• 대한치과의사협회지
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• 제55권9호
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• pp.592-603
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• 2017

Purpose: The effects of various concentrations of ascorbic acid on stem cell spheroids derived from intraoral areas are not known yet. Thus, the purpose of this study is to evaluate the effects of different concentrations of ascorbic acid on the morphology and cellular viability of stem cell spheroids derived from the gingival tissues. Materials and Methods: Stem cells were plated onto silicon elastomer-based concave microwells and grown in the presence of ascorbic acid at concentrations ranging from 0.003% to 0.3%. The morphology of the cells was viewed under an inverted microscope at day 1, 2, 3 and 5. Qualitative live/dead assay and quantitative cellular viability using Cell Counting Kit-8 were performed on day 2 and day 5. Results: Gingiva-derived stem cells formed spheroids irrespective of ascorbic acid concentration in silicon elastomer-based concave microwells. Increase in the diameter of spheroid were seen with higher concentrations of ascorbic acid. Higher cellular viability was seen in higher concentrations of ascorbic acid. Conclusion: Within the experimental setting, the application of ascorbic acid on stem-cell spheroids produced an increase in the size and higher viability with higher dosage. It can be suggested ascorbic acid be applied with stem cell spheroids for tissue engineering purposes.

• 대한치과의사협회지
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• 제55권12호
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• pp.828-840
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• 2017

In recent years, many researchers and clinicians found interest in regenerative medicine using induced pluripotent stem cells (iPSCs) with biomaterials due to their pluripotency, which is able to differentiate into any type of cells without human embryo, which of use is ethically controversial. However, there are limitations to make iPSCs from adult somatic cells due to their low stemness and donor site morbidity. Recently, to overcome above drawbacks, dental tissue-derived iPSCs have been highlighted as a type of alternative sources for their high stemness, easy gathering, and their complex (ectomesenchymal) origin, which easily differentiate them to various cell types for nerve, vessel, and other dental tissue regeneration. In other part, utilizing biomaterials for regenerative medicine using cell is recently highlighted because they can modulate cell adhesion, proliferation and (de)differentiation. Therefore, this paper will convey the overview of advantages and drawbacks of dental tissue-derived iPSCs and their future application with biomaterials.

• International Journal of Oral Biology
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• 제32권1호
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• pp.13-22
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• 2007

The stem cell research is emerging as a cutting edge topic for a new treatment for many chronic diseases. Recently, dental stem cell would be possible for regeneration of tooth itself as well as periodontal tissue. However, the study of the cell characterization is scarce. Therefore, we performed the genetic profiling and the characterization of mouse fetus/neonate derived dental tissue and cell to find the identification during dental development. We separated dental arch from mandibles of 14.5 d fetal mice and neonate 0 d under the stereoscope, and isolated dental cells primarily from the tissues. Then, we examined morphology and the gene expression profiles of the primary cells and dental tissues from fetus/neonate and adult with RT-PCR. Primary dental cells showed heterogeneous but the majority was shown as fibroblast-like morphology. The change of population doubling time levels (PDLs) showed that the primary dental cells have growth potential and could be expanded under our culture conditions without reduction of growth rate. Immunocytochemical and flow cytometric analyses were performed to characterize the primary dental cell populations from both of fetus (E14.5) and neonate. Alpha smooth muscle actin (${\alpha}-SMA$), vimentin, and von Willebrand factor showed strong expression, but desmin positive cells were not detected in the primary dental cells. Most of the markers were not uniformly expressed, but found in subsets of cells, indicating that the primary dental cell population is heterogeneous, and characteristics of the populations were changed during culture period. And mesenchymal stem cell markers were highly expressed. Gene expression profile showed Wnt family and its related signaling molecules, growth factors, transcription factors and tooth specific molecules were expressed both fetal and neonatal tissue. The tooth specific genes (enamelin, amelogenin, and DSPP) only expressed in neonate and adult stage. These expression patterns appeared same as primary fetal and neonatal cells. In this study we isolated primary cells from whole mandible of fetal and neonatal mice. And we investigated the characteristics of the primary cells and the profile of gene expressions, which are involved in epithelial-mesenchymal interactions during tooth development. Taken together, the primary dental cells in early passages or fetal and neonatal mandibles could be useful stem cell resources.

• International Journal of Oral Biology
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• 제33권3호
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• pp.91-95
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• 2008

Embryonic stem cells have a pluripotency and a potential to differentiate to all type of cells. In our previous study, we have shown that embryonic stem cells (ESCs) lines can be generated from murine parthenogenetic embryos. This parthenogenetic ESCs line can be a useful stem cell source for tissue repair and regeneration. The defect in full-term development of parthenogenetic ESCs line enables researchers to avoid the ethical concerns related with ESCs research. In this study, we presented the results demonstrating that parthenogenetic ESCs can be induced into osteogenic cells by supplementing culture media with ascorbic acid and $\beta$-glycerophosphate. These cells showed morphologies of osteogenic cells and it was proven by Von Kossa staining and Alizarin Red staining. Expression of marker genes for osteogenic cells (osteopontin, osteonectin, alkaline phosphatase, osteocalcin, bone-sialoprotein, collagen type1, and Cbfa1) also confirmed osteogenic potential of these cells. These results demonstrate that osteogenic cells can be generated from parthenogenetic ESCs in vitro.

• Journal of Periodontal and Implant Science
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• 제39권2호
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• pp.119-128
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• 2009

Purpose: The aim of this study was to evaluate the stemness of cells from canine dental tissues and bone marrow. Methods: Canine periodontal ligament stem cells (PDLSC), alveolar bone stem cells (ABSC) and bone marrow stem cells(BMSC) were isolated and cultured. Cell differentiations (osteogenic, adipogenic and chondrogenic) and surface antigens (CD146, STRO-1, CD44, CD90, CD45, CD34) were evaluated in vitro. The cells were transplanted into the subcutaneous space of nude mice to assess capacity for ectopic bone formation at 8 weeks after implantation. Results: PDLSC, ABSC and BMSC differentiated into osteoblasts, adipocytes and chondrocytes under defined condition. The cells expressed the mesenchymal stem cell markers differently. When transplanted into athymic nude mice, these three kinds of cells with hydroxyapatite /${\beta}$- tricalcium phosphate (HA/TCP) carrier showed ectopic bone formation. Conclusions: This study demonstrated that canine dental stem cells have stemness like bone marrow stem cells. Transplantation of these cells might be used as a therapeutic approach for dental stem cell-mediated periodontal tissue regeneration.

• Journal of Periodontal and Implant Science
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• 제41권4호
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• pp.192-200
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• 2011

Purpose: The aim of this study is to compare the gene expression profile in mesenchymal stem cells derived from dental tissues and bone marrow for characterization of dental stem cells. Methods: We employed GeneChip analysis to the expression levels of approximately 32,321 kinds of transcripts in 5 samples of bone-marrow-derived mesenchymal stem cells (BMSCs) (n=1), periodontal ligament stem cells (PDLSCs) (n=2), and dental pulp stem cells (DPSCs) (n=2). Each cell was sorted by a FACS Vantage Sorter using immunocytochemical staining of the early mesenchymal stem cell surface marker STRO-1 before the microarray analysis. Results: We identified 379 up-regulated and 133 down-regulated transcripts in BMSCs, 68 up-regulated and 64 down-regulated transcripts in PDLSCs, and 218 up-regulated and 231 down-regulated transcripts in DPSCs. In addition, anatomical structure development and anatomical structure morphogenesis gene ontology (GO) terms were over-represented in all three different mesenchymal stem cells and GO terms related to blood vessels, and neurons were over-represented only in DPSCs. Conclusions: This study demonstrated the genome-wide gene expression patterns of STRO-$1^+$ mesenchymal stem cells derived from dental tissues and bone marrow. The differences among the expression profiles of BMSCs, PDLSCs, and DPSCs were shown, and 999 candidate genes were found to be definitely up- or down-regulated. In addition, GOstat analyses of regulated gene products provided over-represented GO classes. These data provide a first step for discovering molecules key to the characteristics of dental stem cells.

• Restorative Dentistry and Endodontics
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• 제37권1호
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• pp.34-40
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• 2012

Objectives: The aim was to confirm the stem cell-like properties of the dental pulp stromal cells and to evaluate the morphologic changes during in vitro chondrogenesis. Materials and Methods: Stromal cells were outgrown from the dental pulp tissue of the premolars. Surface markers were investigated and cell proliferation rate was compared to other mesenchymal stem cells. Multipotency of the pulp cells was confirmed by inducing osteogenesis, adipogenesis and chondrogenesis. The morphologic changes in the chondrogenic pellet during the 21 day of induction were evaluated under light microscope and transmission electron microscope. TUNEL assay was used to evaluate apoptosis within the chondrogenic pellets. Results: Pulp cells were CD90, 105 positive and CD31, 34 negative. They showed similar proliferation rate to other stem cells. Pulp cells differentiated to osteogenic, adipogenic and chondrogenic tissues. During chondrogenesis, 3-dimensional pellet was created with multi-layers, hypertrophic chondrocyte-like cells and cartilage-like extracellular matrix. However, cell morphology became irregular and apoptotic cells were increased after 7 day of chondrogenic induction. Conclusions: Pulp cells indicated mesenchymal stem cell-like characteristics. During the in vitro chondrogenesis, cellular activity was superior during the earlier phase (within 7 day) of differentiation.

• International Journal of Oral Biology
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• 제43권2호
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• pp.77-82
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• 2018

The mesenchymal stem cells (MSCs) that reside in dental tissues hold a great potential for future applications in regenerative dentistry. In this study, we used human dental pulp cells, isolated from the molars (DPCs), in order to establish the organoid culture. DPCs were established after growing pulp cells in an MSC expansion media (MSC-EM). DPCs were subjected to organoid growth media (OGM) in comparison with human dental pulp stem cells (DPSCs). Inside the extracellular matrix in the OGM, the DPCs and DPSCs readily formed vessel-like structures, which were not observed in the MSC-EM. Immunocytochemistry analysis and flow cytometry analysis showed the elevated expression of CD31 in the DPCs and DPSCs cultured in the OGM. These results suggest endothelial cell-prone differentiation of the DPCs and DPSCs in organoid culture condition.

• 구강회복응용과학지
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• 제25권2호
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• pp.191-200
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• 2009

줄기세포는 미분화 상태의 세포로 지속적인 자가 분열능과 특정 세포로 분화 할 수 있는 분화능을 지니고 있으며 이러한 특성으로 말미암아 난치병의 새로운 장을 열 것이라는 기대감을 얻고 있다. 즉, 세포이식 후 특정 세포로 분화 유도를 시켜 기존의 치료 방법으로는 치료가 되지 않았던 많은 의학적 난제들을 풀 수 있는 열쇠인 것이다. 치아는 평생에 걸쳐 발거되는 조직으로 치아줄기세포는 얻기가 비교적 쉬워서 다른 성체줄기세포들보다 더 높이 평가되고 있다. 과잉치는 전 세계 다양한 인종에서 발견되는 중요한 임상적 질환이다. 과잉치의 유병율은 관련 연구 보고서마다 다양하나 소아치과 의사들의 경우 임상에서 자주 과잉치를 발거하고 있다. 그러나, 현재까지 진행된 줄기세포 관련 연구들을 살펴보면 과잉치의 줄기세포에 관한 보고는 없으며, 이는 줄기세포로 이용 가능한 중요한 자원을 파기하고 있는 것은 아닌지 의문이 생긴다. 그러므로 본 연구는 과잉치에 포함된 세포가 줄기세포의 특징을 가지고 있는지를 밝혀내기 위해 진행되었다.

• 대한소아치과학회지
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• 제46권3호
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• pp.337-342
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• 2019

이 연구의 목적은 발치한 매복 과잉치에서 얻은 치수 유래 세포의 줄기세포 특성을 유세포 분석을 통해 알아보는 것이다. 15명의 환자로부터 채취한 정중 과잉치의 치수 세포를 계대 배양하였고, 3계대와 10계대 세포들을 유세포 분석을 이용하여 분석하였다. 간엽성 줄기세포 표지자 관찰에 사용된 항체 표지자(CD)는 양성 표지자로는 CD 73, CD 90, 그리고STRO-1 와 음성 표지자로는 CD 34, CD 45 이었다. 3계대에서 CD 73, CD 90은 각각 94.82%, 98.86%의 양성반응을, CD 34, CD 45는 각각 2.25%, 2.52%로 음성 반응을 보였으며, STRO-1은 20.93%를 나타냈다. 10계대에서는 CD 73, CD 90은 각각 96.62%, 98.61%의 발현을 보였지만, CD 34, CD 45는 각각 3.86%, 4.14%를 나타냈다. STRO-1은 35.62%로 발현되었다. 이상의 결과에서 과잉치 치수 유래 세포는 간엽성 줄기세포의 특성을 가지며, 3계대와 10계대 모두에서 간엽성 줄기세포의 특성을 유지하고 있다고 사료된다. 이에 빠른 성장 속도와 늦은 계대까지 유지되는 줄기세포능을 고려할 때, 치아 유래 줄기세포의 공여부로서 매복 과잉치의 충분한 활용 가능성을 확인하였다.