Biomaterials (v.31, #19)

The destruction of bone around joint replacements (periprosthetic osteolysis) is an adverse biological response associated with the generation of excessive wear particles. Wear debris from the materials used for joint replacements stimulate a chronic inflammatory and foreign body reaction that leads to increased osteoclast differentiation and maturation, and decreased bone formation. Wear debris induces both local and systemic trafficking of inflammatory cells to the site of particle generation. Recent studies have shown that this effect is mediated primarily by chemotactic cytokines (chemokines) including macrophage chemotactic protein-1 (MCP-1, also known as CCL2), macrophage inhibitory protein-1 (MIP-1), Interleukin-8 (IL-8 or CXCL8) and others. These ligands migrate along a concentration gradient to interact with G-protein-linked transmembrane receptors on the cell surface. Chemokines are involved in the innate and adaptive immune responses, angiogenesis, wound healing and tissue repair. In vitro, in vivo and tissue retrieval studies have shown that chemokine-directed systemic trafficking of polymorphonuclear leukocytes and cells of the monocyte/macrophage lineage to wear particles result in the release of pro-inflammatory factors and subsequent bone loss. Modulation of the chemokine ligand-receptor axis is a potential strategy to mitigate the adverse effects of wear particles from joint replacements.
Keywords: Joint replacement; Osteolysis; Wear; Particles; Chemokines;

The effect of 3D hydrogel scaffold modulus on osteoblast differentiation and mineralization revealed by combinatorial screening by Kaushik Chatterjee; Sheng Lin-Gibson; William E. Wallace; Sapun H. Parekh; Young Jong Lee; Marcus T. Cicerone; Marian F. Young; Carl G. Simon (5051-5062).
Cells are known to sense and respond to the physical properties of their environment and those of tissue scaffolds. Optimizing these cell–material interactions is critical in tissue engineering. In this work, a simple and inexpensive combinatorial platform was developed to rapidly screen three-dimensional (3D) tissue scaffolds and was applied to screen the effect of scaffold properties for tissue engineering of bone. Differentiation of osteoblasts was examined in poly(ethylene glycol) hydrogel gradients spanning a 30-fold range in compressive modulus (≈10 kPa to ≈300 kPa). Results demonstrate that material properties (gel stiffness) of scaffolds can be leveraged to induce cell differentiation in 3D culture as an alternative to biochemical cues such as soluble supplements, immobilized biomolecules and vectors, which are often expensive, labile and potentially carcinogenic. Gel moduli of ≈225 kPa and higher enhanced osteogenesis. Furthermore, it is proposed that material-induced cell differentiation can be modulated to engineer seamless tissue interfaces between mineralized bone tissue and softer tissues such as ligaments and tendons. This work presents a combinatorial method to screen biological response to 3D hydrogel scaffolds that more closely mimics the 3D environment experienced by cells in vivo.
Keywords: Tissue engineering; Hydrogels; Osteoblast; Combinatorial methods; Matrix stiffness; Graded tissues;

The effect of carboxydextran-coated superparamagnetic iron oxide nanoparticles on c-Jun N-terminal kinase-mediated apoptosis in human macrophages by Oleg Lunov; Tatiana Syrovets; Berthold Büchele; Xiue Jiang; Carlheinz Röcker; Kyrylo Tron; G. Ulrich Nienhaus; Paul Walther; Volker Mailänder; Katharina Landfester; Thomas Simmet (5063-5071).
Superparamagnetic iron oxide nanoparticles are frequently used for cell labeling or as diagnostic contrast media, yet studies analyzing their effects on immune cells remain scarce. Here we investigated how nanosized carboxydextran-coated superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) might affect human macrophages. Within 1 h, both SPIO and USPIO were rapidly taken up by macrophages. Confocal microscopy revealed that after 24 h the particles were almost exclusively localized within the lysosomal compartment. Continued cultivation of the macrophages for several days was associated with apoptosis induction caused by a long-lasting activation of the c-Jun N-terminal kinase (JNK) pathway. JNK activation was due to significantly elevated levels of reactive oxygen species, whereas no TNF-α was produced by the macrophages treated with nanoparticles. Compared to SPIO, USPIO induced more pronounced biochemical alterations and cytotoxicity, which could be antagonized by the JNK inhibitor V. Alternatively, treatment of macrophages with Trolox™ or N-acetyl-l-cysteine, two functionally different scavengers of reactive oxygen species, abolished both the JNK activation and the subsequent cytotoxic effects. These data indicate that nanosized superparamagnetic iron oxide-based contrast media exert cytotoxicity in human macrophages that can be functionally antagonized with radical scavengers.
Keywords: Macrophages; Cytotoxicity; Apoptosis; Antioxidant; Dextran;

The influence of hierarchical hybrid micro/nano-textured titanium surface with titania nanotubes on osteoblast functions by Lingzhou Zhao; Shenglin Mei; Paul K. Chu; Yumei Zhang; Zhifen Wu (5072-5082).
Hierarchical hybrid micro/nano-textured titanium surface topographies with titania nanotubes were produced by simple acid etching followed by anodization to mimic the hierarchical structure of bone tissues. Primary rat osteoblasts were used to evaluate the bioactivity. The microtopography formed by acid etching of titanium induced inconsistent osteoblast functions with initial cell adhesion and osteogenesis-related gene expression being dramatically enhanced while other cell behaviors such as proliferation, intracellular total protein synthesis and alkaline phosphatase activity, collagen secretion, and extracellular matrix mineralization being depressed. In comparison, addition of nanotubes to the microtopography led to enhancement of multiple osteoblast functions. Nearly all the cell functions investigated in this study were retained or promoted. Compared to a microtopography, the enhancement of multiple cell functions observed from the hierarchical micro/nano-textured surfaces is expected to lead to faster bone maturation around the titanium implants without compromising the bone mass. In addition, the hierarchical micro/nano-textured surfaces still retain the mechanical interlocking ability of the microtopography thereby boding well for osseointegration. Our study reveals a synergistic role played by the micro and nanotopographies in osteoblast functions and provides insight to the design of better biomedical implant surfaces.
Keywords: Titania; Osseointegration; Osteoblast; Nanotubes; Microtopography;

Biocompatibility of polymer grafted core/shell iron/carbon nanoparticles by Qingxin Mu; Lei Yang; James C. Davis; Raviraj Vankayala; Kuo Chu Hwang; Jincai Zhao; Bing Yan (5083-5090).
For biomedical applications, emerging nanostructures requires stringent evaluations for their biocompatibility. Core/shell iron/carbon nanoparticles (Fe@CNPs) are nanomaterials that have potential applications in magnetic resonance imaging (MRI), magnetic hyperthermia and drug delivery. However, their interactions with biological systems are totally unknown. To evaluate their potential cellular perturbations and explore the relationships between their biocompatibility and surface chemistry, we synthesized polymer grafted Fe@CNPs with diverse chemistry modifications on surface and investigated their dynamic cellular responses, cell uptake, oxidative stress and their effects on cell apoptosis and cell cycle. The results show that biocompatibility of Fe@CNPs is both surface chemistry dependent and cell type specific. Except for the carboxyl modified Fe@CNPs, all other Fe@CNPs present low toxicity and can be used for further functionalization and in a wide range of biomedical applications.
Keywords: Nanomaterials; Core/shell nanoparticles; Biocompatibility; RT-CES; Cytotoxicity;

In the present study, polymer monofilaments were embedded in fibrin seeded with human umbilical vein endothelial cells (HUVEC) to guide HUVEC attachment and migration in order to form oriented vessel-like structures between adjacent monofilaments. Histology and fluorescent fibrin experiments confirmed that microvessel-like structures, which were developing between polymer monofilaments embedded in fibrin, contained a lumen. The effect of human fibroblasts and growth factors (VEGF and bFGF) over the microvessel formation process was tested. The effects of VEGF and bFGF were dose-dependent. The effect of VEGF was optimum at the lower concentration tested (2 ng/mL), while that of bFGF was optimum at the higher tested concentration (20 ng/mL). Furthermore, the use of fibroblasts significantly improved the maturation of the microvessels compared to control and to samples cultured with VEGF and bFGF.
Keywords: Polyethylene terephthalate; Contact guidance; Directional angiogenesis; Microvessel formation; Multi-cellular lumen;

Incorporation of tripolyphosphate nanoparticles into fibrous poly(lactide-co-glycolide) scaffolds for tissue engineering by Shujun Xie; Qin Zhu; Bin Wang; Huijie Gu; Wei Liu; Lei Cui; Lian Cen; Yilin Cao (5100-5109).
Poly(lactide-co-glycolide) (PLGA) has been widely used for scaffolding materials in tissue engineering. It degrades mainly via hydrolysis of the ester bonds into lactic acid and glycolic acid leading to the decrease in pH of the surrounding microenvironment. The current study was designed to quickly neutralize the acidic degradation products of PLGA fibrous scaffolds by incorporating tripolyphosphate (TPP) nanoparticles into PLGA fibers. A homogeneous mixture of PLGA and TPP was first obtained by water-in-oil emulsion–dispersion followed by freeze-drying. The dried blend was melt-spun to yield fibers which were processed into scaffolds and subsequently immersed into phosphate-buffered saline (PBS) to verify the degradation properties. The pH of the saline was monitored for a duration of 80 days. The amount of TPP was optimized to obtain a PLGA based scaffolds without acidic degradation problems. Cellular compatibility of the modified and pristine scaffolds was evaluated using rabbit adipose-derived stem cells (rASCs). It was shown that TPP particles within the fibers were roughly 100 nm in diameter and mainly located inside fibers instead of on the superficial layer. The acidic degradation of PT-16 and PT-64 (PT-X is termed when the monomer molar ratio of TPP to PLGA was 1:X) was significantly improved as the pH values of their respective solutions were maintained in a well neutralized state during the degradation. PT-64 and PT-16 scaffolds could well support the attachment and proliferation of rASCs. Hence, the incorporation of TPP nanoparticles via an emulsion–dispersion method could be an effective strategy to improve/adjust the acidic degradation of PLGA and further pave the way for clinical applications of such polyesters.
Keywords: PLGA; Degradation; Incorporation; Tripolyphosphate; Nanoparticles;

The role of a recombinant fragment of laminin-332 in integrin α3β1-dependent cell binding, spreading and migration by Hironobu Yamashita; Manisha Tripathi; Mark P. Harris; Shanshan Liu; Brandy Weidow; Roy Zent; Vito Quaranta (5110-5121).
The extracellular matrix (ECM) is thought to be an essential component of tissue scaffolding and engineering because it fulfills fundamental functions related to cell adhesion, migration, and three-dimensional organization. Natural ECM preparations, however, are challenging to work with because they are comprised of macromolecules that are large and insoluble in their functional state. Functional fragments of ECM macromolecules are a viable answer to this challenge, as demonstrated by the RGD-based engineered scaffolds, where the tri-peptide, Arg-Gly-Asp (RGD), represents the minimal functional unit of fibronectin and related ECM. Laminins (Ln) are main components of epithelial tissues, since they enter into the composition of basement membranes. Application of Ln to epithelial tissue engineering would be desirable, since they could help mimic ideal functional conditions for both lining and glandular epithelial tissues. However, functional fragments of Ln that could be used in artificial settings have not been characterized in detail. In this paper, we describe the production and application of the recombinant LG4 (rLG4) fragment of laminin-332 (Ln-332), and show that it mimics three fundamental functional properties of Ln-332: integrin-mediated cell adhesion, spreading, and migration. Adhesive structures formed by cells on rLG4 closely resemble those formed on Ln-332, as judged by microscopy-based analyses of their molecular composition. As on Ln-332, focal adhesion kinase (FAK) is phosphorylated in cells adhering to rLG4, and colocalized with other focal adhesion components. We conclude that rLG4 could be a useful substitute to recapitulate, in vitro, the tissue scaffolding properties of Ln-332.
Keywords: Cell adhesion; Laminin; Integrins; Extracellular matrix; Biomimetic substrate;

The development of new biological materials, particularly those capable of serving as permissive substrates for cell growth, differentiation, and biological function, is a key area for advancing medical technology. In this work, we examined the characteristics of a hybrid hydrogel scaffold composed of poloxamer 407 (PO) and the self-assembling oligopeptdide EFK8 in vitro and in vivo. Rheological tests showed that the storage modulus of EFK8-PO increased by 4 orders of magnitude compared to that of EFK8 alone, indicating that EFK8-PO integrates PO’s high and tunable mechanical strength and integrity with the superior bioactivity of EFK8. When human adipose-derived mesenchymal stem cells (hAMSCs) were cultured in PO, we observed severe aggregation. Conversely, almost no aggregation was observed in EFK8 or EFK8-PO after 6 days of culture. hAMSC viability in all 3 hydrogels remained above 80% after 2 weeks of culture. EFK8 and EFK8-PO significantly increased hAMSC proliferation rates. In addition, EFK8- and EFK8-PO- but not PO encapsulated hAMSCs differentiated into adipocytes or osteoblasts when exposed to appropriate induction medium, suggesting EFK8 supports hAMSC multipotency in vitro. Moreover, only EFK8-PO supported hAMSC engraftment and adipogenic differenitiation post-transplantation into nude mice. Immunohistochemical analysis confirmed the new tissue was human in origin. Our studies show that EFK8-PO maintains improved mechanical properties and bioactivity relative to its individual constituents, supporting its potential use as a stem cell scaffold in soft tissue engineering.
Keywords: Hybrid hydrogel; Poloxamer 407; EFK8; Self-assembling peptide; Mesenchymal stem cells; Tissue engineering;

Oxygen mass transfer in a human tissue-engineered trachea by Efrem Curcio; Paolo Macchiarini; Loredana De Bartolo (5131-5136).
On June 2008, the first human tissue-engineered trachea replacement was performed using decellularized (de-antigenised) cadaveric donor trachea, seeded with recipient epithelial cells on the internal surface of the graft and mesenchymal stem-cell-derived chondrocytes on the external. During the follow-up, cytological analysis at 4 postoperative days showed a migration of the stem-cells derived chondrocytes from the outer to the inner surface of the first 2 cm of the graft length. With the aim to rationalize these clinical findings, and under the hypothesis that cellular migration is driven by the oxygen gradients developing from the external part of the construct (exposed to O2 deficiency) towards the better oxygenated epithelial region, an accurate computational model of oxygen transport in the trachea engineered construct was developed and solved using finite element method (FEM). Results confirm that critical limitation to oxygen transport prevalently occurs from proximal to middle section, within the first 2.8 cm of longitudinal length, in good agreement with experimental observation. In the proximal section, recognized as the most critical part of the engineered construct, the severe O2 mass transfer limitation causes a drastic reduction of the diffusive flux within a distance of 650 μm. At cell density of 1 × 107  cells/cm3, the 30% c.a of the total section area is under oxygen deficiency (O2 partial pressure below the critical threshold of 38 mmHg). Along the whole tracheal construct, the Thiele modulus ranges within 2.3 and 3.7 in the external chondrocyte compartment, confirming thus the importance of the mass transfer limitation to oxygen diffusion rate. In general, the efficiency of the O2 transport reduces considerably in the region close to proximal section.
Keywords: Tissue engineering; Airway transplantation; Oxygen; Mass transfer; Computational fluid dynamics;

Defined high protein content surfaces for stem cell culture by Michael R. Doran; Jessica E. Frith; Andrew B.J. Prowse; Jane Fitzpatrick; Ernst J. Wolvetang; Trent P. Munro; Peter P. Gray; Justin J. Cooper-White (5137-5142).
Unlocking the clinical potential of stem cell based therapies requires firstly elucidation of the biological mechanisms which direct stem cell fate decisions and thereafter, technical advances which allow these processes to be driven in a fully defined culture environment. Strategies for the generation of defined surfaces for human embryonic stem cell (hESC) and mesenchymal stem cell (MSC) culture remain in their infancy. In this paper we outline a simple, effective and efficient method for presenting proteins or peptides on an otherwise non-fouling Layer-by-Layer (LbL) self-assembled surface of hyaluronic acid (HA) and chitosan (CHI). We are able to generate a surface that has both good temporal stability and the ability to direct biological outcomes based on its defined surface composition. Surface functionalization is achieved through suspending the selected extracellular matrix (ECM) protein domain or extracted full-length protein in buffer containing a cross-linking agent (N-hydroxysulfosuccinimide/N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) over the LbL HA-CHI surface and then allowing the solvent to evaporate overnight. This simple, but important step results in remarkable protein deposition efficiencies often exceeding 50%, whereas traditional cross-linking methods result in such poor deposition of non-collagenous proteins that a.) quantification of bound amounts of protein is outside the resolution of commonly utilized protein assays, and b.) these surfaces are both unable to support cell attachment and growth. The utility of the protein-modified HA-CHI surfaces is demonstrated through the identification of specific hESC attachment efficiencies and through directing MSC osteogenic outcomes on these fully defined surfaces. This simple and scalable method is shown to enable the development of defined stem cell culture conditions, as well as the elucidation of the fundamental biological processes necessary for the realization of stem cell based therapies.
Keywords: Surface modification; Adhesion molecule; Hyaluronic acid/hyaluronan; Chitin/chitosan; Stem cell;

Nuclear morphology and deformation in engineered cardiac myocytes and tissues by Mark-Anthony P. Bray; William J. Adams; Nicholas A. Geisse; Adam W. Feinberg; Sean P. Sheehy; Kevin K. Parker (5143-5150).
Cardiac tissue engineering requires finely-tuned manipulation of the extracellular matrix (ECM) microenvironment to optimize internal myocardial organization. The myocyte nucleus is mechanically connected to the cell membrane via cytoskeletal elements, making it a target for the cellular response to perturbation of the ECM. However, the role of ECM spatial configuration and myocyte shape on nuclear location and morphology is unknown. In this study, printed ECM proteins were used to configure the geometry of cultured neonatal rat ventricular myocytes. Engineered one- and two-dimensional tissue constructs and single myocyte islands were assayed using live fluorescence imaging to examine nuclear position, morphology and motion as a function of the imposed ECM geometry during diastolic relaxation and systolic contraction. Image analysis showed that anisotropic tissue constructs cultured on microfabricated ECM lines possessed a high degree of nuclear alignment similar to that found in vivo; nuclei in isotropic tissues were polymorphic in shape with an apparently random orientation. Nuclear eccentricity was also increased for the anisotropic tissues, suggesting that intracellular forces deform the nucleus as the cell is spatially confined. During systole, nuclei experienced increasing spatial confinement in magnitude and direction of displacement as tissue anisotropy increased, yielding anisotropic deformation. Thus, the nature of nuclear displacement and deformation during systole appears to rely on a combination of the passive myofibril spatial organization and the active stress fields induced by contraction. Such findings have implications in understanding the genomic consequences and functional response of cardiac myocytes to their ECM surroundings under conditions of disease.
Keywords: Cardiac myocyte; Nucleus; Cytoskeleton; Myofibril; Extracellular matrix; Tissue engineering;

In vitro and in vivo changes to PLGA/sirolimus coating on drug eluting stents by Tingfei Xi; Runlin Gao; Bo Xu; Liang Chen; Tong Luo; Jing Liu; Yan Wei; Shengping Zhong (5151-5158).
The degradation behavior of the absorbable coating may have significant impacts to the short and long-term safety of the drug eluting stent (DES). A poly(lactide-co-glycolide) (PLGA) polymer coating containing sirolimus on stent surface was studied when stents were expanded to 3.0 mm. In vitro and in vivo degradation behavior of coated stents and cast films were characterized by light microscope, gel permeation chromatography (GPC), weight loss, field-emission environmental scanning electron microscope (ESEM-FEG) and SEM combined with energy dispersive spectrometer (EDS). For in vivo study, specially designed chambers with semi-permeable membrane were used to host the stents during the implantation. The data indicated the coating polymer lost 80% molecular weight and 60% of its mass in 60 days. The complete degradation of the coating occurred in 6 months. The degradation of the coating seemed to be homogeneous, and the coating went through a hydration/swelling process before it became completely degraded. It was noted that the coating maintained its integrity and strong adhesion to the strut during the entire degradation process, no fragmentation delaminate were observed. The study also demonstrated similar degradation behaviors of the drug coating in in vitro and in vivo conditions.
Keywords: Biodegradation; In vivo test; Stents; Polylactic acid copolymer;

Bactericidal behaviour of Ti6Al4V surfaces after exposure to UV-C light by Amparo M. Gallardo-Moreno; Miguel A. Pacha-Olivenza; María-Coronada Fernández-Calderón; Ciro Pérez-Giraldo; José M. Bruque; María-Luisa González-Martín (5159-5168).
TiO2-coated biomaterials that have been excited with UV irradiation have demonstrated biocidal properties in environmental applications, including drinking water decontamination. However, this procedure has not been successfully applied towards the killing of pathogens on medical titanium-based implants, mainly because of practical concerns related to irradiating the inserted biomaterial in situ. Previous researchers assumed that the photocatalysis on the TiO2 surface during UV application causes the bactericidal effects. However, we show that a residual post-irradiation bactericidal effect exists on the surface of Ti6Al4V, not related with photocatalysis. Using a combination of staining, serial dilutions, and a biofilm assay, we show a significant and time-dependent loss in viability of different bacterial strains of Staphylococcus epidermidis and Staphylococcus aureus on the post-irradiated surface. Although the duration of this antimicrobial effect depends on the strains selected, our experiments suggest that the effect lasts at least 60 min after surface irradiation. The origin of such phenomena is discussed in terms of the physical properties of the irradiated surfaces, which include the emission of energy and changes in surfaces charge occurring during electron-hole recombination processes. The method here proposed for the preparation of antimicrobial titanium surfaces could become especially useful in total implant surgery for which the antimicrobial challenge is mainly during or shortly after surgery.
Keywords: Titanium dioxide; Titanium alloy; Ultraviolet; Infection; Antimicrobial;

Electrodeposited polypyrrole/carbon nanotubes composite films electrodes for neural interfaces by Yi Lu; Tao Li; Xueqing Zhao; Mei Li; Yuliang Cao; Hanxi Yang; Yanwen Y. Duan (5169-5181).
The search for new electrode materials including new electrode modification methods is crucial for improving long-term performance of neuroprosthetic devices. In this study, an investigation of electrochemically co-deposited polypyrrole/single-walled carbon nanotube (PPy/SWCNT) films for improving the electrode–neural interface was reported. The PPy/SWCNT microelectrodes exhibited a particularly high safe charge injection (Q inj) limit of ∼7.5 mC/cm2 and low electrode impedance at 1 kHz, as well as good stability. Cell attachment and neurite outgrowth of rat pheochromocytoma (PC12) cells on the PPy/SWCNT deposited substrates were clearly observed by Calcein-AM staining and scanning electron microscope (SEM) analysis. Furthermore, tissue response was studied by a 6-week implantation in the cortex of rats. A significantly lower (p  < 0.05) glial fibrillary acidic protein (GFAP) and higher (p  < 0.05) neuronal nuclei (NeuN) immunostaining were found on comparison of the test group (n  = 11) with the control group (n  = 8), in the zone within the distance of 100 μm to the implant interface. All of these characteristics are desirable for chronically implantable neural probes with high density microelectrodes. Importantly, this technique can easily incorporate other modification methods to build a more advanced electrode–neural interface.
Keywords: Neural microelectrodes; Carbon nanotubes (CNTs); Conducting polymers (CPs); Electrode–neural interface; Neural prostheses; Neural stimulation microelectrodes;

A simple and effective approach was introduced to functionalize multi-walled carbon nanotubes (MWNTs) by in situ deposition of hydroxyapatite (HA) to improve their hydrophilicity and biocompatibility. Firstly, we prepared two types of pre-functionalized MWNTs: acid-oxidated MWNTs and covalently modified MWNTs by poly (ethylene glycol) (PEG). The influences of the acid-oxidated time, pre-phosphorylation, and PEGylation of MWNTs on in situ growth of HA were further investigated in simulated body fluid (SBF) with ionic concentration: 2, 5 and 10 times, respectively, at 37 °C for 24 h. The results exhibited that all these factors have positive effects on the HA crystals growth, especially the PEGylation of MWNTs plays a key role during the deposition. Finally, the methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate their cytotoxicity, which showed that the PEGylated MWNTs wrapped by HA crystals have the best biocompatibility.
Keywords: Nanotubes; Biocompatibility; Biomineralization; Hydroxyapatite; In situ;

The incorporation of GALA peptide into a protein cage for an acid-inducible molecular switch by Seung-Hye Choi; Kuiwon Choi; Ick Chan Kwon; Hyung Jun Ahn (5191-5198).
Caged proteins have been utilized as a biological container in a wide range of applications from material science to biomedicine, and GALA peptide has been known to undergo coil-to-helix transition upon the increased acidity. In this study, GALA synthetic peptide is incorporated to cage protein by genetic modification. Our engineered caged scaffold retains intact at the physiological pH but dissociate completely at pH 6.0, and the dissociated subunits are re-assembled simply by neutralization to biological pH. This acid-induced dissociation has the potential as molecular switch in vivo as well as in vitro so that the acid-sensitive caged proteins are applicable to drug delivery system for acidic target sites such as tumor. Since our design depends on the conformational transition of GALA peptide, not on removal of characteristic interface observed only in viral capsid-like protein, non-viral caged proteins can also be engineered to have molecular switching function. Therefore, this design for acid-sensitive scaffold would broaden the width of applications in nanotechnology including biomimetic material synthesis and biomedicine.
Keywords: Cage protein; GALA peptide; Self-assembly; Molecular switch; Disassembly;

The use of submicron/nanoscale PLGA implants to deliver paclitaxel with enhanced pharmacokinetics and therapeutic efficacy in intracranial glioblastoma in mice by Sudhir H. Ranganath; Yilong Fu; Davis Y. Arifin; Irene Kee; Lin Zheng; How-Sung Lee; Pierce K.-H. Chow; Chi-Hwa Wang (5199-5207).
Pharmacokinetics and therapeutic efficacy of submicron/nanoscale, intracranial implants were evaluated for treating malignant glioblastoma in mice. 9.1% (w/w) paclitaxel-loaded polylactide-co-glycolide (PLGA) nanofiber discs (F3) were fabricated and characterized for morphology and size distribution. Along with F3, three other formulations, 9.1% (w/w) paclitaxel-loaded PLGA submicron-fiber discs (F2), 16.7% (w/w) paclitaxel-loaded PLGA microspheres entrapped in hydrogel matrices (H80 and M80) were intracranially implanted in BALB/c mice and the coronal brain sections were analyzed for bio-distribution of paclitaxel on 14, 28 and 42 days post-implantation. BALB/c nude mice with intracranial human glioblastoma (U87 MG-luc2) were used in the therapeutic efficacy study. Animals were randomized to intracranial implantation of F3 and H80 with paclitaxel dose of 10 mg/kg, placebo F3, placebo H80, weekly intratumoral injection of Taxol® (10 mg/kg) or no treatment and the treatment response was analyzed by bioluminescence imaging and histological (H&E, Ki-67) examinations. Enhanced, therapeutic paclitaxel penetration (∼1 μm) in the mouse brain up to 5 mm from the implant site even after 42 days post-implantation from F3 and H80 was confirmed and deduced to be diffusion/elimination controlled. F3 and H80 demonstrated significant (∼30 fold) tumor inhibition and significantly low tumor proliferation index after 41 days of treatment in comparison to sham and placebo controls. The submicron/nanoscale implants are able to demonstrate optimal paclitaxel pharmacokinetics in the brain/tumor with significant tumor inhibition in a glioblastoma xenograft model in mice and hence could be potentially useful to treat highly recurrent GBM.
Keywords: Glioblastoma; Paclitaxel; Chemotherapy; Submicron/nanoscale; Polymers;

A microcomposite hydrogel for repeated on-demand ultrasound-triggered drug delivery by Hila Epstein-Barash; Gizem Orbey; Baris E. Polat; Randy H. Ewoldt; Jameel Feshitan; Robert Langer; Mark A. Borden; Daniel S. Kohane (5208-5217).
Here we develop an injectable composite system based for repeated ultrasound-triggered on-demand drug delivery. An in situ-cross-linking hydrogel maintains model drug (dye)-containing liposomes in close proximity to gas-filled microbubbles that serve to enhance release events induced by ultrasound application. Dye release is tunable by varying the proportions of the liposomal and microbubble components, as well as the duration and intensity of the ultrasound pulses in vitro. Dye is minimal at baseline. The composite shows minimal cytotoxicity in vitro, and benign tissue reaction after subcutaneous injection in rats. Ultrasound application also triggers drug release for two weeks after injection in vivo.
Keywords: On-demand; Triggered-release; Ultrasound; Drug delivery; Hydrogels;

The attenuation of central angiotensin II-dependent pressor response and intra-neuronal signaling by intracarotid injection of nanoformulated copper/zinc superoxide dismutase by Erin G. Rosenbaugh; James W. Roat; Lie Gao; Rui-Fang Yang; Devika S. Manickam; Jing-Xiang Yin; Harold D. Schultz; Tatiana K. Bronich; Elena V. Batrakova; Alexander V. Kabanov; Irving H. Zucker; Matthew C. Zimmerman (5218-5226).
Adenoviral-mediated overexpression of the intracellular superoxide (O2 ·−) scavenging enzyme copper/zinc superoxide dismutase (CuZnSOD) in the brain attenuates central angiotensin II (AngII)-induced cardiovascular responses. However, the therapeutic potential for adenoviral vectors is weakened by toxicity and the inability of adenoviral vectors to target the brain following peripheral administration. Therefore, we developed a non-viral delivery system in which CuZnSOD protein is electrostatically bound to a synthetic poly(ethyleneimine)–poly(ethyleneglycol) (PEI–PEG) polymer to form a polyion complex (CuZnSOD nanozyme). We hypothesized that PEI–PEG polymer increases transport of functional CuZnSOD to neurons, which inhibits AngII intra-neuronal signaling. The AngII-induced increase in O2 ·−, as measured by dihydroethidium fluorescence and electron paramagnetic resonance spectroscopy, was significantly inhibited in CuZnSOD nanozyme-treated neurons compared to free CuZnSOD- and non-treated neurons. CuZnSOD nanozyme also attenuated the AngII-induced inhibition of K+ current in neurons. Intracarotid injection of CuZnSOD nanozyme into rabbits significantly inhibited the pressor response of intracerebroventricular-delivered AngII; however, intracarotid injection of free CuZnSOD or PEI–PEG polymer alone failed to inhibit this response. Importantly, neither the PEI–PEG polymer alone nor the CuZnSOD nanozyme induced neuronal toxicity. These findings indicate that CuZnSOD nanozyme inhibits AngII intra-neuronal signaling in vitro and in vivo.
Keywords: Brain; Superoxide dismutase; Nanotechnology; Drug delivery; Copolymer; Potassium current;

Treatment of osteomyelitis with teicoplanin-encapsulated biodegradable thermosensitive hydrogel nanoparticles by Kuo-Ti Peng; Chin-Fu Chen; I.-Ming Chu; Yu-Min Li; Wei-Hsiu Hsu; Robert Wen-Wei Hsu; Pey-Jium Chang (5227-5236).
Osteomyelitis characterized by an inflammatory response often leads to bone loss and the spread of bacterial infection to surrounding soft tissues. To overcome the side effects induced by the systemic antibiotic treatment for osteomyelitis, recent investigations have explored the use of antibiotic-loaded undegradable or biodegradable delivery implants at the infected bone. Here, we show a novel biodegradable thermosensitive implant composed of poly(ethylene glycol) monomethyl ether (mPEG) and poly(lactic-co-glycolic acid) (PLGA) copolymer as a sol–gel drug delivery system for treating bone infection. The physical properties of a series of mPEG–PLGA nanocomposites, including the critical micelle concentration (CMC), particle size, polyindex (PI), sol–gel transition, viscosity and degradation rate, have been characterized in vitro. This sol-to-gel drug delivery system could provide several advantages in treating osteomyelitis, including easy preparation, 100% encapsulated rate, near-linear sustained release of drugs, injectable design and in situ gelling at the target tissue. Similar to the undegradable teicoplanin-impregnated polymethylmethacylate (PMMA) bone cements, we showed that implantation of the mPEG–PLGA hydrogel containing teicoplanin was effective for treating osteomyelitis in rabbits as detected by the histological staining and immunoblotting analyses. The use of the mPEG–PLGA-based biodegradable hydrogels may hold great promise as a therapeutic strategy for other infected diseases.
Keywords: Biodegradable; Drug delivery; Hydrogel; Infection; Nanoparticle; Sol–gel techniques;

Accelerated Achilles tendon healing by PDGF gene delivery with mesoporous silica nanoparticles by Arnaud Suwalski; Hinda Dabboue; Anthony Delalande; Sabine F. Bensamoun; Francis Canon; Patrick Midoux; Gérard Saillant; David Klatzmann; Jean-Paul Salvetat; Chantal Pichon (5237-5245).
We report the ability of amino- and carboxyl-modified MCM-41 mesoporous silica nanoparticles (MSN) to deliver gene in vivo in rat Achilles tendons, despite their inefficiency to transfect primary tenocytes in culture. We show that luciferase activity lasted for at least 2 weeks in tendons injected with these MSN and a plasmid DNA (pDNA) encoding the luciferase reporter gene. By contrast, in tendons injected with naked plasmid, the luciferase expression decreased as a function of time and became hardly detectable after 2 weeks. Interestingly, there were neither signs of inflammation nor necrosis in tendon, kidney, heart and liver of rat weekly injected with pDNA/MSN formulation during 1.5 months. Our main data concern the acceleration of Achilles tendons healing by PDGF-B gene transfer using MSN. Biomechanical properties and histological analyses clearly indicate that tendons treated with MSN and PDGF gene healed significantly faster than untreated tendons and those treated with pPDGF alone.
Keywords: Silica nanoparticle, MCM-41; Gene therapy; Growth factor; Tendon; Non viral gene delivery;

A leptin derived 30-amino-acid peptide modified pegylated poly-l-lysine dendrigraft for brain targeted gene delivery by Yang Liu; Jianfeng Li; Kun Shao; Rongqin Huang; Liya Ye; Jinning Lou; Chen Jiang (5246-5257).
The blood–brain barrier is the major obstacle that prevents diagnostic and therapeutic drugs being delivered to the central nervous systems in order to exert their effects. Specific ligand-receptor binding mediated endocytosis is one of the possible strategies to cross this barrier. A 30-amino-acid peptide (leptin30) derived from an endogenic hormone—leptin is exploited as brain-targeting ligand as it is reported to possess the same brain accumulation efficiency after intravenous injection. Dendrigraft poly-l-lysine (DGL) is used as non-viral gene vector in this study. DGL–PEG–Leptin30 was complexed with plasmid DNA yielding nanoparticles (NPs). The cellular uptake characteristic and mechanism were explored in brain capillary endothelial cells (BCECs) which express leptin receptors. Furthermore, brain parenchyma microglia cells such as BV-2 cells expressing leptin receptors could promote ligand-receptor mediated endocytosis leading to enhanced gene transfection ability of DGL–PEG–Leptin30/DNA NPs. The targeted NPs were proved to be transported across in vitro BBB model effectively and accumulate more in brains after i.v. resulting in a relatively high gene transfection efficiency both in vitro and in vivo. Besides, the NPs showed low cytotoxicity after in vitro transfection. Thus, DGL–PEG–Leptin30 provides a safe and noninvasive approach for the delivery of gene across the blood–brain barrier.
Keywords: Brain targeting; Gene delivery; Leptin; Non-viral vector; Dendrigraft poly-l-lysine;

Target specific delivery of small interfering RNA (siRNA) has been regarded as one of the most important technologies for the development of siRNA therapeutics. In this work, non-toxic low molecular weight (MW) polyethyleneimine (PEI, 2000 Da) was cross-linked with cystamine bisacrylamide (CBA) to prepare reducible PEI-SS in the body. Then, PEI-SS was conjugated with hyaluronic acid (HA) in the form of block-copolymer to enhance serum stability and facilitate target specific cellular uptake of siRNA by HA receptor mediated endocytosis. The cytotoxicity of (PEI-SS)-b-HA conjugate appeared to be negligible likely due to the degradation of PEI-SS to low MW PEI in the cytosol. Flow cytometric and confocal microscopic analyses confirmed the HA receptor mediated endocytosis of siRNA/(PEI-SS)-b-HA complex. The siRNA/(PEI-SS)-b-HA complex demonstrated an excellent in vitro gene silencing efficiency in the range of 50–80% reducing the mRNA expression level in the absence and presence of 50 vol% serum. Moreover, intra-tumoral injection of vascular endothelial growth factor (VEGF) siRNA/(PEI-SS)-b-HA complex resulted in dramatically inhibited tumor growth with reduced VEGF mRNA and VEGF levels in the tumors.
Keywords: Hyaluronic acid; Polyethyleneimine; siRNA; Target delivery; Gene silencing;

Engineered mesenchymal stem cells with self-assembled vesicles for systemic cell targeting by Debanjan Sarkar; Praveen K. Vemula; Weian Zhao; Ashish Gupta; Rohit Karnik; Jeffrey M. Karp (5266-5274).
Cell therapy has the potential to impact the quality of life of suffering patients. Systemic infusion is a convenient method of cell delivery; however, the efficiency of engraftment presents a major challenge. It has been shown that modification of the cell surface with adhesion ligands is a viable approach to improve cell homing, yet current methods including genetic modification suffer potential safety concerns, are practically complex and are unable to accommodate a wide variety of homing ligands or are not amendable to multiple cell types. We report herein a facile and generic approach to transiently engineer the cell surface using lipid vesicles to present biomolecular ligands that promote cell rolling, one of the first steps in the homing process. Specifically, we demonstrated that lipid vesicles rapidly fuse with the cell membrane to introduce biotin moieties on the cell surface that can subsequently conjugate streptavidin and potentially any biotinylated homing ligand. Given that cell rolling is a pre-requisite to firm adhesion for systemic cell homing, we examined the potential of immobilizing sialyl Lewis X (SLeX) on mesenchymal stem cells (MSCs) to induce cell rolling on a P-selectin surface, under dynamic flow conditions. MSCs modified with SLeX exhibit significantly improved rolling interactions with a velocity of 8 μm/s as compared to 61 μm/s for unmodified MSCs at a shear stress of 0.5 dyn/cm2. The cell surface modification does not impact the phenotype of the MSCs including their viability and multi-lineage differentiation potential. These results show that the transitory modification of cell surfaces with lipid vesicles can be used to efficiently immobilize adhesion ligands and potentially target systemically administered cells to the site of inflammation.
Keywords: Self-assembly; Mesenchymal stem cell; Inflammation;

The nano-morphological relationships between apatite crystals and collagen fibrils in ivory dentine by V. Jantou-Morris; Michael A. Horton; David W. McComb (5275-5286).
In this work, analytical transmission electron microscopy (TEM) was used to study the nanostructure of mineralised ivory dentine, in order to gain a clearer understanding of the relationship between the organic (collagen fibrils) and inorganic (calcium phosphate apatite crystals) components. Thin sections prepared by both focused ion beam (FIB) milling and ultramicrotomy, in the longitudinal and transverse planes, were investigated using electron energy-loss spectroscopy (EELS) in a monochromated field-emission gun scanning TEM (FEI Titan 80-300 FEGSTEM). Both low- and core-loss spectroscopy were used in the investigation, and the signals from phosphorous, carbon, calcium, nitrogen and oxygen were studied in detail. A combination of HAADF (high-angle annular dark-field)-STEM imaging and EELS analysis was used for simultaneous acquisition of both spatial and spectral information pixel by pixel (spectrum imaging). Across the collagen D banding in longitudinal sections, the relative thickness of the bright bands was significantly higher than that of the dark bands. Core-loss spectroscopy showed that the bright bands were richer in apatite than the dark bands. However, no ELNES variation was observed across the D banding. In transverse sections, significant changes in the carbon edge fine structure were observed at the interface between the extra- and intra-fibrillar regions.
Keywords: Ivory dentine; Collagen D banding; Apatite; FIB milling; HAADF-STEM; EELS;