Cell Wall Mannan of Candida Attenuates Osteogenic Differentiation by Human Dental Pulp Cells

IntroductionCandida spp. has recently been introduced to interact with conventional carious bacteria, leading to dental caries progression and virulence ability. Evidence regarding the influence of Candida spp. on human dental pulp cell response remains unknown. This study aimed to investigate the effects of Candida albicans mannans on cytotoxicity, cell proliferation, osteogenic differentiation, and inflammatory-related gene expression in human dental pulp cells (hDPCs).MethodshDPCs were treated with cell wall mannans isolated from C. albicans, Candida krusei, Candida glabrata, Candida tropocalis, Candida parapsilosis, and Candida dubliniensis. Cell viability was performed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Osteogenic differentiation– and inflammatory-related gene expression were determined using a real-time polymerase chain reaction. Mineralization was examined using alizarin red S staining.ResultsThe treatment of mannans isolated from C. albicans, C. krusei, C. glabrata, C. tropocalis, C. parapsilosis, and C. dubliniensis at concentrations ranging from 10–100 μg/mL did not affect cytotoxicity or cell proliferation. Mannans isolated from C. albicans, C. glabrata, and C. tropocalis significantly attenuated mineralization. However, cell wall mannans isolated from C. krusei, C. parapsilosis, and C. dubliniensis did not significantly influence mineral deposition in hDPCs. C. albicans cell wall mannans significantly attenuated osteogenic differentiation–related gene expression (RUNX2, ALP, and ENPP1). Interestingly, IL12 messenger RNA expression was significantly upregulated when treated with C. albicans cell wall mannans. The addition of recombinant IL12 significantly decreased mineralization in hDPCs.ConclusionsC. albicans cell wall mannans attenuated osteogenic differentiation in hDPCs and up-regulated inflammatory-related gene IL12 expression.     

The Effect of Octenidine on Proliferation,Migration, and Osteogenic Differentiation of Human Dental Pulp and Apical Papilla Stem Cells

IntroductionThe research for alternative irrigating solutions is ongoing, since no “ideal” solution has yet been found. Octenidine dihydrochloride (OCT) has been indicated as an endodontic irrigant because it has adequate antimicrobial and biological properties. The present study aimed to assess the effects of OCT on proliferation, migration, and induction of the osteogenic phenotype of stem cells from human dental pulp and apical papilla.MethodsCells were collected from human third molars and exposed to different doses of OCT, chlorhexidine (CHX), sodium hypochlorite (NaOCl), and ethylenediaminetetraacetic acid (EDTA) to determine cell viability by alamarBlue assay; proliferation by bromodeoxyuridine incorporation; migration by the Transwell assay; alkaline phosphatase activity by thymolphthalein release; and production of mineralized nodules by alizarin red staining. The results were analyzed by 1- or 2-way analysis of variance and Tukey (α = .05).ResultsCHX promoted lower cell viability, followed by OCT, NaOCl, and EDTA, especially at intermediate doses (P < .05). Cells exposed to CHX had less proliferation than the other groups (P < .05). The Transwell assay revealed no differences among OCT, EDTA, and culture medium (control group) (P > .05). OCT and EDTA induced greater migration than CHX and NaOCl (P < .05). OCT and EDTA induced higher alkaline phosphatase activity than NaOCl and CHX (P < .05). No difference was detected among the groups using alizarin red staining (P > .05).ConclusionsOCT induced high migration, proliferation, and alkaline phosphatase activity of stem cells from human dental pulp and apical papilla, which could be advantageous for regenerative endodontic procedures.     

Determinants of Dental Pulp Stem Cell Heterogeneity

IntroductionThe aim of this review is to provide a narrative review on the determinants of dental pulp stem cell (DPSC) heterogeneity that may affect the regenerative properties of these cells.MethodsPubMed, Scopus, and MEDLINE (Ovid) literature searches were done on human dental pulp stem cell heterogeneity. The focus was on human dental pulp stem cells with a primary focus on DPSC heterogeneity.ResultsDPSCs display significant heterogeneity as illustrated by the various subpopulations reported, including differences in proliferation and differentiation capabilities and the impact of various intrinsic and extrinsic factors.ConclusionsThe lack of consistent and reliable results in the clinical setting may be due to the heterogeneous nature of DPSC populations. Standardization in isolation techniques and criteria to characterize DPSCs should lead to less variability in results reported and improve comparison of findings between studies. Single-cell RNA sequencing holds promise in elucidating DPSC heterogeneity and may contribute to the establishment of standardized techniques.     

The Role of Small Extracellular Vesicles Derived from Lipopolysaccharide-preconditioned Human Dental Pulp Stem Cells in Dental Pulp Regeneration

IntroductionRegenerative endodontics has created a desirable shift in the treatment paradigm despite current limitations of regenerative outcomes. Mesenchymal stem cells (MSCs) facilitate tissue regeneration and repair in a mild inflammatory environment. Small extracellular vesicles (sEVs) derived from MSCs play an imperative role in the paracrine modulation of regenerative responses modulated by MSCs. However, it remains unknown whether MSCs enhance dental pulp regeneration or whether this enhancement is mediated by sEVs in a mild inflammatory environment. The present study aimed to elucidate the effects of sEVs originated from lipopolysaccharide (LPS)-preconditioned human dental pulp stem cells (hDPSCs) on dental pulp regeneration.MethodsAll sEVs were isolated from hDPSCs cultured with or without LPS (ie, N-sEVs and L-sEVs, respectively). The effect of N-sEVs and L-sEVs on proliferation, migration, angiogenesis, and differentiation of rat bone marrow MSCs was identified in vitro. Moreover, N-sEVs or L-sEVs were implanted into rat pulpless root canal models, and the regenerated tissue in root canals was assessed via hematoxylin-eosin staining, Masson staining, and immunohistochemistry after 30 days of transplantation.ResultsBoth N-sEVs and L-sEVs could modulate BMSC proliferation, migration, angiogenesis, and differentiation. Both kinds of sEVs enhanced the structure of the regenerated tissue closer to that of a normal dental pulp in vivo. L-sEVs had a more significant effect than N-sEVs.ConclusionssEVs released by hDPSCs in a mild inflammatory microenvironment are capable of facilitating the regeneration of dental pulp through functional healing instead of scar healing, which has potential applications in regenerative endodontics.     

人牙髓干细胞的体外培养和鉴定

目的　研究第三恒磨牙来源的人牙髓干细胞的表型和生物学性状。方法　从成人健康阻生牙中获取牙髓,酶消化法分离获得牙髓干细胞,计算细胞克隆形成率(CFU-F);免疫组化、RT-PCR法检测细胞的表面分子表达; 流式细胞仪测定细胞周期;体外分化诱导实验检测细胞的多向分化能力。结果　分离获得的牙髓干细胞在体外具有一定的克隆形成能力,诱导条件下部分牙髓干细胞可向脂肪、肌细胞和成牙本质细胞方向分化,符合干细胞的特征。结论　成功的从人第三恒磨牙牙髓中分离得到牙髓干细胞。     

Effect of EDTA on Attachment and Differentiation of Dental Pulp Stem Cells

Introduction

In regenerative endodontics, it is believed that EDTA induces odontoblast differentiation by releasing growth factors from the dentin matrix. The aim of this study was to evaluate the effect of EDTA on the attachment and differentiation of dental pulp stem cells (DPSCs). We also investigated whether the behavioral changes of DPSCs could be caused by biochemical components released from EDTA-treated dentin.

Methods

Cells were obtained from human third molars, and the stem-like nature of the cells was investigated by flow cytometric analysis. DPSCs were seeded on EDTA-treated and untreated dentin slices. After 3 days of culture, cell attachment was evaluated by cell density, fibronectin 1 gene expression level using quantitative real-time polymerase chain reaction, and scanning electron microscopy. After 21 days of culture, the expression of differentiation genes was investigated by quantitative real-time polymerase chain reaction, and calcification was observed using alizarin red S staining. To investigate the EDTA-induced growth factor release, DPSCs were cultured with or without direct contact with the EDTA-treated dentin surface.

Results

After 3 days of culture, both the cell density and fibronectin expression level were significantly higher in the EDTA-treated dentin group. After 3 weeks, the DPSCs on the EDTA-treated dentin surfaces showed higher expression levels of dentin sialophosphoprotein and dentin matrix protein 1, whereas the DPSCs cultured without direct contact with the EDTA-treated dentin surfaces did not exhibit these findings.

Conclusions

Our results showed that EDTA induced cell attachment and odontoblastic/osteoblastic differentiation, which was observed only in the group in which the DPSCs were placed in direct contact with the EDTA-treated dentin surfaces. These findings suggest that EDTA is beneficial for achieving successful outcomes in regenerative endodontics.     

神经生长因子对人牙髓细胞增殖与分化作用的研究

目的 研究神经生长因子对体外培养的人牙髓细胞增殖和分化作用的影响。方法 组织块法行人牙髓细胞的原代培养后，采用四唑盐比色法和酶动力学方法，测定不同浓度（1、10、100U／mL）的神经生长因子对体外培养的第5-8代人牙髓细胞增殖及碱性磷酸酶活性的作用。结果 与对照组相比，10U／mL的神经生长因子可显著促进人牙髓细胞的增殖（P〈0．05）；100U／mL的神经生长因子可显著促进碱性磷酸酶的活性（P〈0．01）。结论 不同浓度的神经生长因子可显著促进人牙髓细胞的增殖与分化。     

Ionic Extraction of a Novel Nano-sized Bioactive Glass Enhances Differentiation and Mineralization of Human Dental Pulp Cells

Introduction

This study aimed to investigate the effects of a novel nano-sized 58S bioactive glass (nano-58S BG) on the odontogenic differentiation and mineralization of human dental pulp cells (hDPCs) in vitro.

Methods

Extractions were prepared by incubating nano-58S BG, 45S5 BG, or 58S BG particulates in Dulbecco modified Eagle medium at 1% w/v for 24 hours and were filtrated through 0.22-μm filters. The supernatants were used as BG extractions. The hDPCs were cultured in nano-58S BG, 45S5 BG, and 58S BG extractions. The proliferation of hDPCs was evaluated using the methylthiazol tetrazolium assay. Odontogenic differentiation was evaluated based on the real-time polymerase chain reaction of differentiation- and mineralization-related genes, namely, alkaline phosphatase (ALP), collagen type I, dentin sialophosphoprotein (DSPP), and dentin matrix protein 1. The gene expressions were verified using ALP activity assessment, immunocytochemistry staining of osteocalcin and DSPP, and mineralization assay using alizarin red S stain.

Results

All BG extractions up-regulated the expression of odontogenic genes, and the most significant enhancement was in the nano-58S BG group. All BG extractions, especially nano-58S, increased ALP activity, osteocalcin and DSPP protein production, and mineralized nodules formation.

Conclusions

Compared with regular BG, the novel nano-58S BG can induce the differentiation and mineralization of hDPCs more efficiently and might be a better potential candidate for dentin-pulp complex regeneration.     

Evaluation of the Biodistribution of Human Dental Pulp Stem Cells Transplanted into Mice

Introduction

Several studies have attempted to use human dental pulp stem cells (hDPSCs) for pulp-dentin complex regeneration in vitro. However, the safety of such applications should be first evaluated in vivo before their use in clinical trials. The purpose of this study was to investigate the in vivo fate of intrapulpally transplanted hDPSCs.

SHH和bFGF体外诱导人牙髓干细胞向神经细胞分化的研究

目的：研究体外使用音猬因子（SHH）、碱性成纤维生长因子（bFGF）体外诱导人牙髓干细胞（DPSCs）分化为神经细胞的可行性,以优化人牙髓干细胞向神经细胞分化的诱导条件。方法：从因正畸或阻生拔除的第一前磨牙或第三磨牙中提取牙髓,采用酶消化及过滤法得到单细胞悬液,有限稀释法培养分离的原代人牙髓干细胞,并进行克隆化培养,检测间充质干细胞特异性标志物STRO-1的表达。将人牙髓干细胞分别接种于含有不同浓度诱导液,MTT法检测不同时间、两种因子单独或联合对细胞增殖能力的影;免疫荧光法检测抗微管相关蛋白（MAP-2）、神经元烯醇化酶（NSE）、胶质原纤维酸性蛋白（GFAP）的表达。透射电镜观察诱导前后细胞超微结构。结果：克隆来源细胞的间充质干细胞特异性标志物STRO-1表达阳性。100μg/L音猬因子SHH与20μg/L碱性成纤维生长因子bFGF单独作用促增殖作用最强（P〈0.05）,碱性成纤维生长因子bFGF单独作用各组及对照组均未检测出神经元样细胞。音猬因子SHH作用各组检测到阳性细胞。而100μg/L音猬因子SHH与20μg/L碱性成纤维生长因子bFGF联合增殖和分化作用均优于其它组。透射电镜观察到神经元样细胞表现。结论：100μg/L音猬因子和20μg/L碱性成纤维生长因子联合可以在体外有效诱导人牙髓干细胞分化为神经细胞。     

Biological Properties of Dental Pulp Stem Cells Isolated from Inflamed and Healthy Pulp and Cultured in an Inflammatory Microenvironment

IntroductionThe aim of this study was to assess whether the biological characteristics of dental pulp stem cells (DPSCs), such as viability, adhesion to dentin, mineralization, and release of immunomodulatory cytokines, are affected by the inflammatory status of the donor tissue and/or the sustained inflammatory environment.MethodsDPSCs were isolated from pulps from 3 caries-free teeth (healthy or hDPSCs), and from 3 teeth with irreversible pulpitis or deep caries (unhealthy DPSCs or uDPSCs). The cells were cultured in odontogenic and osteogenic media with or without lipopolysaccharides. Viability was analyzed by MTT assay at days 1, 3, 5, and 7; adhesion to dentin was evaluated through an environmental scanning electron microscope after 48 hours and through MTT assay; mineralization was analyzed with alizarin red staining after 21 days; and the release of proinflammatory (interleukin 6) and immunosuppressive cytokines (interleukin 10) was measured with the enzyme-linked immunosorbent assay after 24 hours and 7 days.ResultsThe inflammatory status of the pulp significantly reduced the viability and mineralization capacity of the DPSCs, although it did not affect the adhesion capacity to dentin or the secretion of the proinflammatory interleukin. The inflammatory microenvironment (lipopolysaccharide) only had a significant impact on the secretion of interleukin 6, which was augmented after 7 days.ConclusionsThe inflammatory status of the dental pulp should be taken into account when the use of DPSCs is intended either for research and/or for application in reparative or regenerative therapies.     

人牙髓干细胞在聚乳酸-聚乙醇酸共聚物支架上粘附与增殖的研究

目的：研究成人牙髓干细胞（HDPSCs）在聚乳酸-聚乙醇酸共聚物（PLGA）支架上粘附与增殖的情况。方法：采用酶消化法分离、培养人牙髓干细胞,免疫组化法及体外诱导分化对细胞进行鉴定。将人牙髓干细胞与PLGA支架材料进行复合培养,扫描电镜（SEM）观察支架材料形态,细胞粘附、增殖及基质分泌情况;细胞计数检测其增殖力。结果：细胞接种2、5、10d,扫描电镜及细胞计数均显示HDPSCs与PLGA支架材料粘附紧密,生长状态良好,细胞明显增殖（P〈0.05）,有丰富的细胞外基质形成。结论：PLGA是一种适宜人牙髓干细胞粘附与增殖的支架材料。     

牙髓干细胞在组织再生及细胞治疗中的研究进展

<正>在人体发育过程中,组织器官高度分化,行使不同功能,组成复杂的人体结构。一部分组织器官仍保留着一定再生功能。如上皮组织、肝组织等,而大部分组织器官只具有部分再生能力。如骨、软骨、神经组织。这些组织器官在创伤、感染、肿瘤等疾病破坏后再生能力较弱,给医学带来极大的挑战。近年来,组织再生及细胞治疗的发展为解决上述问题展现出美好的前景。干细胞用于组织再生及细胞治     

Characterization of Dental Pulp Stem Cells Isolated from Canine Premolars

Introduction

Dogs are commonly used animal models for regenerative endodontics research. Although several studies have used stem cells isolated from dog teeth to investigate the dentin/pulp regeneration in vivo, less attention has been paid for the characterization of these cells. Therefore, this study aimed to characterize the dental pulp stem cells isolated from dog teeth (cDPSCs) in order to further define the dog as an animal model for regenerative endodontics.

Methods

Stem cells were isolated from freshly extracted premolars of 10-month-old Beagles. The isolated cells were investigated for their stem cell properties by analysis of their clonogenic and growth characteristics; expression of mesenchymal stem cell markers; and evaluation of their osteo/odontogenic, adipogenic, and neurogenic potential.

Results

A colony formation assay showed the existence of a clonogenic cell population in cDPSCs isolated. The growth curves revealed a higher proliferation rate of cDPSCs compared with hBMMSCs. cDPSCs expressed mesenchymal stem cell markers STRO-1, CD146, and Nanog. However, they were negative for CD73, CD105, and CD45. cDPSCs were able to differentiate into odontoblast-like cells as shown by increased alkaline phosphatase activity, dentin sialoprotein expression, and formation of mineralized nodules. cDPSCs also showed the neurogenic and adipogenic differentiation potential at a lower level compared with those of hDPSCs and hBMMSCs.

Conclusions

The results of this study confirmed the stem cell properties of cDPSCs at a comparable level to those of hDPSCs and hBMMSCs. Overall, the data presented in this study provide evidence in supportive of using cDPSCs and dogs as an animal model in dental tissue engineering via stem cell-based approaches.     

Mesenchymal Stem Cells Derived from Human Inflamed Dental Pulp Exhibit Impaired Immunomodulatory Capacity In Vitro

IntroductionDental pulp stem cells (DPSC) are very attractive in regenerative medicine. In this study, we focused on the characterization of the functional properties of mesenchymal stem cells derived from DPSCs. Currently, it is unknown whether inflammatory conditions present in an inflamed dental pulp tissue could alter the immunomodulatory properties of DPSCs. This study aimed to evaluate the immunomodulatory capacity in vitro of DPSCs derived from healthy and inflamed dental pulp.MethodsDPSCs from 10 healthy and inflamed dental pulps (irreversible pulpitis) were characterized according to the minimal criteria of the International Society for Cell Therapy, proliferation, differential potential, and colony-forming units. Furthermore, the immunomodulatory capacity of DPSCs was tested on the proliferation of T lymphocytes by flow cytometry and the in vitro enzyme activity of indoleamine 2, 3-dioxygenase.ResultsThere were no significant differences in the DPSC characteristics and properties such as immunophenotype, tridifferentiation, colony-forming units, and proliferation of the DPSCs derived from normal and inflamed pulp tissue. Furthermore, there were significant differences in the immunomodulatory capacity of DPSCs obtained from human healthy dental pulp and with the diagnosis of irreversible pulpitis.ConclusionsOur results showed that DPSCs isolated from inflamed dental pulp showed typical characteristics of MSCs and diminished immunosuppressive capacity in vitro in comparison with MSCs derived from healthy dental pulp. Further investigation in vivo is needed to clarify the mechanism of this diminished immunosuppressive capacity.     

Effects of Platelet-rich Plasma and Cell Coculture on Angiogenesis in Human Dental Pulp Stem Cells and Endothelial Progenitor Cells

Introduction

Platelet-rich plasma (PRP) has been described as platelet concentrate. Growth factors released by activated platelets can improve wound vasculogenesis and enhance wound healing. In this study, we used PRP instead of serum to culture human dental pulp stem cells (hDPSCs) and endothelial progenitor cells (EPCs) and investigated revascularization ability. The effect of hDPSC and EPC coculture on vasculogenesis was also studied.

Methods

PRP was prepared by secondary centrifugation. Real-time polymerase chain reaction and Western blotting were used to determine the expression of vasculogenesis-related factors vascular endothelial growth factor, platelet-derived growth factor, fetal liver kinase 1 (Flk-1), and stromal cell-derived factor 1 (SDF-1) in cultured hDPSCs and EPCs. The cells were divided into 4 groups: EPCs + 10% fetal bovine serum (FBS), EPCs + 10% PRP, EPCs + hDPSCs + 10% FBS, and EPCs + hDPSCs + 10% PRP. Then, the formation of vessel-like structures was tested by the tube formation assay.

Results

On day 3, the expression levels of all the markers in the coculture groups were much higher than in the single-culture groups and were also higher in the PRP groups compared with the FBS groups (P < .05), except for SDF-1. Expression levels were significantly higher in the experimental groups (EPCs + 10% PRP, EPCs + hDPSCs + 10% FBS, and EPCs + hDPSCs + 10% PRP) than in the control group (EPCs + 10% FBS) and in the PRP groups/coculture groups compared with the FBS groups/single-culture groups (P < .01). The tube formation assay showed the area of vessel-like structures formed by the PRP group to be larger than in the FBS group (P < .05).

Conclusions

PRP and coculture can both promote vasculogenesis, and PRP can promote EPCs to form vessel-like structures.     

牙髓干细胞对面神经损伤修复研究进展

面神经损伤严重影响患者的身心健康,近些年,利用干细胞治疗神经损伤受到广泛关注。牙髓干细胞因其具有多种优势,在神经损伤的修复治疗中显示出巨大的优势,为面神经损伤的治疗提供了新的思路。本文将概述牙髓干细胞对面神经损伤修复的研究进展和应用前景。     

Human Dental Pulp Stem Cells Suppress Alloantigen-induced Immunity by Stimulating T Cells to Release Transforming Growth Factor Beta

Introduction

Human dental pulp stem cells (hDPSCs) are ideal candidates for regenerating damaged dental tissue. To examine the possibility that hDPSCs may be used to regenerate pulp, we tested their in vitro effects on acute allogeneic immune responses.