Optimizing Collagen Bioprinting for Mechanically Strong and Resilient Tissues

As the main structural protein found in connective tissues, type I collagen constitutes approximately 70-80% of the dry weight in ligaments and tendons.^[1] Ligaments and tendons coordinate and stabilize our body’s movement, but damage to these soft tissues accounts for 30% of musculoskeletal clinical cases every year.^[2] While athletes who play contact sports are most commonly prone to sprains or ruptures of the anterior cruciate ligament (ACL), people can experience ligament and tendon injuries through falls, twists, or overuse. Traditional surgical interventions involve graft replacement from elsewhere in the patient’s body, which often requires months of recovery and fails to achieve enthesis healing or true regenerative capabilities. Promisingly, the development of synthetic biomaterials that mimic the native hierarchical structure of ligaments/tendons can facilitate tissue repair.^[3]<br/>The overarching goal of our work is to elucidate the relationship between printing parameters and mechanical behavior of collagen bioinks to fabricate tunable scaffolds for biocompatible implants. With an eye towards translation and ease-of-use, we utilize the commercially-available CellInk BioX bioprinter and two different concentration collagen bioinks to control the infill density and heating duration for extruded 5x5x3mm³ cubes. Scanning electron microscopy (SEM) is first used to visualize the collagen bioink’s surface morphology, and uniaxial compressive loading is performed post-crosslinking to evaluate the material’s strength and stiffness. Secondly, we aim to create a stiffness gradient by varying incubation time across layer height so that the printed collagen can achieve enhanced integration as a connective tissue. Preliminary results indicate that the higher collagen concentration (70mg/mL) hydrogels require at least 35 minutes of heating time for sufficient crosslinking compared to the recommended 30 minutes for the low collagen concentration (35 mg/mL) samples. Meanwhile, longer heating durations have a greater effect on producing higher strength and stiffness prints compared to changes in infill density alone. Ultimately, optimizing the printing parameters that determine resultant mechanical behavior of collagen bioinks will inform customizable, on-demand printing of ligaments and tendons for regenerative medicine.<br/><br/><br/>[1] Hudson DM, Archer M, Rai J, Weis M, Fernandes RJ, Eyre DR. Age-related type I collagen modifications reveal tissue-defining differences between ligament and tendon. <i>Matrix Biology Plus</i>. 2021;12:1-15. doi:10.1016/j.mbplus.2021.100070<br/>[2] Lim WL, Liau LL, Ng MH, Chowdhury SR, Law JX. Current progress in tendon and ligament tissue engineering. <i>Tissue Eng Regen Med</i>. 2019;16(6):549-571. doi:10.1007/s13770-019-00196-w<br/>[3] Lei T, Zhang T, Ju W, et al. Biomimetic strategies for tendon/ligament-to-bone interface regeneration. <i>Bioactive Materials</i>. 2021;6(8):2491-2510. doi:10.1016/j.bioactmat.2021.01.022<br/>[4] Jin Z, Hu G, Zhang Z, Yu SY, Gu GX. Modeling and analysis of post-processing conditions on 4D-bioprinting of deformable hydrogel-based biomaterial inks. <i>Bioprinting</i>. 2023;33(e00286):1-9. doi:10.1016/j.bprint.2023.e00286