrix (BCol- 3D) and three-dimensional porcine collagen matrix (PCM-3D). HGF were grown for up to 10 days. At 3, 7 and 10 days the following parameters were assessed: HGF number, morphology and distribution by direct fluorescence; cell viability by MTT; cell proliferation and expression of proteins of the extracellular matrix non-collagenous fibronectina and cytoeskeletic - proteins actin and tubulin by indirect immunofluorescence. Quantitative data were submitted to Friedman and Kruskal- Wallis test, and the last one was followed by Tukeys test. Results: Epifluorescence revealed that at 3 days HGF grown on BCol-2D were broader, more flattened and had more cell protrusions and stress fibers in all experimental times. On the other hand, HGF seeded on BCol-3D and PCM-3D displayed a spindle-shaped, elongated, or cylindrical phenotype with fewer protrusions as well as less total cell spread area than on the 2D matrix, with some cells on PCM-3D showing stellate appearance. MTT assay showed cell viability higher in cultures grown on BCol-2D and PCM-3D than in BCol-3D (p<0,001). At 3days a greater number of cells in BCol-2D samples followed by PCM-3D and BCol-3D, respectively, was observed with statistical difference between BCol-2D and PCM-3D and between (p<0,001). At 7 days, there was a decrease in cell number grown on BCol-2D and an increase in BCol-3D and PCM-3D, where the number of cells were significantly higher in BCol-2D in relation to PCM-3D (p=0.002). At the end of the experimental period, there was an increase in total cell number in all of the experimental groups, and it was again significantly greater in BCol-2D than in PCM (p=0.005). Between 3 and 10 days, there was an increase in total cell number in ColB-3D (p<0.001), which showed higher counts within 10 days. Higher percentage of HGF in the cell cycle could be seen in BCol-2D followed by BCol-3D and PCM-3D, respectively, in all of the experimental groups. In PCM-3D there was almost no expression of Ki-67. BCol-2D showed a decrease in HGF cycling between 3 and 10 days (p=0,036). The expression of non-collagenous and cytoskeletal proteins were similar in both matrices during all the period of the study. Conclusion: PCM-3D is suitable for HGF culture, since, within the matrix, important properties were found, among them, morphology, proliferation andprotein expression, similar to those observed in a 3D collagen matrix well established in the literature.

Made available in DSpace on 2014-06-11T19:31:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-09-16Bitstream added on 2014-06-13T19:20:13Z : No. of bitstreams: 1 filadelpho_al_dr_jabo.pdf: 1067546 bytes, checksum: 1640469b38a922f10c7c7709e1696f70 (MD5) / Este trabalho teve por objetivo avaliar a incorporação das matrizes liofilizadas de colágeno no dorso de ratos, testando principalmente a sua integridade e capacidade de servir como suporte para a migração de fibroblastos. Implantou-se no dorso torácico de 60 ratos albinos da variedade Wistar, machos e adultos, uma matriz de colágeno liofilizada. Os animais foram divididos em dois grupos de 30 animais, e cada grupo com três subgrupos de 10 animais. O primeiro grupo de animais, grupo AV, recebeu aplicação tópica de gel de Aloe vera e, no segundo grupo de animais, grupo SF, foi aplicado de modo similar, solução fisiológica a 0,9%. Os animais (10 ratos/subgrupo) foram submetidos à eutanásia aos 15, 30 e 45 dias de pós-operatório. A avaliação microscópica demonstrou aos 15 dias de pós-cirúrgico, nos dois grupos estudados, a ocorrência de uma reação inflamatória aguda, seguida de edema linfático e formação de tecido de granulação. Esse tecido era formado, principalmente, por leucócitos, que migraram para o local da inflamação. Linfócitos e macrófagos foram encontrados em maior número, no material tratado com Aloe vera. Aos 30 dias de pós-implante verificou-se um processo inflamatório moderado, nos dois grupos experimentais. Observou-se ainda, a presença de infiltrado inflamatório mononuclear discreto, associado à proliferação de fibroblastos. E aos 45 dias, no grupo AV, as matrizes encontravam-se menos espessas do que no grupo SF, havendo total bioincorporação dos moldes implantados. Frente aos resultados obtidos, é possível concluir que a matriz liofilizada de colágeno, é substituída por tecido local próximo ao normal e que a aplicação tópica de gel de Aloe vera acelerou o processo de cicatrização das lesões cirúrgicas e de incorporação das matrizes de colágeno liofilizadas / For this study aimed to evaluate the incorporation of lyophilized collagen matrix in the back of rats, mainly by testing their integrity and ability to serve as support for the migration of fibroblasts. Implanted in the thoracic dorsum of 60 albino rats of the Wistar variety, males and adults, a lyophilized collagen matrix. The animals were divided into 2 groups of 30 animals, and each group with 3 subgroups of 10 animals. The first group of animals, the AV group received topical application of Aloe vera gel and in the second group of animals the SF group was applied similarly saline 0.9%. Animals (10 rats / subgroup) were euthanized at 15, 30 and 45 days postoperatively. Microscopic evaluation demonstrated 15 days after surgery in both groups, the occurrence of an acute inflammatory reaction, followed by lymphoedema and formation of granulation tissue. This tissue was formed mainly by leukocytes, which migrated to the site of inflammation. Lymphocytes and macrophages were found in greater numbers, the material treated with Aloe vera. At 30 days post-implantation there was a moderate inflammatory process in both experimental groups. It was also observed, the presence of discrete mononuclear inflammatory infiltrate, associated with the proliferation of fibroblasts. And after 45 days in the AV group, matrixes were thinner than in the SF group, with total bioincorporation the molds in place. Given our results, we conclude that the freeze-dried collagen matrix is replaced by local tissue close to normal and that topical application of Aloe vera gel has accelerated the healing of surgical wounds and incorporation of freeze-dried collagen matrix

Resumo: Este trabalho teve por objetivo avaliar a incorporação das matrizes liofilizadas de colágeno no dorso de ratos, testando principalmente a sua integridade e capacidade de servir como suporte para a migração de fibroblastos. Implantou-se no dorso torácico de 60 ratos albinos da variedade Wistar, machos e adultos, uma matriz de colágeno liofilizada. Os animais foram divididos em dois grupos de 30 animais, e cada grupo com três subgrupos de 10 animais. O primeiro grupo de animais, grupo AV, recebeu aplicação tópica de gel de Aloe vera e, no segundo grupo de animais, grupo SF, foi aplicado de modo similar, solução fisiológica a 0,9%. Os animais (10 ratos/subgrupo) foram submetidos à eutanásia aos 15, 30 e 45 dias de pós-operatório. A avaliação microscópica demonstrou aos 15 dias de pós-cirúrgico, nos dois grupos estudados, a ocorrência de uma reação inflamatória aguda, seguida de edema linfático e formação de tecido de granulação. Esse tecido era formado, principalmente, por leucócitos, que migraram para o local da inflamação. Linfócitos e macrófagos foram encontrados em maior número, no material tratado com Aloe vera. Aos 30 dias de pós-implante verificou-se um processo inflamatório moderado, nos dois grupos experimentais. Observou-se ainda, a presença de infiltrado inflamatório mononuclear discreto, associado à proliferação de fibroblastos. E aos 45 dias, no grupo AV, as matrizes encontravam-se menos espessas do que no grupo SF, havendo total bioincorporação dos moldes implantados. Frente aos resultados obtidos, é possível concluir que a matriz liofilizada de colágeno, é substituída por tecido local próximo ao normal e que a aplicação tópica de gel de Aloe vera acelerou o processo de cicatrização das lesões cirúrgicas e de incorporação das matrizes de colágeno liofilizadas / Abstract: For this study aimed to evaluate the incorporation of lyophilized collagen matrix in the back of rats, mainly by testing their integrity and ability to serve as support for the migration of fibroblasts. Implanted in the thoracic dorsum of 60 albino rats of the Wistar variety, males and adults, a lyophilized collagen matrix. The animals were divided into 2 groups of 30 animals, and each group with 3 subgroups of 10 animals. The first group of animals, the AV group received topical application of Aloe vera gel and in the second group of animals the SF group was applied similarly saline 0.9%. Animals (10 rats / subgroup) were euthanized at 15, 30 and 45 days postoperatively. Microscopic evaluation demonstrated 15 days after surgery in both groups, the occurrence of an acute inflammatory reaction, followed by lymphoedema and formation of granulation tissue. This tissue was formed mainly by leukocytes, which migrated to the site of inflammation. Lymphocytes and macrophages were found in greater numbers, the material treated with Aloe vera. At 30 days post-implantation there was a moderate inflammatory process in both experimental groups. It was also observed, the presence of discrete mononuclear inflammatory infiltrate, associated with the proliferation of fibroblasts. And after 45 days in the AV group, matrixes were thinner than in the SF group, with total bioincorporation the molds in place. Given our results, we conclude that the freeze-dried collagen matrix is replaced by local tissue close to normal and that topical application of Aloe vera gel has accelerated the healing of surgical wounds and incorporation of freeze-dried collagen matrix / Orientador: Silvana Martinez Baraldi Artoni / Coorientador: José Wanderley Cattelan / Banca: Valéria Helena Alves Cagnon Quitete / Banca: Joaquim Coutinho Netto / Banca: José Jurandir Fagliari / Banca: Luiz Francisco Prata / Doutor

Introdução: Fibroblastos gengivais desempenham um importante papel na regeneração de tecidos moles de proteção. Mucograft® (MCS-3D) é um substituto xenógeno novo que vem sendo utilizado na periodontia em técnicas de recobrimento radicular e aumento de tecido queratinizado. O objetivo do presente estudo foi avaliar morfológica e imunohistoquimicamente, se a MCS-3D é uma matriz tridimensional adequada, por meio de sua resposta à cultura de fibroblastos gengivais. Material e Métodos: Fibroblastos gengivais humanos (FGH) foram obtidos pela técnica do explante a partir de tecido conjuntivo gengival de três indivíduos. Os FGH foram cultivados sobre colágeno bovino bidimensional (ColB-2D), colágeno bovino tridimensional (ColB-3D) e matriz colágena suína tridimensional (MCS-3D) por até 10 dias. Em 3, 7 e 10 dias, os seguintes parâmetros foram avaliados: número, morfologia e distribuição de FGH por fluorescência direta; viabilidade celular por MTT; proliferação celular e expressão de proteínas citoesqueléticas: actina e tubulina e proteínas da matriz extracelular não colágena: fibronectina imunofluorescência indireta. Os dados quantitativos foram submetidos aos testes estatísticos de Friedman para comparação intra e intergrupos para as variáveis da análise de expressão das proteínas: fibronectina, actina e tubulina. O teste de Kruskal-Wallis foi utilizado para comparações intra e intergrupos, seguido do teste de Tukey para as variáveis das análises de viabidlidade, proliferação e número total de células. Resultados: A epifluorescência revelou que em 3 dias os FGH apresentavam-se aderidos em todos os grupos experimentais. FGH cultivados sobre ColB-2D exibiram forma menos alongada, ampla e achatada com maior quantidade de projeções e numerosas fibras de estresse em 3, 7 e 10 dias. Por outro lado, os FGH cultivados sobre ColB-3D e MCS-3D demonstraram, em todos os períodos experimentais, fenótipo fusiforme, alongado ou cilíndrico com menor quantidade de projeções do que observado no substrato 2D, com algumas das células cultivadas sobre MCS-3D demonstrando aspecto estrelado. O ensaio de MTT revelou viabilidade celular significantemente superior no ColB-2D e MCS-3D do que no ColB-3D (p<0,001), em todos os períodos experimentais. Em 3 dias, foi observado maior número de FGH cultivados sobre ColB-2D seguido por MCS-3D e ColB-3D respectivamente, havendo diferença estatística entre ColB-2D e MCS-3D (p<0,001) e entre ColB-2D e ColB-3D (p<0,001). No sétimo dia houve redução no número de células no ColB-2D e aumento na MCS-3D e ColB-3D, em que o número de células foi significantemente superior no ColB-2d com relação a MCS-3D (p=0,002). Ao final do período experimental, foi observado aumento no número de células em todos os grupos experimentais, sendo o número de células, mais uma vez, significantemente superior no ColB-2D do que na MCS-3D (p=0,002). Maior porcentagem de FGH no ciclo celular pode ser observado em ColB-2D seguido por ColB-3D e MCS-3D, respectivamente, em todos os períodos experimentais. Na MCS-3D praticamente não houve marcação por Ki-67 e o ColB-2D apresentou redução significativa de FGH ciclando entre o período de 3 e 10 dias (p=0,036). A expressão de proteínas não colágenas e citoesqueléticas foi similar em ambas as matrizes experimentais durante todo o estudo. Conclusão: Podemos concluir que a MCS-3D constitui uma matriz adequada para o cultivo de fibroblastos gengivais humanos, uma vez que, no interior da matriz, importantes propriedades foram verificadas, dentre as quais, morfologia, proliferação, e expressão de proteínas, semelhantes a uma matriz colágena tridimensional já consagrada na literatura. / Introduction: Gingival fibroblasts play a central role in oral soft tissue regeneration. Mucograft® (PCM-3D) is a new xenogeneic substitute that has been used in periodontics in techniques such as root coverage and increasing keratinized tissue. The aim of this investigation was to verify if PCM-3D is a suitable three-dimensional matrix though its in vitro response to the culture of HGF. Methods: Human gingival fibroblasts (HGF) culture was established by the explant technique of gingival connective tissues of three healthy patients. HGF were seeded on bidimensional bovine collagen matrix (BCol-2D), three-dimensional bovine collagen matrix (BCol- 3D) and three-dimensional porcine collagen matrix (PCM-3D). HGF were grown for up to 10 days. At 3, 7 and 10 days the following parameters were assessed: HGF number, morphology and distribution by direct fluorescence; cell viability by MTT; cell proliferation and expression of proteins of the extracellular matrix non-collagenous fibronectina and cytoeskeletic - proteins actin and tubulin by indirect immunofluorescence. Quantitative data were submitted to Friedman and Kruskal- Wallis test, and the last one was followed by Tukeys test. Results: Epifluorescence revealed that at 3 days HGF grown on BCol-2D were broader, more flattened and had more cell protrusions and stress fibers in all experimental times. On the other hand, HGF seeded on BCol-3D and PCM-3D displayed a spindle-shaped, elongated, or cylindrical phenotype with fewer protrusions as well as less total cell spread area than on the 2D matrix, with some cells on PCM-3D showing stellate appearance. MTT assay showed cell viability higher in cultures grown on BCol-2D and PCM-3D than in BCol-3D (p<0,001). At 3days a greater number of cells in BCol-2D samples followed by PCM-3D and BCol-3D, respectively, was observed with statistical difference between BCol-2D and PCM-3D and between (p<0,001). At 7 days, there was a decrease in cell number grown on BCol-2D and an increase in BCol-3D and PCM-3D, where the number of cells were significantly higher in BCol-2D in relation to PCM-3D (p=0.002). At the end of the experimental period, there was an increase in total cell number in all of the experimental groups, and it was again significantly greater in BCol-2D than in PCM (p=0.005). Between 3 and 10 days, there was an increase in total cell number in ColB-3D (p<0.001), which showed higher counts within 10 days. Higher percentage of HGF in the cell cycle could be seen in BCol-2D followed by BCol-3D and PCM-3D, respectively, in all of the experimental groups. In PCM-3D there was almost no expression of Ki-67. BCol-2D showed a decrease in HGF cycling between 3 and 10 days (p=0,036). The expression of non-collagenous and cytoskeletal proteins were similar in both matrices during all the period of the study. Conclusion: PCM-3D is suitable for HGF culture, since, within the matrix, important properties were found, among them, morphology, proliferation andprotein expression, similar to those observed in a 3D collagen matrix well established in the literature.

La greffe de Membrane Amniotique Humaine (MAH) permet la cicatrisation des ulcères pré-perforants de la cornée et de sauver un nombre significatif d'yeux victimes de brûlures chimiques. La MAH est un matériel biologique, son utilisation pour le traitement des maladies de la surface oculaire donne de bons résultats en raison de sa capacité à réduire l'inflammation et promouvoir une épithélialisation rapide. Pour son utilisation en clinique, la MAH doit bien évidemment être stérile, mais aussi facile à transporter du centre préleveur au centre greffeur, et stockable longtemps et facilement. Actuellement en routine à la banque de tissus de Rouen, la membrane amniotique est séparée de l’amnios puis dénudée de leur couche spongieuse. Par la suite cette membrane est conservée par cryopréservation (congélation à –80°C) ce qui complique potentiellement l’acheminement des membranes. Par conséquent, dans le cadre de cette étude avec la Banque Normande de Cornées du CHU de Rouen nous avons développé la lyophilisation des MAH pour faciliter son utilisation et sa distribution. L’étude du mapping de la MAH permettra également de déterminer si le taux de facteurs de croissance est homogène dans la MAH ou s’il dépend sa distance par rapport au cordon ombilical. L’étude de la biocompatibilité in vivo d’un deuxième matériau composé de collagène nous permet également d’envisager une alternative pour une implantation du stroma. Nos analyses protéiques (ELISA et Label-free) des MAH la lyophilisées ne montrent pas de différence significative en terme de quantité et de qualité protéique. L’approche protéomique est complétée par l’analyse de la capacité des cellules épithéliales de cornée humaines (CEC) à se multiplier sur la membrane amniotique lyophilisée in vitro. Nous n’avons pas observé de différence entre la croissance des cellules épithéliales sur la MAH lyophilisée ou congelée. L’analyse du total de protéines extraites montre également que la lyophilisation ne dégrade pas les MAH au niveau protéique.Au niveau structurel les résultats de la microscopie électronique ont montré que la structure du stroma de la MAH est impactée par la lyophilisation. La greffe des MAH a été réalisée sur les ulcères cornéens chez le lapin. Au cours de l’expérimentation les lapins n’ont pas montré de signe d’inflammation, les analyses histologiques ont mis en évidence l’épithélialisation de la surface oculaire. Ce projet est en collaboration avec l‘association ophtalmo sans frontière pour qu’à terme le développement de l’utilisation clinique des MAHL répondant notamment à des besoins humanitaires (Cameroun). Notre étude de la cartographie de la MAH a également montré qu’une variabilité en termes de quantité de protéine existe entre les différents donneurs. Dans cette étude nous avons également montré que la couche spongieuse est une source de facteurs de croissance importante dans le processus de cicatrisation des ulcères cornéens. / The Human Amniotic Membrane (HAM) graft allows the healing of corneal ulcers and rescues a significant number of eyes with chemical burn. HAM is a biological material, its use for the treatment of ocular surface diseases gives good results because of its ability to reduce inflammation and promote rapid epithelialization. For its clinical use, the HAM must of course be sterile, but also easy to transport from the sampling center to the transplant center, and easily storable and for a long time. Currently on routine in the tissue bank of Rouen, the amniotic membrane is separated from the amnion and denuded of its spongy layer. Subsequently this membrane is stored by cryopreservation (freezing at -80 ° C) which potentially complicates the delivery of membranes. Consequently, as part of this study with the Banque Normande de Cornées of Rouen University Hospital, we have developed freeze-drying of HAM to facilitate its use and distribution. The HAM mapping study will also determine whether the level of growth factors is homogeneous in the HAM or whether it depends on its distance from the umbilical cord. The study of the in vivo biocompatibility of a second material composed of collagen also allows us to consider an alternative for implantation at the level of the stroma. Our protein analyzes (ELISA and Label-free) of freeze-dried HAM do not show any significant difference in terms of quantity and protein quality. The proteomic approach is complemented by the analysis of the ability of human corneal epithelial cells (CECs) to multiply on the freeze-dried amniotic membrane in vitro. We did not observe any difference between the epithelial cells growths on freeze-dried or frozen HAM. The analysis of the extracted protein total also shows that freeze-drying does not degrade the HAM at the protein level. At the structural level the electron microscopy results showed that the structure of the MAH stroma is impacted by freeze-drying. The MAH transplant performed on corneal ulcers in rabbits was performed. During the experiment the rabbits did not show any sign of inflammation, the histological analyzes highlighted the epithelialization of the ocular surface.This project is in collaboration with OSF association for the development of the clinical use of MAHL responding in particular to humanitarian needs (Cameroon). Our study of HAM mapping also showed that variability in terms of amount of protein exists between different donors. We have also shown that the spongy layer is an important source of important growth factor in the healing process of corneal ulcers.

Existem poucos ensaios clínicos aleatórios controlados mencionando o impacto da qualidade de vida (QV) e morbidade no tratamento de retrações gengivais múltiplas. O objetivo deste estudo foi comparar a QV e morbidade em dois grupos de pacientes: A. intervenção cirúrgica com enxerto de tecido conjuntivo subepitelial (ETCS) removido do palato; B. intervenção cirúrgica utilizando matriz de colágeno (MC - sem enxerto do palato) no tratamento de retrações gengivais múltiplas. Foi conduzido um ensaio clínico aleatório de boca dividida. Foram selecionados quinze pacientes com retrações gengivais múltiplas envolvendo caninos e pré-molares na maxila. Qualidade de vida foi avaliada através do questionário \"Oral Health Impact Profile\" (OHIP-14) através de regressão multinível. A morbidade incluiu dor, quantidade de medicamentos, capacidade de escovação e mastigação. Os parâmetros foram avaliados no Baseline, 7, 15, 30, e 180 dias após a intervenção. Resultados: OHIP-14 em 7 e 15 dias apresentou melhor QV no grupo MC comparado ao ETCS (p=0,002 e p=0,001 respectivamente). Correlações foram estabelecidas nos diferentes domínios do OHIP-14 após o tratamento com CM: dor física, desconforto psicológico, incapacidade física, incapacidade psicológica e desvantagem social (p<0,05). Houveram diferenças estatísticas significantes para dor pós-operatória e quantidade de medicamentos, favoráveis ao grupo MC (p<0,05). Conclusão: este estudo mostrou que o tratamento cirúrgico com a MC para retrações gengivais múltiplas Classe I de Miller foi positivamente associado ao impacto físico e psicológico na qualidade de vida dos pacientes, quando comparado ao ETCS removido do palato. / There are few randomized controlled clinical trials mentioning the oral health-related quality of life (OHRQoL) effect and morbidity in treatments of multiple gingival recession coverage. This study aims to compare the OHRQoL and morbidity in two groups of patients: A. surgical intervention with connective tissue graft (CTG) removed from the palate; B. surgical intervention using a collagen matrix (CM - no graft from the palate) in the treatment of multiple gingival recessions. Methods: A split-mouth randomized clinical trials was conducted. Fifteen patients with bilateral multiple recessions in maxillary canines and pre-molars were selected. Quality-of-life was assessed by the Oral Health Impact Profile-14 (OHIP-14) questionnaire through multinível regression. The morbidity included pain, quantify of medication, healing and inflammation/edema. Parameters were evaluated at baseline, 7, 15, 30, and 180 days. Results: OHIP-14 in 7 and 15 days showed better OHRQoL in CM group when compared with CTG (p=0,002 and p=0,001 respectively). Correlation was stablished in OHIP-14 subscales after treatment with CM: physical pain, psychological discomfort, physical disability, psychological disability, and handicap (p<0,05). There were statistically significant differences for pain and amount of medication, favoring CM group (p<0,05). Conclusion: This study shows that surgical treatment with CM for multiple gingival recessions Miller Class I was positively associated with psychological and physical impacts on patient\'s quality of life, when compared with CTG removed from the palate.