Cell Mechanobiology

Cellular, Sub-Cellular, & Peri-Cellular Studies

Our goal is to acquire an accurate and quantitative understanding of the physiological stress-strain, fluid flow, and physicochemical environment of cartilage cells (chondrocytes), and the precise roles of these physical factors in the intracellular transduction of mechanical signals.

These studies include:

The Biomechanical Role of The Chondron

The chondrocyte and its immediate pericellular matrix have been identified as a physical hierarchial entity in cartilage tremed "the chondron." It has been postulated that the pericellular matrix plays an important role in mechanical signal transduction to the chondrocyte. To better understand the role of the pericellular matrix, an understanding of the mechanical properties of the chondron is warranted. This is accomplished by combining analytical and computational modeling with experimental measurements of the chondron properties in healthy and osteoarthritic states.
Experimental/Analytical/Computational Approach to Determine the Biomechanical Role of the Chondron

Young's Modulus of Healthy and Diseased Chondrons

References:
- Alexopoulos LG, Haider MA, Vail TP, Guilak F. Alterations in the mechanical properties of the human chondrocyte pericellular matrix with osteoarthritis. Journal of Biomechanical Engineering, 125 (3): 323-333 JUN 2003
- Leonidas G. Alexopoulos, Gregory M. Williams, Maureen L. Upton, Lori A. Setton, and Farshid Guilak, 2003, "Biphasic properties of normal and osteoarthtitic human chondrons", ASME Summer Bioeng Conf., pp. 883
- Leonidas G. Alexopoulos, Mansoor A. Haider, and Farshid Guilak, 2001, "An Axisymmetric elastic layered half-space model for micropipette aspiration of the chondrocyte pericellular matrix" ASME winter meeting, BED-Vol 51, Advances in Bioengineering

Osmotic Stress & Calcium Signaling in Chondrocytes and IVD Cells

Loading of the tissues such as cartilage and the spine alters the osmotic micro-environment in the tissue as interstitial water is expressed from the tissue. Cells within the tissue respond to osmotic stress with altered biosynthesis through a pathway that appears to involve calcium (Ca2+) as a second messenger. These studies suggest that changes in erosmotic stress induce volume change in isolated cells and may initiate [Ca2+]i transients through an actin-dependent mechanism.

Osmotic Stress-Induced Actin Reorganization

References:
- Erickson, G.R., Alexopoulos, L. and Guilak, F.: (2001): Hyper-osmotic stress induces volume change and calcium transients in chondrocytes by transmembrane, phospholipid, and G-protein pathways. Journal of Biomechanics, 34(12):1527-1535.
- Pritchard S. Erickson GR. Guilak F. (2002) Hyperosmotically induced volume change and calcium signaling in intervertebral disk cells: the role of the actin cytoskeleton. Biophysical Journal. 83(5):2502-10
Erickson GR, Northrup DL, Guilak F. (2003) Hypo-osmotic stress induces calcium-dependent actin reorganization in articular chondrocytes. Osteoarthritis and Cartilage, 11 (3): 187-197

Confocal Microscopy of Plated Cells

Three-dimensional confocal scanning laser microscopy was used to quantify the shape and volume of cells adherent on a substrate. A series of registered sections were recorded with fluorescent contrast enhancement using the dye calcein-AM. Confocal Microscopic Reconstruction of Cells on Monolayer

References:
Guilak, F. Volume and surface area measurement of viable chondrocytes in situ using geometric modeling of serial confocal sections. J Microsc, 173:245-256, 1994.
 
Chondrocyte Deformation

It is well accepted that mechanical forces can modulate chondrocyte metabolic activity, although the specific mechanisms of mechanical signal transduction in articular cartilage are still unknown. One proposed pathway through which chondrocytes may perceive changes in their mechanical environment is directly through cellular deformation. An important step towards understanding the role that chondrocyte deformation may play in signal transduction is to determine the changes in chondrocyte shape and volume with applied compression of the tissue. Recently, a technique has been developed for quantitative morphometry of viable chondrocytes within the extracellular matrix using three-dimensional confocal scanning laser microscopy [Guilak, J Microsc, 173:245-256, 1994].In the present study, this method was used to quantify changes in chondrocyte morphology and local tissue deformation in the surface, middle and deep zones in explants of canine articular cartilage under physiological levels of matrix deformation. Deformation of Chondrocytes

Results indicate that at 15% surface-to-surface equilibrium strain in the tissue, a similar magnitude of local tissue strain occurs in the middle and deep zones. In the surface zone, local strains of 19% were observed, indicating that the compressive stiffness of the surface zone is significantly lower than that of the middle and deep zones. At these levels of tissue deformation, significant decreases in cell height of 26%, 19%, and 20% and cell volume of 22%, 16%, and 17% were observed in the surface, middle and deep zones respectively. The deformation of surface zone chondrocytes was anisotropic, with significant lateral expansion occuring in the direction perpendicular to the local split-line direction. With removal of compression, complete recovery of cell morphology was observed in all cases. These observations support the hypothesis that chondrocyte deformation or volume change may occur during in vivo joint loading and could play a role in the mechanical signal transduction pathway of articular cartilage.

References:
- Guilak, F., Ratcliffe, A., and Mow, V.C. (1995): Chondrocyte deformation and local tissue strain in articular cartilage: A confocal microscopy study, Journal of Orthopaedic Research, 13(3), 410-421.

Deformation of the Chondrocyte Nucleus

Changes in cell shape and volume are believed to play a role in the process of mechanical signal transduction by chondrocytes in articular cartilage. One proposed pathway through which chondrocyte deformation may be transduced to an intracellular signal is through cytoskeletally-mediated deformation of intracellular organelles, and more specifically, of the cell nucleus. In this study, confocal scanning laser microscopy was used to perform in situ three-dimensional morphometric analyses of the nuclei of viable chondrocytes during controlled compression of articular cartilage explants from the canine patellofemoral groove. Unconfined compression of the tissue to a 15% surface-to-surface strain resulted in a significant decrease of chondrocyte height and volume by 14.7+/-6.4% and 11.4+/-8.4%, respectively, and of nuclear height and volume by 8.8+/-6.2% and 9.8+/-8.8%, respectively. Disruption of the actin cytoskeleton using cytochalasin D altered the relationship between matrix deformation and changes in nuclear height and shape, but not volume. The morphology and deformation behavior of the chondrocytes were not affected by cytochalasin treatment. These results suggest that the actin cytoskeleton plays an important role in the link between compression of the extracellular matrix and deformation of the chondrocyte nuclei and imply that chondrocytes and their nuclei undergo significant changes in shape and volume in vivo.

Deformation of Cell Nucleus

References:
- Guilak, F. (1995): Compression-induced changes in the shape and volume of the chondrocyte nucleus. Journal of Biomechanics, 28:1529-1541.

Micropipette Aspiration of Cells

Micropipette aspiration is performed to determine the mechanical properties of single cells. In this technique, chondrocytes are isolated from the extracellular matrix using enzymatic digestion. A microscopic pipette (5 µm diameter) is used to apply a small pressure to the cell surface, and the ensuing deformation is recorded through a videomicroscope. In combination with a theoretical analysis, the viscoelastic properties of the cell can be determined with this experiment.

Micropipette Aspiration of Chondrocytes

References:
- Guilak, F., Ting-Beall, H.P., Jones, W.R., Lee, G.M., and Hochmuth, R.M. (1996): Mechanical properties of chondrocytes and chondrons. ASME Advances in Bioengineering, BED33:253-254.
- Jones, W.R., Ting-Beall, H.P., Lee, G.M, Kelley, S.S., Hochmuth, R.M., Guilak, F. (1997): Mechanical properties of human chondrocytes and chondrons from normal and osteoarthritic cartilage. Transactions of the Orthopaedic Research Society, 22:199.
- Jones, W.R., Ting-Beall, H.P., Lee, G.M., Kelley, S.S., Hochmuth, R.M., and Guilak, F. (1999): Alterations in the Young’s modulus and volumetric properties of chondrocytes isolated from normal and osteoarthritic human cartilage. Journal of Biomechanics, 32(2):119-127.
- Trickey, W.R., Lee, G.M., and Guilak, F. (2000): Viscoelastic properties of chondrocytes from normal and osteoarthritic human cartilage. Journal of Orthopaedic Research, 18(6):891-898.
- Guilak, F., Erickson, G.R., and Ting-Beall, H.P. (2002): The effects of the osmotic stress on the viscoelastic and physical properties of articular chondrocytes. Biophysical Journal, 82(2):720-727.
- Trickey W.R., Vail T.P., Guilak F. (2004): The role of the cytoskeleton in the viscoelastic properties of human articular chondrocytes. Journal of Orthopaedic Research, 22(1):131-139.


FE Modeling of Chondrocyte-Matrix Interactions

To determine the nature of the interactions between the chondrocyte, the pericellular matrix (PCM), and the ECM, we developed a model for cell-matrix interactions based upon detailed microscopy of the cell membrane, the PCM, and the ECM. The major obstacle in the analysis of such problems is the large difference in the geometric scales between the level of the tissue explant and the level of the individual cell (i.e., two orders of magnitude) . By dividing the analysis into two separate problems, a multiple scaling algorithm can be used to calculate the stress-strain environment in the vicinity of the cell. This technique was used to examine the effects of several parameters, including relative mechanical properties of the cell, PCM, and ECM, as well as cell shape and intercellular spacing. Results of this parametric finite element model suggest that the mechanical environment around a chondrocyte within the ECM is highly non-uniform and dependent on cell shape and mechanical properties of the chondrocyte and the ECM. The figure above shows the principal solid stress in the vicinity of a chondrocyte during creep compression of a cartilage explant. The presence of a PCM can significantly alter the mechanical response of the cell, implying a functional mechanical role for this region. Slight changes in the PCM mechanical properties, as occurs with aging or disease, may alter the interactions between the chondrocyte and ECM.

FEM of Deformed Cells

References:
- Guilak, F., Ratcliffe, A., and Mow, V.C. (1990): The stress-strain environment around a chondrocyte: a finite element analysis of cell-matrix interactions. Advances in Bioengineering, ASME, BED-17:395-398.
- Guilak, F., Ratcliffe, A., Hunziker, E.B., and Mow, V.C. (1991): Finite element modeling of articular cartilage chondrocytes under physiological loading conditions. Transactions of the Orthopaedic Research Society, 16:366.
- Guilak, F., Ratcliffe, A., and Mow, V.C. (1991): A finite element analysis of cell-matrix interactions in articular cartilage. World Congress on Medical Physics and Biomedical Engineering, 29:88.
- Guilak, F., Ratcliffe, A., and Mow, V.C. (1991): The mechanical environment of the chondrocyte: effects of cell shape and intercellular spacing. Combined Meeting of the Orthopaedic Research Societies of USA, Japan, and Canada, p. 171.
- Guilak, F. and Mow, V.C. (1992): Determination of the mechanical response of the chondrocyte in situ using finite element modeling and confocal microscopy, Advances in Bioengineering, ASME, 22:21-23.
- Mow, V.C., Bachrach, N., Setton, L.A., and Guilak, F. (1994): Stress, strain, pressure, and flow fields in articular cartilage. In: Cell Mechanics and Cellular Engineering, eds. V.C. Mow, F. Guilak, R. Tran-Son-Tay, and R.M. Hochmuth, Springer-Verlag, New York, pp. 345-379.


Deformation-Induced Calcium Mobilization

Cytosolic free calcium concentration was measured in isolated bovine chondrocytes during application of a controlled deformation to the cell membrane. Adult bovine chondrocytes, within two passages of isolation, were plated on coverslips and incubated in fluo-3-AM, a fluorescent indicator of cytosolic Ca2+. Confocal microscope was used to examine the spatial and temporal changes of intracellular fluorescence intensity as deformations of ~10-25% the cell diameter were applied to the chondrocyte using the edge of a glass micropipette (~1 mm o.d.). Mechanical deformation immediately resulted in a transient increase of cytosolic Ca2+ levels which initiated at the site of contact with the micropipette and propagated throughout the cell and to neighboring undeformed cells, presumably through gap junctions. Peak intracellular fluorescence intensity was increased by 102% and returned to baseline levels within 3-4 min. Mechanical deformation did not initiate an increase in Ca2+ levels in chondrocytes bathed in Ca 2+-free media + EGTA, suggesting that the source of Ca2+ was extracellular. Stretch-activated ion channel blockers such as gadolinium (10 uM) or amiloride (1 mM) significantly attenuated the peak increase of fluorescence in deformed cells, suggesting that deformation-induced Ca2+ signaling was regulated, at least in part, through stretch-activated ion channels. These findings support the hypothesis that direct chondrocyte deformation is one potential mechanism by which mechanical loading of the cartilage matrix is immediately transduced to a biochemical signal.

Ca+2 Signaling in Chondrocytes

References:
- Donahue, H.J., Guilak, F., Van der Molen, M., McLeod, K.J., Rubin, C.T., Grande, D.A., and Brink, P.R. (1995): Chondrocytes isolated from mature articular cartilage retain the capacity to form functional gap junctions. Journal of Bone and Mineral Research, 10:1359-1364.
- Guilak, F., Donahue, H.J., Zell, R., Grande, D.A., McLeod, K.J., and Rubin, C.T. (1994): Deformation-induced calcium signaling in articular chondrocytes. In: Cell Mechanics and Cellular Engineering, eds. V.C. Mow, F. Guilak, R. Tran-Son-Tay, and R.M. Hochmuth, Springer-Verlag, New York, pp. 380-397.