A Synthetic Oligopeptide Derived From Enamel Matrix Derivative Promotes the Differentiation of Human Periodontal Ligament Stem Cells Into Osteoblast‐Like Cells With Increased Mineralization

Background: In a previous study, the authors obtained a synthetic peptide (SP) for useful periodontal tissue regeneration. Periodontal ligament stem cells (PDLSCs) have multiple potentiality to contribute to tissue regeneration. The aim of this experiment is to investigate the effect of SP on human PDLSCs. Methods: Periodontal ligament cells were obtained from healthy adult human third molars and used to isolate single PDLSC‐derived colonies. The mesenchymal stem cell nature of the PDLSCs was confirmed by immunohistochemical evaluation of STRO‐1 expression. Proliferation and osteoblastic differentiation were investigated by culturing PDLSCs in normal or osteogenic medium with and without SP (100 ng/mL). Osteoblast differentiation was assessed by measuring alkaline phosphatase (ALP) activity, osteocalcin production, mRNA expression of osteonectin, mineralization, and calcium deposition. Results: Isolated PDLSCs were immunohistochemically positive for vimentin and STRO‐1 and negative for cytokeratin. A greater number of calcified nodules were observed in osteogenic medium culture with SP than without. In the early and later stages of PDLSC culture with SP, osteonectin production and osteocalcin production were increased. SP in culture with osteogenic medium significantly enhanced proliferation of PDLSCs, as well as ALP activity, expression of osteonectin, osteocalcin production, formation of calcified nodules, and mineralization. Conclusions: SP enhances the formation of calcified nodules and osteocalcin production in the culture of PDLSCs into osteoblast‐like cells and is a useful material for periodontal tissue regeneration.     

Stromal cell-derived factor-1 significantly induces proliferation, migration, and collagen type I expression in a human periodontal ligament stem cell subpopulation

Background: The pivotal role of chemokine stromal cell–derived factor‐1 (SDF‐1) in bone marrow mesenchymal stem cells recruitment and tissue regeneration has already been reported. However, its roles in human periodontal ligament stem cells (PDLSCs) remain unknown. PDLSCs are regarded as candidates for periodontal tissue regeneration and are used in stem cell–based periodontal tissue engineering. The expression of chemokine receptors on PDLSCs and the migration of these cells induced by chemokines and their subsequent function in tissue repair may be a crucial procedure for periodontal tissue regeneration. Methods: PDL tissues were obtained from clinically healthy premolars extracted for orthodontic reasons and used to isolate single‐cell colonies by the limited‐dilution method. Immunocytochemical staining was used to detect the expression of the mesenchymal stem cell marker STRO‐1. Differentiation potentials were assessed by alizarin‐red staining and oil‐red O staining. The expression of SDF‐1 receptor CXCR4 was evaluated by real‐time polymerase chain reaction (PCR) and immunocytochemical staining. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and bromodeoxyuridine incorporation assay were used to determine the viability and proliferation of the PDLSC subpopulation. Expression of collagen type I and alkaline phosphatase was detected by real‐time PCR to determine the effect of SDF‐1 on cells differentiation. Results: Twenty percent of PDL single‐cell colonies expressed STRO‐1 positively, and this specific subpopulation was positive for CXCR4 and formed minerals and lipid vacuoles after 4 weeks induction. SDF‐1 significantly increased proliferation and stimulated the migration of this PDLSC subpopulation at concentrations between 100 and 400 ng/mL. CXCR4 neutralizing antibody could block cell proliferation and migration, suggesting that SDF‐1 exerted its effects on cells through CXCR4. SDF‐1 promoted collagen type I level significantly but had little effect on alkaline phosphatase level. Conclusion: SDF‐1 may have the potential of promoting periodontal tissue regeneration by the mechanism of guiding PDLSCs to destructive periodontal tissue, promoting their activation and proliferation and influencing the differentiation of these stem cells.     

High‐power,red‐light‐emitting diode irradiation enhances proliferation,osteogenic differentiation,and mineralization of human periodontal ligament stem cells via ERK signaling pathway

1 Background

Light‐emitting diode (LED) is attracting attention as a new light source for phototherapy. However, its effects on periodontal tissue regeneration remain unknown. The aim of this study was to examine the effects of high‐power, red LED irradiation on human periodontal ligament stem cells (PDLSCs), which play an important role in periodontal tissue regeneration.

2 Methods

PDLSCs were derived from adult human third molars. The light source was red LED (peak wavelength: 650 nm). Energy densities ranging from 0 to 10 J/cm² were tested to determine the optimal dose. PDLSC proliferation was measured using two parameters: live cell protease and ATP levels. After the cells were induced to differentiate, the effect of LED irradiation on osteogenic differentiation and mineralization was examined, with particular focus on the extracellular signal‐regulated kinase (ERK)1/2 signaling pathway using an ERK inhibitor (PD98059).

3 Results

LED irradiation at 8 J/cm² led to a significant increase in PDLSC proliferation and enhanced Runx2 and Osterix mRNA expression, Alkaline phosphatase activity, procollagen type I C‐peptide and osteocalcin production, calcium deposition, and alizarin red S staining. In addition, LED induced the activation of ERK1/2, and the effects of LED on PDLSC proliferation, differentiation, and mineralization could be suppressed by treatment with PD98059.

4 Conclusions

The results of this study show that 650‐nm high‐power, red, LED irradiation increases PDLSCs proliferation, and osteogenic differentiation and mineralization, mediated by ERK1/2 activation. These findings suggest that LED may be a useful tool for periodontal tissue regeneration.     

Physiologic Levels of Endogenous Hydrogen Sulfide Maintain the Proliferation and Differentiation Capacity of Periodontal Ligament Stem Cells

Background: Many invading oral bacteria are known to produce considerable amounts of hydrogen sulfide (H₂S). The toxic activity of exogenous H₂S in periodontal tissue has been demonstrated, but the role of endogenous H₂S in the physiologic function of periodontal tissue remains poorly understood. The purpose of the present study is to investigate the biologic functions of H₂S in the proliferation and differentiation of human periodontal ligament stem cells (PDLSCs). Methods: PDLSCs were isolated from periodontal ligament tissues of periodontally healthy volunteers or patients with periodontitis. Immunocytochemical staining, flow cytometry, and Western blot analysis were used to examine the expression of H₂S‐synthesizing enzymes cystathionine‐β‐synthase (CBS) and cystathionine‐γ‐lyase (CSE). The proliferation capacity of PDLSCs was determined by cell counting kit‐8 assay, carboxyfluorescein succinimidyl ester analysis, and 5‐ethynyl‐2′‐deoxyuridine assay. The osteogenic potential of PDLSCs was tested using alkaline phosphatase staining, Alizarin Red staining, and in vivo transplantation experiments. Oil Red O staining was used to analyze adipogenic ability. Results: The results show that human PDLSCs express both CBS and CSE and produce H₂S. Blocking the generation of endogenous H₂S with CBS inhibitor hydroxylamine significantly attenuated PDLSC proliferation and reduced the osteogenic and adipogenic differentiation capacity of PDLSCs. In contrast, CSE inhibitor dl ‐propargylglycine had no effect on PDLSC function. Exogenous H₂S could inhibit the production of endogenous H₂S and impair PDLSC function in a dose‐dependent manner. Conclusion: Physiologic levels of endogenous H₂S maintain the proliferation and differentiation capacity of PDLSCs, and CBS may be the main source of endogenous H₂S in PDLSCs.     

Exendin-4 relieves the inhibitory effects of high glucose on the proliferation and osteoblastic differentiation of periodontal ligament stem cells

BackgroundWith the impaired regenerative potential in patients with diabetes mellitus (DM), Periodontal ligament stem cells (PDLSCs) are regarded as an attractive source of stem cells for periodontal cytotherapy. Recent studies have shown that Exendin-4 (Ex-4) exerts cell-protective effects and bone remodeling ability on many types of cells. The aim of this study was to investigate whether Ex-4 alleviates the inhibition of high glucose on the proliferation and osteogenic differentiation of PDLSCs.MethodsPDLSCs were incubated in medium supplemented with 5.5 mM d-glucose (NG), 30 mM d-glucose (HG), NG plus Ex-4, and HG plus different concentration (1, 10, 20, 100 nM) of Ex-4 respectively. Cell proliferation was detected by CCK-8 assay and cell cycle analysis. Osteogenesis was assessed by Alizarin Red S staining and evaluation of the mRNA expression of Runx2, ALP and Osx at day 7, 14 and 21. Intracellular level of reactive oxygen species (ROS) was detected using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (CMH2DCF-DA).ResultsThe proliferation ability, mineralized nodules forming capacity and the mRNA expression of Runx2, ALP and Osx of PDLSCs in HG group were decreased, the ROS level was increased compared to NG group. With the treatment of Ex-4, the HG-inhibited proliferation ability and osteogenic differentiation ability of PDLSCs were significantly reversed, the HG-increased ROS level could be down-regulated. Moreover, Ex-4 enhanced the osteogenic differentiation of normal PDLSCs.ConclusionsEx-4 alleviates the inhibitory effect of HG on the proliferation and osteoblastic differentiation of PDLSCs, and has a significant enhance in the osteoblastic differentiation of normal PDLSCs, giving new insights into the possible therapeutic method of diabetic periodontitis.     

DNA from Porphyromonas gingivalis and Tannerella forsythia induce cytokine production in human monocytic cell lines

Toll‐like receptor 9 (TLR9) expression is increased in periodontally diseased tissues compared with healthy sites indicating a possible role of TLR9 and its ligand, bacterial DNA (bDNA), in periodontal disease pathology. Here, we determine the immunostimulatory effects of periodontal bDNA in human monocytic cells (THP‐1). THP‐1 cells were stimulated with DNA of two putative periodontal pathogens: Porphyromonas gingivalis and Tannerella forsythia. The role of TLR9 in periodontal bDNA‐initiated cytokine production was determined either by blocking TLR9 signaling in THP‐1 cells with chloroquine or by measuring IL‐8 production and nuclear factor‐κB (NF‐κB) activation in HEK293 cells stably transfected with human TLR9. Cytokine production (IL‐1β, IL‐6, and TNF‐α) was increased significantly in bDNA‐stimulated cells compared with controls. Chloroquine treatment of THP‐1 cells decreased cytokine production, suggesting that TLR9‐mediated signaling pathways are operant in the recognition of DNA from periodontal pathogens. Compared with native HEK293 cells, TLR9‐transfected cells demonstrated significantly increased IL‐8 production (P < 0.001) and NF‐κB activation in response to bDNA, further confirming the role of TLR9 in periodontal bDNA recognition. The results of PCR arrays demonstrated upregulation of proinflammatory cytokine and NF‐κB genes in response to periodontal bDNA in THP‐1 cells, suggesting that cytokine induction is through NF‐κB activation. Hence, immune responses triggered by periodontal bacterial nucleic acids may contribute to periodontal disease pathology by inducing proinflammatory cytokine production through the TLR9 signaling pathway.     

MAPKs, activator protein-1 and nuclear factor-κB mediate production of interleukin-1β-stimulated cytokines, prostaglandin E₂ and MMP-1 in human periodontal ligament cells

Murayama R, Kobayashi M, Takeshita A, Yasui T, Yamamoto M. MAPKs, activator protein‐1 and nuclear factor‐κB mediate production of interleukin‐1β‐stimulated cytokines, prostaglandin E ₂ and MMP‐1 in human periodontal ligament cells. J Periodont Res 2011; 46: 568–575. © 2011 John Wiley & Sons A/S Background and Objective: Determination of the interleukin‐1 (IL‐1) signaling cascades that lead to the production of various inflammatory mediators and catabolic factors may clarify attractive targets for therapeutic intervention for periodontitis. We comprehensively assessed the involvement of MAPKs, activator protein‐1 (AP‐1) and nuclear factor‐κB (NF‐κB) in IL‐1β‐induced production of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), prostaglandin E₂ (PGE₂) and MMP‐1 in human periodontal ligament cells. Material and Methods: Human periodontal ligament cells were pretreated with an inhibitor for each of the MAPKs or NF‐κB and subsequently treated with IL‐1β. Following treatment, phosphorylation of three types of MAPK (ERK, p38 MAPK and c‐Jun N‐terminal kinase), IκB kinase (IKK) α/β/γ and IκB‐α, as well as the DNA binding activity of AP‐1 and NF‐κB and the production of IL‐6, IL‐8, PGE₂ and MMP‐1, were determined by western blotting, a gel mobility shift assay and ELISA, respectively. Results: The three MAPKs, simultaneously activated by IL‐1β, mediated the subsequent DNA binding of AP‐1 at various magnitudes, while IKKα/β/γ, IκB‐α and NF‐κB were also involved in the IL‐1 signaling cascade. Furthermore, IL‐1β stimulated the production of IL‐6, IL‐8, PGE₂ and MMP‐1 via activation of the three MAPKs and NF‐κB, because inhibitors of these significantly suppressed the IL‐1β‐stimulated production of these factors. Conclusion: Our results strongly suggest that MAPK, AP‐1 and NF‐κB mediate the IL‐1β‐stimulated synthesis of IL‐6, IL‐8, PGE₂ and MMP‐1 in human periodontal ligament cells. Therefore, inhibition of activation of MAPK, AP‐1 and/or NF‐κB may lead to therapeutic effects on progression of periodontitis.     

Aspirin Enhances Osteogenic Potential of Periodontal Ligament Stem Cells (PDLSCs) and Modulates the Expression Profile of Growth Factor–Associated Genes in PDLSCs

Background: This study investigates the effects of aspirin (ASA) on the proliferative capacity, osteogenic potential, and expression of growth factor–associated genes in periodontal ligament stem cells (PDLSCs). Methods: Mesenchymal stem cells (MSCs) from PDL tissue were isolated from human premolars (n = 3). The MSCs’ identity was confirmed by immunophenotyping and trilineage differentiation assays. Cell proliferation activity was assessed through 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Polymerase chain reaction array was used to profile the expression of 84 growth factor–associated genes. Pathway analysis was used to identify the biologic functions and canonic pathways activated by ASA treatment. The osteogenic potential was evaluated through mineralization assay. Results: ASA at 1,000 μM enhances osteogenic potential of PDLSCs. Using a fold change (FC) of 2.0 as a threshold value, the gene expression analyses indicated that 19 genes were differentially expressed, which includes 12 upregulated and seven downregulated genes. Fibroblast growth factor 9 (FGF9), vascular endothelial growth factor A (VEGFA), interleukin‐2, bone morphogenetic protein‐10, VEGFC, and 2 (FGF2) were markedly upregulated (FC range, 6 to 15), whereas pleotropin, FGF5, brain‐derived neurotrophic factor, and Dickkopf WNT signaling pathway inhibitor 1 were markedly downregulated (FC 32). Of the 84 growth factor–associated genes screened, 35 showed high cycle threshold values (≥35). Conclusions: ASA modulates the expression of growth factor–associated genes and enhances osteogenic potential in PDLSCs. ASA upregulated the expression of genes that could activate biologic functions and canonic pathways related to cell proliferation, human embryonic stem cell pluripotency, tissue regeneration, and differentiation. These findings suggest that ASA enhances PDLSC function and may be useful in regenerative dentistry applications, particularly in the areas of periodontal health and regeneration.     

Porphyromonas gingivalis LPS inhibits osteoblastic differentiation and promotes pro-inflammatory cytokine production in human periodontal ligament stem cells

ObjectivePorphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) induces pro-inflammatory cytokines, such as interleukin-1 β (IL-1β), IL-6, and IL-8, which induce periodontal tissue destruction. Periodontal ligament stem cells (PDLSCs) play an important role in periodontal tissue regeneration and are expected to have future applications in cellular therapies for periodontitis. However, no studies have examined the effects of P. gingivalis LPS on PDLSCs. The aim of this study was to investigate how P. gingivalis LPS affects the osteoblastic differentiation and pro-inflammatory cytokine production of PDLSCs.DesignPDLSCs were obtained from healthy adult human mandibular third molars. The identification of PDLSCs was confirmed by immunohistochemical evaluations of the mesenchymal stem cell markers STRO-1 and SSEA-4. Cell proliferation and osteoblastic differentiation were investigated by culturing the PDLSCs in a normal or osteogenic medium with P. gingivalis LPS (0, 1, or 10 μg/mL) and then measuring the alkaline phosphatase (ALP) activity and the production of collagen type 1 Alpha 1 (COL1A1), osteocalcin production, and mineralisation. Additionally, we examined the production of IL-1β, IL-6, and IL-8 in the PDLSCs.ResultsP. gingivalis LPS inhibited the ALP activity, COL1A1 and osteocalcin production, and mineralisation in the PDLSCs, which are positive for STRO-1 and SSEA-4. P. gingivalis LPS also promoted cell proliferation and produced IL-1β, IL-6, and IL-8.ConclusionsThis study provides the first findings that P. gingivalis LPS inhibits osteoblastic differentiation and induces pro-inflammatory cytokines in PDLSCs. These findings will help clarify the relationship between periodontitis and periodontal tissue regeneration.     

Azithromycin may inhibit interleukin-8 through suppression of Rac1 and a nuclear factor-kappa B pathway in KB cells stimulated with lipopolysaccharide

Background: Recent studies have shown that the 15‐member macrolide antibiotic azithromycin (AZM) not only has antibacterial activity, but also results in the role of immunomodulator. Interleukin (IL)‐8 is an important inflammatory mediator in periodontal disease. However, there have been no reports on the effects of AZM on IL‐8 production from human oral epithelium. Therefore, we investigated the effects of AZM on IL‐8 production in an oral epithelial cell line. Methods: KB cells were stimulated by Escherichia coli or Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) lipopolysaccharide (LPS) with or without AZM. IL‐8 mRNA and protein expression and production in response to LPS were analyzed by quantitative polymerase chain reaction, flow cytometry, and enzyme‐linked immunosorbent assay. The activation of nuclear factor‐kappa B (NF‐κB) and Rac1, which is important for IL‐8 expression, was analyzed by enzyme‐linked immunosorbent assay and Western blotting, respectively. Results: IL‐8 mRNA expression, IL‐8 production, and NF‐κB activation in LPS‐stimulated KB cells were inhibited by the addition of AZM. LPS‐induced Rac1 activation was also suppressed by AZM. Conclusions: This study suggests that AZM inhibits LPS‐induced IL‐8 production in an oral epithelial cell line, in part caused by the suppression of Rac1 and NF‐κB activation. The use of AZM might provide possible benefits in periodontal therapy, with respect to both its antibacterial action and apparent anti‐inflammatory effect.     

The role of sirtuin 1 in osteoblastic differentiation in human periodontal ligament cells

Lee Y‐M, Shin S‐I, Shin K‐S, Lee Y‐R, Park B‐H, Kim E‐C. The role of sirtuin 1 in osteoblastic differentiation in human periodontal ligament cells. J Periodont Res 2011; 46: 712–721. © 2011 John Wiley & Sons A/S Background and Objective: Activation of sirtuin 1 (SIRT1) promotes the differentiation of keratinocytes and mesenchymal stem cells, but inhibits the differentiation of muscle and fat cells. However, the involvement of SIRT1 in the differentiation of human periodontal ligament cells into osteoblast‐like cells remains unclear. To identify the role of SIRT1 in human periodontal ligament cells, we measured SIRT1 mRNA and SIRT1 protein levels during the osteoblastic differentiation of human periodontal ligament cells. Additionally, we investigated the effects of overexpressing and underexpressing SIRT1 on the differentiation of human periodontal ligament cells, and the signaling mechanisms involved. Material and Methods: Expression of SIRT1 and osteoblastic differentiation markers was assessed by RT‐PCR, real‐time PCR, Alizarin red staining and western blotting. Results: Marked upregulation of SIRT1 mRNA and SIRT1 protein was observed in cells grown for 3 d in osteogenic induction medium (OM). Activation of SIRT1 using resveratrol and isonicotinamide stimulated osteoblastic differentiation in a dose‐dependent manner, as assessed by the expression of mRNAs encoding alkaline phosphatase, osteopontin, osteocalcin, osterix and Runx2, and induced calcium deposition. In contrast, inhibition of SIRT1 using sirtinol, nicotinamide and gene silencing by RNA interference suppressed mineralization and the expression of osteoblast marker mRNAs. Further mechanistic studies revealed that resveratrol treatment increased the phosphorylation of Akt, adenosine monophosphate kinase (AMPK), Smad 1/5/8 and c‐Jun N‐terminal kinase, but reduced OM‐induced activation of nuclear factor‐κB. Conversely, application of sirtinol suppressed the phosphorylation of Akt, AMPK, Smad 1/5/8, p38, ERK and c‐Jun N‐terminal kinase, and enhanced nuclear factor‐κB activity, in OM‐stimulated cells. Conclusion: These data suggest that SIRT1 is a potent regulator of differentiation of human periodontal ligament cells and may have clinical implications for periodontal bone regeneration.     

Apical tooth germ cell-conditioned medium enhances the differentiation of periodontal ligament stem cells into cementum/periodontal ligament-like tissues

Background and Objective: Limitations of current periodontal regeneration modalities in both predictability and extent of healing response, especially on new cementum and attachment formation, underscore the importance of restoring or providing a microenvironment that is capable of promoting the differentiatiation of periodontal ligament stem cells (PDLSCs) towards cementoblast‐like cells and the formation of cementum/periodontal ligament‐like tissues. The aim of this study was to investigate the biological effect of conditioned medium from developing apical tooth germ cells (APTG‐CM) on the differentiation and cementogenesis of PDLSCs both in vitro and in vivo. Material and Methods: Using the limiting dilution technique, single‐colony‐derived human PDLSCs were isolated and expanded to obtain homogeneous populations of PDLSCs. Morphological appearance, cell cycle analysis, bromodeoxyuridine incorporation, alkaline phosphatase (ALP) activity, mineralization behavior, gene expression of cementoblast phenotype and in vivo differentiation capacities of PDLSCs co‐cultured with APTG‐CM were evaluated. Results: The induced PDLSCs exhibited several characteristics of cementoblast lineages, as indicated by the morphological changes, increased proliferation, high ALP activity, and the expression of cementum‐related genes and calcified nodule formation in vitro. When transplanted into immunocompromised mice, the induced PDLSCs showed tissue‐regenerative capacity to produce cementum/periodontal ligament‐like structures, characterized by a layer of cementum‐like mineralized tissues and associated periodontal ligament‐like collagen fibers connecting with the newly formed cementum‐like deposits, whereas control, untreated PDLSCs transplants mainly formed connective tissues. Conclusion: Our findings suggest that APTG‐CM is able to provide a cementogenic microenvironment and induce differentiation of PDLSCs along the cementoblastic lineage. This has important implications for periodontal engineering.