Biomaterials (v.32, #30)

A computational modeling approach for enhancing self-assembly and biofunctionalisation of collagen biomimetic peptides by Navaneethakrishnan Krishnamoorthy; Magdi H. Yacoub; Sophia N. Yaliraki (7275-7285).
Collagen fibers are essential components of tissues, which are highly conserved across the animal kingdom and could be extremely useful in tissue engineering. The formation of these macromolecular fibers depends on molecular interactions-based self-assembly of the basic building blocks of collagen called tropocollagens. Several attempts to produce biomimetic collagen have been described, however the best method to achieve the optimal material for tissue engineering has not been established. Here, we describe a bottom-up approach to design two computationally mutated molecular models that use non-covalent interactions to cross-link triple helices of tropocollagen molecules and thus promote self-association. Implementing a graph theory approach in the software FIRST reveals the hotspots that are crucial for the overall rigidity of the supramolecular helical structures and the remaining non-hotspots available for mutations. The mutated models were further decorated with GFOGER, a known collagen cell binding motif, to depict a biofunctional model. In addition to their recognized role of cell binding, the charged residues of the binding motif appeared to enhance further the supramolecular helical association. These findings could help to produce biomimetic collagen for biomedical applications.
Keywords: Multiple tropocollagens; Mutational modeling; Graph theory; Molecular dynamics simulation; Self-assembly; Collagen cell binding motif;

The dynamic nature of cell adhesion and detachment is critically important to a variety of physiological and pathophysiological phenomena. Much, however, still remains uncertain and controversial about the mechanochemical players and processes involved in cellular adhesion and detachment. This leads to the need for quantitative characterization of the adhesion and detachment of anchorage-dependent cells. Here, cell adhesion and detachment up to subcellular level are examined using gold surfaces modified with a thiol-functionalized arginine-glycine-aspartic acid (RGD) peptide. A thiol self-assembled monolayer (SAM) on top of the gold surfaces is reductively desorbed with activation potential to spatiotemporally manipulate both cell adhesion and detachment. This method maintains cells of interest living and intact during experiments, making it possible to quantify cell adhesion and detachment as close as possible to in vivo conditions. Experimental characterizations for NIH 3T3 fibroblasts are carried out with a focus on the following issues: the effect of the size and geometric shape of gold surfaces on cell adhesion; the effect of cell confluency, cell shape, and activation potential magnitude on cell detachment; changes in the material properties of cells during cell detachment. The findings of this study should lead to better understanding of cellular dynamics in anchorage-dependent cells.
Keywords: Cell adhesion; Cell detachment; Electrochemistry; Gold; Polyethylene glycol; Thiol-functionalized arginine-glycine-aspartic acid (RGD) peptide;

Enhanced bone-integration capability of alkali- and heat-treated nanopolymorphic titanium in micro-to-nanoscale hierarchy by Takeshi Ueno; Naoki Tsukimura; Masahiro Yamada; Takahiro Ogawa (7297-7308).
This study introduces nanopolymorphic features of alkali- and heat-treated titanium surfaces, comprising of tuft-like, plate-like, and nodular structures that are smaller than 100 nm and determines whether and how the addition of these nanofeatures to a microroughened titanium surface affects bone–implant integration. A comprehensive assessment of biomechanical, interfacial, and histological analyses in a rat model was performed for machined surfaces without microroughness, sandblasted-microroughened surfaces, and micro–nano hybrid surfaces created by sandblasting and alkali and heat treatment. The microroughened surface accelerated the establishment of implant biomechanical fixation at the early healing stage compared with the non-microroughened surface but did not increase the implant fixation at the late healing stage. The addition of the nanopolymorphic features to the microroughened surface further increased implant fixation throughout the healing time. The area of the new bone within 50 μm proximity of the implant surfaces, which was increased 2–3-fold using microroughened surfaces, was further increased 2-fold using nanopolymorphic surfaces. In contrast, the bone area in a 50–200 μm zone was not influenced by either microroughened or nanopolymorphic surfaces. The percentage of bone–implant contact, which was increased 4–5-fold, using microroughened surfaces, was further increased substantially by over 2-fold throughout the healing period. The percentage of soft tissue intervention between bone and implant surfaces, which was reduced to half by microroughened surfaces, was additionally reduced by the nanopolymorphic surfaces to between one-third and one-fourth, resulting in only 5–7% soft tissue intervention compared with 60–75% for the non-microroughened surface. Thus, using an exemplary alkali- and heat-treated nanopolymorphic surface, this study identified critical parameters necessary to describe the process and consequences of bone–implant integration, for which nanofeatures have specific and substantial roles beyond those of microfeatures. Nanofeature-enhanced osteoconductivity, which resulted in both the acceleration and elevation of bone–implant integration, has clearly been demonstrated.► We created nanopolymorphic titanium surfaces using alkali and heat treatment. ► The nanopolymorphic titanium features nanotufts, nanoplates, and nanonodules. ► The nanopolymorphic titanium accelerated and enhanced bone–implant integration.
Keywords: Osseointegration; Dental and orthopedic implant; Nanotechnology; Nanotuft;

Conducting polymers with immobilised fibrillar collagen for enhanced neural interfacing by Xiao Liu; Zhilian Yue; Michael J. Higgins; Gordon G. Wallace (7309-7317).
Conducting polymers with pendant functionality are advantageous in various bionic and organic bioelectronic applications, as they allow facile incorporation of bio-regulative cues to provide bio-mimicry and conductive environments for cell growth, differentiation and function. In this work, polypyrrole substrates doped with chondroitin sulfate (CS), an extracellular matrix molecule bearing carboxylic acid moieties, were electrochemically synthesized and conjugated with type I collagen. During the coupling process, the conjugated collagen formed a 3-dimensional fibrillar matrix in situ at the conducting polymer interface, as evidenced by atomic force microscopy (AFM) and fluorescence microscopy under aqueous physiological conditions. Cyclic voltammetry (CV) and impedance measurement confirmed no significant reduction in the electroactivity of the fibrillar collagen-modified conducting polymer substrates. Rat pheochromocytoma (nerve) cells showed increased differentiation and neurite outgrowth on the fibrillar collagen, which was further enhanced through electrical stimulation of the underlying conducting polymer substrate. Our study demonstrates that the direct coupling of ECM components such as collagen, followed by their further self-assembly into 3-dimensional matrices, has the potential to improve the neural-electrode interface of implant electrodes by encouraging nerve cell attachment and differentiation.
Keywords: Electroactive polymers; Electrical stimulation; Surface modification; Neural cells; Collagen; Neural prosthesis;

G-quadruplex DNAzyme based molecular catalytic beacon for label-free colorimetric logic gates by Jinbo Zhu; Tao Li; Libing Zhang; Shaojun Dong; Erkang Wang (7318-7324).
Efficient and economic DNA nanomaterials that can work as logic components are necessary for the development of DNA computers with high speed and outstanding data storage capacity. A new molecular catalytic beacon (MCB) and a series of label-free colorimetric logic gates based on the formation and dissociation of G-quadruplex DNAzyme were established in this work. These logic gates (NOT, NOR, IMPLICATION, AND, OR and INHIBIT) were realized by the interaction between the special designed oligonucleotide hairpins and the short input single strand complementary DNA. We were able to recognize the logic output signals effortlessly by our naked eyes. It is a simple, economic and safe approach for the design of complex multiple input DNA logic molecular device.
Keywords: Logic gate; Molecular beacon; DNAzyme; G-quadruplexes; Molecular devices;

The creation of a biomimetic interface between boron-doped diamond and immobilized proteins by René Hoffmann; Armin Kriele; Harald Obloh; Norio Tokuda; Waldemar Smirnov; Nianjun Yang; Christoph E. Nebel (7325-7332).
Immobilization of proteins on a solid electrode is to date done by chemical cross-linking or by addition of an adjustable intermediate. In this paper we introduce a concept where a solid with variable surface properties is optimized to mediate binding of the electron-transfer protein Cytochrome c (Cyt c) by mimicking the natural binding environment. It is shown that, as a carbon-based material, boron-doped diamond can be adjusted by simple electrochemical surface treatments to the specific biochemical requirements of Cyt c. The structure and functionality of passively adsorbed Cyt c on variously terminated diamond surfaces were characterized in detail using a combination of electrochemical techniques and atomic force microscopy with single-molecule resolution. Partially oxidized diamond allowed stable immobilization of Cyt c together with high electron transfer activity, driven by a combination of electrostatic and hydrophobic interactions. This surface mimics the natural binding partner, where coarse orientation is governed by electrostatic interaction of the protein’s dipole and hydrophobic interactions assist in formation of the electron transfer complex. The optimized surface mediated electron transfer kinetics around 100 times faster than those reported for other solids and even faster kinetics than on self-assembled monolayers of alkanethiols.
Keywords: Biomimetic material; Protein adsorption; Diamond; AFM (atomic force microscopy); Electrochemistry; Surface treatment;

A hierarchically graded bioactive scaffold bonded to titanium substrates for attachment to bone by Qingshan Fu; Youliang Hong; Xiaoguang Liu; Hongsong Fan; Xingdong Zhang (7333-7346).
In this paper we report a Ti-based, hierarchical porous scaffold anchored to Ti substrates, prepared by synthesizing hydroxyapatite–calcium carbonate-Ti three–layer spheres and combining a modified plasma spraying process and an anodic oxidation treatment. The hierarchical porous scaffolds were composed of 100–350 μm interconnecting macropores, 0.2–90 μm pores and ∼100 nm nanopores with >70% porosity. At the same time, the scaffolds also had the graded structures constructed by bioactive TiO x in surface transforming to metallurgy-bondable Ti in bottom. Mechanical property tests demonstrated that the porous scaffolds had similar Young’s modulus with natural bone and strong bonding strength with the Ti substrates. The simulate body fluid immersion showed that bone-like apatite layer could form rapidly at scaffold surface. The in vitro cell incubation demonstrated that the porous scaffolds had good cellular compatibility and could correctly regulate cascade gene expression of primary osteoblasts. The intramuscular implantations indicated the porous scaffolds had high osteoinductivity and the bone implantations demonstrated that the scaffolds could facilitate new bone growth and have strong bonding strength with surrounding bone.
Keywords: Hierarchical porous scaffolds; Osteoconductivity; Osteoinductivity; Osteointegration; Mechanical properties matching;

Three-dimensional arrayed amino aerogel biochips for molecular recognition of antigens by Yen K. Li; Yun-Chu Chen; Kai-Jen Jiang; Jui-chuang Wu; Yui W. Chen-Yang (7347-7354).
The three-dimensional (3D) biochips prepared in this study are composed of a glass microscopy slide arrayed with amino aerogel dots. The amino aerogel was produced using the sol–gel process, with an ionic liquid as the template followed by a solvent extraction to remove the template and build a three-dimensional mesoporous structure. The FTIR spectrum verified that the major template was removed and the 29Si solid-state NMR spectra recognized the cross-linkages in the SiO2 network structure. SEM images measured the particles at around 100 nm. After grinding, the BET analysis confirmed that the nano-size amino aerogel powders had exhibited specific surface area of 188 m2/g, pore volume of 0.83 cm3/g, and average pore size of 16.2 nm. The as-prepared amino aerogel surface contained amino functional groups capable of performing a sandwich immunoassay. The primary antibody was immobilized on the internal surface of the arrayed amino aerogel to capture its affinity antigen. On the top of the captured antigen, the report antibody was read its labeling fluorescent dye. In comparison to the corresponding two-dimensional (2D) biochip, the 3D amino aerogel biochips were observed to amplify signal intensities more effectively due to their remarkable capturing capability.
Keywords: Amino aerogel; Three-dimensional biochips; Arrayed biochips; Immunoassay; Human interleukin-6;

Creation of mouse embryonic stem cell-derived cardiac cell sheets by Katsuhisa Matsuura; Shinako Masuda; Yuji Haraguchi; Noriko Yasuda; Tatsuya Shimizu; Nobuhisa Hagiwara; Peter W. Zandstra; Teruo Okano (7355-7362).
Research on heart tissue engineering is an exciting and promising area. Although we previously developed bioengineered myocardium using cell sheet-based tissue engineering technologies, the issue of appropriate cell sources remained unresolved. In the present study, we created cell sheets of mouse embryonic stem (ES) cell-derived cardiomyocytes after expansion in three-dimensional stirred suspension cultures. Serial treatment of the suspension cultures with noggin and granulocyte colony-stimulating factor significantly increased the number of cardiomyocytes by more than fourfold compared with untreated cultures. After drug selection for ES cells expressing the neomycin-resistance gene under the control of the α-myosin heavy chain promoter, almost all of the cells showed spontaneous beating and expressed several cardiac contractive proteins in a fine striated pattern. When ES-derived cardiomyocytes alone were seeded onto temperature-responsive culture dishes, cell sheets were not created, whereas cocultures with cardiac fibroblasts promoted cell sheet formation. The cardiomyocytes in the cell sheets beat spontaneously and synchronously, and expressed connexin 43 at the edge of adjacent cardiomyocytes. Furthermore, when the extracellular action potential was recorded, unidirectional action potential propagation was observed. The present findings suggest that stirred suspension cultures with appropriate growth factors are capable of producing cardiomyocytes effectively and easily, and that ES-derived cardiac cell sheets may be a promising tool for the development of bioengineered myocardium.
Keywords: Cardiac tissue engineering; Stem cell; Cell culture; Fibroblast; Electrophysiology; Growth factors;

The components of extracellular matrix (ECM) may substitute for feeder layers that promote the self-renewal pathways in embryonic stem cells. Surface modification of electrospun nanofibrous scaffolds have been studied to closely resemble natural ECMs and support in vitro and in vivo proliferation, pluripotency and differentiation of stem cells. In this study, we analyzed the maintenance of stemness and pluripotency of the mouse embryonic stem cell (mESC) following feeder-free culture on collagen-grafted polyethersulfone (PES-COL) electrospun nanofibrous scaffold. Our results showed that, the mESCs cultured for seven passages on PES-COL scaffolds had a typical undifferentiated morphology, enhanced proliferation, stable diploid normal karyotype, and continued expression of stemness and pluripotency-associated markers, Oct-4, Nanog, SSEA-1, and Alkaline phosphatase (ALP) in comparison with PES scaffolds and gelatin-coated plate. Moreover, these cells retained their in vitro and in vivo pluripotency. Our results indicated the enhanced infiltration and teratoma formation of mESCs in PES-COL. Collagen-grafted polyethersulfone nanofibrous scaffold has potential for feeder-free culture of pluripotent stem cells because of its 3-dimensional structure and bioactivity which enhance pluripotency, proliferation, differentiation, and infiltration of embryonic stem cells.
Keywords: Embryonic stem cell; Feeder-free; Pluripotency; Polyethersulfone; Nanofiber; Surface treatment;

The role of cell surface markers and enamel matrix derivatives on human periodontal ligament mesenchymal progenitor responses in vitro by Philippe Kémoun; Stan Gronthos; Malcolm L. Snead; Jacqueline Rue; Bruno Courtois; Frédéric Vaysse; Jean-Pierre Salles; Gérard Brunel (7375-7388).
Periodontitis is a chronic-, infectious-disease of the human periodontium that is characterized by the loss of supporting tissues surrounding the tooth such as the periodontal ligament (PDL), cementum and alveolar bone. Regeneration of the periodontium is dependent on the participation of mesenchymal stem/stromal cells (MSC) resident in the PDL. Enamel matrix derivative (EMD), an extract from immature porcine enamel rich in amelogenin protein but that also contain bone morphogenetic protein (BMP), is used to treat periodontal defects. The effects of EMD on MSC cells of the PDL are not well characterized. In this in vitro study, we identify PDL progenitor cells from multiple individuals and demonstrate that EMD stimulates them. We show that the effect of EMD on cell proliferation and migration is mediated through the amelogenin it contains, while the differentiation of these progenitor cells to cell types of mineralized tissue is mainly due to BMP signaling.
Keywords: Periodontium; Mesenchymal stem cell; Bone morphogenetic protein; Amelogenin; Enamel matrix derivative (EMD);

Regulation of hepatic stem/progenitor phenotype by microenvironment stiffness in hydrogel models of the human liver stem cell niche by Oswaldo A. Lozoya; Eliane Wauthier; Rachael A. Turner; Claire Barbier; Glenn D. Prestwich; Farshid Guilak; Richard Superfine; Sharon R. Lubkin; Lola M. Reid (7389-7402).
Human livers have maturational lineages of cells within liver acini, beginning periportally in stem cell niches, the canals of Hering, and ending in polyploid hepatocytes pericentrally and cholangiocytes in bile ducts. Hepatic stem cells (hHpSCs) in vivo are partnered with mesenchymal precursors to endothelia (angioblasts) and stellate cells, and reside in regulated microenvironments, stem cell niches, containing hyaluronans (HA). The in vivo hHpSC niche is modeled in vitro by growing hHpSC in two-dimensional (2D) cultures on plastic. We investigated effects of 3D microenvironments, mimicking the liver’s stem cell niche, on these hHpSCs by embedding them in HA-based hydrogels prepared with Kubota’s Medium (KM), a serum-free medium tailored for endodermal stem/progenitors. The KM-HA hydrogels mimicked the niches, matched diffusivity of culture medium, exhibited shear thinning and perfect elasticity under mechanical loading, and had predictable stiffness depending on their chemistry. KM-HA hydrogels, which supported cell attachment, survival and expansion of hHpSC colonies, induced transition of hHpSC colonies towards stable heterogeneous populations of hepatic progenitors depending on KM-HA hydrogel stiffness, as shown by both their gene and protein expression profile. These acquired phenotypes did not show morphological evidence of fibrotic responses. In conclusion, this study shows that the mechanical properties of the microenvironment can regulate differentiation in endodermal stem cell populations.
Keywords: Maturational lineages; Human hepatic stem cells; Human hepatoblasts; Hyaluronans; Microenvironment mechanical properties; Differentiation;

Skeletal stem cell physiology on functionally distinct titania nanotopographies by Laura E. McNamara; Terje Sjöström; Karl E.V. Burgess; Joseph J.W. Kim; Er Liu; Simon Gordonov; Prabhas V. Moghe; R.M. Dominic Meek; Richard O.C. Oreffo; Bo Su; Matthew J. Dalby (7403-7410).
Functionalisation of the surface of orthopaedic implants with nanotopographies that could stimulate in situ osteogenic differentiation of the patient’s stem or osteoprogenitor cells would have significant therapeutic potential. Mesenchymal stem cell (MSC) responses to titanium substrates patterned with nanopillar structures were investigated in this study. Focal adhesions were quantified in S-phase cells, the bone-related transcription factor Runx2 was examined, osteocalcin production was noted, and Haralick computational analysis was used to assess the relatedness of the cell responses to each of the titanium substrata based on cytoskeletal textural features. Metabolomics was used as a novel means of assessing cellular responses to the biomaterial substrates by analysing the global metabolite profile of the cells on the substrata, and shows promise as a technique with high data yield for evaluating cell interactions with materials of different surface chemistry or topography. The cell response to 15 nm high nanopillars was distinct, consistent with a transition from a more quiescent phenotype on the planar substrate, to an ‘active’ phenotype on the pillars. These studies illustrate the potential for clinically relevant titania nanopillared substrata to modulate MSCs, with implications for orthopaedic device design and application.
Keywords: Nanotopography; Titanium; Mesenchymal stem cell; Osteogenesis; Molecular biology; Image analysis;

The knee meniscus: Structure–function, pathophysiology, current repair techniques, and prospects for regeneration by Eleftherios A. Makris; Pasha Hadidi; Kyriacos A. Athanasiou (7411-7431).
Extensive scientific investigations in recent decades have established the anatomical, biomechanical, and functional importance that the meniscus holds within the knee joint. As a vital part of the joint, it acts to prevent the deterioration and degeneration of articular cartilage, and the onset and development of osteoarthritis. For this reason, research into meniscus repair has been the recipient of particular interest from the orthopedic and bioengineering communities. Current repair techniques are only effective in treating lesions located in the peripheral vascularized region of the meniscus. Healing lesions found in the inner avascular region, which functions under a highly demanding mechanical environment, is considered to be a significant challenge. An adequate treatment approach has yet to be established, though many attempts have been undertaken. The current primary method for treatment is partial meniscectomy, which commonly results in the progressive development of osteoarthritis. This drawback has shifted research interest toward the fields of biomaterials and bioengineering, where it is hoped that meniscal deterioration can be tackled with the help of tissue engineering. So far, different approaches and strategies have contributed to the in vitro generation of meniscus constructs, which are capable of restoring meniscal lesions to some extent, both functionally as well as anatomically. The selection of the appropriate cell source (autologous, allogeneic, or xenogeneic cells, or stem cells) is undoubtedly regarded as key to successful meniscal tissue engineering. Furthermore, a large variation of scaffolds for tissue engineering have been proposed and produced in experimental and clinical studies, although a few problems with these (e.g., byproducts of degradation, stress shielding) have shifted research interest toward new strategies (e.g., scaffoldless approaches, self-assembly). A large number of different chemical (e.g., TGF-β1, C-ABC) and mechanical stimuli (e.g., direct compression, hydrostatic pressure) have also been investigated, both in terms of encouraging functional tissue formation, as well as in differentiating stem cells. Even though the problems accompanying meniscus tissue engineering research are considerable, we are undoubtedly in the dawn of a new era, whereby recent advances in biology, engineering, and medicine are leading to the successful treatment of meniscal lesions.► The meniscus is a frequently injured fibrocartilage vital for knee function. ► Meniscus cells are heterogeneous and may be replaced using different cell sources. ► Innovative scaffold and scaffoldless approaches are used to engineer the meniscus. ► Various biochemical agents and mechanical bioreactors may enhance meniscus tissue.
Keywords: Knee meniscus; Meniscus pathology; Meniscal repair; Tissue engineering; Scaffolds;

Vascular endothelial growth factor (VEGF) has been extensively investigated to promote vascularization at damaged or diseased sites and in tissue implants. Here we are interested in determining if the manner in which VEGF is presented from a scaffold to endothelial cells influences the architecture of the blood vessels formed. We bound VEGF to nanoparticles and placed these nanoparticles inside fibrin hydrogels, which contained human umbilical vein endothelial cells (HUVECs) bound to cytodex beads. Fibroblast cells are plated on top of the fibrin gel to further mimic a physiologic environment. In addition, we used a chorioallantoic membrane (CAM) assay to determine the role of VEGF presentation on angiogenesis in vivo. We tested VEGF bound in high density and low density to study differences between growth factor presentation in heterogeneous nanodomains and homogenous distribution. VEGF covalently bound to nanoparticles at high density led to an increase in HUVEC tube branching, thickness, and total vessel network length compared to soluble VEGF. While VEGF bound electrostatically exhibited no significant difference with covalently bound VEGF in the tube formation assay, this method failed to promote host vessel infiltration into the fibrin implant on the CAM. Together our data suggest that the mode of VEGF presentation to endothelial cells influences the vessel architecture and vascularization of implants in vivo.► VEGF is covalently immobilized to heparin nanoparticles. ► VEGF is clustered at high and low densities, and then placed in fibrin gels. ► VEGF conjugated to the particles maintains activity and promotes migration signaling. ► In tube formation assay, high density VEGF significantly improves HUVEC branching. ► In CAM assay, high density VEGF recruits functional host vessels into fibrin gel.
Keywords: Angiogenesis; Growth factors; Heparin; Nanoparticle; Endothelial cell; Fibrin;

The impact of heparin intercalation at specific binding sites in telopeptide-free collagen type I fibrils by Dimitar R. Stamov; T.A. Khoa Nguyen; Heather M. Evans; Thomas Pfohl; Carsten Werner; Tilo Pompe (7444-7453).
Collagen-based biomaterials are currently used as cell culture scaffolds in tissue engineering approaches. These materials are being developed with increased functional complexity, such as the incorporation of glycosaminoglycans. Our study shows the impact of heparin intercalation at specific binding sites in telopeptide-free collagen fibrils in terms of their structure, mechanics, and cell response. We demonstrate that heparin binds specifically and in a competitive manner along the tropocollagen helix at places that are occupied in vivo by telopeptides in fibrillar collagen type I. On the basis of this finding, we elucidate the reason for the in vivo dogma that heparin does not intercalate in fibrillar collagens. We further reveal the direct relationship among structure, mechanics, and function in terms of the effect of incorporation of intercalated heparin on the fibrillar structure, fibrillar bending modulus and flexural rigidity and the dynamic response of adherent cells to collagen scaffolds. This tight relationship is considered particularly important when designing xenogeneic scaffolds based on natural collagen type I to trigger cell proliferation and differentiation.
Keywords: Collagen type I; Telopeptides; Heparin; Biomechanics; Cell adhesion; Biomaterials;

Effectively bridging the lesion gap is still an unmet demand for spinal cord repair. In the present study, we tested our hypothesis if cograft of Schwann cells (SCs) and neural stem cells (NSCs) with genetically enhanced expression of neurotrophin-3 (NT-3) and its high affinity receptor TrkC, respectively, could strengthen neural repair through increased NSC survival and neuronal differentiation at the epicenter after complete T10 spinal cord transection in adult rats. Transplantation of NT-3-SCs + TrkC-NSCs in Gelfoam (1 × 106/implant/rat; n = 10) into the lesion gap immediately following injury results in significantly improved relay of the cortical motor evoked potential (CMEP) and cortical somatosensory evoked potential (CSEP) as well as ameliorated hindlimb deficits, relative to controls (treated with LacZ-SCs + LacZ-NSCs, NT-3-SCs + NSCs, NSCs alone, or lesion only; n = 10/group). Further analyses demonstrate that NT-3-SCs + TrkC-NSCs cografting augments levels of neuronal differentiation of NSCs, synaptogenesis (including inhibitory/type II-like synapses) and myelin formation of SCs, in addition to neuroprotection and outgrowth of serotonergic fibers in the lesioned spinal cord. Compared with controls, the treated spinal cords also show elevated expression of laminin, a pro-neurogenic factor, and decreased presence of chondroitin sulfate proteoglycans, major inhibitors of axonal growth and neuroplasticity. Together, our data suggests that coimplantation of neurologically compatible cells with compensatorily overexpressed therapeutic genes may constitute a valuable approach to study, and/or develop therapies for spinal cord injury (SCI).► Cograft of cells with compensatorily overexpressed genes in the injured spinal cord. ► NT-3-Schwann cells and TrkC-neural stem cells in the transected rat spinal cord. ► NT-3-SCs + TrkC-NSCs cografting augments neuronal differentiation of NSCs. ► Cograft of neural compatible cells is a valuable approach to study neurotrauma.
Keywords: Spinal cord injury; Neurotrophin-3; TrkC; Schwann cells; Neural stem cells; Transection;

The physiological performance of a three-dimensional model that mimics the microenvironment of the small intestine by Jacqueline Pusch; Miriam Votteler; Stella Göhler; Jasmin Engl; Martina Hampel; Heike Walles; Katja Schenke-Layland (7469-7478).
Our focus was to develop a three-dimensional (3D) human dynamic in vitro tissue model that mimics the natural microenvironment of the small intestine. We co-cultured human Caco-2 cells with primary-isolated human microvascular endothelial cells (hMECs) on decellularized porcine jejunal segments within a custom-made dynamic bioreactor system that resembles the apical and basolateral side of the intestine for up to 14 days. The obtained data were compared to results generated using routine static Caco-2 assays. We performed histology and immunohistochemistry. Permeability was measured using directed transport studies. Histological analyses revealed that in tissue-engineered segments, which had been cultured under dynamic conditions, the Caco-2 cells showed a high-prismatic morphology, resembling normal primary enterocytes within their native environment. We further identified that the transport of low permeable substances, such as fluorescein and desmopressin increased within the dynamic bioreactor cultures. Immunohistochemical staining showed a significantly higher expression of the efflux transport p-glycoprotein (p-gp) under dynamic culture conditions when compared to the static cultures. We conclude that the integration of physiological parameters is crucial for the establishment of a reliable 3D intestinal in vitro model, which enables the simulation of drug transport over the gut-blood-barrier in a simplified way.► We established a human 3D dynamic intestinal tissue model. ► In vitro simulation of the physiological microenvironment of small intestine. ► Defined physical signals support intestinal cell growth and transport behavior. ► Serves as a more reliable preclinical test surface for pharmaceutical substances. ► Our system enables the significant reduction of animal experiments.
Keywords: Organ culture; In vitro test; Co-culture; Cell morphology; ECM (extracellular matrix); Epithelial cell;

SAM-based cell transfer to photopatterned hydrogels for microengineering vascular-like structures by Nasser Sadr; Mojun Zhu; Tatsuya Osaki; Takahiro Kakegawa; Yunzhi Yang; Matteo Moretti; Junji Fukuda; Ali Khademhosseini (7479-7490).
A major challenge in tissue engineering is to reproduce the native 3D microvascular architecture fundamental for in vivo functions. Current approaches still lack a network of perfusable vessels with native 3D structural organization. Here we present a new method combining self-assembled monolayer (SAM)-based cell transfer and gelatin methacrylate hydrogel photopatterning techniques for microengineering vascular structures. Human umbilical vein cell (HUVEC) transfer from oligopeptide SAM-coated surfaces to the hydrogel revealed two SAM desorption mechanisms: photoinduced and electrochemically triggered. The former, occurs concomitantly to hydrogel photocrosslinking, and resulted in efficient (>97%) monolayer transfer. The latter, prompted by additional potential application, preserved cell morphology and maintained high transfer efficiency of VE-cadherin positive monolayers over longer culture periods. This approach was also applied to transfer HUVECs to 3D geometrically defined vascular-like structures in hydrogels, which were then maintained in perfusion culture for 15 days. As a step toward more complex constructs, a cell-laden hydrogel layer was photopatterned around the endothelialized channel to mimic the vascular smooth muscle structure of distal arterioles. This study shows that the coupling of the SAM-based cell transfer and hydrogel photocrosslinking could potentially open up new avenues in engineering more complex, vascularized tissue constructs for regenerative medicine and tissue engineering applications.► We combined SAM-based cell transfer and hydrogel photopatterning techniques. ► We identified two SAM desorption mechanisms: photoinduced and electrically triggered. ► Cell transfer was efficient and preserved cell viability and monolayer features. ► Vascular-like constructs lined with an endothelial cell monolayer were generated. ► Double-layered constructs were photopatterned for mimicking arterioles organization.
Keywords: Photocrosslinkable hydrogel; Zwitterionic oligopeptide; Electrochemical cell detachment; Gelatin methacrylate; Vascular microengineering; Endothelial monolayer;

Osteoconductive protamine-based polyelectrolyte multilayer functionalized surfaces by Raymond E. Samuel; Anita Shukla; Daniel H. Paik; Mary X. Wang; Jean C. Fang; Daniel J. Schmidt; Paula T. Hammond (7491-7502).
The integration of orthopedic implants with host bone presents a major challenge in joint arthroplasty, spinal fusion and tumor reconstruction. The cellular microenvironment can be programmed via implant surface functionalization allowing direct modulation of osteoblast adhesion, proliferation, and differentiation at the implant–bone interface. The development of layer-by-layer assembled polyelectrolyte multilayer (PEM) architectures has greatly expanded our ability to fabricate intricate nanometer to micron scale thin film coatings that conform to complex implant geometries. The in vivo therapeutic efficacy of thin PEM implant coatings for numerous biomedical applications has previously been reported. We have fabricated protamine-based PEM thin films that support the long-term proliferation and differentiation of pre-osteoblast cells on non-cross-linked film-coated surfaces. These hydrophilic PEM functionalized surfaces with nanometer-scale roughness facilitated increased deposition of calcified matrix by osteoblasts in vitro, and thus offer the potential to enhance implant integration with host bone. The coatings can make an immediate impact in the osteogenic culture of stem cells and assessment of the osteogenic potential of new therapeutic factors.► Protamine-based thin film with high stiffness and nanoscale roughness. ► Good adhesion and long-term proliferation of osteoblasts on top of multilayers. ► Multilayers are osteoconductive with enhanced deposition of calcified matrix.
Keywords: Osteoconduction; Surface modification; Protamine; Polyelectrolyte multilayers films; Layer-by-layer assembly; Osteoblast;

Stem cell-encapsulating hydrogel microbeads of several hundred microns in size suitable for injection, that could quickly degrade to release the cells, are currently unavailable. The objectives of this study were to: (1) develop oxidized alginate-fibrin microbeads encapsulating human umbilical cord mesenchymal stem cells (hUCMSCs); (2) investigate microbead degradation, cell release, and osteogenic differentiation of the released cells for the first time. Three types of microbeads were fabricated to encapsulate hUCMSCs: (1) Alginate microbeads; (2) oxidized alginate microbeads; (3) oxidized alginate-fibrin microbeads. Microbeads with sizes of about 100–500 μm were fabricated with 1 × 106 hUCMSCs/mL of alginate. For the alginate group, there was little microbead degradation, with very few cells released at 21 d. For oxidized alginate, the microbeads started to slightly degrade at 14 d. In contrast, the oxidized alginate-fibrin microbeads started to degrade at 4 d and released the cells. At 7 d, the number of released cells greatly increased and showed a healthy polygonal morphology. At 21 d, the oxidized alginate-fibrin group had a live cell density that was 4-fold that of the oxidized alginate group, and 15-fold that of the alginate group. The released cells had osteodifferentiation, exhibiting highly elevated bone marker gene expressions of ALP, OC, collagen I, and Runx2. Alizarin staining confirmed the synthesis of bone minerals by hUCMSCs, with the mineral concentration at 21 d being 10-fold that at 7 d. In conclusion, fast-degradable alginate-fibrin microbeads with hUCMSC encapsulation were developed that could start to degrade and release the cells at 4 d. The released hUCMSCs had excellent proliferation, osteodifferentiation, and bone mineral synthesis. The alginate-fibrin microbeads are promising to deliver stem cells inside injectable scaffolds to promote tissue regeneration.
Keywords: Alginate-fibrin microbeads; Fast-degradable hydrogel; Human umbilical cord stem cells; Cell release; Osteodifferentiation and mineralization; Tissue engineering;

A combined cell therapy and in-situ tissue-engineering approach for myocardial repair by Manhal Habib; Keren Shapira-Schweitzer; Oren Caspi; Amira Gepstein; Gil Arbel; Doron Aronson; Dror Seliktar; Lior Gepstein (7514-7523).
Myocardial cell-replacement strategies are hampered by limited sources for human cardiomyocytes and by significant cell loss following transplantation. We tested the hypothesis that a combined delivery of cardiomyocytes with an in-situ polymerizable hydrogel into a post-MI rat heart will result in better functional outcomes than each intervention alone. A photopolymerizable, biodegradable, PEGylated-fibrinogen (PF) hydrogel matrix was used as the carrier for the cardiomyocytes [neonatal rat ventricular cardiomyocytes (NRVCMs) or human embryonic stem cell-derived cardiomyocytes (hESC-CMs)]. Infarcted rat hearts (LAD ligation) were randomized to injection of saline, NRVCMs, biopolymer, or combined biopolymer-cell delivery. Echocardiography revealed typical post-infarction remodeling after 30 days in the saline-injected control group [deterioration of fractional shortening (FS) by 31.0 ± 3.6%]. Injection of NRVCMs or PF alone significantly (p < 0.01) altered this remodeling process (slightly increasing FS by 3.1 ± 6.6% and 0.5 ± 5.3% respectively). Co-injection of the NRVCMs with PF matrix resulted in a significant increase in the cell-graft area (by 144%) and in the highest improvements in FS (by 26.3 ± 6.6%). Finally, feasibility studies were performed with the PF matrix and hESC-CMs. We conclude that an injectable in-situ forming hydrogel can act as a cardiomyocyte cell-carrier and add to the beneficial effects of the grafted cells in preventing unfavorable post-infarction cardiac remodeling.
Keywords: Cardiac tissue engineering; Hydrogel; Cardiomyocyte; Stem cells;

Biomimetic scaffolds have been proposed as a means to facilitate tissue regeneration by multi-potent stromal cells (MSCs). Effective scaffold colonization requires a control of multiple MSC responses including survival, proliferation, differentiation, and migration. As MSC migration is relatively unstudied in this context, we present here a multi-level approach to its understanding and control, integratively tuning cell speed and directional persistence to achieve maximal mean free path (MFP) of migration. This approach employs data-driven computational modeling to ascertain small molecule drug treatments that can enhance MFP on a given materials substratum. Using poly(methyl methacrylate)-graft-poly(ethylene oxide) polymer surfaces tethered with epidermal growth factor (tEGF) and systematically adsorbed with fibronectin, vitronectin, or collagen-I to present hTERT-immortalized human MSCs with growth factor and extracellular matrix cues, we measured cell motility properties along with signaling activities of EGFR, ERK, Akt, and FAK on 19 different substrate conditions. Speed was consistent on collagen/tEGF substrates, but low associated directional persistence limited MFP. Decision tree modeling successfully predicted that ERK inhibition should enhance MFP on collagen/tEGF substrates by increasing persistence. Thus, we demonstrated a two-tiered approach to control MSC migration: materials-based “coarse-graining” complemented by small molecule “fine-tuning”.
Keywords: Mesenchymal stem cells; Cell migration; Extracellular matrix; Epidermal growth factor; Computational modeling;

Tissue engineering of clinical-relevant large tissue constructs remains a big challenge due to the mass transfer limit. A modular approach via the assembling of modular tissues thus eliminating the mass transfer limit holds great promise for fabricating centimeter-sized constructs. In the present study, we investigated the feasibility of using microcarriers seeded with adult mesenchymal stem cells (MSCs) to fabricate a large bone tissue. It was demonstrated that human amniotic MSCs (hAMSCs) were efficiently seeded onto CultiSpher S microcarriers (made of porcine gelatin) in a spinner flask and quickly proliferated while retaining a great viability. Within a total culture period of 28 days, using a two-stage culture strategy, hAMSCs-laden microcarriers with a high cell density were prepared at the first stage and the cells were then directly induced to undergo osteogenic differentiation in the same culture flask. During this cultivation process, the aggregation of cell-laden microcarriers was apparent, which resulted in aggregates of 700–800 μm, a size permissive for maintaining high cell viability. The osteogenic differentiation of hAMSCs on microcarriers was confirmed with increased mineral deposition (Alizarin red S staining and quantification of calcium content), ALP activity as well as gene expression of osteogenic markers (collagen type I and osteocalcin). These modular bone-like tissues were used as building blocks to fabricate a macroscopic bone construct in a cylindrical perfusion culture chamber (2 cm in diameter). After a 7-day perfusion culture, these modular tissues readily assembled into a centimeter-sized construct (diameter × height: 2 cm × 1 cm). Both good cell viability and fairly homogenous distribution of cellular content and bone-characteristic ECM within the macrotissue were elaborated. This paper provided a proof-of-concept study for modularly engineering clinical-relevant large tissue replacements with cell-laden microcarriers.
Keywords: Tissue engineering; Modular approach; Microcarriers; Macrotissue; Mesenchymal stem cells; Osteogenic differentiation;

The performance of BMP-2 loaded TCP/HAP porous ceramics with a polyelectrolyte multilayer film coating by Thomas Crouzier; Frédéric Sailhan; Pierre Becquart; Raphael Guillot; Delphine Logeart-Avramoglou; Catherine Picart (7543-7554).
Delivering rhBMP-2 (recombinant Bone Morphogenic Protein-2) at low but therapeutically efficient dose is one of the current challenges for bone tissue repair. In this context, Polyelectrolyte Multilayer films (PEM) represent an attractive rhBMP-2 carrier due to their ability to protect proteins from denaturation and to coat a wide variety of materials with complex geometry. Herein, we coated macroporous TCP/HAP granules with a biopolymeric PEM film to deliver rhBMP-2 in a “matrix-bound” manner. In vitro release kinetics indicated that the PEM-coated granules sequestered significant amounts of rhBMP-2. The degree of film cross-linking influenced the quantity of rhBMP-2 trapped within the films. Bare (uncoated) TCP/HAP scaffolds were also able to retain rhBMP-2. Bioactivity of rhBMP-2 in the PEM-coated granules was confirmed on two cell markers: luciferase expression on BMP-responsive-element/Luc C2C12 cells and alkaline phosphatase activity induction on C2C12 cells. Promisingly, rhBMP-2 adsorbed onto PEM-coated and on bare granules in a lesser extent, could be stored and remained bioactive over at least 3 weeks. In vivo, both uncoated and PEM-coated TCP/HAP granules loaded with rhBMP-2 exhibited both osteoconductive and osteoinductive properties. This opens perspective for coating these bioactive PEM on other types of implantable materials, including metal alloy that do not exhibit any affinity for rhBMP-2.
Keywords: Bone morphogenetic protein 2 (BMP-2); Polyelectrolyte multiplayer; Controlled drug release; Bone tissue engineering; Polysaccharides; Osteoinduction;

Mechanisms of hepatocyte attachment to keratin biomaterials by Jillian R. Richter; Roche C. de Guzman; Mark E. Van Dyke (7555-7561).
Keratin biomaterials support cellular adhesion, proliferation and migration, which have led to their exploitation in a variety of biomedical applications. The mechanism of cell adhesion to keratin biomaterials, however, is poorly understood. Therefore, the goal of this work was to investigate the mechanisms by which human hair keratin-based biomaterials facilitate cellular adhesion. Hepatocytes were used as a model cell type due to the abundance of published data on cell adhesion mechanisms and their relatively copious attachment to keratin substrates. The roles of β1- and β2-integrins and the hepatic asialoglycoprotein receptor (ASGPR) in hepatocyte adhesion to keratin substrates were studied using attachment assays with and without function blocking antibodies. Blocking of the hepatic integrin subunits did not decrease hepatocyte attachment to keratin. Furthermore, adhesion to keratin did not result in the formation of focal complexes or focal adhesions, nor did it produce an upregulation of phosphorylated-focal adhesion kinase. However, inhibition of hepatic ASGPR decreased the ability of hepatocytes to attach to keratin substrates, which is indicative of the role of this glycoprotein receptor in hepatocyte binding to keratin biomaterials.
Keywords: Keratin; Hepatocyte; Cell adhesion; Integrin; Glycoprotein receptor; Focal adhesion;

The effect of manipulation of silk scaffold fabrication parameters on matrix performance in a murine model of bladder augmentation by Pablo Gomez; Eun Seok Gil; Michael L. Lovett; Danielle N. Rockwood; Dolores Di Vizio; David L. Kaplan; Rosalyn M. Adam; Carlos R. Estrada; Joshua R. Mauney (7562-7570).
Autologous gastrointestinal segments are utilized as the primary option for bladder reconstructive procedures despite their inherent morbidity and significant complication rate. Multi-laminate biomaterials derived from Bombyx mori silk fibroin and prepared from a gel spinning process may serve as a superior alternative for bladder tissue engineering due to their robust mechanical properties, biocompatibility, and processing plasticity. In the present study, we sought to determine the impact of variations in winding (axial slew rate: 2 and 40 mm/s) and post-winding (methanol and lyophilization) fabrication parameters on the in vivo performance of gel spun silk scaffolds in a murine model of bladder augmentation. Three silk matrix groups with distinct structural and mechanical properties were investigated following 10 weeks of implantation including our original prototype previously shown to support bladder regeneration, Group 1 (2 mm/s, methanol) as well as Group 2 (40 mm/s, methanol) and Group 3 (40 mm/s, lyophilization) configurations. Non surgical animals were assessed in parallel as controls. Quantification of residual scaffold area demonstrated that while Group 1 and 2 scaffolds were largely intact, processing parameters utilized for Group 3 led to significantly higher degrees of scaffold degradation in comparison to Group 1. Histological (hematoxylin and eosin, masson’s trichrome) and immunohistochemical (IHC) analyses showed comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within the original defect site throughout all matrix groups similar to controls. Parallel evaluations demonstrated transitional urothelial formation with prominent uroplakin and p63 protein expression supported by Group 1 and 3 scaffolds, while Group 2 variants supported a thin, immature epithelium composed primarily of uroplakin-negative, p63-positive basal cells. Voided stain on paper analysis revealed similar voiding patterns between all matrix groups; however Group 2 animals displayed substantially lower voided volumes with increased frequency in comparison to controls. In addition, cystometric assessments revealed all matrix groups supported comparable degrees of bladder compliance similar to control levels. The results of this study demonstrate that selective alterations in winding and post-winding fabrication parameters can enhance the degradation rate of gel spun silk scaffolds in vivo while preserving their ability to support bladder tissue regeneration and function.
Keywords: Silk; Bladder tissue engineering; Smooth muscle cell; Epithelium; Urinary tract;

Lineage restricted progenitors for the repopulation of decellularized heart by Serina L.J. Ng; Karthikeyan Narayanan; Shujun Gao; Andrew C.A. Wan (7571-7580).
The severe shortage of available donor hearts necessitates the development of other options for heart replacement. Recent results underline the promise of the decellularized organ approach in engineering a functional heart. However, little is known so far regarding the ability of decellularized heart ECM to differentiate embryonic stem cells or committed progenitor cells. In the present work, we compared the differentiation potential of human embryonic stem cells (hESCs) and human mesendodermal cells (hMECs) derived from hESCs, in decellularized hearts under static culture. Expression of various cardiac specific markers such as cTnT, Nkx-2.5, Myl2, Myl7, Myh6 and CD31 was elucidated by gene expression, immunostaining and flow cytometry. Both hMECs and hESCs upregulated expression of cardiac markers upon differentiation, but they exclusively expressed genes for myosin light chain (Myl2, Myl7) and myosin heavy chain (Myh6), respectively. To enhance the differentiation ability of the stem/progenitor cells in the acellular constructs, they were implanted subcutaneously in SCID mice. Immunostaining of the explants revealed the persistence of cardiac marker expressing cells, but which lacked beating function. Our results indicate that the intact extracellular matrix components and preserved mechanical properties of the decellularized heart had directed differentiation of the stem/progenitor cells into the cardiac lineage.
Keywords: ECM (extracellular matrix); Heart; Stem cell; Progenitor cell;

Skin substitutes are the preferred treatment option in the case of extensive skin loss following burns or other injuries. Among skin substitutes, cultured skin substitutes containing autologous fibroblasts and keratinocytes on collagen–glycosaminoglycan (C-GAG) matrix are most preferred for wound repair. A significant negative outcome of wound healing is hypertrophic scarring (HTS), a dermal fibroproliferative disorder, that leads to considerable morbidity. To examine the role of superficial and deep dermal fibroblasts in HTS, and determine if they differentially remodel C-GAG matrices, fibroblasts were isolated from superficial and deep dermis of lower abdominal tissue of abdominoplasty patients and cultured on C-GAG matrices for four weeks. Over time, deep fibroblasts contracted and stiffened the matrices significantly more and decreased their ultimate tensile strength compared to superficial fibroblasts. Differential remodeling of C-GAG matrices by fibroblasts obtained from different locations of the same organ has not been reported before. Deep fibroblasts were found to express significantly more osteopontin, angiotensin-II, peroxisome proliferator-activated receptor (PPAR)-α, and significantly less tumor necrosis factor-α, PPAR-β/δ, PPAR-γ, and the proteoglycan, fibromodulin compared to superficial fibroblasts. These molecular targets could potentially be used in therapeutic strategies for treatment of HTS.
Keywords: Collagen–glycosaminoglycan; Remodeling; Biomechanical; Hypertrophic scar; Deep dermal fibroblast;

Quantum dots-based molecular classification of breast cancer by quantitative spectroanalysis of hormone receptors and HER2 by Chuang Chen; Sheng-Rong Sun; Yi-Ping Gong; Chu-Bo Qi; Chun-Wei Peng; Xue-Qin Yang; Shao-Ping Liu; Jun Peng; Shan Zhu; Ming-Bai Hu; Dai-Wen Pang; Yan Li (7592-7599).
The emerging molecular breast cancer (BC) classification based on key molecules, including hormone receptors (HRs), and human epidermal growth factor receptor 2 (HER2) has been playing an important part of clinical practice guideline. The current molecular classification mainly based on their fingerprints, however, could not provide enough essential information for treatment decision making. The molecular information on both patterns and quantities could be more helpful to heterogeneities understanding for BC personalized medicine. Here we conduct quantitative determination of HRs and HER2 by quantum dots (QDs)-based quantitative spectral analysis, which had excellent consistence with traditional method. Moreover, we establish a new molecular classification system of BC by integrating the quantitative information of HER2 and HRs, which could better reveal BC heterogeneity and identify 5 molecular subtypes with different 5-year prognosis. Furthermore, the emerging 5 molecular subtypes based on simple quantitative molecules information could be as informative as multi-genes analysis in routine practice, and might help formulate a more personalized comprehensive therapy strategy and prognosis prediction.► Hormone receptors and HER2 play an important role in breast cancer classification. ► Quantitative molecular information might be helpful for accurate classification. ► Quantum dots-based techinque allows for molecular quantitative determination. ► Three quantitative molecules information identify 5 distinct molecular subtypes. ► These 5 molecular subtypes might help formulate a more personalized medicine.
Keywords: Quantum dots; Breast carcinoma; Molecular classification; Hormone receptor; HER2; Quantitative analysis;

Targeted multifunctional gold-based nanoshells for magnetic resonance-guided laser ablation of head and neck cancer by Marites P. Melancon; Wei Lu; Meng Zhong; Min Zhou; Gan Liang; Andrew M. Elliott; John D. Hazle; Jeffrey N. Myers; Chun Li; R. Jason Stafford (7600-7608).
Keywords: Theranostics; Gold nanoshells; Magnetic resonance imaging; Laser ablation; Ultrasmall paramagnetic iron oxide (SPIO);

The induction of epigenetic regulation of PROS1 gene in lung fibroblasts by gold nanoparticles and implications for potential lung injury by Cheng-Teng Ng; S. Thameem Dheen; Wai-Cheong G. Yip; Choon-Nam Ong; Boon-Huat Bay; Lin-Yue Lanry Yung (7609-7615).
Advances in nanotechnology have given rise to the rapid development of novel applications in biomedicine. However, our understanding in the risks and health safety of nanomaterials is still not complete and various investigations are ongoing. Here, we show that gold nanoparticles (AuNPs) significantly altered the expression of 19 genes in human fetal lung fibroblasts (using the Affymetrix Human Gene 1.0 ST Array). Among the differentially expressed genes, up-regulation of microRNA-155 (miR-155) was observed concomitant with down-regulation of the PROS1 gene. Silencing of miR-155 established PROS1 as its possible target gene. DNA methylation profiling analysis of the PROS1 gene revealed no changes in the methylation status of this gene in AuNP-treated fibroblasts. At the ultrastructural level, chromatin condensation and reorganization was observed in the nucleus of fibroblasts exposed to AuNPs. The findings provide further insights into the molecular mechanisms underlying toxicity of AuNPs and their impact on epigenetic processes.
Keywords: Gold nanoparticles; miR-155; PROS1; Methylation; Nanotoxicity; Epigenetic;

Visualizing the endocytic and exocytic processes of wheat germ agglutinin by quantum dot-based single-particle tracking by Shu-Lin Liu; Zhi-Ling Zhang; En-Ze Sun; Jun Peng; Min Xie; Zhi-Quan Tian; Yi Lin; Dai-Wen Pang (7616-7624).
Wheat germ agglutinin (WGA) is a paradigm for understanding intracellular transport of lectins. As a protein exploiting the receptor-mediated endocytosis for internalization, WGA is also a valuable model system for exploring the endocytic and exocytic pathway. In this study, quantum dot-based single-particle tracking was performed to investigate the transport of WGA in live cells, revealing firstly that the endocytic and exocytic processes of WGA were both actin- and microtubule-dependent, each including five stages. The vesicle fusion event occurred near the cytomembrane, followed by two destinies with WGA: shedding to the extracellular or reversing to the cytoplasm. These findings suggest a distinct and dynamic scenario for the transport of lectins following a receptor-mediated endo/exocytic pathway in live cells. This is important for the application of lectins as drug carriers and antineoplastic drugs in medicine, and also offers insights into the pathway of endocytosis and exocytosis.
Keywords: Wheat germ agglutinin; Quantum dots; Single-particle tracking; Endocytic process; Exocytic process; Lectin;

MCF10A and MDA-MB-231 human breast basal epithelial cell co-culture in silicon micro-arrays by Mehdi Nikkhah; Jeannine S. Strobl; Eva M. Schmelz; Paul C. Roberts; Hui Zhou; Masoud Agah (7625-7632).
We developed istotropically etched silicon chip micro-arrays for co-culture of metastatic human breast cancer (MDA-MB-231) and non-tumorigenic human breast (MCF10A) cells. The micro-arrays were fabricated using a single-mask, single-etch step process. Each chip contained a 16 × 16 array of cavities 140 μm wide by 60 μm deep separated by planar silicon surfaces. Cells occupied 97–100% of the etched cavities. The cavities were enriched three-fold in MDA-MB-231 cells relative to the seeding ratio of, MDA-MB-231(1): MCF10A(10) cells. Micro co-cultures comprised of both MCF10A and MDA-MB-231 cells formed in 26% of cavities and contained 2–10 cells per cavity. Heterotropic cell interactions were seen in co-culture, and sites of these interactions were enriched with vinculin spikes. A selective morphological response to the histone deacetylase inhibitor (HDI), SAHA (suberoylanilide hydroxamic acid), occurred in MDA-MB-231 cells which was quantified by significant increases in cell length and cell area on flat surfaces and in the number of stretched cells inside the etched cavities. The morphology of MCF10A cells was unaltered in response to SAHA. Real time imaging showed the formation of highly dynamic and randomly orienting cytoplasmic extensions in MDA-MB-231 cells 1 h after adding SAHA; this is the first report of a rapid, morphological response in breast tumor cells to a histone deacetylase inhibitor. The findings demonstrate the utility of etched silicon micro-arrays for the propagation of human breast cell co-cultures and the application of HDI as a potential marker to distinguish metastatic breast cancer cells in a background of normal breast cell types.
Keywords: Micro-array; Silicon; Isotropic; Breast cancer; Co-culture;

The treatment of bladder cancer in a mouse model by epigallocatechin-3-gallate-gold nanoparticles by Dar-Shih Hsieh; Hang Wang; Shan-Wen Tan; Yi-Huei Huang; Cheng-Yuh Tsai; Ming-Kung Yeh; Chang-Jer Wu (7633-7640).
(−)-Epigallocatechin-3-gallate (EGCG), an active ingredient in green tea, was known to effectively inhibit formation and development of tumors. However, excessive uptake of EGCG was also known to cause cytotoxicity to normal cells. In this study, EGCGs that were physically attached onto the surface of nanogold particles (pNG) was confirmed by scanning electron microscopy. The anticancer activity of the EGCG-adsorbed pNG was investigated in C3H/HeN mice subcutaneously implanted with MBT-2 murine bladder tumor cells. EGCG–pNG was confirmed to inhibit tumor cell growing by means of cell apoptosis. The mechanism that EGCG–pNG mediates tumor apoptosis was uncovered to activate the caspase cascade through the Bcl-family proteins in the mitochondrial pathway. Additionally, the mechanism that tumors were suppressed by injecting EGCG–pNG directly into the tumor site was determined to be through downregulation of VEGF, whereas that by oral administration of EGCG was through reversing immune suppression upon cancer progression. In this assessment, the prepared EGCG–pNG was confirmed to be more effective than free EGCG in inhibiting bladder tumor in model mice.
Keywords: EGCG; Nanogold; Bladder cancer; Anti-tumor activity; Immuno-stimulating activity;

The effect of direct translocation across endosomes on the cytotoxicity of the recombinant protein e23sFv-Fdt-casp6 to HER2 positive gastric cancer cells by Jun-Lin Ren; Yan-Ling Meng; Bin Hu; Lin-Tao Jia; Rui Zhang; Yan-Ming Xu; Qiao-Sheng Xie; Ying-Qi Zhang; Bo-Quan Jin; Si-Yi Chen; Tao Wang; An-Gang Yang (7641-7650).
HER2-positive cancers represent a class of malignancies with high metastasis and poor prognosis. We previously generated the e23sFv-PEA II-casp6 recombinant, which contains an anti-HER2 single-chain antibody (e23sFv), a Pseudomonas exotoxin A translocation domain (PEA II), and a constitutively active caspase-6 (casp6), and demonstrated its potent selective anti-tumor activities. In this study, we generated a smaller-sized recombinant e23sFv-Fdt-casp6, in which the PEA II domain was replaced with the furin cleavage sequence from diphtheria toxin (Fdt), and explored its translocation pathway and specific killing mechanism. We found that e23sFv-Fdt-casp6 proteins, following their receptor-mediated endocytosis in HER2-positive gastric cancer cells, underwent furin-mediated cleavage in endosome and engaged in direct translocation of the released C-terminal fragment (active caspase-6) instead of via the trans-Golgi and the endoplasmic reticulum (ER) pathway. The active caspase-6 cleaved its well-documented substrate, Lamin A, and subsequently triggered the apoptosis of cancer cells. The e23sFv-Fdt-casp6 proteins produced from genetically modified cells showed a selective cytotoxicity to cultured HER2-positive gastric cancer cells. Similar to the results of our previous research on e23sFv-PEA II-casp6, the delivery of liposome-encapsulated e23sFv-Fdt-casp6 constructs in tumor-adjacent muscles also inhibited tumor growth and prolonged animal survival in a nude mouse xenograft tumor model. Moreover, e23sFv-Fdt-casp6 proteins were also cytotoxic to trastuzumab-resistant gastric cancer cells characterized by downregulated HER2 expression. Accordingly, e23sFv-Fdt-casp6 recombinant provides a promising therapeutic alternative for HER2-positive and trastuzumab-resistant gastric cancers.
Keywords: HER2; Gastric cancer; Apoptosis; Translocation;

Inflamed leukocyte-mimetic nanoparticles for molecular imaging of inflammation by Xiaoyue Chen; Richard Wong; Ildar Khalidov; Andrew Y. Wang; Jeerapond Leelawattanachai; Yi Wang; Moonsoo M. Jin (7651-7661).
Dysregulated host inflammatory response causes many diseases, including cardiovascular and neurodegenerative diseases, cancer, and sepsis. Sensitive detection of the site of inflammation will, therefore, produce a wide-ranging impact on disease diagnosis and treatment. We hypothesized that nanoprobes designed to mimic the molecular interactions occurring between inflamed leukocytes and endothelium may possess selectivity toward diverse host inflammatory responses. To incorporate inflammation-sensitive molecular interactions, super paramagnetic iron oxide nanoparticles were conjugated with integrin lymphocyte function-associated antigen (LFA)-1 I domain, engineered to mimic activated leukocytes in physiology. Whole body optical and magnetic resonance imaging in vivo revealed that leukocyte-mimetic nanoparticles localized preferentially to the vasculature within and in the invasive front of the tumor, as well as to the site of acute inflammation. This study explored in vivo detection of tumor-associated vasculature with systemically injected inflammation-specific nanoparticles, presenting a possibility of tumor detection by inflamed tumor microenvironment.
Keywords: Integrin; Inflammation; MRI; Nanoparticle;

Intracellular trafficking pathways involved in the gene transfer of nano-structured calcium phosphate-DNA particles by Dana Y.E. Olton; John M. Close; Charles S. Sfeir; Prashant N. Kumta (7662-7670).
Nano-structured calcium phosphate (NanoCaP) particles have been proven to be a powerful means of non-viral gene delivery. In order to better understand the mechanisms through which NanoCaPs-mediated mammalian cell transfection is achieved, we have sought to define the intracellular trafficking pathways involved in the cellular uptake and intracellular processing of these particles. Previous work has indicated that NanoCaP-DNA complexes are most likely internalized via endocytosis, however the subsequent pathways involved have not been determined. Through the use of specific inhibitors, we show that endocytosis of NanoCaP particles is both clathrin- and caveolae-dependent, and suggest that the caveolaer mechanism is the major contributor. We demonstrate colocalization of NanoCaP-pDNA complexes with known markers of both clathrin-coated and caveolar vesicles. Furthermore, through the use of quantitative flow cytometry, we present the first work in which the percent internalization of CaP-DNA complexes into cells is quantified. The overall goal of this research is to foster the continued improvement of NanoCaP-based gene delivery strategies.
Keywords: Nanoparticles; Calcium phosphate; Non-viral gene delivery; Clathrin-mediated endocytosis; Caveolae-mediated endocytosis;

The role of cationic comb-type copolymers in chaperoning DNA annealing by Rui Moriyama; Naohiko Shimada; Arihiro Kano; Atsushi Maruyama (7671-7676).
G-rich oligonucleotides tend to fall into kinetically trapped unstable structures because of their conformational polymorphism. Nucleic acid chaperones accelerate association of nucleic acids assemblies into the thermodynamically most stable conformations by decreasing the energy barrier for breakage or re-assembly of base pairings. Here, we report that an artificial nucleic acid chaperone, a cationic comb-type copolymer, promotes tetramolecular quadruplex assembly from mixtures of two different G-rich sequences, 5′-TGGGGT-3′ (TG4T) and 5′-TGGGGGT-3′ (TG5T). A 1:1 mixture of TG4T and TG5T mainly gave [TG4T▪(TG5T)3], [(TG4T)2▪(TG5T)2] and [(TG4T)3▪TG5T] heteroquadruplexes when the mixture was annealed by cooling from 90 °C to 4 °C at 1.0 °C/min. At a cooling rate of 0.01 °C/min the mixture mostly assembled into [TG4T]4 and [TG5T]4 homoquadruplexes, indicating that homoquadruplexes were thermodynamically more stable than heteroquadruplexes. In the presence of the copolymer, mainly homoquadruplexes were obtained at cooling rate of 1 °C/min, suggesting that the copolymer promoted formation of the thermodynamically most stable structures. We also showed that the copolymer facilitated the recombination of heteroquadruplexes to homoquadruplexes even at 20–30 °C, implying that the copolymer can promote thermodynamically preferred quadruplex assembly from oligonucleotides trapped in metastable structures. These results show that the copolymer works as a DNA annealer that induces proper assembly of stable DNA structures from heterogeneous kinetically trapped mixtures of structures.
Keywords: Cationic comb-type copolymer; G-quadruplex; Interpolyelectrolyte complex; Annealing activity; Nucleic acid chaperone;

Tunable chemistry and morphology of multi-wall carbon nanotubes as a route to non-toxic, theranostic systems by Sławomir Boncel; Karin H. Müller; Jeremy N. Skepper; Krzysztof Z. Walczak; Krzysztof K.K. Koziol (7677-7686).
Nanomedicine is one of the most promising areas of exploitation for multi-walled carbon nanotubes (MWNTs). These ‘needle-like’ nanovehicles are capable of carrying drug molecules via exo- and endohedral functionalisation and are steerable by an external magnetic field due to the presence of ferromagnetic nanoparticles in the nanotube core (up to 7.3 wt.%). These properties make them promising candidates for drug targeting or MRI contrast agents. Particularly, oxidised and nitrogen-doped MWNTs exhibiting enhanced chemical reactivity compared to their unmodified precursors/analogues could be exploited in this field. Here, we assessed the toxicity and intracellular localisation of two different, chemically modified and unmodified nanotubes towards human macrophage cells using a range of toxicity and imaging techniques. Oxidised and N-doped MWNTs were not significantly toxic to HMMs in contrast to unmodified MWNTs. All types of MWNTs entered the cell via active phagocytosis/endocytosis, but also passively by ‘self-injection’ through the plasma membrane, and were ultimately found in the cytoplasm and possibly also the nucleus. The attained results carry hope to utilise functionalised nanotube vectors as non-cytotoxic controllable drug delivery systems.
Keywords: Multi-walled carbon nanotubes; Nitrogen-doped carbon nanotubes; Oxidised carbon nanotubes; Cytotoxicity; Electron microscopy; Confocal microscopy;

Theranostic systems assembled in situ on demand by host-guest chemistry by Hyuntae Jung; Kyeng Min Park; Jeong-A. Yang; Eun Ju Oh; Don-Wook Lee; Kitae Park; Sung Ho Ryu; Sei Kwang Hahn; Kimoon Kim (7687-7694).
Theranostic systems have been explored extensively for a diagnostic therapy in the forms of polymer conjugates, implantable devices, and inorganic nanoparticles. In this work, we report theranostic systems in situ assembled by host-guest chemistry responding to a request. As a model theranostic system on demand, cucurbit[6]uril-conjugated hyaluronate (CB[6]-HA) was synthesized and decorated with FITC-spermidine (spmd) and/or formyl peptide receptor like 1 (FPRL1) specific peptide-spmd by simple mixing in aqueous solution. The resulting (FITC-spmd and/or peptide-spmd)@CB[6]-HA was successfully applied to the bioimaging of its target-specific delivery to B16F1 cells with HA receptors and its therapeutic signal transduction with elevated Ca2+ and phosphor-extracellular signal-regulated kinase (pERK) levels in FPRL1-expressing human breast adenocarcinoma (FPRL1/MCF-7) cells. Finally, we could confirm in vitro and in vivo stability of the highly specific host-guest interaction. The on-demand theranostic platform technology using host-guest chemistry can be exploited for various bioimaging, biosensing, drug delivery, and tissue engineering applications.
Keywords: Hyaluronic acid; Cucurbituril; Multi-functionality; Host-guest chemistry; Theranostic system;

Chondrogenesis of mesenchymal stem cells and dedifferentiated chondrocytes by transfection with SOX Trio genes by Han Na Yang; Ji Sun Park; Dae Gyun Woo; Su Yeon Jeon; Hyun-Jin Do; Hye-Young Lim; Seung Who Kim; Jae-Hwan Kim; Keun-Hong Park (7695-7704).
In this study, bone marrow-derived mesenchymal stem cells (MSCs), adipose-derived mesenchymal stem cells (ASCs) and dedifferentiated chondrocytes were transfected with SOX5, 6, and 9 genes (SOX Trio) and grown under pellet culture conditions (encapsulated in a fibrin hydrogel) to evaluate the chondrogenic potential in vitro and in vivo. RT-PCR, real-time quantitative PCR (qPCR), histology, and immunohistochemical assays were performed to determine the chondrogenic potential of the stem cells and dedifferentiated chondrocytes. Chondrogenic genes and proteins were more highly expressed in SOX Trio-expressing cells than in untransfected cells. In addition, not only specific genes and proteins, but cartilage-forming tissues were observed in nude mice transplanted with fibrin hydrogel encapsulated SOX Trio-expressing MSCs, ASCs, and dedifferentiated chondrocytes. Both in vitro and in vivo analyses revealed that fibrin hydrogel encapsulated cultured or transplanted cells transfected with the SOX Trio successfully differentiated into mature chondrocytes and could be used for the reconstruction of hyaline articular cartilage.
Keywords: Fibrin; hMSC; SOX Trio; Chondrogenesis; Matured chondrocytes;

A high-capacity, hybrid electro-microneedle for in-situ cutaneous gene transfer by Kwang Lee; Jung Dong Kim; Chang Yoel Lee; Song Her; Hyungil Jung (7705-7710).
Cutaneous gene transfer is limited by biological barriers such as skin and cellular membranes; complex approaches are required to overcome these biological barriers, simultaneously. Non-integrated systems that separate cutaneous permeation from intracellular transfection have been used to overcome skin and cellular barriers, respectively, however, do not provide sufficient doses of the gene to local tissue, resulting in inefficient gene transfer in-situ. Although integrated systems for cutaneous gene transfer are available, their safety has been questioned and it is difficult to transfer sufficient amounts of genes due to cumbersome sterilization procedures and the small size of the reservoir. Here, we demonstrate stepwise-aligned cutaneous permeation, cutaneous release, and intracellular transfection using a hybrid electro-microneedle (HEM), which designed as a monolithic hybrid assembly of a dissolving microneedle and an electrode, anomalously. Furthermore, as proof-of-principle, we use the HEM for in-situ cutaneous transfer of p2CMVmIL-12 to successfully treat B16F10 subcutaneous tumors in a mouse model. The HEM described herein holds great promise for cutaneous gene therapy of cancers and for vaccines.
Keywords: Hybrid electro-microneedle; Cutaneous gene transfer; Drawing lithography; Integrated system;

A pH-responsive mesoporous silica nanoparticles-based multi-drug delivery system for overcoming multi-drug resistance by Qianjun He; Yu Gao; Lingxia Zhang; Zhiwen Zhang; Fang Gao; Xiufeng Ji; Yaping Li; Jianlin Shi (7711-7720).
A type of pH-responsive nano multi-drug delivery systems (nano-MDDSs) with uniform particle size (100 ± 13 nm) and excellent monodispersity was developed by in situ co-self-assembly among water-insoluble anti-cancer drug (doxorubicin, DOX), surfactant micelles (CTAB) as chemosensitiver and silicon species forming drugs/surfactant micelles-co-loaded mesoporous silica nanoparticles (drugs@micelles@MSNs or DOX@CTAB@MSNs) via a micelles–MSNs self-assembly mechanism. The nano-MDDS DOX@CTAB@MSNs had a highly precise pH-responsive drug release behavior both in vitro and in vivo, and exhibited high drug efficiencies against drug-resistant MCF-7/ADR cells as well as drug-sensitive MCF-7 cells by the MSNs-mediated transmembrane delivery, the sustained drug release and the high anti-cancer and multi-drug resistance (MDR)-overcoming efficiencies. The MDR-overcoming mechanism was proved to be a synergistic cell cycle arrest/apoptosis-inducing effect resulted from the chemosensitization of the surfactant CTAB. These results demonstrated a very promising nano-MDDS for the pH-responsive controlled drug release and the cancer MDR overcoming.
Keywords: Mesoporous silica; Nanoparticle; Controlled drug release; Multi-drug delivery; One-pot self-assembly; Multi-drug resistance;

Self-assembled biodegradable amphiphilic PEG–PCL–lPEI triblock copolymers at the borderline between micelles and nanoparticles designed for drug and gene delivery by Thomas K. Endres; Moritz Beck-Broichsitter; Olga Samsonova; Thomas Renette; Thomas H. Kissel (7721-7731).
Amphiphilic PEG–PCL–PEI triblock copolymers self-assemble into nano-scaled, positively charged, multifunctional carriers, suitable for drug and gene delivery. A set of block copolymers with varying hydrophilic/hydrophobic ratio (systematically altered at the borderline of micelle and particle forming polymers) was synthesized, characterized and assembled into carriers. A detailed structural characterization in the liquid state of these assemblies was carried out: carrier size was determined using dynamic light scattering, cryogenic scanning electron microscopy and atomic force microscopy. Nuclear magnetic resonance analyses elucidated carrier’s core–shell structure. ζ-potential and thickness of the hydrophilic outer polymer shell were determined by laser Doppler anemometry. Subsequently the impact of carrier’s structure on its features (stability and toxicity) was investigated. Polymers hydrophilic in nature formed small (<40 nm) micelle-like carriers, whilst hydrophobic polymers aggregated to larger particle-like assemblies (>100 nm). Monitoring carrier size as a function of initial polymer concentration clarified different assembly mechanisms. Shell thickness, colloidal stability and toxicity were found to depend on the length of the hydrophilic polymer block. Due to controllable size, charge, stability and toxicity, this class of novel carriers is a promising candidate for prospective co-delivery of drugs and nucleic acids.
Keywords: Cationic triblock copolymer; Self-assembly; Nanoparticle; Micelle; Colloidal stability; Cytotoxicity;

Biodegradable polymer − cisplatin(IV) conjugate as a pro-drug of cisplatin(II) by Haihua Xiao; Ruogu Qi; Shi Liu; Xiuli Hu; Taicheng Duan; Yonghui Zheng; Yubin Huang; Xiabin Jing (7732-7739).
A Pt(IV) complex was covalently conjugated to a new biodegradable amphiphilic tri-block copolymer, MPEG-b-PCL-b-PLL, which contains pendant amino groups, to form a polymeric pro-drug of cisplatin(II), MPEG-b-PCL-b-PLL/Pt(IV). The conjugate was assembled into nano-micelles. The Pt(IV) complex, the polymer carrier and the conjugate were characterized systematically. In vitro release experiments showed that drug release from the polymer-Pt(IV) micelles follows an acid responsive and oxidation-reduction sensitive kinetics. HPLC-ICP-MS analysis revealed that cisplatin(II) can be released from the conjugate under an acidic plus a reductive condition which is available inside a cancerous cell. In vitro MTT assay demonstrated that the polymer-Pt(IV) micelles display higher cytotoxicity against SKOV-3 tumor cells than both cisplatin(II) and Pt(IV) complex. This enhanced cytotoxicity is attributed to effective internalization of the micelles by the cells via endocytosis mechanism, which was observed by fluorescence imaging and by direct determination of the platinum uptake by the cells. This polymer-Pt(IV) conjugate is a promising polymeric pro-drug of cisplatin in micellar form. It can protect the Pt(IV) complex against blood clearance. It can enter cancerous cells via endocytosis mechanism and then cisplatin(II) can be released. Therefore, this polymeric pro-drug of cisplatin is expected to find clinical applications in the future.
Keywords: Cisplatin; Platinum anti-tumor drugs; Polymer-drug conjugate; Cancer therapy; Drug delivery;