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

Abstract

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.

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“…Moreover, the structure and the position of focal adhesions are inherently dynamic, which contribute to the reorganization of cell structure [ 167 ]. Positioned in the ECM proteins, Arg-Gly-Asp (RGD) sequences act pivotally in the process, which induce cellular responses through mechantransduction, including integrin signaling, focal adhesion organization, activation of cytoskeleton-associated molecules, calcium signaling and nuclear translocation of mechanosensitive transcriptional regulators [ 99 , 169 , 170 ].…”

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“…The stiffness of the extracellular matrix, a crucial mechanical cue, significantly impacts osteoclast behavior. Wang et al demonstrated that stiffer substrates (∼4 Mpa) upregulate osteoclast-specific markers and enhance bone resorption capabilities [ 81 ] ( Fig. 6 A).…”

Adipogenesis or osteogenesis: destiny decision made by mechanical properties of biomaterials

“…56 On a soft PDMS matrix, the expression of RhoA and ROCK1/2 decreases in chondrocytes, 57 while the activation of cytoskeleton-related adhesion proteins and cascades of paxillin, FAK, PKC, and RhoA were detected in osteoclasts on the stiffer PDMS substrates. 58 RhoA/ROCK can not only respond to matrix stiffness but also act as intermediary molecules to mediate the cellular localization of YAP/TAZ. MSCs cultured in big fibronectin pattern have been found to entail the activation of the RhoA/ROCK signaling pathway and stimulate the formation of actin bundles, stress fibres and tensile actomyosin structures, which act as signals of the nuclear translocation of YAP/TAZ.…”