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Research Papers

Experiments in Nanomechanical Properties of Live Osteoblast Cells and Cell–Biomaterial Interface

[+] Author and Article Information
Rohit Khanna, Dinesh R. Katti

Department of Civil Engineering,  North Dakota State University, Fargo, ND 58105

Kalpana S. Katti1

Department of Civil Engineering,  North Dakota State University, Fargo, ND 58105kalpana.katti@ndsu.edu

1

Corresponding author.

J. Nanotechnol. Eng. Med 2(4), 041005 (Apr 04, 2012) (13 pages) doi:10.1115/1.4005666 History: Received May 23, 2011; Revised June 20, 2011; Published March 30, 2012; Online April 04, 2012

Characterizing the mechanical characteristics of living cells and cell–biomaterial composite is an important area of research in bone tissue engineering. In this work, an in situ displacement-controlled nanoindentation technique (using Hysitron Triboscope) is developed to perform nanomechanical characterization of living cells (human osteoblasts) and cell–substrate constructs under physiological conditions (cell culture medium; 37 °C). In situ elastic moduli (E) of adsorbed proteins on tissue culture polystyrene (TCPS) under cell culture media were found to be ∼4 GPa as revealed by modulus mapping experiments. The TCPS substrates soaked in cell culture medium showed significant difference in surface nanomechanical properties (up to depths of ∼12 nm) as compared to properties obtained from deeper indentations. Atomic force microscopy (AFM) revealed the cytoskeleton structures such as actin stress fiber networks on flat cells which are believed to impart the structural integrity to cell structure. Load-deformation response of cell was found to be purely elastic in nature, i.e., cell recovers its shape on unloading as indicated by linear loading and unloading curves obtained at 1000 nm indentation depth. The elastic response of cells is obtained during initial cell adhesion (ECell, 1 h, 1000 nm  = 4.4–12.4 MPa), cell division (ECell, 2 days, 1000 nm  = 1.3–3.0 MPa), and cell spreading (ECell, 2 days, 1000 nm  = 6.9–11.6 MPa). Composite nanomechanical responses of cell–TCPS constructs were obtained by indentation at depths of 2000 nm and 3000 nm on cell-seeded TCPS. Elastic properties of cell–substrate composites were mostly dominated by stiff TCPS (EBulk  = 5 GPa) lying underneath the cell.

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Figures

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Figure 3

AFM height image (2 μm × 2 μm) of 24 h cell culture media soaked TCPS sample (a) enclosing 1 μm × 1 μm scratched regions. Surface asperities of unscratched and scratched regions are shown by section analysis (b). Smooth surface profile of unscratched regions indicates uniform distribution of adsorbed components of culture media. Height of adsorbed components is indicated by the height difference between arrows.

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Figure 4

Modulus maps (5 μm × 5 μm) of dry TCPS (a) and 24 h soaked TCPS (b) indicating the spatial distribution of elastic moduli of sample surface. (c) Modulus versus position plot of dry and soaked TCPS is plotted along the line profile in modulus maps. Nanoscale elastic moduli of dry TCPS vary in the range of 4.7–20 GPa and that of adsorbed components of culture media on soaked TCPS fall in the range of 2.3–8 GPa.

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Figure 5

AFM height image (125 μm × 125 μm) of cells on TCPS after 1 h of culture. Cells are observed to be of varying sizes and shapes. Small cell sizes indicate the initial cell adhesion phase of cell cycle (a); 3D AFM height image (50 μm × 50 μm) of single cell indicating the topographical variations within the cell body (b); Cell height varies from ∼200 to 300 nm at edges to a maximum of ∼3 μm observed near the center of cell body.

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Figure 6

AFM height image (35 μm × 35 μm) of cells on TCPS after 2 days of culture. Polygonal cell morphology is indicative of good cell adhesion with the substrate; fine subcellular web-like structures of cytoskeleton are formed of bundles of actin stress fibers running parallel to each other (a); 3D AFM height image (52 μm × 52 μm) indicating the distinct topography and parallel banded structure of stress fibers near the cell periphery (b).

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Figure 7

Elastic moduli of dry (a) and 24 h (b) soaked TCPS plotted as a function of indentation depth. Vertical bars indicate the spread in elastic moduli. Median elastic modulus is indicated on the vertical bar at each depth. The spread in E decreases as indentation depth increases from 40 nm to 500 nm. E40 nm and E500 nm indicate the surface and bulk elastic moduli, respectively. Large spread in elastic moduli of TCPS is obtained at shallow depths. Elastic moduli of soaked TCPS at shallow depths indicate the composite responses of adsorbed components of culture media and TCPS (b).

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Figure 8

Optical micrographs of osteoblasts on TCPS substrate after 1 h (a) and 2 days (b) of culture. Dot-like features are the cells in the initial stages of cell adhesion phase (a). Polygonal morphology is a characteristic of flat osteoblasts, indicating good cell adhesion with TCPS (b). Larger dot-like features (CM ) in (b) seem to be the cells in the mitosis phase of cell cycle. Typical signatures of dividing cells are marked in (b).

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Figure 9

Representative LD curves obtained on live osteoblast cells at 1000 nm displacement after 1 h and 2 days of culture (a). Linear loading and unloading curves on cell indicate complete elastic recovery. Cell–TCPS composite indentation response is described by LD curve obtained at 2000 nm displacement (b). Flat portion of the loading/unloading indicates the cell indentation response and steep loading slope beyond 1250 nm displacement indicates the stiffer response due to TCPS substrate lying underneath the cell. Flat portion of the loading and unloading portions of LD curve in (b) are plotted separately in (c) and (d), respectively. Significant forces have been measured during unloading indicating that cell was not punctured in a cell–substrate indentation experiment.

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Figure 10

Elastic moduli of live osteoblast cells, cell–substrate composites, and virgin TCPS substrates obtained at displacements of 1000 nm at a loading and unloading rate of 100 nm/s after 1 h (a) and 2 days (b) of culture. Elastic moduli are plotted on a log scale. Each bar indicates the elastic modulus of a single nanoindentation experiment. Optical micrographs shown as inset in the plot describe the round cell morphology indicating initial stages of cell adhesion (a) and flat and mitotic cells (b).

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Figure 11

Elastic moduli of live osteoblasts cells, cell–substrate composites, and virgin TCPS substrates obtained at displacements of 2000 nm at a loading and unloading rate of 100 nm/s after 1 h (a) and 2 days (b) of culture. Elastic moduli are plotted on a log scale. Each bar indicates the elastic modulus of a single nanoindentation experiment. Optical micrographs shown as inset in the plot describe the round cell morphology indicating initial stages of cell adhesion (a) and flat and mitotic cells (b).

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Figure 12

Elastic moduli of 1 h and 2 days cultured osteoblasts and cell–TCPS composites are plotted as a function of indentation depths. Vertical bars indicate the spread in elastic moduli. Median elastic modulus is indicated on vertical bar at each depth. Elastic moduli of live osteoblasts decrease with an increase in indentation depth (a, b). Spread in cell–substrate composite elastic moduli indicates the variation in cell–substrate indentation responses due to varying cell height over the cell body (c, d).

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Figure 2

Experimental setup for in situ nanoindentation experiments in a fluid environment using Hysitron Triboscope nanomechanical instrument at Advanced Materials Lab, North Dakota State University, Fargo, ND

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Figure 1

Schematic of sectional view of the submerged indenter tip-sample assembly (a); top view of the submerged sample chamber inside the fluid cell used for in situ nanoindentation tests (b); snapshot of O-ring glued onto the steel disc (c); the sample glued onto the steel disc and the whole sample chamber filled with fluid (d)

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