Biomaterials (v.28, #3)

Macrophages play a critical role in mediating the host response to biomaterials, perhaps most notably by guiding the host inflammatory response through the release of inflammatory molecules such as the cytokine interleukin-1 (IL-1). The extent of the macrophage response following interaction with the biomaterial surface contributes greatly to device efficacy, yet the molecular mechanisms of this interaction are still unclear. The extracellular matrix (ECM) protein fibronectin (FN) is recognized by macrophages and frequently used in biomaterial modification to elicit greater cellular adhesion and tissue integration. Macrophage interaction with FN and other ECM molecules on the biomaterial surface has been shown to induce a variety of inflammatory responses, thus both FN and IL-1 can be utilized as model molecules to better understand the mechanisms of material-mediated macrophage responses. This literature review presents a comprehensive survey of past and current research on the interrelated role of IL-1, FN, and FN-derivatives in determining biomaterial-modulated macrophage function.
Keywords: Monocyte; Alveolar macrophage; Tyrosine phosphorylation; Inflammation; RGD; PHSRN; Cytokine;

Prior studies from our laboratory have shown that RGD peptides increase the attachment of mesenchymal stem cells (MSCs) to hydroxyapatite (HA), however, RGD does not induce cell spreading when coupled to this type of biomaterial. In an effort to improve MSC spreading, and possibly cell attachment, proteoglycan-binding peptides (KRSR or FHRRIKA) were combined with RGD in the current study. It was found that the peptide combinations did not enhance MSC attachment relative to RGD alone, although a slight amount of spreading was elicited by both KRSR and FHRRIKA. Similar results were obtained with proteoglycan-binding peptides modified with a heptaglutamate domain, a motif that improves peptide tethering to HA. To determine whether differentiation status affected cell responses, MSCs were in vitro differentiated into osteoblasts, and evaluated as before. These experiments revealed that, like MSCs, osteoblasts did not adhere in greater numbers to the peptide combinations. Finally, none of the peptides or peptide combinations were able to stimulate the robust amount of cell adhesion and spreading elicited by serum-coated HA surfaces (of note, five different species of serum were tested). Given the propensity of HA to adsorb proadhesive proteins from blood/serum, we question the utility of functionalizing HA with RGD and/or proteoglycan-binding peptides.
Keywords: Cell adhesion; Hydroxyapatite; Mesenchymal stem cell; Protein adsorption; RGD peptide; Bone;

Effect of gypsum on proliferation and differentiation of MC3T3-E1 mouse osteoblastic cells by Áron Lazáry; Bernadett Balla; János P. Kósa; Krisztián Bácsi; Zsolt Nagy; István Takács; Péter P. Varga; Gábor Speer; Péter Lakatos (393-399).
Recently, calcium sulfate dihydrate has been demonstrated as safe biodegradable osteoconductive bone void filler. However, its exact mechanism of action on bone cells is yet unknown. In this study, the influence of gypsum on gene expression and proliferation of MC3T3-E1 mouse pre-osteoblastic cells was investigated. Cells were cultured on gypsum disc, slice, polymethylmethacrylate (PMMA), or plastic culture plate for 15 days. Cell viability, alkaline phosphatase (ALP) activity and expression profile of 15 genes involved in bone metabolism were measured in cultures. Cell proliferation on gypsum was increased by almost 2-fold, while an inhibitory effect of PMMA on proliferation rate of osteoblasts was noted. Cells cultured on gypsum disc surface exhibited an increased ALP activity and markedly different gene expression profile. Quantitative real-time PCR data indicated the expression of genes that might provide a basis for an osteoinductive potential. MC3T3-E1 cells expressed genes typical of bone fracture healing like type II collagen and fibronectin 1. These effects might be related to the calcium content of gypsum and mediated likely via SMAD3. Our results suggest that gypsum can support new bone formation by its calcium content and modulatory effect on gene expression profile of bone cells.
Keywords: Calcium sulfate; Bone graft; Gene expression; Osteoinduction; Bone healing;

Involvement of toll-like receptor 4 in the inflammatory reaction induced by hydroxyapatite particles by Alexia Grandjean-Laquerriere; Olivier Tabary; Jacky Jacquot; Doriane Richard; Patrick Frayssinet; Moncef Guenounou; Dominique Laurent-Maquin; Patrice Laquerriere; Sophie Gangloff (400-404).
Hydroxyapatite (HA) is widely used to coat metal parts in order to improve their biocompatibility. Analysis of retrieved tissues associated with failed implants, suggest that phagocytosis of HA wear debris by monocytes/macrophages might provide a potent stimulus for the release of a variety of cytokines. Phagocytosis involved a large variety of cellular receptors like toll-like receptors that results in activation of the transcriptional nuclear factor-κB (NF-κB) via a cell-signalling pathway. In the present paper, we aimed to evaluate the role of the toll-like receptor 4 (TLR4) in the production of inflammatory cytokines induced by HA particles using TLR4+ and TLR4 peritoneal macrophages. We investigated the production of TNF-α and the activation of the nuclear transcription factor NF-κB. Our data clearly show for the first time that the production of TNF-α by macrophages exposed to HA particles was TLR4 dependent but not the activation of NF-κB. All these results open future therapies to reduce the inflammatory response induced by HA biomaterials.
Keywords: Hydroxyapatite particles; Cytokines; Inflammation; Toll-like receptor 4; Macrophages;

A Vroman-like exchange of different proteins adsorbing from a concentrated mixture to the same hydrophobic adsorbent surface is shown to arise naturally from the selective pressure imposed by a fixed interfacial-concentration capacity (w/v, mg/mL) for which protein molecules compete. A size (molecular weight, MW) discrimination results because fewer large proteins are required to accumulate an interfacial w/v concentration equal to smaller proteins. Hence, the surface region becomes dominated by smaller proteins on a number-or-mole basis through a purely physical process that is essentially unrelated to protein biochemistry. Under certain conditions, this size discrimination can be amplified by the natural variation in protein-adsorption avidity (quantified by partition coefficients P) because smaller proteins (MW<50 kDa) have been found to exhibit characteristically higher P than larger proteins (MW<50 kDa). The standard depletion method is implemented to measure protein-adsorption competition between two different test proteins (i and j) for the same hydrophobic octyl sepharose adsorbent particles. SDS-gel electrophoresis is used as a multiplexing, separation-and-quantification tool for this purpose. Identical results obtained using sequential and simultaneous competition of human immunoglobulin G (IgG, protein j) with human serum albumin (HSA, protein i) demonstrates that HSA was not irreversibly adsorbed to octyl sepharose over a broad range of competing solution concentrations. A clearly observed exchange of HSA for IgG or fibrinogen (Fib) shows that adsorption of different proteins (i competing with j) to the same hydrophobic surface is coupled whereas adsorption among identical proteins (i or j adsorbing from purified solution) is not coupled. Interpretive theory shows that this adsorption coupling is due to competition for the fixed surface capacity. Theory is extended to hypothetical ternary mixtures using a computational experiment that illustrates the profound impact size-discrimination has on adsorption from complex mixtures such as blood.
Keywords: Protein adsorption; Vroman effect; Multi-layers; Adsorption reversibility;

Synthesis of two-component injectable polyurethanes for bone tissue engineering by Ian C. Bonzani; Raju Adhikari; Shadi Houshyar; Roshan Mayadunne; Pathiraja Gunatillake; Molly M. Stevens (423-433).
The advent of injectable polymer technologies has increased the prospect of developing novel, minimally invasive arthroscopic techniques to treat a wide variety of ailments. In this study, we have synthesised and evaluated a novel polyurethane-based injectable, in situ curable, polymer platform to determine its potential uses as a tissue engineered implant. Films of the polymers were prepared by reacting two pentaerythritol-based prepolymers, and characterised for mechanical and surface properties, and cytocompatibility. This polymer platform displayed mechanical strength and elasticity superior to many injectable bone cements and grafts. Cytotoxicity tests using primary human osteoblasts, revealed positive cell viability and increased proliferation over a period of 7 days in culture. This favourable cell environment was attributed to the hydrophilic nature of the films, as assessed by dynamic contact angle (DCA) analysis of the sample surfaces. The incorporation of β-TCP was shown to improve mechanical properties, surface wettability, and cell viability and proliferation, compared to the other sample types. SEM/EDX analysis of these surfaces also revealed physicochemical surface heterogeneity in the presence of β-TCP. Based on preliminary mechanical analysis and cytotoxicity results, these injectable polymers may have a number or potential orthopaedic applications; ranging from bone glues to scaffolds for bone regeneration.
Keywords: Polyurethane; Bone tissue engineering; Cytotoxicity; Mechanical properties; Surface analysis; Wettability;

The MR tracking of transplanted ATDC5 cells using fluorinated poly-l-lysine-CF3 by Junichi Maki; Chiaki Masuda; Shigehiro Morikawa; Masahito Morita; Toshiro Inubushi; Yoshitaka Matsusue; Hiroyasu Taguchi; Ikuo Tooyama (434-440).
Magnetic resonance (MR) imaging using super-paramagnetic iron oxides (SPIOs) is a powerful tool to monitor transplanted cells in living animals. However, since SPIOs are negative contrast agents it is difficult to track transplanted cells in bone and cartilage that originally display low signals. In this study, we examined the feasibility of tracking with fluorescein isothiocyanate (FITC)-labeled poly-l-lysine-CF3 (PLK-CF3) using mouse ATDC5 cells, a stem cell line of bone and cartilage cells. FITC-labeled PLK-CF3 was easily internalized by ATDC5 cells by adding it into culture medium. No acute or long-term toxicities were seen at less than 160 μg/ml. Labeled cells transplanted into the cranial bone of mice were detected for at least 7 days by MR images. FITC-labeled PLK-CF3 is a useful positive contrast agent for MR tracking in bone and cartilage.
Keywords: Bone; Cartilage; Cell therapy; Molecular imaging; Magnetic resonance; Transplantation;

Polylysine-functionalised thermoresponsive chitosan hydrogel for neural tissue engineering by K.E. Crompton; J.D. Goud; R.V. Bellamkonda; T.R. Gengenbach; D.I. Finkelstein; M.K. Horne; J.S. Forsythe (441-449).
Foetal mouse cortical cells were cultured on 2D films and within 3D thermally responsive chitosan/glycerophosphate salt (GP) hydrogels. The biocompatibility of chitosan/GP 2D films was assessed in terms of cell number and neurites per cell. Osmolarity of the hydrogel was a critical factor in promoting cell survival with isotonic GP concentrations providing optimal conditions. To improve cell adhesion and neurite outgrowth, poly-d-lysine (PDL) was immobilised onto chitosan via azidoaniline photocoupling. Increase in PDL concentrations did not alter cell survival in 2D cultures but neurite outgrowth was significantly inhibited. Neurons exhibited a star-like morphology typical of 2D culture systems.The effects of PDL attachment on cell number, cell morphology and neurite outgrowth were more distinct in 3D culture conditions. Neurones exhibited larger cell bodies and sent out single neurites within the macroporous gel. Immobilised PDL improved cell survival up to an optimum concentration of 0.1%, however, further increases resulted in drops in cell number and neurite outgrowth. This was attributed to a higher cell interaction with PDL within a 3D hydrogel compared to the corresponding 2D surface. The results show that thermally responsive chitosan/GP hydrogels provide a suitable 3D scaffolding environment for neural tissue engineering.
Keywords: Chitosan; Hydrogel; Thermally responsive material; Nerve tissue engineering; Nerve; Biocompatibility;

Adipogenesis of murine embryonic stem cells in a three-dimensional culture system using electrospun polymer scaffolds by Xihai Kang; Yubing Xie; Heather M. Powell; L. James Lee; Martha A. Belury; John J. Lannutti; Douglas A. Kniss (450-458).
A mechanistic understanding of adipose tissue differentiation is critical for the treatment and prevention of obesity and type 2 diabetes. Conventional in vitro models of adipogenesis are preadipocytes or freshly isolated adipocytes grown in two-dimensional (2D) cultures. Optimal results using in vitro tissue culture models can be expected only when adipocyte models closely resemble adipose tissue in vivo. Thus the design of an in vitro three-dimensional (3D) model which faithfully mimics the in vivo environment is needed to effectively study adipogenesis. Pluripotent embryonic stem (ES) cells are a self-renewing cell type that can readily be differentiated into adipocytes. In this study, a 3D culture system was developed to mimic the geometry of adipose tissue in vivo. Murine ES cells were seeded into electrospun polycaprolactone scaffolds and differentiated into adipocytes in situ by hormone induction as demonstrated using a battery of gene and protein expression markers along with the accumulation of neutral lipid droplets. Insulin-responsive Akt phosphorylation, and β-adrenergic stimulation of cyclic AMP synthesis were demonstrated in ES cell-derived adipocytes. Morphologically, ES cell-derived adipocytes resembled native fat cells by scanning electron and phase contrast microscopy. This tissue engineered ES cell-matrix model has potential uses in drug screening and other therapeutic developments.
Keywords: Adipose tissue engineering; Stem cell; Electrospinning; Polycaprolactone; Three-dimensional cell culture; Morphogenesis;

Segmental bone regeneration using a load-bearing biodegradable carrier of bone morphogenetic protein-2 by Tien-Min G. Chu; Stuart J. Warden; Charles H. Turner; Rena L. Stewart (459-467).
Segmental defect regeneration has been a clinical challenge. Current tissue-engineering approach using porous biodegradable scaffolds to delivery osteogenic cells and growth factors demonstrated success in facilitating bone regeneration in these cases. However, due to the lack of mechanical property, the porous scaffolds were evaluated in non-load bearing area or were stabilized with stress-shielding devices (bone plate or external fixation). In this paper, we tested a scaffold that does not require a bone plate because it has sufficient biomechanical strength. The tube-shaped scaffolds were manufactured from poly(propylene) fumarate/tricalcium phosphate (PPF/TCP) composites. Dicalcium phosphate dehydrate (DCPD) were used as bone morphogenetic protein-2 (BMP-2) carrier. Twenty-two scaffolds were implanted in 5 mm segmental defects in rat femurs stabilized with K-wire for 6 and 15 weeks with and without 10 μg of rhBMP-2. Bridging of the segmental defect was evaluated first radiographically and was confirmed by histology and micro-computer tomography (μCT) imaging. The scaffolds in the BMP group maintained the bone length throughout the duration of the study and allow for bridging. The scaffolds in the control group failed to induce bridging and collapsed at 15 weeks. Peripheral computed tomography (pQCT) showed that BMP-2 does not increase the bone mineral density in the callus. Finally, the scaffold in BMP group was found to restore the mechanical property of the rat femur after 15 weeks. Our results demonstrated that the load-bearing BMP-2 scaffold can maintain bone length and allow successfully regeneration in segmental defects.
Keywords: Bone morphogenetic protein (BMP); Bone regeneration; Calcium phosphate cement; Bone tissue engineering; Free form fabrication;

The effect of electrochemical functionalization of Ti-alloy surfaces by aptamer-based capture molecules on cell adhesion by Ke-Tai Guo; Dieter Scharnweber; Bernd Schwenzer; Gerhard Ziemer; Hans P. Wendel (468-474).
To improve cell seeding efficiency and cytocompatibility, we designed a new coating material for scaffolds. We used aptamers, highly specific cell binding nucleic acids generated by combinatorial chemistry with an in vitro selection called systematic evolution of exponential enrichment (SELEX). In this study, we functionalized Ti-alloy surfaces to enhance cell adhesion. By coating the material with a cell specific aptamer, working as a capture molecule, we could improve the attachment of cells effectively and avoid the limitations of the currently available materials. Aptamers, immobilized by partial electrochemical entrapment in oxide layers on Ti-alloy surfaces were able to capture cells out of a flowing suspension rapidly. This model proves that surface immobilized aptamers can greatly enhance the attachment of seeded cells. This technology opens new perspectives towards clinical application of stem cell and tissue engineering strategies.
Keywords: Osteoblast; Scaffold;

Performance of functionally graded implants of polylactides and calcium phosphate/calcium carbonate in an ovine model for computer assisted craniectomy and cranioplasty by Harald Eufinger; Christian Rasche; Jutta Lehmbrock; Michael Wehmöller; Stephan Weihe; Inge Schmitz; Carsten Schiller; Matthias Epple (475-485).
Biodegradable functionally graded skull implants on the basis of polylactides and calcium phosphate/calcium carbonate were prepared in an individual mould using a combination of different processing techniques. A geometrically corresponding resection template was designed to enable a craniectomy and cranioplasty with the prepared implant in the same operation. After various preliminary experiments concerning degradation kinetics, pH evolution during degradation, micromorphology, biocompatibility tests in human osteoblast cell cultures and surgery of cadaver heads, a new large-animal model was developed for long-term in vivo studies. In eight 12-months-old sheep, the surgical templates were used to create 4.5×5 cm2 calvarial defects which were then filled with the corresponding degradable implants in the same operation. The animals were sacrificed after 2, 9, 12 and 18 months, and the implants and the surrounding tissues were analysed by computer tomography (CT), macroscopic examination and microscopy. The new animal model proved to be reliable and very suitable for large individual craniectomies and cranioplasties. The formation of new bone from the dural layer of the meninges corresponded well to the degradation of the porous inner layer of the implants whereas the skull contour was stabilised by the compact outer layer over the follow-up period.
Keywords: Composites; Polylactides; Functionally graded materials; Guided bone regeneration; Computer assisted surgery; Animal models;

The premature endothelialization of tissue-engineered grafts had often induced cellular detachment at an early period of implantation in arterial circulation, resulting in occlusion at an early period of implantation. This study was aimed to determine whether gradually increased shear stress applied ex vivo improves cell retention and tissue morphological integrity including cell shape and alignment, actin fiber alignment and expression of vascular endothelial (VE) cadherin. Tissue-engineered grafts used for this study were human umbilical vein endothelial cell (HUVEC)-seeded compliant small-diameter grafts made of poly(l-lactide-co-ε-caprolactone) fiber meshes fabricated by electrospinning. The shear stresses applied to grafts, generated using a custom-designed mock circulatory apparatus, were 3.2, 8.7 and 19.6 dyn/cm2. The grafts completely monolayered prior to shear stress exposure exhibited a polygonal cobblestone morphology with randomly distributed actin fibers and VE cadherin at the continuous peripheral region of adjacent cells. The 24-h-loading of high shear stresses (8.7 and 19.6 dyn/cm2) equivalent to those of the arterial circulatory system resulted in severe cellular damage resulting in the complete loss of cells. However, a gradually increased graded exposure from a low (3.2 dyn/cm2) to a high shear stress (19.6 dyn/cm2) resulted in a markedly reduced cell detachment, a highly elongated cell shape, and orientation or alignment of both cells and actin fibers, which were parallel to the direction of flow. Although VE-cadherin expression was not detected yet, a higher degree of tissue integrity was achieved, which may greatly improve the performance particularly at an early period of implantation.
Keywords: Graded shear stress; Endothelial cell; Small-diameter vascular graft;

Glycosaminoglycan-targeted fixation for improved bioprosthetic heart valve stabilization by Jeremy J. Mercuri; Joshua J. Lovekamp; Dan T. Simionescu; Narendra R. Vyavahare (496-503).
Numerous crosslinking chemistries and methodologies have been investigated as alternative fixatives to glutaraldehyde (GLUT) for the stabilization of bioprosthetic heart valves (BHVs). Particular attention has been paid to valve leaflet collagen and elastin stability following fixation. However, the stability of glycosaminoglycans (GAGs), the primary component of the spongiosa layer of the BHV, has been largely overlooked despite recent evidence provided by our group illustrating their structural and functional importance. In the present study we investigate the ability of two different crosslinking chemistries: sodium metaperiodate (NaIO4) followed by GLUT (PG) and 1-Ethyl-3-(3 dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) followed by GLUT (ENG) to stabilize GAGs within BHV leaflets and compare resulting leaflet characteristics with that of GLUT-treated tissue. Incubation of fixed leaflets in GAG-degrading enzymes illustrated in vitro resistance of GAGs towards degradation in PG and ENG treated tissue while GLUT fixation alone was not effective in preventing GAG loss from BHV leaflets. Following subdermal implantation, significant amounts of GAGs were retained in leaflets in the ENG group in comparison to GLUT-treated tissue, although GAG loss was evident in all groups. Utilizing GAG-targeted fixation did not alter calcification potential of the leaflets while collagen stability was maintained at levels similar to that observed in conventional GLUT-treated tissue.
Keywords: Glutaraldehyde; Periodate; Carbodiimide; Hexosamine; Subdermal implant;

The aim of this study was to synthesize folate-dendrimer conjugates as suitable vehicle for site specific delivery of anti-arthritic drug (indomethacin) to inflammatory regions and to determine its targeting efficiency, biodistribution in adjuvant induced arthritic rats. Folic acid was coupled to the surface amino groups of G4-PAMAM dendrimer (G4D) via a carbodiimide reaction and loaded with indomethacin. The conjugates were characterized by 1H-NMR and IR spectroscopy. The drug content and percent encapsulation efficiency increased with increasing folate content for the dendrimer conjugates. The in vitro release rate was decreased for the folate conjugates when compared with unconjugated dendrimer (DNI). The plasma concentration profile showed a biphasic curve indicating rapid distribution followed by slow elimination. The AUC0−∞, half-life and residence time of indomethacin in inflamed paw was higher for folate-dendrimer conjugates. The time-averaged relative drug exposure (r e) of the drug in paw and overall drug targeting efficiency (T e) were higher for folate conjugate with 21 folate moieties (4.1 and 2.78, respectively) when compared with DNI (1.91 and 1.88, respectively). This study demonstrated the superiority of active targeting over dendrimer mediated passive targeting and also for the first time, folate-mediated targeting of an anti-arthritic drug to the inflammatory tissues.
Keywords: Folate; G4-PAMAM dendrimer; Arthritis; Targeted drug delivery; Biodistribution; Pharmacokinetics;

The effect of polypyrrole with incorporated neurotrophin-3 on the promotion of neurite outgrowth from auditory neurons by Rachael T. Richardson; Brianna Thompson; Simon Moulton; Carrie Newbold; May Ghee Lum; Adrian Cameron; Gordon Wallace; Robert Kapsa; Graeme Clark; Stephen O’Leary (513-523).
This research aims to improve the nerve–electrode interface of the cochlear implant using polymer technology to encourage neuron survival, elongation and adhesion to the electrodes. Polypyrrole (Ppy) doped with p-toluene sulphonate (pTS) is an electroactive polymer into which neurotrophin-3 (NT3) can be incorporated. Ppy/pTS±NT3 was synthesised over gold electrodes and used as a surface for auditory neuron explant culture. Neurite outgrowth from explants grown on Ppy/pTS was equivalent to tissue culture plastic but improved with the incorporation of NT3 (Ppy/pTS/NT3). Electrical stimulation of Ppy/pTS/NT3 with a biphasic current pulse, as used in cochlear implants, significantly improved neurite outgrowth from explants. Using 125I-NT3, it was shown that low levels of NT3 passively diffused from Ppy/pTS/NT3 during normal incubation and that electrical stimulation enhanced the release of biologically active NT3 in quantities adequate for neuron survival. Furthermore, Ppy/pTS/NT3 and its constituents were not toxic to auditory neurons and the Ppy/pTS/NT3 coating on gold electrodes did not alter impedance. If applied to the cochlear implant, Ppy/pTS/NT3 will provide a biocompatible, low-impedance substrate for storage and release of NT3 to help protect auditory neurons from degradation after sensorineural hearing loss and encourage neurite outgrowth towards the electrodes.
Keywords: Controlled drug release; Electroactive polymer; Electrical stimulation; Nerve growth factor; Nerve regeneration;

Development and in vitro evaluation of a thiomer-based nanoparticulate gene delivery system by Thierry Schmitz; Irene Bravo-Osuna; Christine Vauthier; Gilles Ponchel; Brigitta Loretz; Andreas Bernkop-Schnürch (524-531).
Chitosan-thiobutylamidine was developed and evaluated as a novel tool for gene delivery. The conjugate, displaying 299.1±11.5 μmol free thiol groups per gram polymer, formed coacervates with pDNA at a mean size of 125 nm and a zeta potential of +9 mV. Thiol groups, being susceptible for oxidation, were immobilised on the polymeric backbone of chitosan in order to introduce the property of extracellular stability and intracellular pDNA release by forming reversible disulfide bonds. The integrity of the new particles was compared to unmodified chitosan under simulated physiological conditions. Within 10 h, pDNA was completely released from chitosan–DNA particles while only 12% were released from the thiomer-based particles. At pH 7, the amount of thiol groups significantly (p<0.05) decreased by more than 25% within 6 h. In contrast, in a reducing environment as found intracellularly, chitosan-thiobutylamidine–DNA nanoparticles dissociated continuously, liberating approximately 50% of pDNA within 3 h. Transfection studies performed in a Caco2 cell culture evinced the highest efficiency for chitosan-thiobutylamidine–DNA nanoparticles in combination with a glycerol shock solution. The combination of improved stability, enhanced pDNA release under reducing conditions, and higher transfection efficiency identifies chitosan-thiobutylamidine as a promising new vector for gene delivery.
Keywords: Thiomer; Chitosan-thiobutylamidine; Gene delivery; Nanoparticles; Vector;

The potential role of fucosylated cationic liposome/NFκB decoy complexes in the treatment of cytokine-related liver disease by Yuriko Higuchi; Shigeru Kawakami; Fumiyoshi Yamashita; Mitsuru Hashida (532-539).
Cytokine production by Kupffer cells, which is regulated by NFκB, causes severe liver injury in endotoxin syndrome. NFκB decoy has been reported to inhibit NFκB-mediated transcription. The purpose of this study is to inhibit LPS-induced cytokine production by Kupffer cell-targeted delivery of NFκB decoy using fucosylated cationic liposomes (Fuc-liposomes). Cholesten-5-yloxy-N-{4-[(1-imino-2-l-thiofucosyl-ethyl)-amino] butyl-}formamide (Fuc-C4-Chol) was synthesized to prepare Fuc-liposomes. Tissue accumulation, intrahepatic distribution and serum cytokine concentrations were investigated after intravenous injection of Fuc-liposomes/NFκB decoy complexes. Intravenously injected Fuc-liposome complexes rapidly and highly accumulated in the liver while little naked NFκB decoy accumulated in the liver. An intrahepatic distribution study showed that Fuc-liposome complexes are mainly taken up by non-parenchymal cells. The liver accumulation of Fuc-liposome complexes was inhibited by GdCl3 pretreatment, which selectively inhibited Kupffer cell uptake. This result suggested that Kupffer cells contribute to liver accumulation. TNFα, IFNγ, ALT and AST serum levels in LPS-infected mice were significantly attenuated by treatment with Fuc-liposome complexes compared with naked NFκB decoy. Fuc-liposome complexes also reduced the amount of activated NFκB in the liver nuclei. Fuc-liposomes would be a useful carrier for Kupffer cell-selective delivery of NFκB decoy by intravenous injection.
Keywords: Fucosylated cationic liposome; Gene delivery; Gene therapy; Targeting; NFκB decoy; Kupffer cell;

Poor water solubility and low transfection efficiency of chitosan are major drawbacks for its use as a gene delivery carrier. PEGylation can increase its solubility, and folate conjugation may improve gene transfection efficiency due to promoted uptake of folate receptor-bearing tumor cells. The aim of this study was to synthesize and characterize folate-poly(ethylene glycol)-grafted chitosan (FA-PEG-Chi) for targeted plasmid DNA delivery to tumor cells. Gel electrophoresis study showed strong DNA binding ability of modified chitosan. The pH50 values, defined as the pH when the transmittance of a polymer solution at 600 nm has reached 50% of the original value, suggested that the water solubility of PEGylated chitosan had improved significantly. Regression analysis of pH50 value as a function of substitution degree of PEG yielded an almost linear correlation for PEG-Chi and FA-PEG-Chi. The solubility of PEGylated chitosan decreased slightly by further conjugation of folic acid due to the relatively more hydrophobic nature of folic acid when compared to PEG. In addition, the chitosan-based DNA complexes did not induce remarkable cytotoxicity against HEK 293 cells. FA-PEG-Chi can be a promising gene carrier due to its solubility in physiological pH, efficiency in condensing DNA, low cytotoxicity and targeting ability.
Keywords: Chitosan; PEGylation; Folate conjugation; Water solubility; Folate receptor targeting; Gene delivery;

Physicochemically modified silicon as a substrate for protein microarrays by A. Jasper Nijdam; Mark Ming-Cheng Cheng; David H. Geho; Roberta Fedele; Paul Herrmann; Keith Killian; Virginia Espina; Emanuel F. Petricoin; Lance A. Liotta; Mauro Ferrari (550-558).
Reverse phase protein microarrays (RPMA) enable high throughput screening of posttranslational modifications of important signaling proteins within diseased cells. One limitation of protein-based molecular profiling is the lack of a PCR-like intrinsic amplification system for proteins. Enhancement of protein microarray sensitivities is an important goal, especially because many molecular targets within patient tissues are of low abundance. The ideal array substrate will have a high protein-binding affinity and low intrinsic signal. To date, nitrocellulose-coated glass has provided an effective substrate for protein binding in the microarray format when using chromogenic detection systems. As fluorescent systems, such as quantum dots, are explored as potential reporter agents, the intrinsic fluorescent properties of nitrocellulose-coated glass slides limit the ability to image microarrays for extended periods of time where increases in net sensitivity can be attained. Silicon, with low intrinsic autofluorescence, is being explored as a potential microarray surface. Native silicon has low binding potential. Through titrated reactive ion etching (RIE), varying surface areas have been created on silicon in order to enhance protein binding. Further, via chemical modification, reactive groups have been added to the surfaces for comparison of relative protein binding. Using this combinatorial method of surface roughening and surface coating, 3-aminopropyltriethoxysilane (APTES) and mercaptopropyltrimethoxysilane (MPTMS) treatments were shown to transform native silicon into a protein-binding substrate comparable to nitrocellulose.
Keywords: Protein adsorption; Silicon; Surface modification; Surface treatment;

A multicellular spheroid array to realize spheroid formation, culture, and viability assay on a chip by Yu-suke Torisawa; Airi Takagi; Yuji Nashimoto; Tomoyuki Yasukawa; Hitoshi Shiku; Tomokazu Matsue (559-566).
We describe a novel multicellular spheroid culture system that facilitates the easy preparation and culture of a spheroid microarray for the long-term monitoring of cellular activity. A spheroid culture device with an array of pyramid-like microholes was constructed in a silicon chip that was equipped with elastomeric microchannels. A cell suspension was introduced via the microfluidic channel into the microstructure that comprised silicon microholes and elastomeric microwells. A single spheroid can be formed and localized precisely within each microstructure. Since the culture medium could be replaced via the microchannels, a long-term culture (of ∼2 weeks) is available on the chip. Measurement of albumin production in the hepatoma cell line (HepG2) showed that the liver-specific functions were maintained for 2 weeks. Based on the cellular respiratory activity, the cellular viability of the spheroid array on the chip was evaluated using scanning electrochemical microscopy. Responses to four different chemical stimulations were simultaneously detected on the same chip, thus demonstrating that each channel could be evaluated independently under various stimulation conditions. Our spheroid culture system facilitated the understanding of spheroid formation, culture, and viability assay on a single chip, thus functioning as a useful drug-screening device for cancer and liver cells.
Keywords: Biosensor; Microfluidic device; Spheroids; Cytotoxicity; Electrochemistry; Polydimethylsiloxane;