Cellular Response to Bis-GMA/TEGDMA Vinyl Conversion Gradients

Introduction

Experimental Approach

Resins of Bis-GMA and TEGDMA (1.8:1.0 mass ratio) with a camphorquinone-based photoinitiator system were cured (60 s per side) in a Teflon mold (55 mm x 12 mm x 1.5 mm) between two glass slides using a visible light curing unit.

For centered, overlaid fabrication, samples were centered on the sample stage and overlaid with a mask. For off-centered fabrication, samples were placed off-centered on the stage.

Masks to create centered, overlaid conversion gradients were printed on transparency film using a laser printer.

Using near infrared (NIR) spectroscopy, conversion was calculated as the reduction in =C–H absorption (6164 cm^-1) normalized to the aromatic absorption (4623 cm^-1).

Resins were sterilized in 70 % volume fraction ethanol, pre-incubated in growth medium for 24 h, and seeded with 5 x 10⁵ Raw 264.7 macrophages. Control cells were seeded on tissue culture polystyrene (TCPS). After incubating at 37 °C for 24 h, cells were incubated for 5 min with 2 µmol/L ethidium homodimer-1 and 2 µmol/L calcein AM in phosphate buffered saline and imaged using fluorescence microscopy.

Conversion Gradients and Corresponding Cellular Response

NIR spectroscopy (above) confirmed the presence of conversion gradients for both fabrication methods (below). Error bars represent one standard deviation and are the estimate of standard uncertainty. The higher standard deviations in the centered, overlaid group are likely due to printer inconsistencies.

The centered, overlaid technique combined the light source and mask to create resins with the highest conversion in the center. In the off-centered technique, the sample edge was aligned with the center of the stage to result in the highest conversion at that edge and the lowest conversion at the opposite edge. Additional conversion profiles can be produced by adjusting the mask design, cure time, or sample placement.

Preliminary cell studies revealed that macrophage spreading was reduced on the resin, especially at lower conversion levels. Cells on a resin fabricated using the centered, overlaid technique were found to have the highest viability in the center, where the highest vinyl conversion was located (above). Viable cells appeared green, and cells with compromised cell membranes fluoresced red.

Studies to quantify the inflammatory response as a function of resin conversion level are ongoing.

The following funding is gratefully acknowledged: NIST/NIDCR Interagency Agreement (Y1-DE-1021-04), NRC Postdoctoral Research Associateship to N.J.L.

NIST Contributors:

Collaborators: