Global Medical Discovery features paper: Inkjet-Print Micromagnet Array on Glass Slides for Immunomagnetic Enrichment of Circulating Tumor Cells

Significance Statement

To advance cancer studies and enable oncologists to make more accurate diagnosis, prognosis, and develop personalized therapies, Prof. John X.J. Zhang and Dr. Peng Chen led a team of bioengineers from the Thayer School of Engineering at Dartmouth and the University of Texas at Austin in developing an innovative system that combines inkjet-printing, microfluidic technology, and immunoassay for capturing and manipulating circulating tumor cells (CTCs). This miniaturized bio-analytical system can be further integrated with fluorescent microscope for cancer cell imaging, with fluorescent in-situ hybridization (FISH) and PCR for molecular level cancer studies. It opens up a great many possibilities to optimize cancer diagnostic and tumor management.

“This work provides a new angle, other than traditional tissue biopsy and medical imaging, to approach cancer. A fast, simple and less painful blood test may eventually provide equivalently accurate disease information about individual cancer patient in a timely manner, ” said Zhang. circulating tumor cells detach from primary tumor site, shed into the blood circulation system, and may initiate the deadly cancer metastasis process. Through efficient detection of these cells, especially at an early stage, physicians will be able to design the best diagnosis and treatment strategy for each individual cancer patient.

Immunomagnetic assay has been successfully used to separate rare circulating tumor cells from blood. However, traditional immunomagnetic assay is often limited in the low magnetic field gradient and low density of effective magnetic traps. In this work, Dr. Chen developed a novel method using inkjet-printing technology to fabricate microscale magnetic structures that can be easily deposited on an arbitrary substrate.

This inkjet-printing technology is a versatile but cost-effective approach for rapid prototyping with high accuracy and flexibility. Such microscale magnets, when placed in an external magnetic field, largely enhance the magnetic field and the attractive force applied on the target circulating tumor cells, and hence facilitates the detection. In our experiments with COLO205 (a human colorectal cancer cell line) as the separation target, the inkjet-printed micro-magnets integrated assay increased the system sensitivity by 26% compared with using normal glass slide as the substrate.

Dr. Chen’s research is focused on developing miniaturized high-performance bio-analytical systems for point-of-care and globally relevant medical diagnostic applications. “This project is to implement advanced engineering fabrication, sensing, and manipulating techniques with multiplexing immunoassay to separate these rare circulating tumor cells from whole blood.” Zhang said. “The ultimate goal is to deliver a highly sensitive, reliable, affordable, and portable cancer screening platform with high throughput. The next step – bringing the technology from lab bench to clinics is a big challenging step, but is also the most rewarding step.

This work has the potential to revolutionize cancer risk assessment, cancer management, and increase the cure rate for cancers such as breast cancer, lung cancer, and prostate cancer.” This paper is published on Annals of Biomedical Engineering (DOI: 10.1007/s10439-015-1427-z).

      

About The Author

Dr. Peng Chen received his PhD degree in Biomedical Engineering from the University of Texas at Austin. He is now working as a postdoc research associate at the Center for Applied NanoBioscience and Medicine at the University of Arizona. His research interests include (1) miniaturized microfluidic system for detection and analysis of rare circulating tumor cells (CTCs), and (2) vertical flow paper based microfluidic device as point-of-need multiplex diagnostic and surveillance tool for bio-threat detection. He has published 10 peer-reviewed journal papers and 10 conference proceedings. He now serves the editorial board of Scientific Reports (Nature publishing group). Email: pengchen@email.arizona.edu

About The Author

Prof. John Zhang, Ph.D., is a Professor at Thayer School of Engineering at Dartmouth, and a Fellow of American Institute for Medical and Biological Engineering (AIMBE). He received his Ph.D. from Stanford University, and was a Research Scientist at MIT. His key contribution is in developing miniature medical systems to improve global health, through innovations in bio-inspired nanomaterials, lab-on-chip design, and advanced nanofabrication technologies for probing complex biological networks critical to human development and diseases such as cancer. He received the Wallace Coulter Foundation Early Career Award for developing handheld microphotonic imaging scanners and microsystems for early oral cancer detection; NSF CAREER award for the invention of plasmonic scanning probes design for controlled perturbation and imaging at sub-cellular level; and DARPA Young Faculty Award for patterning plasmonic surface on MEMS for biomarker sensing applications. He has published over 120 peer reviewed papers and proceedings, presented over 45 invited seminars worldwide, and filed over 60 US and international patents. He is an alumnus of NAE Frontiers of Engineering programs, an Associate Editor for Biomedical Microdevices, IEEE/ASME Journal of Microelectromechanical Systems, and has published a textbook for undergraduates titled “Molecular Sensors and Nanodevices: Principles, Designs and Applications in Biomedical Engineering”. Email: john.zhang@dartmouth.edu

Reference

Ann Biomed Eng. 2016;44(5):1710-20.

Peng Chen¹, Yu-Yen Huang², Gauri Bhave¹, Kazunori Hoshino³, Xiaojing Zhang^4,5

Show Affiliations

¹Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, 78712, USA.

²Thayer School of Engineering, Dartmouth College, Hanover, NH, 03755, USA.

³Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269, USA.

⁴Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, 78712, USA.

⁵Thayer School of Engineering, Dartmouth College, Hanover, NH, 03755, USA. john.zhang@dartmouth.edu.

We report an inkjet-printed microscale magnetic structure that can be integrated on regular glass slides for the immunomagnetic screening of rare circulating tumor cells . circulating tumor cells detach from the primary tumor site, circulate with the bloodstream, and initiate the cancer metastasis process. Therefore, a liquid biopsy in the form of capturing and analyzing circulating tumor cells may provide key information for cancer prognosis and diagnosis. Inkjet printing technology provides a non-contact, layer-by-layer and mask-less approach to deposit defined magnetic patterns on an arbitrary substrate. Such thin film patterns, when placed in an external magnetic field, significantly enhance the attractive force in the near-field close to the circulating tumor cells to facilitate the separation. We demonstrated the efficacy of the inkjet-print micromagnet array integrated immunomagnetic assay in separating COLO205 (human colorectal cancer cell line) from whole blood samples. The micromagnets increased the capture efficiency by 26% compared with using plain glass slide as the substrate.

Go To Annals of Biomedical Engineering

Global Medical Discovery features paper: Impact of patient characteristics on the clinical efficacy of mongersen (GED-0301), an oral Smad7 antisenseoligonucleotide, in active Crohn’s disease

Significance Statement

Crohn’s disease (CD) is a chronic condition characterized by segmental, transmural inflammation, which, although most common in the terminal ileum and right colon, can affect any part of the alimentary tract. In addition to the commonly observed signs and symptoms of Crohn’s disease, local complications as well as manifestations outside the digestive tract can also occur as a result of Crohn’s disease -associated inflammation, underscoring the importance of effective treatment options.

Subgroup analyses from prospective, randomised, controlled trials in patients with active Crohn’s disease have shown that patient demographics and disease characteristics such as disease duration and treatment history can impact clinical outcomes. Our retrospective study further evaluated the impact of patient baseline clinical and disease characteristics on the efficacy of mongersen, an oral, locally active Smad7 antisense oligonucleotide that targets Smad7 in the ileum and colon. In addition to the impact of human serum C-reactive protein (hsCRP) values, disease duration, and disease activity at baseline on mongersen over 10 weeks, our analysis also looked at how sex, body mass index, smoking status, history of Crohn’s disease-related intestinal resection, steroid status, and immunosuppressant use at baseline impacted treatment efficacy.

Our results showed that patients with Crohn’s Disease Activity Index (CDAI) scores ≤260 at baseline had significantly higher clinical remission rates (defined as a CDAI <150) with mongersen 40 mg/day and 160 mg/day treatment, whereas patients with CDAI scores >260 at baseline achieved clinical remission most frequently with the highest mongersen dose (160 mg/day), suggesting that greater disease activity can impact clinical benefit. Baseline disease characteristics such as hsCRP (<3 mg/L or ≥3 mg/L) and disease duration (<5 or ≥5 years) did not appear to significantly impact efficacy of mongersen treatment in our study.

About The Author

Giovanni Monteleone received his medical degree from the University Magna Graecia of Catanzaro (Italy) and completed his internship, residency, and a fellowship in gastroenterology at the same university. He joined the faculty at the University of Rome Tor Vergata in 2003, becoming an Assistant Professor of Gastroenterology, then Professor of Gastroenterology. He is the Head of Gastroenterology Unit at the Policlinico Tor Vergata in Rome. Dr. Monteleone’s basic research is mostly focused on mechanisms involved in the control of mucosal immune homeostasis and inflammatory signals that sustain and amplify pathological processes in the gastrointestinal tract. In particular, he is interested in understanding how immune cells and non-immune cells cross-talk in the gastrointestinal mucosa and which molecules mediate such an interplay. So far, these studies have contributed to delineate novel pathways of intestinal mucosal damage and paved the way for the development of anti-inflammatory compounds, which are now ready to move into the clinic. Dr. Monteleone is also interested in the immune-inflammatory networks involved in the colitis-associated colon carcinogenesis. He is author of >300 peer-reviewed articles, books, and book chapters, and serves as an editorial board member or a reviewer for several journals.

 

Journal Reference

Aliment Pharmacol Ther. 2016 ;43(6):717-24.

Monteleone G¹, Di Sabatino A², Ardizzone S³, Pallone F¹, Usiskin K⁴, Zhan X⁴, Rossiter G⁴, Neurath MF⁵. 

Show Affiliations

1. Department of Systems Medicine, University of Tor Vergata, Rome, Italy.
2. First Department of Internal Medicine, St. Matteo Hospital Foundation, University of Pavia, Pavia, Italy.
3. Department of Surgery, “L. Sacco” University Hospital, Milan, Italy.
4. Celgene Corporation, Warren, NJ, USA.
5. Department of Medicine, University of Erlangen-Nürnberg, Erlangen, Germany.

Abstract

BACKGROUND:

In a phase 2 study, mongersen, an oral antisense oligonucleotide targeting Smad7, was effective in inducing clinical remission in approximately 60% of patients with active Crohn’s disease (CD).

AIM:

In a post hoc analysis to evaluate those patient disease characteristics that may have influenced the efficacy and safety of mongersen therapy.

METHODS:

Patients with steroid-dependent/resistant, active Crohn’s disease were randomised to mongersen 10, 40 or 160 mg/day or placebo for 2 weeks; patients were followed for 10 weeks. Clinical remission [Crohn’s Disease Activity Index (CDAI) score <150] and clinical response (CDAI score reduction ≥100 points) were assessed at weeks 2, 4 and 12 for these subgroups: disease duration <5/≥5 years, human serum C-reactive protein (hsCRP) <3/≥3 mg/L, and CDAI at baseline ≤260/>260. Additional patient baseline and disease characteristics were explored.

RESULTS:

Clinical remission and response rates were significantly higher in patients receiving mongersen 40 and 160 mg/day but not 10 mg/day vs. placebo and independent of disease duration and hsCRP. Patients with baseline CDAI ≤260 had significantly higher remission rates with 40 and 160 mg/day. In patients with baseline CDAI >260, remission rates were statistically greater with 160 mg/day and numerically better with 40 mg/day vs. placebo. Adverse event rates were similar across treatment groups. Mongersen was safe and well tolerated.

CONCLUSIONS:

Patients with higher CDAI scores achieved clinical remission most frequently with the highest mongersen dose. Disease duration and baseline human serum C-reactive protein did not appear to significantly impact efficacy of mongersen in this study (EudraCT Number: 2011-002640-27.).

© 2016 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd.

Go To Aliment Pharmacol Ther

 

Global Medical Discovery features paper: Nanostructured material formulated acrylic bone cements with enhanced drug release

Significance Statement

Current commercial polymethylmethacrylate (PMMA) based antibiotics-loaded bone cements exhibit very low drug release as most of the antibiotics are embedded inside the polymerized bone cement.  This results in a rapid decrease in antimicrobial activity below the effective therapeutic level within one to several days. To increase the therapeutic window to several weeks for reducing the risk of post-operative joint infection, mesoporous silica nanoparticles (MSN) functional bone cement was developed to enable a sustained release of antibiotics.

Our results show that the novel formulation can be designed to tailor a desired release profile, e.g. up to 70% drug release for several weeks in comparison with commercial bone cement showing 10% or lower drug release and peak concentration only on day one.  MSN with uniform nano-porous channels built up effective diffusion nano-networks which enabled the drug molecules to be released sustainably. Moreover, the mechanical properties of bone cements were well preserved in the presence of MSN in formulation even after drug release.

This formulation is expected to deliver the next generation bone cement with tailor-designed antibiotics release profiles and prolonged antibacterial activity.  The key commercial advantages to potential bone cement manufacturers and end users are sustained antibiotics release, reduction in standard antibiotics dosage, minimised use of intravenous doses and creating new opportunities to use other benefit ingredient for local targeted delivery.  Once commercialized, the socio-economic impact and savings in medical resources and costs are expected to be significant as the risk of post-operative infections can be reduced.       

About The Author

Reginald Tan received PhD degree from University of Cambridge in 1989. He is Director (Research) of the Institute of Chemical and Engineering Sciences (ICES, Singapore) and concurrently a Professor in the Department of Chemical and Bimolecular Engineering, National University of Singapore. His current research is in crystallisation science and modelling, and formulation sciences.

About The Author

Shoucang SHEN obtained PhD degree from National University of Singapore at 2001. Currently, he is a senior scientist in Institute of Chemical and Engineering Sciences (ICES, Singapore). His research interest includes formulation of poorly soluble drugs and controlled release of active ingredients, as well as the exploration of nanotechnology.

About The Author

Ng Wai Kiong obtained PhD degree from National University of Singapore and Diplom Ingenieur from Technical University of Clausthal, Germany. He is Team Leader (Formulation Sciences) in the Crystallisation and Particle Sciences Division, Institute of Chemical and Engineering Sciences (ICES, Singapore).

Reference

Mater Sci Eng C Mater Biol Appl. 2016 ;58:233-41.

Shen SC¹, Ng WK², Dong YC², Ng J², Tan RB³.

Show Affiliations

¹Institute of Chemical and Engineering Sciences, A*STAR (Agency for Science, Technology and Research), 1 Pesek Road, Jurong Island, Singapore 627833, Singapore. Electronic address: shen_shoucang@ices.a-star.edu.sg.

²Institute of Chemical and Engineering Sciences, A*STAR (Agency for Science, Technology and Research), 1 Pesek Road, Jurong Island, Singapore 627833, Singapore.

³Institute of Chemical and Engineering Sciences, A*STAR (Agency for Science, Technology and Research), 1 Pesek Road, Jurong Island, Singapore 627833, Singapore; Department of Chemical and Biomolecular Engineering, The National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore. Electronic address: reginald_tan@ices.a-star.edu.sg.

Abstract

To improve antibiotic properties, poly(methyl methacrylate) (PMMA)-based bone cements are formulated with antibiotic and nanostructured materials, such as hydroxyapatite (HAP) nanorods, carbon nanotubes (CNT) and mesoporous silica nanoparticles (MSN) as drug carriers. For nonporous HAP nanorods, the release of gentamicin (GTMC) is not obviously improved when the content of HAP is below 10%; while the high content of HAP shows detrimental to mechanical properties although the release of GTMC can be substantially increased. As a comparison, low content of hollow nanostructured CNT and MSN can enhance drug delivery efficiency. The presence of 5.3% of CNT in formulation can facilitate the release of more than 75% of GTMC in 80 days, however, its mechanical strength is seriously impaired. Among nanostructured drug carriers, antibiotic/MSN formulation can effectively improve drug delivery and exhibit well preserved mechanical properties. The hollow nanostructured materials are believed to build up nano-networks for antibiotic to diffuse from the bone cement matrix to surface and achieve sustained drug release. Based on MSN drug carrier in formulated bone cement, a binary delivery system is also investigated to release GTMC together with other antibiotics.

Copyright © 2015 Elsevier B.V. All rights reserved.

Go To Materials Science and Engineering: C

Global Medical Discovery features paper: Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents

Significance Statement

Arteries have the remarkable ability to adjust the amount and force of blood that flows through them. Such adjustments are made possible by contraction or relaxation of arterial smooth muscle cells (ASMCs) within the walls of the arteries. Many hormonal and neuronal actions serve to adjust the contraction of ASMCs, to modulate blood flow and pressure. At the cellular level, contraction of ASMCs depends on the flow of calcium ions into the cells through specialized protein pores or “channels” on the cell’s plasma membrane. Opening of voltage-sensitive calcium channels (VSCCs) involves positive changes to the voltage across the plasma membrane. Relaxation of ASMCs occurs when membrane voltage is maintained around -60 millivolts (negative inside compared to outside) by a flux of potassium ions out of the cells through channels that selectively conduct potassium; this negative voltage inhibits the opening of VSCCs. Among the many types of potassium channels on the ASMC plasma membrane, Kv7 channels, are particularly well suited as targets for hormonal and neuronal regulation of ASMC contraction to adjust blood pressure and blood flow.

Membrane voltage, and hence the flux of calcium ions via VSCCs, is very sensitive to the opening (activation) or closing (e.g. blocking) of Kv7 channels. These channels are tetrameric assemblies constituted by four Kv7 channel alpha subunits. There are five different types of Kv7 channel alpha subunits, named Kv7.1 through 7.5. In ASMCs, Kv7 channels are composed of four Kv7.4 subunits, four Kv7.5 subunits, or by some combination of Kv7.4/7.5 subunits (i.e. heterotetrameric channels). Altering the activity of the Kv7 channels with chemicals that bind directly to the channels has been shown to influence arterial contractility and diameter. However, physiological regulation of Kv7 channel activity is still poorly understood. In particular, it is unclear how the subunit composition of the channels influences the regulation of their activity.

In the article by Mani et al. (PMID: 26700561), the activity of ASMC Kv7 channels was monitored in response to activation of cell surface receptors and intracellular signaling regulators to better understand the physiological regulation of these channels. Furthermore, to study how different channel subunits respond to these stimuli, cultured rat aorta ASMCs (A7r5 cells) that naturally express only Kv7.5 subunits were used, and compared with the responses of ASMCs freshly isolated from rat arteries, which predominantly express heterotetrameric Kv7.4/Kv7.5 channels. In addition, human Kv7 channel alpha subunits were artificially introduced into A7r5 cells to compare regulation of human Kv7.4 or human Kv7.5 channels expressed individually or expressed together.

The results indicated that activation of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), a well-known vasodilatory stimulus, robustly increased the activity of Kv7.5 channels. Activation of cAMP/PKA using chemicals, or by engagement of beta adrenergic receptors that activate this signaling pathway, similarly increased the activity of naturally occurring Kv7.5 channels or of artificially introduced human Kv7.5 channels in A7r5 cells. In contrast, activity of freshly isolated rat artery Kv7.4/Kv7.5 channels or artificially introduced human Kv7.4/7.5 channels were only modestly enhanced, and human Kv7.4 channels were insensitive to activation of this signaling pathway. It was further demonstrated that the changes in activity of the channels by the signaling pathway are dependent on temporary addition of a phosphate group to the Kv7.5 channel subunits. No phosphate additions were detectable by activation of the signaling pathway in cells with artificially introduced Kv7.4 channels.

In summary, these results suggest that the responsiveness of arterial smooth muscle Kv7 channel subunits to intracellular cAMP/PKA signal activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4. The differences in Kv7 channel subunit response may have important implications in terms of arterial function, as the Kv7 channel subunit expression patterns may differ among vascular beds and may change during development or with disease.

      

Figure Legend: Phosphorylation of Kv7.5 alpha subunits enhances efflux of potassium ions from arterial smooth muscle cells. Schematic diagram illustrating the signal transduction pathway whereby activation of β-adrenergic receptors (β-Adr) leads to elevation of cytosolic concentrations of cyclic adenosine monophosphate (cAMP) and hence to activation of Protein Kinase A. Protein kinase A can catalyze the transfer of phosphate (P) to Kv7.5 alpha subunits, which increases the opening of channels containing these subunits (Kv7.5 homotetramers or Kv7.4/Kv7.5 heterotetramers; solid arrows). Opening of the channels increases efflux of potassium ions (K+), which promotes smooth muscle relaxation. Kv7.4 alpha subunits are not phosphorylated by Protein Kinase A, and the activity of Kv7.4 homotetramers is not altered by this mechanism (dashed arrow).

 

About The Author

Bharath Mani received his DVM degree in 2001 and MS degree in 2003 from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India, and Ph.D (Pharmacology) degree in 2012 from Loyola University Chicago, USA. His doctoral research examined the role of arterial smooth muscle Kv7 channel function in influencing arterial contraction status.

He is now a postdoctoral fellow at the University of Texas Southwestern Medical Center at Dallas, Texas, USA. His current research investigates neuroendocrine regulation of metabolic and cardiovascular function.

Journal Reference

Mol Pharmacol. 2016; 89(3):323-34.

Mani BK, Robakowski C, Brueggemann LI, Cribbs LL, Tripathi A, Majetschak M, and Byron KL.

Loyola University Chicago, Dept. of Molecular Pharmacology & Therapeutics, Maywood, IL 60153, USA.

Abstract

Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth musclecell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K(+) currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth musclecells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled β-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKA-dependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4.

Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Go To Mol Pharmacol

 

Global Medical Discovery features paper: Single- and repeated-dose toxicity study of bevacizumab, ranibizumab, and aflibercept in ARPE-19 cells under normal and oxidative stress conditions

Journal Reference

Biochem Pharmacol. 2016;103:129-39.

Saenz-de-Viteri M¹, Fernández-Robredo P², Hernández M³, Bezunartea J⁴, Reiter N⁴, Recalde S², García-Layana A⁵.

Show Affiliations

1. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; Department of Ophthalmology, Clínica Universidad de Navarra, School of Medicine, University of Navarra, 36 Pio XII Avenue, 31008 Pamplona, Spain.
2. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain.
3. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain. Electronic address: mahersan@unav.es.
4. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain.
5. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; Department of Ophthalmology, Clínica Universidad de Navarra, School of Medicine, University of Navarra, 36 Pio XII Avenue, 31008 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain.

 

Abstract

We assessed the effect of single and repeated doses of bevacizumab, ranibizumab, and aflibercept on cell viability, proliferation, permeability, and apoptosis of ARPE-19 cells. MTT and BrdU assays were used to determine viability and proliferation after single or repeated doses of anti-VEGF drugs under  normal and oxidative stress conditions. Caspase-3 expression after single and repeated doses of the 3 drugs was assessed using immunofluorescence. Transepithelial-electrical-resistance (TER) was measured to study the effect of anti-VEGFs on retinal pigment epithelium (RPE) permeability under  normal and  oxidative  stress conditions. Flow cytometry was used to detect intracellular accumulation of the drugs. Finally, a wound healing assay was performed to investigate the effect of the drugs on RPE cell migration. Single and multiple doses of anti-VEGF drugs had no effect on cell viability and proliferation. The oxidative effect of H2O2 decreased cell viability and proliferation; however, no difference was observed between anti-VEGF treatments. Immunofluorescence performed after single and repeated doses of the drugs revealed some caspase-3 expression. Interestingly, anti-VEGFs restored the increased permeability induced by H2O2. The 3 drugs accumulated inside the cells and were detectable 5days after treatment. Finally, none of the drugs affected migration.

In conclusion, no measureable toxic effect was observed after single or repeated doses of VEGF antagonists under normal and oxidative stress. Intracellular accumulation of the drugs does not seem to be toxic or affect cell functions. Our study suggests that anti-VEGFs could have a preventive effect on the maintenance of the RPE barrier under oxidative stress.

Copyright © 2016 Elsevier Inc. All rights reserved.

Go To Biochem Pharmacol

 

Global Medical Discovery features paper: Low-density lipoprotein transport through an arterial wall under hyperthermia and hypertension conditions–An analytical solution

About The Author

Professor Kambiz Vafai received his B.S. in Mechanical Engineering from the University of Minnesota at Minneapolis in 1975. He received his M.S. degree in Mechanical Engineering from the University of California at Berkeley in 1977 and his Ph.D. degree in Mechanical Engineering also from the University of California, Berkeley in 1980. He is currently serves as Distinguished Professor of Mechanical Engineering at the University of California, Riverside, USA.

Professor Vafai holds 12 US patents associated with electronic cooling and medical applications. His research interests include transport through porous media, multiphase transport, aircraft brakes, biomedical applications, microcantilever-based biosensors, biofilms, electronic cooling, macromolecule transport through arteries, cooling enhancement investigations, modeling of tissue and organs, natural convection in complex configurations, analysis of porous insulations, heat flux applications, free surface flows, flat-shaped heat pipes, thermal design and modeling, and feasibility, optimization, and parametric studies for various engineering applications and power electronics. He is one of the highest cited in his fields of research.

He is currently Editor in Chief- Journal of Porous Media and Editor in Chief- Special Topics & Reviews in Porous Media

Journal Reference

J Biomech. 2016;49(2):193-204.

Iasiello M¹, Vafai K², Andreozzi A³, Bianco N³.

Show Affiliations

1. Department of Mechanical Engineering, University of California, Riverside, CA 92521, USA; Dipartimento di Ingegneria Industriale, Università degli Studi di Napoli Federico II, P.le Tecchio, 80, Napoli 80125, Italy.
2. Department of Mechanical Engineering, University of California, Riverside, CA 92521, USA. Electronic address: vafai@engr.ucr.edu.
3. Dipartimento di Ingegneria Industriale, Università degli Studi di Napoli Federico II, P.le Tecchio, 80, Napoli 80125, Italy. 

Abstract

An analytical solution for Low-Density Lipoprotein transport through an  arterial wall  under hyperthermia conditions is established in this work. A four-layer model is used to characterize the arterial wall. Transport governing equations are obtained as a combination between Staverman-Kedem-Katchalsky membrane equations and volume-averaged porous media equations. Temperature and solute transport fields are coupled by means of Ludwig-Soret effect.

Results are in excellent agreement with numerical and analytical literature data  under  isothermal conditions, and with numerical literature data for the hyperthermia case. Effects of hypertension  combined with hyperthermia, are also analyzed in this work.

Copyright © 2015 Elsevier Ltd. All rights reserved.

Go To J Biomech

 

Global Medical Discovery features paper: Effect of collagen-glycosaminoglycan scaffold pore size on matrix mineralization and cellular behavior in different cell types

Significance Statement

Bone tissue engineering has emerged as one of the leading fields in tissue engineering and regenerative medicine. Its success relies on understanding the interplay between progenitor cells, regulatory signals, and the biomaterials/scaffolds used to deliver them. Subtle changes in scaffold architecture can have significant effects on cellular activity. Optimising the design of bioactive scaffolds is guided by an understanding of the behaviour and responses of cells to their surrounding environment. Pore size is an essential architectural consideration in construct development; therefore, it is crucial to identify the optimal pore size for augmented tissue formation.

Using a series of collagen-glycosaminoglycan (CG) scaffolds with a homogenous mean pore size ranging from 85 µm up to 325 µm, we identified key differences in osteoblast and mesenchymal stem cell (MSC) behaviour in response to pore size. Scaffolds with the largest pore size (325 µm) facilitated superior osteoblast attachment, migration, scaffold infiltration and matrix deposition. MSC response was similar to osteoblasts but cell motility, proliferation, and scaffold infiltration was reduced. This was associated with differences in the profile of integrin subunits (α2) and collagen receptors (CD44), indicating that osteoblasts have a stronger affinity for collagen-glycosaminoglycan scaffolds compared to MSCs.

This study, for the first time within the literature, compares two very different cell types head to head to investigate individual cell behaviour in response to a single parameter. The findings elucidate fundamental mechanisms underlying the differences between the two cell types and highlight the importance of tailoring scaffold micro-architecture and cell type for cell-specific applications.

About The Author

Dr. Ciara Murphy received her PhD in area of bone tissue engineering from the Royal College of Surgeons in Ireland (RCSI) in 2010. Subsequently, she joined the Orthopaedic & Biotechnology Research (ORB) Group in the Children’s Hospital at Westmead, Sydney, Australia, where she focused her post-doctoral research on developing biologic delivery systems that utilised tissue engineering technologies, including collagen-based scaffolds, as novel therapies for bone healing.

In 2014, she was awarded the New Investigator Recognition Award (NIRA) at the International Orthopaedic Research Society (ORS) for her postdoctoral work. She returned to Ireland in 2015 joining University College Dublin (UCD) as an Assistant Professor in the School of Medicine and a Principal Investigator in the UCD Centre for Biomedical Engineering. Her research focuses on developing advanced biomaterials as innovative platforms for targeted therapeutic delivery, disease model systems and 3-D studies of cell-matrix interactions. 

 

About The Author

A/Prof Garry Duffy leads a multidisciplinary team of biomaterials, stem cell and drug delivery scientists within the Tissue Engineering Research Group (TERG), based in the Royal College of Surgeons in Ireland, with a large focus on chronic diseases. The long-term goal of his lab is to develop advanced biomaterials to facilitate targeted delivery and future clinical translation of cell based therapeutics.  As well as the DRIVE project, Garry also leads the Advanced Materials for Cardiac Regeneration (AMCARE) project, an €8.6 million FP7-funded research programme with the goal of using smart biomaterials and minimally-invasive surgical devices for targeted delivery of stem cells to treat the infarcted myocardium.

About The Author

A/Prof Aaron Schindeler is a Senior Research Scientist at The Children’s Hospital at Westmead and the Director of Basic Research in the Centre for Children’s Bone & Musculoskeletal Health (CCBMH). He joined the orthopaedic research department in 2003 and since then has tackled a range of research questions looking at traumatic bone injuries and genetic diseases affecting. Aaron leads a multidisciplinary team of scientists, engineers, and medical and allied health professionals. Key research areas for him include reducing the risk and impact of fracture and implant infection, cell and genetic therapies for brittle bone disease, studying the metabolic muscle weakness associated with neurofibromatosis type 1, and bone tissue engineering using novel biomaterials and 3D printing. 

About The Author

Prof Fergal O’Brien is a leading innovator in the development of advanced biomaterials for drug delivery and tissue repair. He is Professor of Bioengineering & Regenerative Medicine, Deputy Director for Research and heads the Tissue Engineering Research Group based in the Royal College of Surgeons in Ireland. He is also a PI and Deputy Director of the €58 million SFI-funded Advanced Materials and Bioengineering Research (AMBER) Centre. He is currently a member of the World Council of Biomechanics, Biomaterials Topic Chair for the Orthopaedic Research Society and President of the Section of Bioengineering of the Royal Academy of Medicine in Ireland.

Since his faculty appointment in 2003, he has published over 150 journal articles in leading peer-reviewed international journals and supervised 30 doctoral candidates to completion. He has a current h-index of 47.  Accolades include a Fulbright Scholarship (2001), New Investigator Recognition Award by the Orthopaedic Research Society (2002), Science Foundation Ireland, President of Ireland Young Researcher Award (€1.1. million, 2004), Engineers Ireland Chartered Engineer of the Year (2005), European Research Council (ERC) Investigator Award (€2 million, 2009),  Anatomical Society New Fellow of the Year (2014) and Fellowship of Engineers Ireland (2013) and the European Alliance for Medical & Biological Engineering Science (2016).

 

Journal Reference

J Biomed Mater Res A. 2016;104(1):291-304. 

Murphy CM^1,2,3, Duffy GP^2,3,4, Schindeler A^5,6, O’brien FJ^2,3,4.

Show Affiliations

1. School of Medicine & Medical Science, University College Dublin, Dublin, Ireland.
2. Tissue Engineering Research Group, Department of Anatomy, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
3. Trinity Centre for Bioengineering, Trinity College Dublin (TCD), Dublin, Ireland.
4. Advanced Materials and Bioengineering Research Centre (AMBER) RCSI & TCD, Dublin, Ireland.
5. Orthopaedic Research & Biotechnology Unit the Children’s Hospital at Westmead.
6. Discipline of Paediatrics and Child Health, University of Sydney, Sydney, Australia.

Abstract

We have previously examined osteoblast behavior on porous collagen-glycosaminoglycan (CG) scaffolds with a range of mean pore sizes demonstrating superior cell attachment and migration in scaffolds with the largest pores (325 μm). Scaffolds provide a framework for construct development; therefore, it is crucial to identify the optimal pore size for augmented tissue formation. Utilizing the same range of scaffolds (85 μm – 325 μm), this study aimed to examine the effects of mean pore size on subsequent osteoblast differentiation and matrix mineralization, and to understand the mechanism by which pore size influences behavior of different cell types. Consequently, primary mesenchymal stem cells (MSCs) were assessed and their behavior compared to osteoblasts.

Results demonstrated that scaffolds with the largest pore size (325 μm) facilitated improved osteoblast infiltration, earlier expression of mature bone markers osteopontin (OPN) and osteocalcin (OCN), and increased mineralization. MSCs responded similarly to osteoblasts whereby cell attachment and scaffold infiltration improved with increasing pore size. However, MSCs showed reduced cell motility, proliferation, and scaffold infiltration compared to osteoblasts. This was associated with differences in the profile of integrin subunits (α2) and collagen receptors (CD44), indicating that osteoblasts have a stronger affinity for collagen-glycosaminoglycan scaffolds compared to MSCs.

In summary, these results reveal how larger pores promote improved cell infiltration, essential for construct development, however the optimal scaffold pore size can be cell type specific. As such, this study highlights a necessity to tailor both scaffold micro-architecture and cell-type when designing constructs for successful bone tissue engineering applications.

© 2015 Wiley Periodicals, Inc.

Go To J Biomed Mater Res A.