A Novel and Functional Artificial Bruch’s Membrane Made of Poly (ε-Caprolactone)/Gelatin for Retinal Pigment Epithelium Restoration  

作　　者：López Edith Silva-Bermudez Phaedra Gonzalez-Salinas Roberto Velasquillo Cristina Torres-Romero Abigail López-Colomé Ana María Pérez Armando Gina Prado-Prone Edgar Ramos-Martínez Iván Ramos-Martínez Quiroz-Mercado Hugo Hernández-Zimbrón Luis Fernando López Edith;Silva-Bermudez Phaedra;Gonzalez-Salinas Roberto;Velasquillo Cristina;Torres-Romero Abigail;López-Colomé Ana María;Pérez Armando;Gina Prado-Prone;Edgar Ramos-Martínez;Iván Ramos-Martínez;Quiroz-Mercado Hugo;Hernández-Zimbrón Luis Fernando(Instituto de Fisiologa Celular, Universidad Nacional Autnoma de Mxico, Ciudad de Mxico City, Mxico;Unidad de Ingeniería de Tejidos, Terapia Celular y Medicina Regenerativa, Instituto Nacional de Rehabilitación Luis Guiller-mo Ibarra Ibarra, Ciudad de México, México;Departamento de Investigacin, Asociacin Para Evitar la Ceguera, Ciudad de Mxico, Mxico;Subdireccin de Investigacin Tecnolgica, Instituto Nacional de Rehabilitacin Luis Guillermo Ibarra Ibarra, Ciudad de Mxico, Mxico;Departamento de Biologa Celular y Tisular, Facultad de Medicina, Universidad Nacional Autnoma de Mxico, Ciudad de Mxico, Mxico;Escuela de Ciencias, Universidad Autnoma Benito Jurez de Oaxaca, Oaxaca, Mxico;Departamento de Medicina y Zootecnia de Cerdos, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional utnoma de Mxico, Mxico;Laboratorio de Investigacion Interdisciplinaria, rea de Optometria, Escuela Nacional de Estudios Superiores Unidad Len, Universidad Nacional Autnoma de Mxico, Len Guanajuato, Mxico;Laboratorio Nacional de ptica de la Visin-Centro de Investigaciones en ptica, Len Guanajuato, Mxico)

机构地区：[1]Instituto de Fisiologa Celular, Universidad Nacional Autnoma de Mxico, Ciudad de Mxico City, Mxico [2]Unidad de Ingeniería de Tejidos, Terapia Celular y Medicina Regenerativa, Instituto Nacional de Rehabilitación Luis Guiller-mo Ibarra Ibarra, Ciudad de México, México [3]Departamento de Investigacin, Asociacin Para Evitar la Ceguera, Ciudad de Mxico, Mxico [4]Subdireccin de Investigacin Tecnolgica, Instituto Nacional de Rehabilitacin Luis Guillermo Ibarra Ibarra, Ciudad de Mxico, Mxico [5]Departamento de Biologa Celular y Tisular, Facultad de Medicina, Universidad Nacional Autnoma de Mxico, Ciudad de Mxico, Mxico [6]Escuela de Ciencias, Universidad Autnoma Benito Jurez de Oaxaca, Oaxaca, Mxico [7]Departamento de Medicina y Zootecnia de Cerdos, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional utnoma de Mxico, Mxico [8]Laboratorio de Investigacion Interdisciplinaria, rea de Optometria, Escuela Nacional de Estudios Superiores Unidad Len, Universidad Nacional Autnoma de Mxico, Len Guanajuato, Mxico [9]Laboratorio Nacional de ptica de la Visin-Centro de Investigaciones en ptica, Len Guanajuato, Mxico

出　　处：《Advances in Bioscience and Biotechnology》2024年第8期443-461,共19页生命科学与技术进展（英文）

摘　　要：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 thePurpose: 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

关 键 词：Artificial Bruch’s Membrane ELECTROSPINNING ARPE-19 Cells Age-Related Macular Degeneration 

分 类 号：R73[医药卫生—肿瘤]