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Objectives Saliva can support oral wound healing, a process that requires a temporary inflammatory reaction. We have reported previously that saliva provokes a strong inflammatory response in oral fibroblasts. Bone marrow cells also give rise to macrophages, a heterogeneous subset of cell population involved in wound healing. Lipopolysaccharide (LPS) and interleukin 4 (IL-4) induce activation of pro-(M1), and anti-(M2) inflammatory macrophages, respectively. Yet, the impact of saliva on programming bone marrow cells into either M1 or M2 macrophages remains unclear . Design Herein, we examined whether sterile saliva affects the in vitro process of macrophage polarization based on murine bone marrow cultures and RAW264.7 mouse macrophages. Results We report that sterile saliva, similar to lipopolysaccharides, provoked a robust activation of the M1 phenotype which is characterized by a strong increase of the respective genes IL-12 and IL-6, based on a real-time gene expression analysis, and for IL-6 with immunoassay. Arginase-1 and Ym1, both genes characteristic for the M2 phenotype, were not considerably modulated by saliva. Inhibition of TLR4 signaling with TAK-242, blocking NFκB signaling with Bay 11-7085, but also autoclaving saliva greatly reduced the development of the M1 phenotype. Conclusion These data suggest that saliva activates the TLR4 dependent polarization into pro-inflammatory M1 macrophages in vitro.

Objective Milk is a rich source of transforming growth factor (TGF)-β which supports intestinal mucosal homeostasis of infants. Milk may also have beneficial effects on the integrity of the oral cavity, its being part of the gastrointestinal tract. However, it is unclear if milk and fermented milk products provoke a TGF-β response in oral cells. Material and Methods Human gingival fibroblasts were exposed to pasteurized cow milk, yoghurt, sour milk, buttermilk and whey, followed by a reverse transcriptase polymerase chain reaction of the TGF-β target genes interleukin11 (IL11), proteoglycan4 (PRG4), and NADPH oxidase 4 (NOX4). Immunoassays were performed for IL11 and TGF-β in cell culture supernatant and milk products, respectively. Signaling was investigated with the TGF-β receptor type I kinase inhibitor SB431542. Results We report here that pasteurized cow milk and the aqueous fractions of yoghurt, sour milk, buttermilk and whey enhanced the expression of IL11, NOX4 and PRG4 in gingival fibroblasts. Moreover, IL11 protein levels in the respective supernatant were significantly increased. Cow milk, yoghurt, sour milk and buttermilk contain approximately 1–2 ng TGF-β1, whereas active TGF-β1 is hardly detectable in whey. SB431542 reduced the response of gingival fibroblasts to pasteurized cow milk and fermented milk products based on IL11 release into the supernatant. Conclusions These results demonstrate that gingival fibroblasts respond to pasteurized cow milk and to fermented milk products with an increased expression of TGF-β target genes.

OBJECTIVE Prolyl hydroxylases (PHD) are oxygen sensors and therefore pharmacological targets to stimulate periodontal regeneration. Here we evaluate the release profile of the PHD inhibitors dimethyloxaloylglycine and l-mimosine from bone substitutes. MATERIALS Dimethyloxaloylglycine and l-mimosine were lyophilised onto bone substitutes including bovine bone mineral, beta-tricalcium phosphate, and hydroxyapatite. Release kinetic was evaluated by bioassays with gingival and periodontal ligament fibroblasts. We determined the capacity of PHD inhibitors to provoke VEGF expression and to repress metabolic activity and proliferation as assessed by immunoassay, MTT conversion and (3)[H]thymidine incorporation, respectively. RESULTS We found that the PHD inhibitors are released from bovine bone mineral as indicated by the increase of VEGF production in gingival and periodontal ligament fibroblasts. Supernatants obtained after 1h also decreased metabolic activity and proliferation of the fibroblasts. A fibrin matrix prolonged the release of PHD inhibitors up to 192h. A similar cellular response was found when supernatants from PHD inhibitors loaded beta-tricalcium phosphate and hydroxyapatite embedded in fibrin were assessed. CONCLUSIONS In conclusion bone substitutes can serve as carriers for PHD inhibitors that maintain their capacity to provoke a pro-angiogenic response in vitro. These findings provide the basis for preclinical studies to evaluate if this release kinetic can stimulate periodontal regeneration.

Objective Chronic inflammation in periodontal disease is associated with increased plasminogen activation and elevated levels of chemokines. It is unknown whether chemokines can regulate the activation of plasminogen via modulation of plasminogen activators (PA) and the corresponding plasminogen activator inhibitors (PAI) in periodontal tissue. Design To establish a link between chemokines and activation of plasminogen, human periodontal ligament fibroblasts (PDL) and gingival fibroblasts (GF) were incubated with IL-8, monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and platelet factor-4, either alone or in the presence of the inflammatory mediators TGF-β and IL-1. The potential of the cell lysates to activate plasminogen was based on kinetic studies with the substrate casein. Casein zymography was performed to determine the molecular sizes of the PA. Total PAI-1 in the cell-conditioned medium was quantified by immunoassay. Results We report that the chemokines did not affect activation of plasminogen by PDL and GF. Even in the presence of TGF-β which suppressed, and IL-1 which stimulated plasminogen activation, the chemokines had no direct effect. Inhibition of PA and plasmin, but not of matrix metalloproteinases and cysteine proteinases prevented caseinolysis. The plasminogen activation capacity of the cell lysates was represented by a single band with features of uPA. The immunoassay showed that the release of PAI-1 in PDL and GF remained unaffected by the chemokines, also when stimulated with TGF-β. Conclusions These results suggest that plasminogen activation by PDL and GF is not directly affected by the chemokines even in the presence of the inflammatory mediators TGF-β and IL-1.

Objective Catecholamines released from β-adrenergic neurons upon stress can interfere with periodontal regeneration. The cellular mechanisms, however, are unclear. Here, we assessed the effect of catecholamines on proliferation of periodontal fibroblasts. Methods Fibroblasts from the gingiva and the periodontal ligament were exposed to agonists of the β-adrenergic receptors; isoproterenol (ISO, non-selective β-adrenergic agonist), salbutamol (SAL, selective β2-adrenergic receptor agonist) and BRL 37344 (BRL selective β3-receptor agonist). Proliferation was stimulated with platelet-derived growth factor-BB (PDGF-BB). Pharmacological inhibitors and gene expression analysis further revealed β-adrenergic signalling. Results Gingiva and periodontal ligament fibroblast express the β2-adrenergic receptor. ISO and SAL but not BRL decreased proliferation of fibroblasts in the presence of PDGF-BB. The inhibitory effect of β-adrenergic signalling on proliferation but not protein synthesis in response to PDGF-BB was reduced by propranolol, a non-selective β-adrenergic antagonist. Conclusions These results suggest that β2-receptor agonists can reduce the mitogenic response of periodontal fibroblasts. These data add to the compelling concept that blocking of β2-receptor signalling can support tissue maintenance and regeneration.

Objective Ageing, hypogonadism, malnutrition, and the application of glucocorticoids have adverse effects on skeletal homeostasis. Herein we determined to which extent the periodontium undergoes catabolic changes under these conditions in a sheep model. Methods Six old sheep with a mean age of 7.5 ± 1.0 years were subjected to ovariectomy, calcium/vitamin D-restricted diet, and intramuscular administration of approximately 2 g methylprednisolone. Six adult sheep with a mean age of 3.8 ± 0.9 years remained untreated and served as controls. First and second premolars of both jaws were subjected to histological analysis. The distances from the gingival margin (GM) and from the alveolar bone crest (ABC) to the cemento-enamel junction (CEJ) were determined. Periodontal attachment was given as the ratio between the dimension of the periodontal ligament and the alveolar bone. Clinical data were collected by counting the number of teeth missing, teeth with gingival recession, and teeth with a probing depth > 4 mm. Results We report that distance between GM and CEJ (2.1 ± 1.7 mm and 6.6 ± 2.6 mm maxilla; −0.4 ± 1.4 mm and 3.2 ± 1.5 mm mandible), and between ABC and CEJ (−3.4 ± 1.3 mm and 1.8 ± 2.7 mm maxilla; −3.5 ± 1.1 mm and −0.1 ± 1.4 mm mandible) are significantly lower in test than in control animals. In line with these findings, periodontal attachment was 67% in the maxilla and 86% in the mandible of the test group and almost completely preserved in the control group. Clinical evaluation showed that the overall number of teeth with recessions was significantly higher in the test compared to the control group (4.9 ± 2.4 and 2.3 ± 3.6), but not the number of teeth missing and teeth with a probing depth > 4 mm. Conclusions Together these findings suggest that in sheep, the cumulating effects of ageing, hypogonadism, malnutrition and glucocorticoid application can cause substantial catabolic changes of the periodontium.