N26 - Gut Metabolite Enhanced Cartilage Protection and Regeneration

Introduction: : Osteoarthritis (OA) is a leading cause of disability, affecting over 595 million people worldwide [1]. It is driven by an imbalance of catabolic processes that result in irreversible structural and functional damage to cartilage and osteochondral tissues [2]. As cartilage deteriorates, excessive accumulation of interdependent inflammatory mediators and oxidative stressors activate signaling pathways that perpetuate disease progression [2]. Tissue engineering offers a promising approach for cartilage and osteochondral defect repair and regeneration; however, current strategies often overlook the underlying oxidative and inflammatory mechanisms that drive OA pathogenesis. Therefore, it is critical to develop engineering approaches that not only promote tissue regeneration but also modulate the oxidative and inflammatory microenvironment, with the goal of achieving effective osteochondral repair in the context of OA. In this study, we investigate gut microbial metabolites, particularly Urolithin-A (UA), as a novel immunomodulatory agent for the repair and regeneration of OA cartilage. UA is a potent anti-inflammatory agent derived from ellagitannin-rich foods (e.g., pomegranates and berries) [3]. Our preliminary studies demonstrate that UA exerts strong anti-inflammatory effects while protecting chondrocytes under inflammatory stress in vitro. Building on this, we hypothesize that UA also possesses significant antioxidant activity capable of mitigating oxidative stress and mitochondrial dysfunction, other key drivers of OA pathogenesis. Our objective is to establish UA’s synergetic anti-inflammatory and antioxidant roles to develop an immunomodulatory strategy for cartilage/osteochondral tissue engineering in the context of OA.

Materials and

Methods: : Human articular chondrocytes (C28/I2) were cultured in DMEM media (2.5% FBS and 1% P/S) supplemented with 30µM Hydrogen peroxide (H2O2) to simulate oxidative conditions relevant to OA pathogenesis. Media included H2O2 and UA reintroduced to cultures every 2 days to monitor prolonged exposure of an oxidative stress microenvironment. Our preliminary work established 5µM as an effective dose to attenuate inflammation in chondrocytes and has been studied for this model as well. A dose-dependent study was conducted to assess cell viability (via MTS assay). Nitrite (NO-) production was measured via Greiss assay after prolonged exposure to H2O2. Mitochondrial health was monitored qualitatively via JC-1 Mitochondrial Membrane Potential assay (Cayman Chemicals) as per manufacturer instructions. Quantitative real time PCR (rt-qPCR) was utilized to determine the relative expression of oxidative stress response (Nrf2), crucial chondrogenic (Col2a), pro-inflammatory (IL1B, IL6, IL8, TNFa, PTGS2), and ECM-catabolic (MMP13) genes following 2-, 4-, and 7-day exposure to oxidative conditions. Total RNA isolation utilized Trizol (Invitrogen) and RNeasy Mini kit (Qiagen) following the manufacturer’s protocol. Reverse transcriptase of 1µg RNA was acquired using the iScript cDNA Synthesis Kit (BioRad). RT-qPCR was performed using SsoAdvanced Universal SYBR green (BioRad) and quantification via the 2-ΔΔCT method. GAPDH was utilized as a housekeeping gene and normalized all target genes involved in this study.

Results, Conclusions, and Discussions:: Results and

Discussion: A dose-response study determined that treatment with 30 µM H₂O₂ reduces chondrocyte viability by approximately 50% after 48 hours (Fig 1A). Our optimized UA concentration protects chondrocytes against oxidative stress induced cell death during initial and extended H₂O₂ exposure (Fig 1A-C). Elevated NO- levels contribute to the release of factors that mediate inflammation and apoptosis, thus making it an indicator of damaged cellular microenvironments [4]. Figure 1D shows chondrocytes exposed to H₂O₂ have excessive NO- levels; however, treatment with UA significantly reduced NO⁻ production to levels comparable to healthy, untreated chondrocytes. Cell viability and Griess assay data indicate that UA maintains healthy cell activity following prolonged exposure to oxidative stressors. Accumulation of oxidative stress products suppresses mitophagy, a protective mechanism that removes damaged mitochondria via autophagy-lysosome pathway. JC-1 is a mitochondrial membrane potential (ΔΨM) indicator, where JC-monomers (Green) and JC-aggregates (Red) represent mitochondria depolarization and polarization states. Elevated depolarization indicates mitochondria are dysfunctional. After 4-day exposure to H₂O₂, chondrocytes had increased depolarization as shown by green fluorescence, when compared to control and H₂O₂+UA groups. Alternatively, H₂O₂+UA conditions formed JC-1 aggregates similarly to the control group, showing high ΔΨM polarization (Fig 1E). NRF2 is a chondroprotective gene responsible for suppressing oxidative stress, inflammatory mediators and mitochondrial dysfunction [4]. In this work, H₂O₂+UA treatment preserved NRF2 expression, while maintaining key chondrogenic genes (Col2a) that were otherwise downregulated in the H₂O₂ group (Figure 1F-G). These findings suggest that NRF2 is a significant contributor to UA’s chondroprotective properties, consistent with results from our previously established inflammation model. UA+H₂O₂ treatment also suppressed overproduction of proinflammatory mediators (Fig 2A-E) and ECM-catabolic factors (Fig 2F) in this work. This indicates that our UA treatment acts as an immunomodulator in response to induced inflammation and oxidative stress.

Conclusion: We have developed an oxidative stress model to screen immunomodulators, such as UA. Our data demonstrates UA exerts dual anti-inflammatory and antioxidant effects, protecting chondrocytes from the catabolic microenvironment characteristic of OA. These findings establish a novel tissue engineering strategy leveraging a gut-derived metabolite that effectively promotes tissue protection and regeneration, even within the degenerative environment of OA.

Acknowledgements and/or References (Optional):: Acknowledgements: National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health (R01EB030060 & R01EB020640)

References: (1) Tang et al. Osteoarthritis. Nat Rev Dis Primers 11, 10 (2025).; (2) Yunus et al. Medicina (Kaunas, Lithuania) vol. 56,11 614; (3) Singh et al. Nat Commun 10, 89 (2019).; (4) Ansari et al. Biomed Pharmacother, 2020,129:110452