Evaluation of MSC‐Secretome Effects in an Ex Vivo Compartmentalized Osteochondral Interface Model

Osteoarthritis (OA) represents a significant challenge in both orthopedic research and clinical practice, necessitating the development of effective therapeutic strategies. Here, we describe an ex vivo model based on osteochondral (OCh) explants housed in a three‐dimensional printed device that enables the separation of bone and cartilage compartments. Our model demonstrates effective partitioning, as confirmed by significant differences in measurements of tissue‐specific markers. The markers included matrix metalloproteinase (MMP) activity and sulfated glycosaminoglycan (sGAG) release for cartilage and alkaline phosphatase (ALP) activity, tartrate‐resistant acid phosphatase (TRAP) activity, and osteocalcin (OC) levels for bone. The cartilage compartment of OCh explants was exposed to inflammatory stimuli, to mimic the OA‐related microenvironment, using 10 ng/mL TNFα and 1 ng/mL IL‐1β. Cytokine administration was coupled with secretome (or conditioned medium, CM) treatment obtained from 5 × 10 5 naïve or cytokine‐primed adipose‐derived mesenchymal cells (CM and pCM). After 3 days, inflammatory cytokines induced a significant upregulation of MMP activity, effectively countered by both CM and pCM, alongside a modest increase in sGAG release. No major changes were detected in the bone counterpart. This study holds dual significance: firstly, the development and preliminary assessment of a human‐based ex vivo model in accordance with 3Rs (Replacement, Reduction, Refinement) principles in preclinical research; secondarily, the evidence of an anti‐catabolic potential of the adipose‐derived mesenchymal cell secretome contributes, within a broader research context, to hypothesizing its potentiality in counteracting OA‐associated hallmarks, with possible applications at early onset to mitigate the degenerative processes of this pathology. Trial Registration: ClinicalTrials.gov identifier: NCT04223622