• Restorative Dentistry and Endodontics
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• 제46권2호
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• pp.26.1-26.15
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• 2021

Objectives: The aim of the present systematic review was to investigate the cryopreservation process of dental pulp mesenchymal stromal cells and whether cryopreservation is effective in promoting cell viability and recovery. Materials and Methods: This systematic review was developed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement and the research question was determined using the population, exposure, comparison, and outcomes strategy. Electronic searches were conducted in the PubMed, Cochrane Library, Science Direct, LILACS, and SciELO databases and in the gray literature (dissertations and thesis databases and Google Scholar) for relevant articles published up to March 2019. Clinical trial studies performed with dental pulp of human permanent or primary teeth, containing concrete information regarding the cryopreservation stages, and with cryopreservation performed for a period of at least 1 week were included in this study. Results: The search strategy resulted in the retrieval of 185 publications. After the application of the eligibility criteria, 21 articles were selected for a qualitative analysis. Conclusions: The cryopreservation process must be carried out in 6 stages: tooth disinfection, pulp extraction, cell isolation, cell proliferation, cryopreservation, and thawing. In addition, it can be inferred that the use of dimethyl sulfoxide, programmable freezing, and storage in liquid nitrogen are associated with a high rate of cell viability after thawing and a high rate of cell proliferation in both primary and permanent teeth.

• 한국동물생명공학회지
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• 제34권1호
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• pp.2-9
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• 2019

Because mesenchymal stem cells (MSCs) maintain distinct capacities with respect to self-renewal, differentiation ability and immunomodulatory function, they have been highly considered as the therapeutic agents for cell-based clinical application. Of particular, differentiation condition alters characteristics of MSCs, including cellular morphology, expression of gene/protein and cell surface molecule, immunological property and apoptosis. However, the previous results for differentiation-related apoptosis in MSCs have still remained controversial due to varied outcomes. Therefore, the present study aimed to disclose periodical alterations of pro- and anti-apoptosis in MSCs under differentiation inductions. The human dental pulp-derived MSCs (DP-MSCs) were differentiated into adipocytes and osteoblasts during early (1 week), middle (2 weeks) and late (3 weeks) stages, and were investigated on their apoptosis-related changes by Annexin V assay, qRT-PCR and western blotting. The ratio of apoptotic cell population was significantly (p < 0.05) elevated during the early to middle stages of differentiations but recovered up to the similar level of undifferentiated state at the late stage of differentiation. In the expression of mRNA and protein, whereas expressions of pro-apoptosis-related makers (BAX and BAK) were not altered in any kind and duration of differentiation inductions, anti-apoptosis marker (BCL2) was significantly (p < 0.05) elevated even at the early stage of differentiations. The recovery of apoptotic cell population at the late stage of differentiation is expected to be associated with the response by elevation of anti-apoptotic molecules. The present study may contribute on understanding for cellular mechanism in differentiation of MSCs and provide background data in clinical application of MSCs in the animal biotechnology to develop effective and safe therapeutic strategy.

• 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.

• 한국동물생명공학회지
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• 제34권2호
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• pp.93-99
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• 2019

Telomeres are known as a specialized region in the end of chromosomes to protect DNA destruction, but their lengths are shortened by repetition of cell division. This telomere shortening can be preserved or be elongated by telomerase and TERT expression. Although a certain condition in the cells may affect to the cellular and molecular characteristics, the effect of differentiation induction to telomere length and telomerase activity in mesenchymal stem cells (MSCs) has been less studied. Therefore, the present study aimed to uncover periodical alterations of telomere length, telomerase activity and TERT expression in the dental pulp-derived MSCs (DP-MSCs) under condition of differentiation inductions into adipocytes and osteoblasts on a weekly basis up to 3 weeks. Shortening of telomere was significantly (p < 0.05) identified from early-middle stages of both differentiations in comparison with undifferentiated DP-MSCs by non-radioactive chemiluminescent assay and qRT-PCR method. Telomere length in undifferentiated DP-MSCs was 10.5 kb, but the late stage of differentiated DP-MSCs which can be regarded as the adult somatic cell exhibited 8.1-8.6 kb. Furthermore, the relative-quantitative telomerase repeat amplification protocol or western blotting presented significant (p < 0.05) decrease of telomerase activity since early stages of differentiations or TERT expression from middle stages of differentiations than undifferentiated state, respectively. Based on these results, it is supposed that shortened telomere length in differentiated DP-MSCs was remained along with prolonged differentiation durations, possibly due to weakened telomerase activity and TERT expression. We expect that the present study contributes on understanding differentiation mechanism of MSCs, and provides standardizing therapeutic strategies in clinical application of MSCs in the animal biotechnology.

• International Journal of Oral Biology
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• 제36권3호
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• pp.117-122
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• 2011

Endothelial cells are a vital constituent of most mammalian organs and are required to maintain the integrity of these tissues. These cells also play a major role in angiogenesis, inflammatory reactions, and in the regulation of thrombosis. Angiogenesis facilitates pulp formation and produces the vessels which are essential for the maintenance of tooth homeostasis. These vessels can also be used in bone and tissue regeneration, and in surgical procedures to place implants or to remove cancerous tissue. Furthermore, endothelial cell regeneration is the most critical component of the tooth generation process. The aim of the present study was to stimulate endothelial regeneration at a site of acute cyclophosphamide (CP)-induced endothelial injury by treatment with human umbilical cord-derived endothelial/mesenchymal stem cells (hEPCs). We randomly assigned 16 to 20-week-old female NOD/SCID mice into three separate groups, a hEPC ($1{\times}10^5$ cells) transplanted, 300mg/kg CP treated and saline (control) group. The mice were sacrificed on days 5 and 10 and blood was collected via the abdominal aorta for analysis. The alanine transaminase (ALT), aspartate aminotransferase (AST), serum alkaline phosphatase (s-ALP), and albumin (ALB) levels were then evaluated. Tissue sections from the livers and kidneys were stained with hematoxylin and eosin (HE) for microscopic analysis and were subjected to immunohistochemistry to evaluate any changes in the endothelial layer. CP treatment caused a weight reduction after one day. The kidney/body weight ratio increased in the hEPC treated animals compared with the CP only group at 10 days. Moreover, hEPC treatment resulted in reduced s-ALP, AST, ALT levels compared with the CP only group at 10 days. The CP only animals further showed endothelial injuries at five days which were recovered by hEPC treatment at 10 days. The number of CD31-positive cells was increased by hEPC treatment at both 5 and 10 days. In conclusion, the CP-induced disruption of endothelial cells is recovered by hEPC treatment, indicating that hEPC transplantation has potential benefits in the treatment of endothelial damage.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제29권4호
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• pp.279-288
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• 2007

In the present study, we focused on stem cells in the dental papilla of the tooth germ. The tooth germ, sometimes called the tooth bud, is the primordial structure from which a tooth is formed. The tooth germ consists of the enamel organ, the dental papilla, and the dental follicle. The dental papilla lies below a cellular aggregation of the enamel organ. Mesenchymal cells within the dental papilla are responsible for formation of dentin and pulp of a tooth. Tooth germ disappears as a tooth is formed, but that of a third molar stays in the jawbone of a human until the age of 10 to 16, because third molars grow slowly. Impacted third molar tooth germs from young adults are sometimes extracted for orthodontic treatment. In the present study, we evaluated the osteogenic activity and mineralization of cultured human dental papilla-derived cells. Dental papillas were harvested from mandible during surgical extraction of lower impacted third molar from 3 patients aged 13-15 years. After passage 3, the dental papilla-derived cells were trypsinized and subsequently suspended in the osteogenic induction DMEM medium supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM -glycerophosphate at a density of $1\;{\times}10^6\;cells/dish$ in a 100-mm culture dish. The dental papilla-derived cells were then cultured for 6 weeks and the medium was changes every 3 days during the incubation period. Dental papilla-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 7 of culture period, then decreased in intensity during the culture period. ALP mRNA level was largely elevated at 1 weeks and gradually decreased with culture time. Osteocalcin mRNA expression appeared at day 14 in culture, after that its expression continuously increased in a time-dependent manner up to day 28. The expression remained constant thereafter. Runx2 expression appeared at day 7 with no detection thereafter. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. Osteocalcin secretion was detectable in the culture medium from 1 week. The secretion of osteocalcin from dental papilla-derived cells into the medium greatly increased after 3 weeks although it showed a shallow increase by then. In conclusion, our study showed that cultured human dental papilla-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix.

• 생명과학회지
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• 제21권5호
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• pp.631-646
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• 2011

중간엽 줄기 세포는 다분화능을 가지고 있으며 골수, 지방, 태반, 치아속질, 윤활막, 편도 및 가슴샘 등 인체의 다양한 조직에서 분리된다. 중간엽 줄기세포는 조직의 항상성을 조절하며 다분화능, 분리와 조작의 용이함, 암세포로의 화학주성 및 면역 반응 조절 등의 특징을 가지고 있어서 재생 의학, 암 치료 및 식대주 질환(GVHD) 등에 이용할 수 있는 세포치료제로 주목 받고 있다. 하지만 주위 세포와 조직을 지지하고 조절하는 특징과 관련하여 중간엽 줄기세포가 혈관 생성을 촉진하고 성장인자를 분비하며 암세포를 공격하는 면역 반응을 억제함으로써 암의 진행을 촉진시킨다는 사실 또한 보고 되고 있다. 이러한 사실들로 인해 중간엽 줄기세포의 임상 적용이 제한되고 있다. 본 연구에서는 어떠한 기전을 통해서 중간엽 줄기세포가 암의 진행을 촉진하는 지지 세포로 기능하는지를 밝히기 위해서 인체 지방 조직에서 유래한 중간엽 줄기세포를 두 개의 암세포주(H460, U87MG)와 각각 공동 배양하고 microarray를 이용해서 암세포와 공동 배양되지 않은 중간엽 줄기세포와 유전자의 발현을 비교하였다. 두 암세포주와 공동배양에서 공통적으로 2배 이상 차이 나는 유전자를 DAVID (Database for Annotation, Visualization and Integrated Discovery)와 PANTHER (Protein ANalysis THrough Evolutionary Relationships)를 이용해 분석하였으며 생물학적 과정, 분자적 기능, 세포의 구성 성분, 단백질의 종류, 질병과 인체 조직 그리고 신호전달에 관련된 정보를 획득하였다. 이를 통해서 암세포는 중간엽 줄기세의 분화, 증식, 에너지 대사, 세포의 구조 및 분비기능을 조절하여 유전자의 발현 양상을 암 연관 섬유모세포(cancer associated fibroblast)와 유사한 세포로 변형 시킨다는 사실을 알 수 있었다. 본 연구의 결과는 중간엽 줄기세포를 이용한 임상 치료제의 효과와 안정성을 개선하는데 응용될 수 있을 것이다.