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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 24h 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 (qPCR) and enzyme-linked immunosorbent assay (ELISA), 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. Conclusion 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. This article is protected by copyright. All rights reserved.

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 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. 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. 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. 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. 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.

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. 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-β, phosphoinositide 3-kinase (PI3K)/AKT, and mitogen-activated protein kinase (MAPK) signaling pathway was analyzed. 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-β and pharmacological inhibition of TGF-β signaling did not show pronounced effects. 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. 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.

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)‐1β 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‐1β, 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.

Teeth are the hardest substance of the body and the gatekeeper of the digestive tract. They can survive extreme environmental conditions and they are the last part of the body that is decomposed after death. With these unique features, teeth are often the only remaining body part of a deceased person in a criminal case. Teeth can help investigators to find out who a dead person is, how old the person was, if the person was male or female, what kind of daily habits and lifestyle a person had and how the person died. Thus, the examination of teeth plays a key role in crime scene investigations and can help solve crimes.

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.

BackgroundCollagen 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 methodsPubMed, 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.ResultsA 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.ConclusionGrowth 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.

The aim of this study was to evaluate the effect of bleaching agents containing different concentrations of hydrogen peroxide (HP) on color-change and on enamel-surface in bovine teeth. Furthermore the influence on cell viability and proliferation was investigated. Two hundred and forty teeth were randomly assigned into four groups (home bleaching ≤6%, in-office bleaching ≤6%, in-office bleaching > 6% HP, and control group). Bleaching was performed after artificial staining and the bleached index (BI) as well as the whiteness index (WID) was measured at several time points. Chemical analysis for HP concentrations and the pH of the bleaching products was done. Furthermore, enamel surfaces of randomly selected specimens were analyzed using scanning electron microscopy (SEM) and cytotoxicity of the tested bleaching products was evaluated in vitro using dental pulp cells (DPCs) and L929 cells. A statistically significant whitening effect was observed in almost all products. As expected all investigated products resulted in decreased cell viability, however, with different values of LC50 (median lethal concentration). SEM analysis showed an analog of enamel alterations with decreasing pH, increasing exposure time, and increasing HP concentration. Bleaching agents containing a low HP concentration are considered to be effective and to have less damaging effects on enamel and tested cells.

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.

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.

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.

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.

Calcium hydroxide removal from the root canal by photon induced photoacoustic streaming (PIPS) compared to needle irrigation and irrigation using sonic activation was investigated. Additionally, safety issues regarding apical extrusion were addressed. In endodontic treatment temporary intracanal medication like calcium hydroxide should be completely removed for long term success. For analysis, 60 artificial teeth were prepared, filled with calcium hydroxide, and divided into four groups. The teeth were assigned to needle irrigation, irrigation using a sonic device, PIPS with a lower energy setting (10 mJ, 15 Hz), or PIPS with a higher energy setting (25 mJ/40 Hz). For comparison the weight of each tooth was measured before and after calcium hydroxide incorporation, as well as after removing calcium hydroxide using the four different methods. Regarding safety issues another 24 samples were filled with stained calcium hydroxide and embedded in 0.4% agarose gel. Color changes in the agarose gel due to apical extrusion were digitally analysed using Photoshop. No significant differences were found for calcium hydroxide removal between the two laser groups. Sonic assisted removal and needle irrigation resulted in significant less calcium hydroxide removal than both laser groups, with significantly more calcium hydroxide removal in the ultrasonic group than in the needle irrigation group. For apical extrusion the higher laser (25 mJ/40 Hz) group resulted in significant higher color changes of the periapical gel than all other groups. PIPS with the setting of 10 mJ/15 Hz achieved almost complete removal of calcium hydroxide without increasing apical extrusion of the irrigation solution.

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.

Platelets modulate formation of osteoclasts and osteoblasts, but research with different preparations of platelets remains inconclusive. Here, we assessed whether serum components modulate the effect of platelet preparations. In murine bone marrow cultures, osteoclastogenesis was investigated in the presence of platelet-released supernatant (PRS), serum containing PRS (SC-PRS), and serum. Osteoclastogenesis was quantified by the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, TRAP activity and resorption assays. Also human osteoclastogenesis assays were performed. Viability and proliferation were tested by MTT and (3) [H]thymidine incorporation assays, respectively. Osteoblastogenesis was assessed by histochemical staining for alkaline phosphatase-of murine bone marrow cultures and human MG63 cells. We found PRS to increase the number of TRAP(+) multinucleated cells in the early phase and TRAP activity in the later phase of osteoclastogenesis. SC-PRS and serum decreased the number and activity of TRAP(+) multinucleated cells. Both serum containing preparations reduced viability and proliferation of hematopoietic progenitors. PRS decreased the numbers of alkaline phosphatase-positive colonies while SC-PRS and serum increased osteoblastmarkers in MG63. Proliferation of MG63 was stimulated by all preparations. These results show that activated platelets support osteoclastogenesis, while platelet preparations that contain serum components decrease osteoclastogenesis and increase osteoblastogenesis in vitro, suggesting that serum components modulate the effects of platelets on osteoclastogenesis and osteoblastogenesis.

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., BMC Oral Health, 17, ISSN:1472-6831

Bone regeneration is initiated by the formation of a blood clot. Activated platelets within this fibrin-rich matrix release signaling molecules that can attract mesenchymal progenitor cells. To gain insight into the cellular mechanism by which activated platelets can support the immigration of mesenchymal progenitors, we have tested the hypothesis that platelet-released signaling molecules increase the capacity of bone marrow stromal cells (BMSC) to activate plasminogen. We report herein that platelet-released supernatants (PRS) elevate total urokinase-type plasminogen activator (uPA) and total plasminogen activator inhibitor-1 (PAI-1) levels in BMSC, as assessed by immunoassay. Quantitative polymerase chain reaction showed an upregulation of uPA, uPA receptor, and PAI-1. Zymography and kinetic analysis based on casein hydrolysis revealed enhanced activity of cell-associated uPA upon exposure of BMSC to PRS. Inhibiting c-Jun N-terminal kinase (JNK) and phosphatidylinositol 3-kinase (PI3K) signaling reduced uPA production and decreased plasminogen activation. Corresponding Western blot analysis showed increased phosphorylation of JNK and AKT in BMSC treated with PRS. These results suggest that activated platelets can enhance the plasminogen activation capacity of mesenchymal progenitors through the stimulation of uPA production, requiring JNK and PI3K/AKT signaling. By this mechanism platelets may contribute to the organization of the blood clot during bone regeneration.

Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF) within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF) were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.

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) β1 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, TGFβ1 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.