• Journal of Periodontal and Implant Science
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• v.24 no.2
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• pp.321-339
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• 1994

The cytokines released by osteoblasts induce bone resorption via the differentiation of osteoclast precursors. In this process, $interleukin-1{\beta}$($IL-1{\beta}$)-induced bone resorption is mediated by granulocyte macrophage-colony stimulation factor(GM-CSF), interleukin-6 (IL-6), and tumor necrosis factor ${\alpha}$($TNF-{\alpha}$) released from osteoblasts. Since these cytokines (GM-CSF, IL-6, $TNF-{\alpha}$) are produced by not only osteoblasts but also monocytes, and interleukin-10(I1-10) inhibits the secretion of these cytokines from monocytes, it may be speculated that IL 10 could modulate the production of GM-CSF, IL-6, and $TNF-{\alpha}$ by osteoblasts, then control $IL-1{\beta}-induced$ bone resorption. Therefore, the aims of the present study were to examine the effects of IL-10 on bone resorption. The sixten or seventeen-day pregnant ICR mice were injected with $^{45}Ca$ and sacrificed one day after injection. Then fetal mouse calvaria prelabeled with $^{45}Ca$ were dissected out. In order to confirm the degree of bone resorption, mouse calvaria were treated with Lipopolysaccharide(LPS), $TNF-{\alpha}$, $IL-1{\alpha}$, IL-8, $IL-1{\beta}$, and $IL-1{\alpha}$, Then, IL-10 and $interferon-{\gamma}$ ($IFN-{\gamma}$) were added to calvarial medium, in an attempt to evaluate the effect of $IL-1{\beta}-induced$ bone resorption. In addition, osteoclasts formation in bone marrow cell cultures, and the concentration of IL-6, $TNF-{\alpha}$, and GM-CSF produced from mouse calvarial cells were investigated in response to $IL-1{\beta}$ alone and simultaneously adding f $IL-1{\beta}$ and IL-10. The degree of bone resorption was expressed as the ratio of $^{45}Ca$ release(the treated/the control). The osteoclasts in bone marrow cultures were indentified by tartrate resistant acid phosphatase(TRAP) stain and the concentration of the cytokines was quantified using enzyme linked immunosorbent method. As results of these studies, bone resorption was induced by LPS(1 ng/ml ; the ratio of $^{45}Ca$ release, $1.14{\pm}0.07$). Also $IL-1{\beta}$(1 ng/ml), $IL-1{\alpha}$(1 ng/ml), and $TNF-{\alpha}$(1 ng/ml) resulted in bone resorption(the rations of $^{45}Ca$ release, $1.61{\pm}0.26$, $1.77{\pm}0.03$, $1.20{\pm}0.15$ respectively), but IL-8 did not(the ratio of $^{45}Ca$ release, $0.93{\pm}0.21$). The ratios of $^{45}Ca$ release in response to IL-10(400 ng/ml) and $IFN-{\gamma}$(100 ng/ml) were $1.24{\pm}0.12$ and $1.08{\pm}0.04$ respectively, hence these cytokines inhibited $IL-1{\beta}$(1 ng/ml)-induced bone resorption(the ratio of $^{45}Ca$ release $1.65{\pm}0.24$). While $IL-1{\beta}$(1 ng/ml) increased the number of TRAP positive multinulcleated cells in bone marrow cultures($20{\pm}11$), simultaneously adding $IL-1{\beta}$(1 ng/ml) and IL-10(400 ng/ml) decreased the number of these cells($2{\pm}2$). Nevertheless, IL-10(400 ng/ml) did not affect the IL-6, GM-CSF, and $TNF-{\alpha}$ secretion from $IL-1{\beta}$(1 ng/ml)-activated mouse calvarial cells. From the above results, it may be suggested that IL-10 inhibites $IL-1{\beta}-induced$ osteoclast differntiation and bone resorption. However, the inhibitory effect of IL-10 on the osteoclast formation seems to be mediated not by the reduction of IL-6, GM-CSF, and $TNF-{\alpha}$ production, but by other mechanisms.

• The korean journal of orthodontics
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• v.29 no.3 s.74
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• pp.383-397
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• 1999

Bone is a dynamic tissue which is constantly remodelled by subsequent cycles of bone resorption and formation. Glucocorticoid and vitamine $D_3$ are known as regulating substances in bone metabolism. In vitro experiments using bone tissue, it was suggested that glucocorticoid inhibits bone resorption, whereas the effect of glucocorticoid on bone formation are complex- increasing or decreasing effect. The active form of vitamin $D_3$, 1,25-dihydroxycholecalciferol[1.25-$(OH)_2D_3$], has been reported to stimulate osteoblastic activities including the production of ALP, type I collagen, and osteoclacin. The purpose of this study was to evaluate the effect of admixture of vitamin $D_3$ and dexamethasone, one of glucocorticoids, on osteoblastic cell line(MC3T3-E1). Alkaline phosphatase(ALP) and MTT assay were conducted in the cultivated cells with 1, 10, 100nM/ml of 1,25-$(OH)_2D_3$ and/or 10nM/ml, 100nM/ml, $1{\mu}M/ml$ of dexamethasone. The observed results were as follows. 1. The activity of osteoblastic cells with $1{\mu}M/ml$ of dexamethasone was significantly increased at 1-day cultivation with comparison to control group, but was decreased afterwards. But the activity of ALP was greatest in $1{\mu}M/ml$ of dexamethasone and increased with time lapsed. 2. The activity of osteoblastic cells with vitamin $D_3$ was significantly increased dose-dependently at 1-day cultivation, but was significantly decreased in l00nM/.ml at 2-day cultivation, and was a little increased again at 3-day cultivation. The activity of ALP was increased in 10nM/ml or 100nM/ml at 2-day or 3-day cultivation, and was greatest in 100nM/ml at 3-day cultivation. 3. In case of admixture of dexamethasone and vitamin $D_3$, the cellular activity was decreased in any concentration of vitamin $D_3$ at 2-day cultivation, but was increased again at 3-day cultivation, which was greater than that in control or dexamethasone only group. The activity of ALP was decreased at 1-day cultivation, but was increased in the admixture of 10nM/ml or 100nM/ml of dexamethasone with 100nM/ml of vitamin $D_3$ at 2-day cultivation, and was again decreased at 3-day cultivation.

• Journal of Life Science
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• v.20 no.8
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• pp.1268-1275
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• 2010

This study was carried out to investigate the anti-proliferative activity of solid-state fermented medicinal herbs which include Phellinus baumii. Methanol extracts were prepared from 36 different medicinal herbs and their fermented counterparts. These extracts were used to treat human colorectal HCT116 cell, human embryonic kidney cell HEK-293, pre-adipocyte cell 3T3-L1, and pre-osteoblast cell MC3T3-E1 for 24 hr. At a concentration of 100 ${\mu}g/ml$, the extracts of Amomum villosum, Cnidium officinale Makino, Dendrobium moniliforme, Dictamnus dasycarpus, Diospyros kaki Thunb, Eucommia ulmoides Oliv, Ginkgo biloba L, Magnolia denudata Desrousseaux, Orostachys japonicus, Panax notoginseng, Pharbitis nil Choisy, Polygala tenuifolia and Trichosanthes kirilowii (seed) led to a < 50% decrease in cell proliferation, and mycelium of P. baumii showed a 46.3% decrease in cell proliferation. Meanwhile, the extracts of the 25 fermented herbs showed similar anti-proliferative activities compared to those of individual non-fermented herbs. However, the extracts of the fermented Drynaria fortunei Kunze (1), Lycium chinense Mill (2), Fritillaria thunbergii Miquel (3) and Prunus persica showed increased anti-proliferative activity. The $IC_{50}s$ of (1), (2) and (3) were especially decreased to 28, 85 and 80 ${\mu}g/ml$ from 394, 917 and 149 ${\mu}g/ml$, respectively. Furthermore, the cytotoxicity of the extracts of fermented (1), (2) and (3) against HEK-293, 3T3-L1, and MC3T3-E1was negligible up to 200 ${\mu}g/ml$. These results suggest that solid-state fermentation using the mycellium of P. baumiiproduce potential anti-cancer agents or strengthen the bioactivity of medicinal herbs.

• Journal of Periodontal and Implant Science
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• v.33 no.1
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• pp.27-35
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• 2003

1. 목적 생체재료의 생체친화성을 증진시키고 치유를 촉진하기 위한 목적으로 생체재료의 생화학적 표면개질에 관한 연구가 널리 진행되고 있다. 이와 같은 목적으로 이용되어 온 부착분자에는 아미노산, 펩타이드, 단백질, 효소 및 성장인자들을 들 수 있으며, 이들 분자들을 금속, 골대체물질 및 폴리머와 같은 생체재료의 표면개질에 이용하여 왔다. 이 연구의 목적은 생체적합성이 우수하고 생분해성을 지닌 키토산으로 얇은 막을 제작한 후, 세포외 기질의 구성성분 중 세포부착에 관여하는 RGD 펩타이드를 부착시킨, 표면개질 키토산막의 생물학적 영향을 MG-63 조골양세포를 이용하여 관찰하는 것이다. 2. 방법 2% acetic acid에 키토산 가루를 녹여 만든 2% 키토산 용액으로 24-well 배양접시의 표면을 도포 후 24시간 동안 건조시켜 키토산막을 제작하였다. GRGDS 펩타이드를 cross-linker(EDC, NHS) (Sigma, MO, USA) 용액과 반응시켜서 펩타이드의 카르복실기를 활성화시켰다. 이들을 PBS 완충용액으로 수화시킨 키토산막과 결합시켜 펩타이드의 활성화된 카르복실기와 키토산의 아민기 간에 안정적인 아미드 결합(amide bond)이 형성되도록 하였다. 하루 동안 반응을 일으킨 후 PBS 완충용액과 증류수로 씻어내고 냉동 건조시킴으로써 GRGDS가 결합된 키토산막을 제작하였다. 재료 표면의 화학 성분을 알아보는데 사용되는 방법의 일종인 X-ray photoelectron spectroscopy(XPS) 분석을 통하여 부착분자가 키토산막에 결합된 여부를 확인하였다. GRGDS 펩타이드에 요오드를 결합시킨 후, 이것을 키토산막에 공유 결합시키고 XPS를 통해 요오드가 재료 표면에서 검출되는지를 검사하였다. 요오드가 검출된다면 이것은 키토산막 표면에 실제로 GRGDS 펩타이드가 존재하는 것을 의미하게 된다. 표면개질된 키토산막에 사람조골양세포인 MG-63을 접종하여 이를 실험군으로 하였고, 표면이 개질 되지 않은 키토산막을 대조군으로 하였다. 세포부착의 최적화 농도를 확인하기 위하여 GRGDS를 0.01, 0.05, 0.1, 0.25, 0.5, 1.0mg/ml의 농도로 준비하였다. 배양 후 1일, 7일째에 각 well에서 trypsin EDTA를 이용하여 세포를 분리한 후, 이를 원심 분리하여 세포수측정기를 이용하여 부착 세포의 수를 측정하여 세포의 부착 정도를 비교하였다. 배양 2시간, 24시간 후 주사전자현미경을 이용하여 키토산막에 부착된 세포의 양상을 관찰하였다. 3. 결과 XPS를 통한 표면의 화학 성분 분석 결과 GRGDS 펩타이드를 결합시킨 키토산막에서 요오드가 검출되었으며 펩타이드를 부착하지 않은 대조군에서는 검출되지 않았다. 따라서 cross-linker를 이용한 펩타이드와 키토산막의 공유결합을 확인할 수 있었다. 세포 배양 후 1일째 부착된 세포 수를 측정한 결과 0.1mg/ml 이상의 GRGDS 펩타이드 농도로 공유 결합시킨 키토산막에서 부착 세포 수가 다른 농도에 비해 유의성 있게 많이 관찰되었다. 이 농도 이하에서는 대조군과 실험군간에 세포부착의 유의한 차이가 없었다. 따라서 주사전자현미경을 이용한 부착 세포의 양상에 관한 관찰은 0.1mg/ml 농도의 펩타이드를 이용하였다. 세포 배양 7일째, 부착된 세포 수 측정 결과 GRGDS의 농도에 따른 유의성 있는 차이가 없었으며, 실험군과 대조군간에도 유의성 있는 차이가 없었다. 주사전자현미경 관찰결과 2시간 및 24시간 배양된 실험군 모두에서 별모양의 세포들이 키토산막 표면에 편평하게 잘 부착되어 있으며 많은 위족이 발달된 소견을 보인 반면, 대조군에서는 원형 또는 다각형 모양의 세포들이 실험군에 비해 부착이 덜 되어있는 양상을 보였다. 이 연구를 통하여 기능성 펩타이드를 생체재료의 표면에 공유결합 시키는 방법을 확립할 수 있었으며, RGD 펩타이드의 공유결합으로 표면개질된 키토산막이 조골세포의 부착능을 증진시킬 수 있음을 확인하였다. 표면개질된 생체재료를 소, 중동물에 적용시켜 생체 내에서의 생물학적 영향을 평가할 필요가 있으며, 이 실험의 결과는 향후 다양한 기능성 부착분자를 선발, 고안하여 임플란트용 생체재료의 표면개질에 이용하는 이른바 모방생체재료분야에 널리 활용될 수 있을 것으로 생각된다.

• Journal of Periodontal and Implant Science
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• v.34 no.4
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• pp.781-790
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• 2004

골아세포의 생물학적 기능을 증진시키기 위해 키토산의 표면개질에 대하여 연구하였다. 생체적합성 천연고분자인 키토산은 1차 아미노기를 소유하고 있으므로 적정한 공유결합제를 사용하여 세포성장인자와 같은 생리활성을 지닌 단백질을 키토산의 표면에 고정시킬 수 있다. 본 연구에서는 키토산을 필름형태로 제조하여 세포성장인자 중 형질전환성장인자를 고정하고 골아세포의 부착, 성장 및 분화를 증가시키고자 하였다. 형태전환성장인자의 고정화 효율은 단순한 흡착방법에 비해 높았으며, 표면에 형성된 공유결합은 매우 안정하였다. 골아세포를 배양하여 초기세포부착능에 대한 영향을 연구한 결과, 배양 후 4시간, 1일째, 형질전환성장인자를 고정한 키토산 표면에서 고정하지 않은 키토산의 표면에 비해 더 많은 수의 골아세포가 부착되었고, 더 많이 신장된 부착형태를 보였다. 세포활성정도와 배양 후 4주일째의 칼슘축적량을 측정한 결과, 형질전환성장인자를 고정한 키토산 표면에서 고정하지 않은 키토산의 표면에 비해 더 높았다. 위의 결과는 키토산 표면에 형태전환성장인자의 고정이 성공적으로 이루어졌으며, 또한 실제로 활성이 있는 것이 증명되었다. 위의 연구 결과에서 형질전환성장인자로 고정된 키토산은 골아세포의 초기 부착 및 분화를 촉진시켰음을 알 수 있었던 바 성장인자의 표면고정은 임플란트 및 조직공학용 지지체에도 적용하여 생체적합성과 세포기능을 증진시키는데 이용할 수 있음을 알 수 있었다.

• The korean journal of orthodontics
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• v.32 no.2 s.91
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• pp.129-142
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• 2002

The purpose of this study was to evaluate the effects of caffeine and calcium on the activities of the osteoblastic cell from mouse calvaria. The author cultured osteoblastic cells obtained from the mouse calvaria and were divided into three groups : the caffeine-treated, the calcium-treated and the combine-treated group. In caffeine-treated group, the cell toxicity was measured by MTT assay at 1, 2 and 4 days after treatment of caffeine. In all groups, the densities of the mineralized bone nodules were measured by imaging analyzer after Von Kossa staining. The alkaline phosphotase (ALP) activities were measured at 2, 7, 14, 21 and 28 days and the interleukin-1 ${\beta}$ activities at 48 hours after treatment of caffeine and calcium. The measurements were statistically executed with ANOVA test and the results were as follows. 1. The cellular toxicity of the caffeine increased with the concentration of caffeine during the incubation period. 2. The maximum densities of mineralization were observed at 0.2 mM caffeine-treated group, 1.2 mM calcium-treated group, 0.1 mM caffeine and 1.8 mM calcium-treated group. 3. The activities of ALP were peaked at 14 days at calcium-treated group as no-treated. But, the activities of ALP increased with concentrations of caffeine at caffeine-treated group. At combine-treated group, the act of ALP were peaked at 24 days at 1.2 mM, 1.8 mM calcium-treated group, But decreased at 2.5 mM calcium-treated group. 4. The activites of the IL-1 ${\beta}$ were increased significantly at 0.2 mM caffeine-treated group, 1.8 mM calcium-treated group and 0.1 mM caffeine and 1.8 mM calcium-treated group. But, they were decreased at all groups of high concentration.

• The korean journal of orthodontics
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• v.28 no.2 s.67
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• pp.297-310
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• 1998

The purpose of this study was to investigate the initial tissue change, to repair on the teeth & surrounding tissue under the intrusive orthodontic forces by use of elastic chain, through the microscopic findings. For this study, three young adult mongrel dogs were used, and were divied into three group : the control group was deliveried only casting crown and the experimental group 1 was equipped with energy chain during 1 week and experimental 2 group was deliveried using energy chain during 1 week and 3 weeks observation. All experimental groups and control groups were sacrificed to make the samples for microscopic findings on premolar teeth. All samples were examed and compared the histologic changes through the microscopic with H-E stain. The obtained results were as follows. 1. In hematoxylin-eosin stain of the control group, the periodontal ligament was constant width from apical third to cervical third of the root, and the periodontal fiber arrangement was horizontal or oblique in cervical third, oblique in middle and apical third of the root. 2. In Masson Trichrome stain of the control group, osteoblast and osteoclast appeared in cervical third of root, and bone resorption and new bone formation was observed in middle and apical third of the root. 3. In experimental 1, osteoclasts were increased highly, and hyperemia of blood vessels and new bone formation and bone resorption by reversal line in apical third of the root were seen. PDL width was increased apprarently from crest to apex of the root and more in apical third. 4. In experimental 2, osteoclasts and hyperemia of blood vessels were more increased than control material in apical third of the root. PDL width was increased more than control group in root apex, and was seen less than experimental 1. PDL arrangement was similar to experimental 1 and was mixed only in root apex. Therefore, in premolar intrusion of the young adult dog, there were increased osteoclast, hyperemia and dilation of blood vessel, resorption of alveolar bone and cementum and different arrangement of PDL in initial tissue change. There was not observed complete repair after remove intrusive force.

• The korean journal of orthodontics
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• v.28 no.2 s.67
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• pp.311-327
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• 1998

The purpose of this study was to evaluate the effects of Transforming Growth Factor-${\beta}$ (TGF-${\beta}$) on the viability of human periodontal ligament cells, in-vitro and on the experimental tooth movement in rat, in-vivo. Human periodontal ligaments were cultured from the first premolar tooth extracted for the purpose of the orthodontic treatment. 0.1, 1, 5 and 10ng/m1 of TGF-${\beta}$ was given to the cultured wells, respectively and the viability was evaluated by MTT assay. Twenty Sprague-Dawley rats were divided into 5 experimental groups(4 rats in each) where 100g of force was applied from helical spring across the maxillary incisors. TGF-${\beta}$ was injected via Hamilton syringe into the periodontal ligament at the mesial and the distal surface of a maxillary incisor of 2 rats in each experimental group. Phosphate buffer saline(PBS) was injected in 2 other rats as controls. Experimental groups were sacrificed at 1, 3, 7, 14 and 28 days after force application, respectively. The obtained tissues were evaluated histologically. The obtained results were as follows: 1. The viability of periodontal ligament cells in 0.1ng/ml of TGF-${\beta}$ was not significantly different from that of control at 1-, 2- and 3-day of cultivation. 2. The viability of periodontal ligament cells was significantly increased at 3-day in 1ng/ml or 5ng/ml of TGF-${\beta}$, and at 2-,3-day in 10ng/ml of of TGF-${\beta}$. 3. The zone of hyalinization in periodontal ligament in pressure side was smaller in TGF-${\beta}$ injection group than that in control group at 3-day after the application of experimental force in rat. But no difference was seen after 7-day. 4. Osteoclastic activity and capillary prolieferation in pressure side were greater in TGF-${\beta}$ injection group than that in control group at 3-day to 7-day. 5. Osteoblastic activity and new bone fomation in tension side were greater in TGF-${\beta}$ injection group than that in control group at 3-day to 14-day.

• The Journal of Korean Academy of Prosthodontics
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• v.53 no.3
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• pp.198-206
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• 2015

Purpose: We aimed to investigate the effect of combined various microgrooves and thermal oxidation on the titanium (Ti) and to evaluate various in vitro responses of human periodontal ligament cells (PLCs). Materials and methods: Grade II titanium disks were fabricated. Microgrooves were applied on titanium discs to have $0/0{{\mu}m}$, $15/3.5{{\mu}m}$, $30/10{{\mu}m}$, and $60/10{{\mu}m}$ of respective width/depth by photolithography. Thermal oxidation was performed on the microgrooves of Ti substrata for 3 h at $700^{\circ}C$ in air. The experiments were divided into 3 groups: control group (ST), thermal oxidation group (ST/TO), and combined microgrooves and thermal oxidation group (Gr15-TO, Gr30-TO, Gr60-TO). Surface characterization was performed by field-emission scanning microscopy. Cell adhesion, osteoblastic differentiation, and mineralization were analyzed using the bromodeoxyurdine (BrdU), Alkaline phosphatase (ALP) activity, and extracellular calcium deposition assays, respectively. Statistical analysis was performed using the oneway analysis of variance and Pearson's bivariate correlation analysis (SPSS Version 17.0). Results: In general, the combined microgrooves and thermal oxidation group (Gr15-TO, Gr30-TO, Gr60-TO) showed significantly higher levels compared with the control (ST) or thermal oxidation (ST-TO) groups in the BrdU expression, ALP activity, and extracellular calcium deposition. Gr60-TO group induced highest levels of cell adhesion and osteoblastic differentiation. Conclusion: Within the limitation of this study, we conclude that the Ti surface treatment using combined microgrooves and thermal oxidation is highly effective in inducing the cell adhesion andosteoblastic differentiation. The propose surface is also expected to be effective in inducing rapid and strong osseointegration of Ti oral implants.

• The Journal of Engineering Research
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• v.3 no.1
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• pp.189-197
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• 1998

Hyeroxyapatite(HAp) which has good biocompatibility was made by Wet Chemical Process. The surface of Ti-6Al-4V, coated with HAp by lon Beam Assisted Deposition (IBAD), was treated with 5ppm, 10ppm, 20ppm, and 100ppm of $AgNO_3$ solution. In this Ag impregnation process, $Ca^{2+}$ of HAp was substituted with $Ag^+$ of $AgNO_3$. In this study, the antimicrobial effect and biocompatibility of Ti-6Al-4V alloy which was coated with Ag-HAp were examined. The antimicrobial test was carried out with two kinds of bacteria(P. Aeruginosa, S. Epidermidis), which are highly infectious in a transplanting operation of implant materials. As a result of the test, it was observed that Ti-6Al-4V alloy which was treated by 20ppm of $AgNO_3$ solution has good biocompatibility. In order to observe the antimicrobial mechanism of $Ag^+$, E. coli which is the most common bacterium was treated by Ag-HAp. Then cell morphology of E. coli was observed by the transmission electron microscope(TEM). The destruction of cell wall and cytoplasm of E. coil were observed. A black spot appeared in the cytoplasm was analyzed by energy dispersive analysis X-ray (EDAX) and it showed a small amount of $Ag^+$. Thus, it was proved that $Ag^+$ destroys bacteria effectively and Ti-6Al-4V alloy which was impregnate with Ag ion show antimicrobial effect on infection bacteria.