Biomaterials (v.31, #20)

A fusion protein N-cadherin-Fc as an artificial extracellular matrix surface for maintenance of stem cell features by Xiao-Shan Yue; Yuta Murakami; Toshiyuki Tamai; Masato Nagaoka; Chong-Su Cho; Yoshihiro Ito; Toshihiro Akaike (5287-5296).
N-cadherin is a cell–cell adhesion molecule and plays important roles in neural development. With conventionally used extracellular matrices (ECMs), maintenance of undifferentiated state of stem cells and regulation of their neural differentiation process is very difficult due to the colony formation through intercellular interactions. To overcome the above-mentioned problems, we developed a new artificial ECM to mimic N-cadherin-mediated cell adhesion. In this study, we constructed a chimeric protein (N-cadherin fused to IgG-Fc, abbreviated as N-cad-Fc), which contains extracellular domain of N-cadherin and Fc domain of IgG. We confirmed that N-cad-Fc can stably adsorb to hydrophobic surface. We checked maintenance of undifferentiated state and neural differentiation ability of stem cells cultured on N-cad-Fc-coated surface. Both P19 and MEB5 cells cultured on N-cad-Fc-coated surface showed scattering morphologies without colony formation and higher proliferating potency than conventional culture systems with maintenance of undifferentiated state. Both of two cell lines cultured on N-cad-Fc-coated surface differentiated into neural cells at a single cell level when induced with proper conditions. Furthermore, the expression of neuron-related gene Neurog1 in two cell lines cultured on N-cad-Fc-coated surface was promoted. Therefore, it will be expected that the constructed N-cad-Fc can be used as an artificial ECM for stem cells.
Keywords: Adhesion molecule; N-cadherin; Extracellular matrix; Neural cell; Stem cell;

The significance of crack-resistance curves to the mixed-mode fracture toughness of human cortical bone by Elizabeth A. Zimmermann; Maximilien E. Launey; Robert O. Ritchie (5297-5305).
The majority of fracture mechanics studies on the toughness of bone have been performed under tensile loading. However, it has recently been shown that the toughness of human cortical bone in the transverse (breaking) orientation is actually much lower in shear (mode II) than in tension (mode I); a fact that is physiologically relevant as in vivo bone is invariably loaded multiaxially. Since bone is a material that derives its fracture resistance primarily during crack growth through extrinsic toughening mechanisms, such as crack deflection and bridging, evaluation of its toughness is best achieved through measurements of the crack-resistance or R-curve, which describes the fracture toughness as a function of crack extension. Accordingly, in this study, we attempt to measure for the first time the R-curve fracture toughness of human cortical bone under physiologically relevant mixed-mode loading conditions. We show that the resulting mixed-mode (mode I + II) toughness depends strongly on the crack trajectory and is the result of the competition between the paths of maximum mechanical driving force and “weakest” microstructural resistance.
Keywords: Human cortical bone; Mixed-mode fracture; Fracture toughness; Crack-growth resistance curve;

The performance of ice-free cryopreserved heart valve allografts in an orthotopic pulmonary sheep model by Milan Lisy; Juliane Pennecke; Kelvin G.M. Brockbank; Olaf Fritze; Martina Schleicher; Katja Schenke-Layland; Renate Kaulitz; Iris Riemann; Corinna N. Weber; Josephine Braun; Kerstin E. Mueller; Falko Fend; Torsten Scheunert; Achim D. Gruber; Johannes M. Albes; Agnes J. Huber; Ulrich A. Stock (5306-5311).
Transplantation of cryopreserved heart valves (allografts) is limited by immune responses, inflammation, subsequent structural deterioration and an expensive infrastructure. In previous studies we demonstrated that conventional frozen cryopreservation (FC) is accompanied by serious alterations of extracellular matrix (ECM) structures. As the main culprit of the observed damages ice crystal formation was identified. Objective of this study was the application principles of cryoprotection as observed in nature, occurring in animals or plants, for ice-free cryopreservation (IFC) of heart valves. Using IFC, valves were processed and stored above the glass transition temperature of the cryoprotectant formulation (−124 °C) at −80 °C to avoid any ice formation, tissue-glass cracking and preserving ECM. After implantation in the orthotopic pulmonary position in sheep, we demonstrate that IFC resulted in cell free matrices, while maintaining crucial ECM-components such as elastin and collagen, translating into superior hemodynamics. In contrast, we reveal that FC valves showed ECM damage that was not restored in vivo, and T-cell inflammation of the stroma with significant leaflet thickening. Compared to currently applied FC practice IFC also reduced infrastructural needs for preservation, storage and shipping. These results have important implications for clinical valve transplantation including the promise of better long-term function and lower costs.
Keywords: Allograft heart valves; Transplantation; Cryopreservation; Ice-free;

A nerve graft constructed with xenogeneic acellular nerve matrix and autologous adipose-derived mesenchymal stem cells by Yongjie Zhang; Hailang Luo; Ziqiang Zhang; Yongbo Lu; Xinhui Huang; Lu Yang; Jiajie Xu; Wei Yang; Xiaoju Fan; Bing Du; Peng Gao; Gang Hu; Yan Jin (5312-5324).
Since synthetic nerve conduits do not exhibit the characteristics of regeneration, they are generally inadequate substitutes for autologous nerve graft in the repair of long peripheral nerve defects. To resolve this problem, in this study, we constructed a nerve regeneration characteristics-containing nerve graft through integrating xenogeneic acellular nerve matrix (ANM) with autologous neural differentiated adipose-derived mesenchymal stem cells (ADSCs). Xenogeneic ANM was processed by a protocol removing cells and myelin sheath completely, meanwhile preserving growth factors and extracellular matrix (ECM) microstructure of natural nerve, such as porous and basal lamina tube. Cytocompatibility and immunocompatibility evaluation revealed that ANM could support cell attachment and proliferation, and did not stimulate vigorous host reject response. After inoculation of neural differentiated ADSCs onto ANM, differentiated cells were observed to align along longitudinal axis of ANM, resembling band of büngner, and persistently express NGF, GDNF, and BDNF. In vivo, neural differentiated ADSCs also presented glial cell characteristics and promote nerve regeneration 7 days post transplantation. We repaired 1 cm Sprague Dawley rat sciatic nerve defects using this nerve graft construction and nerve gap regeneration was indicated by electrophysiology, retrograde labeling and histology analysis. Therefore, we conclude that constructed nerve graft, offering nerve regeneration characteristics, hold great promise to replace autologous in repair peripheral nerve defect.
Keywords: Xenogeneic acellular nerve matrix; Adipose-derived mesenchymal stem cells; Nerve graft; Nerve regeneration;

An injectable tissue-engineered bone (ITB) composed of human adipose-derived stromal cells (hADSCs) and platelet-rich plasma (hPRP) was preliminarily constructed, but its osteogenic capability needs improving. This study aimed to evaluate if simvastatin can be applied as a bone anabolic agent for this ITB. We found 0.01 μm, 0.1 μm, and 1 μm simvastatin could induce hADSCs’ osteoblastic differentiation in vitro that accompanied with non-inhibition on cell proliferation, high alkaline phosphatase activity, more mineralization deposition and more expression of osteoblast-related genes such as osteocalcin, core binding factor α1, bone morphogenetic protein-2, vascular endothelial growth factor, and basic fibroblast growth factor. Simvastatin at 1 μm seemed the most optimal concentration due to its high osteocalcin secretion in media (P < 0.01). Quantitative mineralization assay also showed 1 μm SIM had the most obvious synergistic effect on hPRP’s induction for matrix mineralization of hADSCs (P < 0.01). When 1 μm Simvastatin was applied to this ITB to restore the critical-sized calvarial defects in mice, more bone formation was observed in defected regions, and the peripheries just outside the defect margins by X-ray analysis, and H&E staining. These findings indicate that simvastatin at optimal concentrations can be used to promote this ITB’s osteogenesis. However, simvastatin’s effects on this ITB await long-term investigation.
Keywords: Simvastatin; Adipose-derived stromal cells; Platelet-rich plasma; Bone tissue engineering; Critical-sized calvarial defects; Injectable bone;

Tissue response to poly(ether)urethane-polydimethylsiloxane-fibrin composite scaffolds for controlled delivery of pro-angiogenic growth factors by Paola Losi; Enrica Briganti; Angela Magera; Dario Spiller; Chiara Ristori; Barbara Battolla; Michela Balderi; Silvia Kull; Alberto Balbarini; Rossella Di Stefano; Giorgio Soldani (5336-5344).
The development of a scaffold able to mimic the mechanical properties of elastic tissues and to induce local angiogenesis by controlled release of angiogenic growth factors could be applied in the treatment of several ischemic diseases. For this purpose a composite scaffold made of a poly(ether)urethane–polydimethylsiloxane (PEtU–PDMS) semi-interpenetrating polymeric network (semi-IPN) and fibrin loaded growth factors (GFs), such as VEGF and bFGF, was manufactured using spray, phase-inversion technique. To evaluate the contribution of each scaffold component with respect to tissue response and in particular to blood vessel formation, three different scaffold formulations were developed as follows: 1) bare PEtU–PDMS; 2) PEtU–PDMS/Fibrin; and 3) PEtU–PDMS/Fibrin + GFs. Scaffolds were characterized in vitro respect to their morphology, VEGF and bFGF release kinetics and bioactivity. The induction of in vivo angiogenesis after subcutaneous and ischemic hind limb scaffold implantation in adult Wistar rats was evaluated at 7 and 14 days by immunohistological analysis (IHA), while Laser Doppler Perfusion Imaging (LDPI) was performed in the hind limbs at 0, 3, 7, 10 and 14 days. IHA of subcutaneously implanted samples showed that at 7 and 14 days the PEtU–PDMS/Fibrin + GFs scaffold induced a statistically significant increase in number of capillaries compared to bare PEtU–PDMS scaffold. IHA of ischemic hind limb showed that at 14 days the capillary number induced by PEtU–PDMS/Fibrin + GFs scaffolds was higher than that of PEtU–PDMS/Fibrin scaffolds. Moreover, at both time-points PEtU–PDMS/Fibrin scaffolds induced a significant increase in number of capillaries compared to bare PEtU–PDMS scaffolds. LDPI showed that at 10 and 14 days the ischemic/non-ischemic blood perfusion ratio was significantly greater in the PEtU–PDMS/Fibrin + GFs than in the other scaffolds. In conclusion, this study showed that the semi-IPN composite scaffold acting as a pro-angiogenic GFs delivery system has therapeutic potential for the local treatment of ischemic tissue and wound healing.
Keywords: Poly(ether)urethane-polydimethylsiloxane; Fibrin; Vascular endothelial growth factor; Basic fibroblast growth factor; Delivery system; Angiogenesis;

Characterization of the complete fiber network topology of planar fibrous tissues and scaffolds by Antonio D’Amore; John A. Stella; William R. Wagner; Michael S. Sacks (5345-5354).
Understanding how engineered tissue scaffold architecture affects cell morphology, metabolism, phenotypic expression, as well as predicting material mechanical behavior has recently received increased attention. In the present study, an image-based analysis approach that provides an automated tool to characterize engineered tissue fiber network topology is presented. Micro-architectural features that fully defined fiber network topology were detected and quantified, which include fiber orientation, connectivity, intersection spatial density, and diameter. Algorithm performance was tested using scanning electron microscopy (SEM) images of electrospun poly(ester urethane)urea (ES-PEUU) scaffolds. SEM images of rabbit mesenchymal stem cell (MSC) seeded collagen gel scaffolds and decellularized rat carotid arteries were also analyzed to further evaluate the ability of the algorithm to capture fiber network morphology regardless of scaffold type and the evaluated size scale. The image analysis procedure was validated qualitatively and quantitatively, comparing fiber network topology manually detected by human operators (n = 5) with that automatically detected by the algorithm. Correlation values between manual detected and algorithm detected results for the fiber angle distribution and for the fiber connectivity distribution were 0.86 and 0.93 respectively. Algorithm detected fiber intersections and fiber diameter values were comparable (within the mean ± standard deviation) with those detected by human operators. This automated approach identifies and quantifies fiber network morphology as demonstrated for three relevant scaffold types and provides a means to: (1) guarantee objectivity, (2) significantly reduce analysis time, and (3) potentiate broader analysis of scaffold architecture effects on cell behavior and tissue development both in vitro and in vivo.
Keywords: Image analysis; Scaffold morphology; Microstructure; Electrospinning; Collagen gel; Decellularized tissue;

The chondrogenic differentiation of mesenchymal stem cells on an extracellular matrix scaffold derived from porcine chondrocytes by Kyoung-Hwan Choi; Byung Hyune Choi; So Ra Park; Byoung Ju Kim; Byoung-Hyun Min (5355-5365).
Extracellular matrix (ECM) materials have diverse physiological functions by themselves and can also act as reservoirs of cytokines and growth factors, so that they can affect the cell phenotype, attachment, migration and proliferation of cells. In this study, an ECM scaffold derived from porcine cartilage was evaluated for whether it can support and maintain chondrogenesis of rabbit mesenchymal stem cells (rMSCs) in vitro and in the nude mouse model in vivo. The porcine ECM scaffold was compared to a polyglycolic acid (PGA) scaffold and an MSC pellet as a control group. In an in vitro environment until 4 weeks, the ECM scaffold evoked chondrogenic differentiation of rMSCs earlier and produced more cartilaginous tissues than the PGA scaffold. Next, rMSCs in each scaffold were preconditioned with chondrogenic media in vitro for 1 week and implanted in the backs of nude mice for 6 weeks. The initially formed cartilaginous tissues turned into bone matrix with time centripetally from the outside of the region as observed by Safranin-O and von Kossa stains. This phenomenon progressed much more rapidly in the PGA group than in the ECM group. In the ECM group, the chondrogenic phenotypes of rMSCs were also maintained longer than in the PGA group. The loss of chondrogenic phenotypes was accompanied by the calcification of matrix, and hypertrophic changes by immunohistochemistry for osteocalcin and collagen type I and X. Blood vessel invasion took place more deeply and intensively in the PGA group. These results suggest that the ECM scaffold not only strongly supports chondrogenic differentiation of rMSCs, but also helps maintain its phenotype in vivo. We speculate that the ECM scaffold provides rMSCs with a favorable, native cartilage-like environment and therefore can be a promising tool for cartilage tissue engineering.
Keywords: ECM (extracellular matrix) scaffold; Mesenchymal stem cells (MSCs); Chondrogenesis; Cartilage tissue engineering;

Targeted magnetic delivery and tracking of cells using a magnetic resonance imaging system by Johannes Riegler; Jack A. Wells; Panagiotis G. Kyrtatos; Anthony N. Price; Quentin A. Pankhurst; Mark F. Lythgoe (5366-5371).
The success of cell therapies depends on the ability to deliver the cells to the site of injury. Targeted magnetic cell delivery is an emergent technique for localised cell transplantation therapy. The use of permanent magnets limits such a treatment to organs close to the body surface or an implanted magnetic source. A possible alternative method for magnetic cell delivery is magnetic resonance targeting (MRT), which uses magnetic field gradients inherent to all magnetic resonance imaging system, to steer ferromagnetic particles to their target region. In this study we have assessed the feasibility of such an approach for cell targeting, using a range of flow rates and different super paramagnetic iron oxide particles in a vascular bifurcation phantom. Using MRT we have demonstrated that 75% of labelled cells could be guided within the vascular bifurcation. Furthermore we have demonstrated the ability to image the labelled cells before and after magnetic targeting, which may enable interactive manipulation and assessment of the distribution of cellular therapy. This is the first demonstration of cellular MRT and these initial findings support the potential value of MRT for improved targeting of intravascular cell therapies.
Keywords: Magnetism; Transplantation; Nanoparticles; Magnetic resonance imaging (MRI); Magnetic resonance targeting (MRT);

Delivery of paclitaxel from cobalt–chromium alloy surfaces without polymeric carriers by Gopinath Mani; Celia E. Macias; Marc D. Feldman; Denes Marton; Sunho Oh; C. Mauli Agrawal (5372-5384).
Polymer-based carriers are commonly used to deliver drugs from stents. However, adverse responses to polymer coatings have raised serious concerns. This research is focused on delivering drugs from stents without using polymers or any carriers. Paclitaxel (PAT), an anti-restenotic drug, has strong adhesion towards a variety of material surfaces. In this study, we have utilized such natural adhesion property of PAT to attach these molecules directly to cobalt–chromium (Co–Cr) alloy, an ultra-thin stent strut material. Four different groups of drug coated specimens were prepared by directly adding PAT to Co–Cr alloy surfaces: Group-A (PAT coated, unheated, and ethanol cleaned); Group-B (PAT coated, heat treated, and ethanol cleaned); Group-C (PAT coated, unheated, and not ethanol cleaned); and Group-D (PAT coated, heat treated and not ethanol cleaned). In vitro drug release of these specimens was investigated using high performance liquid chromatography. Groups A and B showed sustained PAT release for up to 56 days. A simple ethanol cleaning procedure after PAT deposition can remove the loosely bound drug crystals from the alloy surfaces and thereby allowing the remaining strongly bound drug molecules to be released at a sustained rate. The heat treatment after PAT coating further improved the stability of PAT on Co–Cr alloy and allowed the drug to be delivered at a much slower rate, especially during the initial 7 days. The specimens which were not cleaned in ethanol, Groups C and D, showed burst release. PAT coated Co–Cr alloy specimens were thoroughly characterized using scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. These techniques were collectively useful in studying the morphology, distribution, and attachment of PAT molecules on Co–Cr alloy surfaces. Thus, this study suggests the potential for delivering paclitaxel from Co–Cr alloy surfaces without using any carriers.
Keywords: Drug-eluting stents; Drug delivery; Cobalt–Chromium alloy; Surface treatment; Surface modification;

Ex vivo expansion of human circulating myogenic progenitors on cluster-assembled nanostructured TiO2 by Marzia Belicchi; Silvia Erratico; Paola Razini; Mirella Meregalli; Alessandra Cattaneo; Emanuela Jacchetti; Andrea Farini; Chiara Villa; Nereo Bresolin; Laura Porretti; Cristina Lenardi; Paolo Milani; Yvan Torrente (5385-5396).
Ex vivo expansion of hematopoietic stem cells has been explored in the fields of stem cell biology, gene therapy and clinical transplantation. Recently, we demonstrated the existence of a circulating myogenic progenitor expressing the CD133 antigen. The relative inability of circulating CD133+ stem cells to reproduce themselves ex vivo imposes substantial limitations on their use for clinical applications in muscular dystrophies. Here we report that the use of cluster-assembled nanostructured titanium dioxide (ns-TiO2) substrates, in combination with cytokine enriched medium, enables high-level expansion of circulating CD133+ stem cells in vitro. Furthermore, we demonstrate that expanded circulating CD133+ stem cells retain their in vitro capacity to differentiate into myogenic cells. The exploitation of cluster-assembled ns-TiO2 substrates for the expansion of CD133+ stem cells in vitro could therefore make the clinical application of these stem cells for the treatment of muscle diseases practical.
Keywords: Stem cell; Titanium oxide; Nanotopography; Surface treatment; Cell culture; Muscular dystrophy;

Reducing non-specific binding and uptake of nanoparticles and improving cell targeting with an antifouling PEO-b-PγMPS copolymer coating by Hongwei Chen; Liya Wang; Julie Yeh; Xinying Wu; Zehong Cao; Yongqiang A. Wang; Minming Zhang; Lily Yang; Hui Mao (5397-5407).
One of the major limitations impeding the sensitivity and specificity of biomarker targeted nanoparticles is non-specific binding by biomolecules and uptake by the reticuloendothelial system (RES). We report the development of an antibiofouling polysiloxane containing amphiphilic diblock copolymer, poly(ethylene oxide)-block-poly(γ-methacryloxypropyl trimethoxysilane) (PEO-b-PγMPS), for coating and functionalizing high quality hydrophobic nanocrystals such as iron oxide nanoparticles and quantum dots. These PEO-b-PγMPS-coated nanocrystals were colloidally stable in biological medium and showed low non-specific binding by macromolecules after incubation with 100% fetal bovine serum. Both in vitro experiments with macrophages and in vivo biodistribution studies in mice revealed that PEO-b-PγMPS copolymer-coated nanocrystals have an antibiofouling effect that reduces non-specific cell and RES uptake. Surface functionalization with amine groups was accomplished through co-crosslinking the polysiloxane coating layer and (3-Aminopropyl)trimethoxysilane in aqueous solution. Tumor integrin αvβ3 targeting peptide cyclo-RGD ligands were conjugated on the nanoparticles through a heterobifunctional linker. The resulting integrin αvβ3 targeting nanoparticle conjugates showed improved cancer cell targeting with a stronger affinity to U87MG glioma cells, which have a high expression of αvβ3 integrins, but minimal binding to MCF-7 breast cancer cells with low expression of αvβ3 integrins.
Keywords: Nanoparticles; Copolymer; Antifouling; Non-specific binding; Reticuloendothelial system; Cancer targeting;

Porous nanocrystalline silicon membranes as highly permeable and molecularly thin substrates for cell culture by A.A. Agrawal; B.J. Nehilla; K.V. Reisig; T.R. Gaborski; D.Z. Fang; C.C. Striemer; P.M. Fauchet; J.L. McGrath (5408-5417).
Porous nanocrystalline silicon (pnc-Si) is new type of silicon nanomaterial with potential uses in lab-on-a-chip devices, cell culture, and tissue engineering. The pnc-Si material is a 15 nm thick, freestanding, nanoporous membrane made with scalable silicon manufacturing. Because pnc-Si membranes are approximately 1000 times thinner than any polymeric membrane, their permeability to small solutes is orders-of-magnitude greater than conventional membranes. As cell culture substrates, pnc-Si membranes can overcome the shortcomings of membranes used in commercial transwell devices and enable new devices for the control of cellular microenvironments. The current study investigates the feasibility of pnc-Si as a cell culture substrate by measuring cell adhesion, morphology, growth and viability on pnc-Si compared to conventional culture substrates. Results for immortalized fibroblasts and primary vascular endothelial cells are highly similar on pnc-Si, polystyrene and glass. Significantly, pnc-Si dissolves in cell culture media over several days without cytotoxic effects and stability is tunable by modifying the density of a superficial oxide. The results establish pnc-Si as a viable substrate for cell culture and a degradable biomaterial. Pnc-Si membranes should find use in the study of molecular transport through cell monolayers, in studies of cell-cell communication, and as biodegradable scaffolds for three-dimensional tissue constructs.
Keywords: Porosity; Silicon; Cell culture; Nanoporous; Cell adhesion; Biocompatibility;

Modulation of human vascular endothelial cell behaviors by nanotopographic cues by Sara J. Liliensiek; Joshua A. Wood; Jiang Yong; Robert Auerbach; Paul F. Nealey; Christopher J. Murphy (5418-5426).
Basement membranes possess a complex three-dimensional topography in the nanoscale and submicron range which have been shown to profoundly modulate a large menu of fundamental cell behaviors. Using the topographic features found in native vascular endothelial basement membranes as a guide, polyurethane substrates were fabricated containing anisotropically ordered ridge and groove structures and isotropically ordered pores from 200 nm to 2000 nm in size. We investigated the impact of biomimetic length-scale topographic cues on orientation/elongation, proliferation and migration on four human vascular endothelial cell-types from large and small diameter vessels. We found that all cell-types exhibited orientation and alignment with the most pronounced response on anisotropically ordered ridges ≥800 nm. HUVEC cells were the only cell-type examined to demonstrate a decrease in proliferation in response to the smallest topographic features regardless of surface order. On anisotropically ordered surfaces all cell-types migrated preferentially parallel to the long axis of the ridges, with the greatest increase in cell migration being observed on the 1200 nm pitch. In contrast, cells did not exhibit any preference in direction or increase in migration speed on isotropically ordered surfaces. Overall, our data demonstrate that surface topographic features impact vascular endothelial cell behavior and that the impact of features varies with the cell behavior being considered, topographic feature scale, surface order, and the anatomic origin of the cell being investigated.
Keywords: Biomimetic material; Cell morphology; Cell proliferation; ECM; Endothelial cell; Nanotopography;

Gadolinium hexanedione nanoparticles for stem cell labeling and tracking via magnetic resonance imaging by Ching-Li Tseng; I.-Ling Shih; Leszek Stobinski; Feng-Huei Lin (5427-5435).
The ability to trace transplanted stem cells and monitor their tissue biodistribution is prerequisite to an understanding of cellular migration after transplantation. Therefore, a new magnetic resonance imaging (MRI) contrast agent made of gadolinium hexanedione nanoparticles (GdH-NPs) was developed as a cell tracking agent. The GdH-NPs were fabricated by the microemulsion process. The physical characteristics, biocompatibility, and T1-MRI signal enhancement of these NPs were analyzed and evaluated for stem cell tracking. In this study, the size of the synthesized GdH-NPs was about 140 nm, and it had greater image enhancement ability than commercial gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA). From the biocompability test, we found GdH-NPs were nontoxic for human mesenchymal stem cells (hMSCs). The expression of surface antigens of hMSCs after culture with GdH-NPs was examined, and it showed no difference from the control group. The results of transmission electron microscopy (TEM) imaging for labeled hMSCs showed GdH-NPs were accumulated in the cells by the endocytotic pathway. The accumulation of GdH-NPs in hMSCs was three times higher in comparison to Gd-DTPA. Human MSCs labeled with low concentration of GdH-NPs (10 μg/mL) hold better signals in cellular MR image. We conclude GdH-NPs can be used to label hMSCs in vitro with greater T1 image-enhancing property and without affecting cell quality. Finally, GdH-NPs have great potential as a contrast agent for stem cell tracking by MRI methodology.
Keywords: Gadolinium; Nanoparticle; Stem cell; Magnetic resonance imaging (MRI); Cell tracker;

Targeted near-IR QDs-loaded micelles for cancer therapy and imaging by Md. Nurunnabi; Kwang Jae Cho; Joon Sig Choi; Kang Moo Huh; Yong-kyu Lee (5436-5444).
The use of water-soluble, functionalized quantum dots (QDs) that are highly stable against oxidation for biological and biomedical applications is currently one of the fastest growing fields of nanotechnology. Polymer-based nanoparticles are now widely used for drug delivery and targeted therapy. We modified the surface of near Infrared QDs by the solid dispersion method using PEG–PCDA and PCDA–Herceptin conjugates to demonstrate water-solubility and target-specific properties. Upon UV irradiation, QD cores located within nanoprobes were further stabilized by intramicellar cross-linking between neighboring PCDA–Herceptin moieties. These cross-linked nanoprobes showed higher stability and less toxicity. Near-IR QDs-loaded micelles were spherical with diameters of around 130–150 nm. The anti-tumor effect of near-IR QDs-loaded micelles against MDA-MB-231 tumors was remarkably better than that of control. Mice treated with the near-IR QDs-loaded micelles had a tumor volume of about 285 mm3, indicating shrinkage in initial tumor volume and inhibition of tumor growth by 77.3% compared to that of control group (saline injection). In addition, near-IR QDs-loaded micelles were injected intravenously into tumor-bearing nude mice for simultaneous tumor therapy and imaging. We observed that the targeted near-IR QDs-loaded micelles distributed rapidly throughout the animal body including the tumor in real time. These multi-functional nanoprobes could therefore be used for both active and passive targeting, imaging and treatment of cancers in the early stage.
Keywords: Near-IR QDs; Herceptin; Tumor imaging; Nano-probes;

Hyperbranched poly(amine-ester) based hydrogels for controlled multi-drug release in combination chemotherapy by Hongbin Zhang; Chen Zhao; Hui Cao; Guojie Wang; Li Song; Guoguang Niu; Huai Yang; Jie Ma; Siquan Zhu (5445-5454).
Combination chemotherapy has been a primary management for cancer. Thus a drug delivery system which can administer several drugs simultaneously and control the drug release at the cancer site is desired. Here we synthesized hyperbranched poly(amine-ester) (HPAE) macromers with different degrees of terminal C=C modification to make injectable hydrogels as a multi-drug delivery system. The aqueous solutions of the macromers were fast transformed into hydrogel at body temperature with a low concentration (0.05 wt%) of ammonium persulfate (APS) but no activator for accelerating the polymerization, since the HPAE macromer with tertiary amines and APS themselves formed a redox system as initiator. Three different types of drugs, doxorubicin hydrochloride (Dox), 5-fluorouracil (5FU) and leucovorin calcium (LC), were used as model drugs in this experiment. This system allows locally releasing single and/or combinations of anticancer drugs simultaneously by a controllable way. Behaviors of drug release can be controlled by the drug-loading methods or/and the C=C modification degree of macromers loaded with the drug molecules. The drug release period could be prolonged when the drug was loaded into the macromers with high content of C=C. The HPAE macromers exhibited good biocompatibility which was evaluated in L929 and MCF7 cell lines using MTT cell proliferation assay. The swelling behavior and degradation of HPAE hydrogels in vitro were also examined. These results suggest that the HPAE hydrogels hold great potential for use as injectable systems for locally delivering single and/or multiple drugs in chemotherapy of cancer.
Keywords: Anticancer drugs; Controlled release; Combination therapy; Hydrogel; Hyperbranched poly(amine-ester);

Enhanced effects of low molecular weight heparin intercalated with layered double hydroxide nanoparticles on rat vascular smooth muscle cells by Zi Gu; Barbara E. Rolfe; Zhi P. Xu; Anita C. Thomas; Julie H. Campbell; Gao Q.M. Lu (5455-5462).
Surgical procedures to remove atherosclerotic lesions and restore blood flow also injure the artery wall, promoting vascular smooth muscle cell (SMC) phenotypic change, migration, proliferation, matrix production and ultimately, restenosis of the artery. Hence identification of effective anti-restenotic strategies is a high priority in cardiovascular research, and SMCs are a key target for intervention. This paper presents the in vitro study of layered double hydroxides (LDHs) as drug delivery system for an anti-restenotic drug (low molecular weight heparin, LMWH). The cytotoxicity tests showed that LDH itself had very limited toxicity at concentrations below 50 μg/mL over 6-day incubation. LDH nanoparticles loaded with LMWH (LMWH–LDHs) were prepared and tested on rat vascular SMCs. When conjugated to LDH particles, LMWH enhanced its ability to inhibit SMC proliferation and migration, with greater than above 60% reduction compared with the control (growth medium) over 3 or 7-day incubation. Cellular uptake studies showed that compared with LMWH alone, LMWH–LDH hybrids were internalized by SMCs more rapidly, and uptake was sustained over a longer time, possibly revealing the mechanisms underlying the enhanced biological function of LMWH–LDH. The results suggest the potential of LMWH–LDH as an efficient anti-restenotic drug for clinical application.
Keywords: Layered double hydroxides; Low molecular weight heparin; Rat vascular smooth muscle cells; Proliferation; Migration; Cellular uptake;

Mucosal vaccination using claudin-4-targeting by Hideki Kakutani; Masuo Kondoh; Masahiro Fukasaka; Hidehiko Suzuki; Takao Hamakubo; Kiyohito Yagi (5463-5471).
Mucosa-associated lymphoid tissue (MALT) plays pivotal roles in mucosal immune responses. Efficient delivery of antigens to MALT is a critical issue for the development of mucosal vaccines. Although claudin-4 is preferentially expressed in MALT in the gut, a claudin-4-targeting approach for mucosal vaccination has never been developed. In the present study, we found that claudin-4 is expressed in nasal MALT, and we prepared a fusion protein of ovalbumin (OVA) as a model antigen with a claudin-4-binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) (OVA-C-CPE). Nasal immunization with OVA-C-CPE, but not a mixture of OVA and C-CPE, induced the production of OVA-specific serum IgG and nasal, vaginal and fecal IgA. Deletion of the claudin-4-binding region in OVA-C-CPE attenuated the induction of the immune responses. OVA-C-CPE immunization activated both Th1 and Th2 responses, and nasal immunization with OVA-C-CPE showed anti-tumor activity in mice inoculated with OVA-expressing thymoma cells. These results indicate that the claudin-4-targeting may be a potent strategy for nasal vaccination.
Keywords: Immunomodulation; Mucosa; Drug delivery; Epithelium;

Functional surfaces for high-resolution analysis of cancer cell interactions on exogenous hyaluronic acid by Laura E. Dickinson; Chia Chi Ho; Geoffrey M. Wang; Kathleen J. Stebe; Sharon Gerecht (5472-5478).
Hyaluronic acid, a nonsulfated, linear glycosaminoglycan, is ubiquitously distributed in the extracellular matrix and is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis. Native HA has been attached to substrates to create patterned surfaces resistant to cell adhesion, and has been utilized in a variety of cell adhesion studies using either non covalently bound layers patterned by soft lithography or related methods. We use a new approach to study cell interactions with HA-presenting regions, by covalently linking HA adjacent to PEG-ylated regions, which resist cell adhesion. Colon and breast cancer cells seeded on the patterned HA surfaces adhere preferentially on HA-presenting regions and proliferate there. Furthermore, we demonstrate that cell adhesion is inhibited with the blocking of HA receptor, CD44, and that cellular adhesive processes, through protrusions spreading onto the HA surface, enhance spreading and movement outside the HA-presenting regions. Overall, this approach allows high-resolution analysis of cancer cell attachment, growth, and migration on exogenous native HA.
Keywords: Hyaluronic acid; Micropatterning; Cell adhesion;

On the mineral in collagen of human crown dentine by Anke Märten; Peter Fratzl; Oskar Paris; Paul Zaslansky (5479-5490).
Dentine, the main material of mammalian teeth, contains mineral platelets that are embedded in a collagen fiber mesh. These particles entail stiffness and longevity, which is important for human teeth because these organs do not remodel. By means of small angle X-ray scattering, we mapped 2D and 3D variations in mineral particle characteristics in molar crowns. Our results show that the mean mineral-platelet thickness of 3.2 nm decreases to 2.6 nm within the shallow 300 μm beneath the dentin-enamel junction (DEJ), and that these platelets become still thinner albeit moderately in deep dentine surrounding the pulp. The mineral volume fraction in crown dentine is mostly 50% except for a 250 μm layer beneath the DEJ. Most of the mineral particles are randomly orientated, with about 20% having a preferred orientation that is parallel to the plane of the DEJ. Beneath the cusps and close to the margins of enamel, higher co-alignment is found: 40% of the particles reveal orientations that match expected load trajectories that are imposed on teeth during mastication in the general cusp-root direction. This suggests that variations in mineral platelet arrangements help to locally tune dentine anisotropy and stiffness. The serendipitous finding of incipient caries suggests that at least in early stages of pathological destruction, mineral particle thickness and orientation resemble those of the intact tissue.
Keywords: Carbonated hydroxyl-apatite mineral platelet; Small-angle X-ray scattering; Mineral volume fraction; Dentine attenuation coefficient; Caries;