Speaker
Description
Introduction
3D-bioprinting for cartilage tissue engineering can provide controlled cell localization and complex tissue shapes fabrication to meet clinical needs. Extrusion-based 3D-bioprinting has demonstrated resolution limitations owing to surface tension effects. Therefore, confined printing techniques such as Freeform Reversible Embedding of Suspended Hydrogels (FRESH) have been explored to improve the manufacturing resolution. Common FRESH techniques rely on a gelatin bath and require separate steps of crosslinking or post-processing. To further explore the use of supported bioprinting setups and include in situ crosslinking, here we explore the use of pluronic and CaCl2-based support baths to 3D-bioprint alginate-based constructs for cartilage tissue engineering applications.
Methods
Hydrogel mixes were explored as bioinks; composed of alginate, gelatin and methylcellulose. Firstly, acellular bioink bioprinting optimisation was performed in unsupported and supported setups by assessing the filament resolution.
Secondly, HMSC-based spheroids (1000-1200 cells/spheroid), were embedded in the bioink at various concentrations (1000, 5000, 10000 spheroids/mL) following previous literature1 and 3D-bioprinted using a support bath setup. These constructs were cultured in chondrogenic differentiation medium for 21 days. Post-bioprinting spheroid viability was assessed through LIVE/DEAD staining and chondrogenic differentiation was evaluated through immunofluorescence staining of specific markers (collagen type-I, collagen type-II, SOX-9, RUNX2, collagen type-X) as well as histological assessment (alcian blue and alizarin red).
Results
The explored FRESH-like bioprinting set up, composed of a pluronic and CaCl2 bath, demonstrated an increase in filament resolution of up to 35% (1600 µm vs 600 µm fiber diameter using 22G needle). Furthermore, the in situ crosslinking of the bioprinted constructs was demonstrated by obtaining stable constructs post-bath dissolution without the need of an additional crosslinking step.
Spheroid-based bioprinting of constructs showed bioink stability across the 21 days of chondrogenic differentiation. Furthermore, spheroid viability post-printing was observed to be high, showing a majority of alive cells and minimal cell death on the spheroid surface. Lower spheroid concentrations in the bioink demonstrated a lower spheroid fusion. The highest concentration of printed spheroids was observed to have high levels of spheroid fusion and matrix production.
Immunofluorescence staining of the differentiated spheroids showed the production of collagen type-II around the spheroids and minimal collagen type-I. SOX-9 expression was still observed after 21 days of differentiation with no expression of RUNX2 or collagen type-X. Alcian blue staining showed the high level of glycosaminoglycan production around the spheroids with no calcium deposits visible through alizarin red staining.
Discussion
Acellular bioprinting optimisation demonstrated an improvement of the bioprinting resolution and a quicker manufacturing process. This opens the door to further exploring pluronic based support baths for 3D-bioprinting applications. Moreover, the inclusion of spheroids in the bioprinting process showed the possibility of using these bioprinting support set ups and the subsequent potential to differentiate them into chondrogenic lineage. Future experiments should focus on increasing the number of spheroids and assess the effect this has on cartilage tissue production.
References
Gabriela S. Kronemberger, Francesca D. Spagnuolo, Aliaa S. Karam, Kaoutar Chattahy, Kyle J. Storey, and Daniel J. Kelly ACS Biomaterials Science & Engineering 2024 10 (10), 6441-6450 DOI: 10.1021/acsbiomaterials.4c00819
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