Guiding Osteogenic Lineage Commitment: The Role of Mesenchymal Stem Cell Biology and the Mechanical Microenvironment

Today, bone is the second most implanted tissue in the body and although current technologies have the ability to restore function, there are inherent limitations with many complications. Advancements in the field of bone tissue engineering would alleviate the limitations of modern bone replacement technologies and enable a better quality of life for patients needing craniofacial reconstruction, segmental void replacement, spinal fusions and total joint replacements. Bone is an exceptionally mechanosensitive tissue, with both development and healthy adult tissue homeostasis requiring mechanical loading; however, the role of the mechanical microenvironment in controlling osteogenic differentiation and the molecular mechanisms responsible for transducing a physical signal into a cell fate commitment are currently unknown. The purpose of this dissertation is to broaden the current understanding of mesenchymal stem cell (MSC) biology and the role of the mechanical microenvironment in guiding osteogenic lineage commitment. The first study of this dissertation shows that periosteal derived progenitor cells and homogeneous populations of fibroblasts have a similar potential to commit to the osteogenic and adipogenic lineages suggesting that fibroblasts may be a more plastic cell than previously thought, given the proper environmental cues. The second study demonstrates that oscillatory fluid flow has the potential to induce osteogenic differentiation via Runx2 upregulation, and furthermore activates the small GTPase RhoA as well as its effector, ROCK. RhoA and flow have an additive effect on Runx2 expression, which is similar to their additive effect on actin fibril organization. Additionally, I show that a dynamic actin cytoskeletal under tension is necessary for the transduction of a physical signal into altered gene expression. The third study demonstrates that RhoA activation is dependent on Wnt5a signaling via the tyrosine receptor, ROR2. Additionally, this study shows that flow also induces β-catenin signaling, which may be mediated by adherens junctions. Both Wnt5a signaling and β-catenin signaling are necessary for mechanically induced osteogenic differentiation, indicating multiple pathways must act as switches to initiate cell fate. Finally, the last study investigates the relationship between epigenetic and genetic programming as it relates to osteogenic differentiation. This study shows that a mechanical stimulus is a potent signal capable of epigenetically modifying the chromatin via DNA methylation, which is the only modification that is inherited by future progeny. Furthermore, the decrease in methylation correlates with an increase in Osteopontin gene expression suggesting that the mechanical microenvironment of MSCs has the potential to induce stable alterations in gene expression resulting in osteogenic lineage commitment. Understanding MSC fate potential, the role of the mechanical microenvironment on osteogenic differentiation, and the molecular mechanisms involved in fate commitment will enhance the field of tissue engineering and promote novel approaches in bone regeneration.

Year	Entry
2002	Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, Mitnick MA, Wu D, Insogna K, Lifton RP. High bone density due to a mutation in LDL-receptor–related protein 5. NEJM. May 16, 2002;346(20):1513-1521.
2005	Woods A, Wang G, Beier F. RhoA/ROCK signaling regulates Sox9 expression and actin organization during chondrogenesis. J Biol Chem. March 25, 2005;280(12):11626-11634.
1987	Frost HM. The mechanostat: a proposed pathogenic mechanism of osteoporoses and the bone mass effects of mechanical and nonmechanical agents. Bone Miner. April 1987;2(2):73-85.
2007	Malone AMD. Mechanotransduction Mechanisms in Bone Cells [PhD thesis]. Stanford University; August 2007.
1990	Reich KM, Gay CV, Frangos JA. Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production. J Cell Physiol. April 1990;143(1):100-103.
1999	Wang D, Christensen K, Chawla K, Xiao G, Krebsbach PH, Franceschi RT. Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential. J Bone Miner Res. June 1999;14(6):893-903.
2000	Chen NX, Ryder KD, Pavalko FM, Turner CH, Burr DB, Qiu J, Duncan RL. Ca²⁺ regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts. Am J Physiol Cell Physiol. May 2000;278(5):C989-C997.
2006	Turner CH. Bone strength: current concepts. Annals NY Acad Sci. 2006;1068(1):429-446.
2000	Knothe Tate ML, Steck R, Forwood MR, Niederer P. In vivo demonstration of load-induced fluid flow in the rat tibia and its potential implications for processes associated with functional adaptation. J Exp Biol. September 2000;203(18):2737-2745.
1997	Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of purified, culture‐expanded human mesenchymal stem cells in vitro. J Cell Biochem. February 1997;64(2):295-312.
2001	Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. April 2001;7(2):211-228.
1997	Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsent G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. May 30, 1997;89(5):747-754.
1997	Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell. May 30, 1997;89(5):765-771.
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.
1998	Carter DR, Beaupré GS, Giori NJ, Helms JA. Mechanobiology of skeletal regeneration. Clin Orthop Relat Res. October 1998;355(suppl):S41-S55.
2001	Bakker AD, Soejima K, Klein-Nulend J, Burger EH. The production of nitric oxide and prostaglandin E₂ by primary bone cells is shear stress dependent. J Biomech. May 2001;34(5):671-677.
2004	Li YJ, Batra NN, You L, Meier SC, Coe IA, Yellowley CE, Jacobs CR. Oscillatory fluid flow affects human marrow stromal cell proliferation and differentiation. J Orthop Res. November 2004;22(6):1283-1289.
2005	Batra NN, Li YJ, Yellowley CE, You L, Malone AM, Kim CH, Jacobs CR. Effects of short-term recovery periods on fluid-induced signaling in osteoblastic cells. J Biomech. September 2005;38(9):1909-1917.
1988	Frost HM. Vital biomechanics: proposed general concepts for skeletal adaptations to mechanical usage. Calcif Tiss Int. May 1988;42(3):145-156.
1997	Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao Y-H, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. May 30, 1997;89(5):755-764.
1982	Frost HM. Review article mechanical determinants of bone modeling. Metab Bone Dis Rel Res. 1982;4(4):217-229.
2001	You J, Reilly GC, Zhen X, Yellowley CE, Chen Q, Donahue HJ, Jacobs CR. Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3–E1 osteoblasts. J Biol Chem. April 20, 2001;276(16):13365-13371.
1990	Brown TD, Pedersen DR, Gray ML, Brand RA, Rubin CT. Toward an identification of mechanical parameters initiating periosteal remodeling: a combined experimental and analytic approach. J Biomech. 1990;23(9):893-905.
2004	McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell. April 2004;6(4):483-495.
2001	Gong Y, Slee RB, Fukai N, Rawadi G, Roman-Roman S, Reginato AM, Wang H, Cundy T, Glorieux FH, Lev D, Zacharin M, Oexle K, Marcelino J, Suwairi W, Heeger S, Sabatakos G, Apte S, Adkins WN, Allgrove J, Arslan-Kirchner M, Batch JA, Beighton P, Black GCM, Boles RG, Boon LM, Borrone C, Brunner HG, Carle GF, Dallapiccola B, De Paepe A, Floege B, Halfhide ML, Hall B, Hennekam RC, Hirose T, Jans A, Jüppner H, Kim CA, Keppler-Noreuil K, Kohlschuetter A, LaCombe D, Lambert M, Lemyre E, Letteboer T, Peltonen L, Ramesar RS, Romanengo M, Somer H, Steichen-Gersdorf E, Steinmann B, Sullivan B, Superti-Furga A, Swoboda W, van den Boogaard M-J, Van Hul W, Vikkula M, Votruba M, Zabel B, Garcia T, Baron R, Olsen BR, Warman ML. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell. November 16, 2001;107(4):513-523.
1892	Wolff J. Das Gesetz der Transformation der Knochen. Berlin: Hirschwald; 1892.
1998	Pavalko FM, Chen NX, Turner CH, Burr DB, Atkinson S, Hsieh Y-F, Qiu J, Duncan RL. Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions. Am J Physiol. December 1998;275(6):C1591-C1601.
1999	Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. April 2, 1999;284(5411):143-147.
1989	Carter DR, Orr TE, Fyhrie DP. Relationships between loading history and femoral cancellous bone architecture. J Biomech. 1989;22(3):231-244.
1988	Carter DR, Wong M. The role of mechanical loading histories in the development of diarthrodial joints. J Orthop Res. 1988;6(6):804-816.
1986	Wolff J. The Law of Bone Remodelling. Maquet P, Furlong R, trans. New York, NY: Springer; 1986.
2006	Krishnan V, Bryant HU, MacDougald OA. Regulation of bone mass by Wnt signaling. J Clin Invest. May 2006;116(5):1202-1209.
2007	Wang Y, McNamara LM, Schaffler MB, Weinbaum S. A model for the role of integrins in flow induced mechanotransduction in osteocytes. Proc Natl Acad Sci USA. October 2, 2007;104(40):15941-15946.
1993	Langer R, Vacanti JP. Tissue engineering. Science. May 14, 1993;260(5110):920-926.
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.
1987	Carter DR, Orr TE, Fyhrie DP, Schurmant DJ. Influences of mechanical stress on prenatal and postnatal skeletal development. Clin Orthop Relat Res. June 1987;219:237-250.
2006	Rodda SJ, McMahon AP. Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors. Development. August 15, 2006;133(16):3231-3244.
2002	Little RD, Carulli JP, Del Mastro RG, Dupuis J, Osborne M, Folz C, Manning SP, Swain PM, Zhao S-C, Eustace B, Lappe MM, Spitzer L, Zweier S, Braunschweiger K, Benchekroun Y, Hu X, Adair R, Chee L, FitzGerald MG, Tulig C, Caruso A, Tzellas N, Bawa A, Franklin B, McGuire S, Nogues X, Gong G, Allen KM, Anisowicz A, Morales AJ, Lomedico PT, Recker SM, Van Eerdewegh P, Recker RR, Johnson ML. A mutation in the LDL receptor–related protein 5 gene results in the autosomal dominant high–bone-mass trait. Am J Hum Genet. January 2002;70(1):11-19.
2006	Ingber DE. Cellular mechanotransduction: putting all the pieces together again. FASEB J. May 2006;20(7):811-827.
2008	Mackie EJ, Ahmed YA, Tatarczuch L, Chen K-S, Mirams M. Endochondral ossification: how cartilage is converted into bone in the developing skeleton. Int J Biochem Cell Biol. January 2008;40(1):46-62.
2007	Tan SD, de Vries TJ, Kuijpers-Jagtman AM, Semeins CM, Everts V, Klein-Nulend J. Osteocytes subjected to fluid flow inhibit osteoclast formation and bone resorption. Bone. November 2007;41(5):745-751.
2000	You J, Yellowley CE, Donahue HJ, Zhang Y, Chen Q, Jacobs CR. Substrate deformation levels associated with routine physical activity are less stimulatory to bone cells relative to loading-induced oscillatory fluid flow. J Biomech Eng. August 2000;122(4):387-393.
2006	Isaksson H, Wilson W, van Donkelaar CC, Huiskes R, Ito K. Comparison of biophysical stimuli for mechano-regulation of tissue differentiation during fracture healing. J Biomech. 2006;39(8):1507-1516.
2006	Ruff C, Holt B, Trinkaus E. Who's afraid of the big bad Wolff? “Wolff's law” and bone functional adaptation. Am J Phys Anthropol. April 2006;129(4):484-498.

Year

Entry

2002

Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, Mitnick MA, Wu D, Insogna K, Lifton RP. High bone density due to a mutation in LDL-receptor–related protein 5. NEJM. May 16, 2002;346(20):1513-1521.

2005

Woods A, Wang G, Beier F. RhoA/ROCK signaling regulates Sox9 expression and actin organization during chondrogenesis. J Biol Chem. March 25, 2005;280(12):11626-11634.

1987

Frost HM. The mechanostat: a proposed pathogenic mechanism of osteoporoses and the bone mass effects of mechanical and nonmechanical agents. Bone Miner. April 1987;2(2):73-85.

2007

Malone AMD. Mechanotransduction Mechanisms in Bone Cells [PhD thesis]. Stanford University; August 2007.

1990

Reich KM, Gay CV, Frangos JA. Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production. J Cell Physiol. April 1990;143(1):100-103.