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| Author(s): |
Karina Kamachi Kobashigawa
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Jaboticabal. 2018-02-21. |
| Institution: | Universidade Estadual Paulista (Unesp). Faculdade de Ciências Agrárias e Veterinárias. Jaboticabal |
| Defense date: | 2018-01-24 |
| Advisor: | José Luis Laus; Marcela Aldrovani |
| Abstract | |
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. (AU) | |
| FAPESP's process: | 14/18007-4 - Transplantation of amniotic membrane with limbal epithelial cells cultivated in sandwich configuration: clinical study and viability in rabbit corneas |
| Grantee: | Karina Kamachi Kobashigawa |
| Support Opportunities: | Scholarships in Brazil - Doctorate |