Increasing Collagen to Bioink Drives Mesenchymal Stromal Cells-Chondrogenesis from Hyaline to Calcified Layers

The bioextrusion of mesenchymal stromal cells (MSCs) directly seeded in a bioink enables the production of three-dimensional (3D) constructs, promoting their chondrogenic differentiation. Our study aimed to evaluate the effect of different type I collagen concentrations in the bioink on MSCs' chondrogenic differentiation. We printed 3D constructs using an alginate, gelatin, and fibrinogen-based bioink cellularized with MSCs, with four different quantities of type I collagen addition (0.0, 0.5, 1.0, and 5.0 mg per bioink syringe). We assessed the influence of the bioprinting process, the bioink composition, and the growth factor (TGF-ꞵ1) on the MSCs' survival rate. We confirmed the biocompatibility of the process and the bioinks' cytocompatibility. We evaluated the chondrogenic effects of TGF-ꞵ1 and collagen addition on the MSCs' chondrogenic properties through macroscopic observation, shrinking ratio, reverse transcription polymerase chain reaction, glycosaminoglycan synthesis, histology, and type II collagen immunohistochemistry. The bioink containing 0.5 mg of collagen produces the richest hyaline-like extracellular matrix, presenting itself as a promising tool to recreate the superficial layer of hyaline cartilage. The bioink containing 5.0 mg of collagen enhances the synthesis of a calcified matrix, making it a good candidate for mimicking the calcified cartilaginous layer. Type I collagen thus allows the dose-dependent design of specific hyaline cartilage layers.