Abstract
Purpose: :
The success of filtering surgery is mainly limited by excessive wound healing and bleb scarring, which is currently treated by intra- and/or post-operative use of Mitomycin C (MMC) or 5-fluorouracil (5-FU). Glaucoma drainage devices (GDD) pose an opportunity to introduce a drug-eluting mechanism for long term release of low dose antifibrotics. In previous work we showed that early attachment of fibroblasts to the implant surface may contribute to excessive proliferation. In this study we therefore investigate the effects of a modified PNIPAAm hydrogel on cell adhesion and survival of primary human tenon fibroblasts (HTF) in vitro and assess its ability to take up and release the antiproliferative compound paclitaxel.
Methods: :
Standard culture dishes were coated with a 2µm thick layer of modified PNIPAAm hydrogel. Hydrogels were either allowed to swell with 10-6M paclitaxel or PBS alone. HTF cells were seeded into hydrogel coated and uncoated control dishes and incubated at 37°C for 24 hours. Cells were then transferred to uncoated dishes, where proliferation was then measured by Alamar Blue Assay for 7 days. In a separate experiment the rate of apoptosis was determined by flow cytometry after 24, 48 and 72 hours of exposure to hydrogel. As semi-quantitative indicator for cytotoxicity, photomicrographs of partly coated dishes were taken in order to measure a zone of inhibition.
Results: :
Up to 72 hours after seeding, there was no attachment of cells to either paclitaxel-loaded or pure hydrogel. After transfer to uncoated dishes, cells exposed to hydrogel alone grew at the same rate as control (p=0.71). Paclitaxel-loaded hydrogel led to a significant decrease in proliferation (85.8 ± 5.0% of control, p=0.042). The rate of apoptosis was significantly higher in the pure hydrogel group than in control (21.9 ± 2.8% vs. 4.9 ± 0.9%, p=0.026) with a further increase in the paclitaxel-loaded hydrogels (25.2 ± 2.0%, p=0.001). However, there was no zone of inhibition around the hydrogel in the partly coated dishes.
Conclusions: :
Modified PNIPAAm hydrogel appears to be anti-adhesive to HTF cells. Proliferation assays and the lack of a zone of inhibition indicate that the hydrogel is non-toxic. Fibroblasts are anchorage-dependent cells, the increase of apoptotic cells over time may thus be a result of the inability of HTF to attach to the hydrogel. Both proliferation assays and FACS analysis indicate that the hydrogel is capable of uptake and release of paclitaxel. It may therefore be useful for drug-release applications in GDD.
Keywords: wound healing • drug toxicity/drug effects • apoptosis/cell death