Speaker
Description
Introduction
Extrusion-based bioprinting represents a promising alternative to current cell-based approaches in cartilage regeneration. However, a major challenge in the fabrication of cartilage is still to achieve appropriate mechanical properties which are essential for biological functionality. In current biofabrication approaches bioinks are often combined with PCL scaffolds or other synthetic polymers to mimic the mechanical properties of native cartilage [1]. In this study, we utilize a stand-alone bioink consisting of hyaluronic acid (HA) and polyethylene glycol (PEG) derivatives with a dual-stage crosslinking mechanism that has previously been shown, in principle, to enable the deposition of coherent extracellular matrix (ECM) throughout the construct [2]. Here, we aim to analyze the maturation of 3D bioprinted cartilaginous tissues based on chondrogenically differentiated mesenchymal stromal cells (MSC) over a 42-day period. We determine in detail the development of different ECM components as well as the mechanical properties over time.
Methods
Thiolated HA (HA-SH) and PEG derivatives (PEG-DA, PEG-Allyl) were synthesized and characterized using 1H-NMR spectroscopy and aqueous GPC/SEC. MSCs were embedded in the dual-stage crosslinking bioink and differentiated for a 42-day period post-printing. Chondrogenic differentiation was analyzed at multiple time points during the differentiation period and investigated using histology, immunohistochemistry, and wet chemistry-based quantification of ECM components. The mechanical properties were determined over time using multimodal mechanical tests, analyzing the time-matched mechanical response to compression, tension and torsional shear.
Results
Employing our stand-alone HA-SH-based bioink we could prove that the printability and physicochemical properties of constructs immediately after printing were not influenced by incorporation of MSC. During the 42-day period, a marked increase of collagens, glycosaminoglycans, and construct stiffness was observed. Immunohistochemical staining showed that deposition of aggrecan started within the first week of differentiation, with distribution throughout the constructs from day 14 on, while collagen type II showed substantial distribution at day 21. Our data indicated a clear positive correlation between ECM deposition and the mechanical properties of the constructs. This correlation was demonstrated to be valid even when the collagen production was inhibited by ethyl-3,4-dihydroxybenzoate (EDHB). Furthermore, we observed similar mechanical characteristics of our printed cartilaginous tissues compared to human articular cartilage with respect to nonlinearity, hysteresis, conditioning, and stress relaxation behavior.
Discussion
In this study, we could show a strong correlation between ECM development over time and the mechanical response of our bioprinted cartilaginous tissues. While other biofabrication approaches depend on reinforced hydrogels to show an increase in construct stiffness or to achieve printability [1], we observed a marked increase in construct stiffness based on tissue maturation in a stand-alone HA-based bioink. Moreover, we could demonstrate a mechanical behavior with distinct similarities to native articular cartilage.
References
[1] Sbirkov Y et al., Biomedicines. 12(3):665, 2024
[2] Hauptstein J et al., Macromol. Biosci. 22:e2100331, 2022.
Acknowledgements
This research was funded by the German Research Foundation (DFG), Project 326998133, TRR 225 (subprojects A02, B09).
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