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.

The goal of this study was to develop a system to reliably measure the intrinsic hydraulic permeability of hydrogels and soft hydrated tissues. Such a device can be used to assess the development of functional properties in tissue engineered constructs [1]. The design parameters for such a device include ease of assembly and the ability to measure hydraulic permeability over a range of specimen deformations. To meet these criteria, a device was designed that could quantify the hydraulic permeability of a sample under different levels of deformation, allowing characterization of strain-dependent effects. The device was tested on both agarose and articular cartilage specimens, yielding permeability values consistent with published data [2]. The intrinsic hydraulic permeability of a tissue is an important parameter that governs fluid exudation during deformational loading. The ability of articular cartilage, which exhibits non-linear strain dependent hydraulic permeability [3], to generate and sustain interstitial fluid pressurization is essential to its functional properties (e.g., load bearing and lubrication). This novel device allows for direct and reliable measurement of these physical properties.

A successful tissue engineered articular cartilage construct needs to possess mechanical, biochemical, and histological features similar to that of native cartilage in order to serve its load-bearing function. Agarose is a suitable scaffold material for chondrocyte cultures (1,2), allowing long-term maintenance of cell phenotype and the elaboration of a functional cartilage-like matrix. This culture system facilitates further elucidation of the roles of matrix and cell-matrix interactions in the regulation of chondrocyte response to mechanical loads. We have previously shown (3) that mechanical loading at a physiologic frequency can increase the rate of matrix deposition, increasing mechanical properties of the tissue engineered constructs (∼21 fold increases in HA over day 0 with loading vs. ∼4 fold increases for free swelling controls). We have also shown that dynamic loading of transiently transfected chondrocytes in agarose hydrogels for 1 hour at 10% strain increased aggrecan promoter activity by ∼1.5 fold (4). In this study we sought to further characterize the short term response of chondrocytes to static load (by measuring aggrecan promoter activity) and the effects of dynamic compression on aggrecan gene expression over a longer (3 day) culture period (by monitoring mRNA levels). Monitoring matrix gene expression during early times of culture, when there is little matrix accumulation and the cells deform directly with the matrix (5), may provide insights into cellular responses to strain and allow for the optimization of cartilage bioreactor conditions.