Fig. 5
Mouse PDGFRα reporter+ periosteal progenitors have high osteogenic potential. a Schematic of experiments in which PDGFRα reporter−CD31−CD45−Ter119− and PDGFRα reporter+CD31−CD45−Ter119− periosteal cells were derived by FACS from PDGFRαmT/mG hindlimbs (male, aged 2 mo). b Cell proliferation among PDGFRα reporter− and PDGFRα reporter+ periosteal cells determined by MTS assays at 72 h. c Osteogenic differentiation of PDGFRα reporter−/+ periosteal cells at 16 d determined by Alizarin Red (AR) staining and quantification. Expression of the osteogenic genes, including (d) alkaline phosphatase (Alp), (e) runt-related transcription factor 2 (Runx2), (f) Osterix (Sp7), and (g) osteocalcin (Bglap), in PDGFRα reporter−/+ periosteal cells after 7 d of differentiation. h Osteogenic differentiation of freshly isolated PDGFRα reporter−/+ periosteal cells at 14 d determined by Alizarin Red (AR) staining and quantification. i Schematic of ossicle assay. PDGFRα reporter− or reporter+ cells (3 × 106) were subcutaneously implanted into NOD SCID mice using an HA/β-TCP carrier and harvested after 8 weeks. j Representative micro-CT reconstructions of the implants. k Quantitative analysis of micro-CT images of PDGFRα reporter−/+ periosteal cells. The analysis included the bone volume (BV) and fractional bone volume (BV/TV). l Persistence of PDGFRα reporter+ and PDGFRα reporter− periosteal cells. PDGFRα reporter− cells appeared red, and PDGFRα reporter+ cells appeared green. Nuclei, DAPI (4′,6-diamidino-2-phenylindole, blue). The white dashed lines demarcate the edges of the scaffold. m Representative histologic appearance determined by H&E, including low-magnification (above left) and high-magnification (below left) images, and quantification of the bone area/tissue area (right). n Representative ALP staining. o OCN immunohistochemistry. Positive immunohistochemistry is shown by purple or white. PDGFRα reporter− cells appeared red (Tdtomato+), and PDGFRα reporter+ cells appeared green (GFP+). Red scale bars: 500 μm; White scale bars: 50 μm. B bone, BM bone marrow, S scaffold. The dot plots represent an individual sample or animal, and the whisker plots indicate the mean and one-SD values. In vitro experiments were performed in biological and experimental triplicates (a–h). For subcutaneous implantation, n = 6 animals per group (i–o). *P < 0.05, **P < 0.01 and ***P < 0.001, as assessed by two-tailed Student’s t tests
