Cytocompatible and biodegradable poly(d,l-lactide-coglycolide)/reduced graphene oxide scaffolds.

Applications of graphene in regenerative medicine have attracted the increasing attention of numerous research groups due to the specific properties that confers on biomaterials. In this paper, the degradation behavior of poly(d,l-lactide-co-glycolide (PLGA)/reduced graphene oxide (rGO) scaffolds obtained by thermally induced phase separation (TIPS) and lyophilization was studied in phosphate buffered saline (PBS) solution, at 37 °C during eight weeks. Additionally, the cytotoxicity of the different samples through the metabolic activity of L929 fibroblast cells was also addressed. Scanning electron microscopy tests show that the addition of rGO particles increases the pore size from 60 to 100 µm as well as their morphological definition. Scaffolds with 0.6 and 1% rGO concentrations lost more mass than those with lower filler content, that is, they degraded more quickly. The results obtained by differential scanning calorimetry indicate that the rGO particles restrict the movement of the macromolecular chain segments due to the formation of hydrogen bonds and electrostatic attraction. The electrical conductivity tests show that the addition of rGO leads to a rapid transition from insulating to conductive scaffolds with a percolation value of ≈ 0.5 w/w. All the different PLGA samples with different rGO content up to 1% present no cytotoxic behaviour for L929 fibroblast cells, being therefor suitable for biomedical applications.

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.

Development of Silk Fibroin Scaffolds for Vascular Repair.

Silk fibroin (SF) is a biocompatible natural protein with excellent mechanical characteristics. SF-based biomaterials can be structured using a number of techniques, allowing the tuning of materials for specific biomedical applications. In this study, SF films, porous membranes, and electrospun membranes were produced using solvent-casting, salt-leaching, and electrospinning methodologies, respectively. SF-based materials were subjected to physicochemical and biological characterizations to determine their suitability for tissue regeneration applications. Mechanical analysis showed stress-strain curves of brittle materials in films and porous membranes, while electrospun membranes featured stress-strain curves typical of ductile materials. All samples showed similar chemical composition, melting transition, hydrophobic behavior, and low cytotoxicity levels, regardless of their architecture. Finally, all of the SF-based materials promote the proliferation of human umbilical vein endothelial cells (HUVECs). These findings demonstrate the different relationship between HUVEC behavior and the SF sample's topography, which can be taken advantage of for the design of vascular implants.

Development and evaluation of different electroactive poly(vinylidene fluoride) architectures for endothelial cell culture.

Tissue engineering (TE) aims to develop structures that improve or even replace the biological functions of tissues and organs. Mechanical properties, physical-chemical characteristics, biocompatibility, and biological performance of the materials are essential factors for their applicability in TE. Poly(vinylidene fluoride) (PVDF) is a thermoplastic polymer that exhibits good mechanical properties, high biocompatibility and excellent thermal properties. However, PVDF structuring, and the corresponding processing methods used for its preparation are known to significantly influence these characteristics. In this study, doctor blade, salt-leaching, and electrospinning processing methods were used to produce PVDF-based structures in the form of films, porous membranes, and fiber scaffolds, respectively. These PVDF scaffolds were subjected to a variety of characterizations and analyses, including physicochemical analysis, contact angle measurement, cytotoxicity assessment and cell proliferation. All prepared PVDF scaffolds are characterized by a mechanical response typical of ductile materials. PVDF films displayed mostly vibration modes for the a-phase, while the remaining PVDF samples were characterized by a higher content of electroactive ß-phase due the low temperature solvent evaporation during processing. No significant variations have been observed between the different PVDF membranes with respect to the melting transition. In addition, all analysed PVDF samples present a hydrophobic behavior. On the other hand, cytotoxicity assays confirm that cell viability is maintained independently of the architecture and processing method. Finally, all the PVDF samples promote human umbilical vein endothelial cells (HUVECs) proliferation, being higher on the PVDF film and electrospun randomly-oriented membranes. These findings demonstrated the importance of PVDF topography on HUVEC behavior, which can be used for the design of vascular implants.

Influence of rGO on the Crystallization Kinetics, Cytoxicity, and Electrical and Mechanical Properties of Poly (L-lactide-co-ε-caprolactone) Scaffolds.

Biodegradable scaffolds of poly (L-lactide-co-ε-caprolactone) (PLCL) and reduced graphene oxide (rGO) were prepared by TIPS (thermally induced phase separation). The nonisothermal cold crystallization kinetics were investigated by differential scanning calorimetry (DSC) with various cooling rates. The experimental values indicate that nonisothermal crystallization improves with cooling rate, but the increasing rGO concentration delays crystallization at higher temperatures. The activation energies were calculated by the Kissinger equation; the values were very similar for PLCL and for its compounds with rGO. The electrical conductivity measurements show that the addition of rGO leads to a rapid transition from insulating to conductive scaffolds with a percolation value of ≈0.4 w/w. Mechanical compression tests show that the addition of rGO improves the mechanical properties of porous substrates. In addition, it is an anisotropic material, especially at compositions of 1% w/w of rGO. All of the samples with different rGO content up to 1% are cytotoxic for C2C12 myoblast cells.

Understanding Myoblast Differentiation Pathways When Cultured on Electroactive Scaffolds through Proteomic Analysis.

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 Liquid-Based Materials for Biomedical Applications.

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.

Cytocompatible scaffolds of poly(L-lactide)/reduced graphene oxide for tissue engineering.

Poly(L-lactide)/reduced graphene oxide (PLLA/rGO) scaffolds were studied in the present work. The scaffolds were fabricated by TIPS (thermally induced phase separation). Nonisothermal crystallization study for PLLA/rGO was investigated and revealed the nucleating effect of rGO. rGO effect on cytotoxicity, thermal properties, and hydrolysis resistance of PLLA and PLLA/rGO scaffolds were analysed. In vitro degradation in phosphate-buffered solution at 37 °C is analyzed over twelve weeks. A high crystalline behaviour reduces the speed of hydrolysis and therefore implies less variation in pH, mass loss and water up take. The rGO does not seem to accelerate the degradation process. Finally, rGO contents in PLLA up to 1 wt% dos not lead to cytotoxic effect, the scaffolds supporting cell adhesion and proliferation.

Magnetic Nanoparticles for Biomedical Applications: From the Soul of the Earth to the Deep History of Ourselves.

Precisely engineered magnetic nanoparticles (MNPs) have been widely explored for applications including theragnostic platforms, drug delivery systems, biomaterial/device coatings, tissue engineering scaffolds, performance-enhanced therapeutic alternatives, and even in SARS-CoV-2 detection strips. Such popularity is due to their unique, challenging, and tailorable physicochemical/magnetic properties. Given the wide biomedical-related potential applications of MNPs, significant achievements have been reached and published (exponentially) in the last five years, both in synthesis and application tailoring. Within this review, and in addition to essential works in this field, we have focused on the latest representative reports regarding the biomedical use of MNPs including characteristics related to their oriented synthesis, tailored geometry, and designed multibiofunctionality. Further, actual trends, needs, and limitations of magnetic-based nanostructures for biomedical applications will also be discussed.

Effect of bacterial nanocellulose binding on the bactericidal activity of bovine lactoferrin.

Bovine lactoferrin (bLF) has been extensively described as a wide spectrum antimicrobial protein. bLF bactericidal activity has been mainly attributed to two different mechanisms: environmental iron depletion and cell membrane destabilization. Due to its antimicrobial properties, bLF has been included in the formulation nutraceutical food products and edible active packages. This work comprises the experimental evidence of the requirement of bLF unrestricted mobility ("free bLF") to effectively perform its bactericidal action. To assess the unrestricted and restricted bLF activity, a nontoxic matrix of bacterial nanocellulose (BNC) was used as carrier, and as an anchoring scaffold, respectively. Therefore, BNC was functionalized with bLF through two different methodologies: (i) bLF was embedded within the three-dimensional structure of BNC and; (ii) bLF was covalently bounded to the nanofibrils of BNC. bLF efficiency was tested against two bacteria isolated from clinical specimens, Escherichia coli and Staphylococcus aureus. bLF concentration after covalent binding to BNC was two-fold higher in comparison to the embedding method. Nevertheless, only the embedded bLF exhibited a significant bactericidal activity, due to bLF ability to permeate the BNC matrix and execute its bactericidal action.

Magnetically Activated Electroactive Microenvironments for Skeletal Muscle Tissue Regeneration.

This work reports on magnetoelectric biomaterials suitable for effective proliferation and differentiation of myoblast in a biomimetic microenvironment providing the electromechanical stimuli associated with this tissue in the human body. Magnetoelectric films are obtained by solvent casting through the combination of a piezoelectric polymer, poly(vinylidene fluoride-trifluoro-ethylene), and magnetostrictive particles (CoFe2O4). The nonpoled and poled (with negative and positive surface charge) magnetoelectric composites are used to investigate their influence on C2C12 myoblast adhesion, proliferation, and differentiation. It is demonstrated that the proliferation and differentiation of the cells are enhanced by the application of mechanical and/or electrical stimulation, with higher values of maturation index under mechanoelectrical stimuli. These results show that magnetoelectric cell stimulation is a full potential approach for skeletal muscle tissue engineering applications.

Surface Charge-Mediated Cell-Surface Interaction on Piezoelectric Materials.

Cell-material interactions play an essential role in the development of scaffold-based tissue engineering strategies. Cell therapies are still limited in treating injuries when severe damage causes irreversible loss of muscle cells. Electroactive biomaterials and, in particular, piezoelectric materials offer new opportunities for skeletal muscle tissue engineering since these materials have demonstrated suitable electroactive microenvironments for tissue development. In this study, the influence of the surface charge of piezoelectric poly(vinylidene fluoride) (PVDF) on cell adhesion was investigated. The cytoskeletal organization of C2C12 myoblast cells grown on different PVDF samples was studied by immunofluorescence staining, and the interactions between single live cells and PVDF were analyzed using an atomic force microscopy (AFM) technique termed single-cell force spectroscopy. It was demonstrated that C2C12 myoblast cells seeded on samples with net surface charge present a more elongated morphology, this effect being dependent on the surface charge but independent of the poling direction (negative or positive surface charge). It was further shown that the cell deadhesion forces of individual C2C12 cells were higher on PVDF samples with an overall negative surface charge (8.92 ± 0.45 nN) compared to those on nonpoled substrates (zero overall surface charge) (4.06 ± 0.20 nN). These findings explicitly demonstrate that the polarization/surface charge is an important parameter to determine cell fate as it affects C2C12 cell adhesion, which in turn will influence cell behavior, namely, cell proliferation and differentiation.

3D Cytocompatible Composites of PCL/magnetite.

A study of Magnetite (Fe3O4) as a suitable matrix for the improved adhesion and proliferation of MC3T3-E1 pre-osteoblast cells in bone regeneration is presented. Biodegradable and magnetic polycaprolactone (PCL)/magnetite (Fe3O4) scaffolds, which were fabricated by Thermally Induced Phase Separation, are likewise analyzed. Various techniques are used to investigate in vitro degradation at 37 °C, over 104 weeks, in a phosphate buffered saline (PBS) solution. Magnetic measurements that were performed at physiological temperature (310 K) indicated that degradation neither modified the nature nor the distribution of the magnetite nanoparticles. The coercive field strength of the porous matrices demonstrated ferromagnetic behavior and the probable presence of particle interactions. The added nanoparticles facilitated the absorption of PBS, with no considerable increase in matrix degradation rates, as shown by the Gel Permeation Chromatography (GPC) results for Mw, Mn, and I. There was no collapse of the scaffold structures that maintained their structural integrity. Their suitability for bone regeneration was also supported by the absence of matrix cytotoxicity in assays, even after additions of up to 20% magnetite.

A New Approach for the Fabrication of Cytocompatible PLLA-Magnetite Nanoparticle Composite Scaffolds.

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.

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.

Multifunctional Platform Based on Electroactive Polymers and Silica Nanoparticles for Tissue Engineering Applications.

Poly(vinylidene fluoride) nanocomposites processed with different morphologies, such as porous and non-porous films and fibres, have been prepared with silica nanoparticles (SiNPs) of varying diameter (17, 100, 160 and 300 nm), which in turn have encapsulated perylenediimide (PDI), a fluorescent molecule. The structural, morphological, optical, thermal, and mechanical properties of the nanocomposites, with SiNP filler concentration up to 16 wt %, were evaluated. Furthermore, cytotoxicity and cell proliferation studies were performed. All SiNPs are negatively charged independently of the pH and more stable from pH 5 upwards. The introduction of SiNPs within the polymer matrix increases the contact angle independently of the nanoparticle diameter. Moreover, the smallest ones (17 nm) also improve the PVDF Young's modulus. The filler diameter, physico-chemical, thermal and mechanical properties of the polymer matrix were not significantly affected. Finally, the SiNPs' inclusion does not induce cytotoxicity in murine myoblasts (C2C12) after 72 h of contact and proliferation studies reveal that the prepared composites represent a suitable platform for tissue engineering applications, as they allow us to combine the biocompatibility and piezoelectricity of the polymer with the possible functionalization and drug encapsulation and release of the SiNP.

Development of Magnetically Active Scaffolds for Bone Regeneration.

This work reports on the synthesis, with the thermally induced phase separation (TIPS) technique, of poly (l-lactide) (PLLA) scaffolds containing Fe-doped hydroxyapatite (FeHA) particles for bone regeneration. Magnetization curves and X-ray diffraction indicate two magnetic particle phases: FeHA and magnetite Fe3O4. Magnetic nanoparticles (MNPs) are approximately 30 ± 5 nm in width and 125 ± 25 nm in length, and show typical ferromagnetic properties, including coercivity and rapid saturation magnetization. Scanning electron microscopy (SEM) images of the magnetic scaffolds reveal their complex morphology changes with MNP concentration. Similarly, at compositions of approximately 20% MNPs, the phase separation changes, passing from solid⁻liquid to liquid⁻liquid as revealed by the hill-like structures, with low peaks that give the walls in the SEM images a surface pattern of micro-ruggedness typical of nucleation mechanisms and growth. In vitro degradation experiments, carried out for more than 28 weeks, demonstrated that the MNPs delay the scaffold degradation process. Cytotoxicity is appreciated for FeHA content above 20%.