Osteocalcin Secretion as an Early Marker ofIn VitroOsteogenic Differentiation of Rat Mesenchymal Stem Cells

Abstract

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.

Early Characterization of Blast-related Heterotopic Ossification in a Rat Model

“…A cohort of gene transcripts and key osteogenic-related proteins (RUNX-2, OCN, NOG, SP-1) were identified that correlate to the early histological response and development of ectopic bone. RUNX-2 is the early master regulator of osteogenic/osteoblast differentiation [16], and OCN is a prime marker of bone development produced by osteoblasts [18]. SP-1 is a neuroinflammatory peptide that has been reported to promote the mobilization, proliferation, and osteogenic differentiation of mesenchymal stem cells at sensory nerve structures [25,26].…”

Increased osteogenesis with hydroxyapatite constructs combined with serially-passaged bone marrow-derived mesenchymal stem cells

“…10 mg of each reagent was dissolved in 20 ml of AMP buffer (1.0 mM MgCl 2 , 10 mM p-nitrophenyl phosphate in 0.056 M 2-amino-2-methyl-propanol). Cells were incubated with 2 ml of the substrate solution at room temperature for ~2 minutes and rinsed with dH 2 O. Alizarin Red S (0.5%) was also used to evaluate calcium deposition, following which cells were rinsed with dH 2 O [12,18]. Each experiment contained 3 replicates and was performed in duplicate.…”

“…Cells cultured in 12-well culture plates were used to measure ALP activity, as previously reported [12]. Briefly, P1, P2 and P3 cells, seeded in 12-well plates at a density of 1 × 10 4 cells/cm 2 and cultured for 14 days with or without osteogenic-induced media containing Dex, ascorbic acid phosphate and β-GP, were scraped into 1 ml of 0.05 M sodium phosphate buffer, 2 mM EDTA and 2 M NaCl, following which they were homogenized and sonicated.…”

“…We have previously reported the preparation of HA constructs combined with BMSCs (BMSC/HA construct) [12,17]. Briefly, after release from the substratum, P3 cells were centrifuged and resuspended at a density of 1 × 10 6 cells/ml in standard medium.…”

“…Differentiation into osteoblastic lineage cells after subculture in osteogenic-induced media, containing dexamethasone (Dex), ascorbic acid phosphate and β-glycerophosphate (β-GP), has been shown for rat and human BMSCs [8][9][10][11]. The osteogenic phenotypic markers, alkaline phosphatase (ALP) and osteocalcin (OC), can be detected by days 10 -12 in passage 1 (P1)-cultured cells, including mineralization [12]. The combination of BMSCs with a scaffolding material, such as hydroxyapatite (HA), can promote the formation of new bone tissue in vivo after subcutaneous transplantation with freshly isolated bone marrow cells [13] or culture-expanded BMSCs (HA/ BMSCs construct) [2,[14][15][16][17], Recently, we reported a new cell transplantation technique in which BMSCs were cultured in culture medium containing Dex and ascorbic acid phosphate and transplanted as cell sheets [18].…”