Abstract
Objective
In order to effectively utilize mechanical signals in the clinic as a non-drug-based intervention to improve cartilage defect regeneration after surgical treatment, it is essential to identify crucial components of the cellular response that are typical to the anabolic process. The mechanisms behind the effect of mechanical stimulation are, however, not fully understood and the signaling pathways involved in the anabolic response of chondrocytes to mechano-transduction are not well described. Therefore, a genome-wide identification of mechano-regulated genes and candidate pathways in human chondrocytes subjected to a single anabolic loading episode was performed in this study and time evolution and re-inducibility of the response was characterized.
Design
Osteochondral constructs consisting of a chondrocyte-seeded collagen-scaffold connected to β-tricalcium-phosphate were pre-cultured for 35 days and subjected to dynamic compression (25% strain, 1 Hz, 9×10 minutes over 3h) before microarray-profiling was performed. Proteoglycan synthesis was determined by 35S-sulfate-incorporation over 24 hours. Protein alterations were determined by Western blotting.
Results
Cell viability and hardness of constructs were unaltered by dynamic compression while proteoglycan synthesis was significantly stimulated (1.45-fold, p=0.016). Among 115 significantly regulated genes, 114 were up-regulated, 48 of them ≥ two-fold. AP-1-relevant transcription factors FOSB and FOS strongly increased in line with elevated ERK1/2-phosphorylation and rising MAP3K4 expression. Expression of proteoglycan-synthesizing enzymes CHSY1 and GALNT4 was load-responsive as were factors associated with the MAPK-, TGF-β-, calcium-, retinoic-acid-, Wnt- and Notch-signaling pathway which were significantly altered. SOX9, BMP4 and BMP6 levels rose significantly also after multiple loading episodes at daily intervals even at the 14th cycle with no indication for desensitation. Canonical pSmad2/3 and pSmad1/5/9-signalling was apparently unaltered.
Conclusion
This study associates raising SOX9 protein levels, pERK stimulation and increased CHSY1 expression with anabolic loading of chondrocytes and suggests that more pathways than so far anticipated apparently work together in a complex network of stimulators and feedback-regulators. Knowledge on time evolution of mechanosensitive indicators responding to anabolic loading is crucial to maximize cartilage matrix-deposition for the generation of high-level cartilage replacement tissue.
