Biomimetic ECM collagen hydrogel induces chondrogenic differentiation of BMSCs by activating autophagy through POU5F1-mediated AMPK/mTOR signaling pathway.

Background: As a main component of the tissue microenvironment, the extracellular matrix (ECM) provides an instructive niche that regulates stem cell differentiation. Biomimetic ECM collagen hydrogels have been well regarded as ideal scaffold for cartilage tissue engineering due to their ability to promote chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the molecular mechanisms underlying this inductive effect remain incompletely elucidated. Methods: In the present study, mRNA microarray analysis was performed to investigate the molecular mechanisms involved in collagen hydrogel-mediated chondrogenic induction. Real-time quantitative polymerase chain reaction (RT-qPCR), western blot, immunofluorescence analyses, etc. were used to validate the relevant pathways. Results: The results demonstrated that POU class 5 homeobox 1 (POU5F1), a transcription factor associated with stem cell differentiation and autophagy, was highly expressed in cells induced by collagen hydrogel. Differentially expressed genes (DEGs) were predominantly enriched in the AMP-activated protein kinase (AMPK) / mammalian target of rapamycin (mTOR) signaling pathway. Knockdown of POU5F1 suppressed activation of the AMPK/mTOR pathway and subsequently reduced intracellular autophagic flux, leading to impaired cartilage regeneration. These effects were partially reversed by treatment with rapamycin (RaPa), an mTOR inhibitor. Conclusions: The findings highlight a critical role of autophagy in chondrogenic induction mediated by biomimetic ECM collagen hydrogel and provide mechanistic insight that may inform the rational design optimization of cartilage repair biomaterials.