Hyaline articular cartilage is a non-innervated, non-vascularized stratified tissue with limited repair capacity. Thanks to 3D printing, a specific layer-by-layer reconstruction of cartilage is possible by formulating bio-inks dedicated to the different specific layers of cartilage. We therefore assessed the effect of type I collagen and hyaluronic acid supplementation on the chondrogenic properties of human bone marrow-derived mesenchymal stem cells. Collagen is known for its chondrogenic properties, and combined with alginate, it improves cell adhesion to the support and the expression of chondrogenic markers. Hyaluronic acid is a component of cartilage ECM. It also has interesting chondro-inductive properties. For this purpose, after expansion, MSCs were suspended in an alginate-based bio-ink containing different amounts of soluble collagen I: 0, 0.5, 1 and 5 mg/8mL of bio-ink or in a bio-ink containing or not 16 mg/8mL of hyaluronic acid. The substitutes were produced using a 3D bioextrusion printing system (4 successive layers) and then polymerized. They were then cultured in control or TFG-β1-enriched medium to induce chondrogenesis for 28 and 56 days, at the end of which cell viability, gene expression and matrix synthesis were assessed. No toxicity was observed, whatever the ink or culture conditions. At D28, gene expressions show chondrogenic differentiation of MSCs under the effect of TFG-β1. Low levels of added collagen (0.5 mg) promote the expression of chondrogenic markers, while the high level of 5 mg induces a more osteogenic profile. In substitutes containing hyaluronic acid, we also noted a significant increase in the expression of chondrogenic markers at D56. Histological and immunohistological analyses confirmed the chondrogenic effect of TFG-β1, with type II collagen synthesis potentiated by the lowest collagen quantity (0.5 mg). In contrast, the 5 mg amount of collagen induced more calcifications within the substitutes. However, we found no significant difference in matrix synthesis between bio-inks with and without hyaluronic acid, even though early whitening was observed, associated with superior substitute strength in the presence of hyaluronic acid. Type I collagen therefore offers promising opportunities for reconstructing stratified articular cartilage. The lower amount of collagen added induces a more hyaline matrix synthesis, apt to recreate superficial cartilage layers, rich in type II collagen, while the 5 mg amount induces the production of a more calcified matrix. As hyaluronic acid is known to enhance the biomechanical properties of substitutes, it could enable the reproduction of the deep layer responsible for the compressive strength of articular cartilage. Acknowledgements: This work was supported by « la Direction Générale des Armées (N°ANR-16-ASTR-0021) », « la Fondation de l’Avenir » (N°AP-RM-16-042) » and « la Fondation pour la Recherche Médicale » (N° ECO201906008942).