State of the Art and Current Challenges on Electroactive Biomaterials and Strategies for Neural Tissue Regeneration.

The loss or failure of an organ/tissue stands as one of the healthcare system's most prevalent, devastating, and costly challenges. Strategies for neural tissue repair and regeneration have received significant attention due to their particularly strong impact on patients' well-being. Many research efforts are dedicated not only to control the disease symptoms but also to find solutions to repair the damaged tissues. Neural tissue engineering (TE) plays a key role in addressing this problem and significant efforts are being carried out to develop strategies for neural repair treatment. In the last years, active materials allowing to tune cell-materials interaction are being increasingly used, representing a recent paradigm in TE applications. Among the most important stimuli influencing cell behavior are the electrical and mechanical ones. In this way, materials with the ability to provide this kind of stimuli to the neural cells seem to be appropriate to support neural TE. In this scope, this review summarizes the different biomaterials types used for neural TE, highlighting the relevance of using active biomaterials and electrical stimulation. Furthermore, this review provides not only a compilation of the most relevant studies and results but also strategies for novel and more biomimetic approaches for neural TE.

Electroactive Materials Surface Charge Impacts Neuron Viability and Maturation in 2D Cultures.

Since neurons were first cultured outside a living organism more than a century ago, a number of experimental techniques for their in vitro maintenance have been developed. These methods have been further adapted and refined to study specific neurobiological processes under controlled experimental conditions. Despite their limitations, the simplicity and visual accessibility of 2D cultures have enabled the study of the effects of trophic factors, adhesion molecules, and biophysical stimuli on neuron function and morphology. Nevertheless, the impact of fundamental properties of the surfaces to which neurons adhere when cultured in vitro has not been sufficiently considered. Here, we used an electroactive polymer with different electric poling states leading to different surface charges to evaluate the impact of the net electric surface charge on the behavior of primary neurons. Average negative and positive surface charges promote increased metabolic activity and enhance the maturation of primary neurons, demonstrating the relevance of considering the composition and electric charge of the culture surfaces. These findings further pave the way for the development of novel therapeutic strategies for the regeneration of neural tissues, particularly based on dynamic surface charge variation that can be induced in the electroactive films through mechanical solicitation.

Modulation of myoblast differentiation by electroactive scaffold morphology and biochemical stimuli.

The physico-chemical properties of the scaffold materials used for tissue regeneration strategies have a direct impact on cell shape, adhesion, proliferation, phenotypic and differentiation. Herewith, biophysical and biochemical cues have been widely used to design and develop biomaterial systems for specific tissue engineering strategies. In this context, the patterning of piezoelectric polymers that can provide electroactive stimuli represents a suitable strategy for skeletal muscle tissue engineering applications once it has been demonstrated that mechanoelectrical stimuli promote C2C12 myoblast differentiation. In this sense, this works reports on how C2C12 myoblast cells detect and react to physical and biochemical stimuli based on micropatterned poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE)) electroactive scaffolds produced by soft lithography in the form of arrays of lines and hexagons (anisotropic and isotropic morphology, respectively) combined with differentiation medium. The scaffolds were evaluated for the proliferation and differentiation of C2C12 myoblast cell line and it is demonstrated that anisotropic microstructures promote muscle differentiation which is further reinforced with the introduction of biochemical stimulus. However, when the physical stimulus is not adequate to the tissue, e.g. isotropic microstructure, the biochemical stimulus has the opposite effect, hindering the differentiation process. Therefore, the proper morphological design of the scaffold combined with biochemical stimulus allows to enhance skeletal muscle differentiation and allows the development of advanced strategies for effective muscle tissue engineering.

Piezoelectric and Magnetically Responsive Biodegradable Composites with Tailored Porous Morphology for Biotechnological Applications.

The biomedical area in the scope of tissue regeneration pursues the development of advanced materials that can target biomimetic approaches and, ideally, have an active role in the environment they are placed in. This active role can be related to or driven by morphological, mechanical, electrical, or magnetic stimuli, among others. This work reports on the development of active biomaterials based on poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid), PHBV, a piezoelectric and biodegradable polymer, for tissue regeneration application by tailoring its morphology and functional response. PHBV films with different porosities were obtained using the solvent casting method, resorting to high-boiling-point solvents, as N,N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO), and the combination of chloroform (CF) and DMF for polymer dissolution. Furthermore, magnetoelectric biomaterials were obtained through the combination of the piezoelectric PHBV with magnetostrictive iron oxide (Fe3O4) nanoparticles. Independently of the morphology or filler content, all biomaterials proved to be suitable for biomedical applications.

Patterned Piezoelectric Scaffolds for Osteogenic Differentiation.

The morphological clues of scaffolds can determine cell behavior and, therefore, the patterning of electroactive polymers can be a suitable strategy for bone tissue engineering. In this way, this work reports on the influence of poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE)) electroactive micropatterned scaffolds on the proliferation and differentiation of bone cells. For that, micropatterned P(VDF-TrFE) scaffolds were produced by lithography in the form of arrays of lines and hexagons and then tested for cell proliferation and differentiation of pre-osteoblast cell line. Results show that more anisotropic surface microstructures promote bone differentiation without the need of further biochemical stimulation. Thus, the combination of specific patterns with the inherent electroactivity of materials provides a promising platform for bone regeneration.

Tailoring Electrospun Poly(l-lactic acid) Nanofibers as Substrates for Microfluidic Applications.

Novel microfluidic substrates based on electrospun poly(l-lactic acid) (PLLA) membranes were developed to increase the limited range of commercially available paper substrates, commonly used for the fabrication of microfluidic paper-based analytical devices. PLLA's advantageous properties include biodegradability, biocompatibility, ease of being processed in various tailored morphologies, and cost effectiveness, among others. Oriented and nonoriented electrospun PLLA membranes were fabricated using electrospinning and the influence of fiber orientation, addition of hydrophilic additives, and plasma treatments on the morphology, physicochemical properties, and capillary flow rates were evaluated and compared with the commercial Whatman paper. In addition, a proof-of-concept application based on the colorimetric detection of glucose in printed PLLA and paper-based microfluidic systems was also performed. The results show the potential of PLLA substrates for the fabrication of portable, disposable, eco-friendly, and cost-effective microfluidic systems with controllable properties that can be tailored according to specific biotechnological application requirements, being a suitable alternative to conventional paper-based substrates.

Tuning Myoblast and Preosteoblast Cell Adhesion Site, Orientation, and Elongation through Electroactive Micropatterned Scaffolds.

Electroactive polymers are being increasingly used in tissue engineering applications. Together with the electromechanical clues, morphological ones have been demonstrated to determine cell proliferation and differentiation. This work reports on the micropatterning of poly(vinylidene fluoride-co-trifluoroethylene), P(VDF-TrFE) scaffolds, and their interaction with myoblast and preosteoblasts cell lines, selected based on their different functional morphology. The scaffolds were obtained by soft lithography and obtained in the form of arrays of lines, intermittent lines, hexagons, linear zigzags, and curved zigzags with dimensions of 25, 75, and 150 µm. Moreover, the scaffolds were tested in cell adhesion assays of myoblasts and preosteoblasts cell lines. The results show that more linear surface topographies and dense morphology have a large potential in the regeneration of musculoskeletal tissue, while nonpatterned scaffolds or more anisotropic surface microstructures present largest potential to promote the growth and regeneration of bone tissue. In this way, cell adhesion site, orientation, and elongation can be controlled by choosing properly the topography and morphology of the scaffolds, indicating their suitability and potential for further proliferation and differentiation assays.

Tailored Biodegradable and Electroactive Poly(Hydroxybutyrate-Co-Hydroxyvalerate) Based Morphologies for Tissue Engineering Applications.

Polymer-based piezoelectric biomaterials have already proven their relevance for tissue engineering applications. Furthermore, the morphology of the scaffolds plays also an important role in cell proliferation and differentiation. The present work reports on poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), a biocompatible, biodegradable, and piezoelectric biopolymer that has been processed in different morphologies, including films, fibers, microspheres, and 3D scaffolds. The corresponding magnetically active PHBV-based composites were also produced. The effect of the morphology on physico-chemical, thermal, magnetic, and mechanical properties of pristine and composite samples was evaluated, as well as their cytotoxicity. It was observed that the morphology does not strongly affect the properties of the pristine samples but the introduction of cobalt ferrites induces changes in the degree of crystallinity that could affect the applicability of prepared biomaterials. Young's modulus is dependent of the morphology and also increases with the addition of cobalt ferrites. Both pristine and PHBV/cobalt ferrite composite samples are not cytotoxic, indicating their suitability for tissue engineering applications.