Improved Function of Scaffold-Free Engineered Cartilage Under Combined Compressive and Shear Loads +1Whitney, G A; 1Mansour, J M; 1,2Dennis, J E

+1Case Western Reserve University, Cleveland, OH, 2Benaroya Research Institute, Seattle, WA g.adam.whitney@gmail.com

INTRODUCTION Scaffold-free engineered cartilage sheets are being investigated for their potential in joint resurfacing. We previously observed the failure of these engineered cartilage sheets during in-vitro evaluation under combined compressive and shear (CCS) loads. We also showed improved function after short term culture by medium supplementation with thyroxine, a known collagen stimulating hormone, and by introducing media advection which allows greater seeding density and presumably increases mass transfer.[1] Here we show the effects of thyroxine and advection on the composition, mechanical properties, and damage under CSS loads of these constructs at 2 time points. METHODS Tissue culture: Scaffold-free cartilage sheets were grown from adult rabbit, culture-expanded primary chondrocytes. Chondrocytes were cultured on porous polyester membranes in chondrogenic medium. Constructs were removed from the membrane after 1 month and samples were taken for biochemical and mechanical testing. The remaining cartilage was free-floated for an additional month, and then samples were again taken. Control constructs were grown under static conditions in chondrogenic medium at a cell seeding density of 3.125 million cells/cm2. L-thyroxine (THY; Sigma, St. Louis, MO) was added to test construct cultures at 25 ng/ml during the tissue culture phase. To assess the effect of bulk media motion, advection (ADV) was induced on day 2 of tissue culture, by placing constructs on an orbital shaker at 50 RPM for the remainder of the tissue culture phase. A higher seeding density was possible with advection; “ADV+CELLS” constructs were seeded at 6.25 million cells/cm2 and also placed on the orbital shaker. Mechanical testing: To assess the mechanical properties and the damage of the engineered constructs under CCS loads, 3 to 5 samples per group were first tested by indentation, followed by CCS loading. From indentation testing, mechanical properties were calculated. For CCS load testing, a custom built tribological testing device performed the oscillation, normal load application, and real-time friction force measurements. Tissue damage was assessed visually on a macroscopic scale and also on a microscopic scale. Tissue composition analysis: To verify upregulation of collagen by thyroxine, and to investigate possible causes for differentials in construct properties, tissue composition parameters were measured in 6 samples. Key ECM molecules for load bearing, glycosaminoglycan (GAG) and collagen, were measured by colorimetric assays[2] using hydroxyproline (HYP) as an indicator of collagen[3]. GAG content was normalized to DNA, and HYP content to tissue wet weight. Solid content was calculated as the ratio of dry to wet weight. Morphological assays: After applying CSS loads, samples were collected for histological processing and scanning electron microscopy (SEM). Histological samples were stained with Safranin-o to visualize GAG, and fast green as a counter stain. Statistical analysis: Significance was set at p<0.01 and was determined by Two-way ANOVA with Bonferroni post-tests in GraphPad Prism (GraphPad Software, La Jolla, CA). RESULTS Control samples showed damage under CCS loads at both the 1 and 2 month time points. Surprisingly, damage was more extensive at 2 months, when control samples exhibited complete delamination (Fig. 1B). No sample in any of the three treatment groups exhibited complete delamination. Some damage, either microscopic or macroscopic was observed in all treatment groups (ADV+CELLS shown in Fig. 1C, THY in Fig. 1D), but was minimal compared to the control group.

Biochemical assays (Fig. 2) showed that THY, ADV, and +CELLS treatments had a larger and more immediate effect on collagen content than on GAG content. On average, HYP and GAG increased in all constructs between 1 and 2 months, except for the ADV group. In ADV constructs, surprisingly, GAG decreased and HYP remained nearly constant. A nearly identical trend was observed for solid content. Results from indentation testing showed that the aggregate modulus of both groups treated with advection decreased between 1 and 2 months. Compared to controls, the difference was significantly greater for ADV+CELLS constructs at 1 month only. THY group aggregate modulus showed the opposite trend, increasing between 1 and 2 months, reaching significance compared to controls at 2 months. Permeability was not significantly different in any group compared to controls, however permeability of the ADV+CELLS group neared values reported for native cartilage. The thickness of ADV+CELLS was nearly two times that of all other groups at both time points (Fig. 2), and was significantly different. DISCUSSION ADV, THY and ADV+CELLS all showed significantly increased HYP content over control. The increased damage of control constructs at 2 months indicates that other parameters, such as collagen crosslinking and MMP activity, need to be explored to explain the increased damage at the later time point. Also surprising was the overall decrease in ECM content in the ADV group, and that construct stiffness decreased between 1 and 2 months in both groups treated with advection. Based on the results of the individual treatments, in the future we expect that the combination of the THY and ADV+CELLS treatments will produce superior constructs. Results also suggest that ADV+CELLS or THY treatments may reduce the time needed for in-vitro culture prior to implantation. SIGNIFICANCE We are developing these constructs for arthritis treatment through joint resurfacing. Here we have shown multiple treatments that result in improvements in aggregate modulus at 1 month, and thickness and tribological performance at both 1 and 2 months of culture. REFERENCES 1. Whitney, GA, et al. 2010. TERMIS Annual Meeting. 2. Henderson JH, et al. 2007. Tissue Eng 13(4): 843-853. 3. Ham KD, et al. 2004. Osteoarthritis Cartilage 12(2): 160-168. ACKNOWLEDGEMENTS This research supported by the NIH, grant numbers P01 AR053622, and 5T32AR007505-24.

Figure 2. Compositional and mechanical properties of engineered cartilage as a result of thyroxine, advection, and cell seeding.

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Figure 1. Histology of 2 month engineered cartilage specimen after CCS. A) Untested control specimen. B) Control specimen after CCS C) ADV+CELLS after CCS. D) THY specimen after CCS.

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Poster No. 0733 • ORS 2012 Annual Meeting