L24 - Investigating How Mechanical Properties of the Microenvironment Influence Liver Function in Aging

Introduction: : 1. The liver, the body’s largest internal organ, plays a pivotal role in metabolism, detoxification, and biosynthesis. As individuals age, the liver undergoes various changes that impact its function. These changes include a reduction in metabolic efficiency, the induction of hepatocyte senescence, the accumulation of extracellular matrix (ECM) through fibrosis, and increased mitochondrial dysfunction. Tissue engineering, particularly scaffold-based strategies utilizing materials such as gelatin methacrylate (GelMA), has facilitated the development of three-dimensional liver models that replicate native ECM properties and enable the study of aging processes. Scaffold stiffness, which can be adjusted through UV crosslinking, influences cell behavior through mechanotransduction pathways. As individuals age, liver stiffness increases, and engineered models with tunable mechanics facilitate the investigation of age-related dysfunctions, fibrosis progression, and drug responses. This study employed photocrosslinkable GelMA scaffolds to explore the impact of mechanical properties on the proliferation, morphology, senescence, and function of young and artificially aged hepatocytes.

Materials and

Methods: : 2. To induce the aged phenotype, a scheduled hydrogen peroxide (H2O2) treatment was administered for 7 (200 µM) and 15 (100 µM) days with media changes every 48 h. Cell morphology was observed after each respective treatment period. Soft and stiff scaffolds were then constructed through varying UV exposure time (30 s for soft and 60 s for stiff) of GelMA and compressive mechanical testing using an ADMET machine was used to confirm the results. In this study, compressive moduli were determined to be 7.1 and 13.1 kPa from 0 to 5% strain, with these values serving as the primary indicators of stiffness. Disc-shaped tissue constructs were fabricated through UV irradiation of HepG2 cells encapsulated in 10% GelMA solution. Control (healthy) models were polymerized for 30 s, while models encapsulating aged cells were polymerized for 60 s. The impact of this fabrication method on cell viability was assessed using a live/dead assay. In both 2D and 3D microenvironments, several aging parameters were characterized. Cells were maintained and encapsulated within GelMA for three or ten days to investigate the impact of the respective microenvironments. In 2D, β-galactosidase (SA-β-Gal) histochemical staining was used to confirm the senescent phenotype, while a fluorometric SA-β-Gal assay was conducted exclusively on 3D samples. Additionally, oxidative stress levels were quantified by measuring the mean fluorescent intensity (MFI) of each sample produced by a fluorometric reagent. Furthermore, hepatocyte function was characterized by albumin secretion, where the growth medium from 2D and 3D environments underwent Human albumin ELISA analysis.

Results, Conclusions, and Discussions:: 3. In 2D cultures, untreated HepG2 cells exhibited epithelial morphology, while H2O2-treated cells adopted elongated, spicule-like forms with reduced size and proliferation, particularly after prolonged exposure. These morphological changes correlated with increased senescence, confirmed via β-galactosidase staining. In 3D cultures, both young and aged HepG2 cells formed spheroids by day 5 in both soft and stiff GelMA, exhibiting controlled proliferation. Viability was slightly reduced in aged cells due to UV irradiation but remained high (at 89%). Oxidative stress, quantified by MFI, was elevated in aged samples across both culture systems, particularly after 15 days of treatment, and cells exposed to the 10-day stiff microenvironment exhibited the highest levels of oxidative stress (Figure 1). Surprisingly, some control samples exhibited higher senescence than aged ones, possibly due to the mechanosensitive characteristic of senescence and residual photoinitiator levels from the crosslinking process. Senescence-associated β-galactosidase activity was higher in 2D-aged cells, compared to their controls, confirming the efficacy of H2O2 in inducing senescence. However, in 3D systems, normalized senescence markers were unexpectedly lower in aged samples encapsulated for 10 days in the stiff microenvironment, suggesting additional environmental or biomaterial influences on aging markers. Albumin secretion, a marker of liver-specific function, was paradoxically elevated in treated 2D samples, with no discernible differences observed in 3D microenvironments. This phenomenon was attributed to H2O2-induced intracellular calcium mobilization, which was hypothesized to facilitate secretion rather than inhibit synthesis. In summary, H2O2 effectively induced senescence in HepG2 cells, influencing morphology, viability, and function. While 2D cultures clearly displayed aging signatures, 3D results highlight the complex interplay between mechanical cues, oxidative stress, and biochemical signaling, suggesting the need for more refined models to better replicate liver aging in vivo. However, our findings still present implications for potential future personalized regenerative therapies.

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