Résumé
Extrusion-based 3D bioprinting is a promising technique to produce complex, three-dimensional (3D) structures that mimic the native extracellular matrix (ECM), which is pivotal for developing advanced tissue engineering solutions. Composite hydrogels, which are combinations of two or more distinct materials, can be used as a printing medium that offers several advantages over a single biomaterial due to their tunability, including improved mechanical properties and biocompatibility. These materials can be engineered to have specific physical, chemical, or biological properties, making them useful for a variety of applications. Extrusion-based 3D bioprinting of these hydrogels has generated high expectancy for joint tissue engineering, in particular for cartilage and bone repair. Here, we report the development of a 3D bioprinting process for the generation of biphasic 3D constructs for osteochondral repair of full-thickness joint lesions. The bioink used in this study is based on a natural composite hydrogel developed at 3d.FAB Platform (Villeurbanne, France) comprising of gelatin (5%), alginate (2%), and fibrin (2%) loaded with murine mesenchymal stromal cells (mMSCs) expressing the differentiation factor BMP-2 under inducible conditions – a factor critically involved in both chondrogenesis and osteogenesis. Following printing using the BIO X 3D printer (CELLINK, Sweden) and crosslinking with a solution of transglutaminiase/CaCl2/thrombin, the 3D bioprinted constructs were cultured individually under proliferation, chondrogenic or osteogenic conditions for a period of 28 days in order to optimize the culture conditions for enhanced cell differentiation. Our findings showed: (i) high cell viability (>90%) of mMSCs across all conditions using the Live/Dead assay; (ii) sustained cell proliferation using the PrestoBlue Proliferation Assay; and (iii) cell differentiation towards either the chondrogenic or osteogenic lineages as shown by the up-regulation of specific differentiation markers by RT-qPCR. Cell differentiation was further analyzed at the protein level using immunofluorescence labelling: type II collagen and aggrecan for cartilage; osteocalcin for subchondral bone. Finally, a biphasic construct comprising a cartilage compartment and a subchondral bone compartment was printed in an “all-in-one” strategy and analyzed by immunofluorescence following different culture conditions. It showed the feasibility of printing complex cell-laden 3D constructs with different composition along the depth. Overall, although some challenges remain to be overcome, 3D bioprinting holds great promise for the development of improved strategies for joint tissue engineering by transforming the treatment of joint injuries and diseases in the future.