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 (<i>COL10A1</i>, <i>MMP13</i>, <i>RUNX2</i>, <i>ALP</i>), and reduced glycosaminoglycan production. Of particular interest, we show that P2ce (early short stimulation) was as effective as the two extended stimulation protocols, suggesting that initial mechanical signals are sufficient to trigger cell differentiation toward a hypertrophic chondrocyte phenotype that continues even after stimulation ceases. These in vitro findings provide crucial insights into the cellular basis of endochondral ossification during the early phase of loading and show a beneficial long-term effect of early mechanical stimulation. By demonstrating that the cellular mechanobiology of hypertrophic differentiation responds to brief early stimulation, our findings provide a scientific foundation to guide future in vivo investigations on how rehabilitation protocols could influence fracture healing.