Identification of Individual Target Molecules Using Antibody-Decorated DeepTipTM Atomic-Force Microscopy Probes.

A versatile and robust procedure is developed that allows the identification of individual target molecules using antibodies bound to a DeepTipTM functionalized atomic-force microscopy probe. The model system used for the validation of this process consists of a biotinylated anti-lactate dehydrogenase antibody immobilized on a streptavidin-decorated AFM probe. Lactate dehydrogenase (LDH) is employed as target molecule and covalently immobilized on functionalized MicroDeckTM substrates. The interaction between sensor and target molecules is explored by recording force-displacement (F-z) curves with an atomic-force microscope. F-z curves that correspond to the genuine sensor-target molecule interaction are identified based on the following three criteria: (i) number of peaks, (ii) value of the adhesion force, and (iii) presence or absence of the elastomeric trait. The application of these criteria leads to establishing seven groups, ranging from no interaction to multiple sensor-target molecule interactions, for which force-displacement curves are classified. The possibility of recording consistently single-molecule interaction events between an antibody and its specific antigen, in combination with the high proportion of successful interaction events obtained, increases remarkably the possibilities offered by affinity atomic-force microscopy for the characterization of biological and biomimetic systems from the molecular to the tissue scales.

Results of Palliative Stenting in Malignant Superior Vena Cava Syndrome Analyzing Self-Expanding Stainless Steel and Nitinol Venous Bare Metal Stents.

PURPOSE: The purpose was to analyze the technical, clinical, and survival outcomes of our patients with malignant superior cava vein syndrome (SVCS) treated with endovascular approach and analyze the efficacy of different stent types used. MATERIAL AND METHODS: It is an observational, retrospective, single-center study. From 2006 to 2023, 42 patients (32 male, 10 female, mean age 62 years, age range, 41-87 years) underwent percutaneous stent placement for malignant SVCS. One stainless steel stent (Wallstent) and 2 venous nitinol stent type (Sinus-XL, Venovo) were used. Follow-up mean was 276 days. RESULTS: A total of 53 stents were deployed. Clinical success was 97.6% in less 24 hours. Technical success was achieved in 97.6%. No complications were found except 1 patient died during the procedure due to stent migration and atrial dissociation (2.3%). Overall intraprocedural stent migration rate was 11.9% (18.8% stainless steel stent, 9.6% nitinol stent, p>0.05). Overall survival rates were 87.8%, 41.99%, and 34.12%, and overall primary patency rates were 100%, 93.3%, 91.6% at 1, 6, and 12 months, respectively. CONCLUSIONS: Endovascular treatment is a safe and effective therapeutic option for SVCS with high technical and clinical success rates and low complication and recurrence rates. CLINICAL IMPACT: The malignant superior cava vein syndrome is a rare clinical entity treated classically with radiation and chemotherapy with a slower response, or surgical bypass, which is an aggressive surgical technique. Endovascular treatment offers a low-invasive technique with quick clinical resolution and good permeability results. However, further studies are lacking to deal with procedure technical characteristics, stent type used, technical complications, and medium- and long-term patency studies. This study aims to evaluate all these items, analysing self-expanding stainless steel and nitinol venous bare metal stents, and add value to endovascular treatment, confirming the good results of this technique.

Statistical Study of Low-Intensity Single-Molecule Recognition Events Using DeepTipTM Probes: Application to the Pru p 3-Phytosphingosine System.

The interaction between the plant lipid transfer protein Pru p 3 and phytosphingosine was assessed using an atomic force microscope. Phytosphingosine was covalently immobilized on DeepTipTM probes and Pru p 3 on MicroDeckTM functionalized substrates. Single-molecular interaction events between both molecules were retrieved and classified and the distribution for each one of the identified types was calculated. A success rate of over 70% was found by comparing the number of specific Pru p 3-phytosphingosine interaction events with the total number of recorded curves. The analysis of the distribution established among the various types of curves was further pursued to distinguish between those curves that can mainly be used for assessing the recognition between phytosphingosine (sensor molecule) and Pru p 3 (target molecule) in the context of affinity atomic force microscopy, and those that entail details of the interaction and might be employed in the context of force spectroscopy. The successful application of these functionalized probes and substrates to the characterization of the low-intensity hydrophobic interaction characteristic of this system is a clear indication of the potential of exploiting this approach with an extremely wide range of different biological molecules of interest. The possibility of characterizing molecular assembly events with single-molecule resolution offers an advantageous procedure to plough into the field of molecular biomimetics.

Modulation of Cell Response through the Covalent Binding of Fibronectin to Titanium Substrates.

Titanium (Ti-6Al-4V) substrates were functionalized through the covalent binding of fibronectin, and the effect of the existence of this extracellular matrix protein on the surface of the material was assessed by employing mesenchymal stem cell (MSC) cultures. The functionalization process comprised the usage of the activation vapor silanization (AVS) technique to deposit a thin film with a high surface density of amine groups on the material, followed by the covalent binding of fibronectin to the amine groups using the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) crosslinking chemistry. The biological effect of the fibronectin on murine MSCs was assessed in vitro. It was found that functionalized samples not only showed enhanced initial cell adhesion compared with bare titanium, but also a three-fold increase in the cell area, reaching values comparable to those found on the polystyrene controls. These results provide compelling evidence of the potential to modulate the response of the organism to an implant through the covalent binding of extracellular matrix proteins on the prosthesis.

Application of single cell force spectroscopy (SCFS) to the assessment of cell adhesion to peptide-decorated surfaces.

The adhesion forces of cells to peptide-coated functionalized materials were assessed through the Single Cell Force Spectroscopy (SCFS) technique in order to develop a methodology that allows the fast selection of peptide motifs that favor the interaction between cells and the biomaterial. Borosilicate glasses were functionalized using the activated vapor silanization process (AVS) and subsequently decorated with an RGD- containing peptide using the EDC/NHS crosslinking chemistry. It is shown that the RGD-coated glass induces larger attachment forces on mesenchymal stem cell cultures (MSCs), compared to the bare glass substrates. These higher forces correlate well with the enhanced adhesion of the MSCs observed on RGD-coated substrates through conventional adhesion cell cultures and inverse centrifugation tests. The methodology based on the SCFS technique presented in this work constitutes a fast procedure for the screening of new peptides or their combinations to select candidates that may enhance the response of the organism to the implant of the functionalized biomaterials.

Strategies for the Biofunctionalization of Straining Flow Spinning Regenerated Bombyx mori Fibers.

High-performance regenerated silkworm (Bombyx mori) silk fibers can be produced efficiently through the straining flow spinning (SFS) technique. In addition to an enhanced biocompatibility that results from the removal of contaminants during the processing of the material, regenerated silk fibers may be functionalized conveniently by using a range of different strategies. In this work, the possibility of implementing various functionalization techniques is explored, including the production of fluorescent fibers that may be tracked when implanted, the combination of the fibers with enzymes to yield fibers with catalytic properties, and the functionalization of the fibers with cell-adhesion motifs to modulate the adherence of different cell lineages to the material. When considered globally, all these techniques are a strong indication not only of the high versatility offered by the functionalization of regenerated fibers in terms of the different chemistries that can be employed, but also on the wide range of applications that can be covered with these functionalized fibers.

High-Yield Characterization of Single Molecule Interactions with DeepTipTM Atomic Force Microscopy Probes.

Single molecule interactions between biotin and streptavidin were characterized with functionalized DeepTipTM probes and used as a model system to develop a comprehensive methodology for the high-yield identification and analysis of single molecular events. The procedure comprises the covalent binding of the target molecule to a surface and of the sensing molecule to the DeepTipTM probe, so that the interaction between both chemical species can be characterized by obtaining force-displacement curves in an atomic force microscope. It is shown that molecular resolution is consistently attained with a percentage of successful events higher than 90% of the total number of recorded curves, and a very low level of unspecific interactions. The combination of both features is a clear indication of the robustness and versatility of the proposed methodology.

Comparison of cell mechanical measurements provided by Atomic Force Microscopy (AFM) and Micropipette Aspiration (MPA).

A comparative analysis of T-lymphocyte mechanical data obtained from Micropipette Aspiration (MPA) and Atomic Force Microscopy (AFM) is presented. Results obtained by fitting the experimental data to simple Hertz and Theret models led to non-Gaussian distributions and significantly different values of the elastic moduli obtained by both techniques. The use of more refined models, taking into account the finite size of cells (simplified double contact and Zhou models) reduces the differences in the values calculated for the elastic moduli. Several possible sources for the discrepancy between the techniques are considered. The analysis suggests that the local nature of AFM measurements compared with the more general character of MPA measurements probably contributed to the differences observed.

Enhanced Biological Response of AVS-Functionalized Ti-6Al-4V Alloy through Covalent Immobilization of Collagen.

This study presents the development of an efficient procedure for covalently immobilizing collagen molecules on AVS-functionalized Ti-6Al-4V samples, and the assessment of the survival and proliferation of cells cultured on these substrates. Activated Vapor Silanization (AVS) is a versatile functionalization technique that allows obtaining a high density of active amine groups on the surface. A procedure is presented to covalently bind collagen to the functional layer using EDC/NHS as cross-linker. The covalently bound collagen proteins are characterized by fluorescence microscopy and atomic force microscopy and their stability is tested. The effect of the cross-linker concentration on the process is assessed. The concentration of the cross-linker is optimized and a reliable cleaning protocol is developed for the removal of the excess of carbodiimide from the samples. The results demonstrate that the covalent immobilization of collagen type I on Ti-6Al-4V substrates, using the optimized protocol, increases the number of viable cells present on the material. Consequently, AVS in combination with the carbodiimide chemistry appears as a robust method for the immobilization of proteins and, for the first time, it is shown that it can be used to enhance the biological response to the material.

Straining Flow Spinning of Artificial Silk Fibers: A Review.

This work summarizes the main principles and some of the most significant results of straining flow spinning (SFS), a technology developed originally by the authors of this work. The principles on which the technology is based, inspired by the natural spinning system of silkworms and spiders, are presented, as well as some of the main achievements of the technique. Among these achievements, spinning under environmentally friendly conditions, obtaining high-performance fibers, and imparting the fibers with emerging properties such as supercontraction are discussed. Consequently, SFS appears as an efficient process that may represent one of the first realizations of a biomimetic technology with a significant impact at the production level.

Topographical and mechanical characterization of living eukaryotic cells on opaque substrates: development of a general procedure and its application to the study of non-adherent lymphocytes.

The mechanical behavior of living murine T-lymphocytes was assessed by atomic force microscopy (AFM). A robust experimental procedure was developed to overcome some features of lymphocytes, in particular their spherical shape and non-adherent character. The procedure included the immobilization of the lymphocytes on amine-functionalized substrates, the use of hydrodynamic effects on the deflection of the AFM cantilever to monitor the approaching, and the use of the jumping mode for obtaining the images. Indentation curves were analyzed according to Hertz's model for contact mechanics. The calculated values of the elastic modulus are consistent both when considering the results obtained from a single lymphocyte and when comparing the curves recorded from cells of different specimens.

Spider silk gut: development and characterization of a novel strong spider silk fiber.

Spider silk fibers were produced through an alternative processing route that differs widely from natural spinning. The process follows a procedure traditionally used to obtain fibers directly from the glands of silkworms and requires exposure to an acid environment and subsequent stretching. The microstructure and mechanical behavior of the so-called spider silk gut fibers can be tailored to concur with those observed in naturally spun spider silk, except for effects related with the much larger cross-sectional area of the former. In particular spider silk gut has a proper ground state to which the material can revert independently from its previous loading history by supercontraction. A larger cross-sectional area implies that spider silk gut outperforms the natural material in terms of the loads that the fiber can sustain. This property suggests that it could substitute conventional spider silk fibers in some intended uses, such as sutures and scaffolds in tissue engineering.