Cationic liposome (CL) is a promising vector for nucleic acid therapy. In the present study, we investigated the effect of high hydrostatic pressure (HHP) treatment to lipoplex on the lipoplex-based antisense oligodeoxynucleotides (AS-ODNs) delivery in order to improve the transfection efficacy of lipoplex. Cationic liposome consisting of DOTMA and DOPE was used. AS-ODNs were designed to inhibit the expression of firefly luciferase. The complexes of CL and AS-ODN were prepared at various C/A ratios and then pressurized hydrostatically at various atmospheres (∼10,000 atm) for 10 min (HHP treatment). After removal of pressure, the pressurized lipoplexes were used. The lipoplex with and without the HHP treatment was transferred into HeLa cells expressing firefly luciferase transiently. The luciferase activity using the HHP-treated lipoplex was decreased compared to that of the non-pressurized lipoplex. Also, for HEK293 cells expressing luciferase stably, the lipoplex with the HHP treatment could effectively suppress the luciferase expression. In order to elucidate relationship between the structure and the transfection efficiency of the HHP-treated lipoplex, the properties of the HHPtreated lipoplex were examined by various physicochemical analyses. The different physicochemical properties between the lipoplexes with and without HHP treatment were showed, suggesting that the nature of lipoplex was changed by the HHP treatment. We believe that this change of lipoplex properties by the HHP treatment affected the efficiency of gene suppression. This HHP treatment for lipoplex appears to be a promising contribution to gene and oligonucleotide delivery.

Methotrexate (MTX), is a potent immunomodulating drug and widely used in the treatment of cancer, psoriasis and others disease. Despite its efficacy, the use of MTX is greatly limited due to its toxicity. To solve this problem, we prepared nanoparticles of tetraethyl orthosilicate (NP-TEOS) containing the compound methotrexate (MTX), by the sol-gel method. This drug delivery system (DDS) showed a loading efficiency of 39.7%. Size distribution studies were performed with dynamic light scattering and scanning electron microscopy revealing that these particles were spherical in shape, with a mean diameter between 140-430 nm and a low polydispersity (0.12 – 0.26). Also the particles displayed a low tendency toward aggregation which was confirmed by the low zeta potential -61.4 mV. Profile release showed a slow release loaded with MTX (PBS buffer pH = 7.4). The slow release can be attributed to the low porosity of the NP-TEOS and the extremely low diffusivity of MTX in aqueous media. B16-F10 cells were used to assay the toxicity and uptake of NP-TEOS showing to be nontoxic without MTX making a good candidate for DDS.

We have improved bio-inspired Moth eye nanostructures to enhance the scintillator materials external quantum efficiency significantly. As a proof of concept, we have demonstrated very high light output efficiency enhancement for Lu2SiO5:Ce3+ (LSO:Ce) film in large area. The X-ray mammographic instrument was employed to demonstrate the light output enhancement of the Lu2SiO5:Ce thin film with bio-inspired Moth eye-like nano photonic structures. Our work could be extended to other thin film scintillator materials and is promising to achieve lower patient dose, higher resolution image of human organs and even smaller scale medical imaging.

Bacterial infections are commonly found on paper towels and other paper products leading to the potential spread of bacteria and consequent health concerns. The objective of this in vitro study was to introduce antibacterial properties to paper towel surfaces by coating them with selenium nanoparticles. Scanning electron microscopy was used to measure the size and distribution of the selenium coatings on the paper towels. Atomic force microscopy was used to measure the surface roughness of paper towels before and after coated with selenium nanoparticles. The amount of selenium precipitated on the paper towels was measured by atomic absorption spectroscopy. In vitro bacterial studies with Staphylococcus aureus were conducted to assess the effectiveness of the selenium coating at inhibiting bacterial growth. Results showed that the selenium nanoparticles coated on the paper towel surface were well distributed and semispherical about 50nm in diameter. Most importantly, the selenium nanoparticle coated paper towels inhibited S. aureus growth by 90% after 24 hours and 72 hours compared with the uncoated paper towels. Thus, the study showed that nano-selenium coated paper towels may lead to an increased eradication of bacteria to more effectively clean a wide-range of clinical environments, thus, improving health.

Tissue engineering aims to save lives by producing synthetic organs and bone. This study is attempting to determine what effects a polycaprolactone (PCL) scaffold will have on the blood flow of Rattus norvegicus, as measured by the number of platelets. Prior to experimentation, it was hypothesized that the polycaprolactone scaffold would maintain and/or increase the number of platelets when compared to the control group. This was developed based on prior research that showed polylactic acid (PLA), a polymer being used currently, and polycaprolactone had similar characteristics like boiling point, melting point, and glass transition temperature. To test this hypothesis, the PCL, created from an existing protocol, was used to mold a scaffold in vitro. Three groups of rats were identified, then further split into an “A” and “B” subdivision with 5 members in each. All “A” subdivision members received the scaffold, while the "B" factions lacked it. Each rat underwent surgery to remove 1mm of the right ventricle, which was replaced by the PCL scaffold in the experimental group. The control group did not have the scaffold replacement. Without this piece of the right ventricle, prior research conducted at the University of Virginia in 2006 suggests that the rats would die within one week. However, in the experimental group of rats, the missing piece of the ventricle was replaced with the scaffold, so if it were accepted then the rats would survive beyond 1week. All rats in the experimental group died exactly 1 week after the control group as predicted before experimentation. After all of the rats had a 1-week acclimation period, a 1mm^2 slice of the heart was extracted and then the number of platelets was counted using a phase contrast microscope. The heart extraction was prepared in a petri dish and then placed into a hemocytometer, splitting the dish into smaller sections making it possible to count. The data supports the hypothesis whereby an average 12% increase in the number of platelets in the rats with the PCL scaffold versus the group without it was seen. This increase in platelet count reflects an increase in blood flow. A statistical t-test was conducted on each trial (n=5 per group, n=10 total per trial) comparing experimental versus control group to calculate a p-value. The p-values were 0.034, 0.045, and 0.022, respectively which indicates statistical significance since the value is less than 0.05. After all experimentation, the benefits of using PCL in tissue engineering were examined. For example, PCL costs $80 less to produce per kilogram than polylactic acid. This study suggests that PCL would be a viable candidate for tissue engineering in humans.

Dimethyl sulfoxide (DMSO) has been used for several decades as the most efficient cryoprotective agent (CPA) for many types of cells in spite of its cytotoxicity and its effect on differentiation. Recently we showed that carboxylated poly-L-lysine, which is classified as a polyampholyte, has a cryoprotective effect on cells in solution without any other cryoprotectant. Here we developed high molecular weight polyampholytes with an appropriate ratio of amino and carboxyl groups and evaluated their cryopreservation efficiency. A novel polyampholyte based on naturally available polymer dextran, in which we introduced both amino and carboxyl groups shows an excellent post thaw-survival efficiency of more than 90% of murine L929 cells. It can serve as the sole high molecular weight CPA for tissue engineering applications without animal derived materials.

Human mesenchymal stem cells (hMSCs) were routinely cultured on tissue-culture polystyrene (TCPS) to investigate the in vitro aging and cell stiffening. hMSCs were also cultured on thermoplastic polyurethane (TPU), which is a biocompatible polymer with an elastic modulus of approximately 12.9MPa, to investigate the impact of substrate elastic modulus on cell stiffening and differentiation potential. Cells were passaged over several generations on each material. At each passage, cells were subjected to osteogenic and myogenic differentiation. Local cell elastic modulus was measured at every passage using atomic force microscopy (AFM) indentation. Gene and protein expression was examined using qRT-PCR and immunofluorescent staining, respectively, for osteogenic and myogenic markers. Results show that the success of myogenic differentiation is highly reliant on the elastic modulus of the undifferentiated cells. The success of osteogenic differentiations is most likely somewhat dependent on the cell elastic modulus, as differentiations were more successful in earlier passages, when cells were softer.

Differentiated neurons (dorsal root ganglia and cortical neurons) have been shown to develop longer neurite extensions on softer materials than stiffer ones, but previous studies do not address the ability of neural stem cells to undergo differentiation as a result of material elasticity. In this study, we investigate neuronal differentiation of C17.2 neural stem cells due to growth on polyacrylamide gels of variable elastic moduli. Neurite growth, synapse formation, and mode of division (asymmetric vs. symmetric) were all assessed to characterize differentiation. C17.2 neural stem cells were seeded onto polyacrylamide gels coated with Type I collagen. The cells were then serum starved over a 14 day period, fixed, and analyzed for biochemical markers of differentiation. For division studies, time-lapse imaging of cells on various substrates was performed during serum withdrawal using the Nikon Biostation. Division events were analyzed using ImageJ to quantify sizes of resulting daughter. Data shows that C17.2 cell differentiation (as dictated by number and type of division events) is dependent upon substrate stiffness, with softer polyacrylamide surfaces (140 Pa) leading to increased populations of neurons and increased neurite length. Our data also indicates that the ability of neural stem cells to express synaptic proteins and develop synapses is dependent upon material elasticity.

The objective of this work is to examine the feasibility of electrically conductive hydrogel composites as scaffolds in tissue engineering and tissue regeneration, and to understand the properties of the composites as a growth matrix for clinically relevant cell lines. The composite is comprised of carbon nanobrushes embedded in a biocompatible poloxamer gel. This work assesses the ability of such composite gels to support the growth of fibroblasts and myocytes and eventually serve as a matrix to stimulate wound closure. In such a model, fibroblasts and myocytes are seeded on the hydrogel and bathed in culture medium. The experimental model assesses the ability of fibroblasts and myocytes to grow into and adhere to the gel. The work demonstrates that carbon nanobrushes can be dispersed within poloxamer gels, and that fibroblasts and myocytes can proliferate within homogenously dispersed carbon nanobrush-containing poloxamer gels. This work also examines the effects of carbon nanobrush content on the rheological properties of the poloxamer gel matrix and shows an improvement in several areas in the presence of carbon nanobrushes. Future work will examine the effects of design parameters such as carbon nanobrush content and matrix structure on wound healing, as well as the growth of tendons and other cell lines within the hydrogel composites. This work has relevance for tissue and cellular engineering and tissue regeneration in clinical medicine.

Cartilage defects, which are caused by a variety of reasons such as traumatic injuries, osteoarthritis, or osteoporosis, represent common and severe clinical problems. Each year, over 6 million people visit hospitals in the U.S. for various knee, wrist, and ankle problems. As modern medicine advances, new and novel methodologies have been explored and developed in order to solve and improve current medical problems. One of the areas of investigation is tissue engineering [1, 2]. Since cartilage matrix is nanocomposite, the goal of the current work is to use nanomaterials and nanofabrication methods to create novel biologically inspired tissue engineered cartilage scaffolds for facilitating human bone marrow mesenchymal stem cell (MSC) chondrogenesis. For this purpose, through electrospinning techniques, we designed a series of novel 3D biomimetic nanostructured scaffolds based on carbon nanotubes and biocompatible poly(L-lactic acid) (PLLA) polymers. Specifically, a series of electrospun fibrous PLLA scaffolds with controlled fiber dimension and surface nanoporosity were fabricated in this study. In vitro hMSC studies showed that stem cells prefer to attach in the scaffolds with smaller fiber diameter or suitable nanoporous structures. More importantly, our in vitro differentiation results demonstrated that incorporation of the biomimetic carbon nanotubes and poly L-lysine coating can induce GAG and collagen synthesis that is indicative of chondrogenic differentiations of MSCs. Our novel scaffolds also performed better than controls, which make them promising for cartilage tissue engineering applications.

Adult bone marrow derived mesenchymal stem cells (MSCs) represent an important source of cells for tissue regeneration. Control of MSC migration and homing is still unclear. The goal of this study was to identify potent chemoattractants for MSCs and characterize MSC chemotaxis using a microfluidic device as a model system and assay platform. The three chemokines compared in this study were CXCL7, CXCL12, and AMD 3100.

Microfluidic devices made of polydimethysiloxane (PDMS) were fabricated by soft lithography techniques and designed to generate a stable linear chemokine gradient. Cell movements in response to the gradient were captured by timelapse photos and tracked over 24 hours. Chemokine potency was measured via several chemotaxis parameters including: velocity in the direction of interest (V), center of mass (Mend), forward migration indice (YFMI). The migratory paths of the cells were mapped onto a displacement plot and compared.

The following results were measured in the direction of interest (towards higher concentrations of chemokine): For velocity, only cells exposed to CXCL12 had a statistically significant (p=.014) average velocity (V=0.19 ± 0.07 um/min) when compared to the control condition (V=0.06 ±0 .04 um/min). For the center of mass, where the displacement of cells from their starting positions were compared, again only CXCL12 (Mend= 53.9 ± 10.8 um) stimulated statistically significant (p = .013) displacement of cells compared to the control condition (Mend = 19.3 ± 16.1 um). For the forward migration index, the efficiency of cell movement was measured. Indices in both the CXCL12 (YFIM = 0.19 ± 0.08) and CXCL7 (YFIM = 0.09 ±0.03) conditions were statistically significant (p = .023 for CXCL12 and p = .035 for CXCL7) when compared with the control index (YFIM = .04 ± .02).

This study demonstrated the use of microfluidic devices as a viable platform for chemotaxis studies. A stable linear chemokine gradient was maintained over a long time scale to obtain cell migration results. CXCL12 was quantitatively determined to be the most potent chemoattractant in this research; these chemoattractive properties promote its use in future developments to control MSC homing.

Biofilms are a major source of medical device-associated infections, due to their persistent growth and antibiotic resistance. Recent studies have shown that engineering surface nanoroughness has great potential to create antibacterial surfaces. In addition, stimulation of bacterial metabolism increases the efficacy of antibacterial agents to eradicate biofilms. In this study, we combined the antibacterial effects of nanorough topographies with metabolic stimulation (i.e., fructose metabolites) to further decrease bacterial growth on polyvinyl chloride (PVC) surfaces, without using antibiotics. We showed for the first time that the presence of fructose on nanorough PVC surfaces decreased planktonic bacteria growth and biofilm formation after 24 hours. Most importantly, a 60% decrease was observed on nanorough PVC surfaces soaked in a 10 mM fructose solution compared to conventional PVC surfaces. In this manner, this study demonstrated that bacteria growth can be significantly decreased through the combined use of fructose and nanorough surfaces and thus should be further studied for a wide range of antibacterial applications.

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. 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. Twenty-four hour studies performed in a 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.

The biofilm formation on ETTs 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 on both unmodified and nanomodified tubes appeared to occur at curves in the tube where changes in flow pattern occurred. This emphasizes the need for more accurate models of airflow within pediatric ETTs, suggesting that not only does flow affect 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.

Natural polymers, used for hydrogel fabrication, are generally bioactive and provide good environment for cell growth and proliferation. However, these polymers have low mechanical strength. Several approaches have been attempted to improve their mechanical properties such as fabrication of interpenetrating polymer network (IPN) and semi-IPN hydrogels, and also addition of a nano sized fibers or nano-particles. The aim of this study was to investigate the feasibility of using naturally derived nano-fillers such as cellulose nanocrystallines to enhance the mechanical properties of hydrogels. Gelatin methacrylate (GelMA) was used as a protein model for preparation of photo-crosslinked hydrogel. The effects of concentrations of photo initiator and cellulose nanocrystallines (CNC) on the characteristics of hydrogels were examined. In vitro studies showed negligible cytotoxic effect of CNC on human osteosarcoma cell growth when using less than 20 mg/ml CNC. Therefore, it is viable to use this nano-filler for biomedical applications. It was found that the compression modulus of gelatin hydrogel was increased 1.5 fold by addition of 10 mg/ml of CNC. These results demonstrate the high potential of using CNC for tissue engineering applications to enhance the mechanical strength of hydrogels.

Metallic silver nanoparticles (NPs) have extensively been used in the treatment of disease and purification and heralded the ‘first wave’ of disinfection science, the ‘second wave’ being the nanocomposite of metal-doped TiO2. Recent advances in engineered surfaces have enabled ultrahigh surface area and rapid sterilization via using metal-organic frameworks (MOFs) as the ‘third wave’ disinfectant. MOFs offer the same advantages as colloids but also have ultra high surface area, long term persistence and ultra low doses, applied for water purification.

Anodic aluminum oxide (AAO) membranes were fabricated in a mild two-step anodization procedure. The voltage was varied during both anodization steps to control the pore size and morphology of the AAO membranes. Pore sizes ranged from 34 nm to 117 nm. Characterization of the pore structure was performed by scanning electron microscopy (SEM). To assess the potential of the AAO membranes as a neuronal differentiation platform, C17.2 neural stem cells (NSCs), an immortalized and multipotent cell line, were used. The NSCs were forced to differentiate via serum-withdrawal. Cellular growth was characterized by immunocytochemistry (ICC) and SEM. ImageJ software was used to obtain phenotypic cell counts and neurite outgrowth lengths. Results indicate a highly tunable correlation between AAO nanopore sizes and differentiated cell populations. By selecting AAO membranes with specific pore size ranges, control of neuronal network density and neurite outgrowth length was achieved.

In this study, in order to investigate biocompatibility of nitrogen-doped hydrogenated amorphous carbon (a-C:H:N) film coating segmented polyurethane (SPU) scaffold fiber sheet (a-C:H:N-Scaffold) in in-vitro test, mouse fibroblasts (NIH 3T3) cells were grown on the a-C:H:N-Scaffold. The cell behavior was monitored by time-lapse imaging system. Additionally, the a-C:H:N-Scaffold was implanted at partial aorta descendens of a goat for 35 days. The surface morphology, composition, and wettability of the a-C:H:N-scaffold was estimated by Scanning Electron Microscope (SEM), X-ray photoelectron spectrometer (XPS), and contact angle measurement. In in-vitro test, it was observed that a-C:H:N film coating had a facilitatory effect on cell motility and cell growth. In in-vivo test, it was observed that the a-C:H:N-Scaffold surface was uniformly covered by neointima. The a-C:H:N-Scaffold surface had no thrombus formation as an inflammatory reaction and it was shown that the a-C:H:N film coating had a good blood compatibility. These results suggest that a-C:H:N film coating has good cytocompatibility and blood compatibility and it is a promising approach for improvement of biocompatibility of biomaterial surfaces.

Many advances in nanomaterials synthesis have been recorded during the last 30 years. Bacterial cellulose (BC) produced by bacteria belonging to the genera Acetobacter, Rhizobium, Agrobacterium, and Sarcina is acquiring major importance as one of many eco-friendly materials with great potential in the biomedical field. The shape of BC bulk is sensitive to the container shape and incubation conditions such as agitation, carbon source, rate of oxygenation, electromagnetic radiation, temperature, and pH. The challenge is to control the dimension and the final shape of biosynthesized cellulose, by the optimization of culture conditions. The production of 3D structures based on BC is important for many industrial and biomedical applications such as paper and textile industries, biological implants, burn dressing material, and scaffolds for tissue regeneration. In our work, wild strains of Acetobacter spp. were isolated from homemade vinegar then purified and used for cellulose production. Four media of different initial viscosity were used. Cultures were performed under static conditions at 29°C, in darkness. The dimensions and texture of obtained bacterial cellulose nanofibers were studied using scanning electron microscopy (SEM). X-ray diffraction (XRD) showed that the biosynthesized material has a cellulose I crystalline phase characterized by three crystal planes. fourrier transform infrared spectroscopy (FTIR) data confirmed the chemical nature of the fibers. Thermo-gravimetric analysis (TGA) showed that BC preserves a relatively superior non-degradable fraction compared to microcrystalline cellulose.

In this study the PANI/PHBV blends were prepared and thermal properties, crystallization behavior, microstructure of the blends were investigated. The PANI/PHBV blends were prepared by dissolution of PANI (emeraldine base doped with dodecylbenzenesulphonic acid, DBSA) and PHBV in chloroform and films were obtained by casting. PANI amount in the blend was varied from 0.1 to 1% wt. PANI/PHBV blends were characterized by FTIR spectroscopy and the thermal behavior were analyzed by differential scanning calorimeter (DSC) and thermogravimetric analysis (TGA). FTIR spectra of the pure PHBV and PANI/PHBV blend had similar peaks. However, blends spectra show an enlargement of bands, due interaction of the chain PANI with PHBV matrix. The crystallization behaviors were investigated using DSC, with at a scanning rate of 10oCmin−1. Curve of pure PHBV showed two melting peaks (159.1°C and 172.3°C). With the increase of PANI amount in the PHBV matrix, both of the melting peaks became wider and shifted to lower temperatures. The decrease trend of first and second melting points with increase of PANI amount, suggests a reduction in the crystallinity of the blends.

The aim of this study was to develop a thermo-responsive and bioactive polymer with suitable mechanical properties for musculoskeletal tissue engineering applications. A copolymer was synthesized that comprised of hydrophilic polyethylene glycol, thermo responsive N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacrylate-poly(lactide) (HEMA-PLA) to enhance mechanical strength and an active N-acryloxysuccinimide (NAS) group for conjugation to proteins to enhance biological properties. A model protein such as elastin was used to assess the feasibility of conjugating this polymer to protein. The results of 1HNMR analyses confirmed that random polymerization was viable technique for synthesis of this copolymer. The co-polymers synthesized with PEG content of 3 mol% were water soluble. A hydrogel was created by dissolving the copolymer and elastin below room temperature in aqueous media, followed by rapid gelation at 37°C. The results of Fourier transform infrared analyses confirmed the conjugation of protein to copolymer due to significant reduction of ester group absorption (1735 cm−1). This data confirmed molecular interaction between protein and the temperature responsive co-polymer. Our preliminary results demonstrated that it is viable to tune different properties of this hydrogel by changing the composition of co-polymer.