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

Microtissues as a tool for the evaluation of cellular responses in oral tissue regeneration

Einleitung: Die Komplexität eines dreidimensionalen (3D) in vivo Gewebes kann mit üblichen in vitro Zellkulturen nicht nachgestellt werden. Die Entwicklung von Therapeutika sowie Biomaterialien für regenerative Anwendungen im oralen Tissue Engineering ist daher auf Tierversuche angewiesen. Diese sind ethisch bedenklich, teuer, aufwendig und vor allem keine sichere Quelle für zu erwartende Ergebnisse im menschlichen Patienten. Mikrogewebe als in vitro Kultur versuchen die Lücke zwischen präklinischen und klinischen Studien zu schließen. Unter der Hypothese, dass 3D Zellkulturen ein Modell für in vivo Gewebe sein können, war es Ziel dieser Arbeit Mikrogewebe für die Zahnpulpa und das Parodont zu entwickeln, die der Struktur und Funktionalität des menschlichen oralen Gewebes nachbilden können. Material & Methoden: Zellen aus der humanen Zahnpulpa und dem Parodont wurden in einschichtigen, Sphäroid, Toroid und Zahnscheibenmodellen kultiviert. Zellantworten auf Hypoxie, kleine Moleküle und Biomaterialien wurden getestet. Analysen zur metabolischen Aktivität wurden mit MTT Test, Resazurin basierendem Toxizitätstest und Live-Dead Färbung, Proliferation mit BrdU Test und Ki67 Färbung, Apoptose mit Caspase 3 Färbung, Morphologie mit Hämatoxylin-Eosin Färbung, Adhärenz mit Fluoreszenzfärbung, Gen- und Proteinproduktion mit qPCR sowie ELISA und Western blot durchgeführt. Ergebnisse: Verschiedene Arten von Mikrogeweben konnten mit allen oralen Zellen unter unterschiedlichen Sauerstoffbedingungen gebildet werden. Proliferations- und Apoptoseaktivität unterschieden sich in Lage und Anzahl der Zellen in den verschiedenen Kulturmodellen. Individuelle Morphologien konnten hinsichtlich Form und Verteilung der Zellen in den jeweiligen Kulturmodellen gezeigt werden. Die stärkste Adhärenz wurde in einschichtigen Zellkulturen, die schwächste in Sphäroidzellkulturen nachgewiesen, wobei der Prozess der Adhärenz mit der Zeit Unterschiede zwischen den verschiedenen Modellen zeigte. Hinsichtlich Zellantworten auf Hypoxie, kleine Moleküle und Biomaterialien wurden unterschiedliche Aktivitäten in der mRNA und Protenproduktion von Markern für extrazelluläre Matrix, Entzündung und Angiogenese, je nach Zellkultumodell, gemessen. Conclusio: Jede wissenschaftliche Fragestellung erfordert ein individuell angepasstes Zellkulturmodell, um klinisch relevante Daten produzieren zu können. Bei der Auswahl des Modelles muss festgelegt werden welche Anordnungen und Funktionen untersucht werden sollen und wie diese dem Originalgewebe nachgebaut werden können. Neben der Dreidimensionalität müssen Zellkulturmodelle für das Tissue Engineering in der oralen Regeneration in Zukunft Morphologie und Funktionen des Originalgewebes nachahmen können.Introduction: Traditional in vitro cell cultures fail to reproduce the three-dimensional (3D) complexity of an in vivo tissue. Therefore, therapeutic and biomaterial development for oral tissue engineering and regeneration still requires animal testing, which implies ethical considerations, high costs and, most of all, delivers data that is not always reproducible in human patients. Microtissue cultures attempt to close the in vitro gap between pre-clinical and clinical testing. Hypothesizing that 3D cell culture could mimic oral tissues in vitro, this thesis aims to establish microtissue cultures for dental pulp and periodontal tissues and test their suitability to reflect structure and functionality of respective human patient tissue. Material & Methods: Human dental pulp and periodontal cells were cultured in monolayers, spheroids, toroids and tooth slice models. Cellular responses to hypoxia, small molecules and biomaterials were tested. Oral cells in different culture models were analyzed for metabolic activity by MTT assays, resazurin-based toxicity assays and Live-Dead staining, proliferation by BrdU assay and Ki67 staining, apoptosis by Caspase 3 staining, morphology by hematoxylin-eosin staining, attachment by fluorescence microscopy, gene and protein production by qPCR as well as ELISA and Western blot, respectively. Results: All oral cells were able to form different viable microtissues under normoxia and hypoxia. The location and amount of proliferative and apoptotic cells differed between the culture models. Morphology was individual in each culture model concerning the shape and organization of cells. Attachment was most robust in oral cell monolayers and weakest in oral cell spheroids. Culture models showed different attachment and growth behavior over time. Responsiveness of oral cells to hypoxia, small molecules and biomaterials was different in each culture model regarding mRNA and protein markers for extracellular matrix production, pro-inflammatory and pro-angiogenic activity. Conclusion: In conclusion, each research question requires an individualized cell culture model to deliver clinically relevant data. Depending on the study's aim, the choice of a suitable in vitro system should consider which constructs and functions will be assessed and how to mimic them such that they reflect an original tissue. In future, microtissues and other cell culture systems will have to be developed incorporating features of the original tissue besides three-dimensionality as a pre-clinical source for the development of tissue engineering approaches for oral regeneration.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersArbeit an der Bibliothek noch nicht eingelangt - Daten nicht geprüftMedizinische Universität Wien, Diss., 2020(VLID)491656

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

Common Ground between Biological Rhythms and Forensics

Biological clocks set the timing for a large number of essential processes in the living human organism. After death, scientific evidence is required in forensic investigations in order to collect as much information as possible on the death circumstances and personal identifiers of the deceased victim. We summarize the associations between the molecular mechanisms of biological rhythms and forensically relevant aspects, including post-mortem interval and cause of death, entomological findings, sex, age, ethnicity and development. Given their importance during lifetime, biological rhythms could be potential tools to draw conclusions on the death circumstances and the identity of a deceased person by mechanistic investigations of the different biological clocks in a forensic context. This review puts the known effects of biological rhythms on the functions of the human organism in context with potential applications in forensic fields of interest, such as personal identification, entomology as well as the determination of the post-mortem interval and cause of death

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

European Journal of Oral Sciences / Core circadian clock gene expression in human dental pulpderived cells in response to Lmimosine, 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 pulpderived cells (DPC) and if their expression is modulated by the hypoxia mimetic agent, Lmimosine (LMIM), hypoxia or echinomycin. Dental pulpderived cells in monolayers and spheroids were treated with LMIM, 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 LMIM, 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.(VLID)481807

Do hypoxia and L-mimosine modulate sclerostin and dickkopf-1 production in human dental pulp-derived cells? Insights from monolayer, spheroid and tooth slice cultures

Abstract Background To understand the responses of the dental pulp to hypoxia is of high relevance for regenerative endodontics and dental traumatology. Here, we aimed to reveal the effects of hypoxia and the hypoxia mimetic agent L-mimosine (L-MIM) on the production of sclerostin (SOST) and dickkopf-1 (DKK-1) in human dental pulp-derived cells (DPC). Methods DPC in monolayer, spheroid and tooth slice cultures were treated with L-MIM or hypoxia. Resazurin-based toxicity and MTT assays were performed to determine cell viability. mRNA and protein levels of SOST and DKK-1 were measured with quantitative reverse transcription PCR and ELISA, respectively. To validate the hypoxia-like response, SDF-1, VEGF and IL-8 were assessed. In addition Western blots for HIF-1α, HIF-2α and HIF-3α were done. Results Cells were vital upon treatment procedures and showed increased levels of HIF-1α, and HIF-2α. In monolayer cultures, mRNA levels of SOST and DKK-1 were downregulated by L-MIM and hypoxia, respectively. A significant downregulation of SOST by hypoxia was found at the protein level compared to untreated cells while the effect on DKK-1 and the impact of L-MIM on SOST and DKK-1 did not reach the level of significance at the protein level. In spheroid cultures, mRNA levels of SOST and DKK-1 were downregulated by L-MIM. A significant downregulation of DKK-1 upon hypoxia treatment was found at the protein level while the impact of hypoxia on SOST and the effect of L-MIM on SOST and DKK-1 did not reach the level of significance. SOST and DKK-1 were also produced in tooth slices, but no pronounced modulation by L-MIM or hypoxia was found. Evaluation of SDF-1, VEGF and IL-8 showed a hypoxia-like response in the culture models. Conclusions There is no pronounced influence of hypoxia and L-MIM on DPC viability, SOST and DKK-1 protein production. However, the specific response depends on the culture model and the level of evaluation (mRNA or protein). These results deepen our understanding about the role of hypoxia and the potential impacts of hypoxia-based strategies on dental pulp

The impact of collagen membranes on 3D gingival fibroblast toroids

Abstract Background Development in guided tissue regeneration requires biomaterial testing. 3D cell constructs represent a new approach to bridge the gap between cell culture and animal models. Following the hypothesis that attachment behavior of cells could be observed in toroidal 3D cell constructs, the aim of this study was to evaluate 3D gingival fibroblast (GF) toroids as a simple and feasible in vitro assay to test attachment of oral fibroblasts to collagen membranes. Methods 3D ring-like structures (toroids) were formed from human GF. Hematoxylin-eosin staining was performed with formed GF toroids. Produced GF toroids were seeded onto plastic surfaces or collagen membranes. The morphology was documented at 24 h, 48 h and 72 h after seeding with light and fluorescence microscopy. Toroid vitality was assessed at same time points with a resazurin-based toxicity assay. Results GF showed normal morphology in toroid hematoxylin-eosin staining. Over 72 h, GF toroids on plastic surfaces stayed unchanged, while GF toroids on collagen membranes showed dilatation. GF toroids on plastic surfaces and collagen membranes were metabolically active over the observed period. Conclusions Depending on the surface material, 3D GF toroids show different attachment behavior. Thus, GF toroids are suitable as simple assay to study attachment behavior to various biomaterials