Building an Artificial Stem Cell Niche: Prerequisites for Future 3D-Formation of Inner Ear Structures-Toward 3D Inner Ear Biotechnology.

In recent years, there has been an increased interest in stem cells for the purpose of regenerative medicine to deliver a wide range of therapies to treat many diseases. However, two-dimensional cultures of stem cells are of limited use when studying the mechanism of pathogenesis of diseases and the feasibility of a treatment. Therefore, research is focusing on the strengths of stem cells in the three-dimensional (3D) structures mimicking organs, that is, organoids, or organ-on-chip, for modeling human biology and disease. As 3D technology advances, it is necessary to know which signals stem cells need to multiply and differentiate into complex structures. This holds especially true for the complex 3D structure of the inner ear. Recent work suggests that although other factors play a role, the extracellular matrix (ECM), including its topography, is crucial to mimic a stem cell niche in vitro and to drive stem cells toward the formation of the tissue of interest. Technological developments have led to the investigation of biomaterials that closely resemble the native ECM. In the fast forward moving research of organoids and organs-on-chip, the inner ear has hardly received attention. This review aims to provide an overview, by describing the general context in which cells, matrix and morphogens cooperate in order to build a tissue, to facilitate research in 3D inner ear technology. Anat Rec, 303:408-426, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Nanolayered chemical modification of silicon surfaces with ionizable surface groups for pH-triggered protein adsorption and release: application to microneedles.

The aim of this work was to develop a nanolayered pH sensitive coating method whereby proteins are coated at a suitable pH on the surface of chemically modified biomedical/bioanalytical microdevices and protein release is triggered by a pH-shift upon contact with the physiological environment. In this work such a coating was developed and was applied onto microneedles. First, the surface of microneedle arrays was modified with basic groups with a surface pKa below physiological pH. This modification was a multistep procedure: first the surface was hydroxylated in a piranha mixture, then 3-aminopropyl-triethoxysilane was coupled (yielding a "pH independent" surface with a positive charge over a broad pH range), next 4-pyridinecarboxaldehyde was coupled to the obtained surface amine groups and finally the imine bond was reduced by sodium cyanoborohydride. The obtained pH-sensitive pyridine-modified microneedles were coated with ovalbumin at surface pKa > pH > pI of the protein; thus the surface of the microneedles is positively charged and the protein is negatively charged. The coating efficiency of ovalbumin was 95% for the amine-modified (pH independent) and the pyridine-modified (pH sensitive) surfaces, whereas a non-modified surface had a coating efficiency of only 2%. After the protein-coated microneedle arrays were pierced into the skin, having a pH > surface pKa of the microneedle arrays, 70% of the protein was released within 1 minute, whereas the protein release from pH independent microneedle arrays was only 5%. In conclusion, we developed a procedure to efficiently coat microneedle arrays with proteins that are released upon piercing into human skin.

Fluorescent nanoparticle adhesion assay: a novel method for surface pKa determination of self-assembled monolayers on silicon surfaces.

Since the computer industry enables us to generate smaller and smaller structures, silicon surface chemistry is becoming increasingly important for (bio-)analytical and biological applications. For controlling the binding of charged biomacromolecules such as DNA and proteins on modified silicon surfaces, the surface pK(a) is an important factor. Here we present a fluorescent nanoparticle adhesion assay as a novel method to determine the surface pK(a) of silicon surfaces modified with weak acids or bases. This method is based upon electrostatic interactions between the modified silicon surface and fluorescent nanoparticles with an opposite charge. Silicon slides were modified with 3-aminopropyltriethoxysilane (APTES) and were further derivatized with succinic anhydride. Layer thickness of these surfaces was determined by ellipsometry. After incubating the surfaces with an amine-reactive fluorescent dye, fluorescence microscopy revealed that the silicon surfaces were successfully modified with amine- and carboxyl-groups. Two surface pK(a) values were found for APTES surfaces by the fluorescent nanoparticle adhesion assay. The first surface pK(a) (6.55 ± 0.73) was comparable with the surface pK(a) obtained by contact angle titration (7.3 ± 0.8), and the second surface pK(a) (9.94 ± 0.19) was only found by using the fluorescent nanoparticle adhesion assay. The surface pK(a) of the carboxyl-modified surface by the fluorescent nanoparticle adhesion assay (4.37 ± 0.59) did not significantly differ from that found by contact angle titration (5.7 ± 1.4). In conclusion, we have developed a novel method to determine the surface pK(a) of modified silicon surfaces: the fluorescent nanoparticle adhesion assay. This method may provide a useful tool for designing pH-dependent electrostatic protein and particle binding/release and to design surfaces with a pH-dependent surface charge for (bio-)analytical lab-on-a-chip devices or drug delivery purposes.

Selective modulation of nuclear factor of activated T-cell function in restenosis by a potent bipartite peptide inhibitor.

RATIONALE: Nuclear factor of activated T-cells (NFAT) is importantly implicated in pathological cardiac remodeling and vascular lesion formation. NFAT functionality is mainly regulated by calcineurin, a Ca(2+)-dependent multi-effector phosphatase. Calcineurin inhibitors such as cyclosporine A (CsA) were shown to be effective in the treatment of restenosis and vascular inflammation but with adverse side effects. OBJECTIVE: This prompted the design of more selective inhibitors such as VIVIT and inhibitors of NFAT-calcineurin association, which unfortunately have a poor potency precluding clinical use. METHODS AND RESULTS: Here, we describe the rational design of a potent bipartite inhibitor of NFAT-calcineurin interaction, MCV1, which targets two separate calcineurin docking motifs. Modeling, site-directed mutagenesis, and functional studies demonstrated that MCV1 acts by allosteric modulation of calcineurin. Comparable to CsA, MCV1 prevents NFAT activation at nanomolar potency without impairing calcineurin phosphatase activity, nuclear factor-κB nuclear import, and general cell signaling. In contrast, CsA but not MCV1-activated basal level extracellular signal-regulated kinases activity and prevented nuclear import of calcineurin, independent of NFAT activation. In vivo MCV1 abrogated NFAT-mediated T-cell activation in a model of PMA-elicited peritonitis, whereas topical application of MCV1 markedly reduced neointima formation in a mouse model of restenosis. CONCLUSIONS: We designed a bipartite NFAT inhibitor that is more potent than VIVIT and more selective than CsA. MCV1 constitutes not only a powerful tool to unravel NFAT function but also a potential drug candidate for the treatment of diseases implicating NFAT activation.