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Nanoclay (Nanosilicates, NS) is appearing as an intriguing 2D nanomaterial for bone tissue engineering with multiple proposed functions, e.g., intrinsic osteoinductivity, improving mechanical properties, and drug release capacity. However, the mechanism of NS for in vivo bone regeneration has been hardly defined so far. This knowledge gap will significantly affect the design/application of NS-based biomaterials. To determine the role of NS in osteoblastic differentiation and bone formation, we used the mouse calvarial-derived pre-osteoblas