Impact of degradable macromer content in a poly(ethylene glycol) hydrogel on neural cell metabolic activity, redox state, proliferation, and differentiation

Abstract

Hydrogels that degrade at different rates were prepared by copolymerizing slowly degrading macromer poly(ethylene glycol) (PEG) dimethacrylate with a faster degrading macromer poly(lactic acid)-b-PEG-b-poly(lactic acid) dimethacrylate. A clinically relevant population of neural cells composed of differentiated neurons and multipotent precursor cells was cultured within hydrogels. Within 2 h after encapsulation, metabolic activity was higher in hydrogels prepared with increasing levels of degradable content. This improvement was accompanied by a reduction in intracellular redox state and an increase in the fraction of glutathione in the reduced state, both of which persisted throughout 7 days of culture and which may be the result of radical scavenging by lactic acid. Importantly, an increase in cellular proliferation was observed in gels prepared with increasing degradable macromer content after 7 days of growth without a shift in the cellular composition of the culture toward the glial cell phenotype. The findings of this study provide additional insight into the growth of neural cells in PEG-based hydrogels. Results suggest that lactic acid released during gel degradation may impact the function of encapsulated cells, a finding of general interest to biomaterials scientists who focus on the development of degradable polymers for cell culture and drug delivery devices.

FIG. 1.

Degradation and macroscopic properties of hydrogels prepared with poly(ethylene glycol)-DM (0% degradable) and different levels of poly(ethylene glycol)-poly(lactic acid)-DM (degradable) macromer. Mass loss (a), compressive modulus (b), and swelling ratio (c) vary with degradable macromer content. *Values significantly different from the 0% degradable condition (*p < 0.01). Compressive modulus of 100% condition was immeasurable at 7 days due to extensive degradation.

FIG. 2.

Total ATP content in hydrogels prepared with different levels of degradable macromer expressed as percent of the 0% degradable condition at the 0 h time point. Cells incorporated into higher percent degradable hydrogels do not experience the same loss of viability as cells incorporated into a nondegrading (0%) hydrogel, which decrease in viability over time. Values significantly different from the time-matched 0% degradable condition are indicated as follows: *p < 0.05; ^#p < 0.01.

FIG. 3.

Intracellular redox state in hydrogels prepared with different levels of degradable macromer expressed as percent of the 0% degradable condition at each time point. Increases in degradability reduced the intracellular redox state of cells incorporated into the hydrogel, with a sustained reduction for up to 7 days. Values significantly different from the time-matched 0% degradable condition (p < 0.0001) are indicated with asterisks (*).

FIG. 4.

Fraction of glutathione (GSH) in the reduced state for hydrogel cultures prepared with different levels of degradable macromer. This fraction of GSH in the reduced state for hydrogel cultures prepared with different levels of degradable macromer indicates that the higher levels of degradable hydrogel increase the reduced GSH levels at all time points. Values significantly different from the 0% degradable condition at each time point are indicated with asterisks (*) (p < 0.05).

FIG. 5.

Total DNA content in hydrogel cultures prepared with different levels of degradable macromer after 7 days of growth. DNA content was used as a measure of proliferation as all hydrogels were initially loaded equally. Cell proliferation elevated when cells were cultured with a hydrogel that was at least 30% degradable, as indicated by a higher percent of DNA compared with the 0% degradable cultures. Data are expressed as a percentage of the level present in 0% degradable hydrogels. *Statistical difference from control values (p < 0.05).

FIG. 6.

Cell morphology in hydrogel culture. On day 1 (a) or day 7 (b) cells in degradable hydrogels were observed with fluorescent live (green)–dead (red) indicators. Initially, single cells are present in gel cultures. On day 7 small clusters are apparent (c). Immunocytochemistry performed on gel cultures demonstrates that these clusters are composed of neurons (green) and glial cells (red). Nuclei are counterstained with sytox green (blue). Cell behavior in hydrogels of different degradable content is similar to that pictured here for degradable gels at these time points. Color images available online at www.liebertonline.com/ten.