• 대한치과보철학회지
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• 제54권3호
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• pp.203-209
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• 2016

목적: 치주조직재생을 위해서는 치주인대세포의 증식 및 이주를 촉진시키는 것이 필수적이나 현재까지 이것을 만족시키는 재생술식은 없다. 최근 법랑기질유도체(enamel matrix derivatives)가 치주조직 재생 술식에 적용되고 있으나 그 기전에 대해서는 완전히 알려지지 않았다. 따라서 본 연구의 목적은 법랑기질유도체가 치주인대세포의 증식과 성장인자 발현에 미치는 영향에 대하여 알아보고자 함이다. 재료 및 방법: 건강한 성인의 제 3 대구치로부터 치주인대세포를 추출한 후, 법랑기질유도체(Emdogain (Biora, Malmo, Sweden))의 농도가 각각 0, 12.5, 25, 50, 100, and $200{\mu}g/mL$인 배지에서 배양시켰다. 치주인대 세포증식과 알칼리성 인산분해효소(alkaline phosphatase) 활성도를 측정하고, 발현되는 성장인자를 평가하였다. 결과: 치주인대세포의 세포증식은 $25{\mu}g/mL$이상의 농도의 법랑기질유도체를 첨가한 군에서, 알칼리성 인산분해효소 활성도는 $50{\mu}g/mL$의 법랑기질유도체를 첨가한 군에서 각각 유의하게 증가하였고, $50{\mu}g/mL$의 법랑기질유도체를 첨가한 군에서 치주인대세포의 VEGF (vascular endothelial growth factor)와 TGF(transforming growth factor)-${\beta}$의 발현이 유의하게 증가하였다. 결론: 법랑기질 유도체는 인간 치주인대세포의 세포증식과 알칼리성 인산분해효소 활성을 증진시키고, VEGF와 TGF-${\beta}$등 성장인자의 발현을 촉진함으로써 치주조직 재생에 기여할 수 있을 것이다.

• 대한치주과학회:학술대회논문집
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• 대한치주과학회 2003년도 제43회 종합학술대회 연제초록
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• pp.43-44
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• 2003

• Journal of Periodontal and Implant Science
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• 제35권2호
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• pp.321-333
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• 2005

1. 목적 in vitro 상에서 법랑기질유도체가 치주인대섬유아세포, 불멸화 조골세포와 백악질 유래세포의 증식과 유전자 발현에 미치는 영향을 알아보고자 하였다. 2. 연구방법 및 재료 <세포증식 연구> 교정을 목적으로 발거한 치아에서 분리, 배양한 치주인대섬유아세포와 백악질유래세포, 그리고 $SaOs_2$ 세포를 이용하였다. 법랑기질유도체가 세포 증식에 미치는 영향을 알아보기 위해, 35 mm Petri dish에 dish 당 $5{\times}10^3$ 개의 세포를 접종하였다. 대조군은 1% 항생제와 10% FBS를 포함한 DMEM 배지를 이용했고, 5mM 초산을 첨가한 군과 첨가하지 않은 두 개의 대조군이 이용되었다. 실험군은 100 ${\mu}g/ml$의 법랑기질유도제를 첨가한 군과 100 ${\mu}g/ml$의 법랑기질유도체와 5 mM의 초산을 첨가한 2개의 실험군이 이용되었다. 각 군은 세 개의 배양접시에 행해졌고, 1, 3, 8일에 세포의 수를 각각 측정하였다. 결과는 repeated measures ANOVA로 통계 처리하였다. <유전자 발현 연구> 각 세포의 형질 특성을 알아보기 위해 RT-PCR을 실시하여 조골세포 분화 표식자와 연관된 Human collagen type I(COL I), human osteopontin(OP), human osteocalcin(OC), human alkaline phosphatase(ALP)와 human bone sialoprotein(BSP)의 mRNA 발현을 실험 1, 3, 8일에 걸쳐, 세 군의 차이를 비교 관찰하였다. 3. 결과 <세포증식 연구> 치주인대세포와 백악질유래세포, 그리고 $SaOs_2$ 세포의 증식은 법랑기질유도체에 의해 영향을 받지 않았다. 대조군과 초산이 포함된 대조군 그리고 법랑기질유도체와 초산이 포함된 실험군에서 유의할 만한 세포 수의 차이가 실험 기간 1, 3, 8일에 걸쳐 나타나지 않았다(p<0.05). <유전자 발현 연구> ALP와 COL I은 세 군의 세포에서 모두 발현되었고, 발현 정도는 EMD에 영향을 받지 않았다. OC은 세 군에서 모두 비교적 약하게 발현되었고, 특히 $SaOs_2$ cell과 백악질유래세포에서 약하게 발현되었다. EMD는 OC의 발현정도를 약하게 하였다. OP은 백악질유래세포에서 1, 3, 8일에 걸쳐 EMD 유무에 관련 없이 발현되지 않았다. 그러나 치주인대세포와 $SaOs_2$ cell에서는 강하게 발현되었다. BSP는 치주인대세포와 $SaOs_2$ cell에서 1, 3, 8일에 걸쳐 비교적 고르게 발현되었다. EMD 배지에서 배양된 백악질유래세포는 8일에는 BSP가 발현되지 않았다. 4 결론 이번 실험 결과에 의하면 법랑기질유도체는 치주인대세포, 불멸화 조골세포와 백악질 유래세포의 증식에 있어 유의성 있는 효과를 나타내지 않았다. 그러나, 유전자 발현에 있어서는, 치주인대세포와 백악질유래세포, 그리고 $SaOs_2$ 세포 모두에서 OC mRNA의 발현을 억제하는 효과를 나타내었다. EMD는 세포의 증식에는 영향을 미치지 않지만, 유전자 발현에 있어 일부 영향을 미치는 것으로 보인다. 법랑기질유도체가 세포의 증식과 유전자 발현에 미치는 영향은 배양된 세포의 형질특성, 배양환경, 배양일수 등에 따라 달라질 수 있다. 그러므로 법랑기질유도체가 in vitro 상에서 세포에 미치는 영향은 보다 정량화된 연구가 필요하다.

• 대한치주과학회:학술대회논문집
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• 대한치주과학회 2004년도 제44회 종합학술대회 연제초록
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• pp.149-150
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• 2004

• 대한치과의사협회지
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• 제55권3호
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• pp.230-239
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• 2017

Root coverage can be an efficacious treatment not only in aspect of dental symptoms like root caries and hypersensitivity, but also in aspect of esthetic problem. There are several predictable methods for gaining a connective tissue attachment to an exposed root surface. Among them, this case report will deal with three cases using enamel matrix derivative and connective tissue graft to recover esthetic and physiological periodontal environment and its form. This case report deals with three patients with gingival recession which has occurred by different causes. They were treated with de-epithelialized graft accompanied by application of enamel matrix derivative. 6-12 months later, all three patients showed considerable root coverage and clinically stable condition of healing. In conclusion, within the limitation of this study, de-epithelialized connective tissue graft accompanied by application of enamel matrix derivative shows stable and clinical acceptable results in aspect of root coverage.

• Journal of Periodontal and Implant Science
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• 제38권4호
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• pp.679-690
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• 2008

Purpose: The aim of this study was to investigate the effects of EMD on demineralized root surface using human periodontal ligament cells and compare the effects of root conditioning materials(tetracycline(TCN), EDTA). Material and Methods: Dentin slices were prepared from the extracted teeth and demineralized with TCN and EDTA. Demineralized dentin slices were incubated at culture plate with 25, 50 and $100{\mu}g/ml$ concentration of EMD. Cell attachment, alkaline phosphatase activity test, protein synthesis assay and scanning electronic microscopic examination were done. Results: Cells were attached significantly higher in EMD treated group at 7 and 14 days. Cell numbers were significantly higher in EMD treated group. Alkaline phosphatase activity was significantly higher in EMD treated group at 7 and 14 days. Protein synthesis was significantly higher in EMD treated group at 7 and 14 days. Conclusion: Enamel matrix derivatives enhance the biologic activities of human periodontal ligament cells on demineralized root surface and its effects are dependent on the concentration of EMD.

• Journal of Periodontal and Implant Science
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• 제34권2호
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• pp.393-410
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• 2004

The purpose of this study was to investigate effect of enamel matrix derivative on guided bone regeneration with intramarrow penetration in rabbits. Eight adult male rabbits (mean BW 2Kg) were used in this study. Intramarrow penetration defects were surgically created with round carbide bur(HP long #6) on calvaria of rabbits. Defects were assigned to the control group grafted with mixture of the same quantity of demineralized freeze-dried bone allograft and deproteinized bovine bone mineral. Then, guided bone regeneration was carried out using resorbable membrane and suture. Enamel matrix derivative applied to defects was assigned to the test group. And treated as same manners as the control group. At 1, 2, 3 and 8 weeks after the surgery, animals were sacrificed, specimens were obtained and stained with Hematoxylin-Eosin for light microscopic evaluation. The results of this study were as follows : 1. At 1, 2 and 3 weeks, no differences were observed between the control group and the test group in the aspect of bone formation around bone graft. 2. Proliferation of blood capillary was faster in the test group than in the control group. 3. Bone regeneration in intramarrow penetration was faster in the test group than in the control group. 4. At 8 weeks, new osteoid tissue formation around bone graft was more prominent in the test group than in the control group. From the above results, enamel matrix derivative might be considered as the osteopromotion material and effective in the guided bone regeneration with intramarrow penetration.

• Journal of Periodontal and Implant Science
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• 제33권3호
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• pp.359-372
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• 2003

Among objectives of periodontal therapy. the principal one is the morphological and functional reconstruction of lost periodontal supporting tissues. This includes de novo formation of connective tissue attachment and the regrowth of alveolar bone. The use of enamel matrix derivative(EMD) may be a suitable means of regeneration new periodontal attachment in the infrabony defects. Implant used to replace lost tooth but, implantitis occurred after installation. The purpose of this study was to investigate the effects of EMD on differentiation and growth of osteoblast in titanium disc. Twentyfive millimeter diameter and 1mm thick Ti disc which was coated 25, 50, 100, 200${\mu}g$/ml of EMD(Emdogain(R)) used as experimental group, 25, 50, 100, 200ng/d of rhBMP-2 as positive control group, and no coat as negative control group. A human osteosarcoma cell line Saos-2 was cultured in Ti disc and cell proliferation and Alkaline phosphatase (ALP) activity were measured at 1 and 6 days. PCR was performed at 2 and 8 hours. Semi-quantitative RT-PCR for mRNA expressions of various osteoblastic differentiation markers -type I collagen, ALP, osteopontin, and bone sialoprotein - were performed at appropriate concentrations based upon the results of MTT and ALP assay. Cultured cell-disc complexes were prepared for scanning electron microscopy (SEM) at 2 hour. Data were analyzed using Mann-Whitney and repeated- measures 1-way analysis of variance(SPSS software version 10,SPSS. Chicago. IL). After culture, there was more osteoblast in EMD100${\mu}g$/ml than in EMD50, 200${\mu}g$/ml on day 6. There was significant difference in experimental and positive control group compared control group, as times go by(1 and 6 days). Alkaline phosphatase activity was different significantly in EMD100, 200${\mu}g$/ml and BMP100, 200${\mu}g$/ml on day 6. The results of reverse transcriptase-polymerase chain reaction (RT-PCR) showed that expression of mRNA for ALPase, collagen type I, osteopontin. hone sialoprotein and BMP-2 was detected at 2 hour and 8 hour in EMI 200${\mu}g$/ml subgroup and BMP100ng/ml subgroup. The results of this study suggest that application of enamel matrix derivative on osteoblast attached to titanium surface facilitate the expression of bone specific protein and the differentiation and growth of osteoblast.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제31권4호
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• pp.281-286
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• 2009

Introduction: Enamel matrix derivative (EMD) is a protein which is secreted by Hertwig root sheath and plays a major role in the formation of cementum and attachment of peridontium. Several studies have shown that EMD promoted the proliferation and differentiation of preosteoblasts, osteoblasts and periodontal ligament cells in vitro: however, reports showing the inhibition of osteogenic differentiation by EMD also existed. This study was designed to simultaneously evaluate the effect of EMD on the two cell lines (human mesenchymal stem cells: hMSC, human periodontal ligament derived fibroblasts: hPDLCs) by means of quantitative analysis of some bone related matrices (Alkaline phosphatase : ALP, osteopontin ; OPN, osteocalcin ; OC). Materials and Methods: hMSCs and hPDLCs were expanded and cells in the 4${\sim}$6 passages were adopted to use. hMSc and hPDLCs were cultured during 1,2,7, and 14 days with 0, 50 and 100 ${\mu}g/ml$ of EMD, respectively. ALP activity was assessed by SensoLyte ALP kit and expressed as values of the relative optical density. Among the matrix proteins of the bony tissue, OC and OPN were assessed and quantification of these proteins was evaluated by means of human OC immunoassay kit and human OPN assay kit, respectively. Results: ALP activity maintained without EMD at $1,2^{nd}$ day. The activity increased at $7^{th}$ day but decreased at $14^{th}$ day. EMD increased the activity at $14^{th}$ day in the hPDLCs culture. In the hMSCs, rapid decrease was noted in $7^{th}$ and $14^{th}$ days without regard to EMD concentrations. Regarding the OPN synthesis in hPDLCs, marked decrease of OPN was noted after EMD application. Gradual decrease tendency of OPN was shown over time. In hMSCs, marked decrease of OPN was also noted after EMD application. Overall concentration of OPN was relatively consistent over time than that in hPDLCs. Regarding the OC synthesis, in both of hPDLCs and hMSCs, inhibition of OC formation was noted after EMD application in the early stages but EMD exerted minimal effect at the later stages. Conclusion: In this experimental condition, EMD seemed to play an inhibitory role during the differentiation of hMSCs and hPDLCs in the context of OC and OPN formation. In the periodontium, there are many kinds of cells contributing to the regeneration of oral tissue. EMD enhanced ALP activity in hPDLCs rather than in hMSCs and this may imply that EMD has a positive effect on the differentiation of cementoblasts compared with the effect on hMSCs. The result of our research was consistent with recent studies in which the authors showed the inhibitory effect of EMD in terms of the differentiation of mineral colony forming cells in vitro. This in vitro study may not stand for all the charateristics of EMD; thus, further studies involving many other bone matrices and cellular attachment will be necessary.