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Cartilage damage and osteoarthritis (OA) impose an important burden on society, leaving both young, active patients and older patients disabled and affecting quality of life. In particular, cartilage injury not only imparts acute loss of function but also predisposes to OA. The increase in knowledge of the consequences of these diseases and the exponential growth in research of regenerative medicine have given rise to different treatment types. Of these, cell-based treatments are increasingly applied because they have the potential to regenerate cartilage, treat symptoms, and ultimately prevent or delay OA. Although these approaches give promising results, they require a costly in vitro cell culture procedure. The answer may lie in single-stage procedures that, by using cell combinations, render in vitro expansion redundant. In the last two decades, cocultures of cartilage cells and a variety of (mesenchymal) stem cells have shown promising results as different studies report cartilage regeneration in vitro and in vivo. However, there is considerable debate regarding the mechanisms and cellular interactions that lead to chondrogenesis in these models. This review, which included 52 papers, provides a systematic overview of the data presented in the literature and tries to elucidate the mechanisms that lead to chondrogenesis in stem cell cocultures with cartilage cells. It could serve as a basis for research groups and clinicians aiming at designing and implementing combined cellular technologies for single-stage cartilage repair and treatment or prevention of OA., (©AlphaMed Press.)

An important tenet in designing scaffolds for regenerative medicine consists in mimicking the dynamic mechanical properties of the tissues to be replaced to facilitate patient rehabilitation and restore daily activities. In addition, it is important to determine the contribution of the forming tissue to the mechanical properties of the scaffold during culture to optimize the pore network architecture. Depending on the biomaterial and scaffold fabrication technology, matching the scaffolds mechanical properties to articular cartilage can compromise the porosity, which hampers tissue formation. Here, we show that scaffolds with controlled and interconnected pore volume and matching articular cartilage dynamic mechanical properties, are indeed effective to support tissue regeneration by co-cultured primary and expanded chondrocyte (1:4). Cells were cultured on scaffolds in vitro for 4 weeks. A higher amount of cartilage specific matrix (ECM) was formed on mechanically matching (M) scaffolds after 28 days. A less protein adhesive composition supported chondrocytes rounded morphology, which contributed to cartilaginous differentiation. Interestingly, the dynamic stiffness of matching constructs remained approximately at the same value after culture, suggesting a comparable kinetics of tissue formation and scaffold degradation. Cartilage regeneration in matching scaffolds was confirmed subcutaneously in vivo. These results imply that mechanically matching scaffolds with appropriate physico-chemical properties support chondrocyte differentiation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Animal experiments help to progress and ensure safety of an increasing number of novel therapies, drug development and chemicals. Unfortunately, these also lead to major ethical concerns, costs and limited experimental capacity. We foresee a coercion of all these issues by implantation of well systems directly into vertebrate animals. Here, we used rapid prototyping to create wells with biomaterials to create a three-dimensional (3D) well-system that can be used in vitro and in vivo. First, the well sizes and numbers were adjusted for 3D cell culture and in vitro screening of molecules. Then, the functionality of the wells was evaluated in vivo under 36 conditions for tissue regeneration involving human mesenchymal stem cells (hMSCs) and bovine primary chondrocytes (bPCs) screened in one animal. Each biocompatible well was controlled to contain μl-size volumes of tissue, which led to tissue penetration from the host and tissue formation under implanted conditions. We quantified both physically and biologically the amounts of extracellular matrix (ECM) components found in each well. Using this new concept the co-culture of hMSCs and bPCs was identified as a positive hit for cartilage tissue repair, which was a comparable result using conventional methods. The in vivo screening of candidate conditions opens an entirely new range of experimental possibilities, which significantly abates experimental animal use and increases the pace of discovery of medical treatments.

This report describes a novel system to create rapid prototyped 3-dimensional (3D) fibrous scaffolds with a shell-core fiber architecture in which the core polymer supplies the mechanical properties and the shell polymer acts as a coating providing the desired physicochemical surface properties. Poly[(ethylene oxide) terephthalate-co-poly(butylene) terephthalate] (PEOT/PBT) 3D fiber deposited (3DF) scaffolds were fabricated and examined for articular cartilage tissue regeneration. The shell polymer contained a higher molecular weight of the initial poly(ethylene glycol) (PEG) segments used in the copolymerization and a higher weight percentage of the PEOT domains compared with the core polymer. The 3DF scaffolds entirely produced with the shell or with the core polymers were also considered. After 3 weeks of culture, scaffolds were homogeneously filled with cartilage tissue, as assessed by scanning electron microscopy. Although comparable amounts of entrapped chondrocytes and of extracellular matrix formation were found for all analyzed scaffolds, chondrocytes maintained their rounded shape and aggregated during the culture period on shell-core 3DF scaffolds, suggesting a proper cell differentiation into articular cartilage. This finding was also observed in the 3DF scaffolds fabricated with the shell composition only. In contrast, cells spread and attached on scaffolds made simply with the core polymer, implying a lower degree of differentiation into articular cartilaginous tissue. Furthermore, the shell-core scaffolds displayed an improved dynamic stiffness as a result of a "prestress" action of the shell polymer on the core one. In addition, the dynamic stiffness of the constructs increased compared with the stiffness of the bare scaffolds before culture. These findings suggest that shell-core 3DF PEOT/PBT scaffolds with desired mechanical and surface properties are a promising solution for improved cartilage tissue engineering.

Overexpression of p53 is found in approximately 50% of human cancers, making it an attractive target antigen for immunotherapy of cancer. Research in this area has thus far primarily focused on p53-specific CTLs. Although these CTLs were shown to be highly effective against p53-overexpressing tumors in vivo, immunological tolerance seems to strongly restrict the spectrum of the p53-specific CTL repertoire in p53(+/+) subjects. In view of the emerging role of CD4(+) Th (Th) cells in the antitumor response, we investigated the specificity and antitumor efficacy of the p53-specific Th response in mice. Our data show that high affinity Th cells against the naturally processed epitope p53(108-122) can be elicited in both p53(-/-) and p53(+/+) mice, indicating that the p53-specific T-cell response is not affected by tolerance at the Th level. Furthermore, p53(108-122)-specific Th cells were effective in enabling p53-specific CTLs to control the growth of p53-overexpressing tumors in vivo. Therefore, exploitation of the p53-specific Th response appears to be a highly useful aspect of immunotherapeutic strategies against cancers.

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.

We have shown previously that the PTH/PTHrP (PTH-related peptide) receptor mRNA becomes expressed very early in murine embryogenesis, i.e. during the formation of extraembryonic endoderm. Retinoic Acid (RA) is a potent inducer of extraembryonic endoderm formation and PTH/PTHrP-receptor expression in embryonal carcinoma (EC) and embryonal stem (ES) cells. Using the P19 EC cell line, we have characterized promoter elements of the murine PTH/PTHrP-receptor gene that are involved in this RA-induced expression. The data show that RA-induced expression of the PTH/ PTHrP-receptor gene is mediated by the downstream P2 promoter. Analysis of promoter reporter constructs in transiently transfected P19 cells treated with RA identified an enhancer region between nucleotides -2714 and -2702 upstream of the P2 transcription start site that is involved in the RA effect. This region matches a consensus hormone response element consisting of a direct repeat with an interspacing of 1 bp (R-DR1). The R-DR1 efficiently binds retinoic acid receptor-alpha (RARalpha)-retinoid X receptor-alpha (RXRalpha) and chicken ovalbumin upstream promoter (COUP)-transcription factor I (TFI)-RXRalpha heterodimers and RXRalpha and COUP-TFI homodimers in a bandshift assay using extracts of transiently transfected COS-7 cells. RA differentiation of P19 EC cells strongly increases protein binding to the R-DR1 in a band-shift assay. This is caused by increased expression of RXR (alpha, beta, or gamma) and by the induction of expression of RARbeta and COUP TFI/TFII, which bind to the R-DR1 as shown by supershifting antibodies. The presence of RXR (alpha, beta, or gamma) in the complexes binding to the R-DR1 suggests that RXR homodimers are involved in RA-induced expression of the PTH/PTHrP-receptor gene. The importance of the R-DR1 for RA-induced expression of PTH/ PTHrP-receptor was shown by an inactivating mutation of the R-DR1, which severely impairs RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs. Since this mutation does not completely abolish RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs, sequences other than the R-DR1 might also be involved in the RA effect. Finally, we show that the RA-responsive promoter region is also able to induce expression of a reporter gene in extraembryonic endoderm of 7.5 day-old transgenic mouse embryos.

A number of studies suggest a role for PTHrP and the classical PTH/PTHrP receptor (type I) in one of the first differentiation processes in mouse embryogenesis, i.e. the formation of parietal endoderm (PE). We previously reported that although in type I receptor (-/-) embryos PE formation seemed normal, the embryos were smaller from at least day 9.5 p.c. and 60% had died before day 12.5 p.c. Here we show that the observed growth defect commences even earlier, at day 8.5 p.c. Using two novel antibodies, we show that the expression of the type I receptor protein at this stage is confined to extraembryonic endoderm only. In addition, we show that large amounts of PTHrP protein are present in the adjacent trophoblast giant cells, suggesting a paracrine interaction of PTHrP and the type I PTH/PTHrP receptor in PE formation. The involvement in PE differentiation of other recently described receptors for PTHrP would explain a possible redundancy for the type I receptor in PE formation. However, deletion of the type I PTH/PTHrP receptor in ES cells by homologous recombination completely prevents PTHrP-induced PE differentiation. Based upon these observations, we propose that PTHrP and the type I PTH/PTHrP receptor, although not required for the initial formation of PE, are required for its proper differentiation and/or functioning.