Research Projects

Decreased Adhesion of Staphylococcus aureus to ZnO/PVC Nanocomposites

Lead Student--Benjamin Geilich

In hospitals and clinics worldwide, medical device surfaces have become a rapidly growing source of nosocomial infections. In particular, patients requiring mechanical ventilation (and, thus, intubation with an endotracheal tube) for extended lengths of time are faced with a high probability of contracting ventilator-associated pneumonia. Once inserted into the body, the endotracheal tube provides a surface to which bacteria can adhere and form a biofilm (a robust, sticky matrix that provides protection against the host immune system and antibiotic treatment). Adding to the severity of this problem is the spread of bacterial genetic tolerance to antibiotics, in part demonstrated by the recent and significant increase in the prevalence of multiple drug-resistant Staphylococcus aureus (MRSA). To combat these trends, different techniques in biomaterial design must be explored. Recent research has shown that nanomaterials (materials with at least one dimension less than 100nm) may have the potential to prevent or disrupt bacterial processes that lead to infections. In this study, polyvinyl chloride (PVC) taken from a conventional endotracheal tube was embedded with varying concentrations of zinc oxide (ZnO) nanoparticles. Staphylococcus aureus biofilms were then grown on these nanocomposite surfaces during a 24-hour culture. Following this, biofilms were removed from the surfaces and the number of colony forming units present was assessed. Bacterial proliferation on the samples embedded with the highest concentration of ZnO nanoparticles was 87% less when compared to the control, indicating that this technique is effective in reducing biofilm formation on PVC surfaces without the use of antibiotics.

Anodized and Etched Stainless Steel for Vascular Applications:

Lead Student--Deborah Gorth

Biomaterial compatibility is especially important in vascular implants because poor interactions with the immune system or platelets can create clots rendering the implant not just ineffectual but deleterious. This project explores the possibility of using chemical etching and anodization as methods for introducing nanoscale surfaces features on the surface of stainless steel to reduce platelet adhesion and promote endothelial cell growth to design a more effective implant.

Anodized Nanotubular Titanium as an Orthopedic Implant and the Effect of Electrical Stimulation

Lead Student--Batur Ercan

The current 15-20 year life-span of titanium-based orthopedic implants has been a challenging problem. Limited cytocompatibility properties and osseointegration of implants to surrounding bone has been proposed as one of the leading causes of such limited lifetimes. Over the past decade, nanotechnology has been proposed to improve the lifespan of many biomedical devices, including orthopedic implants. Specifically, to improve the cytocompatibility properties of currently used titanium orthopedic implants, nanotechnology has been used to create nanofeatured thin oxide films (through anodization) on titanium surfaces. In addition to this approach, a theraupedic method to heal bone fractures through electrical stimulation (which is FDA approved) has also been investigated. The results showed that compared to unstimulated conventional titanium, bone forming cell (osteoblast) proliferation and long-term functions (alkaline phosphatase synthesis, collagen type I synthesis and calcium deposition) were improved both upon creation of an anodized nanotubular titanium film and biphasic electrical stimulation. Moreover, anodized nanotubular titanium surfaces showed a decrease in fibroblast cell adhesion/proliferation compared to conventional titanium surfaces, which can reduce implant failures due to fibrous tissue formation around orthopedic implants. One another advantage is that anodized nanotubular titanium also showed enhanced resistance to bacteria (Staphylococcus aureus and Staphylococcus epidermidis) colonization, which can form a biofilm on the implant and potentially lead to implant failure. Upon electrical stimulation, decreased bacteria density was observed on the anodized surfaces compared to conventional ones. Changing the nanotube dimensions further decreased the bacteria colonization on these surfaces. Most importantly, when electrical stimulation was combined with anodized nanotubular titanium features, the beneficial effects (enhancement in bone cell functions and reduction in biofilm formation) were improved the best. Therefore, coupling the positive effects of anodized nanotubular titanium topographies with currently used theraupedic electrical stimulation is a promising method for bone-tissue engineering applications.

Figure: a) Cross-sectional view of anodized nanotubular titanium. b) bone cell adhesion and c) 1day S. aureus culture on nanotubular titanium. Scale bars are a) 30nm, b) 1micron, c) 200nm

Assessment of Nanomodifed Endotracheal Tubes in a Benchtop Airway Model

Lead Student--Mary Machado

Ventilator associated pneumonia (VAP) is a serious and costly clinical problem. Specifically, receiving mechanical ventilation over 24 hours increases the risk of VAP and is associated with high morbidity, mortality and medical costs. Cost effective endotracheal tubes (ETTs) that are resistant to bacterial infection would help to prevent this problem. The objective of this study was to determine differences in bacterial growth on nanomodified and unmodified ETTs under dynamic airway conditions.

The ETTs tested in our system were polyvinyl chloride (PVC), with nano-roughened surfaces created by exposing PVC tubing (Sheridan ETT, Colvidien) to a 0.1% mass solution of lipase from the either the fungi C. cilindracea or R. arrhisus (Sigma Aldrich) dissolved in a potassium phosphate buffer. ETTs placed in the system were modified on both the inner and outer surfaces.

A bench top model based upon the general design of Hartmann et al. (1999) was constructed to test of the effectiveness of nanomodified ETTs under the airflow conditions present in the airway. A sterilization run was performed for 24 hours prior to each use of the system to prevent cross contamination. Cuff pressure was monitored every twelve hours and the upper box was agitated for thirty minutes before each sample was taken. Colony counts were performed on samples taken from the lung box and oropharynx at the 12, and 24 hour time points.

Twenty-four hour studies performed in the dynamic flow chamber showed a marked difference in the biofilm formation on different areas of unmodified tubes. Areas where tubes were curved, such as at the entrance to the mouth and the connection between the oropharynx and the larynx, seemed to collect the largest amount of biofilm. On the nanomodified tubes film formations were markedly different occurring in smaller pieces.

The biofilm formation on ETT in the airflow system after 24 hours showed a large difference depending upon where tubes were oriented within the apparatus. This illustrates the importance of dynamic flow on biofilm formation in pediatric ETTs. It is of particular interest that increased biofilm density in both unmodified and nanomodified tubes appeared to occur at curves in the tube where changes in flow pattern occur. This emphasizes the need for more accurate models of airflow within pediatric ETT, suggesting that not only does flow effect pressure gradients along the tube, but in fact determines the composition of the film itself.

More testing is needed to determine the effects of biofilm formation on the efficiency of ETT under airflow, however this study provides significant evidence for nanomodification alone (without the use of antibiotics) to decrease bacteria function.

Bacteriocidal Ultrasound Therapy is Enhanced on Antibacterial Nanomaterials

Lead Student--Justin Seil

Previous studies have demonstrated the antibacterial effects of zinc oxide nanoparticles when either incorporated into polymer composites or freely floating in media. The mechanism of antibacterial activity, based on additional studies, appears to relate to zinc ion release from the particle surfaces interfering with bacteria adhesion. The relationship between bacteria activity and ultrasonic stimulation is complex. At low intensities, ultrasound can enhance mass transport and disperse bacteria to enhance proliferation. However, ultrasound can also enhance the effectiveness of an antibiotic. At high ultrasound intensity, bacteria can be killed. The present study investigated the use of ultrasound to enhance antibacterial activity of zinc oxide nanoparticles. The proposed mechanism was enhanced release of zinc ions from the particle surfaces due to the mechanical stimulation of the ultrasound. The combination of ultrasonic stimulation and the presence of zinc oxide nanoparticles reduced staphylococcus aureus planktonic growth and surface activity greater than either of the two elements alone. Ultimately, implanted biomaterials, including orthopedic prosthetics, could incorporate an infection-resistant zinc oxide nanoparticle composite coating that could be ultrasonically stimulated at the first sign of infection to locally amplify the antibacterial environment around the implant.

Biocompatibility, Cytotoxicity and Genotoxicity, Evaluation of Hydroxyapatite (HAp)

Lead Student--Sushma Kalmodia

Hydroxyapatite (HA) based biomaterials are widely used class of engineering material, but HA material is not suitable for the load bearing application since it has very poor mechanical properties (strength/toughness). To improve the mechanical properties of HA for load bearing orthopedic application we developed hydroxyapaptite-mullite (HM) composite and sintered by spark plasma sintering (SPS) that shows good mechanical and biological properties. However, some adverse effects associated with the wear debris particles generated by HA based load bearing composite orthopedic implants. Therefore to further evaluate the effects of HM wear debris particles generated at implant site, on apoptotic cell death by regulation of Bcl-2 family gene expression at transcription level. Bcl-2 family proteins regulate and contribute to programmed cell death or apoptosis (act as pro or anti apoptotic regulator) and also involve in cellular activity. Our finding indicates that HM particles have significant role in gene expression of Bcl-2 family with time, concentration and size dependent manner. The apoptotic cell death studied in osteoblast cell (hFOB) by fluorescent activated cell sorter and Bcl-xL and Bax expression at different time and concentration after the HM nano eluates particles treatment, also the ratio of Bcl to Bax determines the cell fate, survival or death, by the HM nanoparticles eluates. Till now, invitro cytotoxicity is predominate test for the biocompatibility evaluation of material before going for animal and clinical trial. However, the cytotoxicity evaluation of the material are only confirmed whether the cells live or dead, without giving insight at molecular level. By using the genome profiling we can annotate the molecular dynamics in response to the biomaterial and synthesized biomaterials composite in a specific way without any adverse effect at molecular level.

Conductivity and Compatibility of Poly Lactic-Co-Glycolic Acid Carbon Nanofiber material for Novel Cardiovascular Patch

Lead Student--David A. Stout

In recent years Poly Lactic-Co-Glycolic Acid (PLGA) has been under investigation for myocardial tissue engineering applications since it has been approved by the Food and Drug Administration (FDA) for therapeutic devices due to its biodegradable and biocompatible properties. Recent research has also shown that carbon nanofibers (CNFs) assist and strengthen polymers. The objective of the present research was to determine if compatibility properties of PLGA can be improved through the addition of CNFs for myocardial tissue engineering applications. For this reason, different PLGA: CNF ratios (100:0, 75:25, 50:50, 25:75, 0:100 wt.%) were created and positioned onto a glass substrate to determine conductivity and sustainability properties. Next, human cardiomyocytes (Celprogen cat#36044-15) were seeded onto the different PLGA:CNF ratio materials. Cell adhesion and proliferation for 1, 3, and 5 days were completed. Results showed that PLGA:CNF materials possess conductivity properties suitable for cardiac applications which increased as more CNFs were added to PLGA. SEM results demonstrated the bonding between the different PLGA:CNF ratio materials and also human cardiomyocyte compatibility. In summary, results indicated that PLGA:CNF materials would be a novel composite biomaterial for myocardial tissue engineering applications due to its biodegradability, conductivity, and cytocompatibility properties, thus, providing an alternative medium for potentially regenerating cardiac tissue.

Decreasing Cancer Cell Functions Using Nano-topography PLGA

Lead Students--Lijuan Zhang and Yongchen Wang

The focus of this project is to prepare PLGA films with various nanometer surface features and determine whether cancer cells respond differently to such topographies. Different size polystyrene beads were used to cast poly(dimethylsiloxane) (PDMS) molds which are used as templates to create nano-featured PLGA films. Cytocompatibility assays such as adhesion, proliferation, apoptosis and VEGF syhthesis are conducted to determine the cancer cell functions. Further, PLGA surface is modified to enhance its anti-cancer property. We hope that modified PLGA with specific nanometer surface features may decrease cancer cell functions, providing an important biomaterial for the treatment of lung cancer with wide range of applications.

Effects of Varying Nanotube Size on Titanium for Antibacterial Properties

Lead Student--Kim Kummer

When varying nanotubular topography on the surface of titanium to we are trying to promote better antibacterial properties for orthopedic applications. Surface topography and chemistry of titanium samples will be altered through the electrochemical process of anodization. The dimensions of these nanotubes can vary based on the anodization conditions. The focus of the initial set of experiments will be to assess the antibacterial properties of each nanotube size obtained. For the bacteria experiments, Staphylococcus Aureus (S. Aureus) growth will be examined.

Investigation of Selenium Nanoparticle Toxicity

Lead Student--Joseph Ramos

Selenium nanoparticles have been shown to have an antibacterial effect in vitro. One particular application for these nanoparticles are medical tubes, such as endotracheal tubes. My project focuses on the behavior of mammalian cells grown on polyvinyl chloride (PVC; the endotracheal tube substrate) coated with selenium nanoparticles to investigate the toxicity as well as identify the ideal biocompatible concentration of the nanoparticles.

Nanocrystalline Diamond for Orthopedic Applications

Lead Student--Lei Yang

Substrate nanotopography has been shown to have a positive impact on promoting osteoblast (bone forming cell) adhesion, proliferation and differentiation regardless of substrate chemistry. However, the mechanisms behind this promotion are still far from clear due to the lack of understanding of how cells sense and respond to nanoscale surface features much smaller than the size of cells. The first part of the study provided experimental results of nanotopography mediated osteoblast functions on nanocrystalline diamond, and consequently a correlation between osteoblast spreading on varied diamond nanotopographies and cellular functions was established. To further understand the altered osteoblast spreading on different nanotopographies, a mathematical model based on actin filaments (fundamental components of the cytoskeleton) was constructed to simulate cell movements on stiff nanotopographies. Simulation results of actin filament extensions on altered substrate topographies suggested that the nanoscale topography increased cell spreading compared to the micron topography. These simulation results also correlated to experimental measurements of cell spreading on different diamond topographies and those results reported by others. The results of the study, thus, suggested an effective experiment/modeling approach to understand cell responses per substrate nanotopographies by examining actin polymerization and filament extension at the nanoscale.

Nano-surface Application for Decreasing Bacterial Adhesion, Ventilator-associated Pneumonia

Lead--Dr. Keiko Tarquinio

It has been quite a dilemma for clinicians to decrease nosocomial infection in pediatric population, since the adult prevention or treatment approaches are not always applicable in children. Focus on ventilator associated pneumonia (which is one of the nosocomial infections), particularly on biofilm formation on endotracheal tubes. We exam what is happening on intubated patients’ endotracheal tubes in pediatric critical care units. At the same time, we look at in vitro research to exam the bacterial adhesion on nano-scale polymer which may be used for medical devises in the future.

Magnetic Nanoparticles for Treating Bone Diseases

Lead Student--Nhiem Tran

Bacterial infection has been causing many problems for biomedical application such as catheters, endotracheal tubes and implants. The goal of the project is to study bactericidal effects of various iron oxide nanoparticles in several forms such as nanoparticles and thin films. In order to study the effects of iron oxide (IO) nanoparticles on Staphylococcus aureus (S. aureus), IO nanoparticles were synthesized via a novel matrix mediated method using polyvinyl alcohol (PVA). The IO nanoparticles were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Further, S. aureus were grown in the presence of three different IO nanoparticle concentrations for 4, 12 and 24 hours. Live/dead assays were performed and the results provide evidence that IO/PVA nanoparticles inhibited S. aureus growth at the highest concentration (3mg/ml) at all time points. The nanoparticles were also electrospun with PVA to produce thin film containing iron oxide. The films were characterized via SEM, Fourier Transformation Infra-red spectrum (FTIR) and X-ray diffraction (XRD). Mechanical properties of these films were investigated to determine stress/strain and Young modulus. Bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa are to be applied on the synthesized thin films. The project is the collaboration between Nanomedicine lab in Brown University (USA) and Biomedical and Material Research lab in National Metallurgical Laboratory (NML – India).

Also, involving the use of hydroxyapatite coated iron oxide nanoparticles as drug delivery system to treat osteoporosis. Hydroxyapatite (HA) has been widely used in the biomedical community, especially for orthopedic applications. In order to use hydroxyapatite nanoparticles in drug delivery systems to treat osteoporosis, here, hydroxyapatite was coated on iron oxide nanoparticles. This current study reports evidence that such composite nanoparticles can enhance bone cell proliferation and long-term bone cell functions. Specifically, magnetite (Fe3O4) were synthesized and coated with hydroxyapatite. The resulting nanoparticles were treated hydrothermally to control the crystalline properties of the coating. Nanoparticles were characterized via transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction (XRD), Zeta potential measurement and vibrating sample magnetometry (VSM). Nanoparticle uptake by osteoblasts (bone forming cells) was also studied using TEM. Osteoblast density was measured after 1, 3 and 5 days in the presence of Fe3O4 nanoparticles alone and HA coated Fe3O4 magnetic nanoparticles. Osteoblast long term experiments showed greater alkaline phosphatase activity, total protein, collagen formation and calcium deposition of osteoblast after 7, 14 and 21 days in the presence of the coated iron oxide nanoparticles. The results of this study showed that hydroxyapatite coated magnetic iron oxide nanoparticles have great potential for orthopedics applications.

Nanoparticle Toxicity

Lead Student--Deborah Gorth

The most exciting characteristic of nanotechnology is that just by changing the size of particles you can also change their reactivity. Nanotechnology has harnessed this fact to create more effective medical treatments for cancer treatment, tissue engineering and regenerative medicine, but the influence of nanoparticle size on toxicity has not been thoroughly addressed to date. This study examined the influence of size of alumina, silica and silver on toxicity in Drosophila by exposing eggs to particles of various sizes at concentrations ranging from 10 ppm-10,000 ppm and quantifying its effect on development.

Preventing Biofilm Formation Using Nanostructured Selenium

Lead Student--Qi Wang (Gavin)

The implanted medical devices provide a surface for bacteria to attach to and grow upon each other resulting in the formation of biofilm. The biofilm structure increases the resistance of bacteria to treatment methods such as antibiotics. Preventing biofilm formation is critical in the fight against healthcare-associated infections (HAIs). In my project, the nanostructured selenium was used to introduce antibacterial properties directly onto the surface of medical devices. Selenium can kill bacteria by depleting their thiol levels. The nano-scale size of selenium nanoparticles increases the surface area available to interact with and kill bacteria, while also changing the surface morphology to ultimately prevent the attachment of bacteria. For this, selenium nanoparticles were synthesized through a simple reaction between glutathione and sodium selenite (4:1 molar mixture) and at the same time were coated on the surface of various medical devices. These substrates included PVC (polyvinyl chloride), polycarbonate, PU (polyurethane), co-polyesters and silicone. SEM (Scanning Electron Microscopy) and AAS (Atomic Absorption Spectroscopy) were used to determine the distribution, adhesion, and the amount of selenium nanoparticles on the surface of substrates. Lastly, experiments with bacteria (specifically, Staphylococcus aureus) were conducted to determine the effectiveness of the selenium coating for killing bacteria or preventing bacteria from attaching.

Superparamagnetic Iron Oxide Nanoparticles for Orthopedic Applications (Decreased Bacteria and Increased Bone Cell Function)

Lead Student--Erik Taylor

Bone related infectious diseases (including osteomyelitis and prosthesis infection) are of great concern to the medical world. These types of deep tissue infection are frequently chronic and always painful for those suffering. Biofilm is one such type of infection whereby bacteria form a robust colony protected by sticky slime matrix from the body’s immune system (or natural clearance) and antibiotic treatment (called antibiotic resistance). Antibiotics available to treat such infectious diseases are often not specifically targeted to the site of the disease and, thus, lack an immediate directed therapeutic effect. It has been previously shown that magnetic nanoparticles can be directed in the presence of a magnetic field to any part of the body, allowing for site-specific drug delivery. Magnetic nanoparticles have also shown promise to enhance bone cell functions and possibly provide an immediate increase in bone density, for example when directed to the site of infection. This review article will explore the multifunctional properties magnetic nanoparticles (termed here superparamagnetic iron oxide nanoparticles, or SPION) having antibacterial activity, bone enhancing properties, and magnetic properties towards the development of a new type of pharmaceutical which could be useful for orthopedic and infection related applications simultaneously.