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.

Which substances loaded onto collagen scaffolds influence oral tissue regeneration?-an overview of the last 15 years.

BACKGROUND: Collagen scaffolds are widely used for guided bone or tissue regeneration. Aiming to enhance their regenerative properties, studies have loaded various substances onto these scaffolds. This review aims to provide an overview of existing literature which conducted in vitro, in vivo, and clinical testing of drug-loaded collagen scaffolds and analyze their outcome of promoting oral regeneration. MATERIALS AND METHODS: PubMed, Scopus, and Ovid Medline® were systematically searched for publications from 2005 to 2019. Journal articles assessing the effect of substances on oral hard or soft tissue regeneration, while using collagen carriers, were screened and qualitatively analyzed. Studies were grouped according to their used substance type-biological medical products, pharmaceuticals, and tissue-, cell-, and matrix-derived products. RESULTS: A total of 77 publications, applying 36 different substances, were included. Collagen scaffolds were demonstrating favorable adsorption behavior and release kinetics which could even be modified. BMP-2 was investigated most frequently, showing positive effects on oral tissue regeneration. BMP-9 showed comparable results at lower concentrations. Also, FGF2 enhanced bone and periodontal healing. Antibiotics improved the scaffold's anti-microbial activity and reduced the penetrability for bacteria. CONCLUSION: Growth factors showed promising results for oral tissue regeneration, while other substances were investigated less frequently. Found effects of investigated substances as well as adsorption and release properties of collagen scaffolds should be considered for further investigation. CLINICAL RELEVANCE: Collagen scaffolds are reliable carriers for any of the applied substances. BMP-2, BMP-9, and FGF2 showed enhanced bone and periodontal healing. Antibiotics improved anti-microbial properties of the scaffolds.

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 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.

In vitro evaluation of experimental light activated gels for tooth bleaching.

Dental bleaching is an important part of aesthetic dentistry. Various strategies have been created to enhance the bleaching efficacy. As one such strategy, light-activated nanoparticles that enable localized generation of reactive oxygen species have been developed. Here, we evaluated the cellular response to experimental gels containing these materials in in vitro models. L-929 cells, 3T3 cells, and gingival fibroblasts were exposed to the gels at 50%, 10%, 2%, 0.4%, 0.08%, 0.016%, and 0.0032%. The gels contained TiO2/Ag nanoparticles, TiO2 nanoparticles, hydrogen peroxide (6% hydrogen peroxide), or no added component and were tested with and without exposure to light. Cells were exposed to gels for 24 h or for 30 min. The latter case mimics the clinical situation of a short bleaching gel exposure. Metabolic activity and cell viability were evaluated with MTT and neutral red assays, respectively. We found a dose-dependent reduction of formazan formation and neutral red staining with gels containing TiO2/Ag nanoparticles or TiO2 nanoparticles in the 24 h setting with and without illumination. The strongest reduction, which was not dose-dependent in the evaluated concentrations, was found for the gel containing hydrogen peroxide. Gels with TiO2 nanoparticles showed a similar response to gel without particles. TiO2/Ag gel showed a slightly higher impact. When the gels were removed by rinsing after 30 min of exposure without light illumination, gel containing TiO2/Ag nanoparticles showed a stronger reduction of formazan formation and neutral red staining than gel containing TiO2 particles. Exposure of cells for 30 min under illumination and consequent rinsing off the gels also showed that Ag-containing particles can have a higher impact on the metabolic activity and viability than particles from TiO2. Overall our results show that experimental bleaching gels containing TiO2/Ag or TiO2 nanoparticles are less cytotoxic than hydrogen peroxide-containing gel. When gels are removed, gel containing TiO2/Ag particles exhibit a stronger reduction of metabolic activity and viability than the gel containing TiO2.

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.

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.

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.

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.

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.

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.

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

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.

Response of human dental pulp cells to a silver-containing PLGA/TCP-nanofabric as a potential antibacterial regenerative pulp-capping material.

BACKGROUND: Damage or exposure of the dental pulp requires immediate therapeutic intervention. METHODS: This study assessed the biocompatibility of a silver-containing PLGA/TCP-nanofabric scaffold (PLGA/Ag-TCP) in two in vitro models, i.e. the material adapted on pre-cultured cells and cells directly cultured on the material, respectively. Collagen saffolds with and without hyaluronan acid (Coll-HA; Coll) using both cell culturing methods and cells growing on culture plates served as reference. Cell viability and proliferation were assessed after 24, 48, and 72 h based on formazan formation and BrdU incorporation. Scaffolds were harvested. Gene expression of interleukin(IL)-6, tumor necrosis factor (TNF)-alpha, and alkaline phosphatase (AP) was assessed 24 h after stimulation. RESULTS: In both models formazan formation and BrdU incorporation was reduced by PLGA/Ag-TCP on dental pulp cells, while no significant reduction was found in cells with Coll and Coll-HA. Cells with PLGA/Ag-TCP for 72 h showed similar relative BrdU incorporation than cells stimulated with Coll and Coll-HA. A prominent increase in the pro-inflammatory genes IL-6 and TNF-α was observed when cells were cultured with PLGA/Ag-TCP compared to the other groups. This increase was parallel with a slight increase in AP expression. Overall, no differences between the two culture methods were observed. CONCLUSIONS: PLGA/Ag-TCP decreased viability and proliferation rate of human dental pulp cells and increased the pro-inflammatory capacity and alkaline phosphatase expression. Whether these cellular responses observed in vitro translate into pulp regeneration in vivo will be assessed in further studies.

Release kinetics and mitogenic capacity of collagen barrier membranes supplemented with secretome of activated platelets - the in vitro response of fibroblasts of the periodontal ligament and the gingiva.

BACKGROUND: Platelet preparations can stimulate the healing process and have mitogenic properties. We hypothesized that collagen barrier membranes (CBM), clinically used in guided bone regeneration and guided tissue regeneration, can serve as carriers for platelet secretome. METHODS: Secretome was generated from washed platelets and unwashed platelets (washed/unwashed PSEC) and lyophilized onto CBM. Overall appearance of CBM was evaluated by scanning electron microscopy. The impact of PSEC on cell attachment was measured based on fluorescence microscopy with DiI-labeled cells. To assess the release kinetics, supernatants of CBM were collected and medium was replaced at hour 1-48. The mitogenic effect was evaluated with periodontal fibroblasts. Furthermore, the release of total protein, platelet-derived growth factor (PDGF)-BB, and transforming growth factor (TGF) ß1 was measured. RESULTS: CBM overall appearance and cell attachment was not modulated by PSEC. Supernatants taken after one hour induced a mitogenic response in fibroblasts and showed the highest levels of total protein, TGFß1 and PDGF-BB. These effects decreased rapidly in subsequent supernatants. While supernatants of CBM loaded with unwashed PSEC induced a stronger mitogenic response than supernatants of CBM loaded with washed PSEC this difference between the PSEC preparations was not observed when cells were seeded on 48-hours-washed CBM. CONCLUSIONS: CBM release platelet-derived factors in continuously declining release kinetics.

Alveolar bone regeneration in response to local application of calcitriol in vitamin D deficient rats.

AIM: Vitamin D deficiency is considered to diminish bone regeneration. Yet, raising the serum levels takes months. A topic application of the active vitamin D metabolite, calcitriol, may be an effective approach. Thus, it becomes important to know the effect of vitamin D deficiency and local application on alveolar bone regeneration. MATERIAL AND METHODS: Sixty rats were divided into three groups; two vitamin depletion groups and a control group. Identical single defects (2 mm diameter) were created in the maxilla and mandible treated with calcitriol soaked collagen in one deficiency group while in the other two groups not. Histomorphometric analysis and micro CTs were performed after 1 and 3 weeks. Serum levels of 25(OH)D3 and PTH were determined. RESULTS: Bone formation rate significantly increased within the observation period in all groups. Bone regeneration was higher in the maxilla than in the mandible. However, bone regeneration was lower in the control group compared to vitamin depletion groups, with no significant effects by local administration of calcitriol (micro CT mandible p = 0.003, maxilla p < 0.001; histomorphometry maxilla p = 0.035, mandible p = 0.18). CONCLUSION: Vitamin D deficiency not necessarily impairs bone regeneration in the rat jaw and a single local calcitriol application does not enhance healing.

In vitro adhesion of fibroblastic cells to titanium alloy discs treated with sodium hydroxide.

OBJECTIVE: Adhesion of osteogenic cells on titanium surfaces is a prerequisite for osseointegration. Alkali treatment can increase the hydrophilicity of titanium implant surfaces, thereby supporting the adhesion of blood components. However, it is unclear if alkali treatment also supports the adhesion of cells with a fibroblastic morphology to titanium. MATERIALS AND METHODS: Here, we have used a titanium alloy (Ti-6AL-4V) processed by alkali treatment to demonstrate the impact of hydrophilicity on the adhesion of primary human gingival fibroblast and bone cells. Also included were the osteosarcoma and fibroblastoma cell lines, MG63 and L929, respectively. Cell adhesion was determined by scanning electron microscopy. We also measured viability, proliferation, and protein synthesis of the adherent cells. RESULTS: Alkali treatment increased the adhesion of gingival fibroblasts, bone cells, and the two cell lines when seeded onto the titanium alloy surface for 1 h. At 3 h, no significant changes in cell adhesion were observed. Cells grown for 1 day on the titanium alloy surfaces processed by alkali treatment behave similarly to untreated controls with regard to viability, proliferation, and protein synthesis. CONCLUSION: Based on these preliminary In vitro findings, we conclude that alkali treatment can support the early adhesion of cells with fibroblastic characteristics to a titanium alloy surface.

Dimethyloxalylglycine lyophilized onto bone substitutes increase vessel area in rat calvarial defects.

AIM: Pharmacological inhibitors of prolyl hydroxylases, also termed hypoxia-mimetic agents (HMAs), when repeatedly injected can support angiogenesis and bone regeneration. However, the possible role of HMA loaded onto bone substitutes to support angiogenesis and bone regeneration under diabetic condition is unknown. The capacity of HMA loaded onto deproteinized bovine bone mineral (DBBM) to support angiogenesis and bone formation was examined in diabetic Wistar rats. METHODS: Diabetes was induced by intraperitoneal injection of streptozotocin. The HMA dimethyloxalylglycine (DMOG) and desferrioxamine (DFO) were lyophilized onto DBBM. Calvarial defects were created with a trephine drill and filled with the respective bone substitutes. After 4 weeks of healing, the animals were subjected to histological and histomorphometric analysis. RESULTS: In this report, we provide evidence that DMOG loaded onto DBBM can support angiogenesis in vivo. Specifically, we show that DMOG increased the vessel area in the defect site to 2.4% ± 1.3% compared with controls 1.1% ± 0.48% (P = 0.012). There was a trend toward an increased vessel number in the defect site with 38.6 ± 17.4 and 31.0 ± 10.3 in the DMOG and the control group (P = 0.231). The increase in angiogenesis, however, did not translate into enhanced bone formation in the defect area with 9.2% ± 7.1% and 8.4% ± 5.6% in DMOG and control group, respectively. No significant changes were caused by DFO. CONCLUSIONS: The results suggest that DMOG loaded onto DBBM can support angiogenesis, but bone formation does not increase accordingly in a type 1 diabetic rat calvarial defect model at the indicated time point.

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.

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.

Collagen barrier membranes decrease osteoclastogenesis in murine bone marrow cultures.

OBJECTIVE: Collagen barrier membranes (CBM) are used for guided bone regeneration to support the process of graft consolidation. It remains, unknown however, whether CBM can affect the consolidation of bone grafts by controlling the differentiation of progenitor cells into bone-resorbing osteoclasts and bone-forming osteoblasts. MATERIAL AND METHODS: To gain an insight into the underlying mechanisms, we performed in vitro bone marrow cultures on CBM (Bio-Gide under conditions that favor osteoclastogenesis and osteoblastogenesis, respectively. Measures of osteoclastogenesis were based on the number of tartrate-resistant acid-phosphatase-positive (TRAP+) multinucleated cells. Resorption assays revealed the activity of mature osteoclasts. Osteoblastogenesis was determined by alkaline-phosphatase activity. Viability was investigated utilizing the MTT assay. RESULTS: Cultivation of murine bone marrow on CBM reduced the number of TRAP+ multinucleated cells compared with cultures on tissue culture plates. Inhibition of osteoclastogenesis was observed on the porous and the dense CBM surfaces. The majority of TRAP+ cells were mononucleated and the decreased osteoclastogenesis was not due to changes in cell viability. Furthermore, CBM are inert regarding the resorptive activity of mature osteoclasts. Moreover, osteoblastogenesis was not reduced when bone marrow cells were grown on the surface of CBM. CONCLUSIONS: These in vitro findings demonstrate that CBM can reduce the formation but not the activity of multinucleated osteoclasts. Our data further reveal that the formation of osteogenic cells from their progenitors is not reduced by the CBM. Overall, our results suggest that the beneficial effects of CBM during graft consolidation may involve their direct impact on osteoclastogenesis.

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.