Lia Stanciu Purdue University
Silvana Andreescu Clarkson University
Thierry Noguer Universite de Perpignan
Baohong Liu Fudan University
Air Force Office of Scientific Research
National Science Foundation
Purdue University, School of Materials Engineering, School of Biomedical Engineering
JJ1: Cell and Protein Adhesion
Tuesday PM, April 26, 2011
Salons 4-6 (Marriott)
11:30 AM - AA2.1
A Novel Galvanic Displacement Reaction as a Rapid, Robust, and Simple Method for SERS Substrate Fabrication Compatible with Microfluidic Device Fabrication Techniques.
Jordan Betz 1 2 , Yi Cheng 2 , Omar Bekdash 1 2 , Gary Rubloff 2 3 Show Abstract
1 Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, United States, 2 Institute for Systems Research, University of Maryland, College Park, Maryland, United States, 3 Department of Materials Science and Engineering, University of Maryland, College Park, Maryland, United States
Surface enhanced Raman spectroscopy (SERS) is a valuable analytical method for the label-free investigation of small molecules, but its use has primarily been limited to ex situ analyses. SERS substrates created by galvanic displacement reactions exhibit excellent enhancement characteristics but the processes often require the use of HF to strip the native oxide layer from the surface to be reacted, rendering this process incompatible with many of the materials used in microfluidics, such as glass and PDMS. A novel galvanic displacement reaction that is compatible with many materials used in the fabrication of microfluidic devices has been developed for fabricating SERS substrates. This method uses a mixture of silver nitrate and dilute ammonium hydroxide, compatible with glass and PDMS, to displace metal from the surfaces of thin films of aluminum, copper, and nickel. Substrates are formed in 30 minutes or less, and the method is so robust that it can even be carried out on the surface of common coins, despite the extreme heterogeneity encountered on coin surfaces. Defects in the native oxide layer are believed to act as nucleation sites, allowing the silver ions to diffuse in and oxidize the surface metal to an ion while simultaneously being reduced to the solid metal state. The composition of the silver features was confirmed by energy dispersive spectroscopy (EDS). The features formed are highly heterogeneous in size, shape, and location due to the random distribution of defect sites and the stochastic nature of diffusion limited reactions, but resemble fractal, dendritic, spherical, and polygonal structures which produce high local electromagnetic fields, enhancing the Raman signal. Analytical enhancement factors of seven orders of magnitude are obtained for the common SERS probe Rhodamine-6G. This rapid, robust, and simple method is readily integrated into microfluidic systems for in situ chemical analysis applications.
11:45 AM - AA2.2
Size-selective Surface Plasmon Resonance-based Biosensors.
Bob Feller 1 2 , Joseph Sly 2 , Victor Lee 2 , Robert Miller 2 , Andre Knoesen 1 Show Abstract
1 Electrical and Computer Engineering, UC Davis, Davis, California, United States, 2 , IBM, San Jose, California, United States
Surface plasmon resonance (SPR)-based biosensors can be used for real time detection of label-free analytes. We examined the use of porous silicate thin films on surface plasmon resonance sensors to introduce a sensitive and size-selective platform for protein binding. Star-shape polymers were used to generate randomly oriented interconnected pores with biologically relevant dimensions. These porous thin film sensors can be used to selectively examine proteins smaller than the dimension of the pore, while enhancing sensitivity due to the increased surface area for binding. Unlike polymer coupling matrices, the rigid inorganic binding platform have a well-defined morphology independent of solvent. Further, the silicate based support provided a generic functionalization scheme using silanol chemistry to passivate the surface against non-specific adsorption and to couple target specific receptors.
12:00 PM - AA2.3
Photonic Crystal Fiber for Efficient Raman Scattering of Thiol-capped Quantum Dots in Aqueous Solution.
Jacky S. W. Mak 1 , Abdiaziz Farah 1 , Feifan Chen 1 , Amr Helmy 1 Show Abstract
1 The Edward S. Rogers Sr. Department of Electrical and Computer Engineering, University of Toronto, Toronto, Ontario, Canada
Aqueous synthesis of colloidal quantum dots (QDs) has gained much popularity in the past two decades due to its simple and cost-effective means of synthesizing very small (2-6 nm), monodisperse, and highly water-soluble QDs. The use of different short-thiol chains as the capping agent is found to enhance different properties of the QDs, including quantum efficiency, size and photoluminescence (PL) tunability, emission decay time, and stability of core-shell structures. Nevertheless, the molecular structure and molecular complex responsible for the different attractive properties are not known. Optical techniques such as Fourier-transform infrared spectroscopy are limited by the strong interference of water bands in the aqueous environment. Raman spectroscopy is also limited by the weak scattering signals from the small amount of QDs dispersed in water. In our study, we demonstrated the use of hollow-core photonic crystal fiber (HC-PCF) to obtain efficient Raman scattering of thiol-capped CdTe QDs in aqueous solution. By employing HC-PCF in Raman spectroscopy, sample solutions can be selectively filled into the central cavity of the fiber while the pump laser is focused and confined inside through both bandgap confinement and total internal reflection. Subsequently, the Raman scattering signal is induced throughout the entire length of the fiber instead of just from the beam waist of the pump laser in the case of conventional Raman spectroscopy. Since the Raman scattering signals are marginally shifted from the wavelength of the pump laser, they can be efficiently collected inside the fiber and directed to the objective for detection. By using the HC-PCF as the interaction medium, strong Raman modes of the CdTe core, thiol agents, and their interfacial structures were successfully observed and compared. According to the best of our knowledge, this is the first time that such strong Raman modes of thiol-capped QDs in aqueous solution have been reported. The vibrational modes in the low Raman shift regime reveal a low crystalline core when QDs are capped with thioglycolic acid. The low crystallinity can be attributed to a large inclusion of Te compounds and surface defects observed in the Raman spectrum. The mid and high Raman shift regime reveal the formation of thiolates, and Cd-S bonds between the thiolate and the CdTe core which stabilized the QD through structured CdSxTe1-x ternary compound. 3-mercaptopropionic acid is also found to promote the formation of unidentate and chelating bidentate complexes between its carboxylate terminuses and the CdTe core. These complexes have further passivated the QD and improved its PL efficiency and stability potentially in the expense of losing solubility and bioconjugating ability. This work substantiates the promise of HC-PCF in enhancing Raman scattering signal of nanoparticles in aqueous solutions and enables possible studies of molecular structures relating to the different properties of QDs.
12:15 PM - AA2.4
Handheld Guided-mode Resonance Biosensor Platform for Label-free Detection of Cardiac Markers in Human Serum.
Gun Yong Sung 1 , Wan-Joong Kim 1 , Bong Kyu Kim 1 , Ansoon Kim 1 2 , Chul Huh 1 , Chil Seong Ah 1 , Kyung-Hyun Kim 1 , Jongcheol Hong 1 , Hyunsung Ko 1 , Sanghoon Song 3 , Junghan Song 3 , Sun Hee Park 1 4 Show Abstract
1 Biosensor Research Team, ETRI, Daejeon Korea (the Republic of), 2 , Hewlett-Packard Laboratories, Palo Alto, California, United States, 3 Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seongnam Korea (the Republic of), 4 , National Research Foundation of Korea, Daejeon Korea (the Republic of)
It represents a viable solution for the realization of a handheld biosensor platform that could screen/diagnose acute myocardial infarction by measuring cardiac marker concentrations such as cardiac troponin I (cTnI), creatine kinase MB (CK-MB), and myoglobin (MYO) for application to u-health monitoring system. The handheld biosensor plarform introduced in this presentation has a more compact structure and a much higher measuring resolution than a conventional spectrometer system.Handheld guided-mode resonance (GMR) biosensor platform was composed of a biosensor chip stage, an optical pick-up module, and a data display panel. Disposable plastic GMR biosensor chips with nano-grating patterns were fabricated by injection–molding. Whole blood filtration and label-free immunoassay were performed on these single chips, automatically. Optical pick-up module was fabricated by using the miniaturized bulk optics and the interconnecting optical fibers and a tunable VCSEL(vertical cavity surface emitting laser). The reflectance spectrum from the GMR biosensor was measured by the optical pick-up module. Cardiac markers in human serum with concentrations less than 0.1 ng/mL were analyzed using a GMR biosensor. cTnI, CK-MB, and MYO were monitored in the serum of both patients and healthy controls. Dose response curves ranging from 0.05 to 10 ng/mL for cTnI, 0.1 to 10 ng/mL for CK-MB, and 0.03 to 1.7 μg/mL for MYO were obtained. The limits of detection for cTnI, CK-MB, and MYO were less than 0.05 ng/mL, 0.1 ng/mL, and 35 ng/mL, respectively. Analysis time was 30 min, which is short enough to meet clinical requirements.1) Our results show that the GMR biosensor will be very useful in developing low-cost portable biosensors that can screen for cardiac diseases.1) W. Kim et al., Anal. Chem. 2010, in press
12:30 PM - AA2.5
Large Area and Cost-effective SERS Active Substrate with MgO Nano-facet Structures.
Jun Ho Son 1 , Hak Ki Yu 1 , Soongweon Hong 2 , Luke Lee 2 , Jong-Lam Lee 1 Show Abstract
1 Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk, Korea (the Republic of), 2 Department of Bioengineering, University of California Berkeley, Berkeley, California, United States
Surface-enhanced Raman spectroscopy (SERS) is one of the most promising methods for biochemical and medical applications and detection of organic chemicals, because it results in the enhancement of Raman scattering as much as 1014 ~ 1015 by molecules adsorbed on rough metal surfaces. The key impediment for the practical use of SERS-based sensors is the lack of robust and facile fabrication strategies for reproducible and uniform large-area and low-cost SERS substrate with high and stable Raman enhancement. Therefore, a lot of fabrication methods have been investigated to form nano-structured SERS substrate including bottom-up and top-down process.In this work, we present the fabrication of MgO nano-facet based SERS substrate with Au metal nano-particles. The MgO nano-facet structures are successfully fabricated on glass substare at room temperature using conventional electron-beam evaporation, resulting in the easy scale-up and high reproducibiltiy. High-resolution TEM images clearly showed the well aligned termination of MgO (200) plane with lattice spacing of 0.21 nm, because the MgO films tend to grow with surface termination by (200) and their family plane to acquire the most stable atomic arrangement. Thin Au layers were deposited on glass/MgO substrate and annealed to form self-assembled Au metal nano-particles for localized surface plasmon resonance (LSPR). The optical extinction spectra and dark-field images of the Au-coated MgO nano-facet/glass substrate confirmed the strong surface plasmon resonance (SPR). Furthermore, by controlling the thickness of MgO layer, the wavelengths for SPR were changed due to the size variations of MgO nano-facet structures. The Au-coated MgO/glass SERS substrate showed highly enhanced Raman intensity for 10-6 M trans-1,2-bi-(4-pyridyl) ethylene (BPE) molecules. Therefore, the MgO nano-facet structure is a good candidate for the highly reproducible and cost-effective SERS active substrate with high electro-magnetic field enhancement, because it can be easily fabricated without lithographic or non-lithographic patterning process.
12:45 PM - AA2.6
Nanotextured Electrical Immunoassays for Ultrasensitive Protein Detection.
Tim Mertz 1 , Krishna Vattipalli 1 , Tom Barrett 2 , John Carruthers 3 , Shalini Prasad 1 Show Abstract
1 Electrical Engineering and Computer Science, Wichita State University, Wichita, Kansas, United States, 2 Department of Medicine, Oregon Health and Sciences University, Portland, Oregon, United States, 3 Department of Physics, Portland State University, Portland, Oregon, United States
Robust diagnosis of a disease can be accomplished by reliable detection of multiple protein biomarkers. Traditional assay methods for protein detection such as enzyme-linked immunosorbent assay (ELISA) have several limitations – need for use of labels, time of detection is several hours, large volume of reagents, multiple proteins cannot be detected simultaneously and they are expensive. We resent the development of nanotextured electrical immunoassays for label-free, sensitive, fast, reliable and cost effective detection of multiple protein biomarkers. Thus, novel nanotechnology has been successfully employed to obtain significant enhancements over ELISA.The biosensors designed by our lab use the electrical and chemical properties of nanoporous alumina membranes to improve the sensitivity and performance of Si-based microdevices for protein sensing. The integration of nanoporous membranes with silicon microdevices results in nanoscale well-like structures, also known as nanowells which exhibit size matching with respect to proteins. The trapping of proteins within the nanowells is an experimental demonstration of “molecular crowding” phenomenon whereby the functionality of the proteins is retained due to confinement in small spaces i.e. nanowells. These alumina nanopores are electrically insulating and isolated resulting in grouping of nanowells for capacitance based detection of proteins. Conjugation of proteins into the nanowells results in a charge perturbation in the electrical double layer at the interface between the biomolecule and the gold electrode, thus causing a measurable change in the capacitance. The device performance has been demonstrated for simultaneous detection of two inflammatory markers – B-type natriuretic peptide (BNP) and troponin T from complex fluids samples i.e. patient human serum. These two proteins are biomarkers of vulnerable coronary vascular plaque rupture and detection of these proteins enables the pre-operative identification of the disease. The performance parameters of the nanomonitors are compared with the traditional assay methods. Apart from being a label-free technique, it was also concluded that the nanomonitors can provide several improvements such as highly increased speed of detection on the order of minutes as compared to several hours for ELISA, significant reduction in volume of reagents to a few µl, large reduction in cost per assay and the reduction in the size of assay thus making it a candidate for a clinical diagnostic “lab-on-a-chip” device.
JJ2: Tissue Engineering
Tuesday PM, April 26, 2011
Salons 4-6 (Marriott)
2:45 PM - **JJ2.1
Hybrid Biomaterials by Freeze Casting.
Ulrike G. K. Wegst 1 Show Abstract
1 Materials Science and Engineering, Drexel University, Philadelphia, Pennsylvania, United States
The functional requirements for synthetic tissue ubstitutes appear deceptively simple: they should provide a porous matrix with interconnecting porosity and surface properties that promote rapid tissue ingrowth; at the same time, they should possess sufficient stiffness, strength and toughness to prevent crushing under physiological loads until full integration and healing are reached. Despite extensive efforts and first encouraging results, current biomaterials for tissue regeneration tend to suffer common limitations: insufficient tissue–material interaction and an inherent lack of strength and toughness associated with porosity. The challenge persists to synthesize materials that mimic both structure and mechanical performance of the natural tissue and permit strong tissue–implant interfaces to be formed. In the case of bone substitute materials, for example, the goal is to engineer high-performance composites with effective properties that, similar to natural mineralized tissue, exceed by orders of magnitude the properties of its constituents. It is still difficult with current technology to emulate in synthetic biomaterials multi-level hierarchical composite structures that are thought to be the origin of the observed mechanical property amplification in biological materials. Freeze casting permits to manufacture such complex, hybrid materials through excellent control of structural and mechanical properties. As a processing technique for the manufacture of biomaterials, freeze casting therefore has great promise.
3:15 PM - JJ2.2
Bone Marrow-Derived Mesenchymal Stem Cells Attachment to the 3D Scaffolds: Functionalized Vertically Aligned Carbon Nanotube Arrays.
Verda Bitirim 1 , Gokce Kucukayan 2 , Donus Tuncel 3 , Erman Bengu 3 , Can Akcali 1 Show Abstract
1 , Bilkent University, Department of Molecular Biology and Genetics, Ankara Turkey, 2 , Bilkent University, Material Science and Nanotechnology Graduate Program, Ankara Turkey, 3 , Bilkent University, Department of Chemistry, Ankara Turkey
Mesenchymal Stem Cells (MSCs) have the potential to provide sources for tissue restoration in regenerative medicine and tissue engineering due to their differentiation potential into adipose, bone, cartilage, muscle, liver and nerve cells and non-immunogenic characteristics. There is no ethical concern in their usage therefore MSCs are promising tool for several cellular therapeutic approaches. A major roadblock in the use of MSCs for cell-based therapies, however, is their rareness and the long duration of their culture. Several established methods are presently available for in vitro isolation and differentiation of MSCs from bone marrow including use of nanostructured scaffolds. In this study, we aimed to mimic the extracellular matrix (ECM) environment by using functionalized vertically aligned carbon nanotube arrays (VANTA) as a 3D scaffold for bone marrow-derived MSC and to investigate the behaviors of cells on these scaffolds. VANTAs were synthesized on pre-oxidized Si (100) surfaces by chemical vapor deposition technique in different lengths using ethanol as carbon source. Synthesized flat VANTA surfaces were functionalized using the conjugate polymers with positively and negatively charged end groups. During the functionalization of VANTA surfaces, 3D cavities were formed by carbon nanotubes where some of them remained only vertically at the domain walls and some of them were completely bent parallel to the substrate surface. These modified surfaces were then seeded with rat bone marrow-derived MSCs after their characterization. We found that MSCs attached and survived according to the modification of the scaffold surfaces. Finally, our data showed that MSCs’ attachment features on VANTA surfaces decreased at the subsequent passages. Our results revealed that the modified and patterned VANTA surfaces would be a good choice as a 3D scaffold for the growth and maintenance of MSCs which may be useful tool for further applications in tissue engineering.
3:30 PM - JJ2.3
Biomimetic Bone: Mineralization of Dense Collagen Matrices.
Douglas Rodriguez 1 , Nadine Nassif 2 , Taili Thula 1 , Yan Wang 2 , Florence Babonneau 2 , Marie-Madeleine Giraud-Guille 2 , Laurie Gower 1 Show Abstract
1 Materials Science & Engineering, University of Florida, Gainesville, Florida, United States, 2 Laboratoire Chimie de la Matière Condensée de Paris , Collège de France, Paris France
Bone is a hierarchically structured composite, which at the nanostructural level consists of an assembly of collagen fibrils that are embedded with uniaxially-aligned nanocrystals of hydroxyapatite. Our studies have revealed that this nanostructure can be reproduced in vitro using a polymer-induced liquid-precursor (PILP) mineralization process. The polymeric additive consists of acidic polypeptides (e.g. polyaspartic acid) that are a simple mimic of the non-collagenous proteins associated with bone and dentin. The high charge density of the polyanionic additive sequesters ion such that liquid-liquid phase separation occurs in the mineralization solution, forming nanodroplets of a hydrated amorphous mineral precursor. Infiltration of the nanodroplets into the collagen fibrils leads to intrafibrillar mineral, which is the foundation of bone nanostructure. Through optimization of various reaction parameters, compositions matching that of bone (over 70wt% mineral) have been achieved in porous reconstituted collagen scaffolds. Dense biogenic collagen matrices, such as rat tail tendon and demineralized bone, and dense reconstituted collagen matrices can also be mineralized by this process; however, there are currently limitations in the depth of mineral penetration that can be attained. The mineralized portion of these matrices, however, does exhibit the interpenetrating organic-inorganic nanostructure characteristic of biogenic bone. Our current studies are directed at examining the mechanism of precursor formation, because stabilization of the liquid-phase precursor may allow for greater penetration depths to be achieved. On-going work includes evaluating the mechanical properties of dense biomimetic bone as well as optimizing the mineralization conditions to create macroscale materials. The long range goal of these studies is use this as a model system to understand bone formation and pathologies, and from an applications perspective, to prepare bioresorbable load-bearing bone substitutes.
3:45 PM - JJ2.4
Bio-inspired Hierarichal Vascular Network Synthesis: Electrohydrodynamic Viscous Fingering.
Kristopher Behler 1 , Eric Wetzel 1 Show Abstract
1 Composite and Hybrid Materials Branch, U.S. Army Research Lab, Aberdeen Proving Ground, Maryland, United States
Vascular networks provide a physical pathway to distribute fluid throughout a system. An uninterrupted and controllable supply of liquid is optimal for many applications such as continual self-healing, drug delivery, chemical and biological agent neutralization, and thermal management. One approach to producing hierarchical vascular networks is electrohydrodynamic viscous fingering (EHVF). Hollow channels or fingers are grown from a traditional viscous fingering approach in which a low viscosity liquid, water, is pumped through a more viscous (1,000 to 100,000 cSt) fluid or polymer. Typically, only a few large fingers are formed and the resulting pattern is governed by the capillary forces which lead to breakup and isolation of the fingers and the viscous forces between the two liquids. The EHVF process deviates from traditional viscous finger by applying a high voltage, 10 – 60 kV to the injected fluid. EHVF harnesses the applied voltage to control the size of fingers in the viscous liquid or polymer thus creating many smaller features and more fingering in systems that more closely resemble natural vasculatures seen in the human circulatory system, river beds and plants. The increase in the number of fingers and the smaller size is a result of the applied voltage, which exerts a force on the system that is greater than the viscous force and capillary force. Relaxation of the fingers is seen once the applied voltage is removed from the system. Thus a method to “freeze” the grown structure is paramount. The addition of spherical particles, aligned fiber mats, random fibers and combinations of the aforementioned additives has shown to reduce the relaxation and allow the grown fingers to retain their shape for extended periods of time. The elimination of relaxation in the finger structure allows for subsequent thermal curing in systems such as poly (dimethylsiloxane), PDMS. Another approach for retaining the fingers is to use an in-situ ultra violet (UV) cure. The UV cure system can be used on both filled and un-filled systems. The subsequent curing of the polymer by either ultra violet (UV) or thermal means creates a durable material in which the grown hollow network of channels can be produced and filled and refilled with various liquids for potential self-healing and self-repairing applications.
4:00 PM - JJ2: Tissue
4:30 PM - **JJ2.5
Irreversible Structural Transition Induced by Mechanical Shear in a Novel Peptide-amphiphile Syst.
Matthew Tirrell 1 Show Abstract
1 Bioengineering, University of California, Berkeley, Berkeley, California, United States
Shear induced, self assembly has been extensively studied using model surfactant systems which form long extended micelles at a critical shear rate. These polymer based extended micelles are able to entangle, giving the system viscoelastic properties. Here we present a peptide amphiphile, a short peptide sequence attached to a fatty acid tail, which transforms into extended worm-like micelles under shear force. Peptide amphiphiles are easily synthesized and modular in design, allowing a range of bioactive peptides to be introduced into the system. The particular peptide sequence used in this study is alanine (A) rich with interspersed lysine (K) residues, which gives it a strongly alpha helical secondary structure. The peptide amphiphile initially forms spherical micelles in aqueous solution and then undergoes a switch to form long cylindrical micelles when the correct shear force is applied. The cylindrical micelles entangle and create a gel with viscoelastic mechanical properties. The physical transition from spherical to worm-like micelle coincides with a secondary structure transition from alpha helix to beta sheet which is monitored using circular dichroism. As in the surfactant system, our peptide amphiphile solution initially behaves as a Newtonian fluid and then transitions to a shear thinning gel once a critical shear rate is surpassed. The resulting gel is self healing, and stable at 4°C on the order of weeks to months. Materials properties can also be tuned to achieve the modulus of various biological tissues. The versatile nature of this system makes it an attractive material for use as an injectable tissue engineering matrix.
5:00 PM - **JJ2.6
Bioactive Membranes and Cell-like Microcapsules.
Samuel Stupp 1 Show Abstract
1 , Northwestern University, Evanston, Illinois, United States
Our laboratory has developed a superfamily of peptide modified with hydrophobic segments that are programmed to self-assemble into filamentous nanostructures. These filaments can have remarkable bioactivity as indicated by various in vivo models for the regeneration of spinal cord, cartilage, bone, enamel, and blood vessels, among others. These peptide amphiphiles are also able to co-assemble with biopolymers to create both open and closed membranes that are highly ordered and have hierarchical architectures. This process of self-assembly can occur in time scales as fast as milliseconds, but it is dynamic and can therefore lead to the growth of membrane structures over longer time scales. The integration of this hierarchical self-assembly and the bioactivity of peptide amphiphiles yields membranes and also filamentous microcapsules of high surface area that are able to signal cells. This lecture will illustrate the use of these hybrid materials to promote angiogenesis through their ability to bind and deliver proteins. The lecture will also discuss the use of the cell-like microcapsules to interact with neurons and promote neurite growth. Another possible function of the cell-like microcapsules to be described is the possibility of using them to mediate the differentiation of stem cells.
5:30 PM - JJ2.7
Multiphase Anistropic Tissue Structures by Microdroplet Based Hydrogel Printing.
Umut Gurkan 1 2 , Feng Xu 1 2 , Yuree Sung 1 , Banupriya Sridharan 1 , Ahmet Yavuz 1 , Utkan Demirci 1 2 Show Abstract
1 Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Harvard-MIT Health Sciences and Technology, Cambrirdge, Massachusetts, United States, 2 Center for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States
Background: In this paper we present generation and preliminary assessment of multiphase anisotropic tissue structures by microdroplet based hydrogel printing method. Current cell/tissue scaffolding methods present shortcomings due to the lack of control over the spatial and temporal control over cell seeding and extracellular matrix composition. Microdroplet based hydrogel bioprinting technology can be used to engineer complex tissue anisotropies with multiple phases by producing scaffolds with controlled micro-scale spatial heterogeneity in extracellular, cellular compositions and physical properties. Therefore, we hypothesized that the microdroplet-based hydrogel bioprinting approach developed in our laboratory will successfully facilitate engineering of the complex tissue anisotropies that are composed of multiple phases. In order to test this hypothesis we printed agarose hydrogel bioinks colored with red, green and blue (RGB) high molecular weight (35-38 kDa) fluorescent dyes and assessed the phase transitions via image processing to evaluate the anisotropy of the resulting multiphase structure by measuring the RGB color intensities.Methods: Microdroplet generation process was performed with multiple ejectors in sterile laminar flow hood under controlled humidity. The inter-droplet distance was determined by the size of the droplets residing on the substrate. The prepared bio-inks (RGB colored hydrogels) were printed in a staggered configuration. The ejector was kept warm (37 degC) to minimize viscosity changes and premature gelation of the hydrogel. Printed staggered phases were gelled by incubation at 4 degC for 5 minutes. The diffusion and integration of the phases was assessed immediately after and 3 hours after printing by taking micographs and analyzing using ImageJ software. RGB color relative intensity values were used analytically to analyze the anisotropic gradient of the phases and phase transitions.Results and Discussion: The printed multiphase hydrogel structure representing an anisotropic tissue unit displayed sharp RGB boundaries between the phases immediately after printing. These sharp boundaries disappeared and smooth transitions emerged within 3 hours. These results suggest that microdroplet based hydrogel printing technology can be used to create highly anisotropic structures with smooth boundaries mimicking the complex cellular and extracellular gradients in the natural tissues. Our long term goal is to develop effective bioprinting methodologies to engineer micro-scale anisotropic complex tissue structures with multiple phases, which can be incorporated into currently available biomaterials to face the challenges of incompatibility at tissue-biomaterial interfaces.
5:45 PM - JJ2.8
Nanoporous Aluminum Oxide for Tissue Engineering.
Andreas Heilmann 1 , Annika Thormann 1 , Andreas Hoess 1 , Andrea Friedmann 1 Show Abstract
1 , Fraunhofer Institute for Mechanics of Materials IWM, Halle (Saale) Germany
Because of their unique highly opened and parallel pore structure, self-supporting membranes made from nanoporous anodic alumina are promising materials for cell cultivation and tissue engineering. Due to the adjustability of the membrane properties (pore diameter, membrane thickness) and additional surface modifications, the cell growth conditions can be varied to a great extent. Anodic oxidation of aluminum was performed to obtain self-supporting membranes with pore diameters between 20 and 450 nm and membrane thick¬nesses between 20 and 60 µm. By using a special mechanical stabilization con¬cept, the membrane thickness can be reduced down to 1 µm. On various porous membranes, proliferation and adhesion behavior of HepG2 hepatoma cells were investigated. The cell growth depends slightly on the pore diameter and much more on the surface properties. Scanning electron microscopy (SEM) was applied to examine cell morphology. The preparation with Focused Ion Beam (FIB) technology was used to study the cell surface interaction. Adhe¬sion points of cells to the underlying porous substrate were located. Especially cells on membranes with pore diameters larger than 220 nm developed small cell extensions, which penetrate into the pores up to 1.5 µm. In addition to the cell growth experiments a bioreactor was designed in order to use nanoporous alumina membranes as an interface between two compart¬ments. With such a concept, co-cultures of different cell types (e.g., mesen¬chymal stem cells and primary hepatocytes) under perfusion conditions were carried out successfully. This seems to be a promising approach for different applications in pharmacological research or tissue engineering.
Lia Stanciu Purdue University
Silvana Andreescu Clarkson University
Thierry Noguer Universite de Perpignan
Baohong Liu Fudan University
Air Force Office of Scientific Research
National Science Foundation
Purdue University, School of Materials Engineering, School of Biomedical Engineering
JJ9: Poster Session: Hybrid Biological Materials III
Thursday PM, April 28, 2011
Salons 7-9 (Marriott)
9:00 PM - JJ9.1
Development of Deformable Ca-Si-Zn Bone Restoration Complexes with Collagen.
Ren-Jei Chung 1 , Huan-Yu Wu 1 , Kai-Shiang Chen 1 Show Abstract
1 Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei Taiwan
This study aimed to develop deformable Ca-Si-Zn bone restoration complex, and focused on these analyses including mechanical properties, crystalline phases, surface properties, thermal analysis, ionic releasing and in vitro tests. This bone restoration complex is a type of “inorganic-organic” composite. The inorganic composition is Zn0.075Ca2.925SiO5, which was calcined at 1400oC by tricalcium silicate (Ca3SiO5) doped with ZnO. The organic composition is commercial deimmunized collagen. Tricalcium silicate(C3S) is one of the main composition of Portland cement. It provides the property of self-setting, after completely setting, deformation is unable. According to the previous studies, few ZnO doping in C3S could inhibit the formation of free CaO during preparation. ZnO not only stabilizes the structure of C3S but also provides antibiotic ability. In addition, collagen could increase plasticity of the composites and absorb tissue fluid and blood during wound healing.Zn0.075Ca2.925SiO5 was prepared through a solid-state reaction and then mixed with collagen and NaH2PO4-2H2O solution as buffer to obtain the complex. This bone restoration complex had a long working time, high plasticity and high compressive strength. So that, the bone restoration complexes are able to be shaped in advance before application and served in tissue engineering as scaffolds.For in vitro study, we used the mouse fibroblast cell line (L929) and human osteoblast cell line (MG63). Investigations including cell attachment observation, PI staining, cell toxicity test (MTT), DNA content evaluation, alkaline phosphatase test (ALP) and flowcytometry detection were carried out to evaluate the biocompatibility of the materials and soaking mediums. The research results showed that the cell numbers increased with cultivation time through OM observation and PI staining. According to cell toxicity test and aptosis analysis using flowcytometry, soaking mediums of different amounts of materials didn’t affect cell growth harmfully. Through alkaline phosphatase test, results showed that the soaking mediums would stimulate the osteoblast and trigger the biomineralization processes leading to higher secretion of ALP during cultivation. In conclusion, these in vitro studies insured the biocompatibility of our hybrid complex for future applications.
9:00 PM - JJ9.11
Development of Novel Nanoparticle/Protein Conjugates for Applications in Nanomedicine.
Valeria Marangoni 1 , Ana Camargo 1 , Leila Beltramini 1 , Valtencir Zucolotto 1 Show Abstract
1 , University of Sao Paulo, Sao Carlos Brazil
Nanobiocomposites have presented a high potential for biomedical applications due to their ability of combining the recognition properties of biomaterials with the unique electronic, photonic, and catalytic features of nanoparticles. In this study we describe a new strategy for conjugating gold nanoparticles and the centrin protein (BeCen1), a member of the calcium-binding EF-hand protein superfamily, usually located at microtubule-organizing centers. BeCen1 also exhibits the ability to form filaments via a nucleation-controlled polymerization. The nanoparticles are formed in the presence of the proteins using diluted acid formic as reducing agent. The protein–nanoparticle binding was confirmed by size exclusion chromatography and Transmission Electron Microscopy (TEM) images. Circular dichroism analyses (CD) revealed that the protein maintained its secondary structure upon conjugation with the nanoparticles. Furthermore, we observed that AuNP/BeCen1 conjugated kept the polymerization ability. The BeCen/AuNPs nanoconjugates exhibit high potential for biotechnological applications including biosensors and catalysis.
9:00 PM - JJ9.12
Direct Oligonucleotide Quantification by Sequence-specific Gold Nanoparticle Assay.
Prayanka Rajendran 1 , Janos Voros 1 , Marcy Zenobi-Wong 1 Show Abstract
1 Laboratory of Biosensors and Bioelectronics, Institut f. Biomedizinische Technik, ETH Zurich, Zurich Switzerland
A sensitive and sequence-based direct detection of oligonucleotides is essential for quantifying the amount of DNA/RNA in single cell. In general, direct oligonucleotide quantification assays are attractive detection systems since they offer the potential to monitor single cell RNA levels without the usage of amplification steps. This method reduces the risk of contamination and eliminates time-consuming intermediate steps. Based on these advantages a spectral based oligonucleotide detection system with thiol-functionalized oligonucleotide-modified 50 nm gold probes is studied. In this system, oligonucleotide-modified gold nanoparticles are dispersed into the microwells and the target analyte is spotted on the coverslip. The coverslip is placed on the microarray structures which contains the gold nanoparticles. The gold nanoparticles then form aggregated polymeric networks via hybridization events with complementary target oligonucleotides that were spotted onto a coverslip. The hybridization events induce aggregation which leads to concomitant change in the extinction spectra. The distinct light scattering properties of the gold nanoparticles can be utilized for the detection and quantification of DNA. The binding of the analyte (target DNA molecule) to the gold nanoparticles during the assay brings the plasmonic nanoparticles in close vicinity to each other. Due to this proximal effect they become optically coupled, creating a more enhanced field and giving a strong resonance depending on the coupling strength or interparticle distance. As a result there is a second extinction peak towards the red region. The assay made in a microwell array structures makes it possible to detect DNA in naturally occurring quantities. The 50 nm gold colloids (7 × 1011 colloids ml-1) (GC50, British Biocell, UK) were tagged with thiolated-DNA (Probe1 and Probe2) (Eurogentec, Belgium). The glass substrate with microwell structures is rendered hydrophilic property and then filled with the mixture of Probe 1 and 2. This is then covered with a coverslip on which the target analyte is dried. The spectral measurements were conducted by a custom built microscope (Axiovert 200, Zeiss, Germany) with a spectrometer (SpectraPro 2150, PIXIS 400, Princeton Instruments, US). The online data analysis and the control of the spectrometer were performed by a custom made program.The microwell array system filled with all components for a biochemical detection assay is advantageous compared to the earlier methods firstly because the dimensions of the microwells define the total volume of the assay so that sample dilution and the reagent consumption is minimized and secondly all the assay components are preloaded to the microwells which makes fluidic components unnecessary. The future directions will be to quantify the RNA from single cells without the need for amplification.
9:00 PM - JJ9.13
Electroactive Peptides via Phage Display for Biosensor Applications.
Ya-Wen Yeh 1 , Chih-Wei Liao 2 , Seonhoo Kim 2 , David Norton 2 , Laurie Gower 2 Show Abstract
1 Department of Biomedical Engineering, University of Florida, Gainesville, Florida, United States, 2 Department of Materials Science and Engineering, University of Florida, Gainesville, Florida, United States
For biosensor devices, functionalizing the surface with covalent linkers is usually employed. However, it is difficult to avoid the loss of activity following the bioreceptor-analyte binding event, which limits the lifetime of the device. The goal of our group is to use phage display to biopan for inorganic binding peptides that are reversible upon applying of an electric field. This can provide dynamic functionalization of surfaces, with applications such as self cleaning devices. For example, when the bioreceptor becomes clogged, the peptides may be released by triggering an electric field to generate a non-binding state. A fresh surface of bioreceptors can then be applied via a flow-through setup. Our group has been panning for peptides that bind strongly to indium zinc oxide (IZO), a transparent semiconducting oxide which makes it an attractive electrode for biochemical sensors. An electro-releasing device is being used to collect the strong binding peptides that are subsequently released. In an alternative method, because a reversible peptide may not necessarily be a strong binding peptide, our group has also developed a novel phage display biopanning protocol with an electro-elution process instead of the regular chemical elution. Our recent works has shown that M13 phage can be electroeluted and the eluted phage that were collected have shown good binding to indium zinc oxide surface. On-going work examines and compares these two approaches for the goal for developing self-cleaning devices and other potential applications of electroactive peptides.
9:00 PM - JJ9.14
Scattering under Shear: Alignment of a Disordered Bicontinuous Mesophase.
Annela Seddon 1 , Adam Squires 2 Show Abstract
1 Physics, University of Bristol, Bristol United Kingdom, 2 Chemistry, University of Reading, Reading United Kingdom
In this work we present evidence that biological lipids can form types of disordered bicontinuous nanostructured phases, known as sponge phases, which until now have been more commonly observed in surfactant / brine systems. By using a combination of small angle x-ray scattering under shear and rheology, we are able to identify these phases and obtain information upon the dynamics of their formation. Understanding the self-assembly behaviour of biological amphiphiles, their rheological properties and their structural response to shear can provide a foundation for the design of nanostructured materials for biological applications. Biological amphiphiles such as lipids can adopt a wide range of mesophases upon self-assembly in water. These mesophases can be classified in terms of their desire for surface curvature and range from flat phases such as the fluid lamellar phase to highly curved phases such as the inverse hexagonal phase. One such phase which is of interest both as a mimic for cell membranes and as a template for nanostructured materials is the L3, or sponge phase. This consists of a series of interconnected lipid bilayers forming a random bicontinuous network of tubules. The inherent long range disorder of this phase has meant that it is more challenging to study with techniques such as small angle x-ray scattering (SAXS) than the more ordered bicontinuous cubic phases. Previous work on surfactants in brine which form sponge phases has shown that the application of shear to the sponge phase leads to the formation of highly orientated lamellar phases. In this work we show that the application of shear to a sponge phase formed by lipids causes the mesophase to form a highly orientated lamellar phase which returns to the sponge phase on cessation of shear. By combining SAXS under shear with rheological measurements, it is possible to identify sponge phase regions for a range of lipid systems under biologically relevant conditions.
9:00 PM - JJ9.15
Enhanced Detection of Cardiovascular Biomarker Proteins: A Detailed Study of Nanoconfinement in Nanoporous Membranes.
Savindra Brandigampala 2 , Paige Feikert 1 , Krishna Vattipalli 1 2 , Shalini Prasad 1 2 Show Abstract
2 Electrical Engineering and Computer Science, Wichita State University, Wichita, Kansas, United States, 1 Bioengineering Program, Wichita State University, Wichita, Kansas, United States
The goal of this work is to understand the role of nano confinement in designing biosensors. We have been investigating silicon based micro devices incorporated with nanoporous membranes in designing sensors. We have observed that nanoporous membranes enable nanoscale size based confinement of biomolecules such as proteins onto micro platforms. This in turn enhances the concentration of the biomolecules which in turn enhances the sensitivity in detecting biomolecules. It is critical that ultralow detection of biomolecules be achieved as they have significant impact in designing diagnostics platforms for early disease diagnosis. Commercially available nano-porous membranes made out of anodized alumina and polycarbonate are evaluated for their role in nano-confinement and enhancing sensitivity of detection. In this biosensor configuration sandwich assay, an electrical double layer is formed between a test protein (C-reactive protein) and the gold surface underneath the porous membrane. Using electrical impedance spectroscopy, the capacitance changes in the electrical double layer, translating to the sensitivity and the linear dose response over a large dynamic range will be analyzed for each of the physical characteristic of the porous membrane – pore densities, height of the pore and diameter of pore.
9:00 PM - JJ9.16
Zinc Oxide Nanorod Films for Electrochemical Urea Biosensor.
Netza Palomera 2 , Marcia Balaguera 3 , Maharaj Tomar 4 , Jaime Ramírez-Vick 1 , Sunil Arya 5 , Surinder Singh 1 Show Abstract
2 Mechanical Engineer, University of Puerto Rico, Mayaguez United States, 3 Chemistry, University of Puerto Rico, Mayaguez United States, 4 Physics, University of Puerto Rico, Mayaguez United States, 1 Engineering Science and Materials, University of Puerto Rico Mayaguez, Mayaguez United States, 5 Electrical Engineer, University of South Florida, Tampa, Florida, United States
Metal oxide nanostructures have shown significant promise for biosensors, gas sensors, photocatalyst and other biomedical applications in recent past. Among these, Zinc oxide (ZnO) nanostructures, exhibiting interesting properties such as high catalytic activity, biocompatibility, high iso-electric point, large surface to volume ratio, make them good candidate for biosensing applications. Here we report the synthesis of ZnO nanorods (ZnONR) on ITO film in aqueous phase and its application in Urea biosensor fabrication. ZnONR have been synthesized by two step method, first seed growth of ZnO by sputtering on ITO films followed by decomposition of zinc nitrate hexahydrate / hexamethylenetetramine (HMT) in aqueous phase. Using high isoelectric point of ZnO, Urs/ZnONR/ITO bioelectrode has been fabricated by physical binding of Urease (Urs) enzyme onto ZnONRs. XRD, FE-SEM, and cyclic voltammetry (CV) has been used to characterize ZnONR and Urs/ZnONR/ITO bioelectrode. The FE-SEM measurements confirm the formation of ZnO nanorods. CV measurements on Urs/ZnONR/ITO biolectrode reveal linearity of 10 – 125 mg/dL with high sensitivity of 10 µAdL/mg (1.66 µA/mM) and relatively low Michaelis-Menten constant (Km) of 2.21 mM for urea. The results indicate the potential of ZnO NR films for fabrication of commercial biosensors.
9:00 PM - JJ9.19
Cytotoxicity of Heterobifunctional Crosslinkers used for Conjugation and Functionalization of Biomaterials.
Nihar Shah 1 , Jill Covert 1 , Rishabh Jain 2 , Ankit Agarwal 2 , Nicholas Abbott 2 , Christopher Murphy 1 Show Abstract
1 Vet Med Surgical and Radiological Sciences, University of California, Davis, California, United States, 2 Chemical and Biological Engineering, University of Wisconsin, Madison, Wisconsin, United States
Heterobifunctional crosslinkers are ubiquitously used in multiple life science fields for a variety of applications, including: biosensors, protein- and cell-surface interaction analysis, protein-protein and DNA/RNA-protein conjugation, immunogen preparation, and biomaterials surface modification. Specifically for tissue- and cell based applications, crosslinkers are used to functionalize material surfaces, devices, and scaffolds, with bioactive molecules that can influence cell behavior. Despite the wide-ranging possibilities for cellular applications, few published reports on the comparative cytotoxicity of crosslinkers exist. Furthermore, studies have been limited to one or two commonly used crosslinkers such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and often within too narrow a concentration range to evaluate their reaction efficiency and relative cytotoxicity. In the development of engineered therapeutic delivery systems, it is critical to assess the potential cellular toxicity of these essential crosslinking reagents.We have determined the relative cytotoxicity of a wide panel of heterobifunctional crosslinkers selected based on multiple criteria including popularity for existing applications, their reactive moieties and functional linking groups. We restricted our selection to water-soluble crosslinkers that could be delivered to cells and tissues in buffered solutions. Specifically, we tested two main families of crosslinkers, commercially available synthetic heterobifunctional crosslinkers and self-reactive catecholamines that were first identified in mussel adhesives. We tested these chemicals immediately after preparation as well as after hydrolysis (crosslinkers) or aggregation (catecholamines). Concentrations spanned from 1 nM up to 10 mM. Cells (primary human dermal microvasculature endothelial cells (HMVECs) and immortalized human keratinocyte cells (HaCaT) were exposed for 1, 4, or 24 hours.Significant findings include: differential responses based on concentration, crosslinker state, treatment time, and cell type. For most crosslinkers tested, cells remain viable from 1 nM up to 100 µM. This large concentration window will provide flexibility for conjugation reaction optimization within cellular viability limits. In some cases, differences were observed between the fresh and modified forms which resulted in either increased (e.g. aggregated catecholamines) or decreased (e.g. hydrolyzed crosslinkers) toxicity. Further segregation of toxicity was delineated by temporal assays, wherein longer treatment durations shifted the onset of toxicity towards lower concentrations. Additionally, for some of the chemicals tested, primary cells (HMVECs) showed greater sensitivity at lower concentrations than immortalized HaCaT cells. The data provided informs the rational selection of crosslinking reagents for cell and tissue applications.
9:00 PM - JJ9.2
Hybridization State Detection of DNA Functionalized Gold Nanoparticles Using Hyperspectral Imaging.
Richard Murdock 1 2 , Omar Khan 2 3 , Nancy Kelley-Loughnane 1 , Thomas Lamkin 1 , Saber Hussain 2 Show Abstract
1 711 HPW/RHPC, Wright Patterson Air Force Base, Dayton, Ohio, United States, 2 711 HPW/RHPB, Wright Patterson Air Force Base, Dayton, Ohio, United States, 3 Neuroscience and Behavioral Biology Department, Emory University, Atlanta , Georgia, United States
The objective of this investigation was to further explore the ability noble metal nanoparticles to produce unique local surface plasmon resonance (LSPR) responses, in order to detect the binding states of particles for sensor applications. Hyperspectral imaging has the unique ability to capture spectral data at multiple wavelengths in each pixel image. With this imaging technique, one is able to distinguish, with certainty, different nanomaterials as well as discriminate between nanomaterials and biological materials. In this study, 4 nm and 13 nm gold nanoparticles (Au NPs) were synthesized, functionalized with complimentary oligonucleotides, and hybridized to form large networks of similar particles. Reflected spectra were collected from each sample (unfunctionalized, functionalized, and hybridized) and evaluated. The spectra showed unique peaks for both sizes of Au NPs, as well as exhibited narrowing and an increase in intensity of the spectra as the NPs were functionalized and subsequently hybridized. This change in the reflected spectrum is different from normal aggregation effects where the absorption peak is red-shifted and broadens, while the reflected spectrum usually is broadened as well. Rather, the DNA linkage of the Au NPs intensifies the LSPR and appears to be dependent on the interparticle distance through oligonucleotide length, which is also explored through the incorporation of a poly-A spacer into the sequence. Also, the hybridized Au NPs were exposed to cells with no adverse affects and still retained their unique spectral signatures. With the ability to distinguish between nearly individual NP binding states, hyperspectral imaging can provide a method of tracking the intracellular actions of nanomaterials as well as have potential biosensing applications.
9:00 PM - JJ9.20
Nucleation Kinetics of Calcium Phosphate on Collagen.
Jinhui Tao 1 , Jim De Yoreo 1 , George Nancollas 2 Show Abstract
1 Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, United States, 2 Department of Chemistry, University at Buffalo, Buffalo, New York, United States
Deciphering the thermodynamic and structural controls underlying nucleation of calcium phosphate on collagen is critical for understanding the organic-inorganic interface of biominerals such as bone and dentine. In bone, apatite crystals are arranged with their  axis parallel to the long axis of collagen and the nucleation of apatite (AP) is believed to be controlled by the collagen fibers. However, the influence of collagen on the thermodynamics and kinetics of the initial stages of apatite nucleation is still unclear. To determine the interfacial energy of calcium phosphate on collagen and the underlying mechanisms of nucleation, in-situ atomic force microscopy (AFM) was used to monitor the nucleation process of calcium phosphate in the presence of collagen. The experiment was divided into two steps. In the first step collagen was physically adsorbed onto mica surfaces in the presence of potassium ions over a range of pH values. The organization of collagen evolved from random fibers to co-aligned fibers to ordered bundles to D-banded micro-ribbons as the K+ ion concentration and pH were varied. In the second step, nucleation experiments were performed at pH 7.40 for a series of supersaturations (S) between 3.08 and 3.47 with respect to AP and the number density of calcium phosphate nuclei as a function of time was measured. High-resolution TEM was used to determine the initial phase of the calcium phosphate nuclei. We found that the initially formed phase was highly dependent on S, with AP forming directly for S ≤ 3.31 and amorphous calcium phosphate (ACP) forming first for S ≥ 3.36 before transforming to octacalcium phosphate (OCP) and then AP. Using classical nucleation theory to analyze the data, the interfacial energies of apatite and ACP were estimated to be 91±1 and 54±0.4 mJ/m2, respectively. With these energies, at the smallest S (S=3.36) for which ACP first forms, the free energy barrier (Gc) for ACP formation should be about 200 times that for AP formation and equal to over 1800kT. Consequently, ACP should not form under these conditions. The failure of the classical expressions for both the barrier and the nucleation rate may be a consequence of the presence of pre-nucleation clusters reported elsewhere. Assuming that these clusters have excess free energy relative to the solution, if nucleation occurs through their aggregation the free energy barrier decreases to several kT. Moreover, while this thermodynamic barrier should still favor AP nucleation, its low value suggests that the unexpected appearance of ACP is a result of significant kinetic barriers to AP formation, such as those associated with the structural rearrangements required to transform the clusters into AP.
9:00 PM - JJ9.21
Analysis of Amelogenin Assembly at the Oil-water Interface: The Role of Hydrophilic C-terminus.
Olga Martinez-Avila 1 , Shenping Wu 2 , Yifan Cheng 2 , Stefan Habelitz 1 Show Abstract
1 Department of Preventive and Restorative Dental Sciences, School of Dentistry, UCSF, San Francisco, California, United States, 2 Department of Biochemistry & Biophysics, UCSF, San Francisco, California, United States
Self-assembly of amelogenin proteins plays a key role in controlling enamel biomineralization. Full length amelogenin protein self-assembles into nanospheres of 20-40 nm under certain conditions and these nanospheres are thought to regulate the growth and organization of nanofibrous apatite crystals through a mechanism still unknown. However, the amphiphilic nature of the bipolar full-length protein provide the characteristics that might allow assembly into supramolecular structures of high order similar to surfactant molecules. Our group recently reported on the use of water-in-oil metastable emulsions to induce the formation of amelogenin nanoribbons that are formed from reverse micelles at the oil-water interface. The hydrophilic C-terminus of the protein may play an essential role in the assembly. In this system, the interactions between the hydrophobic tails of human recombinant full length amelogenin rH174 occur and prevent the formation of amelogenin nanospheres. The purpose of this study was to elucidate the role of the hydrophilic C-terminus by studying the self-assembly of two recombinant MMP-20 proteolytic products, rH163 and rH146 in the water-in-oil system. The effects on protein self-assembly and crystal formation as a function of calcium and phosphate concentration, protein concentration, pH, water-oil ratio and incubation time for rH174, rH163 and rH146 were studied. The gel-matrix was analyzed using Atomic force microscopy, Transmission and Scanning electron microscopy, Energy Dispersive X-ray analysis, Dynamic Light Scattering, Circular Dichroism and helical reconstruction modeling. rH163, lacking the hydrophilic C-terminus, self assemble into regular nanospheres, which do not vary with pH, ion concentration or time, suggesting a clear role of the C-terminus in stabilization of the water-in-oil emulsion and for the generation of reverse micelles that initiates amelogenin self assembly into nanoribbons as shown for full length protein rH174. Surprisingly, rH146, self-assembly into a mixture of helical architectures. Width of rH146 helical structures is similar to untwisted amelogenin nanoribbons formed by rH174 suggesting the twisting of regular nanostrings that may be due to hydrophobic repulsions. Co-existence of few isolated regular nanostrings together with abundant helical structures of amelogenin rH146 highlight the enhanced contribution of the central domain in amelogenin assembly at the oil water-interfaceFunded by NIH-NIDCR R01-DE017529.
9:00 PM - JJ9.23
The Effects of Hydroxyapatite Nanoparticles on Breast Cancer Bone Metastasis in 3-D Scaffolds.
Debra Lin 1 , Siddharth Pathi 2 , Jason Dorvee 1 , Lara Estroff 1 , Claudia Fischbach-Teschl 2 Show Abstract
1 Materials Science & Engineering, Cornell University, Ithaca, New York, United States, 2 Biomedical Engineering, Cornell University, Ithaca, New York, United States
Breast cancer metastasis to bone often leads to the formation of osteolytic lesions, areas with severe mineral density loss. However, the role of cell-material interactions, between cancer cells and the bone mineral hydroxyapatite HA, Ca10(PO4)6(OH)2, in promoting metastatic and osteolytic activity remains unclear. To examine the effects of mineral properties on cancer cell activity, we have developed a method to synthesize a series of well-defined HA particles that vary in size, crystallinity, and composition. Monodispersed HA particles were obtained via a 2-step process of a wet chemical precipitation followed by hydrothermal aging of the precipitate for variable lengths of time to obtain specific particle sizes. The size, crystallinity, and composition of the HA nanoparticles were characterized with X-ray Diffraction (XRD), Transmission Electron Microscopy (TEM), and Fourier Transform Infrared Spectroscopy (FTIR). The particles were then incorporated into 3-D porous poly(lactide-co-glycolide) (PLG)-scaffolds via a gas-foaming salt-leach process. The pore morphology and particle distribution in the scaffolds were characterized via TEM and SEM. The solubility of particles in the presence of media was assessed by analyzed by Inductively Coupled Plasma (ICP). Scaffolds were then seeded with a bone-specific breast cancer cell line, MDA-MB231. Protein adsorption and cell activity were examined for the different scaffolds. Comparing HA-containing scaffolds with control scaffolds, marked increase in cell adhesion and proliferation were observed for scaffolds with HA. Secretion of interleukin-8 (IL-8), an osteolytic and pro-angiogenic cytokine, was also enhanced in HA-containing scaffolds. Scaffolds containing different HA particles exhibited enhanced protein adsorption with decreasing HA particle size and crystallinity, while IL-8 was upregulated with increasing HA particle size and crystallinity. Our results suggest that the nanoscale properties of HA in the bone matrix play a role in regulating metastatic breast cancer cell behavior. These mineralized scaffolds may provide innovative platforms for modeling the interaction of cells with the bone microenvironment.
9:00 PM - JJ9.3
A Soluble Strontium Carbonate Implant Coating for Local and Targeted Cell Stimulation.
Johan Forsgren 1 , Andreas Hoess 1 , Marjam Ott 1 , Maria Strømme 1 , Hakan Engqvist 1 Show Abstract
1 Engineering Sciences, Uppsala University, Uppsala Sweden
A concept for local delivery of strontium ions from prosthetic implants has been developed in order to stimulate and improve the bone formation after implantation. Strontium acts by increasing the differentiation and bone forming activity of osteoblasts while also reducing the bone-resorbing activity of osteoclasts. This is believed to have a positive effect on the bone healing process, reduce convalesce time after surgery and improve implant fixation. In the present work, a coating of strontium carbonate was deposited on titanium substrates via a biomimetic mineralization process where chemically treated titanium was immersed in a strontium acetate solution. Due to electrostatic interactions, ions in the solution accumulate and bind to the surface and eventually form a nanocrystalline film of SrCO3 on the substrate. The final coating has a nanoporous morphology and is soluble in aqueous solutions. The dissolution rate is slow and easily adjusted by simple thermal treatment of the coating, which enables a controlled and sustained effect from the strontium. When immersed in simulated body fluid, the SrCO3 coating interacts with ions in the solution and bone like apatite is formed on the surface. In vitro cell studies with human osteosarcoma osteoblast-like cells demonstrated that the SrCO3 coating promoted cell adhesion and proliferation. In this study, cells were cultured on heat-treated SrCO3 coatings with relatively low solubility for 10 days. No signs of cytotoxicity were observed and the cell viability was equal to that of cells seeded on conventional tissue culture plastics. In addition, alkaline phosphatase (ALP) activity in the cells was increased extensively during the first three days of cultivation on the SrCO3 coated surfaces. At this point the ALP levels in these cells were twice as high as in the control group. The elevation in ALP activity correlated precisely with the amount of strontium released from the surface. The release of strontium ions declined after a couple of days, which resulted in a decreased ALP activity. However, the ALP levels were at all times higher in the cells seeded on SrCO3. Scanning electron microscopy studies confirmed that the cells were thriving on the SrCO3 coating; the cells were stretching out over the porous surface and exhibited well-developed filopodia. The findings from this study are encouraging. Strontium carbonate has to our knowledge never been considered for biomedical applications before. Here it was formed via a biomimetic mineralization method on titanium, which allowed the formation of a nanoporous coating. The coating was evaluated in cell culture experiments where the release of strontium ions increased the activity of osteoblast-like cells. The positive effect on the cells lasted for at least 3 days. By increasing the solubility of the coating through altering the thermal treatment, it is proposed that an even more sustained effect can be achieved.
9:00 PM - JJ9.4
T Cell-specific Delivery of HDAC Inhibitor Targeting Latent HIV Using Antibody-conjugated PLGA Nanoparticles.
Jangwook Lee 1 , Sunmi Choi 1 , Sang Kyung Lee 1 , Kuen Yong Lee 1 Show Abstract
1 , Hanyang University, Seoul Korea (the Republic of)
Drug delivery systems based on nanoparticles, with consideration of excellent pharmaco-kinetics and dynamics as well as targeting effects, have found potential for many biomedical applications. We have developed a novel delivery system using PLGA-based nanoparticles for delivering histone deacetylase inhibitor (HDACi) to human T cells, the major reservoirs for latent HIV. Latent HIV can be activated through inhibition of histone deacetylase (HDAC) using HDACi. However, pleiotropic effects of HDACi include reactivation of other latent viruses due to lack of selectivity, leading to systemic toxicity. In order to restrict the effects of HDACi to human T cells we conjugated a single chain fragment antibody targeting the T cell-specific CD7 molecule (scFvCD7) to the surface of PLGA nanoparticles encasing the HDACi, valproic acid (VPA). These nanoparticles displayed a sustained VPA release over 3 days and a significant association with human T cells with minimal cytotoxicity. Importantly, VPA-loaded scFvCD7-conjugated PLGA nanoparticles successfully activated latent HIV in human T cells in vitro. This approach suggests a new method geared to eradicate HIV through the elimination of HIV reservoirs in supportive HIV therapy.
9:00 PM - JJ9.5
Tree-dimension Imaging of Fibroblast Cells by Scanning Transmission Electron Microscopy with Various Tilting Angles.
Yun-Wen You 1 , Hsun-Yun Chang 1 , Wei-Lun Kao 2 , Guo-Ji Yen 2 , Chi-Jen Chang 2 , Jing-Jong Shyue 1 2 Show Abstract
1 , Academia Sinica , Taipei Taiwan, 2 , National Taiwan University , Taipei Taiwan
Three-dimension (3D) structure information is important in biological research. For the well studied internal structure of organism, the human embryonic kidney cell-line 293T (HEK293T) fibroblast cell is selected in this work for developing 3D imaging techniques by scanning transmission electron microscopy (STEM) and electron tomography (ET). The STEM used here is based on a scanning electron microscope (SEM) operated at 20 kV with home-made specimen holder and a multi-angle solid-state detector behind the sample. Because of the low acceleration voltage, stronger electron-atom scattering yields stronger contrast in the resulting image. While the higher scattering probability leads to diffused background in traditional TEM image and diminish the contrast, the incoherent STEM imaging allowed the observation of thick specimens. In this work, 2D STEM images of 950 nm thick of cell slices with projection angles between ±25° are collected and the 3D volume structure was reconstructed using ART model with the TomoJ plugin in ImageJ. Although the tilting angle is restrained and limits the image resolution, slicing the reconstructed volume generated depth profile of the thick specimen and cross-sectional structure can also be obtained using ImageJ. Comparing with existing systems capable of doing ET, the method presented here yields better contrast on thick biological specimens and SEM-based instrument costs less than a TEM. Using this technique, cellular uptake of self-assembled monolayer modified Au nanoparticles with different surface charge is examined and the final position of these nanoparticles inside the cell is imaged.
9:00 PM - JJ9.6
Carbon Highways in High Surface Area Conducting Paper Membranes for Efficient DNA Separation.
Aamir Razaq 1 , Maria Stromme 1 , Leif Nyholm 2 , Albert Mihranyan 1 Show Abstract
1 Engineering Science, Uppsala University, Uppsala Sweden, 2 Material Chemistry, Uppsala University, Uppsala Sweden
The ability to separate and purify biomolecules, e.g. DNA, from complex biological samples at larger scale is always in demand for biotechnology applications. The interaction of DNA with polypyrrole (PPy) films coated on different substrates has been investigated, and irreversible adsorption of DNA onto PPy has commonly been observed [Misoska et al., Synth. Metals 123 (2001) 279; Pande et al., Biomater 19 (1998) 1657]. It could be hypothesized that this was due to two different phenomena: i) slow diffusion of DNA during the reduction as opposed to migration in an electric field during PPy oxidation; and ii) the PPy films becoming non-conductive during reduction which hinders the electron transport needed for an efficient release of the trapped DNA molecules. To verify these hypotheses, we chemically polymerized a thin ~30-50 nm PPy layer on a high surface area Cladophora-cellulose substrate [Mihranyan et al, J. Phys. Chem. B 112 (2008) 12249]. The resulting composite membrane had the appearance of a black flexible paper sheets with good mechanical strength, large surface area (80 m2/g) and high electron conductivity (~2 S/cm). The latter material was previously utilized for efficient (and fully reversible) extraction of organic and inorganic anions [Razaq et al, J. Phys. Chem. B 113 (2009) 426; Gelin et al, Electrochimica Acta 54 (2009) 3394]. The oxidation of these PPy conducting membranes resulted in the inclusion of [dT]6 hexamer as a counter ion into the film even in the presence of other competitor anions [Rubino et, al J. Phys. Chem. B 114 (2010) 13644]. The goal of the present study was to study the possibilities of obtaining a complete release of [dT]6 hexamers. By exploiting 30-50 nm thick PPy layers, firstly we observed a partially reversible release of [dT]6 hexamers accounting for ~ 30% of the DNA absorbed. In order to increase the conductivity of the membrane in the reduced state, we incorporated chopped carbon fibers (CCFs) into the composite at a 1:1 wt. ratio during the synthesis. A 20% increase in the electrical conductivity of the membrane with CCFs was observed which resulted in a release efficiency of ~ 50%. It was concluded that CCFs acted as conducting highways which facilitated the electron transport in the reduced state and made it possible to access a larger portion of electroactive PPy sites during the reduction. The results, thus, suggest that improved conductivity in the reduced state significantly improves the efficiency of the DNA release. The effects of an application of an external electric field as a driving force for the DNA release will also be discussed as a way of developing a new technology platform for novel electrochemically-controlled separation techniques in biotechnology, nanoanalytical systems, paper-based diagnostic devices, and immunosensors.
9:00 PM - JJ9.7
Culturing Cells in a 3D Ordered Cellular Solid.
Keng-hui Lin 1 5 , Jing-ying Lin 5 , Wan-jung Lin 2 , Wei-jung Lin 2 , Wei-chun Hong 1 , Hsiang-haw Ning 2 , Stephanie Nowotarski 3 , Susana Gouveia 4 , Ines Cristo 6 Show Abstract
1 Physics, Academia Sinica, Taipei Taiwan, 5 Biophysics, National Central University, Chungli Taiwan, 2 Physics, National Taiwan University, Taipei Taiwan, 3 Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States, 4 , Gulbenkian de Ciencia, Oeiras Portugal, 6 , Science and Technology Austria, Vienna Austria
We demonstrated a high throughput method to fabricate gelatin-based ordered cellular solids with tunable pore size and solid fraction. The process involved generating high air fraction and monodisperse liquid foam by a cross-flow microfluidic device. The monodisperse liquid foam was further processed into open-cell solid foam. The solid foams were used as tissue engineering scaffolds where cells were cultured inside. Three different cell types were chosen and they showed physiological morphology and functions. Epithelial cells formed cyst-like structures and were polarized inside pores. Myoblasts formed tubular structure and fused into myotubes. Fibroblasts exhibited wide varieties of morphologies depending on their locations in scaffolds. This ordered cellular solids open doors to study the effect of pore size on cells and the mechanical properties of microscopic foam structures.
9:00 PM - JJ9.9
Behaviour of Two Titanium Alloys in Simulated Body Fluid.
Julia Mirza Rosca 1 , Domingo Herrera 1 , Agustin Santana 1 , Agurtzane Martinez 3 , Jose-Antonio Garcia 3 , David Gonzalez 2 Show Abstract
1 , University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria Spain, 3 , Advanced Surface Engineering Center AIN, Pamplona Spain, 2 , Technological Institute of Canarias, Las Palmas de Gran Canaria Spain
Titanium possesses an excellent corrosion resistance in biological environments because the titanium dioxide formed on its surface is extremely stable. When aluminium and vanadium are added to titanium in small quantities, the alloy achieves considerably higher tensile properties than of pure titanium and this alloy is used in high stress-bearing situations. But these metals may also influence the chemostatic mechanisms that are involved in the attraction of biocells. V presence can be associated with potential cytotoxic effects and adverse tissue reactions. The alloys with aluminium and iron or with aluminium and niobium occur to be more suitable for implant applications: it possesses similar corrosion resistance and mechanical properties to those of titanium-aluminium-vanadium alloy; moreover, these alloys have no toxicity.In this paper, pure Ti, Ti-6Al-7Nb and Ti-6Al-4Fe with a nanostructured surface were studied. Data about mechanical behaviour are presented. The mechanical behaviour was determined using optical metallography, tensile strength and Vickers microhardness. For the electrochemical measurements a conventional three-electrode cell with a Pt grid as counter electrode and saturated calomel electrod (SCE) as reference electrode was used. AC impedance data were obtained at open circuit potential using a PAR 263 A potentiostat connected with a PAR 5210 lock-in amplifier. The ESEM and EDAX observations were carried out with an environmental scanning electron microscope Fei XL30 ESEM with LaB6-cathode attached with an energy-dispersive electron probe X-ray analyzer (EDAX Sapphire). After 3 days of immersion in simulated body fluid the nucleation of the bone growth was observed on the implant surface.It resulted that the tested oxide films presented passivation tendency and a very good stability and no form of local corrosion was detected. The mechanical data confirm the presence of an outer porous passive layer and an inner compact and protective passive layer. EIS confirms the mechanical results. The thicknesses of these layers were measured. SEM photographs of the surface and EDX profiles for the samples illustrate the appearance of a microporous layer made up of an alkaline titanate hydrogel. The apatite-forming ability of the metal is attributed to the amorphous sodium titanate that is formed on the metal during the surface treatment. The results emphasised that the surface treatment increases the passive layer adhesion to the metal surface and improves the biocompatibility of the biomedical devices inducing the bone growth on the implant surface.
Lia Stanciu Purdue University
Silvana Andreescu Clarkson University
Thierry Noguer Universite de Perpignan
Baohong Liu Fudan University
Air Force Office of Scientific Research
National Science Foundation
Purdue University, School of Materials Engineering, School of Biomedical Engineering
JJ10: Biopolymers and Tissue Engineering
Friday AM, April 29, 2011
Salons 4-6 (Marriott)
9:00 AM - **JJ10.1
Sea Urchin Inspired Photonic Solids.
Chekesha Liddell Watson 1 Show Abstract
1 Department of Materials Science and Engineering, Cornell University, Ithaca, New York, United States
Anisotropic shape in biological and natural systems continues to inspire the development of structural complexity in synthetic materials. For instance, during gastrulation the shape of sea urchin embryos undergoes a dramatic systematic change that has been compared to particle models. Controlled deformation morphologies arise from the evacuation of swelling solvent and unreacted monomer through a mildly cross-linked skin layer as colloids contract during seeded polymerization. Deformation is driven by the pressure gradient across the porous shell surface. Isotropic shrinkage transitions to regimes where the inhomogeneous membrane shell flattens, curvature reversal occurs forming a depression, an invagination ensues and its tip deepens until tearing connects to a hollow or solid central core region. Here, to incrementally capture the morphology development a geometric model joining a hemisphere to a torus with varied inner and outer hemi-toroid radii is defined. The consequence of packing and transformation of colloidal building blocks from hemisphere through 'mushroom cap' shaped particles to spheres for the optical properties of photonic crystals will be presented.Self-organizaiton of such nonspherical particles into photonic solids under confinement conditions will also be discussed.
9:30 AM - JJ10.2
Investigation of the Nest Cell Lining of Colletes Inaequalis.
Shannon Taylor 1 , Rebecca Belisle 1 , Debbie Chachra 1 , Christopher Morse 1 Show Abstract
1 , Olin College of Engineering, Needham, Massachusetts, United States
As we look to create new hybrid polymers from renewable resources instead of petroleum, we find inspiration in natural materials. The nest cell lining of Colletes inaequalis, a species of solitary bee native to New England, is a particularly interesting naturally-occurring polymer composite. Waterproof, antibacterial, antifungal, and resistant to degradation, it is a promising case study for a robust, bio-derived polymer. Bees of the genus Colletes create nest cells in soil, lined with a cellophane-like material produced in their enlarged Dufour’s gland. Researchers have confirmed that the nest cell lining is a polyester, composed of polymerized lactones from the Dufour’s gland [1-3]. The bees have been observed to use their brushlike bilobed tongues to spread the gland secretions on the soil walls of their nest cell cavities to form the linings [2, 3]. However, the polymerization mechanism is still unknown, and the mode of construction of the nest cells has not been fully described [2, 4]. We have begun to characterize this thermally and chemically stable polymer and consider its potential for bio-inspiration. We have shown that these cell linings are extremely robust, insoluble, and resistant to hydrolysis by methanolic hydrochloric acid, with high thermal stability (60% to 70% of the material by mass remained intact at 360°C). This work suggests that the polyester characterized by Hefetz et al.  is not the only component of the nest cell lining material. To further characterize the cell lining composition and structure, confocal and scanning electron microscopy were used to investigate the linings of Colletes inaequalis nest cells excavated from a site in Acton, MA. In the images, both a continuous matrix and distinct fibers were observed. This suggests that the linings are a composite material. The fiber diameters range from 1 µm to 10 µm. Combustion analysis of the linings indicated the presence of nitrogen, consistent with a protein content of approximately 9%. Amino acid analysis confirmed the presence of protein, with a composition similar to silks (including 10% serine). Fourier-transform spectrometry with a focal plane array was used to localize the presence of amide bonds in the fibers, suggesting the material is indeed a polyester with a silk supporting structure. Finally, staining with Sypro Red confirmed a higher concentration of protein in the fibers. As a silk-polyester hybrid, this biological material can serve as inspiration for a novel class of biomimetic composites. Further research on the microstructure and the chemical pathways that underlie this unique polymer may lead to the development of innovative processes to create plastics that are both bioderived and robust.1. A Hefetz et al. Science 204, 415, (1979)2. SWT Batra. J Kansas Entomol. Soc. 62:121-4 (1980)3. JH Cane. J Chem Ecology, 7:403-410. (1981)4. PF Torchio et al. Ann Entomol. Soc. Am. 81(4):606-625 (1988)
9:45 AM - JJ10.3
Engineered Biomimetic Polymers as Tunable Agents for Controlling Rates of Crystal Growth and Tissue Mineralization.
Chun-Long Chen 1 , Jiahui Qi 1 , Ronald Zuckermann 1 , James DeYoreo 1 Show Abstract
1 The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, United States
In nature, proteins are known to a play significant role in the control of mineral nucleation and growth, both to create functional tissues and prevent pathological mineralization. The research described here addresses the challenge of developing a class of compounds that mimic the mineralizing functions of proteins for use in the synthesis of functional crystalline materials and as therapeutic agents. Peptoids, or poly-N-substituted glycines, are a novel class of non-natural polymers recently developed to mimic both structures and functionalities of peptides and proteins, and bridge the gap between biopolymers and bulk polymers. As with peptides, sequence-specific peptoids can be efficiently synthesized by using automated solid-phase synthesis starting from a large number of chemically diverse amine building blocks. Moreover, peptoids exhibit much higher protease stability and thermal stability than peptides or proteins. Inspired by recent research that showed low concentrations of acidic peptides and proteins can significantly accelerate calcite growth, we designed and synthesized a suite of anionic peptoids and screened them for control over calcite morphology and growth rate. Results to date demonstrate both a high degree of morphological control and extreme levels of acceleration, with both characteristics observed to be highly dependent on peptoid hydrophobicity, number of carboxylic acid groups, peptoid sequence and concentration. At high concentrations (50μM), calcite crystals formed in the presence of peptoid exhibit various unique shapes ranging from elongated spindles and twisted paddles to crosses and spheres. At low concentrations (<250nM), a number of the peptoids increased calcite growth rates by as much as a factor of 25, with the most effective being strongly amphiphilic. Based on previous research into peptide-induced acceleration, the energetic source of acceleration is likely to be a reduction in the activation barrier that controls the rate of solute addition at atomic steps on the calcite surface. Here we estimate the magnitude of the barrier and present a number of structural scenarios that could result in its reduction, including enhanced cation desolvation rates, disruption of the near-surface solution layer, and displacement of the waters believed to be strongly bound to calcite surfaces.
10:00 AM - JJ10.4
Morphology of Cholesterol-incorporated Poly(ethylene glycol) Polymer Networks via Small Angle X-ray Scattering.
Kristin Engberg 1 , Dale Waters 1 , Rachel Parke-Houben 1 , Laura Hartmann 1 , Michael Toney 2 , Curtis Frank 1 Show Abstract
1 Chemical Engineering, Stanford University, Stanford, California, United States, 2 , Stanford Synchrotron Radiation Lightsource, Menlo Park, California, United States
Cholesterol molecules were incorporated into photopolymerized poly(ethylene glycol) (PEG) hydrogel networks, thus altering the structure and properties of a well-characterized and commonly used biomaterial. Addition of a hydrophobic biomolecule like cholesterol could enhance the functionality of a PEG hydrogel by allowing for attachment of drug-delivering liposomes or by simply altering the properties of the material to allow for greater diffusion of nutrients or shifted mechanical strength. Cholesterol-PEG-acrylamide (Chol-PEG-AAm) molecules were synthesized and added, in molar ratios ranging from 10% to 50% Chol-PEG-AAm, to precursor solutions of PEG-diacrylate macromonomers in both chloroform and deionized water. Photopolymerization resulted in end-linked PEG-co-(PEG-chol) polymer networks that were subsequently washed and swollen in both organic and aqueous solvents. Small angle X-ray scattering (SAXS) was used as a technique to determine the molecular level morphology of the PEG-co-(PEG-chol) gels in order to relate the network structure to the macroscopic properties. Control samples of PEG networks without cholesterol exhibited a peak in the scattering curve regardless of the solvent used during network formation or in swelling afterwards. The observed peak represents an ordered spacing within the network structure and has been shown in previous work to correlate to the spacing of large, hydrophobic crosslink junctions with high functionality. Scattering curves of PEG-co-(PEG-chol) networks polymerized in organic solvent reveal a collapse of the cholesterol into the crosslink junctions upon transference of the networks from an organic to aqueous swelling solvent. This collapse led to a greater spacing between crosslink junctions with increased amounts of incorporated cholesterol. In situ polymerization of PEG-co-(PEG-chol) networks in water using the SAXS X-ray beam was performed. Scattering observed during polymerization demonstrated the presence of micelle-like structures formed by the Chol-PEG-AAm macromonomers in aqueous solution (20% to 50% molar ratios) and showed that they remained present during network formation. Preliminary results of protein diffusion through PEG-co-(PEG-chol) networks showed that the hydrogel network morphology changes caused by the cholesterol incorporation may improve diffusive properties. A solid understanding of the relationship between the nano-scale and macro-scale features of these polymer networks will allow us to design and tune the materials for specific biomedical applications.
10:15 AM - JJ10.5
Design of Polyethylene Glycol-based Multidentate and Multifunctional Oligomers for Biocompatible Semiconductor and Gold Nanocrystals.
Goutam Palui 1 , Hyon Bin Na 1 , Hedi Mattoussi 1 Show Abstract
1 Department of Chemistry and Biochemistry, Chemical Science Laboratories, Florida State University, Tallahassee, Florida, United States
There has been a great interest in developing inorganic nanocrystals as novel probes and platforms for use in bio-inspired applications. They range from tunable fluorescent platforms such as semiconductor quantum dots (QDs), plasmonic probes (including Au nanoparticles, AuNPs) to magnetic nanoparticles. The development of effective and reproducible strategies for making aggregate-free (monodispersed) water-soluble luminescent QDs and AuNPs that are stable and compatible with commonly used biological coupling chemistries is highly sought, since such platforms promise great advances in understanding a variety of biological processes. High quality QDs are generally synthesized using high temperature solution reaction of organometallic precursors, and as-prepared they are dispersible in hydrophobic (organic) solvents. AuNPs are often made via citrate-reduction but require subsequent surface-functionalization to make them biocompatible. Thus, post-synthetic surface modification is required to render these nanocrystals stable in aqueous media and biologically compatible. We have developed a new set of compact multifunctional ligands that contain each an oligomer coupled to several copies of a short poly(ethylene glycol) (PEG)-appended thioctic acid (TA). Reduction of TA (e.g., in the presence of NaBH4) produces dihydrolipoic acid (DHLA) appended-oligomer ligands. Here the insertion of several PEG segments in the ligand structure promotes water solubility, while TA and DHLA groups provide multidentate anchoring onto Au and ZnS-overcoated semiconductor QDs, respectively. Dispersion of QDs and AuNPs that exhibit remarkable colloidal stability over a broad range of pHs and in the presence of added electrolytes and reducing agents have been prepared. Moreover, introducing different functional groups (such as COOH, N3 and NH2) into the oligomers opens the opportunity for effective orthogonal coupling of these platforms to target biomolecules such as proteins and peptides. We will discuss the ligand design, preparation, capping of the nanocrystals, coupling to target biomolecules and use in specific biological investigations such as sensor design.
10:30 AM - JJ10.6
Ocular Drug Delivery Devices Utilizing Nanostructures for Controlled Release.
Daniel Bernards 1 , Mark Steedman 1 , Paula Wynn 2 , On-tat Lee 2 , Robert Bhisitkul 2 , Tejal Desai 1 Show Abstract
1 Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California, United States, 2 Ophthalmology, University of California, San Francisco, San Francisco, California, United States
There is need for sustained drug release devices that are highly efficacious for delivery of protein-based therapeutics ocularly. Many contemporary delivery routes, such as injections, are hampered by patient compliance, discomfort, risk, and inconvenience, as well as limited bioavailability and frequent administration. Alternatively, a therapeutic device capable of delivering a constant rate of drug over time can circumvent these issues. One attractive platform for such a device is the use of nanoporous materials. In porous materials, constant-rate diffusion is possible when the size of a diffusing species is comparable to material pore size. A process often referred to as “single-file” diffusion, this situation can lead to zero-order constant-rate release. For macromolecule delivery this requires pores on the order of a few tens of nanometers. To this end, nanoporous biodegradable polymers have been fabricated for delivery of protein therapeutics within the eye. Utilizing biodegradable poly(caprolactone) (PCL) thin films with transport-controlling nanostructures and structural microstructures, we have fabricated devices for the delivery of protein-based therapeutics. PCL is an excellent candidate material since it biodegrades yet maintains its structural integrity during the majority of the degradation time course: this allows structural degradation to follow the effective therapeutic lifetime of the device. To validate these devices, we characterize the release of Lucentis, an age-related macular degeneration therapeutic, from nanoporous PCL thin film devices over extended periods. Furthermore, PCL films and devices have been implanted or injected into rabbit eyes to establish the long-term biocompatibility, structural integrity, and functionality of these devices.
10:45 AM - JJ10: Hybrid II
11:15 AM - JJ10.7
Self-assembly and Mineralization of Organics on a Template.
Elaine DiMasi 1 Show Abstract
1 , Brookhaven National Laboratory, Upton, New York, United States
Biomineralization depends upon an appropriate template consisting of organic material - proteins, polysaccharides, or similar - being available to nucleate mineral and control crystal growth. The organics in turn require the correct conditions to self-assemble into the appropriate structures. For proteins tertiary structures such as fibrils may be required; for surfactants such as membrane lipids, conditions must permit the formation of sheets or vesicles. When model systems are assembled to study biomimetic mineralization, a properly chosen experimental substrate - gels, hard wafer surfaces, etc - can be crucial. It has been demonstrated across a variety of experimental systems that better molecular order of organic templates can be achieved on surfaces bearing net negative charge. We will review these results and present new observations in surfactant and extracellular matrix protein systems, as well as cultured cell systems, studied with scanning probe and synchrotron x-ray microscopies.
11:30 AM - JJ10.8
Hybrid Nanowire Arrays for Multifunctional Adhesive Surfaces.
Hyunhyub Ko 1 2 3 , Zhenxing Zhang 2 3 4 , Rehan Kapadia 2 3 4 , Ali Javey 2 3 4 Show Abstract
1 Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan Metropolitan City Korea (the Republic of), 2 Electrical Engineering and Computer Sciences, University of California at Berkeley, Berkeley, California, United States, 3 Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States, 4 Berkeley Sensor and Actuator Center, University of California at Berkeley, Berkeley, California, United States
The biological systems with functional micro- and nanostructured surfaces have inspired the fabrication of functional surfaces with unique optical, chemical and mechanical properties. Inspired by the micro- and nanofibrillar structures found in various biological adhesive systems, here, we report the fabrication of hybrid, inorganic/organic nanowire (NW) arrays for multifunctional adhesive surfaces. In particular, inspired by the adhesion systems found in dragonfly and gecko lizard, we demonstrate the first connector (i.e., fasteners to bind things together) concept that utilizes van der Waals interactions rather than mechanical or magnetic interactions as the main binding mechanism. In addition, we demonstrate that hybrid NW connectors can be easily tuned to provide underwater binding operation, self-cleaning superhydrophobic properties, anisotropic adhesion, thermo-responsive smart adhesion, and electrically conductive connection by manipulating the composition of hybrid nanostructures. Since many biological systems have evolved into multi-scale, hierarchical structures with sophisticated and smart functions, we also introduce a simple method for fabricating hierarchical fibrillar arrays based on polymer micropillar arrays (μPLR) decorated with ZnO nanowires on mechanically flexible substrates. The hierarchical μPLR/NW arrays show superhydrophobic surface properties, with the contact angle higher than that of planar surfaces and μPLR arrays without nanostructures. The fabrication strategy suggested here may be potentially extended to fabricate other organic/inorganic hierarchical systems for different applications in biomedical devices, micro-robotics, and in any device that require reversible assembly of components.. Hyunhyub Ko, Zhenxing Zhang, Kuniharu Takei, and Ali Javey, “Hierarchical polymer micropillar arrays decorated with inorganic nanowires”, Nanotechnology, 2010, 21, 295305.. Hyunhyub Ko, Yu-Lun Chueh, Zhenxing Zhang, and Ali Javey, Thermo-responsive smart nanowire connectors, Angew. Chem. Int. Ed., 2010, 49, 616-619.. Hyunhyub Ko, Zhenxing Zhang, Johnny C. Ho, Kuniharu Takei, Rehan Kapadia, Yu-Lun Chueh, Weizhen Cao, Brett A. Cruden, and Ali Javey, Flexible carbon nanofiber connectors with anisotropic adhesion properties, Small, 2010, 6, 22-26.. Hyunhyub Ko, Zhenxing Zhang, Yu-Lun Chueh, Johnny C. Ho, Jongho Lee, Ronald S. Fearing, and Ali Javey, Wet and dry adhesion properties of self-selective, nanowire connectors, Adv. Funct. Mater. 2009, 19, 3098-3102. . Rehan Kapadia, Hyunhyub Ko, Yu-Lun Chueh, Johnny Ho, Toshitake Takahashi, Zhenxing Zhang, and Ali Javey, Hybrid core-multi-shell nanowire forests for electrical connector applications, Appl. Phys. Lett. 2009, 94, 263110.. Hyunhyub Ko, Jongho Lee, Bryan E. Schubert, Yu-Lun Chueh, Paul W. Leu, Ronald S. Fearing and Ali Javey, Hybrid Core-Shell Nanowire Forests as Self-Selective Chemical Connectors, Nano Lett. 2009, 9, 2054-2058.
11:45 AM - JJ10.9
A pH-sensitive Cross-linker based on a Hyperbranched Acetal-polymer.
Hongliang Cao 1 Show Abstract
1 Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Galway Ireland
There are numerous pH gradients that exist in both normal and pathophysiological states in the human body. For example, tumor and inflammed tissue have a pH slightly more acidic than the blood and normal tissue. In addition, the intracellular vesicles of cells involved in the endocytosis mechanism, i.e. endosomes and lysosomes are also acidic. Therefore, pH-responsive systems can potentially be used in a broad range of tissue engineering and drug delivery applications. Acetals compounds and polymer are promising candidates for the development of acid-sensitive systems because the hydrolysis of acetal group is strongly dependant on pH value. In this study, a new hyperbranched polymer with acid-cleavable acetal groups was developed as a pH sensitive cross-linker for protein. This technique involves the formation of covalent bonds between proteins by using multifunctional reagents containing reactive end groups, e.g. N-hydroxysuccinimide (NHS) that react with functional groups—such as primary amines and sulfhydryls—of amino acid residues from protein. The acetal-NHS co-polymer was synthesized by the ATRP method, The 1H NMR spectrum illustrated the double bonds and characteristic acetal proton peak within the acetal-NHS co-polymer. These peaks also provide the integral values required to calculate the final polymer composition, which consist of: 81.8% molar percentage of PEG-A, 10.1% acetal units, 8.0% NHS units, 4.0% vinyl groups and 6.1% branching degree. The pH-dependent hydrolysis of the acetal crosslinker was investigated at pH 3.0, 5.0, and 7.4. At pH 3.0, the polymer was completely hydrolyzed in 24h. At pH 5.0, the polymer was found to be hydrolyzed with half-life of approx. 50 hrs. At neutral pH the hyperbranched polymer remained relatively stable over the same period. It will be hydrolyzed approx. 25% after incubated at room temperature for 120 hrs. Mouse 3T3 fibroblast cells were utilized for the cytotoxicity assessment of the polymer. The results showed the synthesized polymer did not exhibit reduction in cellular metabolic activity at 0.5mg/ml and 1mg/ml concentration. The acetal-NHS hyperbranched polymer was used as a crosslinker for proteinaceous scaffold (collagen type I). Hydrogels made from collagen and the crosslinker were prepared in 96 well plates for 2D and 3D culture. The results of cell viability showed that the hydrogel made from acetal-NHS polymer was not significantly different from star-PEG as cross-linker and cell alone by ADSC and 3T3 cell culture. Acknowledgements. The authors would like to thank the European Commission under the DISC REGENERATION project (NMP3-LA-2008-213904) financial support of this research.
12:00 PM - JJ10.10
Design and Synthesis of Self-oscillatory Hydrogels.
Ye Zhang 1 , Jorge Delgado 1 , Anna Balazs 2 , Irving Epstein 1 , Bing Xu 1 Show Abstract
1 Chemistry, Brandeis University, Waltham, Massachusetts, United States, 2 Chemical Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Inspired by the natural materials that convert chemical energy into mechanical movement, we designed and fabricated chemomechanical hydrogels. By using Belousov-Zhabotinsky (BZ) oscillating chemical reaction as theoretical modeling, we built the catalyst of BZ reaction-ruthenium complex into a copolymer or a supramolecular gelator to form hydrogels. The periodic oxidation and reduction of the anchored ruthenium ion in BZ reaction induce the corresponded hydrating and dehydrating effects in the hydrogels that finally result into its oscillatory volume change. All these efforts make the hydrogels self-oscillate without external stimuli. The overall aim of our research is to explore the possibility of utilizing hererogeneous chemoresponsive gels to produce multifunctional materials with the ability to convert the chemical energy of an oscillating chemical reaction into controllable mechanical forces.
12:15 PM - JJ10.11
Bio-inspired Hierarchical Vascular Network Synthesis: Electrical Treeing.
Kristopher Behler 1 , Zachary Melrose 1 , Eric Wetzel 1 Show Abstract
1 Composite and Hybrid Materials Branch, U.S. Army Research Lab, Aberdeen Proving Ground, Maryland, United States
Vascular networks provide a physical pathway to distribute fluid throughout a system. An uninterrupted and controllable supply of liquid is optimal for many applications such as continual self-healing, drug delivery, chemical and biological agent neutralization, and thermal management. One approach to producing hierarchical vascular networks is electrical treeing (ET). Hollow channels or “trees” are grown from a series of step-wise, partial discharges in a dielectric material resulting from a high local electric field that is greater than the dielectric breakdown strength of the material. Continual breakdown leads to hollow tubules which from an intricate network of branches, ranging in diameter from sub micron to less than 100 microns, within a polymer or epoxy system. Electrical trees have been grown using voltages of 20 kV (AC) and up to 60 kV (DC) resulting in “bush-like”, dense, networks of hollow tubules and less dense “tree-like” structures, respectively. Filling of these structures has been visualized using a dissolved UV sensitive dye pumped into the trees via a syringe pump.
12:30 PM - JJ10.12
Bone-Inspired Multicomponent Bionanocomposites with a Simple Drop-cast Processing Strategy.
Abhijit Biswas 1 , He Zhao 2 , Ilker Bayer 3 , Alexandru Biris 4 Show Abstract
1 Electrical Engineering, Center for Nanoscience and Technology, University of Notre Dame, Notre Dame, Indiana, United States, 2 Aerospace and Mechanical Engineering, Center for Nanoscience and Technology, University of Notre Dame, Notre Dame, Indiana, United States, 3 Smart Materials Group, Italian Institute of Technology, Arnesano Italy, 4 Nanotechnology Center, University of Arkansas at Little Rock, Little Rock, Arkansas, United States
Bone is a specialized form of connective tissue that forms the skeleton of the body and is built at the nano and micro levels as a multicomponent composite material consisting of a hard inorganic phase (minerals) in an elastic, dense organic network. Mimicking bone structure presents an important frontier in the fields of materials science and bone tissue engineering. An ideal bonelike or bone-mimetic biomaterial would replicate the predominant coalignment of the organic and mineral phases of the actual bone tissue architecture. This essentially involves nano- to micro-scale features of both the organization of collagen fibers in a characteristic three dimensional architecture and the coalignment of important mineral such as hydroxyapatite (HAP) crystals within the collagen fibers. It is a significant challenge to achieve such complex and special three dimensional multicomponent bonelike features. We describe an innovative and simple drop-cast processing strategy to create bonelike multicomponent bionanocomposite materials that consist of an organic poly(ε-caprolactone) (PCL) matrix, minerals such as hydroxyapatite (HAP) and CaCO3, and collagen fibers. The process allows morphological and structural control to achieve the desired nanostructure of the bone mimics. The synthesis process involves adding inorganic and organic components sequentially followed by controlling the growth conditions and composition. This enables organization of collagen nanofibers (~ 100 nm) into scaffolds while simultaneously allowing nucleation and co-alignment of hydroxyapatite spheres (~ 100 – 500 nm) within aligned, thermally stable collagen fibers in the porous PCL matrix. We achieved high calcium (26%) and oxygen (17%) within the bioscaffold and adequate phosphorous compositions comparable to the levels of bone tissues. These breakthroughs provide the viable approach to help maintain the required bone mineral density and revascularization for nutrient and compensate for the loss of oxygen delivered to the bone cells. It suggests the huge potential of these bone-inspired materials for bone grafting technologies.