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Biomaterials

Biomaterials - Poster Session A

Poster C7 - Characterization of Rheological Modifiers in Bioinks to Improve 3-Dimensional Bioprints

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Three dimensional (3D) bioprinting is a growing technology in the tissue engineering space, owing to its capability to fabricate 3D tissue constructs with well-defined geometries [1,2,3]. Extrusion bioprinting, one of the most common 3D bioprinting processes, involves extruding bioink through a nozzle and assembling the structure in a layer by layer, additive manufacturing method. It is imperative for the extruded material to remain in its deposited location to retain its intended shape after printing, allowing for high-fidelity prints. Bioinks are commonly composed of materials that exhibit shear-thinning behavior, allowing the material to freely flow through the print nozzle upon applying pressure, while returning to its original viscosity and remaining in place after deposition. Rheological modifiers ensure that the bioink is shear-thinning with common examples including alginate, fibrinogen, collagen, gelatin, xanthan gum (XG), and hyaluronic acid (HA) [3]. This project aimed to determine the optimal weight percent ranges of XG and HA that should be added to cellular bioinks to allow for high-fidelity and stable bioprints. XG was chosen due to its excellent rheological properties and stability over a wide range of pH and temperatures [1], and HA was chosen due to its natural anti-inflammatory properties and its unique ability to support cell differentiation [4,5]. In order to evaluate the shear thinning behavior and stability of the biopolymers across various oscillation frequencies, this project examined the rheological properties of these materials via viscosity-shear rate and frequency sweep experiments at various weight percentages.

Materials and

Methods:  XG and HA solutions were prepared at various weight percentages by dissolving the solids in Hanks balanced salt solution at room temperature. The weight percentages for XG were 0.5%, 1% and 5%, and for HA were 0.5%, 1% and 2.5%. The solutions were centrifuged at 3000 rpm for 45 minutes to remove air bubbles. A C2C12 mouse myoblasts cell-laden fibrinogen bioink, at a concentration of 10^8 cells/mL, was prepared by combining pelleted cells with fibrinogen (72 mg/mL) and 1% XG. A rotary rheometer (TA Instruments, ARES G-2) with a 20-mm parallel-plate geometry was used to characterize the solutions at 21 ºC. Viscosity-shear rate experiments, simulating conditions experienced by bioinks during 3D printing, were conducted with shear rates from 10 s^-1 to 300 s-^1. These rates were calculated using the Hagen-Poiseuille equation and Rabinowitsch relation which accounts for the bioinks’ shear thinning behavior [6]. The effect of cells on viscosity was evaluated by comparing the viscosity-shear rate experiment results for the 1% XG C2C12 bioink with fibrinogen to the 1% XG solution. Next, the viscosities of the XG & HA solutions were compared to collagen type I bioink (35 mg/mL, Advanced Biomatrix’s Lifeink 240), which displays desirable shear thinning behavior during extrusion and post-printing. Three experimental runs were performed for each weight percent. A frequency sweep was also performed at 10% strain (within the linear viscoelastic region identified by a strain-sweep test) from 0.1 rad/s to 100 rad/s, to assess the bioinks’ stability across various oscillation frequencies [1].

Results, Conclusions, and Discussions: The initial comparison between the 1% XG bioinks with and without C2C12 cells showed that XG was the main determining factor for bioink viscosity over the concentrations tested, not the C2C12 cells or fibrinogen (Figure 1a). Increasing the weight percentage of XG increased the viscosity of the solution across all shear rates, along with increasing the degree of shear thinning behavior (Figure 1b). This is likely due to the disentanglement and alignment of the polymer chains along the direction of flow, which increases with shear rate [7]. Similarly, for HA, shear thinning behavior was observed, and increasing the weight percent increased the viscosity of the solutions across the measured shear rates (Figure 1c). Across the two biopolymers at various weight percentages, it was observed that the rheological behavior of 5% XG and 2.5% HA was the most similar to collagen. However, the frequency sweep results indicate that there was a crossover point between the storage and loss modulus at 1 rad/s for 2.5% HA (Figure 1d), which is a common property of non-crosslinked polymers [8]. Conversely, the storage modulus for 5% XG was consistently higher than that of the loss modulus across the various frequencies, indicating predominant solid-like behavior (Figure 1e). It can thus be concluded that 5% XG is the more ideal bioink candidate during extrusion and post-bioprinting. This is due to its consistent solid-like behavior across various frequencies and shear thinning behavior that is comparable to collagen, which is known to display desirable rheological behavior for bioprinting. This ensures ease of extrusion during printing and print fidelity post-bioprinting. For future work, we will perform cyclic strain testing to assess the recoverability of the bioink when a certain stress is applied and removed multiple times, similar to what happens during bioprinting. Cellular viability in bioinks with the desirable range of weight percentages should also be assessed [3], along with print fidelity through 3D print gauging. Overall, this project has enabled us to identify suitable weight concentrations of XG and HA rheological modifiers that should be added to bioinks for desirable shear thinning and stable behavior.

Acknowledgements (Optional):  I would like to thank Professor Adam Feinberg and PhD candidate Samuel Moss for helping me with this project.

References:
[1]: Baniasadi, H., Kimiaei, E., Polez, R. T., Ajdary, R., Rojas, O. J., Österberg, M., & Seppälä, J. (2022). High-resolution 3D printing of Xanthan Gum/nanocellulose bio-inks. International Journal of Biological Macromolecules, 209, 2020–2031. https://doi.org/10.1016/j.ijbiomac.2022.04.183
[2]: Taniguchi Nagahara, M. H., Caiado Decarli, M., Inforçatti Neto, P., Lopes da Silva, J. V., & Moraes, . M. (2022a). Crosslinked alginate‐xanthan gum blends as effective hydrogels for 3d bioprinting of biological tissues. Journal of Applied Polymer Science, 139(28). https://doi.org/10.1002/app.52612
[3]: Teixeira, M. C., Lameirinhas, N. S., Carvalho, J. P., Silvestre, A. J., Vilela, C., & Freire, C. S. (2022a). A guide to polysaccharide-based hydrogel Bioinks for 3D bioprinting applications. International Journal of Molecular Sciences, 23(12), 6564. https://doi.org/10.3390/ijms23126564
[4]: Chen, H., Xue, H., Zeng, H., Dai, M., Tang, C., & Liu, L. (2023, December 24). 3D printed scaffolds based on hyaluronic acid bioinks for tissue engineering: A Review - Biomaterials Research. BioMed Central. https://biomaterialsres.biomedcentral.com/articles/10.1186/s40824-023-00460-0
[5]: Pereira, I., Lopez-Martinez, M. J., Villasante, A., Introna, C., Tornero, D., Canals, J. M., & Samitier, J. (2023, February 15). Hyaluronic acid-based bioink improves the differentiation and network formation of neural progenitor cells. Frontiers. https://doi.org/10.3389/fbioe.2023.1110547
[6]: Jackson, S., & Dickens, T. (2021a). Rheological and structural characterization of 3D-printable polymer electrolyte inks. Polymer Testing, 104, 107377. https://doi.org/10.1016/j.polymertesting.2021.107377
[7]: M, Y. (2018). Studies on rheological behavior of Xanthan Gum Solutions in presence of additives. Petroleum & Petrochemical Engineering Journal, 2(3). https://doi.org/10.23880/ppej-16000165
[8]: Ramli, H., Zainal, N. F., Hess, M., & Chan, C. H. (2022). Basic principle and good practices of rheology for polymers for teachers and beginners. Chemistry Teacher International, 4(4), 307–326. https://doi.org/10.1515/cti-2022-0010