Christopher C.-B. Wang

Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels

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).

A Paradigm for Functional Tissue Engineering of Articular Cartilage via Applied Physiologic Deformational Loading

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 Youngs modulus EY = 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 × 106 cells/ml seeding density agarose constructs, with EY = 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.

An Automated Approach for Direct Measurement of Two-Dimensional Strain Distributions Within Articular Cartilage Under Unconfined Compression

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 Poissons ratio were also determined and found to be smallest at the articular surface and increasing with depth. The apparent Poissons 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 Poissons 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.

Optical determination of anisotropic material properties of bovine articular cartilage in compression.

The precise nature of the material symmetry of articular cartilage in compression remains to be elucidated. The primary objective of this study was to determine the equilibrium compressive Youngs moduli and Poissons ratios of bovine cartilage along multiple directions (parallel and perpendicular to the split line direction, and normal to the articular surface) by loading small cubic specimens (0.9 x 0.9 x 0.8 mm, n =15) in unconfined compression, with the expectation that the material symmetry of cartilage could be determined more accurately with the help of a more complete set of material properties. The second objective was to investigate how the tension-compression nonlinearity of cartilage might alter the interpretation of material symmetry. Optimized digital image correlation was used to accurately determine the resultant strain fields within the specimens under loading. Experimental results demonstrated that neither the Youngs moduli nor the Poissons ratios exhibit the same values when measured along the three loading directions. The main findings of this study are that the framework of linear orthotropic elasticity (as well as higher symmetries of linear elasticity) is not suitable to describe the equilibrium response of articular cartilage nor characterize its material symmetry; a framework which accounts for the distinctly different responses of cartilage in tension and compression is more suitable for describing the equilibrium response of cartilage; within this framework, cartilage exhibits no lower than orthotropic symmetry.

Mitogen-activated protein kinase signaling in bovine articular chondrocytes in response to fluid flow does not require calcium mobilization

In the present study, the role of mitogen-activated protein kinases (MAPKs) in chondrocyte mechanotransduction was investigated. We hypothesized that MAPKs participate in fluid flow-induced chondrocyte mechanotransduction. To test our hypothesis, we studied cultured chondrocytes subjected to a well-defined mechanical stimulus generated with a laminar flow chamber. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) were activated 1.6-3-fold after 5-15 min of fluid flow exposure corresponding to a chamber wall shear stress of 1.6 Pa. Activation of ERK1/2 was observed in the presence of both 10% FBS and 0.1% BSA, suggesting that the flow effects do not require serum agonists. Treatment with thapsigargin or EGTA had no significant effect on the ERK1/2 activation response to flow, suggesting that Ca2+ mobilization is not required for this response. To assess downstream effects of the activated MAPKs on transcription, flow studies were performed using chondrocytes transfected with a chimeric luciferase construct containing 2.4 kb of the promoter region along with exon 1 of the human aggrecan gene. Two-hour exposure of transfected chondrocytes to fluid flow significantly decreased aggrecan promoter activity by 40%. This response was blocked by treatment of chondrocytes with the MEK-1 inhibitor PD98059. These findings demonstrate that, under the conditions of the present study, fluid flow-induced signals activate the MEK-1/ERK signaling pathway in articular chondrocytes, leading to down-regulation of expression of the aggrecan gene.

An analysis of the effects of depth-dependent aggregate modulus on articular cartilage stress-relaxation behavior in compression

An accurate description of the mechanical environment around chondrocytes embedded within their dense extracellular matrix (ECM) is essential for the study of mechano-signal transduction mechanism(s) in explant experiments. New methods have been developed to determine the inhomogeneous strain distribution throughout the depth of the ECM during compression (Schinagl et al., 1996, Annals of Biomedical Engineering 24, 500-512; Schinagl et al 1997. Journal of Orthopaedics Research 15, 499-506) and the corresponding depth-dependent aggregate modulus distribution (Wang and Mow, 1998. Transactions of the Orthopaedics Research Society 23, 484; Chen and Sah, 1999. Transactions of the Orthopaedics Research Society 24, 635). These results provide the motivation for the current investigation to assess the influence of tissue inhomogeneity on the chondrocyte milieu in situ, e.g. stress, strain, fluid velocity and pressure fields within articular cartilage. To describe this inhomogeneity, we adopted the finite deformation biphasic constitutive law developed by Holmes and Mow (1990 Journal of Biomechanics 23, 1145-1156). Our calculations show that the mechanical environment inside an inhomogeneous tissue differs significantly from that inside a homogeneous tissue. Furthermore, our results indicate that the need to incorporate an inhomogeneous aggregate modulus. or an anisotropy, into the biphasic theory to describe articular cartilage depends largely on the motivation for the study.

Determination of Poisson’s Ratios of Bovine Articular Cartilage in Tension and Compression Using Osmotic and Mechanical Loading

The existence of osmotic pressure inside cartilage gives the tissue a propensity to swell. This swelling pressure is balanced by the tensile stresses generated within the solid matrix at free-swelling [1, 2]. Recent studies have shown that cartilage exhibits significant strain-softening when compressed relative to its free-swelling state [3–5]. Such strain-softening behavior has been physically interpreted within the context of osmotic swelling pressure and tension-compression nonlinearity [4, 9]. This has provided the rationale for extracting both the tensile and compressive Young’s moduli from uniaxial compression tests on the same specimen [4, 5]. The goal of the current study is to optically determine another important elastic property, i.e. the equilibrium Poisson’s ratio of young bovine articular cartilage when uniaxially compressed along its three characteristic directions: parallel and perpendicular to the split-line direction (1- and 2-direction, respectively), and in a direction normal to the articular surface (3-direction). Furthermore, the external bath concentration effects on the Poisson’s ratios will be explored at various strain levels.Copyright