Elastic membrane that undergoes mechanical deformation enhances osteoblast cellular attachment and proliferation

Abstract

The main objective of this paper was to investigate the effect of transmission of force on bone cells that were attached to a deformable membrane. We functionalized a silastic membrane that measured 0.005 inches thickness and coated it with an extra cellular matrix (ECM) protein, fibronectin (FN). MC3T3-E1 osteoblast-like cells were cultured on the functionalized FN-coated membrane after which cell attachment and proliferation were evaluated. We observed an immediate attachment and proliferation of the bone cells on the functionalized membrane coated with FN, after 24 hours. Upon application of a mechanical force to cells cultured on the functionalized silicone membrane in the form of a dynamic equibiaxial strain, 2% magnitude; at 1-Hz frequency for 2 h, the osteoblast cells elicited slightly elevated phalloidin fluorescence, suggesting that there was reorganization of the cytoskeleton. We concluded from this preliminary data obtained that the engineered surface transduced applied mechanical forces directly to the adherent osteoblast cells via integrin binding tripeptide receptors, present in the FN molecules, resulting in the enhanced cellular attachment and proliferation.

Figure 1

Schematic diagram showing the four-point bending principle. The principle of low strain (equibiaxial strain) cell stretching, using the four-point bending system.

Figure 2

(a) Characterization of Si membrane using the water contact goniometry method. The figure shows the degree of surface wettability of Si membranes after exposure to UVO radiations at different times. (b) Characterization of Si membrane using Rutherford Backscattering spectrophotometry. Figure shows similar profiles for all the samples of Si membranes after exposure to UVO radiations at 4 different times.

Figure 3

(a) Representative AFM images of a raw and unclean Si membrane surfaces. Roughness analysis done on a 10 μm scan size of the images indicated that the Surface roughness (Ra) on these surfaces was 0.8 nm; while the RMS roughness value was 1.0 nm. (b) Representative AFM images of a non-UVO-activated water-cleaned Si membrane surface. Roughness analysis on a 10 μm scan size of the images indicated that the Surface roughness (Ra) on these surfaces was 1.2 nm; while the RMS roughness value was 1.4 nm. (c) Representative AFM images of a 10 min UVO-activated Si membrane surface. Roughness analysis on a 10 μm scan size of the images indicated that the Surface roughness (Ra) on these surfaces was 1.9 nm; while the RMS roughness value was 2.4 nm. (d) Representative AFM images of a 30 min UVO-activated Si membrane surface. Roughness analysis on a 10 μm scan size of the images indicated that the Surface roughness (Ra) on these surfaces was 4.7 nm; while the RMS roughness value was 5.9 nm.

Figure 3

(a) Representative AFM images of a raw and unclean Si membrane surfaces. Roughness analysis done on a 10 μm scan size of the images indicated that the Surface roughness (Ra) on these surfaces was 0.8 nm; while the RMS roughness value was 1.0 nm. (b) Representative AFM images of a non-UVO-activated water-cleaned Si membrane surface. Roughness analysis on a 10 μm scan size of the images indicated that the Surface roughness (Ra) on these surfaces was 1.2 nm; while the RMS roughness value was 1.4 nm. (c) Representative AFM images of a 10 min UVO-activated Si membrane surface. Roughness analysis on a 10 μm scan size of the images indicated that the Surface roughness (Ra) on these surfaces was 1.9 nm; while the RMS roughness value was 2.4 nm. (d) Representative AFM images of a 30 min UVO-activated Si membrane surface. Roughness analysis on a 10 μm scan size of the images indicated that the Surface roughness (Ra) on these surfaces was 4.7 nm; while the RMS roughness value was 5.9 nm.

Figure 4

Surface density characterisation of FN-coated UVO-activated Si membrane surfaces. Up to 60 min UVO-activated surfaces were coated with 2.5 μg/mL concentrations of FN. 2.5 μg/mL of FN was coated on surfaces for 1 hour incubation at 37°C in order to attain monolayer surface coverage [20].

Figure 5

AFM images of 30 min UVO-activated Si membrane surfaces precoated with 2.5 μg/mL FN. The surface roughness of (RMS) 3.7 nm was obtained. Plane view of the figure (left) shows clusters of FN molecules on the activated Si membrane; while the elevated view shows very rough ridges of clustered FN molecules adhering to the Si surfaces.

Figure 6

Phalloidin-stained actin filaments of osteoblasts proliferated on silicone membranes. MC3T3-E1 osteoblast cells were seeded on FN-coated silicone membrane and subjected to equibiaxial strain for 2 h. Cells were treated with rhodamine-labeled phalloidin (1 : 100) and then visualized by confocal microscopy. (a) Cells on: nonstretched non-UVO-activated FN-coated silicone membrane; (b) Cells on: stretched non-UVO-activated FN-coated silicone membrane. Note the slightly elevated fluorescence of the actin filaments in the stretched sample.

Figure 7

Phalloidin-stained actin filaments of osteoblasts grown on silicone membranes. MC3T3-E1 osteoblast cells were seeded on FN-coated silicone membrane and subjected to equibiaxial strain for 2 h. Cells were treated with rhodamine-labeled phalloidin (1 : 100) and then visualized by confocal microscopy. (a) Cells on: nonstretched UVO-activated FN-coated silicone membrane; (b) Cells on: stretched, 30 min UVO-activated FN-coated silicone membrane. Note the slightly elevated fluorescence of the actin filaments in the stretched sample.

Figure 8

Phalloidin-stained actin filaments of osteoblasts grown on silicone membranes. MC3T3-E1 osteoblasts were seeded on FN-coated silicone membrane and subjected to equibiaxial strain for 2 h. Cells were treated with rhodamine-labeled phalloidin (1 : 100) and then visualized by confocal microscopy. Close up of: (a) Cells on nonstretched 30 min UVO-activated FN-coated silicone membrane; (b) Cells on stretched 30 min UVO-activated FN-coated silicone membrane. Note the slightly elevated fluorescence of the actin filaments in the stretched sample.