RESUMO
PURPOSE: Dysfunction of the retinal pigment epithelium (RPE) causes numerous forms of retinal degeneration. RPE replacement is a modern option to save vision. We aimed to test the results of transplanting cultured RPEs on biocompatible membranes. METHODS: We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse on two types of membranes: commercial polyester scaffolds Transwell (Corning Inc., Kenneburg, ME, USA) with 0.4 µm pore size and prepared Poly (L-lactide-co-DL-lactide) (PDLLA) nanofibrous membranes with an average pore size of 0.4 µm. RESULTS: Five types of assays were used for the analysis: immunocytochemistry (ICC), phagocytosis assay, Western blotting, real-time qPCR (RT-qPCR) and electron microscopy. RT-qPCR demonstrated that RPEs cultured on nanofibrous membranes have higher expressions of BEST1 (bestrophin 1), RLBP1 (retinaldehyde-binding protein 1), RPE65 (retinal pigment epithelium-specific 65 kDa protein), PAX6 (transcription factor PAX6), SOX9 (transcription factor SOX9), DCT (dopachrome tautomerase) and MITF (microphthalmia-associated transcription factor). ICC of the RPEs cultured on nanofibrous membranes showed more intensive staining of markers such as BEST1, MCT1 (monocarboxylate transporter 1), Na+ /K+ ATPase, RPE65 and acetylated tubulin in comparison with commercial ones. Additionally, the absence of α-SMA proved the stability of the RPE polarization state and the absence of epithelial-to-mesenchymal transition. RPE possessed high phagocytic activity. Electron microscopy of both membranes confirmed a confluent layer of RPE cells and their genuine morphological structure, which was comparable to native RPEs. CONCLUSIONS: Retinal pigment epitheliums cultured on polylactide nanofibrous membranes improved the final quality of the cell product by having better maturation and long-term survival of the RPE monolayer compared to those cultured on commercial polyester scaffolds. PDLLA-cultured RPEs are a plausible source for the replacement of non-functioning RPEs during cell therapy.
Assuntos
Nanofibras , Degeneração Retiniana , Animais , Bestrofinas/metabolismo , Células Cultivadas , Nanofibras/química , Poliésteres/metabolismo , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , SuínosRESUMO
PURPOSE: The development of primary human retinal pigmented epithelium (hRPE) for clinical transplantation purposes on biodegradable scaffolds is indispensable. We hereby report the results of the subretinal implantation of hRPE cells on nanofibrous membranes in minipigs. METHODS: The hRPEs were collected from human cadaver donor eyes and cultivated on ultrathin nanofibrous carriers prepared via the electrospinning of poly(L-lactide-co-DL-lactide) (PDLLA). "Libechov" minipigs (12-36 months old) were used in the study, supported by preoperative tacrolimus immunosuppressive therapy. The subretinal implantation of the hRPE-nanofibrous carrier was conducted using general anesthesia via a custom-made injector during standard three-port 23-gauge vitrectomy, followed by silicone oil endotamponade. The observational period lasted 1, 2, 6 and 8 weeks, and included in vivo optical coherence tomography (OCT) of the retina, as well as post mortem immunohistochemistry using the following antibodies: HNAA and STEM121 (human cell markers); Bestrophin and CRALBP (hRPE cell markers); peanut agglutining (PNA) (cone photoreceptor marker); PKCα (rod bipolar marker); Vimentin, GFAP (macroglial markers); and Iba1 (microglial marker). RESULTS: The hRPEs assumed cobblestone morphology, persistent pigmentation and measurable trans-epithelial electrical resistance on the nanofibrous PDLLA carrier. The surgical delivery of the implants in the subretinal space of the immunosuppressed minipigs was successfully achieved and monitored by fundus imaging and OCT. The implanted hRPEs were positive for HNAA and STEM121 and were located between the minipig's neuroretina and RPE layers at week 2 post-implantation, which was gradually attenuated until week 8. The neuroretina over the implants showed rosette or hypertrophic reaction at week 6. The implanted cells expressed the typical RPE marker bestrophin throughout the whole observation period, and a gradual diminishing of the CRALBP expression in the area of implantation at week 8 post-implantation was observed. The transplanted hRPEs appeared not to form a confluent layer and were less capable of keeping the inner and outer retinal segments intact. The cone photoreceptors adjacent to the implant scaffold were unchanged initially, but underwent a gradual change in structure after hRPE implantation; the retina above and below the implant appeared relatively healthy. The glial reaction of the transplanted and host retina showed Vimentin and GFAP positivity from week 1 onward. Microglial activation appeared in the retinal area of the transplant early after the surgery, which seemed to move into the transplant area over time. CONCLUSIONS: The differentiated hRPEs can serve as an alternative cell source for RPE replacement in animal studies. These cells can be cultivated on nanofibrous PDLLA and implanted subretinally into minipigs using standard 23-gauge vitrectomy and implantation injector. The hRPE-laden scaffolds demonstrated relatively good incorporation into the host retina over an eight-week observation period, with some indication of a gliotic scar formation, and a likely neuroinflammatory response in the transplanted area despite the use of immunosuppression.
RESUMO
Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.
Assuntos
Epitélio Pigmentado da Retina , Vitrectomia , Humanos , Animais , Suínos , Porco Miniatura , Cuidados Pós-Operatórios , Vitrectomia/métodos , Epitélio Pigmentado da Retina/cirurgia , Retina/cirurgiaRESUMO
In this study, we propose substrate-independent modification for creating a protein-repellent surface based on dopamine-melanin anchoring layer used for subsequent binding of poly(ethylene oxide) (PEO) from melt. We verified that the dopamine-melanin layer can be formed on literally any substrate and could serve as the anchoring layer for subsequent grafting of PEO chains. Grafting of PEO from melt in a temperature range 70-110 °C produces densely packed PEO layers showing exceptionally low protein adsorption when exposed to the whole blood serum or plasma. The PEO layers prepared from melt at 110 °C retained the protein repellent properties for as long as 10 days after their exposure to physiological-like conditions. The PEO-dopamine-melanin modification represents a simple and universal surface modification method for the preparation of protein repellent surfaces that could serve as a nonfouling background in various applications, such as optical biosensors and tissue engineering.
Assuntos
Materiais Revestidos Biocompatíveis/síntese química , Melaninas/química , Polietilenoglicóis/química , Engenharia Tecidual/métodos , Adsorção , Animais , Técnicas Biossensoriais/métodos , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Bovinos , Cromatografia Líquida de Alta Pressão , Materiais Revestidos Biocompatíveis/análise , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Ligação Proteica , Propriedades de SuperfícieRESUMO
Feasibility of using amphiphilic block copolymers composed of polylactide (PLA) and poly(ethylene oxide) (PEO) blocks for biomimetic surface modification of polylactide-based biomaterials for tissue engineering was investigated. PEO-b-PLA copolymers were deposited on the PLA surface from a solution in PEO-selective solvent. Copolymers with a neutral omega-methoxy end group of the PEO block (mPEO-b-PLA) were used to provide hydrophilic surface of PLLA, which exhibited suppressed nonspecific protein adsorption. Their analogues, containing biotin group at the end of PEO block (bPEO-b-PLA), were used as a model of functional copolymers, carrying a biomimetic group, for example, a cell-adhesion fibronectine-derived peptide sequence. The surface topography of functional groups on the modified surface and their accessibility for interaction with a protein receptor was investigated, taking advantage of specific biotin-avidin interaction, on surfaces modified with a combination of mPEO-b-PLA and bPEO-b-PLA copolymers. The accessibility of model biotin groups for interaction with their protein counterpart was proven through visualization of avidin or avidin-labeled nanospheres with atomic force microscopy.
Assuntos
Materiais Biocompatíveis/química , Poliésteres/química , Polietilenoglicóis/química , Polímeros/química , Espectroscopia de Ressonância Magnética , Microscopia de Força Atômica , Polímeros/síntese química , Propriedades de SuperfícieRESUMO
INTRODUCTION: Combinatory strategies using pharmacology and stem cell therapy have emerged due to their potential in the treatment of retinal pigment epithelium (RPE) cell related diseases, and a variety of different stem cell sources have been evaluated both in animal models and in humans. RPE cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (hiPSCs) are already in clinical trials, holding great promise for the treatment of age-related macular disease (AMD) and hereditary RPE-related retinal dystrophies. Highly efficient protocol for RPE generations have been developed, but they are still time-consuming and laborious. Areas covered: The authors review RPE related diseases, as well as the known functions of RPE cells in retinal homeostasis. The authors also discuss small molecules that target RPE in vivo as well as in vitro to aid RPE differentiation from pluripotent stem cells clinically. The authors base this review on literature searches performed through PubMed. Expert opinion: Using high-throughput systems, technology will provide the possibility of identifying and optimizing molecules/drugs that could lead to faster and simpler protocols for RPE differentiation. This could be crucial in moving forward to create safer and more efficient RPE-based personalized therapies.
Assuntos
Degeneração Macular/terapia , Doenças Retinianas/terapia , Epitélio Pigmentado da Retina/citologia , Animais , Diferenciação Celular/fisiologia , Terapia Combinada , Ensaios de Triagem em Larga Escala , Humanos , Degeneração Macular/fisiopatologia , Células-Tronco Pluripotentes/citologia , Doenças Retinianas/fisiopatologia , Transplante de Células-Tronco/métodosRESUMO
In this study, the adsorption of amphiphilic poly(ethylene oxide)-block-polylactide (mPEO-PLA) copolymers from a selective solvent onto a polylactide surface was studied as a method of polylactide surface modification and its effect on nonspecific protein adsorption was evaluated. A series of well defined mPEO-PLA copolymers was prepared to investigate the effect of copolymer composition on the resulting PEO chain density and on the surface resistance to protein adsorption. The copolymers contained PEO blocks with molecular weights ranging between 5600 and 23,800 and with 16-47 wt% of PLA. The adsorption of both the copolymers and bovine serum albumin was quantified by attenuated total reflection FTIR spectroscopy (ATR-FTIR). In addition to the adsorbed copolymer amount, its actual composition was determined. The PEO chain density on the surface was found to decrease with the molecular weight of the PEO block and to increase with the molecular weight of the PLA block. The adsorbed copolymers displayed the ability to reduce protein adsorption. The maximum reduction within the tested series (by 80%) was achieved with the copolymer containing PEO of MW 5600 and a PLA block of the same MW.
Assuntos
Poliésteres/química , Polietilenoglicóis/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Adsorção , Soroalbumina Bovina/químicaRESUMO
Tissue inhibitors of metalloproteinases (TIMPs) are the major endogenous regulators of metalloproteinase activity in tissues. TIMPs are able to inhibit activity of all known matrix metalloproteinases (MMPs) and thus participate in controlling extracellular matrix synthesis and degradation. We showed previously elevated expressions of MMPs in the rabbit corneal epithelium upon UVB exposure and suggested that these enzymes might be involved in corneal destruction caused by excessive proteolysis. The aim of this study was to investigate TIMPs in the corneal epithelium after UV irradiation using immunohistochemical and biochemical methods. We found that as compared to control rabbit corneas where relatively high levels of TIMPs were present in the epithelium, repeated irradiation of the cornea with UVB rays (not with UVA rays of similar doses) significantly decreased TIMPs in corneal epithelial cells. The results of this study point to the suggestion that the decrease in TIMPs in the corneal epithelium after UVB irradiation contributes to increased proteolytic activity of MMPs in UVB-irradiated corneal epithelium found previously.
Assuntos
Epitélio Corneano/efeitos da radiação , Metaloproteinases da Matriz/metabolismo , Raios Ultravioleta , Animais , Epitélio Corneano/enzimologia , Imuno-Histoquímica , Metaloproteinases da Matriz/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
We report on the design and fabrication of a frame-supported nanofibrous membrane for the transplantation of retinal pigment epithelial (RPE) cells, which is a promising therapeutic option for the treatment of degenerative retinal disorders. The membranous cell carrier prepared from 640 nm-thick poly(DL-lactide) fibres uniquely combines high porosity, large pore size and low thickness, to maximize the nutrient supply to the transplanted cells in the subretinal space and thus to enhance the therapeutic effect of the transplantation. The carrier was prepared by electrospinning, which made it easy to embed a 95 µm-thick circular supporting frame 2 mm in diameter. Implantations into enucleated porcine eyes showed that the frame enabled the ultrathin membrane to be handled without irreversible folding, and allowed the membrane to regain its flat shape when inserted into the subretinal space. We further demonstrated that the minimum membrane thickness compatible with the surgical procedure and instrumentation employed here was as low as 4 µm. Primary porcine RPE cells cultivated on the membranes formed a confluent monolayer, expressed RPE-specific differentiation markers and showed transepithelial resistance close to that of the native RPE. Most importantly, the majority of the RPE cells transplanted into the subretinal space remained viable. The ultrathin, highly porous, and surgically convenient cell carrier presented here has the potential to improve the integration and the functionality of transplanted RPE cells.
Assuntos
Galvanoplastia/métodos , Membranas Artificiais , Nanofibras/química , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/transplante , Alicerces Teciduais , Animais , Proliferação de Células , Sobrevivência Celular , Transplante de Células/instrumentação , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/transplante , Desenho de Equipamento , Análise de Falha de Equipamento , Nanofibras/ultraestrutura , Polímeros/química , Porosidade , Impressão Tridimensional , SuínosRESUMO
Amphiphilic block copolymers are used to create bioactive surfaces on biodegradable polymer scaffolds for tissue engineering. Cell-selective biomaterials can be prepared using copolymers containing peptide sequences derived from extracellular-matrix proteins (ECM). Here we discuss alternative ways for preparation of amphiphilic block copolymers composed of hydrophobic polylactide (PLA) and hydrophilic poly(ethylene oxide) (PEO) blocks with cell-adhesion peptide sequences. Copolymers PLA-b-PEO were prepared by a living polymerisation of lactide in dioxane with tin(II)2-ethylhexanoate as a catalyst. The following approaches for incorporation of peptides into copolymers were elaborated. (a) First, a side-chain protected Gly-Arg-Gly-Asp-Ser-Gly (GRGDSG) peptide was prepared by solid-phase peptide synthesis (SPPS) and then coupled with delta-hydroxy-Z-amino-PEO in solution. In the second step, the PLA block was grafted to it via a controlled polymerisation of lactide initiated by the hydroxy end-groups of PEO in the side-chain-protected GRGDSG-PEO. Deprotection of the peptide yielded a GRGDSG-b-PEO-b-PLA copolymer, with the peptide attached through its C-end. (b) A protected GRGDSG peptide was built up on a polymer resin and coupled with Z-carboxy-PEO using a solid-phase approach. After cleavage of the delta-hydroxy-PEO-GRGDSG copolymer from the resin, polymerisation of lactide followed by deprotection of the peptide yielded a PLA-b-PEO-b-GRGDSG block copolymer, in which the peptide is linked through its N-terminus.
Assuntos
Biopolímeros , Moléculas de Adesão Celular/química , Adesão Celular , Peptídeos/química , Biodegradação AmbientalRESUMO
A simple, versatile, protein-repulsive, substrate-independent biomimetic surface modification is presented that is based on the creation of a PEO brush on a polydopamine anchoring layer and its capacity for selective follow-up modifications with various ligands using a copper-catalyzed alkyne-azide cycloaddition reaction. The desired surface concentration of peptide biomimetic ligands can be controlled by adjusting the peptide concentration in the reaction mixture, then measuring the activity of (125)I-radiolabeled peptides that are immobilized on the substrates. The performance of the prepared substrates is tested in cell cultures with MEF cells and a human ECC line.
Assuntos
Biomimética , Células Cultivadas , Ciclização , Humanos , Propriedades de SuperfícieRESUMO
Electrospun polymeric nanofiber materials doped with 5,10,15,20-tetraphenylporphyrin (TPP) photosensitizer were prepared from four different polymers and were characterized with microscopic methods, steady-state, and time-resolved fluorescence and absorption spectroscopy. The polymers used included polyurethane Larithane™ (PUR), polystyrene (PS), polycaprolactone (PCL), and polyamide 6 (PA6). The antibacterial activity of all nanofiber materials against E. coli was activated by visible light and it was dependent on oxygen permeability/diffusion coefficients and the diameter of the polymeric nanofibers. This activity is based on oxidation ability of singlet oxygen O2(¹Δ(g)) that is generated upon irradiation. All tested nanofiber materials exhibited prolonged antibacterial properties, even in the dark after long-duration irradiation. The post-irradiation effect was explained by the photogeneration of H2O2, which provided the material with long-lasting antibacterial properties.
Assuntos
Antibacterianos/química , Luz , Nanofibras/química , Fármacos Fotossensibilizantes/química , Porfirinas/química , Antibacterianos/farmacologia , Caprolactama/análogos & derivados , Caprolactama/química , Escherichia coli/efeitos dos fármacos , Peróxido de Hidrogênio/química , Teste de Materiais , Oxidantes/química , Poliésteres/química , Polímeros/química , Poliestirenos/química , Poliuretanos/química , Oxigênio Singlete/químicaRESUMO
Label-free monitoring of biomolecular interactions has become of key importance for the emerging proteomics field. Monitoring real time interaction kinetics and high throughput screening of complex samples is of major importance for a variety of applications. We previously reported the use of Silicon-on-Insulator photonics microring resonators for cheap disposable biosensors on chip. Silicon photonics is a platform for micro- and nanoscale integrated devices that can be fabricated at extremely low cost, with standard CMOS processing facilities. Incorporation of a hydrophilic heterobifunctional polymer coating on the silicon chips largely improved the system's response to non-specific binding. We report the chemical coating procedure, the chemical surface characterization and optical measurements for both specific and non-specific interactions. Two heterobifunctional polymer coatings were investigated, alpha-sulfanyl-omega-carboxy-poly(ethylene glycol) and monoprotected diamino-poly(ethylene glycol). Homogenous coatings with thicknesses of 2.3 and 2.5 nm were obtained, corresponding to a surface loading of 99 pm/cm(2) carboxy- and 97 pm/cm(2) aminogroups, respectively. The polymer coated sensor with covalently bound biotin receptor molecules showed very low response to Bovine Serum Albumin (BSA) up to 1 mg/ml in contrast to a high response to avidin with much lower concentrations (2, 10, 87.5 and 175 microg/ml). By extrapolation the detection limit is about 10 ng/ml or 0.37 fg avidin mass. Comparison with the values reported for standard silanization confirms the polymer coating does not deteriorate the system's limit of detection. This makes the optical biosensor chip suitable to be integrated in a microflow system for commercial label-free biosensors and for lab-on-a-chip applications.