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Effective Bone Engineering with Periosteum-derived Cells

¹ Division of Stem Cell Engineering, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan;
² Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan;
³ Department of Regenerative Oral Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; and
⁴ Department of Plastic and Reconstructive Surgery, Kitasato University School of Medicine, Kanagawa, Japan

View larger version (14K): [in this window] [in a new window]   Figure 1. Periosteal cells (closed circles) and marrow stromal cells (open circles) were plated in 12-well plates at a density of 2.0 x 104 cells/well. Cell numbers were counted directly (a). FGF-pre-treated periosteal cells are indicated by closed triangles, and non-pre-treated periosteal cells are indicated by closed circles (b). Values are means ± standard deviation for 3 cultures. Asterisk indicates significant difference in the number of periosteal cells, compared with marrow stromal cells on the same day; p < 0.05.

View larger version (34K): [in this window] [in a new window]   Figure 2. Periosteal cells (black) and marrow stromal cells (white) were plated in 12-well plates at a density of 1.0 x 105 cells/well. Then, the cells were incubated with bFGF (a) or BMP-2 (b) for 6 days. Alkaline phosphatase activity was analyzed on day 7 of culture. In (a), asterisk indicates significant difference (p < 0.05), compared with the control (–). FGF-pre-treated periosteal cells are indicated by black hatched bars; non-pre-treated periosteal cells are indicated by black bars; FGF-pre-treated marrow stromal cells are indicated by white hatched bars; non-pre-treated marrow stromal cells are indicated by white bars (c,d). Values are means ± standard deviation for 3 cultures. In b–d, asterisk indicates significant difference (p < 0.05) between paired conditions.

View larger version (69K): [in this window] [in a new window]   Figure 3. RT-PCR analysis of periosteal cells and marrow stromal cells. For 6 days, each cell type was cultured in serum-conditioned -MEM alone (–) or in serum-conditioned -MEM containing BMP-2 (BMP). To evaluate the effect of FGF pre-treatment, we pre-treated some cells with bFGF for 2 days (Pre-treatment), and treated some cultures with BMP after bFGF pre-treatment (Pre-treatment + BMP).

View larger version (99K): [in this window] [in a new window]   Figure 4. Hematoxylin and eosin staining demonstrating in vivo bone formation. Transplants of untreated control periosteal cells (-) formed little bone (a). Transplants of periosteal cells that received BMP-2 treatment alone (BMP) (b) or received FGF pre-treatment alone (pre-treatment) (c) also formed new bone. Periosteal cells that were treated with BMP-2 after FGF pre-treatment (pre-treatment + BMP) formed significant amounts of new bone (d). Bar indicates 300 µm. Arrow indicates new bone. The difference in new bone volume between periosteal cells (black) and marrow stromal cells (white) was determined by histomorphometric analysis (e). Asterisk indicates significant difference (p < 0.05) between paired conditions. Values are means ± standard deviation for 3 sections of each sample.