Sequential Differentiation of Human Bone Marrow Stromal Cells for Bone Regeneration

In this study we hypothesized that as a simulation of endochondral bone formation, bone marrow stromal cell (BMSC) provide a sequential chondro-osteogenic differentiation potential. A chondrogenic priming of BMSC leads to a spontaneous three-dimensional cell formation. BMSC were chondrogenically differentiated prior to an osteogenic stimulation. Duration of cell culture was 28 days, whereas in group A BMSC were chondrogenically differentiated for 1 day, followed by an osteogenic differentiation for 27 days. In group B BMSC were chondrogenically differentiated for 14 days prior to an osteogenic differentiation of 14 days and group C BMSC were differentiated chondrogenically for 28 days serving as a chondrogenic control group. Chondrogenic priming induced a spontaneous three-dimensional cell formation. To survey the stability of the osteogenic phenotype in the absence of an osteogenic stimulus, investigations were performed in vivo in a specially adapted chorioallantoic membrane model of fertilized White Leghorn eggs. Histology and real time polymerase chain reaction revealed a higher amount of osteogenic extracellular matrix synthesis and significant higher expressions of osteogenic marker genes in group B after 14 days of chondrogenic and 14 days of osteogenic stimulation. Matrix calcification in vivo in the absence of an osteogenic stimulus could be demonstrated. The results of the present study support the theory of a sequential differentiation potential of BMSC. A chondrogenic priming of BMSC stimulated into the osteogenic lineage result in a stable osteogenic phenotype in a scaffold-free, three-dimensional tissue engineering application.