• Restorative Dentistry and Endodontics
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• 제29권4호
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• pp.399-408
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• 2004

Odontoblasts are responsible for the formation and maintenance of dentin. They are known to synthesize unique gene products including dentin sialophosphoprotein (DSPP). Another unique genes of the cells remain unclear. OD314 was isolated from the odontoblasts/pulp cells of rats and partially characterized as an odontoblast-enriched gene (Dey et al., 2001). This study aimed to elucidate the biological function of OD314, relating to odontoblast differentiation and dentinogenesis. After determining the open reading frame (ORP) of OD314 by transient transfection analysis using green fluorescent protein (GPP) expression vector, mRNA in-situ hybridization, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and western analysis were performed. The results were as follows: 1. In in-situ hybridization, OD314 mRNAs were expressed in odontoblasts of developing coronal and root pulp. 2. OD314 was a novel protein encoding 154 amino acids, and the protein was mainly expressed in cytoplasm by transient transfection analysis. 3. Mineralized nodules were associated with multilayer cell nodules in the culture of human dental pulp cells and first detected from day 21 using alizarin-red S staining. 4. In RT-PCR analysis, OD314, osteocalcin (OC) and DSPP strongly expressed throughout 28 days of culture. Whereas, osteonectin (ON) mRNA expression stayed low up to day 14, and then gradually decreased from day 21. 5. Western blots showed an approximately 17 kDa band. OD314 protein was expressed from the start of culture and then increased greatly from day 21. In conclusion, OD314 is considered as an odontoblast-enriched gene and may play important roles in odontoblast differentiation and dentin mineralization.

• Journal of Biosystems Engineering
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• 제38권2호
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• pp.113-120
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• 2013

Purpose: Stem cells provide new opportunities in the regenerative medicine for human or animal tissue regeneration. In this study, we report an efficient method for the modulating behaviors of electro-active stem cells by micro-electric current stimulation (mES) without using chemical agents, such as serum or induction chemicals. Methods: Dental pulp stem cells (DPSCs) were cultured on the tissue culture dish in the mES system. To find a suitable mES condition to promote the DPSC functions, the response surface analysis was used. Results: We found that a working micro-current of 38 ${\mu}A$ showed higher DPSC proliferation compared with other working conditions. The mES altered the expressions of intracellular and extracellular proteins compared to those in unstimulated cells. The mES with 38 ${\mu}A$ significantly increased osteogenesis of DPSCs compared with ones without mES. Conclusions: Our findings indicate that mES may induce DPSC proliferation and differentiation, resulting in applying to DPSCs-based human or animal tissue regeneration.

• International Journal of Stem Cells
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• 제15권3호
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• pp.301-310
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• 2022

Background and Objectives: RUNX2 plays an essential role during the odontoblast differentiation of dental pulp stem cells (DPSCs). RUNX2 Exon 5 is an alternative exon and essential for RUNX2 transcriptional activity. This study aimed to investigate the regulatory mechanisms of RUNX2 exon 5 alternative splicing in human DPSCs. Methods and Results: The regulatory motifs of RUNX2 exon 5 were analyzed using the online SpliceAid program. The alternative splicing of RUNX2 exon 5 in DPSCs during mineralization-induced differentiation was analyzed by RT-PCR. To explore the effect of splicing factor YBX1 on exon 5 alternative splicing, gaining or losing function of YBX1 was performed by transfection of YBX1 overexpression plasmid or anti-YBX1 siRNA in DPSCs. Human RUNX2 exon 5 is evolutionarily conserved and alternatively spliced in DPSCs. There are three potential YBX1 binding motifs in RUNX2 exon 5. The inclusion of RUNX2 exon 5 and YBX1 expression level increased significantly during mineralization-induced differentiation in DPSCs. Overexpression of YBX1 significantly increased the inclusion of RUNX2 exon 5 in DPSCs. In contrast, silence of YBX1 significantly reduced the inclusion of exon 5 and the corresponding RUNX2 protein expression level. Knockdown of YBX1 reduced the expression of alkaline phosphatase (ALP) and osteocalcin (OC) and the mineralization ability of DPSCs, while overexpression of YBX1 increased the expression of ALP and OC and the mineralization ability of DPSCs. Conclusions: Human RUNX2 exon 5 is conserved evolutionarily and alternatively spliced in DPSCs. Splicing factor YBX1 promotes the inclusion of RUNX2 exon 5 and improves the mineralization ability of DPSCs.

• 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.

• 대한소아치과학회지
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• 제43권4호
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• pp.419-426
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• 2016

본 연구는 상악 매복 과잉치를 가지고 전신질환 및 의과 병력이 없는 만 5 - 9세 사이의 남녀 20명으로부터 서면동의를 얻고 상악 전치부에 매복된 과잉치를 발치하고 치수세포를 채취하였다. 채취 후 세포의 1차 배양 동안 오염된 3명(남자 2명, 여자 1명)을 제외하고 총 17명(남자 10명, 여자 7명)을 대상으로 하였다. 이번 연구에서 과잉치 치수조직으로부터 유래한 세포의 계대 배양에 소요된 평균 시간은 $2.91{\pm}0.29$일이었다. 최초 치수조직으로부터 세포를 얻은 후 80% confluency를 얻는데 소요되는 시간이 $4.53{\pm}0.94$일로 가장 많이 걸렸고, 이후 계대부터는 $2.73{\pm}0.32$일이 소요되었다. 남자의 비율은 58.82%, 여자의 비율은 41.18%이었다. 여자의 평균 소요시간은 $2.81{\pm}0.27$일 이었으며, 남자는 $2.98{\pm}0.29$이었다. 과잉치가 역위된 비율은 82.35%, 정위 비율은 17.65%였다. 평균 계대 소요 배양 시간은 역위가 $2.94{\pm}0.30$일, 정위는 $2.80{\pm}0.20$일이었다. 원뿔형(Conical type)은 76.47%였고, 그 외 형태는 23.53%였다. 원뿔형의 평균 소요시간은 $2.92{\pm}0.31$이었고, 그 외 형태의 소요 시간은 $2.88{\pm}0.22$이었다. 매복 방향과 형태에 따른 계대 배향 시간은 통계적으로 유의한 차이를 보이지 않았다. 향후 초기 계대와 후기 계대에서 얻어진 세포의 줄기세포로써의 능력을 평가하는 후속 연구들이 필요하며, 3일 이내의 계대 배양 시간이 소요된 점을 고려할 때, 연구의 효율성과 빠른 배양시간 등은 과잉치 유래세포가 치아유래 줄기세포의 연구에 활용가능성이 충분하리라 판단되었다.

• Restorative Dentistry and Endodontics
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• 제36권5호
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• pp.397-408
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• 2011

연구목적: 이 연구에서는 mineral trioxide aggregate 제재인 white ProRoot MTA (wMTA)와 수산화칼슘 제재인 Dycal을 인간치수세포에 적용한 후 치수세포의 분화와 증식, 석회화, 신생혈관형성(angiogenesis) 그리고 염증에 관여하는 유전자들의 발현 변화를 비교하였다. 연구 재료 및 방법: 실험군은 wMTA와 Dycal을 테플론 튜브(내경 10 mm, 길이 1 mm)에 담아 4시간 경화시킨 후 일차세포배양한 인간치수세포에 적용하였고, 대조군은 빈 튜브만을 적용하였다. 3시간, 6시간, 9시간, 24시간 후 total RNA를 추출하고 oligonucleotide microarray 방법을 통하여 유전자 발현 양상을 분석하였다. 위의 결과를 역전사 중합효소 연쇄반응(reverse transcriptase polymerase chain reaction)으로 재확인하였다. 결과: wMTA를 적용한 실험군에서 24,546개의 유전자 중 43개 유전자의 발현이 2배 이상 증가하였으며(예. BMP2, FOSB, THBS1, EDN1, IL11, COL10A1, TUFT1, HMOX1) 25개 유전자의 발현이 50% 이하로 감소하였다(예. SMAD6, TIMP2, DCN, SOCS2, CEBPD, KIAA1199). Dycal을 적용한 실험군에서 239개 유전자의 발현이 2배 이상 증가하였으며(예. BMP2, BMP6, SMAD6, IL11, FOS, VEGFA, PlGF, HMOX1, SOCS2, CEBPD, KIAA1199) 358개 유전자의 발현이 50% 이하로 감소하였다(예. EDN1, FGF). 결론: wMTA를 적용한 치수세포에서는 분화와 증식 그리고 석회화에 관여하는 유전자들의 변화가 관찰되었다. Dycal을 적용한 치수세포에서는 분화와 증식 그리고 신생혈관형성에 관여하는 유전자들의 변화가 관찰되었다. 또 Dycal이 염증에 관여하는 유전자들을 더 많이 발현시키는 양상을 보였다.

• 대한치과보철학회지
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• 제36권3호
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• pp.483-495
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• 1998

The purpose of this study was to evaluate the human pulpal response to Dentin Bonding Desensitizer. Class V cavities were prepared on the buccal surfaces of the first premolars and Dentin Bonding Desensitizer(ALL-BOND Desensitizer, Bisco, Inc. U.S.A.) was applicated in ten experimental teeth, or ZOE(PROPAC, GC Co. TOKYO, JAPAN) cement in eight control teeth and cavities were filled with light curing glass ionomer(Fuji II LC, GC Co., TOKYO, JAPAN). At 3-day and 25-day postoperative interval. pulpal response was observed and evaluated histologically with light microscope. The results were as follows. ; 1. At 3-day postoperative interval, the control teeth were grade 1 inflammatory cell response and grade 1 connective tissue response. 2. At 25-day postoperative interval, all control teeth were grade 1 inflammatory cell response and in three control teeth grade 1 connective tissue response were observed, and one teeth showed grade 2 connective tissue response. 3. At 3-day postoperative interval, the experimental teeth were grade 1 inflammatory cell response and grade 1 connective tissue response. Below the cavity, a few inflammatory cell(PMNs) in odontoblastic layer, increased blood vessels and pulpal cells were seen and this pulpal response was similar to control teeth. 4. At 25-day postoperative interval, in four experimental teeth grade 1 inflammatory cell response and grade 1 connective tissue response were observed, and one experimental teeth showed mild inflammatory response. 5. At 3-day and 25-day postoperative interval, no reparative dentin deposition was seen. 6. Both experimental and control group, pulpal response showed difference between 3 and 25-day of postoperative interval. In control teeth, increased predentin and pulpal cells were seen and in experimental teeth, congestion of blood vessels and increased pulpal cells were seen. In conclusion, the pulpal irritation due to this Dentin Bonding Desensitizer was not severe, and it was considered that the agent was not harmful to the human pulp.

• Restorative Dentistry and Endodontics
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• 제18권2호
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• pp.261-276
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• 1993

The purpose of this study was to evaluate for the cytotoxicity of glass-ionomer cement liners(GC liningcement, Ketac-bond, Vitrebond and Fuji lining LC) on the fibroblasts cultured from human pulp. The fibroblasts were cultured in DMEM-10% FBS medium. The measurement of pH, succinate dehydrogenase (SDH) activity test and $^{51}Chromium$ release test were performed. Viable cell count and $^{14}C$-leucine incorporation rate were evaluated following culture time of 2, 4 and 6 days. The results of this study were as follows : 1. The pH in all cements was to be neutralized as time elapsed, and Fuji lining LC was the lowest pH value among them. 2. SDH activity was more inhibited in GC lining cement and Vitrebond than Ketac-bond and Fuji lining LC with the setting process, and GC lining cement and Ketac-bond were reduced after 5 minute's setting and then elevated as time elapsed. 3. In SDH activity test following exposure time, the activity in Vitrebond, GC lining cement and Fuji lining LC was inhibited with increased exposure time, but it was fairly constant in Ketac-bond. 4. Overall the liquid component was more inhibited than the powder component of glass-ionomer cement in SDH activity test. 5. In $^{51}Cr$-release test, Fuji lining LC was the most released of all the cements tested and followed by : Vitrebond, Ketac-bond, GC lining cement. 6. In viable cell count, the number of cells increased as the culture day proceeded in Ketac-bond, but they decreased in GC lining cement. Fuji lining LC was only observed after 2 days culture and there was not observed the whole culture days in Vitrebond. 7. In $^{14}C$-leucine incorporation rate test, protein synthesis was decreased with the number of culture days in GC lining cement, Vitrebond and Fuji lining LC, but it was followed that of control in Ketacbond.

• Restorative Dentistry and Endodontics
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• 제25권4호
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• pp.606-618
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• 2000

Dental pulp infection is most commonly caused by extensive dental caries, and some bacterial species invade root canals; bacterial components and products are thought to be associated with the pathogenesis of periapical periodontitis. A principle driving force behind pulpal disease response appears to lie in the host immune system's to bacteria and their products. We examined the production of interleukin $1{\beta}$ (IL-$1{\beta}$) and tumor necrosis factor ${\alpha}$(TNF-${\alpha}$) from human peripheral mononuclear cells, lymphocytes and monocytes stimulated by heat-killed Acitnobacillus actinomycetemcomitans (ATCC 29523), Porphyromonas gingivalis (ATCC 33277) and Prevotella intermedia (ATCC 25611), and also by their sonicated bacterial extracts (SBE), respectively. The effects of three strains of heat-killed bacteria and their SBEs on the morphology of cultured blood cell lines HL-60 (KCLB 10240) and J774A.1 (KCLB 40067) were observed under the inverted microscope. Ultrastructural changes of J774A.1 exposed to heat-killed P. intermedia and its SBE were investigated using transmission electron microscopy. Production of IL-$1{\beta}$ was reduced in human peripheral mononuclear cells after stimulation by sonic bacterial extracts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. Heat-killed and sonic extract of P. gingivalis inhibited the production of TNF-${\alpha}$ in peripheral mononuclear cells. Production of TNF-${\alpha}$ was inhibited in peripheral monocytes after stimulation by sonic extracts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. HL-60 and J 774A.1 cells showed granular degeneration after treatment with heat-killed and sonic extracts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia Chromatin margination and shrinkage were observed in 774A.1 treated with heat-killed P. intermedia. Cell wall structure and organelles were destroyed and vacuoles were formed in cytoplasm in J774A.1 treated with P. intermedia sonic extract. These results suggest that A actinomycetemcomitans, P gingivalis and P intermedia may have an important role in the formation and progression of pulpal diseases via both modulation of production of IL-$1{\beta}$ and TNF-${\alpha}$ from blood mononuclear cells and cytopathic effects.

• Restorative Dentistry and Endodontics
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• 제47권4호
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• pp.42.1-42.11
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• 2022

Objective: This study investigated the effects of various concentrations of sodium hypochlorite (NaOCl) on human whole-blood clotting kinetics, the structure of the blood clots formed, and transforming growth factor (TGF)-β1 release. Materials and Methods: Human whole blood was collected from 5 healthy volunteers and divided into 4 groups: CG (control, 0.5 mL of blood), BN_0.5 (0.5 mL of blood with 0.5 mL of 0.5% NaOCl), BN₃ (0.5 mL of blood with 0.5 mL of 3% NaOCl), and BN_5.25 (0.5 mL of blood with 0.5 mL of 5.25% NaOCl). The effects of NaOCl on clotting kinetics, structure of fibrin and cells, and release of TGF-β1 were assessed using thromboelastography (TEG), scanning electron microscopy (SEM), and enzyme-linked immunosobent assay, respectively. Statistical analysis was conducted using the Kruskal Wallis and Mann-Whitney U tests, followed by the post hoc Dunn test. A p value < 0.05 indicated statistical significance. Results: The blood samples in BN_0.5 and BN₃ did not clot, whereas the TEG of BN_5.25 showed altered clot formation. Samples from the CG and BN₃ groups could only be processed with SEM, which showed that the latter lacked fibrin formation and branching of fibers, as well as clumping of red blood cells with surface roughening and distortion. TGF-β1 release was significantly highest in BN₃ when all groups were compared to CG (p < 0.05). Conclusions: Each concentration of NaOCl affected the release of TGF-β1 from blood clots and altered the clotting mechanism of blood by affecting clotting kinetics and cell structure.