Laminin expression profiles of osteogenic-and chondrogenic-induced dECM sheets

Decellularized extracellular matrix sheets (dECMSs) produced by stem cells have attracted attention because they preserve the natural biological activity of the ECM to direct lineage-specific differentiation with less immunogenicity. As a core ECM protein, laminin modulates cellular phenotype and differentiation. Nevertheless, no studies thus far have explored the distribution and abundance of laminins in diverse dECMSs. Herein, we first compared the differential expression of laminins among dECMSs in osteogenic-induced medium (OI-dECMS), chondrogenic-induced medium (CI-dECMS), and standard medium (dECMS), employing a defined mass spectrometry (MS)-based proteomic analysis. In vitro, dECMSs were verified to be successfully decellularized. Cluster analysis identified a marked fluctuation in the expression of 7 laminins and 17 laminin-associated proteins in OI-dECMS vs dECMS and CI-dECMS vs dECMS. Two significantly changed pathways were selected from the KEGG pathway enrichment analysis: the FAK/ERK pathway and the PI3K/AKT pathway. Moreover, Alkaline Phosphatase (ALP) activity, Alcian blue staining, and RT-qPCR results for recellularization showed that CI-dECMS promotes chondrogenesis while OI-dECMS inhibits osteogenesis compared with dECMS. In vivo experiments were conducted to implant dECMSs in a rat osteochondral defect, demonstrating that dECMS and CI-dECMS promoted bone and cartilage repair. Furthermore, the inhibitory analysis was performed to verify the function of specific laminin isoforms modulating osteogenesis and chondrogenesis, which might be related to FAK/ERK and PI3K/AKT pathways. In summary, this study constructed dECMS, OI-dECMS, and CI-dECMS and uncovered the internal comprehensive molecular regulatory network centralized by laminins, thus proposing a biomimetic substitute for bone and cartilage regeneration.