Intermittent hydrostatic pressure maintains and enhances the chondrogenic differentiation of cartilage progenitor cells cultivated in alginate beads

The objective of this study was to explore the effects of intermittent hydrostatic pressure (IHP) on the chondrogenic differentiation of cartilage progenitor cells (CPCs) cultivated in alginate beads. CPCs were isolated from the knee joint cartilage of rabbits, and infrapatellar fat pad-derived stem cells (FPSCs) and chondrocytes (CCs) were included as the control cell types. Cells embedded in alginate beads were treated with IHP at 5 Mpa and 0.5 Hz for 4 h/day for 1, 2, or 4 weeks. The cells' migratory and proliferative capacities were evaluated using the scratch and Live/Dead assays, respectively. Hematoxylin and eosin staining, safranin O staining, and immunohistochemical staining were performed to determine the effects of IHP on the synthesis of extracellular matrix (ECM) proteins. Real-time polymerase chain reaction analysis was performed to measure the expression of genes related to chondrogenesis. The scratch and Live/Dead assays revealed that IHP significantly promoted the migration and proliferation of FPSCs and CPCs to different extents. The staining experiments showed greater production of cartilage ECM components (glycosaminoglycans and collagen II) by cells exposed to IHP, and the gene expression analysis demonstrated that IHP stimulated the expression of chondrocyte-related genes. Importantly, these effects of IHP were more prominent in CPCs than in FPSCs and CCs. Considering all of our experimental results combined, we conclude that CPCs demonstrated a stronger chondrogenic differentiation capacity than the FPSCs and CCs under stimulation with IHP. Thus, the use of CPCs, combined with mechanical stimulation, may represent a valuable strategy for cartilage tissue engineering.