Isabelle Bataille

Concentrated collagen hydrogels as dermal substitutes

Collagen hydrogels first appeared promising for skin repair. Unfortunately, their extensive contraction and their poor mechanical properties constituted major disadvantages toward their utilization as permanent graft. The present study has investigated a way to correct these drawbacks by increasing the collagen concentration in controlled conditions. Concentrated collagen hydrogels (CCH) at 1.5, 3 and 5mg/ml were obtained. The effect of raised collagen concentration on contraction, cell growth and remodeling activities was evaluated for 21 days in culture. Subsequently, in vivo integration of CCH and normal collagen hydrogels (NCH) was assessed. Compared to NCH, CCH contraction was delayed and smaller. At day 21, surface area of CCH at 3mg/ml was 18 times more important than that of NCH. Whatever the initial fibroblast density, CCH favored cell growth that reached about 10 times the initial cell number at day 21; cell proliferation was inhibited in NCH. Gelatinase A activities appeared lower in CCH than within NCH. In vivo studies in rats revealed a complete hydrolysis of NCH 15 days after implantation. In contrast, CCH at 3mg/ml was still present after 30 days. Moreover, CCH showed cell colonization, neovascularization and no severe inflammatory response. Our results demonstrate that concentrated collagen hydrogels can be considered as new candidates for dermal substitution because they are is easy to handle, do not contract drastically, favor cell growth, and can be quickly integrated in vivo.

Silica-collagen bionanocomposites as three-dimensional scaffolds for fibroblast immobilization.

Silica-collagen bionanocomposite hydrogels were obtained by addition of silica nanoparticles to a protein suspension followed by neutralization. Electron microscopy studies indicated that larger silica nanoparticles (80 nm) do not interact strongly with collagen, whereas smaller ones (12 nm) form rosaries along the protein fibers. However, the composite network structurally evolved with time due to the contraction of the cells and the dissolution of the silica nanoparticles. When compared to classical collagen hydrogels, these bionanocomposite materials showed lower surface contraction in the short term (1 week) and higher viability of entrapped cells in the long term (3 weeks). A low level of gelatinase MMP2 enzyme expression was also found after this period. Several proteins involved in the catabolic and anabolic activity of the cells could also be observed by immunodetection techniques. All these data suggest that the bionanocomposite matrices constitute a suitable environment for fibroblast adhesion, proliferation and biological activity and therefore constitute an original three-dimensional environment for in vitro cell culture and in vivo applications, in particular as biological dressings.

Polysaccharides from the skin of the ray Raja radula. Partial characterization and anticoagulant activity

INTRODUCTION The polysaccharide fraction from the skin of the ray Raja radula was extracted, characterized and assayed for anticoagulant activity. MATERIALS AND METHODS A whole polysaccharidic fraction was extracted from the skin of the ray Raja radula by papain digestion followed by cetylpyridinium chloride and ethanol precipitation and was subjected to gel chromatography and anion exchange chromatography, acetate cellulose electrophoresis and characterized by physicochemical procedures. APTT and anti Xa assays were performed to assess the anticoagulant activity of the polysaccharidic fractions in comparison with unfractionated heparin. RESULTS Gel and anion-exchange chromatography revealed two negatively charged polysaccharidic populations different in both molecular weight and charge. Infrared spectra suggested the occurrence of uronic acids and acetylated hexosamines. The second polysaccharide was highly sulfated, with a sulfate content of approximately 29%. These data suggested that dermatan sulfate (DS) is the sulfate rich polysaccharide whereas hyaluronic acid (HA) is the polysaccharide devoid of sulfate groups. Molecular mass characterization indicated that their average molecular masses were 22 kDa and 85 kDa, respectively. The sulfated polysaccharide, i.e. presumably DS, accounted alone for the observed concentration-dependent anticoagulant activity which was, as measured by APTT, 2 to 3-fold lower than that of heparin. In addition, it had a significant anti-Xa activity. CONCLUSION A major-sulfated polysaccharide, likely a dermatan sulfate, was extracted from the ray Raja radula skin. The results indicated that it exhibited a high anticoagulant activity and suggested that it was mediated by both heparin cofactor II and antithrombin.

In vitro Studies and Preliminary In vivo Evaluation of Silicified Concentrated Collagen Hydrogels

Hybrid and nanocomposite silica-collagen materials derived from concentrated collagen hydrogels were evaluated in vitro and in vivo to establish their potentialities for biological dressings. Silicification significantly improved the mechanical and thermal stability of the collagen network within the hybrid systems. Nanocomposites were found to favor the metabolic activity of immobilized human dermal fibroblasts while decreasing the hydrogel contraction. Cell adhesion experiments suggested that in vitro cell behavior was dictated by mechanical properties and surface structure of the scaffold. First-to-date in vivo implantation of bulk hydrogels in subcutaneous sites of rats was performed over the vascular inflammatory period. These materials were colonized and vascularized without inducing strong inflammatory response. These data raise reasonable hope for the future application of silica-collagen biomaterials as biological dressings.

Mechanical properties of rat thoracic and abdominal aortas

Mechanical properties of abdominal and thoracic arteries of 2mm in diameter were determined from adults Wistar rats. A tensile testing instrument was used to obtain stress/strain curves with arteries immersed in physiological buffer at 37 degrees C. A displacement was applied on all arteries with various frequencies (1-7.5Hz) and strains (5-60%). From each curve a Young modulus was obtained using a mathematical model based on a nonlinear soft tissue model. No influence of frequency on modulus was evidenced in the tested range. Abdominal aortas, which were found slightly thicker than thoracic aortas, were characterized by a higher modulus. Due to the interest of decellularized biological materials, we also used SDS/Triton treated arteries, and found that the chemical treatment increased modulus of thoracic arteries. Tensile tests were also performed on thoracic aortas in the longitudinal and transversal directions. Longitudinal moduli were found higher than transversal moduli and the difference could be related to the longitudinal orientation of collagen fibers. These data and mathematical model seem useful in the design of new vascular synthetic or biological prostheses for the field of tissue engineering.

Influence of polysaccharide composition on the biocompatibility of pullulan/dextran-based hydrogels

The implantation of a biomaterial for tissue engineering requires the presence of a suitable scaffold on which the tissue repair and regeneration will take place. Polymers have been frequently used for that purpose because they show similar properties to that of the natural extracellular matrix. Scaffold properties and biocompatibility are modulated by the composition of the polymers used. In this work four polysaccharide-based hydrogels (PSH) made of dextran and pullulan were synthesized. Their in vitro properties were determined and then tested in vivo in a rat model. As pullulan concentration increased in dextran hydrogels, the glass transition temperature and the maximum modulus decreased. In vitro degradation studies for 30 days demonstrated no significant degradation of PSH except for 100% pullulan hydrogel. In vivo tissue response evaluated 30 days after PSH subcutaneous implantation in rats indicated that all PSH were surrounded by a fibrous capsule. Adding pullulan to dextran induced an increased inflammatory reaction compared to PSH-D(100% dextran) or PSH-D(75)P(25)(75% dextran). This in vitro and in vivo data can be used in the design of hydrogels appropriate for tissue engineering applications.

A biocompatible polysaccharide hydrogel-embedded polypropylene mesh for enhanced tissue integration in rats.

Prosthetic materials are largely used in surgery and tissue engineering. However, many postoperative complications are due to poor integration of the materials, which delays the healing process. The objective of our study was to develop a synthetic scaffold that, according to histopathological and biomechanical criteria, would achieve both tolerance and efficiency. In this study, we evaluated the effect of intramuscular and subcutaneous implantation of a new hybrid mesh (HM) in rats. This HM was composed of clinical grade polypropylene mesh embedded in a polysaccharide hydrogel. Histological and biomechanical studies on the polysaccharide gel alone and on HM were performed 15 and 30 days after implantation, and then compared with two clinically used materials, porcine decellularized small intestinal submucosa and a polypropylene mesh. Results showed that the incorporation of a polypropylene mesh within the polysaccharide hydrogel led to the absence of adverse effects and better tissue organization. Thus, this new synthetic biocompatible HM with suitable properties for tissue repair appears to be a promising material for clinical applications.

Radical Copolymerization of Vinyl Ethers and Cyclic Ketene Acetals as a Versatile Platform to Design Functional Polyesters

Free-radical copolymerization of cyclic ketene acetals (CKAs) and vinyl ethers (VEs) was investigated as an efficient yet simple approach for the preparation of functional aliphatic polyesters. The copolymerization of CKA and VE was first predicted to be quasi-ideal by DFT calculations. The theoretical prediction was experimentally confirmed by the copolymerization of 2-methylene-1,3-dioxepane (MDO) and butyl vinyl ether (BVE), leading to rMDO =0.73 and rBVE =1.61. We then illustrated the versatility of this approach by preparing different functional polyesters: 1) copolymers functionalized by fluorescent probes; 2) amphiphilic copolymers grafted with poly(ethylene glycol) (PEG) side chains able to self-assemble into PEGylated nanoparticles; 3) antibacterial films active against Gram-positive and Gram-negative bacteria (including a multiresistant strain); and 4) cross-linked bioelastomers with suitable properties for tissue engineering applications.

Comparative studies on the mechanisms of action of four polysaccharides on arterial restenosis.

Percutaneous coronary interventions play a major role in the management of patients affected by coronary artery diseases. However, their efficiency is impaired by restenosis, defined as a reduction of the vessel lumen, occurring a few months after the procedure. A low-molecular-weight fraction of fucoidan, a vegetal heparin-like sulphated polysaccharide, was recently shown to greatly reduce in-stent restenosis after angioplasty in rabbits. To better understand the in vivo anti-restenotic effects of this polymer, we used fractions of fucoidan and compared to heparin and dextran of different sizes. We carried out in vitro growth inhibition experiments on vascular smooth muscle cells, performed an in vivo pharmacokinetic study, and locally delivered fluorescently-labeled polysaccharides in rabbit iliac arteries after angioplasty with a non-occlusive catheter. The results indicated that (i) preparation of well-characterized fractions from natural fucoidan is compulsory for in vitro and in vivo studies, (ii) antiproliferative activity of sulphated polysaccharides on cultured smooth muscle cells is not a major predictive factor for the reduction of restenosis in vivo and (iii) pharmacokinetic parameters and binding of low-molecular-weight fucoidan on angioplasty-induced injured vascular walls are important local and general factors controlling its mechanisms of action.