p=0.02 ). Discussion: These results, which expand upon a recently developed chemically defined medium [3], demonstrate that contrary to long-term

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1 Synovial Fluid and Physiologic Levels of Cortisol, Insulin, and Glucose in Media Maintain the Homeostasis of Immature Bovine Cartilage Explants over Long Term Culture Michael B. Albro, PhD, Krista M. Durney, M.S., Jay J. Shim, Akaljot Singh, Alexander D. Cigan, MS, Robert J. Nims, M.S., Brian K. Jones, M.S., Clark T. Hung, PhD, Gerard A. Ateshian, PhD. Columbia University, New York, NY, USA. Disclosures: M.B. Albro: None. K.M. Durney: None. J.J. Shim: None. A. Singh: None. A.D. Cigan: None. R.J. Nims: None. B.K. Jones: None. C.T. Hung: None. G.A. Ateshian: None. Introduction: The ability to maintain articular cartilage explants at homeostasis in long-term culture can serve as an invaluable biomedical tool in basic and clinical sciences by allowing for the examination of tissue behavior in a highly controlled model system that is unable to be achieved in vivo, and by providing for the long-term preservation of cartilage allografts, an important tissue source for repairing osteoarthritic defects [1]. A fundamental challenge of tissue maintenance is the identification of the critical mechanical and chemical stimuli that regulate chondrocyte metabolic activity in the native environment. Immature bovine articular cartilage has served as an important tissue source for cartilage mechanobiology model systems due to its ability to offer large sample sizes and undergo precisely controlled loading regimens. However, long-term culture studies on this tissue with a variety of media formulations have observed excessive levels of tissue swelling and softening in control samples [2], behaviors that can confound interpretations of experimental results. Recently, it has been demonstrated that a serum free chemically defined culture medium supplemented with dexamethasone, prevents tissue degradation [3]. However, this media formulation also significantly increases levels of glycosaminoglycan (GAG) in the explants, leading to a complicating pronounced stiffening of the tissue. It can be surmised that excessive anabolic stimulation may result from a highly supraphysiologic supply of important metabolic mediators in the media formulation (4.5mg/mL glucose & 6μg/mL insulin). Native synovial fluid (SF) possesses far lower levels of glucose (~1mg/mL [4]) and insulin (~0.002 μg/ml [5]). Glucose serves as a precursor for the elaboration of GAG side chains while insulin enhances cellular glucose uptake and at high levels can signal through the anabolic IGF-1 receptor. Further, while corticosteroids naturally function to promote matrix synthesis and inhibit breakdown, SF possesses cortisol (~0.01 μg/ml in unbound form [6]), which is far less potent than the synthetic dexamethasone [7]. The hypothesis of this investigation is that physiologic levels of cortisol, insulin, and glucose in chemically defined media or in SF will lead to an improved homeostatic maintenance of the biochemical and mechanical properties of immature bovine cartilage explants. Surprisingly, investigations of these physiologic culture conditions have not been reported previously. Methods: Cartilage explants ( 3mm 1.7mm) were harvested from immature bovine condyles with their articular surfaces intact. Disks were cultured for up to 28 days in a basal medium (BM; 1 mg/ml glucose DMEM, 1mg/mL BSA, 1X nonessential amino acids, PSAM, 50 μg/ml vitamin-c, and 110 mg/l sodium pyruvate) supplemented with insulin, glucose, or cortisol (1mL per explant, n=6 explants per group). SF was sterilely obtained from adult bovine knees (Animal Tech) within 1 hour of sacrifice. SF samples were found to possess depressed glucose levels, presumably due to depletion in the period between sacrifice and harvest. To overcome this depletion, SF was supplemented with 1mg/mL glucose, 1X amino acids, and 50 μg/ml vitamin-c. At culture completion, explants were analyzed for their compressive Young s modulus and swelling ratio (final volume normalized to day 0 volume). GAG levels were normalized to explant initial wet weight (day 0) to assess the absolute change in explant content and to final wet weight to assess the final concentration in the tissue while accounting for swelling. Insulin and glucose: To assess the effects of these constituents, explants were cultured in BM (supplemented w/ 0.05 μg/ml cortisol) at variable levels of glucose (1 or 4.5 mg/ml) and insulin (6, 0.6, 0.06, μg/ml). Insulin levels were varied through the supplementation of ITS+ premix. Transferrin and selenium in ITS+ has been shown to contribute negligibly to ECM synthesis [8]. Cortisol and SF: To assess the effects of cortisol and SF, explants were cultured in BM (w/ physiologic 0.002μg/mL insulin) or SF as unsupplemented or with physiologic (0.01μg/mL) or supraphysiologic (0.2μg/mL) cortisol levels. Results: After 28 days in high glucose and insulin BM, explants exhibited a large increase in swelling (215%) and GAG content (335%). These measures decreased significantly (p<0.001) with decreasing levels of glucose and insulin. At physiologic levels of glucose (1mg/mL) and insulin (0.002μg/mL), both swelling and GAG content were statistically maintained at their day 0 levels. Explants responded similarly in BM and SF. In the absence of supplemented cortisol, BM and SF led to explant swelling (p<0.001) and a loss in stiffness (SF p=0.001; BM p=0.05). Physiologic cortisol supplementation inhibited swelling and statistically maintained explant stiffness. Supraphysiologic cortisol led to a rise in GAG content (BM p=0.003) and mechanical properties (SF

2 p=0.02 ). Discussion: These results, which expand upon a recently developed chemically defined medium [3], demonstrate that contrary to long-term cultures in serum, which led to tissue degradation, physiologic levels of critical metabolic mediators (insulin, glucose, cortisol) can substantially improve the long-term maintenance of the mechanical and biochemical integrity of live cartilage explants. The presence of physiologic cortisol levels is required to prevent explant swelling and softening, presumably due to its action in inhibiting MMP release and the breakdown of collagen [9], which is required to resist the GAG swelling pressure. Excess levels of cortisol, insulin, and glucose appear to promote excess synthesis and retention of GAG accompanied by explant stiffening. This study also represents the first characterization of cartilage during long-term culture in SF, demonstrating that media and SF similarly maintain tissue homeostasis. The required addition of low cortisol levels to SF in order to maintain immature explant homeostasis is likely due to the use of adult bovine SF; systemic cortisol levels decrease with age [10]. The low degree of residual swelling observed at physiologic cortisol levels is likely consistent with immature tissue behavior in vivo, whereby the cartilage radially expands during joint growth. While this study was performed on immature cartilage, the inhibition of tissue swelling and excessive GAG synthesis may be important for studies on mature tissue as well. Significance: This study is the first to identify serum-free culture conditions capable of maintaining homeostasis in long term cultures of immature bovine cartilage explants, neither producing matrix degradation nor excessive GAG synthesis. It is also the first to demonstrate successful long-term culture in SF. Acknowledgments: This study was supported by NIH AR043628, AR060361, AR References: [1] Williams SK +, J Bone Joint Surg, (11): [2] Sah RL +, J Orthop Res, (1): [3] Bian L +, J Biomech, (6): [4] Dechant JE +, Vet Surg, (7):811-6 [5] Rovensky J +, Clin Expt Rheum, (3):292-6 [6] Ishizaki H +, J Vet Med Sci, (6): [7] Cantrill HL +, J Clin Endocrin Metab, (6): [8] Cigan AD +, Tissue Eng A, (17): [9] Garvican ER +, J Orthop Res (3) [10] Katayama M +, Clin Endocrin, :311-

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