Osteocalcin secretion as an early marker of in vitro osteogenic differentiation of rat mesenchymal stem cells.
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.
Nakamura
Akifumi
A
Department of Orthopedic Surgery, Health Management, and Policy, School of Medicine, Nara Medical University, Kashihara, Nara, Japan. akibun@xj9.so-net.ne.jp
Dohi
Yoshiko
Y
Akahane
Manabu
M
Ohgushi
Hajime
H
Nakajima
Hiroshi
H
Funaoka
Hiroyuki
H
Takakura
Yoshinori
Y
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D017886:Durapatite / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005786:Gene Expression Regulation; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism*; D010012:Osteogenesis / Q000235:genetics; D010641:Phenotype; D051381:Rats; D011916:Rats, Inbred F344; D013997:Time Factors
D015415:Biomarkers; D017077:Culture Media, Conditioned; D015675:Osteocalcin; D017886:Durapatite; D000469:Alkaline Phosphatase; D002118:Calcium