Figure 1.

Overview of experimental design. (A) Workflow scheme of the analysis of the effect of compression on HDAC4 shuttle in chondrocytes cultured in alginate. After isolation, chondrocytes were first cultured in monolayer for 6 days. Passage chondrocytes were infected with GFP-HDAC4. After 20 hours, the transfected cells were embedded in alginate gels and pre-cultured in 3D cell/alginate constructs for 7 days to allow pericellular matrix deposition before being subjected to compression. The cell/alginate constructs were analyzed after compression. (B) Transfection efficiency of HDAC4 in chondrocytes was validated with confocal laser scanning microscope by capturing Green fluorescent protein (GFP). Nuclei were visualized by Hoechst 33342 staining. (C) Approximately 300 cells from 3 independent experiments were scored. Data are expressed as means±SD. Transfection efficiency of HDAC4 was 89.78%±3.70%. (D,E) Real-time PCR results indicated that both aggrecan (D) and type II collagen (E) mRNA expression were elevated at 2 and 3 h of compression, but expression levels were decreased at 4 h of compression. Values are presented as mean±SD (n=3). *=P<0.05 versus the unloaded group. (F) Viability was assessed by Hoechst 33342/PI double staining at 48 h post-compression. The cell/alginate culture constructs frozen at −20°C served as positive controls(F-a to c). There were no visible dead cells at 48 h post-compression (F-d to f). Blue indicates nuclei stained by Hoechst 33342, and red indicates PI staining dead cells.