Behfarnia Parichehr, Fazlalizadeh Sheida, Nasr-Esfahani Mohammad Hossein, Ejeian Fatemeh, Mogharehabed Ahmad
Department of Periodontics, Dental Implant Research Center, School of Dentistry, Dental Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran.
Department of Periodontics, Ardabil University of Medical Sciences, Ardabil, Iran.
Dent Res J (Isfahan). 2023 Oct 26;20:105. eCollection 2023.
The aim of the present study is to determine the possibility of isolation and characterization of the human periodontal ligament stem cells (hPDLSCs) using limited harvested periodontal ligament (PDL) tissue of only one patient's wisdom teeth (2-4 teeth) under the more compatible terms of use in clinical application without using the fetal bovine serum (FBS).
In this pilot study, hPDLSCs were isolated from the impacted third molar, and tissue was scraped from the roots of the impacted third molar of 10 volunteers to enzymatically digest using collagenase. The cells were sub-cultured. The samples of the first seven patients and half of the eighth patient's sample were cultured in alpha modified of Eagle's medium (α-MEM) (-FBS) medium and the other part of the eighth patient's sample was cultured with prior medium supplemented with +FBS 15% as a control of the cultivation protocol. While for the past two patients (9 and 10 the α-MEM medium was supplemented with L-Glutamine, anti/anti 2X, and 20% knock-out serum replacement (KSR). Two more nutritious supplements (N2 and B27) were added to the medium of the tenth sample. Flow-cytometric analysis for the mesenchymal stem cell surface markers CD105, CD45, CD90, and CD73 was performed. Subsequent polymerase chain reaction was undertaken on three samples cultured with two growth media.
Cultivation failed in some of the samples because of the lack of cell adhesion to the culturing dish bottom (floating cells), but it was successful for the 9 and 10 patients, which were cultured in the α-MEM serum supplemented with KSR 20%. Flow cytometry analysis was positive for CD105, CD90, and CD73 and negative for CD45. The PDL stem cells (PDLSCs) expressed CD105, CD45, and CD90 but were poor for CD73.
According to the limited number of sample tests in this study, isolation and characterization of PDLSCs from collected PDL tissue of one patient's wisdom teeth (2-4) may be possible by the proper setup in synthetic FBS-free serum.
本研究的目的是确定在不使用胎牛血清（FBS）且更符合临床应用使用条件的情况下，利用仅一名患者的智齿（2 - 4颗牙）有限的收获牙周膜（PDL）组织分离和鉴定人牙周膜干细胞（hPDLSCs）的可能性。
在这项初步研究中，从阻生第三磨牙中分离hPDLSCs，刮取10名志愿者阻生第三磨牙牙根的组织，用胶原酶进行酶消化。细胞进行传代培养。前7名患者的样本和第8名患者一半的样本在α - 改良伊格尔培养基（α - MEM）（-FBS）中培养，第8名患者另一半样本在添加15% +FBS的先前培养基中培养作为培养方案的对照。而对于最后两名患者（9号和10号），α - MEM培养基添加了L - 谷氨酰胺、双抗2X和20%敲除血清替代物（KSR）。在第10个样本的培养基中添加了另外两种营养补充剂（N2和B27）。对间充质干细胞表面标志物CD105、CD45、CD90和CD73进行流式细胞术分析。随后对用两种生长培养基培养的三个样本进行聚合酶链反应。
由于细胞缺乏对培养皿底部的黏附（漂浮细胞），部分样本培养失败，但9号和10号患者的样本培养成功，它们在添加20% KSR的α - MEM血清中培养。流式细胞术分析显示CD105、CD90和CD73呈阳性，CD45呈阴性。PDL干细胞（PDLSCs）表达CD105、CD45和CD90，但CD73表达较差。
根据本研究中有限数量的样本测试，通过在无FBS的合成血清中进行适当设置，从一名患者的智齿（2 - 4颗）收集的PDL组织中分离和鉴定PDLSCs是可能的。
