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Recovery of Stem Cells from Cryopreserved Periodontal Ligament

¹ Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, Building 30, Room 131, 30 Convent Drive MSC-4320, and ² Comparative Molecular Cytogenetics Core, Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; and ³ Department of Oral and Maxillofacial Surgery, College of Dentistry, Seoul National University, Seoul, Korea;

View larger version (61K): [in a new window]   Figure 1. Isolation of cryopreserved periodontal ligament stem cells. (A) Periodontal ligament stem cells recovered from 6-month-cryopreserved periodontal ligament were capable of forming single-cell clusters after being plated at low density and cultured with regular culture medium for 10 days as described in METHODS. The number of single colonies derived from cryopreserved periodontal ligament (CP) was significantly decreased (*p < 0.05) in comparison with that derived from the fresh non-frozen periodontal ligament (P) when the same number (5000) of cells was plated (CP = 4.7 ± 0.6, P = 24.8 ± 1.5, mean ± SD) (N = 3). (B) The proliferation rates were assessed by bromodeoxyuridine (BrdU) incorporation for 12 hrs. Cryopreserved periodontal ligament stem cells (CP) maintain a high level of proliferation rate, similar to that of the regular periodontal ligament stem cells (P), showing that there is no significant difference between the regular periodontal ligament stem cells and cryopreserved periodontal ligament stem cells (73.8 ± 3.7, 67.4 ± 10.3, respectively, mean ± SD) (N = 3). (C) H&E staining of non-frozen human periodontal ligament tissue. (D,E) H&E staining of periodontal ligament cryopreserved for 6 mos. Most areas of periodontal ligament tissue showed normal histological structures in H&E staining. However, some nuclear anisokaryosis was found in frozen periodontal ligament (E, arrow), indicating that the cryopreservation can cause some tissue damage (scale bar, 200 µm for C-E). (F-M) Cryopreserved periodontal ligament stem cells expressed STRO-1, one of the early progenitor markers for mesenchymal stem cells. The cryopreserved periodontal ligament stem cells may co-express STRO-1 with bone sialoprotein (BSP) and TGFß receptor type I (TGFßRI), as shown on the merged Figs. Some cryopreserved periodontal ligament stem cells may express STRO-1 and BSP separately (scale bar, 50 µm for F–M).

View larger version (80K): [in a new window]   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 TGFß1 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 TGFß1 for 4 wks. (H) The newly generated aggregates were positive for anti-type I collagen antibody staining (scale bar, 50 µm for E-H).

View larger version (155K): [in a new window]   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).