Synthesis and evaluation of PDLs22 recombinant protein / 대한치주과학회지

Periodontal ligament (PDL) is the connective tissue located between the tooth root and alveolar bone. In a previous study, PDLs22 was isolated as a PDL-specific gene by using subtractive hybridization between cultured PDL fibroblasts and gingival fibroblasts. It was also suggested that PDLs22 plays important roles in the development, differentiation and maintenance of periodontal tissues. However, little is known about functional study of PDLs22 using recombinant protein in PDL fibroblast differentiation and periodontium formation. In this study, in order to produce the PDLs22 recombinat protein, PDLs22-expression vector were constructed and expressed its protein in various host cell and temperature conditions. The results were as follows: 1. PDLs22 protein was not strongly expressed in the induction system using pRSET-PDLs22 construct. 2. When the BL21(DE3) pLysS was used as a expression host, PDLS22 protein was strongly expressed in the induction system using pHCEIIBNd-PDLs22 construct. 3. The PDLs22 protein was recognized at a molecular weight of 28 kDa in western blots. 4. Almost of the expressed PDLs22 protein was not soluble and observed like as inclusion body. 5. The protein solubility was not improved after modification of induction time and temperature during PDLs22 protein production. In this study, the system for the PDLs22 protein production was connstructed. However, the results suggest that further studies will be needed to produce the considerable amount of PDLs22 recombinat protein, which can use for the periodontal regeneration.

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.

Immunohistochemical localization of several protein changes in periodontal ligament during tooth eruption and interdental separation of rats / 대한치과교정학회지

In this study, we attempt to investigate the mechanisms by which PDL cells regulate osteoclast formation and also to know whether PDL retained their characteristic phenotype during tooth eruption and interdental separation. Rats were prepared at developmental days 21 (pre-root formation), 27 (root development), 34 (advanced root formation/ eruption) and at later times(adult rats). To induce severe resorption state of alveolar bone and tooth root, interdental separation with brass wire was performed between the lower first and second molars for 2 weeks in adult rats. Rat mandibles were demineralized and embedded in paraffin, and horizontal and frontal section were prepared for immunohistochemical analysis using PDL-specific protein 22 (PDLs22), receptor activator of NFKB ligand (RANKL) and osteoprotegerin (OPG) antibodies. 1. Root formation and eruption stage of tooth development. 1) PDLs22 immunolocalization was observed in tooth follicle/PDL cells and osteoblasts throught out the root formation and eruption stages of tooth development. 2) RANKL expression became stronger at eruption stage than root formation stage of tooth development. 3) Strong expression of OPG was detected in follice/PDL cells of root formation stage but it was decreased with tooth eruption. 2. Interdental separation between lower first and second molar. 1) Comparared to normal animal, multinucleated osteoclasts and odontoclasts were markedly induced in the alveolar bone and tooth root with PDL remodeling in hematoxylin-eosin section. 2) PDLs22 expression was decreased with interdental separation. 3) RANKL expression was increased with interdental separation in PDL fibroblasts, osteoblasts, odontoclasts and it lacunae, resorbing dentin, cementum and bone matrix. 4) OPG expression was slightly decreased in the PDL cells adjacent to the alveolar bone and root surface with interdental separation. These results suggested that during tooth eruption and tooth movement, RANKL and OPG in the periodontal tissues are important determinants regulating balanced alveolar bone and tooth root resorption. And it is also suggested that PDL cells retained their characteristic phenotype during tooth eruption and interdental separation except for the short period of PDL remodeling.

Expression of Periodontal Ligament Fibroblast-specific Gene, PDLs22 During Development of Periodontal Ligament, Alveolar Bone and Cementum / 대한해부학회지

Identifying specific factors and/or mechanism regulating development of periodontal tissue will provide important information as to which molecules and cells are required for regulation of periodontal tissue lost as a consequence of disease. The origin and location of cementoblast and osteoblast precursor cells in adult periodontal tissues is not definitely known but it has been suggested that tooth related periodontal ligament may be the source of cementoblasts and the bone-related periodontal ligament for osteoblasts. However, little is known of the molecular mechanism controlling PDL function. PDL-specific protein; PDLs22 had been previously identified as a novel protein isolated from cultured human PDL fibroblasts using subtraction hybridization between human gingival fibroblasts and PDL fibroblasts. The aim of this study was to examine the functional characterization of PDLs22 in differentiation of periodontal ligament, alveolar bone and cementum. Human osteocalcin (OC), osteonectin (ON) and PDLs22 mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR) in primary cell cultures of periodontal ligament fibroblast during mineral nodule formation in vitro. And the localization of PDLs22 in rat tissues was detected by polyclonal antibody against PDLs22 by means of immunohistochemical staining. The results were as follows: 1. PDL cells were capable of producing mineral-like nodules in vitro. 2. PDLs22 mRNA was expressed in the initial stages whereas it was not expressed in the calcification stage, during mineral nodule formation of PDL cells in vitro. 3. PDLs22 protein was expressed in external dental epithelium and stellate reticulum during crown formation stage, and was continued in external dental epithelium of Hertwig's epithelial sheath. Also PDLs22 protein was strongly expressed in the bone and cementum-related side of the PDL and weakly expressed in the middle of PDL. In the developing bone, PDLs22 protein is only expressed in preosteoblast not osteocyte and osteoblast. The results suggest PDLs22 is important mediator of epithelial-mesenchymal reaction in development of PDL, alveolar bone and cementum and is related to initial differentiation of cementum and alveolar bone.