• 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.

• Journal of Periodontal and Implant Science
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• v.28 no.3
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• pp.453-465
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• 1998

감초산이 인체 치은 섬유모세포에 미치는 영향을 세포의 성장과 증식, 총 교원질 합성 및 인체 치은 섬유모세포 핵내 acridine orange 결합으로 추적조사하였다. 조절이 되지 않는 성장을 해결하기 위하여 세포분화인자인 감초산이 배양 치은 섬유모세포의 활성에 미치는 효과를 검색하였다. 감초산 존재하의 배양 인체 섬유모세포의 세포성장 및 증식, 교원질 합성 및 세포 핵내 acridine orange 결합을 각각 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)법, 4-hydroxyproline, 유식세포분석기를 이용한 acridine orange 결합으로 검색하였다. 형태학적으로 $100\;{\mu}g/ml$의 감초산으로 처리한 섬유모세포는 모양이 둥글게 되었다. 감초산은 $50\;{\mu}g/ml$ 이상의 농도에서 치은 섬유모세포의 성장과 증식을 억제하였다. 감초산 존재 시에 세포내 총 교원질 양이 감소하였고, 세포외배지내의 교원질 총 양이 증가하였다. 인체 치은 섬유모 세포를 $100\;{\mu}g/ml$의 감초산과 함께 24 시간동안 배양하였을 때, 80 채널 이상의 평균형광을 갖는 diploid 세포가 감소하였고, 80 채널 이하의 형광을 갖는 acridine orange결합이 증가하였다. 이러한 연구 결과 감초산은 인체 섬유모세포에서 세포성장 및 증식, 교원질합성 및 DNA 분절화를 유도함이 제시하였다.

• Journal of Periodontal and Implant Science
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• v.24 no.2
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• pp.303-320
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• 1994

Current acceptable methods For promotin gperiodontal regeneration are base on removal of diseased soft tissue, root treatment, guided tissue regeneration, inteoduction of new graft materials and biological mediators. Platelet-derived growth factor(PDGF) is one of polypeptide growth factor. PDGF has been reported as a biological mediator which regulates activities of wound healing process including the cell proliferation, migration and metabolism. The purposes of this study is to evaluate the effects of PDGF-AA, BB on the periodontal ligament cells to use as a regeneration promoting agent of periodontal tissue. Human periodontal ligament cells were prepared from the first premolar tooth extracted for the orthodontic treatment and were cultured in DMEM/10% FBS at the $37^{\circ}C$, 5% $CO_2$ incubator. Author measured the DNA synthesis, total protein, collagen and noncollagenous protein synthesis and alkaline phosphatase activity according to the concentration of PDGF-AA and BB(0, 0.1, 1, 10, 100ng/ml). The results were as follows : The DNA synthetic activity was increased dose dependently by PDGF-AA and BB. The maximum mitogenic effect was at the 100ng/ml of PDGF-AA and 10ng/ml of PDGF-BB. The total protein, collagen and noncollagen systhesis was increased dose dependently by PDGF-AA and BB. The % of collagen was slightly decresed according to the concentration of PDGF-AA and BB. The effect of PDGF-AA and BB were not specific for collagen synthesis since it also increased noncollagenous protein synthesis. The effect of PDGF-AA and BB on alkaline phosphatase activity did not show any significant, meanwhile the alkaline phosphatase activity of 14 days group showed significnat increase. In conclusion, PDGF-AA and BB may have important roles in stimulation of DNA synthesis in human periodontal ligament cells, which means an increase in collagen-synthesizing cells, and may be useful for clinical application in periodontal regenerative procedures.

• 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.

• The korean journal of orthodontics
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• v.23 no.2 s.41
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• pp.229-248
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• 1993

[ $Interferon-\gamma$ ] has been suggested as a cytokine of connective tissue stabilizer. In addition, it has also been demonstrated that this cytokine inhibited bone remodeling activities of the bone derived cells. In order to illuminate the effects of this cytokine in orthodontic force induced bone remodeling, it was administered to primary cultured periodontal ligament cells which have been known to have some osteoblast like characteristics. $Interferon-\gamma$ slightly decreased $[^3H]thymidine$ incorporation rate without a significant change in the total cellular DNA content up to 1000 U/ml, which meant these doses were not cytotoxic to the cell. Total protein synthesis was not influenced by various concentration of interferon-y whether it was determined by the $[^3H]proline$ incorporation rate or by the Lowry smethod. The effect of $interferon-\gamma$ on the individual protein was, however, differential, ie, it increased $[^3H]proline$ incorporation into the noncollagenous protein marginally, while it decreased $[^3H]proline$ incorporation into the collagen, so that it caused dose-dependent suppression of the relative collagen synthesis. On the contrary, the fibronectin synthesis determined by the ELISA was increased by 1000 U/ml of $interferon-\gamma$. The differential effects of the interferon-y on the collagen and fibronectin synthesis exhibited not only their protein level but also the steady state mRNA level. $Interferon-\gamma$ decreased steady state level of ${\alpha}1(I)$ procollagen mRNA significantly, while showing no significant changes in the fibronectin mRNA level. In addition to this, it was also found that indomethacin did not affect on the $interferon-\gamma$ induced collagen decrease in this cell, which meant prostaglandins were not involed in the process of $interferon-\gamma$ induced collagen decrease. So it can be concluded that the incubation of periodontal ligament cells with 1000 U/ml of $interferon-\gamma$ for 24 hr showed differential effects on the type I collagen and fibronectin gene expression. The decrease in relative collagen synthesis in the protein level was related with decrease in the steady state level of mRNA, while the increase in the fibronectin synthesis in the protein level was not correlated with the mRNA level.

• Journal of Periodontal and Implant Science
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• v.32 no.3
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• pp.457-473
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• 2002

The purposes of this study is to evaluate the combination effects of TGF-${\beta}_1$ and PDGF-BB on the periodontal ligament cells to use as a regeneration promoting agent of periodontal tissue. Human periodontal ligament cells were prepared from the first premolar tooth extracted for the orthodontic treatment and were cultured in DMEM/100% FBS at the $37^{\circ}C$, 5% $CO_2$ incubator. Authors measured the DNA synthesis, total protein, collagen and noncollagenous protein synthesis according to the concentration of TGF-${\beta}_1$,(1,5ng/ml) and PDGF-BB (1,10 ng/ml) in combination. To explore further this delayed effect of TGF-${\beta}_1$, we preincubated human periodontal ligament cells with TGF-${\beta}_1$ for 4 or 24 hours before PDGF-BB stimulation. The results were as follows: The DNA synthetic activity was increased dose dependently by TGF-${\beta}_1$, PDGF-BB. The combination of TGF-${\beta}_1$ and PDGF-BB consistently enhanced the DNA synthetic activity to PDGF-BB alone. The ability of TGF-${\beta}_1$ to enhance DNA synthetic activity in PDGF-BB treated periodontal ligament cells was dose dependent. The maximum mitogenic effect was at the 5ng/ml of TGF-${\beta}_1$ and l0ng/ml of PDGF-BB. Preincubation of cell with TGF-${\beta}_1$ resulted in significantly greater response to PDGF-BB at all TGF-${\beta}_1$ concentration studied, and may be useful for clinical application in periodontal regenerative procedures. The total protein, collagen and noncollagen synthesis was increased dose pendently by TGF-${\beta}_1$, PDGF-BB. The % of collagen was slightly decreased according to the concentration of TGF-${\beta}_1$, PDGF-BB. The effect of TGF-${\beta}_1$, PDGF-BB were not specific for collagen synthesis since it also increased noncollagenous protein synthesis. This study demonstrates that PDGF-BB is major mitogens for human periodontal ligament cells in vitro, and supports a role for TGF-${\beta}_1$ as a regulation of the mitogenic and total protein formation to PDGF-BB in these cells.

• The Journal of Korean Physical Therapy
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• v.15 no.4
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• pp.1-12
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• 2003

The purpose of this study was to investigate the effect of high voltage pulsed current stimulation (HVPCS) on the healing rate of a dermal wound in a rat. We also determined the mechanism of promoting healing by HVPCS. Twenty male Sprague-Dawley rats were randomly divided into two group; HVPCS group (n=10) and control group (n=10). The HVPCS rats received electrical stimulation with a current intensity of 50 V at 100 pps for a duration of 30 minutes, while the control group was given the same treatment without electricity for a week. The biopsy specimens were fixed in formalin, embedded in paraffin and stained with Masson's trichrome, hematoxylin and eosin (H&E). The fibroblasts and collagen density were counted using a light microscope and computerized image analysis system and calculated as the density and the percent. A Student t-test showed a significantly higher wound healing rate of the HVPCS group than control (t=-4.161, p<0.001). The fibroblasts in the HVPCS group were higher than in the control group (t=-4.921, p<0.001). The density of collagen in the HVPCS group was also higher than in the control group (t=-4.367, p<0.001). These results indicate that the HVPCS accelerated the rate of healing in dermal wound, and increased fibroblasts and collagen density in the regenerative dermis. These findings suggest that the HVPCS may activate fibroblasts by alteration of the electrical environment, and it may increase collagen synthesis in the regenerative dermal wound.

• Journal of Periodontal and Implant Science
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• v.22 no.1
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• pp.203.2-203.2
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• 1992

• Journal of Periodontal and Implant Science
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• v.28 no.4
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• pp.703-721
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• 1998

In order to observe the effects of Nicotine and NNK on cultured human gingival fibroblast, several factors were examined including mutagenicity, the number of cells attached culture plate surface through MTT test, the abundance of collagen & collagenase in mRNA level and collagenolytic activity in extracellular matrix. The results were as follows; 1. Regardless of the co-existence of S9, Nicotine did not show the mutagenicity by itself and NNK by itself showd the same result; However, dose related mutagenicity was shown in NNK with S9. 2. The number of fibroblasts attached cultured plate surface was measured by MTT procedure. The number of cells in Non-smokers increased at all time periods as compared to those of smoker. 3. Non-smoker's fibroblast treated by NNK or Nicotine was dosedependently dosedependently decreased in the number of cells when compared to untreated control. In higher dose, Nicotine showed the cellular toxicity, but NNK did not. 4. No change in the abundance of mRNA for pro${\alpha}1$ and pro${\alpha}2$ was shown in Nicotine treated group but in gingival fibroblasts following treatment with NNK, the abundance of mRNA for pro${\alpha}1$, but not pro${\alpha}2$ collagen was decreased. 5. The abundance of mRNA for collagenase was decreased when NNK was treated but no change occurred in Nicotine treated group. 6. The effect of NNK and Nicotine in collagenolytic activity showed that, collagenase activity exclusively react to type I collagen, was increased in both group, but gelatinase exclusively react to type IV collagen was not influenced at all. Collagenase activity of smoker's fibroblast was also increased as much as Nicotine and NNK group. The findings suggest that both of Nicotine and NNK lead gingival fibroblast to decrease in the abundance of collagen. And it seems to be that Nicotine and NNK have independent pathway toward the gingival fibroblast.

• Journal of Periodontal and Implant Science
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• v.35 no.1
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• pp.77-85
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• 2005

목적 : 이 연구의 목적은 치과 영역에서 골 재생을 촉진하기 위해, 현재 많이 사용하고 있는 BDX(bovinederived xenograft)와 비교하여 BCP(biphasic calcium phosphate)의 효과를 알아보기 위함이다. 실험 재료 및 방법: 본 연구는 태아골모세포주(hFOB 1.19)를 사용하였으며, 사용된 골 이식재에 따라 2개의 실험군으로 구분하였고, 각 실험에 적절한 농도의 BDX와 BCP를 첨가하였다. 그리고, 세포 증식도 검사, 교원질 합성량 분석, 염기성 인산분해효소 활성도 측정, Western blot 분석을 통한 OC과 BSP의 발현 정도등의 실험을 진행하였다. 결과 : BDX와 BCP는 대조군과 비교하여 세포 증식에서 유의한 차이가 없었지만, 교원질 합성량, 염기성 인산분해효소의 활성, 그리고 OC과 BSP의 발현에 있어 대조군과 비교하여 유의하게 증가를 보였다. 그러나, 두 이식재간의 유의한 차이는 보이지 않았다. 결론 : 본 실험실적 연구에서 BCP는 골모세포분화에 긍정적인 영향을 미침으로써 효과적인 이식재로 사용할 수 있음을 가늠할수 있었다.