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1.
Fig. 2

Fig. 2. From: Vitamin C Treatment Promotes Mesenchymal Stem Cell Sheet Formation and Tissue Regeneration by Elevating Telomerase Activity.

Transmission electron microscopy (TEM) of a vitamin C (Vc)-induced periodontal ligament stem cell sheet. (A) Vc (20.0 µg/mL) was used to induce PDLSCs sheet formation and a monolayer cell sheet was formed, as observed by TEM. (B) Characteristic tight cell-to-cell connections were observed (arrows: junction sites). (C The cytoplasm was microfilament-rich (ellipse), and exocytotic vesicles were observed near the plasma membrane (ellipse). (D) Endogenous extracellular matrix (ECM) (ellipse) was observed between cells.

F.L. Wei, et al. J Cell Physiol. ;227(9):3216-3224.
2.
Fig. 1

Fig. 1. From: Vitamin C Treatment Promotes Mesenchymal Stem Cell Sheet Formation and Tissue Regeneration by Elevating Telomerase Activity.

Vitamin C (Vc)-mediated periodontal ligament stem cell sheet. PDLSCs were induced by different concentrations of Vc (from 0 µg/mL to 50.0 µg/mL) for 10 days. 0 µg/mL (A), 5.0 µg/mL (B), 10.0 µg/mL (C) Vc did not induce PDLSCs to form a morphologically complete cell sheet. 20.0 µg/mL (E) and 50.0 µg/mL (F) Vc induced PDLSCs to form a perfect cell sheet, similar to the sheet obtained by the traditional temperature-responsive culture method (G). H&E staining revealed that the cell sheet was fragmented by 10.0 µg/mL Vc (D), but the cell sheet is complete when 20.0 µg/mL Vc is used (H); the whole PDLSCs sheet was two- or three-layered and the cells contacted each other tightly. Scale bars: 40 µm. Each experiment was conducted in triplicate.

F.L. Wei, et al. J Cell Physiol. ;227(9):3216-3224.
3.
Fig. 6

Fig. 6. From: Vitamin C Treatment Promotes Mesenchymal Stem Cell Sheet Formation and Tissue Regeneration by Elevating Telomerase Activity.

Vitamin C (Vc) induced complete cell sheet formation of bone marrow mesenchymal stem cells (BMMSCs) and umbilical cord mesenchymal stem cells (UCMSCs). Vitamin C (Vc, 20.0 µg/mL) was used to induce sheets of two other mesenchymal stem cell types for 10 days. Morphological observation indicated that complete sheets of BMMSCs (A) and UCMSCs (C) were formed. H&E staining revealed the two- or three-layered, uniformly spread, two-dimensional tissue structure in whole cell sheets of BMMSCs (B) and UCMSCs (D). Scale bars: 20 µm. All experiments were conducted in triplicate.

F.L. Wei, et al. J Cell Physiol. ;227(9):3216-3224.
4.
Fig. 5

Fig. 5. From: Vitamin C Treatment Promotes Mesenchymal Stem Cell Sheet Formation and Tissue Regeneration by Elevating Telomerase Activity.

Regeneration of periodontitis defects mediated by periodontal ligament stem cell sheets in miniature pigs. H&E staining revealed new periodontal tissue regeneration in the periodontal defect area in the Vc-induced PDLSCs sheet group (A), UpCell dish PDLSCs sheet group (D) and dissociated PDLSCs group (G). The alveolar bone heights were much larger in Vc-induced PDLSCs sheet (A) and UpCell dish PDLSCs sheet (D) groups than in the dissociated PDLSCs group (G). Much thicker sulcular epithelia were evident in the dissociated PDLCs group (H) compared with the Vc-induced PDLSCs sheet (B) and UpCell dish PDLSCs sheet (E) groups. Sharpy's fibers formed in the Vc-induced PDLSCs sheet group (C), UpCell dish PDLSCs sheet group (F), and dissociated PDLSCs group (I); however, Sharpy's fibers were irregular in the dissociated PDLSCs group. (J) The percentage of periodontal bone was significantly higher in Vc-induced PDLSCs sheet and UpCell dish PDLSCs sheet groups (55.3%±5.1% and 43.6%±3.3%, respectively) than in the dissociated PDLSCs group (31.4%±4.5%). Abbreviations: SE, sulcular epithelium; CEJ, cemeto-enamel junction; HAB, height of alveolar bone; D, dentin; C, cementum; PDL, periodontal ligament; B, bone.

F.L. Wei, et al. J Cell Physiol. ;227(9):3216-3224.
5.
Fig. 4

Fig. 4. From: Vitamin C Treatment Promotes Mesenchymal Stem Cell Sheet Formation and Tissue Regeneration by Elevating Telomerase Activity.

Periodontal ligament stem cell sheet-based cementum/ PDL-like tissue regeneration in nude mice. After 4 weeks of transplantation, PDLSCs differentiated into odontoblasts/cementoblast-like cells (arrows) that formed much more regular bone/cementum-like matrix (rectangles) in the PDLSCs sheet transplants (A, B). While in dissociated PDLSCs transplants, PDLSCs formed less irregular bone/cementum-like matrix (rectangles) containing odontoblasts/cementoblast-like cells (arrows) and there was plenty of gelfoam scaffolding left (C). Goldner's trichrome staining revealed that bone/cementum-like matrix is blue (rectangles) (D, E, F). Picrosirius-red staining also revealed that there was much more condensed bone/cementum-like matrix generation in PDLSCs sheet transplants (rectangles) (G, H). The same polarized light view indicated that the condensed tissues were full of collagen type I (in red) and type III (in green) (rectangles) (J, K). Picrosirius-red staining showed that there were limited amounts of bone/cementum-like matrix regeneration (rectangles) remaining along the surface of gelfoam carriers in dissociated PDLSCs transplants (I); much less new tissue, including collagen type I (in red) and type III (in green) (rectangles); and plenty of remaining gelfoam carriers (no staining) were found when the samples were examined by polarized light (L). The percentages of bone/cementum-like matrix were significantly higher in the Vc-induced PDLSCs sheet and UpCell dish PDLSCs sheet transplant groups (44.2%±5.9% and 35.7%±4.6%, respectively) than in the dissociated PDLSCs transplant group (12.5%±4.0%); the percentage of bone/cementum-like matrix was significantly higher in the Vc-induced PDLSCs sheet transplant group than in the UpCell dish PDLSCs sheet transplant group. Scale bars: 50 µm.

F.L. Wei, et al. J Cell Physiol. ;227(9):3216-3224.
6.
Fig. 3

Fig. 3. From: Vitamin C Treatment Promotes Mesenchymal Stem Cell Sheet Formation and Tissue Regeneration by Elevating Telomerase Activity.

The effect of vitamin C (Vc) on the periodontal ligament stem cell sheet. (A) Different concentrations of Vitamin improved the proliferation of PDLSCs. (B) Telomerase activity in PDLSCs increased gradually after treatment with ascorbic acid at a concentration of 20.0 µg/mL. HEK293T cells were used as a positive control and heat inactivated 293T cells were used as a negative control. (C) Human telomerase reverse transcriptase (hTHRT) protein level gradually increased in the presence of ascorbic acid. (D) The relative mRNA expression of collagen type I (COLI), integrin β1, and fibronectin were higher in 20 µg/mL Vc-induced PDLSCs sheet compared with PDLSCs sheet obtained from an UpCell dish and dissociated PDLSCs. Oct4, Sox2, Nanog, RUNX2, ALP and OCN also increased in 20 µg/mL Vc-induced PDLSCs sheet compared with PDLSCs sheet obtained from an UpCell dish and dissociated PDLSCs; however, there was no significant difference between PDLSCs sheet obtained from an UpCell dish and dissociated PDLSCs (n = 5). (E) Alizarin red S staining suggested that 20 µg/mL Vc induced osteogenic differentiation. (F) The expression of matrix protein, fibronectin, and integrin β1 were enhanced by treatment with Vc, and this effect was abrogated by telomerase inhibition. *P < 0.05; **P < 0.01; NS, no significance. Scale bars: 100 µm.

F.L. Wei, et al. J Cell Physiol. ;227(9):3216-3224.

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