• Journal of Periodontal and Implant Science
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• 제36권2호
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• pp.385-396
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• 2006

For the regeneration of periodontal tissues, the microenvironment for new attachment of connective tissue fibers should be provided, At this point of view, cementum formation in root surface plays a key role for this new attachment. This study was performed to figure out which factor promotes differentiation of cementoblast Considering anatomical structure of tooth, we selected the cells which may affect the differentiation of cementoblast - Ameloblast, OD11&MDPC23 for odontoblasts, NIH3T3 for fibroblsts and MG63 for osteoblasts. And OCCM30 was selected for cementoblast cell line. Then, the cell lines were cultured respectively and transferred the conditioned media to OCCM30. To evaluate the result, Alizarin red S stain was proceeded for evaluation of mineralization. The subjected mRNA genes are bone sialoprotein(BSP), alkaline phosphate(ALP) , osteocalcin(OC), type I collagen(Col I), osteonectin(SPARC ; secreted protein acidic and rich in cysteine). Expression of the gene were analysed by RT-PCR, The results were as follows: 1. For alizarin red S staining, control OCCM30 didn't show any mineralized red nodules until 14 days. But red nodules started to appear from about 4 days in MDPC-OCCM30 & OD11-OCCM30. 2. For results of RT-PCR, ESP mRNAs of control-OCCM30 and others were expressed from 14 days, but in MDPC23-OCCM30 & OD11-OCCM30 from 4 days. Like this, the gene expression of MDPC23-OCCM30 & OD11-OCCM30 were detected much earlier than others. 3. For confirmation of odontoblast effect on cementoblast, conditioned media of osteoblasts(MG63) which is mineralized by producing matrix vesicles didn't affect on the mineralized nodule formation of cementoblasts(OCCM30). This suggest the possibility that cementoblast mineralization is regulated by specific factor in dentin matrix protein rather than matrix vesicles. Therefore, we proved that the dentin/odontoblast promotes differentiation/mineralization of cementoblasts. This new approach might hole promise as diverse possibilities for the regeneration of tissues after periodontal disease.

• 대한치과보존학회:학술대회논문집
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• 대한치과보존학회 2003년도 제120회 추계학술대회 제 5차 한ㆍ일 치과보존학회 공동학술대회
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• pp.546.2-546
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• 2003

Dental follicle is the mesenchymal tissue which surrounds developing tooth germ. During tooth root development, periodontal components such as cementum, periodontal ligament and alveolar bone are considered to be created by progenitors present in the dental follicle. However, little is known about these progenitors. Previously we observed that cultured bovine dental follicle cells (BDFC) contained putative cementoblast progenitors. To further analyze the biology of these cells, we have attempted to immortalize BDFC by expression of the polycomb group protein Bmi-1 and human telomerase reverse transcriptase (hTERT). The BDFC expressing Bmi-1 and hTERT showed extended life span by 90 population doublings more than normal BDFC, and still contained cells with potential to differentiate into cementoblasts upon implantation into immunodeficiency mice. Among them, we established a clonal cell line designated as BCPb8, which formed cemetum-like mineralized tissue reactive to anti-cementum specific monoclonal antibody, 3G9, and expressed mRNA for bone sialoprotein, osteocalcin, osteopontin and type I collagen upon implantation. Thus with the combination of hTERT and Bmi-1, we succeeded in immortalization of cementoblast progenitor in BDFC without affecting differentiation potential. The BCPb8 progenitor cell line could be a useful tool not only to study cementogenesis but also to develop regeneration therapy for periodontitis.

• 셀메드
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• 제8권3호
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• pp.13.1-13.8
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• 2018

In the field of osteoporosis, there has been growing interest in anabolic agents that enhance bone formation. The purpose of this study was to examine the effects of NNMBS 246 osteoblastic differentiation with associated signaling pathways. NNMBS 246 markedly increased alkaline phosphatase (ALP) activity and calcium nodule formation. Stimulation with NNMBS 246 not only increased the differentiation markers (ALP, OPN, OCN) level and transcription markers (RUNX2, Osterix) mRNA expression but also upregulated the ECM molecules and OPG mRNA expression. Treatments of NNMBS 246 downregulated MMPs (MMP-1, MMP-2, MMP-9), but RANKL mRNA expression. Furthermore, NNMBS 246 activated osteoblastic differentiation markers and formed calcium nodules in human periodontal ligament cells (hPDLCs) and cementoblast cells. NNMBS 246 induced phosphorylation of MAPKs, Akt, nuclear p65 and IkB-${\alpha}$. BMP-2/Smad and ${\beta}$-catenin signaling pathways were activated by NNMBS 246. Sirtinol (SIRT1 inhibitor) inhibited NNMBS 246-induced osteoblastic differentiation markers mRNA expression. These results suggested that NNMBS 246 has the potential to enhance osteoblastogenesis probably through the activation of BMP/Smad and ${\beta}$-catenin signal pathways, and SIRT1 plays as critical mediator in bone anabolic effect of NNMBS 246.

• Journal of Periodontal and Implant Science
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• 제47권5호
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• pp.273-291
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• 2017

Purpose: Although static magnetic fields (SMFs) have been used in dental prostheses and osseointegrated implants, their biological effects on osteoblastic and cementoblastic differentiation in cells involved in periodontal regeneration remain unknown. This study was undertaken to investigate the effects of SMFs (15 mT) on the osteoblastic and cementoblastic differentiation of human osteoblasts, periodontal ligament cells (PDLCs), and cementoblasts, and to explore the possible mechanisms underlying these effects. Methods: Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, mineralized nodule formation based on Alizarin red staining, calcium content, and the expression of marker mRNAs assessed by reverse transcription polymerase chain reaction (RT-PCR). Signaling pathways were analyzed by western blotting and immunocytochemistry. Results: The activities of the early marker ALP and the late markers matrix mineralization and calcium content, as well as osteoblast- and cementoblast-specific gene expression in osteoblasts, PDLCs, and cementoblasts were enhanced. SMFs upregulated the expression of Wnt proteins, and increased the phosphorylation of glycogen synthase $kinase-3{\beta}$ ($GSK-3{\beta}$) and total ${\beta}-catenin$ protein expression. Furthermore, p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), and nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) pathways were activated. Conclusions: SMF treatment enhanced osteoblastic and/or cementoblastic differentiation in osteoblasts, cementoblasts, and PDLCs. These findings provide a molecular basis for the beneficial osteogenic and/or cementogenic effect of SMFs, which could have potential in stimulating bone or cementum formation during bone regeneration and in patients with periodontal disease.

• Journal of Periodontal and Implant Science
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• 제41권1호
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• pp.30-43
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• 2011

Purpose: Under different culture conditions, periodontal ligament (PDL) stem cells are capable of differentiating into cementoblast-like cells, adipocytes, and collagen-forming cells. Several previous studies reported that because of the stem cells in the PDL, the PDL have a regenerative capacity which, when appropriately triggered, participates in restoring connective tissues and mineralized tissues. Therefore, this study analyzed the genes involved in mineralization during differentiation of human PDL (hPDL) cells, and searched for candidate genes possibly associated with the mineralization of hPDL cells. Methods: To analyze the gene expression pattern of hPDL cells during differentiation, the hPDL cells were cultured in two conditions, with or without osteogenic cocktails (${\beta}$-glycerophosphate, ascorbic acid and dexamethasone), and a DNA microarray analysis of the cells cultured on days 7 and 14 was performed. Reverse transcription-polymerase chain reaction was performed to validate the DNA microarray data. Results: The up-regulated genes on day 7 by hPDL cells cultured in osteogenic medium were thought to be associated with calcium/iron/metal ion binding or homeostasis (PDE1A, HFE and PCDH9) and cell viability (PCDH9), and the down-regulated genes were thought to be associated with proliferation (PHGDH and PSAT1). Also, the up-regulated genes on day 14 by hPDL cells cultured in osteogenic medium were thought to be associated with apoptosis, angiogenesis (ANGPTL4 and FOXO1A), and adipogenesis (ANGPTL4 and SEC14L2), and the down-regulated genes were thought to be associated with cell migration (SLC16A4). Conclusions: This study suggests that when appropriately triggered, the stem cells in the hPDL differentiate into osteoblasts/cementoblasts, and the genes related to calcium binding (PDE1A and PCDH9), which were strongly expressed at the stage of matrix maturation, may be associated with differentiation of the hPDL cells into osteoblasts/cementoblasts.

• International Journal of Oral Biology
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• 제36권2호
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• pp.51-57
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• 2011

Runx2 and Osterix, the transcription factors for osteoblast differentiation, are known as fundamental factors to regulate the development of calcified tissues. However, the biological functions of these factors in the development of the periodontal tissues remain unclear. In this study, we investigated the distribution of Runx2 and Osterix during periodontal tissue development of the mice. Mandibles from 14-day-old mice were prepared for paraffin section. Serial sections of the mandible containing $1^{st}$ molar tooth germs were obtained as a thickness of $7\;{\mu}m$. Some sections were stained with hematoxylin and eosin. Others were used for immunohistochemistry for PCNA, Runx2, and Osterix. Epithelial cells in growing end of Hertwig's epithelial root sheath (HERS) and mesenchymal cells adjacent to the growing end of HERS expressed PCNA. Undifferentiated mesenchymal cells and hard tissue forming cells like cementoblasts and osteoblasts in early stage of differentiation expressed Runx2. Fully differentiated cementoblasts and osteoblasts secreting matrix proteins expressed Osterix. However, the cells terminated the matrix formation did not express Osterix. Periodontal ligament cells expressed Runx2 and Osterix. Pulp cells expressed Runx2 only. These results suggest that Runx2 and Osterix might regulate the differentiation of cementoblasts in the same manner as osteoblasts. Runx2 might participate in the process of cementoblast differentiation in early stage, whether Osterix might regulate the maturation and matrix synthesis of the cells.

• Molecules and Cells
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• 제40권8호
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• pp.550-557
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• 2017

The periodontal ligament (PDL) is the connective tissue between tooth root and alveolar bone containing mesenchymal stem cells (MSC). It has been suggested that human periodontal ligament stem cells (hPDLSCs) differentiate into osteo/cementoblast and ligament progenitor cells. The periodontitis is a representative oral disease where the PDL tissue is collapsed, and regeneration of this tissue is important in periodontitis therapy. Fibroblast growth factor-2 (FGF-2) stimulates proliferation and differentiation of fibroblastic MSCs into various cell lineages. We evaluated the dose efficacy of FGF-2 for cytodifferentiation of hPDLSCs into ligament progenitor. The fibrous morphology was highly stimulated even at low FGF-2 concentrations, and the expression of teno/ligamentogenic markers, scleraxis and tenomodulin in hPDLSCs increased in a dose dependent manner of FGF-2. In contrast, expression of the osteo/cementogenic markers decreased, suggesting that FGF-2 might induce and maintain the ligamentogenic potential of hPDLSCs. Although the stimulation of tenocytic maturation by $TGF-{\beta}1$ was diminished by FGF-2, the inhibition of the expression of early ligamentogenic marker by $TGF-{\beta}1$ was redeemed by FGF-2 treatment. The stimulating effect of BMPs on osteo/cementogenesis was apparently suppressed by FGF-2. These results indicate that FGF-2 predominantly differentiates the hPDLSCs into teno/ligamentogenesis, and has an antagonistic effect on the hard tissue differentiation induced by BMP-2 and BMP-4.

• 대한치과교정학회지
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• 제36권4호
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• pp.242-250
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• 2006

기계적 응력은 정상적인 발달과정 동안 조직의 항상성에 있어 중요한 역할을 한다. 기계적 응력은 치아 이동과 저작과 같은 상황을 포함한다. 치아 이동과 저작 중에 치주인대 섬유모세포는 기계적 자극을 감지하고 주위의 세포밖 물질과 생체분자 대사의 변동에 의해 반응을 보인다. 그러나 아직까지 기계적 응력 하에서 치주인대 세포에서 발현된 유전자에 관한 연구는 미비한 실정이다. 최근 기계적 응력이 초파리에 존재하는 UNC-50 유전자에 영향을 줄 수 있다고 보고되었다. 또한 UNC-50은 치은 섬유모세포와 비교해 치주인대 섬유모세포에서만 발현된다고 보고되었다. 본 연구에서는 간헐적 교정력 적용 시에 백서의 치아에서 일어나는 치근 및 치주 조직의 조직학적 변화와, UNC-50의 발현 양상을 면역조직학적 염색으로 조사하여 치주인대에서 기계적 응력과 UNC-50의 관련성을 알아보고자 하였다. Sprague-Dawely계 수컷 백서 12마리를 4마리씩 세 군으로 나누어 상악 우측 구치부에 NiTi closed coil spring을 사용하여 40 g 정도의 견인력이 발생하도록 하여 하루에 1시간씩 간헐적인 교정력을 적용한 후 1, 3, 5일 후 치주인대의 조직학적 변화를 관찰하여 다음과 같은 결과를 얻었다. 조직학적 소견에서 상악 제1대구치 근심구개치근의 치근부 1/3에서 압박측은 안장측보다 더 좁은 치주인대공간을 보였고 교정력을 적용시킨 후 3일 후부터 인장측에서 백악모세포 활성으로 인한 백악질 침착이 관찰되었다. UNC-50은 인장측의 분화 중인 백악모세포에서 강한 발현을 보였다. Osteocalcin은 인장측에서 압박측에 비해 신생 백악질에 존재하는 분화 중인 백악모세포를 따라 강한 발현을 보였다. 이상의 연구결과는 UNC-50이 간헐적 교정력 즉 기계적 응력의 변화에 따른 백악모세포의 분화과정에 중요한 역할을 함을 나타낸다. 그러나 이를 명확히 하기 위해서는 교정력 적용 후 UNC-50의 세포내 신호전달과정에 대한 보완연구가 필요할 것이다.