Biomaterials (v.27, #17)

Calendar (I).

Man as living bioreactor: Fate of an exogenously prepared customized tissue-engineered mandible by Patrick H. Warnke; Jörg Wiltfang; Ingo Springer; Yahya Acil; Hendrik Bolte; Markus Kosmahl; Paul A.J. Russo; Eugene Sherry; Ulf Lützen; Stefan Wolfart; Hendrik Terheyden (3163-3167).
In 2004, we reported a novel method of repairing a human mandible by in vivo tissue engineering. The patient served as his own bioreactor as the exogenously prepared customized mandible replacement was grown inside his latissimus dorsi muscle prior to transplantation to repair the existing defect. Our technique was developed through extensive experience with an animal model. We describe our and the patient's experiences with this procedure. We give details to the benefits and limitations of this technique as it stands and outline issues that should be addressed in future human clinical trials.
Keywords: In vivo tissue engineering; Bone regeneration; Replacement; Bioreactor; Mandible reconstruction; BMP;

Dynamic mechanical studies of hydrolytic degradation in isotropic and oriented Maxon B by Stuart P. Hill; Horacio Montes de Oca; Phil G. Klein; Ian M. Ward; John Rose; David Farrar (3168-3177).
Hydrolytic degradation studies have been undertaken on Maxon B, a bioresorbable block copolymer of polyglycolic acid (PGA) and polytrimethylene carbonate (TMC). Isotropic and oriented samples were studied by dynamic mechanical measurements over a wide range of temperatures. In addition to mechanical tests, water content and mass loss were also determined on the degraded samples. At early stages of degradation water content was the dominant factor and plasticisation lead to reductions in the glass transition temperatures of the PGA and TMC components. Orientation was shown to give significant improvements in the mechanical properties, including overall increases in modulus and an increase in the glass transition temperature of the PGA component, which is important for the behaviour at body temperature (37 °C). Oriented samples also showed significantly less reduction in mechanical properties on degradation. Simple one-dimensional Takayanagi models were used to provide useful insight into the understanding of the mechanical behaviour.
Keywords: Bioresorbables; Maxon; DMA; Degradation; Mechanical properties;

The integrity of welded interfaces in ultra high molecular weight polyethylene: Part 1—Model by C. Paul Buckley; Junjie Wu; David W. Haughie (3178-3186).
The difficulty of eradicating memory of powder–particle interfaces in UHMWPE for bearing surfaces for hip and knee replacements is well-known, and ‘fusion defects’ have been implicated frequently in joint failures. During processing the polymer is formed into solid directly from the reactor powder, under pressure and at temperatures above the melting point, and two types of inter-particle defect occur: Type 1 (consolidation-deficient) and Type 2 (diffusion-deficient). To gain quantitative information on the extent of the problem, the formation of macroscopic butt welds in this material was studied, by (1) modelling the process and (2) measuring experimentally the resultant evolution of interface toughness. This paper reports on the model. A quantitative measure of interface structural integrity is defined, and related to the “maximum reptated molecular weight” introduced previously. The model assumes an idealised surface topography. It is used to calculate the evolution of interface integrity during welding, for given values of temperature, pressure, and parameters describing the surfaces, and a given molar mass distribution. Only four material properties are needed for the calculation; all of them available for polyethylene. The model shows that, for UHMWPE typically employed in knee transplants, the rate of eradication of Type 1 defects is highly sensitive to surface topography, process temperature and pressure. Also, even if Type 1 defects are prevented, Type 2 defects heal extremely slowly. They must be an intrinsic feature of UHMWPE for all reasonable forming conditions, and products and forming processes should be designed accordingly.
Keywords: Adhesion; Fracture toughness; Interface; Joint replacement; Polyethylene; Modelling;

Interfacial energetics of blood plasma and serum adsorption to a hydrophobic self-assembled monolayer surface by Anandi Krishnan; Paul Cha; Yi-Hsiu Liu; David Allara; Erwin A. Vogler (3187-3194).
Interfacial energetics of blood plasma and serum adsorption to a hydrophobic, methyl-terminated self-assembled monolayer (SAM) surface (solid–liquid SL interface) are shown to be essentially the same as to the buffer–air interface (liquid–vapor LV interface). Specifically, spreading pressure (Π a) isotherms scaled on a w/v concentration basis constructed from advancing contact angles (θ a) of serially diluted plasma/serum derived from four different mammalian species (bovine, equine, human, and ovine) on the SAM surface are not resolvable at the 99% confidence level and furthermore are found to be strikingly similar to isotherms of purified human-blood proteins. Maximum advancing spreading pressures Π a max for protein mixtures fall within a relatively narrow 17<Π a max<26 mN/m band, mirroring results obtained at the LV surface. These observations lead to the conclusion that neither depletion of coagulation proteins in the conversion of plasma to serum nor variation in the plasma proteome among species has a substantial affect on adsorption energetics to these test hydrophobic surfaces. Experimental results are rationalized on the basis that there is a generic mechanism controlling adsorption of globular-blood proteins to test hydrophobic surfaces. We conclude that this generic mechanism is the hydrophobic effect by which proteins are expelled from aqueous solution in order to increase hydrogen-bonding (self-association) among water molecules at the expense of less favorable water–protein interactions. Expelled protein readily displaces water within the hydrophobic-surface region and becomes adsorbed. The amount of water displaced per gram of adsorbed protein does not vary greatly among globular proteins because the partial specific volume v 0 of globular proteins is quite conserved (0.70⩽v 0⩽0.75 cm3/g protein). Any single blood protein or mixture of proteins consequently displaces nearly an equivalent amount of interfacial water and hence adsorption is observed to scale similarly with solution concentration expressed in w / v units.
Keywords: Plasma; Serum; Protein adsorption; Hydrophobic solid–liquid interface; SAM;

Influence of the zinc concentration of sol–gel derived zinc substituted hydroxyapatite on cytokine production by human monocytes in vitro by Alexia Grandjean-Laquerriere; Patrice Laquerriere; Edouard Jallot; Jean-Marie Nedelec; Moncef Guenounou; Dominique Laurent-Maquin; Terry M. Phillips (3195-3200).
A possible complication associated with the implantation of hydroxyapatite (HA)-based prosthesis is the release of particles. These particles can be phagocyted by monocytes that are among the first cells to colonize the inflammatory site. The activated monocytes produce inflammatory mediators such as cytokines that cause osteoclasts activation. The present work, describes studies on the effect of sol–gel derived zinc-substituted HA particles with various zinc substitution percentages (0.5–2%) on cytokine production (TNF-α, IL-1β, IL-6, IL-10, and IL-8) by both LPS-stimulated or unstimulated human monocytes. Our data demonstrates that the zinc has an effect on cytokines production. It decreases the production of TNF-α and increases the production of IL-8 by unstimulated cells. Using LPS-stimulated cells, it decreases the production of inflammatory cytokines and increases the production of anti-inflammatory cytokine (IL-10), indicating that zinc-substituted hydroxyapatite has favourable effects on the cytokines production by monocytes.
Keywords: Hydroxyapatite particles; Cytokines; Inflammation; Zinc; Monocytes;

The influence of implantation site on the biocompatibility and survival of alginate encapsulated pig islets in rats by Denis Dufrane; Mathieu van Steenberghe; Rose-Marie Goebbels; Alain Saliez; Yves Guiot; Pierre Gianello (3201-3208).
This work investigated the impact of implantation sites on the biocompatibility of alginate encapsulated pig islets. Non-diabetic rats were implanted with adult pig islets encapsulated in alginate either intraperitoneally (IP; n = 25 ), subcutaneously (SC; n = 37 ) or under the kidney capsule (KC; n = 34 ). Capsule biocompatibility (retrieval rate, capsule diameter, degree of capsule broken and cellular overgrowth, CD68/CD3 staining) as well as islets viability and functionality were assessed until 30 days after transplantation. Implantation site did not significantly influence the biocompatibility of empty alginate capsules after transplantation ( n = 48 ). Most of the empty capsules (>90%) were retrieved after harvesting and were free of cellular overgrowth until day 30 post-transplantation. Three days after implantation, no significant difference for encapsulated pig islets was observed in terms of capsule biocompatibility and islet functionality in peritoneum, KC or subcutaneously. However, between days 5 and 30 after transplantation, explanted capsules from IP demonstrated a higher degree of broken capsules (>13%) and capsules with severe cellular overgrowth (>50%, CD68+ infiltration) than capsules removed from SC and KC ( p < 0.05 ). This was associated with a significant reduction of islet viability, insulin content and insulin secretion.In rats, the peritoneum site seems not appropriate for promoting the engraftment of encapsulated pig islets. Kidney subcapsular and subcutaneous spaces represent an interesting alternative.
Keywords: Islet; Microencapsulation; Biocompatibility; Xenotransplantation;

A highly porous electrospun scaffold was prepared by physically blending Dextran and poly lactide-co-glycolide (PLGA). The interaction between dermal fibroblasts and the Dextran/PLGA scaffold such as viability, proliferation, attachment, migration, extracellular matrix deposition, cytoskeleton organization and the functional gene expressions were characterized. The results indicated that cells interacted favorably with the scaffold. Moreover, cells migrated into the highly porous three dimensional matrix of the scaffold and organized into dense multi-layered structures that resembled dermal structure. The results of collagen gel assay also revealed that gel contraction was enhanced by the presence of the scaffold. The additional mechanical strength provided by the scaffold could enhance the binding of the seeded fibroblasts. These findings suggested that Dextran/PLGA scaffold can potentially be useful in enhancing the healing of chronic or trauma wounds.
Keywords: Bioactivity; Cell culture; Fibroblast; Dextran; Polyglycolic acid; Polylactic acid;

Biocompatibility of cluster-assembled nanostructured TiO2 with primary and cancer cells by Roberta Carbone; Ida Marangi; Andrea Zanardi; Luca Giorgetti; Elisabetta Chierici; Giuseppe Berlanda; Alessandro Podestà; Francesca Fiorentini; Gero Bongiorno; Paolo Piseri; Pier Giuseppe Pelicci; Paolo Milani (3221-3229).
We have characterized the biocompatibility of nanostructured TiO2 films produced by the deposition of a supersonic beam of TiO x clusters. Physical analysis shows that these films possess, at the nanoscale, a granularity and porosity mimicking those of typical extracellular matrix structures and adsorption properties that could allow surface functionalization with different macromolecules such as DNA, proteins, and peptides. To explore the biocompatibility of this novel nanostructured surface, different cancer and primary cells were analyzed in terms of morphological appearance (by bright field microscopy and immunofluorescence) and growth properties, with the aim to evaluate cluster-assembled TiO2 films as substrates for cell-based and tissue-based applications. Our results strongly suggest that this new biomaterial supports normal growth and adhesion of primary and cancer cells with no need for coating with ECM proteins; we thus propose this new material as an optimal substrate for different applications in cell-based assays, biosensors or microfabricated medical devices.
Keywords: Titanium; Biocompatibility; Nanotopography; Biomimetic material; Cell adhesion; Extracellular matrix;

Role of scaffold internal structure on in vivo bone formation in macroporous calcium phosphate bioceramics by Maddalena Mastrogiacomo; Silvia Scaglione; Roberta Martinetti; Laura Dolcini; Francesco Beltrame; Ranieri Cancedda; Rodolfo Quarto (3230-3237).
Purpose of this study was the analysis of the role of density and pore interconnection pathway in scaffolds to be used as bone substitutes. We have considered 2 hydroxyapatite bioceramics with identical microstructure and different macro-porosity, pore size distribution and pore interconnection pathway. The scaffolds were obtained with two different procedures: (a) sponge matrix embedding (scaffold A), and (b) foaming (scaffold B). Bone ingrowth within the two bioceramics was obtained using an established model of in vivo bone formation by exogenously added osteoprogenitor cells. The histological analysis of specimens at different time after in vivo implantation revealed in both materials a significant extent of bone matrix deposition. Interestingly enough, scaffold B allowed a faster occurrence of bone tissue, reaching a steady state as soon as 4 weeks. Scaffold A on the other hand reached a comparable level of bone formation only after 8 weeks of in vivo implantation. Both scaffolds were well vascularised, but larger blood vessels were observed in scaffold A.Here we show that porosity and pore interconnection of osteoconductive scaffolds can influence the overall amount of bone deposition, the pattern of blood vessels invasion and finally the kinetics of the bone neoformation process.
Keywords: Bone tissue engineering; Porous bioceramics; Interconnection pathway; Vascularisation;

Performance of collagen sponge as a 3-D scaffold for tooth-tissue engineering by Yoshinori Sumita; Masaki J. Honda; Takayuki Ohara; Shuhei Tsuchiya; Hiroshi Sagara; Hideaki Kagami; Minoru Ueda (3238-3248).
Tooth structure can be regenerated by seeding dissociated tooth cells onto polyglycolic acid fiber mesh, although the success rate of tooth production is low. The present study was designed to compare the performance of collagen sponge with polyglycolic acid fiber mesh as a 3-D scaffold for tooth-tissue engineering. Porcine third molar teeth at the early stage of crown formation were enzymatically dissociated into single cells, and the heterogeneous cells were seeded onto collagen sponge or the polyglycolic acid fiber mesh scaffolds. Scaffolds were then cultured to evaluate cell adhesion and ALP activity in vitro. An in vivo analysis was performed by implanting the constructs into the omentum of immunocompromised rats and evaluating tooth production up to 25 weeks. After 24 h, there were a significantly higher number of cells attached to the collagen sponge scaffold than the polyglycolic acid fiber mesh scaffold. Similarly, the ALP activity was significantly higher for the collagen sponge scaffold was than the polyglycolic acid fiber mesh scaffold after 7 days of culture. The area of calcified tissue formed in the collagen sponge scaffold was also larger than in the polyglycolic acid fiber mesh scaffold. The results from in vivo experiments show conclusively that a collagen sponge scaffold allows tooth production with a higher degree of success than polyglycolic acid fiber mesh. Taken together, the results from this study show that collagen sponge scaffold is superior to the polyglycolic acid fiber mesh scaffold for tooth-tissue engineering.
Keywords: Collagen sponge; Odontogenesis; Polyglycolic acid; Scaffold; Tissue engineering; Teeth;

Coating of VEGF-releasing scaffolds with bioactive glass for angiogenesis and bone regeneration by J. Kent Leach; Darnell Kaigler; Zhuo Wang; Paul H. Krebsbach; David J. Mooney (3249-3255).
Bioactive glasses are potentially useful as bone defect fillers, and vascular endothelial growth factor (VEGF) has demonstrated benefit in bone regeneration as well. We hypothesized that the specific combination of prolonged localized VEGF presentation from a matrix coated with a bioactive glass may enhance bone regeneration. To test this hypothesis, the capacity of VEGF-releasing polymeric scaffolds with a bioactive glass coating was examined in vitro and in vivo using a rat critical-sized defect model. In the presence of a bioactive glass coating, we did not detect pronounced differences in the differentiation of human mesenchymal stem cells in vitro. However, we observed significantly enhanced mitogenic stimulation of endothelial cells in the presence of the bioactive glass coating, with an additive effect with VEGF release. This trend was maintained in vivo, where coated VEGF-releasing scaffolds demonstrated significant improvements in blood vessel density at 2 weeks versus coated control scaffolds. At 12 weeks, bone mineral density was significantly increased in coated VEGF-releasing scaffolds versus coated controls, while only a slight increase in bone volume fraction was observed. The results of this study suggest that a bioactive glass coating on a polymeric substrate participates in bone healing through indirect processes which enhance angiogenesis and bone maturation and not directly on osteoprogenitor differentiation and bone formation. The mass of bioactive glass used in this study provides a comparable and potentially additive, response to localized VEGF delivery over early time points. These studies demonstrate a materials approach to achieve an angiogenic response formerly limited to the delivery of inductive growth factors.
Keywords: Angiogenesis; Bioactive glass; Bone regeneration; VEGF;

Ectopic bone formation using an injectable biphasic calcium phosphate/Si-HPMC hydrogel composite loaded with undifferentiated bone marrow stromal cells by Christophe Trojani; Florian Boukhechba; Jean-Claude Scimeca; Fanny Vandenbos; Jean-François Michiels; Guy Daculsi; Pascal Boileau; Pierre Weiss; Georges F. Carle; Nathalie Rochet (3256-3264).
We have used a new synthetic injectable composite constituted of hydroxyapatite/tricalcium phosphate (HA/TCP) particles in suspension in a self-hardening Si–hydroxypropylmethylcellulose (HPMC) hydrogel. The aim of this study was to evaluate in vivo the biocompatibility and the new bone formation efficacy of this scaffold loaded with undifferentiated bone marrow stromal cells (BMSCs). This biomaterial was mixed extemporaneously with BMSCs prepared from C57BL/6 mice, injected in subcutaneous and intramuscular sites and retrieved 4 and 8 weeks after implantation. Dissection of the implants revealed a hard consistency and the absence of a fibrous capsule reflecting a good integration into the host tissues. Histological analysis showed mineralized woven bone in the granule inter-space with numerous active osteoclasts attached to the particles as assessed by the presence of multinucleated cells positively stained for TRAP activity and for the a3 subunit of the V-ATPase. Small vessels were homogenously distributed in the whole implants. Similar results were obtained in SC and IM sites and no bone formation was observed in the control groups when cell-free and particle-free transplants were injected. These results indicate that this injectable biphasic calcium phosphate–hydrogel composite mixed with undifferentiated BMSCs is a new promising osteoinductive bone substitute. It also provides with an original in vivo model of osteoclast differentiation and function.
Keywords: Bone induction; Injectable biomaterial; Si–HPMC/BCP composite; Bone marrow stromal cells; Mouse;

Expansion and osteogenic differentiation of bone marrow-derived mesenchymal stem cells on a vitamin C functionalized polymer by Yongzhong Wang; Amarjit Singh; Peng Xu; Mariya A. Pindrus; Dominick J. Blasioli; David L. Kaplan (3265-3273).
In human body ascorbic acid plays an essential role in the synthesis and function of skeletal tissues and immune system factors. Ascorbic acid is also a major physiological antioxidant, repairing oxidatively damaged biomolecules, preventing the formation of excessive reactive oxygen species or scavenging these species. We recently reported the synthesis of ascorbic acid-functionalized polymers in which the antioxidant features of the pendant ascorbic acid groups was preserved. In the present work we demonstrate that ascorbic acid-functionalized poly(methyl methacrylate) (AA-PMMA) can modulate the proliferation and osteogenic differentiation of early and late-passage bone marrow-derived human mesenchymal stem cells (MSCs). The covalently coupled ascorbic acid impacted MSCs differently than when ascorbic acid was presented to the cells in soluble form. At optimal concentration, the covalently coupled ascorbic acid and soluble ascorbic acid synergistically promoted and retained the ability of MSCs to respond to osteogenic stimulation over extensive cell expansions in vitro. In the presence of soluble ascorbic acid, AA-PMMA films prepared at optimal concentrations (0.1 mg/ml in the present study) showed a significant promotive effect over other concentrations and tissue culture plastic (TCP) with respect to osteogenic differentiation of both EP (young) and LP (old) MSCs. These results suggest that the coupled ascorbic acid is acting mainly at the extracellular level and, at optimal concentrations, the immobilized extracellular ascorbic acid and soluble ascorbic acid synergistically promote osteogenic differentiation of MSCs. Importantly, the covalently coupled ascorbic acid on the films of optimal concentration was able to preserve the capacity of MSCs to undergo osteogenic differentiation in vitro. These results suggest an important role for functionalized biomaterials with antioxidant features in control of cell physiology and cell aging phenomena.
Keywords: Mesenchymal stem cell; Vitamin C; Osteogenesis; Antioxidant; Ageing; Collagen;

Platinum in silicone breast implants by Michael A. Brook (3274-3286).
Silicone elastomers are widely used in implantable devices, including silicone breast implants. These rubbers are generally formed/cured using platinum-catalyzed hydrosilylation. The current scientific literature on the chemistry of platinum is reviewed, as it applies to the use of platinum catalysts for cure of silicone elastomers destined for use in silicone breast implants. These discussions serve as a basis to examine the recent literature describing release of platinum into tissues adjacent to silicone breast implants, the chemical nature of the platinum present in breast implants and the possible association between platinum and clinical outcomes.
Keywords: Silicone elastomer; Breast implant; Hydrosilylation; Platinum catalysis; Biological consequences;

Evaluation of the effect of water-uptake on the impedance of dental resins by Bakul Wadgaonkar; Shuichi Ito; Nadia Svizero; David Elrod; Stephen Foulger; Robert Rodgers; Yoshiki Oshida; Kevin Kirkland; Jeremy Sword; Frederick Rueggeberg; Franklin Tay; David Pashley (3287-3294).
Electrical impedance spectroscopy (EIS) offers a quantitative method of measuring the stability of resin films in aqueous solution over time.The purpose of this study was to measure the EIS of five experimental dental adhesive films (ca. 17 μm thick) of increasing hydrophilicity (ranked by their Hoy's solubility parameters), and how much these values change over 3 weeks in aqueous buffer.The resin films were placed in a U-shaped chamber and a pair of Ag–AgCl electrodes was used for EIS. The EIS results were confirmed by immersing the films in 50% AgNO3 for 24 h to trace the distribution of any water absorption into the resins by TEM observations.The resistance (R r) of the resins 1–4 films increased most during the first day, and varied from 1×1011  ohm for resin 1, to 40 Ω for resin 5 at day 1. The day 1 R r values of resins 1–4 were inversely proportional to their Hoy's solubility parameter for hydrogen bonding forces. Electrical impedance values of resins 1–3 and 5 varied widely but were relatively constant over time, while those of resin 4 decreased more than 99% from day 1 to 21 ( p < 0.05 ). Capacitance (C r) of films of resins 1–4 all increased over the first day and then were relatively unchanged over the 20 days (except for resin 4 that continued to increase) and were between 0.01 and 1 nF. Silver uptake by TEM revealed the development of water-filled branching structures that formed in resins 4 and 5 over time.
Keywords: Dental adhesives; Dielectric constant; Electrochemistry; Hydrophilicity; Photopolymerisation;

A percutaneous device with antibacterial activity and good biocompatibility is desired for clinical applications. Three types of antibacterial agent: lactoferrin (LF), tetracycline (TC), and gatifloxacin (GFLX) were immobilized on the surface of an ethylene-vinyl alcohol copolymer (EVOH) using a liquid phase coating process. In this process, an EVOH plate was alternately dipped in calcium and phosphate ion solutions, and then immersed in a metastable calcium phosphate solution supplemented with 4, 40, or 400 μg/mL of the antibacterial agent. As a result, the antibacterial agent was immobilized on the EVOH surface in the form of an antibacterial agent–apatite composite layer. The amount of immobilized antibacterial agent increased with increasing absorption affinity for apatite in the order: GFLX<TC<LF. On the other hand, the release rate of the antibacterial agent from the composite was ordered in the opposite sense; i.e., LF<TC<GFLX. The composites investigated in this study showed antibacterial activity against Escherichia coli and Staphylococcus aureus, and would be useful as materials in percutaneous devices having antibacterial activity and good biocompatibility.
Keywords: Apatite; Antibacterial agent; Lactoferrin (LF); Tetracycline (TC); Gatifloxacin (GFLX); Percutaneous device;

Development of dialyzer with immobilized glycoconjugate polymers for removal of Shiga-toxin by Atsushi Miyagawa; Miho Watanabe; Katsura Igai; Maria Carmelita Z. Kasuya; Yasuhiro Natori; Kiyotaka Nishikawa; Kenichi Hatanaka (3304-3311).
The dialyzer for Shiga-toxin elimination was developed and its performance was established. The dialyzer was prepared by immobilization of multivalent ligands. Glycoconjugate polymers having oligosaccharides and amino groups were synthesized to function as Shiga-toxin adsorbents. The amino group was utilized to immobilize the polymer inside the cellulose hollow fiber of the dialyzer. Cellulose hollow fibers packed in the dialyzer were carboxymethylated under moderate conditions. The glycoconjugate polymers were bound covalently to the hollow fibers of the dialyzer by condensation reaction between the amino group of the polymer and the carboxyl group of the cellulose hollow fiber. Shiga-toxin eliminabilities of the prepared dialyzers were evaluated at various conditions. Even at high concentration of protein such as FCS, the dialyzer showed an excellent performance for Shiga-toxin adsorption.
Keywords: Adsorption; Dialyzer; Glycoconjugate polymer; Immobilization; Shiga-toxin;

Protein stability in the presence of polymer degradation products: Consequences for controlled release formulations by Amy S. Determan; Jennifer H. Wilson; Matt J. Kipper; Michael J. Wannemuehler; Balaji Narasimhan (3312-3320).
When encapsulating proteins in polymer microspheres for sustained drug delivery there are three stages during which the stability of the protein must be maintained: (1) the fabrication of the microspheres, (2) the storage of the microspheres, and (3) the release of the encapsulated protein. This study focuses on the effects of polymer degradation products on the primary, secondary, and tertiary structure of tetanus toxoid, ovalbumin (Ova), and lysozyme after incubation for 0 or 20 days in the presence of ester (lactic acid and glycolic acid) and anhydride (sebacic acid and 1,6-bis(p-carboxyphenoxy)hexane) monomers. The structure and antigenicity or enzymatic activity of each protein in the presence of each monomer was quantified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism, and fluorescence spectroscopy were used to assess/evaluate the primary, secondary, and tertiary structures of the proteins, respectively. Enzyme-linked immunosorbent assay was used to measure changes in the antigenicity of tetanus toxoid and Ova and a fluorescence-based assay was used to determine the enzymatic activity of lysozyme. Tetanus toxoid was found to be the most stable in the presence of anhydride monomers, while Ova was most stable in the presence of sebacic acid, and lysozyme was stable when incubated with all of the monomers studied.
Keywords: Tetanus toxoid; Ovalbumin; Lysozyme; Polyester; Polyanhydride;

This study aims to fabricate biodegradable polymeric particles by electrohydrodynamic atomization (EHDA) for applications in sustained delivery of anticancer drug—paclitaxel to treat C6 glioma in vitro. Controllable morphologies such as spheres, donut shapes and corrugated shapes with sizes from several tens of microns to hundred nanometers of particles were observed by scanning electron microscopy (SEM) and field emission electron microscope (FSEM). The differential scanning calorimetry (DSC) study indicated that paclitaxel could be either in an amorphous or disordered-crystalline phase of a molecular dispersion or a solid solution state in the polymer matrix after fabrication. The X-ray photoelectron spectroscopy (XPS) result suggested that some amount of paclitaxel could exist on the surface layer of the microparticles. The encapsulation efficiency was around 80% and more than 30 days in vitro sustained release profile could be achieved. Cell cycling results suggested that paclitaxel after encapsulation by EHDA could keep its biological function and inhibit C6 glioma cells in G2/M phase. The cytotoxicity of paclitaxel-loaded biodegradable microparticles to C6 glioma cells could be higher than Taxol® in the long-term in vitro tests evaluated by MTS assay. The drug delivery devices developed by EHDA in this study could be promising for the local drug delivery to treat malignant glioma.
Keywords: Electrohydrodynamic atomization; Biodegradable; Microparticle; Paclitaxel; Cytotoxicity;

Modified montmorillonite as vector for gene delivery by Feng-Huei Lin; Chia-Hao Chen; Winston T.K. Cheng; Tzang-Fu Kuo (3333-3338).
Currently, gene delivery systems can be divided into two parts: viral or non-viral vectors. In general, viral vectors have a higher efficiency on gene delivery. However, they may sometimes provoke mutagenesis and carcinogenesis once re-activating in human body. Lots of non-viral vectors have been developed that tried to solve the problems happened on viral vectors. Unfortunately, most of non-viral vectors showed relatively lower transfection rate. The aim of this study is to develop a non-viral vector for gene delivery system.Montmorillonite (MMT) is one of clay minerals that consist of hydrated aluminum with Si–O tetrahedrons on the bottom of the layer and Al–O(OH)2 octahedrons on the top. The inter-layer space is about 12 Å. The room is not enough to accommodate DNA for gene delivery. In the study, the cationic hexadecyltrimethylammonium (HDTMA) will be intercalated into the interlayer of MMT as a layer expander to expand the layer space for DNA accommodation. The optimal condition for the preparation of DNA–HDTMA–MMT is as follows: 1 mg of 1.5CEC HDTMA–MMT was prepared under pH value of 10.7 and with soaking time for 2 h. The DNA molecules can be protected from nuclease degradation, which can be proven by the electrophoresis analysis. DNA was successfully transfected into the nucleus of human dermal fibroblast and expressed enhanced green fluorescent protein (EGFP) gene with green fluorescence emission. The HDTMA–MMT has a great potential as a vector for gene delivery in the future.
Keywords: Composite; DNA; Gene transfer; Silicate;