Differential expression of type X collagen in a mechanically active 3-D chondrocyte culture system: a quantitative study

Abstract

Objective: Mechanical loading of cartilage influences chondrocyte metabolism and gene expression. The gene encoding type X collagen is expressed specifically by hypertrophic chondrocytes and up regulated during osteoarthritis. In this study we tested the hypothesis that the mechanical microenvironment resulting from higher levels of local strain in a three dimensional cell culture construct would lead to an increase in the expression of type X collagen mRNA by chondrocytes in those areas. Methods:Hypertrophic chondrocytes were isolated from embryonic chick sterna and seeded onto rectangular Gelfoam sponges. Seeded sponges were subjected to various levels of cyclic uniaxial tensile strains at 1 Hz with the computer-controlled Bio-Stretch system. Strain distribution across the sponge was quantified by digital image analysis. After mechanical loading, sponges were cut and the end and center regions were separated according to construct strain distribution. Total RNA was extracted from the cells harvested from these regions, and real-time quantitative RT-PCR was performed to quantify mRNA levels for type X collagen and a housing-keeping gene 18S RNA.Results: Chondrocytes distributed in high (9%) local strain areas produced more than two times type X collagen mRNA compared to the those under no load conditions, while chondrocytes located in low (2.5%) local strain areas had no appreciable difference in type X collagen mRNA production in comparison to non-loaded samples. Increasing local strains above 2.5%, either in the center or end regions of the sponge, resulted in increased expression of Col X mRNA by chondrocytes in that region. Conclusion:These findings suggest that the threshold of chondrocyte sensitivity to inducing type X collagen mRNA production is more than 2.5% local strain, and that increased local strains above the threshold results in an increase of Col X mRNA expression. Such quantitative analysis has important implications for our understanding of mechanosensitivity of cartilage and mechanical regulation of chondrocyte gene expression.

The Effect of Altered Loading on Mandibular Condylar Cartilage

“…Numerous in vitro studies in MCC and articular cartilage have shown an increase in cell differentiation and increased proteoglycan synthesis after mechanically loading the articular chondrocytes [ 35 – 39 ]. Similarly, Yang et al in their in vitro study with articular chondrocytes showed a two-fold increase in type X collagen with mechanical loading[ 40 ]. Moreover, Sobue et al showed an increase in Col2a1 and Col10a1 with 0.5N of compressive load on the MCC in mice [ 5 ].…”

Primary cilia modulate Ihh signal transduction in response to hydrostatic loading of growth plate chondrocytes

“…Animal models in which growth was restricted using a wire loop [31–32], physeal stapling [33], or overloading the growth plate using weighted backpacks [34] all demonstrate that the growth plate is biologically responsive to changes in physical forces. In vitro loading experiments using chondrocytes seeded in scaffolds, such as agarose, alginate, poly (L-lactide-co-ε-caprolactone (PLCL), and gelfoam all indicate that compression-induced changes in the chondrocyte’s environment modulates the synthesis of extracellular matrix components at the mRNA and protein levels [3–4, 35–38]. …”

Skeletal stiffening in an amphibious fish out of water is a response to increased body weight

“…This protein is expressed by hypertrophic chondrocytes at the margins of growing endochondral bones, where it provides structural support and promotes mineralization (Jacenko et al, 1993;Rosati et al, 1994). Furthermore, experiments using cultured chondrocytes show that expression of type X collagen increases in response to mechanical strain (Yang et al, 2006). In fishes, type X collagen is similarly associated with bone formation (Renn and Winkler, 2010;Eames et al, 2012).…”

“…Furthermore, dynamic or cyclical mechanical forces are thought to be the predominant signals for bone growth and mineralization, and gravitational loading of inactive fish out of water (e.g. Turko et al, 2014) would be largely static and therefore may not be expected to cause a typical skeletal loading response (Turner, 1998;Yang et al, 2006).…”