Mediterranean diet component oleic acid increases protective lipid mediators and improves trabecular bone in a Porphyromonas gingivalis inoculation model.

AIM: Therapeutic modulation of bacterial-induced inflammatory host response is being investigated in gingival inflammation and periodontal disease pathology. Therefore, dietary intake of the monounsaturated fatty acid (FA) oleic acid (OA (C18:1)), which is the main component of Mediterranean-style diets, and saturated FA palmitic acid (PA (C16:0)), which is a component of Western-style diets, was investigated for their modifying potential in an oral inoculation model of Porphyromonas gingivalis. MATERIALS AND METHODS: Normal-weight C57BL/6-mice received OA- or PA-enriched diets (PA-ED, OA-ED, PA/OA-ED) or normal standard diet for 16 weeks and were inoculated with P. gingivalis/placebo (n = 12/group). Gingival inflammation, alveolar bone structure, circulating lipid mediators, and in vitro cellular response were determined. RESULTS: FA treatment of P. gingivalis-lipopolysaccharide-incubated gingival fibroblasts (GFbs) modified inflammatory activation, which only PA exacerbated with concomitant TNF-α stimulation. Mice exhibited no signs of acute inflammation in gingiva or serum and no inoculation- or nutrition-associated changes of the crestal alveolar bone. However, following P. gingivalis inoculation, OA-ED improved oral trabecular bone micro-architecture and enhanced circulating pro-resolving mediators resolvin D4 (RvD4) and 4-hydroxydocosahexaenoic acid (4-HDHA), whereas PA-ED did not. In vitro experiments demonstrated significantly improved differentiation in RvD4- and 4-HDHA-treated primary osteoblast cultures and reduced the expression of osteoclastogenic factors in GF. Further, P. gingivalis infection of OA-ED animals led to a serum composition that suppressed osteoclastic differentiation in vitro. CONCLUSIONS: Our results underline the preventive impact of Mediterranean-style OA-EDs by indicating their pro-resolving nature beyond anti-inflammatory properties.

Effect of using different component combinations for orthodontic bracket bonding with self-etch primers.

PURPOSE: To evaluate bonding quality for orthodontic bracket bonding with different component combinations of self-etch primers in vitro. METHODS: Metallic brackets were bonded to bovine lower incisors and assigned to groups. Group 1: comparison of self-etch (Transbond™ Plus, 3M™ Unitek, Neuss, Germany, n = 30; BrackFix® primer SE, VOCO®, Cuxhaven, Germany, n = 20) and etch-and-rinse bonding systems (Transbond™ XT, n = 20; BrackFix®, n = 20); group 2: comparison of different self-etch primer (Transbond™ Plus; BrackFix® primer SE) and adhesive (Transbond™ XT, n = 20; BrackFix®, n = 20) product combinations; group 3: testing cyclic fatigue bond strength of self-etch bonding systems (Transbond™ Plus, n = 20; BrackFix® primer SE, n = 20). All teeth were tested for shear bond strength according to the DIN-13990 standard, the adhesive remnant index (ARI) and enamel fractures were determined microscopically (10 נmagnification). RESULTS: The mean shear bond strength of the self-etch (Transbond™ Plus: 16.38 ± 3.68 MPa; BrackFix® primer SE: 16.24 ± 1.73 MPa) and etch-and-rinse bonding systems (Transbond™ XT: 18.45 ± 2.56 MPa; BrackFix®: 17 ± 5.2 MPa) were of a clinically adequate order of magnitude (≥ 6-10 MPa) and were not statistically different. The component combination BrackFix® primer SE/Transbond™ XT adhesive led to a significantly lower shear bond strength (11.99 ± 3.68 MPa). There were no significant differences between static and fatigue shear bond strengths of self-etch bonding systems. Mean ARI scores mostly ranged between 4 and 5. The combination of the self-etch primer Transbond™ Plus with the BrackFix® adhesive led to a significantly increased enamel fracture rate. CONCLUSIONS: Based on the present findings bond strength of self-etch primers was equal to etch-and-rinse primers for bracket bonding. Combining different self-etch bonding systems might alter the clinical performance.

Potential donor-dependent regulative effects of endogenous sclerostin expression and mineralization potential in primary human PDL cells in vitro.

OBJECTIVES: The glycoprotein sclerostin is mostly expressed in osteocytes and plays a central role in human bone metabolism. However, sclerostin and the corresponding SOST gene have been found in periodontal ligament cells under mineralizing conditions as well. The present study aimed to investigate, whether there was a correlation between endogenous SOST expression, the corresponding gene, and mineralization potential in human periodontal ligament cells and to identify different sclerostin expression and secretion patterns in cells derived from individual donors. MATERIAL AND METHODS: Primary human periodontal ligament cells of three different donors were cultivated under control or mineralizing conditions for 6, 13, 15 and 18 days, respectively. Calcium deposits were stained with alizarin red and quantified afterwards. Quantitative expression analysis of the SOST gene encoding sclerostin was performed using quantitative reverse transcription polymerase chain reaction (RT-PCR). Additionally, intracellular sclerostin expression was analyzed using Western blotting and extracellular sclerostin secretion was quantified using Enzyme-linked Immunosorbent Assay (ELISA). RESULTS: Alizarin red staining identified calcium deposits in periodontal ligament cells under mineralizing conditions beginning from day 13, relative SOST expression occurred on day 6. Whereas staining continued to increase in donor 1 on day 15, it remained stable in donors 2 and 3. Conversely, baseline SOST expression was significantly lower in donor 1 compared to donors 2 and 3. Western blotting and ELISA revealed increased intra- and extracellular sclerostin expression at day 13 under mineralizing conditions. Donor 3 exhibited the highest overall sclerostin levels. CONCLUSIONS: Our data emphasize donor-specific characteristics in differentiation potential and sclerostin expression patterns in primary human periodontal ligament cells. Sclerostin might play a central role in modulating osteogenic differentiation in periodontal ligament cells as part of a negative feedback mechanism in avoiding excessive mineralization.

Impact of FGF1 on human periodontal ligament fibroblast growth, osteogenic differentiation and inflammatory reaction in vitro.

PURPOSE: To investigate in vitro the impact of fibroblast growth factor 1 (FGF1) in comparison to ascorbic acid (AscA) on human periodontal ligament fibroblast (HPdLF) growth, their osteogenic differentiation, and modulation of their inflammatory reaction to mechanical stress. METHODS: The influence of different concentrations of FGF1 (12.5-200 ng/mL) on growth and proliferation of HPdLF cells was analyzed over 20 days by counting cell numbers and the percentage of Ki67-positive cells. Quantitative expression analysis of genes encoding the osteogenic markers alkaline phosphatase (ALPL), Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OSP), as well as the fibroblast markers vimentin (VIM) and fibroblast-specific protein 1 (FSP1), was performed after 2 and 20 days of cultivation. Metabolic activity was determined by MTT assay. For comparison with AscA, 50 ng/mL FGF1 was used for stimulation for 2 and 20 days. Cell number, percentage of Ki67-positive cells, and expression of osteoblast- and fibroblast-specific genes were examined. Alkaline phosphatase activity was visualized by NBT/BCIP and calcium deposits were stained with alizarin red. Cytokine (IL­6, IL­8, COX2/PGE2) expression and secretion were analyzed by qPCR and ELISA in 6 h mechanically compressed HPdLF cultured for 2 days with FGF1 or ascorbic acid. RESULTS: Higher concentrations of FGF1 promoted cell proliferation upon short-term stimulation, whereas prolonged treatment induced the expression of osteogenic markers even with low concentrations. AscA promotes cell growth more markedly than FGF1 in short-term cultures, whereas FGF1 induced osteogenic cell fate more strongly in long-term culture. Both factors induced an increased inflammatory response of HPdLF to mechanical compression. CONCLUSION: Our data suggest that FGF1 promotes an osteogenic phenotype of HPdLF and limits inflammatory response to mechanical forces compared to AscA.

Gene expression and phosphorylation of ERK and AKT are regulated depending on mechanical force and cell confluence in murine cementoblasts.

Cementoblasts, located on the tooth root surface covered with cementum, are considered to have tooth protecting abilities. They prevent tissue damage and secure teeth anchorage inside the periodontal ligament during mechanical stress. However, the involvement of cementoblasts in mechanical compression induced periodontal remodeling needs to be identified and better understood. Here, we investigated the effect of static compressive stimulation, simulating the compression side of orthodontic force and cell confluence on a murine cementoblast cell line (OC/CM). The influence of cell confluence in cementoblast cells was analyzed by MTS assay and immunostaining. Furthermore, mRNA and protein expression were investigated by real-time RT-PCR and western blotting at different confluence grades and after mechanical stimulation. We observed that cementoblast cell proliferation increases with increasing confluence grades, while cell viability decreases in parallel. Gene expression of remodeling markers is regulated by compressive force. In addition, cementoblast confluence plays a crucial role in this regulation. Confluent cementoblasts show a significantly higher basal expression of Bsp, Osterix, Alpl, Vegfa, Mmp9, Tlr2 and Tlr4 compared to sub-confluent cells. After compressive force of 48 h at 60% confluence, an upregulation of Bsp, Osterix, Alpl, Vegf and Mmp9 is observed. In contrast, at high confluence, all analyzed genes were downregulated through mechanical stress. We also proved a regulation of ERK, phospho-ERK and phospho-AKT dependent on compressive force. In summary, our findings provide evidence that cementoblast physiology and metabolism is highly regulated in a cell confluence-dependent manner and by mechanical stimulation.

Carnosine synthase deficiency is compatible with normal skeletal muscle and olfactory function but causes reduced olfactory sensitivity in aging mice.

Carnosine (ß-alanyl-l-histidine) and anserine (ß-alanyl-3-methyl-l-histidine) are abundant peptides in the nervous system and skeletal muscle of many vertebrates. Many in vitro and in vivo studies demonstrated that exogenously added carnosine can improve muscle contraction, has antioxidant activity, and can quench various reactive aldehydes. Some of these functions likely contribute to the proposed anti-aging activity of carnosine. However, the physiological role of carnosine and related histidine-containing dipeptides (HCDs) is not clear. In this study, we generated a mouse line deficient in carnosine synthase (Carns1). HCDs were undetectable in the primary olfactory system and skeletal muscle of Carns1-deficient mice. Skeletal muscle contraction in these mice, however, was unaltered, and there was no evidence for reduced pH-buffering capacity in the skeletal muscle. Olfactory tests did not reveal any deterioration in 8-month-old mice lacking carnosine. In contrast, aging (18-24-month-old) Carns1-deficient mice exhibited olfactory sensitivity impairments that correlated with an age-dependent reduction in the number of olfactory receptor neurons. Whereas we found no evidence for elevated levels of lipoxidation and glycation end products in the primary olfactory system, protein carbonylation was increased in the olfactory bulb of aged Carns1-deficient mice. Taken together, these results suggest that carnosine in the olfactory system is not essential for information processing in the olfactory signaling pathway but does have a role in the long-term protection of olfactory receptor neurons, possibly through its antioxidant activity.

Distinguish fatty acids impact survival, differentiation and cellular function of periodontal ligament fibroblasts.

Alveolar bone (AB) remodeling is necessary for the adaption to mechanical stimuli occurring during mastication and orthodontic tooth movement (OTM). Thereby, bone degradation and assembly are strongly regulated processes that can be altered in obese patients. Further, increased fatty acids (FA) serum levels affect bone remodeling cells and we, therefore, investigated whether they also influence the function of periodontal ligament fibroblast (PdLF). PdLF are a major cell type regulating the differentiation and function of osteoblasts and osteoclasts localized in the AB. We stimulated human PdLF (HPdLF) in vitro with palmitic (PA) or oleic acid (OA) and analyzed their metabolic activity, growth, survival and expression of osteogenic markers and calcium deposits. Our results emphasize that PA increased cell death of HPdLF, whereas OA induced their osteoblastic differentiation. Moreover, quantitative expression analysis of OPG and RANKL revealed altered levels in mechanically stimulated PA-treated HPdLF. Furthermore, osteoclasts stimulated with culture medium of mechanical stressed FA-treated HPdLF revealed significant changes in cell differentiation upon FA-treatment. For the first time, our results highlight a potential role of specific FA in the function of HPdLF-modulated AB remodeling and help to elucidate the complex interplay of bone metabolism, mechanical stimulation and obesity-induced alterations.

Selection and validation of reference genes by RT-qPCR for murine cementoblasts in mechanical loading experiments simulating orthodontic forces in vitro.

Different structures and cell types of the periodontium respond to orthodontic tooth movement (OTM) individually. Cementoblasts (OC/CM) located in the immediate vicinity of the fibroblasts on the cement have found way to the centre of actual research. Here, we identify and validate possible reference genes for OC/CM cells by RT-qPCR with and without static compressive loading. We investigated the suitability of 3 reference genes in an in vitro model of cementoblast cells using four different algorithms (Normfinder, geNorm, comparative delta-Ct method and BestKeeper) under different confluences and time. Comparable to our previous publications about reference genes in OTM in rats and human periodontal ligament fibroblasts (hPDLF), Rpl22 in murine OC/CM cells appears as the least regulated gene so that it represents the most appropriate reference gene. Furthermore, unlike to the expression of our recommended reference genes, the expression of additionally investigated target genes changes with confluence and under loading compression. Based on our findings for future RT-qPCR analyses in OC/CM cells, Rpl22 or the combination Rpl22/Tbp should be favored as reference gene. According to our results, although many publications propose the use of Gapdh, it does not seem to be the most suitable approach.

Orthodontic cell stress modifies proinflammatory cytokine expression in human PDL cells and induces immunomodulatory effects via TLR-4 signaling in vitro.

OBJECTIVE: Biomechanical orthodontics loading of the periodontium initiates a cascade of inflammatory signaling events that induce periodontal remodeling and finally facilitate orthodontic tooth movement. Pattern recognition receptors such as toll-like receptors (TLRs) have been well characterized for their ability to induce the activation of inflammatory, immunomodulatory cytokines. Here, we examined whether the cellular response of human periodontal ligament (hPDL) cells to mechanical stress involves TLR-4 signaling in vitro. MATERIALS AND METHODS: Confluent hPDL cells were cultured in the presence of 5 µg/ml TLR-4 antibody (TLR-4ab) for 1 h prior to the induction of compressive forces by the use of round glass plates for 24 h. At harvest, interleukin-6 and interleukin-8 (IL-6, IL-8) mRNA and protein expression were analyzed by real-time PCR and ELISA. The immunomodulatory role of mechanical cell stress and TLR-4 signaling was addressed in co-culture experiments of hPDL and THP-1 cells targeting monocyte adhesion and by culturing osteoclastic precursors (RAW 264.7) in the presence of the conditioned medium of hPDL cells that had been mechanically loaded before. RESULTS: Basal expression of IL-6 and IL-8 was not affected by TLR-4ab, but increased significantly upon mechanical loading of hPDL cells. When cells were mechanically stressed in the presence of TLR-4ab, the effect seen for loading alone was markedly reduced. Likewise, monocyte adhesion and osteoclastic differentiation were enhanced significantly by mechanical stress of hPDL cells and this effect was partially inhibited by TLR-4ab. CONCLUSIONS: The results of the present study indicate a proinflammatory and immunomodulatory influence of mechanical loading on hPDL cells. Intracellular signaling involves a TLR-4-dependent pathway. CLINICAL RELEVANCE: These findings hold out the prospect of interfering with the cellular response to mechanical cell stress in order to minimize undesired side effects of orthodontic tooth movement.

Bone and soft tissue palatal morphology and potential anchorage sides in cleft palate patients.

AIM: The aim of this study was to evaluate palatal vertical bone thickness and density in relation to soft tissue on the hard palate for better selection of adequate bone regions for the insertion of orthodontic mini-implants (MIs) in cleft palate patients. MATERIALS AND METHODS: Cone beam computed tomography scans (CBCT) were obtained from 60 patients (mean age range 9-12). The study population included patients with isolate right side cleft palate formation (n = 20; 6 females; 14 males), left side cleft palate formation (n = 20; 9 females; 11 males) and without cleft formation as control group (n = 20; 15 females; 5 males). Bone and soft tissue measurements were performed vertical at a 90° angle to the bone surface, on previously defined measurement points (n = 88) on the hard palate. Bone density was measured on ten vertical layers in caudo-cranial direction. RESULTS: In non-cleft patient the highest bone thickness was in the anterior palate and decreased significantly in posterior direction. In patients with right and left cleft palate, the highest vertical bone level could be observed at the palatal premaxillary border opposite to the cleft side. Patients in the control group showed a significantly lower vertical soft tissue thickness than patients with palatal cleft formation. The evaluation of bone density showed no significant differences in all three groups. CONCLUSION: The results suggest that the favorable region for orthodontic MI placement is in the similar anatomical region compared to non-cleft patients, but differs from one side in each group. In unilateral cleft palate patients, the highest bone level was found on the anterior palate side opposite to the cleft side, indicating the most effective region for MIs placement.