Biomaterials (v.31, #9)

A biodegradable, injectable, gel system based on MPEG-b-(PCL-ran-PLLA) diblock copolymers with an adjustable therapeutic window by Yun Mi Kang; Sang Hyo Lee; Ju Young Lee; Jin Soo Son; Byung Soo Kim; Bong Lee; Heung Jae Chun; Byoung Hyun Min; Jae Ho Kim; Moon Suk Kim (2453-2460).
In situ-forming gel systems have drawn increasing attention for their potential use in a variety of biomedical applications. Here, we examined an in situ-forming gel system comprised of MPEG-b-PCL and MPEG-b-(PCL-ran-PLLA) diblock copolymers with different PLLA contents (0–10 mol%) in the PCL segment. The crystalline region of the PCL-ran-PLLA segment decreased with increasing PLLA content. The MPEG-b-(PCL-ran-PLLA) diblock copolymer solutions were liquid at room temperature and only MPEG-b-(PCL-ran-PLLA) diblock copolymer solutions with a PLLA content ≤5 mol% in the PCL segment showed a sol-to-gel transition as the temperature was increased. The viscosity change associated with sol-to-gel phase transition depended on the PLLA content in the PCL segment. A MPEG-b-PCL diblock copolymer solution incubated in vitro showed increasing viscosity without degradation, whereas the viscosity of MPEG-b-(PCL-ran-PLLA) diblock copolymer solutions continuously and sharply decreased with increasing PLLA content in the PCL segment. As the amount of PLLA increased, the size of in vivo-formed MPEG-b-(PCL-ran-PLLA) gels after initial injection tended to gradually decrease because of hydrolytic degradation of the PLLA in the PCL-ran-PLLA segment. An immunohistochemical examination showed that in vivo MPEG-b-(PCL-ran-PLLA) diblock copolymer gels provoked only a modest inflammatory response. Collectively, our results show that the MPEG-b-(PCL-ran-PLLA) diblock copolymer gel described here could serve as a minimally invasive, therapeutic, in situ-forming gel system that offers an experimental window adjustable from a few weeks to a few months.
Keywords: In situ-forming gel; Phase transition; MPEG-b-(PCL-ran-PLLA) diblock copolymer; Degradation;

The osteogenic effect of electrosprayed nanoscale collagen/calcium phosphate coatings on titanium by Lise T. de Jonge; Sander C.G. Leeuwenburgh; Jeroen J.J.P. van den Beucken; Joost te Riet; Willeke F. Daamen; Joop G.C. Wolke; Dieter Scharnweber; John A. Jansen (2461-2469).
For orthopedic and dental implants, the ultimate goal is to obtain a life-long secure anchoring of the implant in the native surrounding bone. To this end, nanoscale calcium phosphate (CaP) and collagen-CaP (col-CaP) composite coatings have been successfully deposited using the electrospray deposition (ESD) technique. In order to study to what extent the thickness of these coatings can be reduced without losing coating osteogenic properties, we have characterized the mechanical and biological coating properties using tape tests (ASTM D-3359) and in vitro cell culture experiments, respectively. Co-deposition of collagen significantly improved coating adhesive and cohesive strength, resulting in a remarkably high coating retention of up to 97% for coating thicknesses below 100 nm. In vitro cell culture experiments showed that electrosprayed CaP and col-CaP composite coatings enhanced osteoblast differentiation, leading to improved mineral deposition. This effect was most pronounced upon co-deposition of collagen with CaP, and these coatings displayed osteogenic effects even for a coating thickness of below 100 nm.
Keywords: Calcium phosphate; Collagen; Nanocomposite; Biofilm;

Using the material technique recently developed by us, we prepared a micropattern on poly(ethylene glycol) (PEG) hydrogel to keep background resistant to cell adhesion for a long time, which made examination of differentiation of localized stem cells available. Our micropattern designed in this paper prevented or ensured contact between cells adhering in arginine-glycine-aspartic acid (RGD) microdomains, and thus afforded a unique way to study the effects of cell-cell contact on the lineage differentiation of stem cells while ruling out the interference of soluble factors or cell seeding concentration etc. As demonstration, mesenchymal stem cells derived from rats were examined in this study, and both osteogenic and adipogenic differentiations were found to be regulated by cell-cell contact. Isolated cells exhibited less significant differentiation than paired or aggregated cells. For those stem cells in contact, the extent of differentiation was fairly linearly related to the extent of contact characterized by coordination number. Additionally, we revealed the existence of some unknown cues besides gap junction responsible for such effects of cell-cell contact.
Keywords: Micropattern; Mesenchymal stem cell; Cell-cell contact; Differentiation; Hydrogel;

A nanostructured carbon-reinforced polyisobutylene-based thermoplastic elastomer by Judit E. Puskas; Elizabeth A. Foreman-Orlowski; Goy Teck Lim; Sara E. Porosky; Michelle M. Evancho-Chapman; Steven P. Schmidt; Mirosława El Fray; Marta Piątek; Piotr Prowans; Krystal Lovejoy (2477-2488).
This paper presents the synthesis and characterization of a polyisobutylene (PIB)-based nanostructured carbon-reinforced thermoplastic elastomer. This thermoplastic elastomer is based on a self-assembling block copolymer having a branched PIB core carrying –OH functional groups at each branch point, flanked by blocks of poly(isobutylene-co-para-methylstyrene). The block copolymer has thermolabile physical crosslinks and can be processed as a plastic, yet retains its rubbery properties at room temperature. The carbon-reinforced thermoplastic elastomer had more than twice the tensile strength of the neat polymer, exceeding the strength of medical grade silicone rubber, while remaining significantly softer. The carbon-reinforced thermoplastic elastomer displayed a high T g of 126 °C, rendering the material steam-sterilizable. The carbon also acted as a free radical trap, increasing the onset temperature of thermal decomposition in the neat polymer from 256.6 °C to 327.7 °C. The carbon-reinforced thermoplastic elastomer had the lowest water contact angle at 82° and surface nano-topography. After 180 days of implantation into rabbit soft tissues, the carbon-reinforced thermoplastic elastomer had the thinnest tissue capsule around the microdumbbell specimens, with no eosinophiles present. The material also showed excellent integration into bones.
Keywords: Biomaterials; SIBS; Carbon-reinforced thermoplastic elastomer; Mechanical properties; Hydrolytic stability; In vivo biocompatibility;

Integration of statistical modeling and high-content microscopy to systematically investigate cell–substrate interactions by Wen Li Kelly Chen; Morakot Likhitpanichkul; Anthony Ho; Craig A. Simmons (2489-2497).
Cell–substrate interactions are multifaceted, involving the integration of various physical and biochemical signals. The interactions among these microenvironmental factors cannot be facilely elucidated and quantified by conventional experimentation, and necessitate multifactorial strategies. Here we describe an approach that integrates statistical design and analysis of experiments with automated microscopy to systematically investigate the combinatorial effects of substrate-derived stimuli (substrate stiffness and matrix protein concentration) on mesenchymal stem cell (MSC) spreading, proliferation and osteogenic differentiation. C3H10T1/2 cells were grown on type I collagen- or fibronectin-coated polyacrylamide hydrogels with tunable mechanical properties. Experimental conditions, which were defined according to central composite design, consisted of specific permutations of substrate stiffness (3–144 kPa) and adhesion protein concentration (7–520 μg/mL). Spreading area, BrdU incorporation and Runx2 nuclear translocation were quantified using high-content microscopy and modeled as mathematical functions of substrate stiffness and protein concentration. The resulting response surfaces revealed distinct patterns of protein-specific, substrate stiffness-dependent modulation of MSC proliferation and differentiation, demonstrating the advantage of statistical modeling in the detection and description of higher-order cellular responses. In a broader context, this approach can be adapted to study other types of cell–material interactions and can facilitate the efficient screening and optimization of substrate properties for applications involving cell–material interfaces.
Keywords: Extracellular matrix; Mesenchymal stem cell; Substrate stiffness; Mechanobiology; High-content screening;

The ability of surface characteristics of materials to trigger leukocyte tissue factor expression by Marion Fischer; Claudia Sperling; Pentti Tengvall; Carsten Werner (2498-2507).
Biomaterial-induced thrombosis is usually attributed to blood coagulation initiated by contact phase and platelet-related reactions. Considering the major role of extrinsic initiation in blood coagulation in vivo, we studied the material related-induction of this pathway by investigating the relevance of surface properties for the expression of Tissue Factor (TF), a critical initiator of the extrinsic pathway of coagulation. We incubated materials with self-assembled monolayers of alkylthiols (SAMs) displaying various ratios of –CH3, –OH, and –COOH terminations with fresh heparinized whole human blood in vitro. The transcription of TF-mRNA in leukocytes showed clear differences in relation to surface properties and increased over time. In addition, a positive correlation between TF transcription and its presence on leukocytes, granulocyte activation, and complement activation was found. Cells displaying the highest TF expression after material contact had significantly lower intracellular TF, pointing to previous TF release. Yet under the conditions of our whole blood incubation set-up within the limited time frame the observed initiation of the extrinsic pathway did not trigger blood coagulation.
Keywords: Haemocompatibility; Cell activation; Coagulation; Leukocyte; Platelet adhesion; Blood compatibility;

N-acetyl cysteine directed detoxification of 2-hydroxyethyl methacrylate by adduct formation by Giuseppina Nocca; Vincenzo D'Antò; Claudia Desiderio; Diana Valeria Rossetti; Rosa Valletta; Adriana Marquez Baquala; Helmut Schweikl; Alessandro Lupi; Sandro Rengo; Gianrico Spagnuolo (2508-2516).
Cytotoxicity of the dental resin monomer 2-hydroxyethyl methacrylate (HEMA) and the protective effects of N-acetyl cysteine (NAC) on monomer-induced cell damage are well demonstrated. The aim of our study was to analyze the hypothesis that the protection of NAC from HEMA cytotoxicity might be due to direct NAC adduct formation. To this end, using HPLC we first measured the actual intracellular HEMA concentrations able to cause toxic effects on 3T3-fibroblasts and then determined the decrease in intracellular and extracellular HEMA levels in the presence of NAC. In addition, by capillary electrophoresis coupled with mass spectrometry analysis (CE-MS), we evaluated NAC–HEMA adduct formation. HEMA reduced 3T3 cell vitality in a dose- and time-dependent manner. The concentration of HEMA inside the cells was 15–20 times lower than that added to the culture medium for cell treatment (0–8 mmol/L). In the presence of 10 mmol/L NAC, both intracellular and extracellular HEMA concentrations greatly decreased in conjunction with cytotoxicity. NAC–HEMA adducts were detected both in the presence and absence of cells. Our findings suggest that the in vitro detoxification ability of NAC against HEMA-induced cell damage occurs through NAC adduct formation. Moreover, we provide evidence that the actual intracellular concentration of HEMA able to cause cytotoxic effects is at least one magnitude lower than that applied extracellularly.
Keywords: HEMA; Dental monomers; Cytotoxicity; N-acetyl cysteine;

The biocompatability of mesoporous inorganic–organic hybrid resin films with ionic and hydrophilic characteristics by Gahee Kim; Lan Young Hong; Jungwoon Jung; Dong-Pyo Kim; Heesoo Kim; Ik Jung Kim; Jung Ran Kim; Moonhor Ree (2517-2525).
New mesoporous silicate-titania resin systems hybridized with 4,5-dihydroxy-m-benzenedisulfonic acid and poly(ethylene glycol)-dimethacrylate component were developed. These inorganic-organic hybrid resins were found to reveal highly controlled ionic and hydrophilic surface with excellent durability and adhesion onto various substrates. The resin films revealed high resistance to nonspecific adsorption of fibrinogen and to adherence by several bacterial pathogens such as Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis. Furthermore, excellent biocompatibility of the developed resins was proved by both HEp-2 cell adhesion in vitro and subcutaneous implantation in mice. The inorganic–organic hybrid resins are strongly promising for biomedical applications including biomedical devices and biosensors.
Keywords: Mesoporous inorganic–organic hybrid resins; Ionic characteristics; Hydrophilicity; Physical properties; Biocompatibility;

It is desirable that polymers used for the fabrication of prosthetic implants promote biological functions such as cellular adhesion, differentiation and viability. In this study, we have used plasma immersion ion implantation (PIII) to modify the surface of polytetrafluoroethylene (PTFE), thereby modulating the binding mechanism of collagen. The amount of collagen bound to the polymer surface following PIII-treatment was similar to that bound by non-covalent physisorption. In a manner consistent with previous enzyme and tropoelastin binding data, the collagen bound to the PIII-treated PTFE surface was resistant to sodium dodecyl sulfate (SDS) elution whilst collagen bound to the untreated surface was fully removed. This demonstrates the capability of PIII-treated surfaces to covalently attach collagen without employing chemical linking molecules. Only the collagen bound to the PIII-treated PTFE surface supported human dermal fibroblast attachment and spreading. This indicates that collagen on the PIII-treated surface possesses increased adhesive activity as compared to that on the untreated surface. Cell adhesion was inhibited by EDTA when the collagen was bound to PIII-treated PTFE, as expected for integrin involvement. Additionally this adhesion was sensitive to the conformation of the bound collagen. Increased actin cytoskeletal assembly was observed on cells spreading onto collagen-coated PIII-treated PTFE compared to the collagen-coated untreated PTFE. These data demonstrate the retention of collagen's biological properties following its attachment to PIII-treated PTFE, suggesting advantages for tissue engineering and prosthetic design.
Keywords: Cell adhesion; Collagen; ECM (extracellular matrix); Ion implantation; Polytetrafluoroethylene; Protein adsorption;

General functionalization route for cell adhesion on non-wetting surfaces by Sook Hee Ku; Jungki Ryu; Seon Ki Hong; Haeshin Lee; Chan Beum Park (2535-2541).
We present a versatile route for promoting cell adhesion and viability on various non-wetting surfaces, inspired by mussel adhesion mechanism. The oxidative polymerization of dopamine, a small designer molecule of the DOPA-K motif found in mussels, results in the formation of a poly(dopamine) ad-layer on any material surface. We found that the poly(dopamine) coating can promote cell adhesion on any type of material surfaces including the well-known anti-adhesive substrate, poly(tetrafluoroethylene). According to our results, mammalian cells well adhered and underwent general cell adhesion processes (i.e., attachment to substrate, spreading, and cytoskeleton development) on poly(dopamine)-modified surfaces, while they barely adhered and spread on unmodified non-wetting surfaces. The mussel-inspired surface functionalization strategy is extremely useful because it does not require the time-consuming synthesis of complex linkers and the process is solvent-free and non-toxic. Therefore, it can be a powerful route for converting a variety of bioinert substrates into bioactive ones.
Keywords: Cell adhesion; Non-wetting surfaces; Mussel adhesives; Poly(dopamine); Surface modification;

The goal of this study was to elucidate the control mechanisms by which exogenous proteins regulate keratinocyte growth factor (KGF) expression in fibroblasts adhered to differing substrates and thereby provide insights into both fundamental in vitro cell signaling and cell-biomaterial interaction research. A serum-free culture system in which cells maintained their proliferative capacity was established and employed. The addition of transforming growth factor- α (TGF-α), interleukin-1β (IL-1β) and platelet-derived growth factor-BB (PDGF-BB) individually showed no effect on KGF protein release, however, IL-1β addition led to increased KGF mRNA transcription, intracellular KGF protein synthesis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) release. Intracellular KGF protein synthesis and extracellular release were enhanced when fibroblasts were treated with a combination of IL-1β and PDGF-BB which suggests KGF synthesis and release are largely regulated by synergistic mechanisms. Surface-bound fibronectin-derived ligands and individual exogenous proteins promoted fibroblast adhesion to semi-interpenetrating polymer networks (sIPNs) but did not stimulate KGF release despite enhancement of KGF mRNA transcription. Additionally, serum conditioning was found to have a significant impact on KGF synthesis and the subsequent mechanisms controlling KGF release. This study demonstrates that KGF release from fibroblasts is likely regulated by multiple mechanisms involving post-transcriptional and exocytic controls which may be impacted by the presence of serum and how serum is removed from the in vitro cell environment.
Keywords: Keratinocyte growth factor; Granulocyte-macrophage colony-stimulating factor; Interpenetrating network; Interleukin-1β; Transforming growth factor-α; Platelet-derived growth factor-BB;

Impact of heart valve decellularization on 3-D ultrastructure, immunogenicity and thrombogenicity by Jianye Zhou; Olaf Fritze; Martina Schleicher; Hans-Peter Wendel; Katja Schenke-Layland; Csaba Harasztosi; Shengshou Hu; Ulrich A. Stock (2549-2554).
Decellularized xenogeneic tissue represents an interesting material for heart valve tissue engineering. The prospect objective is removal of all viable cells while preserving extracellular matrix (ECM) integrity. The major concerns of all decellularization protocols remain ECM disruption, immunogenicity and thrombogenicity. Accordingly the aim of this study was visualization of ultrastructural ECM disruption and human immune response and thrombogenicity using different decellularization protocols of porcine heart valves. Porcine pulmonary leaflets were decellularized with four different protocols: sodium deoxycholate, sodium dedecylsulfate, trypsin/EDTA, and trypsin–detergent–nuclease. Then the tissues were processed for histology and two-photon laser scanning microscopy (LSM). For thrombogenicity and immunogenicity testing tissues were incubated with human blood. The histological examination revealed no remaining cells and no significant differences in the ECM histoarchitecture in any group. LSM detected significant ECM alterations in all groups except sodium deoxycholate group with an almost completely preserved ECM. There was no increased immunogenicity between fresh and decellularized tissue. Compared to GA-fixed tissue however significantly increased immune responses and thrombogenicity was observed in all protocols. From our experiment, sodium deoxycholate enables cell removal with almost complete preservation of ECM structures. And all of these four decellularization protocols affected human immunological response and increased thrombogenicity.
Keywords: Heart valve; Biocompatibility; Immune response; Thrombogenicity laser scanning; Ultrastructure;

A hydrogel-based stem cell delivery system to treat retinal degenerative diseases by Brian G. Ballios; Michael J. Cooke; Derek van der Kooy; Molly S. Shoichet (2555-2564).
Regenerative strategies for retinal degenerative diseases are limited by poor cellular survival, distribution and integration after transplantation to the sub-retinal space. To overcome this limitations a stem cell delivery system was developed, taking advantage of the minimally-invasive, injectable and biodegradable properties of a blend of hyaluronan and methylcellulose (HAMC). The physical and biological properties of this unique HAMC formulation were studied. HAMC supported retinal stem-progenitor cell (RSPC) survival and proliferation in vitro. The blend was a viscous solution, exhibiting properties ideal for delivery to the sub-retinal space. In vivo transplantation studies in mice were carried out to investigate both the biodegradability of HAMC in the sub-retinal space over 7 days and the potential of HAMC as a cell delivery vehicle. RSPCs delivered in HAMC were more evenly distributed in the sub-retinal space than those delivered in traditional saline solutions, suggesting that HAMC is a promising vehicle for cellular delivery to the degenerating retina overcoming previously reported barriers to tissue integration in the retina such as cellular aggregation and non-contiguous distribution.
Keywords: Hyaluronan; Hydrogel; Retina; Stem cell; Cell viability; Cell spreading;

During development and regeneration of the mammalian nervous system, directional signals guide differentiating neurons toward their targets. Soluble neurotrophic molecules encode for preferential direction over long distances while the local topography is read by cells in a process requiring the establishment of focal adhesions. The mutual interaction between overlapping molecular and topographical signals introduces an additional level of control to this picture. The role of the substrate topography was demonstrated exploiting nanotechnologies to generate biomimetic scaffolds that control both the polarity of differentiating neurons and the alignment of their neurites. Here PC12 cells contacting nanogratings made of copolymer 2-norbornene ethylene (COC), were alternatively stimulated with Nerve Growth Factor, Forskolin, and 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic (8CPT-2Me-cAMP) or with a combination of them. Topographical guidance was differently modulated by the alternative stimulation protocols tested. Forskolin stimulation reduced the efficiency of neurite alignment to the nanogratings. This effect was linked to the inhibition of focal adhesion maturation. Modulation of neurite alignment and focal adhesion maturation upon Forskolin stimulation depended on the activation of the MEK/ERK signaling but were PkA independent. Altogether, our results demonstrate that topographical guidance in PC12 cells is modulated by the activation of alternative neuronal differentiation pathways.
Keywords: Topographical guidance; Neurons; Neuronal differentiation; PC12; Focal adhesions;

Coating of biomaterial scaffolds with the collagen-mimetic peptide GFOGER for bone defect repair by Abigail M. Wojtowicz; Asha Shekaran; Megan E. Oest; Kenneth M. Dupont; Kellie L. Templeman; Dietmar W. Hutmacher; Robert E. Guldberg; Andrés J. García (2574-2582).
Healing large bone defects and non-unions remains a significant clinical problem. Current treatments, consisting of auto and allografts, are limited by donor supply and morbidity, insufficient bioactivity and risk of infection. Biotherapeutics, including cells, genes and proteins, represent promising alternative therapies, but these strategies are limited by technical roadblocks to biotherapeutic delivery, cell sourcing, high cost, and regulatory hurdles. In the present study, the collagen-mimetic peptide, GFOGER, was used to coat synthetic PCL scaffolds to promote bone formation in critically-sized segmental defects in rats. GFOGER is a synthetic triple helical peptide that binds to the α2β1 integrin receptor involved in osteogenesis. GFOGER coatings passively adsorbed onto polymeric scaffolds, in the absence of exogenous cells or growth factors, significantly accelerated and increased bone formation in non-healing femoral defects compared to uncoated scaffolds and empty defects. Despite differences in bone volume, no differences in torsional strength were detected after 12 weeks, indicating that bone mass but not bone quality was improved in this model. This work demonstrates a simple, cell/growth factor-free strategy to promote bone formation in challenging, non-healing bone defects. This biomaterial coating strategy represents a cost-effective and facile approach, translatable into a robust clinical therapy for musculoskeletal applications.
Keywords: Biomimetic material; Bone regeneration; ECM (extracellular matrix); Integrin; Peptide; Scaffold;

Repair of meniscal cartilage white zone tears using a stem cell/collagen-scaffold implant by Moreica B. Pabbruwe; Wael Kafienah; John F. Tarlton; Sanjay Mistry; Dennis J. Fox; Anthony P. Hollander (2583-2591).
Injuries to the avascular region of knee meniscal cartilage do not heal spontaneously. To address this problem we have developed a new stem cell/collagen-scaffold implant system in which human adult bone marrow mesenchymal stem cells are seeded onto a biodegradable scaffold that allows controlled delivery of actively dividing cells to the meniscus surface. Sandwich constructs of two white zone ovine meniscus discs with stem cell/collagen-scaffold implant in between were cultured in vitro for 40 days. Histomorphometric analysis revealed superior integration in the stem cell/collagen-scaffold groups compared to the cell-free collagen membrane or untreated controls. The addition of TGF-β1 to differentiate stem cells to chondrocytes inhibited integration. Biomechanical testing demonstrated a significant 2-fold increase in tensile strength in all constructs using the stem cell/collagen-scaffold compared to control groups after 40 days in culture. Integration was significantly higher when collagen membranes were used that had a more open/spongy structure adjacent to both meniscal cartilage surfaces, whereas a collagen scaffold designed for osteoinduction failed to induce any integration of meniscus. In conclusion, the stem cell/collagen-scaffold implant is a potential therapeutic treatment for the repair of white zone meniscal cartilage tears.
Keywords: Meniscus repair; Mesenchymal stem cells; Cartilage tissue engineering; Osteoarthritis;

Long term performance of small-diameter vascular grafts made of a poly(ether)urethane–polydimethylsiloxane semi-interpenetrating polymeric network by Giorgio Soldani; Paola Losi; Massimo Bernabei; Silvia Burchielli; Dante Chiappino; Silvia Kull; Enrica Briganti; Dario Spiller (2592-2605).
In the past years considerable research efforts have been directed at developing more suitable synthetic vascular grafts, but small-diameter vascular grafts (SDVGs) perform less well than autogenous arterial or venous grafts. Grafts such as Dacron® and ePTFE have often been used as alternatives to autologous grafts, but they have shown poor patency rates when used in small-diameter sizes or low-flow locations. Nevertheless, despite these efforts no alternative concepts have emerged yet that promises to replace the current generation of synthetic grafts soon. The purpose of this preliminary in vivo study was to assess the blood and tissue compatibility behaviors of a novel compliant SDVGs, fabricated with a poly(ether)urethane–polydimethylsiloxane (PEtU–PDMS) semi-interpenetrating polymeric network (semi-IPN) and featuring two different porous layers in the wall thickness. Grafts were implanted according to anastomotic techniques which emulate the flow conditions clinically adopted for peripheral or aorto-coronary bypass procedures. Relatively long grafts were implanted in the common carotid artery of adult sheep and compared to standard ePTFE grafts of the same size and length implanted controlaterally. The animal experimentation showed superior handling and compliance characteristics, and patency rates of PEtU–PDMS grafts in comparison with a standard ePTFE graft, and the ability of remodelling in vivo while being gradually replaced by a natural tissue with no sign of calcification.
Keywords: Poly(ether)urethane; Polydimethylsiloxane; Semi-interpenetrating polymeric network; Small-diameter vascular grafts; Biodegradation; In vivo test;

A molecular receptor targeted, hydroxyapatite nanocrystal based multi-modal contrast agent by Anusha Ashokan; Deepthy Menon; Shantikumar Nair; Manzoor Koyakutty (2606-2616).
Multi-modal molecular imaging can significantly improve the potential of non-invasive medical diagnosis by combining basic anatomical descriptions with in-depth phenotypic characteristics of disease. Contrast agents with multifunctional properties that can sense and enhance the signature of specific molecular markers, together with high biocompatibility are essential for combinatorial molecular imaging approaches. Here, we report a multi-modal contrast agent based on hydroxyapatite nanocrystals (nHAp), which is engineered to show simultaneous contrast enhancement for three major molecular imaging techniques such as magnetic resonance imaging (MRI), X-ray imaging and near-infrared (NIR) fluorescence imaging. Monodispersed nHAp crystals of average size ∼30 nm and hexagonal crystal structure were in situ doped with multiple rare-earth impurities by a surfactant-free, aqueous wet-chemical method at 100 °C. Doping of nHAp with Eu3+ (3 at%) resulted bright near-infrared fluorescence (700 nm) due to efficient 5 D 07 F 4 electronic transition and co-doping with Gd3+ resulted enhanced paramagnetic longitudinal relaxivity (r 1 ∼12 mM−1 s−1) suitable for T 1 weighted MR imaging together with ∼80% X-ray attenuation suitable for X-ray contrast imaging. Capability of MF-nHAp to specifically target and enhance the signature of molecular receptors (folate) in cancer cells was realized by carbodiimide grafting of cell-membrane receptor ligand folic acid (FA) on MF-nHAp surface aminized with dendrigraft polymer, polyethyleneimine (PEI). The FA-PEI-MF-nHAp conjugates showed specific aggregation on FR+ve cells while leaving the negative control cells untouched. Nanotoxicity evaluation of this multifunctional nHAp carried out on primary human endothelial cells (HUVEC), normal mouse lung fibroblast cell line (L929), human nasopharyngeal carcinoma (KB) and human lung cancer cell line (A549) revealed no apparent toxicity even upto relatively higher doses of 500 μg/mL and 48 h of incubation. Flow-cytometry based reactive oxygen species (ROS) analysis also showed no significant levels of ROS generation in the nHAp treated cells. The tri-modal contrast imaging functionality together with molecular receptor targeting capability and biocompatibility makes MF-nHAp a promising biomineral contrast agent for combinatorial molecular imaging.
Keywords: Hydroxyapatite nanoparticles; Cancer targeting; Molecular imaging; Magnetic resonance imaging; Near-infrared imaging; X-ray imaging;

Photoacoustic imaging of living mouse brain vasculature using hollow gold nanospheres by Wei Lu; Qian Huang; Geng Ku; Xiaoxia Wen; Min Zhou; Dmitry Guzatov; Peter Brecht; Richard Su; Alexander Oraevsky; Lihong V. Wang; Chun Li (2617-2626).
Photoacoustic tomography (PAT) also referred to as optoacoustic tomography (OAT) is a hybrid imaging modality that employs nonionizing optical radiation and ultrasonic detection. Here, we describe the application of a new class of optical contrast agents based on mesoscopic hollow gold nanospheres (HAuNS) to PAT. HAuNS are ∼40 nm in diameter with a hollow interior and consist of a thin gold wall. They display strong resonance absorption tuned to the near-infrared (NIR) range, with an absorption peak at 800 nm, whose photoacoustic efficiency is significantly greater than that of blood. Following surface conjugation with thiolated poly(ethylene glycol), the pegylated HAuNS (PEG-HAuNS) had distribution and elimination half-lives of 1.38 ± 0.38 and 71.82 ± 30.46 h, respectively. Compared with PAT images based on the intrinsic optical contrast in nude mice, the PAT images acquired within 2 h after intravenous administration of PEG-HAuNS showed the brain vasculature with greater clarity and detail. The image depicted brain blood vessels as small as ∼100 μm in diameter using PEG-HAuNS as contrast agents. Preliminary results showed no acute toxicity to the liver, spleen, or kidneys in mice following a single imaging dose of PEG-HAuNS. Our results indicate that PEG-HAuNS are promising contrast agents for PAT, with high spatial resolution and enhanced sensitivity.
Keywords: Hollow gold nanospheres; Photoacoustic imaging; Brain vasculature; Pharmacokinetics; Toxicity;

One of the more substantial hurdles to be overcome in realizing the exciting potential of siRNA molecules as therapeutic agents for a wide range of diseases is the intact delivery of the active molecule into its target cell. Here, we present a platform for in vitro and in vivo delivery and intracellular release of siRNA in peritoneal macrophages (Mϕs). The delivery platform is based on the encapsulation of siRNA in biodegradable poly(d,l-lactide) (PLA) microspheres, which are targeted to Mϕs by the simple principle of size exclusion. Proof of concept was achieved using siRNAs targeting TNFα and CD86 in macrophages. We show that the release of the siRNA in peritoneal-derived macrophages in vitro occurs intracellularly, and is abrogated by cytochalasin B, a phagocytosis inhibitor. Silencing in these cells is potent and lasts for at least one week. In vivo, we prove that siRNA encapsulated in biodegradable PLA microspheres can be delivered to peritoneal-residing Mϕs and can induce potent silencing of TNFα secretion for at least one week. The PLA microspheres hold great potential for in vivo use, due to their biocompatibility and degradability, and can potentially be used for in vivo immunomodulation of Mϕs for treatment of autoimmune and chronic inflammatory conditions.
Keywords: Biodegradable microspheres; siRNA delivery; Immunomodulation; Macrophage;

Synthesis of a family of amphiphilic glycopolymers via controlled ring-opening polymerization of functionalized cyclic carbonates and their application in drug delivery by Fabian Suriano; Russell Pratt; Jeremy P.K. Tan; Nikken Wiradharma; Alshakim Nelson; Yi-Yan Yang; Philippe Dubois; James L. Hedrick (2637-2645).
Polymers bearing pendant carbohydrates have a variety of biomedical applications especially in the area of targeted drug delivery. Here we report the synthesis of a family of amphiphilic block glycopolymers containing d glucose, d galactose and d mannose via metal-free organocatalyzed ring-opening polymerization of functional cyclic carbonates generating narrowly dispersed products of controlled molecular weight and end-group fidelity, and their application in drug delivery. These glycopolymers self-assemble into micelles having a high density of sugar molecules in the shell, a size less than 100 nm with narrow size distribution even after drug loading, and little cytotoxicity, which are important for drug delivery. Using galactose-containing micelles as an example, we demonstrate their strong targeting ability towards ASGP-R positive HepG2 liver cancer cells in comparison with ASGP-R negative HEK293 cells although the galactose is attached to the carbonate monomer at 6-position. The enhanced uptake of DOX-loaded galactose-containing micelles by HepG2 cells significantly increases cytotoxicity of DOX as compared to HEK293. This new family of amphiphilic block glycopolymers has great potential as carriers for targeted drug delivery.
Keywords: Functional cyclic carbonate; Organocatalytic ring-opening; Amphiphilic polycarbonate; Micelles; Galactose; Drug targeting;

Lactose mediated liver-targeting effect observed by ex vivo imaging technology by Ping'an Ma; Shi Liu; Yubin Huang; Xuesi Chen; Liping Zhang; Xiabin Jing (2646-2654).
Two kinds of micelles containing Rhodamine B were prepared by a solvent evaporation method. One (Lac-f-micelles: lactose-free micelles) was from a Rhodamine-containing copolymer, poly(ethylene glycol)-b-poly(l-lactide-co-2,2-dihydroxylmethyl-propylene carbonate/Rhodamine) [PEG5000-b-P(LA4000-co-DHP600/Rhodamine)], and the other (Lac+micelles lactose-containing micelles) was from a mixture of the targeting copolymer lactose-poly(ethylene glycol)-poly (l-Lactide) [Lac-PEG4600-PLA4500] and the Rhodamine-containing copolymer PEG5000-b-P(LA4000-co-DHP600/Rhodamine). ESEM and DLS measurements showed that the two kinds of micelles have similar size (in the range of 60–100 nm) and size distribution. Cellular uptake studies in vitro revealed that the Lac+micelles showed stronger endocytosis ability than Lac-f-micelles in SMMC7221 human liver cancer cells, but the Lac+micelles were rarely internalized by Vero cells. The micelle solutions were administrated into mice via tail intravenous injection. Then, five visceral organs were isolated from the mice at specified time intervals and relative fluorescent intensities of the ex vivo organs and their homogenates were examined by CRI Maestro 500FL in vivo imaging system. The results showed that the Lac+micelles showed more remarkable liver-targeting effect than the Lac-f-micelles. And the liver-targeting effect could be established in ca. 12 h after tail i.v. injection.
Keywords: Ex vivo imaging; Lactose; Liver-targeting; Mixed micelles; Rhodamine B;

Lyophilized HER2-specific PEGylated immunoliposomes for active siRNA gene silencing by Jie Gao; Jing Sun; Huimei Li; Wei Liu; Yang Zhang; Bohua Li; Weizhu Qian; Hao Wang; Jianming Chen; Yajun Guo (2655-2664).
The development of a tumor-specific immunoliposome delivering small interfering RNA (siRNA) represents a practical way in cancer gene therapy. In this study, we developed PEGylated 3β-[N-(N′, N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol)/dioleoylphosphatidyl ethanolamine (DOPE) immunoliposomes conjugated with the Fab' of recombinant humanized anti-HER2 monoclonal antibody (PIL) for siRNA delivery. The results demonstrated that the lyophilized PIL (LPIL) prepared by the lyophilization/rehydration method possessed a significantly enhanced HER1 gene, a model target, silencing ability compared with PIL in HER2-overexpressing SK-BR3 cells. Among a series of LPIL with different PEGylation degree, LPIL containing 2.5%PEG (2.5%PEG LPIL) showed the best HER1 gene silencing activity. Confocal microscope studies demonstrated that 2.5%PEG LPIL could specifically bind to SK-BR3 cells and were sequentially internalized into them. Using RhoA as a cancer therapeutic target, 2.5%PEG LPIL entrapping anti-RhoA siRNA could specifically silence RhoA expression and inhibit cell invasion in SK-BR3 cells. In conclusion, these finding demonstrated the potential use of 2.5%PEG LPIL in specifically delivering siRNA to HER2-overexpressing cancers.
Keywords: siRNA delivery; Immunoliposomes; HER2; Lyophilization; Gene silencing; PEG;

The use of carboxymethylcellulose gel to increase non-viral gene transfer in mouse airways by Uta Griesenbach; Cuixiang Meng; Raymond Farley; Marguerite Y. Wasowicz; Felix M. Munkonge; Mario Chan; Charlotte Stoneham; Stephanie G. Sumner-Jones; Ian A. Pringle; Deborah R. Gill; Stephen C. Hyde; Barbara Stevenson; Emma Holder; Hiroshi Ban; Mamoru Hasegawa; Seng H. Cheng; Ronald K. Scheule; Patrick L. Sinn; Paul B. McCray; Eric W.F.W. Alton (2665-2672).
We have assessed whether viscoelastic gels known to inhibit mucociliary clearance can increase lipid-mediated gene transfer. Methylcellulose or carboxymethylcellulose (0.25–1.5%) was mixed with complexes of the cationic lipid GL67A and plasmids encoding luciferase and perfused onto the nasal epithelium of mice. Survival after perfusion with 1% CMC or 1% MC was 90 and 100%, respectively. In contrast 1.5% CMC was uniformly lethal likely due to the viscous solution blocking the airways. Perfusion with 0.5% CMC containing lipid/DNA complexes reproducibly increased gene expression by approximately 3-fold (n  = 16, p  < 0.05). Given this benefit, likely related to increased duration of contact, we also assessed the effect of prolonging contact time of the liposome/DNA complexes by delivering our standard 80 μg DNA dose over either approximately 22 or 60 min of perfusion. This independently increased gene transfer by 6-fold (n  = 8, p  < 0.05) and could be further enhanced by the addition of 0.5% CMC, leading to an overall 25-fold enhancement (n  = 8, p  < 0.001) in gene expression. As a result of these interventions CFTR transgene mRNA transgene levels were increased several logs above background. Interestingly, this did not lead to correction of the ion transport defects in the nasal epithelium of cystic fibrosis mice nor for immunohistochemical quantification of CFTR expression. To assess if 0.5% CMC also increased gene transfer in the mouse lung, we used whole body nebulisation chambers. CMC was nebulised for 1 h immediately before, or simultaneously with GL67A/pCIKLux. The former did not increase gene transfer, whereas co-administration significantly increased gene transfer by 4-fold (p  < 0.0001, n  = 18). This study suggests that contact time of non-viral gene transfer agents is a key factor for gene delivery, and suggests two methods which may be translatable for use in man.
Keywords: Gene transfer; Gene therapy; Lung; Epithelium;

Cationic fluorine-containing amphiphilic graft copolymers as DNA carriers by Sheng-Dong Xiong; Ling Li; Jiang Jiang; Li-Ping Tong; Shuilin Wu; Zu-Shun Xu; Paul K. Chu (2673-2685).
A series of cationic fluorine-containing amphiphilic graft copolymers P(HFMA-St-MOTAC)-g-PEG comprising poly(hexafluorobutyl methacrylate) (PHFMA) poly(methacryl oxyethyl trimethylammonium chloride) (PMOTAC) polystyrene (PSt) backbones and poly(ethylene glycol) (PEG) side chains are synthesized as a type of non-viral gene vector. The copolymers self-assemble into spherical micelles in the aqueous media and turbidity and cytotoxicity measurements show that those micelles have excellent dispersive stability and low cytotoxicity. The interactions between the copolymers and calf-thymus DNA are studied by fluorescence spectroscopy and viscosity. The former discloses electrostatic interaction, hydrophobic interaction, and hydrogen bonding in the copolymer/DNA system, whereas the latter indicates that these graft copolymers can bind DNA via the electrostatic and classical intercalation modes. The DNA-binding capacity determined by the gel retardation assay and UV–visible spectrophotometry shows that the copolymers have good binding capacity to DNA and a high charge density or HFMA content in the copolymers bode well for DNA-binding. Transmission electron microscopy, photon correlation spectroscopy, and zeta potential data reveal that stable colloidal complexes (particles) can form easily between the copolymer micelles and DNA. Our results suggest that the copolymers are a promising non-viral vector in a gene delivery system.
Keywords: Fluorocarbon; Graft copolymer; Gene vector; Micelle;

Convection-driven generation of long-range material gradients by Yanan Du; Matthew J. Hancock; Jiankang He; Jose L. Villa-Uribe; Ben Wang; Donald M. Cropek; Ali Khademhosseini (2686-2694).
Natural materials exhibit anisotropy with variations in soluble factors, cell distribution, and matrix properties. The ability to recreate the heterogeneity of the natural materials is a major challenge for investigating cell–material interactions and for developing biomimetic materials. Here we present a generic fluidic approach using convection and alternating flow to rapidly generate multi-centimeter gradients of biomolecules, polymers, beads and cells and cross-gradients of two species in a microchannel. Accompanying theoretical estimates and simulations of gradient growth provide design criteria over a range of material properties. A poly(ethylene-glycol) hydrogel gradient, a porous collagen gradient and a composite material with a hyaluronic acid/gelatin cross-gradient were generated with continuous variations in material properties and in their ability to regulate cellular response. This simple yet generic fluidic platform should prove useful for creating anisotropic biomimetic materials and high-throughput platforms for investigating cell–microenvironment interactions.
Keywords: Anisotropic materials; Composite materials; Microfluidics; Gradients;

Honeybee silk: Recombinant protein production, assembly and fiber spinning by Sarah Weisman; Victoria S. Haritos; Jeffrey S. Church; Mickey G. Huson; Stephen T. Mudie; Andrew J.W. Rodgers; Geoff J. Dumsday; Tara D. Sutherland (2695-2700).
Transgenic production of silkworm and spider silks as biomaterials has posed intrinsic problems due to the large size and repetitive nature of the silk proteins. In contrast the silk of honeybees (Apis mellifera) is composed of a family of four small and non-repetitive fibrous proteins. We report recombinant production and purification of the four full-length unmodified honeybee silk proteins in Escherichia coli at substantial yields of 0.2–2.5 g/L. Under the correct conditions the recombinant proteins self-assembled to reproduce the native coiled coil structure. Using a simple biomimetic spinning system we could fabricate recombinant silk fibers that replicated the tensile strength of the native material.
Keywords: Biomimetic material; FTIR; Honeybee; Apis mellifera; Recombinant protein; Silk;