Multimaterial 3D Laser Printing of Cell-Adhesive and Cell-Repellent Hydrogels.

Here, a straightforward method is reported for manufacturing 3D microstructured cell-adhesive and cell-repellent multimaterials using two-photon laser printing. Compared to existing strategies, this approach offers bottom-up molecular control, high customizability, and rapid and precise 3D fabrication. The printable cell-adhesive polyethylene glycol (PEG) based material includes an Arg-Gly-Asp (RGD) containing peptide synthesized through solid-phase peptide synthesis, allowing for precise control of the peptide design. Remarkably, minimal amounts of RGD peptide (< 0.1 wt%) suffice for imparting cell-adhesiveness, while maintaining identical mechanical properties in the 3D printed microstructures to those of the cell-repellent, PEG-based material. Fluorescent labeling of the RGD peptide facilitates visualization of its presence in cell-adhesive areas. To demonstrate the broad applicability of the system, the fabrication of cell-adhesive 2.5D and 3D structures is shown, fostering the adhesion of fibroblast cells within these architectures. Thus, this approach allows for the printing of high-resolution, true 3D structures suitable for diverse applications, including cellular studies in complex environments.

Geminin ablation in vivo enhances tumorigenesis through increased genomic instability.

Geminin, a DNA replication licensing inhibitor, ensures faithful DNA replication in vertebrates. Several studies have shown that geminin depletion in vitro results in rereplication and DNA damage, whereas increased expression of geminin has been observed in human cancers. However, conditional inactivation of geminin during embryogenesis has not revealed any detectable DNA replication defects. In order to examine its role in vivo, we conditionally inactivated geminin in the murine colon and lung, and assessed chemically induced carcinogenesis. We show here that mice lacking geminin develop a significantly higher number of tumors and bear a larger tumor burden than sham-treated controls in urethane-induced lung and azoxymethane/dextran sodium sulfate-induced colon carcinogenesis. Survival is also significantly reduced in mice lacking geminin during lung carcinogenesis. A significant increase in the total number and grade of lesions (hyperplasias, adenomas, and carcinomas) was also confirmed by hematoxylin and eosin staining. Moreover, increased genomic aberrations, identified by increased ATR and γH2AX expression, was detected with immunohistochemistry analysis. In addition, we analyzed geminin expression in human colon cancer, and found increased expression, as well as a positive correlation with ATM/ATR levels and a non-monotonic association with γH2AX. Taken together, our data demonstrate that geminin acts as a tumor suppressor by safeguarding genome stability, whereas its overexpression is also associated with genomic instability. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Melt Electrowriting of Graded Porous Scaffolds to Mimic the Matrix Structure of the Human Trabecular Meshwork.

The permeability of the human trabecular meshwork (HTM) regulates eye pressure via a porosity gradient across its thickness modulated by stacked layers of matrix fibrils and cells. Changes in HTM porosity are associated with increases in intraocular pressure and the progress of diseases such as glaucoma. Engineered HTMs could help to understand the structure-function relation in natural tissues and lead to new regenerative solutions. Here, melt electrowriting (MEW) is explored as a biofabrication technique to produce fibrillar, porous scaffolds that mimic the multilayer, gradient structure of native HTM. Poly(caprolactone) constructs with a height of 125-500 µm and fiber diameters of 10-12 µm are printed. Scaffolds with a tensile modulus between 5.6 and 13 MPa and a static compression modulus in the range of 6-360 kPa are obtained by varying the scaffold design, that is, the density and orientation of the fibers and number of stacked layers. Primary HTM cells attach to the scaffolds, proliferate, and form a confluent layer within 8-14 days, depending on the scaffold design. High cell viability and cell morphology close to that in the native tissue are observed. The present work demonstrates the utility of MEW for reconstructing complex morphological features of natural tissues.

Photodegradable Hydrogels for Cell Encapsulation and Tissue Adhesion.

Hydrogels for wound management and tissue gluing applications have to adhere to tissues for a given time scale and then disappear, either by removal from the skin or by slow degradation for applications inside the body. Advanced wound management materials also envision the encapsulation of therapeutic drugs or cells to support the natural healing process. The design of hydrogels that can fulfill all of these properties with minimal chemical complexity, a stringent condition to favor transfer into a real medical device, is challenging. Herein, we present a hydrogel design with a moderate structural complexity that fulfills a number of relevant properties for wound dressing: it can form in situ and encapsulate cells, it can adhere to tissues, and it can be degraded on demand by light exposure under cytocompatible conditions. The hydrogels are based on starPEG macromers terminated with catechol groups as cross-linking units and contain intercalated photocleavable nitrobenzyl triazole groups. Hydrogels are formed under mild conditions (N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) buffer with 9-18 mM sodium periodate as the oxidant) and are compatible with encapsulated cells. Upon light irradiation, the cleavage of the nitrobenzyl group mediates depolymerization, which enables the on-demand release of cells and debonding from tissues. The molecular design and obtained properties reported here are interesting for the development of advanced wound dressings and cell therapies and expand the range of functionality of current alternatives.

Printability study of metal ion crosslinked PEG-catechol based inks.

In this paper we explore the printability of reversible networks formed by catechol functionalized PEG solutions and metal cations (Al3+, Fe3+ or V3+). The printability and shape fidelity were dependent on the ink composition (metal ion type, pH, PEG molecular weight) and printing parameters (extrusion pressure and printing speed). The relaxation time, recovery rate and viscosity of the inks were analyzed in rheology studies and correlated with thermodynamic and ligand exchange kinetic constants of the dynamic bonds and the printing performance (i.e. shape fidelity of the printed structures). The relevance of the relaxation time and ligand exchange kinetics for printability was demonstrated. Cells seeded on the materials crosslinked with Al3+, Fe3+ ions were viable and revealed well-spread morphologies during 7 day culture, indicating the potential of the formulations to be used as inks for cell encapsulation. The proposed dynamic ink design offers significant flexibility for 3D bioprinting, and enables straightforward adjustment of the printable formulation to meet application-specific needs.

DNA Replication Inhibitor Geminin and Retinoic Acid Signaling Participate in Complex Interactions Associated With Pluripotency.

BACKGROUND/AIM: Several links between DNA replication, pluripotency and development have been recently identified. The involvement of miRNA in the regulation of cell cycle events and pluripotency factors has also gained attention. MATERIALS AND METHODS: In the present study, we used the g:Profiler platform to analyze transcription factor binding sites, miRNA networks and protein-protein interactions to identify novel links among the aforementioned processes. RESULTS AND CONCLUSION: A complex circuitry between retinoic acid signaling, SWI/SNF components, pluripotency factors including Oct4, Sox2 and Nanog and cell cycle regulators was identified. It is suggested that the DNA replication inhibitor geminin plays a central role in this circuitry.