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.

Effect of different polymerization methods on the cytotoxicity of dental composites.

OBJECTIVES: The aim of this study was to compare the cytotoxic effects of various dental composites polymerized with two different curing units. METHODS: Disc-shaped test samples of composites Filtek Z250, Filtek A110, Filtek P60, Filtek Supreme, and SDI Rok were polymerized using one quartz tungsten halogen (QTH) and one light emitting diode (LED) light curing unit (LCU), namely Optilux 501 (QTH) and Elipar Freelight 2 (LED). L-929 mouse fibroblast cultures (3x10(4) cells/ml) were incubated with the samples in 96 well culture plates for evaluation after 8, 24, 48, 72 h. At the end of each period, the cells were counted and examined under a light microscope, and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was performed. The degree of cytotoxicity for each sample was determined according to the reference value represented by the cells in a control group (a culture without sample). RESULTS: A significant 3 factor interaction occurred among LCUs, composites, and time factors (P<.005). In general, the test materials cured with the LED LCU demonstrated higher cell survival rates when compared with those cured with halogen LCUs. CONCLUSIONS: This study shows that polymerization of dental composites with a light emitting diode LCU positively influences the L-929 mouse fibroblast cell viability.

Vascular smooth muscle contraction/relaxation of rat carotid artery is not altered by bone grafting substitutes in vitro.

PURPOSE: The aim of this study was to explore the effects of various bone grafting substitutes (Osteosponge, Perioglas, Tutoplast, and Surgibone) on vascular smooth muscle tonus. METHODS: Bilateral carotid arteries were removed from rats and contraction/relaxation of isolated vessel rings were measured before and after contact with the biomaterials and then, for dose-dependent epinephrine and papaverin administrations, by a force displacement transducer. The data of each biomaterial group were collected by a computerized system and corresponding software at a sample rate of 1,000 kHz and were converted to contraction force. RESULTS: Vascular contraction forces were influenced in response to biomaterials tested except for Osteosponge (P < 0.05), although the differences between groups were insignificant (P > 0.05). There was a dose-dependent vascular response to epinephrine and papaverine administration upon biomaterial contact (P < 0.05). The dose-dependent vascular responses to epinephrine and papaverine administration were almost similar for all biomaterials tested (P < 0.05), suggesting that the biomaterials led to reversible effects on vascular contraction/relaxation behavior, which resulted in recovery. CONCLUSIONS: Osteosponge, Perioglas, Tutoplast, and Surgibone do not alter vascular smooth muscle tonus and vitality and therefore would, presumably, not jeopardize the angiogenesis of fresh blood vessels and full vascularization during tissue healing.

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 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.

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.

Neural response to sandblasted/acid-etched, TiO-blasted, polished, and mechanochemically polished/nanostructured titanium implant surfaces.

The purpose of this study was to explore morphologic, functional, and behavioral effects of rough (sandblasted-large grid/acid-etched (SLA) and TiO2 blasted), mechanically polished, and mechanochemically polished titanium implant surfaces on nerves. The compound action potentials (cAPs) of sciatic nerves of sacrificed Wistar rats (n=10) were quantified at the in vitro level, while contacting disk-shaped test specimens. The test specimens were also implanted directly on the sciatic nerves of another group of animals (n=33), hot-plate tests were undertaken for 10 consecutive days, and then the animals were sacrificed. Quantification of signal transduction speeds and cAPs of the nerves of these animals were undertaken at the in vitro level. Finally, the nerves were processed for histologic analysis. The signal transduction speeds and duration of cAPs of all groups were similar (P>0.05), whereas the amplitudes of cAPs of nerves contacting SLA implants were higher than those of TiO2 blasted and mechanochemically polished surfaces (P<0.05). Response latencies of nerves contacting mechanically polished specimens were slightly higher than the other groups (P>0.05). Histologic evaluations did not reveal any signs of adverse tissue response adjacent to specimens tested. Rough and polished titanium implant surfaces lead to similar neural response in vivo and in vitro that fall into physiologic limits.

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.

Morphologic, functional and behavioral effects of titanium dioxide exposure on nerves.

OBJECTIVES: The purpose of this study was to explore morphologic, functional, and behavioral effects of titanium dioxide (TiO(2)) on nerves. MATERIAL AND METHODS: A total of 17 albino rats were used for nerve conduction experiments, hot-plate tests, and histological evaluation. TiO(2) was implanted unilaterally on the sciatic nerves of five rats. Ten days after surgery, test and control nerves were dissected and their signal transduction speeds were quantified by suction electrodes in a bath containing a Tyrode solution. Twelve rats were divided into three equal groups resulting in equal number of nerves (n=8) for TiO(2) implantation, surgical exposure of the nerves, and for use as controls. One week after surgery, hot-plate tests were undertaken for 10 consecutive days to determine response latencies of the nerves. At the termination of the experiments, the nerves were harvested, processed, and examined under a microscope. RESULTS: The signal transduction speeds of TiO(2)-implanted nerves was similar to control specimens (P>0.05). The avoidance responses of TiO(2)-implanted, surgically exposed, and control nerves were comparable (P>0.05). At the cellular level, TiO(2) did not lead to any signs of adverse reactions on nerves. CONCLUSIONS: TiO(2), the main oxide surrounding endosseous titanium implants, does not alter the structure and the function of myelinated nerves.

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 EMD's 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 L929 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 Dulbecco's modified eagle medium (DMEM) enriched with 10% fetal bovine serum, DMEM supplemented with 0 (Control), 50 microg/mL and 100 microg/mL of EMD was added and cultures maintained for 96h. 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.