PMC

Electrophoresis of PCR products for rat GAPDH and human Alu sequence. PCR products of rat GAPDH and human Alu were analyzed by 2% agarose gel electrophoresis with 100 bp DNA marker and photographed. DNA samples from sections acquired at day 3, 7, and 28 showed similar intensity of rat GAPDH bands (204 bp), while the intensity of the human Alu sequence (282 bp) was slightly decreased. gDNA: genomic DNA.

Periodontal regeneration in rat periodontal defects. (A) Micro-CT images of periodontal defects four weeks post-transplantation. The buccal area of the rat first mandibular molar was demonstrated from five rats in each test group. The yellow area shows the denuded root surface area below the red line, connecting the mesial and distal alveolar bone crests. (B) Quantification of the yellow area in micro-CT images taken at four weeks after transplantation. The yellow area is smaller in the PDLSC-amnion group than in the control, suggesting the enhanced new tissue formation. * p < 0.05, Student’s t test.

Localization of PKH26-labeled PDLSC in regenerated periodontal tissues. Fluorescence microscopic images of periodontal tissues four weeks after transplantation. Lower magnification image of the section (A). Close-up image of the red box area in panel (A)is shown in panel (B). Higher magnified images of the blue, green, and blue boxed areas are demonstrated in panels (C–E), respectively. The yellow and white dotted lines denote the bottom of created periodontal defect and bone surface, respectively. Red fluorescence signals are derived from PKH26, which was used for labeling of transplanted PDLSCs, and blue signals are from DAPI nuclear staining. PKH2-positive cells were observed in limited areas in periodontal tissues, as indicated by the yellow arrows, at the outer surface of bone and incorporated in blood vessel. The center area of regenerated PDL did not contain PKH26-positive cells. Yellow arrow: PKH26-positive cells, B: bone, D: dentin, PDL: periodontal ligament.

Histological analysis of periodontal tissues. Histological observation of periodontal tissues in rat periodontal defects four weeks after transplantation. Histological images are shown using long axial (A–H) and horizontal (I–N) sections at lower (A,B,I,J) and higher magnification (C,D,E–H,K–N). Sections were stained with hematoxylin and eosin (A–F,I–L) or azan (G,H,M,N). The boxed area in panels A,B are demonstrated in panels C,D, respectively. Close-up images of center of denuded root surface are shown in panels E–H. More bone formation was observed in the PDLSC-amnion group compared with control (A–D,I–L), and cementum-like thin layer of hard tissue was formed on the root surface in the PDLSC-amnion group (F,N, yellow arrows). Collagen fibers originating perpendicular to the root surface were obvious in PDLSC-amnion group sections (H, black arrows). The dotted line outlines the periodontal defect in horizontal sections (I,J). New formation of bone and PDL-like structure were prominent in the PDLSC-amnion group, while the defects were filled with collagen fibers in the control (K,M). The PDL space contained many blood vessels (L,N). *: new bone, black arrowhead: bottom of defect, yellow arrow: cementum-like tissue, black arrow: collagen bundles run from root surface, MR: mesial root, BR: buccal root, DR: distal root, LR: lingual root, NB: new bone, PDL: periodontal ligament, V: blood vessel, Bar = 300 μm (A–D,I–N) and 50 μm (E–H).

PDLSC transfer on amnion using the cell transfer technology and transplantation into periodontal defects. (A) Cultured PDLSCs were transferred onto decellularized amnion surface. The yellow area on the transfer base shows the hydrophobic cell nonadhesive surface and the blue area denotes the hydrophilic cell adhesive surface. Trypsinized PDLSCs were seeded onto blue cell adhesive surface, created by ultra violet light irradiation of the tetraethylene glycol (TEG) or polyethylene glycol (PEG) layer. After incubation for 3 h, PDSLCs adherent to the transfer substrate were placed onto amnion with the cell surface oriented downward. After overnight incubation, the transfer base was removed with forceps and PDLSCs were transferred from the transfer base to the amnion surface. (B) Transfer of PDLSC-GFP onto the amnion. GFP-transfected PDLSCs were transferred onto the amnion using the cell transfer technology, and then cut into squares using a surgical knife. PDLSCs were observed in cell sheet format. (C) CT image of the buccal side of a rat mandible. The dotted line outlines the periodontal defect. (D) CT image of a periodontal defect. By removing bone, PDL, cementum, and dentine, a 2 × 3 mm periodontal defect was created. (E) Photograph of a bone surface before periodontal defect creation. The dotted line outlines the periodontal defect. (F) Photograph of a periodontal defect. The defect was created from the mesial root of first mandibular to the mesial root of second molar. Exposure of the mesial, buccal, and distal roots of the first molar is evident in the defect. (G) Photograph of a periodontal defect after PDLSC-amnion placement. PDLSC-amnion was trimmed to cover the defect with a surgical knife and placed onto the periodontal defect with the cell surface oriented to attach to the denuded root.