Suspended Manufacturing of Multi-Layered Biopolymer Hydrogels for Tissue Engineering

Tissue engineering is proving a promising tool as an alternative to relying on donor availability in restorative tissue therapeutics. Tissue engineering concepts entail combining cells and materials in vitro to generate 3D parts that replicate in vivo tissue environments. Much focus has been spent on researching biopolymer hydrogels as scaffolds within tissue engineering constructs as there is vast potential in their tailorability to simulate the extracellular matrices within native human tissues. Although many features within hydrogels may be finely tuned when developing tissue engineering implants, such as water content, mechanical properties, and microarchitecture, current studies in this area are seldom able to recapitulate the physicochemical and mechanical gradients that are exhibited in human tissues, within a single construct. Moreover, the hydrogel precursor solutions used in 3D bioprinting tissue engineering constructs are usually manipulated towards enhancing either print fidelity (by increasing solution viscosity) or to preserve the viability of suspended cells (by decreasing the viscosity of precursor solutions). 3D bioprinted tissue engineering constructs therefore often fail to harbour both optimal print fidelity in conjunction with viable encapsulated cells thus leaving the intended tissue engineering ECM environment poorly reproduced. The work presented in this thesis addresses these problems and demonstrates that the success of a tissue engineering construct need not be compromised by the usual trade-off between print fidelity and cell viability and can in fact possess both of these features that are crucial to the function of the part. This technology uses a supporting gel bed within which multiple cell/hydrogel printing solutions are layered to keep the construct in shape prior to solidifying. The mechanisms behind the function of the supporting gel bed as a suspending agent were first analysed, showing that its shear-thinning behaviour allows material to be deposited within it followed by rapid restructuring to uphold the construct shape. A range of contrasting hydrogels and cell types were then 3D bioprinted into multiple layers and configurations, pushing the boundaries of what can usually be achieved when conventionally 3D printing low viscosity solutions onto a planar surface. As an example of the potential of this technique in becoming commonplace in the clinic, a chronic-depth skin equivalent was 3D printed, complete with hypodermis, dual compartment dermis and epidermis. The construct contained gradients in material chemistry, mechanical properties, microarchitecture, and cell phenotype, and served as an example of the capacity that this system holds in generating highly sophisticated human tissue mimics.