Bone Organ‐on‐a‐Chip Uncovers That TPD52L1 Enhances Osteogenic Differentiation of MSCs and Contributes to Osteoporosis Repair

This study aims to develop a novel therapeutic strategy for osteoporosis (OP) by enhancing the osteogenic differentiation potential of mesenchymal stem cells (MSCs). A three-channel biomimetic bone organ-on-a-chip model was constructed. Through bioinformatics analysis, TPD52L1, a key upregulated gene involved in the osteogenic differentiation of MSCs, was identified. Molecular experiments were conducted to verify the effects of its overexpression on the Wnt/β-catenin pathway. Critically, systematic knockdown experiments were performed to validate its necessity in mechanotransduction. Subsequently, functional experiments were performed to evaluate its role in the osteogenic and adipogenic differentiation of MSCs in a GIOP model. Safety assessments for TPD52L1-overexpressing MSCs covered subcellular localization, proliferation, anchorage-independent growth, oncogene expression, and short-term in vivo tumorigenicity. TPD52L1 overexpression activated the Wnt/β-catenin pathway and promoted osteogenic differentiation of MSCs. Its knockdown blocked mechanical stimulation-induced pathway activation and osteogenic marker upregulation, confirming its necessity in mechano-osteogenic conversion. TPD52L1 upregulated osteoblast markers osteocalcin (OCN) and alkaline phosphatase (ALP), downregulated the osteoclast marker tartrate-resistant acid phosphatase (TRAP), and reversed the osteoporosis phenotype. When combined with cyclic mechanical force stimulation in the organ-on-a-chip system, a synergistic effect was observed, enhancing bone repair. TPD52L1 was localized in the cytoplasm, and its endogenous expression was upregulated by mechanical stimulation, indicating mechanosensitivity. TPD52L1 overexpression did not affect MSCs' proliferation, anchorage-independent growth, or oncogene expression. Subcutaneous transplantation experiments confirmed that it did not induce tumor formation or significant pathological changes, demonstrating favorable biosafety. TPD52L1 serves as a key target for promoting the osteogenic differentiation of MSCs.