• Journal of Periodontal and Implant Science
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• v.39 no.3
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• pp.331-337
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• 2009

Purpose: Interleukin (IL)-8 is one of pro-inflammatory cytokines. Reactive oxygen species (ROS) are reduced metabolites of $O_2$. Aggregatibacter actinomycetemcomitans is one of representative periodontopathogens. To investigate the role of A. actinomycetemcomitans in IL-8 expression of periodontal ligament (PDL) cells, we estimated the production of IL-8 and ROS in A. actinomycetemcomitans treated PDL cells. Methods: The IL-8 production was determined by enzyme-linked immunosorbent assay. The ROS production was estimated using H2DCFDA and FACS. Results: A. actinomycetemcomitans increased the production of IL-8 and ROS at 10, 100, and 500 multiplicity of infection. N-acetylcysteine, an antioxidant of ROS, down-regulated the production of IL-8 induced by A. actinomycetemcomitans. Conclusions: These results suggest that A. actinomycetemcomitans induces IL-8 production and ROS may act as a mediator in this process.

• Journal of Periodontal and Implant Science
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• v.49 no.4
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• pp.215-227
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• 2019

Purpose: To histologically characterize periodontal healing at 8 weeks in surgically created dehiscence defects in beagle dogs that received a collagen matrix with periodontal ligament (PDL) progenitor cells. Methods: The bilateral maxillary premolars and first molars in 6 animals were used. Standardized experimental dehiscence defects were made on the buccal side of 3 premolars, and primary culturing of PDL progenitor cells was performed on the molars. Collagen matrix was used as a scaffold and a delivery system for PDL progenitor cells. The experimental sites were grafted with collagen matrix (COL), PDL progenitor cells with collagen matrix (COL/CELL), or left without any material (CTL). Histologic and histomorphometric analyses were performed after 8 weeks. Results: The defect height from the cementoenamel junction to the most apical point of cementum removal did not significantly differ across the CTL, COL, and COL/CELL groups, at $4.57{\pm}0.28$, $4.56{\pm}0.41$, and $4.64{\pm}0.27mm$ (mean ${\pm}$ standard deviation), respectively; the corresponding values for epithelial adhesion were $1.41{\pm}0.51$, $0.85{\pm}0.29$, and $0.30{\pm}0.41mm$ (P<0.05), the heights of new bone regeneration were $1.32{\pm}0.44$, $1.65{\pm}0.52$, and $1.93{\pm}0.61mm$ (P<0.05), and the cementum regeneration values were $1.15{\pm}0.42$, $1.81{\pm}0.46$, and $2.57{\pm}0.56mm$ (P<0.05). There was significantly more new bone formation in the COL/CELL group than in the CTL group, and new cementum length was also significantly higher in the COL/CELL group. However, there were no significant differences in the width of new cementum among the groups. Conclusions: PDL progenitor cells carried by a synthetic collagen matrix may enhance periodontal regeneration, including cementum and new bone formation.

• International Journal of Oral Biology
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• v.44 no.4
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• pp.182-190
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• 2019

Periodontitis is an inflammatory disease of the supportive tissues surrounding the teeth, and is characterized by irreversible destruction of the gingiva, periodontal ligament (PDL), and alveolar bone, which results in the loss of teeth. In the present study, we elucidated the correlation between periodontitis and apelin (APLN), an adipokine and a regulatory peptide, respectively, which are involved in inflammation and bone remodeling. The expression of APLN is negatively correlated with periodontitis progression in gingival tissue. In addition, treatment with TNF-α downregulated the expression of APLN in PDL cells and gingival fibroblasts, indicating the protective role played by APLN against periodontitis progression. The overexpression of APLN or treatment with exogenous APLN suppressed the TNF-α-mediated catabolic gene expression of MMP1, IL6, and PTGS2 in PDL cells. Moreover, the inhibition of the APLNA-PJ axis by ML221, an APJ inhibitor, induced catabolic gene expression in PDL cells. Thus, the results of this study provided evidence to support APLN as a regulatory factor of the inflammatory response during periodontitis.

• Journal of Periodontal and Implant Science
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• v.26 no.2
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• pp.469-490
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• 1996

The initial events required for periodontal regeneration is the attachment, spreading, and proliferation of appropriated cells at the healing sites. These have been reported that minocycline stimulates the attachment of periodontal ligament cells, and also $TGF-{\beta}1$ enhances the proliferation of periodontal ligament cells. The purpose of the present study was to evaluate the effects of $TGF-{\beta}1$ on the cellular activity of minocycline treated human periodontal ligament cells. Periodontal ligament cells were obtained from the explants of healthy periodontal ligaments of extracted 3rd molars or premolar teeth extracted from the patients for orthodontic treatment. The cells were cultured in minimal essential medium(${\alpha}-MEM$) supplemented with 10.000units/ml penicillin, $10,000{\mu}g/ml$ streptomycin and 10% FBS(fetal bovine serum) at $37^{\circ}C$ in a humidified atmosphere of 5% carbon dioxide and the 5th to the 8th passages of the cells were used. To evaluate the effect of minocycline on cell attachment, the cells were seeded at a cell density of $1.5{\times}10^4$ cells/well in 24-well culture plates and treated with $20{\mu}g/ml$ and $100{\mu}g/ml$ of minocycline for 1.5 h. After trypsinization, the cells were counted with hemocytometer and were taken photographs for observation of cellular morphology. To evaluate the effect of $TGF-{\beta}1$ on minocycline-pretreated periodontal ligament cells, the cells were seeded at a cell density of $1{\times}10^4$ cells/ well in 24-well culture plates and treated with $20{\mu}g/ml$ and $100{\mu}g/ml$ of minocycline for 1.5 h. After incubation, 1 and 10ng/ml of $rh-TGF-{\beta}1$ were also added to the each well and incubated for 1 and 2 days, respectively. Then, MTT assay, DNA synthesis($^3H-thymidine\;assay$), and protein and collagen assay(3H-proline assay) were carried out. In the MIT assay, after 200ul MTT solutionlconeentration of 5mg/ml) were added to the each well of the 24-well plates and incubated for 3 hours, and 200 ul DMSO were added so as to dissolve insoluble blue formazan crystals which was formed in incubated period. Then it read plates on a ELISA reader. For mitogenic assay, 1 uCi/ml $^3H-thymidine$ was added to each well for the final 2 hours of the incubation periods. After labeling, the wells were washed 3 times with ice cold PBS and 4 times with 5% TCA to remove unincorporated label and precipitate the cellular DNA. DNA, with the incorporated $^3H-thymidine$, was solubilized with 500 ul of 0.1% NaOH/0.1% SDS. A 250 ul aliquot was removed from each well and placed in a scintillation vial with 4ml of scintillation cocktail. Using an liguid scintillation counter, counts per minute(CPM) were determined for each samples. 3 uCi/ml $^3H-proline$ was added to each well for the final 4 hours of the incubation periods and total protein and percent collagen synthesis were carried out. The results indicate that minocycline treated group with $100{\mu}g/ml$ concentration for 1.5 hours significantly increased than that of control in cell attachment, and cell process is also evident compared with that of control in cell morphology, and the cellular activity and DNA synthesis rate of cells treated minocycline and $TGF-{\beta}1$ significantly increased than that of control values, but were below to values of the $TGF-{\beta}1$ only treated group in MIT assay and $^3H-thymidine\;assay$, and the total protein synthesis of minocycline and $TGF-{\beta}1$ treated group also significantly increased than that of control values, but the percent collagen synthesis of tested group significantly decreased to compared with control. On the above the findings, the tested group of minocycline and $TGF-{\beta}1$ did not increase the effect on the cell activity than $TGF-{\beta}1$ only tested group and the tested group of minocycline inhibited cell activity. This results indicate that minocycline was effective on cell attachment in early stage, but it is harmful to cell activity, that inhibitory effect of minocycline was compensated with stimulatory effect of $TGF-{\beta}1$.

• Journal of Periodontal and Implant Science
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• v.29 no.3
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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.

• Journal of Periodontal and Implant Science
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• v.29 no.1
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• pp.31-40
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• 1999

Many factors may affect periodontal changes during the physiologic conditions of woman(e.g. puberty, menstrual cycle, pregnancy, menopause). Recently many research has focused on the immunological changes of host, but the exact mechanism is not clear. Collagen is a major constituent of periodontium, and collagenase specifically digests the collagen and plays a role in destruction of periodontal tissue. So, I suppose that it participates with the cytokines in the inflammation of gingiva and vascular response during the changes of female sex hormones. Because there are some evidences of the existence of the receptors of estrogen and progesterone in the gingiva, it may be a target tissue of female sex hormones. In this experiment, gingival fibroblast and periodontal ligament cell were cultured in the presence of various concentrations of estrogen or progesterone corresponding to the menstrual cycle and pregnancy. Collagenase activity of the supernatant of culture media was determined by Spectrophotometric collagenase assay. The enzyme activity was calculated by the % decrease of the coated collagen. 1. The estrogen at both concentrations had no effect on the activity of collagenase of the gingival fibroblast. 2. The progesterone had some effect on the collagenase activity of the gingival fibroblast at low and high concentration of menstrual cycle, and elevated the enzyme activity at all range of pregnancy concentrations. 3. In periodontal ligament cells, estrogen elevated the enzyme activity at the early pregnancy concentration and progesterone elevated at the concentration just before menstruation. In this experiment, pregesterone elevated the collagenase activity of gingival fibroblast and periodontal ligament cells. But the mechanism of the up-regulation of the enzyme activity was not confirmed. The more experiments of direct effect of progesterone on gingival at the molecular level(e.g. northern blot analysis) can reveal the exact mechanism.

• Journal of dental hygiene science
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• v.15 no.4
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• pp.488-494
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• 2015

A recent report showed that nuclear factor of activated T cell (NFATc) 1 is a member of the NFAT family and is strictly implicated osteoblast differentiation and bone formation. Furthermore, the precise expression and function of NFATc1 in periodontal tissue remains unclear. Therefore, the purpose of this study was to investigate the function of NFATc1 in osteoblastic differentiation, and the underlying mechanism regulating periodontal regeneration in human periodontal ligament cells (hPDLCs). NFATc1 messenger RNA (mRNA) and protein levels were accessed by reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay, respectively. Cell proliferation determined using MTT assay. Differentiation was evaluated by alkaline phosphatase activity and formation of calcium nodule with alizarin red S staining. The mRNA expression of osteoblastic differentiation related genes were examined by RT-PCR. Marked upregulation of NFATc1 mRNA and protein was observed in cells grown in osteogenic medium (OS). NFATc1 transactivation was detected in hPDLCs that had been incubated in OS for 14 days. Treatment with $10{\mu}M$ cyclosporine A (CsA), a known calcineurin inhibitor, reduced the proliferation of hPDLCs, while $5{\mu}M$ CsA had no effect. Inhibition of the calcineurin/NFATc1 pathway by CsA, attenuated OS-induced osteoblastic differentiation in hPDLCs. In summary, this study demonstrates for the first time that NFATc1 plays a key role in osteoblastic differentiation of hPDLCs and activation of NFATc1 could provide a novel mechanism for periodontal bone regeneration.

• Journal of Periodontal and Implant Science
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• v.24 no.2
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• pp.219-237
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• 1994

The ultimate goal of clinical periodontal therapy is to achieve regeneration of a healthy connective tissue reattachment. Conventional therapy including scaling, root planing, gingival curettage, gingivectomy and flap procedures of various types results primarily in repair rather than regeneration of the periodontium. In order for periodontal regeneration to occur, progenitor periodontal ligament cells must migrate to the denuded root surface, attach to it, proliferate and mature into an organized and functional fibrous attachment apparatus. Polypeptide growth factors belong to a class of potent biologic mediators which regulate cell differentiation, proliferation, migration and metabolism. Insulin-like growth factor-I (IGF- I ) of these factors appear to have an important role in periodontal wound healing and bone formation. The purpose of this study is to evaluate the effects of IGF- I on the periodontal ligament cells to use as a regeneration promoting agent of periodontal tissue. Human periodontal ligament cells were obtained from periodontal tissue explants culture of the first premolar tooth extracted for the orthodontic treatment. Cells were cultured in Dulbecco's modified Eagle medium(DMEM) with 10% fetal bovine serum. Fourth to seventh passage cells were plated in 24 well tissue culture plates and medium changed to serum-free medium prior to addition of growth factors. Cell proliferation was measured by the incorporation of $[^3H]-thymidine$ into DNA, Protein synthesis was determined by measurement of $[^3H]-proline$ incorporation into collagenase-digestible protein(CDP) and noncollagenous protein(NCP) according to the method of Peterkofsky and Diegelmann (1971), And alkaline phosphatase activity was measured as one parameter of osteoblastic differentiation. The results were as follows : The DNA synthetic activity was increased in a dose-dependent manner with IGF- I except for 0.1ng/ml concentration of IGF- I At the concentration of 10, 100ng/ml, IGF- I significantly increased the DNA synthetic activity(P<0.05) The total protein, collagen and noncollagen synthesis was increased in a dose-dependent manner with IGF- I except for 0.1ng/ml concentration of IGF- I. At the concentration of 1, 10, 100ng/ml, IGF- I significantly increased the total protein, collagen and noncollagen synthesis activity(P<0.95, P<0.001). The % of collagen was not effected according to the concentration of IGF- I. The alkaline phosphatase activity was increased in a dose-, time-dependent manner with IGF- I (10, 100ng/ml). In conclusions, the present study shows that IGF- I has a potentiality to enhance the DNA synthesis of periodontal ligament cells with including the increase of the total protein and collagen synthetic activity. The use of IGF- I to mediate biological stimulation of periodontal ligament cells shows promise for future therapeutic applications.

• Journal of Periodontal and Implant Science
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• v.27 no.4
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• pp.867-882
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• 1997

Safflower(Carthamus tinctorius $Linn\acute{e}$ has been traditionally used for the treatment of blood stasis, and Dipsasi Radix has been used as a drug for fracture in Chinese medicine. The purpose of present study was to examine the biologic effects of safflower extract and Disasi radix extracts on the periodontal. ligament cells and osteoblastic cells and on the wound healing of rat calvarial defect. The ethanolic extract of safflower blossom, safflower seed and Dipsasi Radix(125, 250, and 500 ${\mu}g/ml$) were prepared as test group, and PDGF-BB(lOng/ml) and unsafonifiable fraction of Zea Mays L.(125, 250, and 500 ${\mu}g/ml$) were employed as positive control. The effects of each agents on the growth and survival, ALPase activity, expression of PDGF-BB receptor, chemotactic response of PDL cell and ATCC human osteosarcoma MG63 cells in vitro were examined. The tissue regenerative effect of each extracts was evaluated by histomorphometric measuring of newly formed bone on the 8mm defect in rat calvaria after oral administration of 3 different dosages groups : 0.02, 0.1 and 0.35g/kg, per day. It was also employed the same dosages of unsaponifiable fraction of Zea Mays L. as positive controls. Safflower blossom extract, safflower seed extract, and Dipsasi Radix extract stimulate the cellular activity of MG63 cells in concentration range of $125-500{\mu}g/ml$, and safflower bolssom extract and safflower seed extract stimulate also the cellular activity of periodontal ligament cells in concentration range of $250-500{\mu}g/ml$. In activity of ALPase, $250-500{\mu}g/ml$ of safflower blossom extracts showed significant stimulating effects on MG63 cells, and the same concentration range of safflower seed extracts showed significant effect on periodontal ligament cells. In the recovery on PDGF-BB receptor expression which was depressed by $IL-1{\beta}$, $125-250{\mu}g/ml$ of safflower blossom extracts and $250-500{\mu}g/ml$ of safflower seed extracts showed significant increasing effect on MG63 cells, and $500{\mu}g/ml$ of safflower blossom extract and $250-500{\mu}g/ml$ of safflower seed extracts showed significant effect on periodontal ligament cells. In chemotactic response, among all tested group, safflower seed extracts only were chemotactic to MG63 cells and periodontal ligament cells in concentration range of $125-500{\mu}g/ml$. Also in the view of bone regeneration in rat calvarial defect model, the only group that was orally administrated 0.35g/kg, day of safflower seed extract showed significant new bone formation. These results suggested that safflower extracts might have a potential possibilities as an useful drug for adjunct to treatment for regeneration of periodontal defect.

• The korean journal of orthodontics
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• v.26 no.1 s.54
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• pp.83-94
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• 1996

Substance P is one of the neuropeptide which presents highly in tension site of periodontal ligament during the orthodontic tooth movement. It has bnn also hon as one of the neuropeptides which cause neurogenic inflammation in various tissues and organs. However, there is no report about the effect of substance P on major extracellular matrix protein, collagen production. The purpose of this study was to evaluate the collagen production by substance P in human periodontal ligament cell. The collagenase-digestion method was used to evaluate collagen production and also used Northern blot hybridization for the evaluation of collagen mRNA level. This study also Included in terms of prostanglandins and gelatinase production with respect to collagen production. For the collagen degradation, zymography was used to estimate denatured collagen degradation. Dose-dependent effect of substance P on noncollagen protein, collagen, and percent collagen was that substance P increased noncollagen protein synthesis, but decreased collagen sytnsis. So the percent collagen, which determined by relative collagen production against total protein production, w3s decreased from $7\%\;to\;3.6\%$. This inhibitory effect of substance P on collagen production was disappeared when cells were treated concomitantly with indomethacin. It means that substance P-induced inhibitory effect on collagen production was due at least in part to the production of prostaglandins. To evaluate whether substance P-induced inhibitory effect on collagen production is correspond to the steady-state levels of procollagen mRNA, Northern blot hybridization was performed and it showed that substance P has no effect on the steady-slate level of ${\alpha}1(I)$ procollagen mRNA. It means that the inhibitory effect of substance P on collagen production was due to the change of a certain mechanism after posttranscription. In this context, gelatinase production by substance P in periodontal ligament cells was evaluated by zymography. Zymogram showed that substance P has no effect on gelatinase production in periodontal ligament cells. To explore wheter substance P-induced inhibitory effect on collagen production is selevtive in periodontal ligament cells or not, MC3T3-E1 cells which originated from mouse calvaria was used. It showed that substance P has no effect on collagen production in MC3T3-E1 cells. Taken together, substance P inhibits collagen production in human periodontal ligament cells. This effect was not due to the change of the steady-state level of procollagen mRNA and gelatinase production, but due at least in part to the change of prostaglandins production.