초록 ▼

- 바이오치아 개발을 위한 골수줄기세포 및 치수줄기세포의 분화, 유전자제어 및 전달연구를 위하여 줄기세포의 분화에 따른 유전자와 단백질의 분석 및 발현제어를 위한 기술개발이 요구됨.
- 따라서 본 연구는 다음가 같은 연구 내용 및 범위를 포함한다.
- 골수줄기세포 및 치수줄기세포의 골모세포/상아모세포 분화 특성 규명
- 골수줄기세포/치수줄기세포의 골모세포/상아모세포 분화 관련 유전자의 genome-wide screening 및 분석.
- 골수줄기세포/치수줄기세포의 골모세포/상아모세포 분화 관련 유전자의 검정.<

- 바이오치아 개발을 위한 골수줄기세포 및 치수줄기세포의 분화, 유전자제어 및 전달연구를 위하여 줄기세포의 분화에 따른 유전자와 단백질의 분석 및 발현제어를 위한 기술개발이 요구됨.
- 따라서 본 연구는 다음가 같은 연구 내용 및 범위를 포함한다.
- 골수줄기세포 및 치수줄기세포의 골모세포/상아모세포 분화 특성 규명
- 골수줄기세포/치수줄기세포의 골모세포/상아모세포 분화 관련 유전자의 genome-wide screening 및 분석.
- 골수줄기세포/치수줄기세포의 골모세포/상아모세포 분화 관련 유전자의 검정.
- 치아조직 재생을 위한 분화유도물질 발굴, 세포배양 조건 확립 및 cell-scaffold복합체의 제조/이식방법 확립.
- 치배상피를 대체할 성체상피줄기세포 탐색.
- 상아질모 세포의 기능제어 분자 타겟을 찾기 위해 인간 상아질에 존재하는 단백체 분석를 분석함.
- 인간 치수줄기세포를 추출하여, 상아질 단백질 중 상아질모세포 분화 유도와 관련성 있는 단백질을 코딩하는 유전자의 기능 검정 및 제어 연구.
- 치주인대세포 bank 설립, 교정치료를 목적으로 내원한 환자로부터 동의서 확보 및 발치후, 치주인대세포를 배양함.
- 세포주 저장용기를 확보하고 세포주 Bank database화를 실현함.
- 치주인대세포의 채득방법 확립, 조직 채득 방법과 효소처리 방법으로 치주인대세포 획득하여 증식률과 분화능을 평가하고 stem cell marker의 발현을 검토함.
- 치주인대세포를 골분화 배양환경에서 배양하여 골분화시 발현 되는 광물화 결절 형성 관련 유전자의 변화 패턴 검토함.
- 치주인대세포의 백악아세포 및 섬유아세포로의 분화관련 유전자 규명 및 virus 구축
- 치주인대세포의 생체내 골 형성능 규명을 위하여 치주인대세포의 광물화 결절 형성 관련 유전자가 발현정도 평가함
- C57BL/6 생쥐 유래 배아 줄기 세포를 유도하고 획득한 생쥐 배아 줄기 세포의 germ-line transmission 효율성을 검토함.
- 줄기세포 배양을 위한 새로운 feeder 인 X-1 세포를 사용할 수 있음을 규명함.
- 치아 수 결정 유전자인 Runx2와 Ectodin의 관계 규명
- 사람의 치아결손 질환자에서 유전자 발현조사
- Static tensional force에 의한 치주 인대 세포의 유전자 발현 변화 규명 및 치주 인대 세포에 2시간 또는 12시간 가한 후, 유전자의 발현 변화를 살펴 봄.
- 세포 이식용 3차원 초정밀 바이오 인공지지체 설계 및 제작 기술 개발 과 이에 대한 평가
- 인공지지체 제작용 CAD/CAM 시스템을 위한 의료 영상 데이터 변환 기술 및 성형 정보 생성 기술 개발
- 초정밀 구조체 제작이 가능한 새로운 UV curable 생분해성 생체 재료의 개발
- 조직 재생능 극대화를 위한 인공지지체 재생능력 활성화 기술 개발
- 효과적인 줄기세포-지지체 복합체 배양 기반기술 확립
- 줄기세포-지지체 복합체에 의한 바이오치아 및 치주조직 재생을 위한 동물이식 모델 확립

Abstract ▼

The purpos e of this study was to establish fundamental technologies for regeneration of bio-tooth. To achieve this purpose, during the 1st stage of this project. We focused on development of technologies related to potentiation of function of dental and non-dental mesenchymal stem cells, and to pro

The purpos e of this study was to establish fundamental technologies for regeneration of bio-tooth. To achieve this purpose, during the 1st stage of this project. We focused on development of technologies related to potentiation of function of dental and non-dental mesenchymal stem cells, and to production of materials derived from human tooth. Therefore, we mainly studied on 1) characterization of differentiation characteristics of bone marrow mesenchymal stem cells (BMSCs) and dental pulp stem cells (DPSCs), 2) genome-wide screening and validation of genes that fluctuated during differentiation of BMSCs and DPSCs, 3) characterization of constituents extracted from tooth, 4) preparation of scaffold derived from tooth, and 5) development of techniques for implantation of cell-scaffold composites for in vivo tooth regeneration.
For the human adult stem cells, effective differentiation to odnto/ osteoblastic cells, in vitro culture condition was optimized. We identified several genes such as ABCA8, APOD, CCRL1, MFAP, C7 that were selectively expressed during osontoblast differentiation of DPSCs. Through proteomic analysis, we identified 147 tooth-soluble protein, including prothrombin, serotransferrin, collagens, keratins, and annexins, and these proteins specifically showed odontogenic differentiation potency of DPSCs. We also prepared tooth-derived scaffold and established implantation techniques of cell-scaffold composites in an in vivo model. Therefore, we identified methods that can potentiate function of DPSCs through genomics and proteomics, and developed tooth-derived scaffold. We firmly believe that the technique and information ontained for this project may contribute to development of Bio-tooth in the next stage of this project.
For the successful biotooth regeneration, it is necessary to study the differentiation mechanism from the dental pulp stem cells to odontoblast and the functional control of the related genes. We have analyzed the proteomes in the human dentin to seek the genes related to odontoblast differentiation and found 233 proteins from human dentin. Human dental pulp stem cells were isolated and the growth condition of them are established. Some of the genes coding the dentin proteins were anlayzed for their mRNA expression pattern during odontoblast-like cell differentiation. Lentivirus-mediated gene overexpression and knock-down systems were established for the efficient delivery of the genes to dental pulp stem cells. We also analyzed the secretory proteomes from the dental pulp stem cells culture in the odontogenic/osteogenic media condition. Our results can be used for the regeneration of biotooth.
We were received a patient written consent from patients. After, the PDL tissues were obtained from the periodontal ligament of premolar teeth extracted for orthodontic reasons. Normal impacted premolars were collected from 29 donors at the Department of Periodontology, National University Hospital. We secured 62 hPDL cells bank. And also, we checked the degree of mineralization and the osteoblastic-differentiation related markers in the hPDL cells isolated by explant or enzyme technic. We confirmed that hPDL cells isolated by explant technic were more high ALP activiy, mineralized nodule formation than the hPDL cells isolated by enzyme technic. But, proliferation is higher in hPDL cells isolated by enzyme technic than hPDL cells isolated by explant technic. Also, we confirmed that hPDL cells expressed stem cell marker such as CD146, STRO-1.
Next, we observed the gene expression profile in hPDL cells proliferation, differentiation and mineralization in vitro. At day 1 and 7, any mineralization nodule was not observed. A few, small mineralized nodules started to be developed on day 14, and mineralization was distinct. on day 21. A number of mineralization nodules were formed and mineralization nodule size was also increased on day 21. At day 0 showing confluent monolayer, as a active proliferative stage, C-myc gene expression observed maximal level. At day 7 showing multilayer formation, alkaline phosphatase, BMP-2 and BMP-4 gene expression was increased and followed by maximal expression of osteocalcin on day 14 showing initiation of nodule mineralization. In relation to apoptosis, C-fos gene expression was peaked on day 21 characterized postmineralization stage.
We investigated the up/down-regulated genes in the hPDL cells during mineralization at 7 and 14 days by using DNA microarray. We filtered the genes over the 3-fold change and the signal value 100. At day 7, 111 genes (128 probes) were expressed more than 3 folds in osteogenic medium-hPDL cells, whereas 19 genes (22 probes) were down-regulated less than -3 folds in non-osteogenic medium-hPDL cells. At 14 days, 77 genes (99 probes) were expressed more than 3 folds in osteogenic medium-hPDL cells, whereas 19genes (21 probes) were down-regulated less than -3 folds in non-osteogenic medium-hPDL cell. At both days, 30 genes (45 probes) were up-regulated, whereas 11 genes (14 probes) were down-regulated. We selected top 10 genes that either increased or decreased. And then, we evaluated the response to the highly expressed genes by rhBMP-2 and FGF-2 during mineralization of the hPDL cells. According to concentration of rhBMP-2, PLZF, FKBP5 and FAM107A gene expression showed the increasing tendency, but SAA1, FABP4 and PIP gene expression showed the decreasing tendency. According to exposed time of rhBMP-2, every genes showed the gradually increasing tendency. According to concentration and exposed time of FGF-2, All genes except for RAC3 showed the decreasing tendency.
Thus, these findings demonstrated that the highly expressed genes during mineralization of the hPDL cells were classified by runx-2 activator, NF-kB signaling-related factors and apoptosis-related factors, and responsed reversely to rhBMP-2 and FGF-2. On the base of these results, PLZF, FKBP5, SAA1, FABP4, FAM107A, CORIN, RAC3 and PIP are considered as the osteoblastic/cementoblastic differentiation-related genes in the hPDL cells. When we transfected PLZF in hPDL cells, bone-related genes such as Runx-2, ALP, BSP expressions were more high than normal hPDL cells.
Thus, PLZF is suggested the cementoblastic differentiation related genes. It has to be studied further to evaluate the role of these genes in the osteoblastic/cementoblastic differentiation in the hPDL cells. Next, we observed the osteoblastic-regeneration of hPDL cells in vivo. The Collatape®(Zimmer dental, co, USA) was used for carrier of hPDL cells. The PDL cells were cultured on the Collatape® in osteogenic medium. At 7 days, the collatape with or without cultured hPDL cells was observed through scanning electron microscope(SEM). And then, we observed the expression of osteoblast-related marker such as Runx-2, ALP, BSP, OCN, CP-23, F-spondin, Col I and Scleraxis between day 0 and 7. At 7 days, the expression of osteoblast-related markers observed more high than Day 0. And then, we translocated the cultured hPDL cells with collatape for 7 days in nude mouse calvarial defects. Only soaked Collatape® in medium for 7 days was used for controls. The defects were evaluated in histologic after 8 weeks. At 8 weeks of healing, new bone formation was found in the defect in test group. However in control group, the defect was packed with fibrous connective tissue. There was not inflammatory reaction in the all groups. In conclusion, we can suggested that hPDL cells can increase new bone formation in nude mouse calvarial defects. On the basis of this results, this new strategy may be applied in the treatment of bony defects of periodontal therapy.
Although most mouse embryonic stem (ES) cells were derived from blastocysts of 129/sv strain, it has been known that this strain has many problems including spontaneous teratoma formation and behavioral and immunological responses. Therefore, it is necessary to backcross gene-manipulated mice derived from this ES cell with inbred C57BL/6 strain to overcome these problems. In this study, we successfully derived mouse ES cells from delayed blastocysts after ovariectomy and injection of progesterone. Total 15 different mouse ES cells were derived from 70 female C57BL/6 mice. Characterization of CJ42-1 ES cells, one of derived ES cells, was performed by analysis of the expression of ES cells markers. We also identified various tissues derived from all three germline layers in excised teratoma by injection of ES cells into nude mice. Collectively, derivation of ES cells from C57BL/6 mice was successfully performed by production of delayed blastocysts.
Mouse ES cells need to culture on feeder cells such as mouse embryonic fibroblasts (MEF). MEF secrete many factors including LIF, BMPs and these factors support to maintain self-renewal and pluripotency of ES cells. MEF has disadvantages like limited passage number. In this study, a new feeder X-1 cells supported to maintain self-renewal and pluripotency of ES cells and overcome a disadvantage of limited passage number of MEF.
One of characteristic features of RUNX2 heterozygous patients is supernumerary teeth. But we did not find this phenotype in mouse Runx2 heterozygote. Ectodin-deficient mice have extra teeth. We try to elucidate relation between Runx2 and Ectodin. In situ hybridazation of Ectodin was decreased in Runx2 heterozygote. We also found that Runx2 didn't bind to ectodin promoter, which was confirmed by Ectodin-Luciferase reporter assay. These results indicate that Runx2 does not regulate ectodin expression directly in tooth patterning. However, it may be possible that Runx2 can regulate ectodin expression indirectly in tooth patterning.
Furthermore, gene expression patterns in abnormal tooth eruption and in PDL cells applied with static tensional forces were established. Thes results also can help improve the bio-tooth regeneration research.
The purpose of this research is to develop a base technology for bio-tooth/periodontal tissue regeneration. For achieving the purpose, we need to develop UV curable and biodegradable biomaterials and fabricate a 3D high precision scaffold for transplantation with the stem cells. And we will try to establish animal model for bio-tooth/periodontal tissue regeneration using the stem cells and scaffolds.
Active researches in tissue engineering and regenerative medicine for various tissues are going on. Most existing researches have tended to improve cell cultivation/ differentiation/ proliferation via a biological/ chemical/ material approaches. In practice, tissue engineering requires a cross-disciplinary approach involving many academic fields. In this regard, this approach can efficiently integrate the individual technologies. Successful completion of this endeavor will address the long-standing challenges that have hampered the progress of building tissues for transplantation into patients.

목차 Contents

• 제 출 문...1
• 보고서 요약서...2
• 요 약 문...4
• SUMMARY...11
• 목 차...17
• 제 1 장 연구개발과제의 개요...18
• 제 1절 연구개발의 목적...18
• 제 2절 연구개발의 필요성...20
• 제 2 장 국내외 기술개발 현황...23
• 제 1절 국내외 기술개발 수준...23
• 제 2절 연구결과가 국내.외 기술개발현황에서 차지하는 위치...28
• 제 3 장 연구개발수행 내용 및 결과...29
• 제1절 골수줄기세포 및 치수줄기세포의 골모세포/상아모세포 분화 특성 규명...29
• 제2절 골수줄기세포/치수줄기세포의 골모세포/상아모세포 분화 관련 유전자의 genome-wide screening 및 분석...32
• 제3절 골수줄기세포/치수줄기세포의 골모세포/상아모세포 분화 관련 유전자의 검정...36
• 제4절 치아조직 재생을 위한 분화유도물질 발굴, 세포배양 조건 확립 및 cell-scaffold복합체의 제조/이식방법 확립...43
• 제5절 치배간엽을 대체할 성체상피줄기세포 탐색...60
• 제6절 골이식에 따른 골성 회복양상 확인...63
• 제7절 프로테오믹스 기법을 이용한 상아질 단백체 분석 연구...64
• 제8절 치수로부터 상질모세포 분리, 특성규명 및 분화 연구...71
• 제9절 상아질모세포 분화유도 유전자 기능 제어 연구...74
• 제10절 치주인대세포의 채득방법에 따른 성상비교...78
• 제11절 치주인대세포의 광물화 형성과정 시 발현되는 다양한 유전자 양상의 검토...80
• 제12절 치주인대세포의 광물화과정시 발현되는 유전자들에 대한 상호비교...85
• 제13절 치주인대세포의 골아세포 및 백악아세포로의 분화시 강하게 발현되는 PLZF의 효과...91
• 제14절 rhBMP-2가 고농도 당에서 배양된 치주인대세포의 광물화 형성과정에 미치는 영향...95
• 제15절 누드마우스의 두개골 결손부에 배양된 치주인대 세포 이식이 신생골 형성에 미치는 영향...98
• 제16절 다양한 세포부착 펩타이드로 흡착된 골이식재가 골아세포 활성도와 골형성에 미치는 영향...100
• 제17절 C57BL/6 생쥐 유래 배아 줄기 세포 유도...104
• 제18절 줄기세포 배양을 위한 새로운 feeder 세포 탐색...107
• 제19절 치아수 결정 pathway 규명...109
• 제20절 사람의 치아결손 질환자에서 유전자 발현연구...112
• 제21절 static tensional force에 의한 치주 인대 세포의 유전자 발현 변화 규명...113
• 제22절 Anti-allergic effect of Vitis amurensis on mast cell-mediated allergy model. Exp Biol Med. 233(2):192-199...116
• 제23절 Signaling pathways in bisphenol A-induced apoptosis: role of calcium-induced reactive oxygen species, mitogen-activated protein kinases and nuclear factor-κB...120
• 제24절 Mosla dianthera decreases immediate-type allergic reaction and tumor necrosis factor-α production...124
• 제25절 Motherwort의 항염증 및 항알레르기 효과 발굴...128
• 제26절 수십 마이크로 이내의 정밀도를 가진 3차원 인공지지체 설계 및 제작...131
• 제27절 재생능 활성화를 위한 처리 기술 개발...150
• 제28절 HA 유도체를 이용한 인공 지지체 개발...155
• 제29절 Cell - scaffold 복합체의 골조직 내 이식법 확립...162
• 제30절 3차원 초정밀 바이오 치아/치주조직 연구용 인공지지체 제작 기술...176
• 제31절 인공지지체 재생능력 활성화 및 기능 규명...215
• 제32절 UV curable 생분해성 생체 재료 합성 기술...223
• 제33절 치아/치주 조직 재생을 위한 인공지지체의 생체적 합성 평가...236
• 제34절 3차원 초정밀 인공지지체 제작 기술 개발...239
• 제35절 인공지지체 재생능력 활성화 기술 개발...261
• 제36절 HA 유도체를 이용하여 인공지지체 제작...267
• 제37절 치아/치주조직 재생을 위한 효과적인 cell-인공지지체 복합체 형성 조건 확립...280
• 제38절 연구범위 및 연구수행 방법...289
• 제 4 장 목표달성도 및 관련분야에의 기여도...295
• 제 5 장 연구개발결과의 활용계획...310
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보...315
• 제 7 장 참고문헌...321