Human dermal fibroblast activation under pulsed electrical stimulation via conductive fabrics : signalling pathways and potential benefit for wound healing

Lors de la cicatrisation, plusieurs types cellulaires dont les kératinocytes et les fibroblastes ainsi que plusieurs facteurs de croissance jouent d’importants rôles. La cicatrisation cutanée peut aussi être activée par des facteurs exogènes, dont la stimulation électrique (SE). La SE peut moduler les fonctions fibroblastiques durant la cicatrisation. Le fibroblaste contribue de façon active à la cicatrisation en sécrétant différentes protéines (collagène, fibronectine, élastine) pour favoriser le comblement tissulaire. Les fibroblastes adoptent aussi un phénotype contractile en exprimant l’α-actine contribuant à la fermeture de la plaie. Notre hypothèse est que certaines de ces fonctions fibroblastiques pourraient être modulées par une stimulation électrique. Pour vérifier cette hypothèse nous avons utilisé une membrane biocompatible et conductrice à base de polyethylene terephthalate (PET) recouvert de polypyrrole (PPy). Les fibroblastes dermiques humains ont été cultivés sur ces membranes conducteurs, puis exposés ou non à un courant pulsé (PES) selon deux régimes : soit 10s PES suivi de 1200s de repos, ou 300s PES suivi de 600s de repos, durant 24 h. Deux intensités électriques ont été étudiées, 50 et 100 mV/mm. Nos travaux démontrent que la SE favorise l’adhésion, la prolifération et la migration des fibroblastes dermiques. Ces activités cellulaires sont consolidées par une sécrétion importante de FGF2 et d’α-SMA. Il est important de noter que l’effet de la SE favorise le changement phénotypique des fibroblastes en myo-fibroblastes grâce à la voie des Smad et de TGFβ/ERK. Nous avons aussi démontré que l’effet de la SE est maintenue à long terme et est transférable de la cellule mère vers les cellules filles. En effet après sous-culture les cellules expriment toujours de façon importante l’α-SMA. En conclusion, nous avons démontré que la stimulation électrique pulsée module positivement les fonctions cicatricielles des fibroblastes humains. Ces travaux démontrent pour la première fois les voies de signalisation (Smad et TGFβ/ERK) sollicitées par la SE pour activer les fibroblastes lors de la cicatrisation. Ces travaux suggèrent l’utilisation de la SE pour favoriser la guérison/cicatrisation des plaies.During skin wound healing, cutaneous cells particularly fibroblasts and keratinocytes as well as several growth factors play important roles. Wound healing can be activated by exogenous factors, including electrical stimulation (ES). ES can also modulate fibroblast functions. Fibroblasts contribute to healing by secreting structural proteins (collagen, fibronectin, elastin) to repair the wound area. Fibroblasts also adopt a contractile phenotype expressing α-actin contributing to wound closure. The hypothesis of the thesis is that fibroblasts proliferate and transdifferentiate into myofibroblasts by sensing pulsed electrical signals and adjusting relevant signalling pathways. To test this hypothesis we used biocompatible polyethylene terephthalate (PET) fabrics coated with electrically conductive polypyrrole (PPy). Human dermal fibroblasts were cultured on these conductive fabrics and exposed to the optimized pulsed ES: either 10s PES in a period of 1200s, or 300s PES in 600s period, for a total of 24 hours. Two electric intensities were studied, 50 and 100 mV/ mm. Our work showed that the PES promoted the adhesion, proliferation and migration of dermal fibroblasts. These cellular activities were consolidated by an elevated level of fibroblast growth factor 2 (FGF2) and the high expression of α-smooth muscle actin (α-SMA). Important findings were that PES promoted the phenotypic change of fibroblasts to myofibroblasts, and such change was coordinated through the Smad and TGFβ/ERK pathways. It also demonstrated that the effect of PES was able to maintain for a long period of time after the end of stimulation, and was transferable from the mother cells to the daughter cells. Following subculture, the electrically stimulated fibroblasts still expressed significant amount of α-SMA. In conclusion, this thesis demonstrates that PES through conductive fabrics can activate the wound healing functions in human dermal fibroblasts. This work revealed for the first time that Smad and TGFβ/ERK pathways are required by the PES-induced fibroblasts-to-myofibroblasts differentiation. This work also demonstrated that the PES activated cells can survive in vivo. These studies suggest the application of the PES in promoting tissue regeneration and wound healing

On the Interaction between 1D Materials and Living Cells

One-dimensional (1D) materials allow for cutting-edge applications in biology, such as single-cell bioelectronics investigations, stimulation of the cellular membrane or the cytosol, cellular capture, tissue regeneration, antibacterial action, traction force investigation, and cellular lysis among others. The extraordinary development of this research field in the last ten years has been promoted by the possibility to engineer new classes of biointerfaces that integrate 1D materials as tools to trigger reconfigurable stimuli/probes at the sub-cellular resolution, mimicking the in vivo protein fibres organization of the extracellular matrix. After a brief overview of the theoretical models relevant for a quantitative description of the 1D material/cell interface, this work offers an unprecedented review of 1D nano- and microscale materials (inorganic, organic, biomolecular) explored so far in this vibrant research field, highlighting their emerging biological applications. The correlation between each 1D material chemistry and the resulting biological response is investigated, allowing to emphasize the advantages and the issues that each class presents. Finally, current challenges and future perspectives are discussed

Titania nanotube arrays as potential interfaces for neurological prostheses

2014 Summer.Includes bibliographical references.Neural prostheses can make a dramatic improvement for those suffering from visual and auditory, cognitive, and motor control disabilities, allowing them regained functionality by the use of stimulating or recording electrical signaling. However, the longevity of these devices is limited due to the neural tissue response to the implanted device. In response to the implant penetrating the blood brain barrier and causing trauma to the tissue, the body forms a to scar to isolate the implant in order to protect the nearby tissue. The scar tissue is a result of reactive gliosis and produces an insulated sheath, encapsulating the implant. The glial sheath limits the stimulating or recording capabilities of the implant, reducing its effectiveness over the long term. A favorable interaction with this tissue would be the direct adhesion of neurons onto the contacts of the implant, and the prevention of glial encapsulation. With direct neuronal adhesion the effectiveness and longevity of the device would be significantly improved. Titania nanotube arrays, fabricated using electrochemical anodization, provide a conductive architecture capable of altering cellular response. This work focuses on the fabrication of different titania nanotube array architectures to determine how their structures and properties influence the response of neural tissue, modeled using the C17.2 murine neural stem cell subclone, and if glial encapsulation can be reduced while neuronal adhesion is promoted

Electrospinning Novel Aligned Polymer Fiber Structures for Use in Neural Tissue Engineering

A suitable tissue scaffold to support and assist in the repair of damaged tissues or cells is important for success in clinical trials and for injury recovery. Electrospinning can create a variety of polymer nanofibers and microfibers, and is being widely used to produce experimental tissue scaffolds for neural applications. This dissertation examines various approaches by which electrospinning is being used for neural tissue engineering applications for the repair of injuries to the central nervous system (CNS) and the peripheral nervous system (PNS). Due to the poor regeneration of neural tissues in the event of injury, tissue scaffolds are being used to promote the recovery and restoration of neural function. Next generation scaffolds using bioactive materials, conductive polymers, and coaxial fiber structures are now being developed to improve the recovery of motor functions in in vivo studies. This dissertation includes fabrication techniques, the results of neural cell cultures performed both in vivo and in vitro on electrospun fiber scaffolds, examines barriers to full functional recovery, and future directions for electrospinning and neural tissue engineering. Aligned, free-standing fiber scaffolds using poly-L-lactic acid (PLLA) were developed as an in vitro model to study cell interaction on free-standing fiber scaffolds in vivo. Stages were designed to allow for the formation of free-standing fiber scaffolds that were not supported by an underlying surface. Fibers were spun across the columns of the stages to produce free-standing fiber scaffolds. The scaffolds were then used for in vitro cell culture using chick dorsal root ganglia (DRG). Fiber scaffolds were also spun on a flat substrate and used for in vitro cell studies for comparison. The axonal outgrowth observed for DRG cells cultured on free-standing fiber scaffolds was comparable to those grown on fibers with an underlying surface, indicating that cells follow the alignment of fibers even without an underlying support. Electrospinning coaxial fibers is a more complex application of electrospinning techniques that has been explored here as a method of creating a core-sheath fiber structure to act as a scaffold across glial scar tissue present in spinal cord injuries (SCIs). Here, we looked at altering the basic electrospinning set-up to spin core-sheath fibers. The core was spun with a conductive polymer, poly(3,4-ethyelenedixoythiophene): poly(styrene sulfonate) (PEDOT:PSS) and the sheath was spun PLLA to create coaxial fibers with a conductive core and an insulating sheath. A conductive polymer was used so that electrical stimulation could be applied along the fibers during cell culture to examine if the additional external stimulation would further assist in axonal outgrowth when combined with the topographical cues of the fiber scaffolds. This allows for the combination of electrical stimulation with the topographical guidance provided by aligned fiber scaffolds to improve axonal outgrowth and functional recovery in vivo

Bioprinting Neural Systems to Model Central Nervous System Diseases

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Stimulus responsive graphene scaffolds for tissue engineering

Tissue engineering (TE) is an emerging area that aims to repair damaged tissues and organs by combining different scaffold materials with living cells. Recently, scientists started to engineer a new generation of nanocomposite scaffolds able to mimic biochemical and biophysical mechanisms to modulate the cellular responses promoting the restoration of tissue structure or function. Due to its unique electrical, topographical and chemical properties, graphene is a material that holds a great potential for TE, being already considered as one of the best candidates for accelerating and guiding stem cell differentiations. Although this is a promising field there are still some challenges to overcome, such as the efficient control of the differentiation of the stem cells, especially in graphene-based microenvironments. Hence, this chapter will review the existing research related to the ability of graphene and its derivatives (graphene oxide and reduced graphene oxide) to induce stem cell differentiation into diverse lineages when under the influence of electrical, mechanical, optical and topographic stimulations

유연 전자 소자가 융합된 다기능성 세포 배양 기판 개발과 전기생리학적 활용

학위논문 (박사)-- 서울대학교 대학원 공과대학 화학생물공학부, 2017. 8. 김대형.Compared to conventional electronic devices, soft electronics offer the proper mechanical properties similar to native tissues and cells. This feature provides not only the promotion of cellular activities during culturing cells in vitro, but also the enhancement of biological interfaces between soft electronics and curvilinear surfaces of target organs in vivo. The high-quality interface enables effective monitoring and stimulation of electrophysiological signals. In this thesis, fabrications of three types of cell-culture-platforms and those applications are introduced. Components of all electronics are fabricated based on soft nanomaterials such as inorganic or carbon nanomaterials, and those electronics are transferred onto a biocompatible polymer substrate. Firstly, soft cell-culture-platform is designed to prepare C2C12 myoblasts sheet for transfer printing and treating the damaged muscle tissue. The platform is instrumented with stretchable nanomembrane sensors for in situ monitoring of cellular physiological characteristics during proliferation and differentiation, and with graphene nanoribbon cell aligners for guiding the unidirectional orientation of plated cells, whose system modulus is matched with target tissues. Furthermore, a high-yield transfer printing mechanism can deliver cell sheets for scaffold-free, localized cell therapy in vivo. Secondly, multifunctional cell-sheet-graphene hybrid is developed as stretchable and transparent medical device, which can be implanted in vivo to form a high-quality biotic/abiotic interface. The hybrid is composed of C2C12 myoblasts sheet on buckled, mesh-patterned graphene electrodes. The graphene electrodes monitor and stimulate the C2C12 myoblasts in vitro, serving as a smart cell culture substrate that controls their aligned proliferation and differentiation. This stretchable and transparent cell-sheet-graphene hybrid can be transplanted onto the target muscle tissue, record electromyographical signals, and stimulate implanted sites electrically and/or optically in vivo without any immune responses. Additional cellular therapeutic effect of the cell-sheet-graphene hybrid is obtained by the integrated C2C12 myobalsts sheet. Finally, electronic-cell-culture-platform is fabricated to provide multifunctionalities by integrating various types of electronics for monitoring and stimulating important metabolic conditions of culturing cells. This platform is based on an array of soft electronics composed of four types of sensors and two types of stimulators, which is transferred onto a biocompatible polymer substrate designed by a 3D printer. The sensors and stimulators can monitor and regulate the behaviors and activities of the cells cultured on the large area surface of the platform. The multi-layer system of the platform enable to monitor and stimulate the activities of numerous cells effectively without sacrificing any culturing cells.Chapter 1. Introduction 1 1.1 Soft electronics for cell culturing in vitro 1 1.2 Biointerfaces of soft electronic devices 6 1.3 Soft electronics for three-dimensional cell culturing 12 1.4 References 16 Chapter 2. Soft and instrumented cell-culture-platform for monitoring and transfer printing of cell sheets 28 2.1 Introduction 28 2.2 Results and Discussion 33 2.3 Conclusion 60 2.4 Experimental 62 2.5 References 70 Chapter 3. Soft cell-sheet-graphene hybrid device for electrophysiological applications of skeletal muscles 78 3.1 Introduction 78 3.2 Results and Discussion 84 3.3 Conclusion 119 3.4 Experimental 120 3.5 References 124 Chapter 4. Electronic-cell-culture-platform for real-time monitoring and stimulation of cellular electrophysiology 136 4.1 Introduction 136 4.2 Results and Discussion 140 4.3 Conclusion 166 4.4 Experimental 167 4.5 References 171 Biblography 178 국문 초록 (Abstract in Korean) 179Docto

Technological advances in fibrin for tissue engineering

Fibrin is a promising natural polymer that is widely used for diverse applications, such as hemostatic glue, carrier for drug and cell delivery, and matrix for tissue engineering. Despite the significant advances in the use of fibrin for bioengineering and biomedical applications, some of its characteristics must be improved for suitability for general use. For example, fibrin hydrogels tend to shrink and degrade quickly after polymerization, particularly when they contain embedded cells. In addition, their poor mechanical properties and batch-to-batch variability affect their handling, long-term stability, standardization, and reliability. One of the most widely used approaches to improve their properties has been modification of the structure and composition of fibrin hydrogels. In this review, recent advances in composite fibrin scaffolds, chemically modified fibrin hydrogels, interpenetrated polymer network (IPN) hydrogels composed of fibrin and other synthetic or natural polymers are critically reviewed, focusing on their use for tissue engineering.The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by Programa de Actividades de I + D entre Grupos de Investigación de la Comunidad de Madrid, S2018/BAA-4480, Biopieltec-CM, Programa Estatal de I + D + i Orientada a los Retos de la Sociedad, RTI2018-101627-B-I00, Proyectos de Generación de Conocimiento 2021, PID2021-126523OB-I00, Proyectos en colaboración público-privada 2021, CPP2021-008396, LOLICOMB Project, PID2020-116439GB-I00 and Cátedra Fundación Ramón Areces. Grant PID2021-126523OB-I00 funded by MCIN/AEI/10.13039/501100011033 and, as appropriate, by “ERDF A way of making Europe.” Grant CPP2021-008396 funded by MCIN/AEI/ 10.13039/501100011033 and by the European Union “NextGenerationEU/PRTR.”Publicad