Protein-engineered biomaterials represent a powerful approach to increase biofunctional activity like tissue repair and celular proliferation. Among these materials, integrins and the development of their specific interactions with plasma-polymerized pyrrole (PPPy) are promising biomaterial for tissue regeneration. In this paper, we studied the molecular recognition in the active site of three integrins (α5β1, αvβ3 and αIIbβ3) with PPPy using the structure proposed by Kumar et al . PPPy molecule has three sites to incorporate different species, we worked mainly with the functional groups, –NH 2 and –OH groups according to our IR spectroscopic results. We carried out docking studies to find the better conformational couplings and to determine electrostatic (ΔG elec ) and non-electrostatic (ΔG non-elec ) contributions to the binding free energy (ΔG b ) of these complexes we used Adaptive Poisson-Bolztmann program (APBS). Our results indicated that when incorporating -1H-azirine, -NH 2 or –OH group in PPPy structure, interactions with integrins were favorable, as indicated by correspondent ΔG b values. These interactions were mainly triggered by Coulomb interactions, an important term in the electrostatic component. Furthermore, our studies suggest that some residues of integrins α5β1, αvβ3 and αIIbβ3 like aspartates are important for the binding to PPPy structures. Detailed interactions between integrin α5β1 and PPPy structures were revealed by molecular dynamics simulations. We used this particular integrin structure because of its favorable ΔG b as well as its major cellular receptor for the extracellular matrix protein fibronectin. Clustering analysis allowed us to carry out focused docking studies and to determine the time evolution of the ΔG b values. By incorporating -NH 2 into PPPy structure, ΔG b values were very favorable during the course of the dynamics simulations by the establishment of hydrogen bonds with Asn224 and/or Asp227 residues, which are part of the integrin α5β1 pocket. However, for the integrin α5β1-PPPy-1H-azirine complex and the rest of the functional groups, the ΔG b values were less favorable, although PPPy was found at a distance of less than 5 Å from the active site residues. This work is complementary to the previous studies made employing PPPy nanoparticles for a variety of tissue engineering applications, and were done to enlighten the role played by the amino group of the PPPy in its integrin recognition process.

No abstract

It is well known that, in excitable cells, the dynamics of the ion currents (I i) is extremely important to determine both the magnitude and time course of an action potential (A p). To observe these two processes simultaneously, we cultured NG108-15 cells over a multi-walled carbon nanotubes electrode (MWCNTe) surface and arranged a two independent Patch Clamp system configuration (Bi-Patch Clamp). The first system was used in the voltage or current clamp mode, using a glass micropipette as an electrode. The second system was modified to connect the MWCNTe to virtual ground. While the A p was recorded through the micropipette electrode, the MWCNTe was used to measure the underlying whole-cell current. This configuration allowed us to record both the membrane voltage (V m) and the current changes simultaneously. Images acquired by atomic force microscopy (AFM) and scanning electron microscopy (SEM) indicate that cultured cells developed a complex network of neurites, which served to establish the necessary close contact and strong adhesion to the MWCNTe surface. These features were a key factor to obtain the recording of the whole-cell I i with a high signal to noise ratio (SNR). The experimental results were satisfactorily reproduced by a theoretical model developed to simulate the proposed system. Besides the contribution to a better understanding of the fundamental mechanisms involved in cell communication, the developed method could be useful in cell physiology studies, pharmacology and diseases diagnosis.

Carbon nanotubes have been used as scaffolds for tissue engineering. However, the identification of these nanomaterials disperses in biological solutions and their direct interaction with nerve cells is still controversial. The aim of this work is to characterize the topographic and ultra-structural distribution of the composite made of multi wall carbon nanotubes-polyvinylpyrrolidone MWCNTs-PVP, fixing it in paraformaldehyde at 4% and was observed with SEM and CM. Although the nanotubes were not labeled or stained with fluorescent compounds, the MWCNTs-PVP deposited on glass and on nerve cells, was observed by the confocal microscope in the reflection mode. In SEM studies, it was observed that MWCNTs-PVP was attached to the surface on neurons. Moreover, in CM studies, it was possible to observe that MWCNTs-PVP was attached to the neuronal membrane, crossing the cell membrane and getting into the cytoplasm. These results support the hypothesis that carbon nanotubes interact with the neuronal cell membrane and can be useful for neuronal tissue engineering. In addition, these results open new alternatives for toxicological studies, in order to elucidate the cytotoxicity of MWCNTs-PVP composite in neurons and other excitable cells.

This paper shows the simultaneous recording of electrical activity and the underlying ionic currents by using a gold substrate to culture NG108-15 cells. Cells grown on two different substrates (plastic Petri dishes and gold substrates) were characterized quantitatively through scanning electron microscopy (SEM) as well as qualitatively by optical and atomic force microscopy (AFM). No significant differences were observed between the surface area of cells cultured on gold substrates and Petri dishes, as indicated by measurements performed on SEM images. We also evaluated the electrophysiological compatibility of the cells through standard patch-clamp experiments by analyzing features such as the resting potential, membrane resistance, ionic currents, etc. Cells grown on both substrates showed no significant differences in their dependency on voltage, as well as in the magnitude of the Na+ and K+ current density; however, cells cultured on the gold substrate showed a lower membrane capacitance when compared to those grown on Petri dishes. By using two separate patch-clamp amplifiers, we were able to record the membrane current with the conventional patch-clamp technique and through the gold substrate simultaneously. Furthermore, the proposed technique allowed us to obtain simultaneous recordings of the electrical activity (such as action potentials firing) and the underlying membrane ionic currents. The excellent conductivity of gold makes it possible to overcome important difficulties found in conventional electrophysiological experiments such as those presented by the resistance of the electrolytic bath solution. We conclude that the technique here presented constitutes a solution to the problem of the simultaneous recording of electrical activity and the underlying ionic currents, which for decades, had been solved only partially.