Effect of Enamel Matrix Drivatives application on the expression of PDLs17, PDLs22 of cultured human periodontal ligament cells in vitro / 대한치주과학회지

The enamel matrix derivative (EMD) has been recently used in the periodontal regenerative techniques. The present study was established to investigate the influence of EMD on human periodontal ligament cells using expression of mRNA of periodontal ligament specific gene (PDLs)17, PDLs22, type I collagen when EMD applied to periodontal ligament cells. Periodontal ligament cells were obtained from a healthy periodontium and cultured in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum and beta-glycerophosphate with ascorbic acid. Test groups were two; One adds EMD in culture media and another added EMD and Dexamethasone (DEX) in culture media. Positive control group added DEX in culture media, and negative control group adds niether of EMD nor DEX. Emdogain(R) (Biora, Sweden, 30 mg/ml) was diluted by 75 microgram/ml concentration to culture media. For reverse transcription-polymerase chain reaction (RT-PCR), total RNA isolated on days 0, 7, 14 and 21. mRNA of PDLs17 was expressed on days 14 and 21 in EMD or DEX group, and expressed on days 7, 14 and 21 in EMD plus DEX group, the other side, expressed on days 21 in negative control group. mRNA of PDLs22 expressed on days 7, 14 and 21 in EMD group, and expressed on days 14 and 21 in DEX group, and expressed on days 7, 14 and 21 in EMD plus DEX group. Negative control group expressed on days 14 and 21. Type I collagen was expressed on all days and all groups. These results indicate that EMD promotes differentiation of periodontal ligament cells, and this is considered to offer basis that can apply EMD to periodontal tissue regeneration technique.

The Effect of Interleukin 1-beta, Platelet Derived Growth Factor-BB and Transforming Growth Factor-betaon the expression of PDLs17 mRNA in the Cultured Human Periodontal Ligament Fibroblasts / 대한치주과학회지

The molecular mechanisms control the function of PDL(periodontal ligament) cells and/or fibroblasts remain unclear. PDLs17, PDL-specific gene, had previousely identified the cDNA for a novel protein from cultured PDL fibroblasts using subtraction hybridization between gingival fibroblasts and PDL fibroblasts. The purpose of this study was to determine the regulation by growth factors and cytokines on PDLs17 gene expression in cultured human periodontal ligament cells and observe the immunohistochemical localization of PDLs17 protein in various tissues of mouse. Primary PDL fibroblasts isolated by scraping the root of the extracted human mandibular third molars. The cells were incubated with various concentration of human recombinant IL-1beta, PDGF-BB and TGF betafor 48h and 2 weeks. At each time point total RNA was extracted and the levels of transcription were assessed by reverse transcription-polymerase chain reaction (RT-PCR assay). Polyclonal antiserum raised against PDLs17 peptides, CLSVSYNRSYQINE and SEAVHETDLHDGC, were made, and stained the tooth, periodontium, developing bone, bone marrow and mid-palatal suture of the mouse. The results were as follows. 1. PDLs17 mRNA levels were increased in response to PDGF (10ng/ml) and TGF beta(20ng/ml) after treatment of the IL-1beta, PDGF-BB and TGFbetafor 48 h. 2. PDLs17 was up-regulated only by TGFbeta(20 ng/ml) after treatment of the IL-1beta, PDGF-BB and TGF betafor 2 weeks and unchanged by the other stimulants. 3. PDLs17 was a novel protein coding the 142 amino acid peptides in the ORF and the nucleotide sequences of the obtained cDNA from RT-PCR was exactly same as the nucleotides of the database. 4. Immunohistochemical analysis showed that PDLs17 is preferentially expressed in the PDL, differentiating osteoblast-like cells and stromal cells of the bone marrow in the adult mouse. 5. The expression of PDLs17 protein was barely detectable in gingival fibroblasts, hematopoetic cells of the bone marrow and mature osteocytes of the alveolar bone. These results suggest that PDLs17 might upregulated by PDGF-BB or TGFbetaand acts at the initial stage of differentiation when the undifferentiated mesenchymal cells in the bone marrow and PDL differentiate into multiple cell types. However, more research needs to be performed to gain a better understanding of the exact function of PDLs17 during the differentiation of bone marrow mesenchymal and PDL cells.