No abstract

Hydrogels have played a significant role in many applications of regenerative medicine and tissue engineering due to their versatile properties in realizing design and functional requirements. However, as bioengineered solutions are translated towards clinical application, new hurdles and subsequent material requirements can arise. For example, in applications such as cell encapsulation, drug delivery, and biofabrication, in a clinical setting, hydrogels benefit from being comprised of natural extracellular matrix-based materials, but with defined, controllable, and modular properties. Advantages for these clinical applications include ultraviolet light-free and rapid polymerization crosslinking kinetics, and a cell-friendly crosslinking environment that supports cell encapsulation or in situ crosslinking in the presence of cells and tissue. Here we describe the synthesis and characterization of maleimide-modified hyaluronic acid (HA) and gelatin, which are crosslinked using a bifunctional thiolated polyethylene glycol (PEG) crosslinker. Synthesized products were evaluated by proton nuclear magnetic resonance (NMR), ultraviolet visibility spectrometry, size exclusion chromatography, and pH sensitivity, which confirmed successful HA and gelatin modification, molecular weights, and readiness for crosslinking. Gelation testing both by visual and NMR confirmed successful and rapid crosslinking, after which the hydrogels were characterized by rheology, swelling assays, protein release, and barrier function against dextran diffusion. Lastly, biocompatibility was assessed in the presence of human dermal fibroblasts and keratinocytes, showing continued proliferation with or without the hydrogel. These initial studies present a defined, and well-characterized extracellular matrix (ECM)-based hydrogel platform with versatile properties suitable for a variety of applications in regenerative medicine and tissue engineering.

Organoids, and in particular patient-derived organoids, have emerged as crucial tools for cancer research. Our organoid platform, which has supported patient-derived tumor organoids (PTOs) from a variety of tumor types, has been based on the use of hyaluronic acid (HA) and collagen, or gelatin, hydrogel bioinks. One hurdle to high throughput PTO biofabrication is that as high-throughput multi-well plates, bioprinted volumes have increased risk of contacting the sides of wells. When this happens, surface tension causes bioinks to fall flat, resulting in 2D cultures. To address this problem, we developed an organoid immersion bioprinting method – inspired by the FRESH printing method – in which organoids are bioprinted into support baths in well plates. The bath – in this case an HA solution – shields organoids from the well walls, preventing deformation. Here we describe an improvement to our approach, based on rheological assessment of previous gelatin baths versus newer HA support baths, combined with morphological assessment of immersion bioprinted organoids. HA print baths enabled more consistent organoid volumes and geometries. We optimized the printing parameters of this approach using a cell line. Finally, we deployed our optimized immersion bioprinting approach into a drug screening application, using PTOs derived from glioma biospecimens, and a lung adenocarcinoma brain metastasis. In these studies, we showed a general dose dependent response to an experimental p53 activator compound and temozolomide (TMZ), the drug most commonly given to brain tumor patients. Responses to the p53 activator compound were effective across all PTO sets, while TMZ responses were observed, but less pronounced, potentially explained by genetic and epigenetic states of the originating tumors. The studies presented herein showcase a bioprinting methodology that we hope can be used in increased throughput settings to help automate biofabrication of PTOs for drug development-based screening studies and precision medicine applications.

Nitrate contamination of groundwater is an important issue in rural areas. In this study, an electrolytic method for the denitrification of groundwater was investigated in a laboratory reactor. We used an ion-exchange membrane, employing a titanium oxide anode and five kinds of cathode for the investigation. The nitrate removal efficiencies with Cu, Pb, Ti/Ir/Ru, Ti/Ir/Ta, and Zn cathodes were 30.0%, 59.9%, 73.8%, 23.3%, and 80.6%, respectively. A cation-selective membrane was employed to separate the electrode compartments, and only the proton produced in the anode side was permitted to the cathode side in order to avoid reversible reactions. In terms of the nitrate removal, good experimental results were obtained by using the membrane reactor equipped with the Zn cathode. The nitrate concentration level dropped from 30 to 6 mg/L, which is below the limit for drinking water. The pH was not affected significantly by the current density and it increased up to 9.0 at 100 mA/cm2. The nitrate removal efficiency showed the highest value with 1 mM of NaCl as an electrolyte. The impression was that membrane electrolysis made it possible to avoid the re-oxidation problem. It appears clear that electrolytic denitrification with an ion-exchange membrane is a very effective process with a mean energy consumption of only between 26.9 and 112.9 kWh/kg-NO3.