A targeted rheological bioink development guideline and its systematic correlation with printing behavior

Abstract

Bioprinting for tissue or disease models is a promising but complex process involving biofabrication, cell culture and a carrier material known as bioink. The native extracellular matrix (ECM), which forms the scaffold for cells in vivo, consists of several components including collagen as a gelling agent to confer mechanical stiffness and provide a substrate for cell attachment. Bioprinting therefore needs an artificial ECM that fulfills the same functions as its natural counterpart during and after the printing process. The combination of bioink materials determines the immune response of the host, cell compatibility and adhesion. Here we evaluate multi-material blending with four pre-selected components using a design of experiments approach. Our exemplary designed hydrogel is highly reproducible for the development of artificial ECM and can be expanded to incorporate additional requirements. The bioink displays shear-thinning behavior and a high zero-shear viscosity, which is essential for the printing process. We assessed the printing behavior of our bioink over a wide range of the key process parameters for extrusion-based bioprinting (temperature, pressure, feed rate, and nozzle geometry). Several processing temperatures were linked by rheological measurements directly to the 3D printing process. The printing results were evaluated using a self-developed categoric strand screening process, varying the feed rate and pressure with a fixed nozzle. Accordingly, nozzles differing in size and shape were evaluated and the interactions between printing pressure and feed rate were characterized separately by applying a modified O-R-O test. We tested the short-term cultivation stability of our bioink to mimic the hypothermic and hyperthermic conditions of the human body. As result we present an expandable concept for bioink development and a highly reproducible and well-characterized procedure for printing with the newly developed hydrogel. We provide detailed insights into the relationship between printing parameters, rheological parameters and short-term cultivation stability.

Calculation of Mass Transfer and Cell-Specific Consumption Rates to Improve Cell Viability in Bioink Tissue Constructs

“…The permeation efficiency may also be affected by the average pore size, which can be estimated by rheology [ 37 ] or thermoporometry, using differential scanning methods [ 38 ]. The average pore size of our gel was ~8.94 nm, based on our previous report [ 20 ] and the equations proposed by Devi et al [ 37 ]. This may explain the prolonged diffusion time (and thus smaller diffusion coefficients) for BSA, reflecting the similar size of the BSA molecule and the average pore.…”

“…In addition, it should be noted that the metabolic behavior of the cells can be altered by the surrounding environment [ 12 ], in this particular case the bioink. Furthermore, gel aging during cultivation [ 20 ], which can also influence diffusion, could be included to model the behavior of the gel over longer periods.…”

“…Hydrogels were prepared as previously described [ 20 ]. For diffusion experiments, the bioink (3.5% gelatin, 2.4% cellulose, 1.5% alginate, and 0.5% (w/v ) carrageenan (all Sigma-Aldrich, Steinheim, Germany) was allowed to equilibrate at 37 °C for 1 h, followed by the application of 1 mL into rubber seals (Lux-tools, Wermelskirchen, Germany, inner Ø 25 mm, the specific height was determined experimentally) to fit the diffusion chamber.…”

“…For each substance, a pre-experiment was carried out to estimate the approximate time period needed to achieve a linear mass transfer slope comprising at least five data points (r 2 > 0.9), in order to work in a steady state region. The thickness of the bioink discs was ~1 mm (Ussing) or ~4 mm (Franz), and was determined by gravimetric measurement of the hydrogel after the experiment, assuming a cylindrical shape and the known density of the bioink [ 20 ]. Diffusion coefficients D were calculated as shown in Equation (1): − D = Δ m × Δ t −1 × A −1 × x × Δ c −1 where Δ m × Δ t −1 is the mass of the diffused substance over time, A is the available area for mass transfer, Δ c is the concentration gradient, and x is the thickness of the bioink disc [ 21 ].…”

Enabling Technologies for Obtaining Desired Stiffness Gradients in GelMA Hydrogels Constructs

“…In passing, it was noted that both laws respect the consistency conditions $$\mu _{\rm mix}(1, \dot{\gamma }) = \mu _{\ell }(\dot{\gamma })$$ and $$\mu _{\rm mix}(0, \dot{\gamma }) = \mu _{h}(\dot{\gamma })$$. As regards the modeling of pure Ldof and Hdof hydrogels, the Carreau model was employed to describe their shear thinning behavior, namely: [ 32,38 ] $$\begin{equation} \mu _{\ast }(\dot{\gamma }) = \mu _{\ast }^\infty + \frac{\mu _{\ast }^0 - \mu _{\ast }^{\infty }}{{\left[1 + {\left(K_{\ast }\dot{\gamma }\right)}^{2}\right]}^{\frac{1-n_{\ast }}{2}}}\, , \quad \ast =\ell , h\, \end{equation}$$where $$\mu _{\ell }^0$$, $$\mu _{h}^0$$ and $$\mu _{\ell }^\infty$$, $$\mu _{h}^\infty$$ are limit viscosity values (Pa$$\cdot$$s); $$K_{\ell }$$, …”