Paredes-Puente, J. (Jacobo)
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- 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.
- Development of an in vitro platform for the analysis of contractile and calcium dynamics in single human myotubes(Royal Soc. Chemistry, 2024) Marco-Moreno, P. (Pablo); Mosqueira-Martín, L. (Laura); González-Imaz, C. (Claudia); Rendon-Hinestroza, J.(Jorge); Vallejo-Illarramendi, A. (Ainara); Vesga-Castro, C. (Camila); Paredes-Puente, J. (Jacobo); Ubiria-Urkola, P. (Paul)In vitro myotube cultures are widely used as models for studying muscle pathophysiology, but their limited maturation and heterogeneity pose significant challenges for functional analyses. While they remain the gold standard for studying muscle function in vitro, myotube cultures do not fully recapitulate the complexity and native features of muscle fibers, which may compromise their ability to predict in vivo outcomes. To promote maturation and decrease heterogeneity, we have incorporated engineered structures into myotube cultures, based on a PDMS thin layer with micrometer-sized grooves (mu Grooves) placed over a glass substrate. Different sizes and shapes of mu Grooves were tested for their ability to promote alignment and fusion of myoblasts and enhance their differentiation into myotubes. A 24 hour electrical field stimulation protocol (4 V, 6 ms, 0.1 Hz) was used to further promote myotube maturation, after which several myotube features were assessed, including myotube alignment, width, fusion index, contractile function, and calcium handling. Our results indicate superior calcium and contractile performance in mu Grooved myotubes, particularly with the 100 mu m-width 700 mu m-long geometry (7 : 1). This platform generated homogeneous and isolated myotubes that reproduced native muscle features, such as excitation-contraction coupling and force-frequency responses. Overall, our 2D muscle platform enables robust high-content assays of calcium dynamics and contractile readouts with increased sensitivity and reproducibility compared to traditional myotube cultures, making it particularly suitable for screening therapeutic candidates for different muscle pathologies.
- Application of texture analysis methods for the characterization of cultured meat.(Nature, 2022-03) Cortizo-Lacalle, D. (Diego); Imaz, A.M. (Ane Miren); Paredes-Puente, J. (Jacobo)Mechanical characterization supposes a key step in the development of cultured meat to help mimicking the sensorial properties of already existing commercial products based on traditional meat. This work presents two well stablished methods that can help studying cultured meat mechanical characteristics: texture profile analysis (double compression test) and rheology. These techniques provide data about the elastic and viscous behaviour of the samples but also values about other texture characteristics such as springiness, cohesiveness, chewiness and resilience. In this work, we present a comparison of cultured meat-based samples with commercial of the shelf common meat products (sausage, turkey and chicken breast). Results show that both Young's and Shear modulus in the cultured meat samples can be compared to commercial products in order to understand its properties. The texture characteristics for the cultured meat studied, show values within the range of commercial products. These results demonstrate the applicability of this methodology for the adjustment of mechanical properties of cultured meat products.
- Contractile force assessment methods for in vitro skeletal muscle tissues.(eLIFE Sciences Publ. LTD., 2022) Vallejo-Illarramendi, A. (Ainara); Aldazabal, J. (Javier); Vesga-Castro, C. (Camila); Paredes-Puente, J. (Jacobo)Over the last few years, there has been growing interest in measuring the contractile force (CF) of engineered muscle tissues to evaluate their functionality. However, there are still no standards available for selecting the most suitable experimental platform, measuring system, culture protocol, or stimulation patterns. Consequently, the high variability of published data hinders any comparison between different studies. We have identified that cantilever deflection, post deflection, and force transducers are the most commonly used configurations for CF assessment in 2D and 3D models. Additionally, we have discussed the most relevant emerging technologies that would greatly complement CF evaluation with intracellular and localized analysis. This review provides a comprehensive analysis of the most significant advances in CF evaluation and its critical parameters. In order to compare contractile performance across experimental platforms, we have used the specific force (sF, kN/m2 ), CF normalized to the calculated cross-sectional area (CSA). However, this parameter presents a high variability throughout the different studies, which indicates the need to identify additional parameters and complementary analysis suitable for proper comparison. We propose that future contractility studies in skeletal muscle constructs report detailed information about construct size, contractile area, maturity level, sarcomere length, and, ideally, the tetanusto-twitch ratio. These studies will hopefully shed light on the relative impact of these variables on muscle force performance of engineered muscle constructs. Prospective advances in muscle tissue engineering, particularly in muscle disease models, will require a joint effort to develop standardized methodologies for assessing CF of engineered muscle tissues.
- 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.
- Three-dimensional bioprinting scaffolding for nasal cartilage defects: A systematic review.(Springer, 2021) Garcés, J.P. (Juan P.); Chiesa-Estomba, C.M. (Carlos M.); Delgado, A. (Alba); Hernaez-Moya, R. (Raquel); Aldazabal, J. (Javier); Rodino, C. (Claudia); González-Fernández, I. (Iago); Altuna, X. (Xabier); Izeta, A. (Ander); Aiastui, A. (Ana); Paredes-Puente, J. (Jacobo)In recent years, three-dimensional (3D)-printing of tissue-engineered cartilaginous scaffolds is intended to close the surgical gap and provide bio-printed tissue designed to fit the specific geometric and functional requirements of each cartilage defect, avoiding donor site morbidity and offering a personalizing therapy. METHODS: To investigate the role of 3D-bioprinting scaffolding for nasal cartilage defects repair a systematic review of the electronic databases for 3D-Bioprinting articles pertaining to nasal cartilage bio-modelling was performed. The primary focus was to investigate cellular source, type of scaffold utilization, biochemical evaluation, histological analysis, in-vitro study, in-vivo study, animal model used, length of research, and placement of experimental construct and translational investigation. RESULTS: From 1011 publications, 16 studies were kept for analysis. About cellular sources described, most studies used primary chondrocyte cultures. The cartilage used for cell isolation was mostly nasal septum. The most common biomaterial used for scaffold creation was polycaprolactone alone or in combination. About mechanical evaluation, we found a high heterogeneity, making it difficult to extract any solid conclusion. Regarding biological and histological characteristics of each scaffold, we found that the expression of collagen type I, collagen Type II and other ECM components were the most common patterns evaluated through immunohistochemistry on in-vitro and in-vivo studies. Only two studies made an orthotopic placement of the scaffolds. However, in none of the studies analyzed, the scaffold was placed in a subperichondrial pocket to rigorously simulate the cartilage environment. In contrast, scaffolds were implanted in a subcutaneous plane in almost all of the studies included. CONCLUSION: The role of 3D-bioprinting scaffolding for nasal cartilage defects repair is growing field. Despite the amount of information collected in the last years and the first surgical applications described recently in humans. Further investigations are needed due to the heterogeneity on mechanical evaluation parameters, the high level of heterotopic scaffold implantation and the need for quantitative histological data.
- 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.
- Design of a neurosurgery training simulator with additive manufacturing to practice the suture of the dura mater(Springer, 2021) Unamuno, X. (Xabier); Morer-Camo, P. (Paz); Llorente-Ortega, M. (Marcos); Paredes-Puente, J. (Jacobo)The objective of this work has been the realization of a training simulator for neurosurgery operations using additive manufacturing. This work has been developed in collaboration with two neurosurgeons. The simulator developed contains two parts, a superficial skull inside which is the brain. Between both is the Dura Mater: a layer that cover the brain. The Dura Mater is a fibrous, solid, thick and not very flexible tissue, with a thickness of about 0.5 mm. In any brain operation, after having performed the craniotomy, the Dura Mater must be sutured. This membrane has a special consistency and the suture has to be made very close to the bone. Therefore, this complex skill must be trained. The Dura Mater must be replaced for each of the practices and satisfy with the thickness and consistency of human tissue so that it responds in a similar way to a suture performed in the operating room.
- Ex vivo maturation of 3D-Printed, chondrocyte-laden, polycaprolactone-based scaffolds prior to transplantation improves engineered cartilage substitute properties and integration(Sage, 2022-10) Chiesa-Estomba, C.M. (Carlos M.); Delgado, A. (Alba); Fernández-Blanco, G. (Gonzalo); Hernaez-Moya, R. (Raquel); Aldazabal, J. (Javier); Rodino, C. (Claudia); Izeta, A. (Ander); Aiastui, A. (Ana); Paredes-Puente, J. (Jacobo)The surgical management of nasal septal defects due to perforations, malformations, congenital cartilage absence, traumatic defects, or tumors would benefit from availability of optimally matured septal cartilage substitutes. Here, we aimed to improve in vitro maturation of 3-dimensional (3D)-printed, cell-laden polycaprolactone (PCL)-based scaffolds and test their in vivo performance in a rabbit auricular cartilage model.
- Encapsulation of MSCs and GDNF in an Injectable Nanoreinforced Supramolecular Hydrogel for Brain Tissue Engineering(2022) Blanco-Prieto, M.J. (María José); Sanmartin-Grijalba, C. (Carmen); Campo, R. (Ruben) del; Aldazabal, J. (Javier); Plano-Amatriain, D. (Daniel); Paredes-Puente, J. (Jacobo); Luquin, M.R. (María Rosario); Garbayo-Atienza, E. (Elisa); Santamaria, E. (Enrique); Torres-Ortega, P.V. (Pablo Vicente)The co-administration of glial cell line-derived neurotrophic factor (GDNF) and mesenchymal stem cells (MSCs) in hydrogels (HGs) has emerged as a powerful strategy to enhance the efficient integration of transplanted cells in Parkinson's disease (PD). This strategy could be improved by controlling the cellular microenvironment and biomolecule release and better mimicking the complex properties of the brain tissue. Here, we develop and characterize a drug delivery system for brain repair where MSCs and GDNF are included in a nanoparticle-modified supramolecular guest-host HA HG. In this system, the nanoparticles act as both carriers for the GDNF and active physical crosslinkers of the HG. The multifunctional HG is mechanically compatible with brain tissue and easily injectable. It also protects GDNF from degradation and achieves its controlled release over time. The cytocompatibility studies show that the developed biomaterial provides a friendly environment for MSCs and presents good compatibility with PC12 cells. Finally, using RNA-sequencing (RNA-seq), we investigated how the three-dimensional (3D) environment, provided by the nanostructured HG, impacted the encapsulated cells. The transcriptome analysis supports the beneficial effect of including MSCs in the nanoreinforced HG. An enhancement in the anti-inflammatory effect of MSCs was observed, as well as a differentiation of the MSCs toward a neuron-like cell type. In summary, the suitable strength, excellent self healing properties, good biocompatibility, and ability to boost MSC regenerative potential make this nanoreinforced HG a good candidate for drug and cell administration to the brain.