Glycine Lipids of Porphyromonas gingivalis Are Agonists for Toll-Like Receptor 2.

The serine-glycine dipeptide lipid classes, including lipid 430 and lipid 654, are produced by the periodontal pathogen Porphyromonas gingivalis and can be detected in lipid extracts of diseased periodontal tissues and teeth of humans. Both serine-glycine lipid classes were previously shown to engage human and mouse Toll-like receptor 2 (TLR2) and to inhibit mouse osteoblast differentiation and function through engagement of TLR2. It is not clear if other lipids related to serine-glycine lipids are also produced by P. gingivalis The goal of this investigation was to determine whether P. gingivalis produces additional lipid classes similar to the serine-glycine lipids that possess biological properties. P. gingivalis (ATCC 33277) was grown in broth culture, and lipids were extracted and fractionated by high-performance liquid chromatography (HPLC). Lipids were separated using semipreparative HPLC, and specific lipid classes were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometric approaches. Two glycine lipid classes were identified, termed lipid 567 and lipid 342, and these lipid classes are structurally related to the serine-glycine dipeptide lipids. Both glycine lipid classes were shown to promote TLR2-dependent tumor necrosis factor alpha (TNF-α) release from bone marrow macrophages, and both were shown to activate human embryonic kidney (HEK) cells through TLR2 and TLR6 but not TLR1. These results demonstrate that P. gingivalis synthesizes glycine lipids and that these lipids engage TLR2 similarly to the previously reported serine-glycine dipeptide lipids.

Serine/Glycine Lipid Recovery in Lipid Extracts From Healthy and Diseased Dental Samples: Relationship to Chronic Periodontitis.

Toll-like receptor 2 (TLR2) activation has been implicated in the pathogenesis of periodontal disease but the identity of the TLR2 agonists has been an evolving story. The serine/glycine lipids produced by Porphyromonas gingivalis are reported to engage human TLR2 and will promote the production of potent pro-inflammatory cytokines. This investigation compared the recovery of serine/glycine lipids in periodontal organisms, teeth, subgingival calculus, subgingival plaque, and gingival tissues, either from healthy sites or periodontally diseased sites. Lipids were extracted using the phospholipid extraction procedure of Bligh and Dyer and were analyzed using liquid chromatography/mass spectrometry for all serine/glycine lipid classes identified to date in P. gingivalis. Two serine/glycine lipid classes, Lipid 567 and Lipid 1256, were the dominant serine/glycine lipids recovered from oral Bacteroidetes bacteria and from subgingival calculus samples or diseased teeth. Lipid 1256 was the most abundant serine/glycine lipid class in lipid extracts from P. gingivalis, Tannerella forsythia, and Prevotella intermedia whereas Lipid 567 was the most abundant serine/glycine lipid class recovered in Capnocytophaga species and Porphyromonas endodontalis. Serine/glycine lipids were not detected in lipid extracts from Treponema denticola, Aggregatibacter actinomycetemcomitans, or Fusobacterium nucleatum. Lipid 1256 was detected more frequently and at a significantly higher mean level in periodontitis tissue samples compared with healthy/gingivitis tissue samples. By contrast, Lipid 567 levels were essentially identical. This report shows that members of the Bacteroidetes phylum common to periodontal disease sites produce Lipid 567 and Lipid 1256, and these lipids are prevalent in lipid extracts from subgingival calculus and from periodontally diseased teeth and diseased gingival tissues.

Porphyromonas gingivalis lipids inhibit osteoblastic differentiation and function.

Porphyromonas gingivalis produces unusual sphingolipids that are known to promote inflammatory reactions in gingival fibroblasts and Toll-like receptor 2 (TLR2)-dependent secretion of interleukin-6 from dendritic cells. The aim of the present study was to examine whether P. gingivalis lipids inhibit osteoblastic function. Total lipids from P. gingivalis and two fractions, phosphoglycerol dihydroceramides and phosphoethanolamine dihydroceramides, were prepared free of lipid A. Primary calvarial osteoblast cultures derived from 5- to 7-day-old CD-1 mice were used to examine the effects of P. gingivalis lipids on mineralized nodule formation, cell viability, apoptosis, cell proliferation, and gene expression. P. gingivalis lipids inhibited osteoblast differentiation and fluorescence expression of pOBCol2.3GFP in a concentration-dependent manner. However, P. gingivalis lipids did not significantly alter osteoblast proliferation, viability, or apoptosis. When administered during specific intervals of osteoblast growth, P. gingivalis total lipids demonstrated inhibitory effects on osteoblast differentiation only after the proliferation stage of culture. Reverse transcription-PCR confirmed the downregulation of osteoblast marker genes, including Runx2, ALP, OC, BSP, OPG, and DMP-1, with concurrent upregulation of RANKL, tumor necrosis factor alpha, and MMP-3 genes. P. gingivalis total lipids and lipid fractions inhibited calvarial osteoblast gene expression and function in vivo, as determined by the loss of expression of another osteoblast differentiation reporter, pOBCol3.6GFPcyan, and reduced uptake of Alizarin complexone stain. Finally, lipid inhibition of mineral nodule formation in vitro was dependent on TLR2 expression. Our results indicate that inhibition of osteoblast function and gene expression by P. gingivalis lipids represents a novel mechanism for altering alveolar bone homeostasis at periodontal disease sites.

Examination of mineralized nodule formation in living osteoblastic cultures using fluorescent dyes.

Detecting the formation of mineralized nodules in osteogenic cell culture provides a means of assessing mature osteoblast cell function and the status of culture. In the present study, to continuously monitor the formation of mineralized nodules during the entire culture period, different concentrations of two fluorescent dyes (xylenol orange and calcein blue) were evaluated for their ability to specifically label calcified areas and their toxicity to cells in osteogenic cultures. Results showed that 20 microM xylenol orange and 30 microM calcein blue gave rise to distinct fluorescent staining for mineralized nodules, which were correlated exactly with von Kossa and alizarin red S staining at the same locations in cultures. In the assessment of toxicity, both dyes at the aforementioned concentrations did not alter cell viability or change the total DNA content in cultures. To demonstrate the advantage of using these fluorochromes to monitor mineralized nodules formation, consecutive fluorescent images of each staining were recorded at the same location of individual culture over the entire duration. The result indicates that both xylenol orange and calcein blue can provide contrasting fluorescent staining to continuously monitor mineralized nodules formation in living osteogenic cell cultures without deleterious effects.

Comparison of the Osteogenic Potential of Mineral Trioxide Aggregate and Endosequence Root Repair Material in a 3-dimensional Culture System.

INTRODUCTION: The ability to promote osteoblast differentiation is a desirable property of root-end filling materials. Several in vitro studies compare the cytotoxicity and physical properties between mineral trioxide aggregate (MTA) and Endosequence root repair material (ERRM), but not their osteogenic potential. Three-dimensional cultures allow cells to better maintain their physiological morphology and better resemble in vivo cellular response than 2-dimensional cultures. Here we examined the osteogenic potential of MTA and ERRM by using a commercially available 3-dimensional Alvetex scaffold. METHODS: Mandibular osteoblasts were derived from 3-week-old male transgenic reporter mice where mature osteoblasts express green fluorescent protein (GFP) driven by a 2.3-kilobase type I collagen promoter (Col(I)-2.3). Mandibular osteoblasts were grown on Alvetex in direct contact with MTA, ERRM, or no material (negative control) for 14 days. Osteoblast differentiation was evaluated by expression levels of osteogenic genes by using quantitative polymerase chain reaction and by the spatial dynamics of Col(I)-2.3 GFP-positive mature osteoblasts within the Alvetex scaffolds by using 2-photon microscopy. RESULTS: ERRM significantly increased alkaline phosphatase (Alp) and bone sialoprotein (Bsp) expression compared with MTA and negative control groups. Both MTA and ERRM increased osterix (Osx) mRNA significantly compared with the negative control group. The percentage of Col(I)-2.3 GFP-positive cells over total cells within Alvetex was the highest in the ERRM group, followed by MTA and by negative controls. CONCLUSIONS: ERRM promotes osteoblast differentiation better than MTA and controls with no material in a 3-dimensional culture system. Alvetex scaffolds can be used to test endodontic materials.

Comparison of the action of transient and continuous PTH on primary osteoblast cultures expressing differentiation stage-specific GFP.

UNLABELLED: Primary calvarial osteoblast cultures derived from type I collagen promoter-GFP reporter transgenic mice were used to examine progression of the osteoblast lineage. This system was validated by assessing the effect of PTH on osteoblast growth in real time. The anabolic effect of PTH seemed to be the result of enhanced osteoblast differentiation rather than expansion of a progenitor population. INTRODUCTION: Activation of green fluorescent protein (GFP) marker genes driven by Col1a1 promoter fragments has been associated with the level of osteoblast differentiation. GFP-marked cultures provide an approach to continuously monitor the level of osteoblast differentiation in real time without the termination of cultures. MATERIALS AND METHODS: Neonatal calvarial cells transgenic for pOBCol2.3GFP and pOBCol3.6GFP were used to establish calvarial osteoblast cultures. Parathyroid hormone (PTH) was added either continuous (days 1-21) or transient (days 1-7) to examine its diverse effect on osteoblast differentiation in cultures for 21 days. Three fluorescent markers were used: (1) pOBCol3.6GFP, which is activated in preosteoblastic cells; (2) pOBCol2.3GFP, which is restricted to differentiated osteoblasts; and (3) xylenol orange (XO), which stains the mineralized nodules. Progression of osteoblast differentiation indicated by fluorescent markers was documented throughout the entire period of culture. Recorded fluorescent images were analyzed in the patterns of expression and quantitated in the area of expression. RESULTS: Continuous PTH blocked osteoblast differentiation, which was evident by the attenuation of pOBCol3.6GFP and an absence of pOBCol2.3GFP. In contrast, transient PTH inhibited the initial osteoblast differentiation but ultimately resulted in a culture with more mineralized nodules and enhanced osteoblast differentiation expressing strong levels of pOBCol3.6GFP and pOBCol2.3GFP. Quantitative analysis showed that transient PTH first decreased then later increased areas of GFP expression and XO staining, which correlated with results of Northern blot and alkaline phosphatase activity. Transient PTH caused a decrease in DNA content during the treatment and after the removal of PTH. CONCLUSION: GFP-marked cultures combined with fluorescent image analysis have the advantage to assess the effect of PTH on osteoblast differentiation in real time. Results suggest that the anabolic effect of transient PTH is caused by an enhancement in osteoblast differentiation rather than an increase in the population of progenitor cells.

Serine dipeptide lipids of Porphyromonas gingivalis inhibit osteoblast differentiation: Relationship to Toll-like receptor 2.

Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential to promote TLR2-dependent bone loss as is reported in experimental periodontitis following oral infection with P. gingivalis. These results also support the conclusion that serine dipeptide lipids are involved in alveolar bone loss in chronic periodontitis.

Biologic activity of porphyromonas endodontalis complex lipids.

INTRODUCTION: Periapical infections secondary to pulpal necrosis are associated with bacterial contamination of the pulp. Porphyromonas endodontalis, a gram-negative organism, is considered to be a pulpal pathogen. P. gingivalis is phylogenetically related to P. endodontalis and synthesizes several classes of novel complex lipids that possess biological activity, including the capacity to promote osteoclastogenesis and osteoclast activation. The purpose of this study was to extract and characterize constituent lipids of P. endodontalis and evaluate their capacity to promote proinflammatory secretory responses in the macrophage cell line, RAW 264.7, as well as their capacity to promote osteoclastogenesis and inhibit osteoblast activity. METHODS: Constituent lipids of both organisms were fractionated by high-performance liquid chromatography and were structurally characterized using electrospray mass spectrometry or electrospray-mass spectrometry/mass spectrometry. The virulence potential of P. endodontalis lipids was then compared with known biologically active lipids isolated from P. gingivalis. RESULTS: P. endodontalis total lipids were shown to promote tumor necrosis factor alpha secretion from RAW 264.7 cells, and the serine lipid fraction appeared to account for the majority of this effect. P. endodontalis lipid preparations also increased osteoclast formation from RAW 264.7 cells, but osteoblast differentiation in culture was inhibited and appeared to be dependent on Toll-like receptor 2 expression. CONCLUSIONS: These effects underscore the importance of P. endodontalis lipids in promoting inflammatory and bone cell activation processes that could lead to periapical pathology.

Effect of selected accelerants on setting time and biocompatibility of mineral trioxide aggregate (MTA).

OBJECTIVE: Various additives have been suggested to be used with mineral trioxide aggregate (MTA) to improve its handling characteristics. The purpose of this study was to evaluate the effects of various additives on setting time and cell attachment on gray MTA (GMTA). STUDY DESIGN: Single-rooted caries-free teeth were split, and dentin disks with class I cavity were made and filled with test and control materials. Setting time was measured using Gilmore apparatus. Mouse MC3T3-E1 osteoblasts and L929 mouse fibroblasts were grown on dentin and GMTA disks. Cell attachment was examined under fluorescent microscope. RESULTS: Adding KY liquid, CaCl(2), and NaOCl to GMTA improved the handling properties and decreased setting time. Osteoblasts and fibroblasts attached and spread on GMTA mixed with additives in a manner similar to GMTA mixed with water. CONCLUSIONS: The various additives could be possible substitutes to water to decrease MTA setting time. MTA is biocompatible when mixed with the various additives.

Cytotoxicity evaluation of Gutta Flow and Endo Sequence BC sealers.

OBJECTIVE: This study evaluated the cytotoxicity of GuttaFlow and EndoSequence BC sealers and compared them with AH Plus and Tubli-Seal sealers. STUDY DESIGN: Samples (0.5 mg) of freshly mixed or set BC, GuttaFlow, AH Plus, and Tubli-Seal sealers were eluted with 300, 600, and 1,000 µL cell culture medium for 24 and 72 hours. L929 cells were seeded into 96-well plates at 3 × 10(4) cells/well and cultured with 100 µL eluate from each eluate group. Cells cultured only with culture medium served as control. After 24 hours' incubation, the cytotoxicity was evaluated by MTT assay. Cell viability was calculated as the percentage of the control group, and the results were analyzed with a one-way analysis of variance. RESULTS: For the freshly mixed sealer, cell viability in the AH Plus group was less than in all of the other 3 sealer groups. The Tubli-Seal sealer group had less cell viability than the EndoSequence BC and GuttaFlow sealer groups. For the set sealer, the Tubli-Seal and AH Plus groups had less cell viability than the EndoSequence BC and GuttaFlow sealer groups. There was no cell viability difference between the EndoSequence BC and GuttaFlow sealer groups in the either freshly mixed or set sealer group. CONCLUSIONS: The GuttaFlow and EndoSequence BC sealers have lower cytotoxicity than the AH Plus and Tubli-Seal sealers.

A nondestructive method for evaluating in vitro osteoblast differentiation on biomaterials using osteoblast-specific fluorescence.

Transgenic mice with a Col1a1-promoter-driven transgene pOBCol2.3GFP were previously developed to visually identify mature osteoblasts through fluorescent expression. Our goal was to determine if this technology could be used to nondestructively evaluate the in vitro differentiation of osteoprogenitor cells on biomaterials such as biomimetic carbonated hydroxyapatite (cHA). Primary osteoprogenitor cells were harvested from calvaria of neonatal Col2.3GFP transgenic mice and cultured on cHA and a tissue culture polystyrene (TCPS) control. The distribution of intensities and area percentage of green fluorescent protein (GFP)-positive cells were quantified using fluorimetry and image analysis of fluorescent microscopy. At 14 days, an increased area and higher mean intensity of GFP-positive cells was observed on cHA as compared to TCPS, indicating more rapid differentiation on cHA. Notably, there were large continuous regions of GFP-positive osteoblasts on cHA, in contrast to the sparse, nodules of osteoblasts on TCPS, implying that cHA provides an osteogenic cue to cells. Xylenol orange staining was capable of distinguishing osteoblast-initiated mineral from the cHA substrate. With this method the unique pattern of osteoblast differentiation on cHA was clearly observed for the first time. Importantly, the generalized method can be used for rapid, high-throughput, nondestructive screening of biomaterials intended to enhance osteogenic differentiation.

Evaluation of cytotoxicity of MTA employing various additives.

OBJECTIVE: Various additives have been suggested to be used with MTA to improve its handling characteristics. The objective of this study was to evaluate the effects of various additives on cytotoxicity of MTA. STUDY DESIGN: Freshly mixed or set MTA pellet (1-mm diameter and 1-mm high cylinder) was prepared by mixing MTA with various additives. Additives tested included water, saline, 2% lidocaine, 5% CaCl(2), 3% NaOCl gel, and K-Y liquid. L929 cells were seeded into 96-well plates at 3 x 10(4) cells per well and incubated with MTA pellets for 24 hours, 48 hours, and 72 hours. Cells without treatment served as a control group. Cell viability was evaluated by MTT assay and calculated as the percentage of the control group. The results were analyzed with 1-way ANOVA. RESULTS: For the set MTA, there were no significant cell viability differences (P > .05) among the various additives at each tested time. For the freshly mixed MTA, 3% NaOCl gel has lower cell viability (P < .05) than all the other groups. The cell viability of 3% NaOCl gel group was 29% to 50%. Gray and white MTA have similar results. CONCLUSION: This study shows that the various additives have no effect on the cytotoxicity of MTA when it becomes set. Also, all the tested additives, except 3% NaOCl gel, had no effect on the cytotoxicity of MTA when it was freshly mixed. The cytotoxicity of 3% NaOCl gel probably has no clinical significance considering there is still 29% to 50% of cell viability after cells were treated with MTA pellet in a 0.32-cm(2) culture well. MTA is biocompatible when mixed with the various additives.

Cytotoxicity evaluation of Activ GP and Resilon sealers in vitro.

OBJECTIVE: This study is to evaluate the cytotoxicity of Activ GP and RealSeal sealers in a cell culture system in vitro, and to compare them with traditional AH 26 and Kerr sealers. STUDY DESIGN: Samples of 0.5 mg freshly mixed or set RealSeal, Activ GP, AH 26, and Kerr sealers were eluted with 200, 400, 800, and 1,200 microL cell culture medium for 24 and 72 hours. L929 cells were seeded into 96-well plates at 3 x 10(4) cells/well and cultured with 100 microL eluate from each eluate group. Cells cultured with culture medium only served as a control. After 24 hours' incubation the cytotoxicity was evaluated by MTT assay. Cell viability was calculated as the percentage of the control group, and the results were analyzed with 1-way analysis of variance. RESULTS: For the freshly mixed sealer, cell viability in the AH 26 group was less than in all of the other 3 sealer groups. The Kerr sealer group had greater cell viability than RealSeal and Activ GP groups. For the set sealer, cell viability in the AH 26 group was greater than in all of the other 3 groups. Cell viability in the RealSeal group was less than in the Kerr and Activ GP groups. CONCLUSION: Freshly mixed RealSeal and Activ GP sealers have lower cytotoxicity than AH 26 sealer and more cytotoxicity than Kerr sealer. When sealers are set, RealSeal sealer has more cytotoxicity than AH 26 and Kerr sealer. Activ GP sealer has more cytotoxicity than AH 26 and is similar to Kerr sealer.

Effects of transient PTH on early proliferation, apoptosis, and subsequent differentiation of osteoblast in primary osteoblast cultures.

In primary calvarial osteoblast cultures derived from transgenic mice expressing green fluorescent protein (GFP) under the control of 3.6-kb Col1a1 promoter, the emergence of GFP signal marks the transition of multipotential osteoprogenitors into preosteoblasts. Early transient treatment (days 1-7) of these cultures with parathyroid hormone (PTH) has an anabolic effect that is not associated with an increase in total DNA content or cell number in day 21 cultures. In the present study, the effect of early PTH treatment on cell proliferation and apoptosis was examined in greater detail in GFP(+) and GFP(-) cells using flow cytometry. In preconfluent cultures, PTH significantly reduced the proportion of cells in S phase but increased those in G(0)/G(1) and G(2)+M phases in both GFP(+) and GFP(-) subpopulations. PTH decreased apoptosis only in GFP(-) but not GFP(+) cells, indicating an increased survival of GFP(-) cells. In contrast, PTH did not change the amounts of cell proliferation and apoptosis seen in either compartment after these cultures reached confluence. To further assess the effect of early PTH treatment on osteogenic differentiation, secondary cultures of sorted GFP(+) or GFP(-) cells were obtained from day 7 primary cultures that had been treated for 1 wk with PTH. This treatment resulted in larger areas of GFP expression accompanied by increased xylenol orange/von Kossa staining in the secondary cultures of GFP fractions. Early transient PTH treatment appears to enhance the commitment of progenitor cells to an osteogenic fate and results in a higher proportion of cells that achieve full osteoblast differentiation.