J Periodontal Implant Sci.  2011 Aug;41(4):167-175. 10.5051/jpis.2011.41.4.167.

Gene expression pattern during osteogenic differentiation of human periodontal ligament cells in vitro

• Choi MH
• Noh WC
• Park JW
• Lee JM
• Suh JY

• KMID: 2094716
• DOI: http://doi.org/10.5051/jpis.2011.41.4.167

Abstract

PURPOSE
Periodontal ligament (PDL) cell differentiation into osteoblasts is important in bone formation. Bone formation is a complex biological process and involves several tightly regulated gene expression patterns of bone-related proteins. The expression patterns of bone related proteins are regulated in a temporal manner both in vivo and in vitro. The aim of this study was to observe the gene expression profile in PDL cell proliferation, differentiation, and mineralization in vitro.
METHODS
PDL cells were grown until confluence, which were then designated as day 0, and nodule formation was induced by the addition of 50 microg/mL ascorbic acid, 10 mM beta-glycerophosphate, and 100 nM dexamethasone to the medium. The dishes were stained with Alizarin Red S on days 1, 7, 14, and 21. Real-time polymerase chain reaction was performed for the detection of various genes on days 0, 1, 7, 14, and 21.
RESULTS
On day 0 with a confluent monolayer, in the active proliferative stage, c-myc gene expression was observed at its maximal level. On day 7 with a multilayer, alkaline phosphatase, bone morphogenetic protein (BMP)-2, and BMP-4 gene expression had increased and this was followed by maximal expression of osteocalcin on day 14 with the initiation of nodule mineralization. In relationship to apoptosis, c-fos gene expression peaked on day 21 and was characterized by the post-mineralization stage. Here, various genes were regulated in a temporal manner during PDL fibroblast proliferation, extracellular matrix maturation, and mineralization. The gene expression pattern was similar.
CONCLUSIONS
We can speculate that the gene expression pattern occurs during PDL cell proliferation, differentiation, and mineralization. On the basis of these results, it might be possible to understand the various factors that influence PDL cell proliferation, extracellular matrix maturation, and mineralization with regard to gene expression patterns.

Keyword

Cell differentiation; Gene expression; Periodontal ligament

MeSH Terms

Alkaline Phosphatase
Anthraquinones
Apoptosis
Ascorbic Acid
Biological Processes
Bone Morphogenetic Proteins
Cell Differentiation
Cell Proliferation
Dexamethasone
Durapatite
Extracellular Matrix
Fibroblasts
Gene Expression
Genes, fos
Genes, myc
Glycerophosphates
Humans
Osteoblasts
Osteocalcin
Osteogenesis
Periodontal Ligament
Proteins
Real-Time Polymerase Chain Reaction
Transcriptome
Alkaline Phosphatase
Anthraquinones
Ascorbic Acid
Bone Morphogenetic Proteins
Dexamethasone
Durapatite
Glycerophosphates
Osteocalcin
Proteins

Figure

• 

  Figure 1 Morphology of the various stages and percentage of calcified areas during the formation of mineralized nodule by human periodontal ligament (PDL) cells. (A) shows the visual field at ×200 magnification on days 1(a), 7(b), 14(c), and 21(d). Developed mineralization is observed and size and number of nodules are increased compared to PDL cells on day 14. (B) Measurement of calcified areas by Alizarin Red S stain. Data are shown as the mean±SD of two patients. Percentages of the calcified areas are 5% and 33% of the culture dish on day 14 and 21, respectively.

• 

  Figure 2 c-myc (A), c-fos (B), ALP (C), BMP-2 (D), BMP-4 (E), and OC (F) mRNA expression during mineralization of human periodontal ligament (PDL) cells. The expression of each gene in PDL1 (a) and PDL2 (b) is shown. The graphs show the ratio of mRNA to GAPDH mRNA from the real-time polymerase chain reaction results. Control means the minimal gene expression level during 21 days. Values are means±SD of two cultures.

• 

  Figure 3 Gene expression pattern during mineralization of periodontal ligament cells. Expression of genes was analyzed by real-time PCR and normalized to the levels of GAPDH mRNA. Values are presented as the percent of the maximal expression for each transcript. a)c-myc, b)c-fos, c)ALP, d)BMP-2, e)BMP-4, f)OC. a),c)-f)Statistically significant difference as compared with day 0 (P<0.05). b)(P>0/05).

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