Compressive Strains at Physiological Frequencies Influence the Metabolism of Chondrocytes Seeded in Agarose

David A. Lee and Dan L. Bader

Full text link: http://www3.interscience.wiley.com/cgi-bin/fulltext/109929044/PDFSTART

The experiment presented in the article studies the universality of mechanotransduction pathways for chondrocyte metabolism under the application of static and dynamic mechanical strains. Specifically, the experiment aims to characterize whether chondrocyte deformation under mechanical strain has the capability of stimulating three major markers of chondrocyte metabolism: proteoglycan synthesis, cell division, and protein synthesis.

In the experiment, static and dynamic mechanical strains were applied on agarose-chondrocyte cylinders using a specially designed cell straining apparatus. The level of gross compressive strain used was 15%, which lies in the middle of the 0-30% physiological range for cell strain in intact cartilage subjected to physiological loads. For dynamic loading, the frequencies studied were 0.3 Hz, 1 Hz, and 3 Hz – all three were within the physiological range. The agarose-chondrocyte cylinders were prepared from chondrocytes removed from the metacarpophalangeal joints of 18-month-old steers, embedded in 3% agarose, and cultured under strain for 48 hours in DMEM with 20% fetal calf serum. Unstrained agarose-chondrocyte cylinders were used for control.

Chondrocyte viability was determined for each of the experimental group using trypan blue assay and the results showed that viability ranged from 97.8 to 99.1% of the time-zero viability. Glycosaminoglycan (GAG) synthesis by chondrocytes embedded in agarose was determined using a rapid spectrophotometric procedure that involved papain digestion of tissue or culture medium that provides glycosaminoglycans for assay. The results, normalized to the control cultures, indicated a significant reduction in glycosaminoglycan synthesis for static strain and 0.3 Hz dynamic strain, but a stimulation of glycosaminoglycan synthesis to 40% more than the unstrained control for dynamic strain at 1 Hz. Dynamic strain at 3 Hz did not significantly alter glycosaminoglycan synthesis. Incorporation of [³H]thymidine was analyzed as a marker for cell division and the results showed that its uptake was inhibited in cylinders subjected to static strain as compared to static strain, but was stimulated in cylinders subjected to dynamic strain at all three frequencies. Finally, incorporation of [³H]proline was analyzed as a marker for protein synthesis. The results showed that when compared to unstrained control, [³H]proline uptake was inhibited in cylinders subjected to both static and dynamic strain at all frequencies investigated. Because the three parameters investigated in the study were each influenced by the strain regimens in a distinct manner, it was concluded that that the mechanotransduction pathways involved were likely to be uncoupled.

I chose this article because I feel that this study is particularly important for the development and design of cell-seeded cartilage repair systems for cartilage diseases such as arthritis. As a matter of fact, a balance between cartilage matrix synthesis and degradation is critical both for the maintenance of articular cartilage function and skeletal development and growth. Because the focus of this study is on the mechanical aspects that can alter this balance, it is thus able to provide a general understanding of chondrocyte regulation in cartilage when subjected to various types of compressive strains. Additionally, the results from the study can give insights on how a tissue engineer can design an experiment that closely mimics human physiological conditions. The study presented made this attempt by subjecting the chondrocyte cell culture to dynamic loading at various frequencies so that it is more similar to normal walking. This way, more applicable results can be drawn from the experiment that can be used to generalize the effects of mechanotransduction on articular cartilage under physiological loading in human joints.