Structural and Functional Modulation of Perineuronal Nets: In Search of Important Players with Highlight on Tenascins.

The extracellular matrix (ECM) of the brain plays a crucial role in providing optimal conditions for neuronal function. Interactions between neurons and a specialized form of ECM, perineuronal nets (PNN), are considered a key mechanism for the regulation of brain plasticity. Such an assembly of interconnected structural and regulatory molecules has a prominent role in the control of synaptic plasticity. In this review, we discuss novel ways of studying the interplay between PNN and its regulatory components, particularly tenascins, in the processes of synaptic plasticity, mechanotransduction, and neurogenesis. Since enhanced neuronal activity promotes PNN degradation, it is possible to study PNN remodeling as a dynamical change in the expression and organization of its constituents that is reflected in its ultrastructure. The discovery of these subtle modifications is enabled by the development of super-resolution microscopy and advanced methods of image analysis.

Mechanobiology of cells and cell systems, such as organoids.

Organoids are in vitro 3D self-organizing tissues that mimic embryogenesis. Organoid research is advancing at a tremendous pace, since it offers great opportunities for disease modeling, drug development and screening, personalized medicine, as well as understanding organogenesis. Mechanobiology of organoids is an unexplored area, which can shed light to several unexplained aspects of self-organization behavior in organogenesis. It is becoming evident that collective cell behavior is distinctly different from individual cells' conduct against certain stimulants. Inherently consisting of higher number of degrees of freedom for cell motility and more complex cell-to-cell and cell-to-extracellular matrix behavior, understanding mechanotransduction in organoids is even more challenging compared with cell communities in 2D culture conditions. Yet, deciphering mechanobiology of organoids can help us understand effects of mechanical cues in health and disease, and translate findings of basic research toward clinical diagnosis and therapy.

Hemocompatibility of amyloid and/or brain targeted liposomes.

Targeted liposomes with different combinations of five ligands (for brain/amyloid targeting) were evaluated for hemocompatibility. Results reveal that all liposomes studied, caused minimum hemolysis; targeted liposomes slightly reduced blood coagulation time, but not significantly more than control liposomes; and compliment factors SC5b9 and iC3b increased when compared with the buffer, by most targeted liposomes. However, the specific amounts of both factors were similar with those induced by control liposomes. Thus, the targeted liposomes are unanticipated to cause hypersensitivity problems. Good correlations between vesicle size and produced factor amounts were observed. In conclusion, the current targeted liposomes are not expected to cause serious blood toxicity, if used in vivo.

Standardized Nanomechanical Atomic Force Microscopy Procedure (SNAP) for Measuring Soft and Biological Samples.

We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions.

Effect of Immobilized Thiolated Glycosaminoglycans on Fibronectin Adsorption and Behavior of Fibroblasts.

Glycosaminoglycans (GAGs) chondroitin sulfate, heparin, hyaluronan, and sulfated hyaluronan are lower and higher thiolated to enable a one-step covalent modification of gold or vinyl-terminated surfaces. Measurements of water contact angle and zeta potentials reveal that sulfated GAG-modified surfaces are more wettable and possess a negative surface potential. Additionally, higher thiolated GAGs (tGAGs) exhibit increased wettability and higher surface roughness. Fibronectin (FN) adsorption increases with sulfation degree of tGAGs. The tGAG-functionalized surfaces with higher degree of sulfation promote fibroblast adhesion most under serum-free conditions. The preadsorption of FN allows for more cell adhesion on tGAG surfaces. Metabolic activity measurements show that cell growth is enhanced for tGAGs up to a certain thiolation degree. Overall, thiolation of GAGs does not hamper their bioactivity toward proteins and cells, which make them highly interesting for biomimetic surface modification of implants and tissue engineering scaffolds.

Tuning cell adhesion and growth on biomimetic polyelectrolyte multilayers by variation of pH during layer-by-layer assembly.

Polyelectrolyte multilayers of chitosan and heparin are assembled on glass where heparin is applied at pH = 4, 9 and 4 during the formation of the first layers followed by pH = 9 at the last steps (denoted pH 4 + 9). Measurements of wetting properties, layer mass, and topography show that multilayers formed at pH = 4 are thicker, contain more water and have a smoother surface compared to those prepared at pH = 9 while the pH = 4 + 9 multilayers expressed intermediate properties. pH = 9 multilayers are more cell adhesive and support growth of C2C12 cells better than pH = 4 ones. However, pH 4 + 9 conditions improve the bioactivity to a similar level of pH = 9 layers. Multilayers prepared using pH 4 + 9 conditions form thick enough layers that may allow efficient loading of bioactive molecules.

Haemocompatibility improvement of metallic surfaces by covalent immobilization of heparin-liposomes.

Stainless steel surfaces were processed by means of plasma enhanced chemical vapor deposition (PE-CVD) fed with acrylic acid vapors in order to functionalize them with carboxyl groups, which were subsequently activated for covalent immobilization of heparin-loaded (HEP) NH(2) group-functionalized (Fun) nanoliposomes (NLs). Empty Fun or HEP non-functionalized (control) NLs were used as controls. NLs were characterized for mean diameter, surface charge and heparin encapsulation/release. Different lipid compositions were used for NL construction; PC/Chol (2:1mol/mol) or PC/Chol (4:1mol/mol) (fluid type vesicles) [which allow gradual release of heparin] and DSPC/Chol (2:1mol/mol) (rigid type vesicles). Surface haemocompatibility was tested by measuring blood clotting time. Platelet adhesion on surfaces was evaluated morphologically by SEM and CLSM. The haemocompatibility of plasma-processed surfaces was improved (compared to untreated surfaces); Fun-HEP NL-coated surfaces demonstrated highest coagulation times. For short surface/blood incubation periods, surfaces coated with Fun-HEP NLs consisting of PC/Chol (2:1) had higher coagulation times (compared to DSPC/Chol NLs) due to faster release of heparin. Heparin release rate from the various NL types and surface platelet adhesion results were in agreement with the corresponding blood coagulation times. Concluding, covalent immobilization of drug entrapping NLs on plasma processed surfaces is a potential method for preparation of controlled-rate drug-eluting metallic stents or devices.

P-selectin/ligand unbinding force measured with atomic force microscopy: comparison of two chemical protocols for the tethering of single molecules.

Leukocytes, as an indispensable arm of the immune system, need to be recruited from the flowing blood and transferred to the sites of infection. Their extravasation is feasible due to their ability to tether and roll over the activated endothelium, which is much dependent on the association of their selectin molecules with ligands on the activated endothelial cells. In view of the importance of this interaction for the physiological immune functions as well as for autoimmune diseases, specifying the affinity of selectins to their ligands at the single molecule level appears a challenging task to gain insight into the mechanisms that control leukocyte-endothelial avidity. To this end we functionalized substrates with P-selectin and cantilever probes with its major ligand, the P-selectin glycoprotein ligand-1, and used atomic force microscopy to measure their unbinding force. Two different chemical protocols were used for the tethering of the molecules on the substrates, one based on a homobifunctional poly(ethylene glycol) linker and the other on the use of antibody-specific binding. The unbinding forces measured with the two methods were 312 ± 149 and 230 ± 57 pN, respectively. Measurements on activated endothelials, declaratory of single molecule interactions, gave comparable results.

In vitro adhesion of staphylococci to diamond-like carbon polymer hybrids under dynamic flow conditions.

This study compares the ability of selected materials to inhibit adhesion of two bacterial strains commonly implicated in implant-related infections. These two strains are Staphylococcus aureus (S-15981) and Staphylococcus epidermidis (ATCC 35984). In experiments we tested six different materials, three conventional implant metals: titanium, tantalum and chromium, and three diamond-like carbon (DLC) coatings: DLC, DLC-polydimethylsiloxane hybrid (DLC-PDMS-h) and DLC-polytetrafluoroethylene hybrid (DLC-PTFE-h) coatings. DLC coating represents extremely hard material whereas DLC hybrids represent novel nanocomposite coatings. The two DLC polymer hybrid films were chosen for testing due to their hardness, corrosion resistance and extremely good non-stick (hydrophobic and oleophobic) properties. Bacterial adhesion assay tests were performed under dynamic flow conditions by using parallel plate flow chambers (PPFC). The results show that adhesion of S. aureus to DLC-PTFE-h and to tantalum was significantly (P < 0.05) lower than to DLC-PDMS-h (0.671 ± 0.001 × 10(7)/cm(2) and 0.751 ± 0.002 × 10(7)/cm(2) vs. 1.055 ± 0.002 × 10(7)/cm(2), respectively). No significant differences were detected between other tested materials. Hence DLC-PTFE-h coating showed as low susceptibility to S. aureus adhesion as all the tested conventional implant metals. The adherence of S. epidermidis to biomaterials was not significantly (P < 0.05) different between the materials tested. This suggests that DLC-PTFE-h films could be used as a biomaterial coating without increasing the risk of implant-related infections.

Design of a novel rotating wall bioreactor for the in vitro simulation of the mechanical environment of the endothelial function.

Endothelial cells (ECs), besides being a permeability barrier between the blood and vessel wall, perform many important functions, e.g., cell migration, remodeling, proliferation, and the production, secretion and metabolism of biochemical substances, as well as the regulation of contractility of vascular smooth muscle cells (SMCs). Their function is modulated by chemical ligands as well as mechanical factors. The mechanical stresses acting on the vessel wall include the normal and circumferential stresses that result from the action of blood pressure, the shear stress that acts parallel to the luminal surface of the vessel due to blood flow and the magnitude and orientation of the gravitation field. The aim of this work was to design and construct a novel bioreactor for the stimulation of endothelial cells in vitro with a combination of mechanical factors that simulates their in vivo environment.

Measuring the force of single protein molecule detachment from surfaces with AFM.

Atomic force microscopy (AFM) was used to measure the non-specific detachment force of single fibrinogen molecules from glass surfaces. The identification of single unbinding events was based on the characteristics of the parabolic curves, recorded during the stretching of protein molecules. Fibrinogen molecules were covalently bound to Si(3)N(4) AFM tips, previously modified with 3-aminopropyl-dimethyl-ethoxysilane, through a homobifunctional poly(ethylene glycol) linker bearing two hydroxysulfosuccinimide esters. The most probable detachment force was found to be 210 pN, when the tip was retracting with a velocity of 1400 nm/s, while the distribution of the detachment distances indicated that the fibrinogen chain can be elongated beyond the length of the physical conformation before detachment. The dependence of the most probable detachment force on the loading rate was examined and the dynamics of fibrinogen binding to the surface were found amenable to the simple expression of the Bell-Evans theory. The theory's expansion, however, by incorporating the concept of the rupture of parallel residue-surface bonds could only describe the detachment of fibrinogen for a small number of such bonds. Finally, the mathematical expression of the Worm-Like Chain model was used to fit the stretching curves before rupture and two interpretations are suggested for the description of the AFM curves with multiple detachment events.

Haemolytic activity of liposomes: effect of vesicle size, lipid concentration and polyethylene glycol-lipid or arsonolipid incorporation.

The haemolysis caused by various types of liposomes was measured after incubation of liposomes with human red blood cell (erythrocyte) suspension. Liposomes composed of phospholipids and containing or not arsonolipids (arsonoliposomes) were tested. In some cases liposomes that were coated with polyethylene glycol (MW 2000), which were formulated by including 8 mol% DSPE-PEG2000 in their lipid membrane, were used. Multilamellar vesicles were prepared by the thin film hydration technique (conventional liposomes) or by the one-step technique (arsonoliposomes). Sonicated vesicles were produced by probe sonication of the initial liposome preparations. Phospholipid concentration in the liposome dispersions were measured by the Stewart assay, and adjusted accordingly. Haemolysis was measured after incubating 100 microl of liposome dispersions with 900 microl of red blood cell suspension (blood) for 1 h. The results reveal that the haemolysis caused, when liposomes are incubated in blood at concentrations below 0.16 mg (lipid)/ml (blood), was minimum. Only in case of Pegylated arsonoliposomes, significant haemolysis percents were observed. At higher lipid concentrations, 0.38 or 0.6 mg/ml, the haemolysis caused by arsonoliposomes was substantially increased, even in the cases of non-Pegylated arsonoliposomes. In most cases, especially when arsonolipid-containing liposomes were evaluated, vesicle size also had considerable effect on vesicle-induced haemolysis. Nevertheless, at concentrations which are relevant with liposomal drug administration in humans, all formulations tested demonstrated negligible haemolysis.

Measurement of interaction forces between fibrinogen coated probes and mica surface with the atomic force microscope: The pH and ionic strength effect.

The study of protein-surface interactions is of great significance in the design of biomaterials and the evaluation of molecular processes in tissue engineering. The authors have used atomic force microscopy (AFM) to directly measure the force of attraction/adhesion of fibrinogen coated tips to mica surfaces and reveal the effect of the surrounding solution pH and ionic strength on this interaction. Silica colloid spheres were attached to the AFM cantilevers and, after plasma deposition of poly(acrylic acid), fibrinogen molecules were covalently bound on them with the help of the cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in the presence of N-hydroxysulfosuccinimide (sulfo-NHS). The measurements suggest that fibrinogen adsorption is controlled by the screening of electrostatic repulsion as the salt concentration increases from 15 to 150 mM, whereas at higher ionic strength (500 mM) the hydration forces and the compact molecular conformation become crucial, restricting adsorption. The protein attraction to the surface increases at the isoelectric point of fibrinogen (pH 5.8), compared with the physiological pH. At pH 3.5, apart from fibrinogen attraction to the surface, evidence of fibrinogen conformational changes is observed, as the pH and the ionic strength are set back and forth, and these changes may account for fibrinogen aggregation in the protein solution at this pH.

pH and ionic strength effect on single fibrinogen molecule adsorption on mica studied with AFM.

Although several investigations have been reported on the effect of pH or ionic strength on protein adsorption, most of them have been carried out with protein monolayers and not with single molecules. We have used atomic force microscopy to image, in phosphate buffer, single fibrinogen molecules adsorbed on mica and compare the surface coverage at variable pH (7.4, 5.8, 3.5) or ionic strength (15, 150, 500 mM) conditions. The images obtained and the statistical analysis of the surface coverage indicate adsorption enhancement at the IEP of fibrinogen (pH 5.8) and minimum adsorption at pH 3.5. On the other hand, more protein was adsorbed when the salt concentration of the buffer at pH 7.4 was increased from 15 to 150 mM. However, further increase of salt concentration up to 500 mM resulted in decreased adsorption. To confirm the aforementioned results an approaching bare Si(3)N(4) tip was used as an electrostatic analogue to a protein molecule and interaction force curves between it and the substrate were recorded. The results were in consistence with the double layer theory which justifies the screening of electrostatic repulsion as the salt concentration increases.

Heparin incorporating liposomes as a delivery system of heparin from PET-covered metallic stents: effect on haemocompatibility.

We investigate the possibility of coating polymer-covered stents with heparin-encapsulating liposomes for improving their haemocompatibility. Thin-film hydration (for multilamellar vesicles, MLV), and the dehydration-rehydration vesicle (DRV) methods are used for preparation of low-molecular weight heparin (LMWH)-encapsulating liposomes with varying lipid compositions. Liposomes are characterized for LMWH encapsulation and retention. For measurement of LMWH, a chromogenic technique is adjusted. For evaluation of heparin release from vesicles in platelet poor plasma (PPP) coagulation time is measured in presence of liposomal samples. Results reveal that LMWH encapsulation in liposomes is higher in DRV, however compositions with high encapsulation are leaky during buffer incubation. Most liposomes release LMWH slowly during plasma incubation (retention after 24 h ranges between 74% and 95%). Concerning the haemocompatibility of polyethylene terephthlate-covered stents after coating with LMWH-encapsulating liposomes, there is a marked increase (higher for DRV-coated stents compared to MLV) in plasma recalcification time compared to the control (plain blood) and reference (non-coated stent), which increases with blood-material contact time. This is probably due to LMWH release, demonstrating that encapsulated LMWH retains its biological functionality. Interestingly, the DRV-coated stents retained a high plasma recalcification time and a large number of liposomes on the stents (as proven by SEM studies) even after extensive washing (high shear conditions), proving that this method may be functional under high flow applying in vivo conditions.