Biomaterials (v.26, #16)

Calendar (I).

Correlating crystallinity and reactivity in an α-tricalcium phosphate by C.L. Camiré; U. Gbureck; W. Hirsiger; M. Bohner (2787-2794).
In this study, the effect of how variant milling time affects material characteristics of alpha phase tricalcium phosphate powder (α-TCP) was studied. Two α-TCP batches were separated in small lots and milled for various times for up to 4 h. The resulting milled lots were characterized by measuring their crystallinity, particle size, specific surface area, thermal stability, and heat released during hydration. Mechanical treatment was seen to greatly increase the α-TCP X-ray amorphous fraction and heat release during hydration, almost independently of α-TCP particle size and specific surface area. Therefore, the results suggest that the formation and presence of an X-ray amorphous phase in the α-TCP powder greatly contribute to its reactivity. The exotherm of the powders increases from 103 to 238 kJ/mol after milling.
Keywords: Bone; Bone cement; Bone graft; Calcium phosphate; Calcium phosphate cement; Hydroxyapatite;

Biodegradable polymer networks were prepared from fumaric acid derivatives of oligomeric esters. Photo-crosslinkable macromers were prepared by reacting star-shaped hydroxyl-group terminated lactide, ε-caprolactone and trimethylene carbonate based oligomers and fumaric acid monoethyl ester in the presence of N,N-dicyclohexylcarbodiimide and 4-dimethylamino pyridine at room temperature. The functionalization method is facile and suited for many hydroxyl-terminated oligomers.The reactivity of the fumarate end groups is such that, upon crosslinking by UV radical polymerization, networks with high gel contents (up to 96%) can be obtained without the addition of reactive diluents. The physical properties of the networks can be tuned by adjusting the composition, architecture and molecular weight of the oligomeric precursors. Such networks, built up of non-toxic compounds and designed to release benign degradation products, may find wide application in tissue engineering and other areas of biomedical research.
Keywords: Biodegradable networks; Oligomer functionalization; Fumaric acid monoethyl ester; Photo-crosslinking;

Enzymatic degradation of PLLA-PEOz-PLLA triblock copolymers by Chau-Hui Wang; Kuo-Rong Fan; Ging-Ho Hsiue (2803-2811).
The enzymatic degradation of poly(L-lactide)-block- poly(2-ethyl-2-oxazoline)-block-poly(L-lactide) triblock copolymer (PLLA-PEOz-PLLA) was investigated using efficient enzyme proteinase K. PLLA-PEOz-PLLA solution-cast film lost a considerable amount of hydrophilic copolymers in the first 2 h, and the degradation after 2 h proceeded predominantly by surface erosion. The two faces of the hydrolyzed film exhibited different morphologies following enzymatic degradation. The lower face showed many spherulites, which are the superstructural morphology of polymer crystals. Porous spheres based on crystalline PLLA were observed on the upper face, because they were more resistant to enzymatic attack. The crystallinity of the films increased monotonously with the hydrolysis time, thus, the absorption of water gradually decreased. The analysis of degradation residues revealed that many colloids of poly(2-ethyl-2-oxazoline)-co-polyethylenimine (PEOz-co-PEI) copolymers were dispersed in the buffer solution. The average diameter, 1 μm, of the colloids was reduced to 200 nm by advanced degradation. The proteinase K exhibited remarkable hydrolysis not only at the ester bond but also the amide bond.
Keywords: PLLA-PEOz-PLLA; Proteinase k; Biodegradable; Enzymatic degradation;

In vitro behavior of a porous TiO2/perlite composite and its surface modification with fibronectin by Matthias von Walter; Matthias Rüger; Christian Ragoß; Guy C.M. Steffens; Dirk A. Hollander; Othmar Paar; Horst R. Maier; Willi Jahnen-Dechent; Anja K. Bosserhoff; Hans-Josef Erli (2813-2826).
In this study, we introduce a porous composite material, termed “Ecopore”, and describe in vitro investigation of the material and its modification with fibronectin. The material is a sintered compound of rutile TiO2 and the volcanic silicate perlite with a macrostructure of interconnecting pores. It is both inexpensive and easy to manufacture. We first investigated Ecopore for corrosion and leaching of elements in physiological saline. The corrosion supernatants did not contain critical concentrations of toxic trace elements. In an in vitro model, human primary osteoblasts (HOB) were cultured directly on Ecopore. HOB grew on the composite as well as on samples of its single constituents, TiO2 and perlite glass, and remained vital, but cellular spreading was less than on tissue culture plastic. The pro-inflammatory cytokines IL-1 and TNF-α were below detection limits in HOB culture supernatants, whereas IL-6 was detectable on a low level. To enhance cellular attachment and growth, the surface of the composite was modified by etching, functionalization with aminosilane and coupling of fibronectin. This modification greatly enhanced the spreading of HOB, indicated by vital staining and Sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) metabolism assays. HOB grew on the entire visible surface of porous fibronectin-modified composite, expressing alkaline phosphatase, a mature osteoblast marker. We conclude that Ecopore is non-toxic and sustains HOB growth, cellular spreading being improvable by coating with fibronectin. The composite may be usable in the field of bone substitution.
Keywords: Ceramic; Cytotoxicity; Surface modification; Fibronectin; Osteoblast;

Effect of isothermal annealing on the hydrolytic degradation rate of poly(lactide-co-glycolide) (PLGA) by Say Chye Joachim Loo; Chui Ping Ooi; Siew Hong Elyna Wee; Yin Chiang Freddy Boey (2827-2833).
Isothermal crystallization through annealing at 115 °C was conducted to increase the degree of crystallinity of poly (lactide-co-glycolide) (PLGA). The maximum increase in the degree of crystallinity (∼21%) was achieved after 60 min of annealing. The crystal size/perfection was observed to increase with annealing time. The annealed PLGA films were then hydrolytically degraded in phosphate buffered saline solution of pH 7.4 at 37 °C for up to 150 days. Minimal mass loss was observed throughout the time investigated, suggesting that the samples were still in the first phase of degradation. The increase in the degree of crystallinity of the PLGA samples annealed at 15 and 30 min was found to retard their overall rate of hydrolytic degradation, when compared to those samples with higher initial crystallinity (annealed for 45 and 60 min) that had faster degradation rates. The increased degradation rate at higher crystallinity was associated with the loss of amorphous material and the formation of voids during annealing, which decreases the glass transition temperature and increases the average water uptake in the samples annealed for longer times. Therefore, the increase in degree of crystallinity is found to retard hydrolytic degradation but only to a certain extent, beyond which the formation of voids through annealing increases the rate of hydrolytic degradation.
Keywords: Poly(lactide-co-glycolide); PLGA; Biodegradable; Hydrolytic degradation; Crystallinity; Isothermal annealing;

Nucleation of biomimetic apatite in synthetic body fluids: dense and porous scaffold development by Elena Landi; Anna Tampieri; Giancarlo Celotti; Ratih Langenati; Monica Sandri; Simone Sprio (2835-2845).
The effectiveness of synthetic body fluids (SBF) as biomimetic sources to synthesize carbonated hydroxyapatite (CHA) powder similar to the biological inorganic phase, in terms of composition and microstructure, was investigated. CHA apatite powders were prepared following two widely experimented routes: (1) calcium nitrate tetrahydrate and diammonium hydrogen phosphate and (2) calcium hydroxide and ortophosphoric acid, but using SBF as synthesis medium instead of pure water. The characteristics of the as-prepared powders were compared, also with the features of apatite powders synthesized via pure water-based classical methods. The powder thermal resistance and behaviour during densification were studied together with the mechanical properties of the dense samples. The sponge impregnation process was used to prepare porous samples having morphological and mechanical characteristics suitable for bone substitution.Using this novel synthesis was it possible to prepare nanosized (≈20 nm), pure, carbonate apatite powder containing Mg, Na, K ions, with morphological and compositional features mimicking natural apatite and with improved thermal properties.After sintering at 1250 °C the carbonate-free apatite porous samples showed a surprising, high compressive strength together with a biomimetic morphology.
Keywords: Biomimetic hydroxyapatite; Carbonated hydroxyapatite; Synthetic body fluid; Powders-chemical preparation; Mechanical properties;

Effect of dual ion implantation of calcium and phosphorus on the properties of titanium by D. Krupa; J. Baszkiewicz; J.A. Kozubowski; A. Barcz; J.W. Sobczak; A. Biliński; M. Lewandowska-Szumieł; B. Rajchel (2847-2856).
This study is concerned with the effect of dual implantation of calcium and phosphorus upon the structure, corrosion resistance and biocompatibility of titanium. The ions were implanted in sequence, first Ca and then P, both at a dose of 1017 ions/cm2 at a beam energy of 25 keV. Transmission electron microscopy was used to investigate the microstructure of the implanted layer. The chemical composition of the implanted layer was examined by XPS and SIMS. The corrosion resistance was determined by electrochemical methods in a simulated body fluid (SBF) at a temperature of 37 °C. The biocompatibility tests were performed in vitro in a culture of human-derived bone cells (HDBC) in contact with the tested materials. The viability of the cells was determined by an XTT assay and their activity by the measurements of the alkaline phosphatase activity in contact with implanted and non-implanted titanium samples. The in vitro examinations confirmed that, under the conditions prevailing during the experiments, the biocompatibility of Ca+P ion-implanted titanium was satisfactory. TEM results show that the surface layer formed by the Ca+P implantation is amorphous. The corrosion resistance of titanium, examined by the electrochemical methods, appeared to be increased after the Ca+P ion implantation.
Keywords: Titanium; Ion implantation; Corrosion; Biocompatibility;

Coating porous titanium with calcium phosphate (Ca-P) is an effective way to enhance titanium's osteoinduction capability. This study investigated the effectiveness of two coating methods: biomimetic deposition (BD) and electrochemical deposition (ED) in aqueous solutions. The titanium surfaces were treated by acidic etching and alkaline before coating. Effects of the pre-coating treatments on Ca-P coating were also investigated. Both deposition methods could produce Ca-P coatings on the inner pore surfaces of the titanium. The BD coatings were thicker and more uniform than were the ED coatings. On the other hand, ED was less sensitive to the condition of the titanium surface, and much faster in the coating deposition. However, ED produces less uniform and thinner coating layers on the inner pore surfaces of the titanium than does BD. The crystal structure of the coating is octacalcium phosphate (OCP) regardless of the deposition method. The morphology of flake-like OCP crystals in the deposition layers is similar for both deposition methods, except that the crystal flakes rupture after ED.
Keywords: Porous titanium; Calcium phosphate; Coatings; Biomimetic deposition; Electrochemical deposition;

A self-transformable sulfonated poly(ethylene glycol) acrylate diblock copolymer (PEG-SO3A/OA) entrapped into polysulfone membrane was studied. The asymmetric membrane structure was prepared by a phase inversion process. The induced hydrophilicity by reorientation of diblock copolymer at the interface was evaluated by contact angle measurement, platelet adhesion test, and electron spectroscopy for chemical analysis (ESCA) depth profiling with ion sputtering. Molecular dynamic (MD) simulations as a function of copolymer density were also performed to obtain optimum interfacial structure information. The dependency of water clustering behavior as a hydrophilicity parameter was described in terms of an atom–atom radial distribution function (RDF). The results showed that the sulfonated diblock copolymer enhanced the hydrophilicity and long-term stability more than the copolymer having no hydrophobic block. Also, according to the ESCA, oxygen composition significantly began to decrease along the membrane depth, indicating the reorientation of diblock chains. The copolymer-entrapped surfaces significantly induced the degree of water clustering, and the resulting equilibrium rearrangement of interfacial structure was distinctly dependent upon the density of the copolymer.
Keywords: Hydrophilicity; Self-assembly; Molecular modeling; Polysulfone; Membrane;

A series of segmented multiblock polyurethanes (MPUs) were synthesized by polyaddition reaction using hexamethylene diisocyanate (HDI)/poly(ethylene oxide) (PEO, as a hydrophilic component)/ poly(tetramethylene oxide) (PTMO)/ poly(butadiene diol)(PBD)/1,4-butanediol(BD)/{2-[bis(2-hydroxyethyl) methyl ammonio]ethyl stearyl phosphate}[BESP, as a phospholipids component: 0–42 mol% (0–9 wt%)]. To improve the blood compatibility of biomedical grade polyurethane (Pellethene®), the Pellethene® was blended with MPUs and then crosslinked using dicumyl peroxide as a crosslinking agent. Effects of BESP content [0–42 mol% (0–9 wt%)] in MPUs on the properties of MPUs and blend (Pellethene®/MPUs) films were investigated. The X-ray photoelectron spectra indicated that the BESP moieties were located at the surface of the crosslinked blend (Pellethene®/MPUs) films. As the BESP content in MPUs increased, the water contact angle on the surfaces of corsslinked blend film was decreased but the water absorption and mechanical properties were markedly increased. By the test of platelet adhesion on the surfaces of corsslinked blend film, it was found that the platelet adhesion on the surface was significantly decreased from 70% to 6% by increasing BESP content from 0 to 42 mol% (0–9 wt%) in MPUs. These results suggest that crosslinked blend films may have more potential as a new material for biomedical applications, which are directly in contact with blood.
Keywords: Polyurethane; Phospholipid; Biomaterials; Platelet adhesion;

Phase formation and evolution in the silicon substituted tricalcium phosphate/apatite system by J.W. Reid; A. Pietak; M. Sayer; D. Dunfield; T.J.N. Smith (2887-2897).
The sintering of silicon doped calcium phosphate ceramics prepared from a basic colloidal hydroxyapatite (Ca5(PO4)3OH or HA) precipitate mixed with silica over 800 °C yields a phase mixture of tricalcium phosphate phases (TCP) designated Si–TCP, β-TCP and a silicon substituted dehydrated apatite (Si–Ap). The Si–TCP phase is defined as a combination of a silicon stabilized TCP in which the silicon content attains a saturated value (Ca3(P0.9Si0.1O3.95)2 or Si–TCPsat) and α-TCP (Ca3(PO4)2). Si–TCPsat has the same crystalline space group (P21/a) as α-TCP, but with characteristically different lattice parameters due to the substitution of silicon in tetrahedral phosphorus sites. The nucleation and growth kinetics of Si–TCP in samples of composition 0.2 mol SiO2:mol HA (0.2:1) and 1 mol SiO2:mol HA (1:1) can be understood in terms of the initial growth of α-TCP at a silica-HA interface followed by a transformation to Si–TCPsat or β-TCP. A thermodynamic model for the formation of Si–TCPsat predicts a nucleation temperature of 795 °C, in close agreement with experiment. If sufficient silicon is available, the α-TCP transforms to Si–TCPsat during extended sintering. In the absence of sufficient silicon, the α-TCP transforms to β-TCP.
Keywords: Tricalcium phosphate; Hydroxyapatite; Silica; Phase evolution; XRD;

The purpose of the present study is to compare the high-cycle fatigue behavior of newly developed Ti–7.5Mo alloy with that of c.p. Ti, Ti–13Nb–13Zr and Ti–6Al–4V alloys in their as-cast state. Experimental results indicate that Ti–6Al–4V and c.p. Ti have higher stress-controlled fatigue resistance but lower strain-controlled fatigue resistance than Ti–7.5Mo and Ti–13Nb–13Zr. Among four materials Ti–7.5Mo demonstrates the best strain-controlled fatigue performance. The fracture surfaces of the present materials are comprised of three morphologically distinct zones: crack initiation zone, crack propagation zone, and the final-stage overload zone. The fatigue cracks almost always initiate from casting-induced surface/subsurface pores. A river pattern is observed in the propagation zone. In the overload zone dimples are typically observed. Three factors most significantly affecting the fatigue performance of the present materials are the presence of the casting-induced surface/subsurface pores; the location of the pores; and the inherent mechanical properties of the materials.
Keywords: Titanium alloy; Ti–7.5Mo; Fatigue; Casting;

Design, characterization and testing of Ti-based multicomponent coatings for load-bearing medical applications by D.V. Shtansky; N.A. Gloushankova; A.N. Sheveiko; M.A. Kharitonova; T.G. Moizhess; E.A. Levashov; F. Rossi (2909-2924).
A comparative investigation of multicomponent thin films based on the systems Ti–Ca–C–O–(N), Ti–Zr–C–O–(N), Ti–Si–Zr–O–(N) and Ti–Nb–C–(N) is presented. TiC0.5+10%CaO, TiC0.5+20%CaO, TiC0.5+10%ZrO2, TiC0.5+20%ZrO2, Ti5Si3+10%ZrO2, TiC0.5+10%Nb2C and TiC0.5+30%Nb2C composite targets were manufactured by means of self-propagating high-temperature synthesis, followed by DC magnetron sputtering in an atmosphere of argon or in a gaseous mixture of argon and nitrogen. The films were characterized in terms of their structure, chemical composition, surface topography, hardness, elastic modulus, elastic recovery, surface charge, friction coefficient, and wear rate. The biocompatibility of the films was evaluated by both in vitro and in vivo experiments. In vitro studies involved the investigation of the proliferation of Rat-1 fibroblasts and IAR-2 epithelial cells on the tested films, morphometric analysis and actin cytoskeleton staining of the cells cultivated on the films. In vivo studies were fulfilled by subcutaneous implantation of Teflon plates coated with the tested films in mice and analysis of the population of cells on the surfaces. The films deposited under optimal conditions showed high hardness in the range of 30–37 GPa, significant reduced Young's modulus, low friction coefficient down to 0.1–0.2 and low wear rate in comparison with conventional magnetron-sputtered TiC and TiN films. The surface of all films was negatively charged with an outstanding shift between the Ar and Ar+N2 Zeta potential curves that reaches 5 mV at the highest pH values. We did not detect statistically significant differences in the attachment, spreading and cell shape of cultured IAR-2 and Rat-1 cells on the Ti–Ca–C–O–(N), Ti–Zr–C–O–(N) (TiC0.5+10%ZrO2 target), Ti–Si–Zr–O–(N) films and the uncoated substrata. The adhesion and proliferation of cultured cells in vitro was perfect at all investigated films. Assessment of the population of cells covering on the Teflon plates coated with the Ti–Ca–C–O–(N) and Ti–Zr–C–O–(N) films after 16 weeks of subcutaneous implantation revealed the high biocompatibility level of tested films and absence of inflammatory reactions in mice. Contrary, the epitheliocytes and fibroblasts cultivated on the Ti–Zr–C–O–(N) (TiC0.5+20%ZrO2 target) and Ti–Nb–C–(N) films had disturbing actin cytoskeleton.
Keywords: Multicomponent coatings; Composite targets; Magnetron sputtering; Structure; Physical–mechanical properties; Tribology; Cell proliferation; Cytocompatibility; Biocompatibility;

Preparation and characteristics of a calcium magnesium silicate (bredigite) bioactive ceramic by Chengtie Wu; Jiang Chang; Junying Wang; Siyu Ni; Wanyin Zhai (2925-2931).
In this study, new bredigite (Ca7MgSi4O16) ceramics were prepared by sintering sol–gel-derived bredigite powder compacts at 1350 °C for 8 h. The bending strength, fracture toughness and Young's modulus were about 156 MPa, 1.57 MPa m1/2 and 43 GPa, respectively. The in vitro bioactivity of the bredigite ceramics was evaluated by investigating the apatite-formation ability in simulated body fluid (SBF) and the effect of ionic products from bredigite dissolution on the mouse fibroblasts cell line L929. In addition, the in vitro biocompatibility of the bredigite ceramics was evaluated by osteoblasts adhesion and proliferation assay. The results showed that bredigite ceramics could induce HAp formation in SBF. The products from bredigite dissolution significantly promoted cell growth at a certain concentration range. Furthermore, osteoblasts adhered and spread well on bredigite ceramics, and osteoblasts proliferation on bredigite ceramics was obvious.
Keywords: Bredigite; Bioactivity; Hydroxyapatite; Osteoblasts; Biocompatibility;

Gene expression clustering using self-organizing maps: analysis of the macrophage response to particulate biomaterials by Grant E. Garrigues; David R. Cho; Harry E. Rubash; Steven R. Goldring; James H. Herndon; Arun S. Shanbhag (2933-2945).
The most common cause of total joint replacement failure is peri-implant bone loss causing pain and prosthesis loosening. This process, known as osteolysis or aseptic loosening, is characterized by macrophage phagocytosis of particulate implant wear debris. In an incompletely defined step, particulate biomaterial debris induces macrophages to release a variety of inflammatory mediators and signaling proteins that lead to bone loss. In an in vitro model of this process, we used microarray technology and data analysis techniques, including the use of self-organizing maps (SOMs), to understand the mRNA gene expression changes occurring in macrophages exposed to clinically relevant particles of ultra-high molecular weight polyethylene and TiAlV alloy. Earlier studies have been limited by technology that only allowed analysis of a few genes at a time, but the microarray techniques used in this paper generate the quantitative analysis of over a thousand genes simultaneously.Our microarray analysis utilized an SOM clustering to elucidate general patterns in the data, lists of top up- and down-regulated genes for each time point and genes with differential expression under different biomaterial exposures. The expression levels of the majority of genes ( > 95 % ) did not vary over time or with exposure to different biomaterials, but a few important genes, such as TNF- α , IL- 1 β , IL-6, and MIP 1 α , proved to be highly regulated in response to biomaterial exposure. We also uncovered a novel set of genes, which not only validates and logically extends the current model of the pathogenesis of osteolysis and aseptic loosening, but also provides new targets for further research and therapeutics.
Keywords: Osteolysis; Aseptic loosening; Gene expression; Self-organizing maps; Macrophage; Total joint replacement; Microarrays; Ti-alloy; UHMWPE; Wear debris;

Magnetron co-sputtered silicon-containing hydroxyapatite thin films—an in vitro study by E.S. Thian; J. Huang; S.M. Best; Z.H. Barber; W. Bonfield (2947-2956).
The use of silicon-substituted hydroxyapatite (Si-HA) as a biomaterial has been reported recently. In vivo testing has shown that Si-HA promotes early bonding of the bone/implant interface. In order to extend its usage to major load-bearing applications such as artificial hip replacement implants, it has been proposed that the material could be used in the form of a coating on implant surfaces. This paper reports a preliminary study of the biocompatibility of magnetron co-sputtered silicon-containing hydroxyapatite (Si-HA) coatings on a metallic substrate. Magnetron co-sputtered Si-HA films of thickness 600 nm with a Si content of approximately 0.8 wt% were produced on titanium substrates. X-ray diffraction analysis showed that the as-deposited Si-HA films were either amorphous or made up of very small crystals. The crystallinity of Si-HA films was increased after post-deposition heat treatment at 700 °C for 3 h, and the principal peaks were attributable to HA. The formation of nano-scale silicon–calcium phosphate precipitates was noted on the heat-treated films. In vitro cell culture has demonstrated that human osteoblast-like cells attached and grew well on all films, with the highest cell growth and signs of mineralisation observed on the heat-treated Si-HA films. In addition, many focal contacts were produced on the films and the cells had well-defined actin cytoskeletal organisation. This work shows that as-deposited and heat-treated Si-HA films have excellent bioactivity and are good candidates when rapid bone apposition is required. Furthermore, heat-treated Si-HA films have improved biostability compared to as-deposited films under physiological conditions.
Keywords: Magnetron co-sputtering; Silicon-substituted hydroxyapatite (Si-HA); Thin films/coatings; Human osteoblast-like (HOB) cells; Heat treatment;

Stability and cellular responses to fluorapatite–collagen composites by Byung-Ho Yoon; Hae-Won Kim; Su-Hee Lee; Chang-Jun Bae; Young-Hag Koh; Young-Min Kong; Hyoun-Ee Kim (2957-2963).
Fluorapatite (FA)–collagen composites were synthesized via a biomimetic coprecipitation method in order to improve the structural stability and cellular responses. Different amounts of ammonium fluoride (NH4F), acting as a fluorine source for FA, were added to the precipitation of the composites. The precipitated composites were freeze-dried and isostatically pressed in a dense body. The added fluorine was incorporated nearly fully into the apatite structure (fluoridation), and a near stoichiometric FA–collagen composite was obtained with complete fluoridation. The freeze-dried composites had a typical biomimetic network, consisting of collagen fibers and precipitates of nano-sized apatite crystals. The human osteoblast-like cells on the FA–collagen composites exhibited significantly higher proliferation and differentiation (according to alkaline phosphatase activity) than those on the hydroxyapatite–collagen composite. These enhanced osteoblastic cell responses were attributed to the fluorine release and the reduced dissolution rate.
Keywords: Fluorapatite (FA); Collagen; Biomimetic coprecipitation; Stability; Cellular responses;

Procoagulant stimulus processing by the intrinsic pathway of blood plasma coagulation by Rui Zhuo; Rachel Miller; Karen M. Bussard; Christopher A. Siedlecki; Erwin A. Vogler (2965-2973).
Potentiation of the intrinsic pathway of human blood plasma coagulation in vitro by contact with a solid procoagulant surface leads to bolus release of thrombin (FIIa) in concentration proportion to the intensity of activation as measured by procoagulant surface area or energy (water wettability). This rather remarkable finding is confirmed using two different assays: one triggering coagulation substantially through the intrinsic pathway alone and the second triggering coagulation through the intrinsic pathway in the presence of exogenous FIIa spikes. Similarity of experimental outcomes of these assays strongly suggests that endogenous FIIa production through the intrinsic pathway is independent of the absolute amount of FIIa present in plasma. Furthermore, we corroborate previous work indicating that procoagulant surfaces remain activating after repeated use and are not poisoned or denatured in the process of activating plasma coagulation. It is concluded that the sharp control mechanism that gives rise to bolus-production of FIIa from the intrinsic pathway must occur between surface activation of FXII and the FII→FIIa step, is not related to inhibition by FIIa, and does not involve deactivation of procoagulant surfaces.
Keywords: Blood coagulation; Plasma; Procoagulants; Hageman factor; FXII; Mathematical model of coagulation; Thrombin; FII;

Modulation of protein adsorption and cell adhesion by poly(allylamine hydrochloride) heparin films by Michelle R. Kreke; Anand S. Badami; Joshua B. Brady; R. Michael Akers; Aaron S. Goldstein (2975-2981).
Electrostatic layer-by-layer film assembly is an attractive way to non-covalently incorporate proteins and bioactive moieties into the surface of conventional biomaterials. Selection of polycationic and polyanionic components and deposition conditions can be used to control the interfacial properties, and through them protein adsorption, cell adhesion, and tissue development. In this study the polycation was poly(allylamine hydrochloride) (PAH), which is a weak base and consequently adsorbs at interfaces in a pH-dependent manner, and the polyanion was heparin, which is capable of interacting with many adhesion ligands and growth factors. PAH/heparin multilayer films were formed using PAH solutions of pH 6.4, 7.4, 8.4, and 9.4. Film thickness increased both with the number of PAH/heparin bilayers and the pH of the PAH solution. Films consisting of 10 bilayers with heparin topmost exhibited similar bulk atomic compositions and penetration of PAH into the heparin top layer. Finally, fibronectin adsorption and cell adhesion were maximal at an intermediate pH (pH 8.4>pH 9.4>pH 7.4). These results demonstrate that heparin-containing electrostatic films support cell adhesion and protein adsorption in a manner sensitive to film deposition conditions.
Keywords: Poly(allylamine hydrochloride); Heparin; Fibronectin; Osteoblast; Adhesion;

Nanoscale neuro-integrative coatings for neural implants by Wei He; Ravi V. Bellamkonda (2983-2990).
Silicon microelectrode arrays (Si MEAs) have great potential in enabling chronic in vivo recording of neural activity, but this potential has been hampered by scar tissue formation at the site of implantation. In this study, we report the fabrication and characterization of nanoscale coatings that have the potential of enhancing the biocompatibility of Si electrodes. We use electrostatic layer-by-layer (LbL) assembly to prepare nanoscale bioactive coatings on silicon substrates. We use the response of chick cortical neurons to these coatings to assess potential improvement in biocompatibility in vitro. The coatings are built on oxide covered silicon wafers by alternating polycations, polyethyleneimine (PEI) or chitosan (CH), with polyanions, either gelatin or laminin (LN). We use quartz crystal microbalance (QCM) to characterize the coatings. Our analysis confirms that we achieved ∼30–110 Å scale coatings via LbL assembly. In contrast to bare oxide covered silicon, coated substrates had significantly enhanced chick cortical neuron adhesion and differentiation, with multilayers of PEI–LN showing the greatest improvement. The multilayers of PEI–LN were stable for at least 7 days in physiological conditions, as determined by an enzyme-linked immunosorbent assay (ELISA). In addition, impedance spectroscopy confirmed that multilayers of PEI and LN did not increase the magnitude of impedance of Si MEAs at the biologically relevant frequency of 1 kHz. Our study demonstrates that electrostatic LbL assembly enables nanoscale bioactive coatings, and that PEI–LN multilayers significantly enhance cortical neuronal attachment and differentiation in vitro with no deleterious effects on impedance of the electrodes. Such well-controlled nanoscale coatings have the potential to significantly impact the compatibility and performance of Si MEAs in vivo.
Keywords: Nanoscale coatings; Layer-by-layer assembly; Neuron; Laminin; Silicon; Electrical recording;

A model shed antigen, ovalbumin (OVA), was incorporated into polymeric biomaterial carriers made of poly(lactic-co-glycolic acid) (PLGA) in the form of microparticles (MP) or scaffolds (SC). These polymeric biomaterial carrier vehicles with incorporated antigen were then injected or implanted into mice and the resulting time-dependent systemic humoral immune response towards the controlled released OVA was assessed by following the OVA-specific IgG concentration and isotypes using ELISA. To assess the differential level of enhancement of the immune response depending on the form of carrier vehicle (MP vs. SC), the total amount of polymer and OVA delivered was kept constant as well as the release rate of OVA for both carrier vehicles. The level of the humoral immune response was higher and sustained for OVA released from PLGA SC which were implanted with associated tissue damage, and lower and transient when the same amount of polymer and OVA were delivered from PLGA MP, which were minimally invasively delivered by injection. This immune response was primarily Th2 helper T cell-dependent, although for the strong adjuvant, CFA, and PLGA SC carriers there was both a Th2 and Th1 response contribution. These results implicate ‘danger signals’ associated with the implantation of the scaffolds due to tissue injury which primed the system for an enhanced immune response.
Keywords: Poly (lactic-co-glycolic acid) microparticles and scaffolds; Adjuvant; Immune response; Controlled release; Tissue engineering;

Blood vessel formation after soft-tissue implantation of hyaluronan-based hydrogel supplemented with copper ions by G. Giavaresi; P. Torricelli; P.M. Fornasari; R. Giardino; R. Barbucci; G. Leone (3001-3008).
The possibility of ameliorating bone healing of implanted bone allografts is a field of great interest. Early vascular invasion is a key factor in bone allograft incorporation. It is well known that copper ions (Cu2+) show a proangiogenic action favouring the development of new vessels. In this work a hyaluronan based 50% hydrogel (Hyal-50%) was enriched with (Cu2+) and its proangiogenic activity was evaluated. Fifteen Sprague Dawley female rats were submitted to the subcutaneous implantation of Hyal-50%, freeze-dried bone allografts, Hyal-50%-Cu2+, freeze-dried bone allografts plus Hyal-50% and freeze-dried bone allografts plus Hyal-50%-Cu2+. One month later, histomorphometric analysis evidenced the presence of a fibrous-reactive capsule around all specimens showing significant differences among groups (p<0.0005). The highest thickness of the fibrous capsule was found around the freeze-dried bone implants (p<0.05); as well as the Hyal-50%-Cu2+ plus freeze-dried bone (15.2%, p<0.05) and Hyal 50% plus freeze-dried bone (21.4%, p<0.0005) implants showed a significant higher thickness compared with Hyal 50% and Hyal-50%-Cu2+. Statistical analysis showed a significant (p<0.01) higher vascular density in Hyal- 50%-Cu2+ and Hyal-50%-Cu2+ plus freeze-dried bone group when compared to other groups. The present preliminary results suggest the advantages offered by the combined use of a well-known biocompatible and tissue healing promoting material (Hyal-50%) and a new technique that consists of stimulating tissue vascularization using Cu2+ and that bone allograft incorporation may benefit from this technology.
Keywords: Hyaluronan; Hydrogel; Copper ions; Freeze-dried bone allograft; Vessel formation; Soft-tissue implants;

Osteoconductive modifications of Ti-implants in a goat defect model: characterization of bone growth with SR μCT and histology by Ricardo Bernhardt; Juliette van den Dolder; Sussane Bierbaum; Rene Beutner; Dieter Scharnweber; John Jansen; Felix Beckmann; Hartmut Worch (3009-3019).
In this work the osteoconductive potential of coatings for titanium implants using different extracellular matrix components was evaluated. Cylindrical implants with two defined cavities A and B were coated with collagen type I, type III, or RGD peptide, and placed in the femur of goats together with an uncoated reference state. Bone contact and volume were determined after 5 and 12 weeks implantation, using both histomorphometry and synchrotron radiation micro computed tomography (SR μCT) as the methods complement each other: SR μCT allows for a high precision of bone detection due to the large number of analysed slices per sample, while histology offers a better lateral resolution and the possibility of additionally determining bone contact.Both methods revealed similar tendencies in bone formation for the differently bio-functionalized implants, with the SR μCT data resulting in significant differences. After 5 and 12 weeks, all three coatings showed a significant increase in bone volume over the uncoated reference, with the highest results for the collagen coatings. The coating consisting of just the RGD-sequence to improve cell adhesion showed only a slight improvement compared with the reference material.For uncoated titanium, RGD, and especially collagen type I, the response in cavity A, situated in denser bone, was stronger than in cavity B. Collagen type III, on the other hand, appeared to be the more effective coating in areas of lesser bone density as represented by cavity B. These results indicate that matrix molecules (or combinations thereof) are capable of generating the appropriate signals for the specific microenvironment around implants and can thus accelerate the bone formation process and increase the stability of implants.
Keywords: Titanium; Collagen type I; Collagen type III; RGD peptide; Osseointegration; Histomorphometry; Synchrotron radiation; Micro computed tomography;

The attraction of Mac-1+ phagocytes during acute inflammation by methyl-coated self-assembled monolayers by Judite N. Barbosa; Pedro Madureira; Mário A. Barbosa; Artur P. Águas (3021-3027).
We have used self-assembled monolayers (SAMs) of alkanethiolates on gold to study the contribution of methyl terminal functional groups in implant-triggered inflammation. The CH3-coated biomaterials were inserted in an air-pouch cavity of the BALB/c mouse and the in situ inflammatory response was monitored 4, 24, 48 and 72 h later. Flow cytometry was applied to define surface expression of the adhesion receptor Mac-1 (CD11b/CD18), a marker of activated leukocytes, and also of CD3 and B220 antigens (T and B lymphocytes). The CH3-coated surfaces caused a significant enhancement in the number of Mac-1+ cells in the implant. The only significant change in T and B lymphocytes was a transient increase in T cells detected 48 h after the implantation. Peak numbers of Mac-1+ phagocytes were observed 24 h after implantation. We conclude that if CH3 is present at the surface of implants, this chemical group will trigger a significant enhancement of activated phagocytes involved in the inflammatory reaction, and this phenomenon may extend the local phlogistic event.
Keywords: Self-assembled monolayers; Inflammation; Leukocytes; Mac-1 adhesion receptor; Flow cytometry;

In vivo evaluation of plasma-sprayed titanium coating after alkali modification by Weichang Xue; Xuanyong Liu; XueBin Zheng; Chuanxian Ding (3029-3037).
In this paper, plasma-sprayed titanium coatings were modified by alkali treatment. The changes in chemical composition and structure of coatings were examined by SEM and AES. The results obtained indicated that a net-like microscopic texture feature, which was full of the interconnected fine porosity, appeared on the surface of alkali-modified titanium coatings. The surface chemical composition was also altered by alkali modification. A sodium titanate compound was formed on the surface of the titanium coating and replaced the native passivating oxide layer. Its thickness was measured as about 150 nm which was about 10 times of that of the as-sprayed coating. The bone bonding ability of titanium coatings were investigated using a canine model. The histological examination and SEM observation demonstrated that more new bone was formed on the surface of alkali-modified implants and grew more rapidly into the porosity. The alkali-modified implants were found to appose directly to the surrounding bone. In contrast, a gap was observed at the interface between the as-sprayed implants and bone. The push-out test showed that alkali-modified implants had a higher shear strength than as-sprayed implants after 1 month of implantation. An interfacial layer, containing Ti, Ca and P, was found to form at the interface between bone and the alkali-modified implant by EDS analysis.
Keywords: Surface modification; Akali treatment; Titanium coating; Bone; In vivo;

The recognition of adsorbed and denatured proteins of different topographies by β 2 integrins and effects on leukocyte adhesion and activation by Thomas Brevig; Bjørn Holst; Zahida Ademovic; Noemi Rozlosnik; Jette H. Røhrmann; Niels B. Larsen; Ole C. Hansen; Peter Kingshott (3039-3053).
Leukocyte β 2 integrins Mac-1 and p150,95 are promiscuous cell-surface receptors that recognise and mediate cell adhesion to a variety of adsorbed and denatured proteins. We used albumin as a model protein to study whether leukocyte adhesion and activation depended on the nm-scale topography of a protein adlayer. Albumin adsorbed from the native conformation gave rise to different adlayer topographies and different amounts of adsorbed protein on hydrophobic and relatively hydrophilic polystyrene and silanised silicon-wafer surfaces, whereas adsorption of pre-denatured Alb resulted in similar adlayer topographies and similar amounts of adsorbed protein on these surfaces. All three distinct protein-adlayer topographies supported adhesion of in vitro differentiated, macrophage-like U937 and THP-1 cells, but did not support adhesion of their promonocytic precursors. Human monocytes freshly isolated from peripheral blood did not adhere to adsorbed albumin, not even in the presence of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1α chemokines. Adhesion of the macrophage-like cells to albumin in any of the three topographies was inhibited by antibodies against β 2 integrins, but not by antibodies against β 1 integrins, and did not induce secretion of the proinflammatory cytokine tumour necrosis factor-α.
Keywords: Albumin; Integrin; Macrophage; Monocyte; Nanotopography; Protein adsorption;

Thermosensitive polymers poly(N-isopropylacrylamide) (PNIPAM), poly(N-vinylcaprolactam) (PVCL) and PVCL grafted with amphiphilic poly(ethylene oxide) (PEO) chains (PVCL-graft-C11EO42) were prepared and characterized and their putative cytotoxicity was evaluated. The cytotoxicity of these thermosensitive polymers and their monomers was investigated as a function of polymer concentration, incubation time and incubation temperature by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) cytotoxicity tests in Caco-2 and Calu-3 cell cultures. Also, the influence of the chain end functionality on toxicity was examined. Viability (MTT) and cellular damage (LDH) of the cells were shown to be dependent on the surface properties of the polymers, hydrophilicity or hydrophobicity. Hydrophilic PVCL and PVCL-graft-C11EO42 were well tolerated at all polymer concentrations (0.1–10.0 mg/ml) after 3 h of incubation at room temperature and at physiological temperature (37 °C). The more hydrophobic PNIPAM induced more clear cellular cytotoxicity at 37 °C. The monomers N-isopropylacrylamide and vinylcaprolactam and PEO-macromonomer showed dramatically higher cytotoxicity values with respect to the corresponding polymers. Cell damage was directly dependent on concentration, temperature and incubation time.
Keywords: Cytotoxicity; Thermosensitive polymers; PVCL; PNIPAM; MTT test; LDH test;

Calvarial bone response to a tricalcium phosphate-genipin crosslinked gelatin composite by Chun-Hsu Yao; Bai-Shuan Liu; Shan-Hui Hsu; Yueh-Sheng Chen (3065-3074).
A biodegradable composite which was composed of genipin cross-linked gelatin mixing with tricalcium phosphate ceramic particles (GGT) was developed as a bone substitute. This study was evaluated by the biological response of rabbit calvarial bone to assess the potential of the GGT composite as a biodegradable and osteoconductive bone substitute. Eighteen New Zealand white rabbits were used for cranial implantation. Bone defects (15×15 mm) of nine rabbits were filled with the GGT composites, while the others were filled with the de-proteinized bovine bones as controls. Three rabbits were examined for each group in every time period at 4, 8 and 12 weeks post-surgery. The assessment included serial post-operative gross examinations, radiographic analyses and histological evaluations. This study demonstrated that this composite is: (1) malleable, with easily molded to the calvarial bone defect without fracture; (2) biocompatible, with no evidence of adverse tissue reaction; (3) osteoconductive, with progressive growth of new bone into the calvarial bone defect; (4) biodegradable, with progressive replacement of the composite by new bone. Additionally, results of both radiographic analyses and histological evaluations revealed obviously greater new bone ingrowth in the GGT composite compared with the de-proteinized bovine bone at the same implantation time. Therefore, the GGT composite could serve as a useful bone substitute for repairing bone defects.
Keywords: Genipin; Osteoconductive composite; Rabbit calvarial defect model;

Accelerated chondrocyte functions on NaOH-treated PLGA scaffolds by Grace E. Park; Megan A. Pattison; Kinam Park; Thomas J. Webster (3075-3082).
Compared to conventional poly(lactic-co-glycolic acid) (PLGA), previous studies have shown that NaOH-treated PLGA two-dimensional substrates enhanced functions of osteoblasts (bone-forming cells), vascular and bladder smooth muscle cells, and chondrocytes (cartilage-synthesizing cells). In this same spirit, the purpose of this in vitro study was to fabricate three-dimensional NaOH-treated PLGA scaffolds and determine their efficacy toward articular cartilage applications. To improve functions of chondrocytes including their adhesion, growth, differentiation, and extracellular matrix synthesis, PLGA scaffolds were modified via chemical etching techniques using 1  N NaOH for 10 min. Results demonstrated that NaOH-treated PLGA three-dimensional scaffolds enhanced chondrocyte functions compared to non-treated scaffolds. Specifically, chondrocyte numbers, total intracellular protein content, and the amount of extracellular matrix components (such as glycosaminoglycans and collagens) were significantly greater on NaOH-treated than on non-treated PLGA scaffolds. Underlying material properties that may have enhanced chondrocyte functions include a more hydrophilic surface (due to hydrolytic degradation of PLGA by NaOH), increased surface area, altered porosity (both percent and diameter of individual pores), and a greater degree of nanometer roughness. For these reasons, this study adds a novel tissue-engineering scaffold to the cartilage biomaterial community: NaOH-treated PLGA. Clearly, such modifications to PLGA may ultimately enhance the efficacy of tissue-engineering scaffolds for articular cartilage repair.
Keywords: PLGA; Articular cartilage; Chondrocyte; NaOH treatment; Scaffold;

Structural parameters of collagen nerve grafts influence peripheral nerve regeneration by Felix Stang; Hisham Fansa; Gerald Wolf; Michael Reppin; Gerburg Keilhoff (3083-3091).
Large nerve defects require nerve grafts to allow regeneration. To avoid donor nerve problems the concept of tissue engineering was introduced into nerve surgery. However, non-neuronal grafts support axonal regeneration only to a certain extent. They lack viable Schwann cells which provide neurotrophic and neurotopic factors and guide the sprouting nerve.This experimental study used the rat sciatic nerve to bridge 2 cm nerve gaps with collagen (type I/III) tubes. The tubes were different in their physical structure (hollow versus inner collagen skeleton, different inner diameters). To improve regeneration Schwann cells were implanted. After 8 weeks the regeneration process was monitored clinically, histologically and morphometrically. Autologous nerve grafts and collagen tubes without Schwann cells served as control.In all parameters autologous nerve grafts showed best regeneration. Nerve regeneration in a noteworthy quality was also seen with hollow collagen tubes and tubes with reduced lumen, both filled with Schwann cells. The inner skeleton, however, impaired nerve regeneration independent of whether Schwann cells were added or not. This indicates that not only viable Schwann cells are an imperative prerequisite but also structural parameters determine peripheral nerve regeneration.
Keywords: Collagen; Nerve regeneration; Schwann cells; Nerve tissue engineering;

Recruitment of multiple cell lines by collagen-synthetic copolymer matrices in corneal regeneration by F. Li; M. Griffith; Z. Li; S. Tanodekaew; H. Sheardown; M. Hakim; D.J. Carlsson (3093-3104).
Collagen hydrogel matrices with high optical clarity have been developed from collagen I, cross-linked with a copolymer based on N-isopropylacrylamide, acrylic acid and acryloxysuccinimide. The controlled reaction of collagen amine groups with this copolymer under neutral pH and aqueous conditions gave robust, optically clear hydrogels and prevented the excessive collagen fibrillogenesis that can lead to collagen opacity. These sterile, non-cytotoxic hydrogels allowed epithelial cell overgrowth and both stromal cell and nerve neurite ingrowth from the host tissue. This regenerative ability appeared to result from the high glucose permeability, nanoporosity and the presence of cell adhesion factors, RGD in collagen and the laminin pentapeptide, YIGSR, grafted onto the copolymer. Under physiological conditions, optical clarity superior to the human cornea and tensile performance adequate for suturing were obtained from some formulations.
Keywords: Collagen-copolymer hydrogel; Cell recruitment; Permeability; Porosity; Topography; Optical clarity;

Poly(N-isopropylacrylamide)-grafted gelatin (PNIPAM-gelatin) serves as a temperature-induced scaffold at physiological temperature. This study was aimed at determining the effect of the graft architecture of thermoresponsive PNIPAM-gelatin on the surface topography and elastic modulus of the hydrogels prepared with different architectured PNIPAM-gelatins: the surface topography and elastic modulus were determined by atomic force microscopy (AFM). PNIPAM-gelatin surfaces showed an irregularly concavo-convex structure with a vertical interval of approximately 1 μm regardless of the weight ratio of PNIPAM to gelatin (P/G: 5.8, 12, and 18). The elastic moduli of hydrogels varied at measured sites. The mean elastic moduli of PNIPAM-gelatin with the lowest P/G were low, but increased with increasing P/G. Human umbilical vein endothelial cells adhered and spread on PNIPAM-gelatin hydrogels with the highest P/G, whereas reduced adhesion and nonspreading, round-shaped cells resided on the hydrogels with lower P/Gs. Interrelationship between elastic modulus and cell adhesion and spreading potentials were discussed from physicochemical and cellular biomechanical viewpoints.
Keywords: Poly(N-isopropylacrylamide)-grafted gelatin; Atomic force microscopy; Microscopic structure; Mechanical property; Cell adhesiveness;

Fibrin as a cell carrier in cardiovascular tissue engineering applications by Anita Mol; Marjolein I. van Lieshout; Christa G. Dam-de Veen; Stefan Neuenschwander; Simon P. Hoerstrup; Frank P.T. Baaijens; Carlijn V.C. Bouten (3113-3121).
In cardiovascular tissue engineering approaches, efficient seeding methods are essential. To achieve this and to save time, cells can be encapsulated in gels. Combining the advantages of a gel as a cell carrier with the advantages of a fiber-based scaffold, providing structural integrity to the developing tissue, might offer several advantages. In this study, seeding by using fibrin as a cell carrier is compared to the conventional static seeding method with regard to tissue development. Seeding with fibrin resulted in less loss of soluble collagen into the medium and a more mature extracellular matrix in a shorter period of time. The use of fibrin degradation inhibitors was shown to inhibit extracellular matrix formation, although it did not hamper cell proliferation. The use of fibrin as a cell carrier to seed cells into a fiber-based scaffold may represent a promising, timesaving approach in cardiovascular tissue engineering applications.
Keywords: Scaffold; Non-woven fabric; Fibrin; Cell encapsulation; ECM;

Polydimethylsiloxane (PDMS) is used extensively to study cell–substrate interactions because its mechanical properties are easily tuned in physiologically relevant ranges. However, changes in mechanical properties also modulate surface chemistry and cell response. Here, we correlate the mechanical and surface properties of PDMS to vascular smooth muscle cell (VSMC) behavior. We find that a 5-fold increase in base:crosslinker ratio leads to ∼40-fold decrease in elastic modulus but no significant differences in surface wettability. However, when polyelectrolyte multilayers are adsorbed to promote cell adhesion, wettability varies inversely with substrate stiffness. Despite these differences in hydrophobicity, the amount of adsorbed protein remains the same. In the absence of serum, there is a 39% decrease in cell attachment and a 42% decrease in spreading as the elastic modulus decreases from 1.79 to 0.05 MPa. In the presence of serum or adsorbed fibronectin, the differences in attachment and spreading are diminished. This is not the case for the rate of serum-stimulated cell proliferation, which remains inversely dependent on crosslinker concentration. We conclude that for the range of crosslinker concentrations investigated, the surface properties dominate the initial cell attachment and spreading, whereas the mechanical properties influence the long-term cell growth.
Keywords: Polydimethylsiloxane; Elasticity; Wettability; Cell adhesion; Cell proliferation;

Microgrooved fibrillar collagen membranes as scaffolds for cell support and alignment by Robert B. Vernon; Michel D. Gooden; Stephanie L. Lara; Thomas N. Wight (3131-3140).
For several years, microgrooved substrates have been evaluated as a means to orient cells in engineered tissues. Recently, we fabricated thin (0.1–5.3 μm) planar and tubular collagen membranes (CMs) from air-dried hydrogels of native, fibrillar type I collagen (Vernon et al., Biomaterials 2004;26:1109–17). The CMs were strong, stable, and permeable and, hence, of potential use as scaffolds for tissue engineering. In the present study, planar CMs supported a robust attachment, spreading, and proliferation of human dermal fibroblasts (HDFs) and human umbilical artery smooth muscle cells (HUASMCs). Collagen hydrogels were air-dried onto microgrooved templates and subsequently removed in the form of grooved CMs with the potential to align cells. The grooved CMs were highly effective at inducing HDFs and HUASMCs to elongate and align, as revealed by scanning electron microscopy and by assays of f-actin and nuclear orientation. Alignment of cells was maintained at high cell densities. CMs with grooves of substantially different widths and depths were similarly effective in causing cell alignment; however, cells aligned poorly on CMs that had grooves less than 1 μm in depth. Grooved CMs with the capability to align cells might be of considerable use in the fabrication of tissue substitutes.
Keywords: Cell culture; Collagen; Fibroblast; Hydrogel; Membrane; Microgroove; Micropatterning; Scaffold; Smooth muscle cell; Surface topography;

Cell adhesion to synthetic biomaterials is a prerequisite for anchorage cell culture and tissue engineering. The current study investigated utilization of an avidin–biotin binding system in enhancing chondrocyte adhesion to tissue culture polystyrene (TCPS). Biotinylated chondrocytes adhered to avidin-coated TCPS more quickly than untreated chondrocytes to bare TCPS. Also the avidin–biotin binding system enhanced cell initial spreading. However, the effects were only transient. The growth of biotinylated chondrocytes was first decreased during the first 3 days but increased afterwards. The progeny of biotinylated chondrocytes still maintained the ability in expressing cartilage extracellular matrix proteins such as type II collagen, type IX collagen and aggrecan. These results show potential for the application of the avidin–biotin binding system to cell culture and tissue engineering.
Keywords: Biotin; Avidin; Chondrocytes; Cell adhesion; Cell proliferation; Gene expression;

Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices by Susanne Ng; Ying-Nan Wu; Yi Zhou; Yi-Er Toh; Zi-Zong Ho; Ser-Mien Chia; Jian-Hang Zhu; Hai-Quan Mao; Hanry Yu (3153-3163).
Hepatocytes are anchorage-dependent cells sensitive to microenvironment; the control of the physicochemical properties of the extra-cellular matrices may be useful to the maintenance of hepatocyte functions in vitro for various applications. In a microcapsule-based 3-D hepatocyte culture microenvironment, we could control the physical properties of the collagen nano-fibres by fine-tuning the complex-coacervation reaction between methylated collagen and terpolymer of hydroxylethyl methacrylate–methyl methacrylate–methylacrylic acid. The physical properties of the nano-fibres were quantitatively characterized using back-scattering confocal microscopy to help optimize the physical support for hepatocyte functions. We further enhanced the chemical properties of the collagen nano-fibres by incorporating galactose onto collagen, which can specifically interact with the asialoglycoprotein receptor on hepatocytes. By correlating a range of collagen nano-fibres of different physicochemical properties with hepatocyte functions, we have identified a specific combination of methylated and galactosylated collagen nano-fibres optimal for maintaining hepatocyte functions in vitro. A model of how the physical and chemical supports interplay to maintain hepatocyte functions is discussed.
Keywords: Hepatocyte; ECM (extra-cellular matrix); Collagen; Cell culture; Confocal microscopy;

Electrospun dual-porosity structure and biodegradation morphology of Montmorillonite reinforced PLLA nanocomposite scaffolds by Yun Hui Lee; Jong Hoon Lee; In-Gu An; Chan Kim; Doo Sung Lee; Young Kwan Lee; Jae-Do Nam (3165-3172).
Combining a nanocomposite technique and the electrospinning process, a robust dual-porosity scaffold structure was developed for a facile transport of metabolic nutrients and wastes through the nano-sized pores and for the cell implantation and blood vessel invasion through the micro-sized pores. The montmorillonite (MMT) nano-sized platelets were incorporated into poly(L-lactic acid) (PLLA) solution, which was subsequently electrospun and mechanically entangled by a cold compression molding process for a robust 3-dimensional scaffold structure. Using a salt leaching/gas forming method, micro-sized pores were developed in the electrospun fiber bundles giving a dual-porosity scaffold structure. Compared with the pristine PLLA scaffold, the developed nanocomposite fibrous scaffold structure exhibited increased strength and improved structural integrity during the biodegradation process. The nanocomposite scaffold systems also exhibited many tiny pinholes desirably generated on the scaffold walls without serious fragmentation during biodegradation reactions.
Keywords: Nanocomposite; Scaffold; Polylactic acid; Biodegradation;

In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering by Joshua R. Mauney; Claude Jaquiéry; Vladimir Volloch; Michael Heberer; Ivan Martin; David L. Kaplan (3173-3185).
Mineralized and partially or fully demineralized biomaterials derived from bovine bone matrix were evaluated for their ability to support human bone marrow stromal cell (BMSC) osteogenic differentiation in vitro and bone-forming capacity in vivo in order to assess their potential use in clinical tissue-engineering strategies. BMSCs were either seeded on bone-derived scaffolds and cocultured in direct cell-to-scaffold contact, allowing for the exposure of soluble and insoluble matrix-incorporated factors, or cocultured with the scaffold preparations in a transwell system, exposing them to soluble matrix-incorporated factors alone. Osteoblast-related markers, alkaline phosphatase (ALP) activity and bone sialoprotein (BSP) and osteopontin (OP) mRNA expression were evaluated in BMSCs following 14 days of cocultivation in both systems. The data demonstrate that BMSCs from some donors express significantly higher levels of all osteoblast-related markers following cocultivation in direct cell-to-scaffold contact with mineralized scaffolds in comparison to fully demineralized preparations, while BMSCs from other donors display no significant differences in response to various scaffold preparations. In contrast, BMSCs cocultured independently with soluble matrix-incorporated factors derived from each scaffold preparation displayed significantly lower levels of ALP activity and BSP mRNA expression in comparison to untreated controls, while no significant differences were observed in marker levels between cells cocultured similarly with different biomaterial preparations. In addition, BMSCs were seeded directly on mineralized and partially or fully demineralized biomaterials and implanted in subcutaneous sites of athymic mice for 8 weeks to evaluate their in vivo bone-forming capacity. The ex vivo incorporation of BMSCs into all bone-derived scaffold preparations substantially increased the mean extent and frequency of samples containing de novo bone formation over similar nonseeded controls, as determined by histological and histomorphometrical analysis. No statistically significant differences were observed in the extent or frequency of bone formation between various scaffold preparations seeded with BMSCs from different donors. These results demonstrate that the in vivo osteoinductivity of bone-derived scaffolds can be modulated by ex vivo incorporated BMSCs and the extent of scaffold demineralization plays a significant role in influencing in vitro osteogenic differentiation of BMSCs depending on the coculture system and BMSC donor.
Keywords: Demineralization; Bone tissue engineering; Mesenchymal stem cell;

Fabrication of degradable polymer scaffolds to direct the integration and differentiation of retinal progenitors by E.B. Lavik; H. Klassen; K. Warfvinge; R. Langer; M.J. Young (3187-3196).
Retinal progenitor cells (RPCs) are self-renewing cells capable of differentiating into the different retinal cell types including photoreceptors, and they have shown promise as a source of replacement cells in experimental models of retinal degeneration. We hypothesized that a biodegradable polymer scaffold could deliver these cells to the subretinal space in a more organized manner than bolus injections, while also providing the graft with laminar organization and structural guidance channels. We fabricated highly porous scaffolds from blends of poly(l-lactic acid) and poly(lactic-co-glycolic acid) using a variety of techniques to produce pores oriented normal to the plane of the scaffold. RPCs were seeded on the polymer scaffolds and cultured for 14 days. Seeded scaffolds were then either fixed for characterization or used in an explant or in vivo rat model. The scaffolds were fully covered by RPCs in 3 days. Attachment of RPCs to the polymer scaffold was associated with down-regulation of immature markers and up-regulation of markers of differentiation. This suggests that the scaffold may promote differentiation of RPCs. The seeded cells elaborated cellular processes and aligned in the scaffold in conjunction with degenerating retinal explants. The cells also exhibited morphologies consistent with photoreceptors including a high degree of polarization of the cells. This data suggests that the scaffold may be a means to assist in the promotion of photoreceptor phenotypes. Implantation of the seeded scaffold into the rat eye is associated with increased RPC survival. Taken together, these data suggest that these polymer scaffolds provide a useful means for delivering RPCs to the subretinal space and may assist in the formation of retinal cell phenotypes, although it is clear that more cues are needed to direct the differentiation of RPCs into functional photoreceptors.
Keywords: Polymer; Scaffold; PLGA; Retina; Retinal progenitor cells;

Preparation and characterization of RGD-immobilized chitosan scaffolds by Ming-Hua Ho; Da-Ming Wang; Hsyue-Jen Hsieh; Hwa-Chang Liu; Tzu-Yang Hsien; Juin-Yih Lai; Lein-Tuan Hou (3197-3206).
Chitosan scaffolds were modified with RGDS (Arg–Gly–Asp–Ser) in the present work via an imide-bond forming reaction between amino groups in chitosan and carboxyl groups in peptides. Successful immobilization was verified with FTIR spectroscopy, and the immobilized amount was determined to be on the order of 10−12  mol/cm2 through analysis of the immobilized amino acids. Results of experiments of cell culture with rat osteosarcoma (ROS) cells demonstrated that RGDS immobilization could enhance the attachment of ROS cells onto the chitosan, resulting in higher cell density attached to the RGDS-modified scaffold than to the unmodified scaffold. It should be noted that only RGDS, but not other peptide such as RGES, is effective in enhancing cell attachment and possible proliferation. Experiments of in vitro mineralization indicated that there were more cells on the RGDS-modified scaffold than on the unmodified scaffold, which tended to form bone-like tissues. The results presented in this work suggest that immobilization of RGDS can make chitosan scaffolds more compatible for the culture of osteoblast-like cells and the regeneration of bone-like tissues.
Keywords: Chitosan; Scaffold; RGD; Tissue engineering; Bone regeneration; Biocompatibility;

Conformational analysis of heparin binding peptides by Manuela Vacatello; Gabriella D’Auria; Lucia Falcigno; Monica Dettin; Roberta Gambaretto; Carlo Di Bello; Livio Paolillo (3207-3214).
A properly engineered biomaterial for dental/orthopaedic applications must induce specific responses from the osteoblasts at the implant site. A most desirable response is an efficient adhesion, as it represents the first phase in the cell/material interaction and the quality of this phase will influence the cell's capacity to organize into a new functional tissue. The four osteoblast-adhesive peptides discussed in this paper are mapped on the 339–364 sequence (339MAPRPSLAKKQRFRHRNRKGYRSQRG364) located in the primary heparin-binding site of human vitronectin (HVP). Adsorbed on a polystyrene scaffold, these peptides display different adhesive activities towards osteoblasts. In this paper we report on the structural analysis in solution of the peptides through NMR and computational techniques. We find that the peptides with the highest adhesive activities display a hydrophobic patch opposite to the charged surface candidate to interact with heparin. These findings suggest that the peptides might adsorb on the polystyrene support in a favourable orientation for their activity. Furthermore, molecular models obtained for the four peptides in solution were used in rigid docking simulations with a heparin model. Assuming that the peptide solution conformations are not very different from the polystyrene-adsorbed structures, the simulations reveal that peptide adhesive activity is also affected by the number of ionic interactions and spacing between charged residues.

In vitro degradation of porous poly(propylene fumarate)/poly(dl-lactic-co-glycolic acid) composite scaffolds by Elizabeth L. Hedberg; Charles K. Shih; Jeremy J. Lemoine; Mark D. Timmer; Michael A. K. Liebschner; John A. Jansen; Antonios G. Mikos (3215-3225).
This study investigated the in vitro degradation of porous poly(propylene fumarate) (PPF-based) composites incorporating microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) during a 26-week period in pH 7.4 phosphate-buffered saline at 37 °C. Using a fractional factorial design, four formulations of composite scaffolds were fabricated with varying PEG content of the microparticles, microparticle mass fraction of the composite material, and initial leachable porogen content of the scaffold formulations. PPF scaffolds without microparticles were fabricated with varying leachable porogen content for use as controls. The effects of including PLGA/PEG microparticles in PPF scaffolds and the influence of alterations in the composite formulation on scaffold mass, geometry, water absorption, mechanical properties and porosity were examined for cylindrical specimens with lengths of 13 mm and diameters of 6.5 mm. The composite scaffold composition affected the extent of loss of polymer mass, scaffold length, and diameter, with the greatest loss of polymer mass equal to 15±5% over 26 weeks. No formulation, however, exhibited any variation in compressive modulus or peak compressive strength over time. Additionally, sample porosity, as determined by both mercury porosimetry and micro-computed tomography did not change during the period of this study. These results demonstrate that microparticle carriers can be incorporated into PPF scaffolds for localized delivery of bioactive molecules without altering scaffold mechanical or structural properties up to 26 weeks in vitro.
Keywords: Composite polymer; Injectable scaffold; Degradation; Bone-tissue engineering;

Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration by Solitaire A. DeLong; James J. Moon; Jennifer L. West (3227-3234).
Basic fibroblast growth factor (bFGF) was immobilized to hydrogel scaffolds with retention of mitogenic and chemotactic activity. The bFGF was functionalized in order to incorporate it covalently within polyethylene glycol (PEG) hydrogel scaffolds by reaction with acryloyl-PEG-NHS. Hydrogels were formed by exposing aqueous solutions of PEG diacrylate, acryloyl-PEG-RGDS, and acryloyl-PEG-bFGF to long-wavelength ultraviolet light in the presence of a photoinitiator. These bFGF-modified hydrogels with RGD adhesion sites were evaluated for their effect on vascular smooth muscle cell (SMC) behavior, increasing SMC proliferation by ∼41% and migration by ∼15%. A covalently immobilized bFGF gradient was formed using a gradient maker to pour the hydrogel precursor solutions and then photopolymerizing to lock in the concentration gradient. Silver staining was used to detect the bFGF gradient, which increased linearly along the hydrogel's length. Cells were observed to align on hydrogels modified with a bFGF gradient in the direction of increasing tethered bFGF concentration as early as 24 h after seeding. SMCs also migrated differentially, up the concentration gradient, on bFGF-gradient hydrogels compared to control hydrogels with and without a constant bFGF concentration. These hydrogel scaffolds may be useful for studying protein gradient effects on cell behavior and for directing cell migration in tissue-engineering applications.
Keywords: Tissue engineering; Hydrogel; Basic fibroblast growth factor; Gradient; Migration; Smooth muscle cell;

Residual stresses in bilayer dental ceramics by Burak Taskonak; John J. Mecholsky; Kenneth J. Anusavice (3235-3241).
It is clinically observed that lithia-disilicate-based all-ceramic fixed partial dentures (FPD) can fail because of the fragmentation of the veneering material. The hypothesis of this study is that the global residual stresses within the surface of those veneered FPDs may be responsible for partial fragmentation of the veneering ceramic. Bilayer and monolithic ceramic composites were prepared using a lithia disilicate based (Li2O⋅SiO2) glass–ceramic core and a glass veneer. A four-step fracture mechanics approach was used to analyze residual stress in bilayered all-ceramic FPDs. We found a statistically significant increase in the mean flexural strengths of bilayer specimens compared with monolithic glass specimens ( p ⩽ 0.05 ). There was a statistically significant difference between the mean longitudinal and transverse indentation-induced crack sizes in bilayer specimens ( p ⩽ 0.05 ), which indicates the existence of residual stress. Global residual stresses in the veneer layer, calculated using a fracture mechanics equation, were determined to be responsible for the increased strength and observed chipping, i.e., spallation in bilayer ceramic composites.
Keywords: Residual stress; Indentation; Fracture mechanics; Dental ceramics; Fractography;

A two year in vivo study of polyvinyl alcohol-hydrogel (PVA-H) artificial meniscus by Masanori Kobayashi; Yong-Shun Chang; Masanori Oka (3243-3248).
For the recognized importance of knee meniscus function, the treatment of meniscus injury has been changing from resection to repair. However, depending on the type of injury, meniscectomy sometimes cannot be avoided. In such a case, it is important to anticipate the future problem of degenerative change or osteoarthrosis in the knee joint. In consideration of the prognosis and circumstances in such patients, we have developed an artificial meniscus using polyvinyl alcohol-hydrogel (PVA-H) for salvage.We have already reported the results up to 1 year after animal operation. The present study investigated the results in postoperative 2.0 years to assess further the use of artificial meniscus.In the results, the articular cartilage state of knee joint implanted PVA-H meniscus was good even after 2 years, while Osteoarthrosis (OA) change progressed in meniscectomy knee joint. In addition, neither wear, dislocation nor breakage of PVA-H was observed.These results proved that an artificial meniscus using PVA-H can compensate for meniscal function and might be clinically applicable.
Keywords: Polyvinylalcohol; Arthritis; Knee replacement; In vivo test;

Biomechanical and histomorphometric study on the bone–screw interface of bioactive ceramic-coated titanium screws by Jae Hyup Lee; Hyun-Seung Ryu; Dong-Soo Lee; Kug Sun Hong; Bong-Soon Chang; Choon-Ki Lee (3249-3257).
The purpose of this study was to compare the osseointegration of 4 different kinds of bioactive ceramic-coated screws with uncoated screws by biomechanical and histomorphometric analysis.Calcium pyrophosphate (CPP), apatite-wollastonite 1:3 glass ceramic (W3G), apatite-wollastonite 1:1 glass ceramic (WAG) and bioactive CaO–SiO2–B2O3 glass ceramic (CSG) coatings were prepared and coated by the dipping method. Coated and uncoated titanium screws were inserted into the tibia of 18 adult mongrel male dogs for 2, 4, and 8 weeks. The insertion torques, radiographs, undecalcified histology, histomorphometric analysis, and extraction torques were evaluated.No difference of insertion torque was found among the five screw types. At 2 and 4 weeks after implantation, the extraction torque of ceramic-coated screws was significantly higher than that of uncoated screws (p⩽0.0001). At 8 weeks, the extraction torques of CPP-, W3G-, and WAG-coated screws were significantly higher than those of CSG-coated and uncoated screws (p<0.0001).At 2, 4, and 8 weeks, the extraction torques of 4 different ceramic-coated screws were significantly higher than the corresponding insertion torque. Strong fixation was observed even at 2 weeks in the CPP-, W3G- and WAG-coated screws.The bone–screw contacts of the 4 different coated screws at 2 and 4 weeks were statistically higher than that of the uncoated screws, and the bone–screw contacts of the CPP-, W3G- and WAG-coated screws at 8 weeks were also statistically higher than that of the uncoated screws.The fixation strength was increased by the presence of osteoconductive coating materials, such as CPP, W3G, and WAG, which enabled to achieve higher fixation strength even as early as 2–8 weeks after the insertion.
Keywords: Bioactive ceramic; Coating; Titanium screw; Osseointegration; Biomechanical; Histomorphometric analysis;

Wear behaviour of cross-linked polyethylene assessed in vitro under severe conditions by Saverio Affatato; Gianluca Bersaglia; Mirko Rocchi; Paola Taddei; Concezio Fagnano; Aldo Toni (3259-3267).
The polyethylene (PE) for hip implants presents serious clinical problems; the production of debris may induce adverse tissue reactions that may lead to extensive bone loss around the implant and consequently osteolysis and implant loosening. Several attempts have been made to improve the wear properties of ultra-high molecular weight polyethylene (UHMWPE). More recently the attention of various researchers has been focused on cross-linked polyethylene (XLPE), due to its improved wear resistance with respect to conventional UHMWPE.This study was aimed at comparing the wear performances of clinically available acetabular liners (Zimmer Inc.) made of electron beam XLPE and conventional UHMWPE. To evaluate the influence of the material properties on wear, conventional UHMWPE and XLPE acetabular cups were tested against deliberately scratched CoCrMo femoral heads (R a=0.12–0.14 μm) in a hip joint wear simulator run for 3 million cycles with bovine calf serum as lubricant.Gravimetric measurements revealed significant differences between the wear behaviours of the two sets of acetabular cups: XLPE exhibited a wear rate about 40 times lower than conventional UHMWPE. Raman spectroscopy coupled to partial least-squares analysis was used to evaluate the possible crystallinity changes induced by mechanical stress (and thus the material wear resistance): only the UHMWPE cup which showed the highest weight loss displayed significant crystallinity changes. These results were correlated to the thickness of the plasticity-induced damage layer.The wear debris produced during the tests were isolated according to a validated protocol and imaged by scanning electron microscopy . The wear particles produced by XLPE were smaller than those produced by UHMWPE; the latter were observed as fibrillar and agglomerated particles. The mean equivalent circle diameter was 0.71 and 0.26 μm for UHMWPE and XLPE, respectively.
Keywords: Conventional ultra-high molecular weight polyethylene; Cross-linked polyethylene; Roughened CoCr femoral heads; Hip joint simulator; Wear debris characterisation; Micro-Raman spectroscopy;

Involvement of protein kinase C in chitosan glutamate-mediated tight junction disruption by Jennifer M. Smith; Michael Dornish; Edward J. Wood (3269-3276).
Chitosan has been successfully used as an excipient for trans-epithelial drug delivery systems. It is known to transiently open intercellular tight junctions thus increasing the permeability of an epithelium. In order to investigate the possible role of protein kinases in trans-epithelial delivery, changes in trans-epithelial electrical resistance (‘TEER’) of epithelial (Caco-2) cell monolayers were assessed in response to chitosan glutamate treatment, in the presence and absence of specific protein kinase inhibitors. Changes in subcellular localisation of the tight junction protein ZO-1 observed by immunofluorescence and western blotting of cellular fractions were also assessed. Inhibition of protein kinase C (PKC), but not mitogen activated protein kinase (MAPK) was found to prevent the chitosan-mediated decrease in TEER, and changes in localisation of ZO-1. In order to determine which PKC isozymes were responsible for the chitosan-mediated tight junction disruption, the activation of the PKC isozymes α, β and δ was investigated. A chitosan-mediated translocation of PKC α but not PKC β or δ from the cytosol to the membrane fraction, indicative of PKC α activation was observed. Thus, treatment of Caco-2 cells with chitosan may result in the activation of PKC-dependent signal transduction pathways which affect tight junction integrity.
Keywords: Chitosan; Cell adhesion; Cell signalling; Drug delivery; Epithelium;

The interaction of chitosan with fibroblast growth factor-2 and its protection from inactivation by Kazunori Masuoka; Masayuki Ishihara; Takashi Asazuma; Hidemi Hattori; Takemi Matsui; Bonpei Takase; Yasuhiro Kanatani; Masanori Fujita; Yoshio Saito; Hirofumi Yura; Kyosuke Fujikawa; Koichi Nemoto (3277-3284).
Application of ultraviolet light (UV) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution including fibroblast growth factor-2 (FGF-2) results within 30 s in an insoluble, flexible hydrogel. The retained FGF-2 molecules in the chitosan hydrogel remain biologically active, and are released from the chitosan hydrogel upon the in vivo biodegradation of the hydrogel. In view of these findings, we here tested the interaction of chitosan with FGF-2, thereby modifying and stabilizing the FGF-2 activity from inactivations. The photocrosslinkable chitosan hydrogel has a low affinity for FGF-2 (Kd=6.12×10−7m). Soluble chitosan (CH-LA; Az-CH-LA without photocrosslinkable azide group) substantially prolonged the biological half-life time of FGF-2. Furthermore, CH-LA could protect the FGF-2 activity from inactivation, such as heat, proteolysis, and acid. The effect of chitosan on the FGF-2 activity is of a protective nature, since it had no effect of modifying the FGF-2 activity directly on growth of human umbilical vein endothelial cells (data not shown). Thus, one of the ways by which the chitosan potentiated the FGF-2 activity could be through protecting it from inactivations by the interaction between FGF-2 and chitosan molecules.
Keywords: Chitosan; Fibroblast growth factor-2 (FGF-2); Stabilization; Complex formation;

Anti-fouling hydrogel coatings, copolymers of 2-hydroxyethyl methacrylate, 1-vinyl-2-pyrrolidinone, and polyethylene glycol, were investigated for the purpose of improving biosensor biocompatibility. These coatings were modified to incorporate poly(lactide-co-glycolide) (PLGA) microspheres in order to release dexamethasone (DX) and/or vascular endothelial growth factor (VEGF). DX and VEGF release kinetics from microspheres, hydrogels, and microspheres embedded in hydrogels were determined in 2-week and 1-month studies. Overall, monolithic, non-degradable hydrogel drug release had an initial burst followed by release at a significantly lower amount. Microsphere drug release kinetics exhibited an initial burst followed by sustained release for 1 month. Embedding microspheres in hydrogels resulted in attenuated drug delivery. VEGF release from embedded microspheres, 1.1±0.3 ng, was negligible compared to release from hydrogels, 197±33 ng. After the initial burst from DX-loaded hydrogels, DX release from embedded microspheres was similar to that of hydrogels. The total DX release from hydrogels, 155±35 μg, was greater than that of embedded microspheres, 60±6 μg. From this study, hydrogel sensor coatings should be prepared incorporating VEGF in the hydrogel and DX either in the hydrogel or in DX microspheres embedded in the hydrogel.
Keywords: Hydrogel; Microsphere; Controlled drug release; Biocompatibility;

Fabrication and characterization of a smart drug delivery system: microsphere in hydrogel by Xian-Zheng Zhang; Patti Jo Lewis; Chih-Chang Chu (3299-3309).
In this contribution, a novel smart drug delivery system (DDS) consisting of hydroxyl-functionalized glycerol poly(ε-caprolactone) (PGCL)-based microspheres and poly(N-isopropylacrylamide) (PNIPAAm) hydrogel was developed for prolonged and sustained controlled drug release. Various amounts PGCL-based microspheres were incorporated physically into temperature sensitive poly(N-isopropylacrylamide) (PNIPAAm) hydrogel to form the novel DDSs. Resulting DDSs were characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and compression modulus measurements to investigate the morphological, thermal, and mechanical properties. The temperature dependence of swelling ratio and response kinetics upon heating or cooling were also investigated to understand the smart properties, i.e., temperature sensitive properties of these DDSs. Finally, ovalbumin (OVA), used as the model drug, was loaded into PGCL-based microspheres to examine and compare the effects of controlled release at different temperature (22 and 37 °C) of these novel smart DDSs.
Keywords: Drug delivery system; PNIPAAm hydrogel; PGCL-based microsphere; Temperature sensitive;

Self-curing controlled release systems for steroids. Application of prednisolone-based polymeric systems to ear diseases by Mar Fernandez; Juan Parra; Blanca Vazquez; Antonio Lopez-Bravo; Julio San Román (3311-3318).
An injectable delivery system for prednisolone has been prepared based on a self-curing formulation comprised of poly(methyl methacrylate) particles and hydroxyethyl methacrylate as monomer. The polymerisation reaction was initiated by the redox system 4,4′-bis (dimethylaminobenzydrol)/benzoyl peroxide (BZN/BPO) and followed at 25 °C by measuring the time–temperature profile. A maximum temperature of 53 °C and a setting time of 15 min were obtained, calculated according to standard specifications. The swelling of the cured system was studied in phosphate-buffered saline (PBS) at 37 °C giving a hydration degree at equilibrium of 20%. The swelling kinetics fitted a fickian behaviour at the initial stages of the experiments, with a diffusion coefficient of 0.72×10−7  cm2/s. The release of the drug was sustained from the beginning without an initial drug burst. The study of the wettability showed a rather hydrophilic character of the surface of the loaded system, and the biocompatibility evaluated through MTT assay revealed the absence of cytotoxicity due to the release of toxic substances.
Keywords: Drug delivery; PMMA/PHEMA; Prednisolone; Ear disease;

Controlled release of plasmid DNA from photo-cross-linked pluronic hydrogels by Ki Woo Chun; Jun Bae Lee; Sun Hwa Kim; Tae Gwan Park (3319-3326).
Chemically cross-linked hydrogels composed of PluronicTM, water-soluble tri-block copolymers of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide), were synthesized by a photo-polymerization method to achieve controlled DNA release. Pluronic F127 was di-acrylated to form a macromer and cross-linked to form a hydrogel structure in the presence and absence of vinyl group-modified hyaluronic acid (HA). UV irradiation time and the presence of the vinyl group-modified HA affected the mechanical property of Pluronic hydrogels to a great extent. Swelling ratio, degradation, and rheological behaviors of Pluronic hydrogels were investigated. When plasmid DNA was loaded in the hydrogels for sustained delivery, various release profiles were attained by varying UV irradiation time and modified HA amounts. Entrapped DNA was gradually damaged with increasing the UV exposure time as evidenced by decreasing the transfection efficiency. The DNA fractions released from the HA/Pluronic hydrogels, however, exhibited considerable transfection efficiencies commensurate with the UV exposure time, suggesting that they were not chemically degraded during the release period and substantially maintained functional gene expression activities despite the UV irradiation.
Keywords: Hydrogels; Photo-polymerization; Pluronic; Sustained gene delivery;

Size control of calcium alginate beads containing living cells using micro-nozzle array by Shinji Sugiura; Tatsuya Oda; Yasuhiko Izumida; Yasuyuki Aoyagi; Mitsuo Satake; Atsushi Ochiai; Nobuhiro Ohkohchi; Mitsutoshi Nakajima (3327-3331).
Size-controlled small (i.e. less than 300 μm) polyelectrolyte complex gel beads are urgently desired for wide-spread application, including use in medical, pharmaceutical, and bioengineering fields. However, it was impossible to obtain smaller beads less than 300 μm with conventional apparatuses. We developed a novel microfluidics device that utilizes silicon micro-nozzle (MN) array, enabling to produce 50–200 μm calcium alginate beads with a narrow size distribution. Alginate aqueous solution was extruded through a precisely fabricated thin (30 μm×30 μm) and short (500 μm) MN and was sheared by the viscous drag force of oil flow to form alginate droplets. Alginate droplets were immediately reacted with CaCl2 droplets at the downstream of oil flow to form calcium alginate gel beads. This device enabled us to successfully encapsulate living cells into 162 μm calcium alginate beads with maintaining viability, which was confirmed by the expression of marker protein.
Keywords: Alginate; Cell encapsulation; Microcapsule; Micromachining; Microstructure; Size control;

Atomic force microscopy (AFM) combined with nanoindentation technique was used to definitely, site-specifically, test the nanomechanical properties, including nanohardness and elastic modulus, of the isolated domains within single enamel, the prisms and the surrounding sheaths, of mature human maxillary third molars. In this way, it is for the first time that evident differences of nanomechanical properties were revealed between these domains. The nanohardness and elastic modulus of the sheaths were about 73.6% and 52.7% lower than those of the prisms, respectively. Measuring the residual impressions with AFM supported the similar conclusion. The variations of mechanical properties in these domains are considered to be mainly relative to their different component and fibrils arrangement.
Keywords: Nanohardness; Elastic modulus; Nanoindentation; Enamel prisms;

The effect of functionalized self-assembling peptide scaffolds on human aortic endothelial cell function by Elsa Genové; Colette Shen; Shuguang Zhang; Carlos E. Semino (3341-3351).
A class of designed self-assembling peptide nanofiber scaffolds with more than 99% water content has been shown to be a good biological material for cell culture. Here, we report the functionalization of one of these peptide scaffolds, RAD16-I (AcN–RADARADARADARADA–CONH2), by direct solid phase synthesis extension at the amino terminal with three short-sequence motifs. These motifs are present in two major protein components of the basement membrane, laminin 1 (YIGSR, RYVVLPR) and collagen IV (TAGSCLRKFSTM). These motifs have been previously shown to promote specific biological activities including endothelial cell adhesion, spreading, and tubular formation. Therefore, the generic functionalized peptide developed was AcN–X–GG-RADARADARADARADA–CONH2 with each motif represented by “X”. We show in this work that these tailor-made peptide scaffolds enhance the formation of confluent cell monolayers of human aortic endothelial cells (HAEC) in culture. Moreover, additional assays designed to evaluate endothelial cell function showed that HAEC monolayers obtained on these scaffolds not only maintained LDL uptake activity but also enhanced nitric oxide release and elevated laminin 1 and collagen IV deposition. These results suggest that this new scaffold provide a better physiological substrate for endothelial cell culture and suggest its further application for biomedical research, cancer biology and regenerative biology.
Keywords: Self-assembly; Biomimetic material; Cell proliferation; Extracellular matrix; Endotheliar monolayer;

Natural polyelectrolyte films based on layer-by layer deposition of collagen and hyaluronic acid by Jun Zhang; Bernard Senger; Dominique Vautier; Catherine Picart; Pierre Schaaf; Jean-Claude Voegel; Philippe Lavalle (3353-3361).
The aim of the present work was to assemble extracellular matrix components into polyelectrolyte multilayers using the layer-by-layer deposition method. The films are constructed with type-I collagen and hyaluronic acid. The construction exhibits the general features observed during polyelectrolyte multilayer buildup: alternate positive and negative values of the zeta potential of the film during its construction and regular increase of the film thickness with the number, n, of deposition step. This increase is shown to be linear with n. As expected for a linearly growing film, the confocal microscopy shows that when the film is brought in contact with a collagen solution, collagen does not diffuse into the film but interacts only with its outer layer. However, the films are not constituted of homogeneously distributed polyanion/polycation complexes as it is usually observed, but they are formed of fibers as imaged by AFM. The typical width of these fibers increases with the number of deposition steps. Finally, it is found that chondrosarcoma cells spread well and synthesize extracellular matrix components only on the collagen ending films, whereas no cellular matrix was found for HA ending ones. Such architectures may be further functionalized by inclusion of active drugs, peptides, proteins…, and could be used as tunable biomaterial interfaces.
Keywords: Surface treatment; Extracellular matrix (ECM); Collagen; Multilayered films; Layer-by-layer assembly; Biomimetic material;

Age-related transparent root dentin: mineral concentration, crystallite size, and mechanical properties by J.H. Kinney; R.K. Nalla; J.A. Pople; T.M. Breunig; R.O. Ritchie (3363-3376).
Many fractures occur in teeth that have been altered, for example restored or endodontically repaired. It is therefore essential to evaluate the structure and mechanical properties of these altered dentins. One such altered form of dentin is transparent (sometimes called sclerotic) dentin, which forms gradually with aging. The present study focuses on differences in the structure and mechanical properties of normal versus transparent dentin. The mineral concentration, as measured by X-ray computed microtomography, was significantly higher in transparent dentin, the elevated concentration being consistent with the closure of the tubule lumens. Crystallite size, as measured by small angle X-ray scattering, was slightly smaller in transparent dentin, although the importance of this finding requires further study. The elastic properties were unchanged by transparency; however, transparent dentin, unlike normal dentin, exhibited almost no yielding before failure. In addition, the fracture toughness was lowered by roughly 20% while the fatigue lifetime was deleteriously affected at high stress levels. These results are discussed in terms of the altered microstructure of transparent dentin.
Keywords: Dentin; Aging; Transparent; Sclerotic; Fracture; Fatigue;