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Mesenchymal Stem Cells Acquire Characteristics of Cells in the Periodontal Ligament in vitro
P.R. Kramer1,*,
S. Nares2,
S.F. Kramer1,
D. Grogan1 and
M. Kaiser1
1 Department of Biomedical Sciences, Baylor College of Dentistry, Texas A&M University System Health Science Center, 3302 Gaston Ave., Dallas, TX 75246; and
2 Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, Bethesda, MD 20892;
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Figure 1. Isolation and characterization of periodontal ligament. (A1) Cartoon depiction of a parasagittal cross-section through the jaw and attached tooth. Tooth slices (rectangle) were made in the apical region of the extracted tooth to avoid any contamination with gingival epithelial cells. Tooth slices, termed ‘organotypic tooth explants’, had dimensions of a half-millimeter depth, a width of 1–4 mm, and a height of 2–5 mm; variation was due to topography of the tooth and differences in circumference. (A2) Paraffin section (12 µm) from an organotypic tooth explant stained with hematoxylin and eosin. Organotypic tooth explants always consisted of cementum (C) and periodontal ligament (PDL) and dentin (D). In approximately 15% of the sections, a layer of alveolar bone (B) was present. (B1) Proliferative periodontal cells were then cultured in vitro from organotypic tooth explants. Greater than 80% of the cells cultured have a spindle shape resembling the morphological properties noted for human periodontal ligament cells (black arrows). (B2) Cultured cells from explants stain positive for collagen III (green fluorescence). Thus, periodontal ligament fibroblasts were isolated from the cellular population based on morphology and high levels of collagen III expression. (C1-G3) Staining of periodontal tissues for collagen III, osteopontin, osteocalcin, BMB-2/4, and bone sialoprotein in vivo was completed on 12-µm paraffin or fresh-frozen sections. Collagen III staining (brown, panel C2), osteopontin staining (brown, panel D2), osteocalcin staining (brown, arrows, panel E2), BMP-2/4 staining (brown, panels F1 and F2), and bone sialoprotein staining (brown, arrows, panels G2 and G3) of acute organotypic tooth explants. Panels C1, D1, E1, and G1 are controls where non-immune serum was added, instead of primary antibody, during the immunohistochemical procedure. In panels C1-G2, the bone (b), periodontal ligament (pdl), cementum (c), and dentin (d) tissue layers are indicated. Bars = 50 µm (B2, C1, C2, D1, G1, D2), 100 µm (E1, E2, G2), and 200 µm (F1). Panels F2 and G3 are high-magnification images of regions in panels F1 and G2, respectively.
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Figure 2. Osteocalcin staining and Y-chromosome tagging of human female periodontal ligament and male mesenchymal stem cells. In panels A, C, E, and H, osteocalcin was fluorescently stained with SA-488 (green), and male mesenchymal stem cells were labeled for the Y chromosome (red dots). In panels B and D, osteocalcin staining was completed with DAB (brown). Osteocalcin staining of mesenchymal stem cells is shown in panel A (green) and panel B (brown). Periodontal ligament cells express high levels of osteocalcin in vitro, as shown in panel C (green) and panel D (brown). (E) Co-cultures of mesenchymal stem cells and periodontal ligament (1:1) after 7 days in vitro. Images from panels A, C, and E were from slides run in the same staining experiment and processed through the same solutions. Negative controls included: (F) periodontal ligament cells not treated with osteocalcin antibody (i.e., non-immune serum) and stained; and (G) periodontal ligament cells incubated with antibody but not reacted with biotin-tyramide (reactant necessary for streptavidin SA-488 binding and the fluorescent green staining). (H) Cross-section confocal image of the interior (arrows) of cocultured cells after osteocalcin staining (green). Horizontal and vertical lines indicate the X and Y planes through which the cross-sectioned images were made. Note: Mesenchymal cell is tagged by a red dot. (I) Histogram showing the mean optical density values for various cell groups after fluorescent osteocalcin staining, including mesenchymal stem cells (MSC), periodontal ligament cells (PDL), and co-cultures of MSC/PDL after 7 or 21 days. Optical density values were given as the mean ± SEM. Values were significantly different (P < 0.05), as shown by ANOVA. Asterisk indicates that the OD for MSC staining was significantly less (P < 0.05) than for the other 3 cell groups. N = 3 for MSC and MSC/PDL (7 days), and N = 10 for PDL and MSC/PDL (21 days). Bar = 50 µm (A, C, and E). Panels B, D, F, G, and H have the same magnification as panel A.
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Figure 3. Bone sialoprotein staining of periodontal ligament and male mesenchymal stem cells. In panels A, C, E, and F, cell cultures were stained with bone sialoprotein (green) and labeled for the Y chromosome (red dots). In panels B and D, bone sialoprotein staining was completed using DAB (brown). Bone sialoprotein staining of mesenchymal stem cells is shown in panel A (green) and panel B (brown). Periodontal ligament cells express low levels of bone sialoprotein in vitro, as shown in panel C (green) and panel D (brown). (E) Co-cultures of mesenchymal stem cells and periodontal ligament (1:1) after 7 days in vitro. (F) Mesenchymal stem cells (white arrows) express bone sialoprotein after co-culture (1:1) with periodontal ligament for 21 days. Panels A, C, E, and F are images taken from slides run in the same staining experiment and processed through the same solutions. (G) Negative control showing the DAB staining of periodontal ligament cells incubated with non-immune serum without bone sialoprotein antibody. (H) Histogram showing the mean optical density for mesenchymal stem cells (MSC) and periodontal ligament cells (PDL) and co-cultures after bone sialoprotein fluorescent staining. Optical density values were given as the mean ± SEM. Values were significantly different (P < 0.0001) as shown by ANOVA. Asterisk = P < 0.05, and double asterisk = P < 0.01 as compared with MSC. N  6. Bars = 50 µm (A, B, F) and 100 µm (C, D, E).
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Figure 4. Osteopontin staining of periodontal ligament and male mesenchymal stem cells. In panels A, C, E, and F, cell cultures were stained with osteopontin (green) and labeled for the Y chromosome (red dots). In panels B and D, osteopontin staining was completed with the use of DAB (brown). Osteopontin staining of mesenchymal stem cells is shown in panel A (green) and panel B (brown). Periodontal ligament cells express high levels of osteopontin in vitro, as shown in panel C (green) and panel D (brown). In vitro co-cultures of mesenchymal stem cells and periodontal ligament (1:1) were incubated for 7 days (panel E) and 21 days (panel F). Periodontal ligament, mesenchymal stem cell, and co-culture images were from slides run in the same staining experiment and processed through the same solutions. (G) Negative control shows the DAB staining of periodontal ligament cells incubated with non-immune serum without osteopontin antibody. (H) Histogram showing the mean optical density for mesenchymal stem cells (MSC), periodontal ligament cells (PDL), and co-cultures after osteopontin fluorescent staining. Optical density values were given as the mean ± SEM. Values were significantly different (P < 0.0001), as shown by ANOVA. Double asterisk indicates that the OD for MSC staining was significantly less (P < 0.01) than that of the other 3 cell groups. N  6. Bars = 50 µm (F) and 100 µm (C, D, E). Panel C is the same magnification as A, and panel D is the same magnification as B.
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Journal of Dental Research, Vol. 83, No. 1,
27-34 (2004)
DOI: 10.1177/154405910408300106
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