Biomaterials (v.30, #8)

Biodegradable poly(ethylene glycol)–peptide hydrogels with well-defined structure and properties for cell delivery by Shao Qiong Liu; Pui Lai Rachel Ee; Chyan Ying Ke; James L. Hedrick; Yi Yan Yang (1453-1461).
In this study, biodegradable PEG–peptide hydrogels have been synthesized using Click chemistry. A series of Arg-Gly-Asp (RGD) containing peptides were prepared via a solid phase synthesis approach, which were further functionalized with azide to yield peptide azide or peptide diazide. A tetra-hydroxy terminated 4-arm PEG was functionalized with acetylene and was reacted with peptide azide/diazide and/or PEG diazide to produce hydrogels via a copper mediated 1,3-cycloaddition (Click chemistry) generating a triazole linkage as the networking forming reaction. The gelation time ranged from 2 to 30 min, depending on temperature, catalyst and precursor concentration, as well as peptide structure. The resulting hydrogels were characterized by swelling, viscoelastic properties and morphology as well as their ability for cell attachment and proliferation. Hydrogels cross-linked by peptide diazide yielded higher storage modulus (G′) with shorter spacers between azide groups. As expected, the swelling degree decreased while the G′ increased with increasing the concentration of the precursors as a result of increased cross-linking density. Primary human dermal fibroblasts were used as model cells to explore the possibility of using the RGD peptide hydrogels for cell-based wound healing. The attachment and proliferation of the cells on the hydrogels were evaluated. The RGD peptide hydrogels synthesized with a peptide concentration of 2.7–5.4 mm achieved significantly improved cell attachment and greater cell proliferation rate when compared to the hydrogels without RGD peptides. These hydrogels may provide a platform technology to deliver cells for tissue repair.
Keywords: Peptide hydrogel; Click chemistry; PEG; RGD; Cell attachment;

Phospholipid decoration of microcapsules containing perfluorooctyl bromide used as ultrasound contrast agents by Raquel Díaz-López; Nicolas Tsapis; Danielle Libong; Pierre Chaminade; Carole Connan; Mohamed M. Chehimi; Romain Berti; Nicolas Taulier; Wladimir Urbach; Valérie Nicolas; Elias Fattal (1462-1472).
We present here an easy method to modify the surface chemistry of polymeric microcapsules of perfluorooctyl bromide used as ultrasound contrast agents (UCAs). Capsules were obtained by a solvent emulsification–evaporation process with phospholipids incorporated in the organic phase before emulsification. Several phospholipids were reviewed: fluorescent, pegylated and biotinylated phospholipids. The influence of phospholipid concentration on microcapsule size and morphology was evaluated. Only a fraction of the phospholipids is associated to microcapsules, the rest being dissolved with the surfactant in the aqueous phase. Microscopy shows that phospholipids are present within the shell and that the core/shell structure is preserved up to 0.5 mg fluorescent phospholipids, up to about 0.25 mg pegylated phospholipids or biotinylated phospholipids (for 100 mg of polymer, poly(lactide-co-glycolide) (PLGA)). HPLC allows quantifying phospholipids associated to capsules: they correspond to 10% of pegylated phospholipids introduced in the organic phase. The presence of pegylated lipids at the surface of capsules was confirmed by X-ray photon electron spectroscopy (XPS). The pegylation did not modify the echographic signal arising from capsules. Finally biotinylated microcapsules incubated with neutravidin tend to aggregate, which confirms the presence of biotin at the surface. These results are encouraging and future work will consist of nanocapsule surface modification for molecular imaging.
Keywords: Microcapsules; Perfluorocarbons; Surface modification; Functionalization; Pegylated phospholipids; Biotinylated phospholipids;

The proportions of A and B carbonate ions in a selection of AB carbonate apatites, including hydroxyapatite (CHAP), chlorapatite (CCLAP) and fluorapatite (CFAP), have been obtained using the out-of-plane bend (ν2) bands of Fourier transform infrared (FTIR) spectra. Band area ratios (B/A) are in very good agreement with site occupancies from single-crystal X-ray structure refinement; the correlation is linear (1:1) for B/A values ranging up to three. Most compositions have ν2 spectra with one band for A carbonate (at 878–880 cm−1) and one for B (at 870–872 cm−1). Na-free AB CHAP has a third prominent band at 862 cm−1, which is assigned to the stuffed channel species (A2), and Na-bearing CFAP has a third band at 864 cm−1, which is assigned to a second B carbonate environment (B2). The A2 and B2 assignments are based largely on spectral changes in annealed samples.
Keywords: Apatite structure; Infrared spectrum; Hydroxyapatite; Fluorapatite; Biomineralisation; Bone;

Macrophage phenotype and remodeling outcomes in response to biologic scaffolds with and without a cellular component by Bryan N. Brown; Jolene E. Valentin; Ann M. Stewart-Akers; George P. McCabe; Stephen F. Badylak (1482-1491).
Recently, macrophages have been characterized as having an M1 or M2 phenotype based on receptor expression, cytokine and effector molecule production, and function. The effects of macrophage phenotype upon tissue remodeling following the implantation of a biomaterial are largely unknown. The objectives of this study were to determine the effects of a cellular component within an implanted extracellular matrix (ECM) scaffold upon macrophage phenotype, and to determine the relationship between macrophage phenotype and tissue remodeling. Partial-thickness defects in the abdominal wall musculature of Sprague–Dawley rats were repaired with autologous body wall tissue, acellular allogeneic rat body wall ECM, xenogeneic pig urinary bladder tissue, or acellular xenogeneic pig urinary bladder ECM. At 3, 7, 14, and 28 days the host tissue response was characterized using histologic, immunohistochemical, and RT-PCR methods. The acellular test articles were shown to elicit a predominantly M2 type response and resulted in constructive remodeling, while those containing a cellular component, even an autologous cellular component, elicited a predominantly M1 type response and resulted in deposition of dense connective tissue and/or scarring. We conclude that the presence of cellular material within an ECM scaffold modulates the phenotype of the macrophages participating in the host response following implantation, and that the phenotype of the macrophages participating in the host response appears to be related to tissue remodeling outcome.
Keywords: ECM (extracellular matrix); Biocompatibility; Macrophage; Monocyte; Wound healing;

The influence of Ni(II) on surface antigen expression in murine macrophages by Vincenzo D'Antò; Alexander Eckhardt; Karl-Anton Hiller; Gianrico Spagnuolo; Rosa Valletta; Luigi Ambrosio; Gottfried Schmalz; Helmut Schweikl (1492-1501).
Biomedical alloys may release nickel ions during corrosion phenomena and, in addition to their interaction with oral tissues, these ions may also influence characteristic properties of the immune system cells. The aim of this study was to evaluate the effect of nickel chloride on the expression of functionally distinct surface antigens in murine RAW macrophages. The expression of the surface antigens CD14, CD40, MHC class I, MHC class II, CD80, CD86, CD54 was analyzed by flow cytometry. The bacterial endotoxin lipopolysaccharide (LPS) was used as a positive control to induce antigen expression. Cells were stimulated with NiCl2 (0.1 and 0.5 mm) in the presence and absence of LPS (0.1 or 25 μg/ml). After exposure periods of 6, 24 and 48 h, LPS caused a time- and dose-dependent increase in the expression of all surface antigens. CD14 expression was up-regulated by 0.1 μg/ml LPS by about 10-fold after 24 h and 100-fold after 48 h. After 48 h, NiCl2 alone up-regulated the expression of all surface antigens between 2- and 4-fold, while in cells stimulated by LPS, 0.1 mm NiCl2 was effective only on CD14, CD40 and MHC class I. Moreover, 0.5 mm NiCl2 even inhibited the LPS-induced expression of all surface antigens, except for CD54, which was still significantly up-regulated. These results show that nickel chloride is able to induce an up-regulation of surface antigen expression, but a high concentration may impair essential functions of macrophages stimulated by LPS.
Keywords: Nickel; Biocompatibility; Macrophages; Surface antigens;

The behavior of endothelial cells on polyurethane nanocomposites and the associated signaling pathways by Huey-Shan Hung; Chia-Ching Wu; Shu Chien; Shan-hui Hsu (1502-1511).
A series of nanocomposites from polyurethane (PU) incorporated with various low concentrations (17.4–174 ppm) of gold nanoparticles (approximately 5 nm) (denoted “PU–Au”) were used as a model system to study the mechanisms that influenced endothelial cell (EC) migration on biomaterial surfaces. The migration rate of ECs on the PU–Au nanocomposites was determined by a real-time image system. It was found that ECs had the highest migration rate on the nanocomposite containing 43.5 ppm of gold (“PU–Au 43.5 ppm”). The high EC migration rate was associated with increased levels of endothelial nitric oxide synthase (eNOS) and phosphorylated-Akt (p-Akt) expressed by ECs cultured on PU–Au. The inductions of both eNOS and p-Akt on PU–Au were abolished by the addition of LY294002 (PI3K inhibitor), suggesting that these cellular events may be regulated through the PI3K signaling pathway. Using a biotinylated VEGF-165 that recognizes VEGF receptors and by FACS analysis, slightly higher expression of VEGF receptors for ECs on PU–Au was also demonstrated. Phalloidin staining showed that actin appeared as a circumferential band surrounding each cell on tissue culture polystyrene, whereas on PU–Au, especially on PU–Au 43.5 ppm, the cells had their margin spread out and extend processes with stress fibers in the protruding lamellipodia. Moreover, the higher EC migration rate on PU–Au 43.5 ppm was suppressed by LY294002. The higher protein expression of focal adhesion kinase (FAK) on PU–Au 43.5 ppm was observed in FAK-GFP transfected ECs. It was concluded that PU–Au nanocomposites activated FAK and the PI3K/Akt signaling pathway in ECs, leading to proliferation and migration of ECs on these surfaces.
Keywords: Migration; Polyurethane (PU); Endothelial cell (EC); Endothelial nitric oxide synthase (eNOS); Phosphoinositide 3-kinase (PI3K);

In vitro and in vivo evaluation of the surface bioactivity of a calcium phosphate coated magnesium alloy by Liping Xu; Feng Pan; Guoning Yu; Lei Yang; Erlin Zhang; Ke Yang (1512-1523).
Magnesium has shown potential application as a bio-absorbable biomaterial, such as for bone screws and plates. In order to improve the surface bioactivity, a calcium phosphate was coated on a magnesium alloy by a phosphating process (Ca–P coating). The surface characterization showed that a porous and netlike CaHPO4·2H2O layer with small amounts of Mg2+ and Zn2+ was formed on the surface of the Mg alloy. Cells L929 showed significantly good adherence and significantly high growth rate and proliferation characteristics on the Ca–P coated magnesium alloy (p  < 0.05) in in-vitro cell experiments, demonstrating that the surface cytocompatibility of magnesium was significantly improved by the Ca–P coating. In vivo implantations of the Ca–P coated and the naked alloy rods were carried out to investigate the bone response at the early stage. Both routine pathological examination and immunohistochemical analysis demonstrated that the Ca–P coating provided magnesium with a significantly good surface bioactivity (p  < 0.05) and promoted early bone growth at the implant/bone interface. It was suggested that the Ca–P coating might be an effective method to improve the surface bioactivity of magnesium alloy.
Keywords: Magnesium; Calcium phosphate coating; Bioactivity; Biocompatility; Bone; in vitro test;

Bacterial and mammalian cell response to poly(3-sulfopropyl methacrylate) brushes loaded with silver halide salts by Madeleine Ramstedt; Barbro Ekstrand-Hammarström; Andrey V. Shchukarev; Anders Bucht; Lars Österlund; Martin Welch; Wilhelm T.S. Huck (1524-1531).
This study investigates the antibacterial and cytotoxic effect of surfaces with sulphonate brushes containing silver salts. By using the same type of samples for both cytotoxicity and antibacterial studies, these two parameters could be compared in a controlled way. The silver was incorporated into the brush in four different forms to enable release of silver ions at different concentrations and different rates. It was found that although the surfaces displayed very good antibacterial properties in buffer solutions, this effect disappeared in systems with high protein content. Similarly, the silver-containing surfaces displayed cytotoxic effects in the absence of serum proteins but this effect was reduced in the presence of serum. The speciation of silver in the different solutions is discussed. Cytotoxic and antibacterial effects are compared at the different silver concentrations released. The implications of a concentration range where silver could be used to kill bacterial without harmful effects on mammalian cells are also discussed and questioned.
Keywords: Antibacterial; Cytotoxic; Surface modification; Metal ion release; Silver;

The effect of 3-hydroxybutyrate methyl ester on learning and memory in mice by Xiang-Hui Zou; Hong-Ming Li; Sheng Wang; Michael Leski; Yong-Chao Yao; Xiao-Di Yang; Qing-Jun Huang; Guo-Qiang Chen (1532-1541).
Learning and memory requires energy-demanding cellular processes and can be enhanced when the brain is supplemented with metabolic substrates. In this study, we found that neuroglial cell metabolic activity was significantly elevated when cultured in the presence of polyhydroxybutyrate (PHB) degradation product 3-hydroxybutyrate (3-HB) and derivatives. We demonstrated that the receptor for 3-HB, namely, protein upregulated in macrophages by IFN-γ (PUMA-G), was expressed in brain and upregulated in mice treated with 3-hydroxybutyrate methyl ester (3-HBME). We also affirmed increased expression of connexin 36 protein and phosphorylated ERK2 (extracellular signal-regulated kinase 2) in brain tissues following 3-HBME treatment, although these differences were not statistically significant. Mice treated with 3-HBME performed significantly (p  < 0.05) better in the Morris water maze than either the negative controls (no treatment) or positive controls (acetyl-l-carnitine treatment). Moreover, we found that 3-HBME enhanced gap junctional intercellular communication between neurons. Thus, 3-HB and derivatives enhance learning and memory, possibly through a signaling pathway requiring PUMA-G that increases protein synthesis and gap junctional intercellular communication.
Keywords: PHB; 3-Hydroxybutyrate methyl ester; Learning and memory; PUMA-G; Connexin; Gap junctional intercellular communication;

Mechanical properties of completely autologous human tissue engineered blood vessels compared to human saphenous vein and mammary artery by Gerhardt Konig; Todd N. McAllister; Nathalie Dusserre; Sergio A. Garrido; Corey Iyican; Alicia Marini; Alex Fiorillo; Hernan Avila; Wojciech Wystrychowski; Krzysztof Zagalski; Marcin Maruszewski; Alyce Linthurst Jones; Lech Cierpka; Luis M. de la Fuente; Nicolas L'Heureux (1542-1550).
We have previously reported the initial clinical feasibility with our small diameter tissue engineered blood vessel (TEBV). Here we present in vitro results of the mechanical properties of the TEBVs of the first 25 patients enrolled in an arterio-venous (A-V) shunt safety trial, and compare these properties with those of risk-matched human vein and artery. TEBV average burst pressures (3490 ± 892 mmHg, n  = 230) were higher than native saphenous vein (SV) (1599 ± 877 mmHg, n  = 7), and not significantly different from native internal mammary artery (IMA) (3196 ± 1264 mmHg, n  = 16). Suture retention strength for the TEBVs (152 ± 50 gmf) was also not significantly different than IMA (138 ± 50 gmf). Compliance for the TEBVs prior to implantation (3.4 ± 1.6%/100 mmHg) was lower than IMA (11.5 ± 3.9%/100 mmHg). By 6 months post-implant, the TEBV compliance (8.8 ± 4.2%/100 mmHg, n  = 5) had increased to values comparable to IMA, and showed no evidence of dilation or aneurysm formation. With clinical time points beyond 21 months as an A–V shunt without intervention, the mechanical tests and subsequent lot release criteria reported here would seem appropriate minimum standards for clinical use of tissue engineered vessels.
Keywords: Arterial tissue engineering; Autologous cell; ECM (extracellular matrix); Haemodialysis; Soft tissue biomechanics; Vascular grafts;

Collagen–phosphorylcholine interpenetrating network hydrogels as corneal substitutes by Wenguang Liu; Chao Deng; Christopher R. McLaughlin; Per Fagerholm; Neil S. Lagali; Belinda Heyne; Juan C. Scaiano; Mitchell A. Watsky; Yasuhiro Kato; Rejean Munger; Naoshi Shinozaki; Fengfu Li; May Griffith (1551-1559).
A biointeractive collagen–phospholipid corneal substitute was fabricated from interpenetrating polymeric networks comprising 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxysuccinimide crosslinked porcine atelocollagen, and poly(ethylene glycol) diacrylate crosslinked 2-methacryloyloxyethyl phosphorylcholine (MPC). The resulting hydrogels showed an overall increase in mechanical strength beyond that of either original component and enhanced stability against enzymatic digestion (by collagenase) or UV degradation. More strikingly, these hydrogels retained the full biointeractive, cell friendly properties of collagen in promoting corneal cell and nerve in-growth and regeneration (despite MPC's known anti-adhesive properties). Measurements of refractive indices, white light transmission and backscatter showed the optical properties of collagen–MPC are comparable or superior to those of the human cornea. In addition, the glucose and albumin permeability were comparable to those of human corneas. Twelve-month post-implantation results of collagen–MPC hydrogels into mini-pigs showed regeneration of corneal tissue (epithelium, stroma) as well as the tear film and sensory nerves. We also show that porcine collagen can be substituted with recombinant human collagen, resulting in a fully-synthetic implant that is free from the potential risks of disease transmission (e.g. prions) present in animal source materials.
Keywords: Recombinant collagen; Phospholipid; Cornea substitute; Tissue engineering; Biomedical regeneration;

Correlation of anisotropic cell behaviors with topographic aspect ratio by Adam S. Crouch; D. Miller; Kevin J. Luebke; W. Hu (1560-1567).
In this study, we have used nanoimprinting to create a range of micro- and nanoscale gratings, or their combination, in bulk polystyrene plates to investigate anisotropic cell behaviors of human dermal fibroblasts with respect to the aspect ratio (depth/width) of gratings. The depth and width of the polystyrene gratings both show strong effects individually on cell alignment and elongation that are qualitatively similar to the results of other studies. However, consistent quantitative comparison of these individual parameters with different studies is complicated by the diversity of combinations of width and depth that have been tested. Instead, the aspect ratio of the gratings as a unified description of grating topography is a more consistent parameter to interpret topographic dependence of cell morphology. Both cell alignment and elongation increase with increasing aspect ratio, and even a shallow grating (aspect ratio of ∼0.05) is sufficient to induce 80% cell alignment. Re-plotting data recently published by other groups vs. aspect ratio shows a similar dependence, despite differences in cell types and surface structures. This consistency indicates that aspect ratio is a general factor to characterize cell behaviors. The relationship of cell elongation and alignment with topographic aspect ratio is interpreted in terms of the theory of contact guidance. This model provides simplicity and flexibility in geometry design for devices and materials that interface with cells.
Keywords: Nanoimprint lithography; Topography; Aspect ratio; Human dermal fibroblast; Cell alignment; Cell elongation;

The in vivo degradation of a ruthenium labelled polysaccharide-based hydrogel for bone tissue engineering by Samia Laïb; Borhane H. Fellah; Ahmed Fatimi; Sophie Quillard; Claire Vinatier; Olivier Gauthier; Pascal Janvier; Marc Petit; Bruno Bujoli; Sylvain Bohic; Pierre Weiss (1568-1577).
In this paper we report a new method that permitted for the first time to selectively track a polysaccharide-based hydrogel on bone tissue explants, several weeks after its implantation. The hydrogel, which was developed for bone healing and tissue engineering, was labelled with a ruthenium complex and implanted into rabbit bone defects in order to investigate its in vivo degradation. 1, 2, 3 and 8 weeks after surgery, the bone explants were analyzed by synchrotron X-ray microfluorescence, infrared mapping spectroscopy, scanning electron microscopy, and optical microscopy after histological coloration. The results showed that the labelled polysaccharide-based hydrogel was likely to undergo phagocytosis that seemed to occur from the edge to the center of the implantation site up to at least the 8th week.
Keywords: Bone healing; Hydrogel; Degradation; In vivo test; Fluorescence; Siloxane;

The influence of laminin-derived peptides conjugated to Lys-capped PLLA on neonatal mouse cerebellum C17.2 stem cells by Liumin He; Susan Liao; Daping Quan; Michelle Ngiam; Casey K. Chan; S. Ramakrishna; Jiang Lu (1578-1586).
Chemical guiding cues are being exploited to stimulate neuron adhesion and neurite outgrowth. In this study, an amino-functioned PLLA, lysine-capped PLLA [K-(CH2) n -PLLA (n  = 2, 5, 8)], was synthesized with different length of linking spaces between lysine molecule and PLLA backbone. Drop-cast films were fabricated from K-(CH2) n -PLLA/PLLA blends (10/90, w/w) and amino groups were detected on the surfaces of the resultant films. More amine groups were detected on the surface and the hydrophilicity of the films was obviously improved by annealing the films in water. The representative atomic force microscopy (AFM) images indicated that incorporation of lysine-capped PLLA into PLLA matrix increased the roughness of the films and resulted in a phase separation with distinct two nano-domains which may correspond to the hydrophilic and hydrophobic domains. Furthermore, the laminin-derived peptides, CYIGSR (Cys-Tyr-Ile-Gly-Ser-Arg) and CSIKVAV (Cys-Ser-Ile-Lys-Val-Ala-Val), were jointly tethered to the amine groups of lysine-capped PLLA by a linking reagent sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC). The neonatal mouse cerebellum C17.2 stem cells were seeded on the peptides-grafted K-(CH2) n -PLLA/PLLA (n  = 2, 5, 8) films and pure PLLA films were used as controls. Improved viability and longer neurites were obtained on the peptide-grafted films than PLLA film over the cultivation period, especially for K-(CH2)5-PLLA/PLLA, which had the highest peptide density of 0.28 ± 0.03 μg/cm2. This study highlights the potential of using the lysine-cappeded PLLA with laminin-derived peptides for promoting nerve regeneration.
Keywords: Lysine-capped PLLA; Phase separation; Laminin-derived peptides; Cell adhesion; Neurite outgrowth; Tissue engineering;

Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication by Wonhye Lee; Jason Cushing Debasitis; Vivian Kim Lee; Jong-Hwan Lee; Krisztina Fischer; Karl Edminster; Je-Kyun Park; Seung-Schik Yoo (1587-1595).
We present a method to create multi-layered engineered tissue composites consisting of human skin fibroblasts and keratinocytes which mimic skin layers. Three-dimensional (3D) freeform fabrication (FF) technique, based on direct cell dispensing, was implemented using a robotic platform that prints collagen hydrogel precursor, fibroblasts and keratinocytes. A printed layer of cell-containing collagen was crosslinked by coating the layer with nebulized aqueous sodium bicarbonate. The process was repeated in layer-by-layer fashion on a planar tissue culture dish, resulting in two distinct cell layers of inner fibroblasts and outer keratinocytes. In order to demonstrate the ability to print and culture multi-layered cell–hydrogel composites on a non-planar surface for potential applications including skin wound repair, the technique was tested on a poly(dimethylsiloxane) (PDMS) mold with 3D surface contours as a target substrate. Highly viable proliferation of each cell layer was observed on both planar and non-planar surfaces. Our results suggest that organotypic skin tissue culture is feasible using on-demand cell printing technique with future potential application in creating skin grafts tailored for wound shape or artificial tissue assay for disease modeling and drug testing.
Keywords: Tissue engineering; 3D freeform fabrication; Skin tissue regeneration; Fibroblasts; Keratinocytes; Collagen hydrogel;

Mixed peptide–chitosan membranes to mimic the biological activities of a multifunctional laminin α1 chain LG4 module by Kentaro Hozumi; Natsumi Yamagata; Dai Otagiri; Chikara Fujimori; Yamato Kikkawa; Yuichi Kadoya; Motoyoshi Nomizu (1596-1603).
Laminin α1 chain LG4 module is multifunctional and interacts with syndecans and integrin α2β1 via AG73 (RKRLQVQLSIRT) and EF-I (DYATLQLQEGRLHFMFDLG) sites, respectively. Here, we conjugated the AG73 and EF1zz (ATLQLQEGRLHFXFDLGKGR, X: Nle) peptides on a chitosan membrane in various ratios to develop an LG4 mimic biomaterial. The AG73–chitosan membrane promoted strong cell attachment with membrane ruffling and the EF1zz–chitosan membrane promoted integrin-mediated cell adhesion with well-organized actin stress fibers. When AG73 and EF1zz were conjugated on a chitosan membrane with 1:9 molar ratio, the mixed peptide–chitosan membrane promoted the strong cell attachment and neurite outgrowth similar to that on the recombinant LG4 protein. Well-organized actin stress fibers and vinculin accumulated focal contacts were observed in the cells attached on the AG73:EF1zz (molar ratio = 1:9)–chitosan membrane. These results suggest that the mixed peptide–chitosan membrane interacts with both syndecans and integrin α2β1 and mimics the cell adhesion of a multifunctional LG4 protein. The mixed peptide–chitosan approach has potential as a multifunctional biomaterial for cell and tissue engineering.
Keywords: Cell adhesion; Chitin/chitosan; ECM (extracellular matrix); Integrin; Laminin; Peptide;

Preferential cell attachment to nitrogen-doped diamond-like carbon (DLC:N) for the measurement of quantal exocytosis by Atanu Sen; Syed Barizuddin; Maruf Hossain; Luis Polo-Parada; Kevin D. Gillis; Shubhra Gangopadhyay (1604-1612).
Electrochemical measurement of transmitter or hormone release from individual cells on microchips has applications both in basic science and drug screening. High-resolution measurement of quantal exocytosis requires the working electrode to be small (cell-sized) and located in immediate proximity to the cell. We examined the ability of candidate electrode materials to promote the attachment of two hormone-secreting cell types as a mechanism for targeting cells for to recording electrodes with high precision. We found that nitrogen-doped diamond-like carbon (DLC:N) promoted cell attachment relative to other materials tested in the rank order of DLC:N > In2O3/SnO2 (ITO), Pt > Au. In addition, we found that treating candidate electrode materials with polylysine did not increase attachment of chromaffin cells to DLC:N, but promoted cell attachment to the other tested materials. We found that hormone-secreting cells did not attach readily to Teflon AF as a potential insulating material, and demonstrated that patterning of Teflon AF leads to selective cell targeting to DLC:N “docking sites”. These results will guide the design of the next generation of biochips for automated and high-throughput measurement of quantal exocytosis.
Keywords: INS-1 cells; Cell adhesion; Contact angle; Diamond-like carbon; BioMEMS; Chromaffin cells;

Polymeric drugs based on bioactive glycosides for the treatment of brain tumours by M.L. López Donaire; J. Parra-Cáceres; B. Vázquez-Lasa; I. García-Álvarez; A. Fernández-Mayoralas; A. López-Bravo; J. San Román (1613-1626).
Polymeric drugs carrying glycolipids have been designed as target macromolecules for the treatment of brain tumours. A methacrylate derivative of oleyl 2-acetamido-2-deoxy-α-d-glucopyranoside (OAGMA) has been prepared and the corresponding glycopolymer obtained by free radical polymerisation. To modulate the hydrophobic character of the polymeric drug, the acrylic glycomonomer was copolymerised with vinyl pyrrolidone (VP). Reactivity ratios obtained by performing copolymerisation reactions inside the NMR apparatus were r OAGMA  = 5.94 and r VP  = 0.01, indicating the much higher reactivity of the glycomonomer. The hydrolytical release of oleyl 2-acetamido-2-deoxy-α-d-glucopyranoside (OAG) from the copolymeric drugs was produced in vitro by the ester enzymatic hydrolysis using enzyme/buffered solutions. The cytotoxicity of OAG and OAGMA tested against a human glioblastoma line and normal fibroblasts revealed a concentration dependent selectivity towards tumour cells versus fibroblasts. The antimitotic activity of the copolymeric drugs was also confirmed. The addition of the eluates of the copolymeric systems collected at 1 and 2 days produced a significant decrease in cellular viability of the glioblastoma cells without affecting that of normal fibroblasts. On the contrary, fibroblasts were able to adhere and proliferate onto the copolymeric systems showing normal morphology and revealing a good biocompatibility of the copolymeric drugs against healthy cells.
Keywords: Cell viability; Brain; Controlled drug release; Copolymers;

PLGA–lecithin–PEG core–shell nanoparticles for controlled drug delivery by Juliana M. Chan; Liangfang Zhang; Kai P. Yuet; Grace Liao; June-Wha Rhee; Robert Langer; Omid C. Farokhzad (1627-1634).
Current approaches to encapsulate and deliver therapeutic compounds have focused on developing liposomal and biodegradable polymeric nanoparticles (NPs), resulting in clinically approved therapeutics such as Doxil/Caelyx and Genexol-PM, respectively. Our group recently reported the development of biodegradable core–shell NP systems that combined the beneficial properties of liposomal and polymeric NPs for controlled drug delivery. Herein we report the parameters that alter the biological and physicochemical characteristics, stability, drug release properties and cytotoxicity of these core–shell NPs. We further define scalable processes for the formulation of these NPs in a reproducible manner. These core–shell NPs consist of (i) a poly(d,l-lactide-co-glycolide) hydrophobic core, (ii) a soybean lecithin monolayer, and (iii) a poly(ethylene glycol) shell, and were synthesized by a modified nanoprecipitation method combined with self-assembly. Preparation of the NPs showed that various formulation parameters such as the lipid/polymer mass ratio and lipid/lipid–PEG molar ratio controlled NP physical stability and size. We encapsulated a model chemotherapy drug, docetaxel, in the NPs and showed that the amount of lipid coverage affected its drug release kinetics. Next, we demonstrated a potentially scalable process for the formulation, purification, and storage of NPs. Finally, we tested the cytotoxicity using MTT assays on two model human cell lines, HeLa and HepG2, and demonstrated the biocompatibility of these particles in vitro. Our data suggest that the PLGA–lecithin–PEG core–shell NPs may be a useful new controlled release drug delivery system.
Keywords: Controlled drug release; Drug delivery; Liposome; Nanoparticle; Polyethylene oxide; Self-assembly;

Akt1 silencing efficiencies in lung cancer cells by sh/si/ssiRNA transfection using a reductable polyspermine carrier by Dhananjay Jere; Ji-Eun Kim; Rohidas Arote; Hu-Lin Jiang; You-Kyoung Kim; Yun Jaie Choi; Cheol-Heui Yun; Myung-Haing Cho; Chong-Su Cho (1635-1647).
Efforts directed in ameliorating silencing studies with shRNA, siRNA and ssiRNA (siRNA with sticky overhangs) are faltered mainly due to the lack of efficient carrier system. In the present study, we developed reductable polyspermine (RPS) carrier composed of multiple spermine units with disulfide linkages for gene expression and silencing studies. In gene expression studies, EGFP expression was found to be almost 4 folds higher and 20 folds safer with RPS carrier than with PEI25K. Moreover, on systemic administration, RPS exhibited significantly high EGFP expression in mice lungs. Similarly in gene silencing studies, EGFP silencing achieved was nearly 1.5 times superior with RPS carrier than PEI25K. Also, RPS delivered Akt1 shRNA (shAkt), siRNA (siAkt) and ssiRNA (ssiAkt) efficiently silenced oncoprotein Akt1 and thereby decreased A549 cell survival. The degrees of cell survival, proliferation and metastasis were differed with the nature of siRNA treatment. Further study at different time intervals revealed that ssiAkt treatment, although superior to sh/siAkt, was highly transient while, shAkt treatment was uniform and prolong. These finding demonstrate the potential use of RPS carrier in gene expression and silencing studies, and significance of the nature of siRNA employed in cancer study.
Keywords: Spermine; siRNA delivery; Reductable carrier; Gene delivery; Akt; siRNA comparison;

The role of the silicatein-α interactor silintaphin-1 in biomimetic biomineralization by Matthias Wiens; Melanie Bausen; Filipe Natalio; Thorben Link; Ute Schlossmacher; Werner E.G. Müller (1648-1656).
Biosilicification in sponges is initiated by formation of proteinaceous filaments, predominantly consisting of silicateins. Silicateins enzymatically catalyze condensation of silica nanospheres, resulting in symmetric skeletal elements (spicules). In order to create tailored biosilica structures in biomimetic approaches it is mandatory to elucidate proteins that are fundamental for the assembly of filaments. Silintaphin-1 is a core component of modularized filaments and also part of a spicule-enfolding layer. It bears no resemblance to other proteins, except for the presence of an interaction domain that is fundamental for its function as scaffold/template. In the presence of silicatein silintaphin-1 facilitates the formation of in vitro filaments. Also, it directs the assembly of γ-Fe2O3 nanoparticles and surface-immobilized silicatein to rod-like biocomposites, synthetic spicules. Thus, silintaphin-1 will contribute to biomimetic approaches that pursue a controlled formation of patterned biosilica-based materials. Its combination with γ-Fe2O3 nanoparticles and immobilized silicatein will furthermore inspire future biomedical applications and clinical diagnostics.
Keywords: Biosilicification; Silicatein interactor; Silintaphin-1; Sponges; γ-Fe2O3 nanoparticles;

A unified model has been developed to predict release not only from bulk eroding and surface eroding systems but also from matrices that transition from surface eroding to bulk eroding behavior during the course of degradation. This broad applicability is afforded by fundamental diffusion/reaction equations that can describe a wide variety of scenarios including hydration of and mass loss from a hydrolysable polymer matrix. Together, these equations naturally account for spatial distributions of polymer degradation rate. In this model paradigm, the theoretical minimal size required for a matrix to exhibit degradation under surface eroding conditions was calculated for various polymer types and then verified by empirical data from the literature. An additional set of equations accounts for dissolution- and/or degradation-based release, which are dependent upon hydration of the matrix and erosion of the polymer. To test the model's accuracy, predictions for agent egress were compared to experimental data from polyanhydride and polyorthoester implants that were postulated to undergo either dissolution-limited or degradation-controlled release. Because these predictions are calculated solely from readily attainable design parameters, it seems likely that this model could be used to guide the design controlled release formulations that produce a broad array of custom release profiles.
Keywords: Polyanhydride; Polyorthoester; Biodegradation; Controlled drug release; Modeling;