Clock gene expression in the submandibular glands

Clock genes, which mediate molecular circadian rhythms, are expressed in a circadian fashion in the suprachiasmatic nucleus and in various peripheral tissues. To establish a molecular basis for circadian regulation in the salivary glands, we examined expression profiles of clock-related genes and salivary gland-characteristic genes. Clock-related genes-including Per1, Per2, Cry1, Bmal1, Dec1, Dec2, Dbp, and Reverbalpha-showed robust circadian expression rhythms in the submandibular glands in 12:12-hour light-dark conditions. In addition, a robust circadian rhythm was observed in amylase 1 mRNA levels, whereas the expression of other salivary-gland-characteristic genes examined was not rhythmic. The Clock mutation resulted in increased or decreased mRNA levels of Per2, Bmal1, Dec1, Dec2, and Dbp, and in Cry1-/- background, Cry2 disruption also increased or decreased mRNA levels of these clock-related genes and the amylase 1 gene. These findings indicate that the Clock- and Cry-dependent molecular clock system is active in the salivary glands.     

Core circadian clock gene expression in human dental pulp‐derived cells in response to L‐mimosine,hypoxia and echinomycin

Core circadian clock genes set the pace for a wide range of physiological functions, including regeneration. The role of these genes and their regulation in the dental pulp, in particular under hypoxic conditions, is unknown. Here we investigated if core clock genes are expressed in human dental pulp‐derived cells (DPC) and if their expression is modulated by the hypoxia mimetic agent, L‐mimosine (L‐MIM), hypoxia or echinomycin. Dental pulp‐derived cells in monolayers and spheroids were treated with L‐MIM, hypoxia or echinomycin. mRNA levels of the core circadian clock genes were analysed using quantitative PCR (qPCR) and their protein levels were analysed by western blot. All core clock genes and proteins were produced in DPC monolayer and spheroid cultures. The expression of cryptochrome circadian regulators and period circadian regulators was reduced by L‐MIM, hypoxia and echinomycin at mRNA, but not at protein levels. Time course experiments indicated that modulations were based on alterations in overall mRNA levels of core circadian clock genes. Our results suggest a potential role of the core circadian clock in the response of dental pulp to hypoxia. Future studies need to consider that regulation of the core circadian clock at mRNA levels might not be paralleled by modulation of protein levels.     

Characterization of fibroblasts derived from human periodontal ligament and gingiva

Growth characteristics and macromolecular synthesis of fibroblasts derived from human periodontal ligament (PDLF) and gingiva (GF) have been compared in cell culture. Cells were isolated from explants and plated at 500,000 cells/100 mm culture dish (day 0) with daily changes of culture medium. DNA histograms were obtained by flow microfluorimetric analysis to confirm the growth state of the cell cultures. Human PDLF cultures became confluent at day 6 as determined by cell number and cell cycle analysis while GF were confluent by day 4. Initially, DNA content of logarithmically growing cells was significantly greater in GF cultures; however, when confluent, DNA content and cell number was greater in PDLF cultures. Total protein content in GF was slightly greater than PDLF until day 7 but this difference was not significant. Analysis of collagen and noncollagen protein synthesis revealed a greater trend in noncollagen protein synthesis in the GF cultures compared to PDLF cultures. Analysis of glycosaminoglycans in the culture medium of GF and PDLF revealed similar distributions of components. In the cellular fraction, GF had greater amounts of hyaluronic acid and heparin and lesser amounts of chondroitin sulfates A and C than PDLF cultures. The results indicate that the growth characteristics of PDLF and GF, although similar in many respects, do exhibit specific differences in proliferative rates and macromolecular synthesis. The differences observed in these parameters may be important during in vivo events, such as guided tissue regeneration, where significant functional differences are observed between gingival connective tissue and periodontal ligament connective tissue.     

Titanium dioxide-based scanning powder can modulate cell activity of oral soft tissue - Insights from in vitro studies with L929 cells and periodontal fibroblasts

PurposeTo reveal the impact of titanium dioxide-based scanning powder for intraoral digital impression on the biological activity of oral fibroblasts.MethodsMurine L929 cells and human periodontal ligament (PDLF) and gingival fibroblasts (GF) were treated with ten-fold serial dilutions of scanning powder and the corresponding conditioned medium (filtrate of overnight incubation of powder in medium) starting with 30 mg/ml. Bicinchoninic acid protein assay, formazan- and resazurin-based toxicity assays, live/dead and annexin V/propidium iodide (PI) staining and immunoassays for interleukin (IL)-6 and IL-8 were performed. Powder composition was analyzed using energy dispersive X-ray spectroscopy (EDS).ResultsFormazan and resazurin conversion was lesser in L929 cells than PDLF and GF in the presence of scanning powder. Induction of cell death was caused by 30 mg/ml of powder in L929 cells but not in PDLF and GF. No pronounced impact of the conditioned medium was seen in cytotoxicity assays or live/dead-, and annexin V/PI staining. In PDLF and GF IL-6 expression was increased by the powder, while there was a decrease in IL-8. Powder particles did not deplete protein from medium. EDS showed a heterogeneous mixture consisting predominantly of titanium dioxide.ConclusionsScanning powder decreased cell activity and induced cell death in L929 cells at high concentrations. Human oral fibroblasts showed an increase in IL-6 levels but more resistance to the cytotoxicity of the powder. Within the limitations of an in vitro study our results suggest that proper cleaning after scanning is of clinical relevance to avoid potential unwanted effects of the powder.     

Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis

Scheres N, Laine ML, de Vries TJ, Everts V, van Winkelhoff AJ. Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01229.x © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective: Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth‐supporting tissues in human periodontitis. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can participate in the host immune response in periodontitis. This study aimed to investigate the effects of viable P. gingivalis on the expression of genes associated with inflammation and bone degradation by these fibroblast subsets. Material and Methods: Primary human GF and PDLF from six healthy donors were challenged in vitro with viable P. gingivalis W83 for 6 h. Gene expression of inflammatory cytokines in GF and PDLF was analyzed using real‐time PCR, and protein expression was analyzed using ELISA. Results: Viable P. gingivalis induced a strong in vitro inflammatory response in both GF and PDLF. We found increased gene expression of interleukin (IL)‐1β, IL‐6, IL‐8, tumor necrosis factor‐α, monocyte chemotactic protein‐1 and regulated upon activation, normal T‐cell expressed and secreted (RANTES). Macrophage colony‐stimulating factor was induced and the expression of osteoprotegerin was decreased in GF, but not in PDLF. In nonchallenged cells, a higher level of expression of IL‐6 was observed in GF than in PDLF. Between individual donors there was large heterogeneity in responsiveness to P. gingivalis. Also, in each individual, either GF or PDLF was more responsive to P. gingivalis. Conclusion: Considerable heterogeneity in responsiveness to P. gingivalis exists both between GF and PDLF and between individuals, which may be crucial determinants for the susceptibility to develop periodontitis.     

IGF-1 signaling enhances cell survival in periodontal ligament fibroblasts vs. gingival fibroblasts

The role of insulin-like growth factors (IGFs) in the regulation of apoptosis has been suggested, yet their impact on specific cells such as periodontal ligament fibroblasts (PDLF) and gingival fibroblasts (GF) remains unknown. The purpose of this study was to test the role of IGF-1 signaling in cell survival in PDLF compared with GF. In periodontal tissue sections, a significantly reduced apoptotic rate was first demonstrated in PDLF compared with GF. In vitro, IGF-1 substantially enhanced cell survival in PDLF compared with GF by the up-regulation of anti-apoptotic molecules and the down-regulation of pro-apoptotic molecules. Furthermore, the differential expression of insulin-like growth factor binding protein 5 (IGFBP-5) was observed in vitro, and its differential distribution was confirmed in vivo. Analysis of the present data suggests an enhanced cell survival in PDLF compared with GF by the up-regulation of IGF-1 signaling pathway.     

Specificity of Antimicrobial Peptide LL‐37 to Neutralize Periodontopathogenic Lipopolysaccharide Activity in Human Oral Fibroblasts

Background: The antimicrobial peptide LL‐37 is known to have a potent lipopolysaccharide (LPS)‐neutralizing activity in various cell types. Because of observed heterogeneity within periodontopathogenic LPS, the authors hypothesized that LL‐37 had specificity to neutralize such LPS activity. The present study, therefore, aims to investigate the LPS‐neutralizing activity of LL‐37 to various periodontopathogenic LPS in interleukin‐8 (IL‐8) production after challenging them in human oral fibroblasts. Methods: Human periodontal ligament fibroblasts (PDLF) and gingival fibroblasts (GF) were cultured from biopsies of periodontal ligament and gingival tissues. After cell confluence in 24‐well plates, LPS (10 μg/mL) from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans were added with or without LL‐37 (10 μg/mL). After 18 hours, the supernatant was collected and analyzed in IL‐8 production by enzyme‐linked immunosorbent assay. Results: All periodontopathogenic LPS statistically significantly induced IL‐8 production in both PDLF and GF (P <0.01). After neutralization with LL‐37, both PDLF and GF showed a statistically significant reduction in IL‐8 production compared with LPS‐treated groups without LL‐37 (P <0.01), and the percentage of reduction in IL‐8 production in PDLF appeared to be higher than in GF. In addition, the percentage of reduction in IL‐8 production varied considerably according to each periodontopathogenic LPS. Conclusions: The antimicrobial peptide LL‐37 had an ability to suppress periodontopathogenic LPS‐induced IL‐8 production in both PDLF and GF. Its LPS‐neutralizing activity revealed specificity to periodontopathogenic LPS and seemed to be dependent on the heterogeneity within LPS between different genera.     

In vitro viability, mitogenicity and clonogenic capacities of periodontal ligament fibroblasts after storage in four media supplemented with growth factors

Abstract – The choice of storage medium for preserving traumatically avulsed teeth is important for the success of future replantation. The objective of this study was to evaluate the effectiveness of growth factors (IGF‐1 and PDGF‐BB) when added to storage media in preserving the functional abilities of cultured periodontal ligament fibroblasts (PDLF). The evaluated storage media were: ViaSpan, Hanks’ balanced salt solution (HBSS), α minimal essential medium (α MEM), and α MEM supplemented with FCS and antibiotic (α MEM‐S). PDLF were obtained from explants of human healthy extracted teeth. Plates with confluent PDLF were soaked in the various media supplemented with IGF‐1 (10 ng/ml) and PDGF‐BB (4 ng/ml) for 2, 8 and 24 h at room temperature (24 °C). The control group was incubated with the examined storage media without growth factors at 24 °C. An additional control group was incubated with culture medium at 37 °C without growth factors. After incubation, the viability of the cells was determined by Trypan blue exclusion test. Viable cells were then analyzed for mitogenic (with thymidine) and clonogenic (by culturing one cell/well) capacities. Storage of PDLF with growth factors (GF) for 2, 8 and 24 h decreased their vitality by only 3% (not statistically significant). The mitogenicity of PDLF stored for 2, 8 and 24 h in various media with GF was statistically comparable to that of the control group. Generally, the highest mitogenic capacity of PDLF stored with or without GF was found after 8 h of storage. Increasing the storage period to 24 h decreased the mitogenic capacity of the cells stored with GF by only 10–40% compared to the control group. In contrast, the clonogenic capacity of PDLF stored with GF increased with increasing storage periods by 100–300%, and the highest clonogenic capacity was found in most storage media after 24 h of storage with GF. The highest clonogenic and mitogenic capacities were found in cells stored in HBSS followed by α MEM‐S. The mitogenic and clonogenic capacities of PDLF stored in various media supplemented with GF for 2–8 h were generally lower than without GF supplementation. The mitogenic and clonogenic effects of GF‐supplementation was observed only after 24 h of storage. After 24 h of storage with GF, the clonogenic capacity increased by 8–224% and the mitogenicity by 20–37%, except in cells stored in α MEM (‐1%). However, these differences were generally not statistically significant. In conclusion, the mitogenic and clonogenic effects of GF were observed only after 24 h of storage at room temperature. HBSS and α MEM‐S supplemented with GF were the most effective media for preserving the viability, mitogenicity and clonogenic capacity of PDLF stored for 24 h at room temperature. For short periods of storage (2 and 8 h), HBSS and α MEM‐S without GF were preferable.     

Identification of genes differentially expressed in cultured human periodontal ligament fibroblasts vs. human gingival fibroblasts by DNA microarray analysis

Despite their similar spindle-shaped appearance, periodontal ligament fibroblasts (PDLF) and gingival fibroblasts (GF) appear to display distinct functional activities in the maintenance of tissue integrity and during inflammatory/immune responses. We postulated that different characteristics of PDLF and GF are defined by the differential expression of specific genes. To test this, we investigated the possible variance of gene expression profile between cultured PDLF and GF, using DNA microarray technology. One hundred sixty-three genes were found differentially expressed by at least three-fold between PDLF and GF. Genes encoding transmembrane proteins and cytoskeleton-related proteins tended to be up-regulated in PDLF, whereas genes encoding cell-cycle regulation proteins and metabolism-related proteins tended to be up-regulated in GF. We concluded that PDLF and GF appear to display different gene expression patterns that may reflect intrinsic functional differences of the two cell populations and may well coordinate with their tissue-specific activities.     

Compression and hypoxia play independent roles while having combinative effects in the osteoclastogenesis induced by periodontal ligament cells

Objective:To investigate the isolated and combined effects of compression and hypoxia on the osteoclastogenesis induced by periodontal ligament cells (PDLCs).Materials and Methods:A periodontal ligament tissue model (PDLtm) was established by 3-D culturing human PDLCs on a thin sheet of poly lactic-co-glycolic acid scaffold. The PDLtm was treated with hypoxia and/or compression for 6, 24, or 72 hours. After that, a real-time polymerase chain reaction was used for gene expression analysis. The conditioned media were used for the coculture of osteoblast and osteoclast (OC) precursors; tartrate-resistant acid phosphatase staining was done to examine OC formation.Results:Either compression or hypoxia alone significantly up-regulated the gene expression of pro-osteoclastogenic cytokines in the PDLtm and enhanced osteoclastogenesis in the cocultures, and the combination of the two had significantly stronger effects than either stimulation alone. In addition, comparing the two stimulants, we found that the osteoclastogenic property of the PDLCs peaked earlier (at 6 hours) in the compression group than in the hypoxia group (at 24 hours).Conclusions:Both compressive force and hypoxia may take part in initiating osteoclastogenesis in orthodontic tooth movement and may have combinatory effects, which could update our concepts of the mechanisms involved in the initiation of bone resorption on the pressure side of the tooth in question.     

人牙周膜成纤维细胞与牙龈成纤维细胞差异表达基因扣除文库的构建

目的建立人牙周膜成纤维细胞(periodontalligamentfibroblast,PDLF) 与牙龈成纤维细胞(gin-givalfibroblast,GF)差异表达基因的扣除文库。方法体外培养人PDLC和GF,分别提取mRNA,采用基于PCR和消减杂交的基因克隆技术构建人PDLC与GF差异表达基因的扣除文库。结果成功构建了人PDLF与GF细胞差异表达基因的扣除文库,文库容量为4×l02。结论基于PCR和消减杂交的基因克隆技术是构建细胞间差异表达基因扣除文库的较为简洁而有效的方法。     

Age-related changes of CD4+ T cell migration and cytokine expression in germ-free and SPF mice periodontium

ObjectiveIncreasing age is a potential risk factor for periodontal tissue breakdown, which may be affected by commensal flora. The aim of this study evaluated age-related changes in CD4⁺ T cells, C-C chemokine ligand 5 (CCL5), interleukin (IL)-17A, and receptor activator of nuclear factor-kappa B ligand (RANKL) expression using germ-free (GF) and conventionally reared (SPF) mice.DesignGF and SPF mice at 8 (n = 6/group) and 22 weeks old (n = 6/group) were used. Immunohistochemical analyses were performed to determine the effects of aging on protein expression in periodontal tissues. Age-related changes in alveolar bone were quantified using micro-CT analysis.ResultsSPF mice, but not GF mice, showed an age-related increase in alveolar bone loss (P < 0.01). SPF mice at 22 weeks of age increased expression of CD4⁺ T cells, CCL5, IL-17A, and RANKL compared to those at 8 weeks of age in connective tissue and alveolar bone surface (P < 0.01). Furthermore, there was increased CD4⁺ T cells, which were co-expressed with IL-17A and RANKL in SPF mice at 22 weeks of age. On the other hand, the GF mice did not show any significant differences in CD4⁺ T cells, CCL5, IL-17A and RANKL expression between the two age groups.ConclusionsSPF mice induced an age-related increase in CD4⁺ T cells co- expressed with IL-17A and RANKL, with occurring alveolar bone loss. In contrast, GF mice did not show age-related changes in CD4⁺ T cell migration and cytokine expression.     

Apigenin inhibited hypoxia induced stem cell marker expression in a head and neck squamous cell carcinoma cell line

ObjectiveCancer stem cells contribute to tumor recurrence, and a hypoxic environment is critical for maintaining cancer stem cells. Apigenin is a natural product with anticancer activity. However, the effect of apigenin on cancer stem cells remains unclear. Our aim was to investigate the effect of apigenin on cancer stem cell marker expression in head and neck squamous cell carcinoma cells under hypoxia.DesignWe used three head and neck squamous cell carcinoma cell lines; HN-8, HN-30, and HSC-3. The mRNA expression of cancer stem cell markers was determined by semiquantitative RT-PCR and Real-time PCR. The cytotoxic effect of apigenin was determined by MTT colorimetric assay. Flow cytometry was used to reveal the number of cells expressing cancer stem cell surface markers.ResultsHN-30 cells, a cancer cell line from the pharynx, showed the greatest response to hypoxia by increasing their expression of CD44, CD105, NANOG, OCT-4, REX-1, and VEGF. Apigenin significantly decreased HN-30 cell viability in dose- and time-dependent manners. In addition, 40 μM apigenin significantly down-regulated the mRNA expression of CD44, NANOG, and CD105. Consistent with these results, the hypoxia-induced increase in CD44⁺ cells, CD105⁺ cells, and STRO-1⁺ cells was significantly abolished by apigenin.ConclusionApigenin suppresses cancer stem cell marker expression and the number of cells expressing cell surface markers under hypoxia.