Biomaterials (v.32, #34)

Subtleties of biomineralisation revealed by manipulation of the eggshell membrane by Nan Li; Li-na Niu; Yi-pin Qi; Cynthia K.Y. Yiu; Heonjune Ryou; Dwayne D. Arola; Ji-hua Chen; David H. Pashley; Franklin R. Tay (8743-8752).
Biocalcification of collagen matrices with calcium phosphate and biosilicification of diatom frustules with amorphous silica are two discrete processes that have intrigued biologists and materials scientists for decades. Recent advancements in the understanding of the mechanisms involved in these two biomineralisation processes have resulted in the use of biomimetic strategies to replicate these processes separately using polyanionic, polycationic or zwitterionic analogues of extracellular matrix proteins to stabilise amorphous mineral precursor phases. To date, there is a lack of a universal model that enables the subtleties of these two apparently dissimilar biomineralisation processes to be studied together. Here, we utilise the eggshell membrane as a universal model for differential biomimetic calcification and silicification. By manipulating the eggshell membrane to render it permeable to stabilised mineral precursors, it is possible to introduce nanostructured calcium phosphate or silica into eggshell membrane fibre cores or mantles. We provide a model for infiltrating the two compartmental niches of a biopolymer membrane with different intrafibre minerals to obtain materials with potentially improved structure-property relationships.
Keywords: Apatite; Biomineralisation; Silica; Membrane;

The use of computational fluid dynamic models for the optimization of cell seeding processes by Adebayo A. Adebiyi; Mohammad E. Taslim; Keith D. Crawford (8753-8770).
The seeding of a porous scaffold with stem cells is a fundamental step in engineering sizeable tissue constructs that are clinically viable. However, a key problem often encountered is inhomogeneous seeding of the cells particularly when the cells are delivered through the thickness of the scaffold. The objective of this study was to establish the quantitative relationships between the cell seeding efficiency and the initial vacuum pressure in a compact perfusion seeding device that uses the effect of differential pressure induced by vacuum to seed cells on a porous scaffold. A transient CFD solution of the fluid flow in the device was used to optimize the initial vacuum pressure for efficient cell seeding. Results indicate that the optimal initial vacuum pressure for homogenous cell seeding is approximately −20 kPa for the seeding device. This study presents a 3-D computational model that can be employed in designing and optimizing cell seeding techniques and corresponding technology.
Keywords: Computational fluid dynamics; Cell seeding; Bone tissue engineering; Scaffold; Stem cell;

Hydrogel design for cartilage tissue engineering: A case study with hyaluronic acid by Iris L. Kim; Robert L. Mauck; Jason A. Burdick (8771-8782).
Hyaline cartilage serves as a low-friction and wear-resistant articulating surface in load-bearing, diarthrodial joints. Unfortunately, as the avascular, alymphatic nature of cartilage significantly impedes the body’s natural ability to regenerate, damage resulting from trauma and osteoarthritis necessitates repair attempts. Current clinical methods are generally limited in their ability to regenerate functional cartilage, and so research in recent years has focused on tissue engineering solutions in which the regeneration of cartilage is pursued through combinations of cells (e.g., chondrocytes or stem cells) paired with scaffolds (e.g., hydrogels, sponges, and meshes) in conjunction with stimulatory growth factors and bioreactors. A variety of synthetic and natural materials have been employed, most commonly in the form of hydrogels, and these systems have been tuned for optimal nutrient diffusion, connectivity of deposited matrix, degradation, soluble factor delivery, and mechanical loading for enhanced matrix production and organization. Even with these promising advances, the complex mechanical properties and biochemical composition of native cartilage have not been achieved, and engineering cartilage tissue still remains a significant challenge. Using hyaluronic acid hydrogels as an example, this review will follow the progress of material design specific to cartilage tissue engineering and propose possible future directions for the field.
Keywords: Hydrogel; Mesenchymal stem cells; Hyaluronic acid; Chondrogenesis; Cartilage; Biomaterial;

Biocompatibility and biofilm inhibition of N,N-hexyl,methyl-polyethylenimine bonded to Boston Keratoprosthesis materials by Irmgard Behlau; Koushik Mukherjee; Amit Todani; Ann S. Tisdale; Fabiano Cade; Liqiang Wang; Elizabeth M. Leonard; Fouad R. Zakka; Michael S. Gilmore; Frederick A. Jakobiec; Claes H. Dohlman; Alexander M. Klibanov (8783-8796).
The biocompatibility and antibacterial properties of N,N-hexyl,methyl-polyethylenimine (HMPEI) covalently attached to the Boston Keratoprosthesis (B-KPro) materials was evaluated. By means of confocal and electron microscopies, we observed that HMPEI-derivatized materials exert an inhibitory effect on biofilm formation by Staphylococcus aureus clinical isolates, as compared to the parent poly(methyl methacrylate) (PMMA) and titanium. There was no additional corneal epithelial cell cytotoxicity of HMPEI-coated PMMA compared to that of control PMMA in tissue cultures in vitro. Likewise, no toxicity or adverse reactivity was detected with HMPEI-derivatized PMMA or titanium compared to those of the control materials after intrastromal or anterior chamber implantation in rabbits in vivo.
Keywords: Antibacterial; Polyethylenimine (PEI); Keratoprosthesis; PMMA; Titanium; Corneal cytotoxicity;

Metal-based scaffolds such as stents are the most preferred treatment methods for coronary artery disease. However, impaired endothelialization on the luminal surface of the stents is a major limitation occasionally leading to catastrophic consequences in the long term. Coating the stent surface with relevant bioactive molecules is considered to aid in recovery of endothelium around the wound site. However, this strategy remains challenging due to restrictions in availability of proper bioactive signals that will selectively promote growth of endothelium and the lack of convenience for immobilization of such signaling molecules on the metal surface. In this study, we developed self-assembled peptide nanofibers that mimic the native endothelium extracellular matrix and that are securely immobilized on stainless steel surface through mussel-inspired adhesion mechanism. We synthesized Dopa-conjugated peptide amphiphile and REDV-conjugated peptide amphiphile that are self-assembled at physiological pH. We report that Dopa conjugation enabled nanofiber coating on stainless steel surface, which is the most widely used backbone of the current stents. REDV functionalization provided selective growth of endothelial cells on the stainless steel surface. Our results revealed that adhesion, spreading, viability and proliferation rate of vascular endothelial cells are remarkably enhanced on peptide nanofiber coated stainless steel surface compared to uncoated surface. On the other hand, although vascular smooth muscle cells exhibited comparable adhesion and spreading profile on peptide nanofibers, their viability and proliferation significantly decreased. Our design strategy for surface bio-functionalization created a favorable microenvironment to promote endothelial cell growth on stainless steel surface, thereby providing an efficient platform for bioactive stent development for long term treatment of cardiovascular diseases.
Keywords: Stent; Endothelialization; Peptide; Self assembly; ECM (extracellular matrix); Biomimetic materials;

The performance of primary human renal cells in hollow fiber bioreactors for bioartificial kidneys by Zay Yar Oo; Rensheng Deng; Min Hu; Ming Ni; Karthikeyan Kandasamy; Mohammed Shahrudin bin Ibrahim; Jackie Y. Ying; Daniele Zink (8806-8815).
Bioartificial kidneys (BAKs) containing human primary renal proximal tubule cells (HPTCs) have been applied in clinical trials. The results were encouraging, but also showed that more research is required. Animal cells or cell lines are not suitable for clinical applications, but have been mainly used in studies on BAK development as large numbers of such cells could be easily obtained. It is difficult to predict HPTC performance based on data obtained with other cell types. To enable more extensive studies on HPTCs, we have developed a bioreactor containing single hollow fiber membranes that requires relatively small amounts of cells. Special hollow fiber membranes with the skin layer on the outer surface and consisting of polyethersulfone/polyvinylpyrrolidone were developed. The results suggested that such hollow fiber membranes were more suitable for the bioreactor unit of BAKs than membranes with an inner skin layer. An HPTC-compatible double coating was applied to the insides of the hollow fiber membranes, which sustained the formation of functional epithelia under bioreactor conditions. Nevertheless, the state of differentiation of the primary human cells remained a critical issue and should be further addressed. The bioreactor system described here will facilitate further studies on the relevant human cell type.
Keywords: Bioreactor; Epithelium; Haemocompatibility; Membrane;

The effects of the physical properties of culture substrates on the growth and differentiation of human embryonic stem cells by Sunray Lee; Jihoon Kim; Tae Jung Park; Youngmin Shin; Sang Yup Lee; Yong-Mahn Han; Seongman Kang; Hyun-Sook Park (8816-8829).
The physical factors of cell-culture environment have received little attention despite their anticipated significant role in human embryonic stem cell (hESC) culture optimization. Here we show that hESC culture conditions can be optimized by utilizing polyethylene terephthalate (PET) membranes whose defined pore densities (PDs) determine membrane surface hardness. The PET membranes with 1–4 × 106 pores/cm2 (0.291–0.345 GPa) supported the adherence and survival of hESCs without matrix coating. Furthermore, PET membrane with 4 × 106 pores/cm2 (0.345 GPa) supported optimal hESC self renewal as well as by the increase in cell proliferation. The expression level and activity of Rho-associated kinase (ROCK) were specifically down-regulated in hESCs cultured on the optimal PET membrane. We suggest that PET membranes of a defined PD/hardness provide an excellent culture substrate for the maintenance of uniform and undifferentiated hESCs.
Keywords: Polymer membrane; Hardness; Pore density; Embryonic stem cells; Rho family GTPase;

Anisotropic cell sheets for constructing three-dimensional tissue with well-organized cell orientation by Hironobu Takahashi; Masamichi Nakayama; Tatsuya Shimizu; Masayuki Yamato; Teruo Okano (8830-8838).
Normal human dermal fibroblasts were aligned on micropatterned thermoresponsive surfaces simply by one-pot cell seeding. After they proliferated with maintaining their orientation, anisotropic cell sheets were harvested by reducing temperature to 20 °C. Surprisingly, the cell sheets showed different shrinking rates between vertical and parallel sides of the cell alignment (aspect ratio: approx. 3: 1), because actin fibers in the cell sheets were oriented with the same direction. The control of cell alignment provided not only a physical anisotropy but also biological impacts to the cell sheet. Vascular endothelial growth factor (VEGF) secreted by aligned fibroblasts was increased significantly, whereas transforming growth factor-β1 (TGF-β1) expression was the same level in anisotropic cell sheets as cell sheets having random cell orientations. Furthermore, although the amount of deposited type Ⅰ collagen was different non-significantly onto between cell sheets with and without controlled cell alignment, collagen deposited onto fibroblasts sheets with cell alignment also showed anisotropy, verified by a fluorescence imaging analysis. The physical and biological anisotropies of cell sheets were potentially useful to construct biomimetic tissues that were organized by aligned cells and/or extracellular matrix (ECM) including collagen in cell sheet-based regenerative medicine. Furthermore, due to the unique thermoresponsive property, the anisotropic cell sheets were successfully manipulated using a gelatin-coated plunger and were layered with maintaining their cell alignment. The combined use of the anisotropic cell sheet and cell sheet manipulation technique promises to create complex tissue that requires the three-dimensional control of their anisotropies, as one of the next-generation cell sheet technologies.
Keywords: Cell sheet; Cell alignment; Collagen; Micropatterning; Tissue engineering; Thermoresponsive surface;

Bone response to fast-degrading, injectable calcium phosphate cements containing PLGA microparticles by Rosa P. Félix Lanao; Sander C.G. Leeuwenburgh; Joop G.C. Wolke; John A. Jansen (8839-8847).
Apatitic calcium phosphate cements (CPC) are frequently used to fill bone defects due to their favourable clinical handling and excellent bone response, but their lack of degradability inhibits complete bone regeneration. In order to render these injectable CaP cements biodegradable, hollow microspheres made of poly (d,l-lactic-co-glycolic) acid (PLGA) have been previously used as porogen since these microspheres were shown to be able to induce macroporosity upon degradation as well as to accelerate CPC degradation by release of acid degradation products. Recently, the capacity of PLGA microspheres to form porosity in situ in injectable CPCs was optimized by investigating the influence of PLGA characteristics such as microsphere morphology (dense vs. hollow) and end-group functionalization (acid terminated vs. end-capped) on acid production and corresponding porosity formation in vitro. The current study has investigated the in vivo bone response to CPCs containing two types of microspheres (hollow and dense) made of PLGA with two different end-group functionalizations (end capped and acid terminated). Microspheres were embedded in CPC and injected in the distal femoral condyle of New Zealand White Rabbits for 6 and 12 weeks. Histological results confirmed the excellent biocompatibility and osteoconductivity of all tested materials. Composites containing acid terminated PLGA microspheres displayed considerable porosity and concomitant bone ingrowth after 6 weeks, whereas end capped microspheres only revealed open porosity after 12 weeks of implantation. In addition, it was found that dense PLGA microspheres induced significantly more CPC degradation and bone tissue formation compared to hollow PLGA microspheres. In conclusion, it was shown that PLGA microspheres have a strong capacity to induce fast degradation of injectable CPC and concomitant replacement by bone tissue by controlled release of acid polymeric degradation products without compromising the excellent biocompatibility and osteoconductivity of the CPC matrix.
Keywords: Calcium phosphate cement; PLGA; Microspheres; In vivo; Bone;

Human hepatocytes and endothelial cells in organotypic membrane systems by Simona Salerno; Carla Campana; Sabrina Morelli; Enrico Drioli; Loredana De Bartolo (8848-8859).
The realization of organotypic liver model that exhibits stable phenotype is a major challenge in the field of liver tissue engineering. In this study we developed liver organotypic co-culture systems by using synthetic and biodegradable membranes with primary human hepatocytes and human umbilical vein endothelial cells (HUVEC). Synthetic membranes prepared by a polymeric blend constituted of modified polyetheretherketone (PEEK-WC) and polyurethane (PU) and biodegradable chitosan membranes were developed by phase inversion technique and used in homotypic and organotypic culture systems. The morphological and functional characteristics of cells in the organotypic co-culture membrane systems were evaluated in comparison with homotypic cultures and traditional systems. Hepatocytes in the organotypic co-culture systems exhibit compact polyhedral cells with round nuclei and well demarcated cell–cell borders like in vivo, as a result of heterotypic interaction with HUVECs. In addition HUVECs formed tube-like structures directly through the interactions with the membranes and hepatocytes and indirectly through the secretion of ECM proteins which secretion improved in the organotypic co-culture membrane systems. The heterotypic cell–cell contacts have beneficial effect on the hepatocyte albumin production, urea synthesis and drug biotransformation. The developed organotypic co-culture membrane systems elicit liver specific functions in vitro and could be applied for the realization of engineered liver tissues to be used in tissue engineering, drug metabolism studies and bioartificial liver devices.
Keywords: Organotypic; Membrane; Human hepatocytes; Human endothelial cells; Chitosan; Liver tissue engineering;

Combined chemical and topographical guidance cues for directing cytoarchitectural polarization in primary neurons by Adrienne C. Greene; Cody M. Washburn; George D. Bachand; Conrad D. James (8860-8869).
Chemical and topographical cues can be used to guide dissociated neurons into user-defined network geometries on artificial substrates, yet control of neuron polarity (differentiation into axons and dendrites) remains an elusive goal. We developed a dual guidance cue strategy for directing morphological maturity in neurons in vitro using combined chemical and topographical guidance cues on glass substrates. The surface chemistry provides chemical attraction and repulsion for controlling neuron placement and outgrowth, while the topography provides additional surface area for neuron attachment. Poly-l-lysine (PLL) was adsorbed into etched trenches in glass substrates, and an acetone liftoff process was used to produce bifunctional surfaces with a hydrophobic hexamethyldisilazane (HMDS) background and trench patterns of PLL. We examined the cytoarchitectural polarization of dissociated hippocampal pyramidal neurons on guidance cues designed to promote rapid outgrowth of neurites onto continuous line features and delayed neurite outgrowth onto interrupted line features. An optimum distance of approximately 5 μm between the cell body attachment node and the first interrupted line guidance cue led to specific cytoarchitectural polarization of ≥60% of neurons by 3 days of culture in vitro.
Keywords: Neural cell; Cell polarity; Micropatterning; Neural network;

The influence of surface topography of a porous perfluoropolyether polymer on corneal epithelial tissue growth and adhesion by Margaret D.M. Evans; Hassan Chaouk; John S. Wilkie; Beatrice A. Dalton; Sarah Taylor; Ruo Zhong Xie; Timothy C. Hughes; Graham Johnson; Gail A. McFarland; Hans H. Griesser; John G. Steele; Gordon F. Meijs; Deborah F. Sweeney; Keith M. McLean (8870-8879).
Design principles for corneal implants are challenging and include permeability which inherently involves pore openings on the polymer surface. These topographical cues can be significant to a successful clinical outcome where a stratified epithelium is needed over the device surface, such as with a corneal onlay or corneal repair material. The impact of polymer surface topography on the growth and adhesion of corneal epithelial tissue was assessed using porous perfluoropolyether membranes with a range of surface topography. Surfaces were characterised by AFM and XPS, and the permeability and water content of membranes was measured. Biological testing of membranes involved a 21-day in vitro tissue assay to evaluate migration, stratification and adhesion of corneal epithelium. Similar parameters were monitored in vivo by surgically implanting membranes into feline corneas for up to 5 months. Data showed optimal growth and adhesion of epithelial tissue in vitro when polymer surface features were below a 150 nm RMS value. Normal processes of tissue growth and adhesion were disrupted when RMS values approached 300 nm. Data from the in vivo study confirmed these findings. Together, outcomes demonstrated the importance of surface topography in the design of implantable devices that depend on functional epithelial cover.
Keywords: Cornea; In vitro; In vivo; Fluoropolymer;

The enhanced performance of bone allografts using osteogenic-differentiated adipose-derived mesenchymal stem cells by Thomas Schubert; Daela Xhema; Sophie Vériter; Michaël Schubert; Catherine Behets; Christian Delloye; Pierre Gianello; Denis Dufrane (8880-8891).
Adipose tissue was only recently considered as a potential source of mesenchymal stem cells (MSCs) for bone tissue engineering. To improve the osteogenicity of acellular bone allografts, adipose MSCs (AMSCs) and bone marrow MSCs (BM-MSCs) at nondifferentiated and osteogenic-differentiated stages were investigated in vitro and in vivo. In vitro experiments demonstrated a superiority of AMSCs for proliferation (6.1 ± 2.3 days vs. 9.0 ± 1.9 days between each passage for BM-MSCs, respectively, P  < 0.001). A significantly higher T-cell depletion (revealed by mixed lymphocyte reaction, [MLR]) was found for AMSCs (vs. BM-MSCs) at both non- and differentiated stages. Although nondifferentiated AMSCs secreted a higher amount of vascular endothelial growth factor [VEGF] in vitro (between 24 and 72 h of incubation at 0.1–21% O2) than BM-MSCs (P  < 0.001), the osteogenic differentiation induced a significantly higher VEGF release by BM-MSCs at each condition (P  < 0.001). After implantation in the paraspinal muscles of nude rats, a significantly higher angiogenesis (histomorphometry for vessel development (P  < 0.005) and VEGF expression (P  < 0.001)) and osteogenesis (as revealed by osteocalcin expression (P  < 0.001) and micro-CT imagery for newly formed bone tissue (P  < 0.05)) were found for osteogenic-differentiated AMSCs in comparison to BM-MSCs after 30 days of implantation. Osteogenic-differentiated AMSCs are the best candidate to improve the angio-/osteogenicity of decellularized bone allografts.
Keywords: Adipose mesenchymal stem cells; Bone marrow mesenchymal stem cells; Bone allograft; Immunomodulation; Angiogenesis; Osteogenesis;

Characterization of the effects of x-ray irradiation on the hierarchical structure and mechanical properties of human cortical bone by Holly D. Barth; Elizabeth A. Zimmermann; Eric Schaible; Simon Y. Tang; Tamara Alliston; Robert O. Ritchie (8892-8904).
Bone comprises a complex structure of primarily collagen, hydroxyapatite and water, where each hierarchical structural level contributes to its strength, ductility and toughness. These properties, however, are degraded by irradiation, arising from medical therapy or bone-allograft sterilization. We provide here a mechanistic framework for how irradiation affects the nature and properties of human cortical bone over a range of characteristic (nano to macro) length-scales, following x-ray exposures up to 630 kGy. Macroscopically, bone strength, ductility and fracture resistance are seen to be progressively degraded with increasing irradiation levels. At the micron-scale, fracture properties, evaluated using insitu scanning electron microscopy and synchrotron x-ray computed micro-tomography, provide mechanistic information on how cracks interact with the bone-matrix structure. At sub-micron scales, strength properties are evaluated with insitu tensile tests in the synchrotron using small-/wide-angle x-ray scattering/diffraction, where strains are simultaneously measured in the macroscopic tissue, collagen fibrils and mineral. Compared to healthy bone, results show that the fibrillar strain is decreased by ∼40% following 70 kGy exposures, consistent with significant stiffening and degradation of the collagen. We attribute the irradiation-induced deterioration in mechanical properties to mechanisms at multiple length-scales, including changes in crack paths at micron-scales, loss of plasticity from suppressed fibrillar sliding at sub-micron scales, and the loss and damage of collagen at the nano-scales, the latter being assessed using Raman and Fourier Transform Infrared spectroscopy and a fluorometric assay.
Keywords: Human cortical bone; Deformation; Toughness; X-ray diffraction; Tomography; Collagen;

The role of multifunctional delivery scaffold in the ability of cultured myoblasts to promote muscle regeneration by Cristina Borselli; Christine A. Cezar; Dymitri Shvartsman; Herman H. Vandenburgh; David J. Mooney (8905-8914).
Many cell types of therapeutic interest, including myoblasts, exhibit reduced engraftment if cultured prior to transplantation. This study investigated whether polymeric scaffolds that direct cultured myoblasts to migrate outwards and repopulate the host damaged tissue, in concert with release of angiogenic factors designed to enhance revascularizaton of the regenerating tissue, would enhance the efficacy of this cell therapy and lead to functional muscle regeneration. This was investigated in the context of a severe injury to skeletal muscle tissue involving both myotoxin-mediated direct damage and induction of regional ischemia. Local and sustained release of VEGF and IGF-1 from macroporous scaffolds used to transplant and disperse cultured myogenic cells significantly enhanced their engraftment, limited fibrosis, and accelerated the regenerative process. This resulted in increased muscle mass and, improved contractile function. These results demonstrate the importance of finely controlling the microenvironment of transplanted cells in the treatment of severe muscle damage.
Keywords: Hydrogel; Alginate; Drug delivery; Cell activation; Muscle;

Accelerated mineralization of dense collagen-nano bioactive glass hybrid gels increases scaffold stiffness and regulates osteoblastic function by Benedetto Marelli; Chiara E. Ghezzi; Dirk Mohn; Wendelin J. Stark; Jake E. Barralet; Aldo R. Boccaccini; Showan N. Nazhat (8915-8926).
Plastically compressed dense collagen (DC) gels mimic the microstructural, mechanical, and biological properties of native osteoid. This study investigated the effect of hybridizing DC with osteoinductive nano-sized bioactive glass (nBG) particles in order to potentially produce readily implantable, and mineralizable, cell seeded hydrogel scaffolds for bone tissue engineering. Due to the high surface area of nBG and increased reactivity, calcium phosphate formation was immediately detected within as processed DC-nGB hybrid gel scaffolds. By day 3 in simulated body fluid, accelerated mineralization was confirmed through the homogeneous growth of carbonated hydroxylapatite on the nanofibrillar collagen framework. At day 7, there was a 13 fold increase in the hybrid gel scaffold compressive modulus. MC3T3-E1 pre-osteoblasts, three-dimensionally seeded at the point of nanocomposite self-assembly, were viable up to day 28 in culture. In the absence of osteogenic supplements, MC3T3-E1 metabolic activity and alkaline phosphatase production were affected by the presence of nBG, indicating accelerated osteogenic differentiation. Additionally, no cell-induced contraction of DC-nBG gel scaffolds was detected. The accelerated mineralization of rapidly produced DC-nBG hybrid gels indicates their potential suitability as osteoinductive cell delivery scaffolds for bone regenerative therapy.
Keywords: Nanocomposite hydrogels; Dense collagen scaffolds; Nano-bioactive glass; Mineralization; Hydroxyapatite; Tissue engineering;

Effect of initial cell seeding density on 3D-engineered silk fibroin scaffolds for articular cartilage tissue engineering by Sarmistha Talukdar; Quynhhoa T. Nguyen; Albert C. Chen; Robert L. Sah; Subhas C. Kundu (8927-8937).
The repair of articular cartilage defects poses a continuing challenge. Cartilage tissue engineering through the culture of chondrocytes seeded in 3D porous scaffolds has the potential for generating constructs that repair successfully. It also provides a platform to study scaffold-cell and cell–cell interactions. The scaffold affects the growth and morphology of cells growing on it, and concomitantly, cells affect the properties of the resultant tissue construct. Silk fibroin protein from Antheraea mylitta, a non-mulberry Indian tropical tasar silkworm, is a potential biomaterial for diverse applications due to its widespread versatility as a mechanically robust, biocompatible, tissue engineering material. Analysis of silk fibroin scaffolds seeded with varying initial densities (25, 50 and 100 million cells/ml) and cultured for 2 weeks showed that thickness and wet weight increased by 60–70% for the highest cell density, and DNA, GAG and collagen content of the cartilaginous constructs increased with increasing cell density. Mechanical characterization of the constructs elucidated that the highest density constructs had compressive stiffness and modulus 4–5 times that of cell-free scaffolds. The present results indicate the importance of cell seeding density in the rapid formation of a functional cartilaginous tissue.
Keywords: Silk fibroin; Chondrocyte; 3D scaffold; ECM; Articular cartilage; Tissue engineering;

Growth promoting substrates for human dermal fibroblasts provided by artificial extracellular matrices composed of collagen I and sulfated glycosaminoglycans by Anja van der Smissen; Vera Hintze; Dieter Scharnweber; Stephanie Moeller; Matthias Schnabelrauch; Annett Majok; Jan C. Simon; Ulf Anderegg (8938-8946).
The application of native extracellular matrix (ECM) components is a promising approach for biomaterial design. Here, we investigated artificial ECM (aECM) consisting of collagen I (coll) and the glycosaminoglycans (GAGs) hyaluronan (HA) or chondroitin sulfate (CS). Additionally, GAGs were chemically modified by the introduction of sulfate groups to obtain low-sulfated and high-sulfated GAG derivatives. Sulfate groups are expected to bind and concentrate growth factors and improve their bioactivity. In this study we analyzed the effect of aECM on initial adhesion, proliferation, ECM synthesis and differentiation of human dermal fibroblasts (dFb) within 8–48 h. We show that initial adhesion and cell proliferation of dFb progressively increased in a sulfate dependent manner. In contrast, synthesis of ECM components coll and HA was decreased on high-sulfated aECM coll/HA3.0 and coll/CS3.1. Furthermore, the matrix metallo-proteinase-1 (MMP-1) was down-regulated on coll/HA3.0 and coll/CS3.1 on mRNA and protein level. The fibroblast differentiation marker α-smooth muscle actin (αSMA) is not affected by aECM on mRNA level. Artificial ECM consisting of coll and high-sulfated GAGs proves to be a suitable biomaterial for dFb adhesion and proliferation that induces a “proliferative phenotype” of dFb found in the early stages of cutaneous wound healing.
Keywords: Wound healing; Collagen I; Sulfated glycosaminoglycan; Hyaluronan; Chondroitin sulfate; Human dermal fibroblasts;

Promotion of osteoblast differentiation in 3D biomaterial micro-chip arrays comprising fibronectin-coated poly(methyl methacrylate) polycarbonate by Brigitte Altmann; Thorsten Steinberg; Stefan Giselbrecht; Eric Gottwald; Pascal Tomakidi; Maria Bächle-Haas; Ralf-Joachim Kohal (8947-8956).
Due to the architecture of solid body tissues including bone, three-dimensional (3D) in vitro microenvironments appear favorable, since herein cell growth proceeds under more physiological conditions compared to conventional 2D systems. In the present study we show that a 3D microenvironment comprising a fibronectin-coated PMMA/PC-based micro-chip promotes differentiation of primary human osteoblasts as reflected by the densely-packed 3D bone cell aggregates and expression of biomarkers indicating osteoblast differentiation. Morphogenesis and fluorescence dye-based live/dead staining revealed homogenous cell coverage of the microcavities of the chip array, whereat cells showed high viability up to 14 days. Moreover, Azur II staining proved formation of uniform sized multilayered aggregates, exhibiting progressive intracellular deposition of extracellular bone matrix constituents comprising fibronectin, osteocalcin and osteonectin from day 7 on. Compared to 2D monolayers, osteoblasts grown in the 3D chip environment displayed differential mostly higher gene expression for osteocalcin, osteonectin, and alkaline phosphatase, while collagen type I remained fairly constant in both culture environments. Our results indicate that the 3D microenvironment, based on the PMMA biomaterial chip array promotes osteoblast differentiation, and hereby renders a promising tool for tissue-specific in vitro preconditioning of osteoblasts designated for clinically-oriented bone augmentation or regeneration.
Keywords: 3D in vitro cell culture; Osteoblast; Poly(methyl methacrylate); Scaffold; Microfabrication; Bone tissue engineering;

Identifying the critical molecules associated with “biocompatibility” is a grand challenge. Poly(methacrylic acid -co- methyl methacrylate) (MAA) beads improve wound closure and wound vascularity in vivo, but the mechanism of this phenomenon is unknown. We used quantitative real-time PCR to identify the subtle changes in the expression of a small selection of molecules involved in wound healing and angiogenesis in a macrophage-like cell (dTHP-1) treated with the MAA beads (45 mol% methacrylic acid). MAA beads decreased the expression of osteopontin (OPN) compared to poly(methyl methacrylate) (PMMA) and untreated cells, and increased the expression of IL-1β, IL-6 and TNF-α over the 24–96 h of the experiment. Interestingly, molecules associated with angiogenesis, such as bFGF, CXCL12, HIF-1α, PDGF-B, TGF-β and VEGF, were not significantly affected by MAA beads over the course of the study. MAA beads also increased the gene expression of OPN in HUVEC compared to untreated cells, while PMMA beads did not. MAA beads modified the phenotype (gene expression) of dTHP-1 cells in a subtle yet distinct manner that was different than PMMA. It remains to connect the changes in OPN in dTHP-1 (and HUVEC) and other molecules to the enhanced vascularity seen in vivo with this polymer.
Keywords: Macrophage; Methacrylic acid; Osteopontin; Gene expression; Cytokines; Angiogenesis;

Surface-density gradients of poly(ethylene glycol) (PEG) were fabricated, in order to carry out a systematic study of the influence of PEG chain density on protein adsorption and cell-adhesion behavior, as well as the correlation between them. Gradients with a linear change in coverage of the polycationic polymer Poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) were prepared on titanium dioxide surfaces by a controlled dipping process and characterized by variable-angle spectroscopic ellipsometry and fluorescence microscopy. The adsorption behavior of single proteins (fibrinogen and albumin) generally correlated with semiempirical geometric models, illustrating the effect of the PEG-chain surface distribution on the inhibition of protein adsorption. Distinct differences could be observed between individual adsorbing proteins, attributable to their mode of surface attachment. The single and competitive adsorption of protein solutions containing albumin and fibrinogen was then investigated by fluorescence microscopy, indicating a larger amount of fibrinogen adsorption compared with albumin adsorption (in minutes to hours) along the entire PLL-g-PEG gradient samples. To further elucidate the underlying mechanism of cell adhesion and spreading as a function of PEG coverage and the potential involvement of integrins, cell-adhesion assays were carried out with human foreskin fibroblasts (hFF). The use of surface-gradient samples demonstrated the importance for protein adsorption of PEG conformation, the amount of exposed titanium dioxide surface area (and its distribution), and the structure and chemistry of the proteins involved. Correspondingly the influence of these factors on cell adhesion could be directly observed, and insights gained into the roles of both nonspecific binding and specific integrin binding in cell adhesion.
Keywords: Protein adsorption; Cell adhesion; Poly(ethylene glycol); Surface gradients; Titanium dioxide; β3-integrin;

The influence of elasticity and surface roughness on myogenic and osteogenic-differentiation of cells on silk-elastin biomaterials by Xiao Hu; Sang-Hyug Park; Eun Seok Gil; Xiao-Xia Xia; Anthony S. Weiss; David L. Kaplan (8979-8989).
The interactions of C2C12 myoblasts and human bone marrow stem cells (hMSCs) with silk-tropoelastin biomaterials, and the capacity of each to promote attachment, proliferation, and either myogenic- or osteogenic-differentiation were investigated. Temperature-controlled water vapor annealing was used to control beta-sheet crystal formation to generate insoluble silk-tropoelastin biomaterial matrices at defined ratios of the two proteins. These ratios controlled surface roughness and micro/nano-scale topological patterns, and elastic modulus, stiffness, yield stress, and tensile strength. A combination of low surface roughness and high stiffness in the silk-tropoelastin materials promoted proliferation and myogenic-differentiation of C2C12 cells. In contrast, high surface roughness with micro/nano-scale surface patterns was favored by hMSCs. Increasing the content of human tropoelastin in the silk-tropoelastin materials enhanced the proliferation and osteogenic-differentiation of hMSCs. We conclude that the silk-tropoelastin composition facilitates fine tuning of the growth and differentiation of these cells.
Keywords: Elastin; Silk; Biomaterials; Muscle cells; Stem cells;

The development of collagen-GAG scaffold-membrane composites for tendon tissue engineering by Steven R. Caliari; Manuel A. Ramirez; Brendan A.C. Harley (8990-8998).
Current tissue engineering approaches for tendon defects require improved biomaterials to balance microstructural and mechanical design criteria. Collagen-glycosaminoglycan (CG) scaffolds have shown considerable success as in vivo regenerative templates and in vitro constructs to study cell behavior. While these scaffolds possess many advantageous qualities, their mechanical properties are typically orders of magnitude lower than orthopedic tissues such as tendon. Taking inspiration from mechanically efficient core–shell composites in nature such as plant stems and porcupine quills, we have created core–shell CG composites that display high bioactivity and improved mechanical integrity. These composites feature integration of a low density, anisotropic CG scaffold core with a high density, CG membrane shell. CG membranes were fabricated via an evaporative process that allowed separate tuning of membrane thickness and elastic moduli and were found to be isotropic in-plane. The membranes were then integrated with an anisotropic CG scaffold core via freeze-drying and subsequent crosslinking. Increasing the relative thickness of the CG membrane shell was shown to increase composite tensile elastic modulus by as much as a factor of 36 in a manner consistent with predictions from layered composites theory. CG scaffold-membrane composites were found to support tendon cell viability, proliferation, and metabolic activity in vitro, suggesting they maintain sufficient permeability while demonstrating improved mechanical strength. This work suggests an effective, biomimetic approach for balancing strength and bioactivity requirements of porous scaffolds for tissue engineering.
Keywords: Collagen; Scaffold; Membrane; Composite; Tendon; Mechanical properties;

Superhigh-magnetization nanocarrier as a doxorubicin delivery platform for magnetic targeting therapy by Mu-Yi Hua; Hung-Wei Yang; Hao-Li Liu; Rung-Ywan Tsai; See-Tong Pang; Kun-Lung Chuang; Yu-Sun Chang; Tsong-Long Hwang; Ying-Hsu Chang; Heng-Chang Chuang; Cheng-Keng Chuang (8999-9010).
The aim of this study describes the creation of superhigh-magnetization nanocarriers (SHMNCs) comprised of a magnetic Fe3O4 (SHMNPs) core and a shell of aqueous stable self-doped poly[N-(1-one-butyric acid)]aniline (SPAnH), which have a high drug loading capacity (∼27.1 wt%) of doxorubicin (DOX). The SHMNCs display superparamagnetic property with a magnetization of 89.7 emu/g greater than that of Resovist (a commercial contrast agent used for magnetic resonance imaging; 73.7 emu/g). Conjugating the anticancer drug DOX to these nanocarriers enhances the drug’s thermal stability and maximizes the efficiency with which it is delivered by magnetic targeting (MT) therapy to MGH-U1 bladder cancer cells, in part by avoiding the effects of p-glycoprotein (P-gp) pumps to enhance the intracellular concentration of DOX. The high R2 relaxivity (434.7 mM−1s−1) of SHMNCs not only be a most effective MT carrier of chemotherapeutic agent but be an excellent contrast agent of MRI, allowing the assessment of the distribution and concentration of DOX in various tissues and organs. This advanced drug delivery system promises to provide more effective MT therapy and tumor treatment using lower therapeutic doses and potentially reducing the side effects of cardiotoxicity caused by DOX.
Keywords: P-glycoprotein pump; Bladder cancer; Superhigh-magnetization nanocarriers; Magnetic targeting therapy; Magnetic resonance image;

Fluorophore-labeled nanocapsules displaying IgG Fc-binding domains for the simultaneous detection of multiple antigens by Masumi Iijima; Takashi Matsuzaki; Nobuo Yoshimoto; Tomoaki Niimi; Katsuyuki Tanizawa; Shun’ichi Kuroda (9011-9020).
Simultaneous detection of multiple antigens by conventional immunological methods has been limited by the source of primary antibodies. Each antibody should be derived from a different host species (or subclass of immunoglobulin (Ig)) for suppressing the cross-reactions of secondary antibodies. Here we describe an innovative method for simultaneous, rapid, and sensitive detection of multiple antigens using ∼30-nm bio-nanocapsules (BNCs) displaying IgG Fc-binding Z domains derived from Staphylococcus aureus protein A (ZZ-BNC). When Cy2-labeled ZZ-BNC (Cy2-ZZ-BNC) was used instead of Cy2-labeled secondary antibody in western blot analysis, both sensitivity and signal intensity were significantly increased. The complex of Cy5-ZZ-BNC and mouse IgG2a (which shows moderate affinity to the Z domain) was not dissociated, even in the presence of 8-fold excess of free mouse IgG2a. In addition, crosslinking with BS3 (bis-sulfosuccinimidyl suberate) efficiently stabilized the interaction. The ZZ-BNCs labeled with various Cy dyes facilitated the simultaneous detection of multiple antigens using primary antibodies derived from the same host species, by western blot analysis, immunocytochemistry and flow cytometry, which could expand the possibility of bio-imaging probes in various immunofluorescence techniques.
Keywords: Nanoparticle; Scaffold; Image analysis; Immunochemistry; Flow cytometry; Fluorescence;

Assessment of nanomaterial cytotoxicity with SOLiD sequencing-based microRNA expression profiling by Shuchun Li; Haitao Wang; Yuhua Qi; Jing Tu; Yunfei Bai; Tian Tian; Ningping Huang; Yong Wang; Fei Xiong; Zuhong Lu; Zhongdang Xiao (9021-9030).
The cytotoxicity of nanomaterials has become a major concern in the field of nanotechnology. The key challenge is the lack of reliable methods to examine the overall cellular effects of nanomaterials. Here, a new method is developed to assess the cytological effects of nanomaterial basing on miRNA expression profiling. The SOLiD sequencing is used to acquire the miRNAs expression profiling in NIH/3T3 cells after exposure to Fe2O3 NPs, CdTe QDs and MW-CNTs, respectively. The systematic analysis of miRNAs expression profiling is established by taking account of all miRNAs into their regulatory networks. By affecting the output of targeted mRNAs, miRNAs widely regulated the KEGG pathways and GO biological processes in nanomaterial treated cells. Therefore, the miRNA expression profiling can well reflect the characteristic of nanomaterials, and the method not only provide more evidences to assess biocompatibility of nanomaterials and but also clues to discover new biological effects of nanomaterials.
Keywords: MicroRNA; Cytotoxicity; Nanomaterial; SOLiD sequencing;

The photoluminescence, drug delivery and imaging properties of multifunctional Eu3+/Gd3+ dual-doped hydroxyapatite nanorods by Feng Chen; Peng Huang; Ying-Jie Zhu; Jin Wu; Chun-Lei Zhang; Da-Xiang Cui (9031-9039).
The design and synthesis of multifunctional systems with high biocompatibility are very significant for the future of clinical applications. Herein, we report a microwave-assisted rapid synthesis of multifunctional Eu3+/Gd3+ dual-doped hydroxyapatite (HAp) nanorods, and the photoluminescence (PL), drug delivery and in vivo imaging of as-prepared Eu3+/Gd3+ doped HAp nanorods. The photoluminescent and magnetic multifunctions of HAp nanorods are realized by the dual-doping with Eu3+ and Gd3+. The PL intensity of doped HAp nanorods can be adjusted by varying Eu3+ and Gd3+ concentrations. The magnetization of doped HAp nanorods increases with the concentration of doped Gd3+. The as-prepared Eu3+/Gd3+-doped HAp nanorods exhibit inappreciable toxicity to the cells in vitro. More importantly, the Eu3+/Gd3+-doped HAp nanorods show a high drug adsorption capacity and sustained drug release using ibuprofen as a model drug, and the drug release is governed by a diffusion process. Furthermore, the noninvasive visualization of nude mice with subcutaneous injection indicates that the Eu3+/Gd3+-doped HAp nanorods with the photoluminescent function are suitable for in vivo imaging. In vitro and in vivo imaging tests indicate that Eu3+/Gd3+-doped HAp nanorods have a potential in applications such as a multiple-model imaging agent for magnetic resonance (MR) imaging, photoluminescence imaging and computed tomography (CT) imaging. The Eu3+/Gd3+ dual-doped HAp nanorods are promising for applications in the biomedical fields such as multifunctional drug delivery systems with imaging guidance.
Keywords: Hydroxyapatite; Nanorod; Microwave; Drug delivery; Luminescence; Imaging;

The effect of CdSe–ZnS quantum dots on calcium currents and catecholamine secretion in mouse chromaffin cells by Sara Gosso; Daniela Gavello; Carlo N.G. Giachello; Claudio Franchino; Emilio Carbone; Valentina Carabelli (9040-9050).
Semiconductor nanocrystal quantum dots (QDs) possess an enormous potential of applications in nanomedicine, drug delivery and bioimaging which derives from their unique photoemission and photostability characteristics. In spite of this, however, their interactions with biological systems and impact on human health are still largely unknown. Here we used neurosecretory mouse chromaffin cells of the adrenal gland for testing the effects of CdSe–ZnS core–shell quantum dots (5–36 nM) on Ca2+ channels functionality and Ca2+-dependent neurosecretion. Prolonged exposure (24 h) to commonly used concentrations of CdSe–ZnS QDs (≥16 nM) showed that the semiconductor nanocrystal is effectively internalized into the cells without affecting cell integrity (no changes of membrane resistance and cell capacitance). QDs reduced the size of Ca2+ currents by ∼28% in a voltage-independent manner without affecting channel gating. Correspondingly, depolarization-evoked exocytosis, measured at +10 mV, where Ca2+ currents are maximal, was reduced by 29%. CdSe–ZnS QDs reduced the size of the readily releasable pool (RRP) of secretory vesicles by 32%, the frequency of release by 33% and the overall quantity of released catecholamines by 61%, as measured by carbon fibers amperometry. In addition, the Ca2+-dependence of exocytosis was reduced, whereas the catecholamine content of single granules, as well as the kinetics of release, remained unaltered. These data suggest that exposure to CdSe–ZnS QDs impairs Ca2+ influx and severely interferes with the functionality of the exocytotic machinery, compromising the overall catecholamine supply from chromaffin cells.
Keywords: Semiconductor nanocrystals; Adrenal chromaffin cells; Voltage-gated Ca2+ channels; Membrane capacitance changes; Exocytosis; Amperometry;

We introduce the strategic development of self-assembling peptide/protein fragments based on the far-red fluorescent protein mPlum. The first beta strand (mPlum 1, 18 amino acids) of mPlum was engineered to spontaneously bind with the rest of the protein (mPlum 2–11, next 10 beta strands) and to form a native chromophore. The target beta strand mPlum 1 was separated from mPlum 2–11 and linked via a flexible peptide linker, resulting in fluorescently inactive circularly permuted mPlum protein (CpmPlum). In vitro evolution of this CpmPlum to a fluorescently active form and the subsequent splitting of the engineered mPlum 1 peptide afforded self-assembling mPlum fragments. Recombinantly expressed and synthetically prepared beta strand peptides were successfully assembled with the remaining mPlum protein in vitro and in cells. This developed pair of peptide/protein fragments was effectively used for peptide tag detection of alpha-synuclein in mammalian cells. Sequential expression of self-assembling mPlum fragments offered an entirely genetically encoded sensing system of naturally unfolded alpha-synuclein.
Keywords: Fluorescent protein; Protein engineering; Split protein; Molecular evolution; Biosensors;

Bioimaging and toxicity assessments of near-infrared upconversion luminescent NaYF4:Yb,Tm nanocrystals by Jia-Cai Zhou; Zheng-Lin Yang; Wei Dong; Ruo-Jin Tang; Ling-Dong Sun; Chun-Hua Yan (9059-9067).
In vitro or in vivo bioimaging utilizing the upconversion (UC) luminescence of rare earth fluoride nanocrystals (NCs) has attracted much attention, especially for Yb3+/Tm3+ doped NCs with a near-infrared (NIR) UC emission at 800 nm. Herein, water-soluble NaYF4:Yb,Tm NCs with strong NIR UC emission were synthesized with a solvothermal method. In vitro and in vivo bioimaging and toxicity assessments were carried out with HeLa cell and Caenorhabditis elegans (C. elegans) cases, respectively. NaYF4:Yb,Tm NCs afforded an efficient NIR image of the HeLa cells with an incubation concentration of 10 μg mL−1, and CCK-8 assay revealed a low cytotoxicity. Fed with Escherichia coli (E. coli) and NCs together, the C. elegans showed a NIR image in the gut from the pharynx to the anus. Further, these NCs could be excreted out when those worms were then fed with only E. coli. Toxicity studies were further addressed with protein expression, life span, egg production, egg viability, and growth rate of the worms in comparison with those of the intact ones. The feeding of rare earth fluoride NCs with a dose of 100 μg does not arise obvious toxicity effect from the growth to procreation. The in vitro and in vivo studies confirm that NaYF4:Yb,Tm NCs could be served as an excellent NIR emission bioprobe with low toxicity.
Keywords: Near-infrared emission; NaYF4:Yb,Tm nanocrystals; Bioimaging; Toxicity assessments; In vitro; In vivo;

The reversal of cisplatin-induced nephrotoxicity by selenium nanoparticles functionalized with 11-mercapto-1-undecanol by inhibition of ROS-mediated apoptosis by Yinghua Li; Xiaoling Li; Yum-Shing Wong; Tianfeng Chen; Haobin Zhang; Chaoran Liu; Wenjie Zheng (9068-9076).
Although cisplatin is still one of the most effective chemotherapy agents for human cancers, its clinical use is limited by serious side effects, especially nephrotoxicity. Oxidative stress is an important mediator of cisplatin-induced nephrotoxicity. In the present study, a simple method for functionalization of selenium nanoparticles by self-assembly of 11-mercapto-1-undecanol (Se@MUN) to achieve enhanced antioxidant activity and antagonis against cisplatin-induced nephrotoxicity has been demonstrated. The chemical structure of the nanoparticles was characterized by various microscopic and spectroscopic methods. The results revealed that the spherical nanoparticles were capped with MUN on the surface through formation of Se-S bond. The in vitro protective effects of Se@MUN on HK-2 proximal tubular cells against cisplatin-induced nephrotoxicity and the underlying mechanisms were also investigated. Se@MUN exhibited free radical scavenging activity and higher cellular uptake in human normal cells by comparing with SeNPs. Se@MUN significantly attenuated cisplatin-induced reduction in cell viability, appearance of Sub-G1 peak, nuclear condensation and DNA fragmentation in HK-2 cells. Activation of caspase-3 in cells exposed to cisplatin was also effectively blocked by Se@MUN. Moreover, Se@MUN significantly prevented the cisplatin-induced overproduction of intracellular ROS. Our findings suggest that Se@MUN is a promising selenium species with potential application in prevention of cisplatin-induced renal injury.
Keywords: Nanoparticle; Antioxidant; Apoptosis; Bioactivity; Free radical; In vitro test;

Delivery of Oct4 and SirT1 with cationic polyurethanes-short branch PEI to aged retinal pigment epithelium by Chi-Hsien Peng; Jong-Yuh Cherng; Guang-Yuh Chiou; Yu-Chih Chen; Chen-Hsiu Chien; Chung-Lan Kao; Yuh-Lih Chang; Yueh Chien; Liang-Kung Chen; Jorn-hon Liu; Shih-Jen Chen; Shih-Hwa Chiou (9077-9088).
Cationic polyurethane, a biodegradable non-viral vector, protects DNA from nuclease degradation and helps to deliver genes efficiently. Oct4, a POU-domain transcription factor, is highly expressed in maintaining pluripotency and cellular reprogramming process in stem cells. SirT1, a NAD-dependent histone deacetylase, is an essential mediator of cellular longevity. Herein we demonstrated that both Oct4 and SirT1 (Oct4/SirT1) expression was decreased in an age-dependent manner in retina with aged-related macular degeneration and retinal pigment epithelium cells (RPEs). To investigate the possible rescuing role of Oct4/SirT1, polyurethane-short branch polyethylenimine (PU-PEI) was used to deliver Oct4/SirT1 into aged RPEs (aRPEs) or light-injured rat retinas. Oct4/SirT1 overexpression increased the expression of several progenitor-related genes and the self-renewal ability of aRPEs. Moreover, Oct4/SirT1 overexpression resulted in the demethylation of the Oct4 promoter and enhanced the expression of antioxidant enzymes, which was accompanied by a decrease in intracellular ROS production and hydrogen peroxide-induced oxidative stress. Importantly, PU-PEI-mediated Oct4/SirT1 gene transfer rescued retinal cell loss and improved electroretinographic responses in light-injured rat retinas. In summary, these data suggest that PU-PEI-mediated delivery of Oct4/SirT1 reprograms aRPEs into a more primitive state and results in cytoprotection by regulating the antioxidative capabilities of these cells.
Keywords: Polyurethane-short branch polyethylenimine; Oct4; SirT1; Retinal pigment epithelium cells; Age-related macular degeneration; Reprogramming;

Functional hydrophobin-coating of thermally hydrocarbonized porous silicon microparticles by Luis M. Bimbo; Ermei Mäkilä; Janne Raula; Timo Laaksonen; Päivi Laaksonen; Katharina Strommer; Esko I. Kauppinen; Jarno Salonen; Markus B. Linder; Jouni Hirvonen; Hélder A. Santos (9089-9099).
Porous silicon (PSi) particles have been widely used in modulating the dissolution rate of various types of drugs loaded within its mesopores. This material can be surface treated in order to vary its hydrophobicity and several other properties, such as drug loading degree and release rate. Hydrophobins are a family of self-assembling proteins of fungal origin which have the ability to form layers on hydrophobic materials. This type of protein layer can modify the characteristics and control the binding properties of the surface on which it assembles. In this study, we have developed a procedure to coat thermally hydrocarbonized-PSi microparticles with hydrophobin II (HFBII) in order to modify the particles’ hydrophobicity and to improve their biocompatibility, while maintaining intact the advantageous drug releasing properties of the PSi. The HFBII content adsorbed onto the particles was successfully quantified by a protein assay. Drug dissolution and permeation across Caco-2 cell monolayers were also conducted, together with viability studies in AGS, Caco-2 and HT-29 cells. The characterization and coating stability assessment showed that the HFBII-coating desorbs partially from the particles’ surface as the pH increases. The HFBII coating also improved the biocompatibility of the particles without compromising the enhanced drug permeation or release.
Keywords: Silicon; Microparticle; Hydrophobin; Biocompatibility; Drug release;

The effect of thiol functional group incorporation into cationic helical peptides on antimicrobial activities and spectra by Nikken Wiradharma; Majad Khan; Lin-Kin Yong; Charlotte A.E. Hauser; See Voon Seow; Shuguang Zhang; Yi-Yan Yang (9100-9108).
Antimicrobial peptides (AMP) have been proposed as blueprints for the development of new antimicrobial agents for the treatment of drug resistant infections. A series of synthetic AMPs capable of forming α-helical structures and containing free-sulfhydryl groups are designed in this study ((LLKK)2C, C(LLKK)2C, (LLKK)3C, C(LLKK)3C). In particular, the AMP with 2 cysteine residues at the terminal ends of the peptide and 2 repeat units of LLKK, i.e., C(LLKK)2C, has been demonstrated to have high selectivity towards a wide range of microbes from Gram-positive Bacillus subtilis, Gram-negative Escherichia coli, Pseudomonas aerogenosa, and yeast Candida albicans over red blood cells. At the MIC levels, this peptide does not induce significant hemolysis, and its MIC values occur at the concentration of more than 10 times of their corresponding 50% hemolysis concentrations (HC50). Microscopy studies suggest that this peptide kills microbial cells by inducing pores of ∼20–30 nm in size in microbial membrane on a short time scale, which further develops to grossly damaged membrane envelope on a longer time scale. Multiple treatments of microbes with this peptide at sub MIC concentration do not induce resistance, even up to passage 10. However, the same treatment with conventional antibiotics penicillin G or ciprofloxacin easily develop resistance in the treated microbes. In addition, the peptides are shown not to induce secretion of IFN-γ and TNF-α in human monocytes as compared to lipopolysaccharide, which implies additional safety aspects of the peptides to be used as both systemic and topical antimicrobial agents. Therefore, this study provides an excellent basis to develop promising antimicrobial agents that possess a broad range of antimicrobial activities with less susceptibility for development of drug resistance.
Keywords: Antimicrobial peptides (AMP); α-Helix; Thiol and sulfhydryl; Membrane lysis; Hemolysis; Drug resistance;

Reversibly hydrophobilized 10 kDa polyethylenimine (PEI) based on rapidly acid-degradable acetal-containing hydrophobe was designed for nontoxic and highly efficient non-viral gene transfer. Water soluble PEI derivatives with average 5, 9 and 14 units of pH-sensitive 2,4,6-trimethoxybenzylidene-tris(hydroxymethyl)ethane (TMB-THME) hydrophobe per molecule, denoted as PEI-g-(TMB-THME)n, were readily obtained by treating 10 kDa PEI with varying amounts of TMB-THME-nitrophenyl chloroformate. Gel retardation assays showed that all PEI-g-(TMB-THME)n derivatives could effectively condense DNA at an N/P ratio of 5/1. Notably, polyplexes of PEI-g-(TMB-THME)n derivatives had smaller sizes (about 100∼170 nm) and higher surface charges (+25 ∼ +43 mV) than the parent 10 kDa PEI at the same N/P ratios ranging from 10/1 to 40/1. MTT assays revealed that these PEI-g-(TMB-THME)n derivatives were practically non-toxic at polymer concentrations used in transfection experiments. The acetal degradation of PEI-g-(TMB-THME)9 was shown to be highly pH dependent in which half lives of 1.3, 2.8 and 11 h were determined for pH 4.0, 5.0 and 6.0, respectively, while negligible hydrolysis (<12%) was observed after 24 h at pH 7.4. Gel electrophoresis, dynamic light scattering (DLS) and zeta potential analyses indicated that polyplexes formed at an N/P ratio of 10/1 were dissociated following 5 h incubation at pH 5.0, highlighting the importance of hydrophobic TMB-THME moieties in DNA condensation and supporting that acetal hydrolysis in endosomes would facilitate DNA release. Notably, in vitro transfection experiments performed at N/P ratios of 10/1 and 20/1 in HeLa, 293T, HepG2 and KB cells using plasmid pGL3 expressing luciferase as the reporter gene showed that reversibly hydrophobilized PEIs had superior transfection activity to 25 kDa PEI control. For example, polyplexes of PEI-g-(TMB-THME)14 showed about 235-fold and 175-fold higher transfection efficiency as compared to 10 kDa PEI in HeLa cells in serum-free and 10% serum media, respectively, which were approximately 7-fold and 16-fold higher than 25 kDa PEI formulation at its optimal N/P ratio under otherwise the same conditions. Confocal laser scanning microscope (CLSM) studies confirmed that PEI-g-(TMB-THME)14 efficiently delivered Cy5-labeled DNA to the nuclei of HeLa cells. These endosomal pH-sensitive reversibly hydrophobilized PEIs have great potentials for safe and efficient non-viral gene transfection.
Keywords: pH-sensitive; Cationic polymers; Polyethylenimine; Gene delivery; Plasmid DNA; Degradable polymers;

Hemocompatibility of siRNA loaded dextran nanogels by Broes Naeye; Hendrik Deschout; Magnus Röding; Mats Rudemo; Joris Delanghe; Katrien Devreese; Jo Demeester; Kevin Braeckmans; Stefaan C. De Smedt; Koen Raemdonck (9120-9127).
Although the behavior of nanoscopic delivery systems in blood is an important parameter when contemplating their intravenous injection, this aspect is often poorly investigated when advancing from in vitro to in vivo experiments. In this paper, the behavior of siRNA loaded dextran nanogels in human plasma and blood is examined using fluorescence fluctuation spectroscopy, platelet aggregometry, flow cytometry and single particle tracking. Our results show that, in contrast to their negatively charged counterparts, positively charged siRNA loaded dextran nanogels cause platelet aggregation and show increased binding to human blood cells. Although PEGylating the nanogels did not have a significant effect on their interaction with blood cells, single particle tracking revealed that it is necessary to prevent their aggregation in human plasma. We therefore conclude that PEGylated negatively charged dextran nanogels are the most suited for further in vivo studies as they do not aggregate in human plasma and exhibit minimal interactions with blood cells.
Keywords: Blood compatibility; Haemocompatibility; Blood; Drug Delivery; Nanoparticle; Gene therapy;

mRNA-Lipoplex loaded microbubble contrast agents for ultrasound-assisted transfection of dendritic cells by Marie-Luce De Temmerman; Heleen Dewitte; Roosmarijn E. Vandenbroucke; Bart Lucas; Claude Libert; Jo Demeester; Stefaan C. De Smedt; Ine Lentacker; Joanna Rejman (9128-9135).
In cancer immunotherapy the immune system should be triggered to specifically recognize and eliminate tumor cells in the patient’s body. This could be achieved by loading dendritic cells (DCs) with tumor-associated antigens (TAAs). This can be achieved by transfecting DCs with messenger RNA encoding a tumor-associated antigen. Here we demonstrate transient transfection of dendritic cells by means of mRNA-lipoplexes bound to microbubbles. Microbubble-attached lipoplexes were introduced into the cells by applying ultrasound. Our data demonstrate that ultrasound-mediated delivery of mRNA-complexes led to efficient transfection of DCs. When mRNA encoding luciferase was used, maximal levels of the enzyme activity were detected 8 h after ultrasound application. Upon longer incubation protein expression gradually declined. This treatment did not affect viability of the cells. Intracellular localisation of mRNA-lipoplexes in DCs was determined by flow cytometry using fluorescently labeled lipoplexes. Over 50% of DCs contained fluorescently labeled mRNA-complexes. In the absence of additional maturation signals, transfection of immature DCs with mRNA-lipoplex loaded microbubbles and ultrasound application induced only a minor shift in the expression level of maturation markers (CD40 and CD86). However, in the presence of the activation stimulus (LPS), cells were able to further mature as shown by a significant up-regulation of CD40 expression. Thus, our results demonstrate that mRNA-lipoplex loaded microbubbles can serve as an applicable and safe tool for efficient mRNA-transfection of cultured DCs.
Keywords: mRNA; Microsphere; Gene therapy; Controlled drug release;

Platinum (IV)-coordinate polymers were synthesized by condensation polymerization using diamminedichlorodihydroxyplatinum (DHP) or its dicarboxyl derivative diamminedichlorodisuccinatoplatinum (DSP) as comonomers. Cyclic voltammogram study showed that Pt (IV) in the polymers was much easier reduced to Pt (II), particularly at the acidic pH, than that in the monomer DSP. Thus, these polymers were intracellular reduction-responsive backbone-type polymer conjugates that could be degraded and release Pt (II). These conjugates not only had high and fixed platinum contents (27.7% for P(DSP-EDA) and 29.6% for P(DSP-PA), respectively), but also showed increased cytotoxicity compared with corresponding Pt (IV) monomer DSP toward various tumor cell lines. In vivo, the conjugate showed a longer blood circulation time and better tumor accumulation.
Keywords: Platinum (IV)-conjugate; Platinum (IV) prodrug; Drug delivery; Reduction-responsive;

Lysosomally cleavable peptide-containing polymersomes modified with anti-EGFR antibody for systemic cancer chemotherapy by Jung S. Lee; Tom Groothuis; Claudia Cusan; Daniel Mink; Jan Feijen (9144-9153).
Polymersomes (Ps) based on a biodegradable and biocompatible block copolymer of methoxy poly(ethylene glycol) (mPEG) and poly(d,l-lactide) (PDLLA) in which apeptide sequence, Gly-Phe-Leu-Gly-Phe (GFLGF), was introduced in between the two blocks(mPEG-pep-PDLLA) were developed. The peptide linker is cleavable by the lysosomal enzymecathepsin B (Cath B). Ps containing the peptide linker (Ps(pep)) with an average diameter of about 124 nm were prepared by injecting a THF solution of the block copolymer into DI water. The Ps had a membrane thickness of about 15 nm as determined by transmission electron microscopy (TEM). In order to investigate the enzymatic degradation of the Ps (pep), dynamic light scattering (DLS) measurements of Ps(pep) dispersions with different concentrations of Cath B at pH 5.5 and 7.4 were performed as a function of time. A gradual decrease in kilo counts per second (Kcps) of the Ps (pep) over 7 d was observed after incubation of the Ps (pep) dispersions with 5 units/ml of Cath B at pH 5.5 at 37 °C. The size distribution became also bimodal, indicating that aggregation and precipitation of Ps (pep) occurred by disintegration of the Ps (pep) as a result of cleavage of the peptide. The rate of disintegration of the Ps (pep) was depending on the concentration of Cath Band the pH. No changes by DLS were seen when the dispersions were incubated with the enzyme at pH 7.4. Acridine orange (AO) was encapsulated in Ps (pep)as a model drug and rapid release of AO triggered by Cath B degradation of Ps (pep) was observed at pH 5.5. Anti-epidermal growth factor receptor (anti-EGFR) antibody (abEGFR) was immobilized on the surface of Ps(pep)in order to enhance the cellular uptake of Ps (pep). Fluorescein isothiocyanate labeled dextran (40,000 g/mol) (FD40) was incorporated in the Ps (pep) for the cell study and Ps either without peptide or antibody or without both peptide and antibody were used as negative controls. After 3 d exposure to SKBR3 cells, abEGFR-conjugated Ps (pep) (abEGFR-Ps (pep)) were directly bound to the membrane of the cells and were endocytosed more rapidly as compared to Ps (pep)without abEGFR. Intracellular release of FD40 from Ps (pep) was observed, suggesting that the peptide linker in Ps (pep) was cleaved in the lysosomal compartments of the cells leading to Ps (pep) membrane disruption. An Alexa Fluor® 488 labeled fragment of anti-mouse IgG (F(ab’)2A) was also coupled to Ps (pep). Specific binding of the Ps (pep) coupled IgG (F(ab′)2A) onto SKBR3 cells treated with primary mouse antibody was observed, whereas no binding was found with SKBR3 cells treated with goat antibody.
Keywords: Biodegradable polymersomes; Peptide conjugated block copolymer; Cathepsin B; Anti-epidermal growth factor receptor antibody; Chemotherapy;