Mimicking the Biochemical and Mechanical Extracellular Environment of the Endochondral Ossification Process to Enhance the In Vitro Mineralization Potential of Human Mesenchymal Stem Cells.

” Chondrogenesis and mechanical stimulation of the cartilage template are essential for bone formation through the endochondral ossification process in vivo. Recent studies have demonstrated that in vitro regeneration strategies that mimic these aspects separately, either chondrogenesis or mechanical stimulation, can promote mineralization to a certain extent both in vitro and in vivo. However, to date no study has sought to incorporate both the formation of the cartilage template and the application of mechanical stimulation simultaneously to induce osteogenesis. In this study, we test the hypothesis that mimicking both the biochemical and mechanical extracellular environment arising during endochondral ossification can enhance the in vitro mineralization potential of human mesenchymal stem cells (hMSCs). hMSC aggregates were cultured for 21 days under the following culture conditions; (1) Growth Medium – hydrostatic pressure (HP), (2) Chondrogenic Priming-HP, (3) Growth Medium + HP, and (4) Chondrogenic Priming +HP. Each group was then further cultured for another 21 days in the presence of osteogenic growth factors without HP. Biochemical (DNA, sulfate glycosaminoglycan, hydroxyproline, alkaline phosphatase activity, and calcium), histological (Alcian Blue and Alizarin Red), and immunohistological (Col I, II, and X, and BSP-2) analyses were conducted to investigate chondrogenic and osteogenic differentiation at various time points (14, 21, 35, and 42 days). Our results showed the application of HP-induced chondrogenesis similar to that of chondrogenic priming, but interestingly, there was a reduction in hypertrophy markers (collagen type X) by applying HP alone versus chondrogenic priming alone. Moreover, the results showed that both chondrogenic priming and HP in tandem during the priming period, followed by culture in osteogenic medium, accelerated the osteogenic potential of hMSCs.”

“application of hydrostatic pressure (0.1 MPa – 10 MPa) to human bone marrow stem cells aggregates  or those seeded on a collagen or agarose scaffold can significantly  enhance expression of chondrogenic markers (aggrecan, SOX‐9).  The highest amount of matrix deposition was found when a pressure of 10 MPa was applied for a  minimum of 5 days”

” the  application of HP without any external growth factors resulted in reduced hypertrophy,  whilst allowing for chondrogenesis, however once the HP was removed and the cells were  exposed to osteogenic growth factors the cells began to produce hypertrophic markers but  at a much slower rate than those exposed to chondrogenic growth factors alone”