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 6

Immobilization alters the extracellular environment affecting MPCs.

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Immobilization alters the extracellular environment affecting MPCs. (A) ...
(A) Elastic modulus at site of HO formation immobilized and mobilized mice 1 week after B/T as determined by atomic force microscopy. ####P < 0.0001. (B) Second harmonic generation of collagen fibrils at 1 week after injury with anisotropy quantification (right) (n = 2/group, **P < 0.01). (C) Confocal microscopy images of hind limb cross sections 1 week after injury with indicated antibodies and quantified for cell spread and area (right) (n = 3/group, ####P < 0.0001). (D) Western blots for pSMAD2, SMAD2, PPARγ, and GAPDH on whole-tissue lysate from 1-week post-B/T immobilized and mobilized mice. *P < 0.05. (E) Confocal microscopy images of LST cells on aligned (A) and nonaligned (NA) collagen fiber plates probed with indicated antibodies and quantified for cell spread and area (right) (n = 3/group, n = 5 roi/plate, ####P < 0.0001). (F) Quantification of focal adhesions normalized by cell area of LST cells in aligned and nonaligned plates (****P < 0.0001) (G) Confocal microscopy images of LST cells on aligned and nonaligned collagen fiber plates quantified for cellular pFAK (10–12 cells/n, n = 3/group). (H) Quantification of speed and distance traveled by LSTs on aligned and nonaligned plates: Supplemental Video 1 (####P < 0.0001). (I) Confocal microscopy images of hind limb cross sections at 1 week after injury quantified for number for number of PDGFRα+ cells. **P < 0.01. (J) Confocal microscopy images of LST cells on aligned and nonaligned collagen fiber plates probed for TAZ and quantified for nuclear/cytoplasmic ratio (right) (4–5 images/n, n = 3/group, ***P = 0.0003). (K) Effects of aligned (A) or nonaligned (N) electrospun collagen I coated fibers on Runx2 and Adipoq expression in either DMEM or mixed medium (n = 3/group, *P < 0.05, A vs. N within media type). (L) Confocal microscopy images of LST cells on aligned and nonaligned collagen fiber plates in mixed medium for 7 days and subsequently stained with BODIPY and lipid droplets quantified (right) (5–6 images/n, n = 3/group, ****P < 0.0001). *Calculated using Student’s t test, #Calculated using Mann-Whitney U test. Scale bars: 100 µm.