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    Glycerylphytate as an ionic crosslinker for 3D printing of multi-layered scaffolds with improved shape fidelity and biological features
    (London : Royal Society of Chemistry, 2020) Mora-Boza, A.; Włodarczyk-Biegun, M.K.; Del Campo, A.; Vázquez-Lasa, B.; Román, J.S.
    The fabrication of intricate and long-term stable 3D polymeric scaffolds by a 3D printing technique is still a challenge. In the biomedical field, hydrogel materials are very frequently used because of their excellent biocompatibility and biodegradability, however the improvement of their processability and mechanical properties is still required. This paper reports the fabrication of dual crosslinked 3D scaffolds using a low concentrated (<10 wt%) ink of gelatin methacryloyl (GelMA)/chitosan and a novel crosslinking agent, glycerylphytate (G1Phy) to overcome the current limitations in the 3D printing field using hydrogels. The applied methodology consisted of a first ultraviolet light (UV) photopolymerization followed by a post-printing ionic crosslinking treatment with G1Phy. This crosslinker provides a robust framework and avoids the necessity of neutralization with strong bases. The blend ink showed shear-thinning behavior and excellent printability in the form of a straight and homogeneous filament. UV curing was undertaken simultaneously to 3D deposition, which enhanced precision and shape fidelity (resolution ≈150 μm), and prevented the collapse of the subsequent printed layers (up to 28 layers). In the second step, the novel G1Phy ionic crosslinker agent provided swelling and long term stability properties to the 3D scaffolds. The multi-layered printed scaffolds were mechanically stable under physiological conditions for at least one month. Preliminary in vitro assays using L929 fibroblasts showed very promising results in terms of adhesion, spreading, and proliferation in comparison to other phosphate-based traditional crosslinkers (i.e. TPP). We envision that the proposed combination of the blend ink and 3D printing approach can have widespread applications in the regeneration of soft tissues.
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    Synthetic 3D PEG-Anisogel Tailored with Fibronectin Fragments Induce Aligned Nerve Extension
    (Columbus, Ohio : American Chemical Society, 2019) Licht, Christopher; Rose, Jonas C.; Anarkoli, Abdolrahman Omidinia; Blondel, Delphine; Roccio, Marta; Haraszti, Tamás; Gehlen, David B.; Hubbell, Jeffrey A.; Lutolf, Matthias P.; De Laporte, Laura
    An enzymatically cross-linked polyethylene glycol (PEG)-based hydrogel was engineered to promote and align nerve cells in a three-dimensional manner. To render the injectable, otherwise bioinert, PEG-based material supportive for cell growth, its mechanical and biochemical properties were optimized. A recombinant fibronectin fragment (FNIII9*-10/12-14) was coupled to the PEG backbone during gelation to provide cell adhesive and growth factor binding domains in close vicinity. Compared to full-length fibronectin, FNIII9*-10/12-14 supports nerve growth at similar concentrations. In a 3D environment, only the ultrasoft 1 w/v% PEG hydrogels with a storage modulus of ∼10 Pa promoted neuronal growth. This gel was used to establish the first fully synthetic, injectable Anisogel by the addition of magnetically aligned microelements, such as rod-shaped microgels or short fibers. The Anisogel led to linear neurite extension and represents a large step in the direction of clinical translation with the opportunity to treat acute spinal cord injuries.
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    A novel, low-volume method for organ culture of embryonic kidneys that allows development of cortico-medullary anatomical organization
    (San Francisco, Calif. : Public Library of Science, 2010) Sebinger, D.D.R.; Unbekandt, M.; Ganeva, V.V.; Ofenbauer, A.; Werner, C.; Davies, J.A.
    Here, we present a novel method for culturing kidneys in low volumes of medium that offers more organotypic development compared to conventional methods. Organ culture is a powerful technique for studying renal development. It recapitulates many aspects of early development very well, but the established techniques have some disadvantages: in particular, they require relatively large volumes (1-3 mls) of culture medium, which can make high-throughput screens expensive, they require porous (filter) substrates which are difficult to modify chemically, and the organs produced do not achieve good cortico-medullary zonation. Here, we present a technique of growing kidney rudiments in very low volumes of medium-around 85 microliters-using silicone chambers. In this system, kidneys grow directly on glass, grow larger than in conventional culture and develop a clear anatomical cortico-medullary zonation with extended loops of Henle. © 2010 Sebinger et al.