Aylin Gurpinar

Effect of Enamel Matrix Derivative on Mouse Fibroblasts and Marrow Stromal Osteoblasts

There is increasing evidence that the cells of the epithelial root sheath synthesize enamel matrix proteins and that these proteins play a fundamental role in cementogenesis and periodontal tissue formation. Emdogain, enamel matrix derivative (EMD), is a porcine enamel matrix derived product used to enhance regeneration of the peridontium after inflammatory destruction. Today, little is known about EMDs potential regenerative properties on cell function. The purpose of this study was to investigate the effects of EMD on mouse fibroblasts (L 929 cells) and rat marrow stromal osteoblasts. For effects on cell proliferation, the L 929 cell lines were plated in 24-well culture plates at an initial density of 10,000 cell/mL and allowed to attach. Following a 24-h incubation within Dulbeccos modified eagle medium (DMEM) enriched with 10% fetal bovine serum, DMEM supplemented with 0 (Control), 50 μg/mL and 100 μg/mL of EMD was added and cultures maintained for 96 h. Cell proliferation was measured at 24, 48, 72 and 96 h as the total cell number per well and cell morphology was investigated. Osteoblasts were digested from mouse tibia marrow and were plated in similar manner as with L 929 cells, while the observation periods were 2, 6, 8 and 10 days in this group. Although both cell types were able to maintain their original cell morphology throughout the tests, in both cell groups the number of cells in the EMD groups at each observation period were not significantly different than that in the control group (ANOVA, p > 0.05). Moreover, EMD failed to show any impact on cell growth with higher concentration (ANOVA, p > 0.05). These results suggest that although EMD had no cytotoxic effect on mouse fibroblasts and stromal marrow osteoblasts, the same material failed to enhance the growth of both cell types.

Comparison of cellular proliferation on dense and porous PCL scaffolds.

In this contribution, PCL (poly-epsilon caprolactone) scaffolds were prepared by solvent-casting/particle-leaching technique in the presence of two pore formers, PEG(4000) or sucrose molecules in different quantities (0, 10, 20, 30, 40, 50, 55 w/w% PEG(4000)/PCL; 10, 20 w/w% Sucrose/PCL). The surface and bulk properties of the resulting scaffolds were studied by SEM, DSC and FTIR. SEM photographs showed that, macroporosity was obtained in the PCL structures prepared with sucrose crystals while microporous structure was obtained in the presence of PEG(4000) molecules. Average pore diameters calculated from SEM photographs were 40.1 and 191.2 mum for 40% PEG(4000)/PCL and 10% Sucrose/PCL scaffolds, respectively. The DSC and FTIR results confirmed that there is no any interaction between pore formers and PCL during structural formation, and both pore formers, PEG(4000) and sucrose, remained independently in the scaffolds. L929 mouse fibroblast cells were seeded onto PCL structures and maintained during 7 days to evaluate cell proliferation. Cell culture results showed that, 10% Sucrose/PCL scaffold was the most promising substrate for L929 cell growth due to 3-D architecture and macroporous structure of the scaffold.

Clinical impact and biomaterial evaluation of autologous platelet gel in cardiac surgery

We compared the clinical efficacy of autologous platelet gel (APG) and gelatine (CONT), including biomaterial evaluation. In a prospective, randomized, controlled trial, 64 patients undergoing complex coronary artery bypass graft (CABG) surgery and/or aortic surgery, in whom the surgeon was able to identify a bleeding site for which conventional means to stop bleeding were impractical or proved unsuccessful, were enrolled. Aortic punch biopsy from each patient was harvested in explant cell (EC) culture media. Hemostasis success for the “oozing” category was 89% in APG and 60% in CONT (p< 0.05). For the “heavy bleeding” category, the success rates were 92% in APG and 45% in CONT (p<0.01). Contact of gelatine inhibited EC proliferation and APG increased cell cycling and EC quantity. Phagocytic capacity (PC) was significantly higher in the APG group (p<0.001). APG was significantly better than CONT with respect to hemostatic success rate, effects on wound healing and increased resistance to infection (PC).

In vitro evaluation of casein phosphopeptide‐amorphous calcium phosphate as a potential tooth transport medium: viability and apoptosis in L929 fibroblasts

Casein phosphopeptides (CPP) are derived from casein, which accounts for 80% of the total protein in bovine milk . The purpose of this in vitro study was to evaluate the potential use of a CPP-amorphous calcium phosphate (CPP-ACP) preparation as a transport medium for avulsed teeth. L929 fibroblastic cell line was plated in 24-well culture plates. Following incubation, the cells were treated with 10(-3), 10(-4), 10(-6), 10(-8), 10(-12) dilutions of a water-based CPP-ACP paste (Tooth Mousse, GC Corp., Tokyo, Japan). Untreated cells served as controls. The L929 cells were counted at the 1st, 3rd and 7th days. Propidium iodide/acridine orange staining was used to assess apoptosis of treated cells and of the positive control. For each concentration (dilution), statistical analysis of cell survival within time was performed using two-way analysis of variance (ANOVA, P = 0.05). One way ANOVA and Tukey tests were applied to compare the effect of different concentrations on cell survival at each evaluation day (P = 0.05). Except for the 10(-3) and 10(-4) dilutions, all groups demonstrated an increase in cell numbers at days 1 and 3, followed by a decrease at day 7. Irrespective of the increase or decrease in cell viability, time-dependent changes for each dilution group were significantly different. Cells in the 10(-3) and 10(-4) dilution groups demonstrated a rapid apoptotic response. A relatively few number of apoptotic cells were observed in the 10(-6) and 10(-8) dilution groups, while no sign of apoptosis was evident in the 10(-12) dilution group and control. These results suggest that when highly diluted, the tested CPP-ACP preparation may help preserve L929 cell viability in the short term without inducing apoptosis.

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

BackgroundAtrial 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.MethodsL929 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.ResultsThe 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.ConclusionsVitamin 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.

Clinical and biomaterial evaluation of hyaluronan-based heparin-bonded extracorporeal circuits with reduced versus full systemic anticoagulation in reoperation for coronary revascularization.

Objective This prospective randomized study compares full and reduced heparinization on novel hyaluronan-based heparin-bonded circuits vs. uncoated controls under challenging clinical setting including biomaterial evaluation. Methods 100 patients undergoing reoperation for coronary artery bypass grafting were allocated into two equal groups (n = 50): Group one was treated with hyaluronan-based heparin bonded preconnected circuits (Vision HFOGBS, Gish, California, USA) and Group two with identical uncoated controls (Vision HFO, Gish, USA). In the study group, half of the patients (n = 25) received low-systemic heparin (125 IU/kg, ACT >250 s) or full dose like control group. Blood samples were collected after induction of anesthesia (T1) and heparin administration before cardiopulmonary bypass (CPB) (T2), 15 min after initiation of CPB (T3), before cessation of CPB (T4), 15 min after reversal with protamine (T5), and the first postoperative day at 08: 00 h (T6). Results Platelet counts were preserved significantly better at T5, T6 in hyaluronan groups (P < 0.05 vs. control). Serum IL-2 levels were significantly lower at T4, T5 in both hyaluronan groups and C3a levels at T4 and T5 only in low-dose group (P < 0.05). Troponin-T levels in coronary sinus blood demonstrated well preserved myocardium in hyaluronan groups. No significant differences in thrombin–antithrombin levels were observed between full and low-dose heparin groups at any time point. Amount of desorbed protein was 1.41 ± 0.01 in full and 1.43 ± 0.01 in low dose vs. 1.78 ± 0.01 mg/dl in control (P < 0.05). Conclusion Hyaluronan-based heparin-bonded circuits provided better clinical outcome and less inflammatory response compared with uncoated surfaces. Reduced systemic heparinization combined with hyaluronan-based heparin-bonded circuits is feasible and clinically well tolerated.

Comparison of polymethoxyethylacrylate-coated circuits with leukocyte filtration and reduced heparinization protocol on heparin-bonded circuits in different risk cohorts.

Objectives: The relative benefits of strategic leukofiltration on polymer-coated and low-dose heparin protocol on heparin-coated circuits were studied across EuroSCORE patient risk strata for three different cohorts. Methods: In a prospective, randomized study, 270 patients undergoing coronary artery bypass grafting were allocated into three groups (n = 90): Group 1 -polymethoxyethylacrylate-coated circuits+leukocyte filters; Group 2 -polypeptide-based heparin-bonded circuits with reduced heparinization; and Group 3 -Control: uncoated circuits. Each group was further divided into three subgroups (n = 30), with respect to low- (EuroSCORE 0-2), medium- (3-5), and high- (6+) risk patients. Blood samples were collected at T1: following induction of anesthesia; T2: following heparin administration; T3: 15 min after CPB; T4: before cessation of CPB; T5: 15 min after protamine reversal; and T6: ICU. Results: In high-risk cohorts, leukocyte counts demonstrated significant differences at T4 and T5 in Group 1, and at T4 in Group 2. Platelet counts were preserved significantly better at T4 and T5 in both groups (p <0.05 versus control). Serum IL-2 and C3a levels were significantly lower at T3, T4 and T5 in Group 1, and T4 and T5 in Group 2 (p <0.05). Postoperative bleeding, respiratory support time and incidence of atrial fibrillation were lower in the study groups versus control. Cell counts on filter mesh and heparin-coated fibers/circuits were significantly higher in the high-risk cohorts versus uncoated fibers. Phagocytic capacity increased on filter mesh, especially in high-risk specimens. SEM evaluation demonstrated better preserved coated circuits. Conclusion: Leukofiltration and coating reduced platelet adhesion, protein adsorption, atrial fibrillation and reduced heparinization acted via modulation of systemic inflammatory response in high-risk groups.

Cytotoxicity of Two-step Self- etching Primer/Adhesives on L929 Cells

The cytotoxicity of four self-etching primer/adhesive systems (Clearfil® SE Bond, Clearfil® Protect Bond, Mac Bond® II and FL® Bond) was tested against L929 fibroblasts. The primer or adhesive component of each adhesive system was diluted serially with the culture medium at a ratio of 1:1,000 and 1:4,000 (v/v). Cytotoxicity was identified by adding L929 cells in 24-well culture plates at an initial density of 35,000 cells mL 1. The cells were maintained for 5 days; every 24h, the medium was changed with fresh medium containing specific dilutions of the primer or adhesive components of the test materials. Cytotoxicity was assessed quantitatively at 24, 48, 72, 96 and 120h. Physiological and pathological cellular changes as well as reactions and growth of the cell cultures were examined under an inverted microscope. All self-etching systems were found to be cytotoxic to varying degrees; more pronounced toxic effects were observed at lower dilution (1:1,000 [v/v]). The adhesive components of Mac Bond® II and FL® Bond showed the highest cytotoxicity at 1:1,000 (v/v). The primer and adhesive of Clearfil® SE Bond, the primer of Mac Bond® II and the antibacterial monomer (MDPB)-containing Clearfil® Protect Bond (at 1:4,000 [v/v]) were relatively less cytotoxic.