Bikuna-Izagirre, M. (María)
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- Technological advances in ocular trabecular meshwork in vitro models for glaucoma research(Wiley, 2022) Moreno-Montañes, J. (Javier); Aldazabal, J. (Javier); Extramiana, L. (Leire); Bikuna-Izagirre, M. (María); Paredes-Puente, J. (Jacobo); Carnero, E. (Elena)Glaucoma is the leading cause of irreversible blindness worldwide and ischaracterized by the progressive degeneration of the optic nerve. Intraocularpressure (IOP), which is considered to be the main risk factor for glaucomadevelopment, builds up in response to the resistance (resistance to what?) providedby the trabecular meshwork (TM) to aqueous humor (AH) outflow. Although theTMand its relationship to AH outflow have remained at the forefront of scientificinterest, researchers remain uncertain regarding which mechanisms drive thedeterioration of the TM. Current tissue‐engineering fabrication techniques havecome up with promising approaches to successfully recreate the TM. Nonetheless,more accurate models are needed to understand the factors that make glaucomaarise. In this review, we provide a chronological evaluation of the technologicalmilestones that have taken place in the field of glaucoma research, and we conduct acomprehensive comparison of available TM fabrication technologies. Additionally,we also discuss AH perfusion platforms, since they are essential for the validation ofthese scaffolds, as well as pressure–outflow relationship studies and the discovery ofnew IOP‐reduction therapies.
- Gelatin blends enhance performance of electrospun polymeric scaffolds in comparison to coating protocols.(MDPI, 2022-04) Aldazabal, J. (Javier); Bikuna-Izagirre, M. (María); Paredes-Puente, J. (Jacobo)The electrospinning of hybrid polymers is a versatile fabrication technique which takes advantage of the biological properties of natural polymers and the mechanical properties of synthetic polymers. However, the literature is scarce when it comes to comparisons of blends regarding coatings and the improvements offered thereby in terms of cellular performance. To address this, in the present study, nanofibrous electrospun scaffolds of polycaprolactone (PCL), their coating and their blend with gelatin were compared. The morphology of nanofibrous scaffolds was analyzed under field emission scanning electron microscopy (FE-SEM), indicating the influence of the presence of gelatin. The scaffolds were mechanically tested with tensile tests; PCL and PCL gelatin coated scaffolds showed higher elastic moduli than PCL/gelatin meshes. Viability of mouse embryonic fibroblasts (MEF) was evaluated by MTT assay, and cell proliferation on the scaffold was confirmed by fluorescence staining. The positive results of the MTT assay and cell growth indicated that the scaffolds of PCL/gelatin excelled in comparison to other scaffolds, and may serve as good candidates for tissue engineering applications.
- Nanofibrous PCL-based human trabecular meshwork for aqueous humor outflow studies(2023) Moreno-Montañes, J. (Javier); Aldazabal, J. (Javier); Extramiana, L. (Leire); Bikuna-Izagirre, M. (María); Paredes-Puente, J. (Jacobo); Carnero, E. (Elena)Primary open-angle glaucoma is characterized by the progressive degeneration of the optic nerve, with the high intraocular pressure (IOP) being one of the main risk factors. The human trabecular meshwork (HTM), specifically the juxtacanalicular tissue (JCT), is responsible for placing resistance to the aqueous humor (AH) outflow and the resulting IOP control. Currently, the lack of a proper in vitro JCT model and the complexity of three-dimensional models impede advances in understanding the relationship between AH outflow and HTM degeneration. Therefore, we design an in vitro JCT model using a polycaprolactone (PCL) nanofibrous scaffold, which supports cells to recapitulate the functional JCT morphology and allow the study of outflow physiology. Mechanical and morphological characterizations of the electrospun membranes were performed, and human trabecular meshwork cells were seeded over the scaffolds. The engineered JCT was characterized by scanning electron microscopy, quantitative real-time polymerase chain reaction, and immunochemistry assays staining HTM cell markers and proteins. A pressure-sensitive perfusion system was constructed and used for the investigation of the outflow facility of the polymeric scaffold treated with dexamethasone (a glucocorticoid) and netarsudil (a novel IOP lowering the rho inhibitor). Cells in the in vitro model exhibited an HTM-like morphology, expression of myocilin, fibronectin, and collagen IV, genetic expression, outflow characteristics, and drug responsiveness. Altogether, the present work develops an in vitro JCT model to better understand HTM cell biology and the relationship between the AH outflow and the HTM and allow further drug screening of pharmacological agents that affect the trabecular outflow facility.
- Artificial trabecular meshwork structure combining melt electrowriting and solution electrospinning(MDPI, 2024) Moreno-Montañes, J. (Javier); Aldazabal, J. (Javier); De-Juan-Pardo, E.M. (Elena M.); Bikuna-Izagirre, M. (María); Paredes-Puente, J. (Jacobo); Carnero, E. (Elena)The human trabecular meshwork (HTM) is responsible for regulating intraocular pressure (IOP) by means of gradient porosity. Changes in its physical properties, like increases in stiffness or alterations in the extracellular matrix (ECM), are associated with increases in the IOP, which is the primary cause of glaucoma. The complexity of its structure limits the engineered models to one-layered and simple approaches, which do not accurately replicate the biological and physiological cues related to glaucoma. Here, a combination of melt electrowriting (MEW) and solution electrospinning (SE) is explored as a biofabrication technique used to produce a gradient porous scaffold that mimics the multi-layered structure of the native HTM. Polycaprolactone (PCL) constructs with a height of 20-710 mu m and fiber diameters of 0.7-37.5 mu m were fabricated. After mechanical characterization, primary human trabecular meshwork cells (HTMCs) were seeded over the scaffolds within the subsequent 14-21 days. In order to validate the system's responsiveness, cells were treated with dexamethasone (Dex) and the rho inhibitor Netarsudil (Net). Scanning electron microscopy and immunochemistry staining were performed to evaluate the expected morphological changes caused by the drugs. Cells in the engineered membranes exhibited an HTMC-like morphology and a correct drug response. Although this work demonstrates the utility of combining MEW and SE in reconstructing complex morphological features like the HTM, new geometries and dimensions should be tested, and future works need to be directed towards perfusion studies.
- Technological advances in ocular trabecular meshwork in vitro models for glaucoma research.(Wiley, 2022) Moreno-Montañes, J. (Javier); Aldazabal, J. (Javier); Extramiana, L. (Leire); Bikuna-Izagirre, M. (María); Carnero, E. (Elena)Glaucoma is the leading cause of irreversible blindness worldwide and ischaracterized by the progressive degeneration of the optic nerve. Intraocularpressure (IOP), which is considered to be the main risk factor for glaucomadevelopment, builds up in response to the resistance (resistance to what?) providedby the trabecular meshwork (TM) to aqueous humor (AH) outflow. Although the TMand its relationship to AH outflow have remained at the forefront of scientificinterest, researchers remain uncertain regarding which mechanisms drive thedeterioration of the TM. Current tissue‐engineering fabrication techniques havecome up with promising approaches to successfully recreate the TM. Nonetheless,more accurate models are needed to understand the factors that make glaucomaarise. In this review, we provide a chronological evaluation of the technologicalmilestones that have taken place in the field of glaucoma research, and we conduct acomprehensive comparison of available TM fabrication technologies. Additionally,we also discuss AH perfusion platforms, since they are essential for the validation ofthese scaffolds, as well as pressure–outflow relationship studies and the discovery ofnew IOP‐reduction therapies.
- Mechanical and morphological modulation of electrospun polymeric scaffolds for tissue engineering applications(Servicio de Publicaciones. Universidad de Navarra, 2023-06) Bikuna-Izagirre, M. (María); Aldazabal, J. (Javier); Paredes-Puente, J. (Jacobo)Electrospinning technologies herald the arrival of a new era in which previously unthinkable scaffolds for tissue engineering applications will be solved efficiently. However, electrospinning techniques, like solution electrospinning and melt electrowriting are held down by fabrications parameters, technology limitations, and the application perse. The science of scaffolding fabrication seeks to mimic the extracellular matrix of a particular tissue in ways that mitigates the damage or enables its pathophysiological study. Thenceforce, scaffolds have the primordial role of not only supporting the cells, but to replicate as close as possible the native extracellular matrix, taking into consideration the biocompatibility, biodegradability, morphology and mechanical properties. The last two properties are pivotal in the scaffold ́s outcome, as cells communicate with the environment, and behave in response to external signals. In context of scaffolds ́ assembly, electrospinning fabrication parameters should be correctly modulated, to ensure an appropriate cellular environment. In this dissertation we attempt to tackle this concern relying on solution electrospinning and melt electrowriting techniques. As potential tissue engineering applications, the recreation of an artificial human trabecular meshwork and a skeletal muscle platform are developed. The mechanical and morphological requirements of each tissue are evaluated and fabrication parameters adapted. An in vitro human trabecular meshwork scaffold was developed and validated with human trabecular meshwork cells ́ behavioral studies. With the development of a perfusion bioreactor human trabecular meshwork cells react to medicaments inducing measurable pressure changes. Finally, an attempt for skeletal muscle platform was made. This first approach enabled us the optimization of the process for next attempts.