Normal Human Epithelial Cells Regulate the Size and Morphology of Tissue-Engineered Capillaries

Abstract

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 Effects of Constant Flow Bioreactor Cultivation and Keratinocyte Seeding Densities on Prevascularized Organotypic Skin Grafts Based on a Fibrin Scaffold

“…Fibroblasts play an important role in organizing the clot, which includes the secretion of extracellular matrix and the promotion of angiogenesis. 14,15 To our knowledge, bi-layered prevascularized skin grafts so far have been only engineered based on the so-called ''self-assembly approach,'' [16][17][18] collagenbased scaffolds, 19,20 de-cellularized donor dermis, 21 or use of a de-cellularized porcine jejunal segment as suggested by Groeber et al 22 All of these methods are characterized by specific shortcomings: The self-assembly approach takes approximately 6 weeks to form a prevascularized neo-dermis, which is a significantly longer timeframe than our employed method. Collagen-based scaffolds, de-cellularized donor dermis, or xenogenous templates cannot offer a complete autologous approach; drawbacks such as transmission of infectious diseases, allergic reactions, or intricate experimental setup have to be taken into account as a possible future issue regarding human implementation.…”

Engineering a Bilayered Hydrogel to Control ASC differentiation

“…Since our lab is interested in skin and soft-tissue wound healing, we strategically implemented a bilayer of collagen and fibrin since collagen supports dermal and epidermal regeneration by the host, whereas fibrin naturally induces vascular network formation 9 . Furthermore, it is now understood that cross-talk occurs between proliferating skin keratinocytes, fibroblast, and the underlying vascular network; and this complex mechanism is highly critical for proper wound healing 10 . Without an underlying vascular network, tissue-engineered skin grafts have difficulty inosculating with host tissue.…”

Progress in developing a living human tissue-engineered tri-leaflet heart valve assembled from tissue produced by the self-assembly approach

“…Also, due to free edge rolling on itself during the assembly, we have shown that the use of cloth adhering to the tissue provides ease of use in the process of assembling the valve and helps in the attachment of the leaflets on the wire formed frame. In order to closer mimic heart valve tissues, endothelial cells could also be seeded on the leaflets to favour anti-thrombogenic and immunogenic reaction using autologous microvascular cells harvested from the initial biopsy [73]. Further studies will be required to evaluate the short-term and midterm mechanical properties and function of these living human tissue-engineered heart valves.…”