• Journal of Periodontal and Implant Science
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• 제32권1호
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• pp.1-12
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• 2002

The periodontal ligament(PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms 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 expression pattern and tissue localization of PDLs22 protein in embryonic and various postnatal stages of developing mouse using immunohistochemical staining. Embryos (E18) and postnatal (P1, P4, P5, P15, P18) were decapitated and the heads were fixed overnight in a freshly prepared solution of 4% paraformaldehyde. Some specimens were decalcified for $2{\sim}4$ weeks in a solution containing 10% of the disodium salt of ethylenediamine-tetraacetic acid (EDTA). Next, tissues were dehydrated, embedded in paraffin and sectioned serially at $6{\mu}m$ in thickness. Polyclonal antiserum raised against PDLs22 peptides, ISNKYLVKRQSRD, were made. The localization of PDLs22 in tissues was detected by polyclonal antibody against PDLs22 by means of immunohistochemical staining. The results were as follows; 1. Expression of PDLs22 protein was not detected in the tooth germ of bud and cap stage. 2. At the late bell stage and root formation stage, strong expression of PDLs22 protein was observed in developing tooth follicle, osteoblast-like cells, and subodontoblastic cells in the tooth pulp, but not in gingival fibroblasts, ameloblasts and odontoblasts of tooth germ 3. In erupted tooth, PDLs22 protein was intensely expressed in PDL and osteoblast-like cells of alveolar bone, but not in gingival fibroblasts, mature osteocytes and adjacent salivary glands. 4. In the developing alveolar bone and mid-palatal suture, expression of PDLs22 protein was seen in undifferentiated mesenchymal cells and osteoblast-like cells of developing mid-palatal suture, but not in mature osteocytes and chondrocytes. These results suggest that PDLs22 protein may play an important role in the differentiation of undifferentiated mesenchymal cells in the bone marrow and PDL cells, which can differentiate into multiple cell types including osteoblasts, cementoblasts, and PDL fibroblasts. However, more researches should be performed to gain a better understanding of the exact function of PDLs22 protein which related to the PDL cell differentiation.

• Journal of Periodontal and Implant Science
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• 제43권4호
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• pp.168-176
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• 2013

Purpose: The purpose of the current study was to examine the effect of dexamethasone (Dex) at various concentrations on the apoptosis and mineralization of human periodontal ligament (hPDL) cells. Methods: hPDL cells were obtained from the mid-third of premolars extracted for orthodontic reasons, and a primary culture of hPDL cells was prepared using an explant technique. Groups of cells were divided according to the concentration of Dex (0, 1, 10, 100, and 1,000 nM). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed for evaluation of cellular viability, and alkaline phosphatase activity was examined for osteogenic differentiation of hPDL cells. Alizarin Red S staining was performed for observation of mineralization, and real-time polymerase chain reaction was performed for the evaluation of related genes. Results: Increasing the Dex concentration was found to reduce cellular viability, with an increase in alkaline phosphatase activity and mineralization. Within the range of Dex concentrations tested in this study, 100 nM of Dex was found to promote the most vigorous differentiation and mineralization of hPDL cells. Dex-induced osteogenic differentiation and mineralization was accompanied by an increase in the level of osteogenic and apoptosis-related genes and a reduction in the level of antiapoptotic genes. The decrease in hPDL cellular viability by glucocorticoid may be explained in part by the increased prevalence of cell apoptosis, as demonstrated by BAX expression and decreased expression of the antiapoptotic gene, Bcl-2. Conclusions: An increase in hPDL cell differentiation rather than cellular viability at an early stage is likely to be a key factor in glucocorticoid induced mineralization. In addition, apoptosis might play an important role in Dex-induced tissue regeneration; however, further study is needed for investigation of the precise mechanism.

• Journal of Periodontal and Implant Science
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• 제40권3호
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• pp.111-118
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• 2010

Purpose: The aim of this study was to investigate whether vitrification in the cryopreservation of periodontal ligament (PDL) cells could be useful for tooth banking. Methods: In step 1, primary cultured human PDL cells were cryopreserved in 100% conventional cryopreservation media and 100% vitrification media (ESF40 media) in different temperatures for 2 weeks. In step 2, a series of modified vitrification formulae named T1 (75% vitrification media + 25% F-media), T2 (50% vitrification media + 50% F-media) and T3 (25% vitrification media + 75% F-media) were used to store PDL cells for 2 weeks and 4 weeks in liquid nitrogen. MTT assay was performed to examine the viability of PDL cells. Results: Maximum cell viability was achieved in cells stored in 100% conventional cryopreservation media at $-196^{\circ}C$ (positive control group) in step 1. Compared to the positive control group, viability of the cells stored in 100% vitrification media was very low as 10% in all test conditions. In step 2, as the percentage of vitrification media decreased, the cell viability increased in cells stored for 2 weeks. In 4-week storage of cells in step 2, higher cell viability was observed in the T2 group than the other vitrification formulae while the positive control group had the highest viability. There was no statistically significant difference in the cell viability of 2-week and 4-week stored cells in the T2 group. Conclusions: These observations indicate 100% vitrification media is not successful in PDL cell cryopreservation. Conventional cryopreservation media is currently the most appropriate media type for this purpose while T2 media would be interesting to test for long-term storage of PDL cells.

• Journal of Periodontal and Implant Science
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• 제35권2호
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• pp.289-298
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• 2005

Bone morphogenetic protein-7(BMP-7), a member of the transforming growth factor superfamily, stimulates osteoblast differentiation and bone formation. There are lots of evidences supporting a direct participation of periodontal ligament(PDL) cells on periodontal tissue regeneration. The purpose of this study was to evaluate the effect of recombinant human(rh) BMP-7 on primary rat PDL cells in vitro, with special focus on the ability of bone formation. The PDL cells were cultured with rhBMP-7 at the concentration of 0, 10, 25, 50, 100 and 200ng/ml for MTT assay. We evaluated the alkaline phosphatase activity at 3 and 5 days of incubation and the ability to produce mineralized nodules of rat PDL cells at 14 days of cell culture in concentration of 0, 10, 25, 50 and 100ng/ml. The cell activity was not reduced in cells treated with BMP-7 at $10{\sim}100ng/ml$, whereas the cell activity was reduced in the concentration of 200ng/ml than the control at day 1 and 3(p<0.01). At 3 and 5 day, alkaline phosphatase activity was significantly increased in cells treated with BMP-7 at 50ng/ml and 100ng/ml(p<0.05). The area of mineralized bone nodule was greater in cells treated with BMP-7 at 50 and 100 ng/ml than the control(p<0.01). These results suggest that rhBMP-7 stimulate rat PDL cells to differentiate toward osteoblast phenotype and secretion of the extracellular matrix of rat PDL cells.

• Journal of Periodontal and Implant Science
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• 제29권3호
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• pp.645-654
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• 1999

Interleukin-6(IL-6) stimulate osteoclast differentiation. $17{\beta}$-estradiol, 1,25-dihydroxyvitamin $D_3$(1,25-$(OH)_2D_3$) and interleukin-$1{\beta}$ inhibit or stimulate osteoclast differentiation by decreasing or increasing the synthesis of interleukin-6(IL-6) from stromal/osteoblastic cells, respectively. Periodontal ligament(PDL) cells reside between the alveolar bone and the cementum and have osteoblastic characteristics. To estimate the effect of $17{\beta}$-estradiol and 1,25$(OH)_2D_3$ on IL-6 production of PDL cells, PDL cells were treated with $17{\beta}$-estradiol or 1,25-$(OH)_2D_3$ in the absence or the presence of IL-$1{\beta}$. The concentration of IL-6 produced form PDL cells was determined by enzym linked immunosorbent assay(ELISA). In unstimulated PDL cells, we detected constitutive production of IL-6 at 1st and 2nd day. IL-$1{\beta}$ increased IL-6 synthesis at 1st day and 2nd day. $17{\beta}$-estradiol had no significant effect on the secretion of this cytokine, either constitutively or after stimulation with IL- $1{\beta}$(0.05 ng/ml). 1,25-$(OH)_2D_3$($10^{-8}M$) decreased not only constitutive IL-6 production but also IL-$1{\beta}$-induced IL-6 production at 2nd day. These results suggest that 1,25-$(OH)_2D_3$ may control IL-$1{\beta}$-induced osteoclast differentiation by decreasing IL-$1{\beta}$-induced IL-6 secretion of PDL cells.

• 대한치과교정학회지
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• 제39권4호
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• pp.248-256
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• 2009

이 연구의 목적은 배양된 사람 치주인대 세포에서 파골세포의 형성에 관련된 물질을 합성, 분비할 수 있는지를 알아보고 압박력이 M-CSF, IL-$1{\beta}$, RANKL 및 OPG mRNA의 발현에 미치는 영향을 알아보고자 하였다. 교정치료를 목적으로 발치된 소구치에서 얻은 치주인대세포를 배양한 후 다양한 크기(0.5, 1.0, 2.0, 3.0, $4.0\;g/cm^2$)의 기계적 자극을 다양한 기간(0.5, 1.5, 6, 24, 48 hours) 동안 적용하고, M-CSF, IL-$1{\beta}$, RANKL, OPG mRNA 발현양의 변화를 검사하였다. 각각의 실험군에서 얻어진 mRNA에 대해 역전사 중합효소 연쇄반응검사를 시행하였다. 검사 결과 압박력은 사람 치주인대 세포에서 M-CSF mRNA를 발현시켰으며 M-CSF, IL-$1{\beta}$, RANKL mRNA의 발현양은 자극의 크기와 기간에 따라 증가하였다. 그러나 압박력은 사람 치주인대 세포에서 OPG mRNA의 발현양에 영향을 미치지 않는 것으로 나타났다. 이상의 결과는 기계적 자극이 치주인대 세포에서 M-CSF, IL-$1{\beta}$, RANKL mRNA의 발현양을 조절함으로 파골세포의 분화에 영향을 미칠 수 있음을 시사한다.

• Journal of Periodontal and Implant Science
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• 제35권2호
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• pp.511-524
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• 2005

The regeneration of lost periodontal tissue is a major goal of therapy. Periodontal ligament cell(PDL) is a specialized connective tissue that connects cementum and alveolar bone to maintain and support teeth in situ and preserve tissue homoeostasis. Bone morphogenetic proteins(BMPs) have shown much potential in the reconstruction of the periodontum by stimulate new bone and new cementum formation. Limitiations of BMP administration to periodontal lesions is high dose delivery, BMP transient biological activity, and low bioavailability of factors at the wound site. Gene delivery method can be alternative treatment strategy to deliver BMPs to periodontal tissue. The purpose of this study is to investigate efficiency of BMP-2 gene delivery with cell-based therapy using PDL cells. PDL cell were transduced with adenoviruses encoding either BMP-2 or Lac-Z gene. To evaluate osteogenic activity of expressed BMP-2 on PDL cells, we investigated secreted BMP-2, cellular activity, ALPase, produced mineralized nodules. To evaluate collagen scaffold as carrier for transduced cell delivery, we examined morphology and secreted BMP-2 of transducd PDL cells on it. BMP-2 transducd PDL cells produced higher levels of BMP-2, ALPase, mineralized nodules than non transduced cells. Cellular activity of transduced cells was showed similar activity to non transduced cells. Transduce cells attached on collagen scaffold secreted BMP-2 at 7day and was showed similar morphology to non transduced cells. These results demonstrated that transduced PDL cells produced biologically active BMP-2 and collagen scaffold could be carrier of transducd cells.

• Journal of Periodontal and Implant Science
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• 제31권4호
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• pp.787-801
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• 2001

The molecular mechanisms control the function of PDL(periodonta1 ligament) cells and/or fibroblasts remain unclear. PDLsl7, 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　PDLsl7 gene expression in　cultured human periodontal ligament cells and observe the immunohistochemical　localization of PDLsl7 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 $IL-1{\beta}$, PDGF-BB and TGF\;${\beta}$ for 48h nd 2 weeks. At each time point total RNA was extracted and the levels of transcription ere assessed by reverse transcription-polymerase chain reaction (RT-PCR assay). polyclonal antiserum raised against PDLsl7 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. PDLsl7 mRNA levels were increased in response to PDGF (10ng/ml) and $TGF\;{\beta}$(20ng/ml) after treatment of the $IL-1{\beta}$, PDGF-BB and $TGF{\beta}$for 48 h. 2. PDLsl7 was up-regulated only by $TGF{\beta}$(20 ng/ml) after treatment of the $IL-1{\beta}$, PDGF-BB and $TGF\;{\beta}$ for 2 weeks and unchanged by the other stimulants. 3. PDLsl7 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 PDLsl7 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 PDLsl7 protein was barely detectable in gingival fibroblasts, hematopoetic cells of the bone marrow and mature osteocytes of the alveolar bone. These results suggest that PDLsl7 might upregulated by PDGF-BB or $TGF{\beta}$ and 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 PDLsl7 during the differentiation of bone marrow mesenchymal and PDL cells.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제36권1호
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• pp.7-15
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• 2010

Complex human tissues harbor stem cells and precursor cells, which are responsible for tissue development or repair. Recently, dental tissues such as dental pulp, periodontal ligament (PDL), dental follicle have been identified as easily accessible sources of undifferentiated cells. These tissues contain mesenchymal stem cells that can be differentiate into bone, cartilage, fat or muscle by exposing them to specific growth conditions. In this study, the authors procured the stem cell from pulp, PDL, and dental follicle and differentiate them into osteoblast and examine the bone induction capacity. Dental pulp stem cell (DPSC), periodontal ligament stem cell (PDLSC), and dental follicle precursor cell (DFPC) were obtained from human 3rd molar and cultured. Each cell was analyzed for presence of stem cell by fluorescence activated cell sorter (FACs) against CD44, CD105 and CD34, CD45. Each stem cell was cultured, expanded and grown in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix. Osteogenic pathway was checked by alizarin red staining, alkaline phosphatase (ALP) activity test and RT-PCR for ALP and osteocalcin (OCN) gene expression. According to results from FACs, mesenchymal stem cell existed in pulp, PDL, and dental follicle. As culturing with bone differentiation medium, stem cells were differentiated to osteoblast like cell. Compare with stem cell from pulp, PDL and dental follicle-originated stem cell has more osteogenic effect and it was assumed that the character of donor cell was able to affect on differential potency of stem cell. From this article, we are able to verify the pulp, PDL, and dental follicle from extracted tooth, and these can be a source of osteoblast and stem cell for tissue engineering.

• 한국자원식물학회지
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• 제35권2호
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• pp.385-389
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• 2022

본 연구에서는 솔잎 추출물(PDL)의 항비만활성을 검증하기 위해, PDL이 3T3-L1에서 DMI/insulin에 의해 유도되는 과도한 지질 형성을 억제하는지 평가하였다. 본 결과를 미루어 볼 때, PDL은 지방세포의 adipogenesis과 관련된 PPARγ, CEBPα 그리고 perilipin-1의 발현을 억제하여 과도한 지질형성을 억제하는 것으로 판단되며, PDL은 향후 비만 예방 및 치료를 위한 잠재적 소재로 활용이 가능할 것으로 판단된다. 그러나 본 연구는 PDL이 항비만 활성이 있는지 검증한 초기 단계 연구로 추가적인 기전연구와 동물기반 전임상 항비만활성 검증이 필요하다.