Development of an elastic cell culture substrate for a novel uniaxial tensile strain bioreactor

Abstract

Bioreactors can be used for mechanical conditioning and to investigate the mechanobiology of cells in vitro. In this study a polyurethane (PU), Chronoflex AL, was evaluated for use as a flexible cell culture substrate in a novel bioreactor capable of imparting cyclic uniaxial tensile strain to cells. PU membranes were plasma etched, across a range of operating parameters, in oxygen. Contact angle analysis and X-ray photoelectron spectroscopy showed increases in wettability and surface oxygen were related to both etching power and duration. Atomic force microscopy demonstrated that surface roughness decreased after etching at 20 W but was increased at higher powers. The etching parameters, 20 W 40 s, produced membranes with high surface oxygen content (21%), a contact angle of 66° ± 7° and reduced topographical features. Etching and protein conditioning membranes facilitated attachment, and growth to confluence within 3 days, of MG-63 osteoblasts. After 2 days with uniaxial strain (1%, 30 cycles/min, 1500 cycles/day), cellular alignment was observed perpendicular to the principal strain axis, and found to increase after 24 h. The results indicate that the membrane supports culture and strain transmission to adhered cells.

Keywords: adhesion; bioreactor; osteoblast; polyurethane; strain; surface modification.

FIGURE 1

Diagram showing complex state of strain (dotted arrows) and fluid shear stress (solid arrows) in a porous scaffold under compression. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]

FIGURE 2

The bioreactor, (a), is shown with two assembled banks, each containing three culture wells. A cross-sectional diagram, (b), through a culture well shows the arrangement of the PU membrane. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]

FIGURE 3

Contact angles for unetched PU, with weighted least square linear fit for plasma etched PU membranes. Data show mean ±SD for n = 3.

FIGURE 4

Surface oxygen and C:O ratio of as-fabricated control and plasma-etched PU samples, as determined by XPS. Data show mean ±SD for n = 6.

FIGURE 5

Relative atomic ratios, for carbon, oxygen, and nitrogen as determined by XPS, of PU membrane that was plasma etched at 50 W. Data show mean ±SD for n = 6.

FIGURE 6

RMS roughness (Rq) of PU across a 25 µm sampling length. Data show mean with SEM for n = 20, and statistical significance with respect to the unetched sample is indicated.

FIGURE 7

AFM height maps for: (a) unetched PU, and PU etched for 40 s at (b) 20 W and (c) 50 W. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]

FIGURE 8

Phase contrast micrographs of MG-63 cells on pre-incubated and non pre-incubated PU substrates at 8 days after seeding. Etching was carried out at 20 W for 40 s.

FIGURE 9

MG-63 cultures imaged by phase-contrast microscopy at selected intervals after seeding on (a)–(d) TCPS, (e)–(h) plasma-etched (50 W, 40 s) PU, and (i)–(l) unetched PU.

FIGURE 10

Phase contrast micrograph of MG-63s after 8 days culture in the bioreactor. The monolayer extends to the edge of the well (indicated by dashed line) where they are in contact with the food grade silicone o-ring.

FIGURE 11

Relative metabolic activity of MG-63s after cyclic stimulation in the bioreactor. Data show mean ±SD for n = 6; TCPS vs PU: P < 0.0001.

FIGURE 12

MG-63s stained for f actin (green) and DNA (blue). (a) Fixed 6 h after initial strain; (b) 6 h after second strain application; and (c) 24 h after second strain application. The principal strain axis is indicated by the arrow. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]

FIGURE 13

Alignment of cells relative to the principal strain axis: (a) 6 h after strain initial strain and (b) 6 h after second strain application at 48 h; and (c) 24 h after second strain application.