Biomaterials (v.30, #29)

Strategies for inducing the formation of bands of Büngner in peripheral nerve regeneration by Victor T. Ribeiro-Resende; Brigitte Koenig; Susanne Nichterwitz; Sven Oberhoffner; Burkhard Schlosshauer (5251-5259).
Peripheral human nerves fail to regenerate across longer tube implants (>2 cm), most likely because implants lack the microarchitecture of native nerves, including bands of Büngner. Bands of Büngner comprise longitudinally aligned Schwann cell strands that guide selectively regrowing axons. We aim to optimize tubular implants by integrating artificial bands of Büngner. Three principle strategies for inducing the formation of bands of Büngner were investigated: (a) an aligned extracellular matrix, (b) polarizing differentiation factors, and (c) microstructured biomaterial filaments. In vitro oriented collagen and a combination of differentiation factors (NGF, neuregulin-1, TGF-β) induced Schwann cell alignment to some extent. The most pronounced Schwann cell alignment was evident on ultrathin, endless poly-ɛ-caprolactone (PCL) filaments with longitudinal microgrooves. Precoated PCL filaments proved to be non-cytotoxic, displayed good cell attachment, and supported Schwann cell proliferation as well as guided axonal outgrowth. In vitro on PCL filaments Schwann cells displayed a polarized expression of the cell adhesion molecule L1 similar to that seen in vivo in bands of Büngner after sciatic nerve crush in adult rats. In summary, the integration of bioengineered bands of Büngner based on microstructured polymer filaments in nerve conduits promises to be the most valuable approach to initiating a more efficient regeneration across longer nerve lesions.
Keywords: Bands of Büngner; Collagen; Growth factor; Nerve regeneration; Poly-caprolactone; Rat Schwann cell;

A double-chamber rotating bioreactor for the development of tissue-engineered hollow organs: From concept to clinical trial by M. Adelaide Asnaghi; Philipp Jungebluth; Manuela T. Raimondi; Sally C. Dickinson; Louisa E.N. Rees; Tetsuhiko Go; Tristan A. Cogan; Amanda Dodson; Pier Paolo Parnigotto; Anthony P. Hollander; Martin A. Birchall; Maria Teresa Conconi; Paolo Macchiarini; Sara Mantero (5260-5269).
Cell and tissue engineering are now being translated into clinical organ replacement, offering alternatives to fight morbidity, organ shortages and ethico-social problems associated with allotransplantation. Central to the recent first successful use of stem cells to create an organ replacement in man was our development of a bioreactor environment. Critical design features were the abilities to drive the growth of two different cell types, to support 3D maturation, to maintain biomechanical and biological properties and to provide appropriate hydrodynamic stimuli and adequate mass transport. An analytical model was developed and applied to predict oxygen profiles in the bioreactor-cultured organ construct and in the culture media, comparing representative culture configurations and operating conditions. Autologous respiratory epithelial cells and mesenchymal stem cells (BMSCs, then differentiated into chondrocytes) were isolated, characterized and expanded. Both cell types were seeded and cultured onto a decellularized human donor tracheal matrix within the bioreactor. One year post-operatively, graft and patient are healthy, and biopsies confirm angiogenesis, viable epithelial cells and chondrocytes. Our rotating double-chamber bioreactor permits the efficient repopulation of a decellularized human matrix, a concept that can be applied clinically, as demonstrated by the successful tracheal transplantation.
Keywords: Bioreactor; Tissue engineering; Co-culture; Oxygenation; Stem cells; Airway transplantation;

Nanostructured hybrid hydrogels prepared by a combination of atom transfer radical polymerization and free radical polymerization by Sidi A. Bencherif; Daniel J. Siegwart; Abiraman Srinivasan; Ferenc Horkay; Jeffrey O. Hollinger; Newell R. Washburn; Krzysztof Matyjaszewski (5270-5278).
A new method to prepare nanostructured hybrid hydrogels by incorporating well-defined poly(oligo (ethylene oxide) monomethyl ether methacrylate) (POEO300MA) nanogels of sizes 110–120 nm into a larger three-dimensional (3D) matrix was developed for drug delivery scaffolds for tissue engineering applications. Rhodamine B isothiocyanate-labeled dextran (RITC-Dx) or fluorescein isothiocyanate-labeled dextran (FITC-Dx)-loaded POEO300MA nanogels with pendant hydroxyl groups were prepared by activators generated electron transfer atom transfer radical polymerization (AGET ATRP) in cyclohexane inverse miniemulsion. Hydroxyl-containing nanogels were functionalized with methacrylated groups to generate photoreactive nanospheres. 1H NMR spectroscopy confirmed that polymerizable nanogels were successfully incorporated covalently into 3D hyaluronic acid-glycidyl methacrylate (HAGM) hydrogels after free radical photopolymerization (FRP). The introduction of disulfide moieties into the polymerizable groups resulted in a controlled release of nanogels from cross-linked HAGM hydrogels under a reducing environment. The effect of gel hybridization on the macroscopic properties (swelling and mechanics) was studied. It is shown that swelling and nanogel content are independent of scaffold mechanics. In-vitro assays showed the nanostructured hybrid hydrogels were cytocompatible and the GRGDS (Gly–Arg–Gly–Asp–Ser) contained in the nanogel structure promoted cell–substrate interactions within 4 days of incubation. These nanostructured hydrogels have potential as an artificial extracellular matrix (ECM) impermeable to low molecular weight biomolecules and with controlled pharmaceutical release capability. Moreover, the nanogels can control drug or biomolecule delivery, while hyaluronic acid based-hydrogels can act as a macroscopic scaffold for tissue regeneration and regulator for nanogel release.
Keywords: Hydrogels; Nanogels; Hyaluronic acid; Hybrid; Atom transfer radical polymerization (ATRP); Free radical photopolymerization (FRP);

On the kinetics and impact of tetragonal to monoclinic transformation in an alumina/zirconia composite for arthroplasty applications by Jérôme Chevalier; Sylvie Grandjean; Meinhard Kuntz; Giuseppe Pezzotti (5279-5282).
Latest trends in load-bearing materials for arthroplastic applications involve the development of highly fracture resistant alumina/zirconia composites, as an alternative choice to alumina and zirconia monolithic ceramics. Composite materials are designed from both chemical and microstructural viewpoints in order to prevent environmental degradation and fracture events in vivo, whose shadow yet hampers the full exploitation of ceramic materials in the field of arthroplasty. The aim of this paper is to evaluate the resistance to environmental degradation in an alumina/zirconia composite (Biolox Delta®), which represents a primary candidate for hip and knee joint applications. Our approach consists first in the experimental determination of an activation energy value for environmentally driven tetragonal to monoclinic (t–m, henceforth) polymorphic transformation in the zirconia phase of the material; then, based on such an experimental value, a prediction is given for the long-term in vivo environmental resistance of prostheses made of the composite material. The present evaluation clarifies the in vivo performance of this new composite for orthopedic applications.
Keywords: Alumina; Zirconia; Degradation; Surface modification;

The LCST behavior of poly(ethylene glycol) (PEG) in aqueous sodium sulfate solutions was exploited to fabricate microspheres without the use of other monomers, polymers, surfactants or organic solvents. Reactive PEG derivatives underwent thermally induced phase separation to produce spherical PEG-rich domains that coarsened in size pending gelation, resulting in stable hydrogel microspheres between ≈1 and 100 microns in size. The time required to reach the gel point during the coarsening process and the extent of crosslinking after gelation both affected the final microsphere size and swelling ratio. The gel point could be varied by pre-reaction of the PEG derivatives below the cloud point, or by controlling pH and temperature above the cloud point. Pre-reaction brought the PEG derivatives closer to the gel point prior to phase separation, while the pH and temperature influenced the rate of reaction. Dynamic light scattering indicated a percolation-to-cluster transition about 3–5 min following phase separation. The mean radius of PEG-rich droplets subsequently increased with time to the 1/4th power until gelation. PEG microspheres produced by these methods with controlled sizes and densities may be useful for the production of modular scaffolds for tissue engineering.
Keywords: Poly(ethylene glycol); Microsphere; LCST; Hydrogel; Scaffold; Cloud point;

Skeletal muscle cell proliferation and differentiation on polypyrrole substrates doped with extracellular matrix components by Kerry J. Gilmore; Magdalena Kita; Yao Han; Amy Gelmi; Michael J. Higgins; Simon E. Moulton; Graeme M. Clark; Robert Kapsa; Gordon G. Wallace (5292-5304).
Conducting polymers have been developed as substrates for in vitro studies with a range of cell types including electrically-excitable cells such as nerve and smooth muscle. The goal of this study was to optimise and characterise a range of polypyrrole materials to act as substrates for electrical stimulation of differentiating skeletal myoblasts. Although all of the polymer materials provided suitable substrates for myoblast adhesion and proliferation, significant differences became apparent under the low-serum conditions used for differentiation of primary myoblasts. The significance of the work lies in the design and control of polymer materials to facilitate different stages of skeletal muscle cell proliferation and/or differentiation, opening up opportunities for engineering of this tissue. This paper therefore constitutes not just a biocompatibility assessment but a comprehensive study of how synthesis conditions affect the final outcome in terms of cell response.
Keywords: Skeletal muscle; Conducting polymer; Cell differentiation; Cell adhesion; Tissue engineering; Atomic force microscopy;

Generating substrate bound functional chemokine gradients in vitro by Gertrud M. Hjortø; Morten Hansen; Niels B. Larsen; Thomas N. Kledal (5305-5311).
Microcontact printing (mCP) is employed to generate discontinuous microscale gradients of active fractalkine, a chemokine expressed by endothelial cells near sites of inflammation where it is believed to form concentration gradients descending away from the inflamed area. In vivo, fractalkine is a transmembrane molecule extending its chemokine domain into the vascular lumen. Substrate bound in vitro gradients may thus closely resemble in vivo conditions. Direct mCP of sensitive proteins like fractalkine may cause partial protein denaturation and will not ensure correct orientation of the biologically active part of the molecules. Here, indirect mCP of a capture antibody recognizing a molecular tag on the target protein is successfully used to pattern tagged fractalkine in microscale gradient patterns. Fractalkine functions as an adhesion molecule for leukocytes. Cells expressing the fractalkine receptor are found to attach to the gradient structure at a density correlated with the fractional area covered by fractalkine. This indicates that the patterned fractalkine maintains its biological function. The method can be applied to in vitro studies of cell responses to the wide range of naturally surface-bound chemokines (haptotactic gradients). The use of a capture antibody facilitates control of the orientation of tagged molecules, thereby ensuring a high degree of bio-functionality through correct presentation and reduced protein denaturation.
Keywords: Micropatterning; Gradient; Adhesion molecule; Chemokine; Polydimethyl siloxane; Microcontact printing;

Proinflammatory and osteoclastogenic effects of beta-tricalciumphosphate and hydroxyapatite particles on human mononuclear cells in vitro by Tobias Lange; Arndt F. Schilling; Fabian Peters; Friedrich Haag; Michael M. Morlock; Johannes M. Rueger; Michael Amling (5312-5318).
Particulate wear debris can activate defence cells and osteoclasts at the bone–implant interface possibly leading to bone resorption and implant failure. Cellular responses and inflammatory effects have been reported for particulate hydroxyapatite (HA). However, the immunological effects of particulate beta-tricalciumphosphate (beta-TCP) have not been studied and the question of whether beta-TCP is more biocompatible in this regard as is HA remains to be determined. Therefore the present work investigates effects of endotoxin-free HA and beta-TCP particles of the same size (d 50  = 1 μm) and dose (SAR 10:1) on human peripheral blood mononuclear cells in vitro. The production of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-8) and cytokines connected to osteoclast and dendritic cell differentiation (OPG, RANKL, M-CSF, GM-CSF) was determined by ELISA. After 6 and 18 h of incubation HA and beta-TCP caused a quite similar induction of TNF-alpha, IL-1beta and IL-8. Effects of particles on the production of M-CSF and OPG were not detectable. However, in sharp contrast to HA, beta-TCP caused less induction of GM-CSF and not any of RANKL, both known for promoting dendritic cells and osteoclastogenesis respectively. Therefore these in vitro data suggest that wear debris of beta-TCP poses lesser risk of the detrimental effects of osteoclast induction known from HA.
Keywords: Beta-TCP; Hydroxyapatite; Human PBMC; Inflammation; Osteoclastogenesis; Dendritic cells;

Cellular behavior on TiO2 nanonodular structures in a micro-to-nanoscale hierarchy model by Katsutoshi Kubo; Naoki Tsukimura; Fuminori Iwasa; Takeshi Ueno; Lei Saruwatari; Hideki Aita; Wen-An Chiou; Takahiro Ogawa (5319-5329).
Biological tissues involve hierarchical organizations of structures and components. We created a micropit-and-nanonodule hybrid topography of TiO2 by applying a recently reported nanonodular self-assembly technique on acid-etch-created micropit titanium surfaces. The size of the nanonodules was controllable by changing the assembly time. The created micro-nano-hybrid surface rendered a greater surface area and roughness, and extensive geographical undercut on the existing micropit surface and resembled the surface morphology of biomineralized matrices. Rat bone marrow-derived osteoblasts were cultured on titanium disks with either micropits alone, micropits with 100-nm nodules, micropits with 300-nm nodules, or micropits with 500-nm nodules. The addition of nanonodules to micropits selectively promoted osteoblast but not fibroblast function. Unlike the reported advantages of microfeatures that promote osteoblast differentiation but inhibit its proliferation, micro-nano-hybrid topography substantially enhanced both. We also demonstrated that these biological effects were most pronounced when the nanonodules were tailored to a diameter of 300 nm within the micropits. An implant biomechanical test in a rat femur model revealed that the strength of bone–titanium integration was more than three times greater for the implants with micropits and 300-nm nanonodules than the implants with micropits alone. These results suggest the establishment of functionalized nano-in-microtitanium surfaces for improved osteoconductivity, and may provide a biomimetic micro-to-nanoscale hierarchical model to study the nanofeatures of biomaterials.
Keywords: Bone–titanium integration; Osseointegration; Total hip replacement; Dental implant; Nanotechnology; Self-assembly;

We investigated the morphological effect of phase-separated block copolymer surfaces composed of poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) (PMPC) and poly(dimethylsiloxane) (PDMS) on protein adsorption and cell adhesion behavior. We observed three different types of phase-separated surface morphologies by TEM and AFM. The elemental composition of phosphorus on the surface increases with the PMPC composition. Furthermore, the polymer surface formed by a block copolymer-containing a higher MPC unit composition shows a slightly lower static water contact angle. This result indicates that the elemental surface ratio of the surface depends on the MPC composition in the block copolymer. Protein adsorption tests revealed that only hydrophobic PDMS domains showed selective protein adsorption. Cell adhesion tests revealed that the number of adhered cells increased with increasing hydrophobic PDMS domain size of block copolymers in serum-containing media. In contrast, no cells adhered onto block copolymer surfaces in serum-free media, whereas a large amount of adhered cells were observed on the hydrophobic PDMS surface. This result indicates that segregated hydrophobic domains on a biocompatible PMPC surface strongly affect serum protein adsorption, thereby promoting considerable cell adhesion, although the surface is hydrophilic. Thus, both the composition of MPC units and the segregated hydrophobic surface morphology are important considerations in biomaterial surface design.
Keywords: Phosphorylcholine; Polydimethylsiloxane; Block copolymer; Protein adsorption; Cell adhesion;

Current synthetic vascular grafts possess a significant mechanical mismatch compared to the native vasculature and do not permit endothelialization; both of these deficiencies contribute to the relatively high rate of failure of many synthetic grafts. In this communication, we report the modification of polyurethane (PU)-based materials to impart hemocompatibility, support endothelial growth, and display vascular-appropriate mechanics. This modification was achieved by incorporating branched polyethylenimine (PEI) into the PU backbone, followed by covalent attachment of either hyaluronic acid (HA; 4.7, 64, and 104 kDa), heparin, or poly(ethylene glycol) (PEG; used as a non-adhesive control) to the PEI. This grafting chemistry resulted in comparatively dense immobilization of HA and heparin (0.062 and 2.3 μg/cm2, respectively) to the PU–PEI surfaces. PU materials modified with HA were more effective than either PEG- or heparin-grafted materials with respect to limiting protein adsorption and platelet adhesion. Confluent, morphologically-healthy cultures of endothelial cells were achieved only on materials grafted with low molecular weight HA, but not high MW HA, heparin, or PEG. These modifications in PU chemistry were performed while retaining material mechanics in the range of native vascular tissue. Thus, this study describes the generation of materials that possess the unique ability to display excellent hemocompatibility while simultaneously supporting extensive endothelialization and retaining vascular-appropriate mechanics. The bioactivity of these materials was regulated by the molecular weight of the grafted HA, and their physical and biological properties make them promising for use as vascular grafts.
Keywords: Vascular graft; Polyurethane; Hyaluronic acid; Heparin; Endothelialization;

Time-dependent degradation of titanium osteoconductivity: An implication of biological aging of implant materials by Wael Att; Norio Hori; Masato Takeuchi; Jianyong Ouyang; Yang Yang; Masakazu Anpo; Takahiro Ogawa (5352-5363).
The shelf life of implantable materials has rarely been addressed. We determined whether osteoconductivity of titanium is stable over time. Rat bone marrow-derived osteoblasts were cultured on new titanium disks (immediately after acid-etching), 3-day-old (stored after acid-etching for 3 days in dark ambient conditions), 2-week-old, and 4-week-old disks. Protein adsorption capacity, and osteoblast migration, attachment, spread, proliferation and mineralization decreased substantially on old titanium surfaces in an age-dependent manner. When the 4-week-old implants were placed into rat femurs, the biomechanical strength of bone–titanium integration was less than half that for newly processed implants at the early healing stage. More than 90% of the new implant surface was covered by newly generated bone compared to 58% for 4-week-old implants. This time-dependent biological degradation was also found for machined and sandblasted titanium surfaces and was associated with progressive accumulation of hydrocarbon on titanium surfaces. The new surface could attract osteoblasts even under a protein-free condition, but its high bioactivity was abrogated by masking the surface with anions. These results uncover an aging-like time-dependent biological degradation of titanium surfaces from bioactive to bioinert. We also suggest possible underlying mechanisms for this biological degradation that provide new insights into how we could inadvertently lose, and conversely, maximize the osteoconductivity of titanium-based implant materials.
Keywords: Bone–titanium integration; Hydrocarbon; Osseointegration; Total hip replacement; Dental implant;

Degradable, antibacterial silver exchanged mesoporous silica spheres for hemorrhage control by Chenglong Dai; Yuan Yuan; Changsheng Liu; Jie Wei; Hua Hong; Xiaosheng Li; Xiaohua Pan (5364-5375).
Effective hemorrhage control becomes increasingly significant in today's military and civilian trauma, and current available local hemostatic agents have been reported to have various drawbacks and side effects. Herein in this study, a silver exchanged calcium doped ordered mesoporous silica sphere (AgCaMSS) with good degradability and antibacterial properties was developed for hemorrhage control. The well-ordered and symmetry hexagonal AgCaMSS with pore size of 3.2 nm, BET surface area of 919 m2/g and pore volume of 0.74 m3/g was prepared by one-step based catalyzed self-assembly and subsequent ion-exchange procedures. The degradation behaviors in Tris–HCl solution indicated that the addition of calcium and silver facilitated the dissolution and the weight loss of the prepared AgCaMSS could attain more than 40% after 42 days. The results obtained demonstrated that the optimal AgCaMSS formulation could significantly promote the blood clotting, activate the intrinsic pathway of coagulation cascade, induce platelet adherence. Consequently, effective hemostasis with low exothermic effects was achieved and the mortalities in femoral artery and liver injury models were reduced. The antibacterial experiment using broth culture method revealed that the prepared AgCaMSS had better antibacterial activities against Escherichia coli and Staphylococcus aureus. Based on these results, it can be concluded that the AgCaMSS developed here would be a promising material platform for designing hemostats in more extensive clinical application.
Keywords: Mesoporous silica sphere; Hemostatic efficiency; Degradation; Antibacterial;

Osteoblast: Osteoclast co-cultures on silk fibroin, chitosan and PLLA films by Gemma L. Jones; Antonella Motta; Mike J. Marshall; Alicia J. El Haj; Sarah H. Cartmell (5376-5384).
This study investigates the growth of a co-culture of osteoblasts and osteoclasts on four different types of degradable biomaterials with bone tissue engineering potential. Single or co-cultures of osteoblasts and osteoclasts (used at a ratio of 1:100 osteoblast:osteoclasts) were cultured on vapour stabilised silk fibroin, methanol stabilised silk fibroin, chitosan and poly (l lactic acid) (PLLA) films for 10 days. Osteoclast differentiation was determined by tartrate resistant acid phosphatase (TRAP) staining, total cell number by a picogreen DNA assay, cell morphology by scanning electron microscopy (SEM) and the material topography by atomic force microscopy (AFM). Samples were also monitored for degradation by differential scanning calorimetry (DSC) and fourier transform infrared (FTIR). Results demonstrated that vapour stabilised silk fibroin, methanol stabilised silk fibroin and chitosan all support the growth of osteoblasts and osteoclasts in both single and co-cultures. PLLA showed poor osteoclast differentiation in both single and co-cultures but supported osteoblast attachment and proliferation. Both silk fibroin materials showed sign of early degradation in the ten-day period, but very little change was seen in chitosan and PLLA samples. This study indicates that this novel co-culture approach for bone tissue engineering may be possible if scaffolds are created from silk fibroin or chitosan.
Keywords: Co-culture; Bone tissue engineering; Silk fibroin; Chitosan; PLLA; Osteoblast;

The use of synthetic hydroxyapatite as bone substitute calls for the knowledge of the influence on adjacent cells. The aim of this study was to investigate the proteins with differential protein expression levels in the proteome of human osteoblast cell line incubated separately with various nano sized hydroxyapatite powders with different shapes and chemical compositions using iTRAQ-coupled 2D LC-MS/MS approach. In the present study, we investigated several intracellular signaling molecules involved in calcium regulation to analyze how osteoblast cells respond to dissimilar HA nanoparticles. It was found there was a significant decrease in cell population after adding the HA nanoparticles to the osteoblasts. Our results combining proteomics analysis and RT-PCR validation on targeted genes involved in calcium regulation confirmed the differences in the cellular response to dissimilar HA nanoparticles.
Keywords: Nanoparticles; Hydroxyapatite; Osteoblast; iTRAQ; Protein profile; LC-MS/MS analysis;

The use of defined in vitro systems to study the developmental and physiological characteristics of a variety of cell types is increasing, due in large part to their ease of integration with tissue engineering, regenerative medicine, and high-throughput screening applications. In this study, myotubes derived from fetal rat hind limbs were induced to develop several aspects of mature muscle including: sarcomere assembly, development of the excitation–contraction coupling apparatus and myosin heavy chain (MHC) class switching. Utilizing immunocytochemical analysis, anisotropic and isotropic band formation (striations) within the myotubes was established, indicative of sarcomere formation. In addition, clusters of ryanodine receptors were colocalized with dihydropyridine complex proteins which signaled development of the excitation–contraction coupling apparatus and transverse tubule biogenesis. The myotubes also exhibited MHC class switching from embryonic to neonatal MHC. Lastly, the myotubes survived significantly longer in culture (70–90 days) than myotubes from our previously developed system (20–25 days). These results were achieved by modifying the culture timeline as well as the development of a new medium formulation. This defined model system for skeletal muscle maturation supports the goal of developing physiologically relevant muscle constructs for use in tissue engineering and regenerative medicine as well as for high-throughput screening applications.
Keywords: Cell culture; Silane; Muscle; In vitro test; Surface analysis; Surface modification;

Synthetic neoglycopolymer-recombinant human collagen hybrids as biomimetic crosslinking agents in corneal tissue engineering by Kimberley Merrett; Wenguang Liu; Debbie Mitra; Kenneth D. Camm; Christopher R. McLaughlin; Yuwen Liu; Mitchell A. Watsky; Fengfu Li; May Griffith; Deryn E. Fogg (5403-5408).
Saturated neoglycopolymers, prepared via tandem ROMP-hydrogenation (ROMP = ring-opening metathesis polymerization) of carbohydrate-functionalized norbornenes, are investigated as novel collagen crosslinking agents in corneal tissue engineering. The neoglycopolymers were incorporated into recombinant human collagen type III (RHC III) as collagen crosslinking agents and glycosaminoglycan (GAG) mimics. The purely synthetic nature of these composites is designed to reduce susceptibility to immunological and allergic reactions, and to circumvent the transmission of animal infectious diseases. The collagen-neoglycopolymer biomaterials exhibit higher stability to collagenase-induced biodegradation than the control materials, composites of RHC III crosslinked using EDC/NHS (EDC = 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide; NHS =  N-hydroxysuccinimide). Even at this proof of concept stage, the thermal stability, enzymatic resistance, and permeability of the neoglycopolymer hydrogels are comparable or superior to those of these fully optimized control materials, which have successfully been tested clinically. Tensile strength is adequate for transplantation, but lower than that of the optimized control materials.
Keywords: Biomimetic materials; Crosslinking; Collagen; Cornea; Tissue engineering; Neoglycopolymer;

Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering by Jennifer M. Singelyn; Jessica A. DeQuach; Sonya B. Seif-Naraghi; Robert B. Littlefield; Pamela J. Schup-Magoffin; Karen L. Christman (5409-5416).
Myocardial tissue lacks the ability to significantly regenerate itself following a myocardial infarction, thus tissue engineering strategies are required for repair. Several injectable materials have been examined for cardiac tissue engineering; however, none have been designed specifically to mimic the myocardium. The goal of this study was to investigate the in vitro properties and in vivo potential of an injectable myocardial matrix designed to mimic the natural myocardial extracellular environment. Porcine myocardial tissue was decellularized and processed to form a myocardial matrix with the ability to gel in vitro at 37 °C and in vivo upon injection into rat myocardium. The resulting myocardial matrix maintained a complex composition, including glycosaminoglycan content, and was able to self-assemble to form a nanofibrous structure. Endothelial cells and smooth muscle cells were shown to migrate towards the myocardial matrix both in vitro and in vivo, with a significant increase in arteriole formation at 11 days post-injection. The matrix was also successfully pushed through a clinically used catheter, demonstrating its potential for minimally invasive therapy. Thus, we have demonstrated the initial feasibility and potential of a naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering.
Keywords: Angiogenesis; Biomimetic material; Cardiac tissue engineering; In vivo test; Scaffold;

Dynamic cell culturing and its application to micropatterned, elastin-like protein-modified poly(N-isopropylacrylamide) scaffolds by Nihan Ozturk; Alessandra Girotti; Gamze T. Kose; José C. Rodríguez-Cabello; Vasif Hasirci (5417-5426).
In this study a tissue engineering scaffold was constructed from poly(N-isopropylacrylamide) (pNIPAM) to study the influence of strain on cell proliferation and differentiation. The effect of surface chemistry and topography on bone marrow mesenchymal stem cells was also investigated. Micropatterned pNIPAM films (channels with 10 μm groove width, 2 μm ridge width, 20 μm depth) were prepared by photopolymerization. The films were chemically modified by adsorption of a genetically engineered and temperature sensitive elastin-like protein (ELP). Dynamic conditions were generated by repeated temperature changes between 29 °C and 37 °C. ELP presence on the films enhanced initial cell attachment two fold (Day 1 cell number on films with ELP and without ELP were 27.6 × 104 and 13.2 × 104, respectively) but had no effect on proliferation in the long run. ELP was crucial for maintaining the cells attached on the surface in dynamic culturing (Day 7 cell numbers on the films with and without ELP were 81.4 × 104 and 12.1 × 104, respectively) and this enhanced the ability of pNIPAM films to transfer mechanical stress on the cells. Dynamic conditions improved cell proliferation (Day 21 cell numbers with dynamic and with static groups were 180.4 × 104 and 157.7 × 104, respectively) but decreased differentiation (Day 14 specific ALP values on the films of static and dynamic groups were 6.6 and 3.5 nmol/min/cell, respectively). Thus, a physically and chemically modified pNIPAM scaffold had a positive influence on the population of the scaffolds under dynamic culture conditions.
Keywords: Thermoresponsive; pNIPAM; Mechanical stress; Elastin-like protein; Bone tissue engineering;

Fabrication of transferable micropatterned-co-cultured cell sheets with microcontact printing by Imen Elloumi Hannachi; Kazuyoshi Itoga; Yoshikazu Kumashiro; Jun Kobayashi; Masayuki Yamato; Teruo Okano (5427-5432).
The purpose of the present study is to develop a novel method for the fabrication of transferable micropatterned cell sheets for tissue engineering. To achieve this development, microcontact printing of fibronectin on commercially available temperature-responsive dishes was employed. Primary rat hepatocytes were seeded on the dish surfaces printed with fibronectin. Under serum-free conditions, hepatocytes were attached onto fibronectin domains selectively. Then, a second cell type of endothelial cells was seeded in the presence of serum. Double fluorescent staining revealed that endothelial cells successfully adhered to the intervals of hepatocyte domains. Finally, all the cells were harvested as a single contiguous micropatterned cell sheet upon temperature-reduction. With a cell sheet manipulator having a gelatin layer for the support of harvested cell sheets, harvested micropatterned cell sheets were transferred to new dish surfaces. This technique would be useful for the fabrication of thick tissue constructs having a complex microarchitecture.
Keywords: Micropatterning; Microcontact printing; Co-culture; Cell sheet; Maskless photolithography; Tissue engineering;

Mechanosensitivity of fibroblast cell shape and movement to anisotropic substratum topography gradients by Deok-Ho Kim; Karam Han; Kshitiz Gupta; Keon W. Kwon; Kahp-Yang Suh; Andre Levchenko (5433-5444).
In this report, we describe using ultraviolet (UV)-assisted capillary force lithography (CFL) to create a model substratum of anisotropic micro- and nanotopographic pattern arrays with variable local density for the analysis of cell–substratum interactions. A single cell adhesion substratum with the constant ridge width (1 μm), and depth (400 nm) and variable groove widths (1 –9.1 μm) allowed us to characterize the dependence of cellular responses, including cell shape, orientation, and migration, on the anisotropy and local density of the variable micro- and nanotopographic pattern. We found that fibroblasts adhering to the denser pattern areas aligned and elongated more strongly along the direction of ridges, vs. those on the sparser areas, exhibiting a biphasic dependence of the migration speed on the pattern density. In addition, cells responded to local variations in topography by altering morphology and migrating along the direction of grooves biased by the direction of pattern orientation (short term) and pattern density (long term), suggesting that single cells can sense the topography gradient. Molecular dynamic live cell imaging and immunocytochemical analysis of focal adhesions and actin cytoskeleton suggest that variable substratum topography can result in distinct types of cytoskeleton reorganization. We also demonstrate that fibroblasts cultured as monolayers on the same substratum retain most of the properties displayed by single cells. This result, in addition to demonstrating a more sophisticated method to study aspects of wound healing processes, strongly suggests that even in the presence of adhesive cell–cell interactions, the cues provided by the underlying substratum topography continue to exercise substantial influence on cell behavior. The described experimental platform might not only further our understanding of biomechanical regulation of cell–matrix interactions, but also contribute to bioengineering of devices with the optimally structured design of cell–material interface.
Keywords: Topography; Focal adhesions; Extracellular matrix; Cell migration; Wound healing;

Single-cell hydrogel encapsulation for enhanced survival of human marrow stromal cells by Golnaz Karoubi; Mark L. Ormiston; Duncan J. Stewart; David W. Courtman (5445-5455).
Inadequate extracellular matrix cues and subsequent apoptotic cell death are among crucial factors currently limiting cell viability and organ retention in cell-based therapeutic strategies for vascular regeneration. Here we describe the use of a single-cell hydrogel capsule to provide enhanced cell survival of adherent cells in transient suspension culture. Human marrow stromal cells (hMSCs) were singularly encapsulated in agarose capsules containing the immobilized matrix molecules, fibronectin and fibrinogen to ameliorate cell-matrix survival signals. MSCs in the enriched capsules demonstrated increased viability, greater metabolic activity and enhanced cell-cytoskeletal patterning. Increased cell viability resulted from the re-induction of cell-matrix interactions likely via integrin clustering and subsequent activation of the extracellular signal regulated MAPK (ERK)/mitogen activated protein kinase (MAPK) signaling cascade. Proof of principle in-vivo studies, investigating autologous MSC delivery into Fisher 344 rat hindlimb, depicted a significant increase in the number of engrafted cells using the single-cell encapsulation system. Incorporation of immobilized adhesion molecules compensates, at least in part, for the missing cell-matrix cues, thereby attenuating the initial anoikis stimuli and providing protection from subsequent apoptosis. Thus, this single-cell encapsulation strategy may markedly enhance therapeutic cell survival in targeted tissues.
Keywords: Myocardial ischemia; Peripheral vascular disease; Cell therapy; Encapsulation; Anoikis; Hydrogel;

Multiple factor interactions in biomimetic mineralization of electrospun scaffolds by Parthasarathy A. Madurantakam; Isaac A. Rodriguez; Christopher P. Cost; Ramakrishnan Viswanathan; David G. Simpson; Matthew J. Beckman; Peter C. Moon; Gary L. Bowlin (5456-5464).
One of the major limitations in scaffold-based bone tissue engineering has been the inability to increase the loading of biologically active inorganic mineral. The present study introduces a novel two step strategy to increase overall mineral content of electrospun scaffolds and employs multiple factor interaction as a statistic to identify the combination of factors that yields maximal scaffold mineralization. Different amounts of nHA (0, 10, 25 and 50% by wt. of polymer) were electrospun in combination with polydioxanone (PDO) or poly(glycolide: lactide) to generate composite scaffolds. Successful incorporation of nHA within, on and in between nanofibers was confirmed by transmission and scanning electron microscopy. These scaffolds were immersed in different types (conventional, revised, ionic and modified) of simulated body fluid (SBF), prepared at 1× and 4× concentrations and the incubation was carried out either in static or dynamic setting at biomimetic conditions. At 2 weeks, the total amount of mineral within the scaffold was quantified using a modified Alizarin Red-based assay. Each of the five independent factors was analyzed independently and tested for interaction using random effects ANOVA. Statistics revealed significant higher order interactions among factors and the combination of PDO containing 50% nHA incubated in 1× revised SBF resulted in maximum mineralization.
Keywords: Electrospinning; Nanocrystalline hydroxyapatite; Simulated body fluid; Bone engineering; Composite scaffolds; Biomimetic mineralization;

Fibronectin-mediated endothelialisation of chitosan porous matrices by Isabel F. Amaral; Ronald E. Unger; Sabine Fuchs; Ana M. Mendonça; Susana R. Sousa; Mário A. Barbosa; Ana P. Pêgo; C.J. Kirkpatrick (5465-5475).
Chitosan (Ch) porous matrices were investigated regarding their ability to be colonized by human microvascular endothelial cells (HPMEC-ST1.6R cell line) and macrovascular endothelial cells namely HUVECs. Specifically we assessed if previous incubation of Ch in a fibronectin (FN) solution was effective in promoting endothelial cell (EC) adhesion to Ch matrices with different degrees of acetylation (DAs). Upon FN physiadsorption, marked differences were found between the two DAs investigated, namely DA 4% and 15%. While cell adhesion was impaired on Ch with DA 15%, ECs were able to not only adhere to Ch with DA 4%, but also to spread and colonize the scaffolds, with retention of the EC phenotype and angiogenic potential. To explain the observed differences between the two DAs, protein adsorption studies using 125I-FN and immunofluorescent labelling of FN cell-binding domains were carried out. In agreement with the higher cell numbers found, scaffolds with DA 4% revealed a higher number of exposed FN cell-binding domains as well as greater ability to adsorb FN and to retain and exchange adsorbed FN in the presence of competitive proteins. These findings suggest that the DA is a key parameter modulating EC adhesion to FN-coated Ch by influencing the adsorbed protein layer.
Keywords: Chitin/chitosan; Endothelial cell; Angiogenesis; Fibronectin; Protein adsorption; Scaffold;

Interpenetrating fibrin–alginate matrices for in vitro ovarian follicle development by Ariella Shikanov; Min Xu; Teresa K. Woodruff; Lonnie D. Shea (5476-5485).
In this report, we investigate the fibrin–alginate interpenetrating network (FA-IPN) to provide dynamic cell-responsive mechanical properties, which we apply to the in vitro growth of ovarian follicles. The mechanical properties and polymerization rate of the gels were investigated by rheology, and the fiber structure was imaged by electron microscopy. Using a mouse model, two-layered secondary follicles were encapsulated in FA-IPNs, and growth, morphology, hormone production, fibrin degradation rate and the numbers of competent eggs were assessed. The initial mechanics of the FA-IPN are determined by the composite material, and subsequent degradation of fibrin by the encapsulated cells would produce a material with mechanical properties due to the alginate alone. The rate of meiotically competent oocytes produced by culture in FA-IPN was 82%, which was significantly greater than in alginate alone. This increase in oocyte quality is an important step in identifying 3D culture systems that can provide a fundamental tool to investigate follicle maturation, and may be applied to promote the growth of human follicles, which can be used to provide reproductive options for women facing a cancer diagnosis.
Keywords: Fibrin–alginate; IPN (interpenetrating polymer network); Ovarian follicle; Biodegradation; Dynamic mechanical properties;

The treatment of collagen fibrils by tissue transglutaminase to promote vascular smooth muscle cell contractile signaling by Tighe A. Spurlin; Kiran Bhadriraju; Koo-Hyun Chung; Alessandro Tona; Anne L. Plant (5486-5496).
The enzyme tissue transglutaminase 2 (TG2) appears to play an important role in several physiological processes such as wound healing, the progression of cancer and of vascular disease. Additionally, TG2 has been proposed as a means of stabilizing collagen extracellular matrix (ECM) scaffolds for tissue engineering applications. In this report, we examined the effect of TG2 treatment on the mechanical properties of the ECM, and associated cell responses. Using a model ECM of fibrillar collagen, we quantitatively examined vascular smooth muscle cell (vSMC) response to untreated, or TG2 treated collagen. We show that cells respond to TG2 treated collagen with increased spreading, an increase in contractile response as indicated by elevated F-actin polymerization and myosin light chain phosphorylation, and increased proliferation, without apparent changes in integrin specificity or matrix topography. Comparative atomic force microscopy loading studies indicate that TG2 treated fibrils are 3 times more resistant to shearing force from an AFM tip than untreated fibrils. The data suggest that TG2 treatment of collagen increases matrix mechanical stiffness, which apparently alters the contractile and proliferative response of vSMC.
Keywords: Transglutaminase; Atomic force microscopy; Collagen type I; Vascular smooth muscle cells; Collagen fibrils;

Effect of polyurethane chemistry and protein coating on monocyte differentiation towards a wound healing phenotype macrophage by Joanne E. McBane; Loren A. Matheson; Soroor Sharifpoor; J. Paul Santerre; Rosalind S. Labow (5497-5504).
Tissue regeneration alternatives for peripheral vascular disease are actively being investigated; however, few studies in this area have probed the role of the wound healing monocyte-derived macrophage (MDM). Inflammatory MDMs transition to wound healing MDMs as the relative levels of tumor necrosis factor-alpha (TNF-α) decrease and IL-10 increase. TNF-α has been linked to the regulation of HMGB1 (high mobility group box 1 protein), a nuclear protein that upon macrophage stimulation can be secreted and act as a pro-inflammatory cytokine. This study investigated the influence of a degradable polar hydrophobic ionic polyurethane (D-PHI) on MDM cell expression of pro- versus anti-inflammatory markers, when the material was uncoated or pre-coated with collagen prior to cell studies. Effects were compared to similar groups on tissue culture polystyrene (TCPS). Collagen coated TCPS and D-PHI had significantly more DNA than the uncoated TCPS after 7d (p  = 0.001 and p  = 0.006 respectively); however, there was significantly less esterase activity from cells on D-PHI (±collagen) than for cells on TCPS after 7d (p  = 0.002, p  = 0.0003 respectively). No significant differences in esterase activity were observed between collagen coated and non-coated D-PHI surfaces. Analyses of pro-inflammatory cytokines (TNF-α, IL-1β and HMGB1) secreted from differentiating monocytes adherent to D-PHI demonstrated a decrease whereas anti-inflammatory IL-10 increased over time when compared to TCPS, suggesting that D-PHI was less inflammatory than TCPS. Since D-PHI maintains cell attachment while aiding in the transition of MDM to a wound healing phenotype, this material has qualities suitable to be used in tissue engineering applications where MDM play a key role in tissue regeneration.
Keywords: Collagen; Degradable polyurethane; Esterase; High mobility box group 1 protein; Macrophage; Vascular graft;

The use of injectable spherically symmetric cell aggregates self-assembled in a thermo-responsive hydrogel for enhanced cell transplantation by Wen-Yu Lee; Yu-Hsiang Chang; Yi-Chun Yeh; Chun-Hung Chen; Kurt M. Lin; Chieh-Cheng Huang; Yen Chang; Hsing-Wen Sung (5505-5513).
Typical cell transplantation techniques involve the administration of dissociated cells directly injected into muscular tissues; however, retention of the transplanted cells at the sites of the cell graft is frequently limited. An approach, using spherically symmetric aggregates of cells with a relatively uniform size self-assembled in a thermo-responsive methylcellulose hydrogel system, is reported in the study. The obtained cell aggregates preserved their endogenous extracellular matrices (ECM) and intercellular junctions because no proteolytic enzyme was used when harvesting the cell aggregates. Most of the cells within aggregates (with a radius of approximately 100 μm) were viable as indicated by the live/dead staining assay. After injection through a needle, the cell aggregates remained intact and the cells retained their activity upon transferring to another growth surface. The cell aggregates obtained under sterile conditions were transplanted into the skeletal muscle of rats via local injection. The dissociated cells were used as a control. It was found that the cell aggregates can provide an adequate physical size to entrap into the muscular interstices and offer a favorable ECM environment to enhance retention of the transplanted cells at the sites of the cell graft. These results indicated that the spherically symmetric cell aggregates developed in the study may serve as a cell delivery vehicle for therapeutic applications.
Keywords: Cell transplantation; Cell therapy; Cell aggregate; Intramuscular injection; Mesenchymal stem cell;

This paper describes the formation of poly(N-isopropylacrylamide) (PNIPAAm) and poly[(N-isopropylacrylamide)-r-((3-(methacryloylamino)propyl)-dimethyl(3-sulfopropyl)ammonium hydroxide)] (P(NIPAAm-r-MPDSAH)) films on a glass surface via surface-initiated, atom transfer radical polymerization as a cell-culture platform. The films of PNIPAAm with various thicknesses and of P(NIPAAm-r-MPDSAH) with various ratios of NIPAAm and MPDSAH are formed to investigate the behaviors of cell adhesion and detachment. In the case of the PNIPAAm-grafted glass surfaces, the optimal film thickness, achieving the effective control of both cell adhesion and detachment, is estimated to be 11–13 nm for NIH 3T3 fibroblast cells. The adhesion and detachment behaviors of NIH 3T3 fibroblast cells are further tuned by incorporating the hydrophilic and non-biofouling MPDSAH moiety into the PNIPAAm system. The cell adhesion and detachment are controlled best, when the ratio of NIPAAm and MPDSAH is 75:1 (NIPAAm:MPDSAH).
Keywords: Cell culture; Thermoresponsive polymer; Poly(N-isopropylacrylamide); Atom transfer radical polymerization; Fibroblast; Cell adhesion/detachment;

Intervertebral disc regeneration in an ex vivo culture system using mesenchymal stem cells and platelet-rich plasma by Wei-Hong Chen; Hen-Yu Liu; Wen-Cheng Lo; Shinn-Chih Wu; Chau-Hwa Chi; Hsueh-Yuan Chang; Shih-Hsiang Hsiao; Chih-Hsiung Wu; Wen-Ta Chiu; Bao-Ji Chen; Win-Ping Deng (5523-5533).
An ex vivo degenerative intervertebral disc (IVD) organ culture system was established for the screening of disc regeneration agents. Its application was demonstrated by a stem cell and growth factor-based therapeutic approach for the amelioration of IVD. An ex vivo culture system using chymopapain to partially digest nucleus proposus tissue was established to mimic human IVD degeneration. This system was then used for the evaluation of different therapeutic regimens including: mesenchymal stem cell derived from eGFP-transgenic porcine (MSC-GFP), platelet-rich plasma (PRP) and MSC-GFP/PRP combined treatment, and confirmed in in vivo animal model. Chondrogenic-specific gene products including Col II and aggrecan were found upregulated and chondrogenic matrix deposition increased, as evident by sustained fluorescent signals over 4 weeks, in the MSC-GFP implanted group. Previously, we demonstrated in vitro stage-specific chondrogenesis of MSC by chondrocytic commitment. These same molecules upregulated for chondrogenesis were also observed in MSC-GFP group. PRP that has been shown to promote nucleus pulposus (NP) regeneration also resulted in significant increased levels of mRNA involved in chondrogenesis and matrices accumulation. The ex vivo IVD regeneration results were repeated and supported by in vivo porcine degenerative system. Moreover, the disc height index (DHI) was significantly increased in both in vivo MSC-GFP and PRP regeneration groups. Unexpectedly, the MSC-GFP/PRP combined therapy demonstrated an inclination towards osteogenesis in ex vivo system. The ex vivo degenerative IVD culture system described in this study could serve as an alternative and more accessible model over large animal model. This system also provides a high-throughput platform for screening therapeutic agents for IVD regeneration.
Keywords: Intervertebral disc; Mesenchymal stem cell; Platelet-rich plasma; Nucleus pulposus; Regeneration;

A thermosensitive chitosan-based hydrogel barrier for post-operative adhesions' prevention by Chang-Zheng Wei; Chun-Lin Hou; Qi-Sheng Gu; Li-Xia Jiang; Bin Zhu; Ai-Lian Sheng (5534-5540).
Post-operative peritoneal adhesions are common and serious complications for surgeons. They can cause pelvic pain, infertility, and potentially lethal bowel obstruction. We synthesized injectable hydrogels that formed by chemical modification through grafted hydrobutyl groups to chitosan chains. Gelation of hydroxybutyl chitosan (HBC) occurs in less than 60 s. Once formed, it can also be recovered completely. The residue time of hydrogels can extend to 4 weeks in Kunming mice. HBC hydrogels showed mild cytotoxicity to mice fibroblast cell (L929) and human vascular endothelial cell (ECV-304) in vitro and were biocompatible in the murine muscles, causing no adhesions for 4 weeks. HBC gels can form a durable barrier between defected cecum and abdominal wall. In a mice sidewall defect-bowel abrasion model, HBC gels showed significant efficacy in reducing adhesion formation.
Keywords: Post-operative adhesions; Barriers; Chitin/chitosan; Anti-adhesion; Hydrogel; Thermosensitive;

Protein adsorption and clotting time of pHEMA hydrogels modified with C18 ligands to adsorb albumin selectively and reversibly by Inês C. Gonçalves; M. Cristina L. Martins; Mário A. Barbosa; Buddy D. Ratner (5541-5551).
This work intended to create a nanostructured biomaterial that would bind albumin in a selective and reversible way in order to inhibit the adsorption of other blood proteins and therefore minimize activation of coagulation. Different levels of C18 ligand have been immobilized on poly(2-hydroxyethyl methacrylate) (pHEMA). We hypothesize that samples with intermediate amounts of C18 ligand would allow albumin to recognize them and bind through its hydrophobic pockets specific for long chain fatty acids. Surface characterization has confirmed increasing amounts of C18 ligand on pHEMA as the percentage of C18 in solution increases, with maximum coverage achieved in 10% C18 samples. Adsorption from pure albumin solution revealed a small decrease in albumin adsorption from pHEMA to 1% C18 and 2.5% C18 samples, but on surfaces with 5% or higher C18 the amount of adsorbed albumin increased as the percentage of C18 increased. Competitive adsorption studies in the presence of both albumin and fibrinogen, and in the presence of all plasma proteins showed that 1% C18 and 2.5% C18 were the only surfaces selective for albumin, and that the presence of all plasma proteins may even potentiate albumin adsorption. Reversibility studies demonstrated that both 2.5% C18 and 5% C18 samples exchange 125I-albumin selectively in the presence of both unlabeled albumin and plasma, but 2.5% C18 samples presented higher exchangeability rates (58%). Clotting times using recalcified plasma revealed that samples with none or small amounts of C18 (pHEMA to 5% C18) did not shorten the clotting time compared to the negative control (polystyrene), indicating low activation of the intrinsic coagulation cascade.
Keywords: polyHEMA; Surface modification; Protein adsorption; Human serum albumin; Clotting time;

Synthesis and characterization of iodinated polyurethane with inherent radiopacity by S. Kiran; Nirmala R. James; Roy Joseph; A. Jayakrishnan (5552-5559).
The synthesis and characterization of polyurethane (PU) with excellent radiopacity for medical and allied applications are reported. Bisphenol-A (BPA) was iodinated to obtain 4,4′-isopropylidinedi-(2,6-diiodophenol) (IBPA) which was used as a chain extender for the preparation of a radiopaque PU. The PU was prepared by reacting 4,4′-methylenebis(phenyl isocyanate) (MDI), poly(tetramethylene glycol) (PTMG) and IBPA in 2.2:1.2:1 molecular ratio and is characterized by infrared spectroscopy (IR), thermogravimetry (TGA), dynamic mechanical analysis (DMA), energy dispersive X-ray analysis (EDX), gel permeation chromatography (GPC) and X-radiography. X-ray images showed that the PU prepared using IBPA as the chain extender is highly radiopaque. An in vitro cytotoxicity test using L929 mouse fibroblast cells shows that the PU is non-cytotoxic. The outlined synthesis of a PU with radiocontrast properties opens up the possibility of synthesizing many different kinds of radiopaque PUs with desirable range of physical properties exploiting the versatility in their chemical synthesis.
Keywords: Polyurethane; Radiopacity; Biocompatibility; DMA; Iodination; Cytotoxicity;

Mechanical properties, drug eluting characteristics and in vivo performance of a genipin-crosslinked chitosan polymeric stent by Mei-Chin Chen; Chin-Tang Liu; Hung-Wen Tsai; Wei-Yun Lai; Yen Chang; Hsing-Wen Sung (5560-5571).
A limitation with the use of polymers as stent matrices is their inherent mechanical weakness. In this study, a polymeric stent, made from chitosan-based films fixed by genipin which has a cyclic molecular structure, was developed (the genipin stent). The mechanical properties of the genipin stent were investigated; its counterpart fixed by a linear epoxy compound (the epoxy stent) and a commercially available metallic stent were used as controls. The results indicated that the cyclic crosslinking structures formed within the genipin stent matrix were beneficiary to the improvement of its mechanical property. Additionally, the tolerable compression load of the genipin stent was superior to that of the control metallic stent. The cytotoxicity of the genipin stent was significantly lower than the epoxy stent. The deployment of the genipin stent in rabbit infrarenal abdominal aortas was performed using a French sheath. At 3 months postoperatively, the retrieved arteries remained patent; no thrombosis was observed. A nearly intact layer of endothelial cells was seen on the stent-implanted vessel wall. To evaluate its possibility as a drug delivery vehicle, sirolimus (an anti-proliferative drug) was loaded in the genipin stent. It was found that the genipin stent with heparin coating exhibited a linear sustained-release profile and the released sirolimus still possessed its original activity in inhibiting smooth muscle cell proliferation. These findings suggest that the genipin stent with enhanced mechanical strength can be used as an attractive stent platform for local drug delivery.
Keywords: Crosslinking; Endothelialization; Biocompatibility; Biodegradation; Sirolimus;

Clinical fracture of cross-linked UHMWPE acetabular liners by Jevan Furmanski; Martin Anderson; Sonny Bal; A. Seth Greenwald; David Halley; Brad Penenberg; Michael Ries; Lisa Pruitt (5572-5582).
Highly cross-linked ultrahigh molecular weight polyethylene (UHMWPE) is increasingly used as a bearing material in total hip replacements. Cross-linking of UHMWPE has been shown to increase wear resistance but decrease its fracture resistance. We analyzed the clinical fracture failure of four cross-linked UHMWPE total hip replacement components of four different designs via microscopic observation of the fracture surfaces, and found that in all cases fractures initiated at stress concentrations in an unsupported region of the component (termed the elevated rim). Finite element analyses (FEA) of each individual implant design were then conducted. Results from this analysis demonstrated that the predicted magnitude and orientation of maximum principal stress due to mechanical loading of the elevated rim was sufficient to propagate initiated fatigue cracks in each case. FEA also predicted that cracks may arrest after some amount of growth due to a steep stress gradient near the initiation site. Further, while anatomical positioning of the implant and material properties affect the risk of fracture, we examined whether these failures are strongly related to the notched elevated rim design feature that is common to the four failed cases presented here. We believe that cross-linked UHMWPE remains an excellent bearing material for total hip replacements but that designs employing this material should mitigate stress concentrations or other design features that increase the risk of fracture.
Keywords: Acetabular liner; UHMWPE; Notch fatigue; Fractography; Crosslinking; Fracture;

The binding of pullulan modified cholesteryl nanogels to Aβ oligomers and their suppression of cytotoxicity by Sebastien Boridy; Haruko Takahashi; Kazunari Akiyoshi; Dusica Maysinger (5583-5591).
Among various hydrogels able to form monodisperse and stable nanoparticles (20–30 nm) are those with pullulan-bearing cholesteryl moieties (CHP). These nanoparticles can interact with soluble proteins through hydrophobic bonding. The objectives of this study were to investigate whether CHP nanogels would interact with oligomeric forms of the 42 amino acid variant of β-amyloid (Aβ1–42) and if the formation of CHP-Aβ1–42 oligomer entities will reduce cytotoxicity of Aβ1–42 in primary cortical cells and microglial (N9) cells. By employing fluorescent CHP analogs with different charges we provide evidence that, (i) both neutral and positively charged CHP nanoparticles interact with Aβ1–42 monomers and oligomers, (ii) neutral CHP is non-toxic, but positively charged derivatives (CHPNH2) are toxic, particularly in primary cortical cultures, and (iii) binding of both monomeric and oligomeric Aβ1–42 to CHP significantly reduces Aβ1–42 toxicity in both the primary cortical and microglial cells. These results suggest that CHP nanogels could provide a valid complementary approach to antibody immunotherapy in neurological disorders characterized by the formation of soluble toxic aggregates, such as those in Alzheimer's disease (AD).
Keywords: Nanogel; β-Amyloid (Aβ); Alzheimer's disease (AD); Oligomers;

Synthesis, characterization, and in vivo targeted imaging of amine-functionalized rare-earth up-converting nanophosphors by Li-Qin Xiong; Zhi-Gang Chen; Meng-Xiao Yu; Fu-You Li; Chun Liu; Chun-Hui Huang (5592-5600).
Rare-earth up-converting nanophosphors (UCNPs) have great potential to become a new generation of biological luminescent labels, but their use has been limited by difficulties in obtaining water-soluble UCNPs bearing appropriate functional groups. To address this problem, we report herein a simple and efficient procedure for the preparation of amine-functionalized UCNPs by a modified hydrothermal microemulsion route assisted with 6-aminohexanoic acid. The amine content of the resultant UCNPs has been determined to be (9.5 ± 0.8) × 10−5  mol/g, which not only confers excellent dispersibility in aqueous solution, but also allows further conjugation with targeted molecules such as folic acid (FA) as a ligand. By means of the laser scanning up-conversion luminescence microscopy (LSUCLM) and the in vivo up-conversion luminescence (UCL) imaging under excitation at the CW infrared laser at 980 nm, FA-coupled UCNPs have been demonstrated to be effective in targeting folate-receptor overexpressing HeLa cells in vitro and HeLa tumor in vivo and ex vivo. These results indicated that our UCNPs could be used as whole-body targeted UCL imaging agents.
Keywords: Rare-earth nanophosphors; Up-conversion luminescence; Modified hydrothermal microemulsion synthesis; In vivo imaging; Amine-functionalized;

A bifunctional nanoparticles-based carrier for simultaneous in vivo imaging and photodynamic therapy by encapsulating methylene blue (MB) alone in the phosphonate-terminated silica matrix has been developed. The phosphonate-terminated silica nanoparticles, entrapping water-soluble photosensitizer MB (MB-encapsulated PSiNPs), are synthesized by the controlled synchronous hydrolysis of tetraethoxysilane and trihydroxyl silyl propyl methyl phosphonate in the water-in-oil microemulsion. The resulting MB-encapsulated PSiNPs effectively prevent the leakage of entrapped MB from the particles and provide protection for against reduction by diaphorase. Enough dose of irradiation to the MB-encapsulated PSiNPs under the light of 635 nm results in efficient generation of singlet oxygen and induces photodynamic damage to Hela cells. Furthermore, the non-invasive visualization of MB-encapsulated PSiNPs in mice under the in vivo imaging system confirmed the MB-encapsulated PSiNPs also presents near-infrared luminescence for in vivo imaging. And the effect of the PDT toward the xenograft tumor in vivo is exciting after imaging the MB-encapsulated PSiNPs injected tumor using in vivo optical imaging system. Thus, the single particle platform is effective for simultaneous in vivo imaging and photodynamic therapy without using extra agent, which can provide image-guidance for site-specific photodynamic therapy.
Keywords: Methylene blue; Silica nanoparticles; Photodynamic therapy; In vivo imaging;

Radiopaque iodinated copolymeric nanoparticles for X-ray imaging applications by Hagit Aviv; Sonke Bartling; Fabian Kieslling; Shlomo Margel (5610-5616).
Recently we described iodinated homopolymeric radiopaque nanoparticles of 28.9 ± 6.3 nm dry diameter synthesized by emulsion polymerization of 2-methacryloyloxyethyl(2,3,5-triiodobenzoate) (MAOETIB). The nanoparticle aqueous dispersion, however, was not stable and tended to agglomerate, particularly at weight concentration of dispersed nanoparticles above ∼0.3%. The agglomeration rate increases as the concentration of nanoparticles in aqueous phase rises and prevents the potential in vivo use as contrast agent for medical X-ray imaging. Here we describe efforts to overcome this limitation by synthesis of iodinated copolymeric nanoparticles of 25.5 ± 4.2 nm dry diameter, by emulsion copolymerization of the monomer, MAOETIB, with a low concentration of glycidyl methacrylate (GMA). The surface of resulting copolymeric nanoparticles is far more hydrophilic than that of polyMAOETIB (PMAOETIB) nanoparticles. Therefore, P(MAOETIB-GMA) nanoparticles are significantly more stable against agglomeration in aqueous continuous phase. After intravenous injection of P(MAOETIB-GMA) nanoparticles in rats and mice (including those with a liver cancer model) CT-imaging revealed a significant enhanced visibility of the blood pool for 30 min after injection. Later, lymph nodes, liver and spleen strongly enhanced due to nanoparticle uptake by the reticuloendothelial system. This favorably enabled the differentiation of cancerous from healthy liver tissue and suggests our particles for tumor imaging in liver and lymph nodes.
Keywords: Iodine-containing radiopaque nanoparticles; Emulsion polymerization; Copolymeric iodinated nanoparticles; Radiopacity; Contrast agent; Computed tomography;

Functionalizable and ultra stable nanoparticles coated with zwitterionic poly(carboxybetaine) in undiluted blood serum by Wei Yang; Lei Zhang; Shanlin Wang; Andrew D. White; Shaoyi Jiang (5617-5621).
A new surface chemistry presenting an abundance of functional groups for ligand immobilization in an ultra-low fouling background all in one material for nanoparticles was introduced. This surface platform, as demonstrated by zwitterionic poly(carboxybetaine acrylamide) (polyCBAA) coated nanoparticles, is not only ultra stable in undiluted human blood serum, but also can be conjugated to biomolecules conveniently and effectively. Thus, this surface chemistry is ideal to create multi-functional nanoparticles for targeted delivery and diagnostics. In addition, this work clearly shows that 10% blood serum commonly used to evaluate the stability of nanoparticles is insufficient and a new evaluation criterion with undiluted blood serum is recommended.
Keywords: Zwitterionic; Poly(carboxybetaine); Functionalization; Undiluted blood serum;

Optical and biological sensing capabilities of Au2S/AuAgS coated gold nanorods by Haowen Huang; Xuanyong Liu; Yunlong Zeng; Xianyong Yu; Bo Liao; Pinggui Yi; Paul K. Chu (5622-5630).
Gold nanorods coated with a multiplex component, namely Au2S/AuAgS coated gold nanorods, are produced without precipitation and aggregation among the nanorods. Both the thickness of the shell and size of the core can be readily controlled by this technique allowing one to tune the plasmon resonance of the nanocomposites over a range of several hundred nanometers. These Au2S/AuAgS coated gold nanorods exhibit interesting optical properties and are suitable for many biological sensing applications. Functionalization of the Au2S/AuAgS coated gold nanorods is achieved by manipulating the affinity between the Au2S/AuAgS and thiol compounds. Biomolecules can be covalently attached via the NH2 bond of the antibodies to the NHS-terminated nanorods. The longitudinal peaks of the Au2S/AuAgS coated gold nanorods are extremely sensitive to the refractive index changes induced by target binding, suggesting that they are excellent sensors for target-specific binding events and have the potential to achieve single-molecule sensitivity in microspectroscopy.
Keywords: Biological sensing; Nanorods; Functionalization; Au2S/AuAgS coated gold nanorods Core–shell nanostructure Optical properties;

The effect of electrical stimulation on the differentiation of hESCs adhered onto fibronectin-coated gold nanoparticles by Dae G. Woo; Myung-Sun Shim; Ji S. Park; Han N. Yang; Dong-Ryul Lee; Keun-Hong Park (5631-5638).
To encourage stem cell differentiation, gold nanoparticles (20 nm) were used to deliver electrical stimulation to human embryonic stem cells (hESCs) in vitro. Nano-structured gold nanoparticles were designed by coating the surface of culture dishes with gold nanoparticles using a layer-by-layer (LBL) system. In this method, gold nanoparticles were continuously coated onto dishes by SEM analysis. Evaluation of gene modified hESCs that were subsequently attached onto fibronectin-coated gold nanoparticles revealed that the un-differentiation marker, Oct-4, was no longer present following electrical stimulation. In addition, the osteogenic markers of collagen type I and Cbfa1 increased in response to electrical stimulation, while those of hESCs were not observed without electrical stimulation.
Keywords: Stem cell differentiation; Gold nanoparticles; hESCs; Gene modified hESCs; Electrical stimulation;

Polyelectrolyte-coated gold nanorods and their interactions with type I collagen by Christopher G. Wilson; Patrick N. Sisco; Francis A. Gadala-Maria; Catherine J. Murphy; Edie C. Goldsmith (5639-5648).
Gold nanorods (AuNRs) have unique optical properties for numerous biomedical applications, but the interactions between AuNRs and proteins, particularly those of the extracellular matrix (ECM), are poorly understood. Here the effects of AuNRs on the self-assembly, mechanics, and remodeling of type I collagen gels were examined in vitro. AuNRs were modified with polyelectrolyte multilayers (PEMs) to minimize cytotoxicity, and AuNRs with different terminal polymer chemistries were examined for their interactions with collagen by turbidity assays, rheological tests, and microscopy. Gel contraction assays were used to examine the effects of the PEM-coated AuNRs on cell-mediated collagen remodeling. Polyanion-terminated AuNRs significantly reduced the lag (nucleation) phase of collagen self-assembly and significantly increased the dynamic shear modulus of the polymerized gels, whereas polycation-terminated AuNRs had no effect on the mechanical properties of the collagen. Both polyanion- and polycation-terminated AuNRs significantly inhibited collagen gel contraction by cardiac fibroblasts, and the nanoparticles were localized in intra-, peri-, and extracellular compartments, suggesting that PEM-coated AuNRs influence cell behavior via multiple mechanisms. These results demonstrate the significance of nanoparticle–ECM interactions in determining the bioactivity of nanoparticles.
Keywords: Nanoparticle; Collagen; Mechanical properties; Polymerization; Fibroblast; Gold;

Multimeric peptide-based PEG nanocarriers with programmable elimination properties by Simi Gunaseelan; Shahriar Pooyan; Peiming Chen; Mahta Samizadeh; Matthew S. Palombo; Stanley Stein; Xiaoping Zhang; Patrick J. Sinko (5649-5659).
In the current study, the design, synthetic feasibility and biochemical characterization of biodegradable peptidic PEG-based nanocarriers are described. The components were selected to influence the body elimination pathway upon nanocarrier biodegradation. Two prototypical nanocarriers were prepared using non-PEGylated and PEGylated peptidic cores [CH3CO-(Lys-βAla-βAla) X -Cys-CONH2 (X  = 2, 4)]. A homodimeric nanocarrier with 4 copies of fluorescein-PEG5kDa was synthesized by linking two PEGylated peptidic cores (X  = 2) using a disulfide bond. A dual labeled heterodimeric nanocarrier with 2 copies of fluorescein-PEG5kDa and 4 copies of Texas Red was also synthesized. Optimum conditions for linking imaging agents, PEG, or a peptidic core to a peptidic core were determined. Significantly higher yields (69% versus 30%) of the PEGylated peptidic core were obtained by using 2 copies of β-alanine as a spacer along with increasing DMSO concentrations, which resulted in reduced steric hindrance. Stoichiometric addition of the components was also demonstrated and found to be important for reducing polydispersity. Nanocarrier biodegradation was evaluated in simulated intracellular and extracellular/blood environments using 3 mm and 10 μm glutathione in buffer, respectively. The nanocarrier was 9-fold more stable in the extracellular environment. The results suggest selective intracellular degradation of the nanocarrier into components with known body elimination pathways.
Keywords: Peptide; PEG; Nanocarrier; Disulfide; GSH-dependent biodegradation; Intracellular elimination;

Codelivery of different therapeutics has a potential to efficaciously treat human diseases via their synergetic effects. We have recently developed a new class dendrimers, poly(l-lysine) dendrimers with a silsesquioxane cubic core (nanoglobules). These dendrimers have compact globular and well-defined structures and highly functionalized surfaces, and can be used as versatile carriers for biomedical applications. In this study, a generation-3 (G3) nanoglobular dendrimer was used to conjugate a peptide c(RGDfK) with a PEG spacer for codelivery of doxorubicin (DOX) and siRNA. Doxorubicin (DOX) was coupled to the RGD targeted nanoglobule via a degradable disulfide spacer to give G3-[PEG-RGD]-[DOX]. G3-[PEG-RGD]-[DOX] showed higher cytotoxicity than free DOX at high doses in glioblastoma U87 cells. G3-[PEG-RGD]-[DOX] was further complexed with siRNA and such complexes were readily internalized by U87 cells as shown by confocal microscopy. The siRNA complexes of the targeted conjugate resulted in significantly higher gene silencing efficiency in U87-Luc cells than those of control conjugates G3-[PEG-RGD] and G3-[DOX]. The nanoglobules are promising carriers for the codelivery of nucleic acids and chemotherapeutic agents.
Keywords: Codelivery; Nanoglobules; Doxorubicin; siRNA;

Elaboration of radiopaque iodinated nanoparticles for in situ control of local drug delivery by Damia Mawad; Hanna Mouaziz; Alexandra Penciu; Henri Méhier; Bernard Fenet; Hatem Fessi; Yves Chevalier (5667-5674).
Drug delivery systems can benefit from intrinsic radiopacity as this property will allow following up the diffusion path of the nanoparticles containing the therapeutic drug after their local administration. Herein, we report the synthesis of iodinated derivatives of cellulose acetate (CA) and their formulation into aqueous radiopaque nanoparticle suspensions. Modification and purification of CA with mono- or tri-iodobenzoyl chloride were characterized by NMR spectroscopy and elemental analysis of iodine. In particular, measurements of diffusion coefficients by the DOSY 2D NMR method allowed controlling the complete elimination of non-grafted iodinated materials. Pure radiopaque CA was successfully achieved with an iodine content varying between 14 and 32%. Aqueous suspensions of nanoparticles were successfully formed, characterized by being spherical, <100 nm in size and stable as a suspension over 3 months. The degree of substitution, in particular the triiodo moieties, imparted a good level of radiopacity whether in dry powder form (2627 HU) or as a nanoparticle suspension (298 HU). These values are comparable to radiopacity of systems reported in literature to be in vivo visible. Loading of paclitaxel was successfully attempted, suggesting that the developed radiopaque nanoparticles can ultimately function as a drug delivery system.
Keywords: Nanoparticle; Radiopacity; Cellulose acetate; Iodinated polymers; DOSY; Drug delivery;

Sustained release of antibiotic from poly(2-hydroxyethyl methacrylate) to prevent blinding infections after cataract surgery by Erin M. Anderson; Misty L. Noble; Shai Garty; Hongyan Ma; James D. Bryers; Tueng T. Shen; Buddy D. Ratner (5675-5681).
Intraocular lens implantation after opacified natural lens removal is the primary treatment for cataracts in developed countries. Cataract surgery is generally considered safe, but entails significant risks in countries where sophisticated sterile operating theaters are not widely available. Post-operative infection (endophthalmitis) is a potential blinding complication. Infection often results from bacterial colonization of the new lens implant and subsequent antibiotic-tolerant biofilm formation. To combat this risk, we developed a polymeric hydrogel system that can deliver effective levels of antibiotic over an extended period of time within the globe of the eye. Norfloxacin™ antibiotic was loaded into cross-linked poly(2-hydroxyethyl methacrylate) (pHEMA) gels, which were subsequently surface-modified with octadecyl isocyanate to produce a hydrophobic rate-limiting barrier controlling norfloxacin release. Octadecyl surface modification was characterized using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). A 15-min modification leads to a uniform surface coating and near zero order release of norfloxacin from the matrix. Norfloxacin released from coated pHEMA kills Staphylococcus epidermidis in suspension and on a simulated medical implant surface. With these data, we demonstrate a new and effective system for sustained drug release from a hydrogel matrix with specific application for intraocular lens surgery.
Keywords: Drug delivery; Hydrogel; Intraocular lenses; Endophthalmitis; Infections;

A series of well-defined, fluorescently labelled homopolymers, random and block copolymers based on N-(2-hydroxypropyl)-methacrylamide were prepared by reversible addition–fragmentation chain transfer polymerization (RAFT polymerization). The polydispersity indexes for all polymers were in the range of 1.2–1.3 and the number average of the molar mass (M n) for each polymer was set to be in the range of 15–30 kDa. The cellular uptake of these polymers was investigated in the human multi-drug resistant breast adenocarcinoma cell line MCF7/ADR. The uptake greatly depended on the polymer molecular mass and structure. Specifically, smaller polymers (approx. 15 kDa) were taken up by the cells at much lower concentrations than larger polymers (approx. 30 kDa). Furthermore, for polymers of the same molar mass, the random copolymers were more easily internalized in cells than block copolymers or homopolymers. This is attributed to the fact that random copolymers form micelle-like aggregates by intra- and interchain interactions, which are smaller and less stable than the block copolymer structures in which the hydrophobic domain is buried and thus prevented from unspecific interaction with the cell membrane. Our findings underline the need for highly defined polymeric carriers and excipients for future applications in the field of nanomedicine.
Keywords: Endocytosis; Structure–property relationship; Drug delivery; Polymer microstructure; RAFT polymerization;

Drug permeability and mucoadhesion properties of thiolated trimethyl chitosan nanoparticles in oral insulin delivery by Lichen Yin; Jieying Ding; Chunbai He; Liming Cui; Cui Tang; Chunhua Yin (5691-5700).
Trimethyl chitosan-cysteine conjugate (TMC-Cys) was synthesized in an attempt to combine the mucoadhesion and the permeation enhancing effects of TMC and thiolated polymers related to different mechanisms for oral absorption. TMC-Cys with various molecular weights (30, 200, and 500 kDa) and quaternization degrees (15 and 30%) was allowed to form polyelectrolyte nanoparticles with insulin through self-assembly, which demonstrated particle size of 100–200 nm, zeta potential of +12 to +18 mV, and high encapsulation efficiency. TMC-Cys/insulin nanoparticles (TMC-Cys NP) showed a 2.1–4.7-fold increase in mucoadhesion compared to TMC/insulin nanoparticles (TMC NP), which might be partly attributed to disulfide formation between TMC-Cys and mucin as evidenced by DSC measurement. Compared to insulin solution and TMC NP, TMC-Cys NP induced increased insulin transport through rat intestine by 3.3–11.7 and 1.7–2.6 folds, promoted Caco-2 cell internalization by 7.5–12.7 and 1.7–3.0 folds, and augmented uptake in Peyer's patches by 14.7–20.9 and 1.7–5.0 folds, respectively. Such results were further confirmed by in vivo experiment with the optimal TMC-Cys NP. Biocompatibility assessment revealed lack of toxicity of TMC-Cys NP. Therefore, self-assembled nanoparticles between TMC-Cys and protein drugs could be an effective and safe oral delivery system.
Keywords: Thiolated trimethyl chitosan; Self-assembled nanoparticles; Insulin; Oral delivery; Mucoadhesion; Permeation enhancement;

The assembly of a short linear natural cytosine-phosphate-guanine DNA into dendritic structures and its effect on immunostimulatory activity by Sakulrat Rattanakiat; Makiya Nishikawa; Hisakage Funabashi; Dan Luo; Yoshinobu Takakura (5701-5706).
DNA containing unmethylated CpG dinucleotides, or CpG motifs, (CpG DNA) has been explored as a therapeutic agent, owing to its potent immunostimulatory activity. A previous study showing that Y-shaped (Y-) CpG DNA has a high immunostimulatory activity compared with single- or double stranded CpG DNA suggests the possibility that CpG DNA in a more complicated structure is a stronger activator of the immune system. In the present study, dendrimer-like DNA (DL-DNA) was prepared by ligating Y-DNA monomers. The DL-DNA of the second or third generation with 12 or 24 highly potent CpG motifs in one unit, respectively, were designed and successfully prepared for the first time. These DL-DNAs induced greater amounts of tumor necrosis factor-α and interleukin-6 from RAW264.7 macrophage-like cells than did a mixture of Y-DNA with the same sequences as the corresponding DL-DNA. DL-DNA was more efficiently taken up by RAW264.7 cells than Y-DNA, but the increase was lower than that exhibited by the levels of cytokine release. These results suggest that the dendritic structure formation is a potential approach to increasing the immunostimulatory activity of CpG DNA without any modifications of the chemical structure of the natural phosphodiester DNA.
Keywords: CpG motif; Immunostimulatory activity; Oligodeoxynucleotides; Toll-like receptor 9; Dendrimer;

In this study, we show that degradable particles of a hydrophobic polymer can effectively deliver drugs to tumors after i.v. administration. Free-standing nanoparticles with diameters of 100–300 nm were successfully fabricated from highly hydrophobic, biodegradable poly(ω-pentadecalactone-co-butylene-co-succinate) (PPBS) copolyesters. PPBS copolymers with various compositions (20–80 mol% PDL unit contents) were synthesized via copolymerization of ω-pentadecalactone (PDL), diethyl succinate (DES), and 1,4-butanediol (BD) using Candida antarctica lipase B (CALB) as the catalyst. Camptothecin (CPT, 12–22%) was loaded into PPBS nanoparticles with high encapsulation efficiency (up to 96%) using a modified oil-in-water single emulsion technique. The CPT-loaded nanoparticles had a zeta potential of about −10 mV. PPBS particles were non-toxic in cell culture. Upon encapsulation, the active lactone form of CPT was remarkably stabilized and no lactone-to-carboxylate structural conversion was observed for CPT-loaded PPBS nanoparticles incubated in both phosphate-buffered saline (PBS, pH = 7.4) and DMEM medium for at least 24 h. In PBS at 37 °C, CPT-loaded PPBS nanoparticles showed a low burst CPT release (20–30%) within the first 24 h followed by a sustained, essentially complete, release of the remaining drug over the subsequent 40 days. Compared to free CPT, CPT-loaded PPBS nanoparticles showed a significant enhancement of cellular uptake, higher cytotoxicity against Lewis lung carcinoma and 9L cell lines in vitro, a longer circulation time, and substantially better antitumor efficacy in vivo. These results demonstrate the potential of PPBS nanoparticles as long-term stable and effective drug delivery systems in cancer therapy.
Keywords: Camptothecin; Polymer nanoparticle; Hydrophobic; Controlled release; Antitumor effect;

Nanodiamond–insulin complexes as pH-dependent protein delivery vehicles by Rafael A. Shimkunas; Erik Robinson; Robert Lam; Steven Lu; Xiaoyang Xu; Xue-Qing Zhang; Houjin Huang; Eiji Osawa; Dean Ho (5720-5728).
Enhanced specificity in drug delivery aims to improve upon systemic elution methods by locally concentrating therapeutic agents and reducing negative side effects. Due to their robust physical properties, biocompatibility and drug loading capabilities, nanodiamonds serve as drug delivery platforms that can be applied towards the elution of a broad range of therapeutically-active compounds. In this work, bovine insulin was non-covalently bound to detonated nanodiamonds via physical adsorption in an aqueous solution and demonstrated pH-dependent desorption in alkaline environments of sodium hydroxide. Insulin adsorption to NDs was confirmed by FT-IR spectroscopy and zeta potential measurements, while both adsorption and desorption were visualized with TEM imaging, quantified using protein detection assays and protein function demonstrated by MTT and RT-PCR. NDs combined with insulin at a 4:1 ratio showed 79.8 ± 4.3% adsorption and 31.3 ± 1.6% desorption in pH-neutral and alkaline solutions, respectively. Additionally, a 5-day desorption assay in NaOH (pH 10.5) and neutral solution resulted in 45.8 ± 3.8% and 2.2 ± 1.2% desorption, respectively. MTT viability assays and quantitative RT-PCR (expression of Ins1 and Csf3/G-csf genes) reveal bound insulin remains inactive until alkaline-mediated desorption. For applications in sustained drug delivery and therapy we have developed a therapeutic protein–ND complex with demonstrated tunable release and preserved activity.
Keywords: Nanodiamond; Insulin; Drug delivery; Wound healing; Nanomedicine;

Incorporation of antimicrobial compounds in mesoporous silica film monolith by Isabel Izquierdo-Barba; María Vallet-Regí; Natalia Kupferschmidt; Osamu Terasaki; Artur Schmidtchen; Martin Malmsten (5729-5736).
Incorporation of the antimicrobial peptide LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), as well as low molecular weight antimicrobial chlorhexidine, into mesoporous silica was obtained using an EISA one-pot synthesis method. FTIR confirmed efficient encapsulation of both LL-37 and chlorhexidine into mesoporous silica, while XRD and TEM showed that antimicrobial agent incorporation can be achieved without greatly affecting the structure of the mesoporous silica. The modified mesoporous silica released LL-37 and chlorhexidine slowly, reaching maximum release after about 200 h. The release rate could also be controlled through incorporation of SH groups in the pore walls, adding to pore hydrophobicity and reducing the release rate by about 50% compared to the unmodified mesoporous silica. Mesoporous silica containing either LL-37 or chlorhexidine displayed potent bactericidal properties against both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. While chlorhexidine-loaded mesoporous silica displayed an accompanying high toxicity, as judged from hemolysis, LDH release, and MTT assay, the corresponding material containing LL-37 showed very low toxicity by all these assays, comparable to that observed for mesoporous silica in the absence of antibacterial drug, as well as to the negative controls in the respective assays. Mesoporous silica containing LL-37 therefore holds potential as an implantable material or a surface coating for such materials, as it combines potent bactericidal action with low toxicity, important features for controlling implant-related infections, e.g., for multi-resistant pathogens or for cases where access to the infection site of systemically administered antibiotics is limited due to collagen capsule formation or other factors.
Keywords: Antimicrobial; Chlorhexidine; LL-37; Mesoporous; Silica;

Targeted epidermal growth factor receptor nanoparticle bioconjugates for breast cancer therapy by Sarbari Acharya; Fahima Dilnawaz; Sanjeeb K. Sahoo (5737-5750).
Selective drug delivery is an important approach with great potential for overcoming problems associated with the systemic toxicity and poor bioavailability of antineoplastic drugs. Nanomedicine plays a pivotal role by delivering drugs in a targeted manner to the malignant tumor cells thereby reducing the systemic toxicity of the anticancer drugs. The objective of this study was to prepare and characterize rapamycin loaded polymeric poly(lactide-co-glycolide) (PLGA) nanoparticles (NP) that were surface conjugated with antibodies to epidermal growth factor receptor (EGFR), highly expressed on breast cancer cells, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) mediated cross linking agents. To potentiate the anticancer efficiency of the formulations, in vitro cytotoxicity of native rapamycin, rapamycin loaded nanoparticles and EGFR antibody conjugated rapamycin loaded nanoparticles (EGFR-Rapa-NPs) were evaluated on malignant MCF 7 breast cancer cell lines. IC50 doses as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay showed the superior antiproliferative activity of EGFR-Rapa-NPs over unconjugated nanoparticles and native rapamycin due to higher cellular uptake on malignant breast cancer cells. Cell cycle arrest and cellular apoptosis induced by the above formulations were confirmed by flow cytometry. Molecular basis of apoptosis studied by western blotting revealed the involvement of a cytoplasmic protein in activating the programmed cell death pathway. Thus it was concluded that EGFR-Rapa-NPs provide an efficient and targeted delivery of anticancer drugs, presenting a promising active targeting carrier for tumor selective therapeutic treatment in near future.
Keywords: Apoptosis; Cancer therapy; Drug delivery; Nanoparticles; Surface modification;

The use of PEGylated liposomes to prolong circulation lifetimes of tissue plasminogen activator by Ji-Young Kim; Jin-Ki Kim; Jeong-Sook Park; Youngro Byun; Chong-Kook Kim (5751-5756).
Tissue plasminogen activator (tPA), a widely used thrombolytic agent, has an application limit due to short half-life. To prolong the half-life of tPA, liposomes composed of egg phosphatidylcholine (EPC), cholesterol (CHOL) and sodium cholesterol-3-sulfate (CS) were prepared by lipid film method. In addition, distearolyphosphatidyl ethanolamine-N-poly(ethylene glycol) 2000 (DSPE–PEG 2000) was included to give steric barrier to liposomes. Physicochemical characteristics such as particle size, zeta potential, entrapment efficiency and long-term storage stability at 4 °C were investigated. The fibrinolytic activity of tPA-loaded in liposomes was confirmed by fibrin clot lysis assay. In vivo pharmacokinetic properties of tPA and the effect of PEG on the blood circulation of tPA-loaded in liposomes in circulation were also evaluated. Both conventional liposomes (EPCL) and PEGylated liposomes (EPC–PEGL) were proper as an injectable formulation with small particle size. Loading process of tPA into liposomes did not alter fibrinolytic activity of intact tPA. Encapsulation of tPA into EPCL and EPC–PEGL prolonged half-life of tPA by 16 and 21 folds compared with free tPA, respectively. Therefore, the use of liposomes could prolong the circulation lifetimes and longevity effect of liposomes on tPA was increased by PEG.
Keywords: Tissue plasminogen activator; Half-life; Prolong; Liposomes; PEG; Fibrinolysis;

Amphiphilic multi-arm-block copolymer conjugated with doxorubicin via pH-sensitive hydrazone bond for tumor-targeted drug delivery by Mani Prabaharan; Jamison J. Grailer; Srikanth Pilla; Douglas A. Steeber; Shaoqin Gong (5757-5766).
Folate-conjugated unimolecular micelles based on amphiphilic hyperbranched block copolymer, Boltorn® H40-poly(l-aspartate-doxorubicin)-b-poly(ethylene glycol)/FA-conjugated poly(ethylene glycol) (H40-P(LA-DOX)-b-PEG-OH/FA), were synthesized as a carrier for tumor-targeted drug delivery. The anticancer drug DOX was covalently conjugated onto the hydrophobic segments of the amphiphilic block copolymer arms by pH-sensitive hydrazone linkage. The size of the unimolecular micelles was determined as ∼17–36 and 10–20 nm by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The release profiles of the DOX from the H40-P(LA-DOX)-b-PEG-OH/FA micelles showed a strong dependence on the environmental pH values. The DOX release rate increased in the acidic medium due to the acid-cleavable hydrazone linkage between the DOX and micelles. Cellular uptake of the H40-P(LA-DOX)-b-PEG-OH/FA micelles was found to be higher than that of the H40-P(LA-DOX)-b-PEG-OH micelles because of the folate-receptor-mediated endocytosis, thereby providing higher cytotoxicity against the 4T1 mouse mammary carcinoma cell line. Degradation studies showed that the H40-P(LA-DOX)-b-PEG-OH/FA copolymer hydrolytically degraded into polymer fragments within six weeks. These results suggest that H40-P(LA-DOX)-b-PEG-OH/FA micelles could be a promising nanocarrier with excellent in vivo stability for targeting the drugs to cancer cells and releasing the drug molecules inside the cells by sensing the acidic environment of the endosomal compartments.
Keywords: Drug delivery; pH-sensitive; Unimolecular micelles; Tumor-targeted; Cellular uptake; Cytotoxicity;

Polyethylenimine coating to produce serum-resistant baculoviral vectors for in vivo gene delivery by Yi Yang; Seong-Loong Lo; Jingye Yang; Jing Yang; Sally S.L. Goh; Chunxiao Wu; Si-Shen Feng; Shu Wang (5767-5774).
Recombinant baculoviral vectors efficiently transduce many types of mammalian cells. However, their in vivo applications are hampered by the sensitivity of the virus to complement-mediated inactivation. Based on our observation that the surface charge of baculovirus is negative at neutral pH, we developed a procedure to coat baculoviral vectors with positively charged polyethylenimine 25 kDa, a commonly tested non-viral gene delivery vector, through electrostatic interaction. This coating was effective in protecting baculoviral vectors against human and rat serum-mediated inactivation in vitro, providing transduction efficiencies comparable with that generated by the control virus used under a serum-free condition. Enhanced in vivo gene expression in the liver and spleen was observed after tail vein injection of the coated viruses into mice. When injected directly into human tumor xenografts in nude mice, the coated viruses suppressed tumor development more effectively than uncoated viral vectors. These findings demonstrated the usefulness of using a simple coating method to circumvent a major obstacle to in vivo application of baculoviral vectors. The method may also serve as a flexible platform technology for improved use of the vectors, for example introducing a targeting ligand and minimizing immune responses.
Keywords: Gene therapy; Gene transfer; Nanoparticle; Surface modification;

Bioengineered silk protein-based gene delivery systems by Keiji Numata; Balajikarthick Subramanian; Heather A. Currie; David L. Kaplan (5775-5784).
Silk proteins self-assemble into mechanically robust material structures that are also biodegradable and non-cytotoxic, suggesting utility for gene delivery. Since silk proteins can also be tailored in terms of chemistry, molecular weight and other design features via genetic engineering, further control of this system for gene delivery can be considered. In the present study, silk-based block copolymers were bioengineered with poly(l-lysine) domains for gene delivery. Ionic complexes of these silk-polylysine based block copolymers with plasmid DNA (pDNA) were prepared for gene delivery to human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, atomic force microscopy, and dynamic light scattering. The polymers self-assembled in solution and complexed plasmid DNA through ionic interactions. The pDNA complexes with 30 lysine residues prepared at a polymer/nucleotide ratio of 10 and with a solution diameter of 380 nm showed the highest efficiency for transfection. The pDNA complexes were also immobilized on silk films and demonstrated direct cell transfection from these surfaces. The results demonstrate the potential of bioengineered silk proteins as a new family of highly tailored gene delivery systems.
Keywords: Gene therapy; DNA; Nanoparticle; Silk; Bioengineering;

The use of reactive polymer coatings to facilitate gene delivery from poly (ɛ-caprolactone) scaffolds by Wei-Wen Hu; Yaseen Elkasabi; Hsien-Yeh Chen; Ying Zhang; Joerg Lahann; Scott J. Hollister; Paul H. Krebsbach (5785-5792).
To functionalize biomaterials for bioconjugation, a chemical vapor deposition (CVD) polymerization technique was utilized to modify material surfaces. Poly [(4-amino-p-xylylene)-co-(p-xylylene)] (PPX–NH2) was deposited on inert polycaprolactone (PCL) surfaces to provide a reactive amine layer on the substrate surfaces. The biocompatibility of PPX–NH2 was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and lactate dehydrogenase (LDH) assays. The results demonstrated that cells continuously proliferated on CVD treated PCL surfaces with high survival rates. Biotin was conjugated on modified PCL surfaces to immobilize avidin for binding of biotinylated adenovirus. Scanning electron microscopy (SEM) examination illustrated that adenoviruses were evenly bound on both 2-D films and 3-D scaffolds, suggesting CVD was capable of modifying various substrates with different geometries. Using a wax masking technique, the biotin conjugation was controlled to immobilize avidin on specific sites. Due to the virus binding specificity on CVD-modified surfaces, cell transduction was restricted to the pattern of immobilized virus on biomaterials, by which transduced and non-transduced cells were controlled in different regions with a distinct interface. Because CVD was functional in different hierarchies, this surface modification should be able to custom-tailor bioconjugation for different applications.
Keywords: Gene transfer; Surface modification; Scaffold; Adenovirus; Polycaprolactone; Interface;

The gene transfection efficiency of a folate–PEI600–cyclodextrin nanopolymer by Hong Yao; Samuel S. Ng; Wesley O. Tucker; Yuk-Kai-Tiu Tsang; Kwan Man; Xiao-mei Wang; Billy K.C. Chow; Hsiang-Fu Kung; Gu-Ping Tang; Marie C. Lin (5793-5803).
The success of gene therapy relies on a safe and effective gene delivery system. In this communication, we describe the use of folate grafted PEI600–CyD (H1) as an effective polyplex-forming plasmid delivery agent with low toxicity. The structures of the polymer and polyplex were characterized, and the in vitro transfection efficiency, cytotoxicity, and in vivo transfection of H1 were examined. We found that folate molecules were successfully grafted to PEI600–CyD. At N/P ratios between 5 and 30, the resulting H1/DNA polyplexes had diameters less than 120 nm and zeta potentials less than 10 mV. In various tumor cell lines examined (U138, U87, B16, and Lovo), the in vitro transfection efficiency of H1 was more than 50%, which could be improved by the presence of fetal bovine serum or albumin. The cytotoxicity of H1 was significantly less than high molecular weight PEI-25 kDa. Importantly, in vivo optical imaging showed that the efficiency of H1-mediated transfection (50 μg luciferase plasmid (pLuc), N/P ratio = 20/1) was comparable to that of adenovirus-mediated luciferase transduction (1 × 109  pfu) in melanoma-bearing mice, and it did not induce any toxicity in the tumor tissue. These results clearly show that H1 is a safe and effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA and its application warrants further investigation.
Keywords: Polyethylenimine; β-Cyclodextrin; Folate; Gene therapy; Biodegradation; In vivo test;

A family of bioreducible poly(disulfide amine)s for gene delivery by Mei Ou; Rongzuo Xu; Sun Hwa Kim; David A. Bull; Sung Wan Kim (5804-5814).
A family of bioreducible poly(disulfide amine)s, which differ in the length of polymethylene spacer [–(CH2) n –] in the main chain and the side chain, has been synthesized. These bioreducible poly(disulfide amine)s exhibit local environment specific degradability and are associated with lower cytotoxicity than branched poly(ethylenimine) (bPEI, 25 kDa). These cationic polymers also show higher buffering capacity and protonation degree than bPEI, facilitating the endosomal escape of carried genetic materials. The transfection efficiency of these agents is oligomethylene length dependent. Poly(cystaminebisacrylamide-spermine) [poly(CBA-SP)], poly(cystaminebisacrylamide-bis(3-aminopropyl)-1,3-propanediamine) [poly(CBA-APPD)], and poly(cyxtaminebisacrylamide-bis(3-aminopropyl)-ethylenediamine) [ploy(CBA-APED)] with longer propylene [–(CH2)3–] side spacer, demonstrate higher transfection efficacy than the counterpart poly(cystaminebisacrylamide-bis(2-aminoethyl)-1,3-propanediamine) [poly(CBA-AEPD)] and poly(cystaminebisacrylamide-triethylenetetramine) [poly(CBA-TETA)], which have shorter ethylene [–(CH2)2–] side spacer. The poly(CBA-SP), poly(CBA-APPD), poly(CBA-APED) with the main chain spacer of –(CH2)4–, –(CH2)3–, –(CH2)2– demonstrate similar transfection efficiency, indicating the length of polymer main chain spacer has less influence on transfection efficiency. However, with the same short ethylene [–(CH2)2–] side spacer, poly(CBA-AEPD), with the longer main chain oligomethylene units [–(CH2)3–], showed relatively higher transfection efficiency than poly(CBA-TETA), having shorter main chain oligomethylene units [–(CH2)2–]. Of these polymeric carriers, poly(CBA-SP) demonstrated the highest transfection in the C2C12 cell line, while poly(CBA-APED) showed the highest transfection in the HeLa cell line. All of these agents showed greater transfection activity than commercialized bPEI 25 kDa. The poly(disulfide amine)s are promising safe and efficient non-viral vectors for gene delivery.
Keywords: Poly(disulfide amine)s; Biodegradable; Gene delivery; Gene transfection;

Amine-modified hyperbranched polyesters as non-toxic, biodegradable gene delivery systems by Regina Reul; Juliane Nguyen; Thomas Kissel (5815-5824).
For chronic non-viral gene therapy, biodegradable carriers with low cytotoxicity are essential. To create a series of non-toxic and biodegradable gene carriers, hyperbranched polymers based on 2,2-bis-(methylol)propionic acid (bis-MPA); (Boltorn H®) were modified by introducing tertiary amines. The terminal OH groups were modified with diethylaminopropylamine (DEAPA) by carbonyldiimidazole (CDI) chemistry. The resulting polymers were characterized by 1H, 13C NMR, IR and GPC. Degradability and degradation rate were investigated with respect to the degree of amine substitution. The toxicity of all hyperbranched polyesters was generally very low compared to polyethyleneimine (PEI). Measurements of size and zeta potential showed that small nano-complexes with a positive zeta potential were formed. Dependency of the degree of amine substitution on interaction with DNA was studied by agarose gel retardation assay and ethidium bromide exclusion assay. Influence of the amine substitution on transfection efficiency of the different polymers demonstrated that a certain amine substitution degree was required to achieve transfection efficiency. These carriers provide degradability, very low toxicity and the ability to transfect cells which can be influenced by the degree of amine substitution.
Keywords: Hyperbranched polymer; Polyester; Biodegradability; Cytotoxicity; DNA interaction;

Poly(amidoamine)s with pendant primary amine (polymer 1a1c) were evaluated as in vitro non-viral gene delivery vectors for bone marrow stromal cells (BMSCs). The cytotoxicity of these poly(amidoamine)s, measured by MTT assay, increased with increasing length of side chain, however, they were less toxic than branched polyethylenimine (PEI) 25 kDa. Using pGL-3 and pEGFP-C1 as luciferase gene and green fluorescent protein (GFP) gene, among all polycations including polymer 1a1c and PEI, polymer 1b at optimal N/P ratio showed highest luciferase expression (1.92 × 108 RLU/mg protein) as well as percentage of cells expressing GFP (29.01 ± 2.33%). For all polycations, intracellular trafficking of Cy3-labelled plasmid DNA (pDNA) was similar. Fluorescent particles attached to cell membrane at 0.5 h after adding the polycation/DNA complexes, aggregated in cytoplasm after 2 h, and then stayed around the perinuclear region after 4 h. pDNA nuclear localization appeared at 4 h post-transfection, but much more pDNA entered into nucleus at 24 h. At high N/P ratio, polymer 1a1c could deliver pDNA into 70–80% of BMSCs after 24 h transfection, however, labelled pDNA was observed in only 4–25% of cells at the same time. Compared to PEI, polymer 1b showed comparable or even higher percentage of pDNA uptake and nuclear localization. We concluded that poly(amidoamine)s with pendant primary amine, especially polymer 1b, are new kind of promising candidates of less toxic and highly efficient non-viral gene delivery vectors for BMSCs.
Keywords: Bone marrow stromal cell; Gene delivery; Poly(amidoamine); Polyethylenimine; Intracellular trafficking; Nuclear localization;

For a non-viral gene delivery system to be clinically effective, it should be non-toxic, compatible with biological components, and highly efficient in gene transfection. With this goal in mind, we investigated the gene delivery efficiency of a ternary complex consisting of DNA, an intracellularly degradable polycation, and sodium hyaluronate (DPH complex). Here, we report that the DPH ternary complex achieved significantly higher transfection efficiency than other polymer systems, especially in the presence of serum. The high transfection efficiency and serum tolerance of DPH are attributed to a unique interplay between CLPEI and HA, which leads to (i) the improved stability of DNA in the extracellular environment and at the early stage of intracellular trafficking and (ii) timely dissociation of the DNA–polymer complex. This study reinforces findings of earlier studies that emphasized each step as a bottleneck for efficient gene delivery; yet, it is the first to show that it is possible to overcome these obstacles simultaneously by taking advantage of two distinctive approaches.
Keywords: Non-viral gene delivery; Crosslinked polyethyleneimine; Sodium hyaluronate; Extra- and intracellular stabilities; DNA unpacking;

The suppression of lung tumorigenesis by aerosol-delivered folate–chitosan-graft-polyethylenimine/Akt1 shRNA complexes through the Akt signaling pathway by Hu-Lin Jiang; Cheng-Xiong Xu; You-Kyoung Kim; Rohidas Arote; Dhananjay Jere; Hwang-Tae Lim; Myung-Haing Cho; Chong-Su Cho (5844-5852).
RNA interference (RNAi) represents a promising new approach to the inhibition of gene expression in vitro and in vivo, and has therapeutic potential for human diseases. Efficient delivery of small interfering RNA (siRNA) or small hairpin RNA (shRNA) is a critical concern in RNAi studies. Here we report the development of a new polymeric gene carrier for cancer cell-targeting, designed to enhance the intracellular delivery of shRNA and reduce cytotoxicity. Folate–chitosan-graft-polyethylenimine (FC-g-PEI) copolymer was prepared by an imine reaction between periodate-oxidized folate–chitosan (FC) and low molecular weight polyethylenimine (PEI). FC-g-PEI copolymer was investigated as a potential cancer cell-targeting gene carrier. The composition of FC-g-PEI was characterized using 1H nuclear magnetic resonance (1H NMR), and particle size and zeta potential of FC-g-PEI/shRNA complexes were measured using dynamic light scattering (DLS). FC-g-PEI showed good shRNA condensation ability and high protection of shRNA from nuclease attack. It also exhibited lower cytotoxicity compared to PEI 25K control, and showed good cancer cell-targeting ability. Furthermore, aerosol delivery of FC-g-PEI/Akt1 shRNA complexes suppressed lung tumorigenesis in a urethane-induced lung cancer model mouse through the Akt signaling pathway. Together, these results suggest that FC-g-PEI may be useful for shRNA-based gene therapy.
Keywords: Gene therapy; Non-viral vector; Aerosol delivery; Folate–chitosan-graft-polyethylenimine; Akt1 shRNA; Lung tumorigenesis;

Microsphere-based tracing and molecular delivery in embryonic stem cells by Anestis Tsakiridis; Lois M. Alexander; Nicole Gennet; Rosario M. Sanchez-Martin; Alessandra Livigni; Meng Li; Mark Bradley; Joshua M. Brickman (5853-5861).
Embryonic stem (ES) cells are in vitro cell lines that can differentiate into all lineages of the fetus and the adult. Despite the versatility of genetic manipulation in murine ES cells, these approaches are time-consuming and rely on inefficient transient cellular delivery systems that can only be applied to undifferentiated ES cell cultures. Here we describe a polystyrene microsphere-based system designed to efficiently deliver biological materials into both undifferentiated and differentiating ES cells. Our results demonstrate that these microspheres can be successfully employed for simultaneous cellular labeling and controlled transfer of various cargos such as fluorophores, proteins and nucleic acids into ES cells without any significant toxicity or loss of pluripotency. This versatile delivery system is also effective in other stem cell lines derived from early embryos, trophoblast and neural stem cells.
Keywords: Embryonic stem cell; Differentiation; Transfection; Delivery; Microspheres;

Mucoadhesive liposomes for intranasal immunization with an avian influenza virus vaccine in chickens by Chwei-Jang Chiou; Li-Ping Tseng; Ming-Chung Deng; Pei-Rong Jiang; Shang-Li Tasi; Tze-Wen Chung; Yi-You Huang; Der-Zen Liu (5862-5868).
The aim of this study was to characterize a nasally delivered bioadhesive liposome using an inactivated H5N3 virus as a model antigen. Bioadhesive liposomes were developed using tremella (T) or xanthan gum (XG) as the bioadhesive polysaccharide. Using chickens as the target animal, we evaluated whether delivery of a bioadhesive liposomal influenza vaccine via a mucosal site of infection could improve vaccine effectiveness. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cytotoxicity assays demonstrated that T, XG and liposomes were non toxic to chicken spleen macrophages. Enzyme-linked immunosorbent assay (ELISA) was used to determine the adjuvant effect of the bioadhesive liposomal-vaccines. Chickens immunized with a low dose (200 μL) of bioadhesive liposomal influenza vaccine had significantly higher mucosal and serum antibody levels (P  < 0.05). In addition, liposomes mixed with a low-viscosity bioadhesive gel used for nasal delivery resulted in superior antibody responses compared with liposomes mixed with a high-viscosity gel (P  < 0.05). This suggest that a low-viscosity gel mixed with liposomes is more suitable for nasal delivery, and that chickens elicit higher mucosal secretory immunoglobulin A (s-IgA) and serum IgG after two vaccinations.
Keywords: Liposome; Adjuvant; Mucoadhesive; Intranasal; Newcastle disease virus; s-IgA;

The use of a mutant TNF-α as a vaccine adjuvant for the induction of mucosal immune responses by Hiroyuki Kayamuro; Yasuhiro Abe; Yasuo Yoshioka; Kazufumi Katayama; Tetsuya Nomura; Tokuyuki Yoshida; Kohei Yamashita; Tomoaki Yoshikawa; Yuichi Kawai; Tadanori Mayumi; Takachika Hiroi; Norio Itoh; Kazuya Nagano; Haruhiko Kamada; Shin-ichi Tsunoda; Yasuo Tsutsumi (5869-5876).
Safe and potent adjuvants are required in order to establish effective mucosal vaccines. Cytokines are promising adjuvants because they are human-derived safe biomaterial and display immune-modulating functions. We have created a mutant tumor necrosis factor-α (TNF-α), mTNF-K90R, that exhibits high bioactivity and resistance to proteases. Here, we examined the potential of mTNF-K90R as a mucosal adjuvant. Initially, we showed that intranasal co-administration of mTNF-K90R with ovalbumin (OVA) potently produced OVA-specific Immunoglobulin (Ig) G antibodies (Abs) in serum and IgA Abs both at local and distal mucosal sites compared to co-administration with wild-type TNF-α. The OVA-specific immune response was characterized by high levels of serum IgG1 and increased production of interleukin-4 (IL-4), IL-5 and IL-10 from splenocytes of immunized mice, suggesting a Th2 response. Furthermore, intranasal immunization with an antigen from influenza virus plus mTNF-K90R exhibited mucosal adjuvant activity for induction of both systemic and mucosal immune responses. Importantly, histopathological examination of the nasal tissue of mTNF-K90R treated mice detected no signs of toxicity. These findings suggest that mTNF-K90R is safe and effective mucosal adjuvant and this system may have potential application as a universal mucosal adjuvant system for mucosal vaccines improving the immune response to a variety of viral antigens.
Keywords: Bioactivity; Cytokine; Mucosa; Immunomodulation;

Mixed-mode fracture of human cortical bone by Elizabeth A. Zimmermann; Maximilien E. Launey; Holly D. Barth; Robert O. Ritchie (5877-5884).
Although the mode I (tensile opening) fracture toughness has been the focus of most fracture mechanics studies of human cortical bone, bones in vivo are invariably loaded multiaxially. Consequently, an understanding of mixed-mode fracture is necessary to determine whether a mode I fracture toughness test provides the appropriate information to accurately quantify fracture risk. In this study, we examine the mixed-mode fracture of human cortical bone by characterizing the crack-initiation fracture toughness in the transverse (breaking) orientation under combined mode I (tensile opening) plus mode II (shear) loading using samples loaded in symmetric and asymmetric four-point bending. Whereas in most structural materials, the fracture toughness is increased with increasing mode-mixity (i.e., where the shear loading component gets larger), in the transverse orientation of bone the situation is quite different. Indeed, the competition between the maximum applied mechanical mixed-mode driving force and the weakest microstructural paths in bone results in a behavior that is distinctly different to most homogeneous brittle materials. Specifically, in this orientation, the fracture toughness of bone is markedly decreased with increasing mode-mixity.
Keywords: Human cortical bone; Mixed-mode fracture; Fracture toughness; Fracture mechanisms;

Acute changes in lung capillary permeability continue to complicate procedures such as cardiopulmonary bypass, solid organ transplant, and major vascular surgery and precipitate the more severe disease state Adult Respiratory Distress Syndrome (ARDS). To date there is no treatment targeted directly to the lung microvasculature. We hypothesized that biomimetic polymers could be used to enhance passive barrier function by reducing the porosity of the endothelial glycocalyx and attenuate mechanotransduction by restricting the motion of the glycoproteins implicated in signal transduction. To this end, cationic copolymers containing methacrylamidopropyl trimethylammonium chloride (P-TMA Cl) have been developed as an infusible therapy to target the lung capillary glycocalyx in order to mechanically enhance the capillary barrier and turn off pressure-induced mechanotransduction. Copolymers were tested for functional efficacy by measuring both albumin permeability (P DA) and hydraulic conductivity (L p) across cultured endothelial monolayers. P-TMA Cl significantly decreased P DA in normal and inflamed cells and attenuated pressure-induced increases in L p. Decreases in L p across endothelial monolayers in the presence of P-TMA Cl is evidence of a dampening of mechanotransduction-induced barrier dysfunction. We show the potential for biomimetic polymers targeted to lung endothelium as a viable therapy to enhance endothelial barrier function thereby attenuating a major component of vascular inflammation.
Keywords: Endothelial cell; Vascular inflammation; Mechanotransduction; Hydraulic conductivity;

Ultra-low fouling peptide surfaces derived from natural amino acids by Shengfu Chen; Zhiqiang Cao; Shaoyi Jiang (5892-5896).
This work demonstrated the ultra-low fouling natural peptides composed of certain negatively and positively charged residues such as glutamic acid (E) or aspartic acid (D) and lysine (K), in the form of either alternating or randomly mixed charge. These peptide-based materials are major candidates as biodegradable nonfouling materials since their final metabolized products are natural amino acids. Although hydrophilic materials can generally reduce nonspecific binding to a certain extent, it is very challenging to achieve ultra-low fouling, which is critical for many biomedical applications, such as medical implants, drug delivery carriers, and biosensors. Based on the design principle of uniformly mixed charges and the selection of appropriate amino acid residues, the natural peptides developed exhibit high resistance to nonspecific protein adsorption (<0.3 ng/cm2 adsorbed proteins) comparable to what is achieved by poly(ethylene glycol) (PEG)-based materials. Mixed charged groups, when uniformly distributed at the molecular level, can achieve ultra-low fouling properties similar to zwitterionic groups due to their strong hydration ability.
Keywords: Amino acids; Mixed charge; Peptides; Ultra-low fouling;