Hydrogels are of considerable interest in the development of regenerative therapies for treating disease and injuries of the central nervous system. When prepared from synthetic materials, the rate at which the hydrogel degrades can be controlled, a particularly useful tool as materials that degrade over short or long time scales both have clinical relevance. However, because the rate of hydrogel degradation has been shown to impact the quality of other types of tissue produced within the hydrogel, it is important to first understand how neural tissue develops within hydrogels with different mass loss profiles. Toward this end, the focus of this work was to characterize neural cell growth within hydrogels prepared by combining slow-degrading (nondegradable) macromer with different levels of degradable macromer containing PLA subunits. Over time, degradable cross-links are hydrolyzed, releasing lactic acid until a network composed of only PEG cross-links remains. Consistent with previous reports, as the gel degrades, hydrogels utilized in this work imbibe more water, becoming more open and swollen (mesh size increases) and, as a result, less stiff as reflected by a decrease in compressive modulus ().
In this work, within 2
h of formation an improvement in cell viability and/or metabolic function was observed as degradable content of the hydrogel increased (). In addition, intracellular redox state was more reduced and the level of reduced GSH content was increased in gels containing more degradable macromer. Differences in macroscopic properties are not likely to be responsible for the improvements in cell function observed at this early time point (2
h) as statistical differences in compressive modulus and swelling ratio (thus, hydrogel mesh size) were not present until later (24
h). Instead, the lactic acid product of hydrogel degradation, which is a free radical scavenger47
and can protect cells from damage due to photoinitiator-generated and endogenously present free radicals,38
may positively impact encapsulated cell function. Indeed lactic acid is present during polymerization and is released into the hydrogel culture medium at levels that scavenge radicals and modify intracellular redox state in monolayer culture (>0.005
Effects are likely due only to free lactic acid released from the polymer network. Although the PLA form is too bulky to enter the cell, both lactic acid and the ionized-form lactate are capable of transport into cells. Lactic acid is uncharged and small enough to permeate through the lipid membrane; lactate is capable of entering cells via the monocarboxylate transporter protein shuttle system.48
Once inside the cell, lactic acid is also capable of undergoing oxidation to pyruvate, another potent antioxidant, thus increasing intracellular pyruvate levels. Pyruvate is then capable of decreasing quantities of intracellular ROS, leading to a reduced intracellular redox state and, as a result, an increase in reduced GSH content and ultimately an improvement in cell survival and/or metabolic activity. The transient increase in ATP content observed at the 2
h time point may be the combined effect of lactic acid as well as endogenous upregulation of ATP, as others have documented an increase in ATP content when neural cells are under stress.49
After 7 days of culture, intracellular redox state was more reduced in hydrogels with degradable content () and reduced GSH content () approached preencapsulation levels (46.2
1.0%, data not shown), effects which may be similarly related to the presence of lactic acid. A shift in intracellular redox state has been shown to impact neural precursor cell fate where more oxidized cellular states lead to differentiation and more reduced states lead to self-renewal.42,43
Thus, when exposed to lactic acid released from a biomaterial, neural precursor cells may be protected from damage due to naturally occurring ROS; intracellular redox state may shift to one that is more reduced, thus maintaining cells in a proliferative state.38
At the 7 day time point total DNA content in hydrogels increased with increasing degradable macromer content (). The increase in DNA content may be related to a difference in the number of surviving, proliferative cells present in gels at 24–48
h where conditions with lower degradable content would result in fewer surviving cells and lower DNA content after 7 days of growth. Lactic acid may also directly impact cell proliferation, as entry into the S phase of the cell cycle (DNA replication) is facilitated by the presence of lactate (the ionized form of lactic acid).50
In addition to being exposed to varying levels of lactic acid, at these longer time points cells are gradually exposed to three-dimensional polymer networks of different mechanical strength. To the best of our knowledge, there are no data in the literature to suggest that mechanical properties would directly impact intracellular redox state and reduced GSH content. However, other aspects of neural cell function have been shown to be impacted by mechanical properties. For example, the stiffness of a substrate has been shown to impact cellular proliferation and/or the cellular composition of cultures. In the case of neural cells, when cultured for extended periods of time on two-dimensional surfaces, less stiff surfaces tend to promote the differentiation and/or survival of neurons while discouraging the growth of glia.51
In this work proliferation was improved in gels with reduced stiffness without altering cell composition. Trends similar to what has been reported in the literature were not observed in this work perhaps because the time-scale over which the stiffness of the surrounding polymer network is dramatically lower is confined to a brief window during late stages of culture. Instead, the improvement in proliferation is more likely related to exposure to lactic acid as previously discussed. Importantly the improvement in proliferation is not accompanied by an increase in the growth of glial cells (), which if implanted in the brain tissue could contribute to the formation of a glial scar.
PEG comprised the bulk of the mass of hydrogels prepared in this work with minor mass contributions from the hydrophobic PLA subunits within the degradable crosslinks. Although hydrogels prepared with different levels of degradable macromer may exhibit differences in hydrophobicity, these differences were negligible given the relatively low molar percentage of PLA incorporated (<7
mol%) and the dominant hydrophilicity of the major component, PEG. Thus, differences in hydrophobicity were not likely to substantially underlie the findings reported in this study. When encapsulated in gels with different levels of degradable macromer, the pH of the gel microenvironment is also likely to vary, with more acidic conditions arising in faster degrading gels, as alluded to in previous work.29
In this work, the rate of ATP hydrolysis would have been impacted by a change in pH as ATP has been shown to be rapidly degraded outside of the 6.8 to 7.4
pH range. Because higher levels of ATP were observed in gels containing higher levels of degradable macromer, an increase in the rate of ATP hydrolysis cannot account for trends reported in this study. Thus, hydrogel degradable content must preserve ATP, improving the viability of surrounding neural cells.