Strategies for Guidance and Electrical and Biological Stimulation in a Neural Regeneration Device

Tesis por compendio[ES] Actualmente las lesiones del sistema nervioso periférico que conllevan una pérdida de continuidad de los haces axonales suelen implicar secuelas de tipo permanente. Es cierto que en el sistema nervioso periférico existe una cierta regeneración natural de los tractos axonales dañados, pero solamente cuando el espacio entre ambos extremos de la lesión es pequeño, como máximo de 5 mm. Si el espacio es mayor que esta distancia la regeneración no sucede de forma natural y se crea un neuroma traumático. Por tanto, estas lesiones largas requieren de una intervención quirúrgica para puentear la lesión, normalmente con un nervio autógrafo del propio paciente o un nervio alógrafo de un cadáver. No obstante, su uso presenta diversos inconvenientes, como la morbilidad del sitio donante donde puede ocurrir un neuroma, la necesidad de realizar una segunda cirugía, la diferencia de tamaño entre nervio receptor y donante o la necesidad de inmunosupresión en el caso de los nervios alógrafos. Por ello, la ingeniería de tejidos trabaja en el desarrollo de los conductos de guiado nervioso que incorporan estrategias para guiar topográficamente la regeneración, así como células y moléculas bioactivas. La presente tesis doctoral presenta un nuevo conducto de guiado nervioso con una aproximación multimodular para su aplicación en la regeneración de lesiones nerviosas largas (a partir de 15 mm) que hace uso de conductos tubulares huecos modulares de ácido hialurónico (HA) que contienen en su interior una estructura tubular de microfibras de ácido poliláctico (PLA). La estructura fibrilar aporta un guiado topográfico necesario para guiar el crecimiento axonal durante la regeneración a la vez que mantiene unidos los diferentes módulos de HA. Por su parte, los conductos de HA son un hidrogel que evita adherencias con el tejido circundante. A su vez, proporcionan un soporte sobre el que pueden crecer células presembradas. En concreto se ha optado por presembrar células de Schwann, las cuales son unas células gliales de soporte críticas para la regeneración del sistema nervioso periférico. Se ha observado que dichas células son capaces recubrir por completo las paredes internas de los conductos de HA formando una estructura tipo vaina, así como de recubrir las microfibras de PLA creciendo en dirección longitudinal. Los experimentos in vivo en modelo de nervio ciático de conejo han mostrado que la aproximación multimodular mejora significativamente la regeneración nerviosa gracias a proporcionar una mejor neovascularización. A su vez, gracias a las células de Schwann presembradas se ha logrado una mejora adicional de la regeneración nerviosa gracias a su efecto favorecedor del crecimiento axonal. Además, se han estudiado diferentes mejoras aplicables al conducto de guiado nervioso con el objetivo de mejorar los resultados obtenidos in vivo. Gracias a la incorporación de fibroína de seda a los conductos de HA se ha logrado mejorar sus propiedades mecánicas y biológicas. Asimismo, también se ha desarrollado un sustrato electroconductor de microfibras de PLA recubiertas con el polímero electroconductor Polipirrol gracias al cual se ha observado in vitro que es capaz de mejorar el crecimiento axonal al aplicar una estimulación eléctrica. Además, mediante un sistema de modificación génica de las células de Schwann por electrotransfección se ha logrado aumentar su secreción del factor neurotrófico derivado del cerebro (BDNF), gracias a lo cual se ha observado que se incrementa la velocidad de crecimiento axonal in vitro.[CA] Actualment les lesions del sistema nerviós perifèric que comporten una pèrdua de continuïtat dels feixos axonals solen implicar seqüeles de tipus permanent. És cert que al sistema nerviós perifèric hi ha una certa regeneració natural dels tractes axonals danyats, però només quan l'espai entre ambdós extrems de la lesió és petit, com a màxim de 5 mm. Si l'espai és més gran que aquesta distància la regeneració no succeeix de manera natural i es crea un neuroma traumàtic. Per tant, aquestes lesions llargues requereixen una intervenció quirúrgica per pontejar la lesió, normalment amb un nervi autògraf del pacient o un nervi al·lògraf d'un cadàver. No obstant això, el seu ús presenta diversos inconvenients, com la morbilitat del lloc donant on pot ocórrer un neuroma, la necessitat de fer una segona cirurgia, la diferència de mida entre nervi receptor i donant o la necessitat d'immunosupressió en el cas dels nervis al·lògrafs . Per això, l'enginyeria de teixits treballa en el desenvolupament dels conductes de guiatge nerviós que incorporen estratègies per guiar topogràficament la regeneració, així com cèl·lules i molècules bioactives. Aquesta tesi doctoral presenta un nou conducte de guiatge nerviós amb una aproximació multimodular per a la seva aplicació en la regeneració de lesions nervioses llargues (a partir de 15 mm) que fa ús de conductes tubulars buits modulars d'àcid hialurònic (HA) que contenen al seu interior una estructura tubular de microfibres d'àcid polilàctic (PLA). L'estructura fibril·lar aporta un guiatge topogràfic necessari per guiar el creixement axonal durant la regeneració alhora que manté units els diferents mòduls d'HA. Per part seva, els conductes d'HA són un hidrogel que evita adherències amb el teixit circumdant. Alhora, proporcionen un suport sobre el qual poden créixer cèl·lules presembrades. En concret s'ha optat per presembrar cèl·lules de Schwann, les quals són unes cèl·lules glials de suport crítiques per a la regeneració del sistema nerviós perifèric. S'ha observat que aquestes cèl·lules són capaces de recobrir completament les parets internes dels conductes d'HA formant una estructura tipus beina, així com de recobrir les microfibres de PLA creixent en direcció longitudinal. Els experiments in vivo en model de nervi ciàtic de conill han mostrat que l'aproximació multimodular millora significativament la regeneració nerviosa gràcies a proporcionar una millor neovascularització. Alhora, gràcies a les cèl·lules de Schwann presembrades s'ha aconseguit una millora addicional de la regeneració nerviosa gràcies al seu efecte afavoridor del creixement axonal. A més, s'han estudiat diferents millores aplicables al conducte de guiatge nerviós per tal de millorar els resultats obtinguts in vivo. Gràcies a la incorporació de fibroïna de seda als conductes d'HA s'ha aconseguit millorar les seues propietats mecàniques i biològiques. També s'ha desenvolupat un substrat electroconductor de microfibres de PLA recobertes amb el polímer electroconductor Polipirrol gràcies al qual s'ha observat in vitro que és capaç de millorar el creixement axonal quan s'aplica una estimulació elèctrica. A més, mitjançant un sistema de modificació gènica de les cèl·lules de Schwann per electrotransfecció s'ha aconseguit augmentar la secreció del factor neurotròfic derivat del cervell (BDNF), gràcies a la qual cosa s'ha observat que s'incrementa la velocitat de creixement axonal in vitro.[EN] Currently, lesions of the peripheral nervous system that lead to a loss of continuity of the axonal bundles usually involve permanent sequelae. It is true that in the peripheral nervous system there is some natural regeneration of damaged axonal tracts, but only when the space between the two ends of the lesion is small, at most 5 mm. If the gap is greater than this distance, regeneration does not occur naturally, and a traumatic neuroma is created. Therefore, these long injuries require surgical intervention to bridge the injury, usually with an autograph nerve from the patient or an allograph nerve from a cadaver. However, its use has various drawbacks, such as the morbidity of the donor site where a neuroma can occur, the need to perform a second surgery, the difference in size between the recipient and donor nerves, or the need for immunosuppression in the case of allograft nerves. For this reason, tissue engineering works on the development of nerve guidance conduits that incorporate strategies to topographically guide the regeneration, as well as cells and bioactive molecules. This doctoral thesis presents a new nerve guidance conduit with a multimodular approach for its application in the regeneration of long nerve lesions (from 15 mm) that makes use of modular hollow tubular conduits of hyaluronic acid (HA) that contain in their inside a tubular structure of microfibers of polylactic acid (PLA). The fibrillar structure provides the necessary topographic guidance to guide axonal growth during regeneration while keeping the different HA modules together. For their part, the HA conduits are a hydrogel that prevents adhesions with the surrounding tissue. In turn, they provide a support on which preseeded cells can grow. Specifically, it has been decided to pre-seed Schwann cells, which are glial support cells that are critical for the regeneration of the peripheral nervous system. It has been observed that these cells are capable of completely covering the inner walls of the HA conduits, forming a sheath-like structure, as well as covering the PLA microfibers by growing in a longitudinal direction. In vivo experiments in a rabbit sciatic nerve model have shown that the multimodular approach significantly improves nerve regeneration by providing better neovascularization. In turn, thanks to the pre-seeded Schwann cells, an additional improvement in nerve regeneration has been achieved thanks to its promoting effect on axonal growth. In addition, different improvements applicable to the nerve guidance conduit have been studied with the aim of improving the results obtained in vivo. Thanks to the incorporation of silk fibroin into HA conduits, their mechanical and biological properties have been improved. Likewise, an electroconductive substrate of PLA microfibers coated with the electroconductive polymer Polypyrrole has also been developed, thanks to which it has been observed in vitro that it is capable of improving axonal growth by applying electrical stimulation. In addition, by means of a gene modification system of Schwann cells by electrotransfection, it has been possible to increase their secretion of brain-derived neurotrophic factor (BDNF), thanks to which it has been observed that the speed of axonal growth is increased in vitro.Agradezco la ayuda de los diferentes proyectos del Ministerio de Economía y Competitividad del Gobierno de España que han hecho posible la financiación de esta tesis doctoral: MAT2015-66666-C3-1-R, DPI2015-72863-EXP, AEI RTI2018-095872-B-C21-C22/ERDF y FPU16/01833 del Ministerio de Universidades del Gobierno de España, sin la cual no hubiera podido realizar esta tesis doctoral.Gisbert Roca, F. (2022). Strategies for Guidance and Electrical and Biological Stimulation in a Neural Regeneration Device [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/189937Compendi

Modelado del efecto eléctrico de los fármacos voltaje-dependientes en las células ventriculares de corazón: nuevo marco teórico

[ES] El primer objetivo del programa es aplicar un nuevo modelo de estimación del factor de bloqueo de una corriente iónica que se ha desarrollado para tener en cuenta su dependencia con el voltaje en el caso del uso de un fármaco voltaje-dependiente. Este nuevo modelo busca, por un lado, mejorar el método convencional que no tiene en cuenta la dependencia del factor de bloqueo con el voltaje, y, por otro, presentar un coste computacional menor que el de los modelos de Markov. El segundo objetivo del programa es aplicar esta nueva estimación del factor de bloqueo a un modelo de simulación de la actividad eléctrica de una célula excitable para así mejorar la aproximación del efecto que tiene el uso de un fármaco voltaje-dependiente dado sobre el comportamiento eléctrico de dicha célula. Así, este nuevo programa informático pretende ser una nueva herramienta que ofrecer a las empresas farmacéuticas para mejorar el estudio del efecto de nuevos fármacos voltaje-dependientes sobre la actividad eléctrica de las células que presenten canales iónicos alterados por el fármaco y así aumentar la fiabilidad de las simulaciones computacionales previas al ensayo clínico.[CA] El primer objectiu del programa es aplicar un nou model d’estimació del factor de bloqueig d’una corrent iònica que s’ha desenvolupat per tindre en compte la seua dependència amb el voltatge en el cas d’utilitzar un fàrmac voltatge-dependent. Aquest nou model busca, per un costat, millorar el mètode convencional que no té en compte la dependència del factor de bloqueig amb el voltatge i, per altre, presentar un cost computacional menor que el dels models de Markov. El segon objectiu del programa es aplicar aquesta nova estimació del factor de bloqueig a un model de simulació de l’activitat elèctrica d’una cèl·lula excitable per millorar l’aproximació de l’efecte que té l’ús d’un fàrmac voltatge-dependent donat sobre el comportament elèctric de dita cèl·lula. Així, aquest nou programa informàtic pretén ser una nova ferramenta que oferir a les empreses farmacèutiques per millorar l’estudi de l’efecte de nous fàrmacs voltatgedependents sobre l’activitat elèctrica de les cèl·lules que presenten canals iònics alterats pel fàrmac i així augmentar la fiabilitat de les simulacions computacionals prèvies a l’assaig clínic.[EN] The first goal of the software is to apply the new estimation model of the blocking factor of an ionic current that has been developed in order to consider its voltage dependency when using a voltage-dependent drug. On the one hand, this model seeks to improve the conventional method that disregards the voltage-dependency of the blocking factor and, on the other hand, to present a lower computational cost than the Markov models. The second aim of the software is to apply this new estimation of the blocking factor to an electrical activity simulation model of an excitable cell in order to improve the approach of the effect that a given voltage-dependent drug has on the electrical behaviour of the cell. Thus, this new software intends to be a new tool to offer to pharmaceutics companies in order to improve the study of the effect that new voltage-dependent drugs have on the electrical activity of the cells with ionic channels disturbed by the drug and thereby increase the reliability of the computational simulations previous to the clinical trials.Gisbert Roca, F. (2016). Modelado del efecto eléctrico de los fármacos voltaje-dependientes en las células ventriculares de corazón: nuevo marco teórico. http://hdl.handle.net/10251/67468.TFG

Axonal extension from dorsal root ganglia on fibrillar and highly aligned poly(lactic acid)-polypyrrole substrates obtained by two different techniques: Electrospun nanofibres and extruded microfibres

[EN] The biological behaviour of Schwann cells (SCs) and dorsal root ganglia (DRG) on fibrillar, highly aligned and electroconductive substrates obtained by two different techniques is studied. Mats formed by nanometer-sized fibres of poly(lactic acid) (PLA) are obtained by the electrospinning technique, while bundles formed by micrometer-sized extruded PLA fibres are obtained by grouping microfibres together. Both types of substrates are coated with the electrically conductive polymer polypyrrole (PPy) and their morphological, physical and electrical characterization is carried out. SCs on micrometer-sized substrates show a higher motility and cell-cell interaction, while a higher cell-material interaction with a lower cell motility is observed for nanometer-sized substrates. This higher motility and cell-cell interaction of SCs on the micrometer-sized substrates entails a higher axonal growth from DRG, since the migration of SCs from the DRG body is accelerated and, therefore, the SCs tapestry needed for the axonal growth is formed earlier on the substrate. A higher length and area of the axons is observed for these micrometer-sized substrates, as well as a higher level of axonal sprouting when compared with the nanometer-sized ones. These substrates offer the possibility of being electrically stimulated in different tissue engineering applications of the nervous system.The authors acknowledge financing from the Spanish Government's State Research Agency (AEI) through projects DPI2015-72863-EXP and RTI2018-095872-B-C22/ERDF. FGR acknowledges scholarship FPU16/01833 of the Spanish Ministry of Universities. 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Electrical Stimulation Increases Axonal Growth from Dorsal Root Ganglia Co-Cultured with Schwann Cells in Highly Aligned PLA-PPy-Au Microfiber Substrates

[EN] Nerve regeneration is a slow process that needs to be guided for distances greater than 5 mm. For this reason, different strategies are being studied to guide axonal growth and accelerate the axonal growth rate. In this study, we employ an electroconductive fibrillar substrate that is able to topographically guide axonal growth while accelerating the axonal growth rate when subjected to an exogenous electric field. Dorsal root ganglia were seeded in co-culture with Schwann cells on a substrate of polylactic acid microfibers coated with the electroconductive polymer polypyrrole, adding gold microfibers to increase its electrical conductivity. The substrate is capable of guiding axonal growth in a highly aligned manner and, when subjected to an electrical stimulation, an improvement in axonal growth is observed. As a result, an increase in the maximum length of the axons of 19.2% and an increase in the area occupied by the axons of 40% were obtained. In addition, an upregulation of the genes related to axon guidance, axogenesis, Schwann cells, proliferation and neurotrophins was observed for the electrically stimulated group. Therefore, our device is a good candidate for nerve regeneration therapies.This research was funded by the Spanish Government's State Research Agency (AEI) through project RTI2018-095872-B-C22/ERDF and by RISEUP project FetOpen in H2020 Program: RISEUP 964562 H2020 FetOpen program.Gisbert Roca, F.; Serrano Requena, S.; Monleón Pradas, M.; Martínez-Ramos, C. (2022). Electrical Stimulation Increases Axonal Growth from Dorsal Root Ganglia Co-Cultured with Schwann Cells in Highly Aligned PLA-PPy-Au Microfiber Substrates. International Journal of Molecular Sciences. 23:1-22. https://doi.org/10.3390/ijms231263621222

Solid Polymer Electrolytes Based on Polylactic Acid Nanofiber Mats Coated with Polypyrrole

This is the peer reviewed version of the following article: Gisbert, F., García-Bernabé, A., Compañ, V., Martínez-Ramos, C., Monleón, M., Solid Polymer Electrolytes Based on Polylactic Acid Nanofiber Mats Coated with Polypyrrole. Macromol. Mater. Eng. 2021, 306, 2000584, which has been published in final form at https://doi.org/10.1002/mame.202000584. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] The production of electroconductive nanofiber membranes made from polylactic acid (PLA) coated with polypyrrole (PPy) is investigated, performing a scanning of different reaction parameters and studying their physicochemical and dielectric properties. Depending on PPy content, a transition between conduction mechanisms is observed, with a temperature-dependent relaxation process for samples without PPy, a temperature-independent conduction process for samples with high contents of PPy and a combination of both processes for samples with low contents of PPy. A homogeneous and continuous coating is achieved from 23 wt% PPy, observing a percolation effect around 27 wt% PPy. Higher wt% PPy allow to obtain higher conductivities, but PPy aggregates appear from 34% wt% PPy. The high conductivity values obtained for electrospun membranes both through-plane and in-plane (above 0.05 and 0.20 S cm¿1, respectively, at room temperature) for the highest wt% of PPy, their porous structure with high specific surface area and their thermal stability below 140 °C make them candidates for many potential applications as solid polymer electrolytes in, for example, batteries, supercapacitors, sensors, photosensors, or polymer electrolyte membrane fuel cells (PEMFCs). In addition, the biocompatibility of PLA-PPy membranes expand their potential applications also in the field of tissue engineering and implantable devices.The authors acknowledge financing from the Spanish Government's State Research Agency (AEI) through projects DPI2015-72863-EXP and RTI2018-095872-B-C22/ERDF. FGR acknowledges the scholarship FPU16/01833 of the Spanish Ministry of Universities. 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Chemical Engineering Journal, 340, 9-14. doi:10.1016/j.cej.2018.01.010Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., … Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676-682. doi:10.1038/nmeth.2019Garlotta, D. (2001). Journal of Polymers and the Environment, 9(2), 63-84. doi:10.1023/a:1020200822435Mofokeng, J. P., Luyt, A. S., Tábi, T., & Kovács, J. (2011). Comparison of injection moulded, natural fibre-reinforced composites with PP and PLA as matrices. Journal of Thermoplastic Composite Materials, 25(8), 927-948. doi:10.1177/0892705711423291Chieng, B., Ibrahim, N., Yunus, W., & Hussein, M. (2013). Poly(lactic acid)/Poly(ethylene glycol) Polymer Nanocomposites: Effects of Graphene Nanoplatelets. Polymers, 6(1), 93-104. doi:10.3390/polym6010093Chitte, H. K., Shinde, G. N., Bhat, N. V., & Walunj, V. E. (2011). 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BDNF-Gene Transfected Schwann Cell-Assisted Axonal Extension and Sprouting on New PLA-PPy Microfiber Substrates

[EN] The work here reported analyzes the effect of increased efficiency of brainderived neurotrophic factor (BDNF) production by electroporated Schwann cells (SCs) on the axonal extension in a coculture system on a biomaterial platform that can be of interest for the treatment of injuries of the nervous system, both central and peripheral. Rat SCs are electrotransfected with a plasmid coding for the BDNF protein in order to achieve an increased expression and release of this protein into the culture medium of the cells, performing the best balance between the level of transfection and the number of living cells. Gene-transfected SCs show an about 100-fold increase in the release of BDNF into the culture medium, compared to nonelectroporated SCs. Cocultivation of electroporated SCs with rat dorsal root ganglia (DRG) is performed on highly aligned substrates of polylactic acid (PLA) microfibers coated with the electroconductive polymer polypyrrol (PPy). The coculture of DRG with electrotransfected SCs increase both the axonal extension and the axonal sprouting from DRG neurons compared to the coculture of DRG with nonelectroporated SCs. Therefore, the use of PLA¿PPy highly aligned microfiber substrates preseeded with electrotransfected SCs with an increased BDNF secretion is capable of both guiding and accelerating axonal growth.The authors acknowledge financial support from the Spanish Government's State Research Agency (AEI) through projects DPI2015-72863-EXP and RTI2018-095872-B-C22/ERDF. F.G.R. acknowledges the scholarship FPU16/01833 and the short stay mobility aid EST18/00524 of the Spanish Ministry of Universities. F.G.R. also acknowledges the hosting at the Vectorology and Anti-cancer Therapies Centre (UMR 8203 CNRS). The authors thank the Electron Microscopy Service at the UPV, where the FESEM images were obtained.Gisbert-Roca, F.; André, FM.; Más Estellés, J.; Monleón Pradas, M.; Mir, LM.; Martínez-Ramos, C. (2021). BDNF-Gene Transfected Schwann Cell-Assisted Axonal Extension and Sprouting on New PLA-PPy Microfiber Substrates. Macromolecular Bioscience (Online). 21(5):1-13. https://doi.org/10.1002/mabi.202000391S11321

Conduits based on the combination of hyaluronic acid and silk fibroin: Characterization, in vitro studies and in vivo biocompatibility

[EN] We address the production of structures intended as conduits made from natural biopolymers, capable of promoting the regeneration of axonal tracts. We combine hyaluronic acid (HA) and silk fibroin (SF) with the aim of improving mechanical and biological properties of HA. The results show that SF can be efficiently incorporated into the production process, obtaining conduits with tubular structure with a matrix of HA-SF blend. HA-SF has better mechanical properties than sole HA, which is a very soft hydrogel, facilitating manipulation. Culture of rat Schwann cells shows that cell adhesion and proliferation are higher than in pure HA, maybe due to the binding motifs contributed by the SF protein. This increased proliferation accelerates the formation of a tight cell layer, which covers the inner channel surface of the HA-SF tubes. Biocompatibility of the scaffolds was studied in immunocompetent mice. Both HA and HA-SF scaffolds were accepted by the host with no residual immune response at 8 weeks. New collagen extracellular matrix and new blood vessels were visible and they were present earlier when SF was present. The results show that incorporation of SF enhances the mechanical properties of the materials and results in promising biocompatible conduits for tubulization strategies.The authors acknowledge financing from the Spanish Ministry of Economy and Competitiveness through grants RTI2018-095872-B-C22/ERDF, DPI2015-72863-EXP, MAT2016-79832-R, MAT2016-76847-R and Community of Madrid through grant Neurocentro-B2017/BMD-3760. FGR acknowledges scholarship FPU16/01833 of the Spanish Ministry of Education, Culture and Sports. We thank the Electron Microscopy Service at the UPV, where the FESEM images were obtainedGisbert-Roca, F.; Lozano Picazo, P.; Pérez-Rigueiro, J.; Guinea Tortuero, GV.; Monleón Pradas, M.; Martínez-Ramos, C. (2020). 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Alginate-Agarose Hydrogels Improve the In Vitro Differentiation of Human Dental Pulp Stem Cells in Chondrocytes. A Histological Study

[EN] Matrix-assisted autologous chondrocyte implantation (MACI) has shown promising results for cartilage repair, combining cultured chondrocytes and hydrogels, including alginate. The ability of chondrocytes for MACI is limited by different factors including donor site morbidity, dedifferentiation, limited lifespan or poor proliferation in vitro. Mesenchymal stem cells could represent an alternative for cartilage regeneration. In this study, we propose a MACI scaffold consisting of a mixed alginate-agarose hydrogel in combination with human dental pulp stem cells (hDPSCs), suitable for cartilage regeneration. Scaffolds were characterized according to their rheological properties, and their histomorphometric and molecular biology results. Agarose significantly improved the biomechanical behavior of the alginate scaffolds. Large scaffolds were manufactured, and a homogeneous distribution of cells was observed within them. Although primary chondrocytes showed a greater capacity for chondrogenic differentiation, hDPSCs cultured in the scaffolds formed large aggregates of cells, acquired a rounded morphology and expressed high amounts of type II collagen and aggrecan. Cells cultured in the scaffolds expressed not only chondral matrix-related genes, but also remodeling proteins and chondrocyte differentiation factors. The degree of differentiation of cells was proportional to the number and size of the cell aggregates that were formed in the hydrogels.This work was funded by the Ministry of Economy and Competitiveness of the Spanish Government (PID2019-106099RB-C42, MM) and by the Generalitat Valenciana, Spain (PROMETEO/2020/069, CC). CIBER-BBN and CIBER-ER are financed by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and the Instituto de Salud Carlos III, with assistance of the European Regional Development Fund.Oliver-Ferrándiz, M.; Milián, L.; Sancho-Tello, M.; Martín De Llano, JJ.; Gisbert-Roca, F.; Martínez-Ramos, C.; Carda, C.... (2021). Alginate-Agarose Hydrogels Improve the In Vitro Differentiation of Human Dental Pulp Stem Cells in Chondrocytes. A Histological Study. Biomedicines. 9(7):1-22. https://doi.org/10.3390/biomedicines9070834S1229

A Hyaluronic Acid Demilune Scaffold and Polypyrrole-Coated Fibers Carrying Embedded Human Neural Precursor Cells and Curcumin for Surface Capping of Spinal Cord Injuries

[EN] Tissue engineering, including cell transplantation and the application of biomaterials and bioactive molecules, represents a promising approach for regeneration following spinal cord injury (SCI). We designed a combinatorial tissue-engineered approach for the minimally invasive treatment of SCI¿a hyaluronic acid (HA)-based scaffold containing polypyrrole-coated fibers (PPY) combined with the RAD16-I self-assembling peptide hydrogel (Corning® PuraMatrix¿peptide hydrogel (PM)), human induced neural progenitor cells (iNPCs), and a nanoconjugated form of curcumin (CURC). In vitro cultures demonstrated that PM preserves iNPC viability and the addition of CURC reduces apoptosis and enhances the outgrowth of Nestin-positive neurites from iNPCs, compared to nonembedded iNPCs. The treatment of spinal cord organotypic cultures also demonstrated that CURC enhances cell migration and prompts a neuron-like morphology of embedded iNPCs implanted over the tissue slices. Following sub-acute SCI by traumatic contusion in rats, the implantation of PMembedded iNPCs and CURC with PPY fibers supported a significant increase in neuro-preservation (as measured by greater III-tubulin staining of neuronal fibers) and decrease in the injured area (as measured by the lack of GFAP staining). This combination therapy also restricted platelet-derived growth factor expression, indicating a reduction in fibrotic pericyte invasion. Overall, these findings support PM-embedded iNPCs with CURC placed within an HA demilune scaffold containing PPY fibers as a minimally invasive combination-based alternative to cell transplantation alone.This research was funded by the Science by Women program, Women for Africa Foundation to H.E. and the grants FEDER/Ministerio de Ciencia e Innovacion-Agencia Estatal de Investigacion [RTI2018-095872-B-C21 and -C22/ERDF]; Part of the equipment employed in this work was funded by Generalitat Valenciana and cofinanced with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020). RISEUP project FetOpen in H2020 Program: H2020-FETOPEN-2018-2019-2020-01.Elkhenany, H.; Bonilla, P.; Giraldo-Reboloso, E.; Alastrue Agudo, A.; Edel, MJ.; Vicent, MJ.; Gisbert-Roca, F.... (2021). A Hyaluronic Acid Demilune Scaffold and Polypyrrole-Coated Fibers Carrying Embedded Human Neural Precursor Cells and Curcumin for Surface Capping of Spinal Cord Injuries. Biomedicines. 9(12):1-19. https://doi.org/10.3390/biomedicines9121928S11991

Electric Field Bridging-Effect in Electrified Microfibrils’ Scaffolds

Introduction: The use of biocompatible scaffolds combined with the implantation of neural stem cells, is increasingly being investigated to promote the regeneration of damaged neural tissue, for instance, after a Spinal Cord Injury (SCI). In particular, aligned Polylactic Acid (PLA) microfibrils’ scaffolds are capable of supporting cells, promoting their survival and guiding their differentiation in neural lineage to repair the lesion. Despite its biocompatible nature, PLA is an electrically insulating material and thus it could be detrimental for increasingly common scaffolds’ electric functionalization, aimed at accelerating the cellular processes. In this context, the European RISEUP project aims to combine high intense microseconds pulses and DC stimulation with neurogenesis, supported by a PLA microfibrils’ scaffold. Methods: In this paper a numerical study on the effect of microfibrils’ scaffolds on the E-field distribution, in planar interdigitated electrodes, is presented. Realistic microfibrils’ 3D CAD models have been built to carry out a numerical dosimetry study, through Comsol Multiphysics software. Results: Under a voltage of 10 V, microfibrils redistribute the E-field values focalizing the field streamlines in the spaces between the fibers, allowing the field to pass and reach maximum values up to 100 kV/m and values comparable with the bare electrodes’ device (without fibers). Discussion: Globally the median E-field inside the scaffolded electrodes is the 90% of the nominal field, allowing an adequate cells’ exposure