Modifying oxygen tension affects bone marrow stromal cell osteogenesis for regenerative medicine

Figure 1 Osteocalcin secretion. In cells exposed to hypoxia (5% O₂) for 7 d followed by normoxia (21% O₂) for 7 d (HN), the level of osteocalcin increased rapidly from day 10, peaking at day 12, as compared with cells in the other groups. NN, normoxia for 14 d; NH, normoxia for 7 d followed by hypoxia for 7 d; and HH, hypoxia for 14 d. ^aP < 0.05.

Figure 2 Observations of cell morphology and calcium deposition. Cells treated with normoxia (21% O₂) for 14 d (NN) (A) or hypoxia (5% O₂) for 7 d followed by normoxia for 7 d (HN) (C) were differentiated and cuboidal-shaped, and showed cell nodules with calcium deposits. These changes were not present in cells exposed to normoxia for 7 d followed by hypoxia for 7 d (NH) (B) or hypoxia for 14 d (HH) (D). Arrowheads indicate calcium deposition.

Figure 3 Calcium deposition. Cells were exposed to normoxia (21% O₂) for 14 d (NN), normoxia for 7 d followed by hypoxia (5% O₂) for 7 d (NH), hypoxia for 7 d followed by normoxia for 7 d (HN), or hypoxia for 14 d (HH). Calcium deposition was adjusted to total protein content. Cells in the HN group showed the highest amount of calcium deposition among the four groups. ^aP < 0.05.

Figure 4 Alkaline phosphatase staining. Cells were exposed to normoxia (21% O₂) for 14 d (NN) (A), normoxia for 7 d followed by hypoxia (5% O₂) for 7 d (NH) (B), hypoxia for 7 d followed by normoxia for 7 d (HN) (C), or hypoxia for 14 d (HH) (D) and then stained with alkaline phosphatase staining. The HN group showed the strongest and the broadest alkaline phosphatase staining (C).

Figure 5 Cell proliferation assay. Cell proliferation under hypoxic conditions (H: 5% O₂) was significantly higher than that under normal oxygen conditions (N: 21% O₂). ^aP < 0.05.

Figure 6 Histology of β-tricalcium phosphate discs wrapped with osteogenic matrix cell sheets. Cell sheets were exposed to hypoxia (5% O₂) for 7 d followed by normoxia (21% O₂) for 7 d (B) or normoxia for 14 d (A). Prominent newly formed bone (*) was observed in the HN group. (C) and (D) are higher magnifications of squared areas indicated in (A) and (B), respectively.

Figure 7 Calcium content in β-tricalcium phosphate discs wrapped with osteogenic matrix cell sheet. Cell sheets were exposed to hypoxia (5% O₂) for 7 d followed by normoxia (21% O₂) for 7 d (HN) or normoxia for 14 d (NN). The HN group showed a significantly higher amount of calcium content compared with the NN group. ^aP < 0.05.

Figure 8 Real-time quantitative PCR of β-tricalcium phosphate discs wrapped with osteogenic matrix cell sheet. Increased collagen type I mRNA expression was observed in the HN group exposed to hypoxia (5% O₂) for 7 d followed by normoxia (21% O₂) for 7 d, compared with the NN group exposed to normoxia for 14 d (A). A tendency for higher expression of osteocalcin mRNA was also observed in the HN group (B). ^aP < 0.05.