Greater Cell Viability Research Articles

Abstract 337: Kidney Injury Affects Metabolism and Modification of Apolipoprotein A1 Promoting Renal Lymphangiogenesis

Kidney disease causes ApoAI/HDL to lose their vasculoprotective effects. A likely mechanism for the protective loss is modification of ApoAI/HDL reactive carbonyls which are increased. Kidney injury increases reactive carbonyls and permits more ApoAI to escape into the glomerular ultrafiltrate. We examined how apoAI was metabolized in the injured proteinuric kidney. Nphs1-hCD25 mice (NEP25) expressing human CD25 in podocytes were injured by immunotoxin, LMB2. Six weeks after LMB2, we assessed urinary excretion and intrarenal localization of ApoAI and examined the renal lymphatic network with podoplanin. In vitro, we studied proximal tubular cells (PTC) and lymphatic endothelial cells (LEC) exposed to ApoAI or modified ApoAI (IsoLG-ApoAI). NEP25 mice developed glomerulosclerosis, increased urinary excretion of albumin and ApoAI with high molecular weight forms of ApoAI appearing in urine of injured mice assessed by western blot. Proteinuric kidneys had more ApoAI and VEGFC staining that localized to tubular epithelial cells and showed a dramatically more dense and complex lymphatic network than wild type mice. ApoAI localized to lymphatic vessels. Cultured PTC and LEC responded differently to normal and isolevuglandin (IsoLG)-modified ApoAI. In PTC, IsoLG-ApoAI promoted more ApoAI uptake and VEGFC secretion, a powerful lymphaniogenic stimulus. In LEC, IsoLG-ApoAI led to greater cell viability, migration and ROS production than normal ApoAI. Notably, supernatant of PTC exposed to ApoAI secreted a high molecular weight form of ApoAI. In conclusion, injury that disrupts the glomerular filtration barrier causing proteinuria also leads to a dramatic increase in filtered ApoAI followed by greater uptake by the proximal tubules, interstitium and lymphatic vessels. Proximal tubules can modify ApoAI and activate tubular secretion of VEGFC, which in turn promotes renal lymphangiogenesis and encourages delivery of kidney-modified ApoAI into circulation.

Cytotoxicity of three commercial orthodontic elastomeric ligature brands

This study aimed to evaluate cytotoxicity of three commercial brands of orthodontic elastomeric ligatures. Materials and Methods: The three commercial orthodontic elastomeric ligature brands were C-brand, T-brand and U-brand from different manufacturing countries. The cytotoxic assay was performed using cell cultures (L929) which were subjected to the cell viability test with methyl-tetrazolium (MTT). The L929 cell line was grown in 96-well tissue culture plates (1×10 5 cells/cm 3 ). Elastomeric ligatures (0.2 g) from each brand was placed in 1 ml of Minimum Essential Medium (MEM) at 37°C with 5% CO 2 at 100% humidity in an incubator for 24 hours. After incubation, the cell mitochondrial activity was evaluated by the MTT test for detecting viable cells from optical density according to ISO 10993-5: 2009 (E) test procedure. Cytotoxic effects were determined by the percentage of cell viability. Results: Microscopic investigation showed that all commercial elastomeric ligatures caused alterations in the fibroblast cell morphology from an elongated to a spherical form. The cell viability of C-brand, T-brand and U-brand were 51.10, 73.04 and 93.04%, respectively. Conclusion: T-brand and U-brand orthodontic elastomeric ligatures were non cytotoxic potential. U-brand had the greatest cell viability and C-brand had the lowest cell viability at 24 hours.

Abstract 401: Preservation of cells in blood and culture

Abstract Introduction: Liquid biopsies and cell-based therapies are emerging approaches for the treatment of cancer. Accuracy of diagnosis using liquid biopsies and effectiveness of treatment using cell-based therapies will rely upon the preservation cells for both viability and the information contained in them. Experimental procedures: Biomatrica's proprietary chemical library was used to develop formulations that could preserve cells in multiple formats. Cell preservation was assessed by a variety of cell-based and molecular techniques such as microscopy, flow cytometry and real-time PCR. Results: Blood samples spiked with circulating tumor cells (CTCs) were preserved for several days at room temperature to allow for downstream morphological and transcriptomic analyses. CTC recoveries in preserved samples were as much as 4 times those in non-protected samples over 4 days. Changes in RNA expression of CTC-specific genes in preserved samples ranged from 0 to +/- 2 delta Ct values, while in non-protected samples, changes ranged from 0 to +/-5 delta Ct values over 4 days. In some formulations, CTC viabilities were as much as twice the viabilities of CTCs in non-protected samples. Supporting these viability results was a demonstrated culturability of isolated CTCs. Furthermore, cell-free DNA in plasma of preserved blood samples changed by less than 2 Ct values over 4 days, whereas in control samples, plasma DNA changed by as much as 10 Ct values. Hemolysis in preserved blood samples was also lower by as much as 69% compared to non-protected samples. In a separate set of experiments, activated T-cells were stored in the presence of common buffers (RPMI, PBS) or formulations at room temperature. T-cells cultured with formulations displayed greater 40 to 60% greater cell viability after 3 days compared to those stored in common buffers. Conclusions: Biomatrica has developed a library of cell preservatives that can be used to preserve cells in blood samples and in cell culture. The ability to recover cells in blood samples and determine their genetic information would enable monitoring of a patient's cancer status and personalizing drug treatments. In addition, cell preservation may allow further advances in cell-based therapies as cells could be more reliably recovered from patients, modified (e.g. CAR-T, stem cells), and administered to patients. Citation Format: Cecille Browne, Dan Lu, Daniela Roth, Vasco Liberal. Preservation of cells in blood and culture. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 401.

Long Chain Fatty Acid Esters of Quercetin-3-O-glucoside Attenuate H₂O₂-induced Acute Cytotoxicity in Human Lung Fibroblasts and Primary Hepatocytes.

Cellular oxidative stress causes detrimental effects to macromolecules, such as lipids, nucleic acids and proteins, leading to many pathological conditions. Quercetin-3-O-glucoside (Q3G), a glycosylated derivative of quercetin (Q), is a natural polyphenolic compound known to possess antioxidant activity. The hydrophilic/lipophilic nature of an antioxidant molecule is considered as an important factor governing the accessibility to the active sites of oxidative damages in vivo. Six long chain fatty acid esters of Q3G were evaluated with comparison to Q and Q3G, for their cytoprotective activity under H2O2-induced oxidative stress using cell culture model systems through cell viability, lipid peroxidation and fluorescence microscopy studies. Pre-incubation of α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) esters of Q3G exhibited significantly (p ≤ 0.05) greater cell viability in both human lung fibroblast (WI-38) and human primary hepatocytes upon exposure to H2O2 insult when compared to the control. Cytoprotection due to oleic acid and linoleic acid esters of Q3G was observed only in human primary hepatocytes. All the derivatives, Q3G and quercetin showed ability to significantly (p ≤ 0.05) lower production of lipid hydroperoxides under induced oxidative stress, compared to the control. However, ALA and DHA esters of Q3G resulted in significantly lower lipid hydroperoxidation than Q and Q3G. Based on fluorescence microscopy study, H2O2-induced apoptosis was attenuated by the fatty acid derivatives of Q3G. The fatty acid derivatives of Q3G possess better cytoprotective effect than Q3G against H2O2-induced cytotoxicity in vitro and the concentration should be selected to avoid cytotoxicity.

About electrochemical stability and biocompatibility of two types of CoCr commercial dental alloys

The present paper is an investigation of two commercial Co‐Cr dental alloys, named Wirobond 280 and Wirobond C. The two alloys have similar composition; the difference is made by the small amount of Ga and Mn existing in Wirobond 280. The electrochemical behavior of the alloys was tested in the artificial Erikson saliva at different temperatures between 25 and 55 °C. The electrochemical methods used have been the potentiodynamic polarization and the electrochemical impedance spectroscopy. The investigation have been performed in the presence of two different fluoride ions concentrations and permitted to compute corrosion rate and the energy activation. In order to complete the tests the cell response has been studied in vitro. The stability of Wirobond 280 alloy is correlated with the greater cell viability and with a smaller level of ROS in comparison with the Wirobond C alloy. Regarding fluoride doping effect, based on the experimental data, we can conclude that NaF increases the corrosion rate of the Wirobond 280 and decrease the corrosion rate in the case of the Wirobond C. The study established that the Wirobond 280 alloy is the one with best properties.

Abstract 1444: Docosahexaenoic acid reduces cancer cell migration may link with actin binding proteins and miRNA 17-92 cluster expressions changes

Abstract Metastasis and progression, reflected by higher migration and longer cell survival, are major contributing factors in making cancers the leading cause of death worldwide. Cancer cells exhibit higher migration rates and greater cell viability facilitated by actin binding protein-mediated cytoskeletal remodeling and secondary messengers (cAMP and/or cGMP) signaling. Studies have shown that docosahexaenoic acid (DHA), a poly unsaturated fatty acid, inhibits cancer cell metastatic phenotype. Moreover, miRNA-17∼92 cluster has been reported to be highly expressed in metastasis. Therefore, we put forward a hypothesis that DHA will induce changes in actin binding protein dynamics and miRNA-17∼92 cluster expression to inhibit cancer cell migration and viability. Non-cance (MLE12) and cancer (A549) cells were treated with 8-Br-cAMP and/or DHA for 30 min, 3h, 6h and 24h. Actin binding proteins (profilin, cofilin, vimentin and gelsolin) and miRNA-17∼92 cluster expression were analyzed by western blot and qRT PCR respectively. Cell migration was estimated by wound assay and transwell apparatus. F-actin content and actin binding proteins were measured using confocal microscopy at the leading edges of wound. Proliferation and cell viability were estimated by flow cytometer, IF using ki-67, and cleaved-caspase-3 antibodies. F-actin content, cell migration and proliferation were increased while cell viability was decreased by cAMP. DHA treatment reverses the increase in actin content and cell migration in cancer cells but not in non-cancer cells. These findings correlate with changes in actin binding protein content and miRNA 17∼92 cluster expression. Thus, DHA specifically inhibits cancer cell migration, proliferation, and viability and this effect correlates with decreases in actin binding protein levels and miRNA-17∼92 cluster expression. In conclusion, DHA supplementation suppresses cellular changes related to metastatic potential in cancer cells and could provide therapeutic potential against cancer cell metastasis. Moreover, actin binding proteins and miRNA-17∼92 cluster expression could serve as innovative therapeutic targets and biomarkers for cancer progression. Citation Format: Mehboob Ali, Kathryn M. Heyob, Lynette K. Rogers. Docosahexaenoic acid reduces cancer cell migration may link with actin binding proteins and miRNA 17-92 cluster expressions changes. [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 1444. doi:10.1158/1538-7445.AM2015-1444

Examining the neuroprotective effects of protocatechuic acid and chrysin on in vitro and in vivo models of Parkinson disease

Polypharmacology-based strategies using drug combinations with different mechanisms of action are gaining increasing attention as a novel methodology to discover potentially innovative medicines for neurodegenerative disorders. We used this approach to examine the combined neuroprotective effects of two polyphenols, protocatechuic acid (PCA) and chrysin, identified from the fruits of Alpinia oxyphylla. Our results demonstrated synergistic neuroprotective effects, with chrysin enhancing the protective effects of PCA, resulting in greater cell viability and decreased lactate dehydrogenase release from 6-hydroxydopamine-treated PC12 cells. Their combination also significantly attenuated chemically induced dopaminergic neuron loss in both zebrafish and mice. We examined the molecular mechanisms underlying these collective cytoprotective effects through proteomic analysis of treated PC12 cells, resulting in the identification of 12 regulated proteins. Two were further characterized, leading to the determination that pretreatment with PCA and chrysin resulted in (i) increased nuclear factor-erythroid 2-related factor 2 protein expression and transcriptional activity; (ii) modulation of cellular redox status with the upregulated expression of hallmark antioxidant enzymes, including heme oxygenase-1, superoxide dismutase, and catalase; and (iii) decreased levels of malondialdehyde, a known lipid peroxidation product. Treatment with PCA and chrysin also inhibited activation of nuclear factor-κB and expression of inducible nitric oxide synthase. Our findings suggest that natural products, when used in combination, can be effective potential therapeutic agents for treating diseases such as Parkinson disease. A therapy involving both PCA and chrysin exhibits its enhanced neuroprotective effects through a combination of cellular mechanisms: antioxidant cytoprotection and anti-inflammation.

Evaluation of two human dental pulp stem cell cryopreservation methods.

Dental pulp is a promising source of mesenchymal stem cells for use in cell therapy and regenerative medicine. Methods for storing stem cells with minimum compromise of cell viability, differentiation capacity and function should be developed for clinical and research applications. The aim of this study was to evaluate whether human dental pulp stem cells (hDPSCs) isolated and cryopreserved for 1, 7 and 30 days maintain viability and expression of specific stem cell markers. Human dental pulp stem cells were isolated from 23 healthy patients aged 18 to 31 years. Dental pulp was enzymatically dissociated, and CD105+ cells were separated using the Miltenyi™ system. The hDPSCs were cryopreserved using the Kamath and Papaccio methods. Post-cryopreservation viability was measured by flow cytometry (7AAD) and by the expression of the phenotype markers CD105+/ CD73+, CD34-/CD45-. The Papaccio method showed greater cell viability for cells that had been frozen for 30 days (59.5%) than the Kamath method (56.2%), while the Kamath method provided better results for 1 day (65.5%) and 7 days (56%). Post-cryopreservation expression of the markers CD105+/CD34- was greater after 1 and 7 days with the Kamath method and CD105+/CD45- were expressed after all 3 cryopreservation times. There was greater expression of CD73+ in the hDPSCs after 1 and 7 days with the Kamath method, and after 30 days with the Papaccio method. These results suggest that hDPSCs express mesenchymal stem cell markers after cryopreservation. However, cryopreservation time may affect marker expression, probably by altering the spatialconfiguration of cell membrane proteins or by compromising cells at a certain level of differentiation.

Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.

The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×104 cells/mL (8.9×103 cells/cm2) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).

Electrophoretic deposition of tetracycline modified silk fibroin coatings for functionalization of titanium surfaces

Electrophoretic deposition has been widely used for the fabrication of functional coatings onto metal implant. A characteristic feature of this process is that positively charged materials migrate toward the cathode and can deposit on it. In this study, silk fibroin was decorated with tetracycline in aqueous solution to impart positive charge, and then deposited on negatively titanium cathode under certain electric field. The characterization of the obtained coatings indicated that the intermolecular hydrogen bonds formed between the backbone of silk fibroin and tetracycline molecular. In vitro biological tests demonstrated that osteoblast-like cells achieved acceptable cell affinity on the tetracycline cross-linked silk fibroin coatings, although greater cell viability was seen on pure silk fibroin coatings. The cationic silk fibroin coatings showed remarkable antibacterial activity against gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. Therefore, we concluded that electrophoretic deposition was an effective and efficient technique to prepare cationic silk fibroin coatings on the titanium surface and that cationic silk fibroin coatings with acceptable biocompatibility and antibacterial property were promising candidates for further loading of functional agents.

Processing Technique for Lipofilling Influences Adipose-Derived Stem Cell Concentration and Cell Viability in Lipoaspirate

Autologous fat grafting is a highly used technique in plastic and reconstructive surgery. Several fat-processing techniques have been described, with centrifugation frequently touted as the optimal method. Processing is one factor important for maximizing cell viability and adipose-derived mesenchymal stem cell (ADSC) concentrations. This study compared two methods of fat preparation (centrifugation vs Telfa-rolling) to determine which technique results in the greatest degree of cell viability and ADSC concentration. Abdominal fat was harvested from five patients. Equal aliquots were divided and processed by both centrifugation and Telfa-rolling. Samples were analyzed for ADSC proportions via flow cytometry and cell viability using methylene blue-based cell counting. Paired t tests were performed on all samples, and a P value lower than 0.05 was considered statistically significant. Telfa-rolling processing resulted in a higher percentage of isolated ADSCs (P<0.5 for 3 of 4 parameters) and a significantly higher number of viable cells (P<0.05). Telfa-rolling results in a higher proportion of ADSCs and greater cell viability than centrifugation for donor adipose graft preparation. Further studies are necessary to confirm whether optimal preparation translates to improved augmentation and cell take at the recipient site. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Three-dimensional flow perfusion culture enhances proliferation of human fetal osteoblasts in large scaffold with controlled architecture

To demonstrate the feasibility and benefits of custom designed perfusion bioreactor in conjunction with well-defined three-dimensional (3D) environment for enhanced proliferation and homogeneous distribution of human fetal osteoblasts in large scaffold in vitro. Large-scale β-tricalcium phosphate (β-TCP) scaffolds with tightly controlled architectures were fabricated. And a custom designed perfusion bioreactor was developed. Human fetal osteoblasts were seeded onto the scaffolds, cultured for up to 16 days in static or flow perfusion conditions. At Days 4, 8 & 16 post-incubation, the proliferation and distribution of osteoblasts were determined by daily D-glucose consumption, cell viability (methyl thiazolyl tetrazolium (MTT) assay), histological evaluation and scanning electron microcopy (SEM). Sphere like structures observed in the SEM images were assessed by energy dispersive X-ray (EDX) analysis. In both static and perfusion cultures, the daily D-glucose consumption increased with prolonged time. The daily D-glucose consumption was significantly higher in the perfusion culture than that in static culture (P < 0.05). The increased cell viability with time during the culture was similar to the daily D-glucose consumption under both conditions. There was much greater cell viability under flow perfusion culture compared to static culture (P < 0.05). Flow perfused constructs demonstrated improved cell proliferation and a homogeneous layer composed of cells and extracellular matrix in channels throughout the whole scaffold. However, the cells were biased to periphery in scaffolds culture statically. Sphere like structures present in the matrix were identified as calcium phosphate nodules via EDX analysis. Flow perfusion culture plus well-defined 3D interconnected channel environments enhances the proliferation and improve the distribution of human fetal osteoblasts in large scaffolds. Scaffolds with controlled architecture may be a potential tool of studying the fluid flow configuration and cell behavior inside scaffold in details. And human fetal osteoblasts can be used as a cell source in large bone graft research.

Quantified High-Throughput Screening of Escherichia coli Producing Poly(3-hydroxybutyrate) Based on FACS

Here, we report on a highly sensitive method for the detection of P(3HB) accumulation in Escherichia coli cells based on the automated flow cytometry system using fluorescent dyes. E. coli containing P(3HB) were stained with either BODIPY or Nile red fluorescent dye, and their staining properties were analyzed under a variety of conditions. Compared with Nile red, BODIPY was much more sensitive in staining P(3HB) and overall demonstrated a more rapid staining of cells, a greater resistance to photobleaching, and greater cell viability. In addition, we also successfully monitored heterogeneity in P(3HB) accumulation within a cell population using BODIPY staining and flow cytometry. We believe this optimized staining method using BODIPY in combination with screening by high-speed flow cytometer will be helpful in the engineering of host cells toward an enhanced production of bioplastics.

Chemoresistance in ovarian cancer linked to expression of microRNAs

We evaluated the differential expression of several microRNAs (miRNAs) among malignant cells in ascites and matched omental metastasis in patients with epithelial ovarian cancer (EOC). Ascites and omental tumors were collected prospectively from five patients who were undergoing primary surgical cytoreduction. Patient samples were processed and treated with carboplatin, paclitaxel and combination chemotherapy. Cell viability was evaluated and miRNA profiling was performed on both tumor cells from ascites fluid and omental cake. Quantitative real-time PCR (RT-q-PCR) and western blots were used to evaluate expressions of miRNA-21 and miRNA -214 and associated proteins. Malignant cells in ascites showed greater cell viability when treated with carboplatin compared to omental metastasis. A significant up-regulation of miRNA-21 and miRNA-214 was observed in malignant cells of ascites compared to omental metastasis; this was confirmed by both cell viability assay and RT-q-PCR. Ours is the first report that demonstrates significant up-regulation of miRNA-21 and miRNA-214 in tumor cells from ascites of patients with EOC compared to omental metastasis. This finding has important implications for intrinsic carboplatin resistance in these patients.

Abstract 232: Cancer stem cells and cisplatin.

Abstract Introduction: Cisplatin is an important chemotherapeutic agent, which has resulted in higher overall response rates when combined with 5-FU or taxane combinations in treating squamous cellular carcinoma of the head and neck. However, HNSCC tumor response rates to cisplatin range from 20-30%, a major clinical hurdle today. To address this issue thoroughly, we investigated the resistance to cisplatin in HNSCC under the purview of the cancer stem cell hypothesis, using the widely studied CSC marker combinations of, CD44+ 24- and ALDH-Hi. In order to get a broader perspective on this issue, we worked on multiple HNSCC tumor cell lines with varying degrees of cisplatin resistance, arising from tumors of the pharynx, larynx, tongue, tonsils, alveolus, hypopharynx and supraglottis. Methods: Seven HNSCC cell lines were analyzed for their CSC percentage through FACS, utilizing CD44+ 24- and ALDH-Hi marker systems. Cisplatin IC-50 for each of these seven cell lines was then determined by plotting percent viability vs. drug concentration curves. The remaining viable cells after cisplatin treatment were then analyzed through FACS for their CD44+ 24- and ALDH-Hi percentages in order to compare with the CSC percentage in the untreated HNSCC lines. Results: There was a wide variation in the percentages of CD44+24- and ALDH-Hi cells across these seven HNSCC lines. An inverse correlation between the initial percentages of CD44+24- and ALDH-Hi cells and the corresponding cisplatin IC-50 values was seen. HNSCC cell lines that had lower IC-50 values for cisplatin displayed greater cell viability at higher concentrations of cisplatin. This was evident by a long tail in the sigmoidal dose response curve. The number of viable cells at the end of cisplatin treatment had a linear relationship with the overall percentage of CSCs in the control untreated cell lines. Conclusion: Our results suggest that the resistance offered by the CD44+24- and ALDH1-Hi cells is apparent only once the initial 50% loss of cells occurs. This in vitro biphasic decline in percent viable cells clearly indicates that HNSCC cisplatin resistance is a twofold problem. The bulk of non-cancer stem cells within a tumor can acquire resistance to cisplatin via multiple molecular pathways, but this is only a front line cover of resistance to the resistance offered by the cancer stem cells. Citation Format: Vishnu Modur. Cancer stem cells and cisplatin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 232. doi:10.1158/1538-7445.AM2013-232

Osteoblastic Cell Response and Bone Formation of Phosphate Ion Coated on Plasma Polymerized Ti Surface

This study examined the bone formation ability and cell response on a phosphate (PO3(4-)) ion exchanged amine plasma polymerized titanium (Ti) surface. The enhanced bone-like apatite (hydroxyapatite, HAp)-forming ability was attributed to the PO3(4-) ion exchanged amine plasma polymerized Ti (P/NH2/Ti) surface, which was formed by the reduction of PO3(4-) ions. PO3(4-) ions promote HAp nucleation and growth on Ti in SBF, and PO3(4-) ions improve the crystallinity of the HAp deposited layer. The cell viability tests revealed significantly greater cell viability on the P/NH2/Ti surfaces than on the other surfaces.

209 FIBRIN TG-VEGF IS A SUPPORTER OF EARLY ANGIOGENESIS IN URINARY SPHINCTER ENGINEERING

INTRODUCTION AND OBJECTIVES: A 3-D construct seeded with autologous cells on a biodegradable scaffold could serve as tissue replacement in pelvic floor reconstruction. Our goals are to design a scaffold that incorporates suture suitable for implantation and optimizes the conditions for a seeded implant in a 3-D construct by testing various formulations of collagen and hyaluronan. METHODS: The C2C12 mouse myoblast cell line was used. Scaffold matrix components included collagen and hyaluronan (HystemHP®, Glycosan Biosystems). Constructs were seeded at 3, 4.5, and 6 10 cells/ml and polymerized in a silicone mold. 1:1,10:1 and 20:1 ratios of collagen:hyaluronan (C:H) were tested. Three scaffold designs were tested: constructs with a suture running through the middle, with Nitex mesh at the ends only, and with suture and mesh just inside the edges of the matrix. After 21 days, constructs were tested for passive stiffness. The constructs were then stained for dead cells, cell nuclei and filamentous actin, and confocal imaging was performed. RESULTS: All construct conditions resulted in elongated, differentiated muscle cells with filamentous actin that appeared fused and multinucleated. C:H at 1:1 produced a more cellular construct with a greater proportion of elongated cells and fewer dead cells at 3 weeks than the 20:1 and 10:1 (C:H) samples (Figure 1). Constructs seeded with 6 10 cells/ml and C:H of 1:1 were 2x stiffer than those seeded at lower densities. The suture-only design (tension-free) resulted in random cell aggregation. Mesh alone also resulted in cell aggregation due to lack of integration of cells into the mesh. Combining suture and mesh allowed cells to integrate into the mesh and promoted rearrangement of the cells and matrix with development of uniaxial tension between suture ends which promoted cell elongation and alignment. CONCLUSIONS: Greater cell viability was observed in constructs with an equal ratio of C:H. Combining suture with mesh located inside the matrix promoted the development of uniaxial tension that allowed the cells to align in the direction of tension. A higher seeding density resulted in a stiffer construct. Further testing is needed to determine the viability and functionality of constructs in vivo.

Surface conjugation of triphenylphosphonium to target poly(amidoamine) dendrimers to mitochondria

Dendrimers have emerged as promising carriers for the delivery of a wide variety of pay-loads including therapeutic drugs, imaging agents and nucleic acid materials into biological systems. The current work aimed to develop a novel mitochondria-targeted generation 5 poly(amidoamine) (PAMAM) dendrimer (G(5)-D). To achieve this goal, a known mitochondriotropic ligand triphenylphosphonium (TPP) was conjugated on the surface of the dendrimer. A fraction of the cationic surface charge of G(5)-D was neutralized by partial acetylation of the primary amine groups. Next, the mitochondria-targeted dendrimer was synthesized via the acid-amine-coupling conjugation reaction between the acid group of (3-carboxypropyl)triphenyl-phosphonium bromide and the primary amines of the acetylated dendrimer (G(5)-D-Ac). These dendrimers were fluorescently labeled with fluorescein isothiocyanate (FITC) to quantify cell association by flow cytometry and for visualization under confocal laser scanning microscopy to assess the mitochondrial targeting in vitro. The newly developed TPP-anchored dendrimer (G(5)-D-Ac-TPP) was efficiently taken up by the cells and demonstrated good mitochondrial targeting. In vitro cytotoxicity experiments carried out on normal mouse fibroblast cells (NIH-3T3) had greater cell viability in the presence of the G(5)-D-Ac-TPP compared to the parent unmodified G(5)-D. This mitochondria-targeted dendrimer-based nanocarrier could be useful for imaging as well as for selective delivery of bio-actives to the mitochondria for the treatment of diseases associated with mitochondrial dysfunction.

The role of surface charge on the uptake and biocompatibility of hydroxyapatitenanoparticles with osteoblast cells

The objective of this study is to evaluate the effect of hydroxyapatite (HAP) nanoparticleswith different surface charges on the cellular uptake behavior and in vitro cell viability andproliferation of MC3T3-E1 cell lines (osteoblast). The nanoparticles’ surface charge wasvaried by surface modification with two carboxylic acids: 12-aminododecanoic acid(positive) and dodecanedioic acid (negative). The untreated HAP nanoparticles anddodecanoic acid modified HAP nanoparticles (neutral) were used as the control. X-raydiffraction (XRD) revealed that surface modifications by the three carboxylic acids did notchange the crystal structure of HAP nanoparticles; Fourier transform infrared spectroscopy(FT-IR) confirmed the adsorption and binding of the carboxylic acids on the HAPnanoparticles’ surfaces; and zeta potential measurement confirmed that the chemicalssuccessfully modified the surface charge of HAP nanoparticles in water based solution.Transmission electron microscopy (TEM) images showed that positively charged,negatively charged and untreated HAP nanoparticles, with similar size and shape,all penetrated into the cells and cells had more uptake of HAP nanoparticleswith positive charge compared to those with negative charge, which might beattributed to the attractive or repulsive interaction between the negatively charged cellmembrane and positively/negatively charged HAP nanoparticles. The neutral HAPnanoparticles could not penetrate the cell membrane due to their larger size. MTTassay and LDH assay results indicated that as compared with the polystyrenecontrol, greater cell viability and cell proliferation were measured on MC3T3-E1cells treated with the three kinds of HAP nanoparticles (neutral, positive, anduntreated), among which positively charged HAP nanoparticles showed the strongestimprovement for cell viability and cell proliferation. In summary, the surfacecharge of HAP nanoparticles can be modified to influence the cellular uptake ofHAP nanoparticles and the different uptake also influences the behavior of cells.These in vitro results may also provide useful information for investigations ofHAP nanoparticle applications in gene delivery and intracellular drug delivery.

Human Chondrocyte Viability After Treatment With Local Anesthetic and/or Magnesium: Results From an In Vitro Study

We aimed to assess the effect on chondrocyte viability of adding magnesium to a variety of commonly available local anesthetic agents. Human chondrocytes were grown under standard culture conditions. Cells were exposed to a local anesthetic agent with the addition of magnesium (10%, 20%, or 50%). Cells were also exposed to the varying concentrations of magnesium and 0.9% saline solution. Untreated cells served as controls. The CellTiter 96 AQueous One Solution Cell Proliferation Assay was used to assess for cell viability 24 hours after exposure. One-way analysis of variance was used to test for statistical significance. Magnesium sulfate alone was no more toxic than normal saline solution (P > .3) compared with untreated cells. The addition of magnesium to the local anesthetic agents resulted in greater cell viability than when cells were treated with a local anesthetic alone (lidocaine [P = .033], levobupivacaine [P = .007], bupivacaine [P < .001], and ropivacaine [P < .001]). Our findings support the use of magnesium either alone or in combination with a local anesthetic rather than a local anesthetic alone, and this represents a potential strategy for the reduction of chondrocyte toxicity associated with intra-articular local anesthetic administration after arthroscopy. The addition of magnesium to a local anesthetic results in a reduced toxic effect to the articular chondrocyte. This may represent a potential approach to intra-articular analgesia.