Development, Validation, and Performance of Chitosan-Based Coatings Using Catechol Coupling.

The use of long-lasting polymer coatings on biodevice surfaces has been investigated to improve material-tissue interaction, minimize adverse effects, and enhance their functionality. Natural polymers, especially chitosan, are of particular interest due to their excellent biological properties, such as biocompatibility, non-toxicity, and antimicrobial properties. One way to produce chitosan coating is by covalent grafting with catechol molecules such as dopamine, caffeic acid, and tannic acid, resulting in an attachment ten times stronger than that of simple physisorption. Caffeic acid presents an advantage over dopamine because it allows direct chitosan grafting, due to its terminal carboxylic acid group, without the need of a linking arm, as employed in the dopamine approach. In this study, the grafting of chitosan using caffeic acid, over surfaces or in solution, is compared with dopamine grafting using poly(ethylene glycol) as a linking arm. The following coating properties are observed; covering and homogeneity are assessed by X-ray photoelectron spectroscopy and atomic force microscopy analyses, hydrophilicity with contact angle measurements, stability with aging tests, anticorrosion behavior, and coating non-toxicity. Results show that grafting using caffeic acid/chitosan in solution over a metallic surface may be advantageous, compared to traditional dopamine coating.

Comparison of the linking arm effect on the biological performance of a CD31 agonist directly grafted on L605 CoCr alloy by a plasma-based multistep strategy.

Stents are cardiovascular implants deployed on atherosclerotic arteries that aid in reopening, sustaining, and avoiding their collapse. Nevertheless, postimplantation complications exist, and the risk of the renewal of the plaque subsists. Therefore, enhanced properties are mandatory requirements for clinics. For that purpose, a novel approach allowing the direct-grafting of bioactive molecules on cobalt-chromium devices (L605) has been developed. This original strategy involves the direct plasma functionalization of metallic surfaces with primary amines (-NH2). These groups act as anchor points to covalently graft biomolecules of interest, herein a peptide derived from CD31 (P23) with proendothelialization and antithrombotic properties. However, the biological activity of the grafted peptide could be impacted by its conformation. For this study, glutaric anhydride (GA), a short chain spacer, and polyethylene glycol (PEG) with antifouling properties were used as linking arms (LAs). The covalent grafting of the CD31 agonist on L605 by different LAs (GA-P23 and PEG-P23) was confirmed by XPS and ToF-SIMS analyses. The biological performance of these functionalized surfaces showed that, compared to the electropolished (EP) alloy, grafting the P23 with both LA increases adhesion and proliferation of endothelial cells (ECs) since day 1: EP = 68 ± 10%, GA-P23 = 101 ± 7%, and PEG-P23 = 106 ± 5% of cell viability. Moreover, ECs formed a complete monolayer at the surface, preventing clot formation (hemoglobin-free >80%). The potential of this plasma-based strategy for cardiovascular applications was confirmed by promoting a fast re-endothelialization, by improving the hemocompatibility of the alloy when coupled with the CD31 agonist and by its transfer onto commercial L605 stents, as confirmed by ToF-SIMS.

Heparin-Modified Collagen Gels for Controlled Release of Pleiotrophin: Potential for Vascular Applications.

A fast re-endothelialization, along with the inhibition of neointima hyperplasia, are crucial to reduce the failure of vascular bypass grafts. Implants modifications with molecules capable of speeding up the re-endothelialization process have been proposed over the last years. However, clinical trials of angiogenic factor delivery have been mostly disappointing, underscoring the need to investigate a wider array of angiogenic factors. In this work, a drug release system based on a type I collagen hydrogel has been proposed for the controlled release of Pleiotrophin (PTN), a cytokine known for its pro-angiogenetic effects. Heparin, in virtue of its ability to sequester, protect and release growth factors, has been used to better control the release of PTN. Performances of the PTN drug delivery system on endothelial (ECs) and smooth muscle cells (SMCs) have been investigated. Structural characterization (mechanical tests and immunofluorescent analyses of the collagen fibers) was performed on the gels to assess if heparin caused changes in their mechanical behavior. The release of PTN from the different gel formulations has been analyzed using a PTN-specific ELISA assay. Cell viability was evaluated with the Alamar Blue Cell Viability Assay on cells directly seeded on the gels (direct test) and on cells incubated with supernatant, containing the released PTN, obtained from the gels (indirect test). The effects of the different gels on the migration of both ECs and SMCs have been evaluated using a Transwell migration assay. Hemocompatibility of the gel has been assessed with a clotting/hemolysis test. Structural analyses showed that heparin did not change the structural behavior of the collagen gels. ELISA quantification demonstrated that heparin induced a constant release of PTN over time compared to other conditions. Both direct and indirect viability assays showed an increase in ECs viability while no effects were noted on SMCs. Cell migration results evidenced that the heparin/PTN-modified gels significantly increased ECs migration and decreased the SMCs one. Finally, heparin significantly increased the hemocompatibility of the collagen gels. In conclusion, the PTN-heparin-modified collagen here proposed can represent an added value for vascular medicine, able to ameliorate the biological performance, and integration of vascular grafts.

Increasing Cell Seeding Density Improves Elastin Expression and Mechanical Properties in Collagen Gel-Based Scaffolds Cellularized with Smooth Muscle Cells.

Vascular tissue engineering combines cells with scaffold materials in vitro aiming the development of physiologically relevant vascular models. For natural scaffolds such as collagen gels, where cells can be mixed with the material solution before gelation, cell seeding density is a key parameter that can affect extracellular matrix deposition and remodeling. Nonetheless, this parameter is often overlooked and densities sensitively lower than those of native tissues, are usually employed. Herein, the effect of seeding density on the maturation of tubular collagen gel-based scaffolds cellularized with smooth muscle cells is investigated. The compaction, the expression, and deposition of key vascular proteins and the resulting mechanical properties of the constructs are evaluated up to 1 week of maturation. Results show that increasing cell seeding density accelerates cell-mediated gel compaction, enhances elastin expression (more than sevenfold increase at the highest density, Day 7) and finally improves the overall mechanical properties of constructs. Of note, the tensile equilibrium elastic modulus, evaluated by stress-relaxation tests, reach values comparable to native arteries for the highest cell density, after a 7-day maturation. Altogether, these results show that higher cell seeding densities promote the rapid maturation of collagen gel-based vascular constructs toward structural and mechanical properties better mimicking native arteries.

A Cost-Effective Culture System for the In Vitro Assembly, Maturation, and Stimulation of Advanced Multilayered Multiculture Tubular Tissue Models.

The development of tubular engineered tissues is a challenging research area aiming to provide tissue substitutes but also in vitro models to test drugs, medical devices, and even to study physiological and pathological processes. In this work, the design, fabrication, and validation of an original cost-effective tubular multilayered-tissue culture system (TMCS) are reported. By exploiting cellularized collagen gel as scaffold, a simple moulding technique and an endothelialization step on a rotating system, TMCS allowed to easily prepare in 48 h, trilayered arterial wall models with finely organized cellular composition and to mature them for 2 weeks without any need of manipulation. Multilayered constructs incorporating different combinations of vascular cells are compared in terms of cell organization and viscoelastic mechanical properties demonstrating that cells always progressively aligned parallel to the longitudinal direction. Also, fibroblast compacted less the collagen matrix and appeared crucial in term of maturation/deposition of elastic extracellular matrix. Preliminary studies under shear stress stimulation upon connection with a flow bioreactor are successfully conducted without damaging the endothelial monolayer. Altogether, the TMCS herein developed, thanks to its versatility and multiple functionalities, holds great promise for vascular tissue engineering applications, but also for other tubular tissues such as trachea or oesophagus.

A planar model of the vessel wall from cellularized-collagen scaffolds: focus on cell-matrix interactions in mono-, bi- and tri-culture models.

The acquisition of new thorough knowledge on the interactions existing between vascular cells would represent a step forward in the engineering of vascular tissues. In this light, herein we designed a physiological-like tri-culture in vitro vascular wall model using a planar cellularized collagen gel as the scaffold. The model can be obtained in 24 h and features multi-layered hierarchical organization composed of a fibroblast-containing adventitia-like layer, a media-like layer populated by smooth muscle cells and an intima-like endothelial cell monolayer. After 7 days of static culture, the compaction of the collagen matrix by the vascular cells was achieved, and the deposition of the vascular extracellular matrix components fibronectin, fibrillin-1 and tropoelastin was observed. The blood-compatible functionality of the endothelial cell monolayer was demonstrated by a blood clotting assay: after 7 days of maturation, clotting was prevented on the endothelialized constructs (more than 80% free hemoglobin maintained after 60 min of blood contact) but not at all on non-endothelialized ones (less than 20% free hemoglobin). In addition, western blotting results suggested that in the tri-culture model the loss of smooth muscle cell phenotype was delayed compared to what was observed in the mono-culture model, finally resulting in a behaviour more similar to the in vivo conditions. Overall, our findings indicate that this in vitro model has the potential to be used as an advanced system to examine vascular cell behavioural interactions, as well as for drug testing and the investigation of physiological and pathological processes.

Rotation-based technique for the rapid densification of tubular collagen gel scaffolds.

Type I collagen gel is often used as a tubular scaffold because of its easy molding properties as well as its biocompatibility, low immunogenicity and ability to be remodelled by cells. However, its highly hydrated structure contributes to its weak mechanical properties and reduces its ability to be handled, which is important in tubular tissue engineering. Although cell-driven remodelling of collagen matrices is known to reinforce their mechanical properties, this process can take weeks. This study introduces a novel, simple, and rapid technique using a rotational bioreactor to expel water and densify collagen under sterile conditions to generate denser and stronger collagen gel scaffolds. This process produces a dense tubular-shaped collagen gel which, compared to standard collagen gel scaffolds, shows a decreased wall thickness and a four-fold increase in collagen concentration. A denser collagen fiber network observed by immunofluorescence staining and mechanical characterisation shows a twenty-fold increase in the elastic modulus of the dense constructs which maintain cell viability inside the scaffold. Moreover, by simply modifying the scaffold mold, customised shapes and sizes can be obtained to provide a wide range of applications, including complex tubular geometries and multi-layered scaffolds for the culture of various cell types and tissues.

Specific IgG response against Mycobacterium avium paratuberculosis in children and adults with Crohn's disease.

BACKGROUND AND AIMS: Presence of serum antibodies against Mycobacterium avium paratuberculosis (MAP) in Crohn's Disease (CD) as a disease characteristic remains controversial. In the present work, we assessed antibody reactivity of serum and intestinal fluid against four distinct MAP-antigens, including the recently identified MAP-specific lipopentapeptide (L5P). METHODS: Immunoglobulin concentrations and specificity against 3 non MAP-specific antigens: glycosyl-transferase-d (GSD), purified protein derivative from MAP (Johnin-PPD), heparin binding haemagglutinin (MAP-HBHA) and one MAP-specific antigen: synthetic L5P were determined by ELISA in gut lavage fluids from adult controls or patients with CD, and in sera of children or adult controls or patients with CD, ulcerative colitis or celiac disease. RESULTS: Total IgA and IgG concentrations were increased in sera of children with CD but were decreased in sera of adults with CD, thereof specificity against MAP antigens was assessed by normalizing immunoglobulin concentrations between samples. In CD patients, IgG reactivity was increased against the four MAP antigens, including L5P in gut lavage fluids but it was only increased against L5P in sera. By contrast, anti-L5P IgG were not increased in patients with ulcerative colitis or celiac disease. CONCLUSIONS: A significant increase in anti-L5P IgG is observed in sera of children and adults with CD but not in patients with other intestinal inflammatory diseases. Anti-L5P antibodies may serve as serological marker for CD.