The effect of substrate stiffness, thickness, and cross-linking density on osteogenic cell behavior

Abstract

Osteogenic cells respond to mechanical changes in their environment by altering their spread area, morphology, and gene expression profile. In particular, the bulk modulus of the substrate, as well as its microstructure and thickness, can substantially alter the local stiffness experienced by the cell. Although bone tissue regeneration strategies involve culture of bone cells on various biomaterial scaffolds, which are often cross-linked to enhance their physical integrity, it is difficult to ascertain and compare the local stiffness experienced by cells cultured on different biomaterials. In this study, we seek to characterize the local stiffness at the cellular level for MC3T3-E1 cells plated on biomaterial substrates of varying modulus, thickness, and cross-linking concentration. Cells were cultured on flat and wedge-shaped gels made from polyacrylamide or cross-linked collagen. The cross-linking density of the collagen gels was varied to investigate the effect of fiber cross-linking in conjunction with substrate thickness. Cell spread area was used as a measure of osteogenic differentiation. Finite element simulations were used to examine the effects of fiber cross-linking and substrate thickness on the resistance of the gel to cellular forces, corresponding to the equivalent shear stiffness for the gel structure in the region directly surrounding the cell. The results of this study show that MC3T3 cells cultured on a soft fibrous substrate attain the same spread cell area as those cultured on a much higher modulus, but nonfibrous substrate. Finite element simulations predict that a dramatic increase in the equivalent shear stiffness of fibrous collagen gels occurs as cross-linking density is increased, with equivalent stiffness also increasing as gel thickness is decreased. These results provide an insight into the response of osteogenic cells to individual substrate parameters and have the potential to inform future bone tissue regeneration strategies that can optimize the equivalent stiffness experienced by a cell.

Figure 1

(A) Schematic showing wedge-shaped gel formation. Gels ranged from a height of 0 to 150 μm across a horizontal distance of 50 mm. (B) Gel height was confirmed by recording the change in the vertical position of the microscope relative to the stage required to focus on the top surface of the gel.

Figure 2

(A) Schematic of an MC3T3-E1 cell applying force to a fibrous substrate. The force applied by the cell is transferred through the focal adhesion complexes and resisted by the gel. (B) Boundary conditions placed on micromechanics model. A shear load of 1 N was applied to the nodes to simulate the forces exerted by the cell at the cell-substrate interface. To see this figure in color, go online.

Figure 3

Representative images of MC3T3-E1 cells on polyacrylamide gels of (A) 600 Pa, (B) 9.6 kPa, and (C) 38.4 kPa and collagen gels cross-linked with (D) 0 mM EDAC, (E) 50 mM EDAC, and (F) 150 mM EDAC. White arrows on (A) indicate multiple cells grouped together; and (G) cell spread area of MC3T3-E1 cells cultured on polyacrylamide gels of ∼70 μm. (a) Indicates statistically higher than 0.6 kPa to 4.8 kPa gels. (b) Indicates statistically higher than 0.6 kPa to 9.6 kPa gels. (c) Indicates statistically higher than 0.6 kPa to 19.2 kPa gels. p < 0.05. To see this figure in color, go online.

Figure 4

Cell spread area of MC3T3-E1 cells cultured on wedge-shaped soft (1.2 kPa) polyacrylamide gels of various thicknesses. (a) Indicates statistically difference to cells cultured on 1 μm thick gel. (b) Indicates statistical difference to cells cultured on 3 μm thick gel. p < 0.05.

Figure 5

Cell area (μm²) of MC3T3-E1 cells cultured on wedge-shaped crosslinked collagen substrates. The average cell area at relevant thickness for each gel is presented. The average cell area on flat gels (∼70 μm thick) is represented by the respective markers for each concentration of crosslinking agent. To see this figure in color, go online.

Figure 6

ALP activity normalized to DNA content measured in cells cultured on (A) collagen gels and (B) polyacrylamide gels, for 7 days. (a) Indicates significantly higher activity than cells cultured on PA gels of 4.8 kPa and below. (b) Indicates significantly higher activity than cells cultured on PA gels of 9.6 kPa and below. p < 0.05.

Figure 7

Equivalent shear stiffness of nonfibrous polyacrylamide and fibrous cross-linked collagen gels of different thicknesses as calculated using ABAQUS software. To see this figure in color, go online.

Figure 8

Force is transferred mainly through the fibers, which span the entire gel depth in a noncross-linked fibrous gel (A), whereas force is transferred through multiple adjoined fibers in a fully cross-linked gel (B). To see this figure in color, go online.