Substrate stiffness regulates the differentiation profile and functions of osteoclasts via cytoskeletal arrangement

Objectives Aging and common diseases alter the stiffness of bone tissue, causing changes to the microenvironment of the mechanosensitive bone cells. Osteoclasts, the sole bone‐resorbing cells, play a vital role in bone remodeling. This study was performed to elucidate the mechanism through which osteoclasts sense and react to substrate stiffness signals. Materials and methods We fabricated polydimethylsiloxane (PDMS) substrates of different stiffness degrees for osteoclast formation progressed from osteoclast precursors including bone marrow‐derived macrophages (BMMs) and RAW264.7 monocytes. Osteoclast differentiation in response to the stiffness signals was determined by examining the cell morphology, fusion/fission activities, transcriptional profile, and resorption function. Cytoskeletal changes and mechanosensitive adhesion molecules were also assessed. Results Stiffer PDMS substrates accelerated osteoclast differentiation, firstly observed by variations in their morphology and fusion/fission activities. Upregulation of canonical osteoclast markers (Nfatc1, Acp5, Ctsk, Camk2a, Mmp9, Rela, and Traf6) and the fusion master regulator DC‐stamp were detected on stiffer substrates, with similar increases in their bone resorption functions. Additionally, the activation of cytoskeleton‐associated adhesion molecules, including fibronectin and integrin αvβ3, followed by biochemical signaling cascades of paxillin, FAK, PKC, and RhoA, was detected on the stiffer substrates. Conclusions This is the first study to provide evidence proving that extracellular substrate stiffness is a strong determinant of osteoclast differentiation and functions. Higher stiffness upregulated the differentiation profile and activity of osteoclasts, revealing the mechanical regulation of osteoclast activity in bone homeostasis and diseases.