*Article* **3D Printing of a Reactive Hydrogel Bio-Ink Using a Static Mixing Tool**

**María Puertas-Bartolomé 1,2 , Małgorzata K. Włodarczyk-Biegun <sup>3</sup> , Aránzazu del Campo 3,4 , Blanca Vázquez-Lasa 1,2,\* and Julio San Román 1,2**


Received: 29 July 2020; Accepted: 27 August 2020; Published: 31 August 2020

**Abstract:** Hydrogel-based bio-inks have recently attracted more attention for 3D printing applications in tissue engineering due to their remarkable intrinsic properties, such as a cell supporting environment. However, their usually weak mechanical properties lead to poor printability and low stability of the obtained structures. To obtain good shape fidelity, current approaches based on extrusion printing use high viscosity solutions, which can compromise cell viability. This paper presents a novel bio-printing methodology based on a dual-syringe system with a static mixing tool that allows in situ crosslinking of a two-component hydrogel-based ink in the presence of living cells. The reactive hydrogel system consists of carboxymethyl chitosan (CMCh) and partially oxidized hyaluronic acid (HAox) that undergo fast self-covalent crosslinking via Schiff base formation. This new approach allows us to use low viscosity solutions since in situ gelation provides the appropriate structural integrity to maintain the printed shape. The proposed bio-ink formulation was optimized to match crosslinking kinetics with the printing process and multi-layered 3D bio-printed scaffolds were successfully obtained. Printed scaffolds showed moderate swelling, good biocompatibility with embedded cells, and were mechanically stable after 14 days of the cell culture. We envision that this straightforward, powerful, and generalizable printing approach can be used for a wide range of materials, growth factors, or cell types, to be employed for soft tissue regeneration.

**Keywords:** 3D-bioprinting; static mixer; reactive hydrogel; chitosan; hyaluronic acid
