Terms Of Cellular Viability Research Articles

Effect of polydatin on the viability and odontogenic differentiation of human dental pulp stem cells: An in-vitro study

Background/purposeVarious materials have been used to promote human dental pulp stem cells (hDPSCs) differentiation to produce dentin bridge formation with less-than-optimal results. Polydatin (PD), a naturally present material with osteogenic properties can be a promising material in the pulp regeneration/repair process. The aim of this study was to evaluate the effect of (PD) on the viability and differentiation of human dental pulp stem cells. Materials and methodsPD effect on hDPSCs in terms of cellular viability, alkaline phosphatase (ALP) production, and messenger RNAs (mRNA) of odontogenic markers production using quantitative reverse transcription polymerase chain reaction (RT-qPCR) were evaluated. In addition, mineral deposits were detected with Alizarin red stain. ResultsThe viable hDPSCs in the presence of 0.01 μM and 0.1 μM PD were significantly higher than the control on days 3 and 7, respectively. In addition, ALP activity of hDPSCs was significantly increased with 0.01, 0.1, and 1 μM of PD. In addition, increased expression mRNAs of ALP, osteocalcin (OC), osteonectin (ON), osteopontin (OP), Runt-related transcription factor-2 (RUNX-2), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) was observed after PD treatment, however, the difference was not statistically significant. Furthermore, increased size of mineral deposits was observed with PD. ConclusionPD promoted the expression of markers associated with odontogenic differentiation and mineralized tissue deposition in hDPSCs.

Effects of surface micro-deformation on the cellular response to metallic implant materials

In the current study, it is aimed to get a comprehensive understanding of how cellular response to metallic biomaterial surfaces can be controlled via surface parameter specifics, as well as microstructural modifications. For this purpose, a surface micro-deformation procedure was designed where patterns with different parameters, specifically different indent size and repeating frequencies, were created on 316 L stainless steel sample surfaces by using a Vicker's microhardness testing device. The effects of the changes in surface properties following the formation of the different patterns were characterized via scanning electron microscopy (SEM), optical microscopy and profilometer. It was observed that surface roughness significantly increased as a result of the surface micro-deformation process and additionally, dislocation activities of different levels were triggered depending on the specifics of the formed micro-deformation pattern. The effects of the surface micro-deformation process on cellular response were investigated through in vitro experiments where Saos-2 cell line was used. Significant improvements were obtained in terms of cellular viability, proliferation, attachment and cell differentiation behaviors on the samples with surface micro-deformation. Correlating the effects surface parameters such as surface roughness, dislocation density close to the surface and topographical feature specifics on cellular response, it has been observed that by manipulating the metal microstructure and optimizing the surface micro-deformation patterning parameters, cellular response can be significantly enhanced. It was shown that specific surface patterns with smaller indent depth and narrower spacings, as well as optimum dislocation density levels and surface roughness yields superior cellular response. Overall, the obtained information about the specifics of surface features of metallic biomaterials on cellular response can be used for the design of novel implant surface modification methods to improve osseointegration.

In vitro evaluation of pediatric restorative materials on human dental pulp stem cells

This in vitro study aimed to compare the cellular viability of four different restorative materials on human dental pulp stem cells (hDPSCs). The necessary tissues of isolated hDPSCs were obtained from 10 impacted third molars of healthy individuals 19–25 years old undergoing surgical extraction. The effects of GC Fuji IX GP, GC EQUIA Forte, APA Glass Carbomer, and APA Esthetic Fill eluates on hDPSCs’ viability on the first, third, and seventh days were examined by flow cytometry using an Annexin V binding assay. The cell viability ratios differed significantly between the restorative material groups (p < 0.001). When the restorative materials and the control group were compared, the control group had the highest cell viability on all experiment days, while the Glass Carbomer group had the highest percentage of viable cells of all the restorative material groups. A significant difference between days in terms of cellular viability (p < 0.001) was found. Considering the early apoptotic cell ratios, no significant difference was found between the groups and days (p > 0.05). Furthermore, based on time, except for the first day of the GC Fuji IX GP group, no statistically significant difference was observed in the early apoptotic cell ratios in each group (p > 0.05). When the rates of late apoptotic cells were compared between the groups, no significant difference was observed, except for with the control group, which had the lowest rate of late apoptotic cells (p < 0.001). However, there was a significant difference in necrotic cell ratios between groups and days (p < 0.001; p = 0.001). The lowest rates of necrotic cells were found in the control group and Glass Carbomer groups, while the rates of necrotic cells were similar among the other groups. Therefore, we concluded that APA Glass Carbomer presents the highest cellular viability among the materials.

Chitosan Scaffold Containing Periostin Enhances Sternum Bone Healing and Decreases Serum Level of TNF-α and IL-6 after Sternotomy in Rat.

In the aftermath of bone injuries, such as cranium and sternum, bone wax (BW) is used to control bleeding from the bone surfaces during surgery. Made up of artificial substances, however, it is associated with many complications such as inflammation, increased risk for infection, and bone repair delay. We, therefore, in this study set out to design and evaluate a novel BW without the above-mentioned side-effects reported for other therapies. The pastes (new BW(s)) were prepared in the laboratory and examined by MTT, MIC, MBC, and degradability tests. Then, 60 adult male Wistar rats, divided into six equal groups including chitosan (CT), CT-octacalcium phosphate (OCP), CT-periostin (Post), CT-OCP-Post, Control (Ctrl), and BW, underwent sternotomy surgery. Once the surgeries were completed, the bone repair was assessed radiologically and thereafter clinically in vivo and in vitro using CT-scan, H&E, ELISA, and qRT-PCR. All pastes displayed antibacterial properties and the CT-Post group had the highest cell viability compared to the control group. In contrast to the BW, CT-Post group demonstrated weight changes in the degradability test. In the CT-Post group, more number of osteocyte cells, high trabeculae percentage, and the least fibrous connective tissue were observed compared to other groups. Additionally, in comparison to the CT and Ctrl groups, higher alkaline phosphatase activity, as well as decreased level of serum tumor necrosis factor-α, interleukin-6, and OCN in the CT-Post group was evident. Finally, Runx2, OPG, and RANKL genes' expression was significantly higher in the CT-Post group than in other groups. Our results provide insights into the desirability of pastes in terms of cellular viability, degradability, antibacterial properties, and surgical site restoration compared to the BW group. Besides, Periostin could enhance the osteogenic properties of bone tissue defect site.

A New Generation of Electrospun Fibers Containing Bioactive Glass Particles for Wound Healing.

Chitosan fibers blended with polyethylene oxide (CHIT_PEO) and crosslinked with genipin were fabricated by electrospinning technique. Subsequently, CHIT_PEO bioactive glass composite electrospun mats were fabricated with the aim to achieve flexible structures with adequate mechanical properties and improved biological performance respect to CHIT_PEO fibers, for potential applications in wound healing. Three different compositions of bioactive glasses (BG) were selected and investigated: 45S5 BG, a Sr and Mg containing bioactive glass (BGMS10) and a Zn-containing bioactive glass (BGMS_2Zn). Particulate BGs (particles size < 20 μm) were separately added to the starting CHIT_PEO solution before electrospinning. The two recently developed bioactive glasses (BGMS10 and BGMS_2Zn) showed very promising biological properties in terms of bioactivity and cellular viability; thus, such compositions were added for the first time to CHIT_PEO solution to fabricate composite electrospun mats. The incorporation of bioactive glass particles and their distribution into CHIT_PEO fibers were assessed by SEM and FTIR analyses. Furthermore, CHIT_PEO composite electrospun mats showed improved mechanical properties in terms of Young’s Modulus compared to neat CHIT_PEO fibers; on the contrary, the values of tensile strain at break (%) were comparable. Biological performance in terms of cellular viability was investigated by means of WST-8 assay and CHIT_PEO composite electrospun mats showed cytocompatibility and the desired cellular viability.

Solid Polymeric Nanoparticles of Albendazole: Synthesis, Physico-Chemical Characterization and Biological Activity.

Albendazole is a benzimidazole derivative with documented antitumor activity and low toxicity to healthy cells. The major disadvantage in terms of clinical use is its low aqueous solubility which limits its bioavailability. Albendazole was incorporated into stable and homogeneous polyurethane structures with the aim of obtaining an improved drug delivery system model. Spectral and thermal analysis was used to investigate the encapsulation process and confirmed the presence of albendazole inside the nanoparticles. The in vitro anticancer properties of albendazole encapsulated in polyurethane structures versus the un-encapsulated compound were tested on two breast cancer cell lines, MCF-7 and MDA-MB-231, in terms of cellular viability and apoptosis induction. The study showed that the encapsulation process enhanced the antitumor activity of albendazole on the MCF-7 and MDA-MB-23 breast cancer lines. The cytotoxic activity manifested in a concentration-dependent manner and was accompanied by changes in cell morphology and nuclear fragmentation.

Genomic effects of a nanostructured alumina insecticide in human peripheral blood lymphocytes in vitro

Nanotechnology is providing new tools for precision agriculture, such as agrochemical agents and innovative delivery mechanisms to improve cropping efficiency. Powder nanoinsecticides, such as experimental nanostructured alumina (NSA), show great potential for sustainable agriculture as an alternative to conventional synthetic pesticides because their mechanism of insecticide action is based on physical rather than on biochemical phenomena. However, even in highly non-reactive and hardly soluble substances such as alumina, reduced particle size may lead to an increased toxicity of the material. In order to determine whether NSA induces DNA and chromosomal damage, its toxicity was assessed in human peripheral blood lymphocytes (PBL) and contrasted with commercial nanostructured alumina, natural insecticide powders and a conventional pesticide. PBL from healthy donors were exposed for 24 h to increasing concentrations (50, 100 and 200 μg/mL) of NSA particle agglomerates (<350 nm); positive and negative NSA-particles, respectively; bulk Al2O3 (4.5 μm) or Diatomaceous Earth (SiO2, <4.5 μm). Alkaline comet assay and micronuclei (MNi) test were used to assess DNA damage and chromosomal breakage, respectively. Cell viability was tested with resazurin assay. Comet assay results revealed no significant increase in DNA damage by NSA compared to other natural substances. As expected, DNA breaks were significantly higher in cells exposed to an organophosphate [OPP] control (P < 0.05). No statistically significant differences were found in terms of cellular viability at 50 and 100 μg/mL of NSA but cell survival decreased at 200 μg/mL as well as in OPP group. Positively charged NSA particles significantly reduced cell viability and increased DNA migration and oxidative DNA damage (8-oxoG). NSA as well as the electrically charged NSA particles had no significant effect on MNi induction. Our results indicate that NSA particles are non-cytotoxic and non-genotoxic at the tested doses and do not cause obvious DNA damage in human PBL in vitro.

Antitumor potential of a novel camptothecin derivative, ZBH-ZM-06.

Camptothecin (CPT) is a cytotoxic quinoline alkaloid that is used clinically as an anticancer drug. However, the clinical application of CPT is limited due to its low solubility as well as serious and unfathomable side-effects. In the present study, we created a novel 10-hydroxy CPT prodrug, ZBH-ZM‑06. Its cellular cytotoxic activity was analyzed in terms of cellular viability, acetylcholinesterase (AchE) inhibition, DNA relaxation, cellular cycling and apoptosis properties. Our results showed that the AchE inhibition rate of 10µmol/l ZBH-ZM-06 was 12.5%, compared to 96.5% for carbonyl-oxycamptothecin (CPT-11). In a chemical stability assay, only 4.9% of ZBH-ZM-06 remained after 4h at pH7.4. In addition, 10µmol/l ZBH-ZM-06 significantly inhibited the tumor cell viability of nine tumor cell lines, compared to CPT-11 and the CPT active ingredient, 7-ethyl-10-hydroxy-camptothecin (SN38) (p<0.01-0.05). In the apoptosis assay, ZBH-ZM-06 increased the ratio of annexinV+/propidium iodide (PI)-/+ cells by flow cytometric analysis (p<0.05). Moreover, ZBH-ZM-06 activated caspase-3 and poly(ADP-ribose)polymerase (PARP) expression by immunoblotting. Furthermore, ZBH-ZM-06 induced a greater G2/M phase arrest ratio, compared to CPT-11 and SN38. These results indicated that ZBH-ZM-06 had higher antitumor activity than CPT-11 and SN38, which was shown by its: i) release of the effective ingredient; ii) growth inhibition of a broad spectrum of tumor cells; iii) inhibition of DNA topoisomerase (Topo-1); and iv) promotion of apoptosis through an intrinsic signaling pathway. Thus, ZBH-ZM-06 may be applied in the preclinic study for cancer treatment.

The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance.

Solid tumours naturally grow in 3D wherein the spatial arrangement of cells affects how they interact with each other. This suggests that 3D cell culture may mimic the natural in vivo setting better than traditional monolayer (2D) cell culture, where cells are grown attached to plastic. Here, using HER2-positive breast cancer cell lines as models (BT474, HCC1954, EFM192A), the effects of culturing cells in 3D using the poly-HEMA method compared to 2D cultures were assessed in terms of cellular viability, response/resistance to anti-cancer drugs, protein expression and enzyme activity. Scanning electron microscopy showed the morphology of cells in 3D to be substantially different to those cultured in 2D. Cell viability in 3D cells was substantially lower than that of cells in 2D cultures, while 3D cultures were more resistant to the effects of HER-targeted (neratinib) and classical chemotherapy (docetaxel) drugs. Expression of proteins involved in cell survival, transporters associated with drug resistance and drug targets were increased in 3D cultures. Finally, activity of drug metabolising enzyme CYP3A4 was substantially increased in 3D compared to 2D cultures. Together this data indicates that the biological information represented by 3D and 2D cell cultures is substantially different i.e. 3D cell cultures demonstrate higher innate resistance to anti-cancer drugs compared to 2D cultures, which may be facilitated by the altered receptor proteins, drug transporters and metabolising enzyme activity. This highlights the importance of considering 3D in addition to 2D culture methods in pre-clinical studies of both newer targeted and more traditional anti-cancer drugs.

Abstract 734: Synergistic antitumor effect of vemurafenib and metformin in thyroid cancer

Abstract Advanced thyroid cancer patients such as those with radioiodine-refractory and metastatic differentiated thyroid cancer or anaplastic thyroid cancer lack effective treatment options. Targeted BRAF kinase inhibitors such as vemurafenib have shown promising results in clinical trials and off-label use for patients with BRAFV600E-positive tumors. While targeting BRAFV600E offers particular effectiveness and selectivity in inhibiting cancer cells in the short term, resistance does develop. Prior results in our laboratory confirmed that vemurafenib halts DNA synthesis and induces apoptosis in our mutated thyroid cancer cells. In addition, resistant BCPAP (papillary thyroid cancer) cells were developed in our laboratory by exposing cells to increasing concentrations of vemurafenib. Resistant cells were characterized by hyperactive mitogen-activated protein kinase (MAPK) signaling and resistance to mammalian target of rapamycin (mTOR) inhibition. These additional targets mediating resistance provide an opportunity to devise novel synergistic combination therapies. Since antitumor effects of the widely used antidiabetic agent metformin were observed in thyroid cancer cells and the drug is thought to suppress mTOR, we investigated metformin adjuvant therapy in combination with vemurafenib. Various cell lines including Nthy-ori 3-1 (normal immortalized thyroid cells), BCPAP (papillary thyroid cancer), vemurafenib-resistant BCPAP, and 8505c (anaplastic thyroid cancer) were analyzed in terms of cellular viability, apoptosis and expression of cell signaling molecules after exposure to both drugs. We found that the combination of vemurafenib and metformin caused significantly decreased viability in vemurafenib-resistant BCPAP cells. Furthermore, synergistic apoptosis was observed in BCPAP papillary thyroid cancer cells and 8505c anaplastic thyroid cancer cells in response to the combination treatment. These effects appeared to be mediated by inhibition of both mTOR and MAPK signaling. Results support further investigation into the use of metformin in combination with targeted kinase inhibitors to treat advanced thyroid cancer patients. Citation Format: Elyse K. Hanly, Neha Y. Tuli, Robert B. Bednarczyk, Augustine L. Moscatello, Zbigniew Darzynkiewicz, Jan Geliebter, Raj K. Tiwari. Synergistic antitumor effect of vemurafenib and metformin in thyroid cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 734. doi:10.1158/1538-7445.AM2015-734

Cobalt toxicity: Chemical and radiological combined effects on HaCaT keratinocyte cell line

Cobalt (Co) is an essential trace element well known as a constituent of vitamin B 12, but different compounds of Co are also described as highly toxic and/or radiotoxic for individuals or the environment. In nuclear power plants, 58Co and 60Co are radioactive isotopes of cobalt present as activation products of stable Co and Ni used in alloys. Skin exposure is a current occupational risk in the hard metal and nuclear industries. As biochemical and molecular cobalt-induced toxicological mechanisms are not fully identified, we investigated cobalt toxicity in a model human keratinocyte cell line, HaCaT. In this study, we propose a model to determine the in vitro chemical impact on cell viability of a soluble form of cobalt (CoCl 2) with or without γ-ray doses to mimic contamination by 60Co, to elucidate the mechanisms of cobalt intracellular chemical and radiological toxicity. Intracellular cobalt concentration was determined after HaCaT cell contamination and chemical toxicity was evaluated in terms of cellular viability and clonogenic survival. We investigated damage to DNA in HaCaT cells by combined treatment with chemical cobalt and a moderate γ-ray dose. Additive effects of cobalt and irradiation were demonstrated. The underlying mechanism of cobalt toxicity is not clearly established, but our results seem to indicate that the toxicity of Co(II) and of irradiation arises from production of reactive oxygen species.

Effects of the pesticide clorpyrifos on an in vitro model of intestinal barrier

Clorpyrifos (CPF), one of the most widely used organophosphorothionate pesticide can be detected as residues in food and drinking water; therefore the oral route is the major route of exposure for the general population, including children, following household use of this insecticide. Aim of this work was to investigate the possible acute cytotoxic effects of CPF on intestine and the integrity of the epithelial barrier, using Caco-2/TC7 cells as intestinal in vitro model. High level of CPF found inside the cells, corresponding to about 80% of the nominal concentration tested (30, 50 and 250 μM), chosen as representative of the concentrations attainable in the intestinal lumen after actual levels of human oral exposure. In these conditions, no cytotoxicity in terms of cellular viability was observed. However, at the highest CPF nominal concentration (250 μM) the impairment of barrier integrity was evidenced, due only to the parent compound, since no CPF metabolites could be detected in our experimental conditions. CPF itself was demonstrated to interfere with the tight junction on this in vitro model of epithelial intestinal cells, altering the barrier integrity and very likely the absorption of other co-administered chemicals.

T Lymphocyte Transduction with Herpes Simplex VirusThymidine Kinase (HSV-tk) Gene: Comparison of Four Different Infection Protocols

In this study, we assessed the efficiency of T lymphocyte transduction with a retroviral vector carrying the herpes simplex virus thymidine kinase (HSV-tk) and neomycin phosphotransferase (neo) genes by four different protocols: standard supernatant infection, supernatant infection plus centrifugation steps, supernatant infection on fibronectin fragment-coated wells, and cocultivation. After retrovirus-mediated gene transfer of tk-neo in PHA/IL-2-stimulated primary T lymphocytes and G418 selection, T cells retained their proliferative activity, alloresponsiveness, ability to produce and to respond to IL-2, and ganciclovir (gcv)-specific sensitivity. When the four different transduction techniques were compared, no significant differences were seen in terms of cellular viability, proliferation capacity, and immunophenotyping. tk gene expression was the same in all transduced selected populations, as indicated by gcv sensitivity. Transduction efficiency was evaluated by semiquantitative PCR. Using the standard supernatant infection method, the rate of infection was extremely low (<5%). After adding the centrifugation step or performing supernatant infection on fibronectin fragment-coated wells, PCR analysis showed a 30%-40% rate of transduced cells. After infection by cocultivation, the rate of transduced cells was 30%-40%. These results demonstrate that supernatant infection plus centrifugation, supernatant infection on fibronectin fragment-coated wells, and cocultivation methods provide equivalent rates of transduced cells. The lack of reproducibility and safety indicates that cocultivation is not suitable for clinical studies. In our view, supernatant infection plus centrifugation is easier to perform than using fibronectin fragments, and it is currently the optimal method for clinical studies when large quantities of T lymphocytes are being processed.

Prolonged depletion of ATP because of delayed repletion of the adenine nucleotide pool following reversible myocardial ischemic injury in dogs.

Sixty-five percent of the ATP and 50% of the total adenine nucleotide (sigma Ad) pool is lost from the subendocardial myocardium after 15 min of severe ischemia induced by circumflex artery occlusion in open-chest dogs (12). In the present experiment, we assessed the effects of various periods of arterial reflow following 15 min of ischemic injury on resynthesis of ATP and sigma Ad. The circumflex artery was occluded for 15 min and reperfused for 20 or 60 min or 24 or 96 hr. The mean ATP after 15 min of ischemia was reduced 62% from 5.42 +/- 0.33 to 2.08 +/- 0.21 mumol/g; and the total nucleotide content was reduced by 50%. ATP content recovered slightly during the first 20 min of reperfusion but remained markedly depressed for at least 24 hr because of the initial depletion of adenine nucleotides and because minimal salvage of de novo repletion occurred in the injured muscle during this time period. By 4 days, ATP and total adenine nucleotides were still slightly depressed but had recovered to 88% and 91% of control. Electrolyte changes and an increased inulin-diffusible space, which are characteristic of irreversibly injured myocardium, reperfused for 20 or 60 min, were not observed. Also, tissue necrosis was absent in the hearts reperfused for 24 or 96 hr. These observations indicate that the marked depression of ATP and adenine nucleotides and the slow recovery of these metabolites occurred in myocardium that nevertheless was reversibly injured in terms of cellular viability.

Prolonged depletion of ATP and of the adenine nucleotide pool due to delayed resynthesis of adenine nucleotides following reversible myocardial ischemic injury in dogs

After 15 min of severe ischemia induced by circumflex artery occlusion in open-chest dogs, 65% of the ATP and 50% of the total adenine nucleotide (ΣAd) pool is lost from the subendocardial myocardium [ 12]. Nevertheless, this injury is reversible if the affected tissue is reperfused with coronary arterial blood. In the present experiment, we assessed the effects of various periods of arterial reflow following 15 min of ischemic injury, on resynthesis of ATP and ΣAd. The circumflex artery was occluded for 15 min and reperfused for 20 or 60 min, or 24 or 96 h. Ten seconds prior to excision of the heart, the circumflex artery was reoccluded and the fluorescent dye thioflavine S was injected intravenously in order to identify the ischemic or the reperfused tissue which had been ischemic. The mean ATP after 15 min of ischemia was reduced 62% from 5.42 ± 0.33 to 2.08 ± 0.21 μmol/g; and the total nucleotide content was reduced by 50%. ATP content recovered slightly during the first 20 min of reperfusion but remained markedly depressed for at least 24 h because of the initial depletion of adenine nucleotides and because minimal salvage or de novo synthesis occurred in the injured muscle during this time period. By 4 days, ATP and total adenine nucleotides were still slightly depressed but had recovered to 88% and 91% of control. Electrolyte changes and an increased inulin diffusible space, which are characteristic of irreversibly injured myocardium reperfused for 20 or 60 min, were not observed. Also tissue necrosis was absent in the hearts reperfused for 24 or 96 h. These observations indicate that the marked depression of ATP and adenine nucleotides and the slow recovery of these metabolites occurred in myocardium which nevertheless was reversibly injured in terms of cellular viability.