Odile Damour

Influence of the degree of acetylation on some biological properties of chitosan films

In this study, we investigated in vitro the role of the degree of acetylation (DA) on some biological properties of chitosan films. We noticed that, whatever the DA, all chitosan films were cytocompatible towards keratinocytes and fibroblasts. We also demonstrated that the higher the DA of chitosan, the lower was the cell adhesion on the films. Fibroblasts appear to adhere twice as much as keratinocytes on these materials. We observed that keratinocyte proliferation increases when the DA of chitosan films decreases. Thus, DA influences the cell growth in the same way as cell adhesion. On the other hand, although they remain alive, fibroblasts do not proliferate on chitosan films. This behaviour is related to an extremely high adhesion on this kind of material, which certainly inhibits cell growth. In conclusion, DA plays a key role in cell adhesion and proliferation, but does not change the cytocompatibility of chitosan. In parallel, it is also important to notice the role played by the surface morphology of the material, a second major parameter which influences the mechanism of adhesion.

Chitosan–chondroitin sulfate and chitosan–hyaluronate polyelectrolyte complexes: biological properties

In this work, we compare some biological properties of a highly deacetylated chitosan to those obtained with the materials made from its polyelectrolyte complexes with various GAGs such as chondroitin-sulfates and hyaluronic acid. The hydrolysis of the complexes by means of the specific hydrolytic enzymes is studied. Cell-adhesion and cell-proliferation on these materials is compared to that obtained with a pure chitosan material. Finally, a series of in vivo experiments is performed to test the wound-healing properties of this kind of complexes. All the results agree to show that chitosan has a protective effect against GAGs hydrolysis by their specific enzymes but only at pHs different from the optimal pH of the enzyme considered. In addition, they also agree to confirm that a pure chitosan material gives the best results in connection with cell-attachment and cell-proliferation or wound healing. Nevertheless, whatever the case, no adverse effect was observed with the polyelectrolyte complexes GAGs-chitosan.

Improvement of skin quality after fat grafting: clinical observation and an animal study.

Background: Restoration of volume and contour defects is a challenge in plastic surgery. Autologous fat tissue transfer is gaining increasing popularity in this field. The aim of this study was to investigate the histologic modifications of the skin after fat tissue grafting on an animal model. Methods: Thirty nude mice, divided into three groups, were used in the experiment. All 30 mice received human fat tissue on one side. On the opposite side, 10 mice received nothing (negative control group), 10 mice received cell proliferation medium, and the remaining 10 mice received only subcutaneous tunneling. Eight weeks later, biopsies of the skin and subcutaneous tissue were performed and specimens were analyzed by hematoxylin-phloxin-saffron staining. Dermis thickness was measured. To differentiate human from murine collagen fibers, human and murine collagen type I antibodies were used. The other types of collagen were investigated by immunohistochemistry (immunostaining) using collagen type III, V, and VI antibodies. Results: Fat tissue was found in all animals. Macroscopically, fat tissue presented normal aspects, with abundant peripheral neovascularization. Histologic examination showed abundant extracellular matrix around the injected human fat tissue. This was attributable to increased type I collagen fibers of murine origin as a result of the murine fibroblast stimulation by the grafted human fat tissue. Dermal thickness after fat grafting was significantly greater. This was not attributable to inflammatory reactions, because no modification was detected in our control groups. Conclusions: This study shows that fat tissue grafting stimulates a neosynthesis of collagen fibers at the recipient site and makes the dermis thicker. However, the long-term effects remain undetermined and need further investigation.

Cultured autologous oral mucosal epithelial cell sheet (CAOMECS) transplantation for the treatment of corneal limbal epithelial stem cell deficiency.

PURPOSE Total bilateral corneal limbal epithelial stem cell deficiency (LSCD) cannot be treated with the surgical transplantation of autologous limbus or cultured autologous limbal epithelium. Transplantation of allogenic limbal epithelium is possible but requires immunosuppressive treatments. Cultured autologous oral mucosal epithelial cell sheet (CAOMECS) is a transparent, resistant, viable, and rapidly bioadhesive cell sheet, cultured with the UpCell-Insert technology (CellSeed, Inc., Tokyo, Japan), which allows for grafting onto the patients corneal stroma without suturing. It has therefore been proposed as an alternative treatment for LSCD. METHODS The objectives were to assess the safety and efficacy of CAOMECS, using a prospective Gehans design. Safety was measured in terms of ocular adverse events during the study period, and efficacy was measured using a composite criterion based on epithelial defect, punctate epithelial keratopathy, conjunctival epithelium on the cornea, number of vascular pediculi, and vessel activity. RESULTS CAOMECS was found to be safe and effective. In total, 26 eyes of 25 patients received a graft. Two patients experienced serious adverse events classified as not product related. Twenty-five patients were included in the efficacy analysis, as one patient was lost to follow-up. The treatment was found to be effective in 16 of 25 patients at 360 days after grafting. Of the 23 patients who completed follow-up at 360 days, 22 had no ulcers, and 19 showed a decrease in the severity of the punctate epithelial keratopathy. CONCLUSIONS CAOMECS is a well-tolerated and safe tissue-engineered product. These results suggest its efficacy for reconstructing the ocular surface in patients with total bilateral corneal LSCD.

Collagen synthesis by fibroblasts cultured within a collagen sponge

We prepared a collagen sponge made of type I and III bovine collagen, glycosaminoglycans (GAG) and chitosan. Fibroblasts grown within the collagen sponge express a sixfold increase of their collagen synthesis, compared with fibroblasts embedded in a collagen gel. Moreover, collagen synthesis is twice as high in the collagen sponge than in a monolayer culture. The collagen sponge culture system promotes a dynamic model for us to perform studies on the regulations of collagen synthesis. Increased collagen production within the collagen sponge leads fibroblasts to reconstitute their own extracellular matrix, which should be more physiological than a bovine collagen gel.

A tissue-engineered endothelialized dermis to study the modulation of angiogenic and angiostatic molecules on capillary-like tube formation in vitro.

Summary Background Because angiogenesis is a major feature of different physiological and pathological situations, the identification of factors that stimulate or inhibit this process and the elucidation of their mechanisms of action are most certainly of clinical relevance. We have produced a new model of endothelialized reconstructed dermis that promotes the spontaneous formation of a human capillary‐like network and its stabilization in vitro for a period longer than 1 month.

Characterization of Skin Reconstructed on a Chitosan-Cross-Linked Collagen-Glycosaminoglycan Matrix

Reconstruction of skin requires both the dermal and epidermal equivalent of the skin. We have developed a reconstructed skin composed of two compartments: (1) a dermal equivalent comprising an acellular dermal substrate populated by foreskin fibroblasts and (2) an epidermis regenerated from normal human keratinocytes seeded onto the dermal equivalent. The dermal substrate contains type I and III collagen and glycosaminoglycans (GAGs) cross-linked by chitosan. Fibroblasts seeded into the porous structure of the dermal substrate provide a dermal equivalent suitable to support epidermal cells. Keratinocytes attach quickly, exhibit mitotic activity and form a continuous and stratified epidermis. After 2 weeks of culture, histological sections show a basal layer with cuboidal cells attached to the dermal equivalent and several suprabasal cell layers including the stratum corneum. Transmission electron microscopy revealed the cell membrane densification (hemidesmosomes) at the dermoepidermal junction; however, the lamina densa was found discontinuous at this stage. We noted the presence of lipid vesicles in spinous layer and keratohyalin granules in granular layer. The epidermal differentiation was complete terminal with the stratum corneum containing several layers of corneocytes filled with tonofilaments. Reconstructed skin, based on our chitosan-cross-linked collagen-GAG matrix is morphologically equivalent to normal human skin and should thus provide a useful tool for in vitro toxicological studies as well as a suitable wound covering for the treatment of patients with severe burns.

Collagen fibril network and elastic system remodeling in a reconstructed skin transplanted on nude mice

Wound healing of deep and extensive burns can induce hypertrophic scar formation, which is a detrimental outcome for skin functionality. These scars are characterized by an impaired collagen fibril organization with fibril bundles oriented parallel to each other, in contrast with a basket weave pattern arrangement in normal skin. We prepared a reconstructed skin made of a collagen sponge seeded with human fibroblasts and keratinocytes and grown in vitro for 20 days. We transplanted it on the back of nude mice to assess whether this reconstructed skin could prevent scar formation. After transplantation, murine blood vessels had revascularized one-third of the sponge thickness on the fifth day and were observed underneath the epidermis at day 15. The reconstructed skin extracellular matrix was mostly made of human collagen I, organized in loosely packed fibrils 5 days after transplantation, with a mean diameter of 45 nm. After 40-90 days, fibril bundles were arranged in a basket weave pattern while their mean diameter increased to 56 nm, therefore exactly matching mouse skin papillary dermis organization. Interestingly, we showed that an elastic system remodeling was started off in this model. Indeed, human elastin deposits were organized in thin fibrils oriented perpendicular to epidermis at day 90 whereas elastic system usually took years to be re-established in human scars. Our reconstructed skin model promoted in only 90 days the remodeling of an extracellular matrix nearly similar to normal dermis (i.e. collagen fibril diameter and arrangement, and the partial reconstruction of the elastic system).

Development of a Reconstructed Cornea from Collagen–Chondroitin Sulfate Foams and Human Cell Cultures

PURPOSE To develop an artificial cornea, the ability to coculture the different cell types present in the cornea is essential. Here the goal was to develop a full-thickness artificial cornea using an optimized collagen-chondroitin sulfate foam, with a thickness close to that of human cornea, by coculturing human corneal epithelial and stromal cells and transfected human endothelial cells. METHODS Corneal extracellular matrix was simulated by a porous collagen/glycosaminoglycan-based scaffold seeded with stromal keratocytes and then, successively, epithelial and endothelial cells. Scaffolds were characterized for bulk porosity and pore size distribution. The performance of the three-dimensional construct was studied by histology, immunofluorescence, and immunohistochemistry. RESULTS The scaffold had 85% porosity and an average pore size of 62.1 microm. Keratocytes populated the scaffold and produced a newly synthesized extracellular matrix as characterized by immunohistochemistry. Even though the keratocytes lost their CD34 phenotype marker, the absence of smooth muscle actin fibers showed that these cells had not differentiated into myofibroblasts. The epithelial cells formed a stratified epithelium and began basement membrane deposition. An endothelial cell monolayer beneath the foam was also apparent. CONCLUSIONS These results demonstrate that collagen-chondroitin sulfate scaffolds are good substrates for artificial cornea construction with good resilience, long-term culture capability, and handling properties.

Evolution of three dimensional skin equivalent models reconstructed in vitro by tissue engineering

Since the culture of keratinocytes on feeder layers, research to produce skin equivalents has been motivated by the challenge of treating large burns and chronic wounds and by European regulations which both require proof of the innocuousness and the effectiveness of cosmetic products, and which forbid animal testing. The dynamism in fundamental research, dermocosmetology and the pharmaceutical industry has led to the evolution and complexification of reconstructed skin. The Collagen-GAG-Chitosan sponge, as well as the self-assembly model, allow dermal reconstruction in which the neosynthesized extracellular matrix contains all of the desired macromolecules. It is deposited forming an ultrastructurally organised architecture. The quality of the dermis obtained allows the development and regeneration of a pluristratified and differentiated epidermis firmly anchored by an organised dermal-epidermal junction. Evolution of reconstructed skin into models which are more and more similar to the physiological skin results in higher graft take rates in the treatment of burns and chronic wounds, and brings to research, to dermocosmetology and to the pharmaceutical industry, a wide range of products such as pigmented, endothelialized, immunocompetent, and now adipose reconstructed skins. The present review will mainly concentrate on the latest developments in skin engineering and will mostly concern the studies carried out by our groups.