Christel Barbaud

A substituted dextran enhances muscle fiber survival and regeneration in ischemic and denervated rat EDL muscle

Ischemia and denervation of EDL muscle of adult rat induce a large central zone of degeneration surrounded by a thin zone of peripheral surviving muscle fibers. Muscle regeneration is a complex phenomenon in which many agents interact, such as growth factors and heparan sulfate components of the extracellular matrix. We have shown that synthetic polymers, called RGTA (as regenerating agents), which imitate the heparan sulfates, are able to stimulate tissue repair when applied at the site of injury. In crushed muscles, RGTA were found to accelerate both regeneration and reinnervation. In vitro, RGTA act as protectors and potentiators of various heparin binding growth factors (HBGF). It was postulated that in vivo their tissue repair properties were due in part to an increase of bioavailability of endogenously released HBGF. In the present work, we show that ischemic and denervated EDL muscle treated by a unique injection of RGTA differs from the control after 1 wk in several aspects: 1) the epimysial postinflammatory reaction is inhibited and the area of fibrotic tissue among fibers is reduced; 2) the peripheral zone, as measured by the number of intact muscle fibers, was increased by more than twofold; and 3) In the central zone, RGTA enhances the regeneration of the muscle fibers as well as muscle revascularization. These results suggest that RGTA both protects muscle fibers from degeneration and preserves the differentiated state of the surviving fibers. For the first time it is demonstrated that a functionalized polymeric compound can prevent some of the damage resulting from muscle ischemia. RGTA may therefore open a new therapeutic approach for muscle fibrosis and other postischemic muscle pathologies.—Desgranges, P., Barbaud, C., Caruelle, J.‐P., Barritaoult, D., Gautron, J. A substituted dextran enhances muscle fiber survival and regeneration in ischemic and denervated rat EDL muscle. FASEB J. 13, 761–766 (1999)

Heparan mimetic regulates collagen expression and TGF-β1 distribution in gamma-irradiated human intestinal smooth muscle cells

Radiation‐induced intestinal fibrosis is characterized by collagen accumulation, a process in which TGF‐β1 plays a key role. We analyzed the effects of gamma radiation on collagen expression and TGF‐βl distribution in human intestinal smooth muscle cells (HISM). We investigated the activity of a car‐boxymethylated and sulfated dextran (RG‐1503), exhibiting antifibrotic properties and promoting in vivo intestinal tissue repair, on irradiated HISM. After 60Co irradiation (10 Gy), HISM were labeled with [3H] proline (±RG‐1503). Radiolabeled collagen I, III, and V were quantified by SDS‐PAGE. TGF‐β1 was quantified by ELISA in culture medium, pericellular and intracellular compartments. Irradiation induced a specific 2.85‐fold increase in collagen III production by HISM. Collagen V decreased by 80% 72 h after irradiation. Pericellular TGF‐β1 was increased (up to twofold) in irradiated HISM. RG‐1503 added before or after irradiation reversed both mRNA and protein levels of collagen III and V to control values. RG‐1503 decreased the amount of TGF‐β1 in the cell layer below the control values. Irradiation of HISM induced the development of a fibrotic phenotype in terms of collagen production and TGF‐β1 distribution. The antifibrotic RG‐1503 restored HISM physiological characteristics and may represent a promising therapeutic approach for radiation‐induced intestinal fibrosis.—Alexakis, C., Guettoufi, A., Mestries, P., Strup, C., Mathé, D., Barbaud, C., Barritault, D., Caruelle, J.‐ P., Kern, P. Heparan mimetic regulates collagen expression and TGF‐β1 distribution in gamma‐irradiated human intestinal smooth muscle cells. FASEB J. 15, 1546–1554 (2001)

Chemically modified dextrans modulate expression of collagen phenotype by cultured smooth muscle cells in relation to the degree of carboxymethyl, benzylamide, and sulfation substitutions.

We developed regenerating agents (RGTAs) corresponding to polysaccharides derived from dextran and containing defined amounts of carboxymethyl (CM), carboxymethyl sulfate (CMS), carboxymethyl benzylamide (CMB), or carboxymethyl benzylamide sulfate (CMBS) groups with varying degrees of substitution. These compounds mimicked some effects of heparin on smooth muscle cell (SMC) proliferation and promoted in vivo tissue remodeling. We demonstrated that only RGTAs containing both CM and sulfate groups decreased SMC proliferation, in correlation with increased sulfation level. This effect was amplified by the presence of benzylamide. Independent of this activity on cell proliferation (i.e., with postconfluent cells), RGTAs modulated collagen biosynthesis by SMCs. On the one hand, CMBS more than CMS RGTAs induced a decrease of collagen III synthesis at the level of mRNA steady state and protein production. On the other hand, CMS to a greater extent than CMBS RGTAs increased both collagen V mRNA and protein production. In addition, only benzylamide-containing RGTAs increased accumulation of collagen I and III in the cell layer. In conclusion, RGTA bioactivities required the presence of CM functions, increased with the sulfation level, and varied with benzylamide substitution. RGTAs that modulate cell proliferation and collagen biosynthesis by differential mechanisms may represent potential antifibrotic agents.

Bioactive functionalized polymer of malic acid for bone repair and muscle regeneration.

A bioactive poly(β-hydroxyalkanoate) derived from malic acid was prepared and tested on bone repair and muscle regeneration. This functionalized and hydrolyzable polymer was obtained after several steps, the first one being the anionic copolymerization of three malolactonic acid esters. Chemical modifications were carried out on the terpolymer to turn benzyl-protecting groups into carboxyl groups and allyl groups into sulfonate groups. The resulting polymer bore carboxylate, sulfonate, and sec-butyl pendent groups in 65/25/10 molar proportions and were aimed at interacting with heparan binding growth factors. This polymer did not present any toxic effect in cell viability of HepG2 cells, over a large range of concentrations (0.01-0.25 mg l-1). Its ability to improve wound healing was tested in vivo and positive results are reported. Furthermore, the bioactivity of this polymer was evaluated using the regeneration model of Extensor digitorum longus (EDL) rat muscle. The study displayed a significant increase in the muscle regeneration and maturation.

Synthesis and Characterization of a New Polysaccharide-graft-polymethacrylate Copolymer for Three-Dimensional Hybrid Hydrogels

Hybrid materials constituted by hydrophobic and hydrophilic biocompatible macromolecules are useful for biomedical applications. In this context, a well-known acrylic monomer (methyl methacrylate) was polymerized and grafted onto the polysaccharide dextran by the use of ceric ammonium nitrate as a redox initiator in aqueous nitric acid medium. The effects of concentrations of dextran, acrylic monomer, and ceric ions on the copolymerization yields were investigated in detail. The obtained polymers were studied by solubility measurements, Fourier transform infrared spectrometry, (13)C nuclear magnetic resonance spectroscopy, and viscosimetric analysis. Interestingly, we found conditions to form transparent and homogeneous thin films or 3D structures with hybrid properties. Indeed, the copolymer, but not dextran or PMMA, could be dissolved in water/THF (20/80 v/v). The thermomechanical properties of the resulting copolymer analyzed by differential scanning calorimetry and dynamic mechanical analysis showed the occurrence of a single glass-transition temperature and a marked difference with the two homopolymers. The cytocompatibility of the copolymer with human endothelial cells was evidenced by the normal cell adhesion, proliferation, and morphology after 5 days in culture on these gels. In conclusion, this type of copolymer with hybrid properties of two biocompatible macromolecules could be of great interest as a 3D scaffold or for coating in biomedical applications.

Synthesis of new α, α′, β-trisubstituted β-lactones as monomers for hydrolyzable polyesters

Novel α, α′, β-trisubstituted β-lactones have been prepared by a concise and efficient synthesis in five steps from commercial racemic diethyl oxalpropionate. These lactones with different lateral groups can be polymerized or co-polymerized for obtaining polyesters with high molecular weight. Chemical modification of these functionalized hydrolyzable polymers will be used to adjust their properties to the selected temporary applications.

Films of dextran-graft-polybutylmethacrylate to enhance endothelialization of materials.

We have synthesized new structures obtained from amphiphilic copolymers of dextran and polybutylmethacrylate with the aim of endothelialization of biomaterials. Grafting of butylmethacrylate onto dextran has been carried out using ceric ammonium nitrate as initiator. Three copolymers were obtained (11, 30 and 37 wt.% dextran) and homogeneous thin films were successfully prepared. In contrast to dextran, the resulting films were stable in water, and copolymers characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry and dynamic mechanical analysis showed evidence of hybrid properties between the parent homopolymers. Surfaces of films were smooth when analyzed by atomic force microscopy (roughness 2+/-1 nm) but greatly differed in their hydrophilicity by increasing the dextran content (water contact angle from 99 degrees to 57 degrees). In contrast to polybutylmethacrylate, where the proliferation of vascular smooth muscle cells (VSMCs) was excellent but that of endothelial cells was very low, the copolymer containing 11% of dextran was excellent for endothelial cells but very limited for VSMCs. An in vitro wound assay demonstrated that copolymer with 11% dextran is even more favorable for endothelial cell migration than tissue-culture polystyrene. Increasing the dextran content in the copolymers decreased the proliferation for both vascular cell types. Altogether, these results show that transparent and water-insoluble films made from copolymers of dextran and butylmethacrylate copolymers with an appropriate composition could enhance endothelial cell proliferation and migration. Therefore, a potential benefit of this approach is the availability of surfaces with tunable properties for the endothelialization of materials.

Synthesis of new statistical and block co-polyesters by ROP of α,α,β-trisubstituted β-lactones and their characterizations

The preparation of α,α,β-trisubstituted β-lactones has opened the route to living anionic ring-opening polymerization of these monomers. Therefore, transfer reactions due to hydrogen abstraction were limited and it is now possible to strickly control sample molecular weight. Moreover, living end-groups lead to the preparation of block co-polymers with block controlled lenghts. In the same way, high-molecular-weight statistical co-polymers have been synthesized. Hydrolyzable micelles and nanoparticles will be prepared from these co-polymers after chemical modifications to adjust the hydrophilic/hydrophobic balance.

Synthesis of novel α, α', β-trisubstituted β-lactones

Abstract A concise and efficient synthesis of α,α′,β-trisubstituted β-lactones is presented. These novel lactones are easily obtained in five steps and will be dedicated to anionic ring opening polymerization.

Functionalized and degradable polymers of malic acid stimulate bone repair

A water soluble and hydrolysable polyester derived from malic acid has been synthesized by copolymerization of three different malolactonic acid esters. Functional pendant groups have been selected to interact with and protect heparin binding growth factors (HBGF). Three β-substituted β-lactones have been synthesized by using aspartic acid as a chiral precursor and benzyl, allyl and butyl alcohols in the formation of the ester pendant groups. The terpolymer has been subjected to three consecutive different chemical modifications. This modified terpolymer, able to induce new bone formation in an in vivo model, has the same property as carboxymethyl benzylamide sulfonate dextrans (CMDBS). Consequently, the distribution of the lateral functional groups is more essential than the glucidic nature of the backbone to acquire biological efficiency.