Multilayered extracellular matrix–derived scaffolds direct progenitor cell differentiation in vitro and osteochondral-tissue formation in vivo

Osteochondral repair requires restoration of zonally organised articular cartilage and subchondral bone, yet translatable implants rarely reproduce this spatial complexity. Here, we developed an acellular multilayer scaffold comprising a superficial 2% (w/v) articular cartilage extracellular matrix (AC-ECM) phase, an intermediate 5% AC-ECM phase and a basal 6% bone ECM (BN-ECM) phase. The scaffold formed continuous interfaces, displayed region-dependent pore architecture and showed limited residual deformation after compression. In vitro, constructs seeded with caprine mesenchymal stromal and articular cartilage progenitor cells supported cell expansion and the accumulation of sulfated glycosaminoglycan- and collagen-rich matrix, with regional differences in collagen I, II and X immunoreactivity. Following eight weeks of subcutaneous implantation, cell-seeded scaffolds contained more collagenous matrix than unseeded controls, while vascularisation preferentially localised to the BN-ECM phase. In a six-month caprine osteochondral defect model, scaffold treatment significantly improved macroscopic and histological repair and increased PTA-attenuating, collagen-rich repair-tissue fill within the chondral region (∼60% versus ∼40%). It also limited extension of this tissue into the subchondral region and produced superficial collagen fibres more closely aligned parallel to the articular surface. Repair tissue exhibited greater collagen II immunoreactivity, increased ACAN and COL2A1 expression and reduced COL10A1 expression, whereas deeper mineralised tissue formation was not significantly improved. These findings demonstrate that this acellular multilayer ECM scaffold improves the cartilage component of osteochondral repair without exogenous cells or growth factors and identify subchondral bone regeneration as the principal remaining design challenge.