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Biomaterials (v.31, #7)

Editorial board (pp. ifc).

Bioceramics of calcium orthophosphates by Sergey V. Dorozhkin (pp. 1465-1485).
A strong interest in use of ceramics for biomedical applications appeared in the late 1960's. Used initially as alternatives to metals in order to increase a biocompatibility of implants, bioceramics have become a diverse class of biomaterials, presently including three basic types: relatively bioinert ceramics, bioactive (or surface reactive) and bioresorbable ones. Furthermore, any type of bioceramics could be porous to provide tissue ingrowth. This review is devoted to bioceramics prepared from calcium orthophosphates, which belong to the categories of bioresorbable and bioactive compounds. During the past 30–40 years, there have been a number of major advances in this field. Namely, after the initial work on development of bioceramics that was tolerated in the physiological environment, emphasis was shifted towards the use of bioceramics that interacted with bones by forming a direct chemical bond. By the structural and compositional control, it became possible to choose whether the bioceramics of calcium orthophosphates was biologically stable once incorporated within the skeletal structure or whether it was resorbed over time. At the turn of the millennium, a new concept of calcium orthophosphate bioceramics, which is able to regenerate bone tissues, has been developed. Current biomedical applications of calcium orthophosphate bioceramics include replacements for hips, knees, teeth, tendons and ligaments, as well as repair for periodontal disease, maxillofacial reconstruction, augmentation and stabilization of the jawbone, spinal fusion and bone fillers after tumor surgery. Potential future applications of calcium orthophosphate bioceramics will include drug-delivery systems, as well as they will become effective carriers of growth factors, bioactive peptides and/or various types of cells for tissue engineering purposes.

Keywords: Calcium orthophosphates; Hydroxyapatite; Fluorapatite; Bioceramics; Biomaterials; Grafts

Functionalizable and ultra-low fouling zwitterionic surfaces via adhesive mussel mimetic linkages by Changlu Gao; Guozhu Li; Hong Xue; Wei Yang; Fengbao Zhang; Shaoyi Jiang (pp. 1486-1492).
In this work, a biomimetic polymer (pCB2-catechol2), with two zwitterionic poly(carboxybetaine) (pCB) arms for ultra-low fouling and two adhesive catechol groups for surface anchoring, was developed. Two pCB arms were grown from an initiator with two catechol groups via atom transfer radical polymerization (ATRP). Binding tests of pCB2-catechol2 were performed on a gold surface under a range of conditions such as pH values and solvents. Protein adsorption from single protein solutions of fibrinogen and lysozyme, and complex media of 100% blood plasma and serum was evaluated using a surface plasmon resonance (SPR) sensor. Results are compared with those from two other polymers (i.e., one polymer with one pCB chain and one catechol group, termed as pCB-catechol, and another polymer with one pCB chain and two catechol groups, termed as pCB-catechol2). Furthermore, the direct immobilization of anti-activated leukocyte cell adhesion molecule (anti-ALCAM) was carried out on the pCB2-catechol2 modified surface. Results showed that the antibody-immobilized surface maintained its excellent ultra-low fouling properties. The detection of activated leukocyte cell adhesion molecule (ALCAM) in 100% blood plasma with high sensitivity and specificity was achieved. This work demonstrates an effective and convenient strategy to obtain functionalizable and ultra-low fouling surfaces.

Keywords: Zwitterionic; Catechol; Nonfouling; Functionalization

Toughening mechanisms in iron-containing hydroxyapatite/titanium composites by Q. Chang; D.L. Chen; H.Q. Ru; X.Y. Yue; L. Yu; C.P. Zhang (pp. 1493-1501).
Pure hydroxyapatite (HA) is brittle and it cannot be directly used for the load-bearing biomedical applications. The purpose of this investigation was to develop a new iron-containing HA/titanium composite via pressureless sintering at a relatively low temperature with particular emphasis on identifying the underlying toughening mechanisms. The addition of iron to HA/titanium composites led to a unique and favorable core/shell microstructure of Ti–Fe particles that consisted of outer titanium and inner iron, and good interfacial bonding with HA matrix. While the relative density, hardness and Young's modulus reduced, the flexural strength, fracture toughness, fatigue resistance, and the related fracture surface roughness increased significantly with increasing amount of Ti–Fe particles. Different toughening mechanisms including crack bridging, branching and deflection were observed in the composites, thus effectively increasing the crack propagation resistance and resulting in a substantial improvement in the mechanical properties of the composites.

Keywords: Hydroxyapatite composite; Titanium; Iron; Mechanical properties; Fatigue; Toughening mechanism

A nitric oxide releasing, self assembled peptide amphiphile matrix that mimics native endothelium for coating implantable cardiovascular devices by Meenakshi Kushwaha; Joel M. Anderson; Charles A. Bosworth; Adinarayana Andukuri; William P. Minor; Jack R. Lancaster Jr.; Peter G. Anderson; Brigitta C. Brott; Ho-Wook Jun (pp. 1502-1508).
Cardiovascular disease is the number one cause of death in the United States. Deployment of stents and vascular grafts has been a major therapeutic method for treatment. However, restenosis, incomplete endothelialization, and thrombosis hamper the long term clinical success. As a solution to meet these current challenges, we have developed a native endothelial ECM mimicking self-assembled nanofibrous matrix to serve as a new treatment model. The nanofibrous matrix is formed by self-assembly of peptide amphiphiles (PAs), which contain nitric oxide (NO) donating residues, endothelial cell adhesive ligands composed of YIGSR peptide sequence, and enzyme-mediated degradable sites. NO was successfully released from the nanofibrous matrix rapidly within 48h, followed by sustained release over period of 30 days. The NO releasing nanofibrous matrix demonstrated a significantly enhanced proliferation of endothelial cells (51±3% to 67±2%) but reduced proliferation of smooth muscle cells (35±2% to 16±3%) after 48h of incubation. There was also a 150-fold decrease in platelet attachment on the NO releasing nanofibrous matrix (470±220 platelets/cm2) compared to the collagen-I (73±22×103platelets/cm2) coated surface. The nanofibrous matrix has the potential to be applied to various cardiovascular implants as a self-assembled coating, thereby providing a native endothelial extracellular matrix (ECM) mimicking environment.

Keywords: Self-assembly; Nitric oxide; Endothelium; Vascular grafts; Peptide; Stents

Cytotoxicity and cellular uptake of iron nanowires by Meng-Meng Song; Wen-Jing Song; Hong Bi; Jun Wang; Wei-Lin Wu; Jun Sun; Min Yu (pp. 1509-1517).
The toxicity of nanostructured materials and nanoparticles are very important considerations for many nanotechnology applications. Iron nanowires (NWs), as useful magnetic nanomaterials, may be good candidates for several biomedical applications. Here Fe NWs with an average diameter of about 50 nm were prepared by electrodeposition within the nanopores of anodic aluminum oxide (AAO) templates, and characterized by using scanning electron microcopy (SEM), transmission electron microscopy (TEM) and vibrating sample magnetometer (VSM). The cytotoxicity of the Fe NWs was studied at cell level. Specifically, the influence of concentration and size on the cytotoxicity of Fe NWs to HeLa cells was evaluated by MTT assay combined with direct morphology observations by TEM and phase contrast microscopy. The results clearly showed that the presence of Fe NWs had no significant effect on the cell proliferation and cell viability, even the HeLa cells exposed to Fe NWs at the high concentration of 10 000 per cell for 72 h still showed high cell viability of about 80%. From phase contrast microscopy, confocal laser scanning microscopy (CLSM) and TEM observations, it was found that Fe NWs were indeed internalized by HeLa cells. The cellular uptake process and results of Fe NWs demonstrated that as-prepared Fe NWs had not only a good biocompatibility but also a very low cytotoxicity.

Keywords: Iron nanowires; Biocompatibility; Cellular uptake; MTT assay; Live cell imaging

The influence of the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide on the proliferation, differentiation and mineralization of odontoblast-like cells by Mariko Nishida; Satoshi Imazato; Yusuke Takahashi; Shigeyuki Ebisu; Takuya Ishimoto; Takayoshi Nakano; Yoshiyuki Yasuda; Takashi Saito (pp. 1518-1532).
A dentin primer incorporating an antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) shows strong antibacterial effects, and may provide better prognosis for direct capping of infected pulp exposed by caries removal compared with conventional adhesives. However, influences of MDPB on healing of the pulp have not yet been fully elucidated. The purpose of this study was to compare the influences of unpolymerized MDPB on proliferation, differentiation and mineralization of odontoblast-like MDPC-23 cells with those of other resin monomers, Bis-GMA, MDP, TEGDMA and HEMA. The inhibitory effects of MDPB on the proliferation of MDPC-23 were lower than those of Bis-GMA. While MDPB strongly affected the differentiation compared with the other monomers, it was less inhibitory than Bis-GMA and MDP on the mineralization ability of odontoblast-like cells. These findings indicate that MDPB has superior biocompatibility than Bis-GMA in terms of hard tissue formation by odontoblastic cells, suggesting its possible less negative influences on dentinogenesis.

Keywords: Cell culture; Biocompatibility; Dental resins; Antibacterial; Odontoblasts

The effect of topography of polymer surfaces on platelet adhesion by Li Buay Koh; Isabel Rodriguez; Subbu S. Venkatraman (pp. 1533-1545).
In this study, the effect of surface topography on fibrinogen and platelet adsorption was investigated. High aspect ratio surface features, in the submicron to nanometer range, were constructed on the poly- (lactic- co-glycolic-acid) (PLGA) films. The topographic surfaces were fabricated by solvent-mediated polymer casting on a master template. Fibrinogen adsorption and platelets adhesion on these topographic surfaces were quantified by enzyme linked immunosorbent assay (ELISA) and lactate dehydrogenase (LDH) assay respectively, while the activation of platelets was quantified by flow cytometric analysis using fluorescein isothiocyanate (FITC) tagging. The lowest fibrinogen adsorption amount and platelet activity was observed on surfaces with specific topographical features in the submicron range with a significant reduction in adhesion when compared to the pristine PLGA films. The topographical parameters found to induce low levels of fibrinogen adsorption and platelet response were high aspect ratio structures (>3:1) with reduced interspacing (<200nm) or high density. The results signify that topographical manipulation of thrombogenic surfaces of biodegradable polymers is a feasible approach for reducing their thrombogenicity.

Keywords: Anti-thrombotic materials; Platelet adhesion; Nanotopography; High aspect ratio; Poly(lactic-; co; -glycolic-acid)

Enhancement of bone–titanium integration profile with UV-photofunctionalized titanium in a gap healing model by Takeshi Ueno; Masahiro Yamada; Takeo Suzuki; Hajime Minamikawa; Naoko Sato; Norio Hori; Kazuo Takeuchi; Masami Hattori; Takahiro Ogawa (pp. 1546-1557).
In this study, we tested the potential of UV-photofunctionalized titanium surfaces to overcome compromised bone–titanium integration in a gap healing model. Titanium in rod and disk forms was acid etched and then stored for 4 weeks under dark ambient conditions. Titanium rods with and without UV pretreatment were placed into a rat femur with (contact healing) or without (gap healing) contact with the innate cortical bone. The titanium implants were subjected to a biomechanical push-in test, micro-CT bone morphometry, and surface elemental analysis after 2 weeks of healing. The strength of bone–titanium integration in the gap healing model was one-third of that in the contact healing model. However, UV-treated implants in the gap healing condition produced a strength of bone–titanium integration equivalent to that of untreated implants in the contact healing condition. Bone volume around UV-treated implants was 2- to 3-fold greater than that around the untreated implants in the gap healing model. A bone generation profile drawn along the long axis of the implant exhibited greater contrast between the untreated and UV-treated surfaces in the cortical area than in the bone marrow area. The bone tissue formed on UV-treated implants showed a higher Ca/P ratio than that formed on untreated titanium. The rate of cell proliferation, alkaline phosphatase activity, and calcium deposition in femoral periosteal cells and in bone marrow-derived osteoblasts were greater in cultures on UV-treated titanium disks than in cultures on untreated disks. The UV-enhanced function in periosteal cells was more pronounced when they were co-cultured with bone marrow-derived osteoblasts, indicating a synergistic effect of UV-treated titanium with biological signals from bone marrow-derived osteoblasts. Within the limitation of the model used in this study, UV-photofunctionalized titanium surfaces may overcome the challenging condition of bone–titanium integration without cortical bone support. UV treatment of implants induced marked improvements in the behavior of bone formation and quantity and quality of bone tissue around the implants. These effects may be related to the promoted function of both periosteum- and bone marrow-derived osteogenic cells at the local level around UV-treated titanium surfaces.

Keywords: Orthopedic and dental implant; Osseointegration; Periosteum; Arthroplasty; Total hip replacement

Quantitative analysis of the tissue response to chronically implanted microwire electrodes in rat cortex by Brent D. Winslow; Patrick A. Tresco (pp. 1558-1567).
Several hypotheses have been proposed to explain how the brain tissue reaction to single unit recording electrodes influences biocompatibility including progressive changes in the spatial distribution of reactive astrocytes, and the loss of neurons over the indwelling period. To examine these hypotheses, the spatial distribution of biomarkers associated with the foreign body response to insulated microwires placed in rat cerebral cortex was analyzed 2, 4, and 12 weeks after implantation using quantitative methods. We observed a stereotypical tissue response that was similar in some aspects to that previously reported for penetrating planar silicon microelectrode arrays with some specific differences including an overall lower degree of cortical tissue reactivity. While we found no evidence that reactive gliosis increases over time or that neuronal loss is progressive, we did find evidence of persistent inflammation and enhanced BBB permeability at the electrode brain tissue interface that extended over the 3 month indwelling period and that exhibited more animal to animal variability at 3 months than at 2 and 4 weeks.

Keywords: Electrode; Foreign body response; Neural prosthesis; Inflammation

The effect of poly (ethylene-co-vinyl alcohol) on senescence-associated alterations of human dermal fibroblasts by Pei-Jen Lou; Ming-Yi Chiu; Chi-Chun Chou; Bor-Wu Liao; Tai-Horng Young (pp. 1568-1577).
It is well known that biomaterials play an important role in the regulation of adhesion and growth of a variety of cultured cell types. However, whether biomaterials are associated with the senescence of cultured cells is not known. The present work shows that the decrease of the hydrophobic property of poly (ethylene-co-vinyl alcohol) (EVAL) from 44 mole% to 27 mole% ethylene could induce characteristic senescence-associated phenotypic changes such as larger cell shape, re-organized actin cytoskeleton, lower proliferation capacity, higher levels of senescence-associated β-galactosidase (SA β-gal) activity, and upregulation of the cell-cycle inhibitor p53 and its transcriptional target p21 in the cultured human diploid fibroblasts (HDFs). Furthermore, it was found that the cultured cells recovered their ability to grow when the substrate was reused every passage. It seemed that the extracellular matrix (ECM) proteins adsorbed onto the EVAL surface might have a protective role in the cellular aging process. Therefore, whether a biomaterial strongly influences cellular aging process must be considered in the selection of a biomaterial for the biomedical application.

Keywords: Human diploid fibroblasts (HDFs); Senescence; Poly (ethylene-co-vinyl alcohol) (EVAL); Extracellular matrix (ECM) proteins

The effect of VEGF functionalization of titanium on endothelial cells in vitro by Chye Khoon Poh; Zhilong Shi; Tee Yong Lim; Koon Gee Neoh; Wilson Wang (pp. 1578-1585).
One of the key challenges in bone healing and regeneration is the engineering of an implant with surface properties that can enhance revascularization to meet the metabolic demands of recovery. Successful implant integration into the surrounding tissue is highly dependent on the crucial role of blood supply in driving bone repair and development. Therapeutic application of vascular endothelial growth factor (VEGF) is a promising approach to enhance blood supply and healing through revascularization around an engineered implant in a regulated manner. In this in vitro study, we investigated the effects of immobilized VEGF on titanium alloy substrates coated with thin adherent polydopamine film. X-ray photoelectron spectroscopy (XPS) was used to determine the chemical composition of the surfaces at various stages of surface functionalization to verify the successful deposition of polydopamine and VEGF on the metal surface. Surface topography was evaluated from the surface profile determined by atomic force microscopy (AFM). The functionalized surfaces showed a significant increase in human dermal microvascular endothelial cells (HDMECs) attachment, viability and proliferation compared to the pristine substrate. Furthermore the immobilized VEGF was able to induce the differentiation of human mesenchymal stem cells (hMSCs) into endothelial cells. Therefore utilizing the reactivity of polydopamine films to immobilize VEGF onto metal substrates may provide a promising approach for application in situations where revascularization around implants would be beneficial in improving bone healing and implant integration.

Keywords: Bone implants; Revascularization; Polydopamine; Surface modification; VEGF

Magnetic micro-manipulations to probe the local physical properties of porous scaffolds and to confine stem cells by Damien Robert; Delphine Fayol; Catherine Le Visage; Guillaume Frasca; Séverine Brulé; Christine Ménager; Florence Gazeau; Didier Letourneur; Claire Wilhelm (pp. 1586-1595).
The in vitro generation of engineered tissue constructs involves the seeding of cells into porous scaffolds. Ongoing challenges are to design scaffolds to meet biochemical and mechanical requirements and to optimize cell seeding in the constructs. In this context, we have developed a simple method based on a magnetic tweezer set-up to manipulate, probe, and position magnetic objects inside a porous scaffold. The magnetic force acting on magnetic objects of various sizes serves as a control parameter to retrieve the local viscosity of the scaffolds internal channels as well as the stiffness of the scaffolds pores. Labeling of human stem cells with iron oxide magnetic nanoparticles makes it possible to perform the same type of measurement with cells as probes and evaluate their own microenvironment. For 18 μm diameter magnetic beads or magnetically labeled stem cells of similar diameter, the viscosity was equivalently equal to 20 mPa s in average. This apparent viscosity was then found to increase with the magnetic probes sizes. The stiffness probed with 100 μm magnetic beads was found in the 50 Pa range, and was lowered by a factor 5 when probed with cells aggregates. The magnetic forces were also successfully applied to the stem cells to enhance the cell seeding process and impose a well defined spatial organization into the scaffold.

Keywords: Tissue engineering; Porous scaffolds; Cell seeding; Mechanical properties; Magnetic tweezer; Iron oxide nanoparticles

Polyester based nerve guidance conduit design by Deniz Yucel; Gamze Torun Kose; Vasif Hasirci (pp. 1596-1603).
Nerve conduits containing highly aligned architecture that mimics native tissues are essential for efficient regeneration of nerve injuries. In this study, a biodegradable nerve conduit was constructed by converting a porous micropatterned film (PHBV–P(L-D,L)LA–PLGA) into a tube wrapping aligned electrospun fibers (PHBV–PLGA). The polymers were chosen so that the protective tube would erode slower than the fibrous core to achieve complete healing before the tube eroded. The pattern dimensions and the porosity (58.95 (%) with a maximum pore size of 4–5μm) demonstrated that the micropatterned film would enable the migration, alignment and survival of native cells for proper regeneration. This film had sufficiently high mechanical properties (ultimate tensile strength: 3.13MPa, Young's Modulus: 0.08MPa) to serve as a nerve guide. Electrospun fibers, the inner part of the tubular construct, were well aligned with a fiber diameter of ca. 1.5μm. Fiber properties were especially influenced by polymer concentration. SEM showed that the fibers were aligned parallel to the groove axis of the micropatterned film within the tube as planned considering the nerve tissue architecture. This two component nerve conduit appears to have the right organization for testing in vitro and in vivo nerve tissue engineering studies.

Keywords: Nerve regeneration; Nerve guide; Conduit; Electrospun mat; Micropattern

The use of immobilized osteogenic growth peptide on gradient substrates synthesized via click chemistry to enhance MC3T3-E1 osteoblast proliferation by Nicole M. Moore; Nancy J. Lin; Nathan D. Gallant; Matthew L. Becker (pp. 1604-1611).
In this study, we report the use of surface immobilized peptide concentration gradient technology to characterize MC3T3-E1 osteoblast cell response to osteogenic growth peptide (OGP), a small peptide found naturally in human serum at μmol/L concentrations. OGP was coupled to oxidized self assembled monolayer (SAM) gradients by a polyethylene oxide (PEO) linker using click chemistry. After 4h incubation with MC3T3-E1 cells, OGP functionalized surfaces had higher cell attachment at low peptide concentrations compared to control gradients. By day 3, OGP gradient substrates had higher cell densities compared to control gradients at all concentrations. MC3T3-E1 cell doubling time was 35% faster on OGP substrates relative to SAM gradients alone, signifying an appreciable increase in cell proliferation. This increase in cell proliferation, or decrease in doubling time, due to OGP peptide was reduced by day 7. Hence, immobilized OGP increased cell proliferation from 0 days to 3 days at all densities indicating it may be useful as a proliferative peptide that can be used in tissue engineering substrates.

Keywords: Bone tissue engineering; Osteoblast; Peptide; Cell proliferation

Extraction of high quality RNA from polysaccharide matrices using cetlytrimethylammonium bromide by Limin Wang; Jan P. Stegemann (pp. 1612-1618).
Polysaccharides are increasingly being used as biomaterials for tissue engineering and regenerative medicine. Quantitative analysis of gene expression from cells in three-dimensional (3D) scaffolds requires extraction of messenger RNA, which is complicated in polysaccharide materials by ionic complexing between nucleic acids and the matrix. We used a strongly cationic surfactant, cetyltrimethylammonium bromide (CTAB), to extract RNA from human mesenchymal stem cells embedded in 3D chitosan, agarose and collagen matrices. CTAB extraction was compared to conventional guanidinium thiocyanate-based methods for RNA isolation by assessing RNA yield, purity (A260/A280 and A260/A230) and integrity (28S/18S and RIN). For polysaccharide-based matrices, CTAB extraction yielded significantly more RNA with higher purity than guanidinium thiocyanate-based methods alone. The extracted RNA was largely intact as indicated by 28S/18S ratios and RIN values, while these parameters could not be measured using conventional kits alone. For pure collagen matrices, the CTAB method was comparable or better than guanidinium thiocyanate-based methods in terms of RNA yield and quality. We further validated the CTAB protocol using semi-quantitative and quantitative RT-PCR to amplify both large and small amplicons. Our results show that the CTAB-based method is a facile and effective way to extract abundant, high quality RNA from polysaccharide and protein biomaterials.

Keywords: RNA; PCR; Polysaccharide; Tissue engineering; Chitosan; Collagen

Molecular profiling of single cells in response to mechanical force: Comparison of chondrocytes, chondrons and encapsulated chondrocytes by Qi Guang Wang; Bac Nguyen; Colin R. Thomas; Zhibing Zhang; Alicia J. El Haj; Nicola J. Kuiper (pp. 1619-1625).
A chondrocyte and its surrounding pericellular matrix (PCM) are defined as a chondron. The PCM plays a critical role in enhancing matrix production, protecting the chondrocyte during loading and transducing mechanical signals. Tissue engineering involves the design of artificial matrices which aim to mimic PCM function for mechanical strength and signalling motifs. We compare the mechanical performance and mechanoresponsiveness of chondrocytes with and without PCM, and encapsulated by alternate adsorption of two oppositely charged polyelectrolytes; chitosan and hyaluronan. Zeta potential measurements confirmed the success of the encapsulation. Encapsulation did not influence chondrocyte viability or metabolic activity. Cells were compressed by micromanipulation with final deformations to 30%, 50% and 70%. Force–displacement data showed that the larger the deformation at the end of compression, the greater the force on the cell. Mechanoresponsiveness of cells was studied by combining single cell PCR with dynamic compression at 20% and 40%. Aggrecan and Type II collagen gene expression were significantly increased in encapsulated chondrocytes and chondrons compared to chondrocytes whereas dynamic compression had no effect on SOX9 or lubricin gene expression. Our results demonstrate that although encapsulation can mimic responses of chondrocytes to biomechanical compression the molecular profile did not reach the enhanced levels observed with chondrons.

Keywords: Single cell encapsulation; Chondrocytes; Chondron; Pericellular matrix; Chitosan; Hyaluronan

Physiologic compliance in engineered small-diameter arterial constructs based on an elastomeric substrate by Peter M. Crapo; Yadong Wang (pp. 1626-1635).
Compliance mismatch is a significant challenge to long-term patency in small-diameter bypass grafts because it causes intimal hyperplasia and ultimately graft occlusion. Current engineered grafts are typically stiff with high burst pressure but low compliance and low elastin expression. We postulated that engineering small arteries on elastomeric scaffolds under dynamic mechanical stimulation would result in strong and compliant arterial constructs. This study compares properties of engineered arterial constructs based on biodegradable polyester scaffolds composed of either rigid poly(lactide- co-glycolide) (PLGA) or elastomeric poly(glycerol sebacate) (PGS). Adult baboon arterial smooth muscle cells (SMCs) were cultured in vitro for 10 days in tubular, porous scaffolds. Scaffolds were significantly stronger after culture regardless of material, but the elastic modulus of PLGA constructs was an order of magnitude greater than that of porcine carotid arteries and PGS constructs. Deformation was elastic in PGS constructs and carotid arteries but plastic in PLGA constructs. Compliance of arteries and PGS constructs were equivalent at pressures tested. Altering scaffold material from PLGA to PGS significantly decreased collagen content and significantly increased insoluble elastin content in constructs without affecting soluble elastin concentration in the culture medium. PLGA constructs contained no appreciable insoluble elastin. This research demonstrates that: (1) substrate stiffness directly affects in vitro tissue development and mechanical properties; (2) rigid materials likely inhibit elastin incorporation into the extracellular matrix of engineered arterial tissues; and (3) grafts with physiologic compliance and significant elastin content can be engineered in vitro after only days of cell culture.

Keywords: Arterial tissue engineering; Vascular compliance; Elastin; Poly(glycerol sebacate); Small-diameter vascular grafts

The promotion of endothelial progenitor cells recruitment by nerve growth factors in tissue-engineered blood vessels by Wen Zeng; Wei Yuan; Li Li; Jianhong Mi; Shangcheng Xu; Can Wen; Zhenhua Zhou; Jiaqiang xiong; Jiansen Sun; Dajun Ying; Mingcan Yang; Xiaosong Li; Chuhong Zhu (pp. 1636-1645).
Endothelial progenitor cells (EPCs) mobilization and homing are critical to the development of an anti-thrombosis and anti-stenosis tissue-engineered blood vessel. The growth and activation of blood vessels are supported by nerves. We investigated whether nerve growth factors (NGF) can promote EPCs mobilization and endothelialization of tissue-engineered blood vessels. In vitro, NGF promoted EPCs to form more colonies, stimulated human EPCs to differentiate into endothelial cells, and significantly enhanced EPCs migration. Flow cytometric analysis revealed that NGF treatment increased the number of EPCs in the peripheral circulation of C57BL/6 mice. Furthermore, the treatment of human EPCs with NGF facilitated their homing into wire-injured carotid arteries after injection into mice. Decellularized rat blood vessel matrix was incubated with EDC cross-linked collagen and bound to NGF protein using the bifunctional coupling agent N-succinmidyl3-(2-pyridyldit-hio) propionate (SPDP). The NGF-bound tissue-engineered blood vessel was implanted into rat carotid artery for 1 week and 1 month. NGF-bound blood vessels possessed significantly higher levels of endothelialization and patency than controls did. These results demonstrated that NGF can markedly increase EPCs mobilization and homing to vascular grafts. Neurotrophic factors such as NGF have a therapeutic potential for the construction of tissue-engineered blood vessels in vivo.

Keywords: Endothelial progenitor cell; Tissue-engineered blood vessel; Nerve growth factors; Endothelialization; Anti-thrombosis; Anti-stenosis

Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology by Tadashi Sasagawa; Tatsuya Shimizu; Sachiko Sekiya; Yuji Haraguchi; Masayuki Yamato; Yoshiki Sawa; Teruo Okano (pp. 1646-1654).
To survive three-dimensional (3-D) cell-dense thick tissues after transplantation, the improvements of hypoxia, nutrient insufficiency, and accumulation of waste products are required. This study presents a strategy for the initiation of prevascular networks in a 3-D tissue construct by sandwiching endothelial cells between the cell sheets. For obtaining a stable stacked cell sheet construct, a sophisticated 3-D cell sheet manipulation system using temperature-responsive culture dishes and a cell sheet manipulator was developed. When sparsely cultured human umbilical vein endothelial cells (HUVECs) were sandwiched between two myoblast sheets, the inserted HUVECs sprouted and formed network structures in vitro. Additionally, when myoblast sheets and HUVECs were alternately sandwiched, endothelial cell connections through the layers and capillary-like structures were found in a five-layer construct. Moreover, the endothelial networks in the five-layer myoblast sheet construct were observed to connect to the host vessels after transplantation into the subcutaneous tissues of nude rats, resulted in a neovascularization that allow the graft to survive. These results indicated that the prevascularized myoblast sheet constructs could induce functional anastomosis. Consequently, our prevascularizing method using a cell sheet stacking manipulation technology provides a substantial advance for developing various types of three-dimensional tissues and contributes to regenerative medicine.

Keywords: Cell sheet; Endothelial cell; Co-culture; Prevascularization; Functional anastomosis

Cross-linked open-pore elastic hydrogels based on tropoelastin, elastin and high pressure CO2 by Nasim Annabi; Suzanne M. Mithieux; Anthony S. Weiss; Fariba Dehghani (pp. 1655-1665).
In this study the effect of high pressure CO2 on the synthesis and characteristics of elastin-based hybrid hydrogels was investigated. Tropoelastin/α-elastin hybrid hydrogels were fabricated by chemically cross-linking tropoelastin/α-elastin solutions with glutaraldehyde at high pressure CO2. Dense gas CO2 had a significant impact on the characteristics of the fabricated hydrogels including porosity, swelling ratio, compressive properties, and modulus of elasticity. Compared to fabrication at atmospheric pressure high pressure CO2 based construction eliminated the skin-like formation on the top surfaces of hydrogels and generated larger pores with an average pore size of 78 ± 17 μm. The swelling ratios of composite hydrogels fabricated at high pressure CO2 were lower than the gels produced at atmospheric pressure as a result of a higher degree of cross-linking. Dense gas CO2 substantially increased the mechanical properties of fabricated hydrogels. The compressive and tensile modulus of 50/50 weight ratio tropoelastin/α-elastin composite hydrogels were enhanced 2 and 2.5 fold, respectively, when the pressure was increased from 1 to 60 bar. In vitro studies show that the presence of large pores throughout the hydrogel matrix fabricated at high pressure CO2 enabled the migration of human skin fibroblast cells 300 μm into the construct.

Keywords: Tropoelastin; α-Elastin; Hydrogel; Glutaraldehyde; High pressure CO; 2; Fibroblast

Dose effect of tumor necrosis factor-α on in vitro osteogenic differentiation of mesenchymal stem cells on biodegradable polymeric microfiber scaffolds by Paschalia M. Mountziaris; Stephanie N. Tzouanas; Antonios G. Mikos (pp. 1666-1675).
This study presents a first step in the development of a bone tissue engineering strategy to trigger enhanced osteogenesis by modulating inflammation. This work focused on characterizing the effects of the concentration of a pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α), on osteogenic differentiation of mesenchymal stem cells (MSCs) grown in a 3D culture system. MSC osteogenic differentiation is typically achieved in vitro through a combination of osteogenic supplements that include the anti-inflammatory corticosteroid dexamethasone. Although simple, the use of dexamethasone is not clinically realistic, and also hampers in vitro studies of the role of inflammatory mediators in wound healing. In this study, MSCs were pre-treated with dexamethasone to induce osteogenic differentiation, and then cultured in biodegradable electrospun poly(ɛ-caprolactone) (PCL) scaffolds, which supported continued MSC osteogenic differentiation in the absence of dexamethasone. Continuous delivery of 0.1ng/mL of recombinant rat TNF-α suppressed osteogenic differentiation of rat MSCs over 16 days, which was likely the result of residual dexamethasone antagonizing TNF-α signaling. Continuous delivery of a higher dose, 5ng/mL TNF-α, stimulated osteogenic differentiation for a few days, and 50ng/mL TNF-α resulted in significant mineralized matrix deposition over the course of the study. These findings suggest that the pro-inflammatory cytokine TNF-α stimulates osteogenic differentiation of MSCs, an effect that can be blocked by the presence of anti-inflammatory agents like dexamethasone, with significant implications on the interplay between inflammation and tissue regeneration.

Keywords: Cytokine; Mesenchymal stem cells; Biomineralisation; Osteogenesis; Bone tissue engineering; InflammationAbbreviations; ALP; Alkaline phosphatase; +dex; Supplemented with dexamethasone; MSCs; Mesenchymal stem cells; NF-κB; Nuclear factor-κB; PBS; Phosphate-buffered saline; PCL; Poly(ɛ-caprolactone); SEM; Scanning electron microscopy; TNF-α; Tumor necrosis factor alpha; UV; Ultraviolet light

The effect of extracellular matrix components on the preservation of human islet function in vitro by Jamal Daoud; Maria Petropavlovskaia; Lawrence Rosenberg; Maryam Tabrizian (pp. 1676-1682).
Human islet isolation leads to the loss of the ECM basement membrane which contributes to eventual apoptosis in vitro. The reestablishment of this environment is vital in understanding the mechanism of islet interaction with its surroundings in order to arrive at conditions favourable to islet culture in vitro. In this study, we investigated the effects of the main ECM components collagen I and IV, fibronectin, and laminin on human islet adhesion, survival, and functionality. Results have provided insight into integrin-mediated effects and behaviour. Collagen I/IV and fibronectin induced adhesion, while fibronectin was the only ECM protein capable of maintaining islet structural integrity and insulin content distribution. Furthermore, islet phenotype was eventually lost, but insulin gene expression was highest in islets cultured on collagen I and IV. However, insulin release was highest on fibronectin, along with a decrease in SUR1 expression, while glucose metabolism, along with GLUT2 and GCK expression, was highest on collagen I and IV surfaces. These findings provide a basis for the future establishment of a modified three-dimensional construct for the culture of human pancreatic islets in vitro.

Keywords: Extracellular Matrix; Islet; Diabetes; Surface modification; Islet viability and functionality

Directed growth of fibroblasts into three dimensional micropatterned geometries via self-assembling scaffolds by Mustapha Jamal; Noy Bassik; Jeong-Hyun Cho; Christina L. Randall; David H. Gracias (pp. 1683-1690).
We describe the use of conventional photolithography to construct three dimensional (3D) thin film scaffolds and direct the growth of fibroblasts into three distinct and anatomically relevant geometries: cylinders, spirals and bi-directionally folded sheets. The scaffolds were micropatterned as two dimensional sheets which then spontaneously assembled into specific geometries upon release from the underlying substrate. The viability of fibroblasts cultured on these self-assembling scaffolds was verified using fluorescence microscopy; cell morphology and spreading were studied using scanning electron microscopy. We demonstrate control over scaffold size, radius of curvature and folding pitch, thereby enabling an attractive approach for investigating the effects of these 3D geometric factors on cell behaviour.

Keywords: Tissue engineering; Photolithography; Curvature; Cell culture

Differentiation of human bone marrow mesenchymal stem cells grown in terpolyesters of 3-hydroxyalkanoates scaffolds into nerve cells by Lei Wang; Zhi-Hui Wang; Chong-Yang Shen; Ming-Liang You; Jian-Feng Xiao; Guo-Qiang Chen (pp. 1691-1698).
Polyhydroxyalkanoates, abbreviated as PHA, have been studied for medical applications due to their suitable mechanical properties, blood and tissue tolerance and in vivo biodegradability. As a new member of PHA family, terpolyester of 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxyhexanoate, abbreviated as PHBVHHx, was compared with polylactic acid (PLA), copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) for their respective functions leading to differentiation of human bone marrow mesenchymal stem cell (hBMSC) into nerve cells. Results indicated that 3D scaffolds promoted the differentiation of hBMSC into nerve cells more intensively compared with 2D films. Smaller pore sizes of scaffolds increased differentiation of hBMSC into nerve cells, whereas decreased cell proliferation. PHBVHHx scaffolds with pore sizes of 30–60μm could be used in nerve tissue engineering for treatment of nerve injury. The above results were supported by scanning electron microscope (SEM) and confocal microscopy observation on attachment and growth of hBMSCs on PLA, PHBHHx and PHBVHHx, and by CCK-8 evaluation of cell proliferation. In addition, expressions of nerve markers nestin, GFAP and β-III tubulin of nerve cells differentiated from hBMSC grown in PHBVHHx scaffolds were confirmed by real-time PCR.

Keywords: PHB; PLA; Terpolyester; Polyhydroxyalkanoates; Human bone marrow mesenchymal stem cells; Tissue engineering

Poly(l-lysine) nanostructured particles for gene delivery and hormone stimulation by Xin Zhang; Mustapha Oulad-Abdelghani; Alexander N. Zelkin; Yajun Wang; Youssef Haîkel; Didier Mainard; Jean-Claude Voegel; Frank Caruso; Nadia Benkirane-Jessel (pp. 1699-1706).
In this work, we designed replica particles based on poly (l-lysine) (PLL) polymers crosslinked via a homobifunctional linker to support coadsorption of a plasmid DNA and a peptide hormone for concurrent transfection and induction of a cellular function. PLL replica particles (PLLRP) were prepared by infiltrating polymer into mesoporous silica (MS) particles, crosslinking the adsorbed chains by using a homobifunctional crosslinker and finally removing the template particles. Moreover, we verified their cytotoxicity. Furthermore, based on this PLLRP gene delivery system, we simultaneously evaluated the melanin stimulation and gene expression in these cells by fluorescence microscopy. To further understand the bi-functionality, we labeled the SPT7pTL and PGA-α-MSH with YOYO-1 and Rhodamine, respectively, to follow its intracellular pathway by confocal microscopy. Our data suggests that the PLLRP is a promising vector for gene therapy and hormone stimulation.

Keywords: Mesoporous silica (MS); Poly (; l; -lysine) replica particles (PLL; RP; ); Gene delivery; Hormone stimulation

A specific tumor-targeting magnetofluorescent nanoprobe for dual-modality molecular imaging by Jyun-Han Ke; Jia-Jyun Lin; James R. Carey; Jenn-Shing Chen; Chiao-Yun Chen; Li-Fang Wang (pp. 1707-1715).
Poly(acrylic acid) was decorated onto Fe3O4 resulting in a highly water-soluble superparamagnetic iron oxide. The Poly(acrylic acid) iron oxide (PAAIO) complexes possess specific magnetic properties in the presence of an external magnetic field and are attractive contrast agents for magnetic resonance imaging (MRI). The free carboxylic groups of PAAIO exposed on the surface allow for covalent attachment of a fluorescent dye, Rhodamine 123 (Rh123) to form PAAIO-Rh123, which permits applications in fluorescence imaging. PAAIO-Rh123 is therefore a dual-modality molecular probe. In order to endow specific properties to compounds that target cancer cells and to prevent recognition by the reticuloendothelial system (RES), folic acid-linked poly(ethylene glycol) (FA-PEG) was further conjugated onto PAAIO-Rh123. The amounts of Rh123 and FA-PEG on the modified iron oxides were quantitatively determined by elemental analysis. The iron content was determined by inductively coupled plasma-optical emission spectrometer (ICP-OES). The particle diameters were characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). Superparamagnetism was confirmed by the superconducting quantum interference device (SQUID) magnetometer. The cellular internalization efficacy of the modified iron oxides was realized in folate-overexpressed FR(+) and folate-deficient FR(−) KB cells by flow cytometry and confocal laser scanning microscopy (CLSM). The quantitative amount of iron internalized into different harvested KB cells was measured by ICP-OES. The T2-weighted MR images were tested in FR(+) KB cells.

Keywords: Superparamagnetic iron oxide nanoparticles; Folic acid; Optical fluorescence imaging; Magnetic resonance imaging

Synthesizing and binding dual-mode poly (lactic-co-glycolic acid) (PLGA) nanobubbles for cancer targeting and imaging by Jeff S. Xu; Jiwei Huang; Ruogu Qin; George H. Hinkle; Stephen P. Povoski; Edward W. Martin; Ronald X. Xu (pp. 1716-1722).
Accurate assessment of tumor boundaries and recognition of occult disease are important oncologic principles in cancer surgeries. However, existing imaging modalities are not optimized for intraoperative cancer imaging applications. We developed a nanobubble (NB) contrast agent for cancer targeting and dual-mode imaging using optical and ultrasound (US) modalities. The contrast agent was fabricated by encapsulating the Texas Red dye in poly (lactic-co-glycolic acid) (PLGA) NBs and conjugating NBs with cancer-targeting ligands. Both one-step and three-step cancer-targeting strategies were tested on the LS174T human colon cancer cell line. For the one-step process, NBs were conjugated with the humanized HuCC49ΔCH2 antibody to target the over-expressed TAG-72 antigen. For the three-step process, cancer cells were targeted by successive application of the biotinylated HuCC49ΔCH2 antibody, streptavidin, and the biotinylated NBs. Both one-step and three-step processes successfully targeted the cancer cells with high binding affinity. NB-assisted dual-mode imaging was demonstrated on a gelatin phantom that embedded multiple tumor simulators at different NB concentrations. Simultaneous fluorescence and US images were acquired for these tumor simulators and linear correlations were observed between the fluorescence/US intensities and the NB concentrations. Our research demonstrated the technical feasibility of using the dual-mode NB contrast agent for cancer targeting and simultaneous fluorescence/US imaging.

Keywords: Cancer targeting; Dual-mode imaging; Nanobubbles; Nanoparticles; Fluorescence; Ultrasound

The performance of PEGylated nanocapsules of perfluorooctyl bromide as an ultrasound contrast agent by Raquel Díaz-López; Nicolas Tsapis; Mathieu Santin; Sharon Lori Bridal; Valérie Nicolas; Danielle Jaillard; Danielle Libong; Pierre Chaminade; Véronique Marsaud; Christine Vauthier; Elias Fattal (pp. 1723-1731).
The surface of polymeric nanocapsules used as ultrasound contrast agents (UCAs) was modified with PEGylated phospholipids in order to escape recognition and clearance by the mononuclear phagocyte system and achieve passive tumor targeting. Nanocapsules consisted of a shell of poly(lactide- co-glycolide) (PLGA) encapsulating a liquid core of perfluorooctyl bromide (PFOB). They were decorated with poly(ethylene glycol-2000)-grafted distearoylphosphatidylethanolamine (DSPE-PEG) incorporated in the organic phase before the solvent emulsification–evaporation process. The influence of DSPE-PEG concentration on nanocapsule size, surface charge, morphology, hydrophobicity and complement activation was evaluated. Zeta potential measurements, Hydrophobic interaction chromatography and complement activation provide evidence of DSPE-PEG presence at nanocapsule surface. Electronic microscopy reveals that the core/shell structure is preserved up to 2.64 mg of DSPE-PEG for 100 mg PLGA. In vivo ultrasound imaging was performed in mice bearing xenograft tumor with MIA PaCa-2 cells, either after an intra-tumoral or intravenous injection of nanocapsules. Tumor was observed only after the intra-tumoral injection. Despite the absence of echogenic signal in the tumor after intravenous injection of nanocapsules, histological analysis reveals their accumulation within the tumor tissue demonstrating that tissue distribution is not the unique property required for ultrasound contrast agents to be efficient.

Keywords: Polymeric nanocapsules; PEGylated phospholipids; Ultrasound contrast agent; Tumor distribution

Regulation of osteogenic differentiation of rat bone marrow stromal cells on 2D nanorod substrates by Gagandeep Kaur; Mani T. Valarmathi; Jay D. Potts; Esmaiel Jabbari; Tara Sabo-Attwood; Qian Wang (pp. 1732-1741).
Bone marrow stromal cells (BMSCs) possess multi-lineage differentiation potential and can be induced to undergo differentiation into various cell types with the correct combination of chemical and environmental factors. Although, they have shown great prospects in therapeutic and medical applications, less is known about their behavior on nanosurfaces mimicking the extra cellular matrix (ECM). In this report we have employed 2D substrates coated with tobacco mosaic virus (TMV) nanorods to study the differentiation process of BMSCs into osteoblast like cells. TMV is a rod-shaped plant virus with an average length of 300nm and diameter of 18nm. The osteogenic differentiation of BMSCs on TMV was studied over time points of 7, 14 and 21 days. We examined the temporal gene expression changes during these time points by real-time quantitative PCR (RT-qPCR) analysis. As expected, osteo-specific genes (osteocalcin, osteopontin and osteonectin) were upregulated and showed a maximum change in expression on TMV at 14 days which was 7 days earlier than on tissue culture plastic (TCP). Based on the genes expression profile generated by RT-qPCR experiments, we proposed that the early interaction of cells with TMV triggers on signaling pathways which regulate speedy expression of osteocalcin in turn, resulting in early mineralization of the cells. To further investigate these regulating factors we studied global changes in gene expression (DNA microarray analyses) during osteogenic differentiation on the nanosubstrate. Multitudes of genes were affected by culturing cells on nanorod substrate, which corroborated our initial PCR findings. Microarray analysis further revealed additional targets influenced by the presence of nanorods on the surface, of which, the expression of bone morphogenetic protein 2 (BMP2) was of particular interests. Further investigation into the temporal change of BMP2, revealed that it acts as a major promoter in signaling the early regulation of osteocalcin on TMV coated substrates.

Keywords: Nanoparticle; Nanotopography; Bone marrow stromal cells; Mesenchymal stem cells; Virus substrate; Osteogenic differentiation

Nano-scale control of cellular environment to drive embryonic stem cells selfrenewal and fate by Guillaume Blin; Nassrine Lablack; Marianne Louis-Tisserand; Claire Nicolas; Catherine Picart; Michel Pucéat (pp. 1742-1750).
Embryonic stem cells (ESC) are pluripotent cells capable to give rise to any embryonic cell lineage. In culture, these cells form colonies creating their own niche. Depending upon the molecular and physico-chemical environment, the pluripotent cells oscillate between two metastable states of pluripotency either reminiscent of the inner cell mass of the embryo or the epiblast, a stage of development which give rise to the three embryonic layers, ectoderm, endoderm and mesoderm. Herein, we used PLL/HA nanofilms cross-linked to various degrees to modulate the nanoenvironment of ESCs. Adhesion of ESC on nanofilms increased from native films to highly cross-linked films. The adhesion process was associated with cell proliferation. Expression of genes markers of the ICM decreased with adhesion of cells to cross-linked films. In parallel, genes more reminiscent of the epiblast, were turned on. ESC differentiation within embryoid bodies further revealed that cell pluripotency was better retained when cells did not adhere on native films. We further report that both the stiffness and the chemistry of nanofilms play a key role in modulating the niche of ESC and in turn govern their selfrenewal and fate.

Keywords: Stem cell; Nanofilm; Polyelectrolytes multilayers film; Cell niche; ICM; Epiblast

Design, syntheses and evaluation of hemocompatible pegylated-antimicrobial polymers with well-controlled molecular structures by Shrinivas Venkataraman; Ying Zhang; Lihong Liu; Yi-Yan Yang (pp. 1751-1756).
In this paper, we have designed and synthesized well-defined pegylated-polymers with tertiary amines from readily available commodity monomers 2-(dimethylamino)ethyl methacrylate (DMAEMA) and oligo(ethylene glycol) methyl ether methacrylate (OEGMA, Mn ∼ 475 Da) by reversible addition–fragmentation chain transfer (RAFT) polymerisation. By employing a simple and efficient post-polymerisation functionalization strategy, tertiary amines were quaternized to result in cationic polymers. By the careful selection of the functional halide, X–(CH2) q–R, (where in X=halide; R=the chemical functionality; q=the number of alkyl spacer between the quaternary ammonium group and R), a series of polymers with well-controlled molecular weight, different amphiphilic balance and chemical functionalities (such as alkyl, primary alcohol, primary amine and carboxylic acid) were readily synthesized. The antimicrobial activities of these cationic polymers were determined against Gram-positive bacteria Bacillus subtilis. Minimum inhibitory concentration (MIC), the polymer concentration to completely inhibit the bacterial growth, was found to be dependent both on the nature of functional group and the hydrophobicity of the polymer. Amongst the functional groups, both the alkyl and the alcohol groups were found to be effective, with MIC values in the range of 20–80mg/L. The haemolytic properties of polymers were analyzed against mouse red blood cells. The poymers with a short alkyl or hydroxyl group demonstrated little haemolysis, yet retained strong antimicrobial activity. The overall hydrophobicity of the polymer influenced its haemolytic behavior. These polymers can be promising antimicrobial agents. In addition, the approach proposed in this study to atom-efficient design and synthesis of antimicrobial polymers from the commercially available monomers can also be applied to develop well-defined functional cationic polymers for various biomedical applications.

Keywords: RAFT; Cationic polymers; Antimicrobial; Haemolysis

The utilization of pathogen-like cellular trafficking by single chain block copolymer by Gaurav Sahay; Vivek Gautam; Robert Luxenhofer; Alexander V. Kabanov (pp. 1757-1764).
Amphiphilic triblock copolymer, poly(ethylene oxide)- b-poly(propylene oxide)- b-poly(ethylene oxide), Pluronic® P85, is unexpectedly shown to utilize sophisticated cellular trafficking mechanisms and enter brain microvessel endothelial cells and primary neurons that are poorly penetrable. Though caveolae serve as a primary entry site for the copolymer single chains, in cells devoid of caveolae, the copolymer can still exploit caveolae- and clathrin-independent routes. This parallels the copolymer's trafficking itinerary with that of biological pathogens. The similarity is reinforced since both bypass early endosomes/lysosomes and transport to the endoplasmic reticulum. The copolymer finally reaches the mitochondrion that serves as its final destination. Notably, it also succeeds to gain entry in brain microvessel endothelial cells through caveolae and in primary neurons through caveolae- and clathrin-independent pathway. In neurons the copolymer accumulates in the cell body followed by anterograde trafficking towards the axons/dendrites. Overall, dissecting the trafficking of a synthetic polymer in multiple cell types triggers development of novel delivery systems that can selectively target intracellular compartments and provide entry in cells currently considered impenetrable.

Keywords: Pluronic block copolymer; Endocytosis; Intracellular trafficking; Pathogen; Drug delivery; Synthetic polymer

Polymeric micelles for the pH-dependent controlled, continuous low dose release of paclitaxel by Adam W.G. Alani; Younsoo Bae; Deepa A. Rao; Glen S. Kwon (pp. 1765-1772).
Poly(ethylene glycol)- block-poly(aspartate-hydrazide) (PEG-p(Asp-Hyd)) was modified using either levulinic acid (LEV) or 4-acetyl benzoic acid (4AB) attached via hydrazone bonds. Paclitaxel (PTX) conjugated to the linkers formed PEG-p(Asp-Hyd-LEV-PTX) and PEG-p(Asp-Hyd-4AB-PTX). PEG-p(Asp-Hyd-LEV-PTX) and PEG-p(Asp-Hyd-4AB-PTX) assemble into unimodal polymeric micelles with diameters of 42 nm and 137 nm, respectively. PEG-p(Asp-Hyd-LEV-PTX) and PEG-p(Asp-Hyd-4AB-PTX) at a 1:1 and 1:5 molar ratio assemble into unimodal mixed polymeric micelles with diameters of 85 and 113 nm, respectively. PEG-p(Asp-Hyd-LEV-PTX) micelles release LEV-PTX faster at pH 5.0 than at pH 7.4 over 24 h. At pH 7.4 mixed polymeric micelles at 1:5 ratio show no difference in LEV-PTX release from PEG-p(Asp-Hyd-LEV-PTX) micelles. Mixed polymeric micelles at 1:5 molar ratio gradually release LEV-PTX at pH 5.0, with no release of 4AB-PTX. PEG-p(Asp-Hyd-LEV-PTX) micelles and mixed polymeric micelles exert comparable cytotoxicity against SK-OV-3 and MCF-7 cancer cell lines. In summary, mixed polymeric micelles based on PEG-p(Asp-Hyd-LEV-PTX) and PEG-p(Asp-Hyd-4AB-PTX) offer prospects for pH-dependent release of PTX, offering a novel prodrug strategy for adjusting its pharmacokinetic and pharmacodynamic properties for cancer therapy. If successful this delivery system offers an alternative new mode of delivery for paclitaxel with a new scope for its efficacy along with a minimal synthetic framework needed to accomplish this.

Keywords: Controlled drug release; Hydrazone; Mixed micelle; Paclitaxel; Prodrug

Sustained release of ganciclovir and foscarnet from biodegradable scleral plugs for the treatment of cytomegalovirus retinitis by Yi-Jie Peng; Chin-Wei Wen; Shih-Hwa Chiou; Shih-Jung Liu (pp. 1773-1779).
The purpose of this report was to develop solvent-free biodegradable scleral plugs for simultaneous ganciclovir and foscarnet delivery for cytomegalovirus retinitis treatment. To fabricate a biodegradable plug, polylactide–polyglycolide copolymers were pre-mixed with the drugs. The mixture was then compression molded and sintered to form a compact scleral plug. The drug release features were monitored with HPLC assay both in vitro and in vivo. Both drugs showed a biphasic release curvature with an initial burst and followed by a second sustained release phase and maintained at therapeutic level for 3–4 weeks. As compared to ganciclovir, foscarnet was released faster in initial phase, but later, showed extended retention in vitreous humor. For biocompatibility analysis, dark-adapted flash electroretinography was performed, and the a-wave and b-wave amplitudes were statistically equal before and after the scleral plug implantation. Finally, serial microstructure changes of releasing scleral plugs were evaluated with scanning electron microscope. The scleral plug surface showed progressive transformation from granular solid surface to smoothen and cavitated appearance.

Keywords: Biodegradable scleral plugs; Polylactide–polyglycolide; Ganciclovir; Foscarnet; Intravitreal sustained release; Cytomegalovirus retinitis

Molecular dynamics study of the encapsulation capability of a PCL–PEO based block copolymer for hydrophobic drugs with different spatial distributions of hydrogen bond donors and acceptors by Sarthak K. Patel; Afsaneh Lavasanifar; Phillip Choi (pp. 1780-1786).
Molecular dynamics simulation was used to study the potential of using a block copolymer containing three poly(ɛ-caprolactone) (PCL) blocks of equal length connected to one end of a poly(ethylene oxide) (PEO) block, designated as PEO- b-3PCL, to encapsulate two classes of hydrophobic drugs with distinctively different molecular structures. In particular, the first class of drugs consisted of two cucurbitacin drugs (CuB and CuI) that contain multiple hydrogen bond donors and acceptors evenly distributed on their molecules while the other class of drugs (fenofibrate and nimodipine) contain essentially only clustered hydrogen bond acceptors. In the case of cucurbitacin drugs, the results showed that PEO- b-3PCL lowered the Flory–Huggins interaction parameters ( χ) considerably (i.e., increased the drug solubility) compared to the linear di-block copolymer PEO- b-PCL with the same PCL/PEO (w/w) ratio of 1.0. However, the opposite effect was observed for fenofibrate and nimodipine. Analysis of the intermolecular interactions indicates that the number of hydrogen bonds formed between the three PCL blocks and cucurbitacin drugs is significantly higher than that of the linear di-block copolymer. On the other hand, owing to the absence of hydrogen bond donors and the clustering of the hydrogen bond acceptors on the fenofibrate and nimodipine molecules, this significantly reduces the number of hydrogen bonds formed in the multi-PCL block environment, leading to unfavourable χ values. The findings of the present work suggest that multi-hydrophobic block architecture could potentially increase the drug loading for hydrophobic drugs with structures containing evenly distributed multiple hydrogen bond donors and acceptors.

Keywords: Molecular dynamics simulation; Hydrophobic drugs; Multi-block copolymer; Interaction parameter; Hydrogen bond donors and acceptors

The use of RGDGWK-lipopeptide to selectively deliver genes to mouse tumor vasculature and its complexation with p53 to inhibit tumor growth by Sanjoy Samanta; Ramakrishna Sistla; Arabinda Chaudhuri (pp. 1787-1797).
In vivo selection of phage display libraries have been exploited successfully in the past to isolate various high affinity conformationally strained cyclic peptide ligands (CX5–7C, peptides flanked by a cysteine residue on each side) for integrin receptors capable of selectively homing to tumor vasculatures. Previously, such phase display library studies have shown that integrin α5β1 binds with high affinity to cyclic peptides containing CRGDGWC motif. Herein we show that a lipopeptide with just the RGDGW motif (without the flanking cysteine groups) covalently attached to the lysine residue of a monolysinylated cationic amphiphile (RGDGWK-lipopeptide1) delivers genes to cultured cells preferably via α5β1 integrins. Importantly, remarkable tumor growth inhibition was observed when the electrostatic complex of the RGDGWK-lipopeptide1 and the anti-cancer p53 gene was intravenously administered in C57BL/6J mice bearing the aggressive B16F10 tumor. Immunohistochemical staining of mice tumor cryosections with vasculature markers combined with monitoring expression of the green fluorescence protein in the same tumor cryosections revealed that the RGDGWK-lipopeptide1 targets genes to tumor vasculatures. The colocalization of the TUNEL ( terminal deoxy uridine triphosphate nick- end labeling, a widely used marker of apoptosis) and VE-cadherin (markers of tumor endothelial cells) positive cells in tumor cryosections support the notion that the remarkable tumor growth inhibition property of the RGDGWK-lipopeptide1:p53 complex is initiated through apoptosis of the tumor endothelial cells.

Keywords: Tumor vasculature targeting; Integrin targeting; Tumor growth inhibitionAbbreviations; EDCI; 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; HOBt; 1-Hydroxybenztriazole; BOC; Tert-butyloxy carbonyl; Cbz; Benzyloxycarbonyl; Chol; Cholesterol; DCM; Dichloromethane; DMEM; Dulbecco's Modifid Eagles Medium; DMF; N,N-dimethylformamide; FBS; Fetal bovine serum; PBS; Phosphate buffered saline; GFP; Green fluorescence protein; TUNEL; Terminal deoxynucleotidyl transferase dUTP nick end labeling; FACS; Fluorescence activated cell sorter

A guanidinylated bioreducible polymer with high nuclear localization ability for gene delivery systems by Tae-il Kim; Minhyung Lee; Sung Wan Kim (pp. 1798-1804).
Guanidinylated bioreducible polymer (GBP) was developed for gene delivery systems utilizing cellular penetrating ability of guanidine groups. GBP could retard pDNA from a weight ratio of 5 completely in agarose gel electrophoresis but pDNA was released from GBP polyplexes even at a weight ratio of 20 in reducing condition (2.5mm DTT) due to their biodegradation. GBP also could construct 200 nm-sized and positively charged (∼30mV) polyplex nanoparticles with pDNA. The cytotoxicity of GBP was found to be minimal and GBP showed about 8 folds improved transfection efficiency than a scaffold polymer, poly(cystaminebisacrylamide-diaminohexane) (poly(CBA-DAH)) and even higher transfection efficiency than PEI25k in mammalian cell lines. Its high cellular uptake efficiency (96.1%) and strong nuclear localization ability for pDNA delivery due to the structural advantage of bioreducible polymer and guanidine groups were also identified, suggesting GBP is a promising candidate for efficient gene delivery systems.

Keywords: Bioreducible polymer; Gene transfer; Guanidinylation; Cytotoxicity; Nuclear localization ability

Structure-activity relationships of cationic shell-crosslinked knedel-like nanoparticles: Shell composition and transfection efficiency/cytotoxicity by Ke Zhang; Huafeng Fang; Zhenghui Wang; Zhou Li; John-Stephen A. Taylor; Karen L. Wooley (pp. 1805-1813).
Cationic nanoparticles are a promising class of transfection agents for oligonucleotide and gene delivery, but vary greatly in their effectiveness and cytotoxicity. Recently, we developed a new class of cationic transfection agents based on cationic shell-crosslinked knedel-like nanoparticles (cSCKs) that efficiently transfect mammalian cells with both oligonucleotides and plasmid DNA. In an effort to further improve transfection efficiency without increasing cytotoxicity, we examined the effects of the composition of primary amine (pa), tertiary amine (ta) and carboxylic acid (ca) groups in the shell of these nanoparticles. A series of discrete complexes of the cSCKs with plasmid DNA (pDNA) or phosphorothioate 2′-OMe oliogonucleotides (ps-MeON) were prepared over a broad range of amine to phosphate ratios ( N/ P ratio) of 4:1–40:1. The sizes of the complexes and the ability of the nanoparticles to completely bind ODNs were found to depend on the cSCK amine to DNA phosphate ( N/ P) ratio and the cSCK buffering capacity. The cSCKs were then evaluated for their ability to transfect cells with plasmid DNA by monitoring fluorescence from an encoded EGFP, and for delivery of ps-MeON by monitoring luminescence from luciferase resulting from ps-MeON-mediated splicing correction. Whereas the cationic cSCK-pa25-ta75 was found to be best for transfecting plasmid DNA into HeLa cells at an N/ P ratio of 20:1, cSCK-pa50-ta50, at an N/ P ratio 10:1 was best for ps-MeON delivery. We also found that increasing the proportion of tertiary relative to primary amine reduced the cytotoxicity. These results demonstrate that a dramatic improvement in gene and oligonucleotide delivery efficiency with decreased cytotoxicity in HeLa cells can be achieved by incorporation of tertiary amines into the shells of cSCKs.

Keywords: Nanoparticle; Gene transfer; Cytotoxicity; Endocytosis; Proton sponge effect; Antisense

Ultrapure chitosan oligomers as carriers for corneal gene transfer by Eytan A. Klausner; Zhong Zhang; Robert L. Chapman; Richard F. Multack; Michael V. Volin (pp. 1814-1820).
NOVAFECT chitosans are ultrapure chitosan oligomers that were recently marketed as carriers for non-viral gene therapy. There are no reports on systematic design and improvement of formulations based on NOVAFECT chitosans for gene delivery. Therefore, we have designed and characterized chitosan-DNA nanoparticles based on NOVAFECT. We found that the size of oligomeric chitosan-DNA nanoparticles is small, ≤98.2±4.4nm. Zeta potential measurements of oligomeric chitosan-DNA nanoparticles exhibited a strong positive charge ≥44.1±3.5millivolt. In vitro transfection studies demonstrated the ability of oligomeric chitosan-DNA nanoparticles to effectively transfect COS-7 cells. In rat corneas, injection of a select formulation of oligomeric chitosan-DNA nanoparticles into the stroma showed that (a) luciferase gene expression was 5.4 times greater than following administration of polyethylenimine-DNA nanoparticles; and (b) the cells that express the transgene, green fluorescent protein, were keratocytes (corneal fibroblasts). This study lays the foundation for evaluating oligomeric chitosan-DNA nanoparticles as pharmaceuticals for corneal gene therapy, a promising approach for the treatment of acquired and inherited corneal diseases that otherwise lead to blindness. Oligomeric chitosan-DNA nanoparticles can also be evaluated for the treatment of ocular diseases outside of the cornea, and for various additional gene therapy applications.

Keywords: Ultrapure chitosan oligomers; Chitosan-DNA nanoparticles; Corneal gene therapy; Non-viral gene transfer; Cornea

Efficient siRNA delivery to mammalian cells using layered double hydroxide nanoparticles by Katharina Ladewig; Marcus Niebert; Zhi P. Xu; Peter P. Gray; Gao Q.M. Lu (pp. 1821-1829).
Although siRNAs have surpassed expectations in experiments to alter gene expression in vitro, the lack of an efficient in vivo delivery system still remains a challenge in siRNA therapeutics development and has been recognized as a major hurdle for clinical applications. In this paper we describe an inorganic nanoparticle-based delivery system that is readily adaptable for in vivo systems. Layered double hydroxide (LDH) nanoparticles, a family of inorganic crystals, tightly bind, protect, and release siRNA molecules and deliver them efficiently to mammalian cells in vitro. The uptake of siRNA-loaded LDH nanoparticles occurs via endocytosis, whereby the nanoparticles dissolve due to the low pH in the endosome, thereby aiding endosomal escape into the cytoplasm. The influence of LDH nanoparticles on cell viability and proliferation is negligible at concentrations ≤0.050mgmL−1, and a pronounced down-regulation of protein expression upon LDH mediated siRNA transfection of HEK293T cells is observed.

Keywords: siRNA delivery; Layered double hydroxide nanoparticles

Low molecular weight polyethylenimine cross-linked by 2-hydroxypropyl-γ-cyclodextrin coupled to peptide targeting HER2 as a gene delivery vector by Hongliang Huang; Hai Yu; Guping Tang; Qingqing Wang; Jun Li (pp. 1830-1838).
Gene delivery is one of the critical steps for gene therapy. Non-viral vectors have many advantages but suffered from low gene transfection efficiency. Here, in order to develop new polymeric gene vectors with low cytotoxicity and high gene transfection efficiency, we synthesized a cationic polymer composed of low molecular weight polyethylenimine (PEI) of molecular weight of 600 Da cross-linked by 2-hydroxypropyl-γ-cyclodextrin (HP γ-CD) and then coupled to MC-10 oligopeptide containing a sequence of Met-Ala-Arg-Ala-Lys-Glu. The oligopeptide can target to HER2, the human epidermal growth factor receptor 2, which is often over expressed in many breast and ovary cancers. The new gene vector was expected to be able to target delivery of genes to HER2 positive cancer cells for gene therapy. The new gene vector was composed of chemically bonded HP γ-CD, PEI (600 Da), and MC-10 peptide at a molar ratio of 1:3.3:1.2. The gene vector could condense plasmid DNA at an N/P ratio of 6 or above. The particle size of HP γ-CD-PEI-P/DNA complexes at N/P ratios 40 was around 170–200nm, with zeta potential of about 20 mV. The gene vector showed very low cytotoxicity, strong targeting specificity to HER2 receptor, and high efficiency of delivering DNA to target cells in vitro and in vivo with the reporter genes. The delivery of therapeutic IFN-α gene mediated by the new gene vector and the therapeutic efficiency were also studied in mice animal model. The animal study results showed that the new gene vector HP γ-CD-PEI-P significantly enhanced the anti-tumor effect on tumor-bearing nude mice as compared to PEI (25kDa), HP γ-CD-PEI, and other controls, indicating that this new polymeric gene vector is a potential candidate for cancer gene therapy.

Keywords: Non-viral gene vector; Polyethylenimine; Cyclodextrin; HER2; Cancer gene therapy

Development of pH–responsive nanocarriers using trimethylchitosans and methacrylic acid copolymer for siRNA delivery by V. Dehousse; N. Garbacki; A. Colige; B. Evrard (pp. 1839-1849).
RNA interference-based therapies are dependent on intracellular delivery of siRNA. The release of siRNA from the endosomal compartment may be a rate limiting step in the transfection process. The purpose of this study was to produce pH–responsive nanocarriers made of trimethylchitosan (TMC). To this end, pH-sensitive methacrylic acid (MAA) copolymer was added to TMC–siRNA formulations. Four different TMCs associated or not with MAA were evaluated as siRNA carriers. Nanoparticles were characterized in terms of size, surface charge, morphology and interaction with siRNA. A swelling behaviour due to a decrease in pH was observed and was found to be dependent on MAA content in the complexes. In vitro experiments aimed at evaluating how the capacity of the nanocarriers to transfect siRNA in L929 cells was affected by MAA content. Confocal microscopy experiments showed that fluorescent MAA-containing particles exhibit a different distribution pattern inside the cells comparing to their counterpart without this pH-sensitive polymer. Transfection efficiency was investigated by RhoA mRNA expression inhibition. MAA–TMC–siRNA complexes were able to transfect L929 cells with greater efficiency than corresponding TMC–siRNA complexes. This study gives an insight into the opportunity of pH-sensitive nanocarriers for siRNA delivery. Such formulations may represent an attractive strategy to improve endosomal escape of siRNA.

Keywords: Chitin/chitosan; Drug delivery; Gene therapy; RNA interference

The enhancement of transfection efficiency of cationic liposomes by didodecyldimethylammonium bromide coated gold nanoparticles by Dan Li; Gaiping Li; Peicai Li; Lixue Zhang; Zuojia Liu; Jin Wang; Erkang Wang (pp. 1850-1857).
The development of transfection enhancement of liposomes with attributes of high stability and easy handling in gene therapy is challenging. In this study, we report didodecyldimethylammonium bromide (DDAB, a cationic lipid) coated gold nanoparticles (DDAB-AuNPs), which can enhance the transfection efficiency generated by two kinds of commercially available cationic liposomes: Lipotap and DOTAP. It showed that DDAB-AuNPs at the optimal concentrations could produce more than 2 times increase when measuring the number of cells expressed green fluorescent protein and 48-fold increase for luciferase levels after transfection, respectively. The electrophoretic mobility shift assay (EMSA) and confocal laser scanning microscopy (CLSM) experiments showed that more DNA molecules binding to the lipoplexes after adding DDAB-AuNPs. In addition, the flow cytometry (FCM) results indicated that DDAB-AuNPs increased cellular uptake efficiency of DNA molecules, which might account for the enhancement of transfection efficiency. It has also been found that the DDAB-AuNPs could decrease the cytotoxicity of liposomes to the cells.

Keywords: Trasfection enhancer; Liposome; Gold nanoparticles

Gene delivery using a derivative of the protein transduction domain peptide, K-Antp by Sang-Hyun Min; Dong Min Kim; Mi Na Kim; Jiang Ge; Dong Chul Lee; In Young Park; Kyung Chan Park; Ji-Sook Hwang; Cheong-Weon Cho; Young Il Yeom (pp. 1858-1864).
Due to their intracellular permeability, protein transduction domains (PTDs) have been widely used to deliver proteins and peptides to mammalian cells. However, their performance in gene delivery has been relatively poor. To improve the efficiency of PTD-mediated gene delivery, we synthesized a new peptide, KALA-Antp (K-Antp), which contains the sequences for PTD of the third α-helix of Antennapedia (Antp) homeodomain and the fusogenic peptide KALA. In this configuration, Antp is designed to provide the cell permeation capacity and nuclear localization signal, while the KALA moiety to promote cellular entry of the peptide–DNA complex. An optimal K-Antp/DNA formula was nearly 400–600 fold more efficient than Antp or poly-lysine-Antp (L-Antp) in gene delivery, and comparable or superior to a commercial liposome. The K-Antp-mediated plasmid DNA transfection not only exhibited temperature sensitivity, reflecting the involvement of an endocytosis-mediated gene transfer mechanism similar to other known PTDs, but also temperature insensitivity, suggesting the role of an energy-independent mechanism. Incorporation of an endosomolytic polymer polyethylenimine (PEI) into the system or treatment with chloroquine further increased the efficiency of K-Antp-mediated gene delivery. These results demonstrate the potential of the combinatorial use of KALA, Antp and PEI in the development of efficient PTD-derived gene carriers.

Keywords: KALA-Antp (K-Antp); Protein transduction domain (PTD); Fusogenic peptide; Antennapedia peptide (Antp); KALA; Gene delivery

The effect of surface modification of adenovirus with an arginine-grafted bioreducible polymer on transduction efficiency and immunogenicity in cancer gene therapy by Pyung-Hwan Kim; Tae-il Kim; James W. Yockman; Sung Wan Kim; Chae-Ok Yun (pp. 1865-1874).
Adenoviral vectors offer many advantages for cancer gene therapy, including high transduction efficiency, but safety concerns related to severe immunogenicity and other side effects have led to careful reconsideration of their use in human clinical trials. To overcome these issues, a strategy of generating hybrid vectors that combine viral and non-viral elements as more intelligent gene carriers has been employed. Here, we coated adenovirus (Ad) with an arginine-grafted bioreducible polymer (ABP) via electrostatic interaction. We examined the effect of ABP-coated Ad complex at various ABP molecules/Ad particle ratios. Enhanced transduction efficiency was observed in cells treated with cationic ABP polymer-coated Ad complex compared to naked Ad. We also examined the coating of Ad with ABP polymers at the optimal polymer ratio using dynamic light scattering and transmission electron microscopy. In both high and low coxsackie virus and adenovirus receptor (CAR)-expressing cells, ABP-coated Ad complex produced higher levels of transgene expression than cationic polymer 25K PEI. Notably, high cytotoxicity was observed with 25K PEI-coated Ad complex treatment, but not with ABP-coated Ad complex treatment. In addition, ABP-coated Ad complex was not significantly inhibited by serum, in contrast to naked Ad. Moreover, ABP-coated Ad complex significantly reduced the innate immune response relative to naked Ad, as assessed by interleukin-6 (IL-6) cytokine release from macrophage cells. Overall, our studies demonstrate that Ad complex formed with ABP cationic polymer may improve the efficiency of Ad and be a promising tool for cancer gene therapy.

Keywords: Cancer gene therapy; Adenovirus; Arginine-grafted bioreducible polymer; Hybrid vector; Electrostatic interaction

Probing cellular mechanobiology in three-dimensional culture with collagen–agarose matrices by Theresa A. Ulrich; Amit Jain; Kandice Tanner; Joanna L. MacKay; Sanjay Kumar (pp. 1875-1884).
The study of how cell behavior is controlled by the biophysical properties of the extracellular matrix (ECM) is limited in part by the lack of three-dimensional (3D) scaffolds that combine the biofunctionality of native ECM proteins with the tunability of synthetic materials. Here, we introduce a biomaterial platform in which the biophysical properties of collagen I are progressively altered by adding agarose. We find that agarose increases the elasticity of 3D collagen ECMs over two orders of magnitude with modest effect on collagen fiber organization. Surprisingly, increasing the agarose content slows and eventually stops invasion of glioma cells in a 3D spheroid model. Electron microscopy reveals that agarose forms a dense meshwork between the collagen fibers, which we postulate slows invasion by structurally coupling and reinforcing the collagen fibers and introducing steric barriers to motility. This is supported by time lapse imaging of individual glioma cells and multicellular spheroids, which shows that addition of agarose promotes amoeboid motility and restricts cell-mediated remodeling of individual collagen fibers. Our results are consistent with a model in which agarose shifts ECM dissipation of cell-induced stresses from non-affine deformation of individual collagen fibers to bulk-affine deformation of a continuum network.

Keywords: Hydrogel; Collagen; ECM (extracellular matrix); Mechanical properties; Elasticity; Brain

The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells by Jeffrey C. Mohr; Jianhua Zhang; Samira M. Azarin; Andrew G. Soerens; Juan J. de Pablo; James A. Thomson; Gary E. Lyons; Sean P. Palecek; Timothy J. Kamp (pp. 1885-1893).
The differentiation of human embryonic stem cells (hESCs) into cardiomyocytes (CMs) using embryoid bodies (EBs) is relatively inefficient and highly variable. Formation of EBs using standard enzymatic disaggregation techniques results in a wide range of sizes and geometries of EBs. Use of a 3-D cuboidal microwell system to culture hESCs in colonies of defined dimensions, 100–500 μm in lateral dimensions and 120 μm in depth, enabled formation of more uniform-sized EBs. The 300 μm microwells produced highest percentage of contracting EBs, but flow cytometry for myosin light chain 2A (MLC2a) expressing cells revealed a similar percentage (∼3%) of cardiomyocytes formed in EBs from 100 μm to 300 μm microwells. These data, and immunolabeling with anti-MF20 and MLC2a, suggest that the smaller EBs are less likely to form contracting EBs, but those contracting EBs are relatively enriched in cardiomyocytes compared to larger EB sizes where CMs make up a proportionately smaller fraction of the total cells. We conclude that microwell-engineered EB size regulates cardiogenesis and can be used for more efficient and reproducible formation of hESC-CMs needed for research and therapeutic applications.

Keywords: Human embryonic stem cells; Microwells; Embryoid body; Cardiomyocytes; Differentiation

Hepatitis B virus induced coupling of deadhesion and migration of HepG2 cells on thermo-responsive polymer by Xi Li; Huixing Feng; Wei Ning Chen; Vincent Chan (pp. 1894-1903).
The unique physical property of thermo-responsive polymer (TRP) has recently prompted its increasing applications in tissue engineering. On the other hand, TRP has not been exploited for potential applications in quantitative cell screening against external stimulations. In this study, TRP is applied as a model system for elucidating the effect of HBV replication on the biophysical responses of HepG2 cells transfected by wild type HBV genome. Moreover, mutant HBV genome is designed to assess the specific activity of the SH3-binding domain of HBx during HBV replication. The adhesion contact recession and geometry transformation of HepG2 cells transfected with empty vector (pcDNA3.1 cells), wild type HBV (wtHBV cells) and mutant HBV genome (mHBV cells) are probed during the thermal transformation across lower solution critical temperature of TRP. In comparison with pcDNA3.1 cells and mHBV cells, the initial rate of reduction in degree of deformation and average adhesion energy for wtHBV cells is significantly increased. Interestingly, migration speed and persistence time of cells are found to be correlated with the cell deadhesion kinetics. Immuno-fluorescence microscopy demonstrates that HBV replication reduces the actin concentration and focal adhesions at cell periphery during the initial 30 min cell deadhesion. The results strongly suggested that HBV infection triggers the dynamic responses of HepG2 cells through the cytoskeleton remodeling and subsequent mechanochemical transduction. Overall, it is shown that TRP provides a convenient platform for quantifying biological stimulations on adherent cells.

Keywords: Deadhesion; Migration; Biophysics; HBV

Immobilization of bacteriophages on modified silica particles by Rebecca Cademartiri; Hany Anany; Isabelle Gross; Rahul Bhayani; Mansel Griffiths; Michael A. Brook (pp. 1904-1910).
Bacteriophages are selective anti-bacterial agents, which are receiving increasing acceptance by regulatory agencies for use both in the food industry and in clinical settings for biocontrol. While immobilized phage could be particularly useful to create antimicrobial surfaces, current immobilization strategies require chemical bioconjugation to surfaces or more difficult processes involving modification of their head proteins to express specific binding moieties, for example, biotin or cellulose binding domains; procedures that are both time and money intensive. We report that morphologically different bacteriophages, active against a variety of food-borne bacteria: Escherichia coli; Salmonella enterica; Listeria monocytogenes; and Shigella boydii, will effectively physisorb to silica particles, prepared by silica surface modification with poly(ethylene glycol), carboxylic acid groups, or amines. The phages remain infective to their host bacteria while adsorbed on the surface of the silica particles. The number of infective phage bound to the silica is enhanced by the presence of ionic surfaces, with greater surface charge – to a maximum – correlating with greater concentration of adsorbed phage. Above the maximum charge concentration, the number of active phage drops.

Keywords: Surface modification; Silica; Bacteriophage; Adsorption; Anti-bacterial; Protein adsorption

Sentinel lymph node mapping by a near-infrared fluorescent heptamethine dye by Chao Zhang; Shaojun Wang; Juan Xiao; Xu Tan; Ying Zhu; Yongping Su; Tianmin Cheng; Chunmeng Shi (pp. 1911-1917).
We describe a near-infrared fluorescent heptamethine dye (IR-780 iodide) with unique properties for sentinel lymph node (SLN) mapping in both small and large animals. This dye has a significant photobrightening effect in serum and a long retention time in the lymphatic system which allows to acquire much higher signal-to-noise ratios. Injection of only 10 nmol of this dye permits SLNs to be imaged easily in pigs using excitation fluence rates of only 2 μW/cm2. In addition, this dye has a unique stability property after formalin fixation in tissues which raises the possibility of developing new and sensitive means of detecting lymph nodes in harvested surgical specimens. This dye can be completely cleared from the circulation in a couple of days and does not cause acute systemic toxicity.

Keywords: In vivo test; Fluorescence; Sentinel lymph node; Mapping; Heptamethine dye

Fluorescent oligonucleotide probe based on G-quadruplex scaffold for signal-on ultrasensitive protein assay by Zai-Sheng Wu; Peng Hu; Hui Zhou; Guoli Shen; Ruqin Yu (pp. 1918-1924).
This work reported the G-quadruplex structure of pyrene-labeled G-rich DNA probe and its application in the immunoglobulin E (IgE) detection, providing plausibly an insight into the biological function of human telomere. Based on the intermolecular G-quadruplex, a terminal-single-pyrene-labeled oligonucleotide signaling probe was developed and a novel protein assay strategy was proposed via combining specific DNA cleavage by S1 nuclease with target-recognizing aptamer. This assay platform not only circumvented the optimization of specific sites for reporter attachment and the pyrene monomer fluorescence quenching by flanking nucleotide bases but also presented a signal-on mechanism. Thus, ultrasensitive homogeneous detection of IgE was successfully conducted. A linear dynamic range of 4.72 × 10−12 to 7.56 × 10−9m, a regression coefficient of 0.9941 and a detection limit of 9.45 × 10−14m were given. Additionally, a preliminary concept of the single-pyrene-labeled excimer fluorescence probes associated with G-quadruplex for screening biological markers was described. Importantly, the unexpected structural property of G-quadruplex discovered seems to provide valuable information to allow understanding of the structure and function of human telomere and exploring of useful structure-based anticancer drug.

Keywords: G-quadruplex; Aptamer; Single-pyrene-labeled signaling probe; S1 nuclease; IgE

A collagen-mimetic triple helical supramolecule that evokes integrin-dependent cell responses by Chisato M. Yamazaki; Yuichi Kadoya; Kentaro Hozumi; Hitomi Okano-Kosugi; Shinichi Asada; Kouki Kitagawa; Motoyoshi Nomizu; Takaki Koide (pp. 1925-1934).
Collagen is an abundantly distributed extracellular matrix protein in mammalian bodies that maintains structural integrity of the organs and tissues. Besides its function as a structural protein, collagen has various biological functions which regulate cell adhesion, migration and differentiation. In order to develop totally synthetic collagen-surrogates, we recently reported a basic concept for preparing collagen-like triple helical supramolecules based on the self-assembly of staggered trimeric peptides with self-complementary shapes. In this paper, we add one of the specific cellular functions of the native collagen to the collagen-mimetic supramolecule. We synthesized a self-assembling peptide unit containing the integrin-binding sequence Gly-Phe-Hyp-Gly-Glu-Arg. The supramolecule carrying the sequence exhibited significant binding activity to human dermal fibroblasts. The supramolecular structure was found to be essential for function in in vitro cell culture. Cell adhesion was shown to be comparable to that of native collagen, and was further demonstrated to be mediated solely by integrin α2β1. Well-grown focal contacts and stress fibers were observed in cells spread on the supramolecular collagen-mimetic. The results demonstrate the potential of peptide-based artificial collagen as a biomaterial for regulating specific cellular function and fate.

Keywords: Collagen; Cell adhesion; ECM (extracellular matrix); Integrin; PeptideAbbreviations; Acm; acetamidomethyl; AP; alkaline phosphatase; BSA; bovine serum albumin; CD; circular dichroism; DMEM; Dulbecco's modified Eagle's medium; ECM; extracellular matrix; EDTA; ethylenediaminetetraacetic acid; FAK; focal adhesion kinase; FITC; fluorescein isothiocyanate; Fmoc; 9-fluorenylmethoxycarbonyl; Gdn-HCl; guanidine hydrochloride; HDF; human dermal fibroblast; Hyp or O; 4-hydroxyproline; MALDI-TOF MS; matrix-assisted laser desorption-ionization time-of-flight mass spectrometry; Npys; 3-nitro-2-pyridinesulfenyl; RP-HPLC; reversed phase-high performance liquid chromatography; PBS; phosphate buffered saline; Pys; 2-pyridinesulfenyl; PySSPy; 2,2′-dipyridyldisulfide; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; TFA; trifluoroacetic acid;Standard one-letter abbreviations were used for the common amino acids

Creation of lysine-deficient mutant lymphotoxin-α with receptor selectivity by using a phage display system by Yasuo Yoshioka; Hikaru Watanabe; Tomohiro Morishige; Xinglei Yao; Shinji Ikemizu; Chioko Nagao; Shandar Ahmad; Kenji Mizuguchi; Shin-ichi Tsunoda; Yasuo Tsutsumi; Yohei Mukai; Naoki Okada; Shinsaku Nakagawa (pp. 1935-1943).
The cytokine lymphotoxin-α (LTα) activates various biological functions through its three receptor subtypes, tumor necrosis factor receptor 1 (TNFR1), TNFR2 and herpes virus entry mediator (HVEM), but the relative contribution of each receptor to each function is unclear. Therefore it is important to create mutant LTα with receptor selectivity for optimized cancer therapy and the analysis of receptor function. Here, we attempted to create a lysine-deficient mutant LTα with TNFR1-selective bioactivity using a phage display technique. We obtained the TNFR1-selective mutant LTα R1selLT, which contained the mutations K19N, K28Q, K39S, K84Q, K89V, and K119H. Compared with wild-type LTα (wtLTα), R1selLT showed several-fold higher bioactivity via TNFR1 but 40-fold lower bioactivity via TNFR2. Kinetic association–dissociation parameters of R1selLT with TNFR2 were higher than those of wtLTα, whereas these parameters of R1selLT with TNFR1 were lower than those of wtLTα, suggesting that destabilization of the R1selLT–TNFR2 complex causes the decreased bioactivity of R1selLT on TNFR2. We also showed that the K84Q mutation contributed to the enhanced activity via TNFR1, and K39S lowered activity via TNFR2. R1selLT likely will be useful in cancer therapy and in analysis of the LTα structure–function relationship.

Keywords: Affinity; Bioactivity; Cytokine; Molecular modeling; Recombinant protein

Engineering fibrin matrices: The engagement of polymerization pockets through fibrin knob technology for the delivery and retention of therapeutic proteins by Allyson S.C. Soon; Sarah E. Stabenfeldt; Wendy E. Brown; Thomas H. Barker (pp. 1944-1954).
Engineering extracellular matrices that utilize the body's natural healing capacity enable the progression of regenerative therapies. Fibrin, widely used as a surgical sealant, is one such matrix that may be augmented by the addition of protein factors to promote cell infiltration and differentiation. The thrombin-catalyzed conversion of fibrinogen to fibrin exposes N-terminal fibrinknobs that bind to C-terminalpockets to form the fibrin network. Here, we have created a platform system for the production of therapeutic proteins that capitalize on these nativeknob:pocket interactions for protein delivery within fibrin matrices. This system enables the retention of therapeutic proteins within fibrin without additional enzymatic or synthetic crosslinking factors. Using an integrin-binding fibronectin fragment as a model protein, we demonstrate that engineered knob–protein fusions bind consistently and specifically to fibrin(ogen). Equilibrium dissociation constants ( KD) obtained using surface plasmon resonance indicate that these fusions have μm binding affinities, comparable to the native knob-containing fibrin fragments. The specificity of these interactions was verified by ELISA in the presence of molar excess of competing knob mimics. Release profiles and real-time confocal imaging demonstrate that the fusions were retained within fibrin matrices, even under the stringent continuous perfusion conditions used in the latter. In summary, this work explores the benefits and limitations of engaging native, biologically-inspired, non-covalent knob:pocket interactions within fibrin(ogen) for the retention of therapeutic proteins in fibrin matrices and provides insight into the stability of native knob:pocket interactions within fibrin networks.

Keywords: Fibrin; Fibrinogen; Molecular biology; Recombinant protein; Affinity; Controlled drug release

Size-dependent elastic/inelastic behavior of enamel over millimeter and nanometer length scales by Siang Fung Ang; Emely L. Bortel; Michael V. Swain; Arndt Klocke; Gerold A. Schneider (pp. 1955-1963).
The microstructure of enamel like most biological tissues has a hierarchical structure which determines their mechanical behavior. However, current studies of the mechanical behavior of enamel lack a systematic investigation of these hierarchical length scales. In this study, we performed macroscopic uni-axial compression tests and the spherical indentation with different indenter radii to probe enamel's elastic/inelastic transition over four hierarchical length scales, namely: ‘bulk enamel’ (mm), ‘multiple-rod’ (10's μm), ‘intra-rod’ (100's nm with multiple crystallites) and finally ‘single-crystallite’ (10's nm with an area of approximately one hydroxyapatite crystallite). The enamel's elastic/inelastic transitions were observed at 0.4–17 GPa depending on the length scale and were compared with the values of synthetic hydroxyapatite crystallites. The elastic limit of a material is important as it provides insights into the deformability of the material before fracture. At the smallest investigated length scale (contact radius ∼20 nm), elastic limit is followed by plastic deformation. At the largest investigated length scale (contact size ∼2 mm), only elastic then micro-crack induced response was observed. A map of elastic/inelastic regions of enamel from millimeter to nanometer length scale is presented. Possible underlying mechanisms are also discussed.

Keywords: Enamel; Hydroxyapatite; Compression; Nanoindentation; Elastic/plastic transition; Size-dependent

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