Abstract：
BACKGROUND: The pulp contains a resident population of stem cells which can be stimulated to differentiate in order to repair the tooth by generating a mineralized extracellular matrix. Over recent decades there has been considerable interest in utilizing in vitro cell culture models to study dentinogenesis, with the aim of developing regenerative endodontic procedures, particularly where some vital pulp tissue remains.
OBJECTIVE: The purpose of this review is to provide a structured oversight of in vitro research methodologies which have been used to study human pulp mineralization processes.
METHODS: The literature was screened in the PubMed database up to March 2021 to identify manuscripts reporting the use of human dental pulp cells to study mineralization. The dataset identified 343 publications initially which were further screened and consequently 166 studies were identified and it was methodologically mined for information on: i) study purpose, ii) source and characterization of cells, iii) mineralizing supplements and concentrations, and iv) assays and markers used to characterize mineralization and differentiation, and the data was used to write this narrative review.
RESULTS: Most published studies aimed at characterizing new biological stimulants for mineralization as well as determining the effect of scaffolds and dental (bio)materials. In general, pulp cells were isolated by enzymatic digestion, although the pulp explant technique was also common. For enzymatic digestion, a range of enzymes and concentrations were utilized, although collagenase type I and dispase were the most frequent. Isolated cells were not routinely characterized using either fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) approaches and there was little consistency in terming cultures as dental pulp cells or dental pulp stem cells. A combination of media supplements, at a range of concentrations, of dexamethasone, ascorbic acid and beta-glycerophosphate, were frequently applied as the basis for the experimental conditions. Alizarin Red S (ARS) staining was the method of choice for assessment of mineralization at 21-days. Alkaline phosphatase assay was relatively frequently applied, solely or in combination with ARS staining. Further assessment of differentiation status was performed using transcript or protein markers, with dentine sialophosphoprotein (DSPP), osteocalcin and dentine matrix protein-1 (DMP -1), the most frequent.
CONCLUSIONS: While this review highlights variability among experimental approaches, it does however identify a consensus experimental approach.
CONCLUSIONS: Standardization of experimental conditions and sustained research will significantly benefit endodontic patient outcomes in the future.
OBJECTIVE: The purpose of this review is to provide a structured oversight of in vitro research methodologies which have been used to study human pulp mineralization processes.
METHODS: The literature was screened in the PubMed database up to March 2021 to identify manuscripts reporting the use of human dental pulp cells to study mineralization. The dataset identified 343 publications initially which were further screened and consequently 166 studies were identified and it was methodologically mined for information on: i) study purpose, ii) source and characterization of cells, iii) mineralizing supplements and concentrations, and iv) assays and markers used to characterize mineralization and differentiation, and the data was used to write this narrative review.
RESULTS: Most published studies aimed at characterizing new biological stimulants for mineralization as well as determining the effect of scaffolds and dental (bio)materials. In general, pulp cells were isolated by enzymatic digestion, although the pulp explant technique was also common. For enzymatic digestion, a range of enzymes and concentrations were utilized, although collagenase type I and dispase were the most frequent. Isolated cells were not routinely characterized using either fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) approaches and there was little consistency in terming cultures as dental pulp cells or dental pulp stem cells. A combination of media supplements, at a range of concentrations, of dexamethasone, ascorbic acid and beta-glycerophosphate, were frequently applied as the basis for the experimental conditions. Alizarin Red S (ARS) staining was the method of choice for assessment of mineralization at 21-days. Alkaline phosphatase assay was relatively frequently applied, solely or in combination with ARS staining. Further assessment of differentiation status was performed using transcript or protein markers, with dentine sialophosphoprotein (DSPP), osteocalcin and dentine matrix protein-1 (DMP -1), the most frequent.
CONCLUSIONS: While this review highlights variability among experimental approaches, it does however identify a consensus experimental approach.
CONCLUSIONS: Standardization of experimental conditions and sustained research will significantly benefit endodontic patient outcomes in the future.
摘要：
背景：牙髓含有干细胞的常驻群体，可以通过产生矿化的细胞外基质来刺激其分化以修复牙齿。近几十年来，人们对利用体外细胞培养模型研究牙本质发生产生了相当大的兴趣。为了发展再生牙髓手术，特别是一些重要的牙髓组织仍然存在。
目的：这篇综述的目的是对已用于研究人类牙髓矿化过程的体外研究方法进行结构化监督。
方法：截至2021年3月，在PubMed数据库中筛选了文献，以鉴定报告使用人类牙髓细胞研究矿化的手稿。数据集最初确定了343种出版物，这些出版物经过了进一步的筛选，因此确定了166种研究，并在方法上对其进行了挖掘，以获取以下信息：i）研究目的，ii)细胞的来源和表征，iii)矿化补充剂和浓度，和iv)用于表征矿化和分化的测定和标记，数据被用来撰写这篇叙述性综述。
结果：大多数已发表的研究旨在表征矿化的新生物刺激物以及确定支架和牙科（生物）材料的作用。总的来说,通过酶消化分离牙髓细胞，尽管纸浆外植体技术也很常见。对于酶消化，使用了一系列的酶和浓度，虽然胶原酶I型和分散酶是最常见的。使用荧光激活细胞分选（FACS）和磁激活细胞分选（MACS）方法均未对分离的细胞进行常规表征，并且在将培养物称为牙髓细胞或牙髓干细胞时几乎没有一致性。媒体补充剂的组合，在一定的浓度范围内，地塞米松，抗坏血酸和β-甘油磷酸酯，经常被用作实验条件的基础。茜素红S（ARS）染色是评估21天矿化的首选方法。碱性磷酸酶测定法应用相对频繁，单独或与ARS染色组合。使用转录物或蛋白质标记进行分化状态的进一步评估，与牙本质唾液酸糖蛋白（DSPP），骨钙蛋白和牙本质基质蛋白-1（DMP-1），最频繁的。
结论：虽然这篇综述强调了实验方法之间的差异，然而，它确实确定了一种共识性的实验方法。
结论：实验条件和持续研究的标准化将在未来显著有利于牙髓患者的预后。
目的：这篇综述的目的是对已用于研究人类牙髓矿化过程的体外研究方法进行结构化监督。
方法：截至2021年3月，在PubMed数据库中筛选了文献，以鉴定报告使用人类牙髓细胞研究矿化的手稿。数据集最初确定了343种出版物，这些出版物经过了进一步的筛选，因此确定了166种研究，并在方法上对其进行了挖掘，以获取以下信息：i）研究目的，ii)细胞的来源和表征，iii)矿化补充剂和浓度，和iv)用于表征矿化和分化的测定和标记，数据被用来撰写这篇叙述性综述。
结果：大多数已发表的研究旨在表征矿化的新生物刺激物以及确定支架和牙科（生物）材料的作用。总的来说,通过酶消化分离牙髓细胞，尽管纸浆外植体技术也很常见。对于酶消化，使用了一系列的酶和浓度，虽然胶原酶I型和分散酶是最常见的。使用荧光激活细胞分选（FACS）和磁激活细胞分选（MACS）方法均未对分离的细胞进行常规表征，并且在将培养物称为牙髓细胞或牙髓干细胞时几乎没有一致性。媒体补充剂的组合，在一定的浓度范围内，地塞米松，抗坏血酸和β-甘油磷酸酯，经常被用作实验条件的基础。茜素红S（ARS）染色是评估21天矿化的首选方法。碱性磷酸酶测定法应用相对频繁，单独或与ARS染色组合。使用转录物或蛋白质标记进行分化状态的进一步评估，与牙本质唾液酸糖蛋白（DSPP），骨钙蛋白和牙本质基质蛋白-1（DMP-1），最频繁的。
结论：虽然这篇综述强调了实验方法之间的差异，然而，它确实确定了一种共识性的实验方法。
结论：实验条件和持续研究的标准化将在未来显著有利于牙髓患者的预后。
