Osteocytes are widely regarded as mechanosensors, capable of detecting changes in the mechanical environment of the bone tissue and modifying cellular responses accordingly. Indeed, an intact osteocyte network is required for bone changes in response to unloading, and studies have shown that loading/unloading influences osteocyte expression of proteins that modulate bone turnover, such as sclerostin and receptor activator of nuclear factor kappa B ligand (RANKL). However, mechanisms underlying osteocyte mechanotransduction remain unclear. For instance, one of the earliest responses of bone cells to mechanical stimuli is a rise in intracellular, or cytosolic, calcium (Ca2+cyt), but the mechanisms by which osteocytes generate or utilize Ca2+ signals to direct bone adaptation are largely unknown.
In this thesis, I explored the mechanisms underlying the sustainment of Ca2+cyt oscillations in osteocytes as well as downstream consequences of these patterns. I discovered that Ca2+cyt oscillations are generated in osteocytes by Ca2+ release from the endoplasmic reticulum and that the predominant expression of T-Type voltage sensitive Ca2+ channels in these cells facilitates this behavior. I also explored the role of the actin cytoskeleton – another prominent feature in osteocytes – and found that actin dynamics are important for the generation of Ca2+cyt signals. Furthermore, I confirmed that Ca2+cyt transients subsequently activate actomyosin contractions in osteocytes by monitoring interactions of osteocytes exposed to Ca2+ agonists on micropillar substrates.
With this information, I sought to relate Ca2+cyt signaling and actomyosin contractility in osteocytes to their roles as coordinators of bone adaptation. Ca2+-dependent contractions have been shown to facilitate the release of extracellular vesicles, small membrane-enclosed packages of proteins that cells use for communication, in other cell types. I found that mechanical stimulation increased the production and release of extracellular vesicles in osteocytes, and this was dependent on Ca2+ signaling. These extracellular vesicles contained key bone regulatory proteins and were small enough to plausibly transport through the lacunocanalicular system. Thus, I uncovered a novel mechanotransduction pathway by which osteocytes may coordinate tissue-level adaptation. As an extension of this work, I also characterized these behaviors in new osteocyte cell lines which may better reflect native cell physiology.
The work in this thesis anchors Ca2+ signaling as a critical osteocyte response to mechanical loading and adds to the body of work exploring how and why these signals are generated. The results of these studies add new information to the still limited knowledge of this important bone cell and extend Ca2+ signaling research by connecting early mechanosensation events to subsequent protein responses to mechanical loading. Understanding the mechanisms behind the robust Ca2+cyt oscillations in osteocytes and how they relate to their roles as coordinators of bone adaptation may improve our ability to prevent or treat bone degeneration in diseases like osteoporosis where mechanosensitivity is impaired.
|1997||Owan I, Burr DB, Turner CH, Qiu J, Tu Y, Onyia JE, Duncan RL. Mechanotransduction in bone: osteoblasts are more responsive to fluid forces than mechanical strain. Am J Physiol Cell Physiol. September 1997;273(3):C810-C815.|
|1995||Duncan RL, Turner CH. Mechanotransduction and the functional response of bone to mechanical strain. Calcif Tiss Int. December 1995;57(5):344-358.|
|1994||Weinbaum S, Cowin SC, Zeng Y. A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses. J Biomech. March 1994;27(3):339-360.|
|1998||Jacobs CR, Yellowley CE, Davis BR, Zhou Z, Cimbala JM, Donahue HJ. Differential effect of steady versus oscillating flow on bone cells. J Biomech. November 1998;31(11):969-976.|
|1977||Jones HH, Priest JD, Hayes WC, Tichenor CC, Nagel DA. Humeral hypertrophy in response to exercise. J Bone Joint Surg. March 1977;59A(2):204-208.|
|1977||Piekarski K, Munro M. Transport mechanism operating between blood supply and osteocytes in long bones. Nature. September 1, 1977;270(5623):80-82.|
|2008||Robling AG, Niziolek PJ, Baldridge LA, Condon KW, Allen MR, Alam I, Mantila SM, Gluhak-Heinrich J, Bellido TM, Harris SE, Turner CH. Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/sclerostin. J Biol Chem. February 29, 2008;283(9):5866-5875.|
|2013||Vivanco J, Garcia S, Ploeg HL, Alvarez G, Cullen D, Smith EL. Apparent elastic modulus of ex vivo trabecular bovine bone increases with dynamic loading. Proc Inst Mech Eng Part H-J Eng Med. August 2013;227(8):904-912.|
|2011||Bonewald LF. The amazing osteocyte. J Bone Miner Res. 2011;26(2):229-238.|
|1892||Wolff J. Berlin: Hirschwald; 1892.|
|2008||Bonewald LF, Johnson ML. Osteocytes, mechanosensing and Wnt signaling. Bone. April 2008;42(4):606-615.|
|2002||Ehrlich PJ, Lanyon LE. Mechanical strain and bone cell function: a review. Osteoporos Int. September 2002;13(9):688-700.|
|2007||Tatsumi S, Ishii K, Amizuka N, Li M, Kobayashi T, Kohno K, Ito M, Takeshita S, Ikeda K. Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell Metab. June 2007;5(6):464-475.|
|1990||El Haj AJ, Minter SL, Rawlinson SCF, Suswillo R, Lanyon LE. Cellular responses to mechanical loading in vitro. J Bone Miner Res. September 1990;5(9):923-932.|
|2006||Davies CM, Jones DB, Stoddart MJ, Koller K, Smith E, Archer CW, Richards RG. Mechanically loaded ex vivo bone culture system "Zetos": systems and culture preparation. Eur Cell Mater. January–June 2006;11:57-75.|
|2003||Jones DB, Broeckmann E, Pohl T, Smith EL. Development of a mechanical testing and loading system for trabecular bone studies for long term culture. Eur Cell Mater. January–June 2003;5:48-60.|