Enamel matrix derivative induces connective tissue growth factor expression in human osteoblastic cells.
BACKGROUND
Enamel matrix derivative (EMD) stimulates the production of transforming growth factor-beta (TGF-beta), which has been suggested to play a role in mediating the effects of EMD in periodontal tissue regeneration. Connective tissue growth factor (CTGF) is a mediator of TGF-beta and promotes cell development. The interaction between EMD and CTGF is unknown. This study explored the effects of EMD on CTGF expression in human osteoblastic cells and whether the interaction is modulated by the TGF-beta signaling pathway. Also, the roles of CTGF in cell proliferation, cell cycle progression, and mineralized nodule formation of EMD-induced osteoblastic cultures were examined.


METHODS
Human osteoblastic cells (Saos-2) were treated with 25 to 100 microg/ml EMD with or without the addition of TGF-beta inhibitor. CTGF mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR), and CTGF protein levels were assayed by Western blot analysis. In addition, cell cycle progression and DNA synthesis were determined by flow cytometry and 5-bromo-2'-deoxyuridine (BrdU) incorporation following EMD treatment with or without CTGF antibody. Mineralization was examined by alizarin red staining and quantified by elution with cetylpyridinium chloride.


RESULTS
Western blot and RT-PCR analysis demonstrated a dose-dependent increase of CTGF expression by EMD. EMD-induced CTGF expression was reduced significantly in the presence of TGF-beta inhibitor. Cell cycle and BrdU analysis revealed an increase in cell proliferation following EMD treatment, which was due to an increase in the percentage of cells in the G2/M phase of the cell cycle. No significant effect was found when anti-CTGF antibody was added. Conversely, mineralization was inhibited significantly in EMD-treated cultures in the presence of anti-CTGF antibody.


CONCLUSIONS
EMD stimulates CTGF expression, and the interaction is modulated via TGF-beta in osteoblastic cells. Also, CTGF affects EMD-induced osteoblastic mineralization but not cell proliferation. To our knowledge, these results provide novel insight into EMD-CTGF interaction, two biomodifiers that have therapeutic relevance to tissue engineering and regeneration.
Institute of Periodontology and Synoptic Dentistry, Charité-Medical University of Berlin, Berlin, Germany.
Nora H M
NH
Heng
Philippe D
PD
N'Guessan
Bernd-Michael
BM
Kleber
Jean-Pierre
JP
Bernimoulin
Nicole
N
Pischon
D015153:Blotting, Western; D002113:Calcification, Physiologic / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D003746:Dental Enamel Proteins / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis*; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis*; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D012038:Regeneration; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000502:physiology
C526317:CCN2 protein, human; D003746:Dental Enamel Proteins; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D016212:Transforming Growth Factor beta; C109069:enamel matrix proteins; D055513:Connective Tissue Growth Factor