Silanization Strategies for Tailoring Peptide Functionalization on Silicon Surfaces: Implications for Enhancing Stem Cell Adhesion.

Biomaterial surface engineering and the integration of cell-adhesive ligands are crucial in biological research and biotechnological applications. The interplay between cells and their microenvironment, influenced by chemical and physical cues, impacts cellular behavior. Surface modification of biomaterials profoundly affects cellular responses, especially at the cell-surface interface. This work focuses on enhancing cellular activities through material manipulation, emphasizing silanization for further functionalization with bioactive molecules such as RGD peptides to improve cell adhesion. The grafting of three distinct silanes onto silicon wafers using both spin coating and immersion methods was investigated. This study sheds light on the effects of different alkyl chain lengths and protecting groups on cellular behavior, providing valuable insights into optimizing silane-based self-assembled monolayers (SAMs) before peptide or protein grafting for the first time. Specifically, it challenges the common use of APTES molecules in this context. These findings advance our understanding of surface modification strategies, paving the way for tailoring biomaterial surfaces to modulate the cellular behavior for diverse biotechnological applications.

Peptide grafting on intraosseous transcutaneous amputation prostheses to promote sealing with skin cells: Potential to limit infections.

The long-term success of intraosseous transcutaneous amputation prostheses (ITAPs) mainly relies on dermal attachment of skin cells to the implant. Otherwise, bacteria can easily penetrate through the interface between the implant and the skin. Therefore, infection at this implant/skin interface remains a significant complication in orthopedic surgeries in which these prostheses are required. Two main strategies were investigated to prevent these potential infection problems which consist in either establishing a strong sealing at the skin/implant interface or on eradicating infections by killing bacteria. In this work, two adhesion peptides, either KRGDS or KYIGSR and one antimicrobial peptide, Magainin 2 (Mag 2), were covalently grafted via phosphonate anchor arms to the surface of the Ti6Al4V ELI (extra low interstitials) material, commonly used to manufacture ITAPs. X-ray photoelectron spectroscopy, contact angle, and confocal microscopy analyses enabled to validate the covalent and stable grafting of these three peptides. Dermal fibroblasts cultures on bare Ti6Al4V ELI surfaces and functionalized ones displayed a good cell adhesion and proliferation on all samples. However, cell spreading and viability appeared to be improved on grafted surfaces, especially for those conjugated with KRGDS and Mag 2. Moreover, the dermal sheet attachment, was significantly higher on surfaces functionalized with Mag 2 as compared to the other surfaces. Therefore, the surface functionalization with the antimicrobial peptide Mag 2 seems to be the best approach for the targeted application, as it could play a dual role, inducing a strong skin adhesion while limiting infections on Ti6Al4V ELI materials.

Polyethylene terephthalate textile heart valve: How poly(ethylene glycol) grafting limits fibrosis.

Transcatheter aortic valve replacement (TAVR) is an alternative technique to surgical valve replacement for over 300,000 patients worldwide. The valve material used in the TAVR is made of biological tissues, whose durability remains unknown. The success of the TAVR favors the research toward synthetic valve leaflet materials as an alternative to biological tissues. In particular, polyethylene terephthalate (PET) textile valves have recently proven durability over a 6-month period in animal sheep models. Excessive fibrotic tissue formation remains, however, a critical issue to be addressed. The aim of this work was therefore to investigate the potential of PET textiles covalently conjugated with polyethylene glycol (PEG), known for its antifouling properties, to modulate the fibrosis formation both in vitro and in vivo. For this purpose, the surfaces of heart valves made of PET textiles were functionalized with an atmospheric pressure plasma, leading to the formation of carboxylic acid (COOH) groups, further used for PEG-NH2 conjugation. Surface modification efficiency was assessed by X-ray photoelectron spectroscopy and water contact angle measurements. The biological behavior of the as-modified surfaces was evaluated by in vitro assays, using rat cardiac fibroblast cells. The results show that PEG treated substrates restrained the fibroblasts adhesion and proliferation. The PEG treated valve, implanted in a juvenile sheep model, showed a significant fibrosis reduction. The explant also revealed calcification issues that need to be addressed.

Fibronectin grafting to enhance skin sealing around transcutaneous titanium implant.

Intraosseous transcutaneous amputation prosthesis is a new approach in orthopedic implants that overcomes socket prosthesis problems. Its long-term performance requires a tight skin-implant seal to prevent infections. In this study, fibronectin (Fn), a widely used adhesion protein, was adsorbed or grafted onto titanium alloy. Fn grafting was performed using two different linking arms, dopamine/glutaric anhydride or phosphonate. The characterization of Fn-modified surfaces showed that Fn grating via phosphonate has led to the highest amount of Fn cell-binding site (RGD, arginine, glycine, and aspartate) available on the surface. Interestingly, cell culture studies revealed a strong correlation between the amount of available RGD ligands and cellular behavior, since enhanced proliferation and spreading of fibroblasts were noticed on Fn-grafted surfaces via phosphonate. In addition, an original in vitro mechanical test, inspired from the real situation, to better predict clinical outcomes after implant insertion, has been developed. Tensile test data showed that the adhesion strength of a bio-engineered dermal tissue was significantly higher around Fn-grafted surfaces via phosphonate, as compared to untreated surfaces. This study sheds light on the importance of an appropriate selection of the linking arm to tightly control the spatial conformation of biomolecules on the material surface, and consequently cell interactions at the interface tissue/implant.

Bioactive micropatterning of biomaterials for induction of endothelial progenitor cell differentiation: Acceleration of in situ endothelialization.

Synthetic grafts do not provide an appealing surface for endothelial cells to adhere and colonize the inner surface. To promote in situ endothelialization the following aspect has to be taken into account, endothelial progenitor cells (EPCs) needs to be mobilized on the surface of the graft. The surface of the graft has to be sufficiently biocompatible to create a prone environment for the EPCs to adhere, proliferate and, differentiate to form a layer and subsequently improve graft patency. In this work, two active molecules GRGDS and sitagliptin, were chosen for their abilities to recruit, enhance adhesion and induce differentiation of endothelial progenitor cells. They were grafted on PET surfaces in order to provide restrained cues triggering cell alignment and evaluate the influence of such structuration on EPCs fate. We then analyze cell behavior onto functionalized biomaterials. Their abilities to control EPCs fate were demonstrated via RT-qPCR, immunofluorescence, and enzymatic tests. The GRGDS/sitagliptin 100 × 10 surface enables to reduce the stemness phenotype on EPCs and induce the expression of endothelial lineage markers. These results highlight the importance of spatial patterning cues in guiding EPCs organization and function, which may have clinical relevance in the development of vascular grafts that promote patency.

Effect of linking arm hydrophilic/hydrophobic nature, length and end-group on the conformation and the RGD accessibility of surface-immobilized fibronectin.

In order to stimulate the cellular response to implant materials, extracellular matrix (ECM) proteins, such as collagen and fibronectin (FN), are immobilized on the implant surface. Amongst all ECM proteins used for biomimetic materials for medical applications, FN is one of the most investigated proteins thanks to its ability to promote cell adhesion and its contribution to important physiological processes. However, its conformation and hence its bioactivity strongly depend on the hydrophilic/hydrophobic nature of the surface as well as on immobilization strategies. This work investigates the effect of these two parameters, as well as the effect of the crosslinker length. FN was grafted onto silicon wafers using eights different linking arms presenting different lengths, hydrophilic/hydrophobic characters and binding sites. The protein was linked through either its amino groups (lysine amino acids) or sulfhydryl functionalities (cysteine amino acids). The grafting of each crosslinker and subsequent FN conjugation onto the surfaces was evidenced by X-ray photoelectron spectroscopy, while the surface hydrophilicity was determined by contact angle measurements. Moreover, atomic force microscopy images revealed that the conformation of surface conjugated FN only depends on the hydrophilicity of the linking arm. The FN conformation was also probed by enzyme-linked immunosorbent assays (ELISA). ELISA data demonstrated that all of the three investigated parameters linking arm parameter (length, hydrophobic/hydrophilic character, and terminal end-group) somewhat influence the RGD accessibility.

Design, Degradation Mechanism and Long-Term Cytotoxicity of Poly(L-lactide) and Poly(Lactide-co-ϵ-Caprolactone) Terpolymer Film and Air-Spun Nanofiber Scaffold.

Degradable nanofiber scaffold is known to provide a suitable, versatile and temporary structure for tissue regeneration. However, synthetic nanofiber scaffold must be properly designed to display appropriate tissue response during the degradation process. In this context, this publication focuses on the design of a finely-tuned poly(lactide-co-ϵ-caprolactone) terpolymer (PLCL) that may be appropriate for vascular biomaterials applications and its comparison with well-known semi-crystalline poly(l-lactide) (PLLA). The degradation mechanism of polymer film and nanofiber scaffold and endothelial cells behavior cultured with degradation products is elucidated. The results highlights benefits of using PLCL terpolymer as vascular biomaterial compared to PLLA.

Grafting of a model protein on lactide and caprolactone based biodegradable films for biomedical applications.

Thermoplastic biodegradable polymers displaying elastomeric behavior and mechanical consistency are greatly appreciated for the regeneration of soft tissues and for various medical devices. However, while the selection of a suitable base material is determined by mechanical and biodegradation considerations, it is the surface properties of the biomaterial that are responsible for the biological response. In order to improve the interaction with cells and modulate their behavior, biologically active molecules can be incorporated onto the surface of the material. With this aim, the surface of a lactide and caprolactone based biodegradable elastomeric terpolymer was modified in two stages. First, the biodegradable polymer surface was aminated by atmospheric pressure plasma treatment and second a crosslinker was grafted in order to covalently bind the biomolecule. In this study, albumin was used as a model protein. According to X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM), albumin was efficiently immobilized on the surface of the terpolymer, the degree of albumin surface coverage (ΓBSA) reached ~35%. Moreover, gel permeation chromatography (GPC) studies showed that the hydrolytic degradation kinetic of the synthesized polymer was slightly delayed when albumin was grafted. However, the degradation process in the bulk of the material was unaffected, as demonstrated by Fourier transform infrared (FTIR) analyses. Furthermore, XPS analyses showed that the protein was still present on the surface after 28 days of degradation, meaning that the surface modification was stable, and that there had been enough time for the biological environment to interact with the modified material.

Structure and membrane interactions of the ß-amyloid fragment 25-35 as viewed using spectroscopic approaches.

The ß-amyloid fragment peptide 25-35 (Aß(25-35)) is recognized as the cytotoxic sequence of the parent peptide Aß. However, it remains unclear whether its neurotoxicity originates from its fibrillar form, how it interacts with lipid membranes, and whether cholesterol modulates these interactions. These questions have been addressed at a molecular level using various microscopic and spectroscopic techniques. The data show that Aß(25-35) forms protofilaments at pH 7.4 at a concentration of 5 mM in the absence and presence of DMPC/DMPG model membranes. The peptide adopts a predominant aggregated ß-sheet conformation under these conditions. However, as the peptide concentration decreases, the ß-sheet structure tends to disappear for the benefit of ß-turns, suggesting that the peptide association is reversible. The ß-sheet structure formed by Aß(25-35) appears to be atypical and characterized by the absence of intermolecular dipolar coupling and by a parallel strand configuration. The data show that Aß(25-35)-phospholipid interactions are characterized by an increase in the conformational order of the lipid acyl chains and a change in the fluidity/elasticity of the bilayers. Concomitantly, the peptide seems to lose a few ß-sheet structures, which suggests that the interactions between Aß(25-35) and DMPC/DMPG membranes are partly driven by peptide concentration. Interactions indeed seem to occur when part of the peptides is not involved in protofilaments and increase as the proportion of the free peptide species increases. The interactions are very similar in the presence of cholesterol, except that the concentration effect of Aß(25-35) is cancelled, suggesting that Chol limits the penetration of the peptide inside the bilayers.

Surface modification of gadolinium oxide thin films and nanoparticles using poly(ethylene glycol)-phosphate.

The performance of nanomaterials for biomedical applications is highly dependent on the nature and the quality of surface coatings. In particular, the development of functionalized nanoparticles for magnetic resonance imaging (MRI) requires the grafting of hydrophilic, nonimmunogenic, and biocompatible polymers such as poly(ethylene glycol) (PEG). Attached at the surface of nanoparticles, this polymer enhances the steric repulsion and therefore the stability of the colloids. In this study, phosphate molecules were used as an alternative to silanes or carboxylic acids, to graft PEG at the surface of ultrasmall gadolinium oxide nanoparticles (US-Gd(2)O(3), 2-3 nm diameter). This emerging, high-sensitivity "positive" contrast agent is used for signal enhancement in T(1)-weighted molecular and cellular MRI. Comparative grafting assays were performed on Gd(2)O(3) thin films, which demonstrated the strong reaction of phosphate with Gd(2)O(3) compared to silane and carboxyl groups. Therefore, PEG-phosphate was preferentially used to coat US-Gd(2)O(3) nanoparticles. The grafting of this polymer on the particles was confirmed by XPS and FTIR. These analyses also demonstrated the strong attachment of PEG-phosphate at the surface of Gd(2)O(3), forming a protective layer on the nanoparticles. The stability in aqueous solution, the relaxometric properties, and the MRI signal of PEG-phosphate-covered Gd(2)O(3) particles were also better than those from non-PEGylated nanoparticles. As a result, reacting PEG-phosphate with Gd(2)O(3) particles is a promising, rapid, one-step procedure to PEGylate US-Gd(2)O(3) nanoparticles, an emerging "positive" contrast agent for preclinical molecular and cellular applications.

Ultra-small gadolinium oxide nanoparticles to image brain cancer cells in vivo with MRI.

The majority of contrast agents used in magnetic resonance imaging (MRI) is based on the rare-earth element gadolinium. Gadolinium-based nanoparticles could find promising applications in pre-clinical diagnostic procedures of certain types of cancer, such as glioblastoma multiforme. This is one of the most malignant, lethal and poorly accessible forms of cancer. Recent advances in colloidal nanocrystal synthesis have led to the development of ultra-small crystals of gadolinium oxide (US-Gd(2)O(3), 2-3 nm diameter). As of today, this is the smallest and the densest of all Gd-containing nanoparticles. Cancer cells labeled with a sufficient quantity of this compound appear bright in T(1)-weighted MRI images. Here we demonstrate that US-Gd(2)O(3) can be used to label GL-261 glioblastoma multiforme cells, followed by localization and visualization in vivo using MRI. Very high amounts of Gd are efficiently internalized and retained in cells, as confirmed with TEM and ICP-MS. Labeled cells were visualized in vivo at 1.5 T using the chicken embryo model. This is one more step toward the development of "positively contrasted" cell tracking procedures with MRI.