Mechanistic Insights Into Atg7-Bmsc@cs-Mediated Mitochondrial Autophagy and Its Role in Metabolic Reprogramming to Enhance Osteogenesis

ATG7 encodes a protein that regulates the transport and activation of autophagy-related proteins, helping to remove damaged or aging components, and is essential for bone regeneration. Mesenchymal stem cells(MSCs) are one of the most promising seed cells in bone tissue engineering. This study aims to investigate the role and mechanism of ATG7 in bone formation and bone defects using ATG7 conditional knockout (ATG7cKO) mice in conjunction with stem cell modification and chitosan hydrogels. Herein, we observed that compared to littermate control ATG7f/f DMP Cre-negative mice, ATG7 cKO mice exhibited significantly reduced body length and weight, decreased average femur and tibia lengths, reduced cortical thickness and bone diameter, significantly lower bone density, reduced trabecular number, and increased trabecular spacing in the femur. Meanwhile, overexpression of ATG7 via adenoviral infection in BMSCs significantly upregulated osteogenic markers ALP, OPN, OCN, DMP1, and significantly promoted osteogenic differentiation of BMSCs. Then the thermosensitive hydrogel loaded with Atg7-BMSCs, Atg7-BMSC@CS were constructed, and Atg7-BMSC@CS significantly enhanced repair of the cranial and femoral defects compared to the control group. Primary osteoblasts isolated from Atg7 cKO and Atg7 control mice were subjected to high-throughput RNA sequencing (RNA-seq) analysis. Among the 856 significantly differentially expressed genes, Gene Ontology (GO), KEGG, and Gene Set Enrichment Analysis (GSEA) revealed that the differentially expressed genes in ATG7-deficient osteoblasts were highly enriched in categories related to mitochondrial oxidative phosphorylation, glycolysis, and mitophagy. Observations of mitochondrial ultrastructure showed an increase in mitochondria with abnormal cristae in ATG7-deficient osteoblasts. TMRM and ROS assays indicated a decrease in mitochondrial membrane potential and enhanced oxidative stress in ATG7-deficient osteoblasts, and mRNA expression of energy metabolism-related marker genes, including GLUT-3, GLUT-4, CYTB, CYC1, Ndufs-1, Ndufs-4, Ndufs-5, Ndufs-6, was significantly downregulated in ATG7-deficient osteoblasts. Additionally, overexpression of ATG7 via adenovirus infection in BMSCs activates the expression of mitophagy marker genes P62, Pink1, Parkin, and LC3 I/II. When treated with the mitophagy inhibitor Mdivi-1, ATG7-activated mitophagy was significantly suppressed, along with the downregulation of osteogenic marker genes ALP, OPN, OCN, and DMP1. ALP and ARS staining results demonstrated that Mdivi-1 treatment could significantly inhibit the osteogenic differentiation effect of ATG7 on BMSCs. In conclusion, Atg7-BMSC@CS promotes osteogenesis and bone repair through mitochondrial energy metabolism reprogramming mediated by mitophagy.