배양된 사람 치주인대세포와 골수유래간엽줄기세포의 분화에 미치는 법랑기질유도체 (Enamel Matrix Derivative, EMD)의 영향

박상규,주성숙,권용대,최병준,김영란,이백수,Park, Sang-Gyu,Jue, Seong-Suk,Kwon, Yong-Dae,Choi, Byung-Joon,Kim, Young-Ran,Lee, Baek-Soo 대한악안면성형재건외과학회 2009 Maxillofacial Plastic Reconstructive Surgery Vol.31 No.4

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.

고농도 포도당이 뼈모세포와 치주인대세포의 세포자멸사에 미치는 영향에 관한 연구

박성호,주성숙,홍정표,신제원,Park, Sung-Ho,Jue, Seong-Suk,Hong, Jung-Pyo,Shin, Je-Won 대한안면통증구강내과학회 2007 Journal of Oral Medicine and Pain Vol.32 No.4

This experiment was designed to clarify the effect of extracellular glucose on the osteoblasts and periodontal ligament cells. The cells were incubated for 24 and 48 hours with ${\alpha}$-MEM including 1,000 mg/L (control group) and 4,500 mg/L (experimental group) of glucose. Then, the expressions of caspase-3, p38 MAPK, JNK-1, and ERK-1 were examined using Elisa assay and Western blot. The results were as follows: 1. The expression of caspase-3 and p38 MAPK was increased by the high extracellular glucose in both cells. 2. The expression of caspase-3 and p38 MAPK was increased greatly in the periodontal ligament cells than the E1 cells by the high extracellular glucose. 3. The expression of JNK-1 was increased by the high extracellular glucose in both cells. 4. The expression of ERK-1 was not changed by the high extracellular glucose in both cells. These results suggest that extracellular glucose at high concentrations may inhibit the periodontal regeneration process increasing cellular apoptosis. And p38 MAPK and JNK-1 pathway may be the most responsible intracellular pathway rather than ERK-1.

고농도 포도당이 사람 치주인대세포의 Integrin과 Cathepsin 발현에 미치는 영향에 관한 연구

김방수,신제원,홍정표,주성숙,Kim, Bang-Soo,Shin, Je-Won,Hong, Jung-Pyo,Jue, Seong-Suk 대한안면통증구강내과학회 2008 Journal of Oral Medicine and Pain Vol.33 No.1

This experiment was designed to clarify the effect of high concentrations of extracellular glucose on the periodontal ligament cells. The cells were incubated for 24 and 48 hours with ${\alpha}-MEM$ including 1,000 mg/L(control group) and 4,500 mg/L(experimental group) of glucose. Then, the expressions of ${\alpha}5$ integrin, cathepsin-B and -L were examined using RT-PCR method. The results were as follows: 1. ${\alpha}5$ integrin expression was increased after incubation in high glucose media. 2. Cathepsin-B expression was increased after the 24-H incubation with high glucose media, but was decreased after the 48-H incubation. 3. The expression of cathepsin-L was decreased by the high glucose media. Theses results suggest that extracellular glucose at high concentration may be attributed to delayed wound healing in diabetes patients through increase in ${\alpha}5$ integrin and decrease in cathepsins, which lead to accumulation of extracellular matrices and adhesion molecules.

수종의 골이식재를 이용한 유도재생골의 조직학적 평가

유호철,박준봉,권영혁,허익,정종혁,주성숙,Ryu, Ho-Chul,Park, Joon-Bong,Kwon, Young-Hyuk,Herr, Yeek,Chung, Jong-Hyuk,Jue, Seong-Suk 대한치주과학회 2006 Journal of Periodontal & Implant Science Vol.36 No.2

This study was performed to evaluate the effect of bone graft materials including demineralized freeze-dried bone, freeze-dried bone, deproteinized bovine bone on space-making capacity and bone formation in guided bone regeneration with titanium reinforced ePTFE membrane(TR-ePTFE). Adult male rabbits(mean BW 2kg) were used in this study. Intramarrow penetration defects were surgically created with round bur on calvaria of rabbits. TR-ePTFE membrane was adapted to calvarial defect and bone graft materials were placed. Animals were sacrificed at 2, 8, 12 weeks after surgery. Non-decalcified specimens were processed for histologic analysis and prepared with Villaneuva bone stain. The results of this study were as follows: 1. TR-ePTFE membrane was biocompatible and capable of maintaining the space-making. 2. Tissue integration was not good at TR-ePTFE membrane. Fixation was not enough. so, wound stabilization was not good. 3. In animals using deproteinized bovine bone, demineralized freeze-dried bone, bone formation was little. 4. In animals using freeze-dried bone, bone formation was better. Within the above results, bone formation may be inhibited when wound stabilizafion was not good.

무기인산염이 골재생에 미치는 효과에 대한 조직계측학적인 연구

이영석,박준봉,권영혁,허익,정종혁,주성숙,Lee, Young-Seok,Park, Joon-Bong,Kwon, Young-Hyuk,Herr, Yeek,Chung, Jong-Hyuk,Jue, Seong-Suk 대한치주과학회 2007 Journal of Periodontal & Implant Science Vol.37 No.1

In this study, author examined the effect of the concentration of the inorganic polyphosphate on the process of the bone regeneration by using the 6 weeks old rabbit with the weight of 2.0kg in average. we performed the experiment by using TR-eITFE membrane filled with collagen immersed with 1%, 2%, and 4% of inorganic polyphosphate, respectively, after removing the proper sized cort-ical bones from the calvaria of rabbit. The experimental results were compared with the one of the following four groups: The control group for membrane only, experimental group I for membrane filled with collagen im-mersed with 1% of inorganic polyphosphate, experimental group II for membrane filled with collagen immerse with 2% of inorganic polyphosphate, experimental group III for membrane filled with colla-gen immersed with 4% of inorganic polyphosphate. The fragments of the tissue with membrane were obtained from each group of the sacrificed rab-bits for 4 or 8 weeks sustained after surgery, were then prestained and coated. New bone formation was assessed by histomorphometric and statistical analysis. We may draw the conclusions from these experiments as following: 1. Collagen was an excellent carrier with a minimal inflammatory reaction and sustaining the form. 2. The sample of the 8th week group has shown the best bone regeneration compared with the cases of all groups including the control group. 3. The samples of collagen immersed with 2% and 4% of inorganic polyphosphate have shown more bone regeneration relative to the sample of the 1% inorganic polyphosphate. 4. The new bone regeneration was shown actively in the group for membrane filled with collagen immersed with 4% of inorganic polyphosphate. With above results, it is strongly suggested the use of inorganic polyphosphate with vehicle under TR-eITFE membrane.

Fourier Analysis of Maxillary Dental Arch Forms

Michiko Nakatsuka(나카츠카 미치코),Tohru Tsujibayashi(쯔지바야시 토오루),Shunji Kumabe(쿠마베 슌지),Seong-Suk Jue(주성숙),Je-Won Shin(신제원),Yasutomo Iwai-Liao(이와이 야스토모) 대한체질인류학회 2008 해부·생물인류학 (Anat Biol Anthropol) Vol.21 No.2

사람 위이틀활의 기준점, 기준선, 기준각을 이용한 연구를 통하여 전치부의 형태와 이틀활의 형태 사이에 유의한 관계가 있음을 알게 되었다. 이에 사람 위이틀활 형태 결정에 영향을 미치는 필수요소들 사이의 관계를 밝히기 위하여 사람 위이틀활 모델 62개의 형태를 푸리에 급수를 사용하여 비교 검토하였다. 푸리에 급수의 상수 및 제 1 주파수의 진폭은 각 형태간에 통계적으로 유의성 있는 차이를 관찰할 수 없었지만 제 2 주파수에서 제 4 주파수까지의 진폭에서는 각 형태간에 유의성 있는 차이를 관찰할 수 있었다. 또한 상수는 앞니부분의 폭경, 이틀활의 폭경 및 이틀활의 길이와 각각 정비례 관계를 보였다. 이상의 결과로 보아, 사람 위이틀활의 형태는 앞니부분으로부터 어금니부분으로의 이행부의 형태가 관여하고 있는 것으로 생각된다. Our previous principal component analysis conducting on reference points, lines and angles, and a vector-developed polar coordinate system has elucidated that the components of eigenvectors had positive relationships in the curvature of anterior teeth segment, between the protrusion of canines and degree of arch roundness, and in the length-to-width ratio of 62 maxillary dentitions, which were preliminarily classified with reference to the conventional Thompson’s morphological descriptions for dental arch forms. In the present study on morphological characters of the maxillary dentitions, we conducted a Fourier analysis on the previously obtained data. We observed that the amplitude of 2<SUP>nd</SUP>, 3<SUP>rd</SUP> and 4<SUP>th</SUP> Fourier harmonics were closely correlated with the length-to-width ratio, curvature of the anterior teeth segment, and the curvilinear contour of maxillary dental arches. In addition, the relationships between previously estimated data and the constant value and the amplitude of the Fourier series were examined by analysis of correlation coefficients(p<0.01). The results of the present study suggest that the morphology of maxillary dentitions consists of three essentials-the length-to-width ratio, the curvature of anterior teeth and the curvilinear contour of dental arches.

유성견 정중구개봉합 확장후 봉합 재형성 과정에 대한 조직형태학 및 면역조직화학적 연구

오인섭,주성숙,신제원 慶熙大學校 齒科大學 1995 慶熙齒大論文集 Vol.17 No.1

Sutural regeneration requires the formation of new connective tissues comprised of various extracellular matrix components. The present study investigated the formation and distribution of the major ECM components such as fibronectin (FN), type I collagen (CI) during expanded sutural regeneration. The expansion were created on median palatine suture of young adult dog_ At 0 and 60 days after expansion, dogs were sacrificed by perfusion with 10% neutral buffered formalin The sutural area were cut, demineralized, dehydrated and embedded in paraffin. Immunostaining of FN and CI was achieved by the avidin-biotin complex method. The results were as follows 1. After expansion, much cellular infiltration was observed in the suture. The orientation of the fiber bundles become obscured. 2. At 60 days after expansion, the increase of the fibrous components were seen between both the sutural surfaces. The new bone formation were seen at the regenerated sutural surface. 3. After expansion, intensive staining for FN, CI was observed especially around blood vessels as well as inflammatory cells. This proteins also expressed later in the collagenous bundles. 4. At 60 days after expansion, intensive staining for FN and CI was observed in osteoid, newly formed connective tissue, and its attachment site to the sutural surface. 5. These results suggest that sutural regeneration is achieved through the cell differentiation and formation of Fh and CI by the cells.

치은섬유모세포 및 치주인대세포에 의한 교원질 수축에 관한 연구

홍영안,김현수,주성숙,신제원 慶熙大學校 齒科大學 1995 慶熙齒大論文集 Vol.17 No.2

The purpose of this in vitro study was to evaluate contraction of the human gingival and periodontal ligament fibroblast in the collagen matrix. The cell cultures were prepared using human gingiva and tooth. The collagen gels were observed daily with an inverted microscope and their area was determined by measuring the diameters of the collagen gel. The configurations of human gingival and periodontal ligament fibroblasts were done by SEM observation. The results as follows; 1. The contraction was found to be proportional to seeding cell without seeding cells, the contraction was not occured. 2. Periodontal ligament fibroblast was shown more fast contraction than gingival fibroblast. 3. The collagen matrix was translucent initially, but with fully contraction, the structure become opaque. 4. The collagen matrix with fibroblast were aggregated to form bundles and reorganized collagen fiber.

발치와의 치유과정에서 교원질 및 비교원단백질의 분포에 관한 면역조직화학적 연구

오화탁,김현수,주성숙,신제원 慶熙大學校 齒科大學 1995 慶熙齒大論文集 Vol.17 No.2

During tooth socket healing, the coagulum in the socket is replaced by fibrous connective tissue which undergoes mineralization and eventually becomes bone. Using this model, the healing process of the tooth socket and the role of type I collagen(CI), fibronectin(FN), bone sialoprotein(BSP), and osteopontin(OP) in the process were studied. Thirty Sprague-Dawley rats weighing 130-150gm were fed fi -aminopropionitcille for 5days before extraction of the first maxillary molars, and sacrified by perfusion with 4% paraformaldehyde at 1, 3, 5, 7, and 10days after tooth extraction. The socket and surrounding tissues were cut, demineralized, dehydrated and embedded in paraffin. For morphological observation, the sections were stained by Azan. Immunostaining of the ECM components was achieved by the avidin-biotin complex method. The results as follows; 1. Morphology of the tooth socket 1day after tooth extraction. The socket was filled with blood coagulum which was composed of densly aggregated red blood cells, platelets and fibrin network. 2. Morphology of the tooth socket 3days after tooth exyraction. A large portion of the coagulum was replaced by fibrous connective tissue contains a large number of fibroblasts, come from periphery of the socket. 3. At 5days, the newly formed bone demonstrated the highest proliferation activity. At 7days, the soclet was occupied with new bone. 4. Type I collagen was observed in the newly formed connective tissue and around the new bone. fiber. 5. Intensive staining for fibronectin was observed in granulation tissue, especially around blood vessels as well as inflammatory cells. 6. Strong immunostaining for bone sialoprotein and osteopontin was found in osteoblasts and new bone, while weaker staining was observed on dense connective tissue. 7. These data suggest that collagen and noncollagen proteins (FN, BSP, OP) an important role daring socket healing.

치아재식후 치유과정에서 fibronectin의 분포에 관한 면?조직화학적 연구

한휘철,김현수,주성숙,신제원 慶熙大學校 齒科大學 1995 慶熙齒大論文集 Vol.17 No.2

Periodontalhealing requires the formation of new periodontal tissues comprised of various extracellular components. The present study investigated the formation and distribution of the fibronectin FN), a major adhesive glycoprotein. Rats weighing 130`150 gm were fed 0.4% R -aminopropionitrile for 5 days to achieve gentle tooth extraction. The maxillary first molars were extracted under anesthesia with pentobarbital, washed in sterile distilled water, treated with bacterial collagenase to digest collagen fibers on the root surfaces. After washing in water overnight, the mesial root surfaces were demineralized by application of citric acid, washed, dried and stored at 4 t. Immediately after tooth extraction and bleeding control, the treated molars extracted previously from other rats were reimplanted. Ten animals each were sacrificed 45 min, 1, 3, 6, 10 days after reimplantation of teeth by intracardiac perfusion with 4% paraformaldehyde. The replanted molars and surrounding tissues were cut, demineralized, dehydrated and embedded in paraffin. Imrnunostaining of the FN was achieved by the avidin-biotin complex method. The results as follows; 1. The control group showed gradual healing from 1 day and complete regeneration of the periodontium at 10 days. 2. At early healing stage, intense staining for FN was observed in the newly formed connective tissues, granulation tissue, especially around blood vessels as well as inflammatory cells. 3. At later healing stage, FN was expressed later in the periodontal ligament, cementum, osteoid, and newly formed connective tissue and its attachment site to the root surface. 4. Strong immunolabeling for FN was found in cementoblasts, osteoblasts, and new bone and cementum, which weaken staining was observed on the dentin and alveolar bone. 5. These results suggest that the healing after replantation is achieved through a series of cell differentiation and formation of FN.