Sylvie Ribeiro

This work investigates the role of surface charge and synergistic mechano‐electrical stimulation of piezoelectric poly(vinylidene fluoride) (PVDF) on the behaviour of H9c2 cardiac cells. H9c2 cells preferentially adhered to poled PVDF samples, regardless of polarity, indicating that increased average surface charge creates a favourable environment for initial attachment. Proliferation assays showed that negatively charged PVDF films promote the highest early growth, while dynamic mechanical stimulation (1 h/day) further enhanced proliferation, particularly on positively poled PVDF, highlighting a synergistic effect between charge and mechanical cues. Differentiation assays demonstrated that stimulation applied during either the proliferation or differentiation phase promoted myotube formation, increasing length, width, and alignment, with negatively poled PVDF supporting the most advanced maturation. At the molecular level, qPCR confirmed upregulation of the early cardiomyogenic marker actc1, whereas the late marker Tnnt2 remained unchanged, indicating partial but incomplete cardiomyocyte differentiation. These results reveal that both static polarization and dynamic piezoelectric stimulation cooperatively modulate cell fate decisions, integrating electrical and mechanical signalling pathways. Together, these findings demonstrate that surface polarization and mechano‐electrical cues of piezoelectric biomaterials act in concert to regulate cardiomyoblast adhesion, proliferation, and differentiation, unveiling charge‐dependent design principles for multifunctional electroactive materials aimed at cardiac tissue regeneration.

A promising method for directing cell behavior and tissue regeneration is the use of smart materials that can transform physical inputs into bioelectrical signals. In this study, the mechanoelectrical control of preosteoblast activity was investigated using a piezoelectric smart biointerface based on positively poled poly(vinylidene fluoride) (PVDF). Distinct mechanical regimes, including vibrational and cyclic stretching, were applied through customized bioreactors, enabling controlled mechanoelectrical inputs ranging from 63 to 227 μVpp mm^-2. The biological response of MC3T3-E1 cells was evaluated in terms of metabolic activity, intracellular calcium signaling, alkaline phosphatase (ALP) activity, matrix mineralization, and gene expression (RUNX2, ALP, OPN, and OCN). The results demonstrated that stretching stimulation combined with higher mechano electric inputs (113-227 μVpp mm^-2) enhanced calcium influx and enhanced osteogenic differentiation, while lower impulses (∼63 μVpp mm^-2) under vibrational circumstances increased cell proliferation. These findings highlight the intensity- and mode-dependent nature of mechanoelectrical signaling in regulating osteogenic commitment. All things considered, this study shows how piezoelectric smart materials can be used as bioresponsive platforms to precisely control cell proliferation and differentiation, creating avenues for bone tissue engineering's next-generation regenerative techniques.

This study investigates the potential of porous poly(D,L-lactide) (PDLLA) scaffolds reinforced with graphene oxide (GO) for bone tissue engineering applications. Scaffolds were fabricated using thermally induced phase separation (TIPS) and characterized in terms of morphology, biodegradation, thermal and mechanical properties, and cytocompatibility. The incorporation of GO enhanced both mechanical strength and thermal stability, likely due to hydrogen bonding and electrostatic interactions between GO’s functional groups (carbonyl, carboxyl, epoxide, and hydroxyl) and PDLLA chains. In vitro degradation studies showed that GO accelerated degradation, while scaffolds with higher GO content retained superior mechanical strength. Cytotoxicity assays confirmed the biocompatibility of all scaffold variants, supporting their suitability for biomedical applications. Overall, the findings demonstrate how GO incorporation can modulate scaffold composition and performance. This provides insights for the design of improved systems for bone tissue regeneration.

Electroactive materials allow the modulation of cell-materials interactions and cell fate, leading to advanced tissue regeneration strategies. Nevertheless, their effect at the cellular level is still poorly understood. In this context, the proteome analysis of C2C12 cell differentiation cultured on piezoelectric polymer films with null average surface charge (non-poled), net positive surface charge (poled +), and net negative surface charge (poled -) has been addressed. Protein/pathway alterations for skeletal muscle development were identified comparing proteomic profiles of C2C12 cells differentiated on poly(vinylidene fluoride), with similar cells differentiated on a polystyrene plate (control), using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). Only significantly expressed proteins (P < 0.01, analysis of variance) were used for bioinformatic analyses. A total of 37 significantly expressed proteins were detected on the C2C12 proteome with PVDF "poled -" at 24 h, whereas on the PVDF "poled +", a total of 105 significantly expressed proteins were considered. At 5 days of differentiation, the number of significantly expressed proteins decreased to 23 and 31 in cells grown on negative and positive surface charge, respectively, the influence of surface charge being more explicit in some proteins. In both cases, proteins such as Fbn1, Hspg2, Rcn3, Tgm2, Mylpf, Anxa2, and Anxa6, involved in calcium-related signaling, were highly expressed during myoblast differentiation. Furthermore, some proteins involved in muscle contraction (Acta2, Anxa2, and Anxa6) were detected in the PVDF "poled +" sample. Upregulation of several proteins that enhance skeletal muscle development was detected in the PVDF "poled -" sample, including Ckm (422%), Tmem14c (384%), Serpinb6a (460%), adh7 (199%), and Car3 (171%), while for the "poled +" samples, these proteins were also upregulated at a smaller magnitude (254, 317, 253, 123, and 72%, respectively). Other differentially expressed proteins such as Mylpf (189%), Mybph (168%), and Mbnl1 (168%) were upregulated only in PVDF "poled -" samples, while Hba-a1 levels (581%) were increased in the PVDF "poled +" sample. On the other hand, cells cultured on non-poled samples have no differences with respect to the ones cultured on the control, in contrary to the poled films, with overall surface charge, demonstrating the relevance of scaffold surface charge on cell behavior. This study demonstrates that both positive and negative overall surface charges promote the differentiation of C2C12 cells through involvement of proteins related with the contraction of the skeletal muscle cells, with a more pronounced effect with the negative charged surfaces.

Ionic liquids (ILs) have been extensively explored and implemented in different areas, ranging from sensors and actuators to the biomedical field. The increasing attention devoted to ILs centers on their unique properties and possible combination of different cations and anions, allowing the development of materials with specific functionalities and requirements for applications. Particularly for biomedical applications, ILs have been used for biomaterials preparation, improving dissolution and processability, and have been combined with natural and synthetic polymer matrixes to develop IL-polymer hybrid materials to be employed in different fields of the biomedical area. This review focus on recent advances concerning the role of ILs in the development of biomaterials and their combination with natural and synthetic polymers for different biomedical areas, including drug delivery, cancer therapy, tissue engineering, antimicrobial and antifungal agents, and biosensing.

Tissue engineering (TE) is a strongly expanding research area. TE approaches require biocompatible scaffolds, cells, and different applied stimuli, which altogether mimic the natural tissue microenvironment. Also, the extracellular matrix serves as a structural base for cells and as a source of growth factors and biophysical cues. The 3D characteristics of the microenvironment is one of the most recognized key factors for obtaining specific cell responses in vivo, being the physical cues increasingly investigated. Supporting those advances is the progress of smart and multifunctional materials design, whose properties improve the cell behavior control through the possibility of providing specific chemical and physical stimuli to the cellular environment. In this sense, a varying set of bioreactors that properly stimulate those materials and cells in vitro, creating an appropriate biomimetic microenvironment, is developed to obtain active bioreactors. This review provides a comprehensive overview on the important microenvironments of different cells and tissues, the smart materials type used for providing such microenvironments and the specific bioreactor technologies that allow subjecting the cells/tissues to the required biomimetic biochemical and biophysical cues. Further, it is shown that microfluidic bioreactors represent a growing and interesting field that hold great promise for achieving suitable TE strategies.

Magnetic biomimetic scaffolds of poly(L-lactide) (PLLA) and nanoparticles of magnetite (nFe₃O₄) are prepared in a wide ratio of compositions by lyophilization for bone regeneration. The magnetic properties, cytotoxicity, and the in vitro degradation of these porous materials are closely studied. The addition of magnetite at 50 °C was found to produce an interaction reaction between the ester groups of the PLLA and the metallic cations of the magnetite, causing the formation of complexes. This fact was confirmed by the analysis of the infrared spectroscopy and the gel permeation chromatography test results. They, respectively, showed a displacement of the absorption bands of the carbonyl group (C=O) of the PLLA and a scission of the polymer chains. The iron from the magnetite acted as a catalyser of the macromolecular scission reaction, which determines the final biomedical applications of the scaffolds—it does so because the reaction shortens the degradation process without appearing to influence its toxicity. None of the samples studied in the tests presented cytotoxicity, even at 70% magnetite concentrations.

Biodegradable poly(l‐lactide‐co‐glycolide) (PLGA)/multiwalled carbon nanotubes (MWCNTs) scaffolds produced by thermally induced phase separation (TIPS) are studied for bone regeneration. Their magnetic properties, cytotoxicity, and in vitro degradation are investigated. Certain properties are analyzed at 37 °C over 16 weeks in phosphate buffer saline (PBS) solution, as a function of degradation time: morphology, mass loss, pH value of PBS, and thermal behavior. The presence of small quantities of nanotubes in the scaffolds, ≤0.5 wt %, leads to a weak magnetic response although the PLGA was diamagnetic. The incorporation of MWCNTs in the scaffolds generated a morphology and a very different process of in vitro degradation than might be expected in a PLGA scaffold. The in vitro degradation process started on week 13 and rapidly advanced, although the structural integrity of the scaffolds was maintained and no collapse of the structure occurred. Cytotoxicity tests on the samples showed cytotoxicity behavior at concentrations of over 0.3 wt % MWCNTs. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020, 137, 48439.

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 micro patterning of poly(vinylidene fluoride-co-trifluoroethylene) - P(VDF-TrFE) scaffolds - and their interaction with myoblast and pre-osteoblasts 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 pre-osteoblasts cell lines. The results show that more linear surface topographies and dense morphology have a large potential in the regeneration of musculoskeletal tissue while non-patterned 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.