Biomaterials (v.27, #18)

Calendar (I).

Experimental and theoretical studies have demonstrated that biological and synthetic hydroxyapatites display crystal structure similarities and differences that affect greatly the bioactivity of the synthetic materials. Recent developments in structure research of bioapatites report experimental values of the hydroxyl concentration in biological apatites in the range of 20–0% of the amount in synthetic hydroxyapatite. Theoretical calculations find the hexagonal space groups energetically unfavorable for hydroxyapatite crystallization. Different configurations are proposed for the carbonate substitution in B-type carbonate apatites.
Keywords: Hydroxyapatite; Crystallography; Modeling; Neutron Diffraction;

We have developed cell stimulative system by covalently immobilized signalling molecules on the surface of coverslips on which cells are later cultured. N-(6-maleimidocaproyloxy)sulfo-succinimide (sulfo-EMCS) cross-links the amino-terminal of epidermal growth factors (EGF) with the thiol-modified glass surface without degrading EGF's physiological activity. The glass surface was covered up to about 1.0 EGF molecules nm−2 with uniform density. This density can be controlled by changing concentration of the maleimide-modified EGF in the solution reacting with the thiol-modified glass coverslips. When the density of EGF was only slightly lower than that of EGF receptor dimers, cellular response was dramatically decreased. The EGF receptor molecules bound with the immobilized EGF were prevented from lateral diffusion and internalization and kept their initial position. These properties are useful for quantitative, spatial and temporal control of the input signal stimulating cells in cellular signaling system studies. In addition, the immobility of the EGF in this system makes suitable targets for stable single-molecule observation under total internal reflection fluorescence microscopy (TIR-FM) to study EGF signalling mechanism, preventing lateral diffusion and internalization of EGF receptors. Here we show results of single-molecule observations of the association and dissociation between phosphorylated EGF receptors and Cy3-labeled growth factor receptor-binding protein 2 (Grb2) proteins in A431 cells stimulated by the immobilized EGF and discuss the utility of this method for the study of intracellular signal transduction.
Keywords: Cell activation; Cell signalling; Growth factors; Molecular imaging; Surface treatment;

Effect of cobalt and chromium ions on human MG-63 osteoblasts in vitro: Morphology, cytotoxicity, and oxidative stress by Cyrille Fleury; Alain Petit; Fackson Mwale; John Antoniou; David J. Zukor; Maryam Tabrizian; Olga L. Huk (3351-3360).
Recent studies demonstrated that Co2+ and Cr3+ ions induced cell mortality, TNF-α secretion, and oxidation of proteins in macrophages. However, little is known about the effects of corrosion products on the osteogenic cells, which have a crucial role in controlling bone remodeling. The aim of the present study was to investigate the effect of Co2+ (0–10 ppm) and Cr3+ (0–150 ppm) on human MG-63 osteoblast-like cells in term of cytotoxicity and oxidative stress. Microscopic analysis demonstrated changes in shape, size, and number of cells. Co2+ had a greater effect on these parameters than Cr3+. Cell counting showed a significant decrease in the number of MG-63 osteoblasts in a time- and dose-dependent manner, with Co2+ more toxic than Cr3+. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis also showed a decreased cellular activity in presence of Co2+ and Cr3+ ions. Oxidized and nitrated proteins, two markers of oxidative stress, were detected as single bands and revealed time- and dose-dependent protein modifications. We also studied the expression of three antioxidant enzymes. The expression of heme oxygenase-1 was increased by both ions after 24 h, before decreasing gradually thereafter. Glutathione peroxidase expression was also increased in a concentration- and time-dependent manner by both Co2+ and Cr3+ ions. Co2+ decreased catalase expression while Cr3+ increased it in a dose- and time-dependent manner. In conclusion, this study demonstrated that Cr3+ and Co2+ have a cytotoxic effect on MG-63 osteoblasts and have the potential to modify their redox state.
Keywords: Hip replacement prosthesis; Metal ions; Osteoblast; Cytotoxicity; Oxidation of proteins; Antioxidant enzymes;

Assessment of GaN chips for culturing cerebellar granule neurons by Tai-Horng Young; Chi-Ruei Chen (3361-3367).
In this work, the behaviors of cerebellar granule neurons prepared from 7-day-old Wistar rats on gallium nitride (GaN) were investigated. We believe that this is the first time that the GaN has been used as a substrate for neuron cultures to examine its effect on cell response in vitro. The GaN surface structure and its relationship with cells were examined by atomic force microscopy (AFM), metallography microscopy, scanning electron microscopy (SEM), lactate dehydrogenase (LDH) release and Western blot analysis. GaN is a so-called III–V compound semiconductor material with a wide bandgap and a relatively high bandgap voltage. Compared with silicon used for most neural chips, neurons seeded on GaN were able to form an extensive neuritic network and expressed very high levels of GAP-43 coincident with the neurite outgrowth. Therefore, the GaN structure may spatially mediate cellular response that can promote neuronal cell attachment, differentiation and neuritic growth. The favorable biocompatibility characteristics of GaN can be used to measure electric signals from networks of neuronal cells in culture to make it a possible candidate for use in a microelectrode array.
Keywords: Gallium nitride (GaN); Cerebellar granule neurons;

In vitro and in vivo reactivity of porous, electrosprayed calcium phosphate coatings by Sander C.G. Leeuwenburgh; Joop G.C. Wolke; Marijke C. Siebers; Joop Schoonman; John A. Jansen (3368-3378).
The dissolution and/or precipitation behaviour of porous calcium phosphate (CaP) coatings, deposited using electrostatic spray deposition (ESD), was investigated (a) in vitro after soaking in simulated body fluid (SBF) for several time periods (2, 4, 8, and 12 weeks), and (b) in vivo after subcutaneous implantation of CaP-coated implants in the back of goats for identical time periods. Physical and chemical properties of coatings were characterized before and after in vitro/vivo testing by means of scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and energy dispersive spectroscopy. Moreover, part of the explants was prepared for light microscopical evaluation of the tissue response.In vitro, all apatitic ESD-coatings induced the formation of homogeneous and adherent CaP precipitation layers. Amorphous CaP, however, displayed a delayed precipitation of poorly adherent CaP layers, whereas heterogeneous calcification was observed on top of β-TCP-coated substrates, indicating that β-TCP and amorphous CaP coatings exhibit a poor ability of inducing calcification in SBF as compared to crystalline apatitic coatings.In vivo, no adverse tissue reactions (toxic effects/inflammatory cells) were observed using light microscopy, and all coatings became surrounded by a dense, fibrous tissue capsule after implantation. All ESD-coatings degraded gradually at a dissolution rate depending on the chemical phase (order of relative solubility: amorphous CaP≈carbonate apatite>β-TCP>carbonated hydroxyapatite), thereby enabling synthesis of CaP coatings with a tailored degradation rate.
Keywords: Electrostatic spray deposition; Calcium phosphate coating; Dissolution; Precipitation; Simulated body fluids (SBF); Subcutaneous;

Deleterious tissue reaction to an alkylene bis(dilactoyl)-methacrylate bone adhesive in long-term follow up after screw augmentation in an ovine model by Lars Grossterlinden; Arne Janssen; Niels Schmitz; Matthias Priemel; Pia Pogoda; Michael Amling; Johannes M. Rueger; Wolfgang Linhart (3379-3386).
Biomaterials are designed to support orthopedic surgeons and once implanted they will help the body to heal itself. In this way one of the most attractive substances are biomaterials that allow gluing of bone fragments and implant fixation. Although no bone adhesive is established for practical use in clinical practice yet, there is evidence in vitro and in vivo that a new class of bone adhesives based on alkylene bis(dilactoyl)-methacrylates may meet the requirements to bridge the gap between bench and bedside. The purpose of this experimental study was to investigate the long-term biocompatibility as well as the integration in the remodeling process of a new polymer of this group of substances that was used for both fragment adaptation and implant fixation in a large-scale animal model. In 24 sheep the lateral tibial condyle was osteotomized and refixed by three cortical screws. In 12 of them overdrilling the bone thread of one screw was performed to simulate the poor mechanical properties of osteoporotic bone and the polymer was used in this setting for screw augmentation, furthermore the osteotomy surface was covered with polymer before osteosynthesis to analyze the influence of the material on bone healing. In the other 12 sheep that served as controls osteosynthesis was performed without a polymer. All animals were permitted to walk immediately after surgery under full weight bearing conditions. Six animals of the polymer group and six animals of the control group were analyzed after 6 weeks and 6 months, respectively. Bone healing and implant integration was evaluated by contact X-rays, histology and histomorphometric quantification. After 6 weeks integrity of the healing bone in the polymer group was preserved as compared to the controls, albeit signs of prolonged aseptic inflammation were observed in the polymer group, which is in line with previous reports. In sharp contrast after 6 months, extensive tissue destruction was observed in all animals of the polymer group that was attributed to a massive foreign body reaction at the histological level. These long-term results suggest that (i) short-term observation not always allow valid conclusions regarding the biocompatibility of biomaterials, (ii) that biocompatibility might vary between species, and (iii) that the polymer used in this setting, although previously attributed to be a good candidate for clinical use in patients, does not meet the necessary criteria and tremendously interferes with the physiology of skeletal repair.
Keywords: Osteoporosis;

An in vivo study of a growth-factor enhanced, cell free, two-layered collagen–tricalcium phosphate in deep osteochondral defects by Tobias Gotterbarm; Wiltrud Richter; Martin Jung; Simona Berardi Vilei; Pierre Mainil-Varlet; Takeshi Yamashita; Steffen J. Breusch (3387-3395).
Focal osteochondral defects are still a challenging problem in joint surgery. We have developed a two-layered implant consisting of a basal porous β-tricalcium phosphate (TCP) for bone reconstruction and a superficial fibrous collagen type I/III layer for cartilage regeneration. Fifty-four osteochondral defects in the trochlear groove of 27 Göttinger Minipigs were created and either left untreated, treated with the implant alone, or the implant augmented with an additional growth factor mixture, which was assumed to stimulate cell and tissue differentiation. Follow-up was 6, 12 and 52 weeks with n = 6 for each group. The repair tissue was evaluated for its gross appearance and biomechanical properties. Histological sections were semi-quantitatively scored for their histomorphological structure.Treatment with the two-layered implant improved defect filling and subchondral bone repair at 6 and 12 weeks follow-up. The TCP was replaced by cancellous bone at 52 weeks. Cartilage repair tissue mainly consisted of fibrocartilage and showed a moderate cell density up to the joint surface. Growth factor treatment improved the mechanical and histomorphological properties of the cartilage repair tissue at 12, but not at 52 weeks postoperatively. In conclusion, the two-layered collagen–TCP implant augmented with chondroinductive growth factors seems a promising new option for the treatment of deep osteochondral defects in joint surgery.
Keywords: Osteochondral defect; Two-layered implant; β-TCP; Collagen type I; Growth factors; Indentation test;

The in-vitro bioactivity of mesoporous bioactive glasses by Xiaoxia Yan; Xiaohui Huang; Chengzhong Yu; Hexiang Deng; Yi Wang; Zhendong Zhang; Shizhang Qiao; Gaoqing Lu; Dongyuan Zhao (3396-3403).
Ordered mesoporous bioactive glasses (MBGs) with different compositions were prepared by using nonionic block copolymer surfactants as structure-directing agents through an evaporation-induced self-assembly process. Their in-vitro bioactivities were studied in detail by electron microscopy, Fourier-transform infrared spectroscopy, and inductively coupled plasma (ICP) atomic emission spectroscopy. The ICP element analysis results were further calculated in terms of the total consumption of Ca and P, Δ[Ca]/Δ[P] ratios, and ionic activity product (IP) of hydroxyapatite. Through the above analysis, it is clear that MBGs show a different structure–bioactivity correlation compared to conventional sol–gel-derived BGs. The in vitro bioactivity of MBGs is dependent on the Si/Ca ratio in the network when the other material parameters such as the mesostructure and texture properties (pore size, pore volume) are controlled. MBG 80S15C with relatively lower calcium content exhibits the best in vitro bioactivity, in contrast to conventional sol–gel-derived BGs where usually higher calcium percentage BGs (e.g. 60S35C) show better bioactivity. Calcination temperature is another important factor that influences the in vitro bioactivity. According to our results, MBGs calcined at 973 K may possess the best in vitro bioactivity. The influences of the composition and calcination temperature upon bioactivity are explained in terms of the unique structures of MBGs.
Keywords: Bioactive glass; Porosity; Bioactivity; SBF; Hydroxyapatite;

Interfacial rheology of blood proteins adsorbed to the aqueous-buffer/air interface by Florly S. Ariola; Anandi Krishnan; Erwin A. Vogler (3404-3412).
Concentration-dependent, interfacial-shear rheology and interfacial tension of albumin, IgG, fibrinogen, and IgM adsorbed to the aqueous-buffer/air surface is interpreted in terms of a single viscoelastic layer for albumin but multi-layers for the larger proteins. Two-dimensional (2D) storage and loss moduli G ′ and G ″ , respectively, rise and fall as a function of bulk-solution concentration, signaling formation of a network of interacting protein molecules at the surface with viscoelastic properties. Over the same concentration range, interfacial spreading pressure Π LV ≡ γ lv o - γ lv rises to a sustained maximum Π LV max . Mixing as little as 25 w/v% albumin into IgG at fixed total protein concentration substantially reduces peak G ′ , strongly suggesting that albumin acts as rheological modifier by intercalating with adsorbed IgG molecules. By contrast to purified-protein solutions, serially diluted human blood serum shows no resolvable concentration-dependent G ′ and G ″ .
Keywords: Plasma; Serum; Protein adsorption; Air–water interface; Interfacial rheology;

Fabrication of a phospholipid membrane-mimetic film on the luminal surface of an ePTFE vascular graft by Sumanas W. Jordan; Keith M. Faucher; Jeffrey M. Caves; Robert P. Apkarian; Shyam S. Rele; Xue-Long Sun; Stephen R. Hanson; Elliot L. Chaikof (3473-3481).
A stabilized, membrane-mimetic film was produced on the luminal surface of an ePTFE vascular graft by in situ photopolymerization of an acrylate functonalized phospholipid using a fiber optic diffusing probe. The phospholipid monomer was synthesized, prepared as unilamellar vesicles, and fused onto close-packed octadecyl chains that were components of an amphiphilic terpolymer anchored onto the polyelectrolyte multilayer (PEM) by electrostatic interactions. Scanning electron microscopy (SEM) confirmed that gelatin impregnation of the graft followed by the subsequent biomimetic film coating filled in the fibril and node structure of the luminal surface of the ePTFE graft and was smooth. The lipid film displayed an initial advancing contact angle of 44°, which increased to 55° after being subjected to a wall shear rate of 500 s−1 for 24 h at 37 °C in phosphate buffered saline (PBS). Fourier transform (FT-IR) spectroscopy was used to characterize the stages of biomimetic film assembly and confirmed the stability of the film under shear flow conditions. In vivo assessment using a baboon femoral arteriovenous shunt model demonstrated minimal platelet and fibrinogen deposition over a 1-h blood-contacting period. The results of this study confirm the versatility of a biomimetic film coating system by successfully transferring the methodology previously developed for planar substrates to the luminal surface of an ePTFE vascular graft.
Keywords: Vascular graft; Biomimetics; Membrane-mimetic; Blood-contacting materials; Thrombogenicity;

Lectin-conjugated PEG–PLA nanoparticles: Preparation and brain delivery after intranasal administration by Xiaoling Gao; Weixing Tao; Wei Lu; Qizhi Zhang; Yan Zhang; Xinguo Jiang; Shoukuan Fu (3482-3490).
In order to improve the absorption of nanoparticles in the brain following nasal administration, a novel protocol to conjugate biorecognitive ligands—lectins to the surface of poly (ethylene glycol)–poly (lactic acid) (PEG–PLA) nanoparticles was established in the study. Wheat germ agglutinin (WGA), specifically binding to N-acetyl-d-glucosamine and sialic acid, both of which were abundantly observed in the nasal cavity, was selected as a model lectin. The WGA-conjugated nanoparticles were prepared by incorporating maleimide in the PLA–PEG molecular and taking advantage of its thiol group binding reactivity to conjugate with 2-iminothialane thiolated WGA. Coupling of WGA with the PEG–PLA nanoparticles was confirmed by the existence of gold-labeled WGA-NP under TEM. The retention of biorecognitive activity of WGA after the covalent coupling procedure was confirmed by haemagglutination test. The resulting nanoparticles presented negligible nasal ciliatoxicity and the brain uptake of a fluorescent marker—coumarin carried by WGA functionized nanoparticles was about 2 folds in different brain tissues compared with that of coumarin incorporated in the unmodified ones. Thus, the technique offered a novel effective noninvasive system for brain drug delivery, especially for brain protein and gene delivery.
Keywords: Brain drug delivery; Nanoparticles; Wheat germ agglutinin; Intranasal administration;

Transdermal delivery of a pineal hormone: Melatonin via elastic liposomes by Vaibhav Dubey; Dinesh Mishra; Abhay Asthana; Narendra Kumar Jain (3491-3496).
Melatonin (MT) is a good candidate for transdermal delivery considering its short biological half-life, low molecular weight and a variable oral absorption. The objective of this work was to develop a novel formulation of melatonin for its efficient transdermal delivery. Melatonin loaded elastic liposomal formulation was prepared, characterized and the effect of this developed formulation on the in vitro permeation of melatonin across human cadaver skin was investigated, using a locally fabricated Franz diffusion cell. Skin permeation potential of the developed formulation was assessed using confocal laser scanning microscopy (CLSM), which revealed an enhanced permeation of the formulation to the deeper layers of the skin (up to 180 μm) following channel like pathways. Skin permeation profile of melatonin through elastic liposomal formulations was observed and the investigations revealed an enhanced transdermal flux (51.2±2.21 μg/cm2/h), decreased lag time (1.1 h) and an optimum permeability coefficient (15.06±0.52 cm/h) for melatonin. The obtained flux was nearly 5 and 12.3 times higher than conventional liposomal and plain drug solution, respectively (P<0.005). Our result suggests the feasibility of elastic liposomal system for transdermal delivery of melatonin thereby eliminating the limitations of long lag time and poor skin permeation associated with the drug.
Keywords: Melatonin; Elastic liposomes; Permeation enhancement; Transdermal delivery; Confocal microscopy;

Layer-by-layer assembly of cationic lipid and plasmid DNA onto gold surface for stent-assisted gene transfer by Fumio Yamauchi; Yuichi Koyamatsu; Koichi Kato; Hiroo Iwata (3497-3504).
Intravascular stent-assisted gene transfer is an advanced approach for the therapy of vascular diseases such as atherosclerosis and stenosis. This approach requires a stent that allows local and efficient administration of therapeutic genes to the target cells at the vascular wall. To create such a stent, a method was developed for loading plasmid DNA onto the metal surface. The method involves the formation of self-assembled monolayer on the noble metal surface followed by electrostatic layer-by-layer (LBL) assembly of a cationic lipid/plasmid DNA complex and free plasmid DNA. In this in vitro feasibility study, the thin plainer film and the wire of gold were used as a substrate. The LBL assembly process was characterized by surface plasmon resonance spectroscopy and static contact angle measurement. Plasmid DNA loaded in the multilayer exhibited improved resistance against nuclease digestion. When cultured directly on the DNA-loaded surface, cells were transfected to express exogenous gene in the DNA loading-dependent manner. Plasmid DNA could also be transferred to endothelial cells from its apical side by placing the DNA-loaded gold wire onto the cell layer.
Keywords: Stent; Gene transfer; Layer-by-layer assembly; Multilayer; Plasmid DNA; Self-assembled monolayer;

A novel glass slide-based peptide array support with high functionality resisting non-specific protein adsorption by Mario Beyer; Thomas Felgenhauer; F. Ralf Bischoff; Frank Breitling; Volker Stadler (3505-3514).
Glass slides have been modified with a multifunctional poly(ethylene glycol) (PEG)-based polymer with respect to array applications in the growing field of proteome research. We systematically investigated the stepwise synthesis of the PEG films starting from self-assembled alkyl silane monolayers via monolayer peroxidation and subsequent graft polymerization of PEG methacrylate (PEGMA). Chemical composition was examined by X-ray photoelectron spectroscopy (XPS); infrared spectroscopy provided information about order and composition of the films as well; film thickness was determined by ellipsometry; using fluorescence microscopy and again XPS, the amount of proteins adsorbed on the slides was investigated. The novel support material allows a versatile modification of the amino group surface density up to 40 nmol/cm2 for the linkage of probe molecules. Further on, we carried out standard peptide synthesis based on the well-established 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, which was monitored by UV/Vis quantification of the Fmoc deblocking and mass spectrometry. The polymer coating is stable with respect to a wide range of chemical and thermal conditions, and prevents the glass surface from unspecific protein adsorption. Finally, we applied our modified glass slides in immunoassays and thus examined specific interactions of monoclonal antibodies with appropriate peptide epitopes.
Keywords: Surface grafting; Glass; Biocompatibility; Biopolymer; Peptide array; Immunoassay;