The rheological evaluation of Xanthan Gum compositions for embedding 3D bioprinting

Every year 1000 of patients are in the waiting list for an organ transplant in the Netherlands alone[1]. Other options are researched to reduce the dependence on donors. One of them is tissue engineering. Tissue engineering is the scientific field which aims at fabricating artificial tissue constructs that can mimic the function of native human tissues and organs[2]. In addition to meeting the clinical demand for artificial tissues, the aim is creating models that can be utilized for drug testing and potentially replace clinical trials on animals for a personalized medicine approach[3]. 3D Bioprinting is the branch of tissue engineering that attempts to fabricate tissue constructs[4] by using computer aided design (CAD). The combination of standardization through the machine aided process of 3D printing, and the customization potential by tweaking the g-code commands of the 3D printer, offers a suitable solution for the personalized and massive need of artificial tissues[4]. While there are a lot of different techniques, such as inkjet and laser assisted bioprinting, the most popular techniques are stereolithography (SLA) and extrusion bioprinting[4–8]. In SLA photocurable inks are polymerized under UV-light or Visible light, and either contain cells within them, or the resulting constructs are coated with cells post-print[8]. While the resolution is quite high since it is based on light patterning, the chemical composition of the resins as well as the low cell concentration per ml which are possible with this approach, hinder the fabrication of living tissue analogues[6]. In extrusion bioprinting a material is controllably deposited through a nozzle in a pattern in the x-y-z directions[5]. The material should be shear thinning, in order to liquify upon shear and recover in the absence of shear so it could retain its shape, in essence demonstrating a thixotropic behavior[9]. The material could be a hydrogel with cells, a material that can be coated with cells post-print or even a cell-only suspension, which all are within the category of bioinks[10]. Unfortunately, the bioinks that mimic well the properties of the extracellular matrix (ECM) are either ’too liquid’ to keep their shape after deposition (low viscosity), or they need workarounds to demonstrate thixotropy. This can be done by crosslinking them post-deposition[11], heating or cooling them rapidly, or using photopolymerization where the same problems as in SLA techniques emerge[6]. Embedded bioprinting has come forward as a solution, where a support bath assists inks that are normally too ’weak’ to print[11–13]. The support bath can either be a thixotropic hydrogel, such as xanthan gum (XG)[13], or a granular suspension[12]. In both cases the printing of weak hydrogel inks and their fidelity is improved[13, 14]. However the resolution of these techniques still depends mostly on the nozzle diameter of the 3D printer, and cell only inks present a range of challenges due to their ’particle in suspension’ nature[14]. Recent developments have shown that, when properly formulated, cell only inks can create self-assembling tissues through the mechanism of diffusion packing[13]. Diffusion packing occurs when a water based Newtonian fluid particle suspension is deposited within a hydrogel embedding bath[13]. However the deposition mechanisms, and consequentially the print resolution, are severely affected by the nozzle architecture and the mechanical properties of the embedding bath[15]. Specifically in the case of XG, Salt concentration, liquid phase composition and pH can all affect it’s mechanical properties. Also during cell culture, pH and salt concentration are altered based on the cell type and the corresponding medium[16, 17].