Table 1.
Oligonucleotide primer sequences utilized in the quantitative real-time PCR analysis.
Figure 1.
Culture and identification of DFCs and DPCs.
(A) Primary DFCs and DPCs from human wisdom tooth germ. The dental follicle on the crown surface has visible blood supply. The dental papilla located inside the crown is transparent and jelly-like. Both DFCs and DPCs show typical fibroblast-like spindle morphology under a light microscope. (B) Homogeneous electron-dense granules in DFCs indicated by yellow arrows are featured for DFCs. Both cells were positive for STRO-1 and for Vimentin, but negative for epithelial marker CK-14. Scale bar = 100 µm in (A) and (B).
Figure 2.
Ultrastructural characterization of DFCs and DPCs.
Homogeneous electron-dense granules in DFCs indicated by red arrows are featured for DFCs. RER and mitochondria can be observed in the cytoplasm of DFCs (blue arrows) and DPCs (blue arrows), however, it is more intense in DPCs. A large number of lysosomes (primary lysosomes and secondary lysosomes) were observed in the cytoplasm of DFCs (red arrow heads). Some cell microfilaments were observed in the cytoplasm of DPCs (red arrow heads). Heterochromatin was observed in the nucleus of DFCs and DPCs (blue arrow heads), but less heterochromatin staining was seen in DFCs (blue arrow heads).
Figure 3.
Proliferation potential of DFCs and DPCs.
Both cells show high proliferation potential, however, DFCs show a higher potential than DPCs. (A) BrdU labeling, (B) growth curve and (C) TEP1 evaluation via immunofluorescent assay, CCK-8 assay and western blotting. Scale bars = 100 µm in (A).
Figure 4.
Analysis of cell surface antigens.
Flow cytometry analysis indicated that DFCs and DPCs are of mesenchymal origin (positive for CD29, CD44, CD90 and negative for CD31) and DFCs and DPCs are stem cells (positive for CD146 and STRO-1). DPCs are more capable of embryologic vascalogenesis as confirmed by relatively higher expression of CD146 in DFCs. DPCs were positive for the endothelium antigen, CD106 and DFCs were negative for CD106, suggesting that DPCs have a stronger potential to differentiate into vascular endothelial cells.
Figure 5.
Multipotential differentiation of DFCs and DPCs.
After being cultured in osteogenic medium (Os) for 25 days, mineralized nodules were stained with alizarin red. DFCs were more mineralized compared to DPCs (p<0.05). Oil red O staining was used to assess the formation of lipid droplets in the adipogenic cultures. DFCs formed more lipid droplets compared to DPCs (p<0.05). Scale bars = 100 µm.
Figure 6.
Proteomics analysis of differentially expressed proteins in DFCs and DPCs.
(A) Representative 2-DE maps of human DFCs and DPCs. (B) The listed proteins are localized in the cytoplasm (40%), nucleus (12%), intermediate filaments (8%), cell membrane (24%), endoplasmic reticulum (4%), endosome (4%), basement membrane or centrosome (4%). (C) Expression profile of three significantly altered proteins (#10, #12 and #14). The selected area was enlarged, and arrows indicate each protein spot or its theoretical location. 3D view showing the alteration of the expression of three proteins was generated by PD-Quest software.
Table 2.
Proteins identified by MALDI-TOF-MS.
Figure 7.
SEM examination of the growth of DFCs and DPCs on human TDM.
Cell number was low on day 1; on day 3, dentinal tubules were disappearing due to coverage by cells. On day 5 and 7, no dentinal tubulus were observed and multilayer cells were observed. Scale bars = 100 µm.
Figure 8.
Expression of markers related to odontogenic differentiation in DFCs and DPCs.
The odontogenic genes and proteins were detected in DFCs, iDFCs, DPCs and iDPCs by (A) qRT-PCR and (B) western blotting. * p<0.05. iDFCs:DFCs induced in TDM, iDPCs:DPCs induced in TDM.
Figure 9.
Odontogenic differentiation of DFCs and DPCs in vivo.
Both DFCs and DPCs contribute to the regeneration of (A) pulp-dentin complex inside TDM and (B) of PDL-cementum complex outside TDM. D: dentin, PD: predentin, DP: dental pulp, ND: neo-dentin, DPLT: dental pulp-like tissue, hTDM: human treated dentin matrix, C: cementum, PL: periodontal ligament, PLLT: periodontal ligament-like tissue. Scale bars = 100 µm.