• Molecules and Cells
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• 제39권11호
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• pp.790-796
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• 2016

Dental pulp is a highly vascularized tissue requiring adequate blood supply for successful regeneration. In this study, we investigated the functional role of stem cells from human exfoliated deciduous teeth (SHEDs) as a perivascular source for in vivo formation of vessel-like structures. Primarily isolated SHEDs showed mesenchymal stem cell (MSC)-like characteristics including the expression of surface antigens and in vitro osteogenic and adipogenic differentiation potentials. Moreover, SHEDs were positive for NG2, ${\alpha}$-smooth muscle actin (SMA), platelet-derived growth factor receptor beta ($PDGFR{\beta}$), and CD146 as pericyte markers. To prove feasibility of SHEDs as perivascular source, SHEDs were transplanted into immunodeficient mouse using Matrigel with or without human umbilical vein endothelial cells (HUVECs). Transplantation of SHEDs alone or HUVECs alone resulted in no formation of vessel-like structures with enough red blood cells. However, when SHEDs and HUVECs were transplanted together, extensive vessel-like structures were formed. The presence of murine erythrocytes within lumens suggested the formation of anastomoses between newly formed vessel-like structures in Matrigel plug and the host circulatory system. To understand underlying mechanisms of in vivo angiogenesis, the expression of angiogenic cytokine and chemokine, their receptors, and MMPs was compared between SHEDs and HUVECs. SHEDs showed higher expression of1VEGF, SDF-$1{\alpha}$, and $PDGFR{\beta}$ than HUVECs. On the contrary, HUVECs showed higher expression of VEGF receptors, CXCR4, and PDGF-BB than SHEDs. This differential expression pattern suggested reciprocal interactions between SHEDs and HUVECs and their involvement during in vivo angiogenesis. In conclusion, SHEDs could be a feasible source of perivascular cells for in vivo angiogenesis.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제28권6호
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• pp.520-530
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• 2006

Undifferentiated mesencymal stem cells(UMSCs) have been thought to be multipotent cells that can replicate as undifferentiated cells and that have the potential to differentiate into lineages of mesenchymal tissue including the bone, cartilage, fat, tendon, muscle, and marrow stroma. It can be used to sinus lifting, Guided bone regeneration, other bone graft in dental part. The purpose of this study is to evaluate the effect of mesencymal stem cells on sinus augmentation with autogenous bone, fibrin glue mixture in a rabbit model. 8 New Zealand white rabbits were divided randomly into 4 groups based on their time of sacrifice(1, 2, 4 and 8 weeks). First, undifferentiated mesenchymal stem cells were isolated from iliac crest marrow of rabbits and expanded in vitro. cell culture was performed in accordance with the technique described by Tsutsumi et al. In the present study, The animals were sacrificed at 1, 2, 4 and 8 weeks after transplantation, and the bone formation ability of each sides was evaluated clinically, radiologically, histologically and histomorphologically. According to the histological observations, Stem cell group showed integrated graft bone with host bone from sinus wall. At 2 and 4weeks, It showed active newly formed bone and neovascularization. At 8 weeks, lamella bone was observed in sinus graft material area. Radiologically, autobone with stem cell showed more radiopaque than autobone without stemcell. there were significant differences in bone volume between 2 and 4 weeks (p<0.05). In summary, the autobone with stem cells had well-formed, newly formed bone and neovasculization, compared with the autobone without stem cells (esp. 2 weeks and 4 weeks) The findings of this experimental study indicate that the use of a mixture of mesenchymal stem cell yielded good results in osteogenesis and bone volume comparable with that achieved by autogenous bone. Therefore, this application of this promising new sinus floor elevation method for implants with tissue engineering technology deserves further study.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제29권2호
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• pp.113-122
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• 2007

Purpose: Platelet-rich plasma (PRP) is known to accelerate and/or enhance hard and soft tissue healing and regeneration. As such, PRP has been used in various clinical fields of surgery. Recently there have been several attempts to use PRP in the field of tissue engineering. However, some controversies still exist on exact mechanism and benefits of PRP. Therefore various animal experiments are necessary to reveal the effect of the PRP. However, even if animal experiment is performed, the efficacy of the experiment could not be validated due to absence of an animal PRP model. The purpose of this study is to establish rat PRP model by comparing several PRP fabricating methods, and to assay growth factor concentration in the PRP. Materials and methods: Rat blood samples were collected from nine SD rat (body weight: 600-800g). PRP was prepared using three different PRP fabricating methods according to previously reported literatures. (Method 1: 800 rpm, 15 minute, single centrifuge; Method 2: 1000 rpm, 10 minute, double centrifuge; Method 3: 3000 rpm, 4min and 2500 rpm, 8 min, double centrifuge). Platelet counts were evaluated in an automated machine before and after PRP fabrications. In terms of growth factor assay, prepared PRP were activated with 100 unit thrombin and 10% calcium chloride. Growth factor (PDGF-BB, VEGF) concentrations on incubation time were determined by sandwich-ELISA technique. Results: An average of 3ml (via infraorbital venous plexus) to 15ml (via celiac axis) the rat blood could be collected. By using Method 3 (3000 rpm, 4 min and 2500 rpm, 8 min, double centrifugation), around 1.5ml of PRP could be prepared. This method allowed us to concentrate platelet 3.77-fold on average. PDGF-BB concentration (mean, 1942.10 pg/ml after 1 hour incubation) and VEGF concentration (mean, 952.71 pg/ml after 1 hour incubation) in activated PRP were higher than those in untreated blood. Also PDGF-BB showed constant concentration during 4-hour incubation, while VEGF concentration was decreased after 1 hour. Conclusion: Total 11,000 g minute separation and condensation double centrifuge method can produce efficient platelet-rich plasma. Platelet-rich plasma activated with thrombin has showed higher concentrations of growth factors such as PDGF-BB and VEGF, compared to the control group. Platelet-rich plasma model in a rat model was confirmed in this study.

• Journal of Periodontal and Implant Science
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• 제39권sup2호
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• pp.279-286
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• 2009

Purpose: Gene therapy (ex vivo) has recently been used as a means of delivering bone morphogenetic proteins (BMPs) to sites of tissue regeneration. In the present study, we investigated the effect of co-transduction of adenoviruses expressing BMP-2 and BMP-7 on osteogenesisof C2C12 cells in vitro. Methods: A replication-defective human adenovirus 5 (Ad5) containing a cDNA for BMPs in the E1 region of the virus (Ad5BMP-2 and Ad5BMP-7) was constructed by in vivo homologous recombination. Functional activity of Ad5BMP-2 and Ad5BMP-7 were evaluated in mouse stromal cells (W20-17cells). C2C12 cells are transduced with various MOI (multiplicity of infection) of Ad5BMP-2 and Ad5BMP-7 to assess most effective and stable titer. Based on this result, C2C12 cells were transduced with Ad5BMP-2 and Ad5BMP-7 alone or by combination. BMPs expression, alkaline phosphatase (ALPase) activity, cell proliferation, and mineralization were assessed. Results: Ad5BMP-2 and Ad5BMP-7 are successfully transduced to W20-17 cells, and secreted BMPs stimulated cell differentiation. Also, C2C12 cells transduced with Ad5BMPs showed expression of BMPs and increased ALPaseactivity. In all groups, cell proliferation was observed over times. At 7days, cells co-transduced with Ad5BMP-2 and Ad5BMP-7 showed lower proliferation than the others. C2C12 cells co-transduced with Ad5BMP-2 and Ad5BMP-7 had greater ALPaseactivity than that would be predicted if effect of individual Ad5BMPs were additive. Little mineralized nodule formation was detected in cells transduced with individual Ad5BMPs. In contrast, Ad5BMP-2 and Ad5BMP-7 combination stimulated mineralization after culturing for 10 days in mineralizing medium. Conclusions: Present study demonstrated that adenoviruses expressing BMPs gene successfully produced BMPs protein and these BMPs stimulated cells to be differentiated into osteoblastic cells. In addition, the osteogenic activity of Ad5BMPs can be synergistically increased by co-transduction of cells with Ad5BMP-2 and Ad5BMP-7.

• Journal of Ginseng Research
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• 제44권3호
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• pp.475-482
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• 2020

Background: Active natural ingredients, especially small molecules, have recently received wide attention as modifiers used to treat neurodegenerative disease by promoting neurogenic regeneration of neural stem cell (NSC) in situ. 20(S)-protopanaxadiol (PPD), one of the bioactive ingredients in ginseng, possesses neuroprotective properties. However, the effect of PPD on NSC proliferation and differentiation and its mechanism of action are incompletely understood. Methods: In this study, we investigated the impact of PPD on NSC proliferation and neuronal lineage differentiation through activation of the Wnt/glycogen synthase kinase (GSK)-3β/β-catenin pathway. NSC migration and proliferation were investigated by neurosphere assay, Cell Counting Kit-8 assay, and EdU assay. NSC differentiation was analyzed by Western blot and immunofluorescence staining. Involvement of the Wnt/GSK3β/β-catenin pathway was examined by molecular simulation and Western blot and verified using gene transfection. Results: PPD significantly promoted neural migration and induced a significant increase in NSC proliferation in a time- and dose-dependent manner. Furthermore, a remarkable increase in anti-microtubule-associated protein 2 expression and decrease in nestin protein expression were induced by PPD. During the differentiation process, PPD targeted and stimulated the phosphorylation of GSK-3β at Ser9 and the active forms of β-catenin, resulting in activation of the Wnt/GSK-3β/β-catenin pathway. Transfection of NSCs with a constitutively active GSK-3β mutant at S9A significantly hampered the proliferation and neural differentiation mediated by PPD. Conclusion: PPD promotes NSC proliferation and neural differentiation in vitro via activation of the Wnt/GSK-3β/β-catenin pathway by targeting GSK-3β, potentially having great significance for the treatment of neurodegenerative diseases.

• Journal of Periodontal and Implant Science
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• 제28권2호
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• pp.235-247
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• 1998

Calcium sulfate has a long history of medical use as an implant material. The biocompatibiliry of the material has been clearly established. Bone ingrowth concomitant with resorption occurs rapidly with efficient conduction of bone from particle to particle. Calcium sulfate also has a potential for functioning as a good bamer membrane. The purpose of this study was to compare the biocompatibility of different types of calcium sulfate grafting materials including an expelimental calcium sulfate compound on periodontal ligament cells in vitro as a preliminary test towards the development of a more convenient and useful form of grafting material which could promote regeneration of periodontal tissue. Human periodontal ligament cells were collected from the premolar teeth extracted for orthodontic treatment. cells were cultured in a.MEM culture medium containing 20% FBS, at $37^{\circ}C$ and 100% humidity, in a 5% CO2 incubator. Cells were cultured into 96 well culture plate $1{\times}104$ cells per well with $\alpha$-MEM and incubated for 24 hours. After discarding the medium, those cells were cultured in $\alpha$-MEM contained with 10% FBS alone (control group), in medcal-grade calcium sulfate(MGCS group), in plaster(plaster group), experimental calcium sulfate paste(CS paste group) for 1, 2, 3 day respectively. And then each group was characterized by examining of the cell counting, MTI assay, collagen synthesis. The results \vere as follows. 1. In the analysis of cell proliferation by cell counting, both medical-grdde calcium sulfate group and plaster group showed no stastically significant difference at day 1, 2, 3 accept for plaster group at day 1 compared to control group, but there was stastically significant difference between CS paste group and all other groups at day 1, 2, 3(P<0.05). 2. In the analysis of cytotoxicity by MIT assay, both medical-grade calcium sJlfate group and plaster group showed no stastically significant difference compared to control group at day 1, 2, 3 but there was stastically significant difference between CS paste group and all other groups at day 1, 2, 3(P<0.OS). 3. In the analysis of collagen synthesis by immunoblotting assay, high level was detected for medical-grade calcium sulfate group and plaster group at day 1, 2, 3 compared to CS paste group. On the basis of these results, medical-grade calcium sulfate and plaster was shown to possess biocompatibility whereas the CS paste had unfavourable outcome. This observation shows a need for modification of the materials contained in calcium sulfate paste.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제32권2호
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• pp.97-106
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• 2010

Aim of the study: An alternative source of adult stem cells that could be obtained in large quantities, under local anesthesia, with minimal discomfort would be advantageous. Adipose tissue could be processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). This study was performed to confirm the availability of ATSCs in bone tissue engineering. Materials amp; Methods: In this study, adipose tissue-derived mesenchymal stem cell was extracted from the liposuctioned abdominal fat of 24-old human and cultivated, and the stem cell surface markers of CD 105 and SCF-R were confirmed by immunofluorescent staining. The proliferation of bone marrow mesenchymal stem cell and ATSCs were compared, and evaluated the osteogenic differentiation of ATSCs in a specific osteogenic induction medium. Osteogenic differentiation was assessed by von Kossa and alkaline phosphatase staining. Expression of osteocyte specific BMP-2, ALP, Cbfa-1, Osteopontin and osteocalcin were confirmed by RT-PCR. With differentiation of ATSCs, calcium concentration was assayed, and osteocalcin was evaluated by ELISA (Enzyme-linked immunosorbant assay). The bone formation by 5-week implantation of HA/TCP block loaded with bone marrow mesenchymal stem cells and ATSCs in the subcutaneous pocket of nude mouse was evaluated by histologic analysis. Results: ATSCs incubated in the osteogenic medium were stained positively for von Kossa and alkaline phosphatase staining. Expression of osteocyte specific genes was also detected. ATSCs could be easily identified through fluorescence microscopy, and bone formation in vivo was confirmed by using ATSC-loaded HA/TCP scaffold. Conclusions: The present results show that ATSCs have an ability to differentiate into osteoblasts and formed bone in vitro and in vivo. So ATSCs may be an ideal source for further experiments on stem cell biology and bone tissue engineering.

• Journal of Periodontal and Implant Science
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• 제42권3호
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• pp.73-80
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• 2012

Purpose: The purpose of this study was to investigate the effects of the immunosuppressants FK506 and cyclosporin A (CsA) on the osteogenic differentiation of rat mesenchymal stem cells (MSCs). Methods: The effect of FK506 and CsA on rat MSCs was assessed in vitro. The MTT assay was used to determine the deleterious effect of immunosuppressants on stem cell proliferation at 1, 3, and 7 days. Alkaline phosphatase (ALP) activity was analyzed on days 3, 7, and 14. Alizarin red S staining was done on day 21 to check mineralization nodule formation. Real-time polymerase chain reaction (RT-PCR) was also performed to detect the expressions of bone tissue-specific genes on days 1 and 7. Results: Cell proliferation was promoted more in the FK506 groups than the control or CsA groups on days 3 and 7. The FK506 groups showed increased ALP activity compared to the other groups during the experimental period. The ALP activity of the CsA groups did not differ from the control group in any of the assessments. Mineralization nodule formation was most prominent in the FK506 groups at 21 days. RT-PCR results of the FK506 groups showed that several bone-related genes-osteopontin, osteonectin, and type I collagen (Col-I)-were expressed more than the control in the beginning, but the intensity of expression decreased over time. Runx2 and Dlx5 gene expression were up-regulated on day 7. The effects of 50 nM CsA on osteonectin and Col-I were similar to those of the FK506 groups, but in the 500 nM CsA group, most of the genes were less expressed compared to the control. Conclusions: These results suggest that FK506 enhances the osteoblastic differentiation of rat MSCs. Therefore, FK506 might have a beneficial effect on bone regeneration when immunosuppressants are needed in xenogenic or allogenic stem cell transplantation to treat bone defects.

• 폴리머
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• 제36권6호
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• pp.789-794
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• 2012

생체적합성 및 생분해성의 장점을 지닌 PLGA는 우수한 기계적 성질과 분해속도를 조절할 수 있는 장점을 가지고 있지만 소수성으로 인해 세포의 초기부착률이 낮고, 지지체 내부에 영양액의 공급이 원활하지 않다. 본 연구에서는 이러한 단점을 보완하고자 탈미네랄화된 골분(DBP, demineralized bone particle) 젤을 PLGA 지지체에 함침시켰다. 다양한 형태의 지지체 압축강도를 측정한 결과 PLGA/DBP 지지체가 연골과 비슷한 강도를 가지고 있었으며, 이를 바탕으로 DBP젤을 함침시킨 PLGA/DBP젤 지지체를 제작하였다. 연골세포를 파종하여 in vivo 실험을 진행하였고 조직화 정도를 확인하기 위해 H&E, Safranin-O, Alcian blue, Collagen type I 및 Collagen type II 염색을 실시하였다. 그 결과 DBP를 함침시킨 PLGA 지지체에서 GAG, Collagen type I 및 Collagen type II의 높은 발현과 조직화를 보여 연골조직으로 대체할 수 있는 가능성을 확인하였다.

• 대한기계학회논문집A
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• 제41권7호
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• pp.645-653
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• 2017

골 조직 공학에서 폴리카프로락톤(Polycaprolactone, PCL)은 생분해성 및 생체적합성의 합성고분자로서 인공지지체의 제작에 널리 이용되고 있는 생체재료 중 하나이다. 인공지지체의 제작에서 지지대폭은 생체 내/외 실험에서 공극 크기뿐만 아니라 공극률에도 영향을 미치기 때문에 지지대 폭을 일정하게 유지하는 것이 조직 재생에 중요하게 고려되는 부분이다. 본 연구에서는 온도, 공압, 이송 속도, 그리고 노즐 팁 높이를 이용하여 체계적이고 효율적인 인공지지체 제조 공정이 될 수 있도록 실험 계획법을 통해 최적 공정 조건을 탐색하였다. $150{\mu}m$ 지지대 폭을 가지는 PCL 인공지지체를 제작하는 것이 목표였으며, 한 가지의 공극 패턴이 아니라 다양한 공극 패턴에 따른 PCL 인공지지체를 제작하는 연구를 수행함으로써 모든 실험 그룹에서 지지대 폭이 일정함을 증명하였다.