title: Fabrication of 3D printed Gelatin-Hyaluronic Acid hydrogels based on enzyme-mediated tyramine conjugates and other derivatives
Creators: name: Morata Martinez, Miranda
Publisher: name: University of Glasgow
Year: 2021

description: Extracellular matrices (ECMs) in soft tissues are highly hydrated structures mainly composed of glycoproteins (such as collagen or fibronectin) and glycosaminoglycans (such as hyaluronic acid (HA) or keratan sulfate), each one with a tissue-specific composition (1). Many of these tissues are unable to regenerate themselves or can only repair minor injuries, as is the case of skin (2), heart (3) and cartilage (4). Hydrogels are hydrophilic polymeric networks with high water retention capability which have been frequently proposed as potential candidates for soft tissues regeneration due to their tunable physical, chemical and biological properties, biocompatibility and their ability to mimic the native ECMs (5-7). Besides, they promote phenotype maintenance and induce re-differentiation of different cells such as cardiomyocytes (8), chondrocytes (9) and hepatoblast (10). Most hydrogels need to be chemically cross-linked to not dissolve at body temperature. Conventional cross-linking methods involving chemical reactions are generally cytotoxic. Solvents, initiators or unreacted substances are left behind, often resulting in inflammation and cell death (11-13). In order to prevent any harmful effect on cells, they must be therefore pre-formed under safe lab conditions, thoroughly washed and sterilised before implantation. Cross-linking reactions mediated by enzymes (14-16), also known as enzyme-mediated or enzyme-catalysed cross-linking, have been proposed relatively recently as a less problematic alternative for hydrogel scaffolding. In these systems, aqueous hydrogel precursor solutions are mixed with cellular components and/or desired bioactive agents prior to injection into the defect area. Enzymes, included or subsequently added to the precursor solutions, catalyse the cross-linking reaction immediately upon injection, generating covalent bonds between specific functional groups found within the polymer side chains. These mild in situ reactions, which can take place in a matter of seconds or minutes, do not produce any cytotoxic effects (17,18) and present several advantages (19-21) over pre-created hydrogels: adaptation to the shape of the defect, lower risk of implant migration, easy and effective cell encapsulation and deliverability, and minimally invasive surgical interventions that improve patient compliance and recovery (18,22-27). Gelatin (Gel) is a natural polymer derived from the partial denaturation of collagen that has attracted attention as a hydrogel scaffold into which cells can be embedded. It has accessible functional groups that can react with other molecules and different integrin-binding sites for cell adhesion and differentiation (28). However, its poor mechanical properties limit its applications. This lack of mechanical strength can be overcome by preparing blends of gelatin with other polymers (29) by enzyme-mediated reactions such as hyaluronic acid (HA). HA is well known for its high hydrophilicity, good lubrication, biocompatibility, and low cell and protein adhesive properties (30). Gel-HA hydrogels enzymatically cross-linked by horseradish peroxidase (HRP) and hydrogen peroxide (H202) through the covalent bonding of tyramine (Tyr) have demonstrated their non-cytotoxicity and potential for cell adhesion and spreading (33-35). HA concentration in this system can be modified according to the required stiffness, water sorption, pore size and gelation time (18, 31, 32), which gives rise to potential candidates for several types of soft tissue models, regeneration strategies and applications in minimally invasive procedures. Traditional approaches based on hydrogel or other soft materials for scaffolding are limited in their capacity of producing complex microstructures with accurate biomimetic properties. Three-dimensional (3D) bioprinting technologies to the contrary, offer a novel versatility to co-deliver cells and biomaterials with precise control over their configurations, spatial distributions and pattern exactitude, achieving personalized constructs that mimic the functionality of target tissues and organs (22, 36-42). One of the most appealing applications of 3D bioprinting nowadays is the development of functional 3D tissue models. Current 2D cell cultures, particularly the animal models employed for in vitro drug testing, are shown to respond differently to drug candidates compared to humans, and hence their use as models of human diseases or medical conditions result ineffective and futile (163). However, like any other new and complex technology, the process towards its complete implementation still has a long way to go. The determination and understanding of the parameters involved in a process of hydrogel printing as well as the effects of their combination are paramount for the success of the scaffold and can present a challenge even to the most veteran researchers. In this work, we propose a viable and reproducible cell encapsulation protocol of Gel-Tyr/HA-Tyr hydrogels by means of 3D bioprinting for in vitro drug testing and further use in regenerative medicine, significantly reducing the worker’s laboratory time and facilitating the completion of long laborious tasks in multi-sample hydrogel generation. We also provide an extensive and well-documented description of several parameters directly involved in every process of printing design and protocol optimisation as well as some of their common individual effects on printed scaffolds.