In vitro release of dimethyloxaloylglycine and l-mimosine from bovine bone mineral.

OBJECTIVE Prolyl hydroxylases (PHD) are oxygen sensors and therefore pharmacological targets to stimulate periodontal regeneration. Here we evaluate the release profile of the PHD inhibitors dimethyloxaloylglycine and l-mimosine from bone substitutes. MATERIALS Dimethyloxaloylglycine and l-mimosine were lyophilised onto bone substitutes including bovine bone mineral, beta-tricalcium phosphate, and hydroxyapatite. Release kinetic was evaluated by bioassays with gingival and periodontal ligament fibroblasts. We determined the capacity of PHD inhibitors to provoke VEGF expression and to repress metabolic activity and proliferation as assessed by immunoassay, MTT conversion and (3)[H]thymidine incorporation, respectively. RESULTS We found that the PHD inhibitors are released from bovine bone mineral as indicated by the increase of VEGF production in gingival and periodontal ligament fibroblasts. Supernatants obtained after 1h also decreased metabolic activity and proliferation of the fibroblasts. A fibrin matrix prolonged the release of PHD inhibitors up to 192h. A similar cellular response was found when supernatants from PHD inhibitors loaded beta-tricalcium phosphate and hydroxyapatite embedded in fibrin were assessed. CONCLUSIONS In conclusion bone substitutes can serve as carriers for PHD inhibitors that maintain their capacity to provoke a pro-angiogenic response in vitro. These findings provide the basis for preclinical studies to evaluate if this release kinetic can stimulate periodontal regeneration

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

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

Additive manufacturing is becoming increasingly important in dentistry for the production of surgical guides. The development of cost-effective desktop stereolithography (SLA) printing systems and the corresponding resins makes this novel technique accessible to dental offices and dental laboratories. The aim of the study was to reveal the response of soft tissue cells to Clear and Dental SG resins used in desktop SLA printing systems at different stages of processing. Cell activity of L929 cells and gingival fibroblasts (GF) in response to the materials was examined in indirect and direct monolayer culture models and a direct spheroid culture model based on MTT, resazurin-based toxicity assays, and live-dead staining. Overall we found that the impact of Clear and Dental SG resins on L929 and GF depends on the processing stage of the materials. Liquid Clear resin induced a stronger reduction of cell activity compared to Dental SG resin. Printing and postcuring reduced the impact on cell activity and viability. As in-house 3D printing for surgical guides is getting integrated in the digital workflow, our data suggest that careful adherence to processing guidelinesespecially postcuringis of clinical relevance.(VLID)486244

BMC Oral Health / 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 148. 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) 1 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, TGF1 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 48hours-washed CBM. Conclusions CBM release platelet-derived factors in continuously declining release kinetics.(VLID)484741