Purpose: The primary aim of this paper is to design, create, and improve functional and artificial Bruch’s membranes (BM) using bioengineering techniques, which can be applied in the treatment of maculopathies by sup...Purpose: The primary aim of this paper is to design, create, and improve functional and artificial Bruch’s membranes (BM) using bioengineering techniques, which can be applied in the treatment of maculopathies by supporting the growth and maintenance of retinal pigment epithelium (RPE) cells, thereby potentially enabling subretinal implantation in patients. Methods: We fabricated by electrospinning ultrathin 3D nanofibrous membranes from Polycaprolactone (PCL), and different concentrations of gelatin (5%, 15% and 30%). ARPE-19 cells were seeded onto these artificial membranes. MTT assays were performed in order to evaluate ARPE-19 cell viability and cytotoxicity. IF assays were realized to observe the ARPE-19 cells onto each membrane. Ultrastructure of the modified Bruch’s membrane and ARPE-19 morphology after 25 days of culture were studied with transmission and scanning electron microscopy. To evaluate expression changes in markers of ARPE-19 (RPE65 and ZO-1) qRT-PCR assays were realized. Data from three independent experiments were pooled and expressed as the mean SD. A confidence level of P 0.05 was considered to be statistically significant. Results ARPE-19 cells grew on PCL/Gelatin membranes mainly in PCL/30% gelatin combination, which had not cytotoxic effect. RPE65/cytokeratin-18/ and actin-positive ARPE-19 cells formed a correctly orientated monolayer of polygonal cells with morphological polarity. The apical cell surfaces exhibited abundant protruding microfolds. Moreover, zones of polygonal border look as if ARPE-19 cells fused among, suggesting the presence of tight junctions. The expression of RPE65 and ZO-1 was unchanged. Conclusions: PCL/30% Gelatine membranes may imitate the natural BM to such extent that they support RPE-cells and exhibited RPE-like morphology. The engineering of a human RPE monolayer with these artificial BM, emulating the in vivo retina, arouse their potential subretinal implantation in patients with wet age-related macular degeneration (AMD) where there is a rupture of the Bruch’s membrane.展开更多
文摘Purpose: The primary aim of this paper is to design, create, and improve functional and artificial Bruch’s membranes (BM) using bioengineering techniques, which can be applied in the treatment of maculopathies by supporting the growth and maintenance of retinal pigment epithelium (RPE) cells, thereby potentially enabling subretinal implantation in patients. Methods: We fabricated by electrospinning ultrathin 3D nanofibrous membranes from Polycaprolactone (PCL), and different concentrations of gelatin (5%, 15% and 30%). ARPE-19 cells were seeded onto these artificial membranes. MTT assays were performed in order to evaluate ARPE-19 cell viability and cytotoxicity. IF assays were realized to observe the ARPE-19 cells onto each membrane. Ultrastructure of the modified Bruch’s membrane and ARPE-19 morphology after 25 days of culture were studied with transmission and scanning electron microscopy. To evaluate expression changes in markers of ARPE-19 (RPE65 and ZO-1) qRT-PCR assays were realized. Data from three independent experiments were pooled and expressed as the mean SD. A confidence level of P 0.05 was considered to be statistically significant. Results ARPE-19 cells grew on PCL/Gelatin membranes mainly in PCL/30% gelatin combination, which had not cytotoxic effect. RPE65/cytokeratin-18/ and actin-positive ARPE-19 cells formed a correctly orientated monolayer of polygonal cells with morphological polarity. The apical cell surfaces exhibited abundant protruding microfolds. Moreover, zones of polygonal border look as if ARPE-19 cells fused among, suggesting the presence of tight junctions. The expression of RPE65 and ZO-1 was unchanged. Conclusions: PCL/30% Gelatine membranes may imitate the natural BM to such extent that they support RPE-cells and exhibited RPE-like morphology. The engineering of a human RPE monolayer with these artificial BM, emulating the in vivo retina, arouse their potential subretinal implantation in patients with wet age-related macular degeneration (AMD) where there is a rupture of the Bruch’s membrane.
