Biomaterials (v.26, #29)

Calendar (I).

Although addition of an antioxidant ( α -tocopherol) is reported to prevent delamination in ultrahigh molecular weight polyethylene (UHMWPE) knee components, contribution of α -tocopherol as an antioxidant to the improvement of long-term fatigue performance of UHMWPE is an unknown mechanism. To solve this problem, bi-directional sliding fatigue tests were performed for γ -irradiated (25 kGy), γ -irradiated (25 kGy) with 0.3 wt% α -tocopherol added, and γ -irradiated (25 kGy) with 0.3 wt% tocopheryl acetate added UHMWPE specimens. Internal defect initiation was quantified with scanning acoustic tomography (SAT). Also, oxidation index and crystallinity were obtained from infrared absorption spectra measured using Fourier transform infrared (FT-IR) microscopy. Only γ -irradiated UHMWPE specimens resulted in severe fatigue fractures. α -Tocopherol-added UHMWPE specimens showed significantly lower projected area ratio of defects (1.80±0.82) than did γ -irradiated (7.0±2.29) and tocopheryl acetate-added ones (8.50±2.01). The oxidation index of γ -irradiated UHMWPE specimens (0.111±0.0052) was extremely higher compared to those of doped ones; 0.0179±0.0026 and 0.0144±0.0069 for α -tocopherol-added and tocopheryl acetate-added ones, respectively. The crystallinity of γ -irradiated UHMWPE specimens (57.5±1.16) was lower compared to those of doped ones; 60.3±0.72 and 60.4±1.38 for α -tocopherol-added and tocopheryl acetate-added ones, respectively. The incorporation of α -tocopherol significantly improves the long-term fatigue performance of γ -irradiated UHMWPE with oxidation stability. Also, the addition of α -tocopherol controls macromolecular structures resulting in the improvement of fatigue performance of UHMWPE.
Keywords: UHMWPE; γ -irradiation; Oxidation; Delamination; Tocopherol; Crystallinity;

Hydroxyapatite (Ca5(PO4)3(OH)) forms on pseudowollastonite (psW) (α-CaSiO3) in vitro in simulated body fluid, human parotid saliva and cell-culture medium, and in vivo in implanted rat tibias. We used crystallographic constraints with ab initio molecular orbital calculations to identify the active site and reaction mechanism for heterogeneous nucleation of the earliest calcium phosphate oligomer/phase. The active site is the planar, cyclic, silicate trimer (Si3O9) on the (0 0 1) face of psW. The trimer has three silanol groups (>SiOH) arranged at 60° from each other, providing a stereochemical match for O atoms bonded to Ca2+ on the (0 0 1) face of hydroxyapatite. Calcium phosphate nucleation is modeled in steps as hydrolysis of surface Ca–O bonds with leaching of Ca2+ into solution, protonation of the surface Si–O groups to form silanols, calcium sorption as an inner-sphere surface complex and, attachment of HPO4 2−. Our model explains the experimental solution and high resolution transmission electron microscopy data for epitaxial hydroxyapatite growth on psW in vitro and in vivo. We propose that the cyclic silicate trimer is the universal active site for heterogeneous, stereochemically promoted nucleation on silicate-based bioactive ceramics. A critical active site-density and a point of zero charge of the bioceramic less than physiological pH are required for bioactivity.
Keywords: Bioceramics; Calcium silicate; Calcium phosphate; Silicate trimer; Titania; Alumina; Silica; Bioactivity; Glass;

The effect of crystallinity on the deformation mechanism and bulk mechanical properties of PLLA by Annette C. Renouf-Glauser; John Rose; David F. Farrar; Ruth Elizabeth Cameron (5771-5782).
Poly (l-lactide) is a widely studied biomaterial, currently approved for use in a range of medical devices, however, most in vitro studies have so far focussed upon either the bulk properties during degradation and/or deformation, or on the microstructure of the unloaded material during degradation. This study aimed to combine these approaches through the technique of simultaneous small-angle X-ray scattering and tensile testing at various stages of degradation up to 8 months, on material with a range of induced microstructures. Results showed that the amorphous material deformed by crazing in the dry, wet and degraded states, however, the mechanism by which the craze developed changed significantly on hydration. Despite this difference, there was little change in the bulk mechanical properties. Crystalline materials deformed through crystal-mediated deformation, with contributions from both cavitation and fibrillated shear, but surprisingly, differences in the length scales within the spherulitic structure caused by annealing at different temperatures had very little effect on the mechanism of deformation, though differences were seen in the bulk properties. Furthermore, hydration had little effect on the crystalline materials, though degradation over 8 months resulted in loss of mechanical properties for samples produced at higher annealing temperatures. In conclusion, the introduction of crystallinity had a huge effect on both bulk and microscopic properties of PLLA, but the spherulitic structure of the crystalline material affected the bulk properties significantly more than it did the micromechanism of deformation.
Keywords: Polylactic acid; Degradation; XRD (X-ray diffraction); Mechanical properties; Crystallinity;

Promotion of bone formation by simvastatin in polyethylene particle-induced osteolysis by Fabian von Knoch; Christian Wedemeyer; Anja Heckelei; Guido Saxler; Gero Hilken; Jochen Brankamp; Thomas Sterner; Stefan Landgraeber; Frank Henschke; Franz Löer; Marius von Knoch (5783-5789).
The effects of statins on bone formation in periprosthetic osteolysis have not been determined to date. We investigated the effect of the HMG-CoA reductase inhibitor simvastatin on osteoblastic bone formation under conditions of ultra-high molecular weight polyethylene (UHMWPE) particle-induced osteolysis.The murine calvarial osteolysis model was utilized in 21 C57BL/J6 mice randomized to three groups. Group I underwent sham surgery only, group II received UHMWPE particles, and group III, particles and simvastatin treatment. After 2 weeks, calvaria were processed for histomorphometry and stained with Giemsa dye. New bone formation was measured as osteoid tissue area within the midline suture. Bone thickness was quantified as indicator of net bone growth. Statistical analysis was performed using one-way ANOVA and a Student's t-test.New bone formation and bone thickness were significantly enhanced following simvastatin treatment. New bone formation was 0.008±0.008 mm2 in sham controls (group I), 0.015±0.012 mm2 after particle implantation without further intervention (group II), compared to 0.083±0.021 mm2 with particle implantation and simvastatin treatment (group III) ( p = 0.003 ) . The bone thickness was 0.213±0.007 mm in group I, 0.183±0.005 mm in group II, and 0.238±0.009 mm in group III ( p = 0.00008 ) .In conclusion, simvastatin treatment markedly promoted bone formation and net bone growth in UHMWPE particle-induced osteolysis in a murine calvarial model. These new findings indicate that simvastatin may have favorable osteoanabolic effects on wear debris-mediated osteolysis after total joint arthroplasty, involving local stimulation of osteoblastic bone formation.
Keywords: Osteolysis; Wear debris; Polyethylene; Calvarium; Osteoblast;

Efficient characterisation of human cell–bioceramic interactions in vitro and in vivo by using enhanced GFP-labelled mesenchymal stem cells by Zhidao Xia; Hua Ye; Rachel M. Locklin; David J.P. Ferguson; Zhanfeng Cui; James T. Triffitt (5790-5800).
Human mesenchymal stem cells (hMSCs) were transfected using four retroviral pseudotypes, amphotropic murine leukemia viruses 4070 (MuLV-10A1), a modification of amphotropic pseudotype 4073 (A71G, Q74K, V139M), gibbon ape leukemia virus (GaLV), or feline endogenous virus (RD114) encoding the neomycin resistance (Neor) gene and enhanced green fluorescent protein (eGFP) as genetic markers. It was observed that the MuLV4073 was the most efficient pseudotype for hMSC transfection. The proliferation and differentiation characteristics of eGFP-labelled hMSCs were not significantly different from control hMSCs. G418 selected eGFP-labelled cells were cultured for 3 weeks on two porous, commercially available calcium phosphate bioceramics, a “synthetic hydroxyapatite” and a “deproteinised bone”, before implantation into NOD/SCID mice for up to 4 weeks. The eGFP-labelled hMSCs could be readily visualised by their intense green fluorescence both in vitro and in vivo. In “synthetic hydroxyapatite” implants the cells remained in a monolayer, whereas in “deproteinised bone” implants mineralised tissues were detected by histology, scanning electron microscopy and energy dispersive X-ray spectrometry. From the results, it is concluded that the use of eGFP-labelled hMSCs is an effective tool to trace the fate of hMSCs and evaluate the interactions between cells and ceramics both in vitro and in vivo. This is of great value in prospective assessments of these cell populations for use in tissue engineering applications.
Keywords: Human mesenchymal stem cells (hMSCs); Retroviral transfection; Enhanced green fluorescent protein (eGFP); Murine leukemia viruses (MuLV); Gibbon ape leukemia virus (GaLV); Feline endogenous virus (RD114); Hydroxyapatite ceramics;

Morphological characterization and in vitro biocompatibility of a porous nickel–titanium alloy by Oleg Prymak; Denise Bogdanski; Manfred Köller; Stefan A. Esenwein; Gert Muhr; Felix Beckmann; Tilmann Donath; Michel Assad; Matthias Epple (5801-5807).
Disks consisting of macroporous nickel–titanium alloy (NiTi, Nitinol®, Actipore®) are used as implants in clinical surgery, e.g. for fixation of spinal dysfunctions. The morphological properties were studied by scanning electron microscopy (SEM) and by synchrotron radiation-based microtomography (SRμCT). The composition was studied by X-ray diffractometry (XRD), differential scanning calorimetry (DSC), and energy-dispersive X-ray spectroscopy (EDX). The mechanical properties were studied with temperature-dependent dynamical mechanical analysis (DMA). Studies on the biocompatibility were performed by co-incubation of porous NiTi samples with isolated peripheral blood leukocyte fractions (polymorphonuclear neutrophil granulocytes, PMN; peripheral blood mononuclear leukocytes, PBMC) in comparison with control cultures without NiTi samples. The cell adherence to the NiTi surface was analyzed by fluorescence microscopy and scanning electron microscopy. The activation of adherent leukocytes was analyzed by measurement of the released cytokines using enzyme-linked immunosorbent assay (ELISA). The cytokine response of PMN (analyzed by the release of IL-1ra and IL-8) was not significantly different between cell cultures with or without NiTi. There was a significant increase in the release of IL-1ra ( p < 0.001 ), IL-6 ( p < 0.05 ), and IL-8 ( p < 0.05 ) from PBMC in the presence of NiTi samples. In contrast, the release of TNF-α by PBMC was not significantly elevated in the presence of NiTi. IL-2 was released from PBMC only in the range of the lower detection limit in all cell cultures. The material, clearly macroporous with an interconnecting porosity, consists of NiTi (martensite; monoclinic, and austenite; cubic) with small impurities of NiTi2 and possibly NiC x . The material is not superelastic upon manual compression and shows a good biocompatibility.
Keywords: Shape memory alloys; Biocompatibility; Cytokines; Leukocytes; Porosity; Nickel–titanium alloys;

Morphological and topographic effects on calcification tendency of pHEMA hydrogels by Xia Lou; Sarojini Vijayasekaran; Runi Sugiharti; Terry Robertson (5808-5817).
Poly(2-hydroxyethyl methacrylate) hydrogels were prepared in the presence of varying concentrations of water, or a co-monomer ethoxyethyl methacrylate at different strengths of crosslinking agent ethylene glycol dimethacrylate. Calcification tendency and its correlation with monomer mixture composition, topography and porosity of these materials were investigated.Scanning (SEM) and transmission electron microscopy (TEM) was used to study topography and porosity respectively. Calcification and calcium diffusion ability in to the hydrogels were investigated by light microscopy, SEM and energy dispersive analysis of X-rays (EDAX) after incubation of the materials in a metastable calcifying solution for 48 days. Polymer and solvent volume fractions were also studied to determine if a correlation existed between porosity and calcification.Most of the series of hydrogels showed surface irregularities. Internal structure showed evidence of a porous structure in one of the series. Calcification studies indicated diffusion of calcium ions in some of the series. The diffusion of calcium is limited to 30–40 μm in most calcified specimens. For hydrogels that exhibited substantial surface irregularities and micro channels, the infiltration of calcium up to 200 μm was observed. Attempts to detect porosity by electron microscopy failed in some of the hydrogels due to difficulty in sample processing and sectioning. However, collaboration of the results with different techniques used, indicated that surface defects are the major contributors to calcium deposition. Decrease in porosity reduces the amount of calcium deposits and infiltration with decreasing solvent volume fraction which is associated with crosslinking concentration and initial water content of the polymer.
Keywords: pHEMA; Porosity; Calcification; SEM; TEM; Topography;

The safety and toxicity of nanoparticles are of growing concern despite their significant scientific interests and promising potentials in many applications. The properties of nanoparticles depend not only on the size but also the structure, microstructure and surface coating. These in turn are controlled by the synthesis and processing conditions. The dependence of cytotoxicity on particle size and on the presence of oleic acid as surfactant on nickel ferrite particles were investigated in vitro using the Neuro-2A cell line as a model. For nickel ferrite particles without oleic acid prepared by ball milling, cytotoxicity was independent of particle size within the given mass concentrations and surface areas accessible to the cells. For nickel ferrite particles coated with oleic acid prepared by the polyol method, the cytotoxicity significantly increased when one or two layers of oleic acid were deposited. Large particles (150±50 nm diameter) showed a higher cytotoxicity than smaller particles (10±3 nm diameter).
Keywords: Cytotoxicity; Surface treatment; Particle size; Magnetic nanoparticles;

Transitory oxidative stress in L929 fibroblasts cultured on poly( ε -caprolactone) films by M. Concepción Serrano; Raffaella Pagani; Juan Peña; M. Teresa Portolés (5827-5834).
Poly( ε -caprolactone) (PCL) is considered as a potential substrate for wide medical applications. In previous studies we carried out the in vitro biocompatibility assessment of PCL films using L929 mouse fibroblasts, obtaining good cell behaviour but a transitory stimulation of mitochondrial activity and cell retraction. Reactive oxygen species (ROS), mainly formed in mitochondria, can impair the function of several cellular components and produce cell oxidative stress by changing the normal red-ox status of the major cell antioxidants as glutathione. The aim of this study was to measure intracellular ROS production and glutathione content of L929 fibroblasts cultured on PCL films. Cell size, internal complexity, cell cycle and lactate dehydrogenase release were also evaluated. The films were treated with NaOH before culture to improve the cell–polymer interaction. PCL induces a transitory but significant oxidative stress in L929 fibroblasts. The treatment of PCL films with NaOH reduces this effect. PCL also induces transitory changes on cell size and complexity. Nevertheless, after 7 days in culture, cells reach control levels for all the studied parameters. Neither cell cycle nor membrane integrity appears affected by this oxidative stress respect to control cells at any culture time. These results underline the cytocompatibility of PCL films and, therefore, its potential utility as a suitable scaffold in tissue engineering.
Keywords: Polycaprolactone; Biocompatibility; Oxidative stress; Glutathione; Lactate dehydrogenase; Fibroblast;

The role of osteopontin in foreign body giant cell formation by Annabel T. Tsai; Julie Rice; Marta Scatena; Lucy Liaw; Buddy D. Ratner; Cecilia M. Giachelli (5835-5843).
Foreign body giant cells (FBGCs) are a hallmark of the foreign body reaction caused by biomaterial implantation and are thought to contribute to biomaterial degradation and the duration of the response. Osteopontin (OPN) is a secreted, acidic matricellular protein with multiple phosphorylation sites that is highly expressed at sites of inflammation. OPN wildtype and knockout mice were implanted with poly(vinyl alcohol) sponges and explanted at 14 days. OPN knockout mice had more foreign body giant cells but fewer macrophages surrounding the implants than their wildtype counterparts. In an in vitro human FBGC assay, addition of soluble OPN was found to reduce macrophage fusion to giant cells. These are the first studies to show a direct inhibitory role of OPN in FBGC formation in response to implantation.
Keywords: Osteopontin; Knockout mice; Macrophages; Foreign body reaction; Foreign body giant cells;

The biocompatibility of dibutyryl chitin in the context of wound dressings by Riccardo A.A. Muzzarelli; Mario Guerrieri; Gaia Goteri; Corrado Muzzarelli; Tatiana Armeni; Roberto Ghiselli; Maria Cornelissen (5844-5854).
Dibutyryl chitin (DBC) is a modified chitin carrying butyryl groups at 3 and 6 positions; its peculiarity is that it dissolves promptly in common solvents, while being insoluble in aqueous systems. The high biocompatibility of dibutyryl chitin in the form of films and non-wovens has been demonstrated for human, chick and mouse fibroblasts by the Viability/Cytotoxicity assay, In situ Cell Proliferation assay, Neutral Red Retention assay, Lactate Dehydrogenase Release assay, MTS cytotoxicity assay, and scanning electron microscopy. DBC was hardly degradable by lysozyme, amylase, collagenase, pectinase and cellulase over the observation period of 48 days at room temperature, during which no more than 1.33% by weight of the DBC filaments (0.3 mm diameter) was released to the aqueous medium. DBC non-wovens were incorporated into 5-methylpyrrolidinone chitosan solution and submitted to freeze-drying to produce a reinforced wound dressing material. The latter was tested in vivo in full thickness wounds in rats. The insertion of 4×4 mm pieces did not promote any adverse effect on the healing process, as shown histologically. DBC is therefore suitable for contacting intact and wounded human tissues.
Keywords: Dibutyryl chitin; Methylpyrrolidinone chitosan; Wound dressing; Fibroblast; Biocompatibility;

The reconstruction of soft tissue defects remains a challenge in plastic and reconstructive surgery, and a real clinical need exists for an adequate solution. This study was undertaken in order to differentiate mesenchymal stem cells (MSCs) into adipocytes, and to then assess the possibility of constructing adipose tissue via the attachment of MSCs to injectable PLGA spheres. We also designed injectable PLGA spheres for scar-free transplantation. In this study, MSCs and adipo-MSCs (MSCs cultured in adipogenic medium for 7 days) were attached to PLGA spheres and cultured for 7 days, followed by injection into nude mice for 2 weeks. As a result, the difference between lipid accumulation in adipo-MSCs at 1 and 7 days was much higher in vitro than in the MSCs. Two weeks after injection, a massive amount of new tissue was formed in the APLGA group, whereas only a small amount was formed in the MPLGA group. We verified that the newly formed tissue originated from the injected MSCs via GFP testing, and confirmed that the created tissue was actual adipose tissue by oil red O staining and Western blot (PPARγ and C/EBPα were expressed only in APLGA groups). Therefore, this study presents an efficient model of adipose tissue engineering using MSCs and injectable PLGA spheres.
Keywords: Adipose tissue engineering; PLGA; Mesenchymal stem cell; Injectable scaffold;

Fabrication of viable tissue-engineered constructs with 3D cell-assembly technique by Yongnian Yan; Xiaohong Wang; Yuqiong Pan; Haixia Liu; Jie Cheng; Zhuo Xiong; Feng Lin; Rendong Wu; Renji Zhang; Qingping Lu (5864-5871).
We have recently developed an organ manufacturing technique that enables us to form cell/biomaterial complex three-dimensional (3D) architectures in designed patterns. This technique employs a highly accurate 3D micropositioning system with a pressue-controlled syringe to deposit cell/biomaterial structures with a lateral resolution of 10 μm. The pressure-activated micro-syringe is equipped with a fine-bore exit needle using which a wide variety of 3D patterns with different arrays of channels (through-holes) were created. The channels can supply living cells with nutrients and allow removing the cell metabolites. The embedded cells remain viable and perform biological functions as long as the 3D structures are retained. The new technology has the potential for eventual high-throughput production of artificial human tissues and organs.
Keywords: Cell assembling; Rapid prototyping; Three-dimensional precursor; Implantable bioartificial organs; Hydrogel;

Controlling cell adhesion and degradation of chitosan films by N-acetylation by Thomas Freier; Hui Shan Koh; Karineh Kazazian; Molly S. Shoichet (5872-5878).
As part of our ongoing effort to develop a biodegradable nerve guidance channel based on chitin/chitosan, we conducted a systematic in vitro study on the biodegradation and neural cell compatibility of chitosan and N-acetylated chitosan. The in vitro degradation (pH 7.4, 37 °C) in the presence of 1.5 μg/ml lysozyme showed a progressive mass loss to greater than 50% within 4 weeks for films with 30–70% acetylation. In contrast, the degradation of samples with very low or high acetylation was minimal over the 4-week period. Neural cell compatibility of chitosan and N-acetylated chitosan was tested using primary chick dorsal root ganglion (DRG) neurons. All chitosan-based films showed DRG cell adhesion after 2 days of culture. However, cell viability decreased with increasing acetylation. Chitosan that was 0.5% acetylated had the greatest cell viability, which was approximately 8 times higher than that of chitosan that was 11% acetylated. Chitosan with 0.5% and 11% acetylation showed more and longer neurites than the other samples studied. Thus chitosan amine content can be tuned for optimal biodegradation and cell compatibility, which are important for tissue engineering in the nervous system.
Keywords: Chitosan; Chitin; Enzymatic degradation; Nerve regeneration; Tissue engineering;

Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier by Philip Kasten; Julia Vogel; Reto Luginbühl; Philip Niemeyer; Marcus Tonak; Helga Lorenz; Lars Helbig; Stefan Weiss; Jörg Fellenberg; Albrecht Leo; Hans-Georg Simank; Wiltrud Richter (5879-5889).
Bone substitute materials can induce bone formation in combination with mesenchymal stem cells (MSC). The aim of the current study was to examine ectopic in vivo bone formation with and without MSC on a new resorbable ceramic, called calcium deficient hydroxyapatite (CDHA). Ceramic blocks characterized by a large surface (48 m2/g) were compared with β -tricalcium phosphate ( β -TCP), hydroxyapatite (HA) ceramics (both ca. 0.5 m2/g surface) and demineralized bone matrix (DBM). Before implantation in the back of SCID mice carriers were freshly loaded with 2×105 expanded human MSC or loaded with cells and kept under osteogenic conditions for two weeks in vitro. Culture conditions were kept free of xenogenic supplements. Deposits of osteoid at the margins of ceramic pores occurred independent of osteogenic pre-induction, contained human cells, and appeared in 4 16 MSC/CDHA composites compared to 2 16 MSC/ β -TCP composites. ALP activity was significantly higher in samples with MSC versus empty controls ( p < 0.001 ). Furthermore, ALP was significantly ( p < 0.05 ) higher for all ceramics when compared to the DBM matrix. Compared to previous studies, overall bone formation appeared to be reduced possibly due to the strict human protocol. Ectopic bone formation in the novel biomaterial CDHA varied considerably with the cell pool and was at least equal to β -TCP blocks.
Keywords: Bone healing; Biocompatibility; Bone; Bone regeneration; Bone tissue engineering; Calcium phosphate; Ceramic structure; Composite; Hydroxyapatite; In situ hybridization; In vivo test; Mesenchymal stem cell; Osteoblast; Osteoconduction; Osteogenesis; Platelet; Progenitor cell; Porosity; Surface roughness;

The design and production of Co–Cr alloy implants with controlled surface topography by CAD–CAM method and their effects on osseointegration by John Alan Hunt; Jill T. Callaghan; Chris J. Sutcliffe; Rhys H. Morgan; Ben Halford; Richard A. Black (5890-5897).
Improved fixation and increased longevity are still important performance criteria in the development of orthopaedic prostheses. The osseointegration of a series of implant designs made of conventional cobalt–chromium alloy was investigated, the shape of each implant being the critical variable. The shape was defined by computer-aided design with a view to maximising interdigitation of new bone with the implant. Two different process routes, conventional casting and selective laser sintering were employed, each process yielded implants that had identical surface topology but different microstructures. Hydroxyapatite (HA) was used to coat some samples by plasma spraying. Bone formation associated with each implant design was delineated through the administration of fluorescent vital dyes at three time points following their implantation into New Zealand white rabbits. After one month, specimens were harvested, resin embedded, serial sectioned and examined under fluorescent light microscopy. The amount of bone growth was quantified using image analysis. Plasma spray HA-coated samples promoted better osteogenesis and integration than uncoated samples. The extent of bone growth associated with identically shaped specimens fabricated by the SLS route was markedly greater, attributed to the microstructure of these implants.
Keywords: Athroplasty; Osteoconduction; Cobalt alloy; Laser manufacturing; Hydroxyapatite coating; Rapid prototyping; Surface topography;

Selective targeting of antibody-conjugated nanoparticles to leukemic cells and primary T-lymphocytes by Norbert Dinauer; Sabine Balthasar; Carolin Weber; Jörg Kreuter; Klaus Langer; Hagen von Briesen (5898-5906).
In the present study, surface-modified nanoparticles based on biodegradable material were used for antibody coupling in order to get a selective drug carrier systems. Gelatin nanoparticles were prepared by a desolvation process. Sulfhydryl groups were introduced which enabled the linkage of NeutrAvidin (NAv). Antibodies specific for the CD3 antigen on lymphocytic cells were conjugated to the nanoparticles surface. The binding of biotinylated anti-CD3 antibody was achieved by NAv–biotin-complex formation. Cellular binding and uptake were determined by flow cytometry and confocal laser scanning microscopy (CLSM). Cell-type-specific targeting of anti-CD3-conjugated nanoparticles into CD3-positive human T-cell leukemia cells and primary T-lymphocytes could be shown. Celluar uptake and effective internalization of antibody-conjugated nanoparticles into CD3 expressing cells were demonstrated. Uptake rates of about 84% into T-cell leukemia cells were observed. To confirm selectivity of T-cell targeting, competition experiments were carried out adding excessive free anti-CD3 prior to nanoparticle incubation leading to significantly reduced cellular uptake of antibody-conjugated nanoparticles. Further analysis on the mechanism of uptake confirmed a receptor-mediated endocytotic process. Protein-based nanoparticles conjugated with an antibody against a specific cellular antigen hold promise as selective drug delivery systems for specific cell types.
Keywords: Nanoparticles; Drug targeting; T-lymphocytic leukemia cells; T-lymphocytes; Cellular uptake; Antibodies;

Interaction of various pectin formulations with porcine colonic tissues by LinShu Liu; Marshall L. Fishman; Kevin B. Hicks; Meir Kende (5907-5916).
Pectins of low and high degrees of esterification, as well as pectin derivatives carrying primary amines, were investigate for gel forming ability with mucosal tissues. The combination of scanning electronic microscopy and small deformation dynamic mechanical studies revealed that pectins with higher net electrical charges are more bioadhesive than the less charged ones. Both the negatively charged pectin formulation, P-25, and the positively charged formulation, P-N, were able to synergize with the mucus to produce rheologically strengthened gels. The highly esterified pectin, P-94, also synergized with the mucosal glycoproteins to form a gel structure via coil entanglements. The ex vivo studies further confirmed the microstructures of mucus gel networks with adsorbed pectins. When incubated with porcine intestinal mucus membrane, P-94 gels were found generally bound to the lumen area, P-25 gels were able to penetrate deeply near the wall area, P-N gels interacted with mucins via electrostatic bonding and dispersed into the whole area from the lumen to the wall. Hence, both P-N and P-94, by enhancing the protective barrier properties of mucus systems, may be useful alternatives for the treatment of mucus related irritation and infection. In drug-delivery systems, P-N and P-25 would deliver incorporated drugs mainly by pectin dissolution, while a diffusion mechanism would release drugs from P-94 gels.
Keywords: Pectin; Mucin; Drug delivery; Bioadhesiveness;

An inverted microcontact printing method on topographically structured polystyrene chips for arrayed micro-3-D culturing of single cells by Marc R. Dusseiller; Dominik Schlaepfer; Mirabai Koch; Ruth Kroschewski; Marcus Textor (5917-5925).
With the goal to investigate the relation of shape and function of single cells or clusters of cells in a 3-dimensional (3-D) microenvironment, we present a novel platform technology to create arrays of microwells on polystyrene (PS) chips for hosting cells in a local microenvironment characterized by controlled shape and surface chemistry. The micro-3-D cell culturing combines 2-dimensional chemical patterning with topographical microstructuring presenting to the cells a local 3-D host structure. Microwells of controlled dimensions were produced by a two-step replication process, based on standard microfabrication of Si, replica molding into poly(dimethylsiloxane), and hot embossing of PS. This allowed the production of large numbers of microstructured surfaces with high reproducibility and fidelity of replication. Using inverted micro contact printing, the plateau surface between the microwells was successfully passivated to block adsorption of proteins and prevent cell attachment by transfer of a graft-copolymer, poly(l-lysine)-g-poly(ethylene glycol). The surface inside the microwells was subsequently modified by spontaneous adsorption of proteins or functionalized PLL-g-PEG/PEG-X (X=biotin or specific, cell-interactive peptide) to elicit specific responses inside the wells. Preliminary cell experiments demonstrated the functionality of such a device to host single epithelial cells (MDCK II) inside the functionalized microwells and thus to control their 3-D shape. This novel platform is useful for fundamental cell-biological studies and applications in the area of cell-based sensing.
Keywords: Fast prototyping; Microfabrication; Surface modification; Microstructured surfaces; Single cell analysis;