Trimethylamine N-Oxide Media Supplementation for Cartilage Tissue Engineering of Synovium-Derived Stem Cells Sonal Ravin Sampat, M.S., J. Chloe Bulinski, Gerard A. Ateshian, Clark T. Hung, PhD. Columbia University, New York, NY, USA. Disclosures: S.R. Sampat: None. J. Bulinski: None. G.A. Ateshian: None. C.T. Hung: None. Introduction: The avascular nature of cartilage leads to a poor healing capacity after injury, motivating the development of cellbased therapies for repair. Synovium-derived stem cells (SDSCs) can differentiate down a chondrogenic lineage and are thought to aid in cartilage repair (1). Previously, we cultured SDSC-seeded agarose constructs which attained a Young s modulus and GAG content within native values of immature bovine cartilage after seven weeks, which in a subsequent study was further increased by culturing constructs in media at a physiologic osmolarity (400 mosm) (2,3). A natural osmolyte previously shown to improve the properties of tissue engineered cartilage is trimethylamine N-Oxide (TMAO) (4). TMAO is an organic osmolyte found in shark tissues which counteracts the protein perturbations caused by urea by inducing protein folding and stabilization (5). The skeletal matrix of shark cartilage is comprised of similar proportions of constituents as human articular cartilage (6). Based on our previous studies showing the pronounced effects of TMAO on the mechanical and biochemical properties of chondrocyteseeded agarose hydrogels, in this study, we assess the response of SDSC-seeded constructs to TMAO media supplementation, which has not yet been characterized. We hypothesize that SDSCs will increase their mechanical and biochemical content in response to TMAO supplementation, similarly to chondrocytes (4). Methods: SDSCs were cultured in a method previously found to yield cells that make tissue engineered cartilage equal to chondrocytes. Synovium tissue was harvested aseptically from juvenile bovine knee joints (2-4 weeks), minced and digested in a 37 C, 5% CO 2 incubator using collagenase type IV (295 U/mg) (Fig. 1). Following digestion, SDSCs were seeded on tissue-culture treated plastic (1760 cells/cm 2 ) and expanded for 2 passages with media containing αmem, 10% FBS, pen/strep, and supplemented with 5 ng/ml FGF-2. To obtain a pure population of SDSCs, a negative isolation procedure was performed following the first passage (P1), as previously described (2,3). After passaging, SDSCs were encapsulated in 2% w/v agarose at 60 x 10 6 cells/ml. The gel was maintained in chondrogenic media (DMEM, 1% ITS+ Premix, 50 µg/ml L-proline, 0.1 µm dexamethasone, 0.9 mm sodium pyruvate, antibiotics) overnight and then discs were cored using a sterile disposable punch (Miltex) to final dimensions of 4 mm diameter and 2.34 mm thickness and cultured in serum-free chondrogenic media (330 mosm) plus ascorbate (50 µg/ml) and TGF-β3 (10 ng/ml). TGF-β3 was removed at day 21, as previously established in the lab. Both 5 mm and 50 mm TMAO concentrations were evaluated to determine an ideal concentration for the culture media. TMAO (Sigma-Aldrich) was supplemented fresh into the culture media during the first 21 days of culture to coincide with TGF-β3 release (Fig. 1). Constructs cultured without TMAO served as the control. Mechanical testing (Young s modulus (E Y ) and Dynamic Modulus (G*)) was performed and biochemical composition was assessed for DNA and glycosaminoglycan (GAG) content. Time points for mechanical and biochemical analyses occurred at days 0, 28, 42, 56. Statistical analysis was performed using a twoway ANOVA with α<0.05 and Tukey s HSD post-hoc test, with statistical significance achieved when p 0.05. In addition, a student s t-test with α<0.05 was used to test significance of the slopes of the two independent regression lines in the correlation plot of Young s modulus (kpa) vs. GAG/ww (%). Results: With the supplementation of TMAO to the media for the first 21 days in culture, constructs seeded with SDSCs responded most favorably to culturing with 5 mm TMAO (p<0.05) as compared to both the control and 50 mm TMAO conditions. At day 56, the 5 mm TMAO group exhibited significant increases in Young s Modulus (Fig. 2A) as compared to the control, with an average Young s Modulus of 431±57 kpa. The increases in modulus were accompanied by corresponding increases in GAG content (Fig. 2B). GAG content for both control (6.26±1.24%ww) and 5 mm TMAO (7.19±0.50%ww) constructs were within native levels and while not statistically significant from one another, they were both significantly higher than the 50 mm TMAO condition (3.61±0.68%ww) (p<0.0005) (Fig 2B and 3). A correlation plot of Young s modulus (kpa) vs. GAG/ww (%) for the control and 5 mm groups indicates a steeper regression line for the 5 mm TMAO group, albeit not significant (p=0.256), as compared to the control with both correlation coefficients (r) ~0.84 (Fig. 2C). While the 5 mm TMAO concentration showed beneficial effects, the 50 mm TMAO condition resulted in lower mechanical and biochemical properties at day 56 as compared to day 42, including DNA content (p<0.05) (Fig. 2 and 3). The osmolarity of the media was assessed at day 42 in culture and was found to be between 330 and 333 mosm, regardless of TMAO concentration (Fig. 3). Discussion: In the current study, the potential of using TMAO to culture SDSCs for cartilage replacement was investigated. Our results support the hypothesis that TMAO supplementation of the culture media improves the mechanical and biochemical properties of SDSC-seeded engineered cartilage tissue. The concentrations of TMAO evaluated in this study were chosen because they produced the most favorable mechanical and biochemical properties on chondrocyte-seeded constructs in our
lab s previously published work with TMAO (4). Although TMAO was only added to the culture media for the first 3 weeks, significant differences between all groups were not seen until 8 weeks in culture. The duration of exposure was chosen based on previous results indicating greatest impact on properties with transient application of growth factors (7). Future studies will look at the impact of timing and duration of TMAO exposure to determine optimal conditions, including the influence of continuous supplementation. TMAO is a known osmolyte (4,5), however, at low concentrations as used in this study, there was no impact on media osmolarity. The results of Fig. 2C indicate that while TMAO does not necessarily boost the construct properties by modulating the relationship of mechanical properties on GAG/ww (%), it may implicate the effect on other extracellular matrix molecules, such as collagen content or cartilage oligomeric protein (COMP) and their assembly or organization. Therefore, the increased mechanical properties with TMAO application may be due to its protein stabilization effects. However, these underlying mechanisms remain to be elucidated. In addition, while media osmolarity was not influenced by the TMAO concentration, the higher concentration of TMAO clearly had adverse effects on the cells. The results of this study suggest that supplementation of media with 5 mm TMAO is a straightforward 3D cultivation technique with significant benefits for improving the compressive mechanical properties and increasing extracellular matrix production of tissue engineered cartilage. Significance: This study evaluates the response of SDSCs to TMAO media supplementation. The findings allow for optimizing culture conditions for SDSC-seeded tissue engineered cartilage constructs. Acknowledgments: This work was supported by NIH grants R01 AR46568, R01AR52871, and T32 AR059038. References: (1) Pei, M+, 2008 (2) Sampat, SR+, 2013 (3) Sampat, SR+, 2011 (4) O Connell, GD+, 2012 (5) Yancey, PH, 2005 (6) Porter, ME+, 2006 (7) Byers, BA+, 2008
ORS 2014 Annual Meeting Poster No: 1266