In the present study, we aimed at evaluating the ability of novel PLGA-P188-PLGA-based microspheres to induce the differentiation of mesenchymal stem/stromal cells (MSC) into chondrocytes. To this aim, we tested microspheres releasing TGFβ3 (PAM-T) in vitro and in situ, in a pathological osteoarthritic (OA) environment. We first evaluated the chondrogenic differentiation of human MSCs seeded onto PAM-T in vitro and confirmed the up-regulation of chondrogenic markers while the secretome of the cells was not changed by the 3D environment. We then injected human MSC seeded onto PAM-T in the knee joints of mice with collagenase-induced OA. After 6 weeks, histological analysis revealed that formation of a cartilage-like tissue occurred at the vicinity of PAM-T that was not observed when MSCs were seeded onto PAM. We also noticed that the endogenous articular cartilage was less degraded. The extent of cartilage protection was further analysed by confocal laser microscopy. When MSCs seeded onto PAM-T were injected early after OA induction, protection of cartilage against degradation was evidenced and this effect was associated to a higher survival of MSCs in presence of TGFβ3. This study points to the interest of using MSCs seeded onto PAM for cartilage repair and stimulation of endogenous cartilage regeneration.