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  2. Three-Dimensional Matrix Stiffness Activates the Piezo1-AMPK-Autophagy Axis to Regulate the Cellular Osteogenic Differentiation

Three-Dimensional Matrix Stiffness Activates the Piezo1-AMPK-Autophagy Axis to Regulate the Cellular Osteogenic Differentiation

  • ACS Biomater Sci Eng. 2023 Jul 10. doi: 10.1021/acsbiomaterials.3c00419.
Yanqiu Wu 1 2 3 Xinxin Xu 1 2 3 Fengyi Liu 1 2 3 Zheng Jing 1 2 3 Danfeng Shen 1 2 3 Ping He 1 2 3 Tao Chen 1 2 3 Tianli Wu 1 2 3 Hengji Jia 1 2 3 Dingqiang Mo 1 2 3 Yuzhou Li 1 2 3 He Zhang 1 2 3 Sheng Yang 1 2 3
Affiliations

Affiliations

  • 1 College of Stomatology, Chongqing Medical University, 426 Songshibei Road, Yubei District, Chongqing 401147, China.
  • 2 Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing 400016, China.
  • 3 Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing 400016, China.
Abstract

Extracellular matrix (ECM) stiffness is a key stimulus affecting cellular differentiation, and osteoblasts are also in a three-dimensional (3D) stiff environment during the formation of bone tissues. However, it remains unclear how cells perceive matrix mechanical stiffness stimuli and translate them into intracellular signals to affect differentiation. Here, for the first time, we constructed a 3D culture environment by GelMA hydrogels with different amino substitution degrees and found that Piezo1 expression was significantly stimulated by the stiff matrix with high substitution; meanwhile, the expressions of osteogenic markers OSX, RUNX2, and ALP were also observably improved. Moreover, knockdown of Piezo1 in the stiff matrix revealed significant reduction of the abovementioned osteogenic markers. In addition, in this 3D biomimetic ECM, we also observed that Piezo1 can be activated by the static mechanical conditions of the stiff matrix, leading to the increase of the intracellular calcium content and accompanied with a continuous change in cellular energy levels as ATP was consumed during cellular differentiation. More surprisingly, we found that in the 3D stiff matrix, intracellular calcium as a second messenger promoted the activation of the AMP-activated protein kinase (AMPK) and unc-51-like autophagy-activated kinase 1 (ULK1) axis and modestly modulated the level of Autophagy, bringing it more similar to differentiated osteoblasts, with increased ATP energy metabolism consumption. Our study innovatively clarifies the regulatory role of the mechanosensitive ion channel Piezo1 in a static mechanical environment on cellular differentiation and verifies the activation of the AMPK-ULK1 axis in the cellular ATP energy metabolism and Autophagy level. Collectively, our research develops the understanding of the interaction mechanisms of biomimetic extracellular matrix biomaterials and cells from a novel perspective and provides a theoretical basis for bone regeneration biomaterials design and application.

Keywords

3D cell culture; AMPK; Piezo1; autophagy; extracellular matrix.

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