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Mechanical stimuli are known to regulate the morphology and differentiated function of connective tissue cells. In particular, hydrostatic pressure has been reported to alter cytoskeletal organization in osteoblast-like cells (1) and chondrocytes (2), and to modulate metabolic activity in both chondrocytes (3–5) and intervertebral disc cells (6). The cellular response to continuous hydrostatic pressure is generally catabolic (3) while intermittent hydrostatic pressure at frequencies ranging from 0.25–1.0 Hz (3–5) is anabolic, giving rise to increased expression and biosynthesis of extracellular matrix (ECM) components. Previously, human dermal fibroblasts in monolayer culture were shown to respond to hydrostatic pressure by increasing heat shock protein expression levels (7). In this study, we characterize the effects of intermittent hydrostatic pressure on gene expression in human dermal fibroblasts seeded in three-dimensional polymer scaffolds.Copyright © 2002 by ASME

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.

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.

Articular cartilage is the load-bearing substance that covers the bony surfaces of articulating bones. With its high water content and small pore size, deformation of cartilage induces a very high hydrostatic pressure within the cartilage. This hydrostatic pressure has been shown both theoretically and experimentally to support upwards of 90% of the applied load (1), and can be on the order of 6–12 MPa. Chondrocytes, the cells within cartilage respond to this pressure by altering their rates of biosynthesis. Studies utilizing radionucleotide incorporation in both explant and monolayer cultures (2–4) have shown that in general dynamic pressurization increases synthesis, while static pressurization decreases synthesis. More recently, Smith et al have shown that dynamic pressurization (10MPa, 1 Hz) of cells in monolayer culture can upregulate matrix gene expression (5,6). Further, a study in PGA constructs has shown that long term application of dynamic pressure can increase matrix deposition (7). In this study, we seek to expand on these findings by examining the response in gene expression of articular chondrocytes encapsulated in alginate, a charged, 3D hydrogel.

Cultured chondrocytes exhibit cathodal migration when subjected to applied direct current (DC) electric fields [1]. Our studies showed a 2-fold enhancement in migratory velocity at physiologic temperature (∼37°C) compared to room temperature (∼25°C). The inositol phosphate pathway antagonists, U-73122 and neomycin, were found to inhibit the directed response, indicating that it may be involved in chondrocyte migration. Further understanding of the molecular mechanisms underlying chondrocyte migration and galvanotaxis may contribute to the development of new strategies for cartilage repair.

A major challenge facing the tissue engineering of articular cartilage is the ability to grow tissue constructs that have the proper mechanical and biochemical properties that permit cartilage to serve its load-bearing function. This study tested the hypothesis that physiologic deformational loading enhances the formation of functional material properties in cell-seeded agarose constructs (versus free-swelling constructs).

The generation of a cartilage substitute will require both the mechanical and biochemical properties of the substance to approach that of native tissue. Recent studies (Buschmann et al., 1992, 1995; Bader and Lee, 1997) have shown that agarose can provide a suitable environment for the production of a mechanically functional matrix. Over time cells seeded in this matrix alter the intrinsic mechanical properties of the matrix. Additionally, several studies have included such biochemical factors as vitamin C (ascorbate) to the culture medium. This biological effector has varied effects on chondrocyte matrix biosynthesis. Specifically, it has been shown to promote calcification in chick growth plate chondrocytes (Wu et al., 1989), while it increased both type II and type I procollagen mRNA in 5 day culture (Sandell and Daniel, 1988). Conversely, it has been shown in bovine culture that aggrecan mRNA levels rise steadily in monolayer culture without ascorbate. but rise quickly and then fall off in its presence (Hering et al., 1994). To assess the effect of ascorbate in our system, primary bovine articular chondrocytes seeded in agarose, we initiated a study to examine the effects of ascorbate on both the production of cartilage extracellular matrix and the development of mechanical properties over a 14 day culture period. Three groups were studied: agarose disks without cells (control) and cell-seeded agarose disks maintained in DMEM supplemented daily with and without 50 μg/ml of ascorbate. Glycosaminoglycan (GAG) and hydroxyproline contents of each disk were determined using standard colorimetric assays.

Many studies have demonstrated that articular cartilage is anisotropic in tension, based on tensile tests of tissue strips harvested parallel to the articular surface, along and perpendicular to the local split line direction (e.g., Akizuki et al., 1986; Kempson et al., 1968; Schmidt et al., 1990; Woo et al., 1979). The observed differences in the tensile modulus suggest that the material symmetry of cartilage in tension is no higher than orthotropy, since two orthogonal planes of symmetry (with unit normals parallel and perpendicular to the split line direction) automatically define a third plane of symmetry mutually perpendicular to the other two. However, the properties of articular cartilage differ significantly in tension and compression (Cohen et al., 1998; Soulhat et al., 1998) and it remains to be established whether cartilage is anisotropic in compression as well. Only one previous preliminary study has investigated the compressive modulus of cartilage along two mutually perpendicular directions (Jurvelin et al., 1996), reporting significant differences.

Chemical and mechanical stimuli are known to cause directed movement in a number of different cell types. Less prominently studied, direct current (DC) electric fields are known to induce a similar response. In this study, we report on DC electric field-induced chondrocyte migration and re-orientation. Galvanotaxis and galvanotropism, migration and shape change in response to applied DC electric fields, respectively, have been demonstrated in many cells. For instance, field strengths of 1–10 V/cm have been reported to induce migration in keratinocytes. corneal epithelial cells, bone cells, fibroblasts and neural cells [1,7,8,11]. Recently, we have demonstrated for the first time that chondrocytes exhibit a galvanotactic response, realigning and migrating in response to applied DC electric fields (6 V/cm) [6]. In cartilage, chondrocytes may see electric fields associated with streaming potentials estimated to be up to 15 V/cm with current densities of up to 0.1A/cm2 [2]. The aim of this study was to explore basic science aspects of directed cell migration under applied DC electric fields and to investigate the potential application of this phenomena for tissue engineering, healing and repair of cartilage. The ability to direct cell growth and function will have significant implications on the bioengineering of replacement tissues.

The objective of this study was to determine the intrinsic hydraulic permeability of 2% agarose hydrogels. Two-percent agarose was chosen because it is a concentration typically used for encapsulation of chondrocytes in suspension cultures [3–5], Hydraulic permeability is a measure of the relative ease by which fluid can pass through a material. Importantly, it governs the level of interstitial fluid flow as well as the interstitial fluid pressurization that is generated in a material during loading. Fluid pressurization is the source of the unique load-bearing and lubrication properties of articular cartilage [1,17] and represents a major component of the in vivo chondrocyte environment. We have previously reported that 2% agarose hydrogels can support fluid pressurization, albeit to a significantly lesser degree than articular cartilage [18]. Interstitial fluid flow gives rise to convective transport of nutrients and ions [6,7] and matrix compaction [9] which may serve as important stimuli to chondrocytes. We report for the first time the strain-dependent hydraulic permeability of 2% agarose hydrogels.