Abstract
To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 ± 2.80 μm, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting.
Highlights
Articular cartilage, the connective tissue lining the articular surface of bones within diarthrodial joints, ensures load support, load transmission, and joint lubrication
Spheroids were cultured in a serum-free chondrogenic culture medium comprised of Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Life Technologies), 1 mM sodium pyruvate (Life technologies), 0.5% (v/v) ITS (Sigma-Aldrich), 10 U/mL penicillin, 10 μg/mL streptomycin, 100 μM dexamethasone (Sigma-Aldrich), 200 μM L-ascorbic acid-2-phosphate (Sigma-Aldrich), and 10 ng/mL TGF-β1 (Peprotech), in a low oxygen tension (5% O2) incubator at 37◦C
After seeding of the hBM-mesenchymal stem cells (MSC) suspension on the microwells, cells lowered into the bottom of the pores by gravitational force within 1–2 h and cells were distributed over the entire surface of the pore
Summary
The connective tissue lining the articular surface of bones within diarthrodial joints, ensures load support, load transmission, and joint lubrication. Articular cartilage defects are already being treated with cell-based regenerative therapies, such as autologous chondrocyte implantation (ACI) This two-step surgical procedure includes the isolation of autologous articular chondrocytes from a non-weight bearing region, the in vitro expansion and the reinjection into the defect site (Davies and Kuiper, 2019). The harvesting procedure to obtain chondrocytes can induce donor-site morbidity and the 2D expansion of articular chondrocytes is characterized by long in vitro culture periods and chondrocyte dedifferentiation to a more fibroblast-like phenotype featuring a decrease in collagen II, aggrecan, and glycosaminoglycans (GAG) (Caron et al, 2012; De Moor et al, 2019) This results in the generation of repair tissue which is biochemically and biomechanically inferior compared to the native cartilage (Knutsen et al, 2007). New biofabrication strategies are being explored in the quest for novel regenerative therapies
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