Purpose: To evaluate the ability of human immature dental pulp stem cells, which are mesenchymal stem cells of neural crest origin, to differentiate into the corneal epithelium for purposes of corneal transplantation and tissue engineering when cultured on de-epithelized amniotic membranes. Methods: We compared the immunophenotypes (ABCG2, K3/12, and vimentin) of cells grown on amniotic membranes or plastic surfaces under serum-free conditions or in culture media containing serum or serum replacement components. Results: Immature dental pulp stem cells grown on amniotic membranes under basal conditions are able to maintain their undifferentiated state. Our data also suggest that the culture medium used in the present work can modulate the expression of immature dental pulp stem cell markers, thus inducing epithelial differentiation of these cells in vitro. Conclusions: Our results suggest that the amniotic membrane is a good choice for the growth and transplantation of mesenchymal stem cells, particularly immature dental pulp stem cells, in clinical ocular surface reconstruction.
ABSTRACT Objective: To evaluate the intra and inter variations of eye drops volume dispensed from bottles available on the market. Methods: Five bottles of lubricant eye drops were studied and nineteen volunteers participated in this study. The average mass from 20µl of eye drops was obtained using accuracy micropipette and balance, and used as standard for comparison with the mass of the drops obtained by the volunteers. Five drops of each vial were individually weighed with the tube perpendicular to the balance, using the first and second fingers of the right hand, so that the pressure was applied only in the middle of the flask. The experiments were performed in a room temperature (21±1°C). Results: All eye drops bottles showed a statistically significant variation on masses of the drops obtained by examiners when compared with the standard average weight of 0.0182±0,0014g, except when compared A with D eye drops, with no statistically significant variation. Conclusion: This study demonstrates the lack of uniformity of drops dispensed by eye drops bottles available in the market and its inadequacy to the real need, since the dispensed drops are larger than indicated.This fact becomes a problem when it comes to long treatment period, especially with expensive drops as indicated for glaucoma therapy. In this sense, the standardization of drops of eye drops is necessary.
OBJETIVO: Avaliar a eficácia e aspecto estrutural de células límbicas epiteliais humanas cultivadas sobre membrana amniótica (MA) com e sem epitélio. MÉTODOS: As culturas límbicas foram obtidas a partir de rima corneoescleral remanescentes de transplantes de córnea de 6 diferentes doadores. Cada explante foi cultivado em três diferentes grupos: MA desepitelizada por tripsina (Grupo 1), MA com epitélio íntegro (Grupo 2) e controle (Grupo 3). A migração epitelial foi avaliada por microscopia de contraste de fase. Após 15 dias, as células cultivadas sobre MA foram submetidas à microscopia eletrônica para avaliar migração e adesão epitelial. RESULTADOS: Todas as células do grupo controle cresceram até atingir confluência. Somente uma das culturas em membrana amniótica desepitelizada não apresentou crescimento epitelial. O crescimento de células epiteliais sobre membrana amniótica epitelizada foi observada em apenas uma cultura. CONCLUSÃO: Baseando-se nestes achados, o uso de membrana amniótica desepitelizada aparenta ser o melhor substrato para migração e adesão epitelial comparando com membrana amniótica epitelizada. Remover o epitélio da membrana amniótica demonstra ser um importante passo para estabelecer culturas de células sobre membrana amniótica.
ABSTRACT Purpose: To culture quiescent human keratocytes and evaluate the effects of ultraviolet light and riboflavin on human corneal keratocytes in vitro. Methods: Keratocytes were obtained from remaining corneoscleral ring donor corneas previously used in corneal transplant surgeries and cultured in DMEM/F12 with 2% FBS until confluence. Characterization of cultured cells was performed by immunofluorescence analysis for anti-cytokeratin-3, anti-Thy-1, anti-α-smooth muscle actin, and anti-lumican. Immunofluorescence was performed before and after treatment of cultured cells with either ultraviolet light or riboflavin. Corneal stromal cells were covered with collagen (200 µL or 500 µL) and 0.1% riboflavin, and then exposed to ultraviolet light at 370 nm for 30 minutes. After 24 hours, cytotoxicity was determined using MTT colorimetric assays, whereas cell viability was assessed using Hoechst 33342 and propidium iodide. Results: Cell cultures achieved confluence in approximately 20 days. Expression of the lumican was high, whereas no expression of CK3, Thy-1, and α-SMA was observed. After crosslinking, MTT colorimetric assays demonstrated a low toxicity rate, whereas Hoechst 33342/propidium iodide staining demonstrated a low rate of apoptosis and necrosis, respectively, in all collagen-treatment groups. Conclusion: Keratocytes can be successfully cultured in vitro and characterized by immunofluorescence using lumican. MTT colorimetric assays, and Hoechst 33342, and propidium iodide staining demonstrated a higher rate of cell death in cells cultured without collagen, indicating collagen protects keratocytes from the cytotoxic effects of ultraviolet light.
Objective: To evaluate the intra and inter variations of eye drops volume dispensed from bottles available on the market. Methods: Five bottles of lubricant eye drops were studied and nineteen volunteers participated in this study. The average mass from 20µl of eye drops was obtained using accuracy micropipette and balance, and used as standard for comparison with the mass of the drops obtained by the volunteers. Five drops of each vial were individually weighed with the tube perpendicular to the [...]
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