The survival of thick tissues/organs produced by tissue engineering requires rapid revascularization after grafting. Although capillary-like structures have been reconstituted in some engineered tissues, little is known about the interaction between normal epithelial cells and endothelial cells involved in the in vitro angiogenic process. In the present study, we used the self-assembly approach of tissue engineering to examine this relationship. An endothelialized tissue-engineered dermal substitute was produced by adding endothelial cells to the tissue-engineered dermal substitute produced by the self-assembly approach. The latter consists in culturing fibroblasts in the medium supplemented with serum and ascorbic acid. A network of tissue-engineered capillaries (TECs) formed within the human extracellular matrix produced by dermal fibroblasts. To determine whether epithelial cells modify TECs, the size and form of TECs were studied in the endothelialized tissue-engineered dermal substitute cultured in the presence or absence of epithelial cells. In the presence of normal keratinocytes from skin, cornea or uterine cervix, endothelial cells formed small TECs (cross-sectional area estimated at less than 50 microm(2)) reminiscent of capillaries found in the skin's microcirculation. In contrast, TECs grown in the absence of epithelial cells presented variable sizes (larger than 50 microm(2)), but the addition of keratinocyte-conditioned media or exogenous vascular endothelial growth factor induced their normalization toward a smaller size. Vascular endothelial growth factor neutralization inhibited the effect of keratinocyte-conditioned media. These results provide new direct evidence that normal human epithelial cells play a role in the regulation of the underlying TEC network, and advance our knowledge in tissue engineering for the production of TEC networks in vitro.
The authors' study suggests that the SASS used as a biological dressing is a promising treatment for hard-to-heal chronic venous and mixed ulcers that are unresponsive to compression therapy.
Hbpital du Saint-saCrrment du centm hospitalier affilid, 1050 Chemin S a i n t e F q , Qu.t?bee, QC GlS 4L8, Canada he physico-chemical properties of a dermal-epidermal reconstructed skin provide numerous advantages for their use as permanent skin T replacement over full thickness skin injuries. The reconstructed skin elaborated in our laboratory by the new tissue engineering technique, the self-assembly approach, possesses a network of elastic fibers and collagen fibrils that confers suppleness and strength to the reconstructed tissue (Larouche et al., 2000). This tissue engineered skin has tremendous therapeutic values for autologous grafting of large burn wounds, giant nevi, and the healing of cutaneous ulcers.Our dermal-epidermal living human substitute is composed of skin fibroblasts that secrete their own extracellular matrix and skin keratinocytes that proliferate and differentiate into a cornified, stratified epithelium (Michel et al ., 1999). No synthetic or exogenous extracellular matrix, such as collagen or fibrin, has been added to the organotypic culture. The only extracellular matrix and basement membrane material found in our reconstructed skin has been secreted, and assembled by the autologous cells in culture and is thus fully immunocompatible with the donor. The cells that constitute this reconstructed skin retain the ability to proliferate, differentiate, and heal (Laplante et al ., 2000), which is of crucial importance in the long-term fonctionnality of the graft. The ability to secrete soluble factors enabling wound healing is a key function that is important for the use of our reconstructed skin as a woundhealing device.The keratinocytes are usually isolated from skin biopsy using an enzymatic digestion with thermolysin or dispase and/or trypsin (Cermain et al., 1993; Martinet et al., 1988; Rheinwald et al., 1975; Liu et al ., 1978). The major obstacle in the isolation of epithelial cells is the contamination with fibroblasts which can overgrow the keratinocytes. The fibroblasts can be cultured from explants, but this method is timeconsuming and not always successful. The mesenchymal cells can also be recuperated with a collagenase (enzymatic) treatment of the peeled dermis, and thus generating a more representative cell population. The simultaneous isolation of keratinocytes, fibroblasts, and endothelial cells using trypsin to digest the basal membrane, and explants to isolate fibroblasts has been documented (Normand et al., 1995). Their ability to reconstruct a tissue-engineered skin has not been investigated. ~ *Author to whom correspondence may be addressed. E-mail address: lude.germain8 chg. ulaval.coThe key step in the reconstruction of skin by the self-assembly approach is to use fibroblasts capable of secreting a mature extracellular matrix and keratinocytes that can associate with one another to form a stratified, differentiated epidermis. To determine the most efficient way to extract both cell types from a single cutaneous biopsy, five different enzymatic combinations were tested...
Introduction: Venous ulcers are the most common chronic wounds of the lower limbs. In our laboratory, the self‐assembly method is used to reconstruct autologous skin that can be used as a biological dressing (In Vitro Cell Dev Bio Anim, 1999: 35, 318; J Chem Eng, 2001: 79, 663). Goals and Methods: The aim of this study was to establish if our self‐assembled skin substitute (SASS) allows the closure of nonhealing venous leg ulcers. A case study of a 68‐year‐old woman with a chronic venous leg ulcer present for over one year and measuring 135 cm2 is thus herein presented. The autologous cells were isolated from a cutaneous biopsy (1 cm2) and cultivated in vitro(J Chem Eng, 2001: 79, 663). The autologous SASS was produced and assembled by the cells thus it does not contain any exogenous or synthetic material (In Vitro Cell Dev Bio Anim, 1999: 35, 318). The wound was debrided, covered with grafts of SASS and maintained in place with a compressive dressing (Coban, 3M, St‐Paul, MN). A new SASS was applied weekly until wound closure. The SASS seemed to lessen the pain caused by the ulcer. Seven applications of the SASS were necessary to lead to a full wound closure. Conclusion: Venous leg ulcer quickly reepithelialized after application of our SASS This case indicates that use of the SASS can reduce pain and promote closure of nonhealing leg ulcers Furthermore, we thus hope that our SASS biological dressing maybe clinically useful to treat difficult‐to‐heal leg ulcers. This study was approved by the Health Canada’s Therapeutic Products Directorate and by the Ethic Committee of the CHAUQ, Saint‐Sacrement Hospital. Acknowledgements: Fédération des Pompiers du Québec pour les Grands Brûlés.
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