title: Purinergic Signaling Regulates the Transforming Growth Factor-β3-Induced Chondrogenic Response of Mesenchymal Stem Cells to Hydrostatic Pressure.
abstract: Although hydrostatic pressure (HP) is known to regulate chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs), improved insight into the mechanotransduction of HP may form the basis for novel tissue engineering strategies. Previously, we demonstrated that matrix stiffness and calcium ion (Ca(++)) mobility regulate the mechanotransduction of HP; however, the mechanisms, by which these Ca(++) signaling pathways are initiated, are currently unknown. The purinergic pathway, in which adenosine triphosphate (ATP) is released and activates P-receptors to initiate Ca(++) signaling, plays a key role in the mechanotransduction of compression, but has yet to be investigated with regard to HP. Therefore, the objective of this study was to investigate the interplay between purinergic signaling, matrix stiffness, and the chondrogenic response of MSCs to HP. Porcine bone marrow-derived MSCs were seeded into soft or stiff agarose hydrogels and subjected to HP (10 MPa at 1 Hz for 4 h/d for 21 days) or kept in free swelling conditions. Stiff constructs were incubated with pharmacological inhibitors of extracellular ATP, P2 receptors, or hemichannels, or without any inhibitors as a control. As with other loading modalities, HP significantly increased ATP release in the control group; however, inhibition of hemichannels completely abrogated this response. The increase in sulfated glycosaminoglycan (sGAG) synthesis and vimentin reorganization observed in the control group in response to HP was suppressed in the presence of all three inhibitors, suggesting that purinergic signaling is involved in the mechanoresponse of MSCs to HP. Interestingly, ATP was released from both soft and stiff hydrogels in response to HP, but HP only enhanced chondrogenesis in the stiff hydrogels, indicating that matrix stiffness may act downstream of purinergic signaling to regulate the mechanoresponse of MSCs to HP. Addition of exogenous ATP did not replicate the effects of HP on chondrogenesis, suggesting that mechanisms other than purinergic signaling also regulate the response of MSCs to HP.
authors.affiliation: 1 Bioengineering Graduate Program, Department of Aerospace and Mechanical Engineering, University of Notre Dame , Notre Dame, Indiana.|2 Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute , Trinity College Dublin, Dublin, Ireland .
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Steward
authors.affiliation: 2 Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute , Trinity College Dublin, Dublin, Ireland .|3 Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin , Dublin, Ireland .|4 Advanced Materials and Bioengineering Research Centre (AMBER), Trinity College Dublin , Dublin, Ireland .
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Kelly
authors.affiliation: 5 Department of Mechanical Engineering, Indiana University-Purdue University Indianapolis , Indianapolis, Indiana.|6 Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis , Indianapolis, Indiana.
authors.forename: Diane R
authors.initials: DR
authors.lastname: Wagner
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000187:drug effects*; D005109:Extracellular Matrix / Q000378:metabolism; D006874:Hydrostatic Pressure; D040542:Mechanotransduction, Cellular / Q000187:drug effects; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D011983:Receptors, Purinergic / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D034421:Sus scrofa; D053782:Transforming Growth Factor beta3 / Q000494:pharmacology*; D014746:Vimentin / Q000378:metabolism
chemical_list: D011983:Receptors, Purinergic; D053782:Transforming Growth Factor beta3; D014746:Vimentin; D000255:Adenosine Triphosphate