Osteoarthritis is a significant socioeconomic illness that mainly affects the articular cartilage, a tissue with a low capacity for self-healing, making it an ideal target for regenerative medicine and tissue engineering. Current interventions to treat cartilage injuries may not be completely effective. In this study, we have developed a novel bioreactor that creates viscous shear stress by flow perfusion. This bioreactor could induce ex vivo maturation of biomimetic 3D cartilage scaffolds, providing a potential solution to this problem. Infrapatellar fat pad mesenchymal stem cells (IPFP-MSCs) were used as a cellular source of the functionalized 3D scaffolds made of 1,4-butanediol thermoplastic polyurethane (bTPUe) modified with pyrene butyric acid (PBA). Our results indicate that our bioreactor induced chondrogenic differentiation, as confirmed by DNA quantification, extracellular matrix determination, and metabolic assay, without any conditioned medium. To control the biomechanical stimulation on IPFP-MSCs, a low-intensity ultrasonic transmission system has been developed and embedded in the bioreactor. Combined with a finite element model (FEM), tissue growth and differentiation can be deconvoluted in real-time from the recorded ultrasonic propagation and interaction across the graft. The FEM reconstructs this complex interaction. This is the first time a low-shear stress-based bioreactor has been reported to not only induce chondrogenic evolution but also monitor it in real time.