The expression patterns of RANKL and OPG in murine tooth eruption

Hwang, Kyung-Mun,Kim, Eun-Jung,Kim, Hyun-Jung,,Kim, Young-Jin,Nam, Soon-Hyeun 大韓小兒齒科學會 2006 大韓小兒齒科學會誌 Vol.33 No.2

치아의 맹출은 치아기 (dental organ)와 치조골의 세포와 연관된 매우 복잡한 과정이다. 우선 치아 맹출이 일어나기 전에 파골세포가 치낭으로 집결하게 된다. 이러한 치낭의 역할은 파골세포와 조골세포의 상호작용으로 이루어 지는 골개조와 밀접한 관련이 있는데, 이는 치아 맹출과 연관된 많은 유전자들이 치낭에서 발현되기 때문이다. RANKL는 TNF ligand family로써 조골세포에 존재하며 파골세포의 형성 및 전구세포로 부터의 활성화를 유도한다. 이러한 RANKL은 OPG에 의해 그 작용이 억제되며 RANKL와 OPG의 상대적인 비율이 파골세포의 형성에 영향을 미친다. 또한 Runx2유전자의 변이는 조골세포의 분화와 활성에 차질을 가져오고 결국 RANKL/OPG pathway를 통해 파골세포 형성에 영향을 줄 수 있다. 치아의 발육 및 맹출에 미치는 RANKL 및 OPG의 영향을 알아보고 Runx2와의 연관성을 알아보기 위해 in situ hybridization 방법으로 태생 1, 3, 5, 7, 9, 11일된 쥐의 하악 및 제1대구치를 사용하여 실험을 실시한 결과 RANKL, OPG, Runx2의 mRNA가 태생 1일부터 11일까지 치낭 및 치아주위조직에 특성있게 나타났다. 이중 태생 5일에서 9일 사이에 RANKL 및 Runx2는 치아의 교합면측과 하방 치조골 부위의 발현이 강하게 나타난 반면 OPG는 약한 발현을 보였다. 이는 또한 파골세포의 활성부위를 알아보기 위해 TRAP염색을 실시하여 태생 5일에서 9일 사이에 최대의 활성화를 나타낸 결과와 연광성 있게 나타났다. RANKL, OPG, Runx2의 특성있는 발현양상들을 종합해 볼 때, 치아 맹출은 치낭, 치아기, 치조골 사이의 상호 작용을 통해 이루어 지며, 이는 치낭이 치아 맹출에 있어서 매우 중요하다는 것을 의미한다. 또한, 이러한 유전자들 (RANKL, OPG, Runx2) 이 치아의 맹출에 중요한 역할을 하는 것으로 사료된다. Tooth eruption is a complex and tightly regulated process that involves cells of the tooth organ and the surrounding alveolus. Osteoclast precursors must be recruited into the dental follicle prior to the onset of eruption. This function of dental follicle may be regarded as the ability of bone remodeling characterized by the interaction of osteoclasts and osteoblasts. This is because tooth eruption is a localized event in which many of the genes required for eruption are expressed in the dental follicle. RANKL is a membrane-bound protein that is a member of the TNF ligand family, which is present on bone marrow stromal cells and osteoblasts, and induces osteoclast formation and activation from precursor cell. The biologic effect of RANKL is inhibited by OPG and, in bone, the relative ratio of RANKL and OPG modulates osteoclastogenesis. To evaluate the roles of RANKL and OPG in tooth eruption and the relations with the expression pattern of Runx2, in situ hybridization was performed with mandibles of mice at postnatal stage 1, 3, 5, 7, 9 and 11. mRNA of RANKL, OPG, and Runx2 are expressed in dental follicle and surrounding tissue from P1 to 11. To determine the sites of osteoclastic activity duing tooth eruption, mandibles were dissected. Peak osteoclastic activity in alveolar bone along the occlusal and basal regions was observed from P5 to 9, with osteoclasts in these regions being large and strongly TRAP-positive. The specific spatic-temporal expression patterns of RANKL, OPG and Runx2 in our study suggest that tooth eruption could be progressed through the interactions of molecular signaling among dental follicle, dental organ and alveolar bone, furthermore it means that dental follicle is quite important in tooth eruption. In addition, it indicates that these genes (RANKL, OPG, and Runx2) play critical roles in tooth eruption.

치수, 치주인대 및 치낭에서 얻어진 성체줄기세포의 조골세포로의 분화능력 평가에 관한 연구

이중규(Joong-Kyou Lee),이재훈(Jae-Hoon Lee) 대한구강악안면외과학회 2010 대한구강악안면외과학회지 Vol.36 No.1

Complex human tissues harbor stem cells and precursor cells, which are responsible for tissue development or repair. Recently, dental tissues such as dental pulp, periodontal ligament (PDL), dental follicle have been identified as easily accessible sources of undifferentiated cells. These tissues contain mesenchymal stem cells that can be differentiate into bone, cartilage, fat or muscle by exposing them to specific growth conditions. In this study, the authors procured the stem cell from pulp, PDL, and dental follicle and differentiate them into osteoblast and examine the bone induction capacity. Dental pulp stem cell (DPSC), periodontal ligament stem cell (PDLSC), and dental follicle precursor cell (DFPC) were obtained from human 3rd molar and cultured. Each cell was analyzed for presence of stem cell by fluorescence activated cell sorter (FACs) against CD44, CD105 and CD34, CD45. Each stem cell was cultured, expanded and grown in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix. Osteogenic pathway was checked by alizarin red staining, alkaline phosphatase (ALP) activity test and RT-PCR for ALP and osteocalcin (OCN) gene expression. According to results from FACs, mesenchymal stem cell existed in pulp, PDL, and dental follicle. As culturing with bone differentiation medium, stem cells were differentiated to osteoblast like cell. Compare with stem cell from pulp, PDL and dental follicle-originated stem cell has more osteogenic effect and it was assumed that the character of donor cell was able to affect on differential potency of stem cell. From this article, we are able to verify the pulp, PDL, and dental follicle from extracted tooth, and these can be a source of osteoblast and stem cell for tissue engineering.

Three-Dimensional Spheroid Formation of Cryopreserved Human Dental Follicle-Derived Stem Cells Enhances Pluripotency and Osteogenic Induction Properties

Hyo-Jung Kim,Iel-Yong Sung,Yeong-Cheol Cho,Min-Su Kang,Gyu-Jin Rho,June-Ho Byun,Won-Uk Park,Myeong-Gyun Son,Bong-Wook Park,Hyeon-Jeong Lee,Young-Hoon Kang 한국조직공학과 재생의학회 2019 조직공학과 재생의학 Vol.16 No.6

BACKGROUND: Enhancement and maintenance of the stemness of mesenchymal stem cells (MSCs) is one of the most important factors contributing to the successful in vivo therapeutic application of these cells. In this regard, threedimensional (3D) spheroid formation has been developed as reliable method for increasing the pluripotency of MSCs. Moreover, using a new protocol, we have previously shown that dental tissues of extracted wisdom teeth can be effectively cryopreserved for subsequent use as a source of autologous stem cells. The main purpose of this study is to analyze the stemness and in vitro osteogenic differentiation potential of 3D spheroid dental MSCs compared with conventional monolayer cultured MSCs. METHODS: In this study, MSC-characterized stem cells were isolated and cultured from long-term cryopreserved dental follicles (hDFSCs), and then 2D hDFSCs were cultured under 3D spheroid-forming conditions using a newly designed microchip dish. The spheroids (3D hDFSCs) thus produced were investigated and characterized with respect to stemness, MSC marker expression, apoptosis, cell cycle analysis, extracellular matrix (ECM) production, and osteogenic and adipogenic differentiation properties. RESULTS: In terms of MSC and senescence markers, spheroid cells showed no difference when compared with 2D hDFSCs; however, 3D hDFSCs were observed to have a higher proportion of cell cycle arrest and a larger number of apoptotic cells. Moreover, spheroids showed substantially increased levels of pluripotency marker (early transcription factors) and ECM protein expression. Compared with 2D hDFSCs, there was also a notable enhancement in the osteogenic induction potential of spheroids, although no differences were observed with respect to in vitro adipogenesis. CONCLUSION: To the best of our knowledge, this is the first study to demonstrate the application of a spheroid culture system for dental follicle-derived stem cells using a microchip dish. Although further studies are needed, including in vivo transplantation, the results obtained in this study indicate that spheroid hDFSCs derived from cryopreserved dental follicle tissues could be used as a valuable source of autologous stem cells for bone tissue regeneration.

염증성 치은 상피와 치낭의 표피성장인자 수용체의 발현 및 실험적 치아이동에 미치는 영향에 관한 연구

김영호,배창 대한치과교정학회 1997 대한치과교정학회지 Vol.27 No.2

동소 mRNA 보합결합법과 면역조직 화학적 염색법을 이용하여 정상 치은 상피와 염증성 치은 상피의 표피성장인자 수용체의 발현을 관찰하여 치은 상피의 염증에 있어서 표피성장인자 수용체의 역할을 연구하고, 타액에 노출되지 않는 치낭 조직에 있어서 표피성장인자 수용체의 발현을 관찰하여 다음과 같은 결과를 얻었다. 1. 동소 mRNA 보합결합법상 정상 치은 상피에서 EGFR mRNA는 거의 기저세포층에 국한되어 나타났으며, 극세포층은 약양성을 보였고 과립층과 각화층에서는 발현되지 않았다. 2. 면역조직 화학적 염색법상 정상 치은 상피에서 EGFR 단백은 거의 각화층과 과립층에국한되어 나타났으며, 극세포층은 약양성을 보였고 기저세포층에서는 발현되지 않았다. 3. 동소 mRNA 보합결합법상 염증성 치은 상피에서 EGFR mRNA는 각화층을 제외한 전층에 걸쳐서 균일하게 분포하였다. 4. 면역조직 화학적 염색법상 염증성 치은 상피에서 EGFR 단백은 기저세포층에서 각화층에 걸쳐서 균일하게 분포하였다. 5. 치낭 조직에서는 동소 mRNA 보합결합법과 면역조직 화학적 염색법 모두에서 Malassez 상피세포 잔존물에 강하게 염색이 되었고, 그 외의 주위 조직에서는 발현되지 않았다. 이상의 결과에서 볼 때, 염증성 치은 상피에서 EGFR의 과발현과 치낭 조직의 Malassez 상피세포 잔존물에서 다량의 EGFR이 존재하는 것으로 미루어, 이는 구강 환경에 가해질 수 있는 손상에 대하여 생체의 항상성을 유지하기 위한 반응으로 여겨진다. Epidermal growth factor(EGF), a single chain polypeptide of 53 amino acids with a molecular weight of 6,045 Da, was first isolated from the male mouse submandibular glands. EGF stimulates cellular proliferation and differentiation in several tissues and accelerates the rate of wound healing. EGF is bound to the specific receptor(EGFR) on the cell membrane of its target cell. EGFR is a transmembrane glycoprotein with a molecular weight of 170,000 Da and is detectable on a large variety of cell types. The authors investigated the expression of EGFR in the normal and inflamed human gingival epithelium to study the role of FGFR in the inflammation of the gingival epithelium, and the expression of EGFR in the dental follicle by using in situ mRNA hybridization and immunohistochemistry. The results were as follows; 1. The expression of EGFR mRNA in the normal gingival epithelium on in situ mRNA hybridization was mainly localized on the basal cell layer, and the spinous layer was weakly positive. The granular and cornified layers ere negative. 2. The expression of EGFR protein in the normal gingival epithelium on immunohistochemistry was localized on the cornified and granular layers, and the spinous layer was weakly positive. The basal cell layer was completely negative. 3. The expression of EGFR mRNA in the inflamed gingival epithelium on in situ mRNA hybridization was evenly and homogeneously distributed in the whole layers of the gingival epithelium except the cornified layer. The staining intensity appeared to increase progressively from the basal cell layer to the cornified layer. 4. The expression of EGFR protein in the inflamed gingival epithelium on immunohistochemistry was evenly and homogeneously distributed in the whole layers of the gingival epithelium. The staining intensity appeared to increase progressively from the cornified layer to the basal cell layer. 5. Strong positive reaction was seen in the epithelial cell rests of Malassez, whereas only background staining was seen in other cells of the dental follicle. In conclusion, the up-regulation of EGFR in the inflamed gingival epithelium and the high amounts of EGFR in the epithelial cell rests of Malassez in the dental follicle can be regarded as responses to the possible damages to the oral environment to maintain the homeostatic conditions.

Aldh1a2 is a maker of dental follicle in tooth germs at cap stage

김진선,조성원 대한구강해부학회 2016 대한구강해부학회지 Vol.37 No.1

Retinoic acid is an important signaling molecule in animal development. Deficiency of retinoic acid causes loss of teeth and skull deformity. In the early stages of tooth development, retinoic acid has been reported to regulate tooth morphology. The major enzymes of retinoic acid formation, Aldh1a2 and Aldh1a3, are expressed during tooth development. However, no expression pattern of the two genes was reported for each tooth development stage. In this study, we examined the expression patterns of Aldh1a2 and Aldh1a3 genes in the tooth germ at cap stage. Aldh1a2 showed strong expression only in the dental follicle of the tooth germ, but Aldh1a3 expression was weakly observed throughout the tooth germ. Aldh1a2 could be used as a new marker of dental follicle.

Multiple calcifying hyperplastic dental follicles: A case report

Aydin, Ulkem,Baykul, Timucin,Yildirim, Benay,Yildirim, Derya,Bozdemir, Esin,Karaduman, Ayse Korean Academy of Oral and Maxillofacial Radiology 2013 Imaging Science in Dentistry Vol.43 No.4

This report describes a 31-year-old female patient with six impacted teeth. The crowns of the impacted teeth were surrounded with cyst-like lesions with a mixed internal structure and well-defined cortical borders. Microscopic examination of the specimen obtained from the follicle of the left mandibular third molar tooth revealed loose to moderately dense collagenous connective tissue with abundant calcified material and sparse epithelial islands. A diagnosis of multiple calcifying hyperplastic dental follicles was made.

Multiple calcifying hyperplastic dental follicles: A case report

Ulkem Aydin,Timucin Baykul,Benay Yildirim,Derya Yildirim,Esin Bozdemir 대한영상치의학회 2013 Imaging Science in Dentistry Vol.43 No.4

This report describes a 31-year-old female patient with six impacted teeth. The crowns of the impacted teeth were surrounded with cyst-like lesions with a mixed internal structure and well-defined cortical borders. Microscopic examination of the specimen obtained from the follicle of the left mandibular third molar tooth revealed loose to moderately dense collagenous connective tissue with abundant calcified material and sparse epithelial islands. A diagnosis of multiple calcifying hyperplastic dental follicles was made.

Regulation of Osteoprotegerin and Receptor activator of nuclear factor kappa-Β ligand in Rat Molar Eruption

양동욱,김현수,이경은,김선헌 대한구강해부학회 2021 대한구강해부학회지 Vol.42 No.1

The eruptive movement of tooth germs in the alveolar bone is supposed to be timely regulated by molecules that involve the differentiation of osteoclasts for preparing a tooth eruption pathway. Thus, the detection of the molecules is the first to understand the movement. This study hypothesized that molecules are expressed differentially from tooth germs themselves with follicles in eruptive movement. To verify this, genes were detected in rat molar germs using differential display-PCR. The localization and expression of the detected molecules were evidenced by immunofluorescence, and real-time PCR, and western blot, respectively. Osteoprotegerin was one of the differentially expressed molecules between the cap stage (third molar germs before the eruptive movement) and the root formation stage (2nd molar germs after the eruptive movement) at postnatal day nine. Osteoprotegerin was localized in follicular and perifollicular tissues, which are overlaying developing the third molar germs. The osteoprotegerin expression was downregulated from day three to nine in a time dependent manner, followed by the upregulation at day 12. In contrast, receptor activator of nuclear factor kappa-Β ligand was expressed strongly in follicular tissues overlaying the second molar germs, and up-regulated in time-dependently. The treatment of alendronate, a second-generation bisphosphonate for nine days, revealed the upregulation of osteoprotegerin and downregulation of receptor activator of nuclear factor kappa-Β ligand. These results suggest that dental follicles may control the eruptive movement of tooth germs by regulating receptor activator of nuclear factor kappa-Β ligand and osteoprotegerin expression in dental follicles.

미성숙 매복지치의 치낭, 치수, 치근유두 조직에서 다능성 줄기세포의 분리와 특성화에 대한 연구

송정호,박봉욱,변준호,강은주,노규진,신상훈,김욱규,김종렬,Song, Jung-Ho,Park, Bong-Wook,Byun, June-Ho,Kang, Eun-Ju,Rho, Gyu-Jin,Shin, Sang-Hun,Kim, Uk-Kyu,Kim, Jong-Ryoul 대한구강악안면외과학회 2010 대한구강악안면외과학회지 Vol.36 No.3

Introduction: The first aim of this study was to isolate the dental tissue-derived stem cells from the dental follicle (DF), dental pulp (DP), and root apical papilla (RAP) of the extracted wisdom teeth. Second was to evaluate their characterization with the expressions of transcription factors and cell surface markers. Finally, their ability of the in vitro multi-lineage differentiations into osteogenic and adipogenic cells were compared, respectively. Materials and Methods: Dental tissues, including dental follicle, dental pulp, and root apical papilla, were separated in the extracted wisdom teeth. These three dental tissues were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with supplements, respectively. After passage 3, the homogeneous shaped dental tissue-derived cells were analyzed the expression of transcription factors (Oct-4, Nanog and Sox-2) and cell surface markers (CD44, CD90 and CD105) with reverse transcription polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS) analysis. In order to evaluate in vitro multi-lineage differentiations, the culture media were changed to the osteogenic and adipogenic induction mediums when the dental tissue-derived cells reached to passage 3. The characteristics of these three dental tissue-derived cells were compared with immunohistochemistry. Results: During primary culture, heterogenous and colony formatted dental tissue-derived cells were observed in the culture plates. After passage 2 or 3, homogenous spindle-like cells were observed in all culture plates. Transcription factors and mesenchymal stem cell markers were positively observed in all three types of dental tissue-derived cells. However, the quantity of expressed transcription factors was most large in RAP-derived cells. In all three types of dental tissue-derived cells, osteogenic and adipogenic differentiations were observed after treatment of specific induction media. In vitro adipogenic differentiation was similar among these three types of cells. In vitro osteogenic differentiation was most strongly and frequently observed in the RAP-derived cells, whereas rarely osteogenic differentiation was observed in the DP-derived cells. Conclusion: These findings suggest that three types of human dental tissue-derived cells from extracted wisdom teeth were multipotent mesenchymal stem cells, have the properties of multi-lineage differentiations. Especially, stem cells from root apical papilla (SCAP) have much advantage in osteogenic differentiation, whereas dental follicle cells (DFCs) have a characteristic of easy adipogenic differentiation.

Effect of Monocyte Chemotactic Protein-1 on Osteoprotegerin Gene Expression

Ren, Yi,Wise, Gary E. Korean Academy of Oral Biology and the UCLA Dental 2003 International Journal of Oral Biology Vol.28 No.1

Tooth eruption requires the recruitment of mononuclear cells (osteoclast precursors) into the dental follicle where they fuse to form osteoclasts that resorb the alveolar bone for tooth eruption. In the rat first mandibular molar, the dental follicle expresses the monocyte chemotactic protein-1 (MCP-1) gene at the time of maximal influx of mononuclear cells into the follicle, day 3 postnatally. At this time, the expression of the osteoprotegerin (OPG) gene also is reduced such that osteoclast formation would not be inhibited. Thus, at day 3, a maximal number of osteoclasts are seen on the alveolar bone surrounding the unerupted tooth. In this study, we wished to determine if MCP-1, in addition to its potential chemotactic role, also contributed to the decrease in the gene expression of OPG in cultured dental follicle cells. The results showed that indeed NCP-1 reduced OPG gene expression in both a time-and dosage-dependent manner with maximal inhibition at 3 hrs at a concentration of 10 ng/ml. Immunostaining showed that MCP-1 reduced the protein levels of OPG in the cultured cells. Thus MCP-1 is another molecule produced by the dental follicle that may inhibit OPG gene expression at a critical time to enable osteoclast formation to occur for tooth eruption.