Effects of Lipopolysaccharide on the Proliferation and Osteogenic Differentiation of Stem Cells from the Apical Papilla

Introduction

Stem cells from the apical papilla (SCAPs) were suggested as the stem cell source in regenerative endodontic procedures. However, bone and/or cementum-like structure were observed in root canals. Lipopolysaccharide (LPS) in infected root canals might alter SCAPs' osteogenic differentiation pattern. The objectives of this study were to investigate the effects of LPS on SCAPs' proliferation and osteogenic differentiation.

兔骨髓干细胞和脂肪干细胞体外成骨能力的比较

目的:评价两种不同来源的间充质干细胞,即骨髓干细胞(BMSC)和脂肪干细胞(ADSC)的体外成骨能力,为其将来应用于细胞治疗和组织工程研究提供一定的理论依据。方法:分别取同一只兔的髂骨骨髓和腹股沟脂肪作为BMSC和ADSC的来源,分离培养后比较成骨、成脂分化潜能,细胞表面抗原标记物;成骨诱导后检测碱性磷酸酶(ALP)活性,RT-PCR分析其成骨相关基因的表达情况。结果:BMSC和ADSC都具有向成骨、成脂分化的潜能;在成骨诱导分化后,BMSC有较高的ALP活性和较多的钙结节形成。RT-PCR结果显示:BMSC表达较高的BMP-2、OCN和OPN等成骨基因。结论:BMSC和ADSC都具有向成骨、成脂分化的潜能,ADSC有较好的成脂分化能力,BMSC有较好的成骨分化能力;两者都可以作为组织工程的种子细胞。     

Comparative Effects of Concentrated Growth Factors on the Biological Characteristics of Periodontal Ligament Cells and Stem Cells from Apical Papilla

IntroductionDuring cell-free regenerative endodontic therapy, both stem cells from apical papilla (SCAPs) and periodontal ligament cells (PDLCs) are possible cell sources because of their proximity. Nonetheless, the regenerative ability of PDLCs and SCAPs under the induction of concentrated growth factors (CGFs) remains unclear.MethodsPDLCs and SCAPs were treated with various concentrations of CGF-conditioned medium (CCM). The effects of CCM with or without Porphyromonas gingivalis lipopolysaccharide (LPS) on cell migration, odonto/osteogenic differentiation, and the expression of inflammatory cytokines were assessed. Dentin matrix transplants composed of PDLCs or SCAPs cell sheets coupled with CGF were put subcutaneously in immunocompromised mice for 8 weeks to explore their regenerative characteristics in vivo.ResultsCCM dose dependently enhanced the migration, proliferation, and odonto/osteogenic differentiation of PDLCs and SCAPs. CCM alleviated LPS-inhibited odonto/osteogenic differentiation of PDLCs and SCAPs as well as the LPS-induced up-regulation of inflammatory cytokines. In vivo, the newly regenerated tissue and microvessels formed by PDLCs and SCAPs were significantly increased under the induction of CGF. SCAPs mainly regenerated pulp/dentinlike tissues and a large number of microvessels, whereas PDLCs mainly formed bone/cementumlike structures.ConclusionsOverall, PDLCs excelled in cell proliferation, migration, and osteogenic differentiation, whereas SCAPs outperformed PDLCs in terms of angiogenic and odontogenic differentiation. The biological differences between PDLCs and SCAPs provided a possible theoretical basis for the formation of bone/cementum/periodontal ligament–like tissues after cell-free regenerative endodontic therapy.     

High-temperature Requirement Protein A1 Regulates Odontoblastic Differentiation of Dental Pulp Cells via the Transforming Growth Factor Beta 1/Smad Signaling Pathway

Introduction

Dentinogenesis includes odontoblast differentiation and extracellular matrix maturation as well as dentin mineralization. It is regulated by numerous molecules. High-temperature requirement protein A1 (HtrA1) plays crucial roles in bone mineralization and development and is closely associated with the transforming growth factor beta (TGF-β) signal in osteogenesis differentiation. Simultaneously, the TGF-β1/small mother against decapentaplegic (Smad) signaling pathway is an important signaling pathway in various physiological processes and as a downstream regulation factor of HtrA1. However, the role of HtrA1 and its relationship with the TGF-β1/Smad signaling pathway in dentin mineralization is unknown.

Acetylsalicylic Acid Treatment and Suppressive Regulation of AKT Accelerate Odontogenic Differentiation of Stem Cells from the Apical Papilla

Introduction

Stem cells isolated from the root apical papilla of human teeth (stem cells from the apical papilla [SCAPs]) are capable of forming tooth root dentin and are a feasible source for bioengineered tooth root regeneration. In this study, we examined the effect of acetylsalicylic acid (ASA) on odontogenic differentiation of SCAPs in vitro and in vivo.

Effect of Bioceramic Materials on Proliferation and Odontoblast Differentiation of Human Stem Cells from the Apical Papilla

Introduction

In regenerative endodontic treatment (RET), practitioners favor the placement of bioceramics as sealing materials over blood clots. It is important to understand the interaction between sealing material and cells in the root canal. The purpose of this study was to compare the effectiveness of various bioceramic materials (ProRoot MTA [Dentsply, Tulsa, OK], Biodentine [Septodont, Saint-Maur-des-Fossés, France], and RetroMTA [BioMTA, Seoul, Korea]) as sealing materials in RET for the proliferation and differentiation of stem cells from the apical papilla (SCAPs).

Effects of L-Chg10-Teixobactin on Viability,Proliferation, and Osteo/Odontogenic Differentiation of Stem Cells from Apical Papilla

IntroductionIntracanal medicament is one of the essential steps for ensuring success in regenerative endodontic procedures. _L-Chg₁₀-teixobactin is a novel antimicrobial agent that exhibited potent antibacterial and antibiofilm effects against Enterococcus faecalis at low concentrations compared with ampicillin. At the same time, its cytotoxicity on dental stem cells has not been studied. This study aimed to investigate the effects of _L-Chg₁₀-teixobactin on the viability, proliferation, migration, and osteo/odontogenic differentiation of stem cells from apical papilla (SCAPs).Materials and MethodsSCAPs isolated from immature human third molars were treated with various concentrations of _L-Chg₁₀-teixobactin, calcium hydroxide, and dimethyl sulfoxide. The viability and proliferation of SCAPs were assessed using the LIVE/DEAD Viability/Cytotoxicity Kit and Cell Counting Kit-8. A scratch wound healing test was used to evaluate the lateral migration capacity of SCAPs. Alkaline phosphatase (ALP) activity, calcium mineralization ability tests -ie, ALP staining and alizarin red S staining, and quantitative real-time polymerase chain reaction were performed to assess the osteo /odontogenic differentiation of SCAPs.ResultsThe tested concentrations of _L-Chg₁₀-teixobactin (0.01, 0.02, and 0.03 mg/mL), 1 mg/mL calcium hydroxide, and 0.03% dimethyl sulfoxide had no significant cytotoxic effect on SCAPs at any time point (P > .05). Besides, there were no significant differences between the control and experimental groups in SCAPs’ viability, proliferation, and migration. _L-Chg₁₀-teixobactin upregulated the gene expression of osteo/odontogenic markers in SCAPs, while no significant difference was found in the ALP activity and alizarin red S staining.Conclusions_L-Chg₁₀-teixobactin demonstrated excellent biocompatibility on SCAPs at concentrations from 0.01 to 0.03 mg/mL and potentially enhance the osteo/odontogenic differentiation of SCAPs; suggesting its promising role as root canal medicament for regenerative endodontic procedures.     

Melatonin Treatment Alters Biological and Immunomodulatory Properties of Human Dental Pulp Mesenchymal Stem Cells via Augmented Transforming Growth Factor Beta Secretion

IntroductionMelatonin is an endogenous neurohormone with well-reported anti-inflammatory and antioxidant properties, but the direct biological and immunomodulatory effects of melatonin on human dental pulp stem cells (hDPSCs) has not been fully elucidated. The aim of this study was to evaluate the influence of melatonin on the cytocompatibility, proliferation, cell migration, odontogenic differentiation, mineralized nodule formation, and immunomodulatory properties of hDPSCs.MethodsTo address the melatonin biological effects on hDPSCs, the cytocompatibility, proliferation, cell migration, odontogenic differentiation, mineralized nodule formation, and immunomodulatory properties of hDPSCs after melatonin treatment were evaluated. The statistical differences were evaluated using 1-way analysis of variance with the Tukey multiple comparison test.ResultsWe found that melatonin did not alter hDPSC immunophenotype or cell viability, even at the highest concentrations used. However, using intermediate melatonin concentrations (10–300 μmol/L), a significantly higher proliferation rate (P < .05 and P < .01) and migration of hDPSCs (P < .01) were observed. Importantly, melatonin treatment (100 μmol/L) significantly increased the secretion of the anti-inflammatory cytokine transforming growth factor beta (P < .05 and P < .01) and provoked a more robust antiproliferative effect on mitogen-stimulated T cells (P < .05). Finally, and unlike previous results found with mesenchymal stem cells from other sources, melatonin fails to induce or accelerate the spontaneous osteogenic differentiation of hDPSCs.ConclusionsTogether, these findings provide key data on the bioactivity of melatonin and its effects on hPDSC biological and immunomodulatory properties.     

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.     

Toll-like Receptor Expression Profile of Human Stem/Progenitor Cells Form the Apical Papilla

IntroductionStem/progenitor cells from the apical papilla (SCAPs) demonstrate remarkable regenerative and immunomodulatory properties. During their regenerative events, SCAPs, similar to other stem/progenitor cells, could interact with their local inflammatory microenvironment via their expressed toll-like receptors (TLRs). The present study aimed to describe for the first time the unique TLR expression profile of SCAPs.MethodsCells were isolated from the apical papilla of extracted wisdom teeth (n = 8), STRO-1 immunomagnetically sorted, and cultured to obtain single colony-forming units. The expression of CD14, 34, 45, 73, 90, and 105 were characterized on the SCAPs, and their multilineage differentiation potential was examined to prove their multipotent aptitude. After their incubation in basic or inflammatory medium (25 ng/mL interleukin 1 beta, 10³ U/mL interferon gamma, 50 ng/mL tumor necrosis factor alpha, and 3 × 10³ U/mL interferon alpha), a TLR expression profile for SCAPs under uninflamed as well as inflamed conditions was respectively generated.ResultsSCAPs demonstrated all predefined stem/progenitor cell characteristics. In basic medium, SCAPs expressed TLRs 1–10. The inflammatory microenvironment up-regulated the expression of TLR1, TLR2, TLR4, TLR5, TLR6, and TLR9 and down-regulated the expression of TLR3, TLR7, TLR8, and TLR10 in SCAPs under the inflamed condition.ConclusionsThe present study defines for the first time a distinctive TLR expression profile for SCAPs under uninflamed and inflamed conditions. This profile could greatly impact SCAP responsiveness to their inflammatory microenvironmental agents under regenerative conditions in vivo.     

骨髓间充质干细胞骨向诱导分化过程中破骨细胞分化因子和细胞间粘附分子-1的表达变化

目的　观察破骨细胞分化因子(ODF)和细胞间粘附分子-1(ICAM-1)在不同分化状态成骨细胞的表达变 化,探讨正畸牙移动过程中成骨细胞对破骨细胞分化成熟的诱导机制。方法　分离培养大鼠骨髓间充质干细胞, 成骨定向诱导后获得不同分化状态的成骨细胞,RT-PCR检测不同分化状态下的成骨细胞ODF和ICAM-1的表达变 化。结果　成骨细胞在分化成熟过程中,ICAM-1 mRNA表达水平逐渐升高;ODF mRNA则在诱导后6 d开始表达, 并维持在一较稳定的水平。结论　不同分化状态的成骨细胞对破骨细胞诱导分化的能力有所差异,相对成熟的成 骨细胞的诱导能力可能更强。     

Endocannabinoids Regulate Stem Cells of the Apical Papilla via a Cannabinoid Receptor and TRPV1-Independent Mechanism

IntroductionEndogenous cannabinoids (endocannabinoids [eCBs]) have been shown to have a multitude of functions including neurotransmission and immune modulatory effects. This study aimed to evaluate if stem cells of the apical papilla (SCAP) express the receptors and enzymes of the endocannabinoid system (ECS) and whether eCBs regulate their proliferation and mineralization potential.MethodsGene expression of the main components of the ECS and transient receptor potential vanilloid 1 (TRPV1) was evaluated in SCAP cultures. SCAP were treated with 2 concentrations of eCBs and/or capsazepine, a TRPV1 antagonist. SCAP viability was evaluated after 1, 4, and 7 days. Osteogenic differentiation was assessed after 14 days, and the gene expression of mineralization markers was assessed after 7 days.ResultsThe enzymes of ECS and TRPV1 but not the cannabinoid receptors (cannabinoid receptors 1 and 2) were expressed in SCAP. Anandamide, 2-arachidonoylglycerol, and N-arachidonoylphenolamine (AM-404) reduced SCAP viability in all experimental periods at the highest concentration compared with the group with no treatment. Anandamide and AM-404 did not inhibit SCAP differentiation potential, but 2-arachidonoylglycerol at the highest concentration did. SCAP treated with AM-404 presented a down-regulation in gene expression of alkaline phosphatase (ALP), dentin matrix protein 1 (DMP-1), and dentin sialophosphoprotein (DSPP) compared with the proliferation medium group but not with control group.ConclusionsSCAP expressed the genes of the main components of ECS and TRPV1, and eCBs can affect SCAP viability, mineralization, and gene expression.     

A Comparative Evaluation of Concentrated Growth Factor and Platelet-rich Fibrin on the Proliferation,Migration, and Differentiation of Human Stem Cells of the Apical Papilla

Introduction

Concentrated growth factor (CGF) is considered to be a natural biomaterial that is better than platelet-rich fibrin (PRF) in bone regeneration, but there is little information acquired in regenerative endodontics. Therefore, the purpose of this study was to evaluate their effects on the proliferation, migration, and differentiation of human stem cells of the apical papilla (SCAPs).

软骨上清液与转化生长因子诱导滑膜间充质干细胞向软骨细胞分化的对比研究

目的:对比使用软骨上清液和转化生长因子两种方法诱导滑膜间充质干细胞向软骨细胞分化。方法:分别采用消化法获取SD大鼠滑膜间充质干细胞和软骨细胞。体外扩增。实验组一:通过软骨细胞上清液诱导滑膜间充质干细胞向软骨方向分化。实验组二:通过软骨诱导液(TGF-β1,ITS+ Premix,2-磷酸抗坏血酸等)诱导滑膜间充质干细胞向软骨方向分化。培养21 d后通过形态学、免疫组织化学法检测其生物学特性,RT-PCR检测诱导后产物Ⅱ型胶原RNA含量。结果:2种诱导方法均能诱导滑膜间充质干细胞成软骨细胞方向分化。在形态学可见2组诱导后产物成软骨样结构,呈乳白色,质地韧。免疫组织化学鉴定基质能被Ⅱ型胶原染色,细胞染色呈现棕黄色。RT-PCR结果显示诱导后的微团表达软骨特异性基因Ⅱ型胶原和蛋白聚糖。结论:两组实验组均能诱导滑膜间充质干细胞向软骨方向分化。对比两种实验方法,使用上清液诱导滑膜间充质干细胞向软骨方向分化能力更强。     

White Mineral Trioxide Aggregate Induces Migration and Proliferation of Stem Cells from the Apical Papilla

Introduction

Regenerative endodontic protocols recommend white mineral trioxide aggregate (WMTA) as a capping material because of its osteoinductive properties. Stem cells from the apical papilla (SCAP) are presumed to be involved in this regenerative process, but the effects of WMTA on SCAP are largely unknown. Our hypothesis was that WMTA induces proliferation and migration of SCAP.

Methods

Here we used an unsorted population of SCAP (passages 3–5) characterized by high CD24, CD146, and Stro-1 expression. The effect of WMTA on SCAP migration was assessed by using transwells, and its effect on proliferation was determined by the WST-1 assay. Fetal bovine serum (FBS) and calcium chloride–enriched medium were used as positive controls.

Results

The SCAP analyzed here showed a low percentage of STRO-1+ and CD24+ cells. Both set and unset WMTA significantly increased the short-term migration of SCAP after 6 hours (P < .05), whereas calcium chloride–enriched medium did after 24 hours of exposure. Set WMTA significantly increased proliferation on days 1–5, whereas calcium-enriched medium showed a significant increase on day 7, with a significant reduction on proliferation afterwards. SCAP migration and proliferation were significantly and steadily induced by the presence of 2% and 10% FBS.

Conclusions

Collectively, these data demonstrate that WMTA induced an early short-term migration and proliferation of a mixed population of stem cells from apical papilla as compared with a later and longer-term induction by calcium chloride or FBS.