Transient Trimethylamine N-oxide Supplementation Confers Chondroprotection to Engineered Cartilage Post-Culture Under Stress Conditions

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1 Transient Trimethylamine N-oxide Supplementation Confers Chondroprotection to Engineered Cartilage Post-Culture Under Stress Conditions Eben G. Estell, BS, Sonal Ravin Sampat, M.S., J. Chloe Bulinski, PhD, Gerard A. Ateshian, Ph.D., Clark T. Hung, PhD. Columbia University, New York, NY, USA. Disclosures: E.G. Estell: None. S.R. Sampat: None. J. Bulinski: None. G.A. Ateshian: None. C.T. Hung: None. Introduction: High concentrations of urea in the tissues of sharks play a role in their ability to osmo-regulate, preventing dehydration in the hypertonic environment of seawater. Trimethylamine N-oxide (TMAO) is thought to counteract the degenerative effects of high urea concentrations by stabilizing protein folding in the cartilaginous shark tissues [1,2]. TMAO has been suggested to confer protection against other conditions of cell stress [3]. Previously, our laboratory successfully investigated the incorporation of TMAO as a culture media supplement for cartilage tissue engineering (applied for the first two weeks of growth, a release schedule shown to be effective with other growth factors)[4]. In the current study, we turn our attention to the potential protective effects of TMAO, and hypothesize that TMAO may serve a chondroprotective role, specifically in restoring tissue growth in engineered cartilage cultivated initially under stress conditions. In this experiment we restrict the availability of nutrients for the first two weeks by using a media-to-cell ratio that is sub-optimal (nearly one-half of that normally used) in order to establish a stress culture condition [5]. We further hypothesize that continuous application of TMAO will yield even higher mechanical and biochemical results compared to control and standard release. Applying TMAO supplementation to cartilage in stressed conditions, and beginning the application of TMAO at different times in the culture will offer insight into the mechanism by which TMAO may serve a chondroprotective role. Methods: Articular chondrocytes were harvested from juvenile bovine knee joints (2-4wk old) and digested for 11 hours at 37 C with Collagenase IV (Worthington). The chondrocytes were expanded for one passage in DMEM containing 10% FBS, 1% PSAM, 10 ng/ml PDGF, 5 ng/ml bfgf, and 5 ng/ml TGF-β1 [6]. Confluent cells were passaged and seeded in 2% w/v agarose at 30M cells/ml and punched into constructs of 4mm diameter and 2.34mm thickness. Constructs were split into four groups with different TMAO supplementation schedules: Control (CTL) received no TMAO, Standard Release (SR) repeated the two week TMAO supplementation from d0-14 used in prior studies, Delayed Application (DA) received TMAO from d14-28, and Continuous (CTS) received TMAO for the duration of the study (to d42). TMAO was supplemented at 50 mm in all cases. This concentration was identified as optimal by previous experiments and was shown not to alter the osmolarity of the media outside of native levels [4]. All groups were cultured in serum free, chemically defined DMEM containing 100 nm Dexamethasone (added fresh with each media change), 100 μg/ml Sodium Pyruvate, 50 μg/ml L-Proline, 1% ITS+ Premix, 1%PSAM, and 50 μg/ml ascorbic acid (added fresh with each media change). All groups were supplemented with TGF-β3 for the first 14 days of culture [7]. Each group was started with 50 constructs per dish in 30 ml of media, about half the normal ratio of one mlof medium per construct that has been found to be optimal [5]. After d14 the ratio was restored to one construct per ml of media and maintained for the rest of the study.. Samples from each group were mechanically tested at d0, d14, d28 and d42 (n=5) and subsequently halved and preserved for later biochemical and histological analysis respectively. The equilibrium Young s Modulus (EY) and dynamic modulus were measured under 10% confined compression. Biochemical analyses were conducted to determine the GAG, hydroxyproline (OHP), and DNA content of each sample. The GAG and OHP values were normalized by sample wet weight. A two-way ANOVA (independent factors: day and group) with Tukey post-hoc analysis was performed to evaluate the significance of mechanical and biochemical results, with significance set at α=0.05. Results: Over 42 days of growth the control group s mechanical and biochemical properties reached a plateau after d14 while all TMAO groups achieved higher values. For both Young s and dynamic moduli, the control group plateaued and the SR group showed a logarithmic increase in modulus, both DA and CTS groups increased exponentially throughout the study (Fig1). By day 42, the mechanical properties of the novel DA and CTS supplementation regimens (Fig 1) were significantly higher than control values. The Young s and dynamic moduli followed a similar trend across groups. Both moduli were approximately 2X greater in the CTS group than the control, and more than 1.5X greater in the DA group. The Young s modulus of the DA and CTS groups were significantly higher than the previously implemented SR group. The CTS group also had a significantly higher dynamic modulus than the SR group. The CTS group was not significantly higher than the DA group for either modulus. The normalized GAG content in the CTS group was significantly higher than both the control and SR group (Fig 2). Analysis of gross construct morphology shows continued ECM elaboration throughout culture time (Fig 3). Discussion: The stressed condition applied by limiting nutrient availability is evident from the higher plateau reachef by the

2 control group mechanical and biochemical properties at low values after d14 (Fig 1,2). The continued increase in properties of the TMAO groups over the control in this sub-optimal condition leads us to accept our hypothesis that TMAO plays a chondroprotective role in developing engineered cartilage and enhances matrix elaboration. The underlying mechanisms that govern the ability of TMAO to rescue the biosynthetic response of the chondrocyte-seeded agarose constructs after the stress culture conditions remain to be elucidated. The protective effects of TMAO may be related to its ability to counteract harmful buildup of solutes in cells under stress by strong enhancement of protein activity and stability [1]. Protein stabilization by TMAO has also been shown in trna [8], suggesting it may protect cells by stabilizing metabolism and protein production pathways. The significant increase in Young s modulus of the DA group over the SR group shows that for a fixed duration of 2 weeks of TMAO supplementation, the timing of this application directly influences the degree of benefit conferred, favoring application at day 14. The CTS group showed a significant increase in all mechanical and biochemical data shown, suggesting that duration may play a larger role than timing. The fact that the CTS group is not significantly higher than the DA group in either case suggests that while a longer duration of supplementation provides an increased benefit over the standard supplementation, supplementation during the first two weeks may mitigate the benefit conferred by TMAO supplementation. In the future, we will further optimize supplementation timing while investigating in parallel the effects of TMAO in mitigating the deleterious effects of other cellular stressors, including injury arising from mechanical (e.g., overloading) and chemical stresses (e.g., nitric oxide, interleukin), on chondrocytes. Significance: We speculate that our current findings may have implications on regenerative medicine strategies for cartilage repair, where TMAO may serve as a beneficial supplement for culturing of cells derived from injured or pathologic cartilage (such as PTOA or OA); thereby providing an alternative cell source for cell-based therapies for cartilage repair. Acknowledgments: NIH R01 AR046568, R01 AR T32 AR References: [1] Yancey, PH J Experimental Biology 2005 [2] Meersman, F+ Biophysical J 2009 [3] Strambini, GB+, Biochemistry 2008 [4] O Connell, GD+ J Orthop Res [5] Mauck, R+ Osteoarthritis and Cartilage 2003 [6] Ng, KW+ Tissue Eng, Part A 2010 [7] Lima, EG+ Osteoarthritis Cartilage 2008 [8] Gluick, TC+ J. Am. Chem. 2003

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4 ORS 2014 Annual Meeting Poster No: 1264

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