Accelerating bone healing in femoral defect model using FBXO6-modified bone marrow-derived mesenchymal stem cells on a collagen scaffold.

Bone repair following large defects remains a significant clinical challenge due to limited osteogenic capacity. F-box only protein 6 (FBXO6), an E3 ubiquitin ligase known to promote degradation of downstream proteins, was highly expressed after osteogenic differentiation. Herein, the role of FBXO6 in osteoblastic differentiation and bone remodeling was explored. Rat bone marrow mesenchymal stem cells (BMSCs) were first isolated and characterized. Following 14 days of osteogenic induction, FBXO6 was significantly upregulated (approximately 4-fold increase). BMSCs overexpressing FBXO6 demonstrated enhanced osteogenic potential, evidenced by increased mRNA expression of osteogenic markers (runt-related transcription factor 2, osteocalcin, COL1A1), elevated alkaline phosphatase (ALP) activity, and greater formation of calcium nodules. Conversely, FBXO6-silenced BMSCs exhibited the opposite effects. FBXO6 overexpression activated the Wnt/β-catenin signaling pathway, a known mediator of osteogenic differentiation. This effect was reversed by treatment with the Wnt/β-catenin inhibitor DKK1. Furthermore, β-catenin overexpression rescued the impaired osteogenesis caused by FBXO6 silencing. Label-Free Quantitative Proteomics analysis identified 439 differentially expressed proteins in FBXO6-overexpressing cells (245 upregulated, 194 downregulated). STARD3 N-terminal like protein (STARD3NL) was prioritized for further investigation (log2FC = -0.97, p = 0.02). FBXO6 interacted with STARD3NL and promoted its destabilization. STARD3NL knockdown attenuated the effects of FBXO6 silencing on osteogenesis. In a 3-mm-diameter critical-size femoral defect model, implantation of collagen scaffolds seeded with FBXO6-downregulated BMSCs significantly suppressed osteogenesis. These findings demonstrate that FBXO6-modified BMSC implantation represents a promising therapeutic strategy for bone defect repair.