Transplante de membrana amniótica com células epiteliais limbais cultivadas em sanduíche: estudos clínicos e de viabilidade em córneas de coelhos
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Data
2018-01-24
Autores
Kobashigawa, Karina Kamachi [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Visando ao estudo de técnicas para a expansão “ex-vivo” de células destinadas à reconstrução de superfícies oculares com deficiência de células tronco limbais (LSCD), explantes superficiais obtidos do limbo de coelhos foram cultivados sobre membrana amniótica (MA) humana. Dois grupos, diferindo quanto à configuração do sistema de cultivo celular, foram concebidos: G-mono, contendo células epiteliais expandidas sobre a face de uma camada de MA (sistema de cultivo bidimensional), e G-Sand, composto por células “ensanduichadas” entre duas MAs (sistema de cultivo tridimensional). Seis culturas celulares de cada grupo foram avaliadas por imuno-histoquímica quanto à presença de células progenitoras ou indiferenciadas e a de células em proliferação (avaliação pré-transplante), enquanto 21 foram transplantadas para olhos de coelhos com LSCD (n=10). Os olhos receptores de células cultivadas foram clinicamente avaliados, por até 63 dias. A terapia limbal adotada na pesquisa foi autógena. Após avaliações clínicas, os coelhos foram aleatoriamente distribuídos em parcelas e submetidos à eutanásia, para colheita de córneas (dias 14 e 63 após o transplante) que foram avaliadas quanto à morfometria tecidual e à expressão quali-quantitativa de imunomarcadores de indiferenciação celular (fator de transcrição nuclear delta p63), de proliferação celular (antígeno nuclear da proliferação celular, PCNA) e de apoptose (teste de TUNEL). Diferenças com P < 0.05 foram consideradas significativas. As culturas do G-Sand apresentaram menos células imunopositivas para delta p63 e mais células positivas para PCNA, do que as culturas do G-mono. Relativamente ao tratamento da LSCD, os resultados mostraram que o protocolo adotado em G-Sand induziu neovascularização corneal menos acentuada e houve redução importante das áreas corneais ulceradas. Não se encontraram diferenças em relação à opacidade corneal entre os grupos (P > 0,05). As córneas do G-mono apresentaram menos células progenitoras do que as do G-Sand. A proliferação celular não diferiu entre as córneas do G-Sand e as do G-mono. Células apoptóticas não foram detectadas pelo teste de TUNEL. Os resultados do presente estudo sugerem que modificações na configuração do sistema de cultivo, de bi- para tridimensional, empregando-se MA como subtstrato, elevaram a proliferação celular, mas ensejaram perda de células progenitoras intensamente imunomarcadas para delta p63. Ademais, que elas não são decisivas para o desfecho terapêutico, pois os achados clínicos do G-Sand foram equiparáveis aos do G-mono. Única vantagem do G-Sand é que ele foi associado com maior quantidade de células progenitoras na córnea, o que pode influenciar a viabilidade ou a sobrevida dos transplantes, a longo prazo.
In order to study techniques for the ex vivo expansion of cells for the reconstruction ocular surfaces with limbal stem cell deficiency (LSCD), superficial explants obtained from rabbit limbus were cultured on human amniotic membrane (MA). Two groups, differing in the configuration of the cell culture system, were designed: G-mono, containing expanded epithelial cells on an MA layer (two-dimensional culture system), and G-Sand, composed of cells "sandwiched" between two MAs (three-dimensional culture system). Six cell cultures of each group were processed by immunohistochemistry to identify the presence of progenitor or undifferentiated cells and for proliferating cells (pre-transplant evaluation), and 21 constructs were transplanted onto rabbit eyes with LSCD. The limbal therapy adopted in the research was autogenous and the transplanted eyes were clinically evaluated for up to 63 days. After clinical evaluations, the rabbits were randomly distributed into subgroups and submitted to euthanasia, to harvest corneas (days 14 and 63 post-transplant) that were evaluated for tissue morphometry and the qualitative-quantitative expression of imunnomarkers of indiferentiated cells (delta-p63 transcriptional factor), proliferative cells (proliferating cell nuclear antigen, PCNA) and apoptosis (TUNEL assay). Differences with P <0.05 were considered significant. G-Sand cultures showed fewer cells immunopositives to delta p63 and more PCNA-positive cells than did G-mono cultures. Regarding the treatment of LSCD, the results showed that the protocol adopted in G-Sand induced a less pronounced corneal neovascularization and there was a significant reduction of the ulcerated corneal areas. No differences were found in corneal opacity between G-mono and G-Sand (P > 0.05). The G-mono corneas had fewer progenitor cells than the G-Sand corneas. Cell proliferation did not differ between G-Sand corneas and G-mono corneas. Apoptotic cells were not detected by the TUNEL assay. The results of the present study suggest that modifying the configuration of the culture system from bi- to three-dimensional, with MA as substrate, does not cause increase in cell proliferation but causes loss of progenitor cells highly immunolabelled for delta p63. In addition, the results also suggest that modification in the configuration of the culture system is not decisive for the therapeutic outcome, since the clinical findings of G-Sand were comparable to those of G-mono. The only advantage of G-Sand is that it has been associated with a greater amount of progenitor cells in the cornea, which may influence the viability or survival of transplants in the long term.
In order to study techniques for the ex vivo expansion of cells for the reconstruction ocular surfaces with limbal stem cell deficiency (LSCD), superficial explants obtained from rabbit limbus were cultured on human amniotic membrane (MA). Two groups, differing in the configuration of the cell culture system, were designed: G-mono, containing expanded epithelial cells on an MA layer (two-dimensional culture system), and G-Sand, composed of cells "sandwiched" between two MAs (three-dimensional culture system). Six cell cultures of each group were processed by immunohistochemistry to identify the presence of progenitor or undifferentiated cells and for proliferating cells (pre-transplant evaluation), and 21 constructs were transplanted onto rabbit eyes with LSCD. The limbal therapy adopted in the research was autogenous and the transplanted eyes were clinically evaluated for up to 63 days. After clinical evaluations, the rabbits were randomly distributed into subgroups and submitted to euthanasia, to harvest corneas (days 14 and 63 post-transplant) that were evaluated for tissue morphometry and the qualitative-quantitative expression of imunnomarkers of indiferentiated cells (delta-p63 transcriptional factor), proliferative cells (proliferating cell nuclear antigen, PCNA) and apoptosis (TUNEL assay). Differences with P <0.05 were considered significant. G-Sand cultures showed fewer cells immunopositives to delta p63 and more PCNA-positive cells than did G-mono cultures. Regarding the treatment of LSCD, the results showed that the protocol adopted in G-Sand induced a less pronounced corneal neovascularization and there was a significant reduction of the ulcerated corneal areas. No differences were found in corneal opacity between G-mono and G-Sand (P > 0.05). The G-mono corneas had fewer progenitor cells than the G-Sand corneas. Cell proliferation did not differ between G-Sand corneas and G-mono corneas. Apoptotic cells were not detected by the TUNEL assay. The results of the present study suggest that modifying the configuration of the culture system from bi- to three-dimensional, with MA as substrate, does not cause increase in cell proliferation but causes loss of progenitor cells highly immunolabelled for delta p63. In addition, the results also suggest that modification in the configuration of the culture system is not decisive for the therapeutic outcome, since the clinical findings of G-Sand were comparable to those of G-mono. The only advantage of G-Sand is that it has been associated with a greater amount of progenitor cells in the cornea, which may influence the viability or survival of transplants in the long term.
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Palavras-chave
células tronco, membrana amniótica, reparação corneal, superfície ocular, terapia celular, stem cell, amniotic membrane, corneal repair, ocular surface, cell therapy