(175ay) Cell Compatible Fluid Gels for Applications in Tissue Engineering

Fluid gels are biocompatible, microparticulate hydrogels produced under high shear and are used in 3D bioprinting applications to provide support and structure to a printed conduit before crosslinking can occur. They typically possess key properties such a shear thinning behaviour and self-healing and have been used in processes such as Suspended Layer Additive Manufacture (SLAM) or Freeform Reverse Embedding of Suspended Hydrogels (FRESH). Both approaches use a hydrogel-based biomaterial system (agarose, gelatin, etc) to support the printed hydrogel before crosslinking to a solid structure can take place and the supporting bath can be removed. The main drawbacks of this are the inability to produce delicate structures such as microvasculature without damage or distortion during removal, along with the ability to print on such a small length scale. Alginate fluid gels are produced using a pin stirrer set-up whereby calcium ions are slowly introduced directly into an alginate solution under shear. The gelation is instant and as such requires high shear and a very controlled, slow injection of ions. To overcome this, we developed a competitive ligand exchange (CLEX) based alginate fluid gel which offers much slower and controllable gelation kinetics. In this reaction, two separate alginate solutions containing chelated calcium ions and a chelated exchange ion respectively are mixed together under shear. Upon mixing a competitive ligand exchange reaction occurs which liberates calcium ions to crosslink the alginate over a period of several seconds to minutes. Quiescent gel particles are formed and immediately sheared apart to form a fluid gel. The result is an alginate fluid gel with shear thinning properties in a more time effective and scalable manner than previously reported. The aim of this work is to develop CLEX based fluid gel formulations which can support cell growth and proliferation.

Variations of CLEX modified alginate fluid gel formulations were prepared with different concentrations of hydrogel and crosslinking kinetics. Each sample was characterised using rheological techniques to determine feasibility as supporting baths during 3D bioprinting. Next, each sample was seeded with different cell lines such as human dermal fibroblast (HDFs), or mesenchymal stem cells (MSCs) to evaluate cell attachment, proliferation and cytotoxicity using methods such as immunohistochemistry staining for extracellular matrix (ECM) proteins, microscopy, and metabolic assays. Results show all formulations tested were cell compatible, exhibiting cell viability of above 95%. However, cell attachment and proliferation to produce key ECM components was only observed in certain formulations which therefore offer an exciting avenue for further investigation as a potential tissue engineering substrate biomaterial.

Future work will include deposition of bioink within the alginate fluid gel cell matrix to evaluate the suitability of directing cell growth and formation of microvascular channels. Furthermore, cell viability and morphology long term will be investigated. In conclusion, these gels have demonstrated key properties required for use as a 3D printing supporting bath such as shear thinning allowing deposition of the bioink into the fluid gel before rearranging around the newly printed structure to provide support. Moreover, we have identified key formulations that facilitate cell attachment and the ability to produce native ECM proteins such as collagen I and elastin from adherent cells, which are key to successful application in a wide variety of tissue engineering applications.