Page 101 - IJB-6-4

Silva, et al.
biomanufacturing technologies [3,6] . In the case of important [1-3,18] . The scalability of bioprinted
EBB, the time lag between hydrogel extrusion and constructs toward clinically relevant sizes is often
its subsequent crosslinking is one of the limiting limited by the accessibility of nutrients throughout
factors in the formation of complex geometries [7,8] . the construct, as nutrient access and waste
This is mainly because the viscoelastic properties of removal depend solely on diffusion-mediated
extruded bioinks are often not sufficient to support transport. As a result, perfusable networks within
these geometries before extensive crosslinking bioprinted constructs are imperative to create
is applied [7,9,10] . Therefore, the shape fidelity of tissues of clinically relevant size, as they will
the printed constructs is greatly compromised, allow adequate nutrient availability and prevent
especially in the fabrication of hollow or highly waste accumulation in the innermost regions of
detailed structures. Emerging techniques such the construct [19,20] . This, in turn, will facilitate the
as freeform reversible embedding of suspended maturation of multilayered constructs and shorten
hydrogels (FRESH) and volumetric printing the gap between native and in vitro functionality. In
[12]
[11]
have contributed to alleviating some of these addition, the biomanufacturing of hollow tubular
limitations by allowing the formation of complex structures might also be beneficial for generating
structures. In the FRESH technique, hydrogels are multilayered large and medium-diameter vascular
printed in a support bath of sacrificial microparticles, grafts for use in either transplantation or disease
which provides structural support while the modeling [21-23] .
hydrogel is crosslinked and therefore guarantees Accordingly, here we report the development
a high degree of shape fidelity . Despite the of a triple-layered coaxial extruder system for the
[13]
superior printing resolution achieved in constructs fabrication of layered tubular structures that allow
manufactured with this technique, results have not the simultaneous dispensing of three different
yet been reported when depositing cell-embedded materials using EBB systems. Our aim was to study
hydrogels [3,14] . Similarly, volumetric bioprinting how different design parameters and bioprinting
allows the fabrication of convoluted free-form conditions of this system affect the viability of
geometries with the spatially selective exposition embedded cells upon extrusion. Computational
of cell-laden photocrosslinkable hydrogels to analyses were initially implemented to optimize
ultraviolet (UV) or blue light. In particular, this design parameters of the coaxial extruder system
technique has demonstrated the rapid fabrication of based on predicted pressure distributions, and
anatomically relevant hollow structures with high these findings were then validated experimentally
cell viability . However, the current technique on bioprinting experiments using human cells
[15]
cannot include multiple materials within the same in alginate-based hydrogels. In addition, the
bioprinting process and can lead to unwanted triple-layered design allowed immediate bioink
heterogeneous stiffness of the constructs, which crosslinking upon extrusion by including a
greatly limits its exploitability. crosslinking solution as the outermost layer of the
Despite recent advances on the development printed tubular structures, and the formation of
of techniques that allow an increased structural hollow structures by posterior removal of sacrificial
complexity of constructs and novel hydrogel material contained in the innermost layer.
formulations that support bioprinting and maturation
of tissues, functionality is a challenge that has 2 Materials and methods
not been fully addressed yet [3,16,17] . To engineer 2.1 Triple-layered coaxial nozzle design
functionally relevant tissues in vitro, the current and in silico evaluation
inability to recreate the 3D microenvironments seen
in vivo is a key restriction that must be overcome. A first prototype of a triple-layered coaxial nozzle
Among the attributes that bioprinted constructs was fabricated by assembling commercially
must have to permit appropriate tissue maturation, available nozzle parts (Nordson EFD, Dunstable,
vascular networks appear to be one of the most Bedfordshire, UK). A mixture consisting of a

International Journal of Bioprinting (2020)–Volume 6, Issue 4 97