The role of sclerostin and dickkopf-1 in oral tissues – A review from the perspective of the dental disciplines [version 1; peer review: 2 approved]

Wnt signaling is of high relevance in the development, homeostasis, and regeneration of oral tissues. Therefore, Wnt signaling is considered to be a potential target for therapeutic strategies. The action of Wnt is tightly controlled by the inhibitors sclerostin (SOST) and Dickkopf (DKK)-1. Given the impact of SOST and DKK-1 in hard tissue formation, related diseases and healing, it is of high relevance to understand their role in oral tissues. The clinical relevance of this knowledge is further underlined by systemic and local approaches which are currently in development for treating a variety of diseases such as osteoporosis and inflammatory hard tissue resorption. In this narrative review, we summarize the current knowledge and understanding on the Wnt signaling inhibitors SOST and DKK-1, and their role in physiology, pathology, and regeneration in oral tissues. We present this role from the perspective of the different specialties in dentistry, including endodontics, orthodontics, periodontics, and oral surgery

Contraction Dynamics of Rod Microtissues of Gingiva-Derived and Periodontal Ligament-Derived Cells

Tissue engineering strategies using microtissues as “building blocks” have high potential in regenerative medicine. Cognition of contraction dynamics involved in the in vitro self-assembly of these microtissues can be conceived as the bedrock of an effective periodontal tissue regenerative therapy. Our study was directed at evaluating the shrinkage in the rod-shaped structure of a directed self-assembly of human gingiva-derived cells (GC) and periodontal ligament-derived cells (PDLC) and developing insights into the potential mechanisms responsible for the shrinkage. GC and PDLC were seeded in non-adherent agarose molds to form rod microtissues. Cells used for the experiments were characterized using fluorescence-activated cell sorting (FACS). To assess the viability, resazurin-based cytotoxicity assays, trypan blue dye exclusion assay, MTT and live/dead staining, and histological evaluation of rods based on hematoxylin and eosin staining were performed. Rod contraction was evaluated and measured at 0, 2, 6, and 24 h and compared to L-929 cells. The role of transforming growth factor (TGF)-β signaling, phosphoinositide 3-kinase (PI3K)/AKT, and mitogen activated protein kinase (MAPK) signaling was analyzed. Our results show that the rod microtissues were vital after 24 h. A reduction in the length of rods was seen in the 24 h period. While the recombinant TGF-β slightly reduced contraction, inhibition of TGF-β signaling did not interfere with the contraction of the rods. Interestingly, inhibition of phosphoinositide 3-kinase by LY294002 significantly delayed contraction in GC and PDLC rods. Overall, GC and PDLC have the ability to form rod microtissues which contract over time. Thus, approaches for application of these structures as “building blocks” for periodontal tissue regeneration should consider that rods have the capacity to contract substantially. Further investigation will be needed to unravel the mechanisms behind the dynamics of contraction

Oral tissue regeneration : insights from in vitro models

Biomaterialien, Wachstumsfaktoren und Pharmazeutika werden in der Zahnheilkunde zur Förderung der Geweberegeneration eingesetzt. Neue Behandlungsstrategien nutzen Trägermaterialien wie Carboxymethylcellulose (CMC) und Hydroxypropylmethylcellulose (HPMC), sowie Kollagenmembranen. CMC und HPMC können sowohl für granuläres Knochenersatzmaterial als auch für Wachstumsfaktoren und Pharmazeutika als Trägermaterial dienen. Auch Kollagenmembranen können für Wachstumsfaktoren wie Platelet-derived growth factor (PDGF)-BB und Pharmazeutika wie Bisphosphonate als Trägermaterial fungieren. Derzeit sind aber sowohl die der Regeneration des oralen Gewebes zugrunde liegenden Mechanismen, als auch der Einfluss der Biomaterialien noch unklar. Im Zuge dieser Arbeit testeten wir die genannten Materialien und Pharmazeutika in vitro. Wir untersuchten die Wirkung von CMC, HPMC und der Kollagenmembran Bio-Gide® (Geistlich Biomaterials) auf die Osteoblasto- and Osteoklastogenese. Um die Rolle von aktivierten Thrombozyten in der frühen Phase der Gewebsregeneration zu klären, untersuchten wir den Effekt von Thrombozyten auf mesenchymale Vorläuferzellen sowohl in Normoxie und als auch unter Hypoxie. Um den Effekt des Wachstumsfaktors PDGF-BB und Entzündung auf das orale Weichgewebe zu beleuchten, testeten wir den Effekt von PDGF-BB und Chemokinen auf die katabole Aktivität von parodontalen Fibroblasten. Weiters stellten wir die Frage nach der Wirkung von Bisphosphonaten auf orales Weichgewebe. Um diese Frage zu klären, prüften wir den Einfluss von gelöstem und gebundenem Zoledronat auf parodontale Fibroblasten. Zusammengefasst zeigen unsere in vitro Daten, dass die der Regeneration von oralem Gewebe zugrundeliegenden Mechanismen durch die hier untersuchten Biomaterialien, Wachstumsfaktoren und Pharmazeutika unterschiedlich moduliert werden können.In dentistry, biomaterials, growth factors, and pharmaceutical components are applied to stimulate oral tissue regeneration. New treatment strategies demand the use of carrier materials such as carboxymethylcellulose (CMC), hydroxypropylmethylcellulose (HPMC), and collagen barrier membranes (CBMs). CMC and HPMC can be used together with granular bone substitute materials, growth factors such as platelet-derived growth factor (PDGF)-BB, and pharmacological compounds. CBMs can be coated with growth factors and pharmaceutical components such as bisphosphonates. However, the cellular mechanism of oral tissue regeneration, the impacts of the carriers and pharmaceutical components are not fully understood. In this thesis we evaluated the effect of CMC, HPMC, and a CBM (Bio-Gide®, Geistlich Biomaterials) on osteoblasto- and osteoclastogenesis in vitro. To understand the impact of platelets in the early phase of tissue regeneration we assessed their effect on mesenchymal progenitor cells under normoxic and hypoxic conditions. To understand the impact of PDGF-BB and inflammatory conditions on the oral soft tissues we evaluated the effect of PDGF-BB and chemokines on the catabolic activity of periodontal fibroblasts. To understand the response of the oral soft tissue to the bisphoshonate zoledronate we evaluated the effects of periodontal fibroblasts to soluble and zoledronate bound to calciumphosphate. In summery our in vitro findings suggest that the evaluated biomaterials, growth factors, and pharmaceutical components can differentially modulate the cellular mechanisms involved in oral tissue regeneration.submitted by Hermann AgisZsfassung in dt. SpracheWien, Med. Univ., Diss., 2010OeBB(VLID)171509

Chronodentistry: the role & potential of molecular clocks in oral medicine

Abstract Molecular clocks help organisms to adapt important physiological functions to periodically changing conditions in the environment. These include the adaption of the 24 h sleep-wake rhythm to changes of day and night. The circadian clock is known to act as a key regulator in processes of health and disease in different organs. The knowledge on the circadian clock led to the development of chronopharmacology and chronotherapy. These fields aim to investigate how efficiency of medication and therapies can be improved based on circadian clock mechanisms. In this review we aim to highlight the role of the circadian clock in oral tissues and its potential in the different fields of dentistry including oral and maxillofacial surgery, restorative dentistry, endodontics, periodontics and orthodontics to trigger the evolving field of chronodentistry

L-mimosine and dimethyloxaloylglycine decrease plasminogen activation in periodontal fibroblasts

BACKGROUND The use of prolyl hydroxylase inhibitors such as l-mimosine (L-MIM) and dimethyloxaloylglycine (DMOG) to improve angiogenesis is a new approach for periodontal regeneration. In addition to exhibiting pro-angiogenic effects, prolyl hydroxylase inhibitors can modulate the plasminogen activator system in cells from non-oral tissues. This study assesses the effect of prolyl hydroxylase inhibitors on plasminogen activation by fibroblasts from the periodontium. METHODS Gingival and periodontal ligament fibroblasts were incubated with L-MIM and DMOG. To investigate whether prolyl hydroxylase inhibitors modulate the net plasminogen activation, kinetic assays were performed with and without interleukin (IL)-1. Moreover, plasminogen activators and the respective inhibitors were analyzed by casein zymography, immune assays, and quantitative polymerase chain reaction. RESULTS The kinetic assay showed that L-MIM and DMOG reduced plasminogen activation under basal and IL-1-stimulated conditions. Casein zymography revealed that the effect of L-MIM involves a decrease in urokinase-type plasminogen activator activity. In agreement with these findings, reduced levels of urokinase-type plasminogen activator and elevated levels of plasminogen activator inhibitor 1 were observed. CONCLUSION L-MIM and DMOG can reduce plasminogen activation by fibroblasts from the gingiva and the periodontal ligament under basal conditions and in the presence of an inflammatory cytokine

Bone substitute materials supplemented with prolyl hydroxylase inhibitors decrease osteoclastogenesis in vitro.

BACKGROUND AND OBJECTIVE Inhibition of prolyl hydroxylases stimulates bone regeneration. Consequently, bone substitute materials were developed that release prolyl hydroxylase inhibitors. However, the impact of prolyl hydroxylase inhibitors released from these carriers on osteoclastogenesis is not clear. We therefore assessed the effect of bone substitute materials that release prolyl hydroxylase inhibitors on osteoclastogenesis. MATERIAL AND METHODS Dimethyloxalylglycine, desferrioxamine, and l-mimosine were lyophilized onto bovine bone mineral and hydroxyapatite, and supernatants were generated. Osteoclastogenesis was induced in murine bone marrow cultures in the presence of the supernatants from bone substitute materials. The formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP activity were determined. To test for possible effects on osteoclast progenitor cells, we measured the effect of the supernatants on proliferation and viability. In addition, experiments were performed where prolyl hydroxylase inhibitors were directly added to the bone marrow cultures. RESULTS We found that prolyl hydroxylase inhibitors released within the first hours from bone substitute materials reduce the number and activity of TRAP-positive multinucleated cells. In line with this, addition of prolyl hydroxylase inhibitors directly to the bone marrow cultures dose-dependently reduced the number of TRAP-positive multinucleated cells and the overall resorption activity. Moreover, the released prolyl hydroxylase inhibitors decreased proliferation but not viability of osteoclast progenitor cells. CONCLUSION Our results show that prolyl hydroxylase inhibitors released from bone substitute materials decrease osteoclastogenesis in murine bone marrow cultures

L-mimosine and hypoxia can increase angiogenin production in dental pulp-derived cells

Abstract Background Angiogenin is a key molecule in the healing process which has been successfully applied in the field of regenerative medicine. The role of angiogenin in dental pulp regeneration is unclear. Here we aimed to reveal the impact of the hypoxia mimetic agent L-mimosine (L-MIM) and hypoxia on angiogenin in the dental pulp. Methods Human dental pulp-derived cells (DPC) were cultured in monolayer and spheroid cultures and treated with L-MIM or hypoxia. In addition, tooth slice organ cultures were applied to mimic the pulp-dentin complex. We measured angiogenin mRNA and protein levels using qPCR and ELISA, respectively. Inhibitor studies with echinomycin were performed to reveal the role of hypoxia-inducible factor (HIF)-1 signaling. Results Both, L-MIM and hypoxia increased the production of angiogenin at the protein level in monolayer cultures of DPC, while the increase at the mRNA level did not reach the level of significance. The increase of angiogenin in response to treatment with L-MIM or hypoxia was reduced by echinomycin. In spheroid cultures, L-MIM increased angiogenin at protein levels while the effect of hypoxia was not significant. Angiogenin was also expressed and released in tooth slice organ cultures under normoxic and hypoxic conditions and in the presence of L-MIM. Conclusions L-MIM and hypoxia modulate production of angiogenin via HIF-1 differentially and the response depends on the culture model. Given the role of angiogenin in regeneration the here presented results are of high relevance for pre-conditioning approaches for cell therapy and tissue engineering in the field of regenerative endodontics

Beta2-adrenergic receptor agonists reduce proliferation but not protein synthesis of periodontal fibroblasts stimulated with platelet-derived growth factor-BB

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