Biomaterials (v.26, #10)

Calendar (I).

Protein array method for assessing in vitro biomaterial-induced cytokine expression by Yiwen Li; Robert J. Schutte; Amal Abu-Shakra; W.Monty Reichert (1081-1085).
This study demonstrates the feasibility of a cytokine-based in vitro test for biomaterials. The combination of monocyte culture and protein array technology tested in this study permitted the detection of subtle changes in cytokine expression following an exposure to titanium (Ti) particles. However, a broader range of materials and sample configurations must be examined before these promising results can be translated into a reliable and predictive in vitro biomaterials testing protocol. Modified glass slides were robotically printed with eight identical arrays consisting of capture antibodies against four mouse cytokines [IL-6, TNF-α, MIP-2, TGF-β1] and two positive and two negative detection controls. RAW 264.7 mouse monocytes seeded into six-well plates at 105  cells/well were treated with either sterilized Ti particles (test biomaterial), or lipopolysaccharide (LPS; positive control), or untreated (negative control). Aliquots (80 μl) of culture medium collected at 1, 6, 24, 48, and 72 h were applied to the protein arrays for simultaneous sandwich fluoroimmunoassay, followed by imaging the fluorescent intensities on a conventional microarray scanner. LPS induced the release of all four cytokines between 1 and 6 h treatment periods, whereas Ti induction of cytokines showed a gradual and subtle increase in cytokine expression for >24 h. Among the four cytokines assayed, TNF-α and MIP-2 were most prominently expressed, while IL-6 was slightly elevated and TGF-β1 was undetected above background.
Keywords: Biocompatibility; Cytokine; Monocyte; Titanium; Macrophage;

Characterisation of a duplex TiO2/CaP coating on Ti6Al4V for hard tissue replacement by Boon Sing Ng; Ingegerd Annergren; Andrew M. Soutar; K.A. Khor; Anders E.W. Jarfors (1087-1095).
An initial TiO2 coating was applied on Ti6Al4V by electrochemical anodisation in two dissimilar electrolytes. The secondary calcium phosphate (CaP) coating was subsequently applied by immersing the substrates in a simulated body fluid (SBF) with three times concentration (SBF×3), mimicking biomineralisation of biological bone. Electrochemical impedance spectroscopy and potentiodynamic polarisation assessments in SBF revealed that the anodic TiO2 layer is compact, exhibiting up to four-folds improvement in in vitro corrosion resistance over unanodised Ti6Al4V. X-ray photoelectron spectroscopy analysis indicates that the anodic Ti oxide is thicker than air-formed ones with a mixture of TiO2−x compound between the TiO2/Ti interfaces. The morphology of the dense CaP film formed, when observed using scanning electron microscopy, is made up of linked globules 0.1–0.5 μm in diameter without observable delamination. Fourier transform infrared spectrometry with an attenuated total internal reflection analysis revealed that this film is an amorphous/poorly crystallised calcium-deficient-carbonated CaP system. The calculated Ca:P ratios of all samples (1.14–1.28) are lower than stoichiometric hydroxyapatite (1.67). These results show that a duplex coating consisting of (1) a compact TiO2 with enhanced in vitro corrosion resistance and (2) bone-like apatite coating can be applied on Ti6Al4V by anodisation and subsequent immersion in SBF.
Keywords: Biocompatibility; Corrosion; Titanium oxide; Biomimetic; SBF; Calcium phosphate;

The driving force and nucleation rate of calcium phosphate (Ca-P) precipitation in simulated body fluid (SBF) were analyzed based on the classical crystallization theory. SBF supersaturation with respect to hydroxyapatite (HA), octacalcium phosphate (OCP) and dicalcium phosphate (DCPD) was carefully calculated, considering all the association/dissociation reactions of related ion groups in SBF. The nucleation rates of Ca-P were calculated based on a kinetics model of heterogeneous nucleation. The analysis indicates that the nucleation rate of OCP is substantially higher than that of HA, while HA is most thermodynamically stable in SBF. The difference in nucleation rates between HA and OCP reduces with increasing pH in SBF. The HA nucleation rate is comparable with that of OCP when the pH value approaches 10. DCPD precipitation is thermodynamically impossible in normal SBF, unless calcium and phosphate ion concentrations of SBF increase. In such case, DCPD precipitation is the most likely because of its highest nucleation rates among Ca-P phases. We examined the influences of different SBF recipes, interfacial energies, contact angle and molecular volumes, and found that the parameter variations do not have significant impacts on analysis results. The effects of carbonate incorporation and calcium deficiency in HA were also estimated with available data. Generally, such apatite precipitations are more kinetically favorable than HA.

Native fibrillar collagen membranes of micron-scale and submicron thicknesses for cell support and perfusion by Robert B. Vernon; Michel D. Gooden; Stephanie L. Lara; Thomas N. Wight (1109-1117).
Fibrillar type I collagen is nontoxic, biocompatible, and possesses considerable strength and stability. In a study of scaffolds for use in laminated tissue substitutes, we examined the properties of membranes made from air-dried hydrogels of collagen fibrils that were polymerized from native, monomeric collagen. Planar collagen membranes (CMs) of 0.1–5.3 μm dry thickness were made by variation of the collagen concentration and/or the volume of the hydrogel. The planar CMs, which were comprised of a dense feltwork of long collagen fibrils 70–100 nm in diameter, showed considerable resistance to rupture and retained their membranous character after 6 weeks in tissue culture medium at 37°C. CMs that were relatively thick when dry exhibited a greater proportional increase in rehydrated thickness and a greater diffusivity (when rehydrated) to 4.3 kDa dextran than did CMs that were relatively thin when dry. Hollow, tubular CMs of several configurations were prepared by embedment of solid, removable forms into collagen hydrogels prior to drying. By use of special fixtures, a planar CM that incorporated multiple, parallel tubes was fabricated. In summary, hydrogels of fibrillar collagen can be transformed into membranous structures suitable for tissue engineering applications.
Keywords: Collagen; ECM; Hydrogel; Membrane; Microstructure; Scaffold;

Synergistic interaction of topographic features in the production of bone-like nodules on Ti surfaces by rat osteoblasts by Marco Wieland; Marcus Textor; Babak Chehroudi; Donald M. Brunette (1119-1130).
The objective of this study was to study the responses of osteoblast-like cells to rough Titanium (Ti)-coated epoxy surfaces of differing topographic complexity. Four topographies were studied: polished (PO), coarse-blasted (CB), acid-etched (AE) and coarse-blasted+acid-etched (SLA). Rat osteoblasts were cultured on these surfaces and their morphology, thickness as well as the number and size of bone-like nodules measured. To determine cell shape and cell thickness, fluorescein-5-thiosemicarbazide was used to stain the cell components including the cell membrane, the stained cells were optically sectioned using epifluorescent microscopy and the optical sections were computationally reconstructed to obtain three-dimensional images in which cell volume and cell thickness could be determined. Similarly optical sections of bone-like nodules labeled with tetracycline were also reconstructed to determine their size. The different surface topographies were found to alter the thickness and morphology of osteoblasts cultured on these surfaces. Osteoblasts produced significantly more and larger nodules on SLA compared to other surfaces . Nevertheless and perhaps surprisingly, given the evidence in various cell populations that cell shape can affect cell differentiation, cell thickness was not directly correlated with an increase in bone-like nodule formation. Data were analyzed by factorial analysis of variance. In this way the primary effect of each surface treatment ( i.e. blasting and acid etching) could be assessed as well as their interaction. Both the acid etching and blasting processes significantly affected the number and size of bone-like nodules cultured on Ti surfaces. Moreover there were significant interaction effects indicating that surface topographic features can act synergistically to enhance bone formation. This result suggests that a useful approach to the optimization of surfaces for bone production could involve systematic investigation of combinations of processes each of which produces distinct surface topographical features.
Keywords: Osteoblast; Bone; Dental implant; Surface roughness; Surface topography; Cell Morphology; Cell culture;

Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants inserted at an ectopic site in rabbits by Henriette C. Kroese-Deutman; P.Quinten Ruhé; Paul H.M. Spauwen; John A. Jansen (1131-1138).
Recombinant human bone morphogenetic protein-2 (rhBMP-2) is known for its osteoinductive potential in bone tissue engineering. Calcium phosphate (Ca-P) cements are injectable, osteoconductive ceramic materials in which a macroporous structure can be induced during the setting reaction. In this study, the osteoinductive capability of rhBMP-2 loaded porous Ca-P cement was evaluated.Porous Ca-P cement discs were made and loaded with rhBMP-2 in vitro and implanted subcutaneously in the back of New Zealand white rabbits. The implantation period was either 2 or 10 weeks. Histological analysis of retrieved specimens revealed evident bone formation in the rhBMP-2 loaded Ca-P cement discs (pore fill: 18±6%) after 10 weeks of implantation. Bone formation occurred only in rhBMP-2 loaded porous Ca-P cement discs. Degradation of the Ca-P cement could not be confirmed after 10 weeks of implantation. The scaffold maintained its shape and stability during this time period.We conclude that porous Ca-P cement is a suitable carrier material for ectopic bone engineering.
Keywords: Calcium phosphate cement; Bone morphogenetic protein (BMP); Growth factors; Ectopic bone formation; Osteoinduction; Bone tissue engineering;

Enhanced neovasculature formation in ischemic myocardium following delivery of pleiotrophin plasmid in a biopolymer by Karen L. Christman; Qizhi Fang; Michael S. Yee; Kandice R. Johnson; Richard E. Sievers; Randall J. Lee (1139-1144).
Coronary heart disease is currently the leading killer in the western world. Therapeutic angiogenic agents are currently being examined for treatment of this disease. We have recently demonstrated the effective use of Pleiotrophin (PTN) as a therapeutic agent for treatment of ischemic myocardium. We have also shown that injection of the biopolymer fibrin glue preserves left ventricular geometry and prevents a deterioration of cardiac function following myocardial infarction. Due to the low transfection efficiency of naked plasmid injections, we examined the use of PTN plasmid and the biopolymer as a gene-activated matrix in the myocardium. In this study, we demonstrate that delivery of PTN plasmid in fibrin glue increases neovasculature formation compared to injection of the naked plasmid in saline.
Keywords: Angiogenesis; Fibrin; Gene therapy; Cardiac tissue engineering;

Micro-rotary fatigue of tooth–biomaterial interfaces by J. De Munck; M. Braem; M. Wevers; Y. Yoshida; S. Inoue; K. Suzuki; P. Lambrechts; B. Van Meerbeek (1145-1153).
The bonding effectiveness of restorative materials to tooth tissue is typically measured statically. Clinically tooth/composite bonds are however subjected to cyclic sub-critical loads. Therefore, in vitro fatigue testing of dental adhesives should predict better the in vivo performance of adhesives. The objective of this study was to determine the fatigue resistance of two representative adhesives, a self-etch and an etch&rinse adhesive, bonded to enamel and dentin. Therefore, tooth/composite interfaces were cyclically loaded using a miniaturized version of a rotating beam fatigue testing device. Subsequently, the load at which 50% of the specimens fail after 105 cycles, was determined as the median micro-rotary fatigue resistance (μRFR). For both adhesives, the μRFR was about 30–40% lower than the corresponding micro-tensile bond strength (μTBS) to both enamel and dentin. Analysis of the fracture surfaces by Feg-SEM revealed typical fatigue fracture patterns. It is concluded that resin/tooth interfaces are vulnerable to progressive damage by sub-critical loads, with the 3-step etch&rinse adhesive being more resistant to fatigue than the 2-step self-etch adhesive.
Keywords: Adhesion; Fatigue; Fracture mechanism; Dental adhesive; Enamel; Dentin;

The in vivo failure of implantable glucose sensors is thought to be largely the result of inflammation and fibrosis-induced vessel regression at sites of sensor implantation. To determine whether increased vessel density at sites of sensor implantation would enhance sensor function, cells genetically engineered to over-express the angiogenic factor (AF) vascular endothelial cell growth factor (VEGF) were incorporated into an ex ova chicken embryo chorioallantoic membrane (CAM)-glucose sensor model. The VEGF-producing cells were delivered to sites of glucose sensor implantation on the CAM using a tissue-interactive fibrin bio-hydrogel as a cell support and activation matrix. This VEGF–cell–fibrin system induced significant neovascularization surrounding the implanted sensor, and significantly enhanced the glucose sensor function in vivo. This model system, for the first time, provides the “proof of principle” that increasing vessel density at the sites of implantation can enhance glucose sensor function in vivo, and demonstrates the potential of gene transfer and tissue interactive fibrin bio-hydrogels in the development of successful implants.
Keywords: Biosensor; Glucose sensor; Chick embryo chorioallantoic membrane (CAM); Fibrin; Gene transfer; Vascular endothelial cell growth factor (VEGF); Neovascularization;

Protein-mediated boundary lubrication in arthroplasty by M.P. Heuberger; M.R. Widmer; E. Zobeley; R. Glockshuber; N.D. Spencer (1165-1173).
Wear of articulated surfaces can be a major lifetime-limiting factor in arthroplasty. In the natural joint, lubrication is effected by the body's natural synovial fluid. Following arthroplasty, and the subsequent reformation of the synovial membrane, a fluid of similar composition surrounds the artificial joint. Synovial fluid contains, among many other constituents, a substantial concentration of the readily adsorbing protein albumin. The ability of human serum albumin to act as a boundary lubricant in joint prostheses has been investigated using a pin-on-disc tribometer. Circular dichroism spectroscopy was employed to follow the temperature- and time-dependent conformational changes of human serum albumin in the model lubricant solution. Effects of protein conformation and polymer surface hydrophilicity on protein adsorption and the resulting friction in the boundary lubrication regime have been investigated. Unfolded proteins preferentially adsorb onto hydrophobic polymer surfaces, where they form a compact, passivating layer and increase sliding friction—an effect that can be largely suppressed by rendering the substrate more hydrophilic. A molecular model for protein-mediated boundary friction is proposed to consolidate the observations. The relevance of the results for in vivo performance and ex vivo hip-joint testing are discussed.
Keywords: Albumin; Protein adsorption; Protein conformation; Joint replacement; Arthroplasty; Friction; Interface; Polyethylene; Hydrophilicity;

Citric acid–polyethylene glycol–citric acid (CPEGC) triblock dendrimers as biocompatible compounds containing G1, G2 and G3 were applied as the drug-delivery systems. Some of the small size molecules and drugs are trapped with the above-synthesized dendrimers. The guest molecules, which are hydrophobic when trapped into the suitable sites of dendrimers, are becoming soluble in aqueous solution. The quantity of trapped molecules and drugs such as 5-amino salicylic acid (5-ASA), pyridine, mefenamic acid, and diclofenac was measured. The drug/dendrimer complexes remained in room temperature for about 10 months and after this long time they were stable and the drugs were not released. Also, the controlled release of the above-mentioned molecules and drugs in vitro conditions was investigated. The structure definition and controlled release of the molecules and drugs were carried out using different spectroscopy methods.
Keywords: Dendrimer; Biocompatible; Citric acid; Poly (ethylene glycol); 5-amino salicylic acid (5-ASA); Drug-delivery;

Novel crosslinker, 4-{(E)-[(3Z)-3-(4-(acryloyloxy)benzylidene)-2-hexylidene]methyl}phenyl acrylate (AMA) was synthesized using (2Z, 6E)-2,6-bis(4-hydroxybenzylidene)cyclohexanone (HBC) and acryloyl chloride. Two types of crosslinked polymeric hydrogels were prepared from 2-hydroxyethyl acrylate (HEA) and 2-hydroxypropyl methacrylate (HPMA) monomers using AMA as a crosslinking agent. 2′,4-dichloro-5′-fluoro-1-ene-2-(4-hydroxyphenyl)phenone (EHP) (J. Bio Active Compat. Polym. 18 (2003) 219) was used as a drug molecule for monitoring the releasing behaviour of the hydrogels. Morphology of the hydrogels was characterized using optical microscopy (OM) and Scanning Electron Microscopy (SEM) techniques. Several modifications were made in the experimental sections to study the effect of crosslinking percentage (CLP), drug loading percentage (DLP), monomer type (HEA and HPMA) and the pH. Totally 18 experiments were carried out to study the desired parameters in the hydrogels. The drug-releasing rate was monitored by the absorption appeared at 330.5 nm using UV spectrometer. It was found that the releasing rate of the drug from the polymeric hydrogels was dependent on the crosslinking density, drug loading percentage, monomer type and pH of the medium.
Keywords: Drug release; Monomer; Hydrogel; Copolymer;

Mechanistic aspects of in vitro fatigue-crack growth in dentin by J.J. Kruzic; R.K. Nalla; J.H. Kinney; R.O. Ritchie (1195-1204).
Although the propagation of fatigue cracks has been recognized as a problem of clinical significance in dentin, there have been few fracture mechanics-based studies that have investigated this issue. In the present study, in vitro cyclic fatigue experiments were conducted over a range of cyclic frequencies (1–50 Hz) on elephant dentin in order to quantify fatigue-crack growth behavior from the perspective of understanding the mechanism of fatigue in dentin. Specifically, results obtained for crack extension rates along a direction parallel to the dentinal tubules were found to be well described by the stress-intensity range, Δ K , using a simple Paris power-law approach with exponents ranging from 12 to 32. Furthermore, a frequency dependence was observed for the crack-growth rates, with higher growth rates associated with lower frequencies. By using crack-growth experiments involving alternate cyclic and static loading, such fatigue-crack propagation was mechanistically determined to be the result of a “true” cyclic fatigue mechanism, and not simply a succession of static fracture events. Furthermore, based on the observed frequency dependence of fatigue-crack growth in dentin and observations of time-dependent crack blunting, a cyclic fatigue mechanism involving crack-tip blunting and re-sharpening is proposed. These results are deemed to be of importance for an improved understanding of fatigue-related failures in teeth.