Implementation of Reactive Accelerated Aging as an In Vitro Method for Degradation Studies of Polymers

Hydrogels are considered promising candidates for implant technologies and biomedical sensors due to their easily achievable biocompatibility and wide variety of available base polymers [1]. However, in vivo applications necessitate extensive testing to obtain medical clearance. To reduce time, cost and the need for corresponding animal experiments, novel in vitro methods for material degradation studies need to be developed [2]. A promising approach is reactive accelerated aging (RAA). This method subjects materials to harsher-than-life conditions such as elevated temperatures and reactive oxygen species to simulate in vivo degradation under accelerated conditions. Thus, the effects of extended in vivo exposure can be reproduced in a significantly shorter period using RAA. So far, the RAA approach has mainly been applied to neural implants, where results showed a good correlation to animal studies and the acceleration factor has been established [3,4].
The presented study aims at extending the use of RAA towards polymer materials. As a first step, we investigated whether the aging conditions used for neural probes, namely increased temperatures and hydrogen peroxide, are also effective for polymer degradation. Polyacrylamide (PAM) hydrogel, a synthetic stimulus-responsive material, was selected as the model material due to its ability to swell or shrink in response to external stimuli. This reaction is dependent on the structural integrity of the material, and hence, offers an easy way of monitoring structural changes and material degradation.
We have implemented and characterized an automated reactive accelerated aging (aRAA) setup similar to the one described in [3]. It comprises a sample chamber where a base of phosphate buffered saline (PBS) solution with added hydrogen peroxide (H₂O₂) is continuously heated to 67 °C. This initiates the breakdown of H₂O₂ into reactive oxygen species that in turn cause degradation of the sample. Solution temperature, pH, and H₂O₂ concentration are continuously monitored and adjusted if necessary by automated addition of fresh PBS/H₂O₂-mixture during the aging process. The samples are placed in customized 3D-printed holders in the aging chamber to avoid dislocation and for easy removal of samples from the setup.
In initial experiments, we have aged samples for up to 96 hours in five groups of three. Every 24 hours one group was removed from the setup and characterized by optical microscopy (identification of mechanical damages such as cracks) and gravimetry (evaluation of swelling behavior), while the other groups remained in the setup and continued to be aged. All samples were fabricated from the same batch of precursor solution to ensure comparability.
The results clearly indicate that the combination of elevated temperature and hydrogen peroxide is crucial to induce polymer degradation, and neither condition alone suffices. Furthermore, the changes in the swelling properties of the studied PAM hydrogels show the onset of polymer breakdown, evidenced by increased liquid absorption (more pronounced swelling) with prolonged aging.
The next step involves comparing the aRAA results with existing data from animal and human studies and cell-based assays to assess the method's effectiveness in replicating in vivo processes and determining a potential acceleration factor. This comparison will furthermore provide the basis for future refinement of the method, such as the addition of other reactive components like enzymes, or variation of pH and/or temperature during aging, to enhance the replication of in vivo processes and conditions.

[1] Zhang et al., Front. Chem. 8:615665, 2021; doi:10.3389/fchem.2020.615665
[2] Myers et al., ALTEX 34(4):479, 2017; doi:10.14573/ALTEX.1608081
[3] Caldwell et al., Biomat. 232:119731, 2020; doi:10.1016/j.biomaterials.2019.119731
[4] Street et al., Rev. Sci. Instrum. 89(9):094301, 2018; doi:10.1063/1.5024686