In Vitro Cytotoxicity of Maxillofacial Silicone Elastomers: Effect of Nano-particles.

PURPOSE: Silicone elastomers are generally used for maxillofacial extraoral prostheses. The purpose of this in vitro study was to evaluate the cytotoxicity of different kinds of nanoparticles added to two types of maxillofacial elastomers. MATERIALS AND METHODS: A-2000 and A-2006 silicone elastomers were used. The silicone specimens were divided into eight groups according to the presence of additional nanoparticles (n = 18). The following represents the groups in the study: Group A: A-2000 silicone (control group); Group B: A-2006 silicone (control group); Group C: A-2000 silicone and the addition of titanium dioxide (TiO2 ); Group D: A-2006 silicone and the addition of TiO2 ; Group E: A-2000 silicone and the addition of fumed silica; Group F: A-2006 silicone and the addition of fumed silica; Group G: A-2000 silicone and the addition of silaned silica; Group H: A-2006 silicone and the addition of silaned silica. A paired sample t-test was used to analyze the cytotoxicity of each group after 24, 48, and 72 hours. RESULTS: Based on the results of the 24-hour analysis, the biocompatibility values of the (A-2006) fumed silica group were higher than those of the control groups. There was no statistically significant difference in A-2006 and A-2000 groups. The cytotoxicity values of the control groups and TiO2 (A-2000 silicone) elastomer groups increased at all test times; however, the cytotoxicity values of the TiO2 (A-2006), fumed silica (A-2006), silaned silica (A-2006), fumed silica (A-2000), and silaned silica (A-2000) groups increased significantly only from 24 to 48 hours. CONCLUSION: Nanoparticles of TiO2 , fumed silica, and silaned silica added to a commercial silicone-based elastomer used for fabrication of maxillofacial prostheses are nontoxic.

Design, synthesis and biological evaluation of Nucleosidic CD99 inhibitors that selectively reduce Ewing sarcoma viability.

Ewing Sarcoma (ES) is a cancer of bone and soft tissues affecting mostly children and young adults. Aggressive progression and poor prognosis of this malignancy call for novel and targeted treatments. CD99 is a transmembrane protein that is abundantly expressed on ES cells and is a diagnostic marker for the disease. ES cells are selectively sensitive to CD99 inhibition compared to most normal cells and other tumors. Therefore, CD99 is a good molecular target for ES treatment. Clofarabine and cladribine are two FDA approved drugs that are administered for their inhibitory acts on DNA synthesis to treat relapsed or refractory acute lymphoblastic and myeloid leukemia. They have also been shown to directly bind to CD99 and inhibit ES growth through a distinct mechanism. In the current study, we designed, synthesized and tested new ES specific derivatives of both drugs that would continue to target CD99 but with expected reduction in cellular membrane permeability and rendered unsuitable for inhibiting DNA synthesis. By using commercially available clofarabine and cladribine purine nucleoside analogs, we modified the primary alcohol moiety at the deoxyribose C-5' terminal site to suppress phosphorylation and thus inhibition of subsequent DNA synthesis pathways. In addition, we incorporated a variety of polar groups in the ribose and purine rings to reduce membrane permeability and investigated the effects of configurational changes in the sugar moiety. Among 26 new derivatives, we identified two compounds, BK50164 and BK60106, that cause cell death specifically in ES primarily due to inhibition of CD99 but not via inhibition of DNA synthesis. These findings provide a road map for the future development selective CD99 inhibitors for targeted treatment of ES.

Dendritic Cell Differentiation from Human Induced Pluripotent Stem Cells: Challenges and Progress.

Dendritic cells (DCs) are the major antigen-presenting cells of the immune system responsible for initiating and coordinating immune responses. These abilities provide potential for several clinical applications, such as the development of immunogenic vaccines. However, difficulty in obtaining DCs from conventional sources, such as bone marrow, peripheral blood, and cord blood, significantly hinders routine application. The use of human induced pluripotent stem cells (hiPSCs) is a valuable alternative for generating sufficient numbers of DCs to be used in basic and preclinical studies. Despite the many challenges that must be overcome to achieve an efficient protocol for obtaining the major DC types from hiPSCs, recent progress has been made. In this study, we review the current state of developing DCs from hiPSCs, as well as the key elements required to enable the routine use of hiPSC-derived DCs in preclinical and clinical assays.

Clofarabine induces ERK/MSK/CREB activation through inhibiting CD99 on Ewing sarcoma cells.

Clofarabine, an FDA approved purine analog, is used in the treatment of relapsed or refractory acute lymphoblastic leukemia. Clofarabine acts by inhibiting DNA synthesis. We demonstrated that clofarabine may have a novel function though inhibiting CD99, a transmembrane protein highly expressed on Ewing Sarcoma (ES) cells. CD99 is a validated target in ES whose inhibition may lead to a high therapeutic index for patients. Here we present additional data to support the hypothesis that clofarabine acts on CD99 and regulates key signaling pathways in ES. Cellular thermal shift assay indicated a direct interaction between clofarabine and CD99 in ES cell lysates. Clofarabine induced ES cell death does not require clofarabine's conversion to its active form by deoxycytidine kinase. A phosphokinase array screen with clofarabine and a CD99 blocking antibody identified alterations in signaling pathways. CD99 inhibition with clofarabine in ES cells caused rapid and sustained phosphorylation of ERK, MSK, and CREB. However, activation of this pathway did not correlate with clofarabine induced ES cell death. In summary, we demonstrated that clofarabine may activate ERK, MSK, and CREB phosphorylation through CD99 within minutes, however this paradoxical activation and subsequent ES cell death requires additional investigation.

Cytotoxicity of long-term denture base materials.

INTRODUCTION: The aim of this study was to evaluate the cytotoxic effect of various denture base materials following four different aging periods. METHODS: In total, 48 disc-shaped specimens per each group were prepared: Group I: acrylic resin polymerized in cool water and heated up to 100°C over 45 min and boiled for 15 min; Group II: acrylic resin polymerized under pressure in 40°C-45°C water bath for 10 min; Group III: autopolymerized hard relining resin Cold Liner Rebase; Group IV: autopolymerized hard relining resin Truliner; Group V: soft relining resin DentuSil. Then the specimens were stored in water for 24 h or 15 days, or thermocycled 2500 times or 10,000 times. Cytotoxicity was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using L929 cells after 72-h cell incubation. Cell viability percentages were counted and statistical analyses were performed. The results were also evaluated according to ISO standard 10993-5. RESULTS: All materials showed similar cell viability percentages following 24-h water storage and 2500 and 10,000 thermal cycles. Following 15-day water storage, a statistically significant difference was observed between the materials. Comparisons of the aging periods for each material showed statistically significant differences. Groups III and IV showed moderately cytotoxic effect following 15-day water storage. The remaining groups showed slightly cytotoxic or non-cytotoxic effect. DISCUSSION: Polymerizing acrylic resins under pressure can be an alternative to conventional polymerizing to ensure a faster denture repair while providing similar cell viability values. Heat-cured acrylic resins provide higher cell viability than hard chairside lining materials in a 15-day period.

Bone marrow derived mesenchymal stem cells ameliorate inflammatory response in an in vitro model of familial hemophagocytic lymphohistiocytosis 2.

BACKGROUND: Familial hemophagocytic lymphohistiocytosis 2 (FHL2) is the most common familial type of hemophagocytic lymphohistiocytosis with immune dysregulation. FHL2 patients have mutations in the perforin gene which cause overactivation and proliferation of cytotoxic T lymphocytes and natural killer cells. Perforin is the key component of the cytolytic granule response function of cytotoxic T lymphocytes and natural killer cells. Perforin dysfunction causes a cytotoxic immune deficiency with a clinical outcome of uncontrolled and continuous immune stimulation response. This excessive stimulation leads to continuous systemic inflammation and, ultimately, multiorgan failure. Radical therapy is hematopoietic stem cell transplantation which is limited by the availability of a donor. Exacerbations of inflammatory attacks require a palliative immunosuppressive regimen. There is a need for an alternative or adjuvant therapy to maintain these patients when immunosuppression is ineffective or a donor is not available. Beneficial actions of mesenchymal stem cells (MSCs) have been shown in autoimmune diseases in clinical trials and are attributed to their immune-modulatory properties. This study aimed to assess the immune-modulatory effect of MSCs in an in-vitro model of FHL2. METHODS: We generated a targeted mutation in the perforin gene of NK92 cells to create an in-vitro FLH2 model using Crispr/Cas technology. A coculture setup was employed to assess the immunomodulatory efficacy of MSCs. RESULTS: Engineered NK92 clones did not show PRF1 mRNA expression and failed to secrete perforin upon phorbol myristate acetate-ionomycin stimulation, providing evidence for a valid FHL2 model. Coculture media of the engineered cells were investigated for the abundance of several cytokines. Coculture with MSCs revealed a reduction in major proinflammatory cytokines and an induction in anti-inflammatory and immunomodulatory cytokines compared to the parental NK92 cells. CONCLUSIONS: This study shows the ameliorating effect of MSCs as an adjuvant immune modulator toward the therapy of FHL2 patients. MSCs are supportive therapy candidates for FHL2 patients under circumstances where prolonged immunosuppression is required to gain time before allogeneic hematopoietic stem cell transplantation.

Effect of artificial saliva with different pH levels on the cytotoxicity of soft denture lining materials.

BACKGROUND: The aim of this study was to evaluate the cytotoxic effects of 9 different soft denture liners on the viability of L-929 mouse fibroblast cells at different incubation periods by storing them in artificial saliva (AS) with different pH levels. METHODS: 96 disk samples from each lining material were prepared and divided into 4 groups: GI: No treatment; GII: Stored in artificial saliva with pH 3 for 21 days; Group III: Stored in artificial saliva with pH 7 for 21 days; and Group IV: Stored in artificial saliva with pH 14 for 21 days. The cytotoxicity of the extracts to cultured mouse fibroblasts (L-929) was measured by MTT (tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-dipHnyltetrazolium bromide) assay. Data were analyzed using 1-way analysis of variation (ANOVA). RESULTS: It was found that for the pH 3 values of New Truliner, Trusoft, Mollosil Plus, Dentusil, TDV, and HydroCast®; for the pH 7 values of Ufi Gel P and Elite plus; and for the pH 14 values of HydroCast®, there was a noncytotoxic effect during both the 24-hour and 48-hour incubation periods. In the control group 48-hour incubation period, HydroCast®, TDV, Mollosil, 24-hour incubation period Elite plus, for pH 3 values; Elite Plus 24-hour incubation period, for pH 7 values Trusoft 48-hour incubation period there was a moderately cytotoxic effect. CONCLUSIONS: This in vitro study revealed that storage in artificial saliva with different pH levels can affect the cytotoxicity of soft lining materials.

Heparin toxicity in cell culture: a critical link in translation of basic science to clinical practice.

Heparin is a universal drug used frequently for its anticoagulant effects. The variabilities in distribution and tendency of heparin to accumulate in tissues cause increased tissue concentrations despite normal serum levels. We aimed to underline the toxic effects of heparin in cell culture make projections for clinical applications. L929 mouse fibroblastic cell line was plated in 96-well culture plates at an initial density of 5000 cells/well. Heparin was prepared in 10 different concentrations (10-300 units/well). Following 3 days of incubation, viabilities were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for each concentration in each day and compared. The viability of cells decreased significantly with increasing doses of heparin; at least 50 units/well in the first and second days and at least 20 units/well in the third day (P < 0.05 for each). There was statistically significant difference when the viabilities of cells treated with same heparin concentration in different days were compared (P < 0.05). The authors clearly demonstrated the toxic effects of heparin in cell culture, toxic effects increased as the dose increased. To prevent the unwanted clinical side-effects of heparin further studies should be made and more accurate testing methods should be developed to determine the effective tissue concentration of heparin.

May toxicity of amiodarone be prevented by antioxidants? A cell-culture study.

BACKGROUND: Atrial Fibrillation is the most common arrhythmia encountered following cardiac surgery. The most commonly administered drug used in treatment and prophylaxis is amiodarone which has several toxic effects on major organ functions. There are few clinical data concerning prevention of toxic effects and there is no routinely suggested agent. The aim of this study is to document the cytotoxic effects of amiodarone on cell culture media and compare the cytoprotective effects of commonly used antioxidant agents. METHODS: L929 mouse fibroblast cell line was cultured and 100,000 cells/well-plate were obtained. First group of cells were treated with increasing concentrations of amiodarone (20 to 180 µM) alone. Second and third group of cells were incubated with one-fold equimolar dose of vitamin C and N-acetyl cysteine prior to amiodarone exposure. The viability of cells were measured by MTT assay and the cytoprotective effect of each agent was compared. RESULTS: The cytotoxicity of amiodarone was significant with concentrations of 100 µM and more. The viabilities of both vitamin C and N-acetyl cysteine treated cells were higher compared to untreated cells. CONCLUSIONS: Vitamin C and N-acetyl cysteine are commonly used in the clinical setting for different purposes in context of their known antioxidant actions. Their role in prevention of amiodarone induced cytotoxicity is not fully documented. The study fully demonstrates the cytoprotective role of both agents in amiodarone induced cytotoxicity on cell culture media; more pronounced with vitamin C in some concentrations. The findings may be projectile for further clinical studies.