A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development.

The neural crest is a dynamic progenitor cell population that arises at the border of neural and non-neural ectoderm. The inductive roles of FGF, Wnt, and BMP at the neural plate border are well established, but the signals required for subsequent neural crest development remain poorly characterized. Here, we conducted a screen in primary zebrafish embryo cultures for chemicals that disrupt neural crest development, as read out by crestin:EGFP expression. We found that the natural product caffeic acid phenethyl ester (CAPE) disrupts neural crest gene expression, migration, and melanocytic differentiation by reducing Sox10 activity. CAPE inhibits FGF-stimulated PI3K/Akt signaling, and neural crest defects in CAPE-treated embryos are suppressed by constitutively active Akt1. Inhibition of Akt activity by constitutively active PTEN similarly decreases crestin expression and Sox10 activity. Our study has identified Akt as a novel intracellular pathway required for neural crest differentiation.

A comparison of scaffold-free and scaffold-based reconstructed human skin models as alternatives to animal use.

Tissue engineered full-thickness human skin substitutes have various applications in the clinic and in the laboratory, such as in the treatment of burns or deep skin defects, and as reconstructed human skin models in the safety testing of drugs and cosmetics and in the fundamental study of skin biology and pathology. So far, different approaches have been proposed for the generation of reconstructed skin, each with its own advantages and disadvantages. Here, the classic tissue engineering approach, based on cell-seeded polymeric scaffolds, is compared with the less-studied cell self-assembly approach, where the cells are coaxed to synthesise their own extracellular matrix (ECM). The resulting full-thickness human skin substitutes were analysed by means of histological and immunohistochemical analyses. It was found that both the scaffold-free and the scaffold-based skin equivalents successfully mimicked the functionality and morphology of native skin, with complete epidermal differentiation (as determined by the expression of filaggrin), the presence of a continuous basement membrane expressing collagen VII, and new ECM deposition by dermal fibroblasts. On the other hand, the scaffold-free model had a thicker epidermis and a significantly higher number of Ki67-positive proliferative cells, indicating a higher capacity for self-renewal, as compared to the scaffold-based model.

Tissue engineering of oral mucosa: a shared concept with skin.

Tissue-engineered oral mucosa, in the form of epithelial cell sheets or full-thickness oral mucosa equivalents, is a potential solution for many patients with congenital defects or with tissue loss due to diseases or tumor excision following a craniofacial cancer diagnosis. In the laboratory, it further serves as an in vitro model, alternative to in vivo testing of oral care products, and provides insight into the behavior of the oral mucosal cells in healthy and pathological tissues. This review covers the old and new generation scaffold types and materials used in oral mucosa engineering; discusses similarities and differences between oral mucosa and skin, the methods developed to reconstruct oral mucosal defects; and ends with future perspectives on oral mucosa engineering.

Characterization of cultured multipotent zebrafish neural crest cells.

The neural crest is a unique cell population associated with vertebrate evolution. Neural crest cells (NCCs) are characterized by their multipotent and migratory potentials. While zebrafish is a powerful genetic model organism, the isolation and culture of zebrafish NCCs would provide a useful adjunct to fully interrogate the genetic networks that regulate NCC development. Here we report for the first time the isolation, in vitro culture, and characterization of NCCs from zebrafish embryos. NCCs were isolated from transgenic sox10:egfp embryos using fluorescence activated cell sorting and cultured in complex culture medium without feeder layers. NCC multilineage differentiation was determined by immunocytochemistry and real-time qPCR, cell migration was assessed by wound healing assay, and the proliferation index was calculated by immunostaining against the mitosis marker phospho-histone H3. Cultured NCCs expressed major neural crest lineage markers such as sox10, sox9a, hnk1, p75, dlx2a, and pax3, and the pluripotency markers c-myc and klf4. We showed that the cultured NCCs can be differentiated into multiple neural crest lineages, contributing to neurons, glial cells, smooth muscle cells, melanocytes, and chondrocytes. We applied the NCC in vitro model to study the effect of retinoic acid on NCC development. We showed that retinoic acid had a profound effect on NCC morphology and differentiation, significantly inhibited proliferation and enhanced cell migration. The availability of high numbers of NCCs and reproducible functional assays offers new opportunities for mechanistic studies of neural crest development, in genetic and chemical biology applications.

Feasibility of a porcine oral mucosa equivalent: a preclinical study.

Oral tissue engineering aims to treat and fill tissue deficits caused by congenital defects, facial trauma, or malignant lesion surgery, as well as to study the biology of oral mucosa. The Food and Drug Administration (FDA) and the European Medicines Agency (EMA) require a large animal model to evaluate cell-based devices, including tissue-engineered oral mucosa, prior to initiating human clinical studies. Porcine oral mucosa is non-keratinized and resembles that of humans more closely than any other animal in terms of structure and composition; however, there have not been any reports on the reconstruction of a porcine oral mucosa equivalent, probably due to the difficulty to culture porcine fibroblasts. In this study, we demonstrate the feasibility of a 3D porcine oral mucosa equivalent based on a collagen-GAG-chitosan scaffold, as well as reconstructed porcine epithelium by using an amniotic membrane as support, or without any support in form of epithelial cell sheets by using thermoresponsive culture plates. Explants technique was used for the isolation of the porcine fibroblasts and a modified fibroblast medium containing 20% fetal calf serum was used for their culture. The histological and transmission electron microscopic analyses of the resulting porcine oral mucosa models showed the presence of non-keratinized epithelia expressing keratin 13, the major differentiation marker of non-keratinized oral mucosa, in all models, and the presence of newly synthesized collagen fibers in the lamina propria equivalent of the full-thickness model, indicating the functionality of porcine fibroblasts.

Influence of the mesenchymal cell source on oral epithelial development.

The extent of the influence of mesenchymal tissue on epithelial development is still debated, and elucidation of epithelial-mesenchymal interactions should be of relevance for controlling normal as well as pathological growth and development. The aim of the present study was to elucidate the influence of the mesenchymal cell type on oral mucosa epithelial development in vitro, using tissue-engineering principles, by including three different sources for mesenchymal cell type, viz. oral mucosa, skin and cornea, each of them presenting a distinct type of epithelium in situ. We investigated epithelial-mesenchymal interactions, considering both morphological criteria and protein expression (filaggrin, keratin 10, keratin 12, keratin 13 and laminin 5). The results of the histology, immunohistochemistry and transmission electron microscopy of the three types of tissue-engineered constructs composed of mesenchymal cells of different sources (oral, dermal and corneal fibroblasts) and of the same oral epithelial cells showed that the mesenchymal cell source had a significant influence on the thickness and ultrastructure of the epithelium, but not on the differentiation of oral epithelial cells, which might be an intrinsic property of these cells due to their genetic programming.

The influence of elastin-like recombinant polymer on the self-renewing potential of a 3D tissue equivalent derived from human lamina propria fibroblasts and oral epithelial cells.

Three-dimensional epithelial tissue equivalents tend to lose their self-renewing potential progressively during culture as their epithelial cells lose their proliferative capacity with time. Even though the tissue engineered construct can mimic the native tissue well, it rapidly degrades after implantation due to the insufficient number of proliferating cells in the equivalent. In the present study we demonstrate for the first time that the use of an elastin-like recombinant polymer (ELR) engineered to contain the cell adhesion peptide RGD can result in a 3D tissue equivalent with high self-renewing potential, containing as many proliferative cells as the native tissue itself. The 3D tissue equivalent was reconstructed by the coculture of human lamina propria fibroblasts and oral epithelial cells in the nanofibrous ELR-collagen scaffold. Histological, immunohistological and transmission electron microscopic analyses of this oral mucosa equivalent demonstrated the expression of markers characteristic of epithelial proliferation (Ki67) and differentiation (keratin 13), and also the presence of a pluristratified epithelium and an ultrastructurally well-organized basement membrane expressing laminin 332. The synthesis of new extracellular matrix by the fibroblasts was also demonstrated. The scaffold proposed here presents great potential for tissue engineering applications, and also for studies of epithelial proliferation, and epithelial disorders including carcinogenesis.

A smart bilayer scaffold of elastin-like recombinamer and collagen for soft tissue engineering.

Elastin-like recombinamers (ELRs) are smart, protein-based polymers designed with desired peptide sequences using recombinant DNA technology. The aim of the present study was to produce improved tissue engineering scaffolds from collagen and an elastin-like protein tailored to contain the cell adhesion peptide RGD, and to investigate the structural and mechanical capacities of the resulting scaffolds (foams, fibers and foam-fiber bilayer scaffolds). The results of the scanning electron microscopy, mercury porosimetry and mechanical testing indicated that incorporation of ELR into the scaffolds improved the uniformity and continuity of the pore network, decreased the pore size (from 200 to 20 µm) and the fiber diameter (from 1.179 µm to 306 nm), broadened the pore size distribution (from 70-200 to 4-200 µm) and increased their flexibility (from 0.007 to 0.011 kPa⁻¹). Culture of human fibroblasts and epithelial cells in ELR-collagen scaffolds showed the positive contribution of ELR on proliferation of both types of cells.

Reconstruction of a full-thickness collagen-based human oral mucosal equivalent.

Tissue engineered human oral mucosa has the potential to be applied to the closure of surgical wounds after tissue deficits due to facial trauma, malignant lesion surgery or preposthetic procedure. It can also be used to elucidate the biology and pathology of oral mucosa and as a model alternative to animals for safety testing of oral care products. Using the technology previously developed in our laboratory for the production of a skin equivalent, we were able to reconstruct a nonkeratinized full-thickness human oral mucosal equivalent closely mimicking human native oral mucosa. The successive coculture of human lamina propria fibroblasts and human oral epithelial cells isolated from the nonkeratinized region of oral cavity in a porous collagen-glycosaminoglycan (GAG)-chitosan scaffold gave rise to a lamina propria equivalent (LPE) and then to an oral mucosa equivalent (OME). The results of the histology, immunohistology and transmission electron microscopy of this OME demonstrated the presence of a nonkeratinized pluristratified and differentiated epithelium as in native nonkeratinized human oral mucosa expressing both K13 and K3/76. This epithelium was firmly anchored to the LPE by a continuous and ultrastructurally well-organized basement membrane. In the LPE, fibroblasts synthesized new extracellular matrix where the average collagen fibre diameter was 28.4 nm, close to that of native oral mucosa. The proliferative capacity of the basal cells was demonstrated by the expression of Ki67.

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.