[논문]유치 줄기세포에 대한 다양한 규산칼슘계 재료의 세포독성

윤지혜; 유용욱; 안은숙; 이준; 안소연

doi:10.5933/jkapd.2019.46.1.85

[국내논문] 유치 줄기세포에 대한 다양한 규산칼슘계 재료의 세포독성
Cytotoxicity of Various Calcium Silicate-based Materials with Stem Cells from Deciduous Teeth 원문보기

大韓小兒齒科學會誌 = Journal of the Korean academy of pediatric dentistry, v.46 no.1, 2019년, pp.85 - 92

윤지혜 (원광대학교 치과대학 소아치과학교실) , 유용욱 (원광대학교 치과대학 구강생화학교실) , 안은숙 (경복대학교 치위생과) , 이준 (원광대학교 치과대학 구강악안면외과학교실) , 안소연 (원광대학교 치과대학 소아치과학교실)

초록
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이 연구의 목적은 유치 줄기세포에 대해 다양한 Calcium silicate-based material (CSM)의 세포독성을 비교하고 평가하는 것이다. Retro $MTA^{(R)}$ (RM), $EZ-Seal^{TM}$ (EZ), ENDOCEM $Zr^{(R)}$ (EN)의 powder를 세포 배지를 이용하여 용출시키고 filtering 하였다. 유치 줄기세포가 다양한 농도의 용출액 하 배양되었다. MTS assay를 통해 CSM 용출액이 세포 증식에 미치는 영향을 분석하였고, Flow cytometry analysis를 통해 세포 표현형의 변화 여부를 관찰하였다. 10% 농도의 용출액에서 배양된 유치 줄기세포의 흡광도 값은 RM > EN > EZ 순이었다(p = 0.0439). 그러나 유치 줄기세포는 재료에 관계없이 간엽줄기세포의 표현형을 유지했다. 세 종류의 CSM이 줄기세포 marker를 변형시키진 않았지만, EZ가 RM과 EN보다 세포적합성이 낮음을 알 수 있었다.

Abstract ▼ AI-Helper

The purpose of this study was to compare and evaluate the cytotoxicity of 3 calcium silicate-based materials (CSMs) on stem cells from human exfoliated deciduous teeth (SHEDs). The powder of Retro $MTA^{(R)}$ (RM), $EZ-Seal^{TM}$ (EZ) and ENDOCEM $Zr^{(R)}$ (EN) was eluted with SHED culture media and then filtered. The SHEDs were cultured in the presence of the various concentrations of the eluate. To investigate the effect of the 3 CSMs on SHED proliferation, the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was performed. Flow cytometry analysis was also performed to identify any changes in the cellular phenotype. The absorbance values of the SHEDs cultured in the eluate of samples at a 10% concentration showed the following relation: RM > EN > EZ (p = 0.0439). However, the SHEDs maintained their mesenchymal phenotype regardless of product exposure. Although the 3 CSMs did not alter the SHED stem cell markers, EZ may be a less cytocompatible than RM and EN.

Keyword

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문제 정의

The purpose of this study was to compare and evaluate the cytotoxicity of 3 CSMs with stem cells from human exfoliated deciduous teeth (SHEDs).
However, most of the studies are on pulp-derived cells and periodontal ligament cells, as mentioned above. Therefore, this study is significant as the first study to evaluate the cytotoxicity of RM, EZ and EN with SHEDs.

제안 방법

The supernatant was filtered using a 0.2 μm pore size syringe and diluted to 100%, 50%, 25%, 10%, 5% and 1% for sample preparation.
After the SHEDs were cultured in various concentrations of medium containing CSM for 3 days, the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium) assay was performed using an EZ-Cytox Cell Assay Kit (Daeil Lab Service Co., Seoul, Korea) for 1 hour at 37°C (Fig. 1B).
cells/well. After 24 hours, the solution obtained by eluting the CSM was diluted to 100%, 50%, 25%, 10%, 5% and 1% and used to replace the medium in each well. After the SHEDs were cultured in various concentrations of medium containing CSM for 3 days, the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium) assay was performed using an EZ-Cytox Cell Assay Kit (Daeil Lab Service Co.
[16], the SHEDs expressed CD29, CD105, CD146, STRO-1 and low levels of CD34. In this study, flow cytometry analysis was performed to determine the phenotypical changes caused by exposure to various CSMs. The results of the analysis of cell surface protein markers are as follows (Fig.
The purpose of this study was to analyze the cytotoxicity of RM, EZ and EN powder, which are CSMs used in pulpotomy for primary teeth. RM consists of a hydraulic calcium-zirconia complex and is characterized by a short initial setting time of 150 seconds.
To determine the cytotoxicity of the CSM eluates to SHEDs, MTS assay was performed, and the results are expressed as absorbance (optical density, OD) values.
To evaluate the cytotoxicity of the dental materials, a contact experiment between cells and the experimental materials was performed. The method of contact include direct contact, indirect contact and eluate contact[25].
5 over 3 hours after MTA manipulation and that the continuous release of CH results in the maintenance of a high pH for 78 days[27,28]. It is significant that this study excluded the influence of the pH increase in the setting process of the materials by evaluating the cytotoxicity of the un-hydrated powder only, in the pre-polymerization stage, by the elution method. Additionally, there have been many reports on the cytotoxicity and biocompatibility of various types of MTA on animal and human cells.
All experiments were performed at least in triplicate, and data from the MTS assay were analyzed using GraphPad PRISM® statistical analysis software (GraphPad, San Diego, CA).
#F8775, Sigma, USA). Prepared cell samples were analyzed for cell surface markers using a FACS Calibur^TM flow cytometer (BD Science) and CellQuest^TM Pro program (Fig. 1C).
The MTS assay was performed to test the cytotoxic effect of the 3 CSMs on SHED proliferation. The overall absorbance values of the SHEDs cultured in the eluates of various concentrations showed the following relation: EN > EZ > RM.

이론/모형

statistical analysis software (GraphPad, San Diego, CA). Significant differences among groups were identified by the Kruskal-Wallis test. The above analyses were performed using STATA 14.

후속연구

In addition, EN contains not only aluminum but also iron and magnesium oxide. Therefore, all 3 products are said to cause no discoloration, but further and longer-term studies are needed to examine their potential for tooth discoloration. In addition, zirconium oxide shortens the setting time of the material because zirconium accelerates the hydration of Portland cement[5,23].

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