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The tissue engineering self-assembly approach allows the production of skin substitutes comprising both the dermis and epidermis, using methods promoting the secretion and organization of a dense extracellular matrix by skin cells. In a reconstructed epidermis, all cellular layers of the native tissue are present. An evaluation of the expression and localization of a number of specific protein markers revealed that the self-assembled, tissue-engineered skin substitute shares some common features with normal human skin, such as the expression of Ki-67, keratins 10 and 14, filaggrin, involucrin, transglutaminase, DLK, a3-integrin subunit, laminin-S, and collagens I, II, 1V, and VII. At the ultrastructural level, many differentiation markers can be observed, including desmosomes, as well as an organized basement membrane presenting hemidesmosomes, lamina densa, and lamina lucida. In this chapter, protocols to generate skin substitutes by the self-assembly approach will be presented and the methods including the labeling of the principal skin differentiation markers by immunofluorescence will be examined.

Tissue engineering is a novel sector arising from the biomaterial field, which is developing rapidly as a result of the dramatic cIinicalneed for organ replacement,since there is unfortunately an ever-growing lack of organs for transplantation. Various approaches are presently being developed in different laboratories and companies based on the utilization of biomaterials, extracellular matrix components, and cellsto produce substitutesto aJlowthe replacement of wounded or diseased tissues[1-4]. Theorgan reconstructionby tissue engineering presented in this chapter are of living tissues. This concept entails that the various cells incorporated in our constructs or tissues are not only readily dividing, but also metabolically active. Thus, mesenchymal cells (fibroblasts, smooth muscle cells) incorporated into the stromal component of these substitutes are also significantly involved in the reorganization of the extracellular matrix. Furthermore, the interactions between the mesenchymal cells and the epithelial cells improve the very nature, structure, and function of the resulting organ. Lastly, the presence of living cells, within the in vitro engineered tissues, adds the benefit of tissue remodeling and healing after transplantation in vivo. The source of cells that can be used for tissue reconstruction is dictated by the foreseen application. Autologous cells will be necessary for the production of living tissue substitutes when striving for permanent replacement of organs in order to prevent any histocompatibility mismatch and the ensuing predictable rejection (e.g., skin grafting for full-thickness burns). However, the rejection process has been shown to vary with the type of cells involved, and it may be possible to graft allogeneic engineered tissue under some appropriate conditions. But in such cases as keratinocytes, dentritic cells and endothelial cells that are privileged targets for rejection, autologous ceIls are necessary to permanently replace tissues

The tissue engineering self-assembly approach allows the production of skin substitutes comprising both the dermis and epidermis, using methods promoting the secretion and organization of a dense extracellular matrix by skin cells. In a reconstructed epidermis, all cellular layers of the native tissue are present. An evaluation of the expression and localization of a number of specific protein markers revealed that the self-assembled, tissue-engineered skin substitute shares some common features with normal human skin, such as the expression of Ki-67, keratins 10 and 14, filaggrin, involucrin, transglutaminase, DLK, a3-integrin subunit, laminin-S, and collagens I, II, 1V, and VII. At the ultrastructural level, many differentiation markers can be observed, including desmosomes, as well as an organized basement membrane presenting hemidesmosomes, lamina densa, and lamina lucida. In this chapter, protocols to generate skin substitutes by the self-assembly approach will be presented and the methods including the labeling of the principal skin differentiation markers by immunofluorescence will be examined., 0

The progress in tissue engineering has lead to the development of tri-dimensional tissues that can be used in vitro for various applications. Different methods have been designed to produce reconstructed dermis or skin in vitro. This chapter describes the human skin models and substitutes with respect to the evolution of their complexity as well as some of their potential applications. Dermal fibroblasts or myofibroblasts included in floating collagen gels produce useful wound healing models. Bi-layered human skin constructs comprising both the dermis and the epidermis could serve. for fundamental (eg. cell-matrix interactions) or applied (e.g. dermatoabsorption) studies. Another skin substitute is produced by seeding keratinocytes on fibroblasts. cultured in a collagen-chondroitin 4-6 sulfates and. chitosan sponge. The addition of endothelial cells to this model lead to the formation of capillary-like structure in the dermis. Finally, a method of human reconstructed skin production by the "auto-assembly" approach is presented. This model is developped from cells that produce their own extracellular matrix. No synthetic material or exogenous matrix is added. Thus, it could be completely autologous. Tissue engineered skin is an attractive tissue for gene therapy. Cells could be transplanted safely in vitro, evaluated for gene expression before their incorporation in reconstructed tissue and grafting in vivo. Of particular importance will be skin stem cells that have a long term regeneration potential and that can he cultured in vitro. The progress accomplished in tissue. engineering of skin is now applied to the reconstruction of other tissues and more complex organs such as ligaments, bronchi, bladder, cornea and blood vessels. These tissues could provide therapeutic alternatives in organ transplantation as well as models for varions in vitro applications.

The field of skin tissue engineering is a paradigm for the various efforts towards the reconstruction of other tissues and organ substitutes. As skin replacement, this biotechnological approach has evolved from simple cultured autologous epidermal sheets to more complex bilayered cutaneous substitutes. The various types of such substitutes are herein presented with their intended use. However, two integrative characteristics are analysed more specifically because of their critical role: neovascularization and re-innervation. Furthermore, the in vitro use of these various skin substitutes has shed light on various physiological and pathological phenomena. Thus, not only the in vivo application of these skin substitutes as grafts, but also their in vitro value as skin models, are presented.

Tissue engineering is a rapidly developing field. This technology could offer a new alternative for wound repair and organ replacement. It is based on the ability of living cells, with or without biomaterials, to be assembled as three-dimensional tissues. The in vivo applications extend from specialized dressings that improve host tissue repair (e.g., ulcer) to permanent grafts that restore the function of the tissue (e.g., skin grafting for burn patients).

Collagens XII and XIV localize near the surface of collagen fibrils and may be involved in epithelial-mesenchymal interactions as well as in the modulation of tissue biomechanical properties. Moreover, human skin fibroblasts cultured in monolayer are known to lose their ability to produce collagen XIV and to switch the transcription of collagen XII from the small splice variant (220 kDa) to the large (320 kDa), whereas the small form is the main form found in human skin. We have investigated the expression patterns of these two molecules in human skin as a function of donor age and anatomic site, by using immunohistology with specific monoclonal antibodies. We demonstrated changes in the expression patterns of collagens XII and XIV in human skin after birth. Moreover, in adult scalp skin, very strong staining of collagen XII fibril bundles was observed around hair follicles, in association with very low expression of collagen XW. We also investigated the expression of collagens XII and XIV by fibroblasts and keratinocytes cultured in a reconstructed skin. In these culture conditions, fibroblasts recovered theft ability to produce collagen XIV and re-expressed the small splice variant of collagen XII. These results could be explained by the deposition of large amounts of collagen fibrils by fibroblasts in this culture system. Thus, the re-expression of these collagens suggests that the deposition of banded collagen fibrils is a pre-requisite for the expression of collagen XIV and small variant of collagen XII.

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).

Background: The coverage of deep and extensive burns with autologous tissue-engineered skin is a promising strategy to improve the cosmetic aspect and functionality of the skin, compared to the transplantation of simple epithelial sheets. Indeed, a dermal compartment could markedly help to prevent hypertrophic scar formation and to strengthen the dermal–epidermal junction while increasing skin suppleness and pliability. The Problem: The thickness of the dermis could be a limitation to the survival of the tissue after transplantation, since its vascularization can take up to 2 weeks to occur through neovascularization. This delay could lead to graft necrosis. Basic/Clinical Science Advances: To overcome this problem, the reconstruction of a preformed network of branching capillaries in the dermis before grafting has proven to be an efficient solution in connecting to the host's vasculature in less than 4 days after grafting. The formation of this capillary-like network is achieved by the coculture of human fibroblasts and endothelial cells in a collagen sponge for a 1-month in vitro maturation period. The successful inosculation process between human capillaries and the host's vasculature was demonstrated after grafting onto nude mice. Clinical Care Relevance: In addition to autologous epithelial sheets and split-thickness autografts, this endothelialized reconstructed skin made of the patient's own cells could be a valuable additional strategy to permanently cover deep wounds. Conclusion: The reconstruction in tissue-engineered organs of a capillary-like network made of the patient's own cells before grafting is a promising approach to promote their early vascularization.

The contribution of the cellular and fibrillar microenvironment to angiogenesis still remains unclear. Our purpose was to evaluate the effect of the extracellular matrix deposited by fibroblasts on the capacity of human endothelial cells to form capillaries in vitro. We have drastically decreased the amount of extracellular matrix surrounding fibroblasts in our model of endothelialized-reconstructed connective tissue (ERCT) by culturing it without ascorbate. Under these conditions, the number of capillary-like tubes (CLT) formed by endothelial cells was reduced by up to 10-fold after 31 days of culture compared to controls. This decrease was due neither to a variation of MMP-2 and MMP-9 secretion, nor to a reduction in the number of fibroblasts and/or endothelial cells, or a diminution of fibroblast growth factor 2 (FGF2) synthesis. The secretion of vascular endothelial growth factor (VEGF) by fibroblasts accounted for 25–70% of the capillary-like tube formation when tissues were cultured in the presence or absence of ascorbate, as demonstrated by VEGF-blocking studies. The culture of endothelial cells on a similar extracellular matrix but in the absence of living fibroblasts did not promote the formation of CLT, even when tissues were fed with fibroblast-conditioned medium. Thus, the deposition of a rich extracellular matrix by living fibroblasts appeared necessary, but not sufficient to promote capillary-like formation. Fibroblasts seem to induce endothelial cells to spontaneously form CLT by secreting and organizing an abundant extracellular matrix, which creates a microenvironment around cells that could in turn trap growth factors produced by fibroblasts and promote three-dimensional cell organization.

Combining bovine collagen with chitosan followed by freeze-drying has been shown to produce porous scaffolds suitable for skin and connective tissue engineering applications. In this study collagen extracted from porcine and avian skin was compared with bovine collagen for the production of tissue engineered scaffolds. A similar purity of the collagen extracts was shown by electrophoresis, confirming the reliability of the extraction process. Collagen was solubilized, cross-linked by adding chitosan to the solution and freeze-dried to generate a porous structure suitable for tissue engineering applications. Scaffold porosity and pore morphology were shown to be source dependant, with bovine collagen and avian collagen resulting into the smallest and largest pores, respectively. Scaffolds were seeded with dermal fibroblasts and cultured for 35 days to evaluate the suitability of the different collagen–chitosan scaffolds for long-term tissue engineered dermal substitute maturation in vitro. Cell proliferation and scaffold biocompatibility were found to be similar for all the collagen–chitosan scaffolds, demonstrating their capability to support long-term cell adhesion and growth. The scaffolds contents was assessed by immunohistochemistry and showed increased deposition of extracellular matrix by the cells as a function of time. These results correlate with measurements of the mechanical properties of the scaffolds, since both the ultimate tensile strength and tensile modulus of the cell seeded scaffolds had increased by the end of the culture period. This experiment demonstrates that porcine and avian collagen could be used as an alternative to bovine collagen in the production of collagen–chitosan scaffolding materials.

For patients with extensive burns, wound coverage with an autologous in vitro reconstructed skin made of both dermis and epidermis should be the best alternative to split-thickness graft. Unfortunately, various obstacles have delayed the widespread use of composite skin substitutes. Insufficient vascularization has been proposed as the most likely reason for their unreliable survival. Our purpose was to develop a vascular-like network inside tissue-engineered skin in order to improve graft vascularization. To reach this aim, we fabricated a collagen biopolymer in which three human cell types—keratinocytes, dermal fibroblasts, and umbilical vein endothelial cells—were cocultured. We demonstrated that the endothelialized skin equivalent (ESE) promoted spontaneous formation of capillary-like structures in a highly differentiated extracellular matrix. Immunohistochemical analysis and transmission electron microscopy of the ESE showed characteristics associated with the microvasculature in vivo (von Willebrand factor, Weibel-Palade bodies, basement membrane material, and intercellular junctions). We have developed the first endothelialized human tissue-engineered skin in which a network of capillary-like tubes is formed. The transplantation of this ESE on human should accelerate graft revascularization by inosculation of its preexisting capillary-like network with the patient's own blood vessels, as it is observed with autografts. In addition, the ESE turns out to be a promising in vitro angiogenesis model.

The major limitation for the application of an autologous in vitro tissue-engineered reconstructed skin (RS) for the treatment of burnt patients is the delayed vascularization of its relatively thick dermal avascular component, which may lead to graft necrosis. We have developed a human endothelialized reconstructed skin (ERS), combining keratinocytes, fibroblasts and endothelial cells (EC) in a collagen sponge. This skin substitute then spontaneously forms a network of capillary-like structures (CLS) in vitro. After transplantation to nude mice, we demonstrated that CLS containing mouse blood were observed underneath the epidermis in the ERS in less than 4 days, a delay comparable to our human skin control. In comparison, a 14-day period was necessary to achieve a similar result with the non-endothelialized RS. Furthermore, no mouse blood vessels were ever observed close to the epidermis before 14 days in the ERS and the RS. We thus concluded that the early vascularization observed in the ERS was most probably the result of inosculation of the CLS network with the host's capillaries, rather than neovascularization, which is a slower process. These results open exciting possibilities for the clinical application of many other tissue-engineered organs requiring a rapid vascularization.

Background: The skin flap technique is widely used in reconstructive surgery for the coverage of deep burns of the face, neck, and joints. Facial deformities and joint contractures are avoided by transplanting vascularized full-thickness skin on wounds. The major drawback of this technique is the injury inflicted upon the donor site, which corresponds to a third degree burn. The usual technique to cover the flap donor site is the transplantation of split-thickness autografts. In the case of patients with deep and extensive burns, the harvesting of good quality autografts is often difficult because of multiple scars. In order to avoid additional trauma to the patient by split-thickness skin harvesting, we have experimented the use of a new model ofin vitroreconstructed skin graft for flap donor site coverage in a mouse model. Materials and methods: The reconstructed skin was grafted on the back of nude mice at the skin flap donor site, while flap was used to cover a wound generated on joint of the posterior leg. Results: A 100% graft take was achieved (16 mice were used) and a limited contraction of the reconstructed skin was observed 30 days posttransplantation (78% of the initial surface area of the graft remained). Histological analysis of the graft demonstrated healing of a well differentiated epidermis laying on a dense dermis. Conclusions: Since this technique would prevent additional trauma to the patient while achieving a good healing of the wound, it may be a useful approach in the coverage of skin flap donor site in humans.

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. Objectives: The aim of this work was to describe the three-dimensional structure of the capillary-like network. Thereafter we strove to study, quantitatively and qualitatively, the influence of angiogenic and angiostatic drugs on capillary-like tube (CLT) formation in vitro in the model. Methods: The endothelialized dermis was prepared by coculturing two human cell types, dermal fibroblasts and umbilical vein endothelial cells, in a collagen sponge biomaterial. Results: The visualization by confocal microscopy of the tubes present in the model showed that the endothelial structures were not cord-like but rather CLTs with well-defined lumina. Moreover, these tubes were organized in a complex network of branching structures. When angiogenic factors (vascular endothelial growth factor 10 ng mL-1 or basic fibroblast growth factor 10 ng mL-1) were added to the model, 1.8 and 1.4 times more capillaries, respectively, were observed, whereas the addition of progesterone (10 microg x mL(-1)) reduced by 2.4 times the number of tubes compared with the control. Conclusions: These results suggest that this model is a highly efficient assay for the screening of potentially angiogenic and angiostatic compounds.