Korean J Orthod.  2012 Dec;42(6):307-317. 10.4041/kjod.2012.42.6.307.

Differentiation and characteristics of undifferentiated mesenchymal stem cells originating from adult premolar periodontal ligaments

• Kim SS
• Kwon DW
• Im I
• Kim YD
• Hwang DS
• Holliday L
• Donatelli
• Son WS
• Jun ES

• KMID: 1976683
• DOI: http://doi.org/10.4041/kjod.2012.42.6.307

Abstract

OBJECTIVE
The purpose of this study was to investigate the isolation and characterization of multipotent human periodontal ligament (PDL) stem cells and to assess their ability to differentiate into bone, cartilage, and adipose tissue.
METHODS
PDL stem cells were isolated from 7 extracted human premolar teeth. Human PDL cells were expanded in culture, stained using anti-CD29, -CD34, -CD44, and -STRO-1 antibodies, and sorted by fluorescent activated cell sorting (FACS). Gingival fibroblasts (GFs) served as a positive control. PDL stem cells and GFs were cultured using standard conditions conducive for osteogenic, chondrogenic, or adipogenic differentiation.
RESULTS
An average of 152.8 +/- 27.6 colony-forming units was present at day 7 in cultures of PDL stem cells. At day 4, PDL stem cells exhibited a significant increase in proliferation (p < 0.05), reaching nearly double the proliferation rate of GFs. About 5.6 +/- 4.5% of cells in human PDL tissues were strongly STRO-1-positive. In osteogenic cultures, calcium nodules were observed by day 21 in PDL stem cells, which showed more intense calcium staining than GF cultures. In adipogenic cultures, both cell populations showed positive Oil Red O staining by day 21. Additionally, in chondrogenic cultures, PDL stem cells expressed collagen type II by day 21.
CONCLUSIONS
The PDL contains multipotent stem cells that have the potential to differentiate into osteoblasts, chondrocytes, and adipocytes. This adult PDL stem cell population can be utilized as potential sources of PDL in tissue engineering applications.

Keyword

Histochemistry; Periodontics; Bone biology

MeSH Terms

Adipocytes
Adult
Antibodies
Azo Compounds
Bicuspid
Calcium
Cartilage
Chondrocytes
Collagen Type II
Durapatite
Fibroblasts
Humans
Mesenchymal Stromal Cells
Multipotent Stem Cells
Osteoblasts
Periodontal Ligament
Periodontics
Stem Cells
Tissue Engineering
Tooth
Antibodies
Azo Compounds
Calcium
Collagen Type II
Durapatite

Figure

• 

  Figure 1 Cell culture colony-forming units (CFUs). At day 7 after plating, PDL cells were fixed with 10% formalin and stained with 4% crystal violet. Aggregates of 50 or more cells were considered colonies and counted. A, A representative GF colony (10×). B, A representative PDL cell colony (10×). The black arrows and circles represent the images of 7-day cell population (right panels) at 10× magnification using light microscopy.

• 

  Figure 2 Colony forming units (CFUs) in gingival fibroblasts (GFs) (n = 7) and periodontal ligament (PDL) cells (n = 7). Colony formation was measured as described in the Material and Methods section. PDL cells exhibited a statistically significant increase in CFUs as compared to GFs, the positive control. *p < 0.05.

• 

  Figure 3 Cell proliferation rates. Periodontal ligament (PDL) cells and gingival fibroblasts (GFs) were cultured for 1, 2, 3, 4, 5, and 6 days in 6-well plates with media changes every other day. The cells were harvested by trypsinization and counted with a Coulter counter. The data are from 1 of 7 representative experiments, each yielding similar results. From day 4, PDL cell proliferation was significantly increased as compared to GFs, the positive control. Diamonds correspond to PDL cells, and squares correspond to GFs. *p < 0.05, †p < 0.01.

• 

  Figure 4 Primary digested pooled adult human periodontal ligament cells were immunostained with various antibodies, and cells were analyzed on a FACScan with an automated cell deposition unit equipped with an argon laser. The data were analyzed with Cell Quest software. These representative FACScan cell distributions were collected from a passage 2 cell population from Donor 1. Percentages of CD29-positive (A), CD34-positive (B), CD44-positive (C), and STRO-1-positive (D) cells were identified and are indicated by the circle in each corresponding graph.

• 

  Figure 5 Expression of mineral deposits within periodontal ligament (PDL) cells and gingival fibroblasts (GFs) after long-term culture in osteogenesis-inducing media. A, C, No calcium deposits were seen in PDL cells and GFs cultured in normal media (20×). B, GFs cultured with osteogenesis-inducing media exhibited relatively small calcium deposits at day 21; the distribution was observed as weak foci associated with cell clusters (20×). D, At day 21, PDL cells cultured with osteogenesis-inducing media showed mineral deposits as evidenced by Alizarin red S staining (20×), with deposits distributed throughout the tissue culture well.

• 

  Figure 6 In vitro chrondogenic differentiation of periodontal ligament (PDL) cells and gingival fibroblasts (GFs) as measured by immunohistochemical staining for collagen type II expression. Panels A and B show GFs cultured in normal DMEM or chondrogenic differentiation medium, respectively, after 3 weeks (10×). Panels C and D show PDL cell cultures after 3 weeks of incubation in α-MEM containing ascorbic acid and glutamine and chondrogenic differentiation medium, respectively (10×). In the cell populations shown in panels B and D above, metachromasia and cell morphology corresponding with embryonic stages of cartilage formation were observed.

• 

  Figure 7 Characterization of the adipogenic differentiation of periodontal ligament (PDL) stem cell and gingival fibroblast (GF) cultures (20×). A, Lipid droplets were not seen in PDL cells cultured with normal media. B, Weak lipid droplets were seen in GFs cultured with adipogenesis-inducing media (100×). C, Lipid droplets were not observed in PDL cells cultured for 21 days in normal media, as is shown by negative oil red O staining (20×). D, PDL cells formed lipid droplets after 21 days of culture in adipogenesis-inducing media (100×).

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