Biomaterials (v.31, #27)

The aim of this paper is to clarify at the nanometer scale the relevant factors influencing the hydrothermal resistance to polymorphic transformation of alumina/zirconia composites, primary candidates for artificial joint applications. The topographic distribution of oxygen vacancies and lattice strain on the composite surface were visualized by means of cathodoluminescence spectroscopy and mapped as a function of exposure time in a thermally activated water vapor environment (i.e., simulating the exposure in human body). Systematically monitoring the optical activity of oxygen vacancies in both alumina and zirconia phases also revealed the effect of surface lattice strain accumulation on the kinetics of polymorphic transformation. From the presented data, an explicit role is evinced for surface oxygen vacancy formation in the alumina matrix, an important step in the complex cascade of mechanochemical events determining the superior environmental resistance of the composite.
Keywords: Alumina; Zirconia; Stress analysis; Microstructure;

Mathematically defined tissue engineering scaffold architectures prepared by stereolithography by Ferry P.W. Melchels; Katia Bertoldi; Ruggero Gabbrielli; Aldrik H. Velders; Jan Feijen; Dirk W. Grijpma (6909-6916).
The technologies employed for the preparation of conventional tissue engineering scaffolds restrict the materials choice and the extent to which the architecture can be designed. Here we show the versatility of stereolithography with respect to materials and freedom of design. Porous scaffolds are designed with computer software and built with either a poly(d,l-lactide)-based resin or a poly(d,l-lactide-co-ε-caprolactone)-based resin. Characterisation of the scaffolds by micro-computed tomography shows excellent reproduction of the designs. The mechanical properties are evaluated in compression, and show good agreement with finite element predictions. The mechanical properties of scaffolds can be controlled by the combination of material and scaffold pore architecture. The presented technology and materials enable an accurate preparation of tissue engineering scaffolds with a large freedom of design, and properties ranging from rigid and strong to highly flexible and elastic.
Keywords: Rapid prototyping; Stereolithography; Microstructure; Tissue engineering scaffold; Three-dimensional printing;

The mechanisms of cytotoxicity of urethane dimethacrylate to Chinese hamster ovary cells by Hsiao-Hua Chang; Mei-Chi Chang; Li-Deh Lin; Jang-Jaer Lee; Tong-Mei Wang; Chun-Hsun Huang; Ting-Ting Yang; Hsueh-Jen Lin; Jiiang-Huei Jeng (6917-6925).
Monomers released from resin-containing products may cause various adverse effects. Urethane dimethacrylate (UDMA) is a principal resin monomer and also a major component released from various dental resin materials. Thus the toxic effects and mechanisms should be elucidated for improving of its safety use. Here we investigated the effects of UDMA on the growth, cell cycle progression, reactive oxygen species (ROS) production and glutathione (GSH) alteration in CHO-K1 cells, and the preventive effects by antioxidants (NAC and catalase) were also evaluated. UDMA elicited growth inhibition (>0.025 mm) of CHO-K1 cells in a clearly dose-dependent manner. Cell cycle perturbation and ROS overproduction were also observed. A 0.1 mm UDMA-induced S-phase cell cycle arrest and ROS accumulation. Cell apoptosis and necrosis became significant when UDMA concentration was 0.25 mm. GSH depletion occurred at cells treated with 0.25 mm UDMA, a highly cytotoxic concentration at which point myriad cells were under apoptosis or necrosis. Thus GSH depletion can be crucial for the death of CHO-K1 cells. Furthermore NAC (0.5–10 mm) and catalase (250–1000 U/ml) obviously attenuated the UDMA-induced toxicity by reducing ROS generation and cell cycle disturbance, and the effects were dose-related. These results suggest that UDMA toxicity is associated with ROS production, GSH depletion, cell cycle disturbance and cell apoptosis/necrosis.
Keywords: Apoptosis; Cell cycle; Cytotoxicity; Glutathione; Reactive oxygen species; Urethane dimethacrylate;

Effect of swelling of poly(vinyl alcohol) layers on complement activation by Yusuke Arima; Masako Kawagoe; Masanori Furuta; Mitsuaki Toda; Hiroo Iwata (6926-6933).
Polymers carrying hydroxyl groups have the potential ability to activate the complement system when in contact with blood. However, the effects of their surface structure on complement activation are still not fully understood. In this study, we examined complement activation by poly(vinyl alcohol) (PVA) layers formed on a gold surface modified with aldehyde groups. The complement system was strongly activated by a PVA surface with a dry thickness of 2.9 nm, while it was poorly activated by a PVA surface with a dry thickness of 7.4 nm. Annealing of the latter for 2 h at 150 °C converted the surface into a complement activating surface. The difference in complement activation between PVA layers was associated with the water content of PVA layers. These results suggest that complement activation by hydrated polymers highly depends on the water content of the polymer layers.
Keywords: Blood compatibility; Complement; Protein adsorption; Surface modification; Poly(vinyl alcohol);

Preserved extracellular matrix components and retained biological activity in decellularized porcine mesothelium by David M. Hoganson; Gwen E. Owens; Elisabeth M. O’Doherty; Chris M. Bowley; Scott M. Goldman; Dina O. Harilal; Craig M. Neville; Russell T. Kronengold; Joseph P. Vacanti (6934-6940).
Mesothelium tissues such as peritoneum and pleura have a thin and strong layer of extracellular matrix that supports mesothelial cells capable of rapid healing. Decellularized porcine mesothelium was characterized for strength, composition of the matrix and biological activity. The tensile strength of the material was 40.65 ± 21.65 N/cm. Extracellular matrix proteins collagen IV, fibronectin, and laminin as well as glycosaminoglycans were present in the material. Cytokines inherent in the extracellular matrix were preserved. Vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and transforming growth factor β (TGF-β) were retained and the levels of VEGF and TGF-β in the decellularized mesothelium were higher than those found in decellularized small intestinal submucosa (SIS). The decellularized mesothelium also stimulated human fibroblasts to produce more VEGF than fibroblasts grown on tissue culture plastic. Decellularized mesothelium is a sheet material with a combination of strength and biological activity that may have many potential applications in surgical repair and regenerative medicine.
Keywords: ECM; Cytokine; Surgical mesh; Cell culture;

Directed 3D cell alignment and elongation in microengineered hydrogels by Hug Aubin; Jason W. Nichol; Ché B. Hutson; Hojae Bae; Alisha L. Sieminski; Donald M. Cropek; Payam Akhyari; Ali Khademhosseini (6941-6951).
Organized cellular alignment is critical to controlling tissue microarchitecture and biological function. Although a multitude of techniques have been described to control cellular alignment in 2D, recapitulating the cellular alignment of highly organized native tissues in 3D engineered tissues remains a challenge. While cellular alignment in engineered tissues can be induced through the use of external physical stimuli, there are few simple techniques for microscale control of cell behavior that are largely cell-driven. In this study we present a simple and direct method to control the alignment and elongation of fibroblasts, myoblasts, endothelial cells and cardiac stem cells encapsulated in microengineered 3D gelatin methacrylate (GelMA) hydrogels, demonstrating that cells with the intrinsic potential to form aligned tissues in vivo will self-organize into functional tissues in vitro if confined in the appropriate 3D microarchitecture. The presented system may be used as an in vitro model for investigating cell and tissue morphogenesis in 3D, as well as for creating tissue constructs with microscale control of 3D cellular alignment and elongation, that could have great potential for the engineering of functional tissues with aligned cells and anisotropic function.
Keywords: Tissue engineering; Micropatterning; Cellular alignment; 3D engineered tissue;

The regulation of phenotype of cultured tenocytes by microgrooved surface structure by Ji Zhu; Jie Li; Bin Wang; Wen Jie Zhang; Guangdong Zhou; Yilin Cao; Wei Liu (6952-6958).
To maintain or enhance cell function by controlling its shape is an important consideration in scaffold design. Tenocyte is characterized by its unique elongated cell shape and the role remains unexplored. In this study, primary porcine tenocytes of newborn pigs were cultured respectively on culture dish (Group A), smooth (Group B) or microgroove silicone membrane (Group C, enforcing an elongated morphology) to observe the effect of cell shape on tenocyte phenotype. The results showed that elongated morphology (Group C) could help in vitro passaged tenocytes to retain their phenotype and function by maintaining the expression of tenomodulin (tenocyte marker) and collagen I (functional molecule). By contrast, the spread tenocytes (Groups A and B) lost or significantly reduced the expression of tenomodulin or collagen I respectively. Interestingly, the lost tenomodulin expression of Group B cells could be regained after being switched to microgroove culture condition of Group C. In addition, significantly increased RhoA-ATP level and reduced ROCK activity were found associated with elongated morphology and artificially activating RhoA or inhibiting ROCK could lead to increased tenomodulin expression in spread cells. Collectively, these results confirm that elongated morphology is essential for tenocytes to keep their phenotype and function and can redifferentiate the dedifferentiated tenocytes by the participation of RhoA/ROCK signaling, and these findings may provide insight into the design of advanced scaffold for tendon engineering.
Keywords: Tenocytes; Elongated morphology; RhoA/ROCK; Tenomodulin; Y27632; LPA;

The rapid anastomosis between prevascularized networks on silk fibroin scaffolds generated in vitro with cocultures of human microvascular endothelial and osteoblast cells and the host vasculature by Ronald E. Unger; Shahram Ghanaati; Carina Orth; Anne Sartoris; Mike Barbeck; Sven Halstenberg; Antonella Motta; Claudio Migliaresi; C. James Kirkpatrick (6959-6967).
The survival and functioning of a bone biomaterial upon implantation requires a rapidly forming and stably functioning vascularization that connects the implant to the recipient. We have previously shown that human microcapillary endothelial cells (HDMEC) and primary human osteoblast cells (HOS) in coculture on various 3-D bone biomaterial scaffolds rapidly distribute and self-assemble into a morphological structure resembling bone tissue. Endothelial cells form microcapillary-like structures containing a lumen and these were intertwined between the osteoblast cells and the biomaterial. This tissue-like self-assembly occurred in the absence of exogenously added angiogenic stimuli or artificial matrices. The purpose of this study was to determine whether this in vitro pre-formed microvasculature persists and functions in vivo and to determine how the host responds to the cell-containing scaffolds. The scaffolds with cocultures were implanted into immune-deficient mice and compared to scaffolds without cells or with HDMEC alone. Histological evaluation and immunohistochemical staining with human-specific antibodies of materials removed 14 days after implantation demonstrated that the in vitro pre-formed microcapillary structures were present on the silk fibroin scaffolds and showed a perfused lumen that contained red blood cells. This proved anastomosis with the host vasculature. Chimeric vessels in which HDMEC were integrated with the host’s ingrowing (murine) capillaries were also observed. No HDMEC-derived microvessel structures or chimeric vessels were observed on implanted silk fibroin when precultured with HDMEC alone. In addition, there was migration of the host (murine) vasculature into the silk fibroin scaffolds implanted with cocultures, whereas silk fibroin alone or silk fibroin precultured only with HDMEC were nearly devoid of ingrowing host microcapillaries. Therefore, not only do the in vitro pre-formed microcapillaries in a coculture survive and anastomose with the host vasculature to become functioning microcapillaries after implantation, the coculture also stimulates the host capillaries to rapidly grow into the scaffold to vascularize the implanted material. Thus, this coculture-based pre-vascularization of a biomaterial implant may have great potential in the clinical setting to treat large bone defects.
Keywords: Neovascularization; Bone tissue engineering; Osteoblast; Endothelial cell; Inosculation;

Cartilage repair using hyaluronan hydrogel-encapsulated human embryonic stem cell-derived chondrogenic cells by Wei Seong Toh; Eng Hin Lee; Xi-Min Guo; Jerry K.Y. Chan; Chen Hua Yeow; Andre B. Choo; Tong Cao (6968-6980).
Human embryonic stem cells (hESCs) have the potential to offer a virtually unlimited source of chondrogenic cells for use in cartilage repair and regeneration. We have recently shown that expandable chondrogenic cells can be derived from hESCs under selective growth factor-responsive conditions. In this study, we explore the potential of these hESC-derived chondrogenic cells to produce an extracellular matrix (ECM)-enriched cartilaginous tissue construct when cultured in hyaluronic acid (HA)-based hydrogel, and further investigated the long-term reparative ability of the resulting hESC-derived chondrogenic cell-engineered cartilage (HCCEC) in an osteochondral defect model. We hypothesized that HCCEC can provide a functional template capable of undergoing orderly remodeling during the repair of critical-sized osteochondral defects (1.5 mm in diameter, 1 mm depth into the subchondral bone) in a rat model. In the process of repair, we observed an orderly spatial-temporal remodeling of HCCEC over 12 weeks into osteochondral tissue, with characteristic architectural features including a hyaline-like neocartilage layer with good surface regularity and complete integration with the adjacent host cartilage and a regenerated subchondral bone. By 12 weeks, the HCCEC-regenerated osteochondral tissue resembled closely that of age-matched unoperated native control, while only fibrous tissue filled in the control defects which left empty or treated with hydrogel alone. Here we demonstrate that transplanted hESC-derived chondrogenic cells maintain long-term viability with no evidence of tumorigenicity, providing a safe, highly-efficient and practical strategy of applying hESCs for cartilage tissue engineering.
Keywords: Human embryonic stem cells; Chondrogenic; Cartilage; Differentiation; Hyaluronan; Hydrogel;

Electrically induced contraction of C2C12 myotubes cultured on a porous membrane-based substrate with muscle tissue-like stiffness by Hirokazu Kaji; Takeshi Ishibashi; Kuniaki Nagamine; Makoto Kanzaki; Matsuhiko Nishizawa (6981-6986).
A porous membrane-based cell culture device was developed to electrically stimulate a confluent monolayer of C2C12 myotubes. The device’s cell culture substrate is a microporous alumina membrane-modified by attaching an atelocollagen membrane on the upperside and a hole-spotted poly(dimethylsiloxane) (PDMS) film on the underside. When electric current is generated between the device’s Pt ring electrodes – one of which is placed above the cells and the other below the PDMS layer – the focused current at the PDMS hole can electrically stimulate the cells. C2C12 myoblasts were cultured on the substrate and differentiated into myotubes. When the electrical pulses were applied, myotubes started to contract slightly in and near the hole, and that the continuous stimulation increased both the number of stimuli-responding myotubes and the magnitude of the contraction considerably owing to the underlying atelocollagen membrane with muscle tissue-like stiffness. Also, the generation of contractile myotubes on a wider region of the membrane substrate was possible by applying the electrical pulses through the array of holes in the PDMS film. Using the present system, the glucose uptake by contractile myotubes was examined with fluorescence-labeled glucose, 2-NBDG, which displayed a positive correlation between the contractile activity of myotubes and the uptake of 2-NBDG.
Keywords: Atelocollagen membrane; C2C12 myotube; Contractility; Electrical pulse stimulation; Glucose uptake;

We answered two major questions: (1) does retrograde signaling involve retrograde transport of nerve growth factor (NGF); and (2) is a gradient of immobilized NGF sufficient to promote and guide local axonal growth? To answer these questions, we developed a technique that resulted in stably immobilized NGF and combined this with compartmented chambers. NGF was photochemically-immobilized on a chitosan surface either in the cell body (CB) compartment, distal axon (DA) compartment, or both. Neuron survival and axon outgrowth were found to be insignificantly different from positive controls where soluble NGF was present. When NGF was immobilized on chitosan surfaces in the DA compartment, and in the absence of soluble NGF, neuron survival was observed, likely due to the retrograde signal of the activated TrkA receptor and NGF-induced signals, but not the retrograde signal of NGF itself. Axons were guided towards the higher end of the step concentration gradient of NGF that was photoimmobilized on the chitosan surface in the DA compartment by laser confocal patterning, demonstrating axonal guidance. These studies provide better insight into NGF signaling mechanisms which are important to both understanding developmental disorders and degenerative diseases of the nervous system, as well as improving design strategies to promote nerve regeneration after injury.
Keywords: Chitosan; Nerve growth factor immobilization; Axon guidance; Superior cervical ganglia neurons; Compartment culture;

Influence of micro-patterned PLLA membranes on outgrowth and orientation of hippocampal neurites by Sabrina Morelli; Simona Salerno; Antonella Piscioneri; Bernke J. Papenburg; Anna Di Vito; Giuseppina Giusi; Marcello Canonaco; Dimitrios Stamatialis; Enrico Drioli; Loredana De Bartolo (7000-7011).
In neuronal tissue engineering many efforts are focused on creating biomaterials with physical and chemical pathways for controlling cellular proliferation and orientation. Neurons have the ability to respond to topographical features in their microenvironment causing among others, axons to proliferate along surface features such as substrate grooves in micro-and nanoscales. As a consequence these neuronal elements are able to correctly adhere, migrate and orient within their new environment during growth. Here we explored the polarization and orientation of hippocampal neuronal cells on nonpatterned and micro-patterned biodegradable poly(l-lactic acid) (PLLA) membranes with highly selective permeable properties. Dense and porous nonpatterned and micro-patterned membranes were prepared from PLLA by Phase Separation Micromolding. The micro-patterned membranes have a three-dimensional structure consisting of channels and ridges and of bricks of different widths. Nonpatterned and patterned membranes were used for hippocampal neuronal cultures isolated from postnatal days 1–3 hamsters and the neurite length, orientation and specific functions of cells were investigated up to 12 days of culture. Neurite outgrowth, length plus orientation tightly overlapped the pattern of the membrane surface. Cell distribution occurred only in correspondence to membrane grooves characterized by continuous channels whereas on membranes with interconnected channels, cells not only adhered to and elongated their cellular processes in the grooves but also in the breaking points. High orientation degrees of cells were determined particularly on the patterned porous membranes with channel width of 20 μm and ridges of 17 μm whereas on dense nonpatterned membranes as well as on polystyrene culture dish (PSCD) controls, a larger number of primary developed neurites were distributed. Based on these results, PLLA patterned membranes may directly improve the guidance of neurite extension and thereby enhancing their orientation with a consequently highly ordered neuronal cell matrix, which may have strong bearings on the elucidation of regeneration mechanisms.
Keywords: Hippocampal neurons; Membranes; Orientation; Micropatterning; Neurite growth;

The use of human mesenchymal stem cells encapsulated in RGD modified alginate microspheres in the repair of myocardial infarction in the rat by Jiashing Yu; Kim T. Du; Qizhi Fang; Yiping Gu; Shirley S. Mihardja; Richard E. Sievers; Joseph C. Wu; Randall J. Lee (7012-7020).
The combination of scaffold material and cell transplantation therapy has been extensively investigated in cardiac tissue engineering. However, many polymers are difficult to administer or lack the structural integrity to restore LV function. Additionally, polymers need to be biological friendly, favorably influence the microenvironment and increase stem cell retention and survival. This study determined whether human mesenchymal stem cells (hMSCs) encapsulated in RGD modified alginate microspheres are capable of facilitating myocardial repair. The in vitro study of hMSCs demonstrated that the RGD modified alginate can improve cell attachment, growth and increase angiogenic growth factor expression. Alginate microbeads and hMSCs encapsulated in microbeads successfully maintained LV shape and prevented negative LV remodeling after an MI. Cell survival was significantly increased in the encapsulated hMSC group compared with PBS control or cells alone. Microspheres, hMSCs, and hMSCs in microspheres groups reduced infarct area and enhanced arteriole formation. In summary, surface modification and microencapsulation techniques can be combined with cell transplantation leading to the maintenance of LV geometry, preservation of LV function, increase of angiogenesis and improvement of cell survival.
Keywords: Mesenchymal stem cells; Alginate; Myocardial infarction; RGD peptides;

Human corneal epithelial cell response to epidermal growth factor tethered via coiled-coil interactions by Cyril Boucher; Juan-Carlos Ruiz; Marc Thibault; Michael D. Buschmann; Michael R. Wertheimer; Mario Jolicoeur; Yves Durocher; Gregory De Crescenzo (7021-7031).
The development of new strategies for protein immobilization to control cell adhesion, growth and differentiation is of prime interest in the field of tissue engineering. Here we propose a versatile approach based on the interaction between two de novo designed peptides, Ecoil and Kcoil, for oriented immobilization of epidermal growth factor (EGF) on polyethylene terephthalate (PET) films. After amination of PET surfaces by ammonia plasma treatment, Kcoil peptides were covalently grafted in an oriented fashion using succinimidyl 6-[30-(2-pyridyldithio)-propionamido] hexanoate (LC-SPDP) linker, and the Kcoil-functionalized films were characterized by X-ray photoelectron spectroscopy (XPS). Bioactivity of Ecoil-EGF captured on Kcoil-functionalized PET via coiled-coil interactions was confirmed by EGF receptor phosphorylation analysis following A-431 cell attachment. We also demonstrated cell biological effects where tethered EGF enhanced adhesion, spreading and proliferation of human corneal epithelial cells compared to EGF that was either physically adsorbed or present in solution. Tethered EGF effects were most likely linked to the prolonged activation of both mitogen-activated protein kinase and phosphoinositidine 3-kinase pathways. Taken together, our results indicate that coiled-coil-based oriented immobilization is a powerful method to specifically tailor biomaterial surfaces for tissue engineering applications.
Keywords: Surface modification; Human corneal epithelial cells; Epidermal growth factor (EGF); Coiled-coil interactions; Cell proliferation; Cell adhesion;

The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells by Michel L. Dubois-Dauphin; Nicolas Toni; Stéphanie D. Julien; Igor Charvet; Lars E. Sundstrom; Luc Stoppini (7032-7042).
Embryonic stem cells (ESCs) offer attractive prospective as potential source of neurons for cell replacement therapy in human neurodegenerative diseases. Besides, ESCs neural differentiation enables in vitro tissue engineering for fundamental research and drug discovery aimed at the nervous system. We have established stable and long-term three-dimensional (3D) culture conditions which can be used to model long latency and complex neurodegenerative diseases. Mouse ESCs-derived neural progenitor cells generated by MS5 stromal cells induction, result in strictly neural 3D cultures of about 120-μm thick, whose cells expressed mature neuronal, astrocytes and myelin markers. Neurons were from the glutamatergic and gabaergic lineages. This nervous tissue was spatially organized in specific layers resembling brain sub-ependymal (SE) nervous tissue, and was maintained in vitro for at least 3.5 months with great stability. Electron microscopy showed the presence of mature synapses and myelinated axons, suggesting functional maturation. Electrophysiological activity revealed biological signals involving action potential propagation along neuronal fibres and synaptic-like release of neurotransmitters. The rapid development and stabilization of this 3D cultures model result in an abundant and long-lasting production that is compatible with multiple and productive investigations for neurodegenerative diseases modeling, drug and toxicology screening, stress and aging research.
Keywords: Embryonic stem cells; neural cells; tissue engineering; brain; pharmacology;

Micro- and nanotechnologies are increasingly being applied in cancer research. Here we report the effects of an experimental breast cancer agent, SAHA, on the cytoarchitecture and adherence of MDA-MB-231 metastatic human breast cancer cells on flat silicon surfaces and in three dimensional (3-D) isotropic silicon microstructures. The 3-D silicon microstructure were fabricated using a single mask and single etch step process to yield arrays of star- and circular-shaped microchambers 151–168 μm in diameter and 53–68 μm deep. There was a marked expansion of the microtubule network, an increase in mean cell area and mean cell length in response to SAHA. SAHA also decreased the nuclear-to-cytoplasmic area (N/C). Atomic force microscopy (AFM) showed there was no change in cellular elasticity over the nuclear region in response to SAHA. The alterations in cytoarchitecture produced by SAHA were associated with changes in the mode of adhesion of the cells in silicon microstructures. In contrast to control cells which conformed to the microstructures, SAHA caused cells to stretch and attach to the microstructures through actin-rich cell extensions. We conclude that isotropically etched silicon microstructures comprise microenvironments that discriminate metastatic mammary cancer cells in which cytoskeletal elements reorganized in response to the anti-cancer agent SAHA.
Keywords: Cancer; Cytoskeleton; MDA-MB-231 cells; SAHA; Silicon microstructures; 3-D;

A surface crosslinked UHMWPE stabilized by vitamin E with low wear and high fatigue strength by Ebru Oral; Bassem W. Ghali; Shannon L. Rowell; Brad R. Micheli; Andrew J. Lozynsky; Orhun K. Muratoglu (7051-7060).
Wear particle-induced periprosthetic osteolysis has been a clinical problem driving the development of wear resistant ultrahigh molecular weight polyethylene (UHMWPE) for total joint replacement. Radiation crosslinking has been used to decrease wear through decreased plastic deformation; but crosslinking also reduces mechanical properties including fatigue resistance, a major factor limiting the longevity of joint implants. Reducing UHMWPE wear with minimal detriment to mechanical properties is an unaddressed need for articular bearing surface development. Here we report a novel approach to achieve this by limiting crosslinking to the articular surface. The antioxidant vitamin E reduces crosslinking efficiency in UHMWPE during irradiation with increasing concentration, thus we propose to spatially control the crosslink density distribution by controlling the vitamin E concentration profile. Surface crosslinking UHMWPE prepared using this approach had high wear resistance and decreased crosslinking in the bulk resulting in high fatigue crack propagation resistance. The interface region did not represent a weakness in the material due to the gradual change in the crosslink density. Such an implant has the potential of decreasing risk of fatigue fracture of total joint implants as well as expanding the use of UHMWPE to younger and more active patients.
Keywords: Vitamin E; Arthroplasty; Delamination; Interface; Biomaterial; Implant;

Mononuclear phagocyte system is the first target for injected nanomaterials. Monocytes/macrophages are the central actors for trafficking and clearance of magnetic nanoparticles in vivo. However the fate of nanosized magnetic label is an ongoing issue. Here we demonstrate that the monocyte/macrophage system shows a complex dynamic behavior with respect to iron-oxide nanoparticles uptake. Once internalized by monocytes or macrophages, magnetic nanoparticles can be released by cells upon stress or activation. We identified membrane vesicles shed by activated or apoptotic cells as the carriers transporting magnetic nanomaterials. These vesicles are taken up by naïve macrophages and trigger an intercellular transfer of the magnetic label. They also lead to the redistribution of magnetic label during differentiation of magnetically-labeled monocytes into macrophages. Intercellular transfer of magnetic label, mediated by cell-released vesicles, complicates the picture of nanoparticles outcome in the organism and their use as MRI cell tracers.
Keywords: Macrophage; Monocyte; Magnetic nanoparticles; Membrane vesicles; Apoptosis; Inflammation;

Customizable, multi-functional fluorocarbon nanoparticles for quantitative in vivo imaging using 19F MRI and optical imaging by Mangala Srinivas; Luis J. Cruz; Fernando Bonetto; Arend Heerschap; Carl G. Figdor; I. Jolanda M. de Vries (7070-7077).
Monitoring cell trafficking in vivo noninvasively is critical to improving cellular therapeutics, drug delivery, and understanding disease progression. In vivo imaging, of which magnetic resonance imaging (MRI) is a key modality, is commonly used for such monitoring. 19F MRI allows extremely specific detection and quantification of cell numbers directly from in vivo image data, longitudinally and without ionizing radiation. We used fluorocarbons previously used in blood substitutes and imaging agents for ultrasound and computed tomography to synthesize monodisperse nanoparticles that are stable at 37 °C and can be frozen for storage. These large 19F labeling compounds are insoluble in aqueous environments and often emulsified, typically forming emulsions unsuitable for long-term storage. Instead, we used a non-toxic polymer already in clinical use, poly(d,l-lactide-co-glycolide), to encapsulate a range of 19F compounds. These nanoparticles can be customized in terms of content (imaging agent, fluorescent dye, drug), size (200–2000 nm), coating (targeting agent, antibody) and surface charge (−40 to 30 mV). We added a fluorescent dye and antibody to demonstrate the versatility of this modular imaging agent. These nanoparticles are adaptable to multimodal imaging, although here we focused on MRI and fluorescence imaging. Here, we imaged primary human dendritic cells, as used in clinical vaccines.
Keywords: MRI; 19F MRI; Quantitative cell tracking; In vivo fluorescence imaging; Multimodal imaging agents;

Long-term in vivo biodistribution imaging and toxicity of polyacrylic acid-coated upconversion nanophosphors by Liqin Xiong; Tianshe Yang; Yang Yang; Congjian Xu; Fuyou Li (7078-7085).
Rare-earth upconversion nanophosphors (UCNPs) have become one of the most promising classes of luminescent materials for bioimaging. However, there remain numerous unresolved issues with respect to the understanding of how these nanophosphors interact with biological systems and the environment. Herein, we report polyacrylic acid (PAA)-coated near-infrared to near-infrared (NIR-to-NIR) upconversion nanophosphors NaYF4:Yb,Tm (PAA-UCNPs) as luminescence probes for long-term in vivo distribution and toxicity studies. Biodistribution results determined that PAA-UCNPs uptake and retention took place primarily in the liver and the spleen and that most of the PAA-UCNPs were excreted from the body of mice in a very slow manner. Body weight data of the mice indicated that mice intravenously injected with 15 mg/kg of PAA-UCNPs survived for 115 days without any apparent adverse effects to their health. In addition, histological, hematological and biochemical analysis were used to further quantify the potential toxicity of PAA-UCNPs, and results indicated that there was no overt toxicity of PAA-UCNPs in mice at long exposure times (up to 115 days). The study suggests that PAA-UNCPs can potentially be used for long-term targeted imaging and therapy studies in vivo.
Keywords: Rare-earth nanophosphors; Upconversion luminescence; In vivo imaging; Biodistribution; Toxicity;

Preservation of dendritic cell function upon labeling with amino functionalized polymeric nanoparticles by O. Zupke; E. Distler; D. Baumann; D. Strand; R.G. Meyer; K. Landfester; W. Herr; V. Mailänder (7086-7095).
Dendritic cells (DCs) are key players in eliciting immunity against antigens, therefore making them the focus of many investigations on immune responses in infections, cancer and autoimmune diseases. Nanosized materials have just recently been investigated for their use as carriers of antigens and as labeling agents for DCs. For this later use nanoparticles should be non-toxic and should most importantly not alter the physiological functions of DCs. Here we demonstrate that by the use of polymeric fluorescent nanoparticles as synthesized by the miniemulsion process immature DCs (iDCs) can be efficiently labeled intracellularly. Amino functionalized nanoparticles are more effective than carboxy functionalized ones. Even after 8 days 95% of DCs have retained nanoparticles with a fluorescence intensity of 67% compared to day 1. Nanoparticle labeling does not influence expression of cell surface molecules on mature DCs (mDCs) like HLA-DR, CD80/83/86, CCR7, CD11c nor does it influence the immunostimulatory capacity of mDCs. This procedure does also not impair the capability of DCs for uptake, processing and presentation of viral antigens as demonstrated by interferon-γ ELISPOT on T cells stimulated with viral antigens presented by DCs. Therefore polymeric nanoparticles are a promising tool to study migration and homing of DCs in animal studies.
Keywords: Nanoparticle; Dendritic cell; Cell imaging; Cellular therapy;

Highly temperature-sensitive liposomes based on a thermosensitive block copolymer for tumor-specific chemotherapy by Kenji Kono; Toshiaki Ozawa; Tomohide Yoshida; Fuminori Ozaki; Yukihito Ishizaka; Kazuo Maruyama; Chie Kojima; Atsushi Harada; Sadahito Aoshima (7096-7105).
Recently, we showed that incorporation of poly[2-(2-ethoxy)ethoxyethyl vinyl ether (EOEOVE)], which exhibits a lower critical solution temperature around 40 °C, provides temperature-sensitive properties to stable liposomes. In this study, we applied this thermosensitive polymer for preparation of temperature-sensitive liposomes for tumor-specific chemotherapy with doxorubicin (DOX). We prepared liposomes consisting of PEG-lipid, egg yolk phosphatidylcholine, cholesterol and copoly(EOEOVE-block-octadecyl vinyl ether), which was synthesized as poly(EOEOVE) having anchors for fixation onto liposome membrane. The copolymer-incorporated liposomes were stable and retained DOX in their inside below physiological temperatures. However, they exhibited a significant release of encapsulated DOX above 40 °C and released DOX almost completely within 1 min at 45 °C. The copolymer-modified liposomes exhibited a long circulating property and biodistribution similar to that of PEG-modified liposomes. The copolymer-modified liposomes loaded with DOX were injected intravenously into tumor-bearing mice. Tumor growth was strongly suppressed when the tumor site was heated to 45 °C for 10 min at 6–12 h after injection. However, injection of the liposomes exhibited only slight tumor-suppressive effects as long as mild heating was not applied to the target site. The highly temperature-sensitive properties of the copolymer-incorporated liposomes might contribute to establishment of tumor-selective and effective chemotherapy.
Keywords: Drug delivery system; Temperature-responsive; Doxorubicin; Chemotherapy; Thermosensitive polymer;

Paclitaxel-clusters coated with hyaluronan as selective tumor-targeted nanovectors by Ilia Rivkin; Keren Cohen; Jacob Koffler; Dina Melikhov; Dan Peer; Rimona Margalit (7106-7114).
Paclitaxel (PTX) is a widely used anti-tumor agent in the treatment of solid tumors. Lack of selective strategies to target PTX into tumor cells together with poor solubility necessitating detergent, are severe clinical limitations. To address these hurdles, we devised a strategy that utilized PTX insolubility, mixing it with lipids that self-assemble into nanoparticle-like clusters. These clusters were then coated with hyaluronan, a glycosaminoglycan (GAG), and termed PTX-GAGs. These particles, delivered PTX selectively into tumor cells in a CD44-dependent manner. Injected systemically to mice bearing solid tumors, the PTX-GAGs showed high safety profile and tumor accumulation. Tumor progression was exponential upon treatment with free PTX or PTX in albumin nanoparticles (the FDA-approved Taxol® and Abraxane®, respectively). Under the same conditions, PTX-GAGs induced tumor arrest and were as potent as a 4-fold higher Taxol® dose. Our findings suggest GAGs merit further investigation as vehicles for taxanes, and may be applicable as carriers in other therapeutic settings.
Keywords: Paclitaxel; Drug delivery; Tumor targeting; Hyaluronan; Abraxane;

We reported a precise engineered nanocapsule encapsulating a neovasculature disruption agent, combretastatin A4 (CA4) in a matrix that was made up of paclitaxel (PTX) conjugated amphiphilic polyester. The nanocapsule was able to release CA4 and PTX sequentially for temporal antiangiogenesis and anticancer activities. The nanocapsule has a small particle size at 68 nm with narrow size distribution (∼0.15). Cellular uptake of the nanocapsule was efficient, and detectable at as early as 20 min, and drugs sequestered in the nanocapsule could exert effective therapeutic effects on tumor neovasculature and cancer cells, respectively. Biodistribution experiments demonstrated the long circulation of nanocapsule in body fluid and the preferential accumulation of nanocapsule in tumor. Both in vivo artificial pro-angiogenesis and tumor xenograft assays demonstrated the promising therapeutic effect of the nanocapsule on tumor vasculature disruption, tumor cell proliferation inhibition and tumor cell apoptosis induction. The intrasplenic liver metastasis experiment also confirmed the liver metastatic prevention capacity of this nanocapsule. In summary, the findings indicated that this dual drug loaded nanocapsule with sequential drug delivery capacity is a promising candidate in combinatorial therapy in fighting against cancer, and may open an avenue for cancer therapy and diagnosis.
Keywords: Angiogenesis; Cancer metastasis; Combination therapy; Paclitaxel; Combretastatin A4; Nanocapsule;

Disassemblable micelles based on reduction-degradable amphiphilic graft copolymers for intracellular delivery of doxorubicin by Yong Sun; Xiaoli Yan; Taiming Yuan; Jie Liang; Yujiang Fan; Zhongwei Gu; Xingdong Zhang (7124-7131).
Disassemblable micelles for intracellular delivery of doxorubicin were developed based on a reduction-degradable amphiphilic polyamide amine-g-polyethylene glycol graft copolymer containing disulfide linkages throughout the main chain. The micelles are spherical of less than 50 nm in diameter, and can load doxorubicin in the core with drug loading content up to 20%. The micelles are stable in normal physiological condition, and quickly disassemble in reductive condition due to the cleavage of the disulfide linkages. The drug release of the micelles in normal condition is less than 25% within 24 h, whereas in the presence of reductive agent, DTT, the micelles can quickly release the entire loaded drug within 10 h. CLSM observation showed that the micelles can effectively deliver the drug cargo into nuclei after internalized through endocytosis. Cytotoxicity of the drug-loaded disassemblable micelles was demonstrated using human cervical cancer cell line (HeLa) and human liver carcinoma cell line (HepG2).
Keywords: Micelle; Degradation; Drug delivery; Cytotoxicity; Reduction-degradable;

A direct evaluation of the in vivo release profile of drugs from carriers is a clinical demand in drug delivery systems, because drug release characterized in vitro correlates poorly with in vivo release. The purpose of this study is to demonstrate the in vivo applicability of the dual MR contrast technique as a useful tool for noninvasive monitoring of the stability and the release profile of drug carriers, by visualizing in vivo release of the encapsulated surrogate MR contrast agent from carriers and its subsequent intratumoral distribution profile. The important aspect of this technique is that it incorporates both positive and negative contrast agents within a single carrier. GdDTPA, superparamagnetic iron oxide nanoparticles, and 5-fluorouracil were encapsulated in nano- and microspheres composed of poly(d,l-lactide-co-glycolide), which was used as a model carrier. In vivo studies were performed with orthotopic xenograft of human breast cancer. The MR-based technique demonstrated here has enabled visualization of the delivery of carriers, and release and intratumoral distribution of the encapsulated positive contrast agent. This study demonstrated proof-of-principle results for the noninvasive monitoring of in vivo release and distribution profiles of MR contrast agents, and thus, this technique will make a great contribution to the field.
Keywords: MRI (magnetic resonance imaging); Molecular imaging; Drug release; In vivo test; Nanoparticle;

Curcumin polymers as anticancer conjugates by Huadong Tang; Caitlin J. Murphy; Bo Zhang; Youqing Shen; Edward A. Van Kirk; William J. Murdoch; Maciej Radosz (7139-7149).
Curcumin has been shown highly cytotoxic towards various cancer cell lines, but its water-insolubility and instability make its bioavailability exceedingly low and thus it generally demonstrates low anticancer activity in in vivo tests. Herein, we report a novel type of polymer-drug conjugates — the high molecular weight curcumin polymers (polycurcumins) made by condensation polymerization of curcumin. The polycurcumins as backbone-type conjugates have advantages of high drug loading efficiency, fixed drug loading contents, stabilized curcumin in their backbones, and tailored water-solubility. The polycurcumins may have many potential applications and their antitumor activities are investigated in this work. The polycurcumins are cytotoxic to cancer cells, but a polyacetal-based polycurcumin (PCurc 8) is highly cytotoxic to SKOV-3, OVCAR-3 ovarian cancers, and MCF-7 breast cancer cell lines. It can be quickly taken up by cancer cells into their lysosomes, where PCurc 8 hydrolyzes and releases active curcumin. It arrests SKOV-3 cell cycle at G0/G1 phase in vitro and induces cell apoptosis partially through the caspase-3 dependent pathway. In vivo, intravenously (i.v.) injected PCurc 8 shows remarkable antitumor activity in SKOV-3 intraperitoneal (i.p.) xenograft tumor model.
Keywords: Drug delivery; Curcumin conjugate; Curcumin polymer; In vivo antitumor activity;

Degradable, pH-sensitive, membrane-destabilizing, comb-like polymers for intracellular delivery of nucleic acids by Yen-Ling Lin; Guohua Jiang; Lisa K. Birrell; Mohamed E.H. El-Sayed (7150-7166).
This report describes the design and synthesis of a new series of degradable, pH-sensitive, membrane-destabilizing, comb-like polymers that can enhance the intracellular delivery of therapeutic nucleic acids. These comb-like polymers are based on a diblock polymer backbone where the first block is a copolymer of pH-sensitive ethyl acrylic acid (EAA) monomers and hydrophobic butyl methacrylate (BMA) or hexyl methacrylate monomers. The second block is a homopolymer of N-acryloxy succinimide (NASI) or ß-benzyl l-aspartate N-carboxy-anhydride (BLA-NCA) monomers, which are functionalized to allow controlled grafting of hydrophobic HMA and cationic trimethyl aminoethyl methacrylate (TMAEMA) copolymers via acid-labile hydrazone linkages. These comb-like polymers displayed high hemolytic activity in acidic solutions, which increased with the increase in polymer concentration. All comb-like polymers degraded into small fragments upon incubation in an acidic solution (pH 5.8) due to hydrolysis of the hydrazone linkages connecting the hydrophobic/cationic grafts to the polymer backbone. Comb-like polymers successfully complexed anti-GAPDH siRNA molecules into serum- and nuclease-stable particles, which successfully silenced GAPDH expression at both the mRNA and protein levels. These results collectively indicate the potential of these new comb-like polymers to serve as vehicles for effective intracellular delivery of therapeutic nucleic acids.
Keywords: pH-sensitive polymers; Acid-labile linkage; Comb-like polymers; Endosomal escape; Intracellular siRNA delivery;

Tuning the mechanical properties of bioreducible multilayer films for improved cell adhesion and transfection activity by Jenifer Blacklock; Andreas Vetter; Andreas Lankenau; David Oupický; Helmuth Möhwald (7167-7174).
A simple approach to the mechanical modulation of layer-by-layer (LbL) films is through manipulation of the film assembly. Here, we report results based on altering the salt concentration during film assembly and its effect on film rigidity. Based on changes in film rigidity, cell adhesion characteristics and transfection activity were investigated in vitro. LbL films consisting of reducible hyperbranched poly(amide amine) (RHB) have been implemented along with DNA for investigating fibroblast adhesion on [RHB/DNA] n /2 films with varying rigidities. The rigidity was varied by changing the ionic concentration of the deposition solution between 0.01 m NaCl and 1.0 m NaCl. Molecular force probe (MFP) measurements were performed to measure the apparent Young’s modulus, E APP, of the films in situ. Cell adhesion and stress-fiber characteristics were investigated using total internal reflectance microscopy (TIRF-M). The average cell peripheral area, fiber density and average fiber length during 5 days of cell growth on films with either low (below 2.0 MPa) or high (above 2.0 MPa) film elastic modulus were investigated. Transfection studies were performed using gfpDNA and SEAP-DNA to investigate if changes in cell adhesion affect transfection activity. Furthermore, cell proliferation and cytotoxicity studies were used to investigate cellular viability over a week. The results have shown that surface modification of bioreducible LbL films of controlled thickness and roughness promotes cellular adhesion, stress-fiber growth and increased transfection activity without the need for an additional adhesive protein pre-coating of the surface or chemical cross-linking of the film.
Keywords: Molecular force probe, MFP; Cell adhesion; Transfection; TIRF-M; Stress-fiber orientation; Layer-by-layer, LbL;

The use of microfiber composites of elastin-like protein matrix reinforced with synthetic collagen in the design of vascular grafts by Jeffrey M. Caves; Vivek A. Kumar; Adam W. Martinez; Jeong Kim; Carrie M. Ripberger; Carolyn A. Haller; Elliot L. Chaikof (7175-7182).
Collagen and elastin networks contribute to highly specialized biomechanical responses in numerous tissues and species. Biomechanical properties such as modulus, elasticity, and strength ultimately affect tissue function and durability, as well as local cellular behavior. In the case of vascular bypass grafts, compliance at physiologic pressures is correlated with increased patency due to a reduction in anastomotic intimal hyerplasia. In this report, we combine extracellular matrix (ECM) protein analogues to yield multilamellar vascular grafts comprised of a recombinant elastin-like protein matrix reinforced with synthetic collagen microfibers. Structural analysis revealed that the fabrication scheme permits a range of fiber orientations and volume fractions, leading to tunable mechanical properties. Burst strengths of 239–2760 mm Hg, compliances of 2.8–8.4%/100 mm Hg, and suture retention strengths of 35–192 gf were observed. The design most closely approximating all target criteria displayed a burst strength of 1483 ± 143 mm Hg, a compliance of 5.1 ± 0.8%/100 mm Hg, and a suture retention strength of 173 ± 4 gf. These results indicate that through incorporation of reinforcing collagen microfibers, recombinant elastomeric protein-based biomaterials can play a significant role in load bearing tissue substitutes. We believe that similar composites can be incorporated into tissue engineering schemes that seek to integrate cells within the structure, prior to or after implantation in vivo.
Keywords: Elastin; Collagen; Mechanical properties; Fiber-reinforced composite; Vascular graft; Recombinant protein;

In order to study cellular responses and extracellular matrix protein remodeling mediated by biomaterials coating, we proposed a biomimetic construct containing protein-conjugated supported lipid bilayers (SLBs) as a cell culture platform. Single or multi-component proteins-bound SLBs were fabricated by conjugating type I collagen and/or fibronectin on the N-hydroxysulfosuccinimide-functionalized SLBs. The proposed protein-conjugated systems were quantitatively characterized by the quartz crystal microbalance with dissipation. NIH 3T3 fibroblasts were cultured on the model constructs and on oxygen plasma pretreated polystyrene (PSo) for parallel comparison. The retards of mobility of SLB after protein conjugation and cell culture were estimated by fluorescence recovery after photobleaching. The resulting cell morphology, adsorption kinetics and somatic dynamics were examined microscopically. We found that, on the SLB based cultures, the largest spreading size and cell number counts of 3T3 fibroblasts were found on the fibronectin containing surfaces. However, on the protein-coated PSo surfaces, no such distinguishable differences can be observed on all protein contents. Immunofluorescent staining results revealed that adsorption of endogenously produced fibronectin by 3T3 cells on PSo based surfaces is significantly more than that on SLB based surfaces. This suggests that the anti-fouling nature of underneath SLBs have played an important role in preventing 3T3 cells from effectively remodeling their microenvironment, whereas cells can easily remodel the nonspecific adsorption prone surfaces such as PSo based platforms. In summary, the protein conjugated SLB surfaces can serve as a platform for determining and regulating cell specific binding and subsequent signaling events with extracellular environments.
Keywords: ECM (extracellular matrix); Biomimetic material; Lipid; Cell adhesion; Cell spreading; Cell morphology;

Surface morphology optimization for osseointegration of coated implants by Chaiy Rungsiyakull; Qing Li; Guangyong Sun; Wei Li; Michael V. Swain (7196-7204).
This paper aims to establish a relationship between the surface morphology induced micromechanics and bone remodeling responses to a solid bead coated porous implant and further to develop a multiobjective optimization framework for the coating design of biomaterials. Multiscale modeling and remodeling techniques were developed, where a macroscopic analysis was initially performed to generate a global response to enable a microscopic analysis. The bone remodeling responses of the microscopic models (with a specific surface morphology) were evaluated in terms of the average apparent density developed in the peri-implant region. To explore the proposed multiscale analysis and design methods, a typical dental implantation setting is exemplified in this study. The response surface method (RSM) was utilized to relate the major implant coating parameters to the bone responses. It is found that increasing the volume fraction of the coating beads/particles results in a greater bone density, whereas increasing bead/particle size does not significantly affect the bone’s responses. Several different multiobjective optimization schemes were adopted to optimize the coated bead size and volume fraction, which reveal that the optimal design parameters of particle diameter and volume fraction are 100 μm – 35% and 38 μm – 17.5% for the cortical and cancellous bones respectively, agreeing with clinical data. To maximize the implant/bone interfacial stability, specific surface coating designs for particular locations are recommended.
Keywords: Implant topography; Surface coating; Bone remodeling; Osseointegration; Reponses surface method; Multiobjective optimization;