Biomaterials (v.31, #15)

Tailoring the degradation kinetics of poly(ester carbonate urethane)urea thermoplastic elastomers for tissue engineering scaffolds by Yi Hong; Jianjun Guan; Kazuro L. Fujimoto; Ryotaro Hashizume; Anca L. Pelinescu; William R. Wagner (4249-4258).
Biodegradable elastomeric scaffolds are of increasing interest for applications in soft tissue repair and regeneration, particularly in mechanically active settings. The rate at which such a scaffold should degrade for optimal outcomes, however, is not generally known and the ability to select from similar scaffolds that vary in degradation behavior to allow such optimization is limited. Our objective was to synthesize a family of biodegradable polyurethane elastomers where partial substitution of polyester segments with polycarbonate segments in the polymer backbone would lead to slower degradation behavior. Specifically, we synthesized poly(ester carbonate)urethane ureas (PECUUs) using a blended soft segment of poly(caprolactone) (PCL) and poly(1,6-hexamethylene carbonate) (PHC), a 1,4-diisocyanatobutane hard segment and chain extension with putrescine. Soft segment PCL/PHC molar ratios of 100/0, 75/25, 50/50, 25/75, and 0/100 were investigated. Polymer tensile strengths varied from 14 to 34 MPa with breaking strains of 660–875%, initial moduli of 8–24 MPa and 100% recovery after 10% strain. Increased PHC content was associated with softer, more distensible films. Scaffolds produced by salt leaching supported smooth muscle cell adhesion and growth in vitro. PECUU in aqueous buffer in vitro and subcutaneous implants in rats of PECUU scaffolds showed degradation slower than comparable poly(ester urethane)urea and faster than poly(carbonate urethane)urea. These slower degrading thermoplastic polyurethanes provide opportunities to investigate the role of relative degradation rates for mechanically supportive scaffolds in a variety of soft tissue repair and reconstructive procedures.
Keywords: Elastomer; Polyurethane; Polycaprolactone; Polycarbonate; Biodegradation; Scaffold;

Luminescence variations in hydroxyapatites doped with Eu2+ and Eu3+ ions by Olivia A. Graeve; Raghunath Kanakala; Abhiram Madadi; Brandon C. Williams; Katelyn C. Glass (4259-4267).
We present a detailed analysis of the luminescence behavior of europium-doped hydroxyapatite (HAp) and calcium-deficient hydroxyapatite (Ca-D HAp) nanopowders. The results show that, while both powders are similar in crystallite size, particle size, and morphology, the luminescence behavior differs significantly. For the HAp:Eu powders, the emission is clearly from Eu3+ ions and corresponds to typical 5D0 → 7FJ emissions, whereas for the Ca-D HAp:Eu powders, we also see a broad emission with two peaks at 420 and 445 nm, corresponding to the 4f65d1 → 4f7 (8S7/2) transition of Eu2+. The powders are weakly luminescent in the as-synthesized state, as expected for combustion-synthesized materials and have higher emission intensities as the heat treatment temperature is increased. Luminescence spectra obtained using an excitation wavelength of 254 nm are weak for all samples. Excitation wavelengths of 305, 337, and 359 nm, are better at promoting the Eu3+ and Eu2+ emissions in hydroxyapatites. We propose that fluorescence measurements are an excellent way of qualitatively determining the phase composition of europium-doped hydroxyapatite powders, since powders that exhibit a blue emission contain substantial amounts of Ca-D HAp, allowing the determination of the presence of this phase in mixed-phase hydroxyapatites.
Keywords: Fluorescence; Hydroxyapatite nanoparticles; Particle size distribution; Dynamic light scattering; Charge compensation; Combustion synthesis;

Structure-biocompatibility relationship of dendritic polyglycerol derivatives by Jayant Khandare; Andreas Mohr; Marcelo Calderón; Pia Welker; Kai Licha; Rainer Haag (4268-4277).
Nanocarriers possess advanced physicochemical properties that improve bioavailability, enhance cellular dynamics, and control targetability in drug delivery. In particular, dendritic polyglycerol is a promising new biocompatible scaffold for drug delivery. The present explores the structure-biocompatibility relationship of dendritic polyglycerol (dPG) derivatives possessing neutral, cationic, and anionic charges. The effect of solution pH on the surface charge was studied in buffered aqueous solution between pH 4.8 and 7.4. Surface charge properties of dPG derivatives are discussed in terms of surface functionalities and compared with amine and hydroxyl terminated polyamidoamine (PAMAM) dendrimers. Zeta potential measurements and fluorescence quenching studies address the binding interactions of dPGs to bovine serum albumin in order to explore the applicability of dPG derivatives for systemic delivery. Cellular entry of dPG-dye conjugate was evaluated using A549 lung epithelial cells, while in vitro toxicity was studied for various dPGs and compared to PAMAM dendrimers, polyethyleneimine (PEI), dextran, and linear polyethylene glycol (PEG) using human hematopoietic cell line U-937. Cellular uptake studies of dye labelled dPGs inferred that the charged derivatives (dPG-sulfate and dPG-amine) are more rapidly internalized primarily inside the cytosol of A549 cells compared to the neutral dPG. The cell compatibility results show that the dendritic polyglycerols are as safe as linear PEG polymer or dextran, which indicates the suitability of dPG derivatives in delivering therapeutic agents systemically.
Keywords: Dendritic polyglycerols; Delivery carrier systems; Zeta potential; Bovine serum albumin binding; Cell biocompatibility and cytotoxicity;

PHBV microspheres – PLGA matrix composite scaffold for bone tissue engineering by Wei Huang; Xuetao Shi; Li Ren; Chang Du; Yingjun Wang (4278-4285).
Polymer scaffolds, particularly in the form of microspheres, have been employed to support cells growth and deliver drugs or growth factors in tissue engineering. In this study, we have established a scaffold by embedding poly (β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV) microspheres into poly (l-lactic-co-glycolic acid) (PLGA) matrix, according to their different solubility in acetone, with the aim of repairing bone defects. PLGA/PHBV scaffolds had good pore parameters, for example, the porosity of PLGA/30% PHBV scaffold can reach to 81.273 ± 2.192%. Besides, the pore size distribution of the model was evaluated and the results revealed that the pore size mainly distributed between 50 μm and 200 μm. With increasing the amount of PHBV microspheres, the compressive strength of the PLGA/PHBV scaffold enhanced. The morphology of the hybrid scaffold was rougher than that of pure PLGA scaffold, which had no significant effect on the cell behavior. The in vitro evaluation suggested that the model is suitable as a scaffold for engineering bone tissue, and has the potential for further applications in drug delivery system.
Keywords: PLGA; PHBV; Scaffold; Bone repair; Microsphere; Tissue engineering;

The effect of micronscale anisotropic cross patterns on fibroblast migration by Hojeong Jeon; Hirofumi Hidai; David J. Hwang; Kevin E. Healy; Costas P. Grigoropoulos (4286-4295).
Cell movement on adhesive surfaces is a complicated process based on myriad cell–surface interactions. Although both micron and nanoscale surface topography have been known to be important in understanding cell–materials interactions, typically only simple patterns (e.g., parallel lines or aligned posts) have been used in studying cell morphology, migration, and behavior. This restriction has limited the understanding of the multidirectional aspects of cell–surface response. The present study was performed to investigate cell morphology and motility on micronscale anisotropic cross patterns and parallel line patterns having different aspect ratios (1:2, 1:4, and 1:∞), grid size (12-, 16-, and 24-μm distance neighboring longer side ridges), and height of ridges (3- and 10-μm). The movement characteristics were analyzed quantitatively with respect to cell migration speed, migration angle, persistence time (P) and motility coefficient (μ). A significant effect of the 1:4 grid aspect ratio cross patterns and parallel line patterns on cell alignment and directionality of migration was observed. Cell motility was also dependent on the patterned surface topography: the migration speed was significantly enhanced by the 1:2 and 1:4 cross patterns when the grid size was smaller than the size of individual cells (i.e., ∼16 μm). In addition, the migration speed of cells on lower patterns was greater than on higher ridges. Overall, cell morphology and motility was influenced by the aspect ratio of the cross pattern, the grid size, and the height of ridges.
Keywords: Cell migration; Contact guidance; Haptotaxis; Micropatterning; Laser manufacturing; Two-photon initiated polymerization;

Controlled-size embryoid body formation in concave microwell arrays by Yoon Young Choi; Bong Geun Chung; Dae Ho Lee; Ali Khademhosseini; Jong-Hoon Kim; Sang-Hoon Lee (4296-4303).
Embryonic stem (ES) cells hold great potential as a renewable cell source for regenerative medicine and cell-based therapy. Despite the potential of ES cells, conventional stem cell culture methods do not enable the control of the microenvironment. A number of microscale engineering approaches have been recently developed to control the extracellular microenvironment and to direct embryonic stem cell fate. Here, we used engineered concave microwell arrays to regulate the size and shape of embryoid bodies (EBs)—cell aggregate intermediates derived from ES cells. Murine ES cells were aggregated within concave microwells, and their aggregate sizes were controlled by varying the microwell widths (200, 500, and 1000 μm). Differentiation of murine ES cells into three germ layers was assessed by analyzing gene expression. We found that ES cell-derived cardiogenesis and neurogenesis were strongly regulated by the EB size, showing that larger concave microwell arrays induced more neuronal and cardiomyocyte differentiation than did smaller microwell arrays. Therefore, this engineered concave microwell array could be a potentially useful tool for controlling ES cell behavior.
Keywords: Concave microwell array; Embryonic stem cell differentiation; Neurogenesis; Cardiogenesis;

The goal of this study was to determine material effects on cartilage regeneration for scaffolds with the same controlled architecture. The 3D polycaprolactone (PCL), poly (glycerol sebacate) (PGS), and poly (1,8 octanediol-co-citrate) (POC) scaffolds of the same design were physically characterized and tissue regeneration in terms of cell phenotype, cellular proliferation and differentiation, and matrix production were compared to find which material would be most optimal for cartilage regeneration in vitro. POC provided the best support for cartilage regeneration in terms of tissue ingrowth, matrix production, and relative mRNA expressions for chondrocyte differentiation (Col2/Col1). PGS was seen as the least favorable material for cartilage based on its relatively high de-differentiation (Col1), hypertrophic mRNA expression (Col10) and high matrix degradation (MMP13, MMP3) results. PCL still provided microenvironments suitable for cells to be active yet it seemed to cause de-differentiation (Col1) of chondrocytes inside the scaffold while many cells migrated out, growing cartilage outside the scaffold.
Keywords: Poly (1,8 Octanediol-co-Citrate) (POC); Poly (glycerol sebacate) (PGS); Polycaprolactone (PCL); Chondrogenesis; Controlled scaffold tissue engineering;

Bilayered scaffold for engineering cellularized blood vessels by Young Min Ju; Jin San Choi; Anthony Atala; James J. Yoo; Sang Jin Lee (4313-4321).
Vascular scaffolds fabricated by electrospinning poly(ε-caprolactone) (PCL) and collagen have been designed to provide adequate structural support as well as a favorable adhesion substrate for vascular cells. However, the presence of small-sized pores limits the efficacy of smooth muscle cells (SMC) seeding, as these cells could not adequately infiltrate into the scaffolds. To overcome this challenge, we developed a bilayered scaffolding system that provides different pore sizes to facilitate adequate cellular interactions. Based on the fact that pore size increases with the increase in fiber diameter, four different fiber diameters (ranging 0.27–4.45 μm) were fabricated by electrospinning with controlled parameters. The fabricated scaffolds were examined by evaluating cellular interactions, and the mechanical properties were measured. Endothelial cells (EC) seeded on nanoscaled fibers showed enhanced cellular orientation and focal adhesion. Conversely, fabrication of a larger fiber diameter improved SMC infiltration into the scaffolds. To incorporate both of these properties into a scaffold, bilayered vascular scaffolds were produced. The inner layer yielded small diameter fibers and the outer layer provided large diameter fibers. We show that the bilayered scaffolds permit EC adhesion on the lumen and SMC infiltration into the outer layer. This study suggests that the use of bilayered scaffolds may lead to improved vessel formation.
Keywords: Electrospinning; Polycaprolactone; Collagen; Vascular grafts; Endothelial cell; Smooth muscle cell;

To form tissues with uniform cell distribution and extracellular matrix arrangement is of great relevance to obtain the desirable function and maintain structural integrity. Scaffold configuration is believed to play a critical role in regulating cell spatial distribution and consequently tissue formation. In this study, three types of poly(ethyleneglycol-terephthalate)–poly (butylenes terephthalate) (PEGT/PBT) scaffolds [compression molded scaffold (CM), compression molded scaffold after chloroform/isopropanol reticulation (CMR), 3D rapid prototyped fibrous scaffold (RP)] with various configurations were used to support the tissue formation of adipose stromal cells for up to 21 days. Characterization of the scaffolds with μCT revealed that RP scaffolds were composed of repeating structural units with well controlled interconnected pores, in contrast to the irregular pore morphology in CM or CMR. Cell seeding efficacy onto various scaffolds was comparable (from 67 ± 4% to 82 ± 3%), while only RP scaffold led to even cell attachment onto the inner fibers of the scaffolds. Continuous cell proliferation and deposition of new collagen and glycosaminoglycans (GAG) were measured for all three scaffolds, while with a significant amount measured in RP at 21 days. By 21 days, complete uniform tissue formation was only achieved in RP scaffolds under a dynamic cell culture in spinner flasks. The present study successfully demonstrates the feasibility of controlling uniform tissue formation at a microscale by manipulating the structural configuration of the scaffold.
Keywords: Uniform tissue formation; Tissue engineering; Scaffold with repeating units; Dynamic culture; Microenvironment;

The use of flow perfusion culture and subcutaneous implantation with fibroblast-seeded PLLA-collagen 3D scaffolds for abdominal wall repair by Fanrong Pu; Nicholas P. Rhodes; Yves Bayon; Rui Chen; Gerben Brans; Remco Benne; John A. Hunt (4330-4340).
Highly cellularised 3D-tissue constructs designed to repair large, complex abdominal wall defects were prepared using poly (lactic acid) (PLLA)-collagen scaffolds in vitro using a flow perfusion bioreactor. The PLLA-collagen scaffolds had a unique structure consisting of a collagen sponge formed within the pores of a mechanically stable knitted mesh of PLLA. The effect of the flow perfusion bioreactor culturing conditions was investigated in vitro for 0, 7, 14 and 28 days on scaffolds seeded with dermal fibroblasts. The cultured constructs were subsequently studied subcutaneously (SC) in an in vivo animal model. The results of in vitro studies demonstrated that the perfusion system facilitated increased cell proliferation and homogenous distribution in the PLLA-collagen scaffolds compared to static conditions. A highly cellularised 3D-tissue construct was formed by 7 days incubation under perfusion conditions, with increased cellularity by the 28 day time point. The in vivo model demonstrated that implanting constructs with high cellularity resulted in exceptional cell stabilisation, with the survival of implanted cells and expression of the phenotypically-relevant extracellular matrix proteins collagen types I and III, studied by fluorescence in situ hybridisation (FISH) and immunohistochemistry. The implantation of this porous PPLA-collagen scaffold seeded with dermal fibroblasts following in vitro maturation using a flow perfusion bioreactor system suggests a significant advance over current state-of-the-art procedures for the reconstruction of large, complex abdominal wall tissue defects.
Keywords: Large complex abdominal wall defect; Tissue engineering; Regenerative medicine; Flow perfusion bioreactor; Fibroblasts; PLLA;

The aim of this study was to develop a method for efficient production of folliculoid keratinocyte-dermal papilla (DP) microtissues to facilitate epithelial–mesenchymal interaction. The behavior of DP cells and adult keratinocytes from hairless skin on poly(ethylene-co-vinyl alcohol) (EVAL) surface was investigated. Keratinocytes, poorly adherent both to substrate and between homotypic cells, become suspended disperse cells after homotypic cell seeding. Seeded simultaneously, keratinocytes and DP cells are able to aggregate into spheroidal microtissues. Dynamical analysis shows that DP cells act as a carrier in the process due to the heterotypic intercellular adhesion. DP cells attach faster to EVAL and start to aggregate. Keratinocytes adhere to DP cells and are then carried by DP cells to form initial hybrid aggregates. Due to the high motility of DP cells, these hybrid aggregates move collectively as clusters and merge into larger spheroids which subsequently detach from the substratum and can be easily collected. Compared with random cell distribution in spheroids generated in hanging drops, these hybrid spheroids have a preferential compartmented core-shell structure: an aggregated DP cell core surrounded by a keratinocyte shell. In addition to ameliorated DP signature gene expression, keratinocytes show down-regulated epidermal terminal differentiation and enhanced follicular differentiation. Functionally, these microtissues are able to grow hairs in vivo. This work sheds light on the complex effects and dynamics of cell–cell and cell-substratum interaction in the patterning of heterotypic cells into tissue forms and is of potential to be applied to mass generation of other epithelial organ primordia in vitro.
Keywords: Hair follicle regeneration; Epithelial organ; Collective movement; Aggregation; Organ germ;

Phosphatidylserine immobilization of lentivirus for localized gene transfer by Seungjin Shin; Hannah M. Tuinstra; David M. Salvay; Lonnie D. Shea (4353-4359).
Localized and efficient gene transfer can be promoted by exploiting the interaction between the vector and biomaterial. Regulation of the vector–material interaction was investigated by capitalizing on the binding between lentivirus and phosphatidylserine (PS), a component of the plasma membrane. PS was incorporated into microspheres composed of the copolymers of lactide and glycolide (PLG) using an emulsion process. Increasing the weight ratio of PS to PLG led to a greater incorporation of PS. Lentivirus, but not adenovirus, associated with PS-PLG microspheres, and binding was specific to PS relative to PLG alone or PLG modified with phosphatidylcholine. Immobilized lentivirus produced large numbers of transduced cells, and increased transgene expression relative to virus alone. Microspheres were subsequently formed into porous tissue engineering scaffolds, with retention of lentivirus binding. Lentivirus immobilization resulted in long-term and localized expression within a subcutaneously implanted scaffold. Microspheres were also formed into multiple channel bridges for implantation into the spinal cord. Lentivirus delivery from the bridge produced maximal expression at the implant and a gradient of expression rostrally and caudally. This specific binding of lentiviral vectors to biomaterial scaffolds may provide a versatile tool for numerous applications in regenerative medicine or within model systems that investigate tissue development.
Keywords: Lentivirus; Phosphatidylserine; Poly(lactide-co-glycolide); Regenerative medicine;

Direct differentiation of human embryonic stem cells into selective neurons on nanoscale ridge/groove pattern arrays by Man Ryul Lee; Keon Woo Kwon; Hosup Jung; Hong Nam Kim; Kahp Y. Suh; Keesung Kim; Kye-Seong Kim (4360-4366).
Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to be used for tissue engineering and regenerative medicine. Repairing nerve injury by differentiating hESCs into a neuronal lineage is one important application of hESCs. Biochemical and biological agents are widely used to induce hESC differentiation. However, it would be better if we could induce differentiation of hESCs without such agents because these factors are expensive and it is difficult to control the optimal concentrations for efficient differentiation with reduced side effects. Moreover, the mechanism of differentiation induced by these factors is still not fully understood. In this study, we present evidence that nanoscale ridge/groove pattern arrays alone can effectively and rapidly induce the differentiation of hESCs into a neuronal lineage without the use any differentiation-inducing agents. Using UV-assisted capillary force lithography, we constructed nanoscale ridge/groove pattern arrays with a dimension and alignment that were finely controlled over a large area. Human embryonic stem cells seeded onto the 350-nm ridge/groove pattern arrays differentiated into neuronal lineage after five days, in the absence differentiation-inducing agents. This nanoscale technique could be used for a new neuronal differentiation protocol of hESCs and may also be useful for nanostructured scaffolding for nerve injury repair.
Keywords: Human embryonic stem cells; Capillary force lithography; Neuronal differentiation; Nanotopography;

The proliferation and differentiation of placental-derived multipotent cells into smooth muscle cells on fibrillar collagen by Mou-Tsy Chou; Sheng-Nan Chang; Chieh Ke; Hsin-I Chang; Mao-Lin Sung; Hsing-Chun Kuo; Cheng-Nan Chen (4367-4375).
Type I collagen constitutes a major portion of the extracellular matrix (ECM) in arterial wall and it is the major substrate for cell growth and differentiation. The goal of this study was to evaluate the differentiation and proliferation of placenta-derived multipotent cells (PDMCs) on polymerized type I collagen fibrils and monomer collagen. PDMCs grown on both polymerized collagen and monomer collagen with transforming growth factor (TGF)-β treatment increases the expression of smooth muscle cell (SMC)-specific markers, including calponin, α-smooth muscle actin (α-SMA) and smooth muscle-myosin heavy chain (SM-MHC). Polymerized collagen increased the expressions of p21CIP1 and p27KIP1; decreased cyclin A, cyclin D1, cyclin-dependent protein kinase 2 (Cdk2); and led to G0/G1 arrest in PDMCs. Furthermore, PDMC–differentiated SMCs exhibited significant collagen contractility in the presence or absence of endothelin-1 (ET-1) stimulation. By using specific inhibitors and small interfering RNA (siRNA), we demonstrated that p38 MAPK pathway and serum response factor (SRF)–DNA binding activity is critical for the polymerized collagen-induced PDMC differentiation into SMCs. Thus, polymerized collagen exhibits the great potential in inducing PDMCs differentiation into SMCs, and exerts anti-proliferative effect on PDMC–differentiated SMCs.
Keywords: Placenta-derived multipotent cells; Smooth muscle cells; Type I collagen; Differentiation; p38 mitogen-activated protein kinase;

An in vitro regenerated functional human endothelium on a nanofibrous electrospun scaffold by Xing Zhang; Vinoy Thomas; Yuanyuan Xu; Susan L. Bellis; Yogesh K. Vohra (4376-4381).
The capacity of the luminal layer of an electrospun bi-layer scaffold composed of gelatin, elastin, polycaprolactone (PCL), and poliglecaprone (PGC) to promote endothelial regeneration was investigated using human aortic endothelial cells (HAECs). HAECs of different densities were cultured on a thin film of the luminal layer of the scaffold mounted on a cell crown for desired periods. Fluorescent images showed that HAECs formed a mono-layer within 24 h after having successfully adhered to the scaffold's surface. Scanning electron microscopy (SEM) revealed a satisfactory coverage by the HAECs. Death rates of HAECs populations determined by fluorescent staining were below 5% within the initial 3 days while the profile of proliferation exhibited an exponential increase within 11 days as determined by the 3-[4,5-dimethyl(thiazol-2yl)-3,5-diphery] tetrazolium bromide (MTT) assay. The functionalities of the endothelial mono-layer were probed by ZO-1 staining for tight junction formation, by 6-keto-PGF assay for prostacyclin (PGI2) secretion, and by human platelets for its anti-thrombotic capability. The results indicated that the regenerated endothelium possessed normal functions associated with native endothelium. This study suggests that this electrospun bi-layer scaffold is a promising candidate for cardiovascular grafting for its capability of promoting the regeneration of a functional endothelium to prevent blood clotting in small diameter grafts.
Keywords: Electrospun scaffold; Endothelial regeneration; Anti-thrombotic vascular graft;

In vitro and in vivo performance of a dual drug-eluting stent (DDES) by Yingying Huang; Subbu S. Venkatraman; Freddy Y.C. Boey; Eeva M. Lahti; P.R. Umashankar; Mira Mohanty; Sabareeswaran Arumugam; Laxmikant Khanolkar; Sudhir Vaishnav (4382-4391).
This study reports on a dual drug-eluting stent (DDES) that has an anti-proliferative and an anti-thrombotic in a biodegradable polymer-coated onto a cobalt–chromium stent. The DDES was prepared by spray coating the bare metal stent with a biodegradable polymer loaded with sirolimus and triflusal, to treat against restenosis and thrombosis, respectively. The 2-layered dual-drug coated stent was characterized in vitro for surface properties before and after expansion, as well as for possible delamination by cross-sectioning the stent in vitro. The in vitro anti-platelet behavior of the triflusal-loaded films was investigated by using dynamic platelet adhesion measurements. Additionally, the in vitro degradation and release study of the films and the stents w/single sirolimus and dual sirolimus–triflusal in different formulations were examined. Finally, in vivo studies (in a porcine carotid artery model) were performed for acute thrombosis, inflammation and restenosis at 30 days. The in vitro results show DDES can sustain release both anti-proliferation drug (sirolimus) and anti-thrombosis drug (triflusal), two drugs were controlled in different rates to effectively reduce thrombosis and proliferation at the same time. In vivo results show a significant reduction in restenosis with dual-drug eluting stent compared with the controls (a bare metal stent, a sirolimus coated and a pure polymer-coated stent). The reduction in restenosis with a dual sirolimus–triflusal eluting stent is associated with an inhibition of inflammation, especially thrombus formation, suggesting that such dual-drug eluting stents have a role to play for the treatment of coronary artery disease.
Keywords: Thrombosis; Restenosis; Stent; Drug release; Cobalt alloy;

The effect of static magnetic fields and tat peptides on cellular and nuclear uptake of magnetic nanoparticles by Carol-Anne M. Smith; Jesus de la Fuente; Beatriz Pelaz; Edward P. Furlani; Margaret Mullin; Catherine C. Berry (4392-4400).
Magnetic nanoparticles are widely used in bioapplications such as imaging (MRI), targeted delivery (drugs/genes) and cell transfection (magnetofection). Historically, the impermeable nature of both the plasma and nuclear membranes hinder potential. Researchers combat this by developing techniques to enhance cellular and nuclear uptake. Two current popular methods are using external magnetic fields to remotely control particle direction or functionalising the nanoparticles with a cell penetrating peptide (e.g. tat); both of which facilitate cell entry. This paper compares the success of both methods in terms of nanoparticle uptake, analysing the type of magnetic forces the particles experience, and determines gross cell response in terms of morphology and structure and changes at the gene level via microarray analysis. Results indicated that both methods enhanced uptake via a caveolin dependent manner, with tat peptide being the more efficient and achieving nuclear uptake. On comparison to control cells, many groups of gene changes were observed in response to the particles. Importantly, the magnetic field also caused many change in gene expression, regardless of the nanoparticles, and appeared to cause F-actin alignment in the cells. Results suggest that static fields should be modelled and analysed prior to application in culture as cells clearly respond appropriately. Furthermore, the use of cell penetrating peptides may prove more beneficial in terms of enhancing uptake and maintaining cell homeostasis than a magnetic field.
Keywords: Nanoparticles; Fibroblast; Tat peptide; Magnetism; Endocytosis; Microarray;

Synthesis, biocompatibility and cell labeling of l-arginine-functional β-cyclodextrin-modified quantum dot probes by Mei-Xia Zhao; Qing Xia; Xu-Dong Feng; Xu-Hui Zhu; Zong-Wan Mao; Liang-Nian Ji; Kui Wang (4401-4408).
A series of quantum dots (QDs), CdSe, CdSe/CdS and CdSe/ZnSe, coated with l-arginine-modified β-cyclodextrin (β-CD-l-Arg) were prepared in a solution of H2O and hexane by ultrasonic method and characterized using PL, UV–vis, TEM, EDX and FTIR techniques. We observed that β-CD-l-Arg-coated QDs are water-soluble and stable with high colloidal properties in water. Their photophysical properties are similar to those of trioctylphosphine oxide (TOPO)-coated nanocrystals. The quantum yield (QY) of β-CD-l-Arg/CdSe/ZnSe QDs in water is 68%, which is much higher than those of β-CD-l-Arg/CdSe/CdS (26%) and β-CD-l-Arg/CdSe (13%). The in vitro cytotoxicity of these QDs was evaluated in ECV-304, SH-SY5Y and Hela cells and low cytotoxicity was observed. In particular, the β-CD-l-Arg/CdSe/ZnSe QDs presented lower cytotoxicity to these cells (CC50 value is 173 μg/mL in ECV-304 cells for 48 h). This may be due to the presence of the ZnSe and β-CD-l-Arg outlayer, which may improve the biocompatibility of QDs. The QDs were further investigated for biological labeling in ECV-304 cells using confocal laser scanning fluorescence microscopy. We found that these QDs were capable of localing to the cytoplasm of cells. These results demonstrate that the β-CD-l-Arg-coated QDs could be used as a potential photoluminescent nanocrystal probing agent with good biocompatibility.
Keywords: QDs; β-CD-l-Arg; Biocompatibility; Cytotoxicity; Bio-imaging;

To enable selective cell-kill, we designed functionalized lipid vesicles with pH-triggered heterogeneous membranes and encapsulated doxorubicin that exhibit tunable surface topography. These vesicles “hide” (mask) the targeting ligands from their surface during circulation in the blood, and only progressively “expose” these ligands as they gradually penetrate deeper into the tumor interstitium, where after endocytosis they burst release their contents. The stimulus to activate the binding reactivity is the pH gradient between the blood stream (pH 7.4–7.0) and the increasingly acidic pH inside the tumor interstitium (pH 6.7–6.5). Doxorubicin release is activated at the endosomal pH 5.5–5.0. We show that tunable functionalized vesicles exhibit environmentally-dependent (pH-dependent) association with cancer cells resulting in high cell-kill selectivity. When lowering the extracellular pH from 7.4 to 6.5, tunable functionalized vesicles deliver doxorubicin to cancer cells that increases from 41% to 93% of maximum resulting in cancer cell killing that increases from 23 to 71% of maximum, respectively. This proof-of-concept shows the potential of tunable targeted liposomal chemotherapy to selectively kill cancer cells in an environmentally-dependent way.
Keywords: Liposomes; Chemotherapy; Targeted liposomal chemotherapy;

Decontamination of chemical and biological warfare agents with a single multi-functional material by Gabi Amitai; Hironobu Murata; Jill D. Andersen; Richard R. Koepsel; Alan J. Russell (4417-4425).
We report the synthesis of new polymers based on a dimethylacrylamide-methacrylate (DMAA-MA) co-polymer backbone that support both chemical and biological agent decontamination. Polyurethanes containing the redox enzymes glucose oxidase and horseradish peroxidase can convert halide ions into active halogens and exert striking bactericidal activity against gram positive and gram negative bacteria. New materials combining those biopolymers with a family of N-alkyl 4-pyridinium aldoxime (4-PAM) halide-acrylate co-polymers offer both nucleophilic activity for the detoxification of organophosphorus nerve agents and internal sources of halide ions for generation of biocidal activity. Generation of free bromine and iodine was observed in the combined material resulting in bactericidal activity of the enzymatically formed free halogens that caused complete kill of E. coli (>6 log units reduction) within 1 h at 37 °C. Detoxification of diisopropylfluorophosphate (DFP) by the polyDMAA MA-4-PAM iodide component was dose-dependent reaching 85% within 30 min. A subset of 4-PAM-halide co-polymers was designed to serve as a controlled release reservoir for N-hydroxyethyl 4-PAM (HE 4-PAM) molecules that reactivate nerve agent-inhibited acetylcholinesterase (AChE). Release rates for HE 4-PAM were consistent with hydrolysis of the HE 4-PAM from the polymer backbone. The HE 4-PAM that was released from the polymer reactivated DFP-inhibited AChE at a similar rate to the oxime antidote 4-PAM.
Keywords: Bioactive polymer; Antimicrobial; Chemical warfare; Biological warfare; Multifunctional materials; Enzymes;

Chitosan and its derivatives have shown great potential as non-viral vectors for gene delivery therapy. Folic acid receptor (FR) is an important anti-cancer therapy target that is applicable to many cancer types. In this study, we developed an efficient and targeted delivery of antisense oligodeoxynucleotides asODNs, using folic acid (FA) conjugated hydroxypropyl-chitosan (HPCS). These nanoparticles were designed to reduce production of P-gp, in order to overcome tumor drug resistance. Nanoparticles prepared were found to be 181 nm in diameter. Spectrofluorimetry was utilized to evaluate the effect of charge ratio of the nanoparticles on loading efficiency. In PBS buffer, 40% of asODNs were released from the nanoparticles at first 24 h. However, just another 15% was released between 24 and 48 h. The antitumor effect of the nanoparticles was evaluated in KB-A-1 cells implanted in Balb/c-nu/nu mice. They inhibited the growth of tumor by 35% compared to the bare asODNs. The FA–HPCS–asODNs nanoparticles demonstrated significantly inhibition of the multi drug resistance (MDR) 1 gene levels and P-gp levels in vitro and in vivo, respectively, related with bare asODNs and HPCS–asODNs ones. During in vivo studies, FA–HPCS–asODNs nanoparticles were also found to bind specifically and efficiently to FR high-expressing cancer cells. These results suggested that the use of targeted, antisense agent nanoparticles would be potential approach to overcome tumor drug resistance.
Keywords: Hydroxypropyl-chitosan nanoparticles; Tumor-targeted; Antisense oligodeoxynucleotides; Multidrug resistance;

A pH-responsive interface derived from resilin-mimetic protein Rec1-resilin by My Y. Truong; Naba K. Dutta; Namita R. Choudhury; Misook Kim; Christopher M. Elvin; Anita J. Hill; Benjamin Thierry; Krasimir Vasilev (4434-4446).
In this investigation, for the first time we report the effects of pH on the molecular orientation, packing density, structural properties, adsorption characteristics and viscoelastic behaviour of resilin-mimetic protein rec1-resilin at the solid–liquid interface using quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) spectroscopy. QCM-D and SPR data confirm that the binding ability of rec1-resilin on a substrate is strongly pH-dependent the protein packing density on a gold surface is calculated to be 4.45 × 1013 per cm2 at the isoelectric point (IEP ∼ 4.9), 8.79 × 1011 per cm2 at pH 2 and 9.90 × 1011 per cm2 at pH 12, respectively. Our findings based on the thickness, dissipation and viscoelasticity of the rec1-resilin adlayer also indicate that it is adsorbed onto the gold substrate with different orientation depending on pH, such as back-on adsorption at acidic pH of 2, compact end-on bilayer adsorption at the IEP and side-on at high alkaline pH of 12. When rec1-resilin is ‘pinned’ to the substrate at IEP and subsequently exposed to an electrolyte solution adjusted to different pH, it switches from a compact globular conformation of the bio-macromolecule at the IEP to a coil conformation at pH between IEP to IED (IED = pKa value of tyrosine amino acid residue) and an extended coil conformation at pH > IED. This transformation from globule to coil to extended coil conformation is kinetically fast, robust and completely reversible. Such responsive surfaces created using ‘smart’ biomimetic rec1-resilin have the potential to find applications in many areas including biotechnology, medicine, sensors, controlled drug delivery systems and engineering.
Keywords: Biomimetic material; Recombinant protein; Protein adsorption; Resilin; Responsive interface; Quartz crystal microbalance;

The use of injectable forms of fibrin and fibronectin to support axonal ingrowth after spinal cord injury by Von R. King; Alla Alovskaya; Diana Y.T. Wei; Robert A. Brown; John V. Priestley (4447-4456).
Many studies have described biomaterial devices (conduits and scaffolds) that can be implanted into experimental lesions and which support axonal growth. However, a disadvantage of such pre-formed devices is that tissue needs to be excised to allow their insertion. In this study we have therefore examined four biomaterials that can be injected into an injury site and which gel in situ; namely collagen, viscous fibronectin, fibrin, and fibrin + fibronectin (FB/FN). The materials were tested in an experimental knife-cut cavity in the rat spinal cord, and evaluated at 1 week and 4 weeks survival for their biocompatibility, neuroprotective efficacy, and permissiveness for axonal growth. At one week, all four materials showed good integration with the host spinal cord and supported some degree of axonal ingrowth, which was associated with infiltration of Schwann cells and deposition of laminin. However axon growth in the collagen implants was uneven because implants contained dense inclusions which were not penetrated by axons. At 4 weeks, axon growth was greatest in the fibronectin and FB/FN implants, however the fibronectin implants had large cavities at the interface between the implant and host spinal cord. The fibronectin implants also had fewer surviving neurons in the intact spinal cord adjoining the implant site. The FB/FN mixture thus had the best combination of properties in that it was easy to handle, integrated with the host spinal cord tissue, and supported robust growth of axons. It therefore has promise as an injectable biomaterial for filling cavities at spinal cord injury sites.
Keywords: Fibrin; Fibronectin; Spinal surgery; Nerve tissue engineering;