Biomaterials (v.30, #30)

On the nature of biomaterials by David F. Williams (5897-5909).
The situations in which biomaterials are currently used are vastly different to those of just a decade ago. Although implantable medical devices are still immensely important, medical technologies now encompass a range of drug and gene delivery systems, tissue engineering and cell therapies, organ printing and cell patterning, nanotechnology based imaging and diagnostic systems and microelectronic devices. These technologies still encompass metals, ceramics and synthetic polymers, but also biopolymers, self assembled systems, nanoparticles, carbon nanotubes and quantum dots. These changes imply that our original concepts of biomaterials and our expectations of their performance also have to change. This Leading Opinion Paper addresses these issues. It concludes that many substances which hitherto we may not have thought of as biomaterials should now be considered as such so that, alongside the traditional structural biomaterials, we have substances that have been engineered to perform functions within health care where their performance is directly controlled by interactions with tissues and tissue components. These include engineered tissues, cells, organs and even viruses. This essay develops the arguments for a radically different definition of a biomaterial.
Keywords: Medical devices; Scaffolds; Gene vectors; Drug delivery; Engineered tissues; Nanostructured materials;

Scaffold-free vascular tissue engineering using bioprinting by Cyrille Norotte; Francois S. Marga; Laura E. Niklason; Gabor Forgacs (5910-5917).
Current limitations of exogenous scaffolds or extracellular matrix based materials have underlined the need for alternative tissue-engineering solutions. Scaffolds may elicit adverse host responses and interfere with direct cell–cell interaction, as well as assembly and alignment of cell-produced ECM. Thus, fabrication techniques for production of scaffold-free engineered tissue constructs have recently emerged. Here we report on a fully biological self-assembly approach, which we implement through a rapid prototyping bioprinting method for scaffold-free small diameter vascular reconstruction. Various vascular cell types, including smooth muscle cells and fibroblasts, were aggregated into discrete units, either multicellular spheroids or cylinders of controllable diameter (300–500 μm). These were printed layer-by-layer concomitantly with agarose rods, used here as a molding template. The post-printing fusion of the discrete units resulted in single- and double-layered small diameter vascular tubes (OD ranging from 0.9 to 2.5 mm). A unique aspect of the method is the ability to engineer vessels of distinct shapes and hierarchical trees that combine tubes of distinct diameters. The technique is quick and easily scalable.
Keywords: Tissue engineering; Three dimensional printing; Vascular grafts; Self assembly;

The use of glandular-derived stem cells to improve vascularization in scaffold-mediated dermal regeneration by José T. Egaña; Sandra Danner; Mathias Kremer; Daniel H. Rapoport; Jörn A. Lohmeyer; Julian F. Dye; Ursula Hopfner; Sergio Lavandero; Charli Kruse; Hans-Günther Machens (5918-5926).
Clinical success in tissue regeneration requires improvements in vascularization capacity of scaffolds. Several efforts have been made in this field including cellular and acellular technologies. In this work we combined the use of stem cells derived from pancreas or submandibular glands expressing green fluorescent protein (GFP+) with a commercially available scaffold for dermal regeneration. Cells were isolated, characterized and seeded in a scaffold for dermal regeneration. Scaffolds containing cells were used to induce dermal regeneration in a full skin defect model. After 3 weeks of in vivo regeneration, tissues were harvested and vascularization was analyzed. Results showed that gland-derived stem cells displayed stem cell features and presented multipotential differentiation capacity because they were able to differentiate in cell types representing the 3 different germ layers. After seeding, cells were homogeneously distributed and formed focal adhesions with the scaffold. Metabolic assays showed that cells can be cultured for at least 3 weeks in the scaffold. In vivo, the presence of pancreatic or submandibular stem cells significantly enhanced the vascularization compared to empty scaffolds. Presence of gland-derived stem cells in the regenerating tissue was confirmed by the detection of GFP expression in the wound area. In order to explore the possible mechanisms behind the improvement in vascular regeneration, in vitro experiments were performed, showing that gland-derived stem cells could contribute by angiogenic and vasculogenic mechanisms to this process. Our results suggest that the combined use of stem cells derived from glands and scaffold for dermal regeneration could be a rational alternative to improve vascularization in scaffold-mediated dermal regeneration.
Keywords: Tissue engineering; Tissue regeneration; Stem cells; Dermis; Vascularization;

Laminar-flow immediate-overlay hepatocyte sandwich perfusion system for drug hepatotoxicity testing by Lei Xia; Susanne Ng; Rongbin Han; Xiaoye Tuo; Guangfa Xiao; Hwa Liang Leo; Tianming Cheng; Hanry Yu (5927-5936).
Drug hepatotoxicity testing requires in vitro hepatocyte culture to maintain the long-term and stable liver specific functions. We developed a drug testing platform based on laminar-flow immediate-overlay hepatocyte sandwich perfusion culture. The immediate-overlay sandwich (collagen-coated porous polymeric membrane as top overlay) protects the cells and integrity of the top collagen matrix from the impact of flow. A bioreactor was designed that allowed proper control of shear stress and mass transfer. The culture parameters such as the optimal perfusion initiation time and flow rate were systematically and mechanistically determined. The optimized system could re-establish hepatocyte polarity to support biliary excretion and to maintain other liver specific functions, such as the biotransformation enzyme activities, for two weeks that extended the usable in vitro hepatocyte-based drug testing window. When the perfusion cultured hepatocytes from days 7 or 14 were used for drug testing, the APAP-induced hepatotoxicity measurements were more sensitive and consistent over time than the static culture control, enabling further exploitations in large-scale drug testing applications.
Keywords: Sandwich culture; Perfusion bioreactor; Mass transfer; Polarity; Shear stress; Stable functions;

Enzymatically fabricated and degradable microcapsules for production of multicellular spheroids with well-defined diameters of less than 150 μm by Shinji Sakai; Sho Ito; Yuko Ogushi; Ichiro Hashimoto; Natsuko Hosoda; Yoshinori Sawae; Koei Kawakami (5937-5942).
Microcapsules with a single, spherical hollow core less than 150 μm in diameter were developed to obtain multicellular spheroids with well-defined sizes of less than 150 μm in diameter. An aqueous solution of phenolic hydroxyl derivative of carboxymethylcellulose (CMC-Ph) containing human hepatoma cell line (HepG2) cells and horse radish peroxidase (HRP) was injected into a coflowing stream of liquid paraffin, containing H2O2, resulting in cell-enclosing CMC-Ph microparticles, 135 μm in diameter, via a peroxidase-catalyzed crosslinking reaction. The CMC-Ph microparticles were then coated with a phenolic hydroxyl derivative of alginate (Alg-Ph) gel membrane several dozen micrometers in thickness, crosslinked via the same enzymatic reaction process, followed by further crosslinking between the carboxyl groups of alginate by Sr2+. A hollow core structure was achieved by immersing the resultant microcapsules in a medium containing cellulase, which degrades the enclosed CMC-Ph microparticles. The HepG2 cells in the microcapsules then grew and completely filled the hollow core. Multicellular spheroids the same size as the CMC-Ph microparticles, with living cells at their outer surface, were collected within 1 min by soaking them in a medium containing alginate lyase to degrade the Alg-Ph gel microcapsule membrane.
Keywords: Alginate; Biodegradation; Cell encapsulation; Microcapsule; Microencapsulation; Enzyme;

Bioengineering of a functional sheet of islet cells for the treatment of diabetes mellitus by Hirofumi Shimizu; Kazuo Ohashi; Rie Utoh; Kazuya Ise; Mitsukazu Gotoh; Masayuki Yamato; Teruo Okano (5943-5949).
The present study was designed to establish a novel tissue engineering approach for diabetes mellitus (DM) by fabricating a tissue sheet composed of pancreatic islet cells for in vivo transplantation. Pancreatic islet cell suspensions were obtained from Lewis rats, and plated onto temperature-responsive culture dishes coated with extracellular matrix (ECM) proteins. After the cells reached confluency, islet cells cultured on laminin-5 coated dishes were successfully harvested as a uniformly spread tissue sheet by lowering the culture temperature to 20 °C for 20 min. The functional activity of the islet cell sheets was confirmed by histological examination and Insulin secretion assay prior to in vivo transplantation. Histological examination revealed that the harvested islet cell sheet was comprised of insulin- (76%) and glucagon- (19%) positive cells, respectively. In vivo functionality of the islet cell sheet was maintained even 7 days after transplantation into the subcutaneous space of Lewis rats. The present study describes an approach to generate a functional sheet of pancreatic islet cells on laminin-5 coated temperature-responsive dishes, which can be subsequently transplanted in vivo. This study serves as the foundation for the creation of a novel cell-based therapy for DM to provide patients an alternative method.
Keywords: Islet transplantation; Cell sheet; Laminin-5; Monolayer culture;

The growth and differentiation of mesenchymal stem and progenitor cells cultured on aligned collagen matrices by Babette Lanfer; Friedrich P. Seib; Uwe Freudenberg; Dimitar Stamov; Thomas Bley; Martin Bornhäuser; Carsten Werner (5950-5958).
Cell-matrix interactions are paramount for the successful repair and regeneration of damaged and diseased tissue. Since many tissues have an anisotropic architecture, it has been proposed that aligned extracellular matrix (ECM) structures in particular could guide and support the differentiation of resident mesenchymal stem and progenitor cells (MSCs). We therefore created aligned collagen type I structures using a microfluidic set-up with the aim to assess their impact on MSC growth and differentiation. In addition, we refined our aligned collagen matrices by incorporating the glycosaminoglycan (GAG) heparin to demonstrate the versatility of the applied methodology to study multiple ECM components in a single system. Our reconstituted, aligned ECM structures maintained and allowed multilineage (osteogenic/adipogenic/chondrogenic) differentiation of MSCs. Most noticeable was the observation that during osteogenesis, aligned collagen substrates choreographed ordered matrix mineralization. Likewise, myotube assembly of C2C12 cells was profoundly influenced by aligned topographic features resulting in enhanced myotube organization and length. Our results shed light on the regulation of MSCs through directional ECM structures and demonstrate the versatility of these cell culture platforms for guiding the morphogenesis of tissue types with anisotropic structures.
Keywords: Multipotent mesenchymal stromal cell; Extracellular matrix; Collagen type I; Microfluidic alignment; Shear flow deposition;

Rapid three-dimensional quantification of VEGF-induced scaffold neovascularisation by microcomputed tomography by Christian Schmidt; Deon Bezuidenhout; Michael Beck; Elizabeth Van der Merwe; Peter Zilla; Neil Davies (5959-5968).
Microcomputed tomography (micro-CT) is increasingly being used to analyze the three-dimensional structure and architecture of microvascular networks. Therefore we have evaluated a micro-CT analysis of VEGF-induced vessel ingrowth into a porous polyurethane scaffold through comparison with analyses by CD31 immunohistochemistry, vascular perfusion by intravital Lycopersicon esculentum lectin perfusion and vascular corrosion casting. Micro-CT scanning found a similar level of vascularisation within the VEGF treated scaffolds to that determined by the other analytical methods. However, although the relative increase in vascularisation (17 fold above PBS controls p  < 0.05) induced by VEGF determined by micro-CT was similar to the perfusion based analyses (20.1 and 10.4 fold for lectin perfusion and vascular corrosion respectively p  < 0.05), it differed substantially from that determined by CD31 immunohistochemistry (3.2 fold p  < 0.05). This difference was due to a large proportion of unperfused vessels in the PBS control that were not present in the VEGF group. The increase in perfusion probably resulted in part from an increase in average vessel diameter. Though this increase was detected by micro-CT, the actual diameters were overestimated by 60–90% most likely as a consequence of a merging effect for juxtaposed vessels. Thus whilst micro-CT gives an accurate three-dimensional quantification of the VEGF-induced increase in perfused vessels, resolution needs to be maximized for accurate sizing of a microvascular network's components.
Keywords: Microcomputed tomography; VEGF; Vascularisation; Scaffold;

Antibiofilm activity of nanosized magnesium fluoride by Jonathan Lellouche; Edith Kahana; Sivan Elias; Aharon Gedanken; Ehud Banin (5969-5978).
The ability of bacteria to develop antibiotic resistance and colonize abiotic surfaces by forming biofilms is a major cause of medical implant-associated infections and results in prolonged hospitalization periods and patient mortality. This raises the urgent need to develop compounds that can inhibit bacterial colonization of surfaces. In this study, we present an unreported microwave-based synthesis of MgF2 nanoparticles (Nps) using ionic liquid. We demonstrate the antimicrobial activity of these fluoride nanomaterials and their ability to restrict biofilm formation of common bacterial pathogens. Scanning and transmission electron microscopic techniques indicated that the MgF2·Nps attach and penetrate into the cells. Flow cytometry analysis revealed that the Nps caused a disruption in the membrane potential. The MgF2·Nps also induced membrane lipid peroxidation and once internalized can interact with chromosomal DNA. Based on these findings we further explored the possibility of using the MgF2·Nps to coat surfaces and inhibit biofilm formation. A microwave synthesis and coating procedure was utilized to coat glass coupons. The MgF2 coated surfaces effectively restricted biofilm formation of the tested bacteria. Taken together these results highlight the potential for developing MgF2 nanoparticles in order to inhibit bacterial infections.
Keywords: Nanoparticles; Magnesium fluoride; Biofilms; Antimicrobial properties; Sterile surfaces;

The disruption of bacterial membrane integrity through ROS generation induced by nanohybrids of silver and clay by Hong-Lin Su; Chih-Cheng Chou; Da-Jen Hung; Siou-Hong Lin; I-Chuan Pao; Jun-Hong Lin; Fang-Liang Huang; Rui-Xuan Dong; Jiang-Jen Lin (5979-5987).
Nanohybrids, synthesized via silver nitrate reduction in the presence of silicate clay, exhibit a high potency against bacterial growth. The plate-like clay, due to its anionic surface charges and a large surface area, serves as the support for the formation of silver nanoparticles (AgNPs) ∼30 nm in diameter. The nanohybrid consisting of Ag/silicate at a 7/93 weight ratio inhibited the growth of dermal pathogens including Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa and Streptococcus pyrogens, as well as the methicillin- and oxacillin-resistant S. aureus (MRSA and ORSA). Scanning electron microscope revealed that these nanohybrids were adherent on the surface of individual bacteria. The thin silicate plates provide a surface for immobilizing AgNPs in one highly concentrated area but prevent them from entering the cell membrane. Subsequent cytotoxicity studies indicated that surface contact with the reduced AgNPs on clay is sufficient to initiate cell death. This toxicity is related to a loss in membrane integrity due to reactive oxygen species (ROS) generation. The hybridization of AgNPs on clay surface is viable for generating a new class of nanohybrids exhibiting mild cytotoxicity but high efficacy for battling drug-resistant bacteria.
Keywords: Silver nanoparticles; Reactive oxygen species; Clay;

Cholesterol-mediated anchoring of enzyme-loaded liposomes within disulfide-stabilized polymer carrier capsules by Rona Chandrawati; Brigitte Städler; Almar Postma; Luke A. Connal; Siow-Feng Chong; Alexander N. Zelikin; Frank Caruso (5988-5998).
Polymer capsules containing multiple liposomes, termed capsosomes, are a promising new concept toward the design of artificial cells. Herein, we report on the fundamental aspects underpinning the assembly of capsosomes. A stable and high loading of intact liposomal cargo into a polymer film was achieved by non-covalently sandwiching the liposomes between a tailor-made cholesterol-modified poly(l-lysine) (PLLc) precursor layer and a poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMAc) capping layer. The film assembly, optimized on planar surfaces, was successfully transferred onto colloidal substrates, and a polymer membrane was subsequently assembled by the alternating adsorption of poly(N-vinyl pyrrolidone) (PVP) and thiol-modified poly(methacrylic acid) (PMASH) onto the pre-adsorbed layer of liposomes. Upon removal of the silica template, stable capsosomes encapsulating the enzyme luciferase or β-lactamase within their liposomal sub-compartments were obtained at both assembly (pH 4) and physiological conditions (pH 7.4). Excellent retention of the liposomes and the enzymatic cargo within the polymer carrier capsules was observed for up to 14 days. These engineered capsosomes are particularly attractive as autonomous microreactors, which can be utilized to repetitively add smaller reactants to cause successive distinct reactions within the capsosomes and simultaneously release the products to the surrounding environment, bringing these systems one step closer toward constructing artificial cells.
Keywords: Polymer capsules; Liposome; Cholesterol; Enzyme;

An electrochemical glucose biosensor exploiting a polyaniline grafted multiwalled carbon nanotube/perfluorosulfonate ionomer–silica nanocomposite by Anantha I. Gopalan; Kwang P. Lee; Dhanusuraman Ragupathy; Se H. Lee; Jong W. Lee (5999-6005).
A glucose biosensor was fabricated with loading of glucose oxidase (GOx) into a new organic–inorganic hybrid nanocomposite. The preparation involves formation of silica network into a Nafion (perfluorosulfonate ionomer) and subsequent loading of polyaniline grafted multiwalled carbon nanotubes (MWNT-g-PANI) onto Nafion–silica nanocomposite. Field emission scanning electron microscopy (FE-SEM) of Nafion–silica/MWNT-g-PANI composite reveals the presence of spherical silica particles (sizes in the range 250 nm–1 μm) and tubular MWNT-g-PANI particles. Chronoamperometry and cyclic voltammetry were used to evaluate the performance of biosensor towards glucose. The Nafion–silica/MWCNT-g-PANI/GOx biosensor exhibited a linear response to glucose in the concentration range of 1–10 mm with a correlation coefficient of 0.9972, good sensitivity (5.01 μA/mm), a low response time (∼6 s), repeatability (R.S.D value of 2.2%) and along-term stability. The presence of silica network within Nafion and MWNT-g-PANI synergistically contributes to the performance of the biosensor towards the electrochemical detection of glucose.
Keywords: Biosensor; Nanocomposite; Silica; Immobilization; Glucose oxidase;

A self-assembling nanoparticle for paclitaxel delivery in ovarian cancer by Kai Xiao; Juntao Luo; Wiley L. Fowler; Yuanpei Li; Joyce S. Lee; Li Xing; R. Holland Cheng; Li Wang; Kit S. Lam (6006-6016).
Paclitaxel (PTX) is one of the most effective chemotherapeutic drugs for the treatment of a variety of cancers. However, it is associated with serious side effects caused by PTX itself and the Cremophor EL emulsifier. In the present study, we report the development of a well-defined amphiphilic linear-dendritic copolymer (named as telodendrimer) composed of polyethylene glycol (PEG), cholic acid (CA, a facial amphiphilic molecule) and lysine, which can form drug-loaded core/shell micelles when mixed with hydrophobic drug, such as PTX, under aqueous condition. We have used PEG5k-CA8, a representive telodendrimer, to prepare paclitaxel-loaded nanoparticles (PTX-PEG5k-CA8 NPs) with high loading capacity (7.3 mg PTX/mL) and a size of 20–60 nm. This novel nanoformulation of PTX was found to exhibit similar in vitro cytotoxic activity against ovarian cancer cells as the free drug (Taxol®) or paclitaxel/human serum albumin nanoaggregate (Abraxane®). The maximum tolerated doses (MTDs) of PTX-PEG5k-CA8 NPs after single dose and five consecutive daily doses in mice were approximately 75 and 45 mg PTX/kg, respectively, which were 2.5-fold higher than those of Taxol®. In both subcutaneous and orthotopic intraperitoneal murine models of ovarian cancer, PTX-PEG5k-CA8 NPs achieved superior toxicity profiles and anti-tumor effects compared to Taxol® and Abraxane® at equivalent PTX doses, which were attributed to their preferential tumor accumulation, and deep penetration into tumor tissue, as confirmed by near infrared fluorescence (NIRF) imaging.
Keywords: Biocompatibility; Amphiphilic linear-dendritic copolymer; Micelle; Drug delivery; Chemotherapy; Ovarian cancer;

Transdermal immunization with low-pressure-gene-gun mediated chitosan-based DNA vaccines against Japanese encephalitis virus by Han-Ning Huang; Tsung-Lin Li; Yi-Lin Chan; Chien-Lung Chen; Chang-Jer Wu (6017-6025).
DNA vaccine is a milestone in contemporary vaccine development. It has considerably offset many shortcomings in conventional vaccines. Although DNA vaccines applied through ‘traditional’ high-pressure gene guns generally elicit high titers of protective immunity, such a practice however requires enormous investment in daunting instruments that often discourage vaccines due to an inevitable pain-eliciting effect. In this study, we exploited a less expensive yet low-pressure-gene-gun that can alleviate such phobia of pain. DNA vaccines were prepared by using the associative feature of cationic chitosan and anionic DNAs. The optimized N/P ratio is 3. The formulized complex sizes to nano-scale. The vaccine complexes were tested in C3H/HeN mice. The expression of GFP reporter gene was observable and traceable in epidermis and spleen over 3 days. The expressions of GFP and the activation of dendritic cells (DCs) were evident and co-localized in hair follicles and epidermis. C3H/HeN mice immunized with the developed chitosan-JEV DNA vaccines can elicit desired JEV specific antibodies, whereby the mice maintained high survival rates against 50 × LD50 JEV challenge. The low-pressure-gene-gun mediated chitosan-based JEV DNA vaccines have proven to be convenient and efficacious, thereby with high capacity in deployment for future prophylaxis against JEV outbreaks.
Keywords: Japanese encephalitis virus; DNA vaccine; Transdermal immunization; Gene carrier; Chitosan/DNA complex; Low-pressure-gene-gun;

Anti-Flt1 peptide of GNQWFI has been reported to inhibit vascular endothelial growth factor receptor 1 (VEGFR1) – mediated endothelial cell migration and tube formation. In this work, a protocol to synthesize anti-Flt1 peptide–hyaluronate (HA) conjugate was successfully developed for the treatment of corneal neovascularization. Using tetrabutyl ammonium salt of HA (HA-TBA), water-insoluble anti-Flt1 peptide could be conjugated with HA in dimethyl sulfoxide (DMSO) by the amide bond formation between carboxyl groups of HA and N-terminal amine groups of GGNQWFI. The formation of anti-Flt1 peptide–HA conjugate was confirmed by 1H NMR and fluorometric analyses. The average number of grafted peptide molecules in anti-Flt1 peptide–HA conjugates could be controlled from 3 to 30 per single HA chain by changing the feeding amount of peptide for the conjugation reaction. According to in vitro biological activity tests, anti-Flt1 peptide–HA conjugate exhibited a significant inhibition effect on the binding of Flt1-Fc to VEGF165 coated on the well. Furthermore, in vivo biological activity of anti-Flt1 peptide–HA conjugate was confirmed from the inhibitory effect on corneal neovascularization in silver nitrate cauterized corneas of SD rats. The VEGF receptor 2 expression was also reduced after treatment with anti-Flt1 peptide–HA conjugate. The water-soluble anti-Flt1 peptide–HA conjugate was thought to have a potential to be developed as anti-angiogenic therapeutics for the treatment of corneal neovascularization.
Keywords: Hyaluronate; Anti-Flt1 peptide; Conjugation; Drug delivery; Corneal neovascularization;

The antimicrobial activity of liposomal lauric acids against Propionibacterium acnes by Darren Yang; Dissaya Pornpattananangkul; Teruaki Nakatsuji; Michael Chan; Dennis Carson; Chun-Ming Huang; Liangfang Zhang (6035-6040).
This study evaluated the antimicrobial activity of lauric acid (LA) and its liposomal derivatives against Propionibacterium acnes (P. acnes), the bacterium that promotes inflammatory acne. First, the antimicrobial study of three free fatty acids (lauric acid, palmitic acid and oleic acid) demonstrated that LA gives the strongest bactericidal activity against P. acnes. However, a setback of using LA as a potential treatment for inflammatory acne is its poor water solubility. Then the LA was incorporated into a liposome formulation to aid its delivery to P. acnes. It was demonstrated that the antimicrobial activity of LA was not only well maintained in its liposomal derivatives but also enhanced at low LA concentration. In addition, the antimicrobial activity of LA-loaded liposomes (LipoLA) mainly depended on the LA loading concentration per single liposomes. Further study found that the LipoLA could fuse with the membranes of P. acnes and release the carried LA directly into the bacterial membranes, thereby killing the bacteria effectively. Since LA is a natural compound that is the main acid in coconut oil and also resides in human breast milk and liposomes have been successfully and widely applied as a drug delivery vehicle in the clinic, the LipoLA developed in this work holds great potential of becoming an innate, safe and effective therapeutic medication for acne vulgaris and other P. acnes associated diseases.
Keywords: Acne vulgaris; Antimicrobial; Free fatty acid; Lauric acid; Drug delivery; Liposome;

Targeted delivery and controlled release of doxorubicin to cancer cells using modified single wall carbon nanotubes by Xiaoke Zhang; Lingjie Meng; Qinghua Lu; Zhaofu Fei; Paul J. Dyson (6041-6047).
A targeted drug delivery system that is triggered by changes in pH based on single wall carbon nanotubes (SWCNTs), derivatized with carboxylate groups and coated with a polysaccharide material, can be loaded with the anticancer drug doxorubicin (DOX). The drug binds at physiological pH (pH 7.4) and is only released at a lower pH, for example, lysosomal pH and the pH characteristic of certain tumor environments. By manipulating the surface potentials of the modified nanotubes through modification of the polysaccharide coating, both the loading efficiency and release rate of the associated DOX can be controlled. Folic acid (FA), a targeting agent for many tumors, can be additionally tethered to the SWCNTs to selectively deliver DOX into the lysosomes of HeLa cells with much higher efficiency than free DOX. The DOX released from the modified nanotubes has been shown to damage nuclear DNA and inhibit the cell proliferation.
Keywords: Single wall carbon nanotubes (SWCNTs); Targeted drug delivery; Chitosan; Alginate sodium; Nanomedicine;

Degradable hydrogels have been extensively used in biomedical applications such as drug delivery, and recent interest has grown in hydrogels that degrade in recognition of a cellular response. This contribution describes a poly(ethylene glycol) (PEG) hydrogel platform with human neutrophil elastase (HNE) sensitive peptide cross-links formed using thiol-ene photopolymerization rendering the gel degradable at sites of inflammation. Further, protein therapeutics can be physically entrapped within the network and selectively released upon exposure to HNE. HNE-responsive hydrogels exhibited surface erosion where the degradation kinetics was influenced by changes in peptide k cat, concentration of HNE, and concentration of peptide within the gel. Using this platform, we were able to achieve controlled, zero-order release of bovine serum albumin (BSA) in the presence of HNE, and release was arrested in the absence of HNE. To further exploit the advantages of surface eroding delivery systems, a smaller protein (carbonic anhydrase) was delivered at the same rate as BSA and only dependent on gel formulation and environmental conditions. Also, protein release was predicted from a 3-layered hydrogel device using mass loss data. Lastly, the bioactivity of lysozyme was maintained above 90% following the exposure to thiol-ene photopolymerization conditions.
Keywords: Hydrogel; Drug delivery; Photopolymerization; Inflammation;

During endocytosis, pH-triggered release of encapsulated therapeutics from delivery carriers may accelerate their intracellular trafficking increasing therapeutic efficacy. To improve the therapeutic potential of targeted immunochemotherapy using anti-HER2/neu liposomal doxorubicin, we exploit the formation of leaky heterogeneities on rigid lipid bilayers to extensively release doxorubicin during endocytosis. We have previously demonstrated that pH-dependent formation of phase-separated lipid heterogeneities on the plane of a bilayer membrane increases the permeability of bilayers when they are composed of lipid pairs with rigid non-matching acyl chain lengths. This was suggested to be due to defective packing among lipids residing at the interfaces of lipid domains. Here we design nanometer-size antiHER2/neu-labeled PEGylated vesicles composed of lipid pairs with longer non-matching acyl chain lengths (n  = 18 and 21). These vesicles exhibit superior killing efficacy of cancer cells compared to established liposome formulations, and their killing efficacy is similar to the effect of combined free doxorubicin and free antiHER2/neu antibody. Other transport-related properties such as liposome blood circulation times, and specific binding and internalization by cancer cells are unaffected. These results demonstrate the potential of vesicles with pH-triggered leaky heterogeneities to increase the therapeutic potential of targeted immunochemotherapy.
Keywords: Liposome; pH-triggered; Immunochemotherapy; Heterogeneous membranes;

Gold nanoparticles with a monolayer of doxorubicin-conjugated amphiphilic block copolymer for tumor-targeted drug delivery by Mani Prabaharan; Jamison J. Grailer; Srikanth Pilla; Douglas A. Steeber; Shaoqin Gong (6065-6075).
Gold (Au) nanoparticles (NPs) stabilized with a monolayer of folate-conjugated poly(l-aspartate-doxorubicin)-b-poly(ethylene glycol) copolymer (Au-P(LA-DOX)-b-PEG-OH/FA) was synthesized as a tumor-targeted drug delivery carrier. The Au-P(LA-DOX)-b-PEG-OH/FA NPs consist of an Au core, a hydrophobic poly(l-aspartate-doxorubicin) (P(LA-DOX)) inner shell, and a hydrophilic poly(ethylene glycol) and folate-conjugated poly(ethylene glycol) outer shell (PEG-OH/FA). The anticancer drug, doxorubicin (DOX), was covalently conjugated onto the hydrophobic inner shell by acid-cleavable hydrazone linkage. The DOX loading level was determined to be 17 wt%. The Au-P(LA-DOX)-b-PEG-OH/FA NPs formed stable unimolecular micelles in aqueous solution. The size of the Au-P(LA-DOX)-b-PEG-OH/FA micelles were determined as 24–52 and 10–25 nm by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The conjugated DOX was released from the Au-P(LA-DOX)-b-PEG-OH/FA micelles much more rapidly at pH 5.3 and 6.6 than at pH 7.4, which is a desirable characteristic for tumor-targeted drug delivery. Cellular uptake of the Au-P(LA-DOX)-b-PEG-OH/FA micelles facilitated by the folate-receptor-mediated endocytosis process was higher than that of the micelles without folate. This was consistent with the higher cytotoxicity observed with the Au-P(LA-DOX)-b-PEG-OH/FA micelles against the 4T1 mouse mammary carcinoma cell line. These results suggest that Au-P(LA-DOX)-b-PEG-OH/FA NPs could be used as a carrier with pH-triggered drug releasing properties for tumor-targeted drug delivery.
Keywords: Gold nanoparticles; pH-sensitive; Drug delivery; Tumor-targeted; Cellular uptake; Cytotoxicity;

The current investigation aimed to develop a biomimetic, three-dimensional (3D) culture system for poorly adherent bone metastatic prostate cancer cells (C4-2B) for use as an in vitro platform for anti-cancer drug screening. To this end, hyaluronic acid (HA) derivatives carrying complementary aldehyde (HAALD) and hydrazide (HAADH) groups were synthesized and characterized. In situ encapsulation of C4-2B cells was achieved by simple mixing of HAALD and HAADH in the presence of the cells. Unlike two-dimensional (2D) monolayer culture in which cells adopt an atypical spread morphology, cells residing in the HA matrix formed distinct clustered structures which grew and merged, reminiscent of real tumors. Anti-cancer drugs added to the media surrounding the cell/gel construct diffused into the gel and killed the embedded cells. The HA hydrogel system was used successfully to test the efficacy of anti-cancer drugs including camptothecin, docetaxel, and rapamycin, alone and in combination, including specificity, dose and time responses. Responses of cells to anti-neoplastics differed between the 3D HA hydrogel and 2D monolayer systems. We suggest that the data obtained from 3D HA systems is superior to that from conventional 2D monolayers as the 3D system better reflects the bone metastatic microenvironment of the cancer cells.
Keywords: Prostate cancer; Bone metastasis; Hyaluronic acid; Hydrogels; 3D; Drug selection;

Prolonged local antibiotics delivery from hydroxyapatite functionalised with cyclodextrin polymers by Stéphane Leprêtre; Feng Chai; Jean-Christophe Hornez; Guillaume Vermet; Christel Neut; Michel Descamps; Hartmut F. Hildebrand; Bernard Martel (6086-6093).
Per-operative infection is a common complication for bone-graft surgery. Combining antiseptic agents with graft materials may offer a solution by increasing local drug concentration at target sites. Aiming to achieve a sustained local antibiotic (ATB) delivery for a widely applied bone substitute material - hydroxyapatite (HA), we attempted incorporating hydroxypropyl-β-cyclodextrin polymer (polyHPβCD) into microporous HA via impregnating either in a CD monomers mixture solution or a pre-synthesized CD polymer solution, followed by thermal fixation processing. In such functionalised material (CD-HA), polyHPβCD could entrap ATBs and release them progressively. Infrared-spectroscopic analysis confirmed the presence of polyHPβCD in functionalised HA via both processing pathways; polyHPβCD functionalisation yields were quantitated by thermogravimetric analysis for optimising the processing regime. Ciprofloxacin (CFX) and vancomycin (VCM), commonly applied in orthopaedics, have been respectively loaded on CD-HA by dip-coating. For both ATBs, kinetic release test in phosphate buffered saline showed significantly increased initial-burst amount and prolonged release from CD-HA compared with those from non-functionalised HA. Encouragingly, ATBs loaded CD-HA also revealed a prolonged bacteriostatic activity against Staphylococcus aureus and progressively increased cytocompatibility to osteoblasts (MC3T3-E1). Overall, polyHPβCD functionalisation on HA could be an effective drug-delivery model for loading different drug molecules in prevention of infection.
Keywords: Hydroxyapatite; Antibacterial; Surface modification; Bone graft; Drug delivery;

Biomimetic apatite-coated alginate/chitosan microparticles as osteogenic protein carriers by Min Lee; Weiming Li; Ronald K. Siu; Julie Whang; Xinli Zhang; Chia Soo; Kang Ting; Benjamin M. Wu (6094-6101).
Bone morphogenetic proteins (BMPs) are currently approved for spinal fusion, tibial fracture repair, and maxillofacial bone regeneration. However, BMP pleiotropism, paradoxical activities on precursor cells, and unexpected side effects at local and ectopic sites may limit their usage. Thus, the need remains for alternative osteoinductive factors that provide more bone-specific activities with fewer adverse effects. Nell-1 [Nel-like molecule-1; Nel (a protein highly expressed in neural tissue encoding epidermal growth factor like domain)] is a novel osteogenic protein believed to specifically target cells committed to the osteogenic lineage. The objective of this project is to incorporate Nell-1 into a moldable putty carrier that can adapt to bony defects and deliver Nell-1 to the local microenvironment. We show here that moldability can be achieved by mixing hyaluronan hydrogel with two types of particles: demineralized bone powder for osteoconductivity, and biomimetic apatite-coated alginate/chitosan microparticles for controlled Nell-1 delivery. Besides enhancing overall osteoconductivity of the carrier, the biomimetic apatite coating also provides a more sustained release (∼15% cumulative release over 30 days) and greatly reduces the initial burst release that is observed with non-coated alginate/chitosan microparticles (∼40% release after 1 day). The efficacy of Nell-1 delivery from these carriers was evaluated in a rat spinal fusion model against Nell-free carriers as controls. At 4 weeks post-implantation, Nell-1 enhanced spinal fusion rates as assessed by manual palpation, radiographs, high-resolution micro-computerized tomography (μCT), and histology. This moldable putty carrier system appears to be a suitable carrier for promoting osteogenesis, and will be further evaluated in larger animal models over longer periods to follow the remodeling of the regenerated bone.
Keywords: Biomimetic apatite; Alginate; Chitosan; Microparticles; Nell-1; Controlled release;

The effect of osteoprotegerin gene modification on wear debris-induced osteolysis in a murine model of knee prosthesis failure by Tao Zhang; Haiying Yu; Weiming Gong; Laibo Zhang; Tanghong Jia; Paul H. Wooley; Shang-You Yang (6102-6108).
Using an in vivo adeno-associated virus (AAV)-mediated gene transfer technique, this study evaluated the therapeutic effects of an osteoprotegerin (OPG) transgene against orthopaedic wear debris-induced osteolysis in a long-term murine model. A titanium pin was surgically implanted into proximal tibia of Balb/c mice to mimic a weight-bearing knee arthroplasty, followed by an intra-articular challenge with Ti particles to provoke periprosthetic inflammation and osteolysis. rAAV-hOPG or AAV-LacZ vectors were injected into the prosthetic joint at 3 weeks post-op. The tissues were harvested at 2, 4, 12 and 24 weeks after transduction for histological and molecular analyses. Successful transgene expression at the local site was confirmed by real-time PCR and ELISA. Inflammatory pseudo-membranes were ubiquitously present at the interface between the Ti implant and the surrounding bone in both LacZ and virus-free control groups, while soft tissue was only observed sporadically at the bone–implant interface in the OPG group. A significant reduction in TRAP+ osteoclast numbers was observed in the OPG treatment group. MicroCT assessment indicated a marked reversal in the loss of peri-implant bone mineral density (BMD) in the OPG-transduced group, when compared with the LacZ and virus-free controls. Further, OPG gene modification appeared to reduce local bone collagen loss by a mean of 40%. Real-time PCR examination confirmed that in vivo OPG gene transfer dramatically influenced the periprosthetic tissue gene expression profiles by diminishing the mRNA expression of TNF, IL-1, CPK and RANKL. There were no transgene-associated toxic effects apparent during the experiment, and the PCR detection of transgenes in remote organs such as lungs, kidneys, liver, and muscle of contralateral limb were consistently negative. Overall, rAAV-mediated OPG gene transfer effectively reversed Ti-particle-induced bone resorption in this experimental model. The therapeutic effects may be due to the blockage of local osteoclastogenesis and possibly the down-regulation of RANKL expression.
Keywords: Aseptic loosening; Gene therapy; Wear debris; Osteolysis; Adeno-associated viral vectors;

PAMAM–Triamcinolone acetonide conjugate as a nucleus-targeting gene carrier for enhanced transfer activity by Kun Ma; Min-Xin Hu; Yan Qi; Ji-Hong Zou; Li-Yan Qiu; Yi Jin; Xiao-Ying Ying; Hong-Ying Sun (6109-6118).
The excellent transfection efficiency and viability are essential for successful gene therapy. It suggested that when bound to its glucocorticoid receptor, glucocorticoid steroid can dilate the nuclear pore complexes and facilitated the transport of pDNA into the nucleus. In this research, the two different degrees of substitution of PAMAM–triamcinolone acetonide (PAMAM–TA) conjugates were synthesised for efficient translocation of pDNA into the nucleus. The physicochemical properties of the polyplexes were investigated by agarose gel electrophoresis, Zeta-sizer and TEM. They both could form nano-size polyplexes with pDNA. The polyplexes were very stable and showed excellent buffering capacities, facilitating endosomal escape, and no obvious difference was found between them. The TA-conjugated PAMAM-mediated transfection of luciferase and EGFP genes showed better transfer activity than native PAMAM and was comparable to the PEI 25K (polyethylenimine), and lower cytotoxicity in HEK 293 and HepG 2 cells. Even with 10% serum, their transfer activity was still high relatively. In addition, confocal microscopy examination confirmed that the enhancing mechanism for enhanced gene transfer activity of PAMAM–TA conjugate may involve the nuclear translocation of the polyplex. The low substituted degree of TA to 0.22 did not interrupt its nuclear localization potency. These findings demonstrated that the TA-grafted PAMAM dendrimer is a potential candidate as a safe and efficient gene delivery carrier for gene therapy.
Keywords: Gene delivery; Non-viral vector; PAMAM dendrimer; Triamcinolone acetonide; Nuclear translocation;

Semisynthetic network alginate polymer (SNAP), synthesized by acetalization of linear alginate with di-aldehyde, is a pH-responsive tetrafunctionally linked 3D gel network, and has potential application in oral delivery of protein therapeutics and active biologicals, and as tissue bioscaffold for regenerative medicine. A constitutive polyelectrolyte gel model based on non-Gaussian polymer elasticity, Flory–Huggins liquid lattice theory, and non-ideal Donnan membrane equilibria was derived, to describe SNAP gel swelling in dilute and ionic solutions containing uni-univalent, uni-bivalent, bi-univalent or bi-bi-valent electrolyte solutions. Flory–Huggins interaction parameters as a function of ionic strength and characteristic ratio of alginates of various molecular weights were determined experimentally to numerically predict SNAP hydrogel swelling. SNAP hydrogel swells pronouncedly to 1000 times in dilute solution, compared to its compact polymer volume, while behaving as a neutral polymer with limited swelling in high ionic strength or low pH solutions. The derived model accurately describes the pH-responsive swelling of SNAP hydrogel in acid and alkaline solutions of wide range of ionic strength. The pore sizes of the synthesized SNAP hydrogels of various crosslink densities were estimated from the derived model to be in the range of 30–450 nm which were comparable to that measured by thermoporometry, and diffusion of bovine serum albumin. The derived equilibrium swelling model can characterize hydrogel structure such as molecular weight between crosslinks and crosslinking density, or can be used as predictive model for swelling, pore size and mechanical properties if gel structural information is known, and can potentially be applied to other point-link network polyelectrolytes such as hyaluronic acid gel.
Keywords: Alginate; Hydrogel; Crosslinking; Modeling; Swelling; Pore size;

Poly (N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate) (P(VP-co-HEMA)) hydrogel system with a composition of VP:HEMA = 37:13 was studied using molecular dynamics simulations in order to investigate the effect of the water content on the equilibrium structures and the mechanical properties. The degree of randomness of the monomer sequence for the random and the blocky copolymers, were 1.170 and 0.104, respectively, and the degree of polymerization was fixed at 50. The equilibrated density of the hydrogel was found to be larger for the random sequence than for the blocky sequence at low water contents (<40 wt%), but this density difference decreased with increasing water content. The pair correlation function analysis shows that VP is more hydrophilic than HEMA and that the random sequence hydrogel is solvated more than the blocky sequence hydrogel at low water content, which disappears with increasing water content. Correspondingly, the water structure is more disrupted by the random sequence hydrogel at low water content but eventually develops the expected bulk water-like structure with increasing water content. From mechanical deformation simulations, stress–strain analysis showed that the VP is found to relax more efficiently, especially in the blocky sequence, so that the blocky sequence hydrogel shows less stress levels compared to the random sequence hydrogel. As the water content increases, the stress level becomes identical for both sequences. The elastic moduli of the hydrogels calculated from the constant strain energy minimization show the same trend with the stress–strain analysis.
Keywords: Hydrogels; Poly (N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate); Molecular dynamics; Simulation; Mechanical properties;

Finite element study of scaffold architecture design and culture conditions for tissue engineering by Andy L. Olivares; Èlia Marsal; Josep A. Planell; Damien Lacroix (6142-6149).
Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexagonal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.
Keywords: Tissue engineering; Scaffold; Rapid prototyping; Computational fluid dynamics; Finite element;