Biomaterials (v.26, #26)

Calendar (I).

Immobilization of hemoglobin at the galleries of layered niobate HCa2Nb3O10 by Lu Gao; Qiuming Gao; Qigang Wang; Shuge Peng; Jianlin Shi (5267-5275).
Hemoglobin (Hb) was intercalated at the galleries of layered niobate HCa2Nb3O10 (HCNO). Two different kinds of layered phases of Hb-CNO composites Hb-CNO-1 and Hb-CNO-2 were obtained with the interlayer distances of 7.2 and 10.3 nm in correspondence with the monolayer and bilayer arrangements of proteins between the niobate layers, respectively, based on the powder XRD pattern, HRTEM, UV-vis spectra and CHN analyses. FTIR spectra of Hb-CNO composites show that amide I and amide II bands were actually the same as those of the native Hb, which indicates that there is almost no structural change after immobilization. Michaelis–Menten model methods were used to study the peroxidatic activity of the reaction of 2-methoxyphenol and H2O2 for the entrapped Hb in the galleries of HCNO. Compared to that of free Hb, the kinetic parameters of Hb-CNO k cat , K M and k cat / K M were affected by the immobilization process. The immobilized Hb showed a higher relative activity than that of free Hb after incubated in phosphate buffer (pH=7) at 80 °C for a period of time. The environments between the layers of HCNO are hydrophilic which will bind water tightly and help to stabilize the ‘essential water’ layer around the protein. So, immobilization of Hb between the layers of HCNO enhanced the activity of Hb in water-DMSO mixture.
Keywords: Enzyme; Composite; Bioactivity;

Preparation and characterization of porous β-tricalcium phosphate/collagen composites with an integrated structure by Chao Zou; Wenjian Weng; Xuliang Deng; Kui Cheng; Xinggang Liu; Piyi Du; Ge Shen; Gaorong Han (5276-5284).
Porous β -tricalcium phosphate (TCP)/collagen composites with different β -TCP/collagen weight ratio were prepared. The influences of the preparation conditions on the microstructure of porous composite and the joint status of β -TCP particles with collagen fibrils were characterized by X-ray diffractometer, scanning electron microscopy and transmission electron microscopy. The results showed: (1) an acid treatment could effectively disassemble collagen fibrils; (2) in the resulting porous composites, β -TCP particles homogenously existed on the skeleton of the collagen fibril network and bonded tightly to both the fibrils and themselves. The tight bonding formation could be due to the reaction between Ca ions in the particles and carboxyl groups in collagen polypeptide chains and due to the reprecipitation of partially dissolved β -TCP during synthesis. The tight bonding between β -TCP particles and collagen fibrils in the composites demonstrated an integrated structure, which was reproducible when β -TCP/collagen ratio ranged from 2 to 4. Such integrated structure would make significant contributions in reliably tailoring properties of the porous composites by varying β -TCP content. In addition, the porous composites had large porosity (∼95%) and appropriate pore size (∼100 μm), showed no negative impact in cytotoxicity assay and complete bone tissue regeneration after 12 weeks in animal test.
Keywords: β-tricalcium phosphate; Collagen; Porous composite; Preparation; Microstructure; Scaffold;

Platelet interactions with calcium-phosphate-coated surfaces by Lena Kikuchi; Jun Y. Park; Charles Victor; John E. Davies (5285-5295).
Many studies have shown that calcium-phosphate (CaP)-coated endosseous implants exhibit more peri-implant bone formation and bone contact at early healing times than uncoated implants. Since the rate of healing is influenced by blood/implant interactions and possibly the degree of blood platelet activation, the aim of this study was to determine whether the topography, microtopography, or the presence of calcium (Ca) and phosphate (PO4) ions in the implant surface plays a predominant role in platelet activation. We define the threshold between topography and microtopography as the limit of the scale range of platelets themselves; thus, a microtopographic surface is defined by one which exhibits features ⩽3 μm. With the help of four international collaborating laboratories, we prepared 11 titanium and CaP-modified titanium surfaces each with different (micro)topographies and interrogated these surfaces with both platelet adhesion (lactate dehydrogenase activity) and platelet activation (microparticle formation and P-selectin expression) assays. Our results show that: calcium (Ca)- and phosphate (PO4)-containing surfaces of increasing surface microtopographical complexity exhibit increasing platelet activation; surfaces with similar surface microtopographies show similar levels of platelet activation regardless of the presence of Ca and PO4 in the surface; and that surface microtopography is responsible for platelet activation rather than the presence of Ca and PO4 in the surface.
Keywords: Calcium phosphate; Coating; Surface topography; Microtopography; Platelet activation; LDH assay; P-selectin; Platelet microparticles; Flow cytometry; Bone wound healing;

The quantification of cellular viability and inflammatory response to stainless steel alloys by LeeAnn O. Bailey; Sherry Lippiatt; Frank S. Biancanello; Stephen D. Ridder; Newell R. Washburn (5296-5302).
The biocompatibility of metallic alloys is critical to the success of many orthopedic therapies. Corrosion resistance and the immune response of the body to wear debris products ultimately determine the performance of these devices. The establishment of quantitative tests of biocompatibility is an important issue for biomaterials development. We have developed an in vitro model to measure the pro-inflammatory cytokine production and in this study investigated the cellular responses induced by nitrogenated and 316L stainless steel alloys in both particulate and solid form. We utilized a murine macrophage cell line, RAW 264.7, to characterize and compare the mRNA profiles of TNF- α and IL-1 β in these cells using real time-polymerase chain reaction (RT-PCR). Fluorescence microscopy and flow cytometry were used to probe the viability of the population and to examine the apoptotic pathway. The goals of this work were to develop improved measurement methods for the quantification of cellular inflammatory responses to biomaterials and to obtain data that leads to an enhanced understanding of the ways in which the body responds to biomaterials. Using these techniques, we observed evidence for an association between the upregulation of IL-1 β and reversible apoptosis, and the upregulation of TNF- α and irreversible apoptosis.
Keywords: Real-time PCR; Biocompatibility; 316L stainless steel; Cytokines;

In vitro bioactivity of MOEP grafted ePTFE membranes for craniofacial applications by Shuko Suzuki; Lisbeth Grøndahl; David Leavesley; Edeline Wentrup-Byrne (5303-5312).
The bioactivity of three methacryloyloxyethyl phosphate (MOEP) grafted expanded polytetrafluoroethylene (ePTFE) membranes with varying surface coverage as well as unmodified ePTFE was investigated through a series of in vitro tests: calcium phosphate (CaP) growth in simulated body fluid (SBF), serum protein adsorption, and a morphology and attachment study of human osteoblast-like SaOS-2 cells. The graft copolymers were prepared by means of gamma irradiation induced grafting and displayed various surface morphologies and wettabilities depending on the grafting conditions used. Unmodified ePTFE did not induce nucleation of CaP minerals, whereas all the grafted membranes revealed the growth of CaP minerals after 7 days immersion in SBF. The sample with lowest surface grafting yield (24% coverage), a smooth graft morphology and relatively high hydrophobicity ( θ adv = 120 ° , θ rec = 80 ° ) showed carbonated hydroxyapatite growth covering the surface. On the other hand, the samples with high surface grafting yield (76% and 100%), a globular graft morphology and hydrophilic surfaces ( θ adv = 60 ° and 80°, θ rec = 25 ° and 15°, respectively) exhibited irregular growth of non-apatitic CaP minerals. Irreversibly adsorbed protein measured after a 1 h immersion in serum solution was quantified by the amount of nitrogen on the surface using XPS, as well as by weight increase. All grafted membranes adsorbed 3–6 times more protein than the unmodified membrane. The sample with the highest surface coverage adsorbed the most protein. Osteoblast-like SaOS-2 cells cultured for 3 h revealed significantly higher levels of cell attachment on all grafted membranes compared to unmodified ePTFE. Although the morphology of the cells was heterogeneous, in general, the higher grafted surfaces showed a much better cell morphology than both the low surface-grafted and the control unmodified sample.The suite of in vitro tests confirms that a judicious choice of grafted monomer such as the phosphate-containing methacrylate monomer (MOEP) significantly improves the bioactivity of ePTFE in vitro.
Keywords: Surface modification; Simulated body fluid; Protein adsorption; Osteoblast SaOS-2 cell attachment; FTIR spectroscopy;

The inflammatory potential of biphasic calcium phosphate granules in osteoblast/macrophage co-culture by Judith M. Curran; James A. Gallagher; John Alan Hunt (5313-5320).
Some biological properties of a range of biphasic calcium phosphate (BCP) granules were quantified using a co-culture model of primary unstimulated human osteoblasts and macrophages. The BCP granules were classified in terms of diameter and the ratio of tricalcium phosphate (TCP): hydroxyapatite (HA). All granules were 50% porous and sterilised by γ -irradiation. Primary unstimulated human osteoblasts and macrophages were cultured in contact with the range of BCP granules for 1, 7 and 14 days. The response of the cells was determined and quantified by the assessment of viable cell adhesion to the substrate, using lactate dehydrogenase assay, and the production and release of the cytokines; interleukin 1 β , (IL-1 β ), tumour necrosis factor α (TNF- α ) and prostaglandin E2 (PGE2). Throughout the test period viable cell adhesion on all BCP granules was significantly lower than the tissue culture polystyrene control. Higher content TCP materials, (80% and 100% TCP) did not support viable cell adhesion after 1 day, lower content TCP materials, (20% and 50% TCP) granules did support viable cell adhesion throughout the time period. The percentage content of TCP was a more significant factor than granule size within the test conditions at all time points.
Keywords: Co-culture; Macrophages; Osteoblasts; Tricalcium phosphate granules; Cytokines;

Previous investigations have found that visible-light (VL)-irradiated camphorquinone (CQ), in the presence of a tertiary amine (e.g., N,N-dimethyl-p-toluidine, DMT), generates reactive oxygen species and causes oxidative DNA damage in vitro. In this study, oxidative DNA damage produced by VL-irradiated CQ/DMT, in the presence and absence of antioxidants (glutathione, N-acetyl-l-cysteine (NAC), mannitol, vitamin C, and vitamin E), was measured by the conversion of ΦX-174 RF I supercoiled (SC) double-stranded plasmid DNA into open and linear forms. VL-irradiated CQ/DMT, lacking antioxidant, damaged 99.4±1% of the ΦX-174 RF I SC double-stranded plasmid DNA. Our results revealed that glutathione (10.0, 5.0, 2.5, 1.0, and 0.5 mm) and NAC (10.0, 5.0, and 2.5 mm) significantly ( p < 0.02 ) reduced oxidative DNA damage produced by VL-irradiated CQ/DMT. Vitamin E, vitamin C, and mannitol were ineffective at reducing oxidative DNA damage produced by VL-irradiated CQ/DMT. Furthermore, vitamin E (10.0 and 5.0 mm) and vitamin C (10.0, 5.0, 2.5, 1.0, 0.5 mm) treatment significantly ( p < 0.02 ) enhanced VL-irradiated CQ/DMT-induced oxidative DNA damage and caused significant ( p < 0.001 ) DNA damage following VL-irradiation in the absence of CQ/DMT. As a result, future studies should evaluate whether glutathione and NAC effectively reduce or prevent oxidative damage induced by VL-irradiated CQ/DMT in vivo.
Keywords: Camphorquinone; Reactive oxygen species; Oxidative DNA damage; Antioxidants;

Electrospun fine-textured scaffolds for heart tissue constructs by Xinhua Zong; Harold Bien; Chiung-Yin Chung; Lihong Yin; Dufei Fang; Benjamin S. Hsiao; Benjamin Chu; Emilia Entcheva (5330-5338).
The structural and functional effects of fine-textured matrices with sub-micron features on the growth of cardiac myocytes were examined. Electrospinning was used to fabricate biodegradable non-woven poly(lactide)- and poly(glycolide)-based (PLGA) scaffolds for cardiac tissue engineering applications. Post-processing was applied to achieve macro-scale fiber orientation (anisotropy). In vitro studies confirmed a dose–response effect of the poly(glycolide) concentration on the degradation rate and the pH value changes. Different formulations were examined to assess scaffold effects on cell attachment, structure and function. Primary cardiomyocytes (CMs) were cultured on the electrospun scaffolds to form tissue-like constructs. Scanning electron microscopy (SEM) revealed that the fine fiber architecture of the non-woven matrix allowed the cardiomyocytes to make extensive use of provided external cues for isotropic or anisotropic growth, and to some extent to crawl inside and pull on fibers. Structural analysis by confocal microscopy indicated that cardiomyocytes had a preference for relatively hydrophobic surfaces. CMs on electrospun poly(l-lactide) (PLLA) scaffolds developed mature contractile machinery (sarcomeres). Functionality (excitability) of the engineered constructs was confirmed by optical imaging of electrical activity using voltage-sensitive dyes. We conclude that engineered cardiac tissue structure and function can be modulated by the chemistry and geometry of the provided nano- and micro-textured surfaces. Electrospinning is a versatile manufacturing technique for design of biomaterials with potentially reorganizable architecture for cell and tissue growth.
Keywords: Electrospinning; Poly(glycolide) and poly(lactide)-based (PLGA) scaffold; Cardiomyocytes; Cardiac tissue engineering;

Chitosan–RGDSGGC conjugate as a scaffold material for musculoskeletal tissue engineering by Tatsuya Masuko; Norimasa Iwasaki; Shintaro Yamane; Tadanao Funakoshi; Tokifumi Majima; Akio Minami; Noriko Ohsuga; Takashi Ohta; Shin-Ichiro Nishimura (5339-5347).
In the present study, we have developed a novel and versatile method for the preparation of chitosan-peptide complex based on the selective reaction of chitosan with 2-iminothiolane. The new type of SH-chitosan derivative showed an excellent solubility to aqueous solution even in the alkaline conditions. This characteristic greatly facilitated further modification study of chitosan with a variety of bioactive substances. A synthetic peptide, RGDSGGC containing RGDS moiety that is known as one of the most important cell adhesive peptides, was readily coupled by disulfide bonds formation with sulfhydryl groups of SH-chitosan in the presence of dimethyl sulfoxide. Next, the effect of the introduction of RGDSGGC moiety to chitosan on cell adhesion and proliferation activity of chondrocytes and fibroblasts were evaluated. As a result, it was suggested that this polysaccharide-peptide conjugate exhibited excellent capacities for both cell adhesion and cell proliferation of chondrocytes and fibroblasts. Considering the growing importance of the biocompatible scaffolds in the recent tailored tissue engineering technique, these results indicate that the present strategy of 2-iminothiolane-based conjugation of polysaccharides with biologically active peptides will become a key and potential technology to develop desirable scaffold materials for the tissue regenerations.
Keywords: Adhesion; Chondrocyte; Fibroblast; Peptide; Polysaccharide;

The influence of GFP-actin expression on the adhesion dynamics of HepG2 cells on a model extracellular matrix by Zhiqin Feng; Wei Ning Chen; Peter Vee Sin Lee; Kin Liao; Vincent Chan (5348-5358).
Integrins belong to a family of important cell surface receptors which mediate the adhesion of most anchorage-dependent cells to nature extracellular matrix (ECM) and biomaterials. It is known that the binding of integrin with ECM proteins triggers mechanochemical responses of cytoskeleton. To date, the intricate interplay between integrin-ECM interaction and cytoskeleton dynamics leading to the regulation of cell morphogenesis on biomaterials remains largely unknown. In this study, green fluorescence protein (GFP)-actins were expressed in HepG2 cells for the temporal visualization of cytoskeletal structure of adherent cells on naturally derived materials. By combining confocal reflectance contrast microscopy and fluorescence microscopy, the adhesion contact dynamics, cytoskeleton remodeling and two-dimensional spreading of intact and GFP-actin expressing HepG2 cells on collagen and fibronectin-coated substrates are simultaneously probed during the initial cell seeding. First of all, our results show that the evolution of adhesion contact of HepG2 cells upon integrin–collagen or integrin–fibronectin interaction is impaired by GFP-actin expression. Also, the initial rate of cell deformation is reduced by 70% and 43% on fibronectin and collagen, respectively, upon GFP-actin expression. Interestingly, the steady-state adhesion energy of HepG2 cells remains unchanged and increases on fibronectin- and collagen-coated substrate, respectively, upon GFP-actin expression. Our highly integrated biophysical approach demonstrates that GFP-actins diffusively concentrate in the cytoplasmic cortex during initial cell seeding while adhesion contact evolves and cell spreads. Kinetics analysis on the adhesion contact formation demonstrates the intricate interplay between cytoskeleton property and ECM proteins in cell adhesion.
Keywords: GFP-actin expression; Adhesion contact dynamics; Extracellular matrix;

Behaviour of human endothelial cells on surface modified NiTi alloy by Stuart D. Plant; David M. Grant; Lopa Leach (5359-5367).
Intravascular stents are being designed which utilise the shape memory properties of NiTi alloy. Despite the clinical advantages afforded by these stents their application has been limited by concerns about the large nickel ion content of the alloy. In this study, the surface chemistry of NiTi alloy was modified by mechanical polishing and oxidising heat treatments and subsequently characterised using X-ray photon spectroscopy (XPS). The effect of these surfaces on monolayer formation and barrier integrity of human umbilical vein endothelial cells (HUVEC) was then assessed by confocal imaging of the adherens junctional molecule VE-cadherin, perijunctional actin and permeability to 42 kDa dextrans. Dichlorofluoroscein assays were used to measure oxidative stress in the cells. XPS analysis of NiTi revealed its surface to be dominated by TiO2. However, where oxidation had occurred after mechanical polishing or post polishing heat treatments at 300 and 400 °C in air, a significant amount of metallic nickel or nickel oxide species (10.5 and 18.5 at%) remained on the surface. Exposure of HUVECs to these surfaces resulted in increased oxidative stress within the cells, loss of VE-cadherin and F-actin and significantly increased paracellular permeability. These pathological phenomena were not found in cells grown on NiTi which had undergone heat treatment at 600 °C. At this temperature thickening of the TiO2 layer had occurred due to diffusion of titanium ions from the bulk of the alloy, displacing nickel ions to sub-surface areas. This resulted in a significant reduction in nickel ions detectable on the sample surface (4.8 at%). This study proposes that the integrity of human endothelial monolayers on NiTi is dependent upon the surface chemistry of the alloy and that this can be manipulated, using simple oxidising heat treatments.
Keywords: Biocompatibility; Endothelial cell; Intravascular stent; Metal ion toxicity; Nickel titanium alloy; XPS;

Pharmacokinetic behaviour of ACP gel, an autocrosslinked hyaluronan derivative, after intraperitoneal administration by Davide Renier; Pierangelo Bellato; Davide Bellini; Alessandra Pavesio; Daniele Pressato; Anna Borrione (5368-5374).
Autocrosslinked polysaccharide (ACP) gel is a fully biocompatible cross-linked derivative of hyaluronic acid, which has prolonged in vivo residence time and improved mechanical properties with respect to native hyaluronan for use in various surgical applications. The objective of this study was to assess the pharmacokinetic behaviour of ACP gel in dogs after intraperitoneal administration. Seven beagle dogs received intraperitoneal injections of tritium-labelled ACP gel. Blood samples were taken, and urine and faeces were collected until sacrifice, scheduled at various time points from 3 to 192 h after administration. Organs were removed from the animals at autopsy. Bodily fluid and organ samples were analysed for total and non-volatile radioactivity.Non-volatile radioactivity slowly appeared in plasma, with a median T max of 12 h, and then declined with a mean half-life of 69 h. Total radioactivity in plasma peaked later and declined more slowly, consistent with the formation of tritiated water. Little non-volatile radioactivity was found in any organs except the liver, where about 16% of the dose was present 72 h after administration, and the intestines, where the presence of radioactivity was probably due to a retention effect. A minor amount of non-volatile radioactivity was also found in the bone marrow.In summary, ACP gel administered into the peritoneal cavity is removed slowly by active initial catabolism at the injection site, and is then catabolised by well described physiological pathway of hyaluronan degradation with final release of simple molecules such as CO2 and H2O. Given its in vivo residence time, ACP gel may be considered an ideal implantable surgical device.
Keywords: Hyaluronan; Autocrosslinked polysaccharide; Animal model; Blood; Plasma;

Biodegradable dextran-based microspheres for delivery of anticancer drug mitomycin C by Richard Y. Cheung; Yuming Ying; Andrew M. Rauth; Norman Marcon; Xiao Yu Wu (5375-5385).
The purpose of this work was to develop a biodegradable microsphere (MS) system for delivering mitomycin C (MMC). Various dextran-based MS systems were investigated for their loading and release characteristics, including nonionic MS, sulfopropyl dextran microspheres (SP-MS) with low or high cross-linking density, oxidized SP-MS (Ox-MS), and hydrophobically modified SP-MS. SP-MS were chemically modified by oxidation with sodium periodate or by reaction with anhydride. The chemical structure of modified SP-MS and MMC-loaded MS (MMC-MS) were examined using Fourier transform infrared (FTIR) and solid-state nuclear magnetic resonance (NMR) spectrophotometry. Drug release was conducted at 37 °C in aqueous solutions of 0.15  m phosphate buffer solution. The kinetics of drug absorption and release and the stability of MMC after loading and release were determined by spectrophotometry and high-performance liquid chromatography. Ionic SP-MS exhibited a higher drug-loading rate and capacity when compared to nonionic MS, while hydrophobically modified SP-MS showed an even greater loading capacity than SP-MS. These results suggest that both ionic complexation and hydrophobic interaction were important factors in MMC loading. The Ox-MS system demonstrated higher drug-loading capacity, more fractional drug release and a longer time to reach release equilibrium as compared to other investigated MS systems. Under optimized reaction and loading conditions, MMC released from Ox-MS was found to be unaltered. This work demonstrates that the Ox-MS system is a potentially useful system for the delivery of MMC.
Keywords: Biodegradable MS; Dextran derivatives; Drug loading and release; Kinetics and mechanism; Cytotoxicity;

Local perivascular delivery of anti-restenotic agents from a drug-eluting poly( ε -caprolactone) stent cuff by Nuno M.M. Pires; Barend L. van der Hoeven; Margreet R. de Vries; Louis M. Havekes; Bart J. van Vlijmen; Wim E. Hennink; Paul H.A. Quax; J. Wouter Jukema (5386-5394).
The introduction of drug-eluting stents (DES) to prevent in-stent restenosis is one of the major advances in interventional cardiology. Currently many types of DES are under evaluation for effectiveness and safety, a time-consuming and difficult procedure in humans. An animal model that allows rapid evaluation of the present and upcoming therapeutic approaches to prevent in-stent restenosis is most valuable and still lacking.Here, a perivascular cuff to induce restenosis was constructed of a poly( ε -caprolactone) (PCL) formulation suitable for the controlled delivery of drugs. Placing the PCL cuff around the femoral artery, in vivo, resulted in reproducible restenosis-like lesions containing predominantly smooth muscle-actin positive cells. Loading the cuff with the anti-restenotic compounds paclitaxel and rapamycin resulted, in vitro, in a sustained and dose-dependent release for at least 3 weeks. Paclitaxel- and rapamycin-eluting PCL cuffs placed around the femoral artery of mice in vivo significantly reduced intimal thickening by 76±2% and 75±6%, respectively, at 21 days. Perivascular sustained release of both anti-restenotic agents is restricted to the cuffed vessel segment with no systemic adverse effects or effect on cuffed contralateral femoral arteries.Drug-eluting PCL cuffs provide an easy and rapid tool to evaluate anti-restenotic agents to be used in combination with the DES strategies.
Keywords: Controlled drug release; Local delivery; Drug-eluting stents; Poly( ε -caprolactone); Restenosis; Animal model;

Fabrication of a cell array on ultrathin hydrophilic polymer gels utilising electron beam irradiation and UV excimer laser ablation by Shintaroh Iwanaga; Yoshikatsu Akiyama; Akihiko Kikuchi; Masayuki Yamato; Kiyotaka Sakai; Teruo Okano (5395-5404).
Most of the surface patterning methods currently applied are based on lithography techniques and microfabrication onto silicon or glass substrates. Here we report a novel method to prepare patterned surfaces on polystyrene substrates by grafting ultrathin cell-repellent polymer layers utilising both electron beam (EB) polymerisation and local laser ablation techniques for microfabrication. Polyacrylamide was grafted onto tissue culture polystyrene (TCPS) dishes using EB irradiation. Water contact angles for these PAAm-grafted TCPS surfaces were less than 10° ( cos θ = 0.99 ) with PAAm grafted amounts of 1.6 μg/cm2 as determined by ATR/FT-IR. UV excimer laser (ArF: 193 nm) ablation resulted in the successful fabrication of micropatterned surfaces composed of hydrophilic PAAm and hydrophobic basal polystyrene layers. Bovine carotid artery endothelial cells adhered only to the ablated domains after pretreatment of the patterned surfaces with 15 μg/mL fibronectin at 37 °C. The ablated domain sizes significantly influenced the number of cells occupying each domain. Cell patterning functionality of the patterned surfaces was maintained for more than 2 months without loss of pattern fidelity, indicating that more durable cell arrays can be obtained compared to those prepared by self-assembled monolayers of alkanethiols, as described in previous reports. The surface fabrication techniques presented here can be utilised for the preparation of cell-based biosensors as well as tissue engineering constructs.
Keywords: Cell patterning; Cell array; Non-fouling surface; Polyacrylamide; Laser ablation; Fibronectin;

Nanopattern-induced changes in morphology and motility of smooth muscle cells by Evelyn K.F. Yim; Ron M. Reano; Stella W. Pang; Albert F. Yee; Christopher S. Chen; Kam W. Leong (5405-5413).
Cells are known to be surrounded by nanoscale topography in their natural extracellular environment. The cell behavior, including morphology, proliferation, and motility of bovine pulmonary artery smooth muscle cells (SMC) were studied on poly(methyl methacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) surfaces comprising nanopatterned gratings with 350 nm linewidth, 700 nm pitch, and 350 nm depth. More than 90% of the cells aligned to the gratings, and were significantly elongated compared to the SMC cultured on non-patterned surfaces. The nuclei were also elongated and aligned. Proliferation of the cells was significantly reduced on the nanopatterned surfaces. The polarization of microtubule organizing centers (MTOC), which are associated with cell migration, of SMC cultured on nanopatterned surfaces showed a preference towards the axis of cell alignment in an in vitro wound healing assay. In contrast, the MTOC of SMC on non-patterned surfaces preferentially polarized towards the wound edge. It is proposed that this nanoimprinting technology will provide a valuable platform for studies in cell-substrate interactions and for development of medical devices with nanoscale features.
Keywords: Nanotopography; Smooth muscle cells; Bionanotechnology; Nanoimprinting; Nanomedicine; Cell-substrate interaction;

Size-controlled hydroxyapatite nanoparticles as self-organized organic–inorganic composite materials by Viorel Marin Rusu; Chuen-How Ng; Max Wilke; Brigitte Tiersch; Peter Fratzl; Martin G. Peter (5414-5426).
This paper presents some results concerning the size-controlled hydroxyapatite nanoparticles obtained in aqueous media in a biopolymer matrix from soluble precursors salts. Taking the inspiration from nature, where composite materials made of a polymer matrix and inorganic fillers are often found, e.g. bone, shell of crustaceans, shell of eggs, etc., the feasibility on making composite materials containing chitosan and nanosized hydroxyapatite was investigated. A stepwise co-precipitation approach was used to obtain different types of composites by means of different ratio between components. The synthesis of hydroxyapatite was carried out in the chitosan matrix from calcium chloride and sodium dihydrogenphosphate in alkaline solutions at moderate pH of 10–11 for 24 h. Our research is focused on studying and understanding the structure of this class of composites, aiming at the development of novel materials, controlled at the nanolevel scale. The X-ray diffraction technique was employed in order to study the kinetic of hydroxyapatite formation in the chitosan matrix as well as to determine the HAp crystallite sizes in the composite samples. The hydroxyapatite synthesized using this route was found to be nano-sized (15–50 nm). Moreover, applying an original approach to analyze the (0 0 2) XRD diffraction peak profile of hydroxyapatite by using a sum of two Gauss functions, the bimodal distribution of nanosized hydroxyapatite within the chitosan matrix was revealed. Two types of size distribution domains such as cluster-like (between 200 and 400 nm), which are the habitat of ‘‘small’’ hydroxyapatite nanocrystallites and scattered-like, which are the habitat of ‘‘large’’ hydroxyapatite nanocrystallites was probed by TEM and CSLM. The structural features of composites suggest that self-assembly processes might be involved. The composites contain nanosized hydroxyapatite with structural features close to those of biological apatites that make them attractive for bone tissue engineering applications.
Keywords: Nanosized hydroxyapatite; Chitosan; Organic–inorganic composite; Bimodal distribution; Self-assembly behavior;