Advanced optical assessment and modeling of extrusion bioprinting

Abstract

In the context of tissue engineering, biofabrication techniques are employed to process cells in hydrogel-based matrices, known as bioinks, into complex 3D structures. The aim is the production of functional tissue models or even entire organs. The regenerative production of biological tissues adheres to a multitude of criteria that ultimately determine the maturation of a functional tissue. These criteria are of biological nature, such as the biomimetic spatial positioning of different cell types within a physiologically and mechanically suitable matrix, which enables tissue maturation. Furthermore, the processing, a combination of technical procedures and biological materials, has proven highly challenging since cells are sensitive to stress, for example from shear and tensile forces, which may affect their vitality. On the other hand, high resolutions are pursued to create optimal conditions for subsequent tissue maturation. From an analytical perspective, it is prudent to first investigate the printing behavior of bioinks before undertaking complex biological tests. According to our findings, conventional shear rheological tests are insufficient to fully characterize the printing behavior of a bioink. For this reason, we have developed optical methods that, complementarily to the already developed tests, allow for quantification of printing quality and further viscoelastic modeling of bioinks.

Introduction

The aim of biofabrication in the context of tissue engineering and regenerative medicine is to produce living, bio-functional tissue. This can be used, for instance, as tissue models for testing medical drugs or for tissue replacement and tissue reconstruction in cases of organ damage1. To achieve this, three-dimensional structures with highly defined, spatially organized properties made from cell-containing biomaterials need to be created2. This requires bottom-up processes, which are currently implemented using 3D printing techniques. The methods include, among others, stereolithography, inkjet, laser-assisted, and micro-extrusion fabrication. Fused filament printing is often chosen due to its simplicity, flexibility, and high material throughput3,4. The materials used in this process are cell-laden hydrogels or their precursors, which are directly linked after extrusion through various possible mechanisms such as ions, light, or chemical crosslinking agents to form stable networks5. The core criteria for producing functional tissue models conducting bioprinting can be simplified into four phases:

During printing: Cell survival must be ensured. Critical factors include shear and tensile forces in the nozzle, and the key-parameters are the rheology of the bio-ink and the nozzle’s geometry.
Immediately after printing: The scaffold’s shape fidelity must be ensured, the print resolution must be sufficient to allow diffusion of nutrients and oxygen to the cells inside and prevent hypoxia. Critical factors are crosslinking kinetics of the polymer network and porosity of the hydrogel.
Mid-term: The hydrogel must mechanically match the physiological requirements of the target tissue, and cell proliferation and migration must be ensured. Critical factors are structural composition of the fibrillar network, cell density, and organization.
Long-term: Tissue maturation must occur. Critical factors are cell mobility and performance, control of tissue maturation by signaling molecules, biodegradation of the artificial matrix, and substitution by the natural extracellular matrix.

Thus, the initial steps in analyzing new bio-inks involve biological testing (biocompatibility of the matrix) on one hand, and printing trials on the other, to evaluate the ink’s suitability for processing into a three-dimensional construct. Both factors are equally crucial and act as knockout criteria for the respective system. Since cell biological testing is time consuming and associated with high costs for cell culture, as well as for the required agents like staining kits, it is advisable to first check the printability and define the process-related conditions of the bio-ink system—for instance, the concentration-dependent printing behavior of the ink and the respective required printing parameters. Some essential information about the printability of a hydrogel can already be obtained from shear rheological tests6. Schwab et al. mention parameters like flow behavior, yield stress, elastic recovery, shear stress, and damping factor tanδ7. The shear-thinning behavior of a bio-ink, for example, affects not only the required pressure but also the internal shear forces to which the cells are exposed. Elastic recovery provides insight into the time-dependent response of the material after shear-induced deformation and, combined with yield stress and the damping factor, plays a crucial role in shape stability. Meanwhile, shear stress influences cell behavior and print resolution. Nonetheless, additional printing trials are necessary to test and possibly optimize a bio-ink for biofabrication. To carry this out systematically, quality indicators must be established to quantitatively categorize the printing results. It must be considered that there are many different types of bio-inks, and as many of them as possible should be analytically captured5,8. Moreover, even within extrusion printing, there are various methods like Embedded Bioprinting, Co-axial Bioprinting, Multi-nozzle Multi-material Bioprinting, Single-nozzle Multi-material Bioprinting, etc9,10. In addition to categorizing the inks with their respective process, such tests should also provide insights into achievable scaffold designs and assist in optimizing, for example, G-codes for printing with a specific ink8. In the past, several coherent tests have been established in this regard2,6,9. Some frequently applied tests are schematically illustrated in Fig. 1.

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Methods

Alginates (Alginate PH176, Vivapharm, JRS PHARMA GmbH & Co. KG, Germany) at concentrations of 2%, 3%, and 4% (w/v) in H2O were printed using an Inkredible + bioprinter (Cellink Bioprinting AB, Gothenburg, Sweden) equipped with a 25G syringe nozzle (Nordson EFD, Ohio, USA). Extrusion pressures of 16 kPa, 90 kPa, and 130 kPa were respectively applied, calculated to achieve a mass flow of approximately 0.250 mg/s. Similarly, the 5% (w/v) GelMA in PBS (synthesized as described by Loessner et al.43 using ~ 300 g Bloom, Type A, Merck KGaA, Darmstadt, Germany), was printed using a BioX (Cellink Bioprinting AB, Gothenburg, Sweden) with the ink cartridge at 37 °C and a mass flow of 0.250 mg/s. The samples were printed in a strut spreading test meander pattern, utilizing the new imaging setup, onto a glass slide at a printing speed of 5 mm/s. These prints were recorded using a phone camera for at least 60 s to achieve temporal resolution of the strut spreading. The diameter of the strut was then evaluated in triplicate

Lamberger, Z., Schubert, D.W., Buechner, M. et al. Advanced optical assessment and modeling of extrusion bioprinting. Sci Rep 14, 13972 (2024). https://doi.org/10.1038/s41598-024-64039-y