Saline-polyethylene glycol blends preserve in vitro annulus fibrosus hydration and mechanics: An experimental and finite-element analysis

Precise control of tissue water content is essential for ensuring accurate, repeatable, and physiologically relevant measurements of tissue mechanics and biochemical composition. While previous studies have found that saline and polyethylene glycol (PEG) blends were effective at controlling tendon and ligament hydration levels, this work has yet to be extended to the annulus fibrosus (AF). Thus, the first objective of this study was to determine and validate an optimal buffer solution for targeting and maintaining hydration levels of tissue-level AF specimens in vitro. This was accomplished by measuring the transient swelling behavior of bovine AF specimens in phosphate-buffered saline (PBS) and PEG buffers across a wide range of concentrations. Sub-failure, failure, and post-failure mechanics were measured to determine the relationship between changes in tissue hydration and tensile mechanical response. The effect of each buffer solution on tissue composition was also assessed. The second objective of this study was to assess the feasibility and effectiveness of using multi-phasic finite element models to investigate tissue swelling and mechanical responses in different external buffer solutions. A solution containing 6.25%w/v PBS and 6.25%w/v PEG effectively maintained tissue-level AF specimen hydration at fresh-frozen levels after 18 h in solution. Modulus, failure stress, failure strain, and post-failure toughness of specimens soaked in this solution for 18 h closely matched those of fresh-frozen specimens. In contrast, specimens soaked in 0.9%w/v PBS swelled over 100% after 18 h and exhibited significantly diminished sub-failure and failure properties compared to fresh-frozen controls. The increased cross-sectional area with swelling contributed to but was not sufficient to explain the diminished mechanics of PBS-soaked specimens, suggesting additional sub-tissue scale mechanisms. Computational simulations of these specimens generally agreed with experimental results, highlighting the feasibility and importance of including a fluid-phase description when models aim to provide accurate predictions of biological tissue responses. As numerous previous studies suggest that tissue hydration plays a central role in maintaining proper mechanical and biological function, robust methods for controlling hydration levels are essential as the field advances in probing the relationship between tissue hydration, aging, injury, and disease.