Biomaterials (v.26, #15)

Calendar (I).

The present study describes the synthesis of spheroidized hydroxyapatite (HA) powders using a radio frequency (rf) inductively coupled plasma (ICP) torch. The spheroidized powders were consolidated through a spark plasma sintering (SPS) system. The microstructure and crystallographic phases in the synthesized powders were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffractometry (XRD) and Raman spectrometry. Results showed that the HA feedstock decomposed after rf plasma processing. Crystalline HA, α-tri-calcium phosphate (α-TCP), tetra-calcium phosphate (TTCP) and calcium oxide (CaO) were detected in the plasma-spheroidized powders. Raman spectra results indicated strong presence of amorphous calcium phosphate (ACP) in the spheroidized powders. The particle size distribution and specific surface area were influenced through the rf plasma working plate power levels. The sintering behavior of the rf plasma synthesized powders was analyzed through the SPS process and the results indicated that the spheroidized powders commence sintering at approximately 900 °C and through to 1150 °C. After sintering above 1100 °C for 3 min, the relative densities of the SPS compacts reached 96% of the theoretical value. The SPS compacts were immersed in simulated body fluids (SBF) for different durations and the results confirmed their bioactivities.

A series of polycaprolactone and ternary-based (Na2O)0.55− x (CaO) x (P2O5)0.45 glass composites were created, each containing 20% volume percentage of glass with various calcium compositions. A short-term degradation study was carried out to investigate the physical and ion release behaviour of these composites, utilising analytical techniques such as dynamical mechanical analysis, and ion chromatography.All the composites experienced significant loss of weight and stiffness throughout the study, with the 24 mol% calcium composites losing the greatest amount of weight and stiffness. The pH profile of the aqueous solutions in which the composites were placed were initially acidic, but began to neutralise mid-way through the study, with the 36 mol% solution achieving the most acidic conditions. The ion release behaviour mirrored the mass loss behaviour of the glass component of the composites. The cations (sodium and calcium ions) release was comparable with the initial stages of composite mass degradation, both of which exhibited almost immediate release when placed into solution. The 24 mol% composites underwent rapid rates of cation release, while the 36 mol% experienced the slowest rates of release. By contrast, anion (phosphates and polyphosphates) release showed a dissimilar trend, with rapid release of the P2O7 and P3O10 occurring during the first few hours in solution, whilst the P3O9 structure released steadily during the first 48 h in solution. Finally, PO4 release was at a constant rate over the duration of the study, releasing up to 300 ppm from the 32 and 36 mol% samples by the end of 200 h. To summarise, these results show that by combining phosphate glasses with biodegradable polymer, it is possible to create composites whose rate of degradation can be controlled to meet the needs of their end application.
Keywords: Glass; Composite; Degradation; Polycaprolactone;

Amphiphilic conetwork polymers were prepared and studied as substrates in the culture of dermal fibroblasts. Both block and random conetworks polymers were produced by radical polymerization of either low-molecular weight monomers or oligomeric macromonomers. The oligomeric macromonomers were prepared by methacrylation of biscarboxy oligo(butyl methacrylates) (OBMA). The latter were synthesized by ozononolysis of poly(butyl methacrylate-co-butadiene) materials. The hydrophilic component was derived from copolymerization with 2,3 propandiol-1-methacrylate and cross-linking was provided by inclusion of ethandiol dimethacrylate (EDMA). None of the synthesized materials showed indications of cytotoxicity to human dermal fibroblasts. All of the block conetworks were highly phase separated and possessed pores on the micron length scale. The equilibrium water contents of the latter could be controlled by addition of EDMA. Block conetworks that did not contain EDMA were highly swollen and had smoother surfaces than those that contained EDMA. The former were poor substrates for cell proliferation (as measured by monitoring DNA content) whilst the latter class gave increasing levels of DNA during culture; an indicator proliferation. The performance of these materials in cell culture was also dependent on the fraction of OBMA in the formulation. Increasing the fractions of BMA, either in the random terpolymer or block networks, system had the effect of increasing both cell proliferation and viability (as measured by the Alamar Blue assay).
Keywords: Ampiphilic network; Hydrogel; Fibroblast; Cell proliferation; Methacrylate;

Preparation of ferrimagnetic magnetite microspheres for in situ hyperthermic treatment of cancer by Masakazu Kawashita; Masashi Tanaka; Tadashi Kokubo; Yoshiaki Inoue; Takeshi Yao; Sunao Hamada; Teruya Shinjo (2231-2238).
Ferrimagnetic microspheres 20–30 μm in diameter are useful as thermoseeds for inducing hyperthermia in cancers, especially for tumors located deep inside the body. The microspheres are entrapped in the capillary bed of the tumors when they are implanted through blood vessels and heat cancers locally by their hysteresis loss when placed under an alternating magnetic field. In the present study, preparation of magnetite (Fe3O4) microspheres 20–30 μm in diameter was attempted by melting powders in high-frequency induction thermal plasma, and by precipitation from aqueous solution. The microspheres prepared by melting powders in high-frequency induction thermal plasma were composed of a large amount of Fe3O4 and a small amount of wustite (FeO), and those subsequently heat treated at 600 °C for 1 h under 5.1×103  Pa were fully composed of Fe3O4 1 μm in size. The saturation magnetization and coercive force of the heat-treated microspheres were 92 emu g–1 and 50 Oe, respectively. The heat generation of the heat-treated microspheres was estimated to be 10 W g–1, under 300 Oe and 100 kHz. The microspheres prepared by precipitation from aqueous solution consisted of β-FeOOH, and those subsequently heat treated at 400 °C for 1 h in a 70% CO2+30% H2 atmosphere consisted of Fe3O4 crystals 50 nm in size. The saturation magnetization and coercive force of the heat-treated microspheres were 53 emu g–1 and 156 Oe, respectively. The heat generation of the heat-treated microspheres was estimated to be 41 W g–1, under 300 Oe and 100 kHz. The latter microspheres are believed to be promising thermoseeds for hyperthermic treatment of cancer.
Keywords: Ferrimagnetism; Magnetite; Microsphere; Hysteresis loss; Hyperthermia;

In this study, we examined the corrosion behaviors of pure titanium, the alloys Ti–6Al–4V and Ti–6Al–7Nb, and the new experimental alloys Ti–Pt and Ti–Pd using anodic polarization and corrosion potential measurements in an environment containing fluoride. Before and after immersion in the test solutions, we made observations using a scanning electron microscope. The test solutions included an artificial saliva containing 0.2% NaF (corresponding to 905 ppm F) and an artificial saliva with a low concentration of oxygen. Although the surfaces of the Ti–Pt and Ti–Pd alloys were not affected by an acidic environment containing fluoride, the surfaces of the pure titanium, the Ti–6Al–4V alloy, and the Ti–6Al–7Nb alloy were markedly roughened by corrosion. The surfaces of the pure titanium, the Ti–6Al–4V alloy, and the Ti–6Al–7Nb alloy were microscopically damaged by corrosion when they were immersed in the solution containing a low concentration of dissolved oxygen, even with a fluoride concentration included in the commercial dentifrices. In this situation, however, the surfaces of the new Ti–Pt and Ti–Pd alloys were not affected. These alloys are expected to be of use in dental work as new titanium alloys with high corrosion resistances.

Characterisation of antibacterial copper releasing degradable phosphate glass fibres by E.A. Abou Neel; I. Ahmed; J. Pratten; S.N. Nazhat; J.C. Knowles (2247-2254).
Phosphate-based glass fibres (PGF) of the general formula Na2O–CaO–P2O5 are degradable in an aqueous environment, and therefore can function as antibacterial delivery systems through the inclusion of ions such as copper. In this study, PGF with varying amounts of copper oxide (CuO) were developed for potential uses in wound healing applications. PGF with 0, 1, 5 and 10 mol% CuO were produced with different diameters and characterised in terms of structural and antibacterial properties. The effect of CuO and fibre pulling speed on the glass properties were investigated using rapid differential scanning calorimetry, differential thermal analysis and X-ray diffraction. The effect of two fibre diameters on short-term (3 h) attachment and killing against Staphylococcus epidermidis were investigated and were related to their rate of degradation in deionised water, as well as copper ion release measured using ion chromatography.Thermal analysis showed that there was a significant increase in the PGF glass transition temperature as the CuO content increased. There was a significant decrease in the rate of degradation with increasing CuO content and an increase in fibre diameter. Over 6 h, both the amount and rate of copper ions released increased with CuO content, as well as a reduction in fibre diameter thus increasing the surface area to volume ratio. There was a decrease in the number of viable staphylococci both attached to the CuO-containing fibres and in the surrounding environment.
Keywords: Phosphate glass fibres; Copper ions; Antimicrobial; DSC; DTA; Ion chromatography; Wound healing; Sutures;

A new model formulation of the SiO2–Al2O3–B2O3–MgO–CaO–Na2O–F glass-ceramics by Simeon Agathopoulos; Dilshat U. Tulyaganov; Patricia Valério; José M.F. Ferreira (2255-2264).
Mono-phase glass-ceramics of akermanite were successfully produced from a Ca-mica and wollastonite via low-temperature sintering and crystallization. Doping with P2O5 considerably improves sintering behaviour since P2O5 increases the stability of glass against crystallization at the temperature of sintering onset. The resulting glass-ceramics feature good in vitro acceptance from osteoblasts, and moderate bioactivity due to the enrichment of the glassy phase with Ca and Si. The good quality of the white colour at the surface and throughout the bulk, the matching of microhardness with tooth enamel, and the possibility to coat other biomaterials such as ZrO2, Ti or hydroxyapatite make these materials promising for medical applications.
Keywords: Akermanite; Glass; Glass-ceramics; Properties; Biocompatibility; In vitro;

Carbon plasma immersion ion implantation of nickel–titanium shape memory alloys by R.W.Y. Poon; K.W.K. Yeung; X.Y. Liu; P.K. Chu; C.Y. Chung; W.W. Lu; K.M.C. Cheung; D. Chan (2265-2272).
Nickel–titanium (NiTi) shape memory alloys possess super-elasticity in addition to the well-known shape memory effect and are potentially suitable for orthopedic implants. However, a critical concern is the release of harmful Ni ions from the implants into the living tissues. We propose to enhance the corrosion resistance and other surface and biological properties of NiTi using carbon plasma immersion ion implantation and deposition (PIII&D). Our corrosion and simulated body fluid tests indicate that either an ion-mixed amorphous carbon coating fabricated by PIII&D or direct carbon PIII can drastically improve the corrosion resistance and block the out-diffusion of Ni from the materials. Our tribological tests show that the treated surfaces are mechanically more superior and cytotoxicity tests reveal that both sets of plasma-treated samples favor adhesion and proliferation of osteoblasts.
Keywords: NiTi shape memory alloys; Orthopedic implants; Corrosion resistance; Mechanical properties; Osteoblasts;

Immobilization of laminin peptide in molecularly aligned chitosan by covalent bonding by Atsushi Matsuda; Hisatoshi Kobayashi; Soichiro Itoh; Kazunori Kataoka; Junzo Tanaka (2273-2279).
We developed a new biomaterial effective for nerve regeneration consisting of molecularly aligned chitosan with laminin peptides bonded covalently. Molecularly aligned chitosan was prepared from crab (Macrocheira kaempferi) tendons by ethanol treatment and 4 wt%-NaOH aqueous solutions to remove proteins and calcium phosphate, followed by deacetyl treatment using a 50 wt%-NaOH aqueous solution at 100 °C. Molecularly aligned tendon chitosan was chemically thiolated by reacting 4-thiobutyrolactone with the chitosan amino group. The introduction of thiol groups and their distribution to tendon chitosan and chitosan cast film were confirmed using ATR FT-IR, 1H-NMR, and EDS. The 1.24 μmol/g of thiol groups introduced on the surface of tendon chitosan and the chitosan cast film was confirmed using ultraviolet (UV) spectra. Thiol groups of cysteine located at the end of synthetic laminin peptides were then reacted chemically with thiolated chitosan to form chitosan-S-S-laminin peptide. YIGSR estimated at 0.92 μmol/g and IKVAV estimated at 0.28 μmol/g on thiolated tendon chitosan were confirmed using UV spectra. YIGSR was estimated at 0.85 μmol/g and IKVAV was estimated at 0.34 μmol/g on the thiolated chitosan cast film.
Keywords: Crab tendon; Chitosan; 4-thiobutyrolactone; YIGSR; IKVAV; Immobilization;

Preparation of poly(ε-caprolactone)/continuous bioglass fibre composite using monomer transfer moulding for bone implant by G. Jiang; M.E. Evans; I.A. Jones; C.D. Rudd; C.A. Scotchford; G.S. Walker (2281-2288).
Poly(ε-caprolactone) (PCL)/continuous bioglass fibre composite was prepared using the monomer transfer moulding technique coupled with a surface initiated polymerisation. The bioglass fibres were surface treated with an amine ended silane in order to initiate polymerisation of ε-caprolactone from the fibre surface. Surface initiated polymerisation significantly improved the Young's modulus and flexural strength and water resistance of the composite. Initial in vitro biocompatibility assessment suggests that amine ended silane treatment of bioglass fibres before their inclusion in the composite does not have a negative effect on the biological responses in terms of macrophage activation as measured by IL-1β release and craniofacial osteoblast attachment.
Keywords: Poly(ε-caprolactone); Surface-initiated polymerisation; Monomer transfer moulding;

High-strength apatitic cement by modification with superplasticizers by E. Fernández; S. Sarda; M. Hamcerencu; M.D. Vlad; M. Gel; S. Valls; R. Torres; J. López (2289-2296).
This study reports on a novel method to improve the strength of apatitic bone cements. The liquid phase of Biocement-H© was modified with commercial superplasticizers. The results showed that small additions, i.e. 0.5 vol%, in the aqueous liquid phase improved the maximum compressive strength of Biocement-H © (35 MPa) by 71%, i.e. 60 MPa. Moreover, the addition of high amounts of superplasticizers, i.e. 50 vol.%, allowed for a significant reduction of the liquid-to-powder ratio from 0.32 to 0.256 mL/g, without affecting the maximum strength and/or the workability of the cement. These results open up new ways to develop injectable and high-strength apatitic bone cements for load-bearing applications.
Keywords: Calcium phosphate cement; Bone tissue engineering; Mechanical properties; Osteoporosis; Superplasticizer;

Designing biodegradable multiblock PCL/PLA thermoplastic elastomers by D. Cohn; A. Hotovely Salomon (2297-2305).
A series of poly(ε-caprolactone)/poly(L-lactic acid) (PCL/PLA) biodegradable poly(ester-urethane)s, was synthesized and characterized. The first step of the synthesis consisted of the ring opening polymerization of L-lactide, initiated by the hydroxyl terminal groups of the PCL chain, followed by the chain extension of these PLA-PCL-PLA triblocks, using hexamethylene diisocyanate (HDI). The trimers comprised PCL2000 flexible segments, while the length of each PLA block covered the 550–6000 molecular weight range. The morphology of the copolymers gradually changed, as the length of the PLA blocks increased. The multiblock copolymers produced displayed enhanced mechanical properties, with ultimate tensile strength values around 32 MPa, Young's modulus as low as 30 MPa and elongation at break values well above 600%. The longer the PLA block, the slower the in vitro degradation of the material, with all copolymers degrading faster than the respective homopolymers.
Keywords: Poly(caprolactone); Poly(lactic acid); Biodegradation; Elastomer; Copolymer;

Laser interference lithography as a new and efficient technique for micropatterning of biopolymer surface by Fayou Yu; Ping Li; Hao Shen; Sanjay Mathur; Claus-Michael Lehr; Udo Bakowsky; Frank Mücklich (2307-2312).
Laser interference lithography (LIL) is a straightforward technique to prepare linear micropatterns for regulating cellular adhesion behaviors on polymer substratum. This process is based on selective laser ablation directly duplicating the interference patterns of two or more coherent laser beams onto the polymer surface. Micropatterns prepared by LIL on poly(ethylene terephthalate) and Thermanox® were characterized using atomic force microscopy (AFM) and white light interferometer while the chemical surface modification induced by laser was analyzed by X-ray photoelectron spectroscopy (XPS). The AFM photographs show that the micropatterns are well-defined and of great consistency. Polymer properties and laser parameters related to LIL as well as laser ablation mechanisms are discussed in this technical note.
Keywords: Laser interference lithography; Micropatterning; Poly(ethylene terephthalate); Thermanox®; Cell adhesion; X-ray photoelectron microscopy;

Self-assembled biomimetic monolayers using phospholipid-containing disulfides by Yi Chang Chung; Yi Hong Chiu; Yin Wei Wu; Yu Tai Tao (2313-2324).
Several phospholipid-based disulfide molecules were synthesized and attached onto the gold-coated silicon wafer using the self-assembling method. The syntheses of these surface-modifying agents were conducted by introducing bromoethylphosphorate (PBr), phosphorylcholine (PC) or phosphorylethanolamine (PE) groups on the terminals of a dialkyl disulfide. After disulfides adsorption onto gold substrate surfaces, the composition, the film thickness, and the conformational order of self-assembled monolayer surfaces were explored and discussed in detail based on reflection–absorption infrared spectroscopy, contact angle measurement, Auger electron spectroscopy, X-ray photoelectron spectroscopy, and so on. The monolayer having the PBr end group could also be converted to a PC surface by treating with trimethylamine. The model functional surfaces of Au-SC11-PC, -PE, -PBr, -OH or corresponding mixed layers were used to mimic biomembrane surfaces. The monolayer having PC groups was found to reduce fibrinogen adsorption as evaluated from protein adsorption experiments using quartz crystal microbalance. It also showed relatively low platelet adherence compare to the glass, PBr and PE surfaces. The cell viability test also revealed that the PC surface displayed lower cytotoxicity than other surfaces.
Keywords: Self-assembled monolayer; Disulfides; Biocompatibility; Phosphorylcholine;

Osseointegration of alumina with a bioactive coating under load-bearing and unloaded conditions by Anita Ignatius; Manfred Peraus; Sandra Schorlemmer; Peter Augat; Wolfgang Burger; Stefan Leyen; Lutz Claes (2325-2332).
The aim of the study was to evaluate the osseointegration of Al2O3 coated with a bioactive glass ceramic (Bioverit®I), in a load-bearing implant model in sheep in comparison to uncoated Al2O3 and to a minimally loaded situation. Both types of implants were inserted into the proximal tibia (load-bearing model) and in a drill hole defect into the tibia diaphysis (minimally loaded model). Under load-bearing conditions, the coating resulted in significantly higher interfacial shear strength and a high amount of mineralized bone in direct contact to the implant surface. In contrast, the uncoated Al2O3 was surrounded by a thick connective tissue layer corresponding to low interfacial shear strength. In the minimally loaded model, however, there was rather a tendency of lower interfacial shear strength in the case of the coated implants. This finding corresponds to the histological results, which showed mineralized bone in the interface of uncoated Al2O3, whereas in the case of the coated implants a thin layer of osteoid was observed. It was suggested that the osseointegration of Al2O3 could be improved by the coating under load-bearing conditions, under which uncoated Al2O3 ceramics cannot directly bind to bone.
Keywords: Coated alumina; Glass ceramics; Bioactive; Osseointegration; Load-bearing model; Interface shear strength;

Mediating specific cell adhesion to low-adhesive diblock copolymers by instant modification with cyclic RGD peptides by E. Lieb; M. Hacker; J. Tessmar; L.A. Kunz-Schughart; J. Fiedler; C. Dahmen; U. Hersel; H. Kessler; M.B. Schulz; A. Göpferich (2333-2341).
One promising strategy to control the interactions between biomaterial surfaces and attaching cells involves the covalent grafting of adhesion peptides to polymers on which protein adsorption, which mediates unspecific cell adhesion, is essentially suppressed. This study demonstrates a surface modification concept for the covalent anchoring of RGD peptides to reactive diblock copolymers based on monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) (H2N-PEG-PLA). Films of both the amine-reactive (ST-NH-PEG2PLA20) and the thiol-reactive derivative (MP-NH-PEG2PLA40) were modified with cyclic αvβ3/αvβ5 integrin subtype specific RGD peptides simply by incubation of the films with buffered solutions of the peptides. Human osteoblasts known to express these integrins were used to determine cell–polymer interactions. The adhesion experiments revealed significantly increased cell numbers and cell spreading on the RGD-modified surfaces mediated by RGD–integrin-interactions.
Keywords: Cell adhesion; RGD peptide; Integrin; Osteoblast; Surface modification; Biomimetic material; Poly(lactic acid); Poly(ethylene oxide); Diblock copolymer;

In vitro apatite formation and its growth kinetics on hydroxyapatite/polyetheretherketone biocomposites by Shucong Yu; Kithva Prakash Hariram; Rajendra Kumar; Philip Cheang; Khor Khiam Aik (2343-2352).
The formation of biologically equivalent carbonate-containing apatite on the surface of synthetic hydroxyapatite (HA) is an important step leading to good bone healing. In this study, HA-reinforced polyetheretherketone (PEEK) composites were prepared by homogeneous mixing of HA and PEEK powders, compaction, and pressureless sintering. The bioactivity of HA/PEEK composite with 10, 20, 30 and 40 vol% HA was evaluated by immersing the composite disks in the simulated body fluid (SBF) for up to 4 weeks. The surface of composite with 40 vol% HA was covered by a layer of bone-like apatite just after 3 days of immersion, while 10 vol% HA was covered only after 28 days. This apatite layer was characterized by SEM, thin film X-ray diffractometer, attenuated total reflectance–Fourier transform infrared spectrometer (FTIR)/FTIR. Introducing a concept called apatite-forming capacity of SBF, growth kinetics of the apatite layer on the surface of the composite disks was carried out. The growth rate constant increased with HA volume fraction of the composite, suggesting that the bioactivity of the HA/PEEK composite increases with increasing HA volume fraction in the composite.
Keywords: Hydroxyapatite; Polyetheretherketone; Carbonated-apatite; Simulated body fluid; Growth kinetic analysis;

Deletion of the tissue response against alginate-pll capsules by temporary release of co-encapsulated steroids by C.M. Bünger; B. Tiefenbach; A. Jahnke; C. Gerlach; Th. Freier; K.P. Schmitz; U.T. Hopt; W. Schareck; E. Klar; P. de Vos (2353-2360).
Transplantation of encapsulated living cells is a promising approach for the treatment of a wide variety of diseases. Large-scale application of the technique, however, is hampered by inflammatory responses against the capsules. In the present study, we investigate whether tissue responses against alginate-PLL-alginate capsules can be modulated by co-encapsulation and temporary release of immunomodulating factors such as dexamethasone. Such an approach may be mandatory in order to increase the function and survival of encapsulated tissue since it has been shown that the tissue response can be caused by many, insurmountable factors. In an in vitro assay, we demonstrated an antiproliferative effect of dexamethasone-containing capsules on L929-mouse-fibroblasts. Subsequently, capsules prepared of purified alginate with or without solved dexamethasone were implanted in the peritoneal cavity of rats and retrieved one month later for histological evaluation. Most of the capsules without dexamethasone proved to be overgrown and adherent to the abdominal organs whereas with co-encapsulated dexamethasone the majority of the capsules were found freely floating in the peritoneal cavity without overgrowth. We conclude that co-encapsulation of dexamethasone has a profound effect on fibroblasts and macrophages adherence to immunoisolating capsules.
Keywords: Alginate; Microcapsules; Dexamethasone; Biocompatibility;

Importance of the surface area ratio on cytokines production by human monocytes in vitro induced by various hydroxyapatite particles by Alexia Grandjean-Laquerriere; Patrice Laquerriere; Moncef Guenounou; Dominique Laurent-Maquin; Terry M. Phillips (2361-2369).
A possible complication associated with the implantation of hydroxyapatite (HA)-based prosthesis is the release of particles. Those particles can be phagocyted by monocytes that are among the first cells to colonize the inflammatory site. The activated monocytes produce inflammatory mediators, such as cytokines, which cause osteoclasts activation. It has previously been demonstrated using a surface area ratio (ratio of the total surface of the given particles to the surface area of cells) of 1 to 1 that there was a correlation between the expression and production of cytokines induced by HA. The present work studies the effect of physical characteristics of HA particles on the production of various inflammatory cytokines (tumour necrosis factor-alpha, interleukin (IL)-6, and IL-8) and anti-inflammatory cytokine (IL-10). However, the experiments were performed using a surface area ratio of 10 to 1. Our data demonstrate that all the particles, whatever their characteristics, induced a high expression of cytokines but the production was different, meaning that there was a post-transcriptional regulation. The size and sintering temperature seemed to be a characteristics that were less important compared to the shape; the needle particles appeared to induce the most important production of all the cytokines studied.
Keywords: Hydroxyapatite; Cytokines; Inflammation; Physical characteristics;

Molecular basis of osteoclastogenesis induced by osteoblasts exposed to wear particles by Donatella Granchi; Ilaria Amato; Luca Battistelli; Gabriela Ciapetti; Stefania Pagani; Sofia Avnet; Nicola Baldini; Armando Giunti (2371-2379).
In this study, we investigate the molecular mechanisms by which human osteoblasts (HOB) challenged with wear debris promote the differentiation of osteoclast precursors. HOB were obtained from trabecular bone and exposed to alumina (Al2O3) or ‘ultra-high molecular weight polyethylene’ (UHMWPE) particles for 24 h. The supernatant (HOB-CM) was used for the immunoenzymatic detection of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), as well as for inducing the osteoclast differentiation from peripheral blood mononuclear cells (PBMC). The OPG-to-RANKL ratio was significantly decreased in the conditioned medium of UHMWPE-challenged HOB. Morphological and cytochemical analysis showed that HOB-CM induced by itself the osteoclast formation, but a large amount of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive giant cells were obtained when PBMCs were cultured with 1 μg/mL UHMWPE HOB-CM. The expression of genes involved in osteoclast differentiation and activation was evaluated, i.e. c-fms, RANK, c-src, c-fos, cathepsin-K (CATK), TRAP, and calcitonin R (CTR). The UHMWPE HOB-CM increases c-src expression, suggesting that polyethylene debris favour the paracrine activity of HOB in inducing the pathway involved in osteoclast polarization and adhesion. On the contrary, Al2O3 HOB-CM downregulates c-fos expression, suggesting that the passage from macrophages into the osteoclast lineage is deviated. These results show that Al2O3 wear debris is less active than UHMWPE in inducing osteoclast differentiation. Moreover, they provide new insight into the molecular basis of particle-induced osteoclastogenesis, that is the starting point for planning mode-specific targeting of periprosthetic osteolysis.
Keywords: Osteoblast; Osteoclast; Gene expression; Wear debris;

Human osteoblast response to pulsed laser deposited calcium phosphate coatings by A. Bigi; B. Bracci; F. Cuisinier; R. Elkaim; M. Fini; I. Mayer; I.N. Mihailescu; G. Socol; L. Sturba; P. Torricelli (2381-2389).
Octacalcium phosphate (OCP) and Mn2+-doped carbonate hydroxyapatite (Mn-CHA) thin films were deposited on pure, highly polished and chemically etched Ti substrates with pulsed laser deposition. The coatings exhibit different composition, crystallinity and morphology that might affect their osteoconductivity. Human osteoblasts were cultured on the surfaces of OCP and Mn-CHA thin films, and the cell attachment, proliferation and differentiation were evaluated up to 21 days. The cells showed a normal morphology and a very good rate of proliferation and viability in every experimental time. Alkaline phosphatase activity was always higher than the control and Ti groups. From days 7 to 21 collagen type I production was higher in comparison with control and Ti groups. The level of transforming growth factor beta 1 (TGF-β1) was lower at 3 and 7 days, but reached the highest values during following experimental times (14 and 21 days). Our data demonstrate that both calcium phosphate coatings favour osteoblasts proliferation, activation of their metabolism and differentiation.
Keywords: Calcium phosphate coating; Octacalcium phosphate; Mn2+-doped carbonated hydroxyapatite; Laser ablation; Human osteoblasts; Differentiation;

Protein repellant silicone surfaces by covalent immobilization of poly(ethylene oxide) by Hong Chen; Zheng Zhang; Yang Chen; Michael A. Brook; Heather Sheardown (2391-2399).
Polydimethylsiloxane elastomers were surface modified with passivating polyethylene oxide (PEO) polymers of different molecular weights, both monofunctional and bifunctional. Following the introduction of Si–H groups on the surfaces by acid-catalyzed equilibration in the presence of polymethylhydrosiloxane, the PEO was linked by platinum-catalyzed hydrosilylation. ATR-FTIR, X-ray photoelectron spectroscopy (XPS) and water contact angle results confirmed that the PEO was successfully grafted to the silicone rubber. Atomic force microscopy and XPS suggested that surface coverage with PEO was very high on the modified surfaces but not complete. The protein-resistant properties of the PEO-modified surfaces were demonstrated by measuring the adsorption of fibrinogen from both buffer and plasma. Fibrinogen adsorption from buffer to the PEO-modified surfaces was reduced by more than 90% compared with controls.
Keywords: PEO; PDMS; Hydrosilylation; Protein adsorption;

Synthesis of photoreactive pullulan for surface modification by Hirokazu Hasuda; Oh Hyeong Kwon; Inn-Kyu Kang; Yoshihiro Ito (2401-2406).
Photoreactive pullulan was prepared, the polymer was photoimmobilized on polymeric or organic surfaces, and its interactions with a protein and a cell type were investigated. The photoreactive pullulan was synthesized by a coupling reaction with 4-azidobenzonic acid. Surface modification was carried out in the presence or absence of a micropatterned photomask containing 100 μm transparent stripes with 150 μm gaps, making it easy to confirm the immobilization. By the micropatterning method, immobilization of the photoreactive pullulan on polystyrene, polyethylene, and silane-coupled glass was confirmed. Contact angles were measured on the unpatterned surfaces. Although the original surfaces have different contact angles, the contact angle on Az-pullulan-immobilized surface was the same on all surfaces. This result demonstrated that photoimmobilization completely covered the surface with Az-pullulan. Protein adsorption was investigated using fluorescently labeled albumin applied to the micropatterned surface: fluorescence microscopy demonstrated that adsorption was reduced on the pullulan-immobilized regions. Culture of RAW264 cells, derived from mouse leukemic monocytes, on the micropatterned surface for 22 h showed that cells did not adhere to the immobilized pullulan regions. In conclusion, photoreactive pullulan was covalently immobilized on various surfaces and tended to reduce interactions with proteins and cells.
Keywords: Photoimmobilization; Photolithography; Polysaccharide; Pullulan; Protein adsorption; Cell adhesion;

Simple surface modification of poly(ε-caprolactone) for apatite deposition from simulated body fluid by Ayako Oyane; Masaki Uchida; Cleo Choong; James Triffitt; John Jones; Atsuo Ito (2407-2413).
Poly(ε-caprolactone) (PCL) with a bone-like apatite layer bound to its surface could be useful as a scaffold for tissue engineering applications. In the present study, the surface of PCL was treated with aqueous NaOH to introduce carboxylate groups onto the surface. The NaOH-treated material was subsequently dipped in aqueous CaCl2 and K2HPO4·3H2O alternately three times to deposit apatite nuclei on the surface. The surface-modified material successfully formed a dense and uniform bone-like surface apatite layer after incubation for 24 h in simulated body fluid with ion concentrations approximately equal to those of human blood plasma.
Keywords: Apatite; Polycaprolactone; Simulated body fluid (SBF); Bioactivity; Scaffold; Biomimetic material;

A degradation study of PLLA containing lauric acid by Annette C. Renouf-Glauser; John Rose; David Farrar; Ruth E. Cameron (2415-2422).
Addition of lauric acid to poly (L-lactide) (PLLA) has resulted in a new family of enhanced degradation biomaterials. Presented is PLLA4.5 (PLLA containing 4.5 wt% lauric acid), the fastest degrading of the family. Degradation was studied via mass changes, gel-permeation chromatography, wide- and small-angle X-ray scattering (WAXS and SAXS), simultaneous SAXS and tensile testing, and visual observation. The undegraded PLLA4.5 deformed by crazing, recognisable from the characteristic shape of the SAXS pattern. As water up-take and degradation proceeded, samples crystallised, decreasing the SAXS long period, until by 4 days the deformation mechanism had become that of crystal-mediated deformation. This resulted in a ‘peanut–lemon’-shaped SAXS pattern, interpreted in terms of cavitation and fibrillated shear. Further degradation up to 12 days resulted in the same deformation mechanism at different sample displacements, with samples failing earlier during tensile testing until a ductile–brittle transition occurred. At 30–40 days water up-take and mass-loss increased significantly and global whitening of samples occurred, while the crystallinity and long period stabilised. Complete degradation had not occurred by the end of the study at 73 days. Through an understanding of how the changes in morphology during degradation affect the micromechanisms of deformation, it may be possible to design microstructures to give a tailored evolution of mechanical response in the body.
Keywords: Polylactic acid; Degradation; Mechanical properties; X-ray diffraction (XRD); Microstructure;

The influence of surface roughness of titanium on β1- and β3-integrin adhesion and the organization of fibronectin in human osteoblastic cells by Frank Lüthen; Regina Lange; Petra Becker; Joachim Rychly; Ulrich Beck; J.G. Barbara Nebe (2423-2440).
Mechanisms of cell adhesion and extracellular matrix formation are primary processes in the interaction with the material surface of an implant which are controlled by integrin receptors. The aim of our study was to find out whether β1- and β3-integrins of osteoblastic cells sense the surface topography of titanium, and if structural alterations of integrin adhesions were involved in the organization of fibronectin. Pure titanium surfaces were modified by polishing (P), machining (NT), blasting with glass spheres (GB), and blasting with corundum particles (CB) resulting in increasing roughness. Confocal microscopic investigations revealed fibrillar adhesions of β1- and α5-integrins on P, NT, and GB, but on CB with its sharp edges these integrin subunits did not form fibrillar adhesions. β3 generally appeared in focal adhesions. We observed aligned fibrillar structures of fibronectin on NT not only on the basal site but interestingly, also on the apical cell surface. In contrast, on CB, fibronectin appeared apically clustered. We suggest that this alignment of fibronectin fibrils depends on the directed actin cytoskeleton and in particular, on the capability of the β1-integrins to form fibrillar adhesions, which is affected by the surface roughness of titanium.
Keywords: Osteoblast; Integrin; Actin; Fibronectin; Titanium; Surface roughness;

Quantitative analysis of macrophage apoptosis vs. necrosis induced by cobalt and chromium ions in vitro by Isabelle Catelas; Alain Petit; Hojatollah Vali; Cathy Fragiskatos; Réal Meilleur; David J. Zukor; John Antoniou; Olga L. Huk (2441-2453).
The potential toxicity of metal ions in tissues surrounding metal–metal hip replacements is a cause for concern. Previous studies conducted in our laboratory demonstrated that Co2+ and Cr3+ induce TNF-α secretion in macrophages, as well as cell mortality. However, the degree of apoptosis and necrosis remained to be investigated. The aim of the present study was to quantify the rate of macrophage mortality by apoptosis vs. necrosis induced by Co2+ and Cr3+. J774 mouse macrophages were incubated in growth medium containing 0–10 ppm Co2+ and 0–500 ppm Cr3+ for 24 and 48 h under conventional cell culture conditions. Transmission electron microscopy, flow cytometry (Annexin-V fluorescein isothiocyanate/propidium iodide assay) and a specific cell death detection ELISA were used to illustrate cell death and differentiate between apoptotic and necrotic cells. Cell culture exposed to low concentrations of Co2+ (0–6 ppm) revealed a low degree of mortality. In contrast, at the highest concentrations (8–10 ppm), late apoptosis occurred within 24 h. After 48 h, however, there was a clear evidence for an increase in the rate of necrosis while apoptosis occurred at much lower rate. Macrophages exposed to Cr3+ demonstrated a predominance of apoptosis after 24 h. At concentrations lower than 250 ppm, early and late apoptosis occurred at the same rate. At higher concentrations (250–500 ppm), the number of early apoptotic cells decreased in favor of late apoptosis. After 48 h, lower concentrations of Cr3+ (⩽150 ppm) induced a higher degree of early apoptosis than after 24 h, and some necrosis. At higher concentrations, the percentage of early apoptotic cells decreased, while necrosis became predominant over late apoptosis. In conclusion, this study demonstrates that macrophage mortality induced by metal ions depends on the type and concentration of metal ions as well as the duration of their exposure. Overall, apoptosis was predominant after 24 h with both Co2+ and Cr3+ ions, but high concentrations induced mainly necrosis at 48 h. These results point to the potential for these ions of inducing tissue damage by necrosis if present in large concentrations in vivo.
Keywords: CoCrMo alloys; Metal ions; Macrophages; Apoptosis; Necrosis; In vitro;

Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution by Tanyarut Boontheekul; Hyun-Joon Kong; David J. Mooney (2455-2465).
Degradability is often a critical property of materials utilized in tissue engineering. Although alginate, a naturally derived polysaccharide, is an attractive material due to its biocompatibility and ability to form hydrogels, its slow and uncontrollable degradation can be an undesirable feature. In this study, we characterized gels formed using a combination of partial oxidation of polymer chains and a bimodal molecular weight distribution of polymer. Specifically, alginates were partially oxidized to a theoretical extent of 1% with sodium periodate, which created acetal groups susceptible to hydrolysis. The ratio of low MW to high MW alginates used to form gels was also varied, while maintaining the gel forming ability of the polymer. The rate of degradation was found to be controlled by both the oxidation and the ratio of high to low MW alginates, as monitored by the reduction of mechanical properties and corresponding number of crosslinks, dry weight loss, and molecular weight decrease. It was subsequently examined whether these modifications would lead to reduced biocompatibility by culturing C2C12 myoblast on these gels. Myoblasts adhered, proliferated, and differentiated on the modified gels at a comparable rate as those cultured on the unmodified gels. Altogether, this data indicates these hydrogels exhibit tunable degradation rates and provide a powerful material system for tissue engineering.

Tissue engineering scaffolds are fabricated from either biological materials, which provide biofunctional signals and interact well with cells, or from synthetic polymers, which provide precise control over their structural properties. We describe a biosynthetic hybrid scaffold comprised of a fibrinogen backbone and crosslinked with difunctional polyethylene glycol (PEG) side chains. Denatured fibrinogen fragments are PEGylated with PEG-diacrylates, mixed with photoinitiator and exposed to UV light to form a hydrogel material in the presence of a cell suspension. This unique hydrogel material provides a distinct advantage over other scaffold materials because its mechanical properties are highly malleable while the biological functionality is maintained by the backbone of the polymeric network. The elastic modulus of the PEG-fibrinogen hydrogel is dependent on the molecular weight of the PEG constituent and proportional to the percent polymeric composition. The biological domains in the fibrinogen backbone provide attachment motifs for endothelial cell and smooth muscle cell adhesion as well as proteolytic sensitivity for biodegradation. Smooth muscle cells demonstrate the ability to proteolytically penetrate through the hydrogel material and form interconnecting networks of cells. Our efforts to develop novel biodegradable scaffolds for cultivating cells in a 3D environment are beneficial for tissue regeneration therapies.

Effects of scaffold composition and architecture on human nasal chondrocyte redifferentiation and cartilaginous matrix deposition by Sylvie Miot; Tim Woodfield; Alma U. Daniels; Rosemarie Suetterlin; Iman Peterschmitt; Michael Heberer; Clemens A. van Blitterswijk; Jens Riesle; Ivan Martin (2479-2489).
We investigated whether the post-expansion redifferentiation and cartilage tissue formation capacity of adult human nasal chondrocytes can be regulated by controlled modifications of scaffold composition and architecture. As a model system, we used poly(ethylene glycol)-terephthalate–poly(butylene)-terephthalate block copolymer scaffolds from two compositions (low or high PEG content, resulting in different wettability) and two architectures (generated by compression molding or three-dimensional (3D) fiber deposition) with similar porosity and mechanical properties, but different interconnecting pore architectures. Scaffolds were seeded with expanded human chondrocytes and the resulting constructs assessed immunohistochemically, biochemically and at the mRNA expression level following up to 4 weeks of static culture.For a given 3D architecture, the more hydrophilic scaffold enhanced cell redifferentiation and cartilaginous tissue formation after 4 weeks culture, as assessed by higher mRNA expression of collagen type II, increased deposition of glycosaminoglycan (GAG) and predominance of type II over type I collagen immunostain. The fiber-deposited scaffolds, with a more accessible pore volume and larger interconnecting pores, supported increased GAG deposition, but only if a more hydrophilic composition was used.By applying controlled and selective modifications of chemico-physical scaffold parameters, we demonstrate that both scaffold composition and architecture are instructive for expanded human chondrocytes in the generation of 3D cartilaginous tissues. The observed effects of composition and architecture were likely to have been mediated, respectively, by differential serum protein adsorption and efficiency of nutrient/waste exchange.
Keywords: Cartilage tissue engineering; Nasal chondrocytes; Extracellular matrix; Wettability; Porosity; Scaffold;

Three-dimensional, nano-structured PLGA scaffolds for bladder tissue replacement applications by Megan A. Pattison; Susan Wurster; Thomas J. Webster; Karen M. Haberstroh (2491-2500).
In total, approximately 400 million people worldwide suffer from urinary bladder cancer (Nat Biotechnol 17 (1999) 149). When radical cysectomy is required as treatment, a replacement material is clearly necessitated. For this purpose, three-dimensional poly(lactic-co-glycolic acid) (PLGA) scaffolds were constructed using solvent casting and salt leaching processes. These scaffolds were manipulated to possess nano-dimensional surface features by soaking in sodium hydroxide at select concentrations and for various periods of time. Human bladder smooth muscle cells were then seeded onto these nano-dimensional scaffolds; adhesion and longer-term cell growth experiments were performed for either 4 h, or 1, 3, and 5 days, respectively. Additionally, collagen and elastin production was quantified following each experiment. In all cases, control cells were placed in an incubator and subjected to normal atmospheric pressure, while experimental cells were placed in a pressure chamber and subjected to a sustained pressure of 10 cm H2O. Results of this study provided evidence that porous, nano-dimensional polymeric scaffolds enhanced cell adhesion and growth, while also promoting increased elastin and collagen production. Moreover, in general, exposure to pressure did not alter cellular adhesion, growth, or extracellular matrix protein production, which suggests that the scaffolds and their resident cells will fair well in the complex mechanical environment of the bladder wall. In combination, these results provide evidence that the nano-dimensional PLGA scaffolds created in this research are promising as the next generation of bladder wall replacement materials.
Keywords: Bladder; Nano-dimensional; Scaffolds; PLGA; Smooth muscle cells;

Ectopic bone formation associated with recombinant human bone morphogenetic proteins-2 using absorbable collagen sponge and beta tricalcium phosphate as carriers by Chang-Sung Kim; Joon-Il Kim; Jin Kim; Seong-Ho Choi; Jung-Kiu Chai; Chong-Kwan Kim; Kyoo-Sung Cho (2501-2507).
The ectopic bone formation of recombinant human bone morphogenetic protein-2(rhBMP-2) was evaluated using absorbable collagen sponges (ACS) and beta tricalcium phosphate (β-TCP) as carriers in a rat subcutaneous assay model. Subcutaneous pockets were created on the back of rats. The pockets were implanted with rhBMP-2/ACS, rhBMP-2/β-TCP, ACS alone, and β-TCP alone. The rats were sacrificed at 2 or 8 weeks for histological and immunohistochemical evaluation. At 2 weeks, bone formation was evident in both the rhBMP-2/ACS and rhBMP-2/β-TCP sites. At 8 weeks, the quantity of the new bone with a more advanced stage of remodeling had increased further in the rhBMP-2/β-TCP sites. However, the newly formed bone observed at 2 weeks was not found in the rhBMP-2/ACS sites. On immunohistochemical observation, osteopontin staining was observed on both the rhBMP-2/ACS (2 weeks) and rhBMP-2/β-TCP (2 and 8 weeks) sites. Osteocalcin was not detected in any of the samples. The lack of space-providing capacity of ACS may be one of the major factors responsible for its failure to maintain the newly induced bone. Therefore, a carrier for BMPs should provide space for bone formation and maturation during the more advanced healing stages.
Keywords: Bone morphogenetic proteins; Ectopic bone formation; Carrier; Bone tissue engineering;

An organic–inorganic hybrid scaffold for the culture of HepG2 cells in a bioreactor by Ken Kataoka; Yoshitaka Nagao; Takamasa Nukui; Ichiro Akiyama; Kanji Tsuru; Satoshi Hayakawa; Akiyoshi Osaka; Nam-ho Huh (2509-2516).
Much interest has recently been shown in the potential utility of bioartificial liver (BAL) as a bridge support for patients and as a module for experimental purposes. A radial-flow bioreactor (RFB), one of the perfused bed/scaffold-type bioreactors, enables a highly functional three-dimensional culture as BAL. The functional capacity of bioreactors depends not only on their mechanistic structures but also on scaffolds packed in them. In the present study, we examined the possible utility of a new porous organic–inorganic-hybrid scaffold in an RFB. The scaffold was made from tetraethoxysilane (TEOS) and polydimethylsiloxane (PDMS) by a sol–gel method using sieved sucrose particles as a porogen. In the porous TEOS–PDMS hybrid scaffold, human hepatocellular carcinoma cells (HepG2) proliferated actively and formed cell clusters more efficiently than they did in a polyvinyl-alcohol scaffold. When cultivated in PDMS–TEOS, HepG2 cells secreted a ∼three-fold greater amount of albumin than that secreted in a monolayer culture. For potential application of BAL to pharmacological studies and future clinical use, it is essential to develop a method to propagate liver cells that maintain highly specific functions. The present results indicate that PDMS–TEOS may be a promising scaffold for developing such functional culture methods.
Keywords: Three-dimensional culture; Organic–inorganic hybrid; Scaffold; Bioreactor; HepG2; TEOS–PDMS;

Crosslinked hyaluronan scaffolds as a biologically active carrier for valvular interstitial cells by Kristyn S. Masters; Darshita N. Shah; Leslie A. Leinwand; Kristi S. Anseth (2517-2525).
Hyaluronic acid (HA), a major component of the cardiac jelly during heart morphogenesis, is a polysaccharide that upon modification can be photopolymerized into hydrogels. Previous work in our lab has found that photopolymerizable HA hydrogels are suitable scaffolds for the culture and proliferation of valvular interstitial cells (VICs), the most prevalent cell type in native heart valves. The physical properties of HA gels are easily modified through alteration in material crosslink density or by copolymerizing with other reactive macromolecules. Degradation products of HA gels and the starting macromers significantly increased VIC proliferation when added to cell cultures. With low molecular weight HA (<6700 Da) exhibiting greatest stimulation of VIC proliferation. Low molecular weight HA degradation products added to VIC cultures also resulted in a four-fold increase in total matrix production and a two-fold increase in elastin production over untreated controls. VIC internalization of HA, as shown by cellular uptake of fluorescently labeled HA, likely activates signaling cascades resulting in the biological responses seen here. Lastly, VICs encapsulated within HA hydrogels remained viable, and significant elastin production was observed after 6 weeks of culture. This work shows promise for the creation of a tissue-engineered heart valve utilizing the synergistic relationship between hyaluronic acid and VICs.
Keywords: Hyaluronic acid; Hydrogel; Photopolymerization; Heart valve; Extracellular matrix;

Non-woven polyethylene terephthalate nanofiber mats (PET NFM) were prepared by electrospinning technology and were surface modified to mimic the fibrous proteins in native extracellular matrix towards constructing a biocompatible surface for endothelial cells (ECs). The electrospun PET NFM was first treated in formaldehyde to yield hydroxyl groups on the surface, followed by the grafting polymerization of methacrylic acid (MAA) initiated by Ce(IV). Finally, the PMAA-grafted PET NFM was grafted with gelatin using water-soluble carbodiimide as coupling agent. Plane PET film was also surface modified and characterized for basic understanding of the surface modification process. The grafting of PMAA and gelatin on PET surface was confirmed by XPS spectroscopy and quantitatively analyzed by colorimetric methods. ECs were cultured on the original and gelatin-modified PET NFM and the cell morphology, proliferation and viability were studied. Three characteristic surface makers expressed by ECs were studied using immuno-florescent microscopy. The gelatin grafting method can obviously improve the spreading and proliferation of the ECs on the PET NFM, and moreover, can preserve the EC's phenotype.
Keywords: Electrospinning; Nanofiber; PET; Surface modification; Vascular graft; Blood vessel; Tissue engineering;

Stable immobilization of rat hepatocyte spheroids on galactosylated nanofiber scaffold by Kian-Ngiap Chua; Wei-Seng Lim; Pengchi Zhang; Hongfang Lu; Jie Wen; Seeram Ramakrishna; Kam W. Leong; Hai-Quan Mao (2537-2547).
Primary rat hepatocytes self-assemble into multi-cellular spheroids and maintain differentiated functions when cultured on a two-dimensional (2-D) substrate conjugated with galactose ligand. The aim of this study is to investigate how a functional nanofiber scaffold with surface-galactose ligand influences the attachment, spheroid formation and functional maintenance of rat hepatocytes in culture, as compared with the functional 2-D substrate. Highly porous nanofiber scaffolds comprising of fibers with an average diameter of 760 nm were prepared by electrospinning of poly(ε-caprolactone-co-ethyl ethylene phosphate) (PCLEEP), a novel biodegradable copolymer. Galactose ligand with a density of 66 nmol/cm2 was achieved on the nanofiber scaffold via covalent conjugation to a poly(acrylic acid) spacer UV-grafted onto the fiber surface. Hepatocytes cultured on the galactosylated PCLEEP nanofiber scaffold exhibited similar functional profiles in terms of cell attachment, ammonia metabolism, albumin secretion and cytochrome P450 enzymatic activity as those on the functional 2-D substrate, although their morphologies are different. Hepatocytes cultured on galactosylated PCLEEP film formed 50–300 μm spheroids that easily detached from surface upon agitation, whereas hepatocytes cultured on galactosylated nanofiber scaffold formed smaller aggregates of 20–100 μm that engulfed the functional nanofibers, resulting in an integrated spheroid-nanofiber construct.
Keywords: Cell morphology; Electrospinning; Function maintenance; Galactosylated surface; Hepatocyte; Nanofiber scaffold; Spheroid culture; Surface grafting polymerization;

Synaptic plasticity in micropatterned neuronal networks by Angela K Vogt; Günter Wrobel; Wolfgang Meyer; Wolfgang Knoll; Andreas Offenhäusser (2549-2557).
Synaptic plasticity is thought to be of central importance for information processing by the nervous system. Additionally, specific neuronal connectivity patterns in the brain are implicated to play a role in the perception, processing and storage of incoming signals. Experimental control over connectivity within functional neuronal networks is therefore a promising approach in research on signal transduction and processing by the nervous system.A cell culture system is presented that allows experimental determination of neuronal connectivity patterns in an in vitro network. Rat embryonic cortical neurons were grown on patterns of extracellular matrix proteins applied to polystyrene substrates by microcontact printing. Cells comply well with the pattern and form synaptic connections along the experimentally defined pathways. Chemical synapses identified by double patch-clamp measurement showed paired pulse depression as well as frequency-dependent depression in response to trains of stimuli. This type of short-term plasticity has similarly been reported by others in brain slices. Thus, the system reproduces features central for neuronal information processing while the architecture of the network is experimentally manipulable. The ability to tailor the geometry of functional neuronal networks offers a valuable tool both for fundamental questions in neuroscientific research and a wide range of biotechnological applications.
Keywords: Neural network; Micropatterning; ECM (extracellular matrix); Cell adhesion; SEM (scanning electron microscopy);

Culturing of skin fibroblasts in a thin PLGA–collagen hybrid mesh by Guoping Chen; Takashi Sato; Hajime Ohgushi; Takashi Ushida; Tetsuya Tateishi; Junzo Tanaka (2559-2566).
A thin biodegradable hybrid mesh of synthetic poly(DL-lactic-co-glycolic acid) (PLGA) and naturally derived collagen was used for three-dimensional culture of human skin fibroblasts. The hybrid mesh was constructed by forming web-like collagen microsponges in the openings of a PLGA knitted mesh. The behaviors of the fibroblasts on the hybrid mesh and PLGA knitted mesh were compared. The efficiency of cell seeding was much higher and the cells grew more quickly in the hybrid mesh than in the PLGA mesh. The fibroblasts in the PLGA mesh grew from the peripheral PLGA fibers toward the centers of the openings, while those in the hybrid mesh also grew from the collagen microsponges in the openings of the mesh resulting in a more homogenous growth. The proliferated cells and secreted extracellular matrices were more uniformly distributed in the hybrid mesh than in the PLGA mesh. Histological staining of in vitro cultured fibroblast/mesh implants indicated that the fibroblasts were distributed throughout the hybrid mesh and formed a uniform layer of dermal tissue having almost the same thickness as that of the hybrid mesh. However, the tissue formed in the PLGA mesh was thick adjacent to the PLGA fibers and thin in the center of the openings. Fibroblasts cultured in the hybrid mesh were implanted in the back of nude mouse. Dermal tissues were formed after 2 weeks and became epithelialized after 4 weeks. The results indicate that the web-like collagen microsponges formed in the openings of the PLGA knitted mesh increased the efficiency of cell seeding, improved cell distribution, and therefore facilitated rapid formation of dermal tissue having a uniform thickness. PLGA–collagen hybrid mesh may be useful for skin tissue engineering.
Keywords: Tissue engineering; Hybrid mesh; Collagen sponge; Skin; Dermis; Fibroblast;

Homologous muscle acellular matrix seeded with autologous myoblasts as a tissue-engineering approach to abdominal wall-defect repair by Maria Teresa Conconi; Paolo De Coppi; Silvia Bellini; Gabriella Zara; Morena Sabatti; Maurizio Marzaro; Giovanni Franco Zanon; Pier Giorgio Gamba; Pier Paolo Parnigotto; Gastone Giovanni Nussdorfer (2567-2574).
Myoblasts were obtained by culturing in vitro, single muscle fibers, isolated by enzymatic digestion from rat flexor digitorum brevis, and their phenotype was confirmed by myogenic differentiation factor, myogenic factor-5, myogenin and desmin. Cultured myoblasts were harvested and seeded on patches of homologous acellular matrix, obtained by detergent-enzymatic treatment of abdominal muscle fragments. Myoblast-seeded patches were inserted between obliqui abdominis muscles on the right side of 1-month-old rats, while non-seeded patches were implanted on the left side. Thirty days after surgery, non-seeded patches were completely replaced by fibrous tissue, while the structure of myoblast-seeded patches was well preserved until the 2nd month. Seeded patches displayed abundant blood vessels and myoblasts, and electromyography evidenced in them single motor-unit potentials, sometimes grouped into arithmic discharges. Ninty days after implantation, the thickness of myoblast-seeded patches and their electric activity decreased, suggesting a loss of contractile muscle fibers. In conclusion, the present results indicate that autologous myoblast-homologous acellular muscle matrix constructs are a promising tool for body-wall defect repair, and studies are under way to identify strategies able to improve and maintain the structural and functional integrity of implants for longer periods.
Keywords: Bioprosthesis; Abdominal wall defects; Satellite cells; Acellular matrix;

This animal study (domestic pig) examined the bone formation after filling defined defects with autogenous bone or a collagen lyophilisat in combination with Platelet-rich-plasma (PRP) by evaluating bone matrix proteins. Six groups, both materials with and without PRP in two concentrations (+1, +2) were compared to untreated bone by means of immunohistochemistry at 2, 4, 12 and 26 weeks.BMP-2 expression was increased at 2 weeks in the collagen+1 group and after 4 weeks in the collagen+1 and +2 group. Collagen-I expression was increased at 2 weeks in all collagen groups. After 4 weeks raised levels were observed after adding the higher concentrated PRP to bone and the collagen material. Osteocalcin expression was enhanced at 2 weeks in all collagen groups and the autogenous bone+PRP1 group, after 4 weeks in the bone and collagen +2 groups. At 12 weeks higher values were observed after adding higher concentrated PRP to bone. Osteonectin and especially osteopontin were confirmed to be effective markers of early bone formation in all specimens.The described setting allows to combine established techniques (microradiography, light microscopy) with approaches to explore the underlying biology (immunohistochemistry) on the same specimen.
Keywords: Platelet-rich plasma; Bovine collagen; Bone regeneration; Prospective study; Bone substitutes; Bone matrix proteins;

Fabrication of well-defined PLGA scaffolds using novel microembossing and carbon dioxide bonding by Yong Yang; Shubhayu Basu; David L. Tomasko; L. James Lee; Shang-Tian Yang (2585-2594).
A novel biologically benign technique was developed to produce three-dimensional tissue engineering scaffolds with well-defined structure. Photolithography was used to design and pattern a planar scaffold skeletal structure on a photoresist (SU-8), and a variety of microembossing processes including sacrificial layer embossing and bilayer embossing were developed to transfer the skeletal pattern to the poly(dl-lactide-co-glycolide) substrate as scaffold skeletons. Subcritical carbon dioxide was then introduced to assemble these skeletons to a three-dimensional scaffold at a low temperature. Compared with conventional scaffolds, which have a broad pore size distribution and varying pore geometry, these microfabricated scaffolds have a uniform and well-defined geometry and structure. This uniformity of structural parameters allows for the studies of cell attachment, spreading, and proliferation in scaffolds in a controlled and logical manner. The cytocompatibility of these microfabricated scaffolds was tested by seeding three different cell lines with different morphologies and growth patterns into these scaffolds. All three cell lines attached well to the scaffolds and grew to high densities as observed with scanning electron microscopy. This study demonstrates a controllable method to fabricate tissue scaffolds with a well-defined 3D architecture that can be used to better elucidate the effect of structure parameters such as pore geometry and pore size on tissue growth in 3D scaffolds.
Keywords: Tissue scaffold; Microembossing; Carbon dioxide bonding; Poly(dl-lactide-co-glycolide); PLGA;

Hepatocyte growth factor (HGF) is a growth factor that promotes angiogenesis (tissue vascularization), cell motility, and cell differentiation, making it a potentially beneficial coating for bone implants. However, very little is known about maximizing HGF attachment to surfaces of tissue-engineered scaffolds. Here, we examine methods and kinetics of HGF adsorption onto a dense hydroxyapatite (HA) surface (used in bone implants) and determine the influence of HGF coating on osteoblast phenotype/differentiation. We demonstrate that incubating HA with HGF in solution (and not allowing the solution to dry) resulted in maximal surface adsorption that was not enhanced by extending incubation time beyond 2 days. Daily shaking of the coated HA surface did not remove adsorbed HGF. To further examine the effect of HA on osteoblast phenotype, MC3T3-E1 preosteoblasts were seeded onto HA or HGF-HA surfaces. Gene expression analyses indicate that HGF coating enhanced osteoblast differentiation as demonstrated by increased runx2 (a transcription factor important for osteoblast lineage and differentiation), alkaline phosphatase (marker of mid stage differentiation) and osteocalcin (marker of late stage differentiation) mRNA levels. Taken together, our results demonstrate that HGF can serve as an excellent bone implant coating based on its ability to readily adsorb to HA surfaces, maintain integrity over time, and enhance osteoblast differentiation.

Efficacy of aligned poly(l-lactic acid) (PLLA) nano/micro fibrous scaffolds for neural tissue engineering is described and their performance with random PLLA scaffolds is compared as well in this study. Perfectly aligned PLLA fibrous scaffolds were fabricated by an electrospinning technique under optimum condition and the diameter of the electrospun fibers can easily be tailored by adjusting the concentration of polymer solution. As the structure of PLLA scaffold was intended for neural tissue engineering, its suitability was evaluated in vitro using neural stem cells (NSCs) as a model cell line. Cell morphology, differentiation and neurite outgrowth were studied by various microscopic techniques. The results show that the direction of NSC elongation and its neurite outgrowth is parallel to the direction of PLLA fibers for aligned scaffolds. No significant changes were observed on the cell orientation with respect to the fiber diameters. However, the rate of NSC differentiation was higher for PLLA nanofibers than that of micro fibers and it was independent of the fiber alignment. Based on the experimental results, the aligned nanofibrous PLLA scaffold could be used as a potential cell carrier in neural tissue engineering.
Keywords: Electrospinning; Poly(l-lactic acid); Nanofiber; Alignment; Contact guidance; Neural stem cell; Neural tissue engineering;

Biologically derived, chemically modified collagenous tissues are being increasingly used to fabricate cardiac valve prostheses and as biomaterials in cardiovascular repair. A stress-free state during chemical modification has been shown to preserve the collagen fiber architecture of the native tissue, potentially preserving native mechanical properties and improving prostheses durability. However, it is not known if the native collagen fiber architecture is stable during long-term in vivo operation. To address this question, we obtained porcine aortic valves chemically treated at (i) 0 mmHg transvalvular pressure (with 40 mmHg aortic pressure) and (ii) 4 mmHg transvalvular pressure, then subjected the valves to 0 , 1 × 10 6 , 50 × 10 6 , and 200 × 10 6 in vitro accelerated wear testing (AWT) cycles. The resulting changes in collagen fiber architecture were quantified using small angle light scattering analysis (SALS). SALS measurements indicated that collagen fibers in the 0 mmHg pressure-fixed leaflets became more aligned between 1 × 10 6 and 50 × 10 6 AWT cycles. In contrast, only minor changes (not statistically significant) in collagen fiber orientation occurred in the 4 mmHg pressure-fixed valvular tissue with cycling. It was also noted that although the 0 mmHg group was fixed without transvalvular pressure, distention of the root induced significant changes in collagen structure of the leaflets. Overall, our observations suggest that the native collagen fiber crimp of the 0 mmHg pressure-fixed leaflets were rapidly lost after only 50 × 10 6 AWT cycles (equivalent to ∼ 1.6 patient years) and thus may not be maintained over a sufficient period of time to be clinically beneficial. Further, the collagen structure of the native aortic valve is exquisitely sensitive to dimensional change in the aortic root—independent of the presence of transvalvular pressure. Our findings also suggest that without in vivo remodeling, any collagenous tissue used to fabricate BHV may undergo similar degenerative, irreversible changes in vivo.
Keywords: Heart valves; Collagen; Chemical fixation; Cyclic loading; Fatigue;

Polyelectrolyte multilayers functionalized by a synthetic analogue of an anti-inflammatory peptide, α-MSH, for coating a tracheal prosthesis by Philippe Schultz; Dominique Vautier; Ludovic Richert; Nadia Jessel; Youssef Haikel; Pierre Schaaf; Jean-Claude Voegel; Joelle Ogier; Christian Debry (2621-2630).
Polyelectrolyte multilayer films made of poly (l-lysine) (PLL) and poly (l-glutamic acid) (PGA) have been functionalized by covalent binding of a synthetic analogue of the anti-inflammatory peptide, α-melanocyte-stimulating hormone (α-MSH) to PGA to create biologically active coatings for tracheal prostheses. The morphology and in vivo stability of the films were investigated by atomic force microscopy and confocal laser scanning microscopy, respectively. For the in vivo evaluation, 87 rats were implanted and examined for a period superior to 3 months. Histological analysis, performed 1 month after implantation, showed a fibroblast colonization of the periprosthetic side and a respiratory epithelium type on the endoluminal side of the implant for all the polyelectrolyte coatings tested. However, for prostheses modified by PGA ending multilayer films, a more regular and less obstructive cell layer was observed on the endoluminal side compared to those modified by PLL ending films. Systemic anti-inflammatory IL-10 production was only detected in rats implanted with prostheses functionalized by α-MSH, demonstrating, in vivo, the anti-inflammatory activity of the embedded peptide into multilayer architectures.
Keywords: Tracheal prosthesis; Polyelectrolyte multilayers; Anti-inflammatory activities;

Knoop hardness of ten resin composites irradiated with high-power LED and quartz-tungsten-halogen lights by Richard B.T. Price; Corey A. Felix; Pantelis Andreou (2631-2641).
This study compared a high-power light-emitting-diode (LED) curing light (FreeLight 2, 3M ESPE) with a quartz-tungsten-halogen (QTH) light (TriLight, 3M ESPE) to determine which was the better at photo-polymerising 10 resin composites. Class I preparations were prepared 4-mm deep into human teeth and filled with 10 different composites. The composites were irradiated for 50% or 100% of their recommended times using the LED light, and for 100% of their recommended times with the QTH light on either the high or medium power setting. Fifteen minutes later, the Knoop hardness of the composites was measured to a depth of 3.5 mm from the surface.When irradiated by the LED light for their recommended curing times, the Knoop hardness of all 10 composites stayed above 80% of the maximum hardness of the composite to a depth of at least 1.5 mm; three composites maintained a Knoop hardness that was more than 80% of their maximum hardness to a depth of 3.5 mm. Repeated measurements analysis of variance indicated that all the two-way and three-way interactions between the curing light, depth, and composite were significant (p<0.01). To eliminate the choice of composite as a factor, an overall comparison of the lights was performed using the Kruskal–Wallis test and distribution free multiple comparisons of the ranked hardness values. The LED light, used for the composite manufacturer's recommended time, was ranked the best at curing the composites to a depth of 3 mm (p<0.01). The LED light used for 50% of the recommended time was not significantly different from the QTH light used for 100% of the recommended time on the high power setting.

A biomechanical and histological evaluation of a bioresorbable lumbar interbody fusion cage by Yoshihiro Hojo; Yoshihisa Kotani; Manabu Ito; Kuniyoshi Abumi; Tsuyoshi Kadosawa; Yasuo Shikinami; Akio Minami (2643-2651).
Novel spinal interbody fusion cages made of bioactive and bioresorbable composites by a unique forging process were developed. Previous in vitro study demonstrated that these cages marked excellent biomechanical values. The purpose of the present in vivo study was to evaluate the viability and advantage of this forged composite of uncalcined hydroxyapatite/poly L-Lactide (F-u-HA/PLLA) cage radiographically, biomechanically, and histologically, when compared to conventional autologous iliac bone (AIB) and carbon fiber cage (CFC). Twenty-five mature sheep underwent posterior lumbar interbody fusion at L2-3 level with pedicle screws system made of titanium. Three types of interbody fusion implants were grafted: AIB (n=7), CFCs (n=9), F-u-HA/PLLA cages (n=9). Two types of cages were packed with autologous fragmented cancellous bone harvested locally. All animals were euthanized at 120 days after surgery. The fusion scoring using the coronal view CT scans was designed to three-dimensionally evaluate fusion quality within and around cages. The mean CT scores of three groups were 33.3 points, 35.0 points, and 33.6 points in AIB, CFC, and F-u-HA/PLLA cage groups, respectively (full-score: 56 points). Statistical differences were not detected among the three groups. The mean range of motion values among fused groups had no significant difference under all pure loadings. The range of motion showed strong and significant correlation with the CT fusion scores. Histologic results demonstrated that F-u-HA/PLLA cages contacted with the surrounding bone directly, and CFC was encircled with thick fibrous tissue layers without any sign of inflammation around cages. The fusion quality of fused spinal segment using F-u-HA/PLLA cages was equal to that of AIB or CFCs both radiographically and biomechanically. In the histological observation, biocompatibility of F-u-HA/PLLA cage was obviously superior to CFC. It has been confirmed that the novel bioactive and bioresorbable cages had valuable advantages over existing CFC for use in spinal reconstructive surgery.

Efficacy of varying the NMEP concentrations in the NMGly–NMEP self-etching primer on the resin-tooth bonding by Norihiro Nishiyama; Kou Fujita; Takuji Ikemi; Takahide Maeda; Kazuomi Suzuki; Kimiya Nemoto (2653-2661).
It is well understood that the application of a self-etching primer enhances the bonding at the resin-teeth interface. In this study, we designed a self-etching primer consisting of N-methacryloyl glycine (NMGly) and N-methacryloyl-2-aminoethyl phosphonic acid (NMEP). The demineralization effects on the hydroxyapatite or dentin by the carboxylic acid in the NMGly and by the phosphonic acid in the NMEP and their effects on the bond strength of the resin to the tooth were examined. The application of the NMGly–NMEP solution to the enamel resulted in an increase in the bond strength when additional amounts of NMEP were added to the NMGly aqueous solution. This increase was due to the phosphonic acid in the NMEP demineralizing the enamel. Conversely, the addition of the NMEP to the NMGly solution resulted in a decrease in the bond strength to the dentin. The optimal concentration of the NMEP in the NMGly–NMEP solution resulted in bond strengths of over 20 MPa for both the enamel and dentin.
Keywords: Self-etching primer; N-methacryloyl-glycine; N-methacryloyl-2-aminoethyl phosphonic acid; Interaction; 13C NMR;

In a total of 15 knees from 15 patients undergoing medial patellofemoral ligament reconstruction using an artificial substitute and a medial retinaculum slip coverage for recurrent patellar dislocation, the reconstructed ligaments were histologically evaluated using hematoxilin-eosin and elastic van Gieson stains. The artificial substitute was a mesh-type Leeds–Keio ligament. The mean age of the patients at the time of surgery was 20 years (range; 13–38 years). The mean interval from initial reconstruction to gathering was 53 months (range; 11–109 months). In the tissue over the artificial ligament, longitudinally aligned collagen fiber bundles with spindle-shaped nuclei were formed in all specimens except one specimen of 11 months postsurgery, but it seemed to be mature ligament only in specimens more than 60 months postsurgery. The tissue inside the artificial ligament was immature as a whole in all specimens, although 13 out of the 15 specimens had regularly aligned collagen fiber bundles slightly or in some portions.
Keywords: Medial patellofemoral ligament; Ligament prosthesis; Collagen; Fibroblast; Remodeling;

Corrosion behavior of copper/LDPE nanocomposites in simulated uterine solution by Shuizhou Cai; Xianping Xia; Changsheng Xie (2671-2676).
We prepared copper/low-density polyethylene (LDPE) nanocomposites with various mass fractions of copper nanoparticles and investigated the Cu2+ release rate of them for 220 days in a simulated uterine solution .The influences of copper nanoparticle mass fraction on Cu2+ release and volume resistivity confirm that continuous network of copper nanoparticles can be obtained in composites when copper nanoparticle mass fraction ranges from 30% to 35%. Scanning electron microscopy with energy dispersive X-ray microanalysis (SEM/EDX) mapping technique was employed to investigate the copper distribution and corrosion depth of 30 wt% copper/LDPE composite before and after incubation. Besides, elements and phases were also analyzed by EDX and X-ray diffraction, respectively. The results suggest that copper/LDPE nanocomposites should be more efficient and feasible than conventional IUD materials.
Keywords: LDPE; Copper; Nanocomposites; IUD; Elemental mapping;

Nanocrystalline hydroxyapatite and calcium sulphate as biodegradable composite carrier material for local delivery of antibiotics in bone infections by Michael A. Rauschmann; Thomas A. Wichelhaus; Volker Stirnal; Elvira Dingeldein; Ludwig Zichner; Reinhard Schnettler; Volker Alt (2677-2684).
The use of polymethylmetacrylate beads for local delivery of antibiotics requires a second surgical procedure for their removal and resorbable calcium sulphate exhibits cytotoxic effects. In this work, a bioresorbable composite of calcium sulphate and nanoparticulate hydroxyapatite (PerOssal®) was studied regarding its antibiotic release properties and biocompatibility.Material characteristics of plain PerOssal® and pure calcium sulphate pellets were studied using scanning and electron microscopy and X-ray methods. Pellets were soaked with gentamicin and vancomycin, respectively. Release properties of both antibiotics from both materials were investigated over 10 days. Quantitative and qualitative cytotoxic assays were performed for biocompatibility testing.Specific surface was 106 m2/g for PerOssal® and 2.2 m2/g for pure calcium sulphate. Almost complete elution of gentamicin was found for both carrier materials (94.7% for PerOssal® vs. 95.8% for calcium sulphate) within 10 days, whereas vancomycin release was higher for PerOssal® (96.3% vs. 74.8%). PerOssal® showed higher initial and lower release after approximately 5 days compared to calcium sulphate. No significant in vitro cytotoxic differences were found between PerOssal® and nontoxic cell culture medium. Calcium sulphate showed cytotoxic effects in two out of four tests. PerOssal® exhibits excellent properties regarding resorption, biocompatibility, and antibiotic release.
Keywords: Antibacterial; Biocompatibility; Cytotoxicity; Drug release; Hydroxyapatite composite;

Development of functionalized superparamagnetic iron oxide nanoparticles for interaction with human cancer cells by A. Petri-Fink; M. Chastellain; L. Juillerat-Jeanneret; A. Ferrari; H. Hofmann (2685-2694).
Our goal is to develop, characterize and optimize functionalized super paramagnetic iron oxide nanoparticles (SPION) demonstrating the capacity to be internalized by human cancer cells. SPION (mean diameter 9 nm) were coated with various ratios to iron oxide of either polyvinyl alcohol (PVA), carboxylate-functionalized PVA, thiol-functionalized PVA and amino-functionalized PVA (amino-PVA). The interaction with cells and cytotoxicity of the SPION preparations were determined using human melanoma cells. From the four functionalized SPION preparations, only the amino-PVA SPION demonstrated the capacity to interact with, and were not cytotoxic to, human melanoma cells. This interaction with melanoma cells was dependent on the amino-PVA to iron oxide ratio, was an active and saturable mechanism displayed by all cells in a culture. These functionalized SPION were characterized by transmission electron microscopy and electrophoretic mobility. The physical comportment of SPION changed at specific PVAs to iron oxide ratios, and this ratio corresponded to the ratio of optimal interaction with cells. In conclusion, the successful development of functionalized SPION displaying potential cellular uptake by human cancer cells depends both on the presence of amino groups on the coating shell of the nanoparticles and of its ratio to the amount of iron oxide.
Keywords: Melanoma; Human; Iron oxide nanoparticles; Particle size-cell uptake; PVA;

Adsorption of a therapeutic enzyme to self-assembled monolayers: effect of surface chemistry and solution pH on the amount and activity of adsorbed enzyme by Cristina C. Barrias; Ma Cristina L. Martins; Ma Clara Sá Miranda; Mário A. Barbosa (2695-2704).
The adsorption of a therapeutic enzyme to self-assembled monolayers (SAMs) of different functionalities (X=CH3-, OH- and COOH-) was evaluated as a function of solution pH. Radiolabelling studies showed that the enzyme has higher affinity for hydrophobic surfaces than for hydrophilic surfaces, and that the highest adsorption was obtained at the more acidic pH values (4.5 and 5.5), despite the type of surface. IRAS and XPS measurements confirmed this tendency. Dye-binding studies and fluorescence quenching were used to investigate if a pH variation induces any conformational changes on the enzyme. Both methods suggest that lowering the pH from physiological to acidic values triggers an increased exposure of non-polar sites in the enzyme, which may modulate its adsorption behaviour to the more hydrophobic surfaces. At pH 4.5, the enzyme carries a substantial positive net charge and therefore relatively low native-state stability. As a consequence, surface binding may be favoured, irrespective of the type of surface, by providing increased conformational entropy to the enzyme. The specific activity (SA) of the adsorbed enzyme was strongly dependent on the conditions used. A decrease in SA (ca. 30% of control) was observed after adsorption on CH3-SAMs for all the pH tested. Adsorption on gold and on the more hydrophilic SAMs (OH- and COOH-) resulted in different degrees of inactivation at the more acidic pH (4.5), and in enzyme activation (up to ca. 230% of control) at higher pH (7–8), near the isoelectric point of the enzyme.
Keywords: Adsorpotion; Enzyme; Self-assembled monolayers (SAMs); Enzyme activation;

An investigation on the physicochemical properties of chitosan/DNA polyelectrolyte complexes by Wenguang Liu; Shujun Sun; Zhiqiang Cao; Xin Zhang; Kangde Yao; William W. Lu; K.D.K. Luk (2705-2711).
In this work, to eliminate the effect of the hydrophobicity of N-acetyl groups in chitosan on the interaction between chitosan and DNA, a water soluble chitosan with molecular weight of 5000 and deacetylated degree of 99% was selected to complex with DNA at varied charged ratios. The physicochemical properties of chitoplexes were investigated by means of FTIR, circular dichroism (CD), static fluorescence spectroscopy, and atomic force microscopy (AFM). The results indicated that upon interacting with chitosan, the DNA molecules saved a B conformation, and the binding affinity of chitosan to DNA was dependent on pH of media. At pH 5.5, highly charged chitosan had a strong binding affinity with DNA; whereas in pH 12.0 medium, only weak interactions existed. The CD spectra of Hoechst 33258 competitive displacement revealed that chitosan was partially bound to the minor groove of DNA. The morphology of chitosan/DNA complexes was strongly dependent upon the charge ratios. At charge ratio (+/−) of 1:4, not all DNA could be entrapped in the complex; at ratio of 8:1, the spherical complexes with mean size of nanoscale were formed without free DNA, but no typical toroid patterns were observed, which might stem from the strong compact of DNA caused by highly charged chitosan. It was supposed that the strong interaction of chitosan with DNA possibly prevented gene unpacking from chitosan vector, consequently restraining gene expression in nucleus.
Keywords: Chitosan; DNA; Gene delivery; Polyelectrolyte complexes;

This study evaluated cellular uptake of polymeric nanoparticles by using Caco-2 cells, a human colon adenocarcinoma cell line, as an in vitro model with the aim to apply nanoparticles of biodegradable polymers for oral chemotherapy. The feasibility was demonstrated by showing the localization and quantification of the cell uptake of fluorescent polystyrene nanoparticles of standard size and poly(lactic-co-glycolic acid) (PLGA) nanoparticles coated with polyvinyl alcohol (PVA) or vitamin E TPGS. Coumarin-6 loaded PLGA nanoparticles were prepared by a modified solvent extraction/evaporation method and characterized by laser light scattering for size and size distribution, scanning electron microscopy (SEM) for surface morphology, zeta-potential for surface charge, and spectrofluorometry for fluorescent molecule release from the nanoparticles. The effects of particle size and particle surface coating on the cellular uptake of the nanoparticles were quantified by spectrofluorometric measurement. Cellular uptake of vitamin E TPGS-coated PLGA nanoparticles showed 1.4 folds higher than that of PVA-coated PLGA nanoparticles and 4-6 folds higher than that of nude polystyrene nanoparticles. Images of confocal laser scanning microscopy, cryo-SEM and transmission electron microscopy clearly evidenced the internalization of nanoparticles by the Caco-2 cells, showing that surface modification of PLGA nanoparticles with vitamin E TPGS notably improved the cellular uptake. It is highly feasible for nanoparticles of biodegradable polymers to be applied to promote oral chemotherapy.
Keywords: Chemotherapy; Confocal laser scanning microscopy (CLSM); Cryo-SEM; Drug delivery; Poly(lactic-co-glycolic acid) (PLGA); Transmission electron microscopy (TEM);

Preparation and characterisation of antibody modified gelatin nanoparticles as drug carrier system for uptake in lymphocytes by Sabine Balthasar; Kerstin Michaelis; Norbert Dinauer; Hagen von Briesen; Jörg Kreuter; Klaus Langer (2723-2732).
Established methods of protein chemistry can be used for the effective attachment of drug targeting ligands to the surface of protein-based nanoparticles. In the present work gelatin nanoparticles were used for the attachment of biotinylated anti-CD3 antibodies by avidin–biotin-complex formation. These antibody modified nanoparticles represent a promising carrier system for the specific drug targeting to T-lymphocytes. The objective of this work was the comprehensive quantification of every chemical reaction step during the preparation procedure of these cell specific nanoparticles.Gelatin nanoparticles were formed by a two-step desolvation process. After the first desolvation step the remaining sediment and the supernatant were analysed for molecular weight distribution by size exclusion chromatography (SEC). Nanoparticles then were formed using the high molecular gelatin fraction and subsequently were stabilised by glutaraldehyde crosslinking. A part of the detectable amino groups on the particle surface was reacted with 2-iminothiolane in order to introduce reactive sulfhydryl groups. The thiolated nanoparticles were coupled to NeutrAvidin™ (NAv) which previously was activated with the heterobifunctional crosslinker sulfo-MBS. All these reaction steps were quantified by photometry or gravimetry. The functionality of NAv after covalent conjugation was confirmed by a biotin-4-fluorescein assay. The NAv-modified nanoparticles then were used for the binding of biotinylated anti-CD3 antibodies by avidin–biotin-complex formation. A highly effective attachment of the ligand was ascertained by different, indirect methods: immunoblotting and fluorimetry. Therefore, a well-defined nanoparticle system with drug targeting ligand modification was established that holds promise for further effective preclinical testing.
Keywords: Gelatin; Nanoparticle; Surface modification; Anti-CD3 antibody; Immunoblotting; Biotin-4-fluorescein;

Novel pH-sensitive interpolymer polyelectrolyte complex was synthesized by gamma radiation-induced copolymerization of acrylic acid (AAc) and dimethyl aminoethyl methacrylate (DMAEMA). pH-dependent swelling showed different phase transitions depending on the copolymer composition and also showed the interpolymer polyelectrolyte complex formation at pH values ranged from pH 3 to pH 4. FT-IR and TGA was employed to study the complex formation. The influence of copolymer composition and pH value of the surrounding medium on the type of water diffusion in the glassy polymer was discussed. The ability of the prepared copolymer to be used as drug carrier for colon-specific drug delivery system was estimated using ketoprofen as a model drug.
Keywords: Dimethyl aminoethyl methacrylate; PH-sensitive; Interpolymer complex; Colon-specific; Drug release;

Endotoxin adsorbent using dimethylamine ligands by Zhi Yuan; Mei Yu; Jihong Li; Guanghui Hou; Huiyan Wang (2741-2747).
Various adsorbents have been investigated for removing endotoxin from protein solutions. It is believed that electrostatic interaction and hydrophobic intermolecular interaction are the main interactions in adsorption of endotoxin. In this work, a series of novel molecular recognition adsorbents for removal of endotoxin with dimethylamine ligand were prepared by coupling ligands on polymethyl methacrylate. We found that its adsorption capacity of endotoxin increased almost 8 times in the presence of a hydroxyl group at β-site of ligand. The computer simulation showed that the hydroxyl group at β-site could form H bond with endotoxin, as a result an octatomic ring was formed. The spacer in adsorbent and the long alkyl chain in endotoxin were located at the same side of the octatomic ring. In this situation, electrostatic interaction, H bond, cooperative effect of octatomic ring and hydrophobic intermolecular interaction effected simultaneously. The combination of endotoxin with adsorbent was tight and adsorption capacity was effectually increased.
Keywords: Endotoxin; Molecular recognition; Adsorbent; Hemoperfusion;

Templates for DNA-templated Fe3O4 nanoparticles by Dorjderem Nyamjav; Albena Ivanisevic (2749-2757).
Two different strategies are reported that result in the site-specific placement of long DNA molecules, templated with Fe3O4 nanoparticles, on SiO x surfaces. The strategies combine solution templating of inorganic nanoparticles onto biomolecules, molecular combing, the layer-by-layer method, and dip-pen nanolithography (DPN). In one methodology, the first step is to place and stretch DNA molecules via a DPN template containing features of poly(allylamine hydrochloride) (PAH). In the second step the elongated DNA is templated with Fe3O4 nanoparticles allowing them to assemble onto the DNA molecules based on electrostatic interactions. In the second methodology, DPN templates composed of PSS patterns are used to comb in a site-specific manner DNA molecules that have been pre-tempated in solution with the Fe3O4 nanoparticles. The templates generated by the two methods can serve as building blocks for the fabrication of future higher order assemblies.
Keywords: Lithography; Nanoparticles; DNA;

An artificial receptor for creatinine was synthesized by the method of molecularly imprinted polymer (MIP). β-Cyclodextrin was used as a monomer cross-linked with epichlorohydrin in the presence of creatinine, which was a template for the imprinting. Different molar ratios of monomer to template were used to synthesize the polymers so that better specific adsorption ability towards creatinine could be achieved. The results showed that to carry out the polymerization with a molar ratio of monomer to template of 3:2 and monomer to cross-linking agent of 1:10 was proper. N-hydroxysuccinimide and 2-pyrrolinidone were used as the analogues of creatinine in the adsorption experiments of multi-component solutions to reveal the specific recognition ability of the molecularly imprinted poly(β-cyclodextrin) (poly(β-CD)) for the template molecule, creatinine. One such detection interference of creatinine was creatine, which also co-exists in serum. Hence, adsorption experiments of creatinine/creatine binary mixture were also carried out to investigate and confirm the specific binding of the creatinine-imprinted poly(β-cyclodextrin) towards creatinine. The hydroxyl groups of the imprinted poly(β-CD) was further capped by chlorotrimethylsilane (CTMS) to investigate the interaction between creatinine and the imprinted poly(β-CD). The adsorption resulting from the mixture solution by MIPs suggested that the creatinine-imprinted poly(β-CD) demonstrated superior binding effect for the target molecule, creatinine, rather than creatine, N-hydroxysuccinimide and 2-pyrrolinidone.
Keywords: Molecularly imprinted poly(β-cyclodextrin); Creatinine; N-hydroxysuccinimide; 2-pyrrolinidone; Creatine; Chlorotrimethylsilane;

Nacre/bone interface changes in durable nacre endosseous implants in sheep by S. Berland; O. Delattre; S. Borzeix; Y. Catonné; E. Lopez (2767-2773).
Raw nacre implants persist even after 9 months of implantation into bone tissue in sheep. However the nacre surface undergoes a limited biodegradation process. Smooth-surfaced nacre implants were seen to become microporous after implantation. The results of these long-term, in vivo studies show that the overall process involves bone-resorbing cells, relies on a two-phase mechanism and may correspond to a regulation process. The rate of surface change depends on the bone implantation site and the nacre/bone interaction. The in vivo biodegradability of nacre is a highly variable parameter. The size and shape of the implanted nacre and the cellular environment of the implant are key factors in determining the biodegradation kinetics of the nacre in a living system.

Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin by Ung-Jin Kim; Jaehyung Park; Hyeon Joo Kim; Masahisa Wada; David L. Kaplan (2775-2785).
A new all-aqueous process is described to form three-dimensional porous silk fibroin matrices with control of structural and morphological features. The result of this process are scaffolds with controllable porosity and pore sizes that fully degrade in the presence of proteases, unlike prior methods to generate silk-based biomaterials that required the use of organic solvent treatments to impart control of structure and stability in aqueous environments, with low rates of proteolytic hydrolysis. A mechanism is proposed for this novel process that imparts physical stability via hydrophobic interactions. Adjusting the concentration of silk fibroin in water, and the particle size of granular NaCl used in the process, leads to the control of morphological and functional properties of the scaffolds. The aqueous-derived scaffolds had highly homogeneous and interconnected pores with pore sizes ranging from 470 to 940 μm, depending on the mode of preparation. The scaffolds had porosities >90% and compressive strength and modulus up to 320±10 and 3330±500 KPa, respectively, when formed from 10% aqueous solutions of fibroin. The scaffolds fully degraded upon exposure to protease during 21 days, unlike the scaffolds prepared from organic solvent processing. These new silk-based three-dimensional matrices provide useful properties as biomaterial matrices due to the all-aqueous mode of preparation, control of pore size, connectivity of pores, degradability and useful mechanical features. Importantly, this process offers an entirely new window of materials properties when compared with traditional silk fibroin-based materials.
Keywords: Silk; Fibroin; Scaffold; Salt leaching; Biodegradation; Biomaterial;