Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair. Although much of the tissue-engineered cartilage in existence has been successful in mimicking the morphological and biochemical appearance of hyaline cartilage, it is generally mechanically inferior to the natural tissue. In this study, we tested the hypothesis that the application of dynamic deformational loading at physiological strain levels enhances chondrocyte matrix elaboration in cell-seeded agarose scaffolds to produce a more functional engineered tissue construct than in free swelling controls. A custom-designed bioreactor was used to load cell-seeded agarose disks dynamically in unconfined compression with a peak-to-peak compressive strain amplitude of 10 percent, at a frequency of 1 Hz, 3 x (1 hour on, 1 hour off)/day, 5 days/week for 4 weeks. Results demonstrated that dynamically loaded disks yielded a sixfold increase in the equilibrium aggregate modulus over free swelling controls after 28 days of loading (100 +/- 16 kPa versus 15 +/- 8 kPa, p < 0.0001). This represented a 21-fold increase over the equilibrium modulus of day 0 (4.8 +/- 2.3 kPa). Sulfated glycosaminoglycan content and hydroxyproline content was also found to be greater in dynamically loaded disks compared to free swelling controls at day 21 (p < 0.0001 and p = 0.002, respectively).

An optimal strategy for the functional tissue engineering of articular cartilage, particularly to accelerate construct development, may incorporate sequential application of different growth factors and applied deformational loading.

Taken together, these results point to the utility of dynamic deformational loading in the mechanical preconditioning of engineered articular cartilage constructs and the necessity for increasing feed media volume and serum supplementation with increasing cell seeding densities.

Chondrocytes cultured in agarose hydrogels develop a functional extracellular matrix. Application of dynamic strain at physiologic levels to these constructs over time can increase their mechanical properties. In this study, the effect of seeding density (20 and 60 x 10(6) cells/ml) on tissue elaboration was investigated. Higher seeding densities increased tissue properties in free-swelling culture, with constructs seeded at 20 and 60 x 10(6) cells/ml reaching maximum values over the 63 day culture period of aggregate modulus HA: 43 +/- 15 kPa, Young's modulus EY: 39 +/- 3 kPa, and glycosaminglycan content [GAG]: 0.96% +/- 0.13% wet weight; and HA: 58 +/- 12 kPa, EY: 60 +/- 5 kPa, and [GAG]: 1.49% +/- 0.26% wet weight, respectively. It was further observed that the application of daily dynamic deformational loading to constructs seeded at 20 x 10(6) cells/ml enhanced biochemical content (approximately 150%) and mechanical properties (approximately threefold) compared to free-swelling controls by day 28. However, at a concentration of 60 x 10(6) cells/ml, no difference in mechanical properties was found in loaded samples versus their free-swelling controls. Multiple regression analysis showed that the mechanical properties of the tissue constructs depend more strongly on collagen content than GAG content; a finding that is more pronounced with the application of daily dynamic deformational loading. Our findings provide evidence for initial cell seeding density and nutrient accessibility as important parameters in modulating tissue development of engineered constructs, and their ability to respond to a defined mechanical stimulus.

It has previously been demonstrated that dynamic deformational loading of chondrocyte-seeded agarose hydrogels over the course of 1 month can increase construct mechanical and biochemical properties relative to free-swelling controls. The present study examines the manner in which two mediators of matrix biosynthesis, the growth factors TGF-beta1 and IGF-I, interact with applied dynamic deformational loading. Under free-swelling conditions in control medium (C), the [proteoglycan content][collagen content][equilibrium aggregate modulus] of cell-laden (10 x 10(6) cells/mL) 2% agarose constructs reached a peak of [0.54% wet weight (ww)][0.16% ww][13.4 kPa]c, whereas the addition of TGF-beta1 or IGF-I to the control medium led to significantly higher peaks of [1.18% ww][0.97% ww][23.6 kPa](C-TGF) and [1.00% ww][0.63% ww][19.3 kPa](C-IGF), respectively, by day 28 or 35 (p<0.01). Under dynamic loading in control medium (L), the measured parameters were [1.10% ww][0.52% ww][24.5 kPa]L, and with the addition of TGF-beta1 or IGF-I to the control medium these further increased to [1.49% ww][1.07% ww][50.5 kPa](L-TGF) and [1.48% ww][0.81% ww][46.2 kPa](L-IGF), respectively (p<0.05). Immunohistochemical staining revealed that type II collagen accumulated primarily in the pericellular area under free-swelling conditions, but spanned the entire tissue in dynamically loaded constructs. Applied in concert, dynamic deformational loading and TGF-beta1 or IGF-I increased the aggregate modulus of engineered constructs by 277 or 245%, respectively, an increase greater than the sum of either stimulus applied alone. These results support the hypothesis that the combination of chemical and mechanical promoters of matrix biosynthesis can optimize the growth of tissue-engineered cartilage constructs.

These results are representative of the functional properties of articular cartilage under physiological load magnitudes and frequencies. The viscoelasticity and nonlinearity of the tissue helps to maintain the compressive strains below 20% under the physiological compressive stresses achieved in this study. These findings have implications for our understanding of cartilage metabolism and chondrocyte viability under various loading regimes. They also help establish guidelines for cartilage functional tissue engineering studies.

An automated approachfor measuring in situ two-dimensional strain fields was developed and validated for its application to cartilage mechanics. This approach combines video microscopy, optimized digital image correlation (DIC), thin-plate spline smoothing (TPSS) and generalized cross-validation (GCV) techniques to achieve the desired efficiency and accuracy. Results demonstrate that sub-pixel accuracies can be achieved for measuring tissue displacements with this methodology with a measurement uncertainty ranging from 0.25 to 0.30 pixels. The deformational gradients (from which the strains are determined) can be evaluated directly using the optimized DIC, with a measurement uncertainty of 0.017 to approximately 0.032. In actual measurements of strain in cartilage, TPSS and differentiation can be used to achieve a more accurate measurement of the gradients from the displacement data. Using this automated approach, the two-dimensional strain fields inside immature bovine carpometacarpal joint cartilage specimens under unconfined compression were characterized (n=21). The depth-dependent apparent elastic modulus and Poisson's ratio were also determined and found to be smallest at the articular surface and increasing with depth. The apparent Poisson's ratio is found to decrease with increasing compressive strain, with values as low as 0.01 observed near the articular surface at 25% compression. The variation of the apparent Poisson's ratio with depth is found to be consistent with a theoretical model of cartilage which accounts for the disparity in its tensile and compressive moduli.

Deformational loading represents a primary component of the chondrocyte physical environment in vivo. This review summarizes our experience with physiologic deformational loading of chondrocyte-seeded agarose hydrogels to promote development of cartilage constructs having mechanical properties matching that of the parent calf tissue, which has a Young's modulus E(Y) = 277 kPa and unconfined dynamic modulus at 1 Hz G* = 7 MPa. Over an 8-week culture period, cartilage-like properties have been achieved for 60 x 10(6) cells/ml seeding density agarose constructs, with E(Y) = 186 kPa, G* = 1.64 MPa. For these constructs, the GAG content reached 1.74% ww and collagen content 2.64% ww compared to 2.4% ww and 21.5% ww for the parent tissue, respectively. Issues regarding the deformational loading protocol, cell-seeding density, nutrient supply, growth factor addition, and construct mechanical characterization are discussed. In anticipation of cartilage repair studies, we also describe early efforts to engineer cylindrical and anatomically shaped bilayered constructs of agarose hydrogel and bone (i.e., osteochondral constructs). The presence of a bony substrate may facilitate integration upon implantation. These efforts will provide an underlying framework from which a functional tissue-engineering approach, as described by Butler and coworkers (2000), may be applied to general cell-scaffold systems adopted for cartilage tissue engineering.