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Osteocalcin (OC) is a bone-specific protein synthesized by osteoblasts that represents a good marker for osteogenic maturation. We examined whether in vitro osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (MSCs) could be simply assessed at earlier stages by monitoring OC secretion into the conditioned medium, rather than measuring OC deposition on the extracellular matrix (ECM), using a sandwich enzyme immunoassay system involving a specific anti-rat OC monoclonal antibody. During a 16-day culture, OC was secreted into the medium of MSCs from day 8 and increased substantially until day 16. In contrast, OC deposition on the ECM was low, even at day 13, when calcium deposition was at high levels. The histological expression pattern of OC messenger RNA provided in situ evidence that osteoblastic cells appeared at the early stages of 6 to 9 days and matured over time in vitro. Furthermore, the temporal expression of osteogenesis-specific genes, such as the transcriptional factors core-binding factor 1 and osterix, followed by increases in secretory OC proved the commitment of MSCs to osteoblastic differentiation. These results revealed that biomineralization followed secretion of OC, which may reflect early osteoblastic differentiation of cultured MSCs under osteoinductive conditions. We ascertained the osteogenic differentiation capacity of cultured MSCs in a non-destructive manner by monitoring OC secretion into the culture medium and proved that secretory OC could represent a reliable marker for predicting in vivo osteogenic potential in bone tissue engineering.

Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

We previously reported that in vivo bone formation could be observed in composites of porous hydroxyapatite (HA) scaffolds and cultured mesenchymal stem cells (MSCs). In the present study, we developed a new method for transplantation of cultured MSCs without the necessity of using a scaffold to form bone tissue. MSCs were culture-expanded and lifted as cell sheet structures. These cell sheets, designated osteogenic matrix sheets, showed positive alkaline phosphatase (ALP) staining, high ALP activities and high osteocalcin (OC) contents, indicating their osteogenic potential. We transplanted these sheets into subcutaneous sites in rats to assess whether they possessed in vivo bone-forming capability. The transplanted sheets showed mineralized matrix together with osteocytes and an active osteoblast lining, indicating new bone formation, at 6 weeks after transplantation. HA scaffolds were also wrapped with the sheets to make HA/sheet composites and implanted into subcutaneous sites in rats. Histological sections of the composites revealed bone formation in the HA pores at 4 weeks after implantation. Our present results indicate that MSCs can be cultured as sheet structures, and the resulting sheets themselves or HA-sheet composites represent osteogenic implants that can be used for hard tissue reconstruction.

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