Biomaterials (v.26, #8)

Calendar (I).

Corrosion behavior and surface characterization of titanium in solution containing fluoride and albumin by Shinji Takemoto; Masayuki Hattori; Masao Yoshinari; Eiji Kawada; Yutaka Oda (829-837).
The objective of this study was to demonstrate the role of albumin on the corrosion behavior of titanium in a solution containing 2.0 g/l fluoride and either 0.1 or 1.0 g/l albumin. The corrosion behavior and surface characterization of passive films on titanium immersed in such a solution were examined. In addition, the change in pH and the concentration of dissolved titanium in the solution were examined. The results showed that the corrosion of titanium in a solution containing fluoride was distinct, and that adding albumin to the solution containing fluoride suppressed corrosion. Fluorine was detected on the titanium surface immersed in the solution containing fluoride, and dissolution of the titanium was confirmed. The titanium immersed in a solution containing both fluoride and albumin had an albumin film regardless of the albumin concentration. In addition, the amount of dissolved titanium from the titanium immersed in the solution was less than when the solution contained no albumin. It was suggested that the formation of adsorbed albumin films on or in the passive film acted to not only protect the titanium from attack by the fluoride but also suppressed dissolution of the titanium-fluoride compounds.
Keywords: Titanium; Albumin; Fluoride; Corrosion; X-ray photoelectron spectroscopy;

Potential of biomimetic surfaces to promote in vitro osteoblast-like cell differentiation by S. Hattar; A. Asselin; D. Greenspan; M. Oboeuf; A. Berdal; J.-M. Sautier (839-848).
Bioactive glasses, osteoproductive materials, have received considerable attention as bone graft substitutes in the treatment of bony defects. More recent strategies for achieving a predictable periodontal regeneration include the use of enamel matrix proteins, due to their role in the formation of bone tissue. The aim of our study is to examine the effects of these materials on the proliferation and differentiation of the mouse preosteoblastic cell line MC3T3-E1. Cells were cultured up to 28 days in contact with three types of granules: Bioglass 45S5® granules (BG), 45S5® granules coated with enamel matrix proteins (Emdogain®) (BG/EMD), and a less reactive glass used as a control (60S).Phase contrast microscopic observations have shown that all substrates supported the growth of osteoblastic cells. Zones of differentiation were observed at an earlier stage in cultures of BG and BG/EMD. TEM observations revealed ultrastructural features very close to what is observed in vivo during intramembranous ossification with a direct bone apposition on the bioactive glasses. Total protein production was higher in the cultures with BG and BG/EMD. Northern Blot analysis revealed a stimulation of the transcription factor Cbfa1/Runx2 at day 13 in cultures of BG when compared to the two other cultures. Bone sialoprotein (early marker of differentiation) and osteocalcin (marker of late-stage differentiation) expression was increased in cultures with BG and BG/EMD when compared to 60S.Taken together, our findings indicate that Bioglass® alone or combined with Emdogain®, have the ability to support the growth of osteoblast-like cells in vitro and to promote osteoblast differentiation by stimulating the expression of major phenotypic markers. In addition, we noticed that the bioactive granules coated with Emdogain revealed significantly higher protein production than the bioactive granules alone at day 20.
Keywords: Bioactive glass; Enamel matrix protein; Osteoblast; In vitro;

Quantitative assessment of the response of osteoblast- and macrophage-like cells to particles of Ni-free Fe-base alloys by G. Ciapetti; J.L. González-Carrasco; L. Savarino; M.A. Montealegre; S. Pagani; N. Baldini (849-859).
In the present study, the effect of mechanically alloyed particles of new FeAlCr alloys developed for potential applications as surgical implants has been tested on osteoblast- and macrophage-like cells and compared to particles of the Ti6Al4V alloy, for which there is a good clinical experience. After microstructural characterisation of the particles, cells were cultured with particles for 24–48 h using three different concentrations of particles, and the response of cells was quantified by assessment of viability, proliferation, and morphology. Mineralisation by osteoblasts was verified after 21 days. The amount of aluminium and chromium ions in the culture medium of macrophages was measured by graphite furnace atomic absorption and phagocytosis of particles assessed by light microscopy. Viability and proliferation of osteoblast- and macrophage-like cells were substantially unaffected by the presence of particles of the new alloys, which were phagocytosed according to their size. Aluminium and chromium ions were released in the culture medium, but no direct correlation with the cell behaviour was found. In vitro mineralisation was achieved by osteoblasts in due time. The new alloys are well tolerated in in vitro systems, and, due to their chemical and mechanical characteristics, they are under development for surgical implants.
Keywords: Ferritic steels; Osteoblasts; Macrophages; Metal ion release;

Whole blood coagulation on protein adsorption-resistant PEG and peptide functionalised PEG-coated titanium surfaces by Kenny M. Hansson; Samuele Tosatti; Joakim Isaksson; Jonas Wetterö; Marcus Textor; Tomas L. Lindahl; Pentti Tengvall (861-872).
The aim of this study was to investigate whole blood coagulation on low blood plasma protein adsorbing surfaces. For this purpose, the polycationic graft copolymer poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), PLL-g-PEG grafted with a cell adhesive peptide containing the amino acid sequence -Arg-Gly-Asp- (RGD), and PLL-g-PEG with a control peptide -Arg-Asp-Gly- (RDG) were adsorbed onto titanium (oxide), forming stable monomolecular adlayers through electrostatic attraction. Free oscillation rheometry and complementary techniques were used to measure the coagulation time (CT) and other interactions of the surfaces with native whole blood, recalcified platelet-rich plasma (PRP), and recalcified citrated platelet-free plasma (PFP). The results show that the uncoated titanium surfaces (reference) activated platelets and quickly triggered the coagulation cascade via the intrinsic pathway, whereas the PLL-g-PEG surfaces displayed a prolonged CT, approximately 2–3 times longer compared to uncoated titanium. We hypothesise that blood coagulates outside the vascular system independent of low protein adsorption to or activation by surfaces, due to the absence of an active down-regulation of procoagulative processes by the vascular endothelium.
Keywords: Coagulation inhibition; Free oscillation rheometry; Blood compatibility; Poly(ethylene glycol); Artifical surface; Protein adsorption;

Macrophages play a central role in the normal healing process after tissue injury and the host response to foreign objects such as biomaterials. The process leading to macrophage adhesion and activation on protein-adsorbed substrates is complex and unresolved. While the use of primary cells offers clinical relevancy, macrophage cell lines offer unique advantages such as availability and relatively homogeneous phenotype as models to probe the molecular mechanism of cell–surface interaction. Our goal was to better characterize the effect of the culture condition and surface-associated ligands on the extent of U937 adhesion. Tyrosine phosphorylation of intracellular proteins was surveyed as a basis to seek a greater understanding of the molecular mechanism involved in mediating U937 adhesion on various ligand-adsorbed surfaces. U937 viability and adhesion on tissue culture polystyrene (TCPS) increased with (i) increasing serum level, (ii) decreasing tyrosine phosphorylation inhibitor AG18 concentration, or (iii) increasing culture time. The adsorption of various adhesion proteins such as fibronectin and peptide ligands (i.e., RGD, PHSRN) on TCPS did not significantly increase the adherent density of U937 when compared with albumin and PBS ligand controls. However, ligand identity and the presence of phorbol myristate acetate dramatically affected the extent (i.e., increase or decrease) and the identity (i.e., molecular weight) of phosphotyrosine proteins in adherent U937 in a time-dependent manner. The extent and identity of phosphotyrosine proteins did not exhibit a clear AG18 dose dependency, rather the level of tyrosine phosphorylation for a distinct group of proteins was either increased or decreased for a given AG18 concentration.
Keywords: AG18; RGD; PHSRN; PMA;

Patterned networks of mouse hippocampal neurons on peptide-coated gold surfaces by Daniel A. Heller; Veronika Garga; Keith J. Kelleher; Tai-Chou Lee; Sunil Mahbubani; Laura A. Sigworth; T.Randall Lee; Michael A. Rea (883-889).
Patterned networks of hippocampal neurons were generated on peptide-coated gold substrates prepared by microscope projection photolithography and microcontact printing. A 19 amino acid peptide fragment of laminin A (PA22-2) that includes the IKVAV cell adhesion domain was used to direct patterns of cell adhesion in primary culture. Microscale grid patterns of peptide were deposited on gold-coated glass cover slips by soft lithography using “stamps” fashioned from polydimethylsiloxane. Strong coordination bonding between gold atoms on the surface and the sulfur atoms of the N-terminal cysteine residues supported stable adhesion of the peptide, which was confirmed by immunofluorescence using anti-IKVAV antiserum. Dispersed hippocampal cells isolated from neonatal mouse pups were grown on peptide-patterned gold substrates for 7 days. Neurons preferentially adhered to peptide-coated regions of the gold surface and restricted their processes to the peptide patterns. Whole cell recordings of neurons grown in patterned arrays revealed an average membrane potential of −50 mV, as well as the presence of voltage-gated ion conductances. Peptide-modified gold surfaces serve as convenient and effective substrates for growing ordered neural networks that are compatible with existing multi-electrode array recording technology.
Keywords: Microcontact printing; Hippocampus; Neuron; Neural network; PA22-2; Gold;

Adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) surface by Wee Jin Tan; Gilbert P. Teo; Kin Liao; Kam W. Leong; Hai-Quan Mao; Vincent Chan (891-898).
The design of bioartificial liver assist device requires an effective attachment of primary hepatocytes on polymeric biomaterials. A better understanding of this cell-surface interaction would aid the optimal choice of biomaterials. In this study, the adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) (PET) surface with grafted poly(acrylic acid) (PAA) and coated collagen is probed with confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy. An increase of acrylic acid density from 0 to 12 nmole/cm2 raises both the root-mean-square surface roughness and amount of adsorbed collagen of PET surface. C-RICM demonstrates that hepatocytes form tight adhesion contacts upon seeding on both plain PET and PAA-grafted PET (both with collagen coating) despite the insignificant two-dimensional cell spreading. At two hours after cell seeding, the normalized contact area and adhesion energy of hepatocytes on 12 nmole/cm2 PAA-grafted-PET (with collagen coating) is 27% and 114% higher, respectively, than that on collagen coated plain PET. Interestingly, the growth kinetics of adhesion patch for hepatocyte on PAA-grafted PET with collagen coating is best fitted by Rt 0.5 and is significantly different from that on collagen coated plain PET, which is best fitted by Rt 0.25. Overall, this study demonstrates the modulation of biophysical response of adherent hepatocytes through the control of the biomaterial surface properties.
Keywords: Polyacrylic acid; Biophysics; Adhesion; Kinetics;

Hydroxyapatite (HAP) was blended into poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) to make films and scaffolds. After HAP blending, mechanical properties of PHB including compressive elastic modulus and maximum stress showed improvement and osteoblast responses including cell growth and alkaline phosphatase activity were also strengthened. On the other hand, scaffolds made of PHBHHx blended with HAP had an adverse effect. No remarkable change on degradation of PHB or PHBHHx blended with HAP, respectively, was observed in simulated body fluid. Scanning electron microscopy examination revealed that osteoblast responses to HAP incorporation may be related to surface morphology and to the exposed HAP particles on polymer surface. All these results indicated that the blending of HAP particles into PHBHHx scaffolds fabricated by salt leaching was unable to either strengthen its mechanical properties or enhance osteoblast responses. Although HAP is bioactive and osteoconductive, its blending with PHBHHx did not generate a better performance on bone reconstruction.
Keywords: PHB; Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate); PHBHHx; Hydroxyapatite; Osteoblast;

Medical grade ultra high molecular weight polyethylene (UHMWPE) has been used as the bearing surface of total joint replacements for over four decades. These polymeric devices are susceptible to accumulated cyclic damage in vivo. Wear debris formation that ultimately leads to a need for revision surgery is linked to the plasticity, fatigue and fracture mechanisms of UHMWPE. This paper examines the deformation, yielding, fracture and fatigue behavior of conventional and highly cross-linked medical grade UHMWPE. Such properties play an important role in determining the long-term success of orthopedic devices. The mechanical properties discussed include the deformation behavior of UHMWPE, the yielding associated with quasi-static tension and compression, fracture toughness, cyclic loading, and fatigue resistance.
Keywords: UHMWPE; Deformation; Yield; Fracture; Fatigue;

Nitric oxide-releasing sol–gels as antibacterial coatings for orthopedic implants by Brian J. Nablo; Aaron R. Rothrock; Mark H. Schoenfisch (917-924).
To assess the benefits of nitric oxide (NO)-releasing sol–gels as potential antibacterial coatings for orthopedic devices, medical-grade stainless steel is coated with a sol–gel film of 40% N-aminohexyl-N-aminopropyltrimethoxysilane and 60% isobutyltrimethoxysilane. Upon converting the diamine groups in these films to diazeniumdiolate NO donors, the NO release from the sol–gel-coated stainless steel is evaluated at both ambient and physiological temperature. Sol–gel films incubated at 25°C have a lower NO flux over the first 24 h compared to those at 37°C, but release more than five times longer. The bacterial adhesion resistance of NO-releasing coatings is evaluated in vitro by exposing bare steel, sol–gel, and NO-releasing sol–gel-coated steel to cell suspensions of Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis at 25°C and 37°C. Cell adhesion to bare and sol–gel-coated steel is similar, while NO-releasing surfaces have significantly less bacterial adhesion for all species and temperatures investigated.
Keywords: Nitric oxide; Sol–gel; Stainless steel; Pseudomonas aeruginosa; Staphylococcus aureus; Staphylococcus epidermidis;

Surface characteristics of holmium-loaded poly(l-lactic acid) microspheres by S.W. Zielhuis; J.F.W. Nijsen; R. Figueiredo; B. Feddes; A.M. Vredenberg; A.D. van het Schip; W.E. Hennink (925-932).
Radioactive holmium-166-loaded poly(l-lactic acid) microspheres (Ho-PLLA-MS) are promising systems for the treatment of liver malignancies. The surface characteristics of Ho-PLLA-MS before and after both neutron and gamma irradiation were investigated in order to get insight into their suspending behaviour and to identify suitable surfactants for clinical application of these systems. X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were used for surface characterization. The residual amounts of poly(vinyl alcohol) (PVA) of the microspheres, which was used as an emulsifier during the solvent evaporation process, were determined using a colorimetric iodine-borate method and the wettability of microspheres and PLLA films with and without holmium (Ho) loading was tested using suspending experiments and contact angle measurements.XPS showed that the surface of Ho-PLLA-MS mainly consisted of PLLA, less than 10% of the surface was covered with PVA after several washing and sieving steps. A colorimetric assay showed that the microspheres contained 0.2–0.3% (w/w) PVA. Combined with XPS data, this assay demonstrates that the PVA is likely dissolved in the core of the microspheres. XPS analysis also showed that after neutron irradiation, some holmium appeared on the surface. Moreover, Ho-loaded PLLA films had a much higher contact angle (85°) than non-loaded films (70°). Therefore, the Ho on the surface of neutron-irradiated Ho-PLLA-MS is probably the reason for their poor suspending behaviour in saline. No surface changes were seen with XPS after gamma irradiation.Based on their surface characteristics, a pharmaceutically acceptable solvent (1% Pluronic F68 or F127 in 10% ethanol) was formulated with which a homogeneous suspension of radioactive Ho-PLLA-MS could be easily obtained, making these systems feasible for further clinical evaluation.
Keywords: PLLA; PVA; Microspheres; XPS; Holmium; Liver malignancies; Irradiation;

Physico-chemical studies of the gelation of chitosan in a hydroalcoholic medium by Alexandra Montembault; Christophe Viton; Alain Domard (933-943).
The formation of chitosan physical hydrogels without any external cross-linking agent was studied. This gelation took place in an acetic acid-water-propanediol solution. Static light scattering was used to detect the gel point and then, to study the gelation for different initial conditions. Thus, we investigated the influence of the degree of acetylation, the gelation temperature and the nature of the initial solvent. The variation of the solvent composition during gelation was determined from a simple weighting, and the ionisation state of the polymer at the gel point, by pH titrations. This work showed that it was possible to form a chitosan physical-hydrogel, whatever the degree of acetylation provided typical conditions were observed. The mechanism of gelation simply consisted in the modification of the hydrophilic/hydrophobic balance allowing the formation of both hydrophobic interactions and hydrogen bonding. Several parameters had an important role on this mechanism: 1—the apparent charge density of chitosan, modified by the degree of neutralisation, 2—the dielectric constant of the solvent, related to the composition of the medium, 3—the degree of acetylation, 4—temperature, playing a role on the interactions responsible for the physical cross-linking and the molecular mobility, and, 5—the molecular mobility depending on possible changes of conformation, steric hindrance and viscosity of the medium.
Keywords: Chitin/chitosan; Physical hydrogel; Sol–gel transition; Degree of acetylation; Charge density; Molecular mobility;

Modeling of dispersed-drug release from two-dimensional matrix tablets by Y. Zhou; J.S. Chu; T. Zhou; X.Y. Wu (945-952).
A mathematical model was developed and analytical solutions were obtained for dispersed-drug release from two-dimensional matrix tablets in a perfect sink. This model can be used to describe kinetics of solute release from matrices with isotropic or anisotropic properties. Moving boundaries of dispersed-drug in both radial and axial directions and release kinetics were predicted by the model. Various factors influencing release kinetics were analyzed including the ratio of initial solute loading (C 0) to solute solubility (C s), the anisotropy of the matrix and the aspect ratio of tablet radius to the half-thickness. The model is also applicable to 1-D planar or 1-D cylindrical geometries when R/H is larger than 100 or smaller than 0.01.
Keywords: Modelling; Drug release; Dispersed-solute; Two-dimensional matrix tablets; Isotropic/anisotropic materials;