Osteogenic differentiation of human mesenchymal stem cells on electroactive substrates.

This study investigates the effect of electroactivity and electrical charge distribution on the biological response of human bone marrow stem cells (hBMSCs) cultured in monolayer on flat poly(vinylidene fluoride), PVDF, substrates. Differences in cell behaviour, including proliferation, expression of multipotency markers CD90, CD105 and CD73, and expression of genes characteristic of different mesenchymal lineages, were observed both during expansion in basal medium before reaching confluence and in confluent cultures in osteogenic induction medium. The crystallisation of PVDF in the electrically neutral α-phase or in the electroactive phase ß, both unpoled and poled, has been found to have an important influence on the biological response. In addition, the presence of a permanent positive or negative surface electrical charge distribution in phase ß substrates has also shown a significant effect on cell behaviour.

Electroactive calcium-alginate/polycaprolactone/reduced graphene oxide nanohybrid hydrogels for skeletal muscle tissue engineering.

Graphene derivatives such as reduced graphene oxide (rGO) are used as components of novel biomaterials for their unique electrical properties. Electrical conductivity is a crucial factor for muscle cells, which are electrically active. This study reports the development of a new type of semi-interpenetrated polymer network based on two biodegradable FDA-approved biomaterials, sodium alginate (SA) and polycaprolactone (PCL), with Ca2+ ions as SA crosslinker. Several drawbacks such as the low cell adhesion of SA and weak structural stability can be improved with the incorporation of PCL. Furthermore, this study demonstrates how this semi-IPN can be engineered with rGO nanosheets (0.5% and 2% wt/wt rGO nanosheets) to produce electroactive nanohybrid composite biomaterials. The study focuses on the microstructure and the enhancement of physical and biological properties of these advanced materials, including water sorption, surface wettability, thermal behavior and thermal degradation, mechanical properties, electrical conductivity, cell adhesion and myogenic differentiation. The results suggest the formation of a complex nano-network with different interactions between the components: bonds between SA chains induced by Ca2+ ions (egg-box model), links between rGO nanosheets and SA chains as well as between rGO nanosheets themselves through Ca2+ ions, and strong hydrogen bonding between rGO nanosheets and SA chains. The incorporation of rGO significantly increases the electrical conductivity of the nanohybrid hydrogels, with values in the range of muscle tissue. In vitro cultures with C2C12 murine myoblasts revealed that the conductive nanohybrid hydrogels are not cytotoxic and can greatly enhance myoblast adhesion and myogenic differentiation. These results indicate that these novel electroactive nanohybrid hydrogels have great potential for biomedical applications related to the regeneration of electroactive tissues, particularly in skeletal muscle tissue engineering.

Fluctuations of conformational mobility of macromolecules around the glass transition.

The heterogeneity of local dynamics in disordered systems is behind some key features of glass transition. In order to improve our understanding of the molecular dynamics in disordered systems in the vicinity of the glass transition, different parameters have been proposed to quantitatively describe dynamical heterogeneity. In the case of polymers, free volume models relate the macromolecular mobility to the free or accessible volume. The relationship between dynamic heterogeneity and fluctuations of accessible volume seems straightforward. In the present work, the heterogeneity of local dynamics in polymeric systems is analyzed by computer simulation with the bond fluctuation model. The value of the accessible volume around each polymer chain is evaluated from a snapshot or static structure at each system state, resulting in a distribution of accessible volume that reflects system heterogeneity. The relationship between the average value and the standard deviation of free volume distributions at different temperatures fits a master curve for different systems, regardless of the specific inter- and intramolecular interaction potentials that define each material. The dynamic slowdown around the glass transition is accompanied by a clear evolution of the mean value and shape of the accessible free volume distribution. The relative fluctuation of the dynamically accessible volume has been used as a parameter to quantitatively describe heterogeneity. The fluctuation varies with temperature with remarkable differences between the liquid and glassy states of the systems studied, presenting a peak at the glass transition temperature, which can be interpreted as a reflection of the distribution of local glass transition temperatures.

Human Mesenchymal Stem Cells Growth and Osteogenic Differentiation on Piezoelectric Poly(vinylidene fluoride) Microsphere Substrates.

The aim of this work was to determine the influence of the biomaterial environment on human mesenchymal stem cell (hMSC) fate when cultured in supports with varying topography. Poly(vinylidene fluoride) (PVDF) culture supports were prepared with structures ranging between 2D and 3D, based on PVDF films on which PVDF microspheres were deposited with varying surface density. Maintenance of multipotentiality when cultured in expansion medium was studied by flow cytometry monitoring the expression of characteristic hMSCs markers, and revealed that cells were losing their characteristic surface markers on these supports. Cell morphology was assessed by scanning electron microscopy (SEM). Alkaline phosphatase activity was also assessed after seven days of culture on expansion medium. On the other hand, osteoblastic differentiation was monitored while culturing in osteogenic medium after cells reached confluence. Osteocalcin immunocytochemistry and alizarin red assays were performed. We show that flow cytometry is a suitable technique for the study of the differentiation of hMSC seeded onto biomaterials, giving a quantitative reliable analysis of hMSC-associated markers. We also show that electrosprayed piezoelectric poly(vinylidene fluoride) is a suitable support for tissue engineering purposes, as hMSCs can proliferate, be viable and undergo osteogenic differentiation when chemically stimulated.

Relaxation dynamics of poly(vinylidene fluoride) studied by dynamical mechanical measurements and dielectric spectroscopy.

The aim of this study is to analyze the mobility of polymer chains in semicrystalline poly(vinylidene fluoride) (PVDF). PVDF crystallizes from the melt in the α crystalline phase. The transformation from the α phase to the electroactive ß phase can be induced by stretching at temperatures in the range between 80 and 140 °C. The spherulitic structure of the crystalline phase is deformed during stretching to form fibrils oriented in the direction of the strain. The amorphous phase confined among the crystalline lamellae is distorted as well and some degree of orientation of the polymer chains is expected. Dynamic-mechanical and dielectric spectroscopy measurements were performed in PVDF films stretched to strain ratios up to 5 at temperatures between 80 and 140 °C. Dynamic-mechanical measurements were conducted between -60 °C and melting and in this temperature range the relaxation spectra show the main relaxation of the amorphous phase (called ß-relaxation) and at higher temperatures a relaxation related to crystallites motions (α (c)-relaxation). Although the mean relaxation times of the ß-relaxation are nearly equal in PVDF before and after crystal phase transformation, a significant change of shape of the relaxation spectrum proves the effect of chain distortion due to crystal reorganization. In stretched PVDF the elastic modulus of the polymer in the direction of deformation is significantly higher than in the transversal one, as expected by chain and crystals fibril orientation. The recovery of the deformation when the sample is heated is related with the appearance of the α (c)-relaxation. Dielectric spectroscopy spectrum shows the main relaxation of the amorphous phase and a secondary process (γ-relaxation) at lower temperatures. Stretching produces significant changes in the relaxation processes, mainly in the strength and shape of the main relaxation ß. The Havriliak-Negami function has been applied to analyze the dielectric response.

Glass transition and dynamics in BSA-water mixtures over wide ranges of composition studied by thermal and dielectric techniques.

Protein-water dynamics in mixtures of water and a globular protein, bovine serum albumin (BSA), was studied over wide ranges of composition, in the form of solutions or hydrated solid pellets, by differential scanning calorimetry (DSC), thermally stimulated depolarization current technique (TSDC) and dielectric relaxation spectroscopy (DRS). Additionally, water equilibrium sorption isotherm (ESI) measurements were performed at room temperature. The crystallization and melting events were studied by DSC and the amount of uncrystallized water was calculated by the enthalpy of melting during heating. The glass transition of the system was detected by DSC for water contents higher than the critical water content corresponding to the formation of the first sorption layer of water molecules directly bound to primary hydration sites, namely 0.073 (grams of water per grams of dry protein), estimated by ESI. A strong plasticization of the T(g) was observed by DSC for hydration levels lower than those necessary for crystallization of water during cooling, i.e. lower than about 0.3 (grams of water per grams of hydrated protein) followed by a stabilization of T(g) at about -80°C for higher water contents. The α relaxation associated with the glass transition was also observed in dielectric measurements. In TSDC a microphase separation could be detected resulting in double T(g) for some hydration levels. A dielectric relaxation of small polar groups of the protein plasticized by water, overlapped by relaxations of uncrystallized water molecules, and a separate relaxation of water in the crystallized water phase (bulk ice crystals) were also recorded.

Molecular mobility in biodegradable poly(ε-caprolactone)/poly(hydroxyethyl acrylate) networks.

Poly(ε-caprolactone)/poly(hydroxyethyl acrylate) networks have been investigated by thermally stimulated depolarization currents (TSDC) and differential scanning calorimetry (DSC). The introduction of hydrophilic units (HEA) in the system aiming at tailoring the hydrophilicity of the system results in a series of copolymer networks with microphase separation into hydrophobic/hydrophilic domains. Polycaprolactone (PCL) crystallization is prevented by the topological constraints HEA units imposed in such heterogeneous domains. Moreover, the mobility of the amorphous PCL chains is enhanced as revealed by the main relaxation process which becomes faster. The glass transition of PHEA-rich domains shifts to lower temperatures, as the total amount of PCL in the copolymer increases, due to the presence of PCL units within the same region. The behaviour of the copolymer networks swollen with different content of water has been investigated to analyze the interaction between water molecules and hydrophobic/hydrophilic domains and provide further insights into the molecular structure of the system.

Dielectric relaxation spectrum of poly (epsilon-caprolactone) networks hydrophilized by copolymerization with 2-hydroxyethyl acrylate.

The dielectric relaxation spectrum of polycaprolactone (PCL) networks hydrophilized with different amounts of 2-hydroxyethyl acrylate (HEA) is investigated. PCL is a semicrystalline polyester with a complex relaxation spectrum that includes the main alpha relaxation and two secondary modes (beta, gamma) at lower temperatures. The overlapping of the different relaxational modes was split by using several Havriliak-Negami functions. Crosslinking the material modifies the dynamics of the main relaxation process as reflected by the parameters that characterize the Vogel behavior of the process and the dynamic fragility. The incorporation of HEA units in the network results in a material with microphase separation: two alpha processes are detected, the one corresponding to the PCL chains and the new one associated to nanometric regions that contain different amount of both comonomers. The incorporation of the HEA units in the system involves the presence of a new beta(sw) relaxation due to the link of two side chains by water molecules through hydrogen bonding.