Gross and imaging assessment for PDLSC-mediated periodontal tissue regeneration. (A–C): Gross manifestations showed that 12 weeks after transplantation, PDLSC-mediated periodontal tissue regeneration was close to normal level ([A], dotted line, arrow). Only limited periodontal tissue was regenerated in HA/TCP group ([B], dotted line, arrow) and the control group ([C], dotted line). (D–I): Computed tomography imaging showed that the alveolar bone defect was obvious and at the same level prior to the PDLSCs transplantation (D, F, H). Height between the two arrows = 7 mm; bracket = 10 mm. Twelve weeks post-PDLSC transplantation, PDLSCs mediated a proximately complete periodontal tissue regeneration ([E], arrows), but limited regeneration and HA/TCP particles were noted in the HA/TCP group ([G], arrows). Very little alteration was found in the untreated control group in the same period ([I], arrows). Bracket = 10 mm. Abbreviations: HA/TCP, hydroxyapatite/ tricalcium phosphate; PDLSC, periodontal ligament stem cell.

Characterization of miniature pig periodontal ligament stem cells (PDLSCs). (A): The cultured PDLSCs from single colonies showed typical fibroblast-like cells under a light microscope. (B–D): Immunocytochemical staining using STRO-1 (B), Nestin (C), and ALP (D) in PDLSCs showed positive staining. Approximately 5.6% of the third-passage PDLSCs stained positive for STRO-1 (E), 12.5% for Nestin (F), and 15.0% for ALP through flow cytometric analysis (G). (H–K): Ultrastructural observation of collagen produced by PDLSCs. Morphologically, PDLSC was fusiform-shaped, with many short and long ramifications under a scanning electron microscope, and the secreted extracellular matrix were around them (H). (I): The cells contained abundant organelles, such as mitochondria, ribosome (arrowheads) and rough endoplasm (arrow), as assessed by transmission electron microscopy. The miniature pig PDLSCs produced abundant collagen fibers ([J], arrows). The quantity of collagens produced by PDLSCs was much greater than that produced by dental papilla stem cells from the same animal ([K], arrows) under the same culture conditions. Scale bars = 100 μm. Abbreviations: ALP, alkaline phosphatase; N, nucleus.

Generation of periodontitis model in miniature pigs. (A): Schematic illustration of the timeline of the procedures conducted for this study. Three separate cohorts of miniature pigs were tested in this manner. (B): A 3-mm-wide, 7-mm-long, and 5-mm-deep bone defect was created in the mesial region of the maxilla or mandibular first molar (dotted line, arrow). (C): Four weeks after creating the bone defect by surgery and subsequent silk ligament, the inflammation of periodontal tissues was observed (arrows). (D, E): Typical periodontitis clinical findings presented in this model. Twelve weeks after the surgery, the gingival was red and swollen. A calculus could be seen around the margin of gingiva ([D], arrow), the inflammation was extended to the furcation area, and the whole mesial-buccal root of the first permanent molar was exposed ([E], arrows). (F–H): Imaging manifestations of this periodontitis model. X-ray image showed that the mineral density of the alveolar bone was decreased at the regions of furcation and the mesial side of the first permanent molar ([G], arrows) compared with control density of the alveolar bone ([F], arrows). CT coronal image showed obvious bone defect in the buccal alveolar region ([H], arrow). (I): Histological photomicrography showing typical periodontitis histopathological manifestations, including marked periodontal tissue reduction and inflammatory infiltration. Scale bar = 100 μm. Abbreviations: AL, attachment loss; CT, computed tomography; GR, gingival recession; HA/TCP, hydroxyapatite/tricalcium phosphate; PD, probing depth; PDLSC, periodontal ligament stem cell; SBI, sulcus bleeding index; Wk, week.

New bone regeneration assessment of PDLSC-mediated treatment for periodontitis in miniature pigs. (A–C): Quantitative assessment of regenerated alveolar bone. Originally, a bone defect with 3 mm wide, 7 mm long, and 5 mm deep was created (A). At 12 weeks after transplantation, the samples were harvested and fixed with 4% formaldehyde. Then, the soft tissues were removed from experimental regions, and the heights of new bone regeneration were measured by using Williams periodontal probe. The distances from the top of newly formed bone to the notch-shaped mark made during the operation were scaled. Each sample was measured in three different positions from buccal to lingual side. Mean values were recorded, and the heights of new bone regeneration were 7 mm minus mean values (B). The results showed that the height of regenerated alveolar bone was significantly higher in the PDLSC-mediated group than in the HA/TCP and control groups (F = 125.917; p = .000) (C). To identify whether the green fluorescent protein (GFP)-labeled PDLSCs implanted into the bone defects had differentiated to osteoblasts, nondecalcified slices were observed under the light microscope first, and osteoblasts and bone lacunas could clearly be seen in the new bone ([D], yellow and pink arrows). At the same visual field, the GFP+ PDLSCs were observed using a fluorescence microscope (E). When (D) and (E) were overlapped, the positions of some GFP+ cells were superposed on the osteoblasts and bone lacunas ([F], yellow arrow showing GFP− osteoblasts, red arrows showing GFP+ osteoblasts), which indicated that the GFP+ cells derived from PDLSCs had differentiated to osteoblasts. Abbreviations: B, bone; HA/TCP, hydroxyapatite/tricalcium phosphate; PDLSC, periodontal ligament stem cell.

Whole view of histopathological assessment for periodontal tissue regeneration by hematoxylin and eosin staining. A buccal-lingual direction histopathological section showed junctional epithelia attached on the cemento-enamel junction in normal periodontal tissues (A). The sulcular epithelium was thin and flat, and less infiltration of inflammatory cells could be observed in the connective tissues of the normal periodontal tissues (B). Sharpey's fibers were typically characteristic in the structure of normal periodontal tissues (C). Although the position of junctional epithelium was below the cemento-enamel junction, the periodontal tissue regeneration in periodontal ligament stem cell (PDLSC)-mediated group (D) was much better than that in HA/TCP group. The sulcular epithelium in PDLSC-mediated group was thicker, and the epithelial pegs and dermal papillae were short and blunt, with fewer inflammatory cells (E). New bone, cementum, and periodontal ligament were regenerated in the periodontal defect area in the PDLSC-mediated group (F), and the height of the new alveolar bone was much higher than that in HA/TCP group (G) but still lower than the normal level (A). Histopathological photomicrography showed that plenty of new bone and periodontal tissues, including cementum and periodontal ligament, were regenerated in the periodontal defective area, and the newly formed Sharpey's fibers plugged into the new regenerated cementum in the PDLSC-mediated group (F). Typical periodontitis, including marked periodontal tissue reduction (GR, AL), and infiltration of inflammatory cells were still found in HA/TCP group (G). The epithelial pegs and dermal papillae were long and slender, with infiltration of inflammatory cells underlying connective tissue (H). Fibers lacking the typical structure of Sharpey's fibers filled in the periodontal defect in the HA/TCP group (I). Scale bars = 1 mm. Yellow arrows show the top of the new bone. CEJ (blue arrows) is covered by junctional epithelium in normal periodontal tissue. GR is the height from CEJ to the margin of gingival; PD is the depth of periodontal pocket; AL is the length from CEJ to the bottom of the pocket (in this study, the bottom of the pocket was below CEJ, so AL is equal to the GR plus PD). Abbreviations: AL, attachment loss; B, bone; C, cementum; CEJ, cemento-enamel junction; D, dentin; GR, gingival recession; PD, probing depth; PDL, periodontal ligament; SE, sulcular epithelium.