Klara Janjić

Effects of Prolyl Hydroxylase Inhibitor L-mimosine on Dental Pulp in the Presence of Advanced Glycation End Products.

INTRODUCTION Proangiogenic prolyl hydroxylase (PHD) inhibitors represent a novel approach to stimulate tissue regeneration. Diabetes mellitus involves the accumulation of advanced glycation end products (AGEs). Here we evaluated the impact of AGEs on the response of human pulp tissue to the PHD inhibitor L-mimosine (L-MIM) in monolayer cultures of dental pulp-derived cells (DPCs) and tooth slice organ cultures. METHODS In monolayer cultures, DPCs were incubated with L-MIM and AGEs. Viability was assessed based on formazan formation, live-dead staining, annexin V/propidium iodide, and trypan blue exclusion assay. Vascular endothelial growth factor (VEGF), interleukin (IL)-6, and IL-8 production was evaluated by quantitative polymerase chain reaction and immunoassays. Furthermore, expression levels of odontoblast markers were assessed, and alizarin red staining was performed. Tooth slice organ cultures were performed, and VEGF, IL-6, and IL8 levels in their supernatants were measured by immunoassays. Pulp tissue vitality and morphology were assessed by MTT assay and histology. RESULTS In monolayer cultures of DPCs, L-MIM at nontoxic concentrations increased the production of VEGF and IL-8 in the presence of AGEs. Stimulation with L-MIM decreased alkaline phosphatase levels and matrix mineralization also in the presence of AGEs, whereas no significant changes in dentin matrix protein 1 and dentin sialophosphoprotein expression were observed. In tooth slice organ cultures, L-MIM increased VEGF but not IL-6 and IL-8 production in the presence of AGEs. The pulp tissue was vital, and no signs of apoptosis or necrosis were observed. CONCLUSIONS Overall, in the presence of AGEs, L-MIM increases the proangiogenic capacity, but decreases alkaline phosphatase expression and matrix mineralization.

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

BackgroundAngiogenin 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.MethodsHuman 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.ResultsBoth, 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.ConclusionsL-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.

Collagen barrier membranes do not adsorb hypoxia mimetic activity‐Activity of gingival fibroblasts cultured directly on collagen barrier membranes loaded with hypoxia mimetic agents

Hypoxia-based strategies for applications in oral surgery and periodontology have been proposed where collagen barrier membranes (CBM) are loaded with hypoxia mimetic agents (HMA) to induce a pro-angiogenic response. While it was found that CBM release HMA, it remained unclear if CBM adsorb HMA activity. Here we evaluated the response of oral cells cultured on CBM, supplemented with the HMA dimethyloxalylglycine (DMOG), desferrioxamine (DFO), and l-mimosine (l-MIM). Gingival fibroblasts (GF) were cultured on unwashed CBM as well as on CBM that had been washed with serum-free medium for 48 hours. The pro-angiogenic response was measured based on vascular endothelial growth factor (VEGF) production. Viability and proliferation were assessed based on MTT and BrdU assays. We found that GF seeded onto CBM loaded with DFO and l-MIM, but not DMOG, showed an increase in VEGF to 6.1-fold and 7.7-fold compared to unloaded CBM, respectively. Cells remained vital, but a trend for decreased proliferation was observed on DMOG and DFO-loaded CBM which did not reach the level of significance. Evaluation of washed CBM revealed no difference between the unloaded CBM and CBM supplemented with DMOG, DFO, or l-MIM. In conclusion, our results suggest that CBM do not adsorb hypoxia mimetic activity but release HMA within the first hours.

L-mimosine and hypoxia enhance angiopoietin-like 4 production involving hypoxia-inducible factor-1alpha: Insights from monolayer and spheroid cultures of dental pulp-derived cells and tooth slice cultures

OBJECTIVE Angiopoietin-like 4 (Angptl4) is an angiogenesis modulating signaling factor and as such involved in blood vessel formation but also in hard tissue resorption. Here we hypothesized that the hypoxia mimetic agent L-mimosine (L-MIM) and hypoxia stimulate the production of Angptl4 in the dental pulp. MATERIAL AND METHODS Monolayer and spheroid cultures of primary human dental pulp-derived cells (DPC) were treated with L-MIM or hypoxia. Furthermore, tooth slice cultures were performed. The production of Angptl4 was assessed at mRNA and protein levels using reverse transcription qPCR and immunoassays, respectively. To assess the involvement of hypoxia inducible factor (HIF)-1α (HIF-1signaling, inhibitor studies with echinomycin and Western Blot analysis for HIF-1α were performed in DPC monolayer cultures.(HIF-1 RESULTS: L-MIM and hypoxia increased production of Angptl4 at mRNA and protein levels in monolayer cultures of DPC. The increase of Angptl4 was paralleled by an increase of HIF-1α and inhibited by echinomycin. Angptl4 protein levels were also elevated in spheroid cultures. In tooth slice cultures, the pulp tissue expressed and released Angptl4 under normoxic and hypoxic conditions and in the presence of L-MIM. There was a trend for an increase in Angptl4 mRNA levels and a trend for a decrease in the protein levels of the supernatants. CONCLUSIONS Our results suggest that the hypoxia mimetic agent L-MIM and hypoxia can increase Angptl4 production in DPC involving HIF-1α. However, the increase in the cell culture supernatants does not translate in an increased release in tooth slice organ cultures.

Hypoxia-based strategies for regenerative dentistry—Views from the different dental fields

The understanding of the cell biological processes underlying development and regeneration of oral tissues leads to novel regenerative approaches. Over the past years, knowledge on key roles of the hypoxia-based response has become more profound. Based on these findings, novel regenerative approaches for dentistry are emerging, which target cellular oxygen sensors. These approaches include hypoxia pre-conditioning and pharmacologically simulated hypoxia. The increase in studies on hypoxia and hypoxia-based strategies in regenerative dentistry highlights the growing attention to hypoxias role in regeneration and its underlying biology, as well as its application in a therapeutic setting. In this narrative review, we present the current knowledge on the role of hypoxia in oral tissues and review the proposed hypoxia-based approaches in different fields of dentistry, including endodontics, orthodontics, periodontics, and oral surgery.

Relevance of the plasminogen system in physiology, pathology, and regeneration of oral tissues – From the perspective of dental specialties

Plasmin is a proteolytic enzyme that is crucial in fibrinolysis. In oral tissues, the plasminogen system plays an essential role in physiological and pathological processes, which in addition to fibrinolysis include degradation of extracellular matrix, inflammation, immune response, angiogenesis, tissue remodeling, cell migration, and wound healing. Oral tissues reveal a change in the plasminogen system during pathological processes such as periodontitis, peri-implantitis, or pulpitis, as well as in response to mechanical load. The plasminogen system is also a key element in tissue regeneration. The number of studies investigating the plasminogen system in dentistry have grown continuously in recent years, highlighting its increasing relevance in dental medicine. In this review, we present the diverse functions of the plasminogen system in physiology and its importance for dental specialists in pathology and regeneration. We thus provide an overview of the current knowledge on the role of the plasminogen system in the different fields of dentistry, including endodontics, orthodontics, periodontics, and oral surgery.

Expression of circadian core clock genes in fibroblasts of human gingiva and periodontal ligament is modulated by L-Mimosine and hypoxia in monolayer and spheroid cultures

OBJECTIVE The circadian clock is involved in a plethora of physiological processes including bone formation and tooth development. While expression of circadian core clock genes was observed in various tissues, their role in the periodontium is unclear. We hypothesized that periodontal cells express circadian core clock genes and that their levels are modulated by hypoxia mimetic agents and hypoxia. MATERIAL AND METHODS Fibroblasts of human gingiva (GF) and periodontal ligament (PDLF) in monolayer and spheroid cultures were treated with the hypoxia mimetic agent L-Mimosine (L-MIM) or hypoxia. Reverse transcription and quantitative PCR were performed to assess the impact on mRNA levels of the circadian core clock genes Clock, Bmal1, Cry1, Cry2, Per1, Per2, and Per3. RESULTS GF and PDLF expressed Clock, Bmal1, Cry1, Cry2, Per1, Per2, and Per3 in monolayer and spheroid cultures. In monolayer cultures, L-MIM significantly reduced Clock, Cry2, and Per3 mRNA expression in GF and Clock, Cry1, Cry2, Per1, and Per3 in PDLF. Hypoxia significantly reduced Clock, Cry2, and Per3 in GF and Cry1, Cry2, and Per3 in PDLF. In spheroid cultures, L-MIM significantly decreased Clock, Cry1, Cry2, and Per3 in GF and PDLF. Hypoxia significantly decreased Cry2 and Per3 in GF and Clock and Per3 in PDLF. CONCLUSIONS GF and PDLF express circadian core clock genes. The hypoxia mimetic agent L-MIM and hypoxic conditions can decrease the expression of Clock, Cry1-2 and Per1 and Per3. The specific response depends on cell type and culture model. Future studies will show how this effect contributes to periodontal health and disease.

Effect of prolyl hydroxylase inhibitor-loaded collagen barrier membranes on osteoclastogenesis and osteoblastogenesis

Prolyl hydroxylase inhibitors induce a proangiogenic response and are therefore proposed to optimize regenerative approaches in periodontics and oral surgery. Here the effect of the prolyl hydroxylase inhibitors dimethyloxalylglycine and deferoxamine, released from collagen barrier membranes, on osteoclastogenesis and osteoblastogenesis was evaluated. Collagen barrier membranes were loaded with dimethyloxalylglycine and deferoxamine. Release studies were performed and supernatants were taken after 1, 3, 6, 24, and 48 h. The effect of these supernatants on osteoblast- and osteoclast-precursor cells was evaluated. Furthermore, dose response studies for dimethyloxalylglycine and deferoxamine were performed. Osteoclastogenesis was evaluated with RAW 264.7 cells based on the number of multinuclear tartrate-resistant acid phosphatase positive cells. Osteoblastogenesis was evaluated with MC3T3-E1 cells based on alkaline phosphatase. Metabolic activity and cell proliferation were assessed based on MTT and BrdU assays. Vascular endothelial growth factor production was evaluated using an immunoassay. We found that supernatants taken in the first hour from collagen barrier membranes loaded with dimethyloxalylglycine or deferoxamine reduced osteoclastogenesis. Osteoblastogenesis was not reduced significantly. Cell proliferation and metabolic activity of RAW 264.7 and MC3T3-E1 cells were inhibited by supernatants of collagen barrier membranes loaded with deferoxamine but not dimethyloxalylglycine. In RAW 264.7 cell culture, vascular endothelial growth factor production was increased only by supernatants of collagen barrier membranes loaded with dimethyloxalylglycine, but not deferoxamine. In MC3T3-E1 cell culture, supernatants of collagen barrier membranes loaded with dimethyloxalylglycine and deferoxamine both increased vascular endothelial growth factor production. Direct measurements showed that the majority of dimethyloxalylglycine and deferoxamine is released in the first hours. Dose-response studies supported the divergent effects of deferoxamine and dimethyloxalylglycine in RAW 264.7 and MC3T3-E1 cultures. Our findings show diverse effects of dimethyloxalylglycine- and deferoxamine-loaded collagen barrier membranes during osteoclastogenesis and osteoblastogenesis. Preclinical studies will reveal if the increase in vascular endothelial growth factor together with the inhibitory effect on osteoclasts can stimulate oral tissue regeneration.

Evaluation of Resins for Stereolithographic 3D-Printed Surgical Guides: The Response of L929 Cells and Human Gingival Fibroblasts

Additive manufacturing is becoming increasingly important in dentistry for the production of surgical guides. The development of cost-effective desktop stereolithography (SLA) printing systems and the corresponding resins makes this novel technique accessible to dental offices and dental laboratories. The aim of the study was to reveal the response of soft tissue cells to Clear and Dental SG resins used in desktop SLA printing systems at different stages of processing. Cell activity of L929 cells and gingival fibroblasts (GF) in response to the materials was examined in indirect and direct monolayer culture models and a direct spheroid culture model based on MTT, resazurin-based toxicity assays, and live-dead staining. Overall we found that the impact of Clear and Dental SG resins on L929 and GF depends on the processing stage of the materials. Liquid Clear resin induced a stronger reduction of cell activity compared to Dental SG resin. Printing and postcuring reduced the impact on cell activity and viability. As in-house 3D printing for surgical guides is getting integrated in the digital workflow, our data suggest that careful adherence to processing guidelines—especially postcuring—is of clinical relevance.

Synthetic Clay–based Hypoxia Mimetic Hydrogel for Pulp Regeneration: The Impact on Cell Activity and Release Kinetics Based on Dental Pulp–derived Cells In Vitro

Introduction: Thixotropic synthetic clays have been successfully used for tissue engineering in regenerative medicine. The impact of these clays on the dental pulp, in particular in combination with hypoxia‐based approaches using hypoxia mimetic agents (HMAs), is unknown. Our aim was to reveal the response of dental pulp–derived cells (DPCs) to a synthetic clay–based hydrogel and evaluate the release of HMAs. Methods: Using resazurin‐based toxicity assays, live‐dead staining, and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide staining, the viability of human DPCs seeded onto a synthetic clay–based hydrogel of 5%–0.15% as well as onto the hydrogels loaded with the HMAs dimethyloxalylglycine (DMOG), desferrioxamine, L‐mimosine, and CoCl2 was evaluated. Furthermore, supernatant of the hydrogels loaded with HMAs were generated. Vascular endothelial growth factor (VEGF) production of DPCs in response to the supernatant was measured to reveal the cellular response to the HMAs. Results: We found that the synthetic clay–based hydrogel did not impair the viability of DPCs. Cell monolayer and cell cluster formations were observed on the hydrogel. No significant increase of VEGF levels was observed in the supernatant when DPCs were cultured on hydrogels loaded with HMAs. Supernatant of DMOG‐loaded hydrogels stimulated VEGF production in DPCs in the first hour, whereas the effect of desferrioxamine, L‐mimosine, and CoCl2 did not reach a level of significance. Conclusions: The synthetic clay–based hydrogel represents a promising biomaterial that does not induce prominent toxic effects in DPCs. It can be loaded with DMOG to induce hypoxia mimetic activity. Overall, we provided first insights into the impact of synthetic clays on DPCs for tissue engineering purposes in regenerative endodontics.