Biomechanical Adaptation of Cells: A Microstructural Approach November 9, 2002

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Biomechanical Adaptation of Cells A
Microstructural ApproachNovember 9, 2002

• Wesley Jackson
• Michael Jaasma
• Tony Keaveny

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Introduction

• Bone is capable of adapting in response to
  mechanical stimulus
• Bone remodeling physiology is well characterized
• It is not well understood how this process is
  regulated

Bone is resorbed
New bone is added
Osteoblasts
Osteoclast
Osteocytes
Diagram adapted from Marks et al. (1988) Am J
Anat 1831
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Tissue Level Response

• Adaptation of bone is determined by a feedback
  loop Frost (1987) Anat Rec 2191

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Tissue Level Response

• Habitual strain levels are not uniform for all
  bone tissue
• Different regions of bone tissue may have
  different strain set-points Turner (1999) Calcif
  Tissue Int 65466
• How is the set-point determined at each region?

Adapted from Hillam (1996)
e - eset ( x) Detissue ( x)
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Proximal Stimuli due to Loading

• Tissue level strains will produce stimuli
  perceptible to bone cells
• Bone cells likely integrate multiple stimuli to
  determine a response

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Cellular Mechanoreception

• Cells contain specialized structures to detect
  mechanical stimuli
• Most mechanoreceptors require cellular
  deformation to transduce the signal

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Cellular Response to Loading

• Cellular functions to remodel the bone is
  controlled by a feedback loop

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Cellular Adaptation Hypothesis

• Cell deformation depends on cell siffness, K
• Mechanoreception also depends on cell stiffness

d(K) - dmin Ddcell (K)
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Cellular Adaptation Hypothesis

• Cells may tune their stiffness to match the local
  habitual loads
• Cells may determine the tissue level strain
  set-point by adapting to their mechanical
  environment

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Evidence of Mechanical Adaptation

• Endothelial cells stiffen in response to shear
  stress Sato et al. (1999) J Biomech 33127
• Fibroblasts functionally adapt in response to
  loading Glogauer (1997) J Cell Sci 11011

Dt
30 min
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Goals

• To investigate the mechanical adaptation of
  osteoblasts to habitual loads in their mechanical
  environment.
• 1. Determine how cell mechanical properties
  change under varying habitual loads
• 2. Develop a microstructural model to describe
  how the mechanical adaptation is correlated to
  cytoskeletal rearrangement

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Approach

• Using two methods to investigate the mechanism of
  cellular adaptation

AFM Experiments Measure mechanical properties of
osteoblasts
Develop microstructural model for the cell
Phenomenological models of mechanical behavior
and adaptation
Parameter studies
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Use of AFM

• Cells loaded with the AFM
• Collect force-deformation data
• Describe mechanical behavior with
  phenomenological model

Diagram adapted from Marks et al. (1988)
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Cytoskeletal Microstructure
Scale bar represents 5mm
Hartwig
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Cytoskeletal Components
Actin Microfilaments
Intermediate Filaments
Microtubules
Idown et al. The Histochemical Journal (2000)
32165 Scale bar represents 5mm
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Actin Microfilaments

• Thin, flexible filaments 7nm in diameter
• Highly dynamic
• Present in a 3-D gel throughout the cytoplasm
• Organized by over 60 accessory proteins
• Primarily concentrated in structures such as
  stress fibers and cytoskeletal cortex

Diagrams from Alberts (2002) Mol Biol Cell
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Microtubules

• Thick, rigid tube 25 nm in diameter
• Highly dynamic
• Present throughout the cytoplasm
• Higher order structures are not observed

Diagrams from Alberts (2002) Mol Biol Cell
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Intermediate Filaments

• Semi-flexible filaments 10nm in diameter
• Very stable proteins
• Forms a 3-D gel throughout the cytoplasm
• Protects the cell from overloading

Diagrams from Alberts (2002) Mol Biol Cell
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Comparison of Mechanical Properties
The concentration of each filament was 2mg/mL
Graph adapted from Janmey (1991) J Cell Biol
113155l
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Cross-linkers

• The filaments of the cytoskeleton are connected
  by cross-linking proteins
• Some cross-linkers are stable, others are dynamic

Svitkina et al. (1996) J Struct Bio 115290 Scale
bar represents 100nm
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Classes of Cross-linkers

• Cross-linking proteins may be loosely classified
  into 2 categories

Network Forming
Bundling
Arp 2/3 filamin sepectrin
a-actinin fascin
1
1
1
2
Scale bars represent 100nm
1 Svitkina et al. (1996) J Struct Bio 115290 2
Wagner et al. (2001) Biochem J 355771
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Comparison of Mechanical Properties

• Cross-linkers enhance the mechanical properties
  of cytoskeletal filaments
• Cross-linkers interact synergistically
• In situ, functions of most cross-linkers are
  redundant

Chart adapted from Tseng at al. (2001) J Mol Biol
310351 and Tseng et al. (2002) J Biol Chem
27725609
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Spatial Distribution

• The cytoskeleton is a heterogeneous structure

F-actin in sparse region of cytoskeleton
Dense region of the cytoskeleton
Scale bar represents 100nm
Scale bar represents 200nm
Photos Svitkina et al. (1996) J Struct Bio
115290
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Current Models

• Several microstructural models exist to describe
  the mechanical properties of the cell
• Tensegrity Igber (1993) J Cell Sci 104613
• Cellular Solid Satcher et al. (1996) Biophys J
  71109
• Percolation Theory Forgacs (1995) J Cell Sci
  1082131
• Each model captures certain aspects of the cells
  mechanical behavior
• None are specifically designed to describe
  mechanical adaptation

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General Modeling Strategy

• Develop a constitutive model using physical
  chemistry to describe filament interactions
• The biological parameters can change to reflect
  mechanical adaptation

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Statistical Mechanics

• Use a model for semi-dilute, semi-flexible
  polymer networks in solution Panyukov and Rabin
  (1996) Phys Rep 2691
• Model inputs
• Averages of filament length, flexibility and
  orientation
• Average cross-link density
• Constitutive equation is based on filament and
  solvent interactions

Diagram adapted from Panyukov et al. (1996)
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Future Models

• Validate model using data from AFM experiments
• Use the model to perform parameter studies to
  determine
• the effects of heterogeneity
• the contribution of cytoskeletal filaments
• the effect of cross-linkers
• Predict the cytoskeletal biology that contributes
  to mechanical adaptation
• Predict cell stiffness in vivo to determine the
  role of cells in regulate bone remodeling

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Acknowledgements

• David Steigmann, PhD.
• Michael Yu
• National Science Foundation BES-0201951
• Whitaker Foundation