Medical 3D printing with polyjet technology: effect of material type and printing orientation on printability, surface structure and cytotoxicity.

Due to its high printing resolution and ability to print multiple materials simultaneously, inkjet technology has found wide application in medicine. However, the biological safety of 3D-printed objects is not always guaranteed due to residues of uncured resins or support materials and must therefore be verified. The aim of this study was to evaluate the quality of standard assessment methods for determining the quality and properties of polyjet-printed scaffolds in terms of their dimensional accuracy, surface topography, and cytotoxic potential.Standardized 3D-printed samples were produced in two printing orientations (horizontal or vertical). Printing accuracy and surface roughness was assessed by size measurements, VR-5200 3D optical profilometer dimensional analysis, and scanning electron microscopy. Cytotoxicity tests were performed with a representative cell line (L929) in a comparative laboratory study. Individual experiments were performed with primary cells from clinically relevant tissues and with a Toxdent cytotoxicity assay.Dimensional measurements of printed discs indicated high print accuracy and reproducibility. Print accuracy was highest when specimens were printed in horizontal direction. In all cytotoxicity tests, the estimated mean cell viability was well above 70% (p < 0.0001) regardless of material and printing direction, confirming the low cytotoxicity of the final 3D-printed objects.

Differential gene expression and protein-protein interaction networks of human periodontal ligament stromal cells under mechanical tension.

Orthodontic treatment is based on complex strategies and takes up to years until a desired therapeutic outcome is accomplished, implying long periods of high costs and discomfort for the patient. Choosing the optimal settings for force intensities in the initial phase of orthodontic tooth movement is the key to successful orthodontic treatment. It is known that orthodontic tooth movement is mainly mediated by tensile and compressive forces that are communicated to the alveolar bone via the periodontal ligament. While the revelation of the complex molecular network was already approached by transcriptomic analysis of compressed periodontal ligament cells, the entity of molecular key players activated by tensile forces remains elusive. Therefore, the aim of this study was to assess the effect of mechanical tensile forces on the gene expression profile of human primary periodontal ligament stromal cells, mimicking the initial phase of orthodontic tooth movement. A transcriptomic analysis of tension-treated and untreated periodontal ligament stromal cells yielded 543 upregulated and 793 downregulated differentially expressed genes. Finally, six highly significant genes were found in the transcriptome that are related to biological processes with relevance to orthodontic tooth movement, including apelin, fibroblast growth factor receptor 2, noggin, sulfatase 1, secreted frizzled-related protein 4 and stanniocalcin 1. Additionally, differences of gene expression profiles between individual cell donors showed a high effect size. Closer understanding of the roles of the identified candidates in the initial phase of orthodontic tooth movement could help to clarify the underlying mechanisms, which will be essential for the development of personalized treatment strategies in orthodontics.

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.

Effects of collagen membranes and bone substitute differ in periodontal ligament cell microtissues and monolayers.

BACKGROUND: Barrier membranes and bone substitute are major tools of guided tissue regeneration (GTR) after periodontal disease. Integrity of the periodontal ligament plays a key role in periodontal health, but its functionality fails to be fully re-established by GTR after disease or trauma. Microtissue models suggest an in vivo-like model to develop novel GTR approaches due to its three-dimensionality. This study aims to assess the effects of collagen membranes and bone substitute on cell viability, adhesion and gene expression of regenerative and inflammatory biomarkers by periodontal ligament cell (PDLC) microtissues. METHODS: Human PDLC microtissues and monolayers were cultured on collagen membranes or bone substitute. After 24 hours incubation, metabolic activity, focal adhesion, mRNA and protein production of collagen-type-I (COL1A1), periostin (POSTN), vascular endothelial growth factor (VEGF), angiogenin (ANG), interleukin (IL)6 and IL8 were measured by resazurin-based toxicity assay, focal adhesion staining, quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: PDLC microtissues and monolayers were viable on collagen membranes and bone substitute, but microtissues were less metabolically active. Dominant staining of actin filaments was found in PDLC microtissues on collagen membranes. COL1A1, POSTN, VEGF, ANG and IL6 were modulated in PDLC microtissues on bone substitute, while there were no significant changes on collagen membranes. PDLC monolayers showed a different character of gene expression changes. CONCLUSIONS: PDLC microtissues and monolayers react diversely to collagen membranes and bone substitute. Further descriptive and mechanistic tests will be required to clarify the potential of PDLC microtissues as in vivo-like model for GTR.

A Novel Quantitative Method for Tooth Grinding Surface Assessment Using 3D Scanning.

Sleep bruxism is an oral parafunction that involves involuntary tooth grinding and clenching. Splints with a colored layer that gets removed during tooth grinding are a common tool for the initial diagnosis of sleep bruxism. Currently, such splints are either assessed qualitatively or using 2D photographs, leading to a non-neglectable error due to the 3D nature of the dentition. In this study we propose a new and fast method for the quantitative assessment of tooth grinding surfaces using 3D scanning and mesh processing. We assessed our diagnostic method by producing 18 standardized splints with 8 grinding surfaces each, giving us a total of 144 surfaces. Moreover, each splint was scanned and analyzed five times. The accuracy and repeatability of our method was assessed by computing the intraclass correlation coefficient (ICC) as well reporting means and standard deviations of surface measurements for intra- and intersplint measurements. An ICC of 0.998 was computed as well as a maximum standard deviation of 0.63 mm2 for repeated measures, suggesting an appropriate accuracy of our proposed method. Overall, this study proposes an innovative, fast and cost effective method to support the initial diagnosis of sleep bruxism.

The impact of 3D-printed LAY-FOMM 40 and LAY-FOMM 60 on L929 cells and human oral fibroblasts.

OBJECTIVES: LAY-FOMM is a promising material for FDA-approved Fused Deposition Modeling (FDM) applications in drug delivery. Here we investigated the impact on oral cells. MATERIALS AND METHODS: We evaluated the impact of 3D-printed LAY-FOMM 40, LAY-FOMM 60, and biocompatible polylactic acid (PLA) on the activity of murine L929 cells, gingival fibroblasts (GF), and periodontal ligament fibroblasts (PDLF) using indirect (samples on cells), direct monolayer culture models (cells on samples), and direct spheroid cultures with resazurin-based toxicity assay, confirmed by MTT and Live-dead staining. The surface topography was evaluated with scanning electron microscopy. RESULTS: The materials LAY-FOMM 40 and LAY-FOMM 60 led to a reduction in resazurin conversion in L929 cells, GF, and PDLF, higher than the impact of PLA in indirect and direct culture models. Fewer vital cells were found in the presence of LAY-FOMM 40 and 60 than PLA, in the staining in both models. In the direct model, LAY-FOMM 40 and PLA showed less impact on viability in the resazurin-based toxicity assay than in the indirect model. Spheroid microtissues showed a reduction of cell activity of GF and PDLF with LAY-FOMM 40 and 60. CONCLUSION: Overall, we found that LAY-FOMM 40 and LAY-FOMM 60 can reduce the activity of L292 and oral cells. Based on the results from the PLA samples, the direct model seems more reliable than the indirect model. CLINICAL RELEVANCE: A material modification is desired in terms of biocompatibility as it can mask the effect of drugs and interfere with the function of the 3D-printed device.

The response of gingiva monolayer, spheroid, and ex vivo tissue cultures to collagen membranes and bone substitute.

Collagen membranes and bone substitute are popular biomaterials in guided tissue regeneration for treatment of traumatized or diseased periodontal tissue. Development of these biomaterials starts in monolayer cell culture, failing to reflect in vivo tissue organization. Spheroid cultures potentially mimic in vivo tissues in structure and functionality. This study aims to compare gingiva cell (GC) monolayers and spheroids to ex vivo gingiva. Human GC monolayers, spheroids and gingiva ex vivo tissues were cultured on plastic surfaces, collagen membranes or bone substitute. Hematoxylin-eosin (HE) staining, immunohistochemistry for KI67 and caspase 3 (CASP3), resazurin-based toxicity assays, quantitative polymerase chain reaction for collagen I (COL1A1), vascular endothelial growth factor (VEGF), angiogenin (ANG), interleukin (IL)6 and IL8 and ELISA for COL1A1, VEGF, ANG, IL6 and IL8 were performed in all cultures. Morphology was different in all culture set-ups. Staining of KI67 was positive in monolayers and staining of CASP3 was positive in spheroids. All culture set-ups were viable. COL1A1 production was modulated in monolayers and ex vivo tissues at mRNA levels, VEGF in monolayers and ex vivo tissues at mRNA levels and in spheroids at protein levels, ANG in spheroids at mRNA levels and in monolayers and spheroids at protein levels, IL6 in monolayers and spheroids at mRNA levels and in spheroids and ex vivo tissues at protein levels and IL8 in monolayers and ex vivo tissues at mRNA levels. Modulations were surface-dependent. In conclusion, each culture model is structurally and functionally different. Neither GC monolayers nor spheroids mimicked gingiva ex vivo tissue in all measured aspects.

The response of periodontal cells to kaolinite.

OBJECTIVES: The impact of kaolinite on human periodontal cells is yet unknown. The aim of the study was to assess the response of human periodontal cells to kaolinite. METHODS: Human periodontal cells were treated with kaolinite at reducing concentrations from 30 to 0.0015 mg/mL and with conditioned medium, which was depleted of kaolinite. Cell viability was evaluated with a resazurin-based toxicity assay, Live-Dead staining, and MTT assay and staining. The pro-angiogenic factors vascular endothelial growth factor (VEGF) and interleukin (IL)-6 and IL-8 were quantified via ELISA in periodontal fibroblasts. L-929, a standard cell-line used for cytotoxicity studies, served as control cell line. Composition of kaolinite was verified using energy-dispersive X-ray spectroscopy. RESULTS: Kaolinite in suspension but not in conditioned medium impaired cell viability dose-dependently. VEGF, IL-6, and IL-8 production was not substantially modulated by kaolinite or the conditioned medium in periodontal cells. CONCLUSION: Overall, kaolinite can decrease cell viability dose-dependently while conditioned medium showed no toxic effect. No pronounced impact of kaolinite on VEGF, IL-6, and IL-8 production was observed. This study provided first insights into the impact of kaolinite on human periodontal cells thereby inferring to the basis for the evaluation of kaolinite as a carrier in regenerative dentistry. CLINICAL RELEVANCE: Kaolinite, a clay mineral, is successfully used in medicine due to its favorable properties. Also, applications in conservative dentistry are described. However, the response of oral cells to kaolinite is still unclear. Here, we assessed the impact of kaolinite on human periodontal cells.

Contraction dynamics of dental pulp cell rod microtissues.

OBJECTIVES: The factors that contribute to the morphological changes of dental pulp cell-derived microtissues are unknown. Here, we investigated the contraction dynamics of rod-shaped microtissues derived from dental pulp cells and examined the underlying cell signaling pathways. METHODS: Human dental pulp cells were seeded into agarose molds to assemble into rod-shaped microtissues. Resazurin- and tetrazolium-based cytotoxicity assays, Live/Dead staining, and hematoxylin and eosin staining for histological evaluation of rods were performed. Rod contraction was evaluated and measured for a period of 10 days. The role of TGF-ß, phosphoinositide 3-kinase (PI3K)/AKT, and mitogen-activated protein kinase (MAPK) signaling pathway was analyzed. RESULTS: Dental pulp cells readily assembled into rods, maintaining the geometric shape for 48 h. Following this period, they condensed to form stable spheroidal structures that remained vital for 10 days from seeding. Inhibition of phosphoinositide 3-kinase signaling pathway by LY294002 significantly prolonged the diminution in the length of rods formed by dental pulp cells. TGF-ß and pharmacological inhibition of TGF-ß signaling did not show pronounced effects. CONCLUSION: Overall, dental pulp cells readily formed rod-shaped patterns of microtissues which, over a period of time, condensed into more stable spheroidal structures. Hence, technologies like bioprinting, using direct fabrication of microtissues need to consider the contraction dynamics. CLINICAL RELEVANCE: The field of regenerative endodontology will benefit from our findings as it can be applied as a novel platform to test the impact of pharmacological agents, biomaterials, and regenerative approaches including bioprinting.

The role of sclerostin and dickkopf-1 in oral tissues - A review from the perspective of the dental disciplines.

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.

The impact of collagen membranes on 3D gingival fibroblast toroids.

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.

Angiopoietin-like 4 production upon treatment with hypoxia and L-mimosine in periodontal fibroblasts.

BACKGROUND AND OBJECTIVE: A key factor in the modulation of angiogenesis as well as in bone resorption is angiopoietin-like 4. However, the role of angiopoietin-like 4 in periodontal tissue is unknown. Here, we hypothesized that hypoxia and the hypoxia mimetic agent L-mimosine can induce the production of angiopoietin-like 4 in periodontal fibroblasts. METHODS: Human periodontal ligament fibroblasts (PDLF) were cultured in monolayer and spheroid cultures. The cultures were incubated in the presence of hypoxia or L-mimosine. Angiopoietin-like 4 mRNA and protein levels were measured by qPCR and ELISA, respectively. Also, the impact of Lipopolysaccharides of E. coli and P. gingivalis, interleukin (IL)-1ß and tumor necrosis factor (TNF)α was evaluated. Furthermore, we tested dependency on hypoxia-inducible factor (HIF)-1 activity by Western blotting for HIF-1 and inhibitor studies with echinomycin. Potential autocrine effects were assessed by exposure of PDLF to recombinant angiopoietin-like 4 in full length, C-terminal and N-terminal fragments. The impact on viability, DNA synthesis, alkaline phosphatase, and matrix mineralization was evaluated. RESULTS: Both hypoxia and L-mimosine elevated angiopoietin-like 4 mRNA and protein levels in monolayer cultures of PDLF. HIF-1 was elevated after both hypoxia and L-mimosine treatment. LPS, IL-1ß, and TNFα did not modulate angiopoietin-like 4 levels significantly. Addition of echinomycin in the cultures inhibited the production of angiopoietin-like 4. In spheroid cultures of PDLF, the increase did not reach the level of significance at mRNA and protein levels. Angiopoietin-like 4 in full length, C-terminal, and N-terminal fragments did not modulate viability, DNA synthesis, alkaline phosphatase, and matrix mineralization. CONCLUSION: Overall, we found that hypoxia and the hypoxia mimetic agent L-mimosine can stimulate angiopoietin-like 4 production in monolayer cultures of PDLF. This increase depends on HIF-1 activity. Future studies will reveal how the modulation of angiopoietin-like 4 in the periodontium contributes to periodontal disease and regeneration.

The impact of clay-based hypoxia mimetic hydrogel on human fibroblasts of the periodontal soft tissue.

Thixotropic clays have favorable properties for tissue regeneration. Hypoxia mimetic agents showed promising results in pre-clinical models for hard and soft tissue regeneration. It is unclear if clays can be used as carrier for hypoxia mimetic agent in a periodontal regenerative setting. Here, we tested the response of human fibroblasts of the periodontal soft tissue to synthetic clay hydrogels and assessed hypoxia mimetic agent release. Cells were cultured on synthetic clay hydrogels (5.00%-0.15%). We assessed viability and differentiation capacity with resazurin-based toxicity assays, MTT staining, Live-Dead staining, and alkaline phosphatase staining. To reveal the response of fibroblasts to hypoxia mimetic agent-loaded clay hydrogels, cells were exposed to clay supplemented with dimethyloxalylglycine, deferoxamine, l-mimosine, and CoCl2. Supernatants from hypoxia mimetic agent-loaded clay hydrogels were harvested and replaced with medium at hour 1, 3, 6, 24, 48, and 72. To reveal the hypoxia mimetic capacity of supernatants, vascular endothelial growth factor production in the fibroblasts was assessed in the culture medium. Our data show that clay did not induce relevant toxic effects in the fibroblasts which remained capable to differentiate into alkaline phosphatase-positive cells at the relevant concentrations. Fibroblasts cultured on clay hydrogel loaded with dimethyloxalylglycine, deferoxamine, l-mimosine, and CoCl2 remained vital, however, no significant increase in vascular endothelial growth factor levels was found in the culture medium. Only dimethyloxalylglycine-loaded clay supernatants taken in the first hours stimulated vascular endothelial growth factor production in fibroblasts. In conclusion no pronounced toxic effects of synthetic clay were observed. Supplementation with dimethyloxalylglycine leads to hypoxia mimetic activity. This pilot study provides first insights into the impact of synthetic clay on periodontal tissue.

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

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.

Angiogenin production in response to hypoxia and l-mimosine in periodontal fibroblasts.

BACKGROUND: A major mediator of angiogenesis is angiogenin, which is expressed in the early phase of healing in oral tissue engineering strategies. It is unclear how angiogenin is regulated in the periodontal tissue. The objective of this study was to reveal the regulation of angiogenin in response to hypoxia and the hypoxia mimetic agent l-mimosine in periodontal fibroblasts. METHODS: Human fibroblasts of the periodontal ligament (PDLF) and the gingiva (GF) in monolayer and spheroid cultures were exposed to hypoxia or l-mimosine. The production of angiogenin was evaluated at mRNA and protein levels with reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. Echinomycin, an inhibitor of hypoxia-inducible factor (HIF)-1 activity, was used to test the involvement of HIF-1. RESULTS: Our data show that hypoxia and l-mimosine can increase angiogenin mRNA and protein levels in PDLF monolayer cultures. In GF monolayer cultures, we found an increase of angiogenin at the mRNA level in response to hypoxia. The increase of angiogenin can be blocked by inhibition of HIF-1 signaling via echinomycin. In PDLF and GF spheroid cultures, the impact of hypoxia and l-mimosine did not reach the level of significance. CONCLUSION: Hypoxia and the hypoxia mimetic agent l-mimosine can increase the production of angiogenin via HIF-1 signaling in PDLF monolayer cultures but not in spheroid cultures. GF were less sensitive to the impact of hypoxia and l-mimosine. Overall, these results suggest a link between hypoxia, HIF-1 signaling and angiogenin in the periodontium.

The Influence of Pro-Inflammatory Factors on Sclerostin and Dickkopf-1 Production in Human Dental Pulp Cells Under Hypoxic Conditions.

Sclerostin (Sost) and dickkopf (Dkk)-1 are inhibitors of the Wnt signaling pathway that plays a role in regenerative processes. Hypoxia-based strategies are used for regenerative approaches, but the influence of hypoxia on Sost and Dkk-1 production in a pro-inflammatory environment is unclear. The aim of this study was to assess if pro-inflammatory molecules have an influence on Sost and Dkk-1 production in dental pulp cells (DPC) under normoxia and hypoxia. Human DPC were treated with interleukin (IL)-1ß, tumor necrosis factor (TNF)α or transforming growth factor (TGF)ß, with L-mimosine (L-MIM) or hypoxia or a combination. Sost and Dkk-1 mRNA and protein levels were measured with qPCR and western blot, respectively. TNFα, TGFß, L-MIM, or combined treatment did not modulate Sost and Dkk-1. IL-1ß downregulated Sost at the mRNA level. Hypoxia alone and together with inflammatory markers downregulated Dkk-1 at the mRNA level. Sost and Dkk-1 protein production was below the detection limit. In conclusion, there is a differential effect of hypoxia and IL-1ß on the mRNA production of Sost and Dkk-1. Pro-inflammatory molecules do not further modulate the effects of L-MIM or hypoxia on Sost and Dkk-1 production in DPC.

3D Printing-Encompassing the Facets of Dentistry.

This narrative review presents an overview on the currently available 3D printing technologies and their utilization in experimental, clinical and educational facets, from the perspective of different specialties of dentistry, including oral and maxillofacial surgery, orthodontics, endodontics, prosthodontics, and periodontics. It covers research and innovation, treatment modalities, education and training, employing the rapidly developing 3D printing process. Research-oriented advancement in 3D printing in dentistry is witnessed by the rising number of publications on this topic. Visualization of treatment outcomes makes it a promising clinical tool. Educational programs utilizing 3D-printed models stimulate training of dental skills in students and trainees. 3D printing has enormous potential to ameliorate oral health care in research, clinical treatment, and education in dentistry.

Deferoxamine but Not Dimethyloxalylglycine, L-Mimosine, or Cobalt Dichloride Can Interfere with the MTT Assay.

Hypoxia mimetic agents (HMAs) have been shown to have a positive influence on cellular functions in a multitude of tissue regenerative strategies. Novel experimental approaches use biomaterials as carriers for controlled delivery of these HMAs. Here, the cytotoxic aspects of biocompatibility are of key relevance. The MTT assay is widely used to evaluate cytotoxicity and proliferation. Based on the implications from the proceeding research we hypothesized that specific HMAs such as deferoxamine at high concentrations can interfere with the MTT assay. Thus, the aim of this study was to test the repercussions of the HMAs dimethyloxalylglycine, deferoxamine, L-mimosine, and CoCl2 on the validity of the MTT assay. Murine MC3T3-E1 cells were cultured in serum-free alphaMEM and in alphaMEM supplemented with 10 % fetal bovine serum with the HMAs dimethyloxalylglycine, deferoxamine, L-mimosine, and CoCl2, respectively, at 3 mM-0.3 mM for 24 h (experimental groups). Cells without HMAs served as control (control groups). The same experiments were performed with medium and phosphate buffered saline (PBS) without cells. In all settings MTT solution was added to PBS-washed or unwashed culture plates for the last two hours of the incubation period. Then MTT solution was removed and dimethyl sulfoxide was added to dissolve the formazan crystals and absorption was measured. Our data show that the presence of deferoxamine can interfere with the MTT assay if not removed before the addition of MTT. This is particularly important when evaluating cell viability in setups where deferoxamine-loaded biomaterials are used.

Core circadian clock gene expression in human dental pulp-derived cells in response to L-mimosine, 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 pulp-derived cells (DPC) and if their expression is modulated by the hypoxia mimetic agent, L-mimosine (L-MIM), hypoxia or echinomycin. Dental pulp-derived cells in monolayers and spheroids were treated with L-MIM, 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 L-MIM, 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.

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.