Immobilization after injury alters extracellular matrix and stem cell fate
Amanda K. Huber, Nicole Patel, Chase A. Pagani, Simone Marini, Karthik R. Padmanabhan, Daniel L. Matera, Mohamed Said, Charles Hwang, Ginny Ching-Yun Hsu, Andrea A. Poli, Amy L. Strong, Noelle D. Visser, Joseph A. Greenstein, Reagan Nelson, Shuli Li, Michael T. Longaker, Yi Tang, Stephen J. Weiss, Brendon M. Baker, Aaron W. James, Benjamin Levi
Amanda K. Huber, Nicole Patel, Chase A. Pagani, Simone Marini, Karthik R. Padmanabhan, Daniel L. Matera, Mohamed Said, Charles Hwang, Ginny Ching-Yun Hsu, Andrea A. Poli, Amy L. Strong, Noelle D. Visser, Joseph A. Greenstein, Reagan Nelson, Shuli Li, Michael T. Longaker, Yi Tang, Stephen J. Weiss, Brendon M. Baker, Aaron W. James, Benjamin Levi
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Research Article Bone biology

Immobilization after injury alters extracellular matrix and stem cell fate

Abstract

Cells sense the extracellular environment and mechanical stimuli and translate these signals into intracellular responses through mechanotransduction, which alters cell maintenance, proliferation, and differentiation. Here we use a mouse model of trauma-induced heterotopic ossification (HO) to examine how cell-extrinsic forces impact mesenchymal progenitor cell (MPC) fate. After injury, single-cell (sc) RNA sequencing of the injury site reveals an early increase in MPC genes associated with pathways of cell adhesion and ECM-receptor interactions, and MPC trajectories to cartilage and bone. Immunostaining uncovers active mechanotransduction after injury with increased focal adhesion kinase signaling and nuclear translocation of transcriptional coactivator TAZ, inhibition of which mitigates HO. Similarly, joint immobilization decreases mechanotransductive signaling, and completely inhibits HO. Joint immobilization decreases collagen alignment and increases adipogenesis. Further, scRNA sequencing of the HO site after injury with or without immobilization identifies gene signatures in mobile MPCs correlating with osteogenesis, and signatures from immobile MPCs with adipogenesis. scATAC-seq in these same MPCs confirm that in mobile MPCs, chromatin regions around osteogenic genes are open, whereas in immobile MPCs, regions around adipogenic genes are open. Together these data suggest that joint immobilization after injury results in decreased ECM alignment, altered MPC mechanotransduction, and changes in genomic architecture favoring adipogenesis over osteogenesis, resulting in decreased formation of HO.

Authors

Amanda K. Huber, Nicole Patel, Chase A. Pagani, Simone Marini, Karthik R. Padmanabhan, Daniel L. Matera, Mohamed Said, Charles Hwang, Ginny Ching-Yun Hsu, Andrea A. Poli, Amy L. Strong, Noelle D. Visser, Joseph A. Greenstein, Reagan Nelson, Shuli Li, Michael T. Longaker, Yi Tang, Stephen J. Weiss, Brendon M. Baker, Aaron W. James, Benjamin Levi

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Figure 2

MPCs at the extremity injury site demonstrate increased mechanotransductive signaling before aberrant cell fate change.

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MPCs at the extremity injury site demonstrate increased mechanotransduct...
(A) Confocal microscopy images of injured and uninjured mouse hind limbs immunologically stained with anti-PDGFRα and anti-FAK, anti-pFAK, or anti-TAZ after 1 week BT injury compared with uninjured control. Nuclei are stained with Hoechst 33342. Tilescan images (left) of HO anlagen with tendon encircled by white dotted outline and red dotted square showing ×20 image (middle). Image overlay at ×20 magnification with individual channels (right). Blue-dotted square shows ×63 magnification. Image overlay at ×63 magnification with individual channels (right). Image overlay at ×20 magnification of uninjured mouse hind limb with individual channels (right). Quantification of ×63 magnification comparing number of PDGFRα+ cells expressing FAK, pFAK, and nuclear TAZ, respectively in injured and uninjured hind limbs by independent samples t test (n = 3/group, ***P < 0.001). (B) FAK, pFAK, and TAZ immunofluorescent stains at 3 weeks postinjury (n = 3–4/group) of injured and uninjured mouse hind limbs immunologically stained with anti-PDGFRα and anti-FAK, anti-pFAK, or anti-TAZ. Scale bars: 100 µm. ###P < 0.001, ****P < 0.0001. (C) Immunohistochemical stains of FAK, pFAK, and PDGFRα of human uninjured bone and HO. Original magnifications, left to right: ×40, ×20, ×40, and ×20.