Cytocompatibility and bioactive potential of AH Plus Bioceramic Sealer: An in vitro study
AH Plus 生物陶瓷封闭剂的细胞相容性和生物活性潜力：一项体外研究

\author{ \作者{
José Luis Sanz | Sergio López-García | Francisco Javier Rodríguez-Lozano |
何塞·路易斯·桑斯 | 廖佩斯-加西亚 | 弗朗西斯科·哈维尔·罗德里格斯-洛萨诺 |

Department d'Estomatologia, Facultat de Medicina I Odontologia, Universitat de València, Valencia, Spain
口腔科学系，瓦伦西亚大学医学与牙科学院，西班牙瓦伦西亚

Department of Dermatology, Stomatology, Radiology and Physical Medicine, Faculty of Medicine, Morales Meseguer Hospital, University of Murcia, Murcia, Spain
西班牙穆尔西亚大学莫拉莱斯·梅塞格尔医院医学院皮肤科、口腔科、放射科和物理医学系

Francisco Javier Rodríguez-Lozano, School of Dentistry, Hospital Morales Meseguer 2 pl., Av. Marqués de los Vélez, s/n, 30008, University of Murcia, Murcia, Spain.
西班牙穆尔西亚大学，西班牙穆尔西亚市 Marqués de los Vélez 大道，Morales Meseguer 2 号医院牙科学院，Francisco Javier Rodríguez-Lozano。

Email: fcojavier@um.es 电子邮件：fcojavier@um.es

Instituto de Salud Carlos III, Grant/ Award Number: RD21/0001/0022; Ministerio de Ciencia, Innovacion y Universidades, Grant/Award Number: FPU19/03115
卡洛斯三世健康研究所，资助/奖励编号：RD21/0001/0022；科学、创新和大学部，资助/奖励编号：FPU19/03115

Results: A higher peak of was detected from ESbcs compared with AHPbcs and AHP in SEM-EDX. Both AHPbcs and ESbcs showed significantly positive results in the cytocompatibility assays (cell viability, migration/proliferation, attachment and morphology) compared with a negative control group, whilst AHP showed significant negative results. Both AHPbcs and ESbcs exhibited an upregulation of at least one osteo/odonto/cementogenic marker compared with the negative and positive control groups. Both ESbcs and AHPbcs showed a significantly higher calcified nodule formation than the negative and positive control groups, indicative of their biomineralization potential and were also significantly higher than AHP group.
结果：与 AHPbcs 和 AHP 相比，SEM-EDX 检测到 ESbcs 的 峰值较高。与阴性对照组相比，AHPbcs 和 ESbcs 在细胞相容性试验（细胞存活率、迁移/增殖、附着和形态）中显示出显著正面结果，而 AHP 显示出显著负面结果。与阴性和阳性对照组相比，AHPbcs 和 ESbcs 都表现出至少一种骨/牙/水泥发生标记物的上调。与阴性和阳性对照组相比，ESbcs 和 AHPbcs 显示出明显更高的钙化结节形成，表明它们具有生物矿化潜力，并且也明显高于 AHP 组。

Conclusion: AH Plus Bioceramic Sealer exhibited a significantly higher cytocompatibility and bioactive potential than Plus and a similar cytocompatibility to that of Endosequence BC Sealer. Endosequence BC Sealer exhibited a significantly higher mineralization potential than the other tested sealers. The results from this in vitro study act as supporting evidence for the use of AH Plus Bioceramic Sealer in root canal treatment.
结论：AH Plus 生物陶瓷封闭剂表现出明显更高的细胞相容性和生物活性潜力，比 Plus 更高，并且与 Endosequence BC 封闭剂的细胞相容性相似。Endosequence BC 封闭剂的矿化潜力明显高于其他测试的封闭剂。这项体外研究的结果作为支持 AH Plus 生物陶瓷封闭剂在根管治疗中使用的证据。

Root canal treatment involves the chemical-mechanical disinfection of the root canal system and its subsequent filling to ensure an adequate seal (Li et al., 2014). The materials placed inside the root canal should be dimensionally stable, biocompatible and present adequate handling properties (Donnermeyer et al., 2019). The most commonly used materials for such purpose are gutta-percha and root canal sealers (Kishen et al., 2016; Vishwanath & Rao, 2019).
根管治疗涉及根管系统的化学机械消毒及随后的填充，以确保充分密封（Li 等，2014）。放置在根管内的材料应具有尺寸稳定性、生物相容性和适当的操作性能（Donnermeyer 等，2019）。用于此目的的最常用材料是橡胶树脂和根管封闭剂（Kishen 等，2016；Vishwanath 和 Rao，2019）。

A wide variety of root canal sealer compositions are available, such as zinc oxide-eugenol, epoxy resin and calcium silicate cement sealers (Sfeir et al., 2021). These materials differ in terms of their setting reactions, which take place by chelate formation, polymer formation by addition and hydration, respectively (Komabayashi et al., 2020). The physicochemical and biological properties of these sealers also differ (Silva et al., 2019, 2021).
根管封闭剂有多种不同的组成，例如氧化锌丁酚、环氧树脂和硅酸钙水泥封闭剂（Sfeir 等，2021 年）。这些材料在其固化反应方面有所不同，分别通过螯合形成、加成聚合和水合反应进行（Komabayashi 等，2020 年）。这些封闭剂的物理化学和生物学性质也有所不同（Silva 等，2019 年，2021 年）。

Endodontic sealers are placed inside the root canal and may extrude to a variable extent during root canal treatment through the apical and/or secondary foramina into the surrounding supporting tissues (Aminoshariae & Kulild, 2020). Therefore, sealers should exhibit an adequate biocompatibility, i.e. they should not induce an adverse reaction or response from biological tissues upon contact (Ferreira et al., 2021). The same applies on a cellular level, wherein surrounding cellular populations should not experience a decrease in their viability, migration/proliferation or differentiation (da Silva et al., 2017). In other words, root canal sealers should exhibit an adequate cytocompatibility and absence of cytotoxicity.
根管封闭剂被放置在根管内，在根管治疗过程中可能会通过根尖和/或次要孔道挤出到周围的支持组织中（Aminoshariae＆Kulild，2020）。因此，封闭剂应该表现出足够的生物相容性，即它们不应在接触时引起生物组织的不良反应或反应（Ferreira 等，2021）。在细胞水平上也是如此，周围的细胞群体不应该经历其生存能力、迁移/增殖或分化的降低（da Silva 等，2017）。换句话说，根管封闭剂应该表现出足够的细胞相容性和无细胞毒性。

Periodontal ligament stem cells (PDLSCs), a subgroup of dental stem cells (DSCs) with a mesenchymal phenotype (Bartold & Gronthos, 2017), are in contact with root canal sealers. These cells possess a multilineage differentiation potential and may play a crucial role in the healing process of existing periapical lesions (Gay et al., 2007; Seo et al., 2004). Consequently, the extrusion of a root canal sealer with adequate biological properties should not hinder these cells.
牙周膜干细胞（PDLSCs）是牙齿干细胞（DSCs）的一个亚群，具有间充质表型（Bartold＆Gronthos，2017），与根管封闭剂接触。这些细胞具有多向分化潜力，可能在现有根尖周病变的愈合过程中发挥关键作用（Gay 等，2007；Seo 等，2004）。因此，具有足够生物学特性的根管封闭剂的挤出不应妨碍这些细胞。

Previous evidence has demonstrated that resin-based or resin-containing sealers and cements often are cytotoxic toward various cell subpopulations (Collado-González, Tomás-Catalá, et al., 2017; Manaspon et al., 2021). The well-known AH Plus sealer (Dentsply DeTrey GmbH) has shown a negative effect on periodontal ligament stem cell viability, migration/proliferation and attachment in various in vitro studies (Oh et al., 2020; Rodríguez-Lozano et al., 2019) compared with a negative control group.
先前的证据表明，基于树脂或含树脂的密封剂和水泥通常对各种细胞亚群具有细胞毒性（Collado-González，Tomás-Catalá等，2017 年；Manaspon 等，2021 年）。众所周知的 AH Plus 密封剂（Dentsply DeTrey GmbH）在各种体外研究中与阴性对照组相比，对牙周膜干细胞的存活率、迁移/增殖和附着产生了负面影响（Oh 等，2020 年；Rodríguez-Lozano 等，2019 年）。

Conversely, calcium silicate cement-based sealers have adequate biocompatibility and bioactive properties when cultured together with PDLSCs (Rodríguez-Lozano et al., 2017; Zheng et al., 2020). These sealers have shown the ability to induce the precipitation of a layer of hydroxyapatite on their surface (Kim et al., 2015). which may form a mineral attachment to dentin tissue (Vallittu et al., 2018). The same term is used to describe their positive influence on cell plasticity, e.g. by favouring the osteo/ odonto/cementogenic differentiation of PDLSCs which, in turn, may result in an enhanced repair process and resolution of periapical lesions (Sanz, Guerrero-Gironés, et al., 2021).
相反，当与 PDLSCs 一起培养时，硅酸钙水泥基封闭剂具有足够的生物相容性和生物活性特性（Rodríguez-Lozano 等，2017 年；Zheng 等，2020 年）。这些封闭剂已显示出诱导在其表面沉淀一层羟基磷灰石的能力（Kim 等，2015 年），这可能形成对牙本质组织的矿物附着（Vallittu 等，2018 年）。同一术语用于描述它们对细胞可塑性的积极影响，例如通过促进 PDLSCs 的成骨/牙本质/水泥发生分化，从而可能导致增强的修复过程和根尖周病变的解决（Sanz，Guerrero-Gironés 等，2021 年）。

AH Plus Bioceramic Sealer (Maruchi) was introduced into the market as a pre-mixed tricalcium silicate cementbased sealer. According to its distributor (Dentsply Sirona USA), this new sealer presents a faster setting time, lower solubility, lower film thickness and higher radiopacity than the Endosequence BC Sealer (Brasseler). However, to the authors' knowledge, the biological properties of the new AH Plus Bioceramic Sealer towards PDLSCs have not been elucidated.
AH Plus 生物陶瓷封闭剂（Maruchi）作为一种预混三钙硅酸盐水泥基封闭剂进入市场。根据其分销商（Dentsply Sirona USA）的说法，这种新封闭剂比 Endosequence BC 封闭剂（Brasseler）具有更快的凝固时间、较低的溶解度、较低的膜厚度和更高的射线密度。然而，据作者所知，新的 AH Plus 生物陶瓷封闭剂对 PDLSCs 的生物学特性尚未阐明。

Accordingly, the aim of the present in vitro study is to assess the cytocompatibility and bioactive properties of AH Plus Bioceramic Sealer on hPDLSCs compared with the classic AH Plus and Endosequence BC Sealer.
因此，本次体外研究的目的是评估 AH Plus 生物陶瓷封闭剂对人牙周膜干细胞的细胞相容性和生物活性特性，与经典的 AH Plus 和 Endosequence BC 封闭剂进行比较。

The manuscript of this in vitro study has been written in accordance with the Preferred Reporting Items for Laboratory studies in Endodontology (PRILE) 2021' guidelines (Nagendrababu et al., 2021a).
本体外研究的手稿已根据《根管治疗学实验室研究首选报告项目(PRILE) 2021》指南编写（Nagendrababu 等，2021a）。

Sample discs were prepared for each of the tested materials ( in total): AH Plus Bioceramic Sealer (AHPbcs), AH Plus (AHP) and Endosequence BC Sealer (ESbcs). The number of discs was based on the protocol from a previous study with similar methodology (Sanz, López-García, et al., 2021). Material data (composition, manufacturer and batch number) are listed in Table 1. Materials were placed into diameter and
为每种被测试材料准备了样品圆盘（共 个）：AH Plus 生物陶瓷封闭剂（AHPbcs）、AH Plus（AHP）和 Endosequence BC 封闭剂（ESbcs）。圆盘数量基于一项具有类似方法的先前研究的方案（Sanz，López-García 等，2021 年）。材料数据（成分、制造商和批号）列在表 1 中。材料被放入直径为 和 的圆盘中。
high sterile (ultraviolet radiation, ) cylindrical rubber molds with Hank's balanced salt solution (HBSS; H6648; Sigma Aldrich) and set in an incubator at , and humidity for 48 hours. AHP is presented in a two-component paste/paste format. Accordingly, pastes A and B were mixed following its manufacturer's instructions. Both AHPbcs and ESbcs are presented in an injectable syringe (pre-mixed) format and were directly placed into the rubber moulds.
高度无菌（紫外线辐射， ）圆柱形橡胶模具，使用 Hank's 平衡盐溶液（HBSS; H6648; Sigma Aldrich）填充，并在 、 和 湿度的孵育箱中孵育 48 小时。AHP 呈两组分膏状/膏状格式呈现。因此，根据制造商的说明，将膏 A 和膏 B 混合。AHPbcs 和 ESbcs 均以可注射的注射器（预混合）格式呈现，并直接放入橡胶模具中。

To simulate clinical conditions, where cells are in contact with the silicate cement-based sealers, extracts or eluates were obtained from each of the materials, following the International Standard ISO 10993-5 guidelines (ISO, 2009). The eluates of the different materials were extracted in sterile conditions, using Dulbecco Modified Eagle's Medium (DMEM) (Gibco) with of foetal bovine serum (FBS) as an extraction vehicle. The extraction procedure was as follows: the tested materials were immersed in the culture medium for at in a humid atmosphere containing . In accordance with the ISO standard, the ratio between the surface of the sample and the volume of the medium was . The extraction medium was collected at the end of this period and filtered through a 0.22 syringe filter (Merck Millipore). Thereafter, in order to study the effect of the concentration of each material, various dilutions ( and ) of these extraction media were prepared using fresh complete DMEM medium (Rodríguez-Lozano et al., 2019).
为了模拟临床条件，其中细胞与硅酸盐水泥基封闭剂接触，从每种材料中获得了提取物或洗脱物，遵循国际标准 ISO 10993-5 指南（ISO，2009）。不同材料的洗脱物在无菌条件下提取，使用含有 胎牛血清（FBS）的 Dulbecco 改良的鹰的培养基（DMEM）（Gibco）作为提取载体。提取过程如下：测试材料在含有 的湿润大气中的 处浸泡在培养基中 。根据 ISO 标准，样品表面与培养基体积之间的比例为 。在此期间结束时收集提取培养基，并通过 0.22 注射器过滤器（Merck Millipore）过滤。随后，为了研究每种材料浓度的影响，使用新鲜完整的 DMEM 培养基（Rodríguez-Lozano 等人，2019）制备了各种稀释液（ 和 ）这些提取介质。

The previously prepared material sample discs were selected for the analysis ( per material). After the incubation period, the set material discs were coated with carbon under a CC7650 SEM Carbon Coater Unit (Quorum Technologies Ltd.). The superficial element distribution of the coated discs was then individually examined in a scanning electron microscopy (SEM) unit (Jeol 6100 EDAX; Jeol Inc.) attached to an energy dispersive spectroscopy (EDS) system (INCA 350 EDS; Oxford Instruments) for the elemental analysis.
先前准备的材料样品圆盘被选中进行分析（每种材料 个）。孵育期后，设定的材料圆盘在 CC7650 SEM 碳镀层单元（Quorum Technologies Ltd.）下被涂覆碳。然后，涂层圆盘的表面元素分布分别在连接到能谱分析系统（INCA 350 EDS；Oxford Instruments）的扫描电子显微镜（SEM）单元（Jeol 6100 EDAX；Jeol Inc.）中进行检查，以进行元素分析。

The human PDLSC (hPDLSC) extraction protocol had been approved by the Human Research Ethics Committee from Universidad de Murcia (ID: 2199/2018), following the Helsinki Declaration guidelines. HPDLSCs were isolated from healthy third molars from 18-30-year-old patients , which had been extracted for orthodontic or periodontal reasons; with written informed consent. The molar sample size was selected in accordance with a previous study with similar methodology (Sanz, LópezGarcía, et al., 2021).
人类 PDLSC（hPDLSC）提取方案已获得穆尔西亚大学人类研究伦理委员会批准（ID：2199/2018），遵循赫尔辛基宣言指南。HPDLSCs 从 18-30 岁患者的健康第三磨牙中分离出来，这些患者因正畸或牙周疾病而拔牙；并获得书面知情同意。磨牙样本量的选择符合一项具有类似方法的先前研究（Sanz，LópezGarcía 等，2021 年）。

Extracted molars were immediately placed in Minimum Essential Medium with Alpha modifications ( -MEM; Gibco, Invitrogen) supplemented with penicillin/streptomycin (Sigma Aldrich) and amphotericin B (Fungizone; Sigma Aldrich) and stored at . The teeth were rinsed thrice with phosphate-buffered saline (PBS) (Gibco), and the periodontal tissues were scraped from the surface of the middle and apical thirds of their roots. Periodontal tissues were sliced into smaller fragments and digested with Collagenase type I solution ( ; Gibco) for at . The periodontal cells were seeded in -MEM supplemented with foetal bovine serum
提取的磨牙立即放入含有α改良的最低必需培养基（ -MEM；Gibco，Invitrogen）的培养基中，添加 青霉素/链霉素（Sigma Aldrich）和两性霉素 B（Fungizone；Sigma Aldrich），并存放在 。牙齿用磷酸盐缓冲盐水（PBS）（Gibco）冲洗三次，牙周组织从其根部的中部和尖端的表面刮除。牙周组织切成较小的碎片，并用胶原酶 I 溶液（ ；Gibco）在 的 内消化。牙周细胞被播种在含有 胎牛血清的 -MEM 中。

TABLE 1 Data on the tested materials
表 1 测试材料数据

(FBS; Sigma Aldrich) and 1% penicillin/streptomycin (Sigma Aldrich).
（FBS; Sigma Aldrich）和 1% 青霉素/链霉素（Sigma Aldrich）。

Before their use in the in vitro experimentation, hPDLSC characterization was performed following the International Society of Cellular Therapy (ISCT) guidelines (Dominici et al., 2006), to confirm their mesenchymal nature. The process was as follows: cells were analysed under flow cytometry (FACSCalibur Flow Cytometry System; BD Biosciences), and the high expression of the mesenchymal stem cell (MSC)-specific surface markers CD73, CD90 and CD105 and low expression of the hematopoietic markers CD34, CD45, CD14 and CD20 were confirmed. This was performed in accordance with similar studies in the field (Collado-González, GarcíaBernal, et al., 2017; Oh et al., 2020). Additionally, the resultant characterized hPDLSCs were cultured in different media (osteogenic/adipogenic/chondrogenic) (Miltenyi Biotec) to confirm their trilineage mesenchymal differentiation. Both the mesenchymal nature and trilineage differentiation potential of the cells used were confirmed by a previous study performed by the present research group (Rodríguez-Lozano et al., 2019). For the subsequent in vitro experimentation, cells from passages 2-4 were used, as performed in previous similar studies (Sanz, LópezGarcía, et al., 2021).
在体外实验中使用之前，根据国际细胞治疗学会（ISCT）的指南（Dominici 等，2006 年）对 hPDLSC 进行表征，以确认其间充质性质。过程如下：细胞在流式细胞术（FACSCalibur 流式细胞术系统；BD 生命科学）下进行分析，确认了间充质干细胞（MSC）特异性表面标记物 CD73、CD90 和 CD105 的高表达，以及血液系统标记物 CD34、CD45、CD14 和 CD20 的低表达。这是根据该领域类似研究进行的（Collado-González，GarcíaBernal 等，2017 年；Oh 等，2020 年）。此外，所得到的表征的 hPDLSC 在不同培养基（成骨/脂肪/软骨）（Miltenyi Biotec）中培养，以确认其三向间充质分化。细胞的间充质性质和三向分化潜力均由本研究小组之前的研究证实（Rodríguez-Lozano 等，2019 年）。 对于随后的体外实验，使用了 2-4 代细胞，与之前类似研究中所做的一样（Sanz，LópezGarcía 等，2021 年）。

Material cytotoxicity was assessed for the different eluates (1:1, 1:2 and 1:4) of AHP, AHPbcs and ESbcs cultured with hPDLSCs (test groups) and compared with hPDLSCs cultured in unconditioned growth medium (negative control group). This analysis was performed via a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli um bromide (MTT) assay, as previously reported by similar studies (Rathinam et al., 2021). In brief, hPDLSCs were seeded onto 96 -well plates with l of DMEM and stored for at and humidity. The material eluates were placed in the culture medium with hPDLSCs . An MTT reagent (Sigma Aldrich) was added for , following its manufacturer's instructions. When a purple precipitate was detectable, Dimethylsulfoxide (DMSO) (Sigma-Aldrich) was added to each well ( well), and plates were covered and kept in dark conditions for to solubilize the formazan crystals produced by viable cells, after reducing the MTT reagent. After 24, 48 and of culture, light absorbance per well was recorded by means of a microplate reader (ELx800; Bio-Tek Instruments) at wavelength. Culture media with fresh eluates from the respective groups were replaced every 3 days.
材料细胞毒性对 AHP、AHPbcs 和 ESbcs 的不同洗脱物（1:1、1:2 和 1:4）进行评估，这些洗脱物与 hPDLSCs（实验组）培养，并与培养在未经处理的生长培养基中的 hPDLSCs（阴性对照组）进行比较。该分析是通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物（MTT）测定法进行的，这是由类似研究（Rathinam 等，2021 年）先前报道的。简而言之，hPDLSCs 被播种在 96 孔板上，加入 升 DMEM，并存放 在 和 湿度下。将材料洗脱物放入培养基中与 hPDLSCs 。添加 MTT 试剂（Sigma Aldrich） ，按照其制造商的说明。当可以检测到紫色沉淀时，向每个孔中加入二甲基亚砜（DMSO）（Sigma-Aldrich），并将板盖好，放在黑暗条件下 以溶解 MTT 试剂还原后由存活细胞产生的甲烷蓝晶体。在培养 、48 和 24 小时后，通过微孔读数器（ELx800；Bio-Tek Instruments）在 波长处记录每孔的光吸收率。 使用来自各组的新鲜洗脱物更换培养基，每 3 天更换一次。

HDPLSC migration/proliferation was assessed after culture in growth medium with the eluates (1:1, 1:2 and 1:4) of AHP, AHPbcs and ESbcs and compared with the cells cultured in unconditioned growth medium (negative control group) via a wound healing assay. HPDLSCs were seeded onto 6 -well plates cells per well; for each experimental condition) and left to proliferate until cell confluency was reached. Then, a superficial scratch wound was made on each cell monolayer using a sterilized pipette tip, and each well was rinsed thrice to remove any remaining cell debris. Wound closure/healing was assessed for all experimental conditions in triplicate (test groups and negative control) at 24,48 and . At each time-point, the percentage of open wound area was quantified for each of the samples by means Image software (National Institutes of Health). Migration rates were presented as percentage areas of relative wound closure (RWC) to account for width variations amongst the scratch wounds. RWC values were calculated as follows: RWC (wound closure area [in pixels]/total number of pixels) . Results are expressed as the percentage of the total wound area thrice relative to the total wound area at for each respective well.
HDPLSC 迁移/增殖在培养基中与 AHP、AHPbcs 和 ESbcs 的洗脱物（1:1、1:2 和 1:4）一起培养后进行评估，并通过创伤愈合实验与在未经处理的培养基中培养的细胞（阴性对照组）进行比较。将 HPDLSCs 种植在 6 孔板上（每孔 个细胞；每个实验条件 个），并让其增殖直到细胞充分密集。然后，使用 个消毒的吸管尖在每个细胞单层上制造一个浅表划痕，然后每个孔洗涤三次以去除任何残留的细胞碎片。在 24、48 和 时评估所有实验条件的创伤闭合/愈合情况。在每个时间点，通过图像 软件（美国国立卫生研究院）对每个样本的开放创伤区域百分比进行定量化。迁移速率以相对创伤闭合区域（RWC）的百分比面积呈现，以考虑划痕创伤之间的宽度变化。RWC 值计算如下：RWC （创伤闭合区域[以像素为单位]/总像素数） 。 结果以相对于每个相应孔位 处的总伤口面积三倍的百分比表示。

Sealer discs were made using the previously described methods ( for each sealer). The surface of the discs was seeded with and cultured in normal growth medium for . Cells were fixed with glutaraldehyde (Sigma-Aldrich) in PBS for . The cells were dehydrated using a series of gradually increasing ethanol dilutions ( 30 to ) and treated with hexamethyldisilazane (Sigma-Aldrich) for . Finally, cells were air-dried, sputter-coated with gold and palladium and examined using a SEM (Jeol 6100 EDAX; Jeol Inc.) at 100×, and magnifications.
密封盘是使用先前描述的方法制作的（每个密封盘 ）。盘的表面被播种 并在正常生长培养基中培养 。细胞用 戊二醛（Sigma-Aldrich）在 PBS 中固定 。细胞经过一系列逐渐增加的乙醇稀释液（30 至 ）脱水处理，并用六甲基二硅氮烷（Sigma-Aldrich）处理 。最后，细胞被空气干燥，镀金和钯，并在 SEM（Jeol 6100 EDAX; Jeol Inc.）下以 100×， 和 倍放大率检查。

The osteo/cemento/odontogenic marker expression of hPDLSCs cultured together with the materials was assessed via real-time quantitative polymerase chain reaction (RT-qPCR), as a measurement of cell differentiation. Twenty-thousand hPDLSCs per well were
与材料一起培养的 hPDLSCs 的骨/水泥/牙源性标记表达通过实时定量聚合酶链反应（RT-qPCR）进行评估，作为细胞分化的测量。每个孔中有两万个 hPDLSCs。
seeded onto 12 -well plates and incubated for 3,7 , 14 and 21 days with undiluted (1:1) sealer-conditioned medium from the two calcium silicate-cements (test groups: AHPbcs or ESbcs), in unconditioned culture medium (negative control groups) or in osteogenic differentiation medium (positive control; OsteoDiff media; Miltenyi Biotec). Culture media with fresh eluates from the respective groups were replaced every 3 days. The undiluted sealer-conditioned medium was prepared by immersing the previously conditioned standardized sealer discs in culture medium (DMEM; Gibco) for . AHP was excluded from the marker expression assay because of its negative results in the hPDLSC viability, migration/proliferation and attachment assays. Total RNA was extracted from hPDLSCs using the RNeasy Mini Kit (Qiagen). One of RNA was reverse transcribed for first-strand complementary DNA (cDNA) synthesis via iScript Reverse Transcription Supermix for RTqPCR (Bio-Rad Laboratories Inc.). Both processes were performed following their respective manufacturers' kit instructions.
将种子植入 12 孔板中，并在未稀释的（1:1）密封剂调节培养基中孵育 3、7、14 和 21 天，分别来自两种硅酸钙水泥（实验组：AHPbcs 或 ESbcs），在未调节的培养基（阴性对照组）或成骨分化培养基（阳性对照；OsteoDiff 培养基；Miltenyi Biotec）中。每 3 天更换一次来自各组的新溶出物的培养基。未稀释的密封剂调节培养基是通过将先前调节过的标准密封剂圆盘浸入培养基（DMEM；Gibco）中制备的。由于 AHP 在 hPDLSC 存活、迁移/增殖和附着实验中的负面结果，AHP 被排除在标记物表达分析之外。使用 RNeasy Mini Kit（Qiagen）从 hPDLSC 中提取总 RNA。将 1μg RNA 反转录为第一链互补 DNA（cDNA），通过 iScript Reverse Transcription Supermix for RTqPCR（Bio-Rad Laboratories Inc.）进行合成。这两个过程均按照各自制造商的说明书进行。

The primer sequences for the differentiation markers used for the assay were as follows ( ): Cementum attachment protein or CAP (forward: TTTTTCTGGTCGCGTGGACT, reverse: TCACCAGCA ACTCCAACAGG), cementum protein 1 or CEMP1 (forward: GGGCACATCAAGCACTGACAG, reverse: CCCTTAGGAAGTGGCTGTCCAG), alkaline phosphatase or ALP (forward: TCAGAAGCTCAACACCAACG, reverse: TTGTACGTCTTGGAGAGGGC), runt-related transcription factor 2 or RUNX2 (forward: TCCAC ACCATTAGGGACCATC, reverse: TGCTAATGCTTCGT GTTTCCA), bone sialoprotein or BSP (forward: TGCC TTGAGCCTGCTTCCT, reverse: CTGAGCAAAATTAA AGCAGTCTTCA), amelogenin or AMELX (forward: CACCCTGCAGCCTCATCACC, reverse: GTGTT GGATTGGAGTCATGG).
用于检测的分化标记物的引物序列如下（ ）：水泥附着蛋白或 CAP（前向：TTTTTCTGGTCGCGTGGACT，反向：TCACCAGCAACTCCAACAGG），水泥蛋白 1 或 CEMP1（前向：GGGCACATCAAGCACTGACAG，反向：CCCTTAGGAAGTGGCTGTCCAG），碱性磷酸酶或 ALP（前向：TCAGAAGCTCAACACCAACG，反向：TTGTACGTCTTGGAGAGGGC），RUNX2 或 RUNX2（前向：TCCACACCATTAGGGACCATC，反向：TGCTAATGCTTCGTGTTTCCA），骨唾液蛋白或 BSP（前向：TGCC TTGAGCCTGCTTCCT，反向：CTGAGCAAAATTAAAGCAGTCTTCA），牙本质蛋白 X 或 AMELX（前向：CACCCTGCAGCCTCATCACC，反向：GTGTTGGATTGGAGTCATGG）。

Differentiation marker expression was measured relative to the expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), with the following sequence : (forward: TCAGCAATGCCTCCTGCAC, reverse: TCTGGG TGGCAGTGATGG). To calculate the relative gene expression, the standardized CT method was used (Livak & Schmittgen, 2001)
差异标记表达相对于 housekeeping 基因甘油醛-3-磷酸脱氢酶（GAPDH）的表达进行测量，其序列如下：（前向：TCAGCAATGCCTCCTGCAC，反向：TCTGGG TGGCAGTGATGG）。为计算相对基因表达量，采用了标准化 CT 方法（Livak & Schmittgen, 2001）。

An Alizarin Red S Staining (ARS) assay was performed to assess hPDLSC calcified nodule formation in contact with the tested sealers (AHPbcs, AHP and ESbcs), as a measurement of their biomineralization potential. Twenty-thousand hPDLSCs per well were seeded onto 12-well plates and left to proliferate until confluency was reached. The cells were then transferred into undiluted (1:1) sealer-conditioned medium and cultured for 21 days. After the culture period, the samples were rinsed with foetal bovine serum and fixed with ethanol for . Then, samples were stained with Alizarin Red solution (Sigma Aldrich) for in controlled conditions (dark ambient and room temperature) and solubilized using cetylpyridinium chloride monohydrate solution (Sigma-Aldrich). Lastly, absorbance values of the samples were measured using Synergy H1 multi-mode microplate reader (BioTek) at . For this assay, both a negative control (hDPSCs cultured in unconditioned growth medium [DMEM; Gibco]) and a positive control (hDPSCs cultured in osteogenic medium (OsteoDiff; Miltenyi Biotec) were used for reference.
进行了茜素红 S 染色（ARS）实验，以评估与测试密封剂（AHPbcs，AHP 和 ESbcs）接触时 hPDLSC 钙化结节形成，作为其生物矿化潜力的测量。每个孔中种植了两万个 hPDLSCs，并让其增殖直到达到充分密度。然后将细胞转移到未稀释（1:1）的密封剂处理培养基中，并培养 21 天。培养期结束后，用胎牛血清冲洗样品，并用乙醇固定 。然后，在控制条件下（黑暗环境和室温）用 茜素红溶液（Sigma Aldrich）染色 ，并使用 十六烷基吡啶盐单水合物溶液（Sigma-Aldrich）溶解。最后，使用 Synergy H1 多模式微孔板读数器（BioTek）测量样品的吸光度值 。对于这个实验，使用了阴性对照（在未处理生长培养基[DMEM; Gibco]中培养的 hDPSCs）和阳性对照（在成骨培养基（OsteoDiff; Miltenyi Biotec）中培养的 hDPSCs）作为参考。

All the experimental conditions and measurements were performed in triplicate for each of the tested sealers (AHPbcs, AHP and ESbcs). Data are expressed as mean standard deviations (SD). The normality in the distribution of the data was previously confirmed via a Q-Q plot. Data were analysed using one-way ANOVA and Tukey's post hoc test using Graph-Pad Prism v8.1.0 (GraphPad Software). To perform the one-way ANOVA test, we grouped the data by time ( and ) and analysed them independently. Each dilution was considered an independent treatment. Statistical significance was considered at .
所有实验条件和测量均对每种被测试密封剂（AHPbcs、AHP 和 ESbcs）进行了三次重复。数据以平均值 标准偏差（SD）表示。通过 Q-Q 图先前确认了数据的正态分布。数据使用单因素方差分析和图形帕德棱镜 v8.1.0（GraphPad Software）进行图基的图基后检验进行分析。为了进行单因素方差分析检验，我们按时间（ 和 ）对数据进行分组并独立分析。每种稀释被视为独立处理。统计显著性在 处考虑。

Data (mean and standard deviations) from the biological assays are presented in the Supplementary Material, as follows: Table S1 (MTT assay), Table S2 (wound healing assay), Table S3 (Alizarin Red S staining) and Table S4 (RT-qPCR).
生物测定的数据（均值和标准差）列在补充资料中，具体如下：表 S1（MTT 测定），表 S2（创伤愈合测定），表 S3（Alizarin Red S 染色）和表 S4（RT-qPCR）。

SEM-EDS analysis revealed the superficial element distributions of the root canal sealers (ESbcs, AHPbcs and AHP; Figure 1). ESbcs and AHPbcs displayed a superficial crystalline structure, whilst particles on the smoother AHP surface were spherical. The elements O, and
SEM-EDS 分析显示了根管封闭剂（ESbcs，AHPbcs 和 AHP）的表面元素分布（图 1）。 ESbcs 和 AHPbcs 显示出表面结晶结构，而 AHP 表面上的颗粒是球形的。元素 O， 和

FIGURE 1 Results from the SEM-EDS analysis for the tested sealers (ESbcs [column a], AHPbcs [column b], AHP [column c]). The first row illustrates SEM images of each material (scale bar: ). The second row shows the EDS elemental spectra. The third row lists the elements present per sealer by weight and atomic weight.
图 1 SEM-EDS 分析结果，用于测试密封剂（ESbcs [列 a]，AHPbcs [列 b]，AHP [列 c]）。第一行展示了每种材料的 SEM 图像（比例尺： ）。第二行显示了 EDS 元素光谱。第三行列出了每种密封剂中存在的元素的重量和原子量。

were detected in all samples. Interestingly, a higher peak of was detected in ESbcs compared with AHPbcs and AHP, whereas a higher peak of zirconium ( ) was observed in AHPbcs compared with the other material samples. Tungsten (W) was detected only in the AHP samples.
所有样本中都检测到了。有趣的是，与 AHPbcs 和 AHP 相比，ESbcs 中检测到了更高的 峰值，而与其他材料样本相比，AHPbcs 中检测到了锆（ ）的更高峰值。钨（W）仅在 AHP 样本中检测到。

The MTT assay revealed an adequate cell viability from all eluates of ESbcs and AHPbcs at all the tested time points (24, 48 and of culture), similar to that of the control group; the 1:1 AHPbcs-treated cells, however, exhibited a significantly lower viability than the control group ( ). AHP-treated cells exhibited a significantly lower viability compared with the control group after 24 , 48 and of culture ( ; Figure 2).
MTT 实验显示，在所有 ESbcs 和 AHPbcs 的洗脱物中，在所有测试时间点（24、48 和 天）细胞存活率均良好，与对照组相似；然而，经过 1:1 AHPbcs 处理的细胞存活率明显低于对照组（ ）。与对照组相比，AHP 处理的细胞在 24、48 和 天的培养后存活率显著降低（ ；图 2）。

Human periodontal ligament stem cells cultured with all the eluates of AHPbcs and ESbcs exhibited similar migration to that of the control group at all time-points (24, 48 and ) in the wound healing assay. Similar to the cytotoxicity assay, cells cultured with AHP exhibited a significantly lower migration compared with the control group after 24,48 and of culture ( ; Figure 3).
人牙周膜干细胞与 AHPbcs 和 ESbcs 的所有洗脱物培养在伤口愈合实验的所有时间点（24、48 和 ）表现出与对照组相似的迁移。与细胞毒性试验类似，与 AHP 培养的细胞在培养 24、48 和 小时后显示出明显较低的迁移，与对照组相比（ ；图 3）。

SEM images revealed differing hPDLSC morphologies and attachment on the surface of the root canal sealer discs (ESbcs, AHPbcs and AHP). HPDLSCs seeded onto the surface of ESbcs and AHPbcs sample discs exhibited a spindle-like elongated morphology, intense growth and spread. It should be highlighted that a higher number of attached cells were visible on the surface of ESbcs compared with that of AHPbcs. Conversely, the surface of AHP sample discs had a low quantity of cells and debris, indicating cell death (Figure 4).
扫描电子显微镜显示了不同的 hPDLSC 形态和附着在根管封闭剂盘（ESbcs，AHPbcs 和 AHP）表面上。种植在 ESbcs 和 AHPbcs 样品盘表面上的 HPDLSC 表现出类似纺锤形的细胞形态，生长和扩散强烈。值得强调的是，与 AHPbcs 相比，ESbcs 表面上可见更多附着的细胞。相反，AHP 样品盘表面上的细胞和碎片数量较少，表明细胞死亡（图 4）。

The RT-qPCR assay for the assessment of osteo/odonto/ cementogenic marker expression from hPDLSCs cultured with the tested materials (ESbcs or AHPbcs) produced a wide variety of results (Figure 5).
用于评估与测试材料（ESbcs 或 AHPbcs）培养的 hPDLSCs 中骨/牙/水泥发生标记表达的 RT-qPCR 检测产生了各种各样的结果（图 5）。

AHPbcs-treated cells exhibited a significantly higher early expression ( 3 and 7 days of culture) of ALP compared with the negative control group and a significantly higher late expression (14 and 21 days of culture) of CEMP1, CAP, ( at 21 days), RUNX2 and BSP. Compared with the positive control group, AHPbcs-treated cells exhibited a significantly higher early expression of ALP and RUNX2 and a late expression of ALP and AMELX.
AHPbcs 处理的细胞在早期表达（培养 3 和 7 天）ALP 方面与阴性对照组相比显著更高，并在晚期表达（培养 14 和 21 天）CEMP1、CAP、 （ 在 21 天）RUNX2 和 BSP 方面显著更高。与阳性对照组相比，AHPbcs 处理的细胞在 ALP 和 RUNX2 的早期表达以及 ALP 和 AMELX 的晚期表达方面显著更高。

FIG URE 2 Results from MTT assay for the different eluates (1:1, 1:2, 1:4) of the tested sealers (ESbcs, AHPbcs and AHP) after 24, 48 and of culture with hPDLSCs. Data are presented absorbance values ) at the different measurement time-points, compared with the negative control group. (One-way ANOVA analysis).
图 2 测试密封剂（ESbcs、AHPbcs 和 AHP）不同洗脱物（1:1、1:2、1:4）与 hPDLSCs 培养 24、48 和 小时后 MTT 测定结果。数据以吸光度值 ）在不同测量时间点呈现，与阴性对照组比较。 （单因素方差分析）。

FIGURE 3 Results from the wound healing assay for the different eluates (1:1, 1:2, 1:4) of the tested sealers (ESbcs, AHPbcs and AHP) after 24, 48 and of culture with hPDLSCs. Graphical results are presented as percentages of open wound areas at the different measurement time points, compared with the negative control group. (One-way ANOVA analysis). Images: Scale bar .
图 3 测试密封剂（ESbcs、AHPbcs 和 AHP）的不同洗脱物（1:1、1:2、1:4）在与 hPDLSCs 培养 24、48 和 后伤口愈合实验结果。图形结果以不同测量时间点的开放伤口面积百分比呈现，与阴性对照组进行比较。 （单因素方差分析）。图像：比例尺 。

ESbcs-treated cells exhibited a significantly higher early expression of all the tested markers compared with the negative and positive control groups, and a significantly higher late expression of CEMP1, CAP, ALP, BSP and AMELX compared with the negative control group. ESbcs-treated cells also exhibited a significantly higher late expression of ALP and BSP compared with the positive control group.
ESbcs 处理的细胞在所有测试标记的早期表达方面与阴性和阳性对照组相比显著更高，并且在 CEMP1、CAP、ALP、BSP 和 AMELX 的晚期表达方面与阴性对照组相比显著更高。ESbcs 处理的细胞在 ALP 和 BSP 的晚期表达方面也与阳性对照组相比显著更高。
When comparing the two calcium silicate cement sealers, ESbcs-treated cells exhibited a significantly higher early expression of all the tested markers and a significantly higher late expression of ALP, BSP and AMELX. On the other hand, AHPbcs-treated cells exhibited a significantly higher late expression of CEMP, CAP and RUNX2.
将两种硅酸钙水泥封闭剂进行比较时，ESbcs 处理的细胞显示出所有测试标记的早期表达显著更高，以及 ALP、BSP 和 AMELX 的晚期表达显著更高。另一方面，AHPbcs 处理的细胞显示出 CEMP、CAP 和 RUNX2 的晚期表达显著更高。

FIGURE 4 Results from the SEM visualization after of culture of hPDLSCs seeded onto the surface of the tested material sample discs (ESbcs, AHPbcs and AHP). Magnifications: 100x, and . Scale bars: , and .
图 4 hPDLSCs 种植在被测试材料样品盘（ESbcs，AHPbcs 和 AHP）表面上 培养后的 SEM 可视化结果。放大倍数：100x， 和 。比例尺： ， 和 。

Results from the cell mineralization assay are presented in Figure 6. Both AHPbcs and ESbcs-treated hPDLSCs exhibited a significantly higher mineralized nodule formation than the negative and positive control groups ( ). The AHP-treated hPDLSCs exhibited a significantly lower mineralization compared with the negative and positive control groups ( and ; respectively). As expected, cells cultured in osteogenic medium (Osteodiff; positive control) showed a significantly higher mineralization ( ) than those cultured in unconditioned medium (negative control). ESbcs-treated cells showed a significantly higher calcified nodule formation than those treated with AHPbcs ( ).
细胞矿化实验的结果见图 6。AHPbcs 和 ESbcs 处理的 hPDLSCs 显示出明显更高的矿化结节形成，比阴性和阳性对照组（ ）高。与阴性和阳性对照组相比，AHP 处理的 hPDLSCs 显示出明显较低的矿化（ 和 ；分别）。预期地，在成骨培养基中培养的细胞（Osteodiff；阳性对照）显示出明显更高的矿化（ ），比在未处理培养基中培养的细胞（阴性对照）高。ESbcs 处理的细胞显示出明显更高的钙化结节形成，比 AHPbcs 处理的细胞（ ）高。

New biomaterial formulations are constantly being introduced into the market for clinical use in the field of endodontics (Camilleri et al., 2022). Currently, calcium silicate cement-based materials are increasing in use amongst clinicians (Careddu et al., 2021). The recently introduced AH Plus Bioceramic Sealer is presented as a potential alternative to the classic AH Plus and other calcium silicate cement-based sealers such as Endosequence BC Sealer. Accordingly, the aim of the present study was to assess its cytocompatibility and bioactive properties on hPDLSCs and compare them with the aforementioned sealers.
新的生物材料配方不断被引入市场，用于根管治疗领域的临床应用（Camilleri 等，2022 年）。目前，基于硅酸钙水泥的材料在临床医生中的使用正在增加（Careddu 等，2021 年）。最近推出的 AH Plus 生物陶瓷封闭剂被提出作为经典 AH Plus 和其他基于硅酸钙水泥的封闭剂（如 Endosequence BC Sealer）的潜在替代品。因此，本研究的目的是评估其对 hPDLSCs 的细胞相容性和生物活性，并将其与上述封闭剂进行比较。

In vitro study designs like the present one offer a consistent analysis of the main biological properties of dental materials cultured together with cellular subpopulations with which they will come into contact during their clinical use and thus may predict their clinical behaviour (Pedano et al., 2020). However, as a limitation of the present work, several variables could affect the differences between the results observed under laboratory and clinical conditions, such as variations in , variations in oxygen levels or the patients' immune response (Sanz, Guerrero-Gironés, et al., 2021). Nevertheless, as a strength of the present work, the use of standardized material sample preparation and biological assay procedures (ISO 10993-5, 2009) results in an increased reproducibility of the study design and consequently an increased homogeneity between studies. Lastly, following specific reporting guidelines enhances the comparability between studies with similar methodologies. In the present study, the recently introduced PRILE guidelines were followed for such purpose (Nagendrababu et al., 2021b). Accordingly, the main steps of this work have been depicted in the PRILE 2021 flowchart (Figure 7).
在 vitro 研究设计，如目前的研究，提供了对牙科材料的主要生物学特性进行一致分析的机会，这些材料与细胞亚群一起培养，这些细胞亚群在临床使用过程中将与之接触，因此可能预测它们的临床行为（Pedano 等，2020 年）。然而，作为目前工作的局限性，一些变量可能影响实验室和临床条件下观察到的结果之间的差异，例如 的变化，氧气水平的变化或患者的免疫反应（Sanz，Guerrero-Gironés 等，2021 年）。然而，作为目前工作的优势，使用标准化的材料样品制备和生物学分析程序（ISO 10993-5，2009 年）导致研究设计的可重复性增加，从而增加了研究之间的同质性。最后，遵循特定的报告指南增强了具有类似方法的研究之间的可比性。在本研究中，最近引入的 PRILE 指南被用于此目的（Nagendrababu 等，2021b 年）。 因此，这项工作的主要步骤已在 PRILE 2021 流程图（图 7）中描述。

Biocompatibility and cytocompatibility assays are useful to confirm the positive or negative response of biological tissues and cellular populations. In the present study three cytocompatibility assays were performed: MTT assay, as a measure of cell viability; wound healing assay, as a measure of cell proliferation/migration; and SEM visualization, as a measure of cell morphology and attachment. All the cytocompatibility assays produced concordant results. Both calcium silicate cement-based sealers (ESbcs and AHPbcs) exhibited an adequate cytocompatibility compared with a negative control group. These results are in accordance with previous studies on the biological properties of calcium silicate cement-based sealers as a group of dental materials (Mann et al., 2022; Park et al., 2021; Zordan-Bronzel et al., 2019). The main components tricalcium silicate and a radiopacifying agent (zirconium oxides) have been shown to be biocompatible in previous studies (Campi et al., 2022).
生物相容性和细胞相容性试验对确认生物组织和细胞群体的积极或消极反应非常有用。在本研究中进行了三项细胞相容性试验：MTT 试验，作为细胞存活率的衡量；伤口愈合试验，作为细胞增殖/迁移的衡量；以及扫描电镜可视化，作为细胞形态和附着的衡量。所有细胞相容性试验产生了一致的结果。与阴性对照组相比，基于硅酸钙水泥的封闭剂（ESbcs 和 AHPbcs）表现出了良好的细胞相容性。这些结果与关于硅酸钙水泥基封闭剂作为一类牙科材料的生物学特性的先前研究相一致（Mann 等，2022 年；Park 等，2021 年；Zordan-Bronzel 等，2019 年）。主要成分三钙硅酸盐和一种增白剂（氧化锆）已在先前的研究中显示出具有生物相容性（Campi 等，2022 年）。

ALP/GAPDH Relative gene expression
ALP/GAPDH 相对基因表达

RUNX2/GAPDH Relative gene expression
RUNX2/GAPDH 相对基因表达

BSP/GAPDH Relative gene expression
BSP/GAPDH 相对基因表达

AMELX/GAPDH Relative gene expression
AMELX/GAPDH 相对基因表达

CAP/GAPDH Relative gene expression
CAP/GAPDH 相对基因表达

ALP/GAPDH Relative gene expression
ALP/GAPDH 相对基因表达

BSP/GAPDH Relative gene expression
BSP/GAPDH 相对基因表达

AMELX/GAPDH Relative gene expression
AMELX/GAPDH 相对基因表达

14 days 14 天

CEMP1/GAPDH Relative gene expression
CEMP1/GAPDH 相对基因表达

ALP/GAPDH Relative gene expression
ALP/GAPDH 相对基因表达

BSP/GAPDH Relative gene expression
BSP/GAPDH 相对基因表达

AMELX/GAPDH Relative gene expression
AMELX/GAPDH 相对基因表达

ALP/GAPDH Relative gene expression
ALP/GAPDH 相对基因表达

RUNX2/GAPDH Relative gene expression
RUNX2/GAPDH 相对基因表达

BSP/GAPDH Relative gene expression
BSP/GAPDH 相对基因表达

FIGURE 5 Results from the analysis of hPDLSCs osteo/odonto/cementogenic marker expression via RT-qPCR after 3, 7, 14 and 21 days of culture with DMEM (negative control), ESbcs, AHPbcs, or Osteodiff (postive control). (Two-way ANOVA analysis). Asterisks above the bars indicate a significant difference with the negative control group. Asterisks above the lines indicate a significant difference between the groups which the line is connecting.
图 5 hPDLSCs 骨/牙/水泥发生标记物表达分析结果，经过 3、7、14 和 21 天与 DMEM（阴性对照）、ESbcs、AHPbcs 或 Osteodiff（阳性对照）培养后通过 RT-qPCR。 （双向 ANOVA 分析）。在柱状图上方的星号表示与阴性对照组有显著差异。在连接线上方的星号表示连接线连接的组之间有显著差异。

FIGURE 6 Results from the Alizarin Red S staining of hPDLSCs after 21 days of culture with DMEM (negative control), ESbcs, AHPbcs, AHP or Osteodiff (positive control). (Two-way ANOVA analysis). Asterisks above the bars indicate a significant difference with the negative control group. Asterisks above the lines indicate a significant difference between the groups which the line is connecting.
图 6 hPDLSCs 经过 21 天与 DMEM（阴性对照）、ESbcs、AHPbcs、AHP 或 Osteodiff（阳性对照）培养后的 Alizarin Red S 染色结果。 （双向 ANOVA 分析）。在柱状图上方的星号表示与阴性对照组有显著差异。在连接线上方的星号表示连接线连接的组之间有显著差异。

The epoxy resin-based sealer AHP showed signs of cytotoxicity on hPDLSCs, evidenced by their significant decrease in cell viability and proliferation and aberrant morphology and attachment. These results are in accordance with previous studies on several cell subpopulations (Saygili et al., 2017; Rodríguez-Lozano et al., 2019). The resinous component in endodontic sealers or in resinmodified calcium silicate cements has been associated with the increased cytotoxicity (Bakir et al., 2022; Sanz, Soler-Doria, et al., 2021; Silva et al., 2013).
基于环氧树脂的密封剂 AHP 对 hPDLSCs 显示出细胞毒性迹象，表现为它们的细胞活力和增殖显著降低，形态和附着异常。这些结果与先前对几种细胞亚群的研究结果一致（Saygili 等，2017 年；Rodríguez-Lozano 等，2019 年）。树脂成分在根管封闭剂或树脂改性硅酸钙水泥中的增加与细胞毒性增加有关（Bakir 等，2022 年；Sanz，Soler-Doria 等，2021 年；Silva 等，2013 年）。

SEM is useful for the evaluation of the superficial morphology and texture of materials. Regarding calcium silicate cement-based materials, it can also be suitable for the evaluation of their hydration (Anthrayose et al., 2021). Both ESbcs and AHPbcs displayed a crystalline surfaces, unlike the epoxy resin sealer. AHPbcs exhibited a denser and more homogeneous microstructure. The hydration extent can influence the properties of cements (Camilleri, 2007) and thus may account for the differences observed in the mineralization assay.
SEM 对材料的表面形态和纹理的评估是有用的。关于硅酸钙水泥基材料，它也可以用于评估它们的水化作用（Anthrayose 等，2021 年）。ESbcs 和 AHPbcs 都显示出结晶表面，不像环氧树脂封闭剂。AHPbcs 表现出更密集和更均匀的微观结构。水化程度可以影响水泥的性质（Camilleri，2007 年），因此可能解释了矿化试验中观察到的差异。

Energy dispersive spectroscopy was used to examine the elements present on the surface of the samples. However, EDS only exhibits the distribution of elements on the sample's surface. Other complementary techniques such as X-ray diffraction (XRD) analysis or attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy are needed to identify calcium hydroxide peaks and other crystalline phases in hydraulic cements after setting (Ferreira et al., 2021).
能量色散光谱学被用来检查样品表面上存在的元素。然而，EDS 只展示样品表面上元素的分布。需要其他补充技术，如 X 射线衍射（XRD）分析或衰减全反射-傅里叶变换红外（ATR-FTIR）光谱学，以识别水泥凝固后的钙羟基峰和其他结晶相（Ferreira 等人，2021 年）。

Differences in the elemental composition of the tested biomaterials may account for their differing biological properties. For example, both calcium silicate cementbased sealers disclosed tricalcium silicate in their composition, but ESbcs also incorporates dicalcium silicate. Additionally, tricalcium silicate only represents in weight in AHPbcs, whilst ESbcs contain of tricalcium silicate and of dicalcium silicate. Differences in the composition of calcium silicate cementbased materials affect their behaviour (Watson et al., 2014). This explains the significantly higher mineralization exhibited by ESbcs-treated cells compared with those treated with AHPbcs in the ARS assay.
测试生物材料的元素组成差异可能解释它们不同的生物学特性。例如，基于硅酸钙水泥的封闭剂中披露了三钙硅酸盐的组成，但 ESbcs 还包含二钙硅酸盐。此外，三钙硅酸盐在 AHPbcs 中仅占 的重量，而 ESbcs 含有 的三钙硅酸盐和 的二钙硅酸盐。钙硅酸盐水泥基材料组成的差异影响其行为（Watson 等，2014 年）。这解释了与用 AHPbcs 处理的细胞相比，用 ESbcs 处理的细胞在 ARS 测定中表现出显著更高的矿化。

As expected, both ESbcs and AHPbcs contained more calcium and oxygen than AHP. Calcium and hydroxyl ion release after hydration has been associated with the favourable biological properties of calcium silicate cement-based endodontic biomaterials (Khalil et al., 2016). The observed peak in Plus can be explained by the inclusion of calcium tungstate as a radiopacifier in its composition. The observed in AHPbcs compared with
预期地，ESbcs 和 AHPbcs 中的钙和氧含量均高于 AHP。水化后的钙和羟基离子释放与硅酸钙水泥基根管生物材料的有利生物学特性相关联（Khalil 等，2016）。在 Plus 中观察到的 峰可以通过其成分中包含钨酸钙作为增白剂来解释。与 AHP 相比，AHPbcs 中观察到的 。

FIG URE 7 Preferred Reporting Items for Laboratory studies in Endodontology (PRILE 2021)-based flowchart.
图 7 基于 2021 年牙髓学实验室研究的首选报告项目（PRILE 2021）流程图。

ESbcs is supported by the differences in their percentage by weight (WT%) listed in their respective Material Safety Data Sheets ( vs. ). The higher proportion of radiopacifier in the AHPbcs correlated with differences in in the biological properties of endodontic sealers, as others have noted (Queiroz, Torres, Rodrigues, Viola, Bosso-Martelo, Chavez-Andrade, et al., 2021b). Thus, in future research, it could be interesting to study
ESbcs 受其各自材料安全数据表中按重量百分比（WT％）列出的差异支持（ vs. ）。 AHPbcs 中放射剂 的比例较高与根管封闭剂的生物学性质的差异相关，正如其他人所指出的（Queiroz，Torres，Rodrigues，Viola，Bosso-Martelo，Chavez-Andrade 等，2021b）。因此，在未来的研究中，研究这一点可能会很有趣。
the biological properties of new biomaterial compositions from the perspective of the differences in radiopacifying agents and concentrations, as performed by a recent study (Queiroz, Torres, Rodrigues, Viola, Bosso-Martelo, Chavez-Andrade, et al., 2021a).
最近一项研究（Queiroz，Torres，Rodrigues，Viola，Bosso-Martelo，Chavez-Andrade 等，2021a）从放射增白剂和浓度的差异的角度探讨了新生物材料组合的生物学特性。

It should be highlighted that the inability to assess the influence of fillers, thickening agents, additives and/or vehicles can act as a limitation of the analysis of the biological properties of the tested materials from the perspective of the differences in their composition. The presence and proportion of these components in the composition of the tested materials are often regarded as confidential business information. For example, of calcium hydroxide is included as a non-confidential additive in Endosequence Sealer. This contributed to the observed peak in the SEM-EDS analysis. Thus, other confidential additives could explain other differences in the materials' biological properties.
应强调的是，无法评估填料、增稠剂、添加剂和/或载体的影响可能会限制从其组成差异的角度分析所测试材料的生物性能。所测试材料的组成中这些成分的存在和比例通常被视为机密商业信息。例如，氢氧化钙的 作为 Endosequence Sealer 中的非机密添加剂。这导致了 SEM-EDS 分析中观察到的 峰。因此，其他机密添加剂可能解释材料生物性能的其他差异。

Results from the RT-qPCR assay are varied but follow a general pattern. In brief, hPDLSCS cultured with both ESbcs or AHPbcs exhibited a significant upregulation of at least one cementogenic, osteogenic and odontogenic marker compared with the negative and positive control group. These markers were assessed, based on similar studies in the field (Rodríguez-Lozano et al., 2020; Zheng et al., 2020): CEMP1, CAP, ALP, RUNX2, BSP, and AMELX.
RT-qPCR 检测结果各异，但遵循一般模式。简而言之，与阴性和阳性对照组相比，与 ESbcs 或 AHPbcs 一起培养的 hPDLSCS 表现出至少一种水泥发生、成骨和牙源性标记物的显著上调。这些标记物是根据领域内类似研究（Rodríguez-Lozano 等，2020 年；Zheng 等，2020 年）进行评估的：CEMP1、CAP、ALP、RUNX2、BSP 和 AMELX。

Cementogenic markers such as CEMP and CAP are important indicators of hPDLSC activity since they play a crucial role in the regeneration and repair of the periodontum. Specifically, an overexpression of CAP is seen during cell recruitment and differentiation during the formation of cementum tissue, whilst CEMP-1 is involved in the regulation of the differentiation of periodontal cells (Arzate et al., 2015; Pitaru et al., 1995). Thus, the overexpression of CEMP and CAP exhibited by ESbcs and AHPbcs-treated hPDLSCs may indicate their positive influence in cell plasticity and enhancement of the healing process of periodontal defects from lesions of endodontic origin.
水泥生成标记物如 CEMP 和 CAP 是 hPDLSC 活性的重要指标，因为它们在牙周组织再生和修复中发挥关键作用。具体来说，在水泥组织形成过程中，CAP 的过度表达在细胞招募和分化期间可见，而 CEMP-1 参与调节牙周细胞的分化（Arzate 等，2015 年；Pitaru 等，1995 年）。因此，ESbcs 和 AHPbcs 处理的 hPDLSCs 表现出 CEMP 和 CAP 的过度表达，可能表明它们对细胞可塑性和增强牙周缺损愈合过程的积极影响，这些缺损源于根管治疗。

BSP is a mineralized tissue-specific marker that is highly expressed during the initial formation of bone tissue and is synthesized by osteoblasts and osteoclasticlike cells in culture (Garcia et al., 2003; Ogata, 2008). ALP promotes bone formation by degrading inorganic pyrophosphate and generating inorganic phosphate, a crucial molecule in differentiation and mineralization of osteoblasts (Osathanon et al., 2009; Seltzer et al., 1962). Therefore, the overexpression of these markers exhibited by ESbcs and AHPbcs-treated hPDLSCs is a complementary indicator of the positive influence of these materials in cell plasticity and differentiation into an osteoblast-like lineage. This may reflect their potential enhancement of the process of bone tissue repair or regeneration.
BSP 是一种矿化组织特异性标记物，在骨组织初始形成过程中高度表达，并由成骨细胞和培养中的类骨吸收细胞合成（Garcia 等，2003 年；Ogata，2008 年）。ALP 通过降解无机焦磷酸盐并生成无机磷酸盐来促进骨形成，这是成骨细胞分化和矿化的关键分子（Osathanon 等，2009 年；Seltzer 等，1962 年）。因此，ESbcs 和 AHPbcs 处理的 hPDLSCs 表现出这些标记物的过表达，是这些材料对细胞可塑性和分化成类似成骨细胞谱系的积极影响的补充指标。这可能反映了它们增强骨组织修复或再生过程的潜力。
RUNX2 has been reported to be essential for the later stages of tooth formation, since it is involved in the development of mineralized dental tissue (Camilleri & McDonald, 2006). Additionally, it has been reported that RUNX2 is essential for osteoblast differentiation (Bruderer et al., 2014), and that its overexpression enhances the osteogenic activity of bone marrow stromal cells (Zhao et al., 2005). AMELX encodes for amelogenin, a structural modeling protein involved in the biomineralization process of amelogenesis (Green et al., 2019). Amelogenesis results in the formation and growth of hydroxyapatite crystals (Guo et al., 2015). Thus, the upregulation of these markers adds to the evidence on the enhancement of these materials of the process of mineralized tissue formation.
RUNX2 已被报道为牙齿形成后期至关重要，因为它参与了矿化牙齿组织的发育（Camilleri & McDonald，2006）。此外，有报道称 RUNX2 对成骨细胞分化至关重要（Bruderer 等，2014），其过表达增强了骨髓基质细胞的成骨活性（Zhao 等，2005）。AMELX 编码 amelogenin，这是一种参与牙釉质生物矿化过程的结构建模蛋白（Green 等，2019）。牙釉质生成导致了羟基磷灰石晶体的形成和生长（Guo 等，2015）。因此，这些标记物的上调增加了有关矿化组织形成过程中这些材料增强的证据。

An Alizarin Red S staining assay was performed as a complementary measure of the influence of the tested materials on hPDLSC mineralized tissue formation. The significantly higher calcified nodule formation exhibited by ESbcs and AHPbcs-treated cells, compared with the negative and positive controls, provided further support to their biomineralization ability. Similar results have been obtained in previous studies on other calcium silicate cement-based endodontic sealers (Rodríguez-Lozano et al., 2019; Sanz, López-García, et al., 2021). Contrarily, AHP-treated cells showed negative results on this assay, as observed in the aforementioned studies.
进行了 Alizarin Red S 染色实验，作为对测试材料对 hPDLSC 矿化组织形成影响的补充措施。与阴性和阳性对照相比，ESbcs 和 AHPbcs 处理的细胞表现出明显更高的钙化结节形成，进一步支持它们的生物矿化能力。与其他基于硅酸钙水泥的根管封闭剂相关的先前研究（Rodríguez-Lozano 等，2019 年；Sanz，López-García 等，2021 年）获得了类似的结果。相反，AHP 处理的细胞在这个实验中表现出负面结果，与前述研究中观察到的情况相同。

Altogether, the results from the cytocompatibility and bioactivity assays point towards the positive influence of ESbcs and AHPbcs on hPDLSC viability, migration, morphology, attachment, differentiation and biomineralization; and the negative influence of AHP on the same parameters. To the authors' knowledge, this is the first study to elucidate the biological properties of AHPbcs in controlled laboratory conditions. Further studies of interest in testing the material's behaviour in animal models or clinical trials.
总的来说，细胞相容性和生物活性测定的结果表明 ESbcs 和 AHPbcs 对 hPDLSC 的存活率、迁移、形态、附着、分化和生物矿化具有积极影响；而 AHP 对相同参数具有负面影响。据作者所知，这是第一项在受控实验室条件下阐明 AHPbcs 生物学特性的研究。进一步感兴趣的研究包括在动物模型或临床试验中测试材料的行为。

The new calcium silicate cement-based sealer AH Plus Bioceramic Sealer exhibited a significantly higher cytocompatibility and bioactive potential than the epoxy resin-based sealer Plus on human periodontal ligament stem cells. The cytocompatibility of AH Plus Bioceramic Sealer was comparable with that of the calcium silicate cement-based sealer Endosequence BC Sealer. Both calcium silicate-based sealers exhibited a significantly higher bioactive potential compared with a negative control group. However, Endosequence BC Sealer exhibited a significantly higher mineralization
新的基于硅酸钙水泥的封闭剂 AH Plus 生物陶瓷封闭剂在人牙周膜干细胞上表现出明显更高的细胞相容性和生物活性潜力，相比之下，环氧树脂基封闭剂 Plus 则较低。AH Plus 生物陶瓷封闭剂的细胞相容性与基于硅酸钙水泥的封闭剂 Endosequence BC Sealer 相当。两种基于硅酸钙的封闭剂在生物活性潜力方面均显著高于阴性对照组。然而，Endosequence BC Sealer 在矿化方面表现出明显更高的水平。
potential than AH Plus Bioceramic Sealer and AH Plus. The results from this in vitro study act as supporting evidence for the use of AH Plus Bioceramic Sealer in root canal treatment.
潜力比 AH Plus 生物陶瓷封闭剂和 AH Plus 更大。这项体外研究的结果作为支持 AH Plus 生物陶瓷封闭剂在根管治疗中使用的证据。

Investigation and methodology: Sergio López-García, Francisco Javier Rodríguez Lozano, José Luis Sanz; supervision, visualization, conceptualization and data curation: María Melo, Leopoldo Forner, Carmen Llena; investigation, methodology and writing-original draft: José Luis Sanz, Francisco Javier Rodríguez-Lozano; conceptualization, formal analysis, project administration, supervision, validation and writing-review and editing: Leopoldo Forner, Carmen Llena, María Melo; investigation, methodology, project administration, resources, writingoriginal draft, and writing-review and editing: José Luis Sanz, Francisco Javier Rodríguez-Lozano, Leopoldo Forner. All authors have read and agreed to the published version of the manuscript.
调查和方法学：Sergio López-García，Francisco Javier Rodríguez Lozano，José Luis Sanz；监督，可视化，概念化和数据整理：María Melo，Leopoldo Forner，Carmen Llena；调查，方法学和撰写原始草稿：José Luis Sanz，Francisco Javier Rodríguez-Lozano；概念化，形式分析，项目管理，监督，验证和撰写审查和编辑：Leopoldo Forner，Carmen Llena，María Melo；调查，方法学，项目管理，资源，撰写原始草稿和撰写审查和编辑：José Luis Sanz，Francisco Javier Rodríguez-Lozano，Leopoldo Forner。所有作者已阅读并同意发表的手稿版本。

JL Sanz received a grant from the Spanish Ministry of Science, Innovation, and Universities (FPU19/03115). S López-García received a contract financed by subsidies for the hiring of personnel researcher in post-doctoral phase (APOSTD) from the European Social Fund (ESF) and Generalitat Valenciana. This work was supported by the Spanish Network of Avanced Therapies (TERAV), RICORS project 'RD21/0001/0022' funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union - NextGenerationEU. Recovery, Transformation and Resilience Plan.
JL Sanz 收到了西班牙科学、创新和大学部（FPU19/03115）的资助。S López-García 获得了由欧洲社会基金（ESF）和瓦伦西亚自治区提供资助的博士后研究人员聘用补贴（APOSTD）的合同。本工作得到了西班牙先进疗法网络（TERAV）的支持，由卡洛斯三世健康研究所（ISCIII）资助的 RICORS 项目“RD21/0001/0022”，并得到了欧盟-下一代欧盟的共同资助。复苏、转型和韧性计划。

The authors declare no conflicts of interest related to this study.
作者声明与本研究无利益冲突。

The cell extraction protocol was approved by the Human Research Ethics Committee from the University of Murcia (reference: ).
细胞提取方案已获得穆尔西亚大学人类研究伦理委员会批准（参考： ）。

The data that support the findings of this study are available from the corresponding author upon reasonable request.
本研究结果的支持数据可根据合理要求从相应作者处获取。

José Luis Sanz © https://orcid.org/0000-0002-5438-8085
何塞·路易斯·桑斯 © https://orcid.org/0000-0002-5438-8085

Francisco Javier Rodriguez-Lozano © https://orcid. org/0000-0002-0623-740X

Carmen Llena © https://orcid.org/0000-0002-3942-2820
卡门·列纳 © https://orcid.org/0000-0002-3942-2820

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How to cite this article: Sanz, J.L., López-García, S., Rodríguez-Lozano, F.J., Melo, M., Lozano, A. & Llena, C. et al. (2022) Cytocompatibility and bioactive potential of AH Plus Bioceramic Sealer: An in vitro study. International Endodontic Journal, 55, 1066-1080. Available from: https://doi.org/10.1111/ iej. 13805
如何引用本文：Sanz, J.L., López-García, S., Rodríguez-Lozano, F.J., Melo, M., Lozano, A. & Llena, C. 等（2022 年）AH Plus 生物陶瓷封闭剂的细胞相容性和生物活性潜力：一项体外研究。国际根管治疗杂志，55，1066-1080。可从：https://doi.org/10.1111/ iej. 13805