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.

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.

Variation in the Elastic Modulus and Increased Energy Dissipation Induced by Cyclic Straining of Argiope bruennichi Major Ampullate Gland Silk.

The trends exhibited by the parameters that describe the mechanical behaviour of major ampullate gland silk fibers spun by Argiope bruennichi spiders is explored by performing a series of loading-unloading tests at increasing values of strain, and by the subsequent analysis of the true stress-true strain curves obtained from these cycles. The elastic modulus, yields stress, energy absorbed, and energy dissipated in each cycle are computed in order to evaluate the evolution of these mechanical parameters with this cyclic straining. The elastic modulus is observed to increase steadily under these loading conditions, while only a moderate variation is found in the yield stress. It is also observed that a significant proportion of the energy initially absorbed in each cycle is not only dissipated, but that the material may recover partially from the associated irreversible deformation. This variation in the mechanical performance of spider silk is accounted for through a combination of irreversible and reversible deformation micromechanisms in which the viscoelasticity of the material plays a leading role.

Axonal Guidance Using Biofunctionalized Straining Flow Spinning Regenerated Silk Fibroin Fibers as Scaffold.

After an injury, the limited regenerative capacity of the central nervous system makes the reconnection and functional recovery of the affected nervous tissue almost impossible. To address this problem, biomaterials appear as a promising option for the design of scaffolds that promote and guide this regenerative process. Based on previous seminal works on the ability of regenerated silk fibroin fibers spun through the straining flow spinning (SFS) technique, this study is intended to show that the usage of functionalized SFS fibers allows an enhancement of the guidance ability of the material when compared with the control (nonfunctionalized) fibers. It is shown that the axons of the neurons not only tend to follow the path marked by the fibers, in contrast to the isotropic growth observed on conventional culture plates, but also that this guidance can be further modulated through the biofunctionalization of the material with adhesion peptides. Establishing the guidance ability of these fibers opens the possibility of their use as implants for spinal cord injuries, so that they may represent the core of a therapy that would allow the reconnection of the injured ends of the spinal cord.

The Spider Silk Standardization Initiative (S3I): A powerful tool to harness biological variability and to systematize the characterization of major ampullate silk fibers spun by spiders from suburban Sydney, Australia.

The true stress-true strain curves of 11 Australian spider species from the Entelegynae lineage were tensile tested and classified based on the values of the alignment parameter, α*, in the framework of the Spider Silk Standardization Initiative (S3I). The application of the S3I methodology allowed the determination of the alignment parameter in all cases, and were found to range between α* = 0.03 and α* = 0.65. These data, in combination with previous results on other species included in the Initiative, were exploited to illustrate the potential of this approach by testing two simple hypotheses on the distribution of the alignment parameter throughout the lineage: (1) whether a uniform distribution may be compatible with the values obtained from the studied species, and (2) whether any trend may be established between the distribution of the α* parameter and phylogeny. In this regard, the lowest values of the α* parameter are found in some representatives of the Araneidae group, and larger values seem to be found as the evolutionary distance from this group increases. However, a significant number of outliers to this apparent general trend in terms of the values of the α* parameter are described.

Differences in the Elastomeric Behavior of Polyglycine-Rich Regions of Spidroin 1 and 2 Proteins.

Two different polyglycine-rich fragments were selected as representatives of major ampullate gland spidroins (MaSp) 1 and 2 types, and their behavior in a water-saturated environment was simulated within the framework of molecular dynamics (MD). The selected fragments are found in the sequences of the proteins MaSp1a and MaSp2.2a of Argiope aurantia with respective lengths of 36 amino acids (MaSp1a) and 50 amino acids (MaSp2.2s). The simulation took the fully extended ß-pleated conformation as reference, and MD was used to determine the equilibrium configuration in the absence of external forces. Subsequently, MD were employed to calculate the variation in the distance between the ends of the fragments when subjected to an increasing force. Both fragments show an elastomeric behavior that can be modeled as a freely jointed chain with links of comparable length, and a larger number of links in the spidroin 2 fragment. It is found, however, that the maximum recovery force recorded from the spidroin 2 peptide (Fmax ≈ 400 pN) is found to be significantly larger than that of the spidroin 1 (Fmax ≈ 250 pN). The increase in the recovery force of the spidroin 2 polyglycine-rich fragment may be correlated with the larger values observed in the strain at breaking of major ampullate silk fibers spun by Araneoidea species, which contain spidroin 2 proteins, compared to the material produced by spider species that lack these spidroins (RTA-clade).

Aging is accompanied by T-cell stiffening and reduced interstitial migration through dysfunctional nuclear organization.

Age-associated changes in T-cell function play a central role in immunosenescence. The role of aging in the decreased T-cell repertoire, primarily because of thymic involution, has been extensively studied. However, increasing evidence indicates that aging also modulates the mechanical properties of cells and the internal ordering of diverse cell components. Cellular functions are generally dictated by the biophysical phenotype of cells, which itself is also tightly regulated at the molecular level. Based on previous evidence suggesting that the relative nuclear size contributes to variations of T-cell stiffness, here we examined whether age-associated changes in T-cell migration are dictated by biophysical parameters, in part through nuclear cytoskeleton organization and cell deformability. In this study, we first performed longitudinal analyses of a repertoire of 111 functional, biophysical and biomolecular features of the nucleus and cytoskeleton of mice CD4+ and CD8+ T cells, in both naive and memory state. Focusing on the pairwise correlations, we found that age-related changes in nuclear architecture and internal ordering were correlated with T-cell stiffening and declined interstitial migration. A similarity analysis confirmed that cell-to-cell variation was a direct result of the aging process and we applied regression models to identify biomarkers that can accurately estimate individuals' age. Finally, we propose a biophysical model for a comprehensive understanding of the results: aging involves an evolution of the relative nuclear size, in part through DNA-hypomethylation and nuclear lamin B1, which implies an increased cell stiffness, thus inducing a decline in cell migration.

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.

Unexpected high toughness of Samia cynthia ricini silk gut.

Silk gut fibers were produced from the silkworm Samia cynthia ricini silk glands by the usual procedure of immersion in a mildly acidic solution and subsequent stretching. The morphology of the silk guts was assessed by scanning electron microscopy, and their microstructure was assessed by infrared spectroscopy and X-ray diffraction. It was found that both naturally spun and Samia silk guts share a common semicrystalline microstructure. The mechanical characterization of the silk guts revealed that these fibers show an elastomeric behavior when tested in water, and exhibit a genuine ground state to which the fiber may revert independently of its previous loading history. In spite of its large cross-sectional area compared with naturally spun silk fibers, Samia silk guts show values of work to fracture up to 160 MJ m-3, much larger than those of most of their natural counterparts, and establish a new record value for this parameter in silk guts.

Improved cell adhesion to activated vapor silanization-biofunctionalized Ti-6Al-4V surfaces with ECM-derived oligopeptides.

Titanium implants are widely used in traumatology and various orthopedic fields. Titanium and other metallic-based implants have limited structural and functional integration into the body, which translates into progressive prosthesis instability and the need for new surgical interventions that have enormous social and economic impacts. To enhance the biocompatibility of titanium implants, numerous biofunctionalization strategies have been developed. However, the problem persists, as more than 70% of implant failures are due to aseptic loosening. In this study we addressed the problem of improving the physiological engraftability and acceptability of titanium-based implants by applying a robust and versatile functionalization method based on the covalent immobilization of extracellular matrix (ECM)-derived oligopeptides on Ti-6Al-4V surfaces treated by activated vapor silanization (AVS). The feasibility of this technique was evaluated with two oligopeptides of different structures and compositions. These oligopeptides were immobilized on Ti-6Al-4V substrates by a combination of AVS and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) crosslinking chemistry. The immobilization was shown to be stable and resistant to chemical denaturing upon sodium dodecyl sulfate treatment. On Ti-6Al-4V surfaces both peptides increased the attachment, spreading, rearrangement and directional growth of mesenchymal stem and progenitor cells (MSC) with chondro- and osteo-regenerative capacities. We also found that this biofunctionalization method (AVS-EDC/NHS) increased the attachment capacity of an immortalized cell line of neural origin with poor adhesive properties, highlighting the versatility and robustness of this method in terms of potential oligopeptides that may be used, and cell lineages whose anchorage to the biomaterial may be enhanced. Collectively, this novel functionalization strategy can accelerate the development of advanced peptide-functionalized metallic surfaces, which, in combination with host or exogenously implanted stem cells, have the potential to positively affect the osteoregenerative and osteointegrative abilities of metallic-based prostheses.

Expression of spidroin proteins in the silk glands of golden orb-weaver spiders.

The expression of spidroins in the major ampullate, minor ampullate, flagelliform, and tubuliform silk glands of Trichonephila clavipes spiders was analyzed using proteomics analysis techniques. Spidroin peptides were identified and assigned to different gene products based on sequence concurrence when compared with the whole genome of the spider. It was found that only a relatively low proportion of the spidroin genes are expressed as proteins in any of the studied glands. In addition, the expression of spidroin genes in different glands presents a wide range of patterns, with some spidroins being found in a single gland exclusively, while others appear in the content of several glands. The combination of precise genomics, proteomics, microstructural, and mechanical data provides new insights both on the design principles of these materials and how these principles might be translated for the production of high-performance bioinspired artificial 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.

Basic Principles in the Design of Spider Silk Fibers.

The prominence of spider silk as a hallmark in biomimetics relies not only on its unrivalled mechanical properties, but also on how these properties are the result of a set of original design principles. In this sense, the study of spider silk summarizes most of the main topics relevant to the field and, consequently, offers a nice example on how these topics could be considered in other biomimetic systems. This review is intended to present a selection of some of the essential design principles that underlie the singular microstructure of major ampullate gland silk, as well as to show how the interplay between them leads to the outstanding tensile behavior of spider silk. Following this rationale, the mechanical behavior of the material is analyzed in detail and connected with its main microstructural features, specifically with those derived from the semicrystalline organization of the fibers. Establishing the relationship between mechanical properties and microstructure in spider silk not only offers a vivid image of the paths explored by nature in the search for high performance materials, but is also a valuable guide for the development of new artificial fibers inspired in their natural counterparts.

Regenerated Silk Fibers Obtained by Straining Flow Spinning for Guiding Axonal Elongation in Primary Cortical Neurons.

The recovery of injured nervous tissue, one of the main goals for regenerative therapeutic approaches, is often hindered by the limited axonal regeneration ability of the central nervous system (CNS). In this regard, the identification of scaffolds that support the reconstruction of functional neuronal tissues and guide the alignment of regenerating neurons is a major challenge in tissue engineering. Ideally, the usage of such scaffolds would promote and guide the axonal growth, a crucial phase for the restoration of neuronal connections and, consequently, the nerve function. Among the materials proposed as scaffolds for CNS regeneration, silk has been used to exploit its outstanding features as a biomaterial to promote axonal regeneration. In this study, we explore, for the first time, the possibility of using high-performance regenerated silk fibers obtained by straining flow spinning (SFS) to serve as scaffolds for inducing and guiding the axonal growth. It is shown that SFS fibers promote the spontaneous organization of dissociated cortical primary cells into highly interconnected cellular spheroid-like tissue formations. Neuronal projections (i.e., axons) from these cellular spheroids span hundreds of microns along the SFS fibers that act as guides and allow the connection of distant spheroids. In addition, it is also shown that SFS fibers serve as scaffolds for neuronal migration covering short and long distances. As a consequence, the usage of high-performance SFS fibers appears as a promising basis for the development of novel therapies, leading to directed axonal regeneration.

Application of the Spider Silk Standardization Initiative (S3I) methodology to the characterization of major ampullate gland silk fibers spun by spiders from Pantanos de Villa wetlands (Lima, Peru).

Spider silk is a natural material with unique properties and a great potential for engineering and biomedical applications. In spite of its simple composition and highly conserved and stereotypical production, spider silks show a wide range of variability in their mechanical properties which, for long, have defied their classification and standardization. Here we propose to launch the Spider Silk Standardization Initiative (S3I), a methodology based on the definition of the α* parameter, in an attempt to define a systematic procedure to classify the tensile properties exhibited by major ampullate gland silk (MAS) spun by Entelegynae spiders. The α* parameter is calculated from the comparison of the true stress-true strain curve of any MAS fiber after being subjected to maximum supercontraction, with the true stress-true strain curve of the species Argiope aurantia, which is set as a reference curve. This work presents the details of the S3I methodology and, as an example, shows its application to an assemblage of Entelegynae spiders from different families collected at the Pantanos de Villa wetlands (Lima, Peru). The systematic and objective classification of the tensile properties of MAS fibers allowed by the S3I will offer insights into key aspects of the biological evolution of the material, and address questions such as how history and adaptation contributed to shape those properties. In addition, it will surely have far reaching consequences in fields such as Materials Science, and Molecular and Evolutionary Biology, by organizing the range of tensile properties exhibited by spider silk fibers.

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.

Preparation and characterization of Nephila clavipes tubuliform silk gut.

Tubuliform silk glands were dissected from Nephila clavipes spiders, and silk gut fibers were produced by immersing the glands in a mild acid solution and subsequent stretching. The tensile properties of the as produced fibers were obtained through tensile tests, and the stress-strain curves were compared with those of naturally spun tubuliform silk fibers. The influence on the mechanical properties of the fibers after immersion in water and drying was also discerned. The microstructure of the silk guts was obtained by X-ray diffraction (XRD) and infrared spectroscopy (FTIR). It was found that the stress-strain curves of the stretched tubuliform silk guts concur with those of their natural counterparts (tubuliform silk fibers).

Emergence of supercontraction in regenerated silkworm (Bombyx mori) silk fibers.

The conditions required for the emergence of supercontraction in regenerated silkworm (Bombyx mori) silk fibers are assessed through an experimental approach that combines the spinning of regenerated fibers with controlled properties and their characterization by 13C solid-state nuclear magnetic resonance (NMR). Both supercontracting and non-supercontracting regenerated fibers are produced using the straining flow spinning (SFS) technique from 13C labeled cocoons. The short-range microstructure of the fibers is assessed through 13C CP/MAS in air and 13C DD/MAS in water, and the main microstructural features are identified and quantified. The mechanical properties of the regenerated fibers and their microstructures are compared with those of natural silkworm silk. The combined analysis highlights two possible key elements as responsible for the emergence of supercontraction: (1) the existence of an upper and a lower limit of the amorphous phase compatible with supercontraction, and (2) the existence of two ordered phases, ß-sheet A and B, which correspond to different packing arrangements of the protein chains.