Primary Human Periodontal Ligament Fibroblasts Research Articles

Nonsense-mediated mRNA decay affects hyperactive root formation in oculo-facio-cardio-dental syndrome via up-frameshift protein 1

ObjectivesOculo-facio-cardio-dental (OFCD) syndrome is a rare X-linked genetic disorder caused by mutations in the BCL6 co-repressor (BCOR) and is mainly characterized by radiculomegaly (elongated dental roots). All BCOR mutations reported to date have been associated with premature termination codons, indicating that nonsense-mediated mRNA decay (NMD) might play a vital role in the pathogenesis of OFCD syndrome. However, the molecular mechanisms underlying NMD remain unclear. In this study, we investigated the involvement of up-frameshift protein 1 (UPF1), which plays a central role in NMD, in the hyperactive root formation caused by BCOR mutations. MethodsPeriodontal ligament cells, isolated from a Japanese woman with a c.3668delC frameshift mutation in BCOR, and primary human periodontal ligament fibroblasts (HPdLFs) were used for an RNA immunoprecipitation assay to confirm the binding of UPF1 to mutated BCOR. Additionally, the effects of UPF1 on the BCOR transcription levels and corresponding gene expression were determined by performing relative quantitative real-time polymerase chain reactions. ResultsRNA immunoprecipitation revealed that UPF1 binds to exon 9 of mutated BCOR. Additionally, UPF1 knockdown via siRNA upregulated the transcription of BCOR, whereas overexpression of wild-type and mutated BCOR with the same frameshift mutation in HPdLFs altered bone morphogenetic protein 2 (BMP2) expression. ConclusionsOur findings indicate that BCOR mutations regulate the transcription of BCOR via UPF1, which may in turn regulate the expression of BMP2. NMD, caused by a c.3668delC mutation, potentially leads to an OFCD syndrome phenotype, including elongated dental roots.

Evaluation of the anti-biofilm effect of poloxamer-based thermoreversible gel of silver nanoparticles as a potential medication for root canal therapy

The purpose of this study was to design silver nanoparticles (AgNPs) poloxamer thermoreversible gel (AgNPs-PL) and investigate whether this gel could provide sustained antibacterial activity against Enterococcus faecalis (E. faecalis) in the root canal. The gels fabricated were characterized in terms of gelatin temperature, particle size, in-vitro Ag+ release, and elemental content. Cytotoxicity of AgNPs-PL on primary human periodontal ligament fibroblasts (HPDLFs) was examined by CCK-8 assay. Characterization of AgNPs-PL gel revealed that it contained particles existing as large clumps/fused aggregates of different shapes, with a mean diameter of 21.624 ± 14.689 nm, exhibited sustained release of Ag+ for 9 days, and non-toxic to HPDLFs at a low dose (4–32 μg/mL) through 24, 48, and 72 h exposures. The antibacterial effect of 16 and 32 μg/mL concentrations of AgNPs-PL was compared with blank poloxamer gel (PL) and calcium hydroxide (CH) using three methods: (I) agar counting plate, (II) scanning electron microscope (SEM) observations, and (III) confocal laser scanning microscope (CLSM) analysis. AgNPs-PL at the two doses above was more effective than PL and CH in removing E. faecalis biofilm at 1, 3, 9 days. Thus, AgNPs-PL exhibits strong activity against E. faecalis and is easy to produce, with a continuous release profile of Ag+. AgNPs-PL gel may be a candidate for a new root canal disinfection.

A Human Periodontal Ligament Fibroblast Cell Line as a New Model to Study Periodontal Stress.

The periodontal ligament (PDL) is exposed to different kinds of mechanical stresses such as bite force or orthodontic tooth movement. A simple and efficient model to study molecular responses to mechanical stress is the application of compressive force onto primary human periodontal ligament fibroblasts via glass disks. Yet, this model suffers from the need for primary cells from human donors which have a limited proliferative capacity. Here we show that an immortalized cell line, PDL-hTERT, derived from primary human periodontal ligament fibroblasts exhibits characteristic responses to glass disk-mediated compressive force resembling those of primary cells. These responses include induction and secretion of pro-inflammatory markers, changes in expression of extracellular matrix-reorganizing genes and induction of genes related to angiogenesis, osteoblastogenesis and osteoclastogenesis. The fact that PDL-hTERT cells can easily be transfected broadens their usefulness, as molecular gain- and loss-of-function studies become feasible.

Cannabinoid type-2 receptor agonist, inverse agonist, and anandamide regulation of inflammatory responses in IL-1β stimulated primary human periodontal ligament fibroblasts.

The aim of this study is to understand the role of cannabinoid type 2 receptor (CB2R) during periodontal inflammation and to identify anti-inflammatory agents for the development of drugs to treat periodontitis (PD). Cannabinoid type 2 receptor is found in periodontal tissue at sites of inflammation/infection. Our previous study demonstrated anti-inflammatory responses in human periodontal ligament fibroblasts (hPDLFs) via CB2R ligands. Anandamide (AEA), HU-308 (agonist), and SMM-189 (inverse agonist) were tested for effects on IL-1β-stimulated cytokines, chemokines, and angiogenic and vascular markers expressed by hPDLFs using Mesoscale Discovery V-Plex Kits. Signal transduction pathways (p-c-Jun, p-ERK, p-p-38, p-JNK, p-CREB, and p-NF-kB) were investigated using Cisbio HTRF kits. ACTOne and Tango™ -BLA functional assays were used to measure cyclic AMP (cAMP) and β-arrestin activity. IL-1β stimulated hPDLF production of 18/39 analytes, which were downregulated by the CB2R agonist and the inverse agonist. AEA exhibited pro-inflammatory and anti-inflammatory effects. IL-1β increased phosphoproteins within the first hour except p-JNK. CB2R ligands attenuated p-p38 and p-NFĸB, but a late rise in p-38 was seen with HU-308. As p-ERK levels declined, a significant increase in p-ERK was observed later in the time course by synthetic CB2R ligands. P-JNK was significantly affected by SMM-189 only, while p-CREB was elevated significantly by CB2R ligands at 180minutes. HU-308 affected both cAMP and β-arrestin pathway. SMM-189 only stimulated cAMP. The findings that CB2R agonist and inverse agonist may potentially regulate inflammation suggest that development of CB2R therapeutics could improve on current treatments for PD and other oral inflammatory pathologies.

Development and application of a 3D periodontal in vitro model for the evaluation of fibrillar biomaterials

BackgroundPeriodontitis is a chronic inflammation of the tooth supporting structures that finally can lead to tooth loss. As chronic periodontitis is associated with systemic diseases multiple approaches have been followed to support regeneration of the destructed tissue. But very few materials are actually used in the clinic. A new and promising group of biomaterials with advantageous biomechanical properties that have the ability to support periodontal regeneration are self-assembling peptides (SAP). However, there is still a lack of 3D periodontal models that can evaluate the migration potential of such novel materials.MethodsAll experiments were performed with primary human periodontal ligament fibroblasts (HPLF). Migration capacity was assessed in a three-dimensional model of the human periodontal ligament by measuring the migration distance of viable cells on coated (Enamel Matrix Protein (EMP), P11–4, collagen I) or uncoated human dentin. Cellular metabolic activity on P11–4 hydrogels was assessed by a metabolic activity assay. Deposition of ECM molecules in a P11–4 hydrogel was visualized by immunostaining of collagen I and III and fibrillin I.ResultsThe 3D periodontal model was feasible to show the positive effect of EMP for periodontal regeneration. Subsequently, self-assembling peptide P11–4 was used to evaluate its capacity to support regenerative processes in the 3D periodontal model. HPLF coverage of the dentin surface coated with P11–4 increased significantly over time, even though delayed compared to EMP. Cell viability increased and inclusion of ECM proteins into the biomaterial was shown.ConclusionThe presented results indicate that the 3D periodontal model is feasible to assess periodontal defect coverage and that P11–4 serves as an efficient supporter of regenerative processes in the periodontal ligament.Clinical relevanceThe establishment of building-block synthetic polymers offers new opportunities for clinical application in dentistry. Self-assembling peptides represent a new generation of biomaterials as they are able to respond dynamically to the changing environment of the biological surrounding. Especially in the context of peri-implant disease prevention and treatment they enable the implementation of new concepts.

Effect of ILK on small-molecule metabolism of human periodontal ligament fibroblasts with mechanical stretching.

Mechanical stimuli can cause periodontal tissue reconstruction. Studies have found that changes in metabolites can be the terminal effect of integrin-mediated mechanical signaling. As a key kinase in integrin regulation, integrin-linked kinase (ILK) mediates mechanical signal transduction, which may contribute to metabolite changes. Defining the components of small-molecule metabolites can optimize mechanical stimuli and periodontal tissue reconstruction. Our purpose is to detect the effect of ILK-mediated mechanical signaling on intracellular small-molecule metabolites (amino acids and organic acids) in human periodontal ligament fibroblasts (HPDLFs). Primary HPDLFs were isolated by enzyme digestion method. Tensile stresses were applied on HPDLFs in vitro using a Flexcell system. ILK gene in HPDLFs was knocked down by RNA interference (RNAi). Twenty common amino acids and seven organic acids in HPDLFs were analyzed by gas chromatography/mass spectrometry technique. Five amino acids (ie, alanine, glutamine, glutamate, glycine, and threonine) and three organic acids (ie, pyruvate, lactate, and citric acid) were found to be changed remarkably after mechanical stretching. In addition, baseline levels of four amino acids (ie, glutamate, glutamine, threonine, and glycine) and two organic acids (ie, lactate and citric acid) were significantly different in ILK knockdown compared with wild-type HPDLFs. This study suggests that five amino acids (ie, alanine, glutamine, glutamate, glycine, and threonine) and three organic acids (ie, pyruvate, lactate, and citric acid) may act as cellular mediators for mechanical signals in HPDLFs. Among them, four amino acids (ie, glutamate, glutamine, threonine, and glycine) and two organic acids (ie, lactate and citric acid) may be closely linked to ILK.

Anti-inflammatory activity of cannabinoid receptor 2 ligands in primary hPDL fibroblasts

ObjectivesApproximately 65 million adults in the US have periodontitis, causing tooth loss and decreased quality of life. Cannabinoids modulate immune responses, and endocannabinoids are prevalent during oral cavity inflammation. Targets for intervention in periodontal inflammation are cannabinoid type 1 and 2 receptors (CB1R, CB2R), particularly CB2R because its levels increase during inflammation. We previously demonstrated that SMM-189 (CB2R inverse agonist) decreased pro-inflammatory cytokine production in primary microglial cells. The hypothesis of this study was that cannabinoids anandamide (AEA), HU-308 (CB2R selective agonist), and SMM-189 decrease pro-inflammatory IL-6 and MCP-1 production by primary human periodontal ligament fibroblasts (hPDLFs) stimulated with P. gingivalis LPS, TNF-α, or IL-1β. DesignCytotoxic effects of cannabinoid compounds (10−4–10−6.5 M), LPS (1–1000 ng/ml), TNFα (10 ng/ml) and IL-1β (1 ng/ml) were assessed by measuring effects on cellular dehydrogenase activity. IL-6 and MCP-1 production were measured using Mesoscale Discovery (MSD) Human Pro-Inflammatory IL-6 and MSD Human Chemokine MCP-1 kits and analyzed using MSD Sector 2400 machine. ResultsEC50 values for AEA, SMM-189, and HU-308 were 16 μM, 13 μM, and 7.3 μM respectively. LPS (1 μg/ml), TNF-α (10 ng/ml), and IL-1β (1 ng/ml) increased IL-6 and MCP-1 production, which were inhibited by AEA, SMM-189, and HU-308. AEA alone significantly increased IL-6, but not MCP-1 levels, but the other cannabinoids alone had no effect. ConclusionThe effective inhibition of LPS, TNF-α, IL-1β stimulated IL-6 and MCP-1 production by CB2R ligands in hPDLFs suggests that targeting the endocannabinoid system may lead to development of novel drugs for periodontal therapy, aiding strategies to improve oral health.

MicroRNA-200c Represses IL-6, IL-8, and CCL-5 Expression and Enhances Osteogenic Differentiation.

MicroRNAs (miRs) regulate inflammation and BMP antagonists, thus they have potential uses as therapeutic reagents. However, the molecular function of miR-200c in modulating proinflammatory and bone metabolic mediators and osteogenic differentiation is not known. After miR-200c was transduced into a human embryonic palatal mesenchyme (HEPM) (a cell line of preosteoblasts), using lentiviral vectors, the resulting miR-200c overexpression increased osteogenic differentiation biomarkers, including osteocalcin (OCN) transcripts and calcium content. miR-200c expression also down-regulated interleukin (IL)-6, IL-8, and chemokine (C-C motif) ligand (CCL)-5 under lipopolysaccharide (LPS) stimulation and increased osteoprotegerin (OPG) in these cells. miR-200c directly regulates the expression of IL-6, IL-8 and CCL-5 transcripts by binding to their 3’UTRs. A plasmid-based miR-200c inhibitor effectively reduces their binding activities. Additionally, miR-200c delivered using polyethylenimine (PEI) nanoparticles effectively inhibits IL-6, IL-8 and CCL-5 in primary human periodontal ligament fibroblasts and increases the biomarkers of osteogenic differentiation in human bone marrow mesenchymal stem cells (MSCs), including calcium content, ALP, and Runx2. These data demonstrate that miR-200c represses IL-6, IL-8 and CCL-5 and improves osteogenic differentiation. miR-200c may potentially be used as an effective means to prevent periodontitis-associated bone loss by arresting inflammation and osteoclastogenesis and enhancing bone regeneration.

Effects of IL-10 and glucose on expression of OPG and RANKL in human periodontal ligament fibroblasts.

The effects of interleukin-10 (IL-10) and glucose on mRNA and protein expression of osteoprotegerin (OPG), and its ligand, receptor activator of nuclear factor-κB ligand (RANKL), were investigated in human periodontal ligament fibroblasts (HPDLFs). Primary HPDLFs were treated with different concentrations of IL-10 (0, 1, 10, 25, 50, and 100 ng/mL) or glucose (0, 5.5, 10, 20, 30, and 40 mmol/L). Changes in mRNA and protein expression were examined using the reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. After IL-10 treatment, mRNA and protein levels of OPG were increased, while mRNA and protein levels of RANKL were decreased (P<0.05), both in a concentration-dependent manner. Glucose stimulation had the opposite concentration-dependent effect to that of IL-10 on OPG and RANKL expression. IL-10 upregulated OPG expression and downregulated RANKL expression, whereas high glucose upregulated RANKL and downregulated OPG in HDPLFs. Abnormal levels of IL-10 and glucose may contribute to the pathogenesis of periodontal disease.

An Autologous Platelet‐Rich Plasma Stimulates Periodontal Ligament Regeneration

Regeneration of periodontal tissues is one of the most important goals for the treatment of periodontal disease. The technology of plasma rich in growth factors provides a biologic approach for the stimulation and acceleration of tissue healing. The purpose of this study is to evaluate the biologic effects of this technology on primary human periodontal ligament fibroblasts. The authors studied the response of periodontal ligament cells to this pool of growth factors on cell proliferation, cell migration, secretion of several biomolecules, cell adhesion, and expression of α2 integrin. Cell proliferation and adhesion were evaluated by means of a fluorescence-based method. Cell migration was performed on culture inserts. The release of different biomolecules by periodontal ligament fibroblasts was quantified through enzyme-linked immunosorbent assay. The α2 integrin expression was assessed through Western blot. This autologous technology significantly stimulated cell proliferation, migration, adhesion, and synthesis of many growth factors from cells including vascular endothelial growth factor, thrombospondin 1, connective tissue growth factor, hepatocyte growth factor, and procollagen type I. The α2 integrin expression was lower in plasma rich in growth factor-treated cells compared to non-stimulated cells, although no statistically significant differences were observed. This plasma rich in growth factors exerts positive effects on periodontal ligament fibroblasts, which could be positive for periodontal regeneration.

A Novel GuttaFlow Sealer Supports Cell Survival and Attachment

IntroductionThe purpose of this in vitro study was to compare the biocompatibility of a novel formulation of a silicone-based endodontic sealer GuttaFlow 2 (GF2; Coltène/Whaledent, Langenau, Germany) with the original (GFO) and fast-set (GFF) formulations of GuttaFlow and with an epoxy resin sealer, AHPlus Jet (AH+J; Dentsply, York, PA). MethodsSealers were set into 3 × 5.5 mm discs. Cell culture media was used to extract leachable products at 24 hours and 1, 2, and 4 weeks. Primary human periodontal ligament fibroblasts were incubated with sealer elutes for 24 hours and evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and the calcein AM assay. Cell attachment was evaluated on set sealer that was either rinsed or unrinsed with cell media for 1 week. Statistical analysis was performed using the Student t test. ResultsBoth calcein and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays revealed that periodontal ligament cell viability was reduced on AH+J at 1, 2, and 4 weeks compared with all GuttaFlow sealers. There were no differences in cell viability between the GuttaFlow samples, and all displayed high rates of cell survival at all time periods. After 2 hours, cell attachment to the rinsed GFO and GFF samples exceeded the control, and at 24 hours cell attachment on all GuttaFlow samples exceeded the control. AH+J sealers supported significantly less cell attachment when compared with all GuttaFlow sealers. Cell attachment to set sealers showed better cell attachment when rinsed compared with unrinsed. ConclusionsGuttaFlow sealers were more biocompatible than AHJ in vitro. The novel GF2 displayed comparable biocompatibility with GFF and GFO.

Lysophosphatidic acid modulates the healing responses of human periodontal ligament fibroblasts and enhances the actions of platelet-derived growth factor.

Platelet-derived growth factor (PDGF) has been used to promote healing in many in vitro and in vivo models of periodontal regeneration. PDGF interacts extensively with lysophosphatidic acid (LPA). We recently showed that LPA modulates the responses of human gingival fibroblasts to PDGF. The objectives of this study were as follows: 1) to evaluate the basic interactions of LPA with primary human periodontal ligament fibroblasts (PDLFs) alone and with PDGF-BB for promoting PDLF growth and migration; 2) to determine the effects in an in vitro oral wound-healing model; and 3) to identify the LPA receptors (LPARs) expressed by PDLF. PDLF regenerative responses were measured using 1 and 10 microM LPA in the absence or presence of 1 or 10 ng/ml PDGF. Cell proliferation was determined by 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry and by cell counting. Migration responses were measured using a microchemotaxis chamber. PDLFs were grown to confluence on glass slides, a 3-mm-wide wound was mechanically inflicted, and wound fill on days 4, 6, and 9 was reported. PDLF LPAR expression was determined using Western blotting. PDLFs exhibited proliferative and chemotactic responses to LPA; these responses were enhanced when LPA and PDGF were present together. LPA plus PDGF elicited complete wound fill. PDLFs express the LPARs LPA(1), LPA(2), and LPA(3). To our knowledge, this study provides the first evidence that LPA stimulates human PDLF wound healing responses and interacts positively with PDGF to regulate these actions. These results suggest that LPA and its receptors play important modulatory roles in PDLF regenerative biology.

Elevated expression of genes assigned to NF-κB and apoptotic pathways in human periodontal ligament fibroblasts following mechanical stretch

There is growing evidence that apoptosis involves the nuclear transcription factor NF-kappaB in conjunction with related genes. However, in the context of mechanical orthodontic forces, force-sensing target genes assigned to pathways of NF-kappaB and apoptosis have not been fully characterised. To contribute to the identification of putative target genes, we used cDNA arrays specific for NF-kappaB and apoptotic pathways and analysed elevated gene expression in primary human periodontal ligament fibroblasts (PDL-F) after a 6 h application of mechanical force. Among several identified genes (including several caspases), interleukin-1 beta (IL-1 beta) and NF-kappaB displayed significantly higher expression on the NF-kappaB array, whereas higher expression was obtained for BCL2-antagonist of cell death (BAD), member 6 of the TNF-receptor superfamily (FAS) and CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD) on the apoptosis array. Based on a defined cut-off level of a more than 1.5-fold higher expression, this significance in elevated gene expression was corroborated by reverse transcription/polymerase chain reaction (RT-PCR). Here, semi-quantitative (sq) PCR revealed a more pronounced elevation of mRNA gene expression in PDL-F after 6 h of stretch, when compared with 12 h. Moreover, the elevation after 6 h as observed by sq-PCR was convergent with quantitative PCR (q-PCR). q-PCR yielded levels of 5.8-fold higher relative gene expression for IL-1 beta and 1.7-fold for NF-kappaB, whereas that computed for BAD indicated a 5.2-fold, for CRADD a 2.1-fold and for FAS a 2.0-fold higher expression. The data obtained from the expression analysis thus indicate a stretch-induced transcriptional elevation of genes assigned to the NF-kappaB and apoptotic pathways. This elevation may render them target candidates for being addressed by mechanical orthodontic forces.

Establishing and characterizing human periodontal ligament fibroblasts immortalized by SV40T-antigen and hTERT gene transfer

The periodontal ligament (PDL) is a highly specialized tissue connecting the cementum with the tooth socket bone and affects the life span of the tooth. However, little is known about the precise characteristics and regenerative mechanism of PDL cells because of the absence of specific markers and cell lines. Therefore, we aimed to establish three immortalized human PDL fibroblast cell lines by using simian virus40 T-antigen (SV40T-Ag) and human telomerase reverse transcriptase (hTERT) transfection, expecting these cells to have the characteristics of primary cells. The transfected cells were named STPLF. The expression of SV40T-Ag and hTERT in all STPLF lines was verified by using the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method, stretch PCR analysis, or Western blotting analysis. All STPLF showed stable proliferation at more than 120 population doublings (PD), whereas primary human PDL fibroblasts (HPLF) stopped at 10-20 PD. Characterization by RT-PCR analysis revealed that all STPLF genes mimicked the expression of their respective original HPLF genes. STPLF expressed runt-related transcription factor-2, osterix, alkaline phosphatase, osteopontin, osteocalcin, periostin, receptor activator of NF-kappa B ligand, osteoprotegerin, epidermal growth factor receptor, alpha-smooth muscle actin, and type XII collagen. STPLF stimulated with 50 micro g/ml ascorbic acid and 2 mM beta-glycerophosphate for 4 weeks produced more calcified deposits than did HPLF cultured with the same reagents. These results suggest that each STPLF line retained the characteristics of the respective original HPLF, that STPLF gained increased calcification activity, and that STPLF are helpful tools for studying the biology and regenerative mechanisms of human PDL.

Morphogenesis and proliferation in mono- and organotypic co-cultures of primary human periodontal ligament fibroblasts and alveolar bone cells.

Cells of the periodontal ligament and the alveolar bone lie in close vicinity in the periodontium. The goal of this study was to create an in vitro model to facilitate the study of the morphogenesis and proliferation of these two cell types under more in-vivo-like conditions. This was accomplished by the generation of organotypic co-cultures of primary human periodontal ligament fibroblasts (PDL) and alveolar bone cells (BC) and matched mono-cultures after 1, 2 and 3 weeks. Indirect immunofluorescence (IIF) for vimentin indicated that PDL cells exhibited sustained stratification only in the presence of BC cells, suggesting an important role for BCs in maintaining the stratification of PDL cells. In mono-cultures, only BC cells showed progressing stratification. They also displayed the most pronounced contraction of the cell culture matrix. Moreover, Ki-67 antigen detection by IIF revealed that these features coincided with cell proliferation localized on the matrix surface at the onset of cell stratification. These findings suggest that, in addition to proliferation, a further prerequisite for stratification may be cell migration. Furthermore, the maintained cell stratification, proliferation, and compartmentalization noted for PDL cells in organotypic co-cultures and BCs in mono-cultures can only be observed in a three-dimensional culture system. Thus, our system represents a novel experimental tool to further elucidate the underlying mechanisms of the growth and differentiation of PDL and bone tissue.

The Cellular Compatibility of Five Endodontic Sealers during the Setting Period

The purpose of this study was to evaluate the cellular compatibility of five endodontic sealers in the first 24 h after mixing. Specimens of N2, Endomethasone, Apexit, AH Plus, and Ketac Endo were extracted with cell culture medium 0, 1, 5, and 24 h after mixing. Eluates were tested for cytotoxicity with immortal 3T3 cells and primary human periodontal ligament fibroblasts using XTT-assays. Data were analyzed for statistically significant differences by means of Dunnett's t tests (p < 0.05). All extracts of N2 completely inhibited cell metabolism (p < 0.05). Similar effects were provoked by the first three eluates of Endomethasone, but the 24-h extract irritated cells significantly less (p < 0.05). Severe cytotoxicity was also observed with all Ketac Endo extracts (p < 0.05). A significant inhibition of mitochondrial activity was induced by the first (3T3) or the first and second eluate (periodontal ligament fibroblasts) of AH Plus (p < 0.05). The subsequent eluates of this sealer and all extracts of Apexit did not reveal any cytotoxic potency.

Long-Term Cytocompatibility of Various Endodontic Sealers Using a New Root Canal Model

It was the purpose of our study to determine the cytotoxicity of several types of root canal sealers in vitro over the period of 1 yr by using a new test model. Roots of extracted human teeth were filled with N2, Apexit, Roekoseal, AH Plus, Ketac Endo, Endomethasone, and one gutta-percha point. In addition, roots filled with laterally condensed gutta-percha/N2. Teeth filled with one gutta-percha point only were controls. All specimens were consecutively extracted with distilled water for a total period of 1 yr. Extracts were investigated for cytotoxicity by using immortalized 3T3 fibroblasts and primary human periodontal ligament fibroblasts. Results were statistically analyzed with Dunnett's t tests (p < 0.05). Pronounced cytotoxic effects were only caused by N2-extracts in both cell cultures (p < 0.05). Furthermore, statistically significant cytotoxic alterations were also induced by 10-week eluates of Endomethasone (p < 0.05). All other investigated materials did not significantly alter cell metabolism.

Compatibility of resorbable and nonresorbable guided tissue regeneration membranes in cultures of primary human periodontal ligament fibroblasts and human osteoblast-like cells.

The purpose of this study was (a) to evaluate the cytocompatibility of three resorbable and nonresorbable membranes in fibroblast and osteoblast-like cell cultures and (b) to observe the growth of those cells on the various barriers by scanning electron microscopy (SEM). Primary human periodontal ligament fibroblasts (HPLF) and human osteoblast-like cells (SAOS-2) were incubated with nonresorbable polytetrafluoroethylene (ePTFE) barriers and resorbable polylactic acid as well as collagen membranes. Cytotoxic effects were determined by XTT (mitochondrial metabolic activity) and sulforhodamine B assays (cellular protein content). In addition, HPLF and SAOS-2 grown for 21 days on the investigated barriers were evaluated by SEM. Data were analyzed statistically by ANOVA using the Wilcoxon-Mann-Whitney test (P < 0.05). No changes were established in the periodontal ligament fibroblasts and human osteoblast-like cells after incubation with the collagen membrane. Cytotoxic effects, however, were induced by the polylactic acid barrier which slightly inhibited cell metabolism of the periodontal fibroblasts (XTT: 90.1% +/- 3.6 of control value). Moderate cytotoxic reactions were caused by the nonresorbable ePTFE membrane in HPLF-cultures (XTT: 82.7% +/- 3.5) and osteoblast-like cell monolayers (XTT: 80.0% +/- 0.6%). Mitochondrial activity in both cell cultures was significantly reduced by ePTFE barriers in comparison to non-incubated control cells (P = 0.028). SEM analysis of cell behavior on barriers demonstrated the differences between these materials: collagen barriers were densely populated with HPLF and SAOS-2, whereas only few or no cells were seen to adhere to the ePTFE and polylactic acid membranes. Our findings indicate that the collagen barrier investigated is very cytocompatible and may be integrated into connective tissue well. On the contrary, the ePTFE and polylactic acid membranes induced slight to moderate cytotoxic reactions which may reduce cellular adhesion. Thus, gap formation between the barrier surface and the connective tissue may be promoted which may facilitate epithelial downgrowth and microbial accumulation. Consequently, these effects may reduce the potential gain in periodontal attachment.

Genotoxicity and cytotoxicity of the epoxy resin-based root canal sealer AH plus

Previous studies with four rapid in vitro and in vivo test systems have shown that the epoxy resin-based root canal sealer AH26 may be genotoxic and cytotoxic (9). The purpose of this study was to determine the cytotoxic and genotoxic effects of the new resinous root canal sealer AH Plus by means of the growth inhibition test with primary human periodontal ligament fibroblasts and permanent 3T3 monolayers, the procaryotic umu test, the eucaryotic DNA synthesis inhibition test, and the in vivo alkaline filter elution test. In addition, Ames tests were performed with extracts from AH Plus. AH Plus caused only slight or no cellular injuries. Furthermore, no genotoxicity and mutagenicity were revealed by AH Plus. These data should be taken into consideration when deciding about a root canal sealer.

Cytotoxicity of four root canal sealers in permanent 3T3 cells and primary human periodontal ligament fibroblast cultures

Objectives. The purposes of this study were to determine the cytocompatibility of four endodontic sealers and gutta-percha using various extracts and to compare the cellular injuries resulting from these materials in permanent 3T3 monolayers and primary human periodontal ligament fibroblasts. Study design. Set specimens from four sealers (AH26, Apexit, Sealapex, N2) as well as gutta-percha were eluted with cell culture medium for 24 hours, 5 days, 5 days, and 24 hours, respectively. Cytotoxic effects due to these extracts were evaluated by determining proliferation of permanent 3T3 cells and primary human periodontal ligament fibroblasts by means of the fluorochrome propidiumiodide. Results. No statistically significant cellular alterations were induced by gutta-percha and Apexit, whereas various extracts of AH26 and Sealapex caused significant moderate or severe growth inhibition. Severe cellular damage was documented for all extracts of N2, which was statistically significantly different from the other materials in both cell lines ( p < 0.05). Conclusions. Our results confirm that various root canal sealers constantly segregate substances after being exposed to an aqueous environment for extended periods, possibly causing moderate or severe cytotoxic reactions and possibly contributing to failure.