Technical Brief

Differences in Morphology and Traction Generation of Cell Lines Representing Different Stages of Osteogenesis

[+] Author and Article Information
Michael J. Poellmann

Department of Mechanical Science and Engineering,
University of Illinois at Urbana-Champaign,
Urbana, IL 61801

Jonathan B. Estrada

School of Engineering,
Brown University,
Providence, RI 02912

Thomas Boudou

Laboratory of Materials and Physical Engineering,
Grenoble Institute of Technology,
Grenoble 38016, France

Zachary T. Berent

Department of Mechanical Science and Engineering,
University of Illinois at Urbana-Champaign,
Urbana, IL 61801

Christian Franck

School of Engineering,
Brown University,
Providence, RI 02912
e-mail: franck@brown.edu

Amy J. Wagoner Johnson

Department of Mechanical Science and Engineering,
University of Illinois at Urbana-Champaign,
1206 W. Green Street,
Urbana, IL 61801
e-mail: ajwj@illinois.edu

1Corresponding author.

Manuscript received April 10, 2015; final manuscript received October 10, 2015; published online November 3, 2015. Assoc. Editor: Carlijn V. C Bouten.

J Biomech Eng 137(12), 124503 (Nov 03, 2015) (5 pages) Paper No: BIO-15-1158; doi: 10.1115/1.4031848 History: Received April 10, 2015; Revised October 10, 2015

Osteogenesis is the process by which mesenchymal stem cells differentiate to osteoblasts and form bone. The morphology and root mean squared (RMS) traction of four cell types representing different stages of osteogenesis were quantified. Undifferentiated D1, differentiated D1, MC3T3-E1, and MLO-A5 cell types were evaluated using both automated image analysis of cells stained for F-actin and by traction force microscopy (TFM). Undifferentiated mesenchymal stem cell lines were small, spindly, and exerted low traction, while differentiated osteoblasts were large, had multiple processes, and exerted higher traction. Size, shape, and traction all correlated with the differentiation stage. Thus, cell morphology evolved and RMS traction increased with differentiation. The results provide a foundation for further work with these cell lines to study the mechanobiology of bone formation.

Copyright © 2015 by ASME
Topics: Traction , Shapes , Microscopy
Your Session has timed out. Please sign back in to continue.


Robling, A. G. , Castillo, A. B. , and Turner, C. H. , 2006, “ Biomechanical and Molecular Regulation of Bone Remodeling,” Ann. Rev. Biomed. Eng., 8(1), pp. 455–498. [CrossRef]
Aubin, J. E. , 2008, “ Mesenchymal Stem Cells and Osteoblast Differentiation,” Principles of Bone Biology, 3rd ed., Elsevier, New York, pp. 85–107.
Boskey, A. L. , and Roy, R. , 2008, “ Cell Culture Systems for Studies of Bone and Tooth Mineralization,” Chem. Rev., 108(11), pp. 4716–4733. [CrossRef] [PubMed]
Diduch, D. R. , Coe, M. R. , Joyner, C. , Owen, M. E. , and Balian, G. , 1993, “ Two Cell Lines From Bone Marrow That Differ in Terms of Collagen Synthesis, Osteogenic Characteristics, and Matrix Mineralization,” J. Bone Jt. Surg. Am., 75(1), pp. 92–105.
Quarles, L. D. , Yohay, D. A. , Lever, L. W. , Caton, R. , and Wenstrup, R. J. , 1992, “ Distinct Proliferative and Differentiated Stages of Murine MC3T3-E1 Cells in Culture: An In Vitro Model of Osteoblast Development,” J. Bone Miner. Res., 7(6), pp. 683–692. [CrossRef] [PubMed]
Wang, D. , Christensen, K. , Chawla, K. , Xiao, G. , Krebsbach, P. H. , and Franceschi, R. T. , 1999, “ Isolation and Characterization of MC3T3-E1 Preosteoblast Subclones With Distinct In Vitro and In Vivo Differentiation/Mineralization Potential,” J. Bone Miner. Res., 14(6), pp. 893–903. [CrossRef] [PubMed]
Kato, Y. , Boskey, A. , Spevak, L. , Dallas, M. , Hori, M. , and Bonewald, L. F. , 2001, “ Establishment of an Osteoid Preosteocyte-Like Cell MLO-A5 That Spontaneously Mineralizes in Culture,” J. Bone Miner. Res., 16(9), pp. 1622–1633. [CrossRef] [PubMed]
Cui, Q. , Wang, G. H. , and Balian, G. , 1997, “ Steroid-Induced Adipogenesis in a Pluripotential Cell Line From Bone Marrow,” J. Bone Jt. Surg. Am., 79(7), pp. 1054–1063.
Franck, C. , Maskarinec, S. A. , Tirrell, D. A. , and Ravichandran, G. , 2011, “ Three-Dimensional Traction Force Microscopy: A New Tool for Quantifying Cell-Matrix Interactions,” PLoS One, 6(3), p. e17833. [CrossRef] [PubMed]
Toyjanova, J. , Bar-Kochba, E. , Lopez-Fagundo, C. , Reichner, J. S. , Hoffman-Kim, D. , and Franck, C. , 2014, “ High Resolution, Large Deformation 3D Traction Force Microscopy,” PLoS One, 9(4), p. e90976. [CrossRef] [PubMed]
Poellmann, M. J. , and Wagoner Johnson, A. J. , 2013, “ Characterizing and Patterning Polyacrylamide Substrates Functionalized With N-Hydroxysuccinimide,” Cell. Mol. Bioeng., 6(3), pp. 299–309. [CrossRef]
Wang, N. , Naruse, K. , Stamenovic, D. , Fredberg, J. J. , Mijailovich, S. M. , Toli-Norrelykke, I. M. , Polte, T. , Mannix, R. , and Ingber, D. E. , 2001, “ Mechanical Behavior in Living Cells Consistent With the Tensegrity Model,” Proc. Natl. Acad. Sci. U.S.A., 98(14), pp. 7765–7770. [CrossRef] [PubMed]
Farid, H. , and Simoncelli, E. P. , 2004, “ Differentiation of Discrete Multidimensional Signals,” IEEE Trans. Image Process., 13(4), pp. 496–508. [CrossRef] [PubMed]
Bar-Kochba, E. , Toyjanova, J. , Andrews, E. , Kim, K.-S. , and Franck, C. , 2014, “ A Fast Iterative Digital Volume Correlation Algorithm for Large Deformations,” Exp. Mech., 55(1), pp. 261–274. [CrossRef]
Hsiong, S. X. , Carampin, P. , Kong, H. J. , Lee, K. Y. , and Mooney, D. J. , 2007, “ Differentiation Stage Alters Matrix Control of Stem Cells,” J. Biomed. Mater. Res. A, 85(1), pp. 145–156.
Docheva, D. , Padula, D. , Popov, C. , Mutschler, W. , Clausen-Schaumann, H. , and Schieker, M. , 2008, “ Researching Into the Cellular Shape, Volume and Elasticity of Mesenchymal Stem Cells, Osteoblasts, and Osteosarcoma Cells by Atomic Force Microscopy,” J. Cell Mol. Med., 12(2), pp. 537–552. [CrossRef] [PubMed]
Zouani, O. F. , Rami, L. , Lei, Y. , and Durrieu, M. C. , 2013, “ Insights Into the Osteoblast Precursor Differentiation Towards Mature Osteoblasts by Continuous BMP-2 Signaling,” Biol. Open, 2(9), pp. 872–881. [CrossRef] [PubMed]
Fu, J. , Wang, Y. K. , Yang, M. T. , Desai, R. A. , Yu, X. , Liu, Z. , and Chen, C. S. , 2010, “ Mechanical Regulation of Cell Function With Geometrically Modulated Elastomeric Substrates,” Nat. Methods, 7(9), pp. 733–736. [CrossRef] [PubMed]
Harris, A. K. , Stopak, D. , and Wild, P. , 1981, “ Fibroblast Traction as a Mechanism for Collagen Morphogenesis,” Nature, 290(5803), pp. 249–251. [CrossRef] [PubMed]
Rape, A. D. , Guo, W. H. , and Wang, Y. L. , 2011, “ The Regulation of Traction Force in Relation to Cell Shape and Focal Adhesions,” Biomaterials, 32(8), pp. 2043–2051. [CrossRef] [PubMed]
Oakes, P. W. , Banerjee, S. , Marchetti, M. C. , and Gardel, M. L. , 2014, “ Geometry Regulates Traction Stresses in Adherent Cells,” Biophys. J., 107(4), pp. 825–833. [CrossRef] [PubMed]
Han, S. J. , Bielawski, K. S. , Ting, L. H. , Rodriguez, M. L. , and Sniadecki, N. J. , 2012, “ Decoupling Substrate Stiffness, Spread Area, and Micropost Density: A Close Spatial Relationship Between Traction Forces and Focal Adhesions,” Biophys. J., 103(4), pp. 640–648. [CrossRef] [PubMed]
Ye, G. J. C. , Aratyn-Schaus, Y. , Nesmith, A. P. , Pasqualini, F. S. , Alford, P. W. , and Parker, K. K. , 2014, “ The Contractile Strength of Vascular Smooth Muscle Myocytes is Shape Dependent,” Integr. Biol., 6(2), pp. 152–163. [CrossRef]
Hampe, N. , Jonas, T. , Wolters, B. , Hersch, N. , Hoffmann, B. , and Merkel, R. , 2014, “ Defined 2-D Microtissues on Soft Elastomeric Silicone Rubber Using Lift-Off Epoxy-Membranes for Biomechanical Analysis,” Soft Matter, 10(14), pp. 2431–2443. [CrossRef] [PubMed]
Winer, J. P. , Chopra, A. , Yasha Kresh, J. , and Janmey, P. A. , 2011, “ Substrate Elasticity as a Probe to Measure Mechanosensing at Cell-Cell and Cell-Matrix Junctions,” Mechanobiology of Cell-Cell and Cell-Matrix Interactions, Springer, Berlin, pp. 11–22.
Salasznyk, R. M. , Williams, W. A. , Boskey, A. , Batorsky, A. , and Plopper, G. E. , 2004, “ Adhesion to Vitronectin and Collagen I Promotes Osteogenic Differentiation of Human Mesenchymal Stem Cells,” J. Biomed. Biotechnol., 1, pp. 24–34. [CrossRef]
Engler, A. J. , Sen, S. , Sweeney, H. L. , and Discher, D. E. , 2006, “ Matrix Elasticity Directs Stem Cell Lineage Specification,” Cell, 126(4), pp. 677–689. [CrossRef] [PubMed]
Kong, H. J. , Polte, T. R. , Alsberg, E. , and Mooney, D. J. , 2005, “ FRET Measurements of Cell-Traction Forces and Nano-Scale Clustering of Adhesion Ligands Varied by Substrate Stiffness,” Proc. Natl. Acad. Sci. U.S.A., 102(12), pp. 4300–4305. [CrossRef] [PubMed]
Khatiwala, C. B. , Peyton, S. R. , and Putnam, A. J. , 2006, “ Intrinsic Mechanical Properties of the Extracellular Matrix Affect the Behavior of Pre-Osteoblastic MC3T3-E1 Cells,” Am. J. Physiol. Cell Physiol., 290(6), pp. C1640–C1650. [CrossRef] [PubMed]
Trappmann, B. , Gautrot, J. E. , Connelly, J. T. , Strange, D. G. T. , Li, Y. , Oyen, M. L. , Cohen Stuart, M. A. , Boehm, H. , Li, B. , Vogel, V. , Spatz, J. P. , Watt, F. M. , and Huck, W. T. S. , 2012, “ Extracellular-Matrix Tethering Regulates Stem-Cell Fate,” Nat. Mater., 11(7), pp. 642–649. [CrossRef] [PubMed]
Wen, J. H. , Vincent, L. G. , Fuhrmann, A. , Choi, Y. S. , Hribar, K. C. , Taylor-Weiner, H. , Chen, S. , and Engler, A. J. , 2014, “ Interplay of Matrix Stiffness and Protein Tethering in Stem Cell Differentiation,” Nat. Mater., 13(10), pp. 979–987. [CrossRef] [PubMed]
Gellynck, K. , Shah, R. , Deng, D. , Parker, M. , Liu, W. , Knowles, J. C. , and Buxton, P. , 2013, “ Cell Cytoskeletal Changes Effected by Static Compressive Stress Lead to Changes in the Contractile Properties of Tissue Regenerative Collagen Membranes,” Eur. Cells Mater., 25, pp. 317–325.
Murshid, S. A. , Kamioka, H. , Ishihara, Y. , Ando, R. , Sugawara, Y. , and Takano-Yamamoto, T. , 2007, “ Actin and Microtubule Cytoskeletons of the Processes of 3D-Cultured MC3T3-E1 Cells and Osteocytes,” J. Bone Miner. Metab., 25(3), pp. 151–158. [CrossRef] [PubMed]
Ruiz, S. A. , and Chen, C. S. , 2009, “ Emergence of Patterned Stem Cell Differentiation Within Multicellular Structures,” Stem Cells, 26(11), pp. 2921–2927. [CrossRef]
Nelson, C. M. , Jean, R. P. , Tan, J. L. , Liu, W. F. , Sniadecki, N. J. , Spector, A. A. , and Chen, C. S. , 2005, “ Emergent Patterns of Growth Controlled by Multicellular Form and Mechanics,” Proc. Natl. Acad. Sci. U.S.A., 102(33), pp. 11594–11599. [CrossRef] [PubMed]
Maruthamuthu, V. , Sabass, B. , Schwarz, U. S. , and Gardel, M. L. , 2011, “ Cell-ECM Traction Force Modulates Endogenous Tension at Cell-Cell Contacts,” Proc. Natl. Acad. Sci. U.S.A., 108(12), pp. 4708–4713. [CrossRef] [PubMed]
Mertz, A. F. , Che, Y. , Banerjee, S. , Goldstein, J. M. , Rosowksi, K. A. , Revilla, S. F. , Niessen, C. M. , Marchetti, M. C. , Dufresne, E. R. , and Horsley, V. , 2013, “ Cadherin-Based Intercellular Adhesions Organize Epithelial Cell-Matrix Traction Forces,” Proc. Natl. Acad. Sci. U.S.A., 110(3), pp. 842–847. [CrossRef] [PubMed]


Grahic Jump Location
Fig. 2

RMS traction increased with differentiation stage. Representative traction field contour maps of (a) uD1, (b) dD1, (c) MC3T3-E1, and (d) MLO-A5 cells shown with logarithmic scale. Color available in online version. (e) The RMS traction increased differentiation stage according to the Spearman rank analysis, with a monotonic and statistically significant correlation (Table1). Statistically significant pairwise differences (p < 0.05) are indicated by brackets.

Grahic Jump Location
Fig. 1

Shape factor evolved with differentiation stage. Representative fluorescent images of (a) uD1, (b) dD1, (c) MC3T3-E1, and (d) MLO-A5 cells. Differentiation stage correlated with (e) area, (f) circularity, and (g) inverse aspect ratio (Table 1). All measures increased differentiation stage, with a monotonic and statistically significant correlation according to the Spearman rank analysis (Table 1). Statistically significant pairwise differences determined by ANOVA and Tukey means analysis (p < 0.05) are indicated by brackets.



Some tools below are only available to our subscribers or users with an online account.

Related Content

Customize your page view by dragging and repositioning the boxes below.

Related Journal Articles
Related eBook Content
Topic Collections

Sorry! You do not have access to this content. For assistance or to subscribe, please contact us:

  • TELEPHONE: 1-800-843-2763 (Toll-free in the USA)
  • EMAIL: asmedigitalcollection@asme.org
Sign In