AVS2012 Session BI-TuP: Biomaterial Interfaces Poster Session

Proliferation of stem cells has been observed to be affected by surface roughness in the micro and nanometer range. Furthermore, when cultured on line patterns with dimensions in the sub-micrometer regime these have been found to adopt elongated morphologies and align with respect to patterns, often called contact guidance. Contact guidance has been observed to induce stem cell differentiation towards neurogenic and myogenic lineages. We have investigated the effect of rippled silicon substrates with different height and periodicity in the nanometer range on the behavior of human derived adult stem cells.

The substrates were prepared by irradiating silicon substrates with xenon ions with different energies and fluxes at angles of either 65 or 67º with respect to the surface normal. From this, substrates with nanoripples of different heights (h) and periodicities (λ) were obtained. As determined by AFM measurements the prepared substrates had ripple features ranging from h=3 nm with λ=50 nm up to h=70 nm with λ=650 nm. The cellular response towards these surfaces was investigated using human dental pulp stem cells (hDPSC). The cells (2,500 cell/cm²) were cultured for periods of 1, 3 and 4 days, fixed and used for assessing cellular proliferation, morphology, alignment with respect to the ripple structures and expression of the osteogenic markers Runx2 and ALPL and the myogenic markers GATA4 and MyoD1.

It is found that the ripple structures influenced cellular proliferation. An increase in proliferation was observed up until ripple structures with h=8 nm and λ=170 nm followed by a decrease as the ripples structures further increased in size. The decrease in proliferation for larger ripple structures was found to correlate with an increasing number of cells undergoing contact guidance. Furthermore, it was found that the cells cultured on the ripple surfaces with features larger than h=8 nm and λ=170 nm had up-regulated expression of the myogenic markers.

The increasing ripple size is associated with larger RMS roughness values. Hence, the increase in cellular proliferation as the ripples grows in size, correlate well with literature on cellular behavior on rough samples. The decrease in proliferation observed with the larger ripple structures correlates with the increasing degree of contact guidance observed for these samples. We speculate that it could be related to changes in the cellular expression profiles. This is supported by the data from the immunohistochemistry. Especially the finding that the expression of MyoD1 is up-regulated with the larger ripple structures as this regulatory protein is known to be associated with cell cycle arrest.

Poly(N-isopropyl acrylamide) (pNIPAM) is a thermoresponsive polymer that undergoes a conformation change in a physiologically relevant temperature range. Above its lower critical solution temperature (LCST, ~32^oC), pNIPAM is relatively hydrophobic, and cells can be easily cultured on pNIPAM-grafted surfaces. When the temperature is lowered, the polymer’s chains extend and cells detach in intact sheets. It has previously been demonstrated that the NIPAM monomer is toxic; however, there are conflicting opinions as to whether the polymerized form of NIPAM is toxic. Since the cell sheets detached from pNIPAM could ultimately be used on humans, it is crucial to assess the cytotoxicity of surfaces coated with pNIPAM. Very few (<10) studies exist that investigate the cytotoxicity of pNIPAM, and their results are conflicting. Furthermore, the published studies are not comprehensive. Instead, they focus on isolated cell lines cultured on pNIPAM films generated using different methods, and use different assays to determine the degree of cytotoxicity. In this work, we present a comprehensive investigation of the cytotoxicity of pNIPAM-grafted surfaces. The cytotoxicity of pNIPAM is evaluated using different cell lines (endothelial, epithelial, smooth muscle, and fibroblasts), polymerization (free radical and commercially available pNIPAM) and deposition (spin coating and plasma polymerization) techniques, and cytotoxicity tests (MTS, Live/Dead, plating efficiency). The pNIPAM-coated surfaces are evaluated using X-ray photoelectron spectroscopy and goniometry. We find that there is lower cell viability on pNIPAM surfaces when compared to controls. The viability also seems to be deposition type dependent. This work will have valuable insights into the cytotoxicity of pNIPAM-coated surfaces, and therefore into the applicability of cells grown on these surfaces for use in human subjects.

Cerium oxide nanoparticles (CNP’s) are a promising catalytic antioxidant in biological systems, exhibiting superoxide dismutase and catalase mimetic, and nitric oxide radical scavenging activity. Nanoceria exhibits redox activity by switching between Ce 3+ and 4+ depending on environment. CNPs have also been shown to protect cells against oxidative stress. Specific formulation of cerium oxide nanoparticle is non-toxic, non-immunogenic and well tolerated both in vitro and in vivo model, which provide the rational/platform for its biological applications. Recently CNPs have become increasingly popular in biological work, both in vivo and in vitro. We have previously shown these CNPs have some potential to treat wound care, cancer therapy, retinal protection and neurodegenerative diseases. However there are several factors to consider, one being its interaction with biological molecules in different buffers, media, and serum. The common anions are phosphate, sulfate and carbonate. In our previous work, CNPs interaction with sulfate and carbonate are proven not to alter the surface chemistry of CNPs, whereas phosphate anions do. The CNPs properties are surface dependent and phosphate anions are shown to modify the surface. Current study focuses on preventing the CNPs surface modification by phosphate buffer through functionalization. Dextran and polyethylene glycol (PEG) were used to functionalize CNPs. The functionalized CNPs were incubated with phosphate ions and changing their absorbance and emission characteristic was analyzed by Ultraviolet-visible spectroscopy (UV) and photoluminescence spectroscopy (PL). The results show that CNPs functionalized with Dextran prevented interaction with PBS (phosphate ions) and preserved the redox property of the nanoparticle. However, PEG coating fails to do so. Varying pH levels in the range of 6-8 had no significant effect on the phosphate ion interaction with CNPs surface. We further investigated the surface interaction of PEG-CNPs with phosphate, while varying the concentration (5% to 40%) and the chain length (300 molecular weight to 6000 molecular weight) of PEG. The results showed that increasing the concentration or chain length of PEG did not have any effect on phosphate and cerium surface interaction. Looking into the surface charge and morphology of PEG and Dextran will allow us to gain further insight into what is occurring on the surface of these nanoparticles with phosphate ions. This basic study will help to engineer CNPs, which will be effective in biological applications and overall to prevent modification of CNPs surface.

Research advances in nanoparticles constructs intended for biomedical applications have proved to be of major importance. This often presents serious challenges in terms of imaging or tracking of the nanoparticles. Our research aims at developing a system that has the effective characteristics of therapeutics and imaging modality. It is well known that Reactive oxygen species (ROS) and nitrogen species play a critical role in many oxidative stress associated disorders like cancer, neurodegeneration, radiation induced tissue damage. Ceria nanoparticles (CNPs) have proved to be potential redox active radical scavenging agents which also exhibit superoxide dismutase and catalase mimetic activity. These nanoparticles can potentially act as antioxidant which is attributed to its redox nature of switching the oxidation states from +3 to +4 mediated at the oxygen vacancies on the surface. Recent research has shown progress in the study of enhancing the contrast in imaging using gold nanoparticles by various microscopy techniques such as TEM, Computed tomography (CT) etc. Its marker ability is attributed to the strong plasmon enhanced absorption and increased light scattering ability which gives detailed information of the location of gold particles by combining optical and electronic microscopy. It is also proven to be non toxic and biocompatible in vivo. Our research attempts on providing a formulated coupling of the above notions to form a CNP-Chitosan-Gold integrated system. Addition of Chitosan helps in the reduction of HAuCl₄ to form gold nanoparticles and this polymer also enables the biocompatibility of the imaging agent. The activity of CNPs can be improved by surface modification through selective functionalization thereby enhancing the redox behavior and stability of the system.

Malignant melanoma is the sixth most common cancer diagnosed in the United States. Surgery, chemotherapy and radiation are some of the successful techniques in killing tumor cells. However, in these techniques, it is not easy to distinguish tumor cells from the healthy once which inadvertently get exposed to chemical agent/radiation. Therefore it is required to develop an anti-cancer agent which selectively kills the cancer cells, while still protecting the normal tissues. In our preliminary work, we have shown that Dextran (1000Da) coated Cerium oxide nanoparticles (Dex-CNPs) selectively kills the cancer cells (50% killing at a concentration of 150µM) without inducing toxicity to normal cells. However, the mechanism involved on how CNPs/Dex-CNPs attain the selectivity and efficiently kill the tumor cells is still unknown. In this study we have synthesized Dextran coated ceria nano particles (Dex- CNPs) with different surface oxidation state ratio (Ce⁴⁺/Ce³⁺) but similar shape and size. This will provide an in depth understanding of the key chemical and physical properties of the system that can improve its efficacy. The varied surface oxidation of the particles is achieved by exposing Dex-CNPs to light which initiates a color change from dark to pale yellow indicating the reduction of Ce⁴⁺ to Ce³⁺. Interestingly we have found that the Dex-CNPs exposed to light have reduced cytotoxicity towards squamous cell carcinoma cell line (CCL30) compared to the protected once. Characterization of the same revealed that Dex- CNPs exposed to light have decreased Ce⁴⁺ /Ce³⁺ surface oxidation ratio compared to the other. This provides more insight in useful synthesis of Dex-CNPs in terms of storage and handling. In summary, higher Ce⁴⁺ /Ce³⁺ surface oxidation ratio is more efficient in hindering tumor growth by effectively hindering the tumor-stoma interaction.

The objective of this study is to ascertain the role of different molecular weights polyethylene glycol (PEG) solvents on the redox property of cerium nanoparticles. PEG with molecular weight of 300, 600, 1500, 3400 and 6000 were selected in this study for preparing 5mM cerium oxide nanoparticles (CNPs) in 20% PEG medium. The size and morphology of the particles were analyzed using TEM. Interestingly, the size and shapes of the nanoparticles were observed different in different chain length of the PEG nanoparticles from round to star shaped. The red-ox state of the samples was accessed at regular intervals, until stability was observed, using UV-Vis spectroscopy. Absorbance of each sample was recorded in the range of 250nm to 600nm. All the PEG-CNPs sample revealed stable peak at 298nm (characterstic of Ce³⁺) with additional minor peak observed at 380nm for 1500, 3400 and 6000 PEG-CNPs sample. The biological activity measured by superoxide dismutase mimetic assay was found to be similar for all the PEG-CNPs. The current research suggests that by changing the chain length of the PEG it is possible to synthesize different size and shape of the PEG-CNPs with similar redox activity for specific applications.

In addition, the protein-coated particles were investigated by transmission electron microscopy to get information about the proteins structure and their layer thickness on the particle surfaces. It was possible to distinguish the protein coatings by their molecule thicknesses and to evaluate the different particle agglomeration influenced by the used proteins by the use of scanning transmission electron microscopy.

The potential of polysaccharides for fouling-resistant coatings lies in their chemical structure: due to the presence of ether- and hydroxyl-groups, they are highly hydropihilic and able to form water-storing hydrogels. In this study, the free carboxyl-groups of two surface-tethered polysaccharides, hyaluronic acid (HA) and chondroitin sulfate (CS) were postmodified with the hydrophobic trifluoroethylamine. This strategy was chosen to study different effects: a blocking of free carboxyl groups to prevent complexation of bivalent ions and to preserve the resistance of these coatings in the marine environment, a shifting of the contact angle towards the minimum in the Baier curve and the introduction of amphiphilic properties due to the hydrophobic fluoro-groups. The coatings were tested towards their protein resistance and with different fouling relevant species to evaluate their resistance properties. Settlement and adhesion strength of the marine bacteria Cobetia marina and the two algae species Ulva linza and Navicula perminuta were reduced by the modification in case of HA based coatings. However, in case of CS coatings, the adverse effect was observed.

Proteins are able to express catalytic and sensing functions that current technologies are unable to reproduce. Instead, efforts have been focused on properly integrating this functionality with non-biological materials. Unfortunately, proteins are generally observed to lose function because of unfolding, aggregation, and overall loss of structure that occurs when a soft, solution-phase material is placed in the harsh structural and electrostatic environment that occurs on and near surfaces. To improve protein-surface interactions, we create a surface that is composed of peptides, which can be tailored for specific protein attachment. Here, we present scanning tunneling microscopy (STM) images of peptide-terminated monolayers on a gold surface created by functionalizing alkanethiol self-assembled monolayers. A Huisgen cycloaddition “click” reaction is used to tether the peptides to the surface at reactive locations that line up with modified residues on the peptide. STM is used to image the surface at each reaction step with molecular resolution. A complete surface reaction would generate a peptide density of approximately 0.6 peptides/nm², based on the distance between reactive azide functional groups and the theoretical size of our peptide. We estimate 0.4 peptides/nm² based on the area covered by peptides in our images.