Figure 2. In vitro characterization of cryopreserved periodontal ligament stem cells. (A,B) Alizarin red staining showed mineralized nodule formation (A). In the regular culture conditions, the cryopreserved periodontal ligament stem cells were unable to form mineralized nodules (B). (C,D) Cryopreserved periodontal ligament stem cells were able to form Oil red O-positive lipid clusters (C). Regular culture medium could not induce any Oil red O-positive lipid clusters in cryopreserved periodontal ligament stem cells (D) (scale bar, 100 µm for A-D). (E) When periodontal ligament stem cells were cultured with 10 ng/mL TGFβ1 for 4 wks, they formed distinct collagen fibers in vitro (open arrows). (F) The in vitro-generated fibers were positive for anti-type I collagen antibody staining (open arrows). (G) Cryopreserved periodontal ligament stem cells were also able to generate collagen aggregates in vitro when cultured with 10 ng/mL of TGFβ1 for 4 wks. (H) The newly generated aggregates were positive for anti-type I collagen antibody staining (scale bar, 50 µm for E-H).