Biomaterials (v.26, #14)

Calendar (I).

Palladium dental casting alloys are alternatives to gold alloys. The aim of this study was to determine the electrochemical behaviour and the corrosion mechanism of binary silver–palladium alloys. Seven binary silver–palladium alloys and pure palladium and silver were tested in a model saliva solution. Electrochemical tests included corrosion potential, polarization resistance, and potentiodynamic polarization measurements. The corrosion products, which may be theoretically formed, were determined by thermodynamic calculation.The behaviour of silver and silver-rich alloys was dominated by the preferential formation of a thiocyanate surface layer, which controlled the free corrosion potential. Palladium dissolved in the form of a thiocyanate complex, but the surface became passivated by either palladium oxide or solid palladium thiocyanate layer, the thermodynamic calculations indicating preference for the oxide. Palladium-rich alloys showed evidence of silver depletion of the surface, resulting in behaviour similar to palladium.Examination of binary silver–palladium alloys has made possible determination of the role of the components of the alloys and model saliva in the corrosion behaviour. The findings are applicable to the more complex commercial dental alloys containing silver and palladium as major components.
Keywords: Silver–palladium alloys; Model saliva; Corrosion; Mechanism;

In the present work, constitutive models for densification and grain growth were employed to investigate the sintering behavior of pure hydroxyapatite ceramics. For densification study, lattice diffusion, grain-boundary diffusion, and interface reaction mechanisms, and for grain-growth study, surface diffusion mechanism, were considered respectively. Hydroxyapatite ceramics were pressurelessly sintered. The sintering results were discussed and compared with the modeling results. Based on the constitutive models employed and the experimental results obtained, grain-boundary diffusion was identified as the dominant mechanism for the densification of the investigated hydroxyapatite. The grain-growth model provided a good prediction to the grain growth of the investigated hydroxyapatite. The activation energies for densification and grain growth of hydroxyapatite ceramics were evaluated as 1150±40 and 1020±40 KJ mol−1, respectively.
Keywords: Constitutive modeling; Densification; Grain growth; hydroxyapatite;

A composite coating that is composed of collagen protein and calcium phosphate minerals is considered to be bioactive and may enhance bone growth and fixation of metallic orthopedic implants. In this study, we have successfully developed a uniform collagen fibril/octacalcium phosphate composite coating on silicon substrate by electrolytic deposition (ELD). The coating deposition was done through applying a constant potential to the cathode in a three-electrode electrochemistry cell that contain a mild acidic (pH 4.8–5.3) aqueous solution of collagen molecules, calcium and phosphate ions. The coating process involved self-assembly of collagen fibrils and the deposition of calcium phosphate minerals as a result of cathode reaction and local pH increase. The two steps could be synchronized to form a bone-like composite at nanometer scale through proper adjustment of the solution and deposition parameters. Coating morphology, crystal structure and compositions were analyzed by optical and fluorescence microscopy, scanning and transmission electron microscopy, energy dispersive X-ray analysis, inductively coupled argon plasma optical emission spectrophotometry, and Fourier-transformed infrared spectroscopy. Under typical deposition conditions, the cathode (Si) surface formed a thin (100 nm) layer of calcium phosphate coating, on top of which a thick (∼100 μm) composite layer formed. The porous composite layer consists of a collagen fibril network on which clusters of octacalcium phosphate crystals nucleate and grow. By combining photolithography and ELD, we were also able to pattern the composite coating into regular arrays of squares. Preliminary results by nanoindentation tests showed that properly prepared composite coating may have higher elastic modulus and scratch resistance than monolithic porous calcium phosphate coating. The results not only provide a novel bioactive coating for biomedical implants, but also establish a new experimental protocol for studying biomineralization mechanisms of collagen based biological tissues.
Keywords: Electrolytic deposition; Composite coating; Self-assembly; Collagen; Octacalcium phosphate;

Rheometric study of the gelation of chitosan in a hydroalcoholic medium by Alexandra Montembault; Christophe Viton; Alain Domard (1633-1643).
The formation of chitosan physical hydrogels without any external cross-linking agent was studied. The gelation took place in an acetic acid-water-propanediol solution. The time to reach the gel point was determined by rheometry and gelations from different initial conditions could be compared. The influence of different parameters on gelation such as the polymer concentration, the degree of acetylation (DA) of chitosan and the composition of the initial solvent were investigated. The fractal morphology of the sample was not affected by the composition of the system. The number of junctions per unit volume at the gel point varied only with the initial number of chain entanglements per unit volume. Then, below an initial concentration of 1.5% (w/w), physical chain entanglements were insufficient and more junctions had to be formed to induce gelation. Over this value, only the kinetics allowing to replace entanglements by stable physical junctions played a key parameter. This kinetics was influenced by several parameters such as DA, temperature or the initial proportion water/alcohol. The acetyl groups played an important role in the formation of hydrophobic interactions, mainly responsible for gelation. The study of the influence of the gelation media revealed two critical points at 40% and 70% of water in the initial solvent, probably due to conformational changes and then to different modes of gelation. These physical hydrogels being used for cartilage regeneration, their final rheological properties were studied as a function of their degree of acetylation, the polymer concentration and the solvent composition in the initial solvent. Our results allowed us to define an optimal gelation condition for our application, corresponding to: DA=40%, a proportion water/alcohol of 50/50 and a polymer concentration of 1.5%.

Laser-induced crystallization of calcium phosphate coatings on polyethylene (PE) by Bastiaan Feddes; Arjen M. Vredenberg; Martin Wehner; Joop C.G. Wolke; John A. Jansen (1645-1651).
Calcium phosphate (CaP) coatings are used for obtaining a desired biological response. Usually, CaP coatings on metallic substrates are crystallized by annealing at temperatures of at least 400–600 °C. For polymeric substrates, this annealing is not possible due to the low melting temperatures. In this work, we present a more suitable method for obtaining crystalline coatings on polymeric substrates, namely laser crystallization. We were successful in obtaining hydroxyapatite coatings on polyethylene. Because of the UV transmission characteristics of the CaP coatings, the use of a low wavelength (157 nm) F2 laser was necessary for this. As a result of the laser treatment, the CaP coating broke up into islands. The cracks between the islands became larger and the surface became porous with increasing laser energy. The mechanism behind the formation of this morphology did not become clear. However, the fact that crystalline CaP coatings can be obtained on polymeric substrates in an easy way, possibly allows for the development of new products.

Highly active, lipase silicone elastomers by Amro M. Ragheb; Michael A. Brook; Michael Hrynyk (1653-1664).
Lipase Candida rugosa was entrapped in silicone rubber via condensation-cure room temperature vulcanization of silanol-terminated poly(dimethylsiloxane) with tetraethyl orthosilicate as a crosslinker, to give a highly active silicone-enzyme elastomer. The effect on enzyme activity of addition of water and hydrophilic polymeric moieties based on poly(ethylene oxide) 2 was examined, as were crosslinker concentration, enzyme concentration, and elastomer thickness. It was demonstrated that lipase is most active in silicone elastomers and more active in silicone oils than simple hydrocarbons. Crosslink density in these elastomers was not an important factor in the reactivity of the rubber. However, the addition of hydrophilic species prior to elastomer formation decreased the efficiency both of the dispersion of the enzyme and the resulting activity of the elastomer. This effect could be moderated by prior exposure of the lipase to silicone oil. Thus, hydrophobic silicones play a protective/activating role for lipase.
Keywords: Immobilized enzyme; Lipase; Silicone elastomer; Poly(ethylene oxide);

Effect of pH and ionic strength on the reactivity of Bioglass® 45S5 by Marta Cerruti; David Greenspan; Kevin Powers (1665-1674).
Bioglass® 45S5 is a silica-based melt-derived glass, used in medical field as a bone regenerative material because of the deposition of a layer of hydroxy carbonate apatite (HCA) on the surface of the glass when immersed in body fluid.The present paper studies the early steps of reaction of 2-μm sized particles of Bioglass®, in solutions buffered with TRIS at different pH, by means of ICP-ES and FTIR spectroscopy. Only at pH 8 could a total reconstruction of the glass be observed, with the formation of both a silica and a calcium phosphate rich layers. At higher pH, selective dissolution of the glass was hindered by the immediate precipitation of a layer of calcium phosphate, whereas at lower pH a total breakdown of the glass occurred and no calcium phosphate precipitation was noted. The use of the ATR-liquid cell allowed the observation of the reaction in real time, and this showed that the process of silica formation is not separable from cation leaching from the glass, as well as the formation of the calcium phosphate rich layer.
Keywords: Bioactive glass; Micron-size particles; TRIS dissolution; FTIR-ATR; ICP-ES;

Precision microarchitectural constructs made of acrylated trimethylene carbonate (TMC)-based liquid prepolymers were photo-polymerized using a custom-designed microstereolithographic apparatus. In this study, three different photo-polymerizable liquid prepolymers were prepared by the polymerization of TMC with a low molecular weight poly(ethylene glycol) (PEG) (mol. wt. 200 or 1000); designated as PEG200 or PEG1000, respectively or trimethylolpropane (TMP) as an initiator, and subsequently end-capped with an acrylate group. As a result of layer-by-layer photo-irradiation of the prepolymer with a movable ultraviolet light pen driven by computer-aided design, a three-dimensional (3D) micropillar array, a microbank array, a microcone array, and multi-microtunnels formed on a platform plate or a glass plate were precisely fabricated. The PEG-based polymers exhibited a very low cell adhesion potential, whereas the TMP-based hydrophobic polymer exhibited high cell adhesion and proliferation potentials. The microbank array, which consisted of a plate made of the TMP-based polymer and microbanks made of the PEG200-based polymer, caused cell adhesion and proliferation only on the plate. Upon the implantation of microcone arrays under the subcutis of rats, the photo-polymerized construct made of the poorly swellable PEG200-based polymer exhibited only surface erosion and limited drug loading and releasing potentials. On the other hand, the photo-polymerized construct made of the highly swellable PEG1000-based polymer exhibited not only surface erosion but also bulk erosion and high drug loading and releasing potentials. In this paper, we discuss their potential biomedical applications.

The current state-of-the-art with respect to the preparation, characterization and biomedical applications of novel nitric oxide (NO) releasing or generating polymeric materials is reviewed. Such materials show exceptional promise as coatings to prepare a new generation of medical devices with superior biocompatiblity. Nitric oxide is a well-known inhibitor of platelet adhesion and activation, as well as a potent inhibitor of smooth muscle cell proliferation. Hence, polymers that release or generate NO locally at their surface exhibit greatly enhanced thromboresistivity and have the potential to reduce neointimal hyperplasia caused by device damage to blood vessel walls. In this review, the use of diazeniumdiolates and nitrosothiols as NO donors within a variety polymeric matrixes are summarized. Such species can either be doped as discrete NO donors within polymeric films, or covalently linked to polymer backbones and/or inorganic polymeric filler particles that are often employed to enhance the strength of biomedical polymers (e.g., fumed silica or titanium dioxide). In addition, very recent efforts to create catalytic polymers possessing immobilized Cu(II) sites capable of generating NO from endogenous oxidized forms of NO already present in blood and other physiological fluids (nitrite and nitrosothiols) are discussed. Preliminary literature data illustrating the efficacy of the various NO release/generating polymers as coatings for intravascular sensors, extracorporeal blood loop circuits, and arteriovenous grafts/shunts are reviewed.
Keywords: Nitric oxide; Blood compatible polymers; Anti-platelet activity;

Expression of inflammatory cytokines, RANKL and OPG induced by titanium, cobalt-chromium and polyethylene particles by Tetsuo Masui; Shinji Sakano; Yukiharu Hasegawa; Hideki Warashina; Naoki Ishiguro (1695-1702).
Bone resorption is regulated by cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and by the balance of a receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the mechanism of particle-induced osteolysis in murine calvariae by assessing the extent of osteolysis and the expression of inflammatory cytokines, RANKL and OPG after implantation of metal and polyethylene particles. The murine calvariae implanted with Ti6Al4V, CoCr or high-density polyethylene (HDP) particles showed significantly more extensive osteolysis and elevated levels of inflammatory cytokines. The ratio between RANKL and OPG was high in the mice implanted with Ti6Al4V and HDP particles, but not in the mice implanted with CoCr particles. These observations suggested that CoCr particle-induced osteoclastogenesis may be caused directly by inflammatory cytokines rather than by the RANKL-RANK pathway. There might be different mechanisms at work in particle-induced osteolysis between Ti6Al4V, HDP and CoCr.
Keywords: Cobalt alloy; Cytokine; Osteolysis; Polyethylene; Titanium alloy;

The response of healing corneal epithelium to grooved polymer surfaces by M.D.M. Evans; G.A. McFarland; S. Taylor; X.F. Walboomers (1703-1711).
Corneal epithelial wounds heal rapidly by the inwards growth of tissue with a contracting wound front. A synthetic polymer lens to correct refractive error (an implantable contact lens) could be incorporated into the cornea using this wound healing process. Topographical cues on the polymer surface may facilitate epithelial tissue migration over the anterior device surface. Here, silicone discs with a defined surface geometry of parallel grooves (groove and ridge widths of 1, 2, 5 and 10 μm; groove depths of 1 and 5 μm) were implanted into corneas and maintained in organ culture. The nature and rate of epithelial tissue migration over the test surfaces was monitored for 8 days and evaluated using microscopy and histology. Irrespective of the pitch, deep groove geometries directed tissue migration laterally along the grooves but this prevented contraction of the wound front and retarded migration rates. No guidance occurred on any of the shallow groove geometries but these allowed inwards radial migration with a contracting wound front and supported migration rates equivalent to a flat surface. None of the geometries tested promoted tissue migration above a flat polymer surface and data suggested that parallel grooves may not be optimal for this application.
Keywords: Cornea; Wound closure; Implant; Surface topography; Silicone; Organ culture;

Cytotoxic and mutagenic effects of dental composite materials by Helmut Schweikl; Karl-Anton Hiller; Carola Bolay; Marion Kreissl; Wetscheslaw Kreismann; Agathe Nusser; Stefanie Steinhauser; Janusz Wieczorek; Rudolf Vasold; Gottfried Schmalz (1713-1719).
Mutagenicity of single compounds of dental resinous materials has been investigated on many occasions before, but the induction of mutagenic effects by extracts of clinically used composites is still unknown. Here, cytotoxic effects and the formation of micronuclei were determined in V79 fibroblasts after exposure to extracts of modern composite filling materials (Solitaire, Solitaire 2, Tetric Ceram, Dyract AP, Definite). For cytotoxicity testing, test specimens were aged for various time periods (0, 24, and 168 h), and V79 cells were then exposed to dilutions of the original extracts for 24, 48, and 72 h. The ranking of the cytotoxic effects of the composites according to EC50 values after a 24-h exposure period was as follows: Solitaire (most toxic)=Solitaire 2<Tetric Ceram<Dyract AP<Definite (least toxic). Cytotoxicity was independent of the period of aging for each composite, but varied with exposure periods. The cytotoxic effect of Solitaire increased about two-fold between exposure periods of 24, 48, and 72 h, no changes were observed with Solitaire 2, and cytotoxicity of Tetric Ceram, Dyract AP, and Definite was reduced. Even eight-fold diluted original extracts of freshly mixed Solitaire test specimens increased the numbers of micronuclei about 10-fold, and Solitaire 2 was slightly less effective. The mutagenic effects of these materials were reduced in the presence of a rat liver homogenate (S9). Weak increases of the numbers of micronuclei were detected only with undiluted extracts of Tetric Ceram and Dyract AP, but Definite was not effective. Our findings suggest that mutagenic components of biologically active composite resins should be replaced by more biocompatible substances to avoid risk factors for the health of patients and dental personnel.
Keywords: Dental resin; Cytotoxicity; Mutagenicity; TEGDMA;

The effect of non-specific interactions on cellular adhesion using model surfaces by Mark H. Lee; David A. Brass; Ronit Morris; Russell J. Composto; Paul Ducheyne (1721-1730).
The contribution of non-specific interactions between cells and model functional surfaces was measured using a spinning disc apparatus. These model functional surfaces were created using self-assembled monolayers (SAM) of alkylsilanes terminated with epoxide, carboxyl (COOH), amine (NH2), and methyl (CH3) groups. These SAMs were characterized using ellipsometry, atomic force microscopy, contact angle goniometry, and X-ray photoelectron spectroscopy to confirm the presence of well-formed monolayers of expected physicochemical characteristics. All substrates also demonstrated excellent stability under prolonged exposure (up to 18 h) to aqueous conditions. The adhesion strength of K100 erythroleukemia cells to the functional substrates followed the trend: CH3 < COOH ≈ epoxide ≪ NH2. The NH2 SAM surface exhibited nearly an order of magnitude greater adhesion strength than the other SAMs and this non-specific effect exceeded the adhesion measured when RGD tri-peptides were also immobilized on the surface. These findings illustrate the importance of substrate selection in quantitative studies of peptide-mediated cellular adhesion.
Keywords: Cell adhesion; Adhesion mechanism; Non-specific interaction; Self-assembled monolayers; RGD peptide; Surface modification;

The corline heparin surface (CHS) used in the extracorporeal circuit during coronary artery bypass grafting is shown to decrease the activation of inflammation and coagulation. Synchrotron radiation studies have shown that a single layer of the CHS may not completely cover the substrate surface. However, a double layer of CHS results in a uniform surface. We investigated the effect of surfaces with different surface concentrations of heparin on cell activation and coagulation compared to an uncoated surface.The CHS is prepared by a conditioning layer of polymeric amine onto which a macromolecular heparin conjugate is attached. We used PVC tubing, uncoated or modified with a single or double layer of the CHS, and circulated fresh whole blood from healthy volunteers in a loop model system at 37°C up to 4 h. Blood was drawn from the loops at different times and activation of inflammation and coagulation was studied by real-time PCR, flow cytometry and ELISA. The activation of leukocytes and platelets and formation of leukocyte–platelet aggregates were reduced by use of the single-layered CHS compared to the uncoated surface. Use of double-layered CHS resulted in significantly reduced cell activation and thrombin generation. Development of the CHS obtained by the double layer of the coating has improved the biocompatibility of the surface.
Keywords: Heparin; Biomaterial; Biocompatibility; Cell activation;

Long-term in vivo biomechanical properties and biocompatibility of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) nerve conduits by Jason S. Belkas; Catherine A. Munro; Molly S. Shoichet; Miles Johnston; Rajiv Midha (1741-1749).
Artificial grafts are promising alternatives to nerve grafts for peripheral nerve repair because they obviate the complications and disadvantages associated with autografting such as donor site morbidity and limited tissue availability. We have synthesized poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (PHEMA-MMA) porous tubes and studied their efficacy in vivo. Specifically, we studied the short- and long-term stability and biocompatibility of 12 mm long tubes for the repair of surgically created 10 mm nerve gaps in rat sciatic nerves.Prior to implantation, tubes were analyzed in vitro using a micro-mechanical tester to measure displacement achieved with load applied. These results served as a calibration curve, y=6.8105×−0.0073 (R 2=0.9750,n=28), for in vivo morphometric tube compression measurements.In vivo, most of the PHEMA-MMA conduits maintained their structural integrity up to 8 weeks, but 29% (4/14) of them collapsed by 16 weeks. Interestingly, the tube wall area of collapsed 16-week tubes was significantly lower than those of patent tubes.Tubes were largely biocompatible; however, a small subset of 16-week tubes displayed signs of chronic inflammation characterized by “finger-like” tissue extensions invading the inner tube aspect, inflammatory cells (some of which were ED1+macrophages) and giant cells. Tubes also demonstrated signs of calcification, which increased from 8 to 16 weeks. To overcome these issues, future nerve conduits will be re-designed to be more robust and biocompatible.
Keywords: Nerve repair; Nerve guidance channel; Compression; Inflammation; Sciatic nerve; Rat;

Changes in the distribution of laminin-5 during peri-implant epithelium formation after immediate titanium implantation in rats by Ikiru Atsuta; Takayoshi Yamaza; Masao Yoshinari; Satoya Mino; Tetsuya Goto; Mizuho A Kido; Yoshihiro Terada; Teruo Tanaka (1751-1760).
Laminin-5 (Ln-5), a component of the basement membrane (BM), regulates epithelial cell migration and adhesion. This study used anti-Ln-5 (γ2chain) antibody to investigate the distribution of Ln-5 during the formation of peri-implant epithelium (PIE) in rats, and compared it to the distribution of Ln-5 during oral mucosa formation after tooth extraction. One day after extraction, the junctional epithelium (JE) had disappeared. After 3 days, new epithelium formed from the oral sulcular epithelium (OSE) and extended horizontally over the wound with Ln-5-positive cells at the leading edge. After 5 days, the epithelium extending from the OSE on each side of the wound joined and formed additional new epithelium. The new epithelium expressed Ln-5 in the BM. After 1–2 weeks, the oral epithelium (OE) extending from the sides of the wound joined in the center. Thereafter, OSE and new epithelium disappeared, and only OE remained covering the wound. Three days after implantation (titanium), no JE remained. New epithelium formed from the keratinized OSE extending apically with Ln-5-positive cells. After 1–2 weeks, the new epithelium became the PIE and spread further apically facing the implant surface. Ln-5 was expressed at the PIE-connective tissue interface, but not at the implant-PIE interface. Finally, after 4 weeks, Ln-5 was expressed at the implant-PIE interface, and the PIE was non-keratinized epithelium. These findings suggest that Ln-5 induces cell migration during PIE formation, and that PIE originates from OSE. Furthermore, they support the hypothesis that Ln-5 contributes to the attachment of PIE to titanium, regardless of the delay in the synthesis and deposition of Ln-5 at the titanium-PIE interface.
Keywords: Titanium dental implant; Laminin-5; Peri-implant epithelium; Oral mucosa formation; Cell migration and attachment;

Hydrogels are frequently employed as medical device biomaterials due to their advantageous biological properties, e.g. resistance to infection and encrustation, biocompatibility; however, their poor mechanical properties generally limit the scope of application to coatings of medical devices. To address this limitation, this study described the formulation of sequential interpenetrating polymer networks (IPN) of poly(ε-caprolactone) (PCL) and poly(hydroxyethylmethacrylate) (p(HEMA)). IPN containing 20% w/w PCL, p(HEMA), both in the presence or absence of ethyleneglycol dimethacrylate (EGDMA 1% w/w), were prepared by free radical polymerisation. Following preparation the degradation and the mechanical and surface properties of the biomaterials and, in addition, the resistances to microbial adherence and encrustation in vitro were examined. In comparison to p(HEMA) the various IPN exhibited substantially greater tensile properties (ultimate tensile strength, % elongation, Young's modulus) that were accredited to the discrete distribution of PCL within the hydrogel network. The IPN exhibited two glass transition temperatures that were statistically similar to those of the individual components, thereby providing evidence of the immiscible nature of the two polymers. The IPN possessed higher receding contact angles and lower equilibrium water contents in comparison to p(HEMA), whereas the limited degradation of the IPN at both pH 7 and 9 was deemed suitable for clinical usage for periods of at least 4 weeks. The resistances of the various IPN to bacterial adherence and urinary encrustation were examined using in vitro models. Importantly the resistance of the IPN to encrustation was, in general, similar to that of p(HEMA) but greater than that of PCL whereas, the resistance of the IPN to bacterial adherence was frequently greater than that of p(HEMA) and PCL. Therefore, this study has shown that the mechanical properties of p(HEMA) may be substantially increased by the formation of IPN with PCL whilst maintaining other appropriate physicochemical properties and resistances to urinary encrustation and bacterial adherence. It is suggested that these IPN may be suitable for device fabrication thereby expanding the manufacturing application of hydrogels without compromising their potential clinical efficacy.
Keywords: Poly(HEMA); Polycaprolactone; Interpenetrating polymer networks; Mechanical properties; Bacterial adherence; Encrustation;

Retention of transforming growth factor β1 using functionalized dextran-based hydrogels by Marion Maire; Delphine Logeart-Avramoglou; Marie-Christelle Degat; Frédéric Chaubet (1771-1780).
Functionalized dextrans (FD) are anionic water-soluble polymers bearing carboxylate, benzylamide and sulfate groups, which exhibit binding capacity to transforming growth factor-β1 (TGF-β1). In this paper, we have investigated the ability of dextran-based hydrogels containing FD, to bind and release recombinant human TGF-β1. Hydrogels were prepared by chemical crosslink native dextran and FD with sodium trimetaphosphate in 1  m NaOH at 50°C. A wide range of hydrogels were prepared as particles ranging of 1–1.6 mm of diameter and characterized with various amounts of FD and with different crosslinker feeding ratios (CFR). Dried particles were soaked with recombinant human transforming growth factor-β1 (rhTGF-β1) to determine their capacity to deliver the growth factor. Results indicated that the in vitro kinetics releases of rhTGF-β1 were related to FD and CFR. Retention capacity of rhTGF-β1 increases with an increase of negative charges of the matrices brought by both phosphate linkages and FD as demonstrated by an additional release of growth factor in high ionic strength solution. Highly crosslinked hydrogels that contained the highest amount of FD (18% (w/w)) retained up to 88% of rhTGF-β1. Bioactivity of released growth factor was confirmed in a cell assay. These functionalized hydrogels may have important uses for the stabilization and the protection of rhTGF-β1 as entrapment systems and could be applied to other proteins of clinical interest.
Keywords: Hydrogel; Functionalized dextran; TGF-β1; Drug delivery; Sodium trimetaphosphate;

Tissue response to partially in vitro predegraded poly-L-lactide implants by Wim H. De Jong; J. Eelco Bergsma; Joke E. Robinson; Ruud R.M. Bos (1781-1791).
The in vivo local reaction of as-polymerized poly-L-lactide composed of 96% L-lactide and 4% D-lactide (PLA96) was investigated by histology at 2, 13 and 26 weeks after subcutaneous implantation in rats. In order to simulate possible end stage reactions the PLA96 was also predegraded in vitro until approximately 50% weight loss. The local reaction of predegraded PLA (PLA96168) was compared to the local reaction of polyethylene (PE) and non-predegraded PLA (PLA96).For PE and PLA96 a mild local reaction was observed at all time points consisting of a minimal layer of macrophage like cells with incidentally multinucleated giant cells at the implant interface, surrounded by a mild connective tissue capsule. For PLA96 at weeks 13 and 26 some minimal alterations in terms of degradation and ingrowth of cells was noted. The in vitro incubation (90 °C for 168 h) of PLA96168 resulted for the thin 0.2 mm samples in complete degradation. Predegraded 0.5, 1.0 and 2.0 mm PLA96168 samples were implanted and evaluated. The 1.0 and 2.0 mm samples could be evaluated for all time points investigated, but some 0.5 mm PLA96168 samples were already completely resorbed at week 2 after implantation. In general, responses found for the predegraded PLA96168 at weeks 2, 13 and 26 were similar with a pronounced macrophage infiltrate containing birefringent material, encapsulation of polymer fragments, and the presence of a debris area consisting of polymer and cellular remnants. In lymph nodes foamy macrophages with birefringent material were only observed in lymph nodes draining sites with predegraded PLA96168.Immunohistochemistry was performed for further characterization of the cellular infiltrate. At the implant interface of the non-degrading PE and PLA96, ED1 and OX6 (MHC class II) positive cells were identified. In the capsule macrophage like cells expressed all three macrophage markers ED1, ED2, and ED3. CD4 and CD8 positive cells, indicating T helper and T supressor/cytotoxic cells, respectively, could be observed in low numbers, CD4 more than CD8. Both CD4 and CD8 were occasionally observed within the degrading PLA96168 implant. Polymorphonuclear neutrophilic granulocytes were mainly observed at 2 weeks after implantation.We showed that predegradation could be used as a means to study late tissue reactions to polymers. Complete degradation may be studied with relatively thin implants, but this may lead to rather optimistic interpretation of resorption periods. When materials are intended to be used for screws and/or plates for bone fixation, implants of at least 1.0–2.0 mm thickness should be used as these may show a more realistic representation of the resorption characteristics of the material under investigation.
Keywords: PLA implant; Histology; Immunohistochemistry; Predegradation; Biocompatibility;

Comparative study of osteoconduction on micromachined and alkali-treated titanium alloy surfaces in vitro and in vivo by Xiong Lu; Yang Leng; Xingdong Zhang; Jinrui Xu; Ling Qin; Chun-wai Chan (1793-1801).
This study sought to evaluate osteoconduction of Ti-6Al-4 V surfaces under various conditions, including micro-patterned, alkali-treated, micro-patterned plus alkali-treated, and surfaces without any treatment as the control. The through-mask electrochemical micromachining (EMM) was used to fabricate micro-hole arrays on the titanium alloy surface. In vitro calcium phosphate formation on titanium surfaces was in static and dynamic simulated body fluid (SBF). In vivo comparison was conducted in the medullary cavity of dog femur using the implant cages which could provide the same physiological environment for specimens with different surface conditions. In vitro experiments indicate good conduction of calcium phosphate on the alkali-treated surfaces, and also better calcium phosphate deposition on the micro-hole surface than on the flat surfaces in dynamic SBF. In vivo experiments confirm the beneficial effect of alkaline treatment on osteoconduction. The results of in vivo experiments also indicate a synergistic effect of the alkaline treatment and the topographic pattern on osteoconduction.
Keywords: Osteoconduction; Micro-pattern; Alkaline treatment; Simulate body fluid; Animal model;

The decrease of particle-induced osteolysis after a single dose of bisphosphonate by Marius von Knoch; Christian Wedemeyer; Andreas Pingsmann; Fabian von Knoch; Gero Hilken; Christoph Sprecher; Frank Henschke; Bertram Barden; Franz Löer (1803-1808).
The most common cause of implant failure in joint replacement is aseptic loosening due to particle-induced osteolysis. Bisphosphonates have been shown to be effective against particle-induced osteolysis when administered daily. We investigated the effect of a single subcutaneous dose of a more potent third generation bisphosphonate on particle-induced osteolysis.We utilized the murine calvaria osteolysis model in C57BL/J6 mice. Bone resorption was measured as resorption within the midline suture using Giemsa staining. Twenty-eight mice were used, seven per group. Seven animals were treated with a single dose of zoledronic acid (ZA) directly after surgery and seven animals were treated four days postoperatively. For statistical analysis one-way ANOVA and a Student's t-test were used.Bone resorption was 0.26±0.09 mm2 in animals with particle implantation, 0.14±0.05 mm2 in animals with particle implantation and ZA treatment directly after surgery (p=0.0047), and 0.15±0.05 mm2 in animals with particle implantation and ZA treatment on the fourth postoperative day (p=0.006).In conclusion, particle-induced bone resorption was markedly decreased by a single s.c. dose of a third generation bisphosphonate. This important new finding holds great promise, because single dose treatment of particle-induced osteolysis may reduce side effects compared to repeated application of bisphosphonates.
Keywords: Osteolysis; Wear debris; Polyethylene; Particulates; Osteoclast;

Spatially resolved photopolymerization kinetics and oxygen inhibition in dental adhesives by Teresa G. Nunes; L. Ceballos; R. Osorio; M. Toledano (1809-1817).
A comparative study of three commercial dental adhesives was performed by 1H nuclear magnetic resonance spectroscopy and Stray-Field magnetic resonance imaging. Spectroscopic evidence was found for the presence of solvent and unreacted methacrylate groups in photopolymerized adhesives. Spatially resolved photopolymerization kinetics and volumetric contraction (solvent evaporation and polymerization shrinkage) were obtained without solvent removal and in the presence of oxygen from the atmosphere. The oxygen and solvent inhibitor effects in the photopolymerization were found to be higher for water/ethanol based adhesives. However, was one of these adhesives that exhibited less spatially dependent irradiation time to start vitrification, higher concentration and a more uniform spatial distribution of rigid domains at the end of the photopolymerization.
Keywords: Dental adhesive; Photopolimerization kinetics; Oxygen inhibition; Volumetric contraction;

Plasma transglutaminase factor XIII induces microvessel ingrowth into biodegradable hydroxyapatite implants in rats by Olaf Kilian; R. Fuhrmann; V. Alt; T. Noll; S. Coskun; E. Dingeldein; R. Schnettler; R.P. Franke (1819-1827).
Coagulation factor XIII is a member of the transglutaminase-family. Transgluaminases cross-link either fibrin monomers in blood coagulation or extracellular proteins in extracellular matrix formation. In early stages of bone healing migration and proliferation of endothelial cells lead to formation of new vessels. The aim of this study was to investigate the angiogenetic activity of plasma factor XIII in bone defects filled with nanoparticulate hydroxyapatite paste. A critical size defect was created in the tibial head of rats which was not filled in group I. In group II the defect was filled with hydroxyapatite paste, and in group III with hydroxyapatite paste enriched with factor XIII. Ten days after surgery angiogenesis in the defects was assessed using immunohistochemistry and confocal laser scanning microscopy. Ac16 antibody was used to detect activation of factor XIII into factor XIIIA. In defects without biomaterial (group I) vessel-rich connective tissue and diffuse distribution of capillaries was observed. In defects filled with pure hydroxyapatite (group II) formation of capillaries was limited to the host bone-hydroxyapatite interface. In contrast, addition of plasma factor XIII to hydroxyapatite (group III) stimulated formation of vessels within the biomaterial. The current study reveals that factor XIII can improve angiogenesis in hydroxyapatite.
Keywords: Plasma factor XIII; Hydroxyapatite; Confocal laser scanning microscopy; Alpha smooth muscle actin antigen; CD34 antigen; Angiogenesis;

Ectopic bone formation in rats: the importance of the carrier by Ed H.M. Hartman; Johan W.M. Vehof; Paul H.M. Spauwen; John A. Jansen (1829-1835).
Much research has been done to develop the ideal bone graft substitute (BGS). One approach to develop this ideal BGS is the use of growth factors, but for this approach osteoprogenitor cells are needed at the site of reconstruction. An alternative is a cell-based approach, where enough cells are provided to form bone in a carrier material. In previous studies of our group, titanium (Ti) carriers have been used, because of the excellent mechanical properties and the bone-compatibility of this material. On the other hand, calcium phosphate (CaP) ceramics are known for their excellent osteoconductivity. The aim of this study is to investigate the influence of the carrier in a cell-based bone regeneration approach, whereby we hypothesize that CaP-ceramic implants will induce more bone formation than Ti-fiber implants, in the same animal model as our previous experiment. Ti-fiber mesh implants and ceramic implants were seeded with rat bone marrow cells (RBM) and implanted subcutaneously. Histological analysis after one, three and six weeks showed differences in the way of bone formation in the two groups: bone appeared to grow from the center to the periphery of the implant in the titanium group, while bone formation in the ceramic group occurred through the whole implant. Histomorphometrical analysis after one week showed very limited bone formation for both the titanium and ceramic group. At three weeks, the amount of bone formation was increased till about 10% for the titanium group and 18% for the ceramic group. No significant difference between the two groups could be observed. In the six week group, the bone formation was 6% (Ti) and 23% (CaP), respectively (P<0.0001). Further, bone formation started earlier in the CaP-ceramic scaffolds than in the Ti scaffolds. Our hypothesis could be confirmed: ceramic implants induce more bone formation than titanium implants.
Keywords: Bone tissue engineering; Cell culture; Osteogenesis; Titanium; Animal model; Ceramics; Hydroxyapatite; Tricalcium phosphate;

Differential regulation of osteoblasts by substrate microstructural features by O. Zinger; G. Zhao; Z. Schwartz; J. Simpson; M. Wieland; D. Landolt; Barbara Boyan (1837-1847).
Microtextured titanium implant surfaces enhance bone formation in vivo and osteoblast phenotypic expression in vitro, but the mechanisms are not understood. To determine the roles of specific microarchitectural features in modulating osteoblast behavior, we used Ti surfaces prepared by electrochemical micromachining as substrates for MG63 osteoblast-like cell culture. Cell response was compared to tissue culture plastic, a sand-blasted with large grit and acid-etched surface with defined mixed microtopography (SLA), polished Ti surfaces, and polished surfaces electrochemically machined through a photoresist pattern to produce cavities with 100, 30 and 10 μm diameters arranged so that the ratio of the microscopic-scale area of the cavities versus the microscopic-scale area of the flat region between the cavities was equal to 1 or 6. Microstructured disks were acid-etched, producing overall sub-micron-scale roughness (Ra=0.7 μm). Cell number, differentiation (alkaline phosphatase; osteocalcin) and local factor levels (TGF-β1; PGE2) varied with microarchitecture. 100 μm cavities favored osteoblast attachment and growth, the sub-micron-scale etch enhanced differentiation and TGF-β1 production, whereas PGE2 depended on cavity dimensions but not the sub-micron-scale roughness.
Keywords: Titanium; Microarchitecture; Microstructure; Roughness; Photolithography; Osteoblasts; MG63 cells; PGE2; Surface; Electrochemical micromachining;

Cyclooxygenase-2 (COX-2) is an inducible enzyme believed to be responsible for prostaglandin synthesis at the site of inflammation. Recently, the activation of COX-2 expression may be one of the important pathogenesis of root canal sealers-induced periapical inflammation. However, little is known about whether chemical interaction can modulate the epoxy resin-based root canal sealers-induced cytotoxicity as well as COX-2 expression. The aim of the present study was to investigate the effects of antioxidants catalase, superoxide dismutase (SOD), and N-acetyl-L-cysteine (NAC) on AH26- and Topseal-induced COX-2 mRNA gene and cytotoxicity in human osteoblastic cell line U2OS cells. The results showed that both epoxy resin-based root canal sealers were cytotoxic to U2OS cells in a concentration-dependent manner (p<0.05). AH26 and Topseal were found to induce COX-2 mRNA gene expression in U2OS cells. The addition of glutathione (GSH) precursor NAC led to decrease the induction of COX-2 mRNA gene expression and cytotoxicity by both AH26 and Topseal (p<0.05). However, catalase and SOD lacked the ability to prevent AH26-and Topseal-induced cytotoxicity and COX-2 mRNA gene expression (p>0.05). Taken together, the activation of COX-2 mRNA gene expression may be one of the pathogenesis of epoxy resin-based root canal sealers-induced periapical inflammation. In addition, GSH depletion, but not the attack of oxygen free radicals, could be the mechanism for epoxy resin-based root canal sealers-induced cytotoxicity and COX-2 mRNA gene expression. Factors that induce GSH synthesis may appear useful in preventing cell damage mediated by epoxy resin-based root canal sealers.
Keywords: Root canal sealers; Epoxy resin; Cyclooxygenase-2; Glutathione; Antioxidants;

The roles of tissue engineering and vascularisation in the development of micro-vascular networks: a review by Ruben Y. Kannan; Henryk J. Salacinski; Kevin Sales; Peter Butler; Alexander M. Seifalian (1857-1875).
The construction of tissue-engineered devices for medical applications is now possible in vitro using cell culture and bioreactors. Although methods of incorporating them back into the host are available, current constructs depend purely on diffusion which limits their potential. The absence of a vascular network capable of distributing oxygen and other nutrients within the tissue-engineered device is a major limiting factor in creating vascularised artificial tissues. Though bio-hybrid prostheses such as vascular bypass grafts and skin substitutes have already been developed and are being used clinically, the absence of a capillary bed linking the two systems remains the missing link. In this review, the different approaches currently being or that have been applied to vascularise tissues are identified and discussed.

Growth of human cells on polyethersulfone (PES) hollow fiber membranes by R.E. Unger; Q. Huang; K. Peters; D. Protzer; D. Paul; C.J. Kirkpatrick (1877-1884).
A novel material of porous hollow fibers made of polyethersulfone (PES) was examined for its ability to support the growth of human cells. This material was made in the absence of solvents and had pore diameters smaller than 100 μm. Human cell lines of different tissue and cell types (endothelial, epithelial, fibroblast, glial, keratinocyte, osteoblast) were investigated for adherence, growth, spread and survival on PES by confocal laser microscopy after staining of the cells with Calcein-AM. Endothelial cell attachment and growth required pre-coating PES with either fibronectin or gelatin. The other cell types exhibited little difference in growth, spread or survival on coated or uncoated PES. All the cells readily adhered and spread on the outer, inner and cut surfaces of PES. With time confluent monolayers of cells covered the available surface area of PES and in some cases cells grew as multilayers. Many of the cells were able to survive on the PES for up to 7 weeks and in some cases growth was so extensive that the underlying PES was no longer visible. Scanning electron microscope observations of cells on the materials correlated with the confocal morphometric data. Thus, PES is a substrate for the growth of many different types of human cells and may be a useful scaffolding material for tissue engineering.
Keywords: Polyethersulfone; PES; Human cells; Biocompatibility; Cell compatibility;

The use of patterned dual thermoresponsive surfaces for the collective recovery as co-cultured cell sheets by Yukiko Tsuda; Akihiko Kikuchi; Masayuki Yamato; Aiko Nakao; Yasuhisa Sakurai; Mitsuo Umezu; Teruo Okano (1885-1893).
Heterotypic cell interactions are critical to achieve and maintain specific functions in many tissues and organs. We have focused on patterned structure surfaces to enable co-culture of heterotypic cells and recovery of patterned co-cultured cell sheets for applications in tissue engineering. Thermoresponsive polymers exhibiting different transition temperatures in water comprise both poly(N-isopropylacrylamide) (PIPAAm) and n-butyl methacrylate (BMA) co-grafted as side chains to PIPAAm main chains. These copolymers were surface-grafted in patterns to obtain patterned dual thermoresponsive cell culture surfaces using electron beam polymerisation method and porous metal masks. On patterned surfaces, site-selective adhesion on and growth of rat primary hepatocytes (HCs) and bovine carotid endothelial cells (ECs) allowed patterned co-culture, exploiting hydrophobic/hydrophilic surface chemistry regulated by culture temperature as the sole variable. At 27°C, seeded HCs adhered exclusively onto hydrophobic, dehydrated P(IPAAm–BMA) co-grafted domains (1-mm area), but not onto neighbouring hydrated PIPAAm domains. Sequentially seeded ECs then adhered exclusively to hydrophobised PIPAAm domains upon increasing culture temperature to 37°C, achieving patterned co-cultures. Reducing culture temperature to 20°C promoted hydration of both polymer-grafted domains, permitting release of the co-cultured, patterned cell monolayers as continuous cell sheets with heterotypic cell interactions. Recovered co-cultured cell sheets can be manipulated, moved and sandwiched with other structures, providing new useful constructs both for basic cell biology research and preparation of tissue-mimicking multi-layer materials through overlaying co-cultured cell sheets.
Keywords: Thermoresponsive surfaces; Surface patterning; Poly(N-isopropylacrylamide); n-Butyl methacrylate; Co-culture; Cell culture; Cell sheet;

Activation of membrane receptors by a neurotransmitter conjugate designed for surface attachment by Tania Q. Vu; Sarwat Chowdhury; Niraj J. Muni; Haohua Qian; Robert F. Standaert; David R. Pepperberg (1895-1903).
The derivatization of surfaces with bioactive molecules is a research area of growing importance for cell and tissue engineering. Tetherable molecules used in such applications must contain an anchoring moiety as well as the biofunctional group, typically along with a spacer to prevent steric clashes between the target molecule and the tethering surface. Post-synaptic membrane receptors at chemical synapses in neural tissue mediate signaling to the post-synaptic neuron and are activated by the binding of diffusible neurotransmitter molecules released by the pre-synaptic neuron. However, little attention has been directed at developing neurotransmitter analogs that might retain functionality when tethered to a surface that could be interfaced with post-synaptic receptor proteins. Muscimol (5-aminomethyl-3-hydroxyisoxazole), an analog of GABA (γ-aminobutryic acid), is a known potent agonist of GABAA and GABAC post-synaptic receptors found in retina and other central nervous system tissue. The present paper reports experiments testing the electrophysiological activity of “muscimol-biotin” on cloned GABA receptors expressed in Xenopus oocytes. This compound, which is potentially suitable for tethering at avidin-coated surfaces, consists of muscimol conjugated through an N-acyl linkage to a 6-aminohexanoyl chain that is distally terminated by biotin. We find that muscimol-biotin, as well as a structurally similar compound (muscimol-BODIPY®) containing a bulky fluorophore at the distal end of the aminohexanoyl chain, exhibits substantial agonist activity at GABAA and GABAC receptors. Muscimol-biotin and other similarly biotinylated neurotransmitter analogs, in combination with surface functionalization using avidin-biotin technology, may be useful in applications involving the controlled activation of neuronal post-synaptic receptors by surface-attached molecules.
Keywords: Neurotransmitter; GABA receptor; Muscimol; Biotin; BODIPY®; Nerve tissue engineering; Electrophysiology;

Construction of varying porous structures in acellular bovine pericardia as a tissue-engineering extracellular matrix by Hao-Ji Wei; Huang-Chien Liang; Meng-Horng Lee; Ya-Chun Huang; Yen Chang; Hsing-Wen Sung (1905-1913).
In the study, a cell extraction process was used to remove the cellular components from bovine pericardia. Varying pore sizes and porosities of the acellular tissues were then created using acetic acid and collagenase and subsequently fixed with genipin. Biochemical analyses found that these acellular tissues with distinct porous structures consisted primarily of insoluble collagen, elastin, and tightly bound glycosaminoglycans. The thermal stability, mechanical properties, and capability against enzymatic degradation of the bovine pericardial tissue remained unaltered after cell extraction. However, following further treatment with acetic acid and collagenase, the thermal stability and capability against enzymatic degradation of the acellular tissues declined. The porous structures of the implanted samples seem to determine whether successful microvessel-ingrowth takes place. The acetic-acid- and collagenase-treated tissues, due to their high pore size and porosity, showed a large number of microvessels infiltrating into the interstices of the implanted samples. In contrast, a low density of microvessels was observed infiltrating into the acellular tissue and penetration of microvessels into the cellular tissue was never encountered.
Keywords: Acellular tissue; Porosity; Biochemical property; Mechanical property; Angiogenesis;

Vascular patches tissue-engineered with autologous bone marrow-derived cells and decellularized tissue matrices by Seung-Woo Cho; Hee Jung Park; Ju Hee Ryu; Soo Hyun Kim; Young Ha Kim; Cha Yong Choi; Min-Jae Lee; Jong-Sung Kim; In-Sung Jang; Dong-Ik Kim; Byung-Soo Kim (1915-1924).
Synthetic polymer vascular patches used in cardiovascular surgery have shortcomings such as thrombosis, intimal hyperplasia, calcification, infection, and no growth potential. Tissue-engineered vascular patches using autologous vascular cells may solve these problems. In this study, we developed a tissue-engineered vascular patch using autologous bone marrow-derived cells (BMCs) and decellularized tissue matrices. Vascular smooth muscle cells and endothelial cells were differentiated from bone marrow mononuclear cells in vitro. Tissue-engineered vascular patches were fabricated by seeding these cells onto decellularized canine inferior vena cava matrices and implanted into the inferior vena cava of dogs. Three weeks after implantation, the tissue-engineered vascular patches were patent with no sign of thrombus formation. Histological, immunohistochemical, and electron microscopic analyses of the vascular patches retrieved 3 weeks after implantation revealed regeneration of endothelium and smooth muscle and the presence of collagen and elastin. BMCs labeled with a fluorescent dye prior to implantation were detected in the retrieved vascular patches, indicating that the BMCs survived after implantation and contributed to the vascular tissue regeneration. This study demonstrates that vascular patches can be tissue-engineered with autologous BMCs and decellularized tissue matrices.
Keywords: Vascular patch; Bone marrow-derived cell; Decellularized tissue matrix; Tissue engineering;

Hyaluronic acid modified biodegradable scaffolds for cartilage tissue engineering by Hyuk Sang Yoo; Eun Ah Lee; Jun Jin Yoon; Tae Gwan Park (1925-1933).
Hyaluronic acid (hyaluronan, HA) was immobilized onto the surface of macroporous biodegradable poly(d,l-lactic acid-co-glycolic acid) [PLGA] scaffolds to enhance the attachment, proliferation, and differentiation of chondrocytes for cartilage tissue engineering. The PLGA scaffolds were prepared by blending PLGA with varying amounts of amine-terminated PLGA-PEG di-block copolymer. They were fabricated by a gas foaming/salt leaching method. HA was chemically conjugated to the surface-exposed amine groups on the pre-fabricated scaffolds. The amount of surface exposed free amine groups was quantitatively determined by conjugating an amine-reactive fluorescent dye to the PLGA blend films. The extent of HA immobilization was also confirmed by measuring water contact angles. When chondrocytes were seeded within HA modified PLGA scaffolds, enhanced cellular attachment was observed compared to unmodified PLGA scaffolds. Furthermore, glycosaminoglycan and total collagen synthesis increased substantially for HA modified PLGA scaffolds. RT-PCR result and histological examination of the resultant cartilage tissue revealed that HA modified scaffolds excelled in inducing cartilage tissue formation in terms of collagen type II expression and tissue morphological characteristics.
Keywords: Scaffold; Surface modification; Hyaluronic acid; Chondrocyte; Polylactic acid;

Tissue engineering offers a promising new approach to bone tissue grafting. One material that has received attention in this regard is the polymer poly (lactic-co-glycolic acid) (PLGA). It has the advantage of controllable bioresorption and ease of processing. Another material of interest is bioactive glass (BG), which shows the ability to stimulate osteoblastic differentiation of osteoprogenitor cells. In this study, we reported on the optimal synthesis parameters and the kinetics of formation of calcium phosphate (Ca-P) phase at the surface of PLGA/BG composites. The formation of calcium phosphate layer was confirmed using scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDXA). PLGA-30%BG microspheres based porous scaffolds for bone tissue engineering were examined for their ability to promote osteogenesis of marrow stromal cells (MSC). This porous scaffold supported both MSC proliferation and promoted MSC differentiation into cells expressing the osteoblast phenotype. It therefore demonstrates significant potential as a bone replacement material.
Keywords: PLGA; Bioactive glass; Calcium phosphate; Marrow stromal cells; Tissue engineering;

Poly(lactide-co-glycolide) microspheres as a moldable scaffold for cartilage tissue engineering by Nichole R. Mercier; Henry R. Costantino; Mark A. Tracy; Lawrence J. Bonassar (1945-1952).
This study demonstrates the use of biodegradable poly(lactide-co-glycolide) (PLG) microspheres as a moldable scaffold for cartilage tissue engineering. Chondrocytes were delivered to a cylindrical mold with or without PLG microspheres and cultured in vitro for up to 8 weeks. Cartilagenous tissue formed using chondrocytes and microspheres maintained thickness, shape, and chondrocyte collagen type II phenotype, as indicated by type II collagen staining. The presence of microspheres further enhanced total tissue mass and the amount of glycosaminoglycan that accumulated. Evaluation of microsphere composition demonstrated effects of polymer molecular weight, end group chemistry, and buffer inclusion on tissue-engineered cartilage growth. Higher molecular weight PLG resulted in a larger mass of cartilage-like tissue formed and a higher content of proteoglycans. Cartilage-like tissue formed using microspheres made from low molecular weight and free carboxylic acid end groups did not display increases in tissue mass, yet a modest increased proteoglycan accumulation was detected. Microspheres comprised of PLG with methyl ester end groups yielded a steady increase in tissue mass, with no real increase in matrix accumulation. The microencapsulation of Mg(OH)2 had negative effects on tissue mass and matrix accumulation. The data herein reflect the potential utility of a moldable PLG-chondrocyte system for tissue-engineering applications.

Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human platelets are a rich source of many growth factors including transforming growth factor-β and platelet-derived growth factor. To investigate the effect of human platelet supernatant (hPS) on chondrocyte proliferation and differentiation, human articular biopsies obtained from three healthy donors. Chondrocytes were isolated and expanded separately in monolayer cultures and seeded in alginate beads in the presence and absence of hPS of 1% or 10% v/v concentration. Transcript levels of genes encoding chondrogenic factors were determined by quantitative reverse transcriptase-polymerase chain reaction. The deposition of types I and II collagen as well as proteoglycan was detected by indirect immunocytochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced a dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and bone morphogenetic protein-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. Differences (1% vs. 10% group, 1% vs. control, 10% vs. control) in the quantitative gene expression of types I and II collagen or of aggrecan were statistically significant ( p < 0.001 ). We conclude that addition of hPS may accelerate chondrocyte expansion but can lead to their dedifferentiation.

Study of gelatin-containing artificial skin V: fabrication of gelatin scaffolds using a salt-leaching method by Sang Bong Lee; Yong Han Kim; Moo Sang Chong; Seung Hwa Hong; Young Moo Lee (1961-1968).
Porous gelatin scaffolds were prepared using a salt-leaching method and these were compared to scaffolds fabricated using a freeze-drying method. The salt-leached gelatin scaffolds were easily formed into desired shapes with a uniformly distributed and interconnected pore structure with an average pore size of around 350 μm. The mechanical strength and the biodegradation rate of the scaffolds increased with the porosity, and were easily modulated by the addition of salt. After 1 week of in vitro culturing, the fibroblasts in salt-leached scaffolds were mainly attached on the surface of the pores in the scaffold, whereas cells seeded on freeze-dried scaffolds were widely distributed and aggregated on the top and the bottom of the scaffold. After 14 d of culturing, the fibroblasts showed a good affinity to, and proliferation on, the gelatin scaffolds without showing any signs of biodegradation. An in vivo study of cultured artificial dermal substitutes showed that an artificial dermis containing the fibroblasts enhanced the re-epithelialization of a full-thickness skin defect when compared to an acellular scaffold after 1 week.

Peptide-immobilized nanoporous alumina membranes for enhanced osteoblast adhesion by Erin E. Leary Swan; Ketul C. Popat; Tejal A. Desai (1969-1976).
Bone tissue engineering requires the ability to regulate cell behavior through precise control over substrate topography and surface chemistry. Understanding of the cellular response to micro-environment is essential for biomaterials and tissue engineering research. This research employed alumina with porous features on the nanoscale. These nanoporous alumina surfaces were modified by physically adsorbing vitronectin and covalently immobilizing RGDC peptide to enhance adhesion of osteoblasts, bone-forming cells. X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were used to characterize the modified nanoporous alumina surface. Survey and high-resolution C1s scans suggested the presence of RGDC and vitronectin on the surface and SEM images confirmed the pores were not clogged after modification. Cell adhesion on both unmodified and modified nanoporous alumina was compared using fluorescence microscopy and SEM. RGDC was found to enhance osteoblast adhesion after 1 day of culture and matrix production was visible after 2 days. Cell secreted matrix was absent on unmodified membranes for the same duration. Vitronectin-adsorbed surfaces did not show significant improvement in adhesion over unmodified membranes.
Keywords: Alumina; Bone tissue engineering; Nanotopography; Osteoblast; RGD peptide; XPS;

Bone tissue engineering on patterned collagen films: an in vitro study by S. Ber; G. Torun Köse; V. Hasırcı (1977-1986).
This study aimed at guiding osteoblast cells from rat bone marrow on chemically modified and patterned collagen films to study the influence of patterns on cell guidance. The films were stabilized using different treatment methods including crosslinking with carbodiimide (EDC) and glutaraldehyde, dehydrothermal treatment (DHT), and deposition of calcium phosphate on the collagen membrane.Mesenchymal osteoprogenitor cells were differentiated into osteoblasts and cultured for 7 and 14 days on micropatterned (groove width: 27 μm, groove depth: 12 μm, ridge width: 2 μm) and macropatterned (groove width: 250 μm, groove depth: 250 μm, ridge width: 100 μm) collagen films to study the influence of pattern dimensions on osteoblast alignment and orientation.Fibrinogen was added to the patterned surfaces as a chemical cue to induce osteoblast adhesion. Cell proliferation on collagen films was determined using MTS assay. Deposition of calcium phosphate on the surface of the film increased surface hydrophilicity and roughness and allowed a good cell proliferation. Combined DHT and EDC treatment provided an intermediate wettability, and also promoted cell proliferation. Glutaraldehyde crosslinking was found to lead to the lowest cell proliferation but fibrinogen adsorption on glutaraldehyde treated film surfaces increased the cell proliferation significantly. Macropatterns were first tested for alignment and only microscopy images were enough to see that there is no specific alignment. As a result of this, micropatterned samples with the topography that affect cell alignment and guidance were used. Osteoblast phenotype expression (ALP activity) was observed to be highest in calcium phosphate deposited samples, emphasizing the effect of mineralization on osteoblast differentiation. In general ALP activity per cell was found to decrease from day 7 to day 14 of incubation. SEM and fluorescence microscopy revealed good osteoblast alignment and orientation along the axis of the patterns when micropatterned films were used.This study shows that it is possible to prepare cell carriers suitable for tissue engineering through choice of appropriate surface topography and surface chemistry. Presence of chemical cues and micropatterns on the surface enhance cell orientation and bone formation.
Keywords: Tissue engineering; Micropatterned surfaces; Cell orientation; Osteoblasts; Collagen;

De novo engineering of reticular connective tissue in vivo by silk fibroin nonwoven materials by Ilaria Dal Pra; Giuliano Freddi; Jasminka Minic; Anna Chiarini; Ubaldo Armato (1987-1999).
Biologically tolerated biomaterials are the focus of intense research. In this work, we examined the biocompatibility of three-dimensional (3D) nonwovens of sericin-deprived, Bombyx mori silk fibroin (SF) in β-sheet form implanted into the subcutaneous tissue of C57BL6 mice, using sham-operated mice as controls. Both groups of mice similarly healed with no residual problem. Macroarray analysis showed that an early (day 3) transient expression of macrophage migration inhibitory factor (MIF) mRNA, but not of the mRNAs encoding for 22 additional proinflammatory cytokines, occurred solely at SF-grafted places, where no remarkable infiltration of macrophages or lymphocytes subsequently happened. Even an enduring moderate increase in total cytokeratins without epidermal hyperkeratosis and a transient (days 10–15) upsurge of vimentin occurred exclusively at SF-grafted sites, whose content of collagen type-I, after a delayed (day 15) rise, ultimately fell considerably under that proper of sham-operated places. By day 180, the interstices amid and surfaces of the SF chords, which had not been appreciably biodegraded, were crammed with a newly produced tissue histologically akin to a vascularized reticular connective tissue, while some macrophages but no lymphocytic infiltrates or fibrous capsules occurred in the adjoining tissues. Therefore, SF nonwovens may be excellent candidates for clinical applications since they both enjoy a long-lasting biocompatibility, inducing a quite mild foreign body response, but no fibrosis, and efficiently guide reticular connective tissue engineering.
Keywords: Silk fibroin; Biocompatibility; Foreign body response; Tissue engineering; Reticular connective tissue; Angiogenesis;

Chondrocytes were seeded on either agarose or polyglycolic acid (PGA) unwoven meshes at 10 million cells/ml of scaffold volume to evaluate the effect that these two biomaterials have on the low-density culture of chondrocytes in a rotating-wall bioreactor. For both static and bioreactor culture, agarose constructs contained more glycosaminoglycan than their PGA counterparts. However, the PGA constructs contained more collagen for both culture conditions when compared to agarose. For the low seeding density of this study, PGA constructs cultured in the bioreactor did not outperform static cultures when comparing collagen content after 8 weeks. The mechanical properties of the PGA constructs also did not improve with culture time. Similar results were observed with the agarose culture, though both static- and bioreactor-culture agarose constructs exhibited increases in aggregate modulus at the end of the culture period. As in PGA culture, chondrocytes cultured in agarose may require a higher density to reap the benefits of the bioreactor environment.

In this study, the resistance of biodegradable biomaterials, composed of blends of poly(ε-caprolactone) (PCL) and the polymeric antimicrobial complex, polyvinylpyrrolidone–iodine (PVP-I) to the adherence of a clinical isolate of Escherichia coli is described. Blends of PCL composed of a range of high (50,000 g mol−1) to low (5000 g mol−1) molecular weight ratios of polymer and either devoid of or containing PVP-I (1% w/w) were prepared by solvent evaporation. Following incubation (4 h), there was no relationship between m. wt. ratio of PCL in films devoid of PVP-I and adherence of E. coli. Conversely, microbial adherence to PCL containing PVP-I decreased as the ratio of high:low m. wt. polymer was decreased and was approximately 1000 fold lower than that to comparator films devoid of PVP-I. Following periods of immersion of PVP-I containing PCL films under sink conditions in phosphate buffered saline, subsequent adherence of E. coli was substantially reduced for 2 days (40:60 m. wt. ratio) and 6 days (100:0 m. wt. ratio). Concurrent exposure of PCL and E. coli to sub-minimum inhibitory concentrations (sub-MIC) of PVP-I significantly reduced microbial adherence to the biomaterial; however, the molecular weight ratio of PCL did not affect this outcome. Pretreatment of PCL with similar sub-MIC of PVP-I prior to inclusion within the microbial adherence assay significantly decreased the subsequent adherence of E. coli. Greatest reduction in adherence was observed following treatment of PCL (40:60 m. wt. ratio) with 0.0156% w/w PVP-I. In conclusion, this study has illustrated the utility of PVP-I as a suitable therapeutic agent for incorporation within PCL as a novel biomaterial. Due to the combined antimicrobial and biodegradable properties, these biomaterials offer a promising strategy for the reduction in medical device related infection.
Keywords: Poly(ε-caprolactone); Povidone–iodine; Microbial adherence; Drug release;

Long term assessment of axonal regeneration through polyimide regenerative electrodes to interface the peripheral nerve by Natalia Lago; Dolores Ceballos; Francisco J Rodrı́guez; Thomas Stieglitz; Xavier Navarro (2021-2031).
Polyimide sieve electrodes were implanted between the severed ends of the sciatic nerve in rats. The degree of axonal regeneration through the electrode was examined by physiological and histological methods from 2 to 12 months postimplantation. Regeneration was successful in the 30 animals implanted. Functional reinnervation of hindlimb targets progressed to reach maximal levels at 6 months. Comparatively, the reinnervation of distal plantar muscles was lower than that of proximal muscles and of digital nerves. The number of regenerated myelinated fibers increased from 2 to 6 months, when it was similar to control values. The majority of myelinated fibers crossing the via holes and regenerated through the distal nerve had a normal appearance. However, in a few cases decline of target reinnervation and loss of regenerated nerve fibers was found from 6 to 12 months postimplantation. Motor axons labeled by ChAT immunoreactivity regenerated scattered within minifascicles, although they were found at higher density at the periphery of the regenerated nerve. The number of ChAT-positive axons was markedly lower distally than proximally to the sieve electrode.
Keywords: Interface; Motor axons; Nerve regeneration; Neural prosthesis; Peripheral nerve; Polyimide; Regenerative electrode;

Effective use of optimized, high-dose (50 kGy) gamma irradiation for pathogen inactivation of human bone allografts by Teri A. Grieb; Ren-Yo Forng; Richard E. Stafford; Jack Lin; Jamie Almeida; Simon Bogdansky; Chad Ronholdt; William N. Drohan; Wilson H. Burgess (2033-2042).
The safety of tissue allografts has come under increased scrutiny due to recent reports of allograft-associated bacterial and viral infections in tissue recipients. We report that 50 kGy of gamma irradiation, nearly three times the dose currently used, is an effective pathogen inactivation method when used under optimized conditions that minimize damage to the tissue. Cancellous bone dowels treated with a radioprotectant solution and 50 kGy of optimized irradiation had an ultimate compressive strength and modulus of elasticity equal to conventionally irradiated (18 kGy) and non-irradiated control bone grafts. We subjected bone dowels treated with this pathogen inactivation method to an in vitro cytotoxicity test using three different mammalian cell lines and concluded that the treated grafts were not cytotoxic. The log reduction of nine pathogens spiked into radioprotectant-treated bone irradiated to 50 kGy was also tested. We achieved 4.9 logs of inactivation of a model virus for HIV and hepatitis C and 5 logs inactivation of a model virus for human parvovirus B-19. Complete inactivation (6.0–9.2 logs) of seven clinically relevant microorganisms was demonstrated. The results show that a combination of radioprotectants and optimized, high-dose gamma irradiation is a viable method for producing safer cancellous bone grafts that have the mechanical strength of existing grafts.
Keywords: Bone grafts; Pathogen inactivation; Gamma irradiation; Biomechanical strength; Biocompatibility;

Characterisation of resin–dentine interfaces by compressive cyclic loading by Roland Frankenberger; David H. Pashley; Sven M. Reich; Ulrich Lohbauer; Anselm Petschelt; Franklin R. Tay (2043-2052).
The aims of this in vitro study were to evaluate the ultra-morphological changes in resin–dentine interfaces after different amounts of thermomechanical load (TML), and to determine the corresponding microtensile bond strengths (μTBS). Enamel/dentine discs with a thickness of 2 mm were cut from 24 human third molars and bonded with four adhesives involving different adhesion approaches: Syntac (Ivoclar Vivadent; used as multi-step etch-and-rinse adhesive), Clearfil SE Bond (Kuraray; two-step self-etch adhesive), Xeno III (Dentsply DeTrey; mixed all-in-one self-etch primer adhesive system), and iBond (Heraeus Kulzer; non-mixed all-in-one self-etch adhesive). The resin–dentine discs were cut into beams (width 2 mm; 2 mm dentine, 2 mm resin composite) and subsequently subjected to cyclic TML using ascending amounts of mechanical/thermal cycles (20 N at 0.5 Hz of mechanical load and 5–55 °C of thermal cycles: for 0/0, 100/3, 1,000/25, 10,000/250, 100,000/2,500 cycles). Loaded specimens were either cut perpendicularly in order to measure μTBS ( n = 20 ; crosshead speed: 1 mm/min) or were immersed in an aqueous tracer solution consisting of 50 wt% ammoniacal silver nitrate and processed for ultra-morphological nanoleakage examination using transmission electron microscopy (TEM). μTBS were significantly decreased by increasing amounts of TML for all adhesives (p<0.05). Bond strengths after 0 vs. 100,000 thermomechanical cycles were: Syntac: 41.3/30.1 MPa; Clearfil SE Bond 44.8/32.5 MPa; Xeno III 27.5/13.7 MPa; iBond 27.0/6.2 MPa. Relatively early, a certain amount of nanoleakage was observed in all groups by TEM, which was more pronounced for Xeno III and iBond. The incidence of nanoleakage remained stable or was even reduced with increasing load cycles for all adhesives except iBond, where exact failure origins were detected within the adhesive and at the top of the hybrid layer.

Failure analysis of explanted sternal wires by Chun-Ming Shih; Yea-Yang Su; Shing-Jong Lin; Chun-Che Shih (2053-2059).
To classify and understand the mechanisms of surface damages and fracture mechanisms of sternal wires, explanted stainless steel sternal wires were collected from patients with sternal dehiscence following open-heart surgery. Surface alterations and fractured ends of sternal wires were examined and analyzed. Eighty fractured wires extracted from 25 patients from January 1999 to December 2003, with mean implantation interval of 55±149 days (range 5–729 days) after cardiac surgery, were studied by various techniques. The extracted wires were cleaned and the fibrotic tissues were removed. Irregularities and fractured ends were assayed by a scanning electron microscopy. After stereomicroscopy and documentation, the explants were cleaned with 1% sodium hypochlorite to remove the blood and tissues and was followed by cleaned with deionized water and alcohol. The explants were examined by stereomicroscopy, and irregularities on surface and fracture surfaces of sternal wires were assayed by scanning electron microscopy, energy dispersive X-ray analysis (EDAX) and X-ray mapping. The explants with surrounding fibrotic tissue were stained and examined with stereomicroscopy and transmission electronic microscopy. Corrosion pits were found on the surface of explanted sternal wires. EDAX and X-ray mapping examinations revealed diminution of nickel concentration in the severely corroded pits on sternal wires. A feature of transgranular cracking was observed for stress corrosion cracking and striation character for typical corrosion fatigue was also identified. TEM examination of tissue showed the metallic particles in phagolysosomes of macrophages inside the surrounding sternal tissue. The synergic effect of hostile environment and the stress could be the precursors of failures for sternal wires.

Epithelial internalization of superparamagnetic nanoparticles and response to external magnetic field by Dormer Kenneth; Seeney Charles; Lewelling Kevin; Lian Guoda; Gibson Donald; Johnson Matthew (2061-2072).
Superparamagnetic magnetite nanoparticles (MNP) coated with silica were synthesized and chronically implanted into the middle ear epithelial tissues of a guinea pig model ( n = 16 ) for the generation of force by an external magnetic field. In vivo limitations of biocompatibility include particle morphology, size distribution, composition and mode of internalization. Synthesis of MNP was performed using a modified precipitation technique and they were characterized by transmission electron microscopy, X-ray diffractometry and energy dispersive spectroscopy, which verified size distribution, composition and silica encapsulation. The mechanism for internalizing 16±2.3 nm diameter MNP was likely endocytosis, enhanced by magnetically force. Using sterile technique, middle ear epithelia of tympanic membrane or ossicles was exposed and a suspension of particles with fluoroscein isothiocyanate (FITC) label applied to the surface. A rare earth, NdFeBo magnet (0.35 T) placed under the animal, was used to pull the MNP into the tissue. After 8 days, following euthanasia, tissues were harvested and confocal scanning laser interferometry was used to verify intracellular MNP. Displacements of the osscicular chain in response to an external sinusoidal electromagnetic field were also measured using laser Doppler interferometry. We showed for the first time a physiologically relevant, biomechanical function, produced by MNP responding to a magnetic field.
Keywords: Nanoparticles; Middle ear; Biomechanics; Hearing; Endocytosis;

Novel biomaterials for bisphosphonate delivery by Solen Josse; Corinne Faucheux; A. Soueidan; Gaël Grimandi; Dominique Massiot; Bruno Alonso; Pascal Janvier; Samia Laı̈b; Paul Pilet; Olivier Gauthier; Guy Daculsi; Jérôme Guicheux; Bruno Bujoli; Jean-Michel Bouler (2073-2080).
One type of gem-bisphosphonate (Zoledronate) has been chemically associated onto calcium phosphate (CaP) compounds of various compositions. For that purpose, CaP powders of controlled granulometry have been suspended in aqueous Zoledronate solutions of variable concentrations. Using mainly 31P NMR spectroscopy, two different association modes have been observed, according to the nature of the CaP support and/or the initial concentration of the Zoledronate solution. β-tricalcium phosphate (β-TCP) and mixtures of hydroxyapatite and β-TCP (BCPs) appear to promote Zoledronate-containing crystals formation. On the other hand, at concentrations <0.05 mol l−1 CDAs (calcium deficients apatites) seem to undergo chemisorption of the drug through a surface adsorption process, due to PO3 for PO4 exchange, that is well described by Freundlich equations. At concentrations >0.05 mol l−1, crystalline needles of a Zoledronate complex form onto the CDAs surface. The ability of such materials to release Zoledronate, resulting in the inhibition of osteoclastic activity, was shown using a specific in vitro bone resorption model.
Keywords: Apatite structure; Calcium phosphate; Drug release; Osteoclast;

Silver ion release from antimicrobial polyamide/silver composites by Radhesh Kumar; Helmut Münstedt (2081-2088).
Silver ion (Ag+) the versatile antimicrobial species was released in a steady and prolonged manner from a silver-filled polyamide composite system. Metallic silver powder having varying specific surface area (SSA) has been used as a resource of biocide in polyamide. Strong evidences are found showing the release of the antimicrobial species from the resulting composite upon soaking it in water due to the interaction of the diffused water molecules with the dispersed silver powder within the matrix. The Ag+ release was observed as increasing with time and concentration of the silver powder and is found to be influenced by the SSA of the silver powder, changes in the physical state of the composite specimen as a result of the water diffusion and the composite morphology. It is observed that the Ag+ release increases initially which is followed by a marginal increase between day 4 and 6. Composites containing higher amounts of silver (4 and 8 wt%) exhibit a further rise in Ag+ release from the sixth day of storage in water. Composite containing silver particles with the lowest specific surface area (0.78 m2/g) showed highest Ag+ release. SEM shows a finer dispersion of the silver powder (4 wt%) having lowest SSA. However particles with higher (1.16 and 2.5 m2/g) SSA possess an agglomerated morphology leading to lower Ag+ release. The composites are found to release Ag+ at a concentration level capable of rendering an antimicrobial efficacy.
Keywords: Silver; Antimicrobial; Plasticisation;

ABA-type triblock copolymers poly (trimethylene carbonate)–poly (ethylene glycol)–poly (trimethylene carbonate) were synthesized by ring-opening polymerization of trimethylene carbonate initiated by dihydroxyl poly (ethylene glycol). The critical micelle concentration of amphiphilic triblock copolymers in aqueous solution was determined by fluorescence spectroscopy using 9-chloromethyl anthracene as fluorescence probe. Core-shell-type nanoparticles were prepared by the dialysis technique. Transmission electron microscopy images showed that these nanoparticles were regularly spherical in shape. Micelle size determined by dynamic light scattering is 50–160 nm. Anticancer drug methotrexate (MTX) as model drug was loaded in the polymeric nanoparticles. X-ray powder diffraction spectra showed that model drugs were molecularly dispersed in the core. In vitro release behavior of MTX was investigated.
Keywords: Amphiphilic triblock copolymers; Poly (ethylene glycol); Poly (trimethylene carbonate); Nanoparticles; Methotrexate;

The safety and efficacy of gentamycin sulphate (GS)- or vancomycin hydrochloride (VCl)-loaded polymer devices based on poly(acrylic acid) and gelatin crosslinked selectively using 0.3 mol % N,N′-methylene bisacrylamide and 1 wt% glutaraldehyde were evaluated by varying the drug concentration onto the devices. The placebo and drug-loaded device of AxGx (acrylic acid:gelatin: 1:1 w/w) were employed for the treatment of experimental osteomyelitis in rabbit. Rabbits were categorized into four groups. Twelve rabbits in each group were treated with 12±1 mg of AxGx-1a (22% w/w GS), 12±1 mg of AxGx-1b (44% w/w GS), 16±1 mg of AxGx-1b (44% w/w GS) and 16±1 mg of AxGx-1c (44% w/w VCl). The drug concentration was measured following implantation in the adjacent tissue of femoral cavity, and serum. In femoral cavity maximum drug concentration was found on the 7th day with all the four types of devices. No drug was found after 21 days, at the local site with devices AxGx-1a and AxGx-1b (12±1 mg), whereas it was detected after 6 weeks with 16±1 mg device (44% w/w GS or VCl). Macroscopic evaluation after treatment revealed that swelling, redness, local warmth and drainage decreased depending upon the drug loading of the implants. Sequential radiographs, histology, microbiologic assay and scanning electron micrography demonstrated devices AxGx-1b and AxGx-1c (16±1 mg of 44% w/w drug loading) to be the most suitable device, which heals the infection after 6 weeks of treatment. No significant difference (p>0.05) in the rate of healing was observed between GS- and VCl-loaded devices. None of the implant showed toxic level of drug in serum at any given time.
Keywords: Interpenetrating polymer network; Gentamycin sulphate; Vancomycin hydrochloride; Biodegradation; Osteomyelitis;

Physically crosslinked alginate/N,O-carboxymethyl chitosan hydrogels with calcium for oral delivery of protein drugs by Yu-Hsin Lin; Hsiang-Fa Liang; Ching-Kuang Chung; Mei-Chin Chen; Hsing-Wen Sung (2105-2113).
In the study, a complex composed of alginate blended with a water-soluble chitosan (N,O-carboxymethyl chitosan, NOCC) was prepared to form microencapsulated beads by dropping aqueous alginate–NOCC into a Ca2+ solution. These microencapsulated beads were evaluated as a pH-sensitive system for delivery of a model protein drug (bovine serum albumin, BSA). The main advantage of this system is that all procedures used were performed in aqueous medium at neutral environment, which may preserve the bioactivity of protein drugs. The swelling characteristics of these hydrogel beads at distinct compositions as a function of pH values were investigated. It was found that the test beads with an alginate-to-NOCC weight ratio of 1:1 had a better swelling characteristic among all studied groups. With increasing the total concentration of alginate–NOCC, the effective crosslinking density of test beads increased significantly and a greater amount of drug was entrapped in the polymer chains (up to 77%). The swelling ratios of all test groups were approximately the same (∼3.0) at pH 1.2. At pH 7.4, with increasing the total concentration of alginate–NOCC, the swelling ratios of test beads increased significantly (20.0–40.0), due to a larger swelling force created by the electrostatic repulsion between the ionized acid groups (–COO). It was shown that BSA was uniformly distributed in all test beads. At pH 1.2, retention of BSA in hydrogels may be improved by rinsing test beads with acetone (the amount of BSA released was below 15%). At pH 7.4, the amounts of BSA released increased significantly (∼80%) as compared to those released at pH 1.2. With increasing the total concentration of alginate–NOCC, the release of encapsulated proteins was slower. Thus, the calcium–alginate–NOCC beads with distinct total concentrations developed in the study may be used as a potential system for oral delivery of protein drugs to different regions of the intestinal tract.
Keywords: Alginate; N,O-carboxymethyl chitosan; Hydrogel; pH sensitivity; Oral drug delivery;

A novel non-toxic camptothecin formulation for cancer chemotherapy by M. Berrada; A. Serreqi; F Dabbarh; A. Owusu; A. Gupta; S. Lehnert (2115-2120).
The use of a novel injectable biocompatible and biodegradable camptothecin-polymer implant for sustained intra-tumoral release of high concentrations of camptothecin is described. The drug delivery vehicle is an in situ thermogelling formulation, which is based on the natural biopolymer chitosan. This formulation, containing homogeneously dispersed camptothecin, was implanted intra-tumorally into a sub-cutaneous mouse tumor model (RIF-l). The effectiveness of treatment was measured in terms of tumor growth delay (TGD). Animals treated with the polymer implants containing camptothecin had significantly longer TGDs compared to untreated animals as well as to animals treated systemically with camptothecin by intra-peritoneal injection with no evidence of toxicity in terms of loss of body weight. The results indicate that this novel biodegradable polymer implant is an effective vehicle for the sustained intra-tumoral delivery of camptothecin which might also be suitable to deliver other insoluble anti-cancer drugs such as taxol.
Keywords: Drug delivery; Thermally responsive material; Biodegradation; Intra-tumoral; Chitosan; BST-gel;

Synthesis and characterization of the paclitaxel/MPEG-PLA block copolymer conjugate by Xuefei Zhang; Yuxin Li; Xuesi Chen; Xiuhong Wang; Xiaoyi Xu; Qizhi Liang; Junli Hu; Xiabin Jing (2121-2128).
A paclitaxel/MPEG-PLA block copolymer conjugate was prepared in three steps: (1) hydroxyl-terminated diblock copolymer of monomethoxy-poly(ethylene glycol)-b-poly(lactide) (MPEG-PLA) was synthesized by ring-opening polymerization of l-lactide using MPEG as a maroinitiator; (2) it was converted to carboxyl-terminated MPEG-PLA by reacting with mono-t-butyl ester of diglycolic acid and subsequent deprotecting the t-butyl group with TFA; (3) the latter was reacted with paclitaxel in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine. Structures of the polymers synthesized were confirmed by 1H NMR, and their molecular weights were determined by gel permeation chromatography. The antitumor activity of the conjugate against human liver cancer H7402 cells was evaluated by MTT method. The results showed that paclitaxel can be released from the conjugate without losing cytotoxicity.
Keywords: Paclitaxel; Prodrug; Ring-opening polymerization; Diglycolic anhydride;

Self-gelling hydrogels based on oppositely charged dextran microspheres by Sophie R. Van Tomme; Mies J. van Steenbergen; Stefaan C. De Smedt; Cornelus F. van Nostrum; Wim E. Hennink (2129-2135).
This paper presents a novel self-gelling hydrogel potentially suitable for controlled drug delivery and tissue engineering. The macroscopic gels are obtained by mixing dispersions of oppositely charged crosslinked dextran microspheres. These microspheres in turn were prepared by crosslinking of dextran derivatized with hydroxyethyl methacrylate emulsified in an aqueous poly(ethylene glycol) solution. Negatively or positively charged microspheres were obtained by addition of methacrylic acid (MAA) or dimethylaminoethyl methacrylate (DMAEMA) to the polymerization mixture. Rheological analysis showed that instantaneous gelation occurred when equal volumes of oppositely charged microspheres, dispersed in buffer solutions of pH 7, were mixed. The shear modulus of the networks could be tailored from 30 to 6500 Pa by varying the water content of the system. Moreover, controlled strain and creep experiments showed that the formed networks were mainly elastic. Importantly for application of these systems, e.g. as controlled matrix of pharmaceutically active proteins, it was demonstrated that the hydrogel system has a reversible yield point, meaning that above a certain applied stress, the system starts to flow, whereas when the stress is removed, gel formation occurred. Further it was shown that the network structure could be broken by either a low pH or a high ionic strength of the medium. This demonstrates that the networks, formed at pH 7 and at low ionic strength, are held together by ionic interactions between the oppositely charged dextran microspheres. This system holds promise as injectable gels that are suitable for drug delivery and tissue engineering applications.

Size and temperature effects on poly(lactic-co-glycolic acid) degradation and microreservoir device performance by Amy C. Richards Grayson; Michael J. Cima; Robert Langer (2137-2145).
The component materials of controlled-release drug delivery systems are often selected based on their degradation rates. The release time of a drug from a system will strongly depend on the degradation rates of the component polymers. We have observed that some poly(lactic-co-glycolic acid) polymers (PLGA) exhibit degradation rates that depend on the size of the polymer object and the temperature of the surrounding environment. In vitro degradation studies of four different PLGA polymers showed that 150 μm thick membranes degraded more rapidly than 50 μm thick membranes, as characterized by gel permeation chromatography and mass loss measurements. Faster degradation was observed at 37 °C than 25 °C, and when the saline media was not refreshed. A biodegradable polymeric microreservoir device that we have developed relies on the degradation of polymeric membranes to deliver pulses of molecules from reservoirs on the device. Earlier molecular release was seen from devices having thicker PLGA membranes. Comparison of an in vitro release study from these devices with the degradation study suggests that reservoir membranes rupture and drug release occurs when a membrane threshold molecular weight of 5000–15000 is reached.

The use of chitosan as a condensing agent to enhance emulsion-mediated gene transfer by Mi-Kyung Lee; Soo-Kyung Chun; Woo-Jeong Choi; Jin-Ki Kim; Sung-Hee Choi; Adele Kim; Kwunchit Oungbho; Jeong-Sook Park; Woong Shick Ahn; Chong-Kook Kim (2147-2156).
Previously we have formulated a new cationic emulsion, composed of 3β [N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol and dioleoylphosphatidyl ethanolamine, castor oil and Tween 80, and it efficiently delivered plasmid DNA into various cancer cells with low toxicity. Chitosan is a natural cationic polysaccharide and is able to form polyelectrolyte complexes with DNA, in which the DNA is condensed and protected against nuclease degradation. Based on these facts, chitosan was used as a condensing agent to enhance the transfection efficiency of cationic emulsion-mediated gene delivery vehicle. The particle size, zeta potential and transmission electron micrographs of DNA/emulsion complexes were observed before and after condensation by chitosan. In vitro transfection efficiency of naked or precondensed DNA/emulsion (pcDNA/E) complexes was investigated in human hepatoma cells (HepG2) using flow cytometer, confocal microscope and western blot. In addition, in vivo gene transfer was also evaluated as GFP mRNA expression by reverse transcriptase-polymerase chain reaction. The size of transfection complexes was reduced after the condensation of DNA by chitosan. Moreover, when the pcDNA/E complexes were administered into the mice, the GFP mRNA expression was prolonged in liver and lung until day 6. These results suggest that the use of chitosan enhance the in vitro transfection efficiency and extend in vivo gene transfer.
Keywords: Gene therapy; Non-viral vectors; Chitosan; Emulsion; Precondensed DNA;

Nanoparticles of Mg and Mn (II) phosphates encapsulating pDNA were prepared. The sizes of these DNA loaded particles in aqueous dispersion were about 100–130 nm diameter, and they aggregated with the progression of time. Although magnesium phosphate nanoparticles were crystalline, the manganous phosphate nanoparticles were found to be amorphous in nature. Nanoparticle dissolution and pDNA release were studied using atomic absorption spectroscopy and gel electrophoresis experiments. These inorganic phosphate nanoparticles dissolved in mild acidic pH (∼5) releasing pDNA indicating that DNA release in the endosomal compartment is possible. In vitro transfection in HeLa cells demonstrated that while magnesium phosphate nanoparticles showed 100% efficiency, manganous phosphate nanoparticles exhibited about 85% transfection efficiency compared to that of ‘polyfect’, as control.
Keywords: Magnesium phosphate; Manganous phosphate; Nanoparticles; Non-viral vectors; Gene delivery; Transfection efficiency;

Multi-channel 3-D cell culture device integrated on a silicon chip for anticancer drug sensitivity test by Yu-suke Torisawa; Hitoshi Shiku; Tomoyuki Yasukawa; Matsuhiko Nishizawa; Tomokazu Matsue (2165-2172).
A novel three-dimensional cell culture system was constructed with an array of cell panels (4×5) in a silicon chip, together with multi-channel drug containers. Human breast cancer (MCF-7) cells were embedded in a collagen–gel matrix and entrapped in a pyramidal-shaped silicon hole. Each cell panel can be isolated by a channel composed of a microfluid part and a reservoir. A cell panel was exposed to 200 mm KCN for 2 days to demonstrate that each cell panel could be independently evaluated under various stimulation conditions. Based on the cellular respiration activity, the proliferation behavior was continuously monitored on the silicon-based cell array for 5 days using scanning electrochemical microscopy (SECM). The cells entrapped in the device (3-D culture) proliferated normally, and the proliferation rate was lower than that of cells grown in a monolayer cell culture (2-D culture). The effects of three anticancer drugs measured simultaneously on the cell chip were in good agreement with those obtained by a conventional colorimetric assay. Our results suggest that the silicon-based device for 3D culture is appropriate for a chemosensitivity assay involving multi-chemical stimulation.
Keywords: Biosensor; Collagen; Cytotoxicity; Electrochemistry; Polydimethylsiloxane;

The enhancement of recombinant protein production by polymer nanospheres in cell suspension culture by Ju Hee Ryu; Min Soo Kim; Gyun Min Lee; Cha Yong Choi; Byung-Soo Kim (2173-2181).
Recombinant Chinese hamster ovary (rCHO) cells are being increasingly used in industry for the production of recombinant therapeutic proteins. Three-dimensional suspension culture is preferred to two-dimensional monolayer culture for the efficient large-scale culture of rCHO cells and subsequent mass production of recombinant proteins. Previously, we have demonstrated that the use of plain polymer nanospheres enhances the growth of anchorage-dependent animal cells (human embryonic kidney 293 cells) in suspension culture in serum-containing medium. Vitronectin and fibronectin were adsorbed onto poly(lactic-co-glycolic acid) (PLGA) nanospheres (696 nm in average diameter) by immersing the nanospheres in fetal bovine serum. In this study, we investigated if the use of vitronectin/fibronectin-adsorbed polymer nanospheres enhances recombinant protein production in rCHO cell suspension culture in serum-free medium. Cell aggregate formation may be critical for the survival and growth of anchorage-dependent animal cells in suspension culture, and cells in single cell suspension may result in cell death. The addition of vitronectin/fibronectin-adsorbed nanospheres to rCHO cell suspension culture promoted the rate and efficiency of cell aggregate formation. The nanospheres enhanced cell growth (2.9 folds on day 10) and, importantly, recombinant antibody production (1.8 folds on day 14), compared to suspension culture without nanospheres. The viability of cells in the aggregates in the nanosphere-added culture was high for the entire culture period of 14 days. Apoptotic activity of cells was much lower in the nanosphere-added culture than in the culture without nanospheres on day 5. The nanosphere suspension culture method developed in this study may be useful for the mass production of recombinant proteins through large-scale suspension culture of anchorage-dependent animal cells.
Keywords: Suspension culture; Recombinant protein production; Nanosphere; Recombinant chinese hamster ovary cells;

Aspects of in vitro fatigue in human cortical bone: time and cycle dependent crack growth by R.K. Nalla; J.J. Kruzic; J.H. Kinney; R.O. Ritchie (2183-2195).
Although fatigue damage in bone induced by cyclic loading has been recognized as a problem of clinical significance, few fracture mechanics based studies have investigated how incipient cracks grow by fatigue in this material. In the present study, in vitro cyclic fatigue experiments were performed in order to quantify fatigue-crack growth behavior in human cortical bone. Crack-growth rates spanning five orders of magnitude were obtained for the extension of macroscopic cracks in the proximal-distal direction; growth-rate data could be well characterized by the linear-elastic stress-intensity range, using a simple (Paris) power law with exponents ranging from 4.4 to 9.5. Mechanistically, to discern whether such behavior results from “true” cyclic fatigue damage or is simply associated with a succession of quasi-static fracture events, cyclic crack-growth rates were compared to those measured under sustained (non-cyclic) loading. Measured fatigue-crack growth rates were found to exceed those “predicted” from the sustained load data at low growth rates (∼3×10−10 to 5×10−7 m/cycle), suggesting that a “true” cyclic fatigue mechanism, such as alternating blunting and re-sharpening of the crack tip, is active in bone. Conversely, at higher growth rates (∼5×10−7 to 3×10−5 m/cycle), the crack-growth data under sustained loads integrated over the loading cycle reasonably predicts the cyclic fatigue data, indicating that quasi-static fracture mechanisms predominate. The results are discussed in light of the occurrence of fatigue-related stress fractures in cortical bone.
Keywords: Cortical bone; Fatigue; Fracture; Life prediction;