• Journal of Periodontal and Implant Science
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• v.37 no.1
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• pp.35-44
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• 2007

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 hybrid-ization 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 ex-pressed 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 re-results suggest that further studies will be needed to produce the considerable amount of PDLs22 re-combinat protein, which can use for the periodontal regeneration.

• Restorative Dentistry and Endodontics
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• v.25 no.2
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• pp.170-179
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• 2000

The rh-BMP-4 is a subgroup of TGF-${\beta}$ superfamily. The application of rh-BMP in alveolar bony defect was reported to new alveolar bone and new cementum formation. For minimized complications following tooth replantation, a operator must replant a tooth fast at the pertinent position. This study was to evaluate the effect of rh-BMP-4 on periodontal regeneration and root resorption following tooth replantation in rats. The 50 Sprague-Dawley rats weighting about 130gm were used in this study. The animals were divided into three groups. Group 1 ; immediate replantation after extraction : Group 2 ; replantation stored teeth extraction of first molar, the removal of periodontal ligament with collagenase, and etching with citric acid : Group 3 ; replantation stored teeth with treated rh-BMP-4 in mesial root. Experimental animals were sacrificed 3, 7, 14 days after replantation by heart infusion. The maxillae were removed, fixed, demineralized, dehydrated, infiltrated and embedded with JB-4 mixture. For light microscopic observation, 5 micron sections were cut and stained with toluidine blue. The results of this study were as follows : 1. After experimental 3 days, all groups were observed dead space between periodontum and root. 2. After experimental 7 days, group 1 and group 3 were observed filling periodontal fibers between alveolar bone and root but group 2 were not. 3. After experimental 7 days, group 3 were observed appearance of attached cementoblast like cell on root surface. Group 1 were observed regular arrangement of fibroblasts and collagen fibers at ${\times}400$ observation. 4. After experimental 14 days, all group were observed filling periodontal fibers between alveolar bone and root. Group 1 were observed normal arrangement of periodontal fibers. Group 3 were observed less abnormal arrangement of periodontal fibers. Group 2 were not observed functional normal arrangement of periodontal fibers. 5. After experimental 14 days, group 2 and 3 were observed several root resorption and irregular root surface but group 1 were not. These results suggest that the rh-BMP-4 can stimulate cementogenesis and enhance to attach collagen fibers.

• Restorative Dentistry and Endodontics
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• v.32 no.3
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• pp.191-197
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• 2007

Mineral trioxide aggregate (MTA) would influence healing of periapical tissues by modulating the production of growth factors and cytokines from PDL fibroblasts, however, the studies are insufficient. Therefore, the purpose of this study was to monitor the expression of transforming growth factor-beta1 $(TGF-\beta1)$, fibroblast growth factor-2 (FGF-2), and interleukin-6 (IL-6) from PDL fibroblasts in the presence of MTA. The human PDL fibroblasts were seeded onto the set MTA or IRM at a level of $1\times10^5$ cells per unit well, and further incubated for 6, 12, 24, and 48 hours. The levels of $TGF-\beta1$, FGF-2 and IL-6 from the supernatant were measured by enzyme-linked immunosorbent assay (ELISA) The data were analyzed using one-way ANOVA. The level of $TGF-\beta1$ was down-reg ulated when the cells were grown in the presence of MTA except at 6 hours. The levels of FGF-2 release were significantly suppressed when PDL fibroblasts were grown in the presence of MTA or IRM at all time intervals (p < 0.05). The expressions of IL-6 from MTA treated co)Is were comparable to those of untreated control cells throughout the observation periods. We presume that this material inhibits the stimulatory function of growth factors on granulation tissue formation and in turn, it promotes the healing process modulated by other bone-remodeling cells.

• The korean journal of orthodontics
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• v.36 no.4
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• pp.242-250
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• 2006

Objective: Periodontal ligament fibroblasts have an ectomesenchymal origin and are thought to play a crucial role for not only homeostasis of periodontal tissues but also bone remodeling, wound healing and regeneration of tissues. Recently, it has been reported that UNC-50 is not expressed in gingival fibroblasts but in PDL fibroblasts. The purpose of this study was to examine the expression of UNC-50 and osteocalcin in the periodontium after application of intermittent force. Methods: Twelve rats had 40 grams of mesially-directed force applied at the upper molar for 1 hour/day. Four rats were sacrificed at 1, 3 and 5 days. Immunohistochemical localization of UNC-50 and osteocalcin antibody was carried out. The results showed apposition of new cellular cementum and a slight increase in periodontal space at the tension side. Results: Strong UNC-50 expression was observed in the differentiating cementoblasts close to PDL fibroblasts in the tension side whereas it was barely expressed at the compression side. Expression was strong at day 3, and decreased at day 5. Osteocalcin immunoreactivity expression was strong in differentiating cementoblasts at the tension side. Conclusion: It can be suggested that UNC-50 is related to the differentiation of cementoblasts, and may be responsible for the molecular event in PDL cells under mechanical stress.

• The korean journal of orthodontics
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• v.27 no.3 s.62
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• pp.503-513
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• 1997

This study was undertaken to investigate the effect of oxygen tension on the activity and function of the cells derived from human periodontal ligament by measuring cell activity, total protein synthesis, collagen synthesis, $IL-1{\beta},\;IL-6,\;TNF-{\alpha}$ Human periodontal ligament fibroblasts were collected from premolars extracted for orthodontic treatment and incubated in the environment of $37^{\circ}C,\;5\%\;CO_2,\100\%$ humidity. After the fifth to sixth passage they were used for the experiment. Gaspack system to which $0.2{\mu}m$ Millipore filter was attached was connected to mixed-gas tanks. The mixed gases were composed of $10\%\;O_2,\;5\%\;CO_2,\;85\%\;N_2$ in hyoxic group or $90\%\;O_2,\;5\%\;CO_2,\;5\%\;N_2$ in hyperoxic group and $5\%\;CO_2,\;95\%$ air for control. After incubation in $37^{\circ}C$ for 2, 4, 6 days, cell activity was determined by tetrazolium(MTT) assay and total protein synthesis was assayed using sulforhodamine B(SRB). And measurement of 4-hydroxyproline was performed to assess collagen synthesis md $IL-1{\beta},\;IL-6,\;and\;TNF-{\alpha}$ were measured by enzymeimmunoassay. The results were as follows. 1. The cell activity and total protein synthesis in hypoxic group were a little higher than or almost the same with those in control group. 2. In hyperoxic group, the cell activity was lower than that in control group and total protein synthesis was decreased. 3. Collagen synthesis was significantly decreased initially in both hypoxic and hyperoxic group and increased nearly to the level of control group as the duration of cell incubation was longer 4. As a result of enzymeimmunoassay, the amount of cytokines was $IL-6,\;TNF-{\alpha}\;and\;IL-1{\beta}$ in order. 5. $IL-6,\;TNF-{\alpha}\;and\;IL-1{\beta}$ were increased more rapidly in both hypoxic and hyperoxic group than in control group as the duration of cell incubation was longer. 6. There were more $IL-6\;and\;TNF-{\alpha}$ in hyperoxic group than in control group after 6 days, and there were more $IL-6\;and\;TNF-{\alpha}$ after 6 days than after 2 or 4 days in hyperoxic group. These results suggested that oxygen tension might modulate the production of extracellular matrix and cytokines in the cells derived from human periodontal ligament.

• The korean journal of orthodontics
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• v.42 no.6
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• pp.307-317
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• 2012

Objective: The purpose of this study was to investigate the isolation and characterization of multipotent human periodontal ligament (PDL) stem cells and to assess their ability to differentiate into bone, cartilage, and adipose tissue. Methods: PDL stem cells were isolated from 7 extracted human premolar teeth. Human PDL cells were expanded in culture, stained using anti-CD29, -CD34, -CD44, and -STRO-1 antibodies, and sorted by fluorescent activated cell sorting (FACS). Gingival fibroblasts (GFs) served as a positive control. PDL stem cells and GFs were cultured using standard conditions conducive for osteogenic, chondrogenic, or adipogenic differentiation. Results: An average of $152.8{\pm}27.6$ colony-forming units was present at day 7 in cultures of PDL stem cells. At day 4, PDL stem cells exhibited a significant increase in proliferation (p < 0.05), reaching nearly double the proliferation rate of GFs. About $5.6{\pm}4.5%$ of cells in human PDL tissues were strongly STRO-1-positive. In osteogenic cultures, calcium nodules were observed by day 21 in PDL stem cells, which showed more intense calcium staining than GF cultures. In adipogenic cultures, both cell populations showed positive Oil Red O staining by day 21. Additionally, in chondrogenic cultures, PDL stem cells expressed collagen type II by day 21. Conclusions: The PDL contains multipotent stem cells that have the potential to differentiate into osteoblasts, chondrocytes, and adipocytes. This adult PDL stem cell population can be utilized as potential sources of PDL in tissue engineering applications.

• Journal of Periodontal and Implant Science
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• v.25 no.1
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• pp.99-110
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• 1995

치주치료의 궁극적 목표인 치주조직 재생에 대한 관심이 높아지는 가운데 치주조직 재생에 주된 역할을 하는 치주인대세포와 치근면 탈회에 의한 신부착 효과에 관한 많은 연구가 시도되고 있다. 그러나 광범위한 연구에도 불구하고 어떤 치근면 처리가 신부착 형성에 가장 효과적인지는 아직 논란의 대상으로 남아있다. 이에 저자는 결합조직 부착에 의한 재생에 필수적인 치은 및 치주인대 섬유아세포의 부착 및 증식을 tetracycline-HCL(TC)과 citric acid(CA)로 각각 처리된 상아질 표면에서 비교관찰하여 치주조직 재생에의 기여도를 추정하기 위해 본 연구를 시행하였다. 교정치료를 위하여 본원에 내원한 환자의 제일 소구치의 건강한 치은조직을 채취하고, 발치 후 효소처리에 의한 치주인대세포를 얻은 후 각각 세포배양하였다. 상아질 절편은 $3{\times}3{\times}0.2mm^3$로 준비하고 TC과 CA처리군으로 나눈 후 각각 치은 및 치주인대 섬유아세포를 부착시키고 초기부착은 8시간까지, 증식은 7일까지 고나찰하여 다음의 결과를 얻었다. 1. 각 군의 세포 부착은 시간이 지남에 따라 증가되었다. 2. 모든 군에서 7일째 빠른 증식상을 보였다. 3. 초기 부착시 치은과 치주인대 섬유아세포의 반응에는 유의한 차이가 없었다. 4. 반면에 치은과 치주인대 섬유아세포는 증식속도에서 차이를 나타내었으며 치은 섬유아세포가 좀 더 빠른 성장을 보였다. 5. 치근면 탈회에 따라 초기부착과 증식상에서 모두 유의성 있는 증가를 보였다. 6. CA는 초기부착에서, TC는 증식장에서 더욱 효과적으로 작용했다.

• Journal of Periodontal and Implant Science
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• v.37 no.sup2
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• pp.311-323
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• 2007

Receptor activation of nuclear factor ${\kappa}$ B ligand (RANKL)은 파골세포의 분화와 기능에 중요한 역할을 하는 단백질로 이들 물질의 조절에는 p38 MAP kinase가 관여한다. 그러나 치주인대 섬유모세포에서 RANKL 발현 시 p38 MAP kinase의 역할은 잘 알려져 있지 않다. 이에 이번 연구는 마우스 치주인대 섬유모세포의 $IL-1{\beta}-induced$ RANKL 발현과정에서 p38의 역할을 규명하고자 하여 다음과 같은 결과를 얻었다. 마우스 치주인대 섬유모세포에 $IL-1{\beta}$ (1ng/ml)의 자극은 수용성 RANKL의 합성을 증가시켰다. 수용성 RANKL의 합성은 p38 MAP kinase 억제제인 SB203580에 의해 농도 의존적으로 억제되었으나 다른 MAP kinase 억제제인 SP600125, JNK 억제제와 PD98059, ERK 억제제에 의해서는 수용성 RANKL의 합성이 조절되지 않았다. NF-kB 억제제에 의해서도 수용성 RANKL의 합성이 억제되지 않았다. RANKL 유전자의 발현은 $IL-1{\beta}$로 자극 시에는 대조군에 비해 약 5배의 발현 증가를 보였으나 SB203580으로 전처치 시 $IL-1{\beta}$ (1ng/ml)로 자극시보다 약 1.5배의 감소를 보였다. 그러나 SP600125, PD98059, 및 NF-kB 억제제로 전처치한 경우에는 $IL-1{\beta}$로 자극한 경우와 비슷한 수준을 보였다. $IL-1{\beta}$로 자극 시 RANKL 유전자의 반감기가 90분 이었으나 SB203580로 전처치 후 $IL-1{\beta}$로 자극 시 RANKL 유전자의 반감기는 60분으로 감소하였다. Cycloheximide 전처리 시 SB203580에 의한 RANKL 유전자 발현 억제가 관찰되지 않았다. 단백질 분석결과 p38 MAP kinase의 인산화 활성은 30분까지 증가하였으나 그 이후 감소하여 2시간째에는 그 발현이 미약하였다. SB203580로 전처치 후 $IL-1{\beta}$로 자극 시 p38 MAP kinase의 인산화 활성이 감소하였다. 이상의 결과는 p38 MAP kinase가 RANKL 유전자 조절에 중요한 역할을 담당하고 있음을 시사한다.

• Restorative Dentistry and Endodontics
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• v.46 no.2
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• pp.20.1-20.11
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• 2021

Objectives: The aim of this study was to evaluate bacterial nanocellulose (BNC) membranes incorporated with antimicrobial agents regarding cytotoxicity in fibroblasts of the periodontal ligament (PDLF), antimicrobial activity, and inhibition of multispecies biofilm formation. Materials and Methods: The tested BNC membranes were BNC + 1% clindamycin (BNC/CLI); BNC + 0.12% chlorhexidine (BNC/CHX); BNC + nitric oxide (BNC/NO); and conventional BNC (BNC; control). After PDLF culture, the BNC membranes were positioned in the wells and maintained for 24 hours. Cell viability was then evaluated using the MTS calorimetric test. Antimicrobial activity against Enterococcus faecalis, Actinomyces naeslundii, and Streptococcus sanguinis (S. sanguinis) was evaluated using the agar diffusion test. To assess the antibiofilm activity, BNC membranes were exposed for 24 hours to the mixed culture. After sonicating the BNC membranes to remove the remaining biofilm and plating the suspension on agar, the number of colony-forming units (CFU)/mL was determined. Data were analyzed by 1-way analysis of variance and the Tukey, Kruskal-Wallis, and Dunn tests (α = 5%). Results: PDLF metabolic activity after contact with BNC/CHX, BNC/CLI, and BNC/NO was 35%, 61% and 97%, respectively, compared to BNC. BNC/NO showed biocompatibility similar to that of BNC (p = 0.78). BNC/CLI showed the largest inhibition halos, and was superior to the other BNC membranes against S. sanguinis (p < 0.05). The experimental BNC membranes inhibited biofilm formation, with about a 3-fold log CFU reduction compared to BNC (p < 0.05). Conclusions: BNC/NO showed excellent biocompatibility and inhibited multispecies biofilm formation, similarly to BNC/CLI and BNC/CHX.

• The korean journal of orthodontics
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• v.34 no.1 s.102
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• pp.71-81
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• 2004

In this study, we attempt to investigate the mechanisms by which PDL cells regulate osteoclast formation and also tc know whether PDL retained their characteristic phenotype during tooth eruption and interdental separation. Rats were prepared at developmental days 21 (pre-root formation), 27(toot 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 immuno-histochemical 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 toot 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, resorting 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.