Transient Early Mechanical Loading Induces Hypertrophic Chondrocyte Differentiation of Human Mesenchymal Stromal Cells.

Optimal mechanical parameters for successful bone-healing remain unclear despite their critical influence on fracture outcomes, and the timing of post-surgery mobilization is still controversial despite many clinical observations and pre-clinical studies. In this bioreactor in vitro work, we investigate the effect of fundamental parameters such as timing, duration, and frequency of mechanical stimulation on the endochondral bone-healing paths, specifically on the hypertrophic chondrocyte differentiation of naïve human mesenchymal stromal cells (hMSCs). Human MSCs encapsulated in Gelatin-Methacryloyl hydrogels (GelMa) were subjected to three different 10% strain protocols: P1 (168 long-break cycles spread over 14 days), P2ce (cycle equivalent: 168 short-break cycles condensed in 42-min stimulation followed by 14 days free swelling), and P2te (time equivalent-14 days continuous stimulation, 80'640 short-break cycles). In the free-swelling control group, samples were cultured for 14 days without any mechanical stimulation. Our results confirmed that 10% strain induces a robust hypertrophic chondrocyte differentiation of naïve MSCs in all three tested protocols, as demonstrated by enlarged cell size, rounded morphology, robust upregulation of hypertrophic markers (