Bioinspired scaffold recapitulating chondrogenic ontogeny and microenvironment for functional cartilage regeneration

Focal articular cartilage defects often progress to osteoarthritis, imposing a substantial global health burden. Current neglect of cartilage developmental regulation and cartilage microenvironment compromises therapeutic efficacy. We developed an innovation CE-SKP/CPH/P2G3 scaffold which effectively repairs focal cartilage defects and emulates native cartilage ontogeny: the superficial CE-SKP hydrogel layer recruits SMSCs and promotes chondrogenesis; the middle CPH hydrogel layer induces chondrocyte hypertrophic calcification, forming cartilage calcified layer; and the basal P2G3 nanofiber membrane isolates subchondral cells, enforcing a top-down developmental sequence and preserving a localized hypoxic niche. <i>In vitro</i> characterization confirms that the porosity, swelling ratio, biodegradation rate, and biocompatibility are optimal for sequential SMSC recruitment, cartilage differentiation, hypertrophic mineralization, and cells isolation. <i>In vivo</i>, the biomimetic tri-layer scaffold promotes regeneration of both cartilage and calcified cartilage by recapitulating the native ontogenetic progression from cartilage to calcified cartilage within the <i>in vivo</i> microenvironment, successfully restoring the normal physiological structure of articular cartilage by 24 weeks post-implantation. ScRNA-seq revealed SMSCs and a novel chondrocyte subpopulation CHON_5 as key repair populations, SMSCs mediated early repair via hypoxia response and migration, while CHON_5 promoted ECM remodeling, synergistically enhancing regeneration in late repair stage. Furthermore, we identified FGF signaling (FGF2-FGFR1/2 and FGF18-FGFR1/2 pairs) was crucial for MSC-CHON_5 communication during sequential cartilage regeneration. Overall, by recapitulating native developmental dynamics and microenvironmental cues, this scaffold offers a novel and effective strategy for functional cartilage regeneration and osteoarthritis treatment.