RESOURCES

Abstract
The emergence of embedded three-dimensional (3D) bioprinting has revolutionized the biofabrication of free-form constructs out of low-viscosity and slow-crosslinking hydrogels. Using gel-based support baths has limitations including lack of proper oxygenation and nutrition and complications with bath removal. Herein, a novel-embedded 3D bioprinting technique is developed with an albumin foam support bath as a promising substitute. The proposed technique, in-foam bioprinting, offers excellent printability and convenience in bath removal while providing cells with easy access to oxygen and nutrients. The foam-based support bath is characterized through foam stability and rheological tests. The bubble size in the foam is measured to study the change in the structure of the bath due to the coalescence of the bubbles over time. Free-form structures are successfully 3D printed with thermoresponsive chitosan-based bioinks to demonstrate the capability of the in-foam bioprinting technique. The viability of bioprinted fibroblast L929 cells is studied over a seven-day period, showing high cell viability of over 97%, which is attributed to the abundance of oxygen and nutrition in the foam support bath. Importantly, in-foam bioprinting is beneficial for biofabricating large samples with a long printing time without jeopardizing cell viability.

Abstract
Thermosensitive chitosan (CH)-based hydrogels prepared with a mix of sodium bicarbonate and β-glycerophosphate as gelling agents rapidly pass from a liquid at room temperature to a mechanically strong solid at body temperature without any crosslinker. They show excellent potential for tissue engineering applications and could be interesting candidates for bioprinting. Unfortunately, since gelation is not instantaneous, formulations compatible with cell encapsulation (chitosan concentrations around 2% or lower) lead to very poor resolution and fidelity due to filament spreading. Here, we investigate the FRESH bioprinting approach with a warm sacrificial support bath, to overcome these limitations and enhance their bioprintability. First, a support bath, made of Pluronic including sodium chloride salt as a rheology modifier agent, was designed to meet the specific physical state requirements (solid at 37 °C and liquid at room temperature) and rheological properties appropriate for bioprinting. This support bath presented yield stress of over 100 Pa, a shear thinning behavior, and fast self-healing during cyclic recovery tests. Three different chitosan hydrogels (CH2%w/v, CH3%w/v, and a mixture of CH and gelatin) were tested for their ability to form filament and 3D structures, with and without a support bath. Both the resolution and mechanical properties of the printed structure were drastically enhanced using the FRESH method, with an approximate four fold decrease of the filament diameter which is close to the needle diameter. The printed structures were easily harvested without altering their shape by cooling down the support bath, and do not swell when immersed in PBS. Live/dead assays confirmed that the viability of encapsulated mesenchymal stem cells was highest in CH2% and that the support bath-assisted bioprinting process did not adversely impact cell viability. This study demonstrates that using a warm FRESH-like approach drastically enhances the potential for bioprinting of the thermosensitive biodegradable chitosan hydrogels and opens up a wide range of applications for 3D models and tissue engineering.