Free-radical polymerization is widely used in the field of tissue engineering to fabricate cell-encapsulating hydrogels. This method enables gelation of a solution of polymer and photoinitiator in the presence of cells via exposure to light. Photoinitiators could have use as models of oxidative stress, as their ability to initiate reactive oxygen species (ROS) production upon light exposure ensures control over the temporal concentration of free radicals. Here we characterize the toxicity and utility, as oxidative stress models, of photoinitiators on oligodendrocyte progenitor cells (OPCs). OPCs are resident cells of the brain that mature into myelinating oligodendrocytes. OPC viability decreased dose-dependently in response to the photoinitiators LAP and I2959 (Figure 1A). The LC
50 of lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) and Irgacure 2959 (I2959) was 0.42 and 4.0 mM, respectively. Pre-treatment with N-acetylcysteine (NAC) protects OPC viability and morphology from LAP and I2959 toxicity (Figure 1B). LAP-exposed OPCs exhibited a low value of process extensions per cell of 0.4, which was held steady from 6 to 48 hours post-photoinitiation. Nuclear particles per cell, a measure of nuclear fragmentation, increased from 1.2 to 1.5 from 24 to 48 hours post-photoinitiation. NAC rescued the LAP-mediated temporal loss of OPC process extensions and nuclear fragmentation, yielding a 63 percent increase and a 13 percent decrease in processes per cell and nuclear fragmentation after 48-hour LAP exposure, respectively (Figure 1C). Thus, NAC rescue treatment attenuates the photoinitiator-mediated temporal changes in OPC growth, morphology and apoptosis. To model continuous oxidative stress, photoinitiated LAP was dosed once daily for two days. Pre-treatment with NAC microparticles increased OPC viability and decreased nuclear fragmentation after continuous LAP exposure. This work shows that photoinitiated I2959 and LAP model oxidative stress. NAC protects OPC viability, morphology and nuclear fragmentation from LAP exposure. NAC, a cheap water-soluble antioxidant, can be used to protect cells from harmful photoinitiators, commonly used by biomaterials engineers.
Figure 1. NAC protects OPCs against photoinitiator-mediated toxicity. (A) OPCs were grown for 24 hours then exposed to 0.01-50 mM Irgacure or LAP with a 10-minute UV light exposure. OPCs were lysed and ATP/DNA was measured 24 hours after photoinitiation. (B) OPCs were grown for 24 hours, incubated for 30 minutes with 1 mM free NAC, then administered 0.55 mM LAP with a 10-minute UV light exposure. OPCs were lysed and ATP/DNA was measured 24 hours after photoinitiation. (C) Photoinitiated 0.55 mM LAP was incubated with OPCs for 48 hours with or without 25 mM NAC treatment administered 15 minutes after photoinitiation. OPCs were fixed, stained with 4′,6-diamidino-2-phenylindole (DAPI), and mounted at 6, 24, and 48 hours after photoinitiation.
