Figure 3. In vivo characterization of cryopreserved periodontal ligament stem cells. (A) After 8 wks of transplantation, cryopreserved periodontal ligament stem cells were capable of forming a cementum-like structure (C) on the surfaces of the hydroxyapatite/tricalcium phosphate (HA) carrier, which was connected to periodontal-ligament-like tissue (PDL). (B) The cells responsible for cementum (C) formation were positive for anti-human specific mitochondria antibody staining (black arrows). Analysis of the immunohistochemical staining data indicated that transplanted cryopreserved periodontal ligament stem cells differentiated into cementoblasts/cementocytes and generated cementum in vivo. (C,D) Transplanted cryopreserved periodontal ligament stem cells were able to form cementum (C) on the surfaces of HA/TCP particles (HA) and were able to generate Sharpey’s fibers (black arrows) inserted into cementum and which were continuous with periodontal-ligament-like tissue (PDL), shown by H&E (C) and Trichrome staining (D). (E,F) Of 6 selected single-colony-derived cryopreserved periodontal ligament stem cell strains, only 4 (67%) were capable of generating a cementum/periodontal-ligament-like structure (E). Newly formed cementum (C) was found to be adjacent to the surfaces of the HA/TCP carrier (HA) and was connected with periodontal-ligament-like tissue (PDL) that was the same as Sharpey’s fibers (back arrows). The remaining 33% (2 of 6) single-colony-derived cryopreserved periodontal ligament stem cell strains were unable to generate cementum in vivo (F). (G,H) Newly formed cementum (C) was positive for anti-type I collagen antibody staining (G), and cementogenic cells were positive for anti-bone sialoprotein (BSP) antibody staining (open arrows in H). (I) Pre-immunoserum control was negative for immunohistochemical staining of type I collagen and BSP antibodies (scale bar, 50 µm for A-I).