Applied Dynamic Loading Enhances Mechanical Properties of Engineered Cartilage using Adult Chondrocytes

*Bian, L.; *Ng, K.W.; *Lima, E.G.; *Xu, D.; **Jayabalan, P.S.; *Ateshian, G.A.; **Stoker, A.M.; **Cook, J.L.; +*Hung, C.T. +*Columbia University, New York, NY

cth6@columbia.edu INTRODUCTION. Previous studies have shown that mature chondrocytes, a more clinically relevant cell source for cartilage repair, exhibit diminished capacity to produce a mechanically functional cartilage extracellular matrix [1]. It has been shown that the mitotic and synthetic activities of human articular cartilage chondrocytes decline with age [2, 3], and an age-related decline in response to anabolic factors like insulin-like growth factor I (IGF-I) is known to occur [4]. Furthermore, in tissue engineering studies using immature primary chondrocytes, it has been demonstrated that dynamic loading may significantly enhance the functional properties of constructs[5]. Therefore, the hypothesis of this study is that mechanical loading can enhance the functional properties of adult (canine) chondrocyte-seeded tissue-engineered cartilage. Since recent studies have shown that application of tissue shear enhances matrix synthesis in cartilage explants [6, 7], this study compares the effects of two modes of mechanical loading, dynamic compression and sliding, on the functional properties of tissue-engineered cartilage.

Figure 1. Modular bioreactor for dynamic loading of constructs. MATERIALS AND METHODS. Using cell isolation protocols previously described, chondrocytes were harvested from adult canine stifle knee joints (3 mongrel dogs) and passaged twice in DMEM with 10% FBS, 1 ng/mL TGF-β1, 5 ng/mL FGF-2, and 10 ng/mL PDGF-ββ [8, 9]. Passaged chondrocytes were encapsulated in type VII agarose (2% w/v) at 30×106 cells/mL. Cell-seeded agarose disks (∅4.0×2.34mm) were cultured in 35 mL of chondrogenic media supplemented with TGF-β3 (10 ng/ml) at 37°C and 5% CO2 [10]. Two modes of dynamic loading were applied to constructs using a custom bioreactor: unconfined axial compressive deformational loading (DL) or sliding loading (SLIDE) (Figure 1). Loading for 3 hours daily was initiated on day 0 (DL0) or day 14 (DL14 or SLIDE14). Dynamic compressive loading was applied at 1 Hz, 10% deformation). Oscillatory sliding was applied at 0.5 Hz, 5% compression). Young’s modulus (EY) and dynamic modulus (G*) of samples (n=4-5 per group) was calculated from static and dynamic (0.5Hz) unconfined compression testing on selected time points. Statistical comparisons were performed using ANOVA and Tukey HSD post hoc analyses and α=0.05. RESULTS. Constructs with applied loading (both DL14 and SLIDE14) exhibited significant increases in Young’s modulus compared to free-swelling (FS) controls as early as day 28 in culture, p<0.05 (Figure 2). The modulus values attained for engineered constructs compare favorably with (and exceed in some cases) those for native canine knee (trochlear groove: 200±68kPa and condyle: 325±79kPa) cartilage (Figure 2). Despite the difference in Young’s modulus there was no significant difference in the overall GAG and collagen content of the constructs among different groups (data not shown). The equilibrium friction coefficient of the constructs subjected to sliding contact loading (Slide14) was significantly lower than that of constructs from the FS and DL14 groups (Figure 3). DISCUSSION. In this study we have successfully demonstrated that dynamic mechanical loading is able to enhance the mechanical properties of tissue-engineered cartilage to native levels using mature chondrocytes. Agarose hydrogel is currently being evaluated as a scaffold component of a third generation autologous chondrocyte implantation product in human clinical trials [11]. These results using continuous growth factor supplementation are in contrast to our previously reported studies with immature chondrocytes where the

sequential application of dynamic loading after transient TGFβ3 application was found to be the optimal culture protocol [12]. In this study sliding was applied after Day 14 of the culture because previous study showed that applying early mechanical loading from Day 0 can be detrimental to the development of the engineered cartilage using immature bovine chondrocytes[5]. However, in this study the negative effect of early loading was not observed in mature canine chondrocytes. More studies are needed to explain the different responses by cells of different species to early mechanical loading. The constructs subjected to sliding contact loading exhibited a lower equilibrium friction coefficient than other groups, which may be attributed to a higher content of boundary lubricants such as superficial zone protein (SZP/lubricin). Previous studies have shown that dynamic shear can increase the production of SZP/lubricin [7, 13]. Future work is needed to analyze the synthesis of lubricin quantitatively using ELISA or qPCR assays. The ability to cultivate engineered cartilage with native mechanical properties is clinically significant. To survive joint loading, engineered cartilage constructs should possess substantially similar mechanical properties to the surrounding host tissue before implantation, especially if they have to support most of the articular contact stresses as a result of the local congruence of the articular surfaces.

Figure 2. Young’s modulus as a function of culture time. *significantly different from FS group for same culture day; + significantly different from groups without + for same culture day. Average modulus values for canine knee condyle (red) and patellar groove (blue) are indicated for reference. (n=4-9 constructs/group).

Figure 3. (A) Representative plot of the friction coefficient with respect to time. (B) Equilibrium modulus of the constructs from the FS, DL14 and Slide14 group on Day 56, ♦p<0.05 vs. FS and DL14, n=5. ACKNOWLEDGEMENT. This work was supported by NIH grants AR46568 and AR53530. REFERENCES. [1]Tran-Khanh, N., et al., J Orthop Res, 23(6): p. 1354-62, 2005. [2]Bolton, M.C., et al., Biochem J, 337 ( Pt 1): p. 77-82, 1999. [3]Martin, J.A., et al., J Gerontol A Biol Sci Med Sci, 56(4): p. B172-9, 2001. [4]Martin, J.A., et al., J Orthop Res, 15(4): p. 491-8, 1997. [5]Lima, E.G., et al., Osteoarthritis Cartilage, 2007. [6]Jin, M., et al., Arch Biochem Biophys, 395(1): p. 41-8, 2001. [7]Nugent, G.E., et al., Arthritis Rheum, 54(6): p. 1888-96, 2006. [8]Barbero, A., et al., Arthritis Rheum, 48(5): p. 1315-25, 2003. [9]Capito, R.M., et al., Osteoarthritis Cartilage, 14(12): p. 1203-13, 2006. [10]Byers, B.A., et al., Tissue Eng Part A, 2008. [11]Selmi, T.A., et al., J Bone Joint Surg Br, 90(5): p. 597-604, 2008. [12]Lima, E.G., et al., Osteoarthritis Cartilage, 15(9): p. 1025-33, 2007. [13]Frank, E.H., et al., J Biomech, 33(11): p. 1523-7, 2000.

** University of Missouri, Columbia, MO

Paper No. 382 • 56th Annual Meeting of the Orthopaedic Research Society