Crosslinking Strategies for 3D Bioprinting of Polymeric Hydrogels

Abstract

Three‐dimensional (3D) bioprinting has recently advanced as an important tool to produce viable constructs that can be used for regenerative purposes or as tissue models. To develop biomimetic and sustainable 3D constructs, several important processing aspects need to be considered, among which crosslinking is most important for achieving desirable biomechanical stability of printed structures, which is reflected in subsequent behavior and use of these constructs. In this work, crosslinking methods used in 3D bioprinting studies are reviewed, parameters that affect bioink chemistry are discussed, and the potential toward improving crosslinking outcomes and construct performance is highlighted. Furthermore, current challenges and future prospects are discussed. Due to the direct connection between crosslinking methods and properties of 3D bioprinted structures, this Review can provide a basis for developing necessary modifications to the design and manufacturing process of advanced tissue‐like constructs in future.

Effects of Processing Parameters of 3D Bioprinting on the Cellular Activity of Bioinks

“…Hence, this system acts as a pre‐crosslinking strategy and enhances cell viability of the overall constructs. [ 241 ] Li et al. reported cell viability of >96% (mouse myoblasts cells C2C12) up to day 2, utilizing this strategy with extrusion‐based printing.…”

“…However, this crosslinking method has a short shelf life, and oxidation of disulfide bonds causes poor thermal stability. [ 241 ] Some notable crosslinking examples include thiol‐norbornene (mouse fibroblast L929 with 81 ± 4.4% viability and chondrogenic cell line (ATDC 5) with 93 ± 2.8% viability at day 1); [ 251 ] thiol‐allyl (human and equine MSC with cell viability and differentiability upto 21 days), [ 252 ] (human bone marrow derived MSC with cell viability of 78.9 ± 13.5% at day 1) etc.…”

“…Human fibroblasts cells can survive freeze‐thaw cycles and fibroblast laden hydrogels can withstand thermal gelation (hydrogen bond formation) with freezing and thawing. [ 241 ] Similarly, DNA and polypeptide containing bioink containing AtT‐20 anterior pituitary cells can be crosslinked with hydrogen bonds while maintaining excellent cell viability of 98.8 ± 1.4%. [ 259 ]…”

“…Hence, this system acts as a pre-crosslinking strategy and enhances cell viability of the overall constructs. [241] Li et al reported cell viability of >96% (mouse myoblasts cells C2C12) up to day 2, utilizing this strategy with extrusion-based printing. [242] The addition of silicate nanoparticles also allows electrostatic interactions, and during extrusion-based bioprinting, it shields the cells against shear stresses.…”

Mechanical Considerations of Bioprinted Tissue

“…Furthermore, bioprinting, as a type of additive manufacturing, is amenable to scaled up tissue fabrication and manufacturing, an important consideration as clinical use of fabricated tissues increases (Wu et al, 2017;Skylar-Scott et al, 2019;Castilho et al, 2020). The development of materials compatible with cells and other biologics, so called "bioinks" has also exploded involving a variety of artificial and native material types (Chimene et al, 2016;Chan et al, 2020;Cui et al, 2020), material sourcing (Abaci and Guvendiren, 2020), curing features (GhavamiNejad et al, 2020), and utilities (Whitford and Hoying, 2016;Unagolla and Jayasuriya, 2020). While the practical aspects and applications involving bioprinting are actively explored (as indicated by the recent burst of informative reviews on bioprinting and bioinks), the integration of biological responses and dynamics in bioprinted systems are less addressed.…”