Biomaterials (v.26, #3)

Calendar (I).

Poly (dimethyl siloxane) (PDMS) has been widely used as a biomaterial in ophthalmic and other applications due to its good compatibility, high mechanical strength, excellent oxygen permeability and transparency. However, for use as an artificial cornea, contact lens and in other applications, modifications with hydrophilic functional groups or polymers are necessary to improve wettability for tear protein and mucin interactions and to improve glucose permeability for cellular health. Poly (N-isopropyl acrylamide) (PNIPAAM) is a biocompatible and hydrophilic polymer that has been extensively studied on controlled drug release applications due to its lower critical solution temperature (LCST) phenomenon. In the current work, a composite interpenetrating network (IPN) of PDMS and PNIPAAM was formed to generate polymers with oxygen and glucose permeability as well as improved wettability compared to PDMS homopolymers and greater mechanical strength than PNIPAAM homopolymers. Transparent vinyl and hydroxyl terminated PDMS/PNIPAAM IPNs (PDMS-V and PDMS-OH IPNs, respectively) were successfully synthesized. Transmission electron microscopy images verified the structure of the IPNs. Surface analysis suggested that PNIPAAM was present on the surface as well as in the bulk material. PDMS-OH IPNs generated from a PDMS-OH matrix cured in the presence of solvent had the highest glucose permeability at 10−7  cm2/s, comparable to that of the native cornea. The LCST phenomenon remained in these materials, although changes were not as abrupt as with pure PNIPAAM. These results suggest that these materials may be further developed as ophthalmic biomaterials or for controlled drug-release applications.
Keywords: Poly (dimethyl siloxane); Poly (N-isopropylacrylamide); Interpenetrating network; Glucose permeation; Artificial cornea;

This paper describes the effect of temperature on the nucleation of corrosion pits on titanium microelectrodes in Ringer's physiological solution. The results are shown for potentials far below the pitting potential, and describe breakdown of passivity with no permanent propagation of pits. Nucleation events could be observed at all the temperatures used, although they were very rare events at 20°C. The frequency of breakdown rises significantly with increase in temperature. Examples are shown of current transients due to both pit nucleation and to metastable pit propagation, the latter being rare events. Analysis shows that these events constitute a significant fraction of the passive corrosion rate of titanium.
Keywords: Corrosion; Pitting corrosion; Titanium; Microelectrode; Passivation; Breakdown;

A 3D analysis of mechanically stressed dentin–adhesive–composite interfaces using X-ray micro-CT by Roberto De Santis; Francesco Mollica; Davide Prisco; Sandro Rengo; Luigi Ambrosio; Luigi Nicolais (257-270).
Dentin bonding systems (DBS) have been developed in order to bond restorative materials (i.e. composite) to tooth tissues when function and integrity have to be re-established. Adhesion to dentin results from the penetration of DBS into the demineralised substrate constituted by a conditioned collagen network. The long-term stability of a restored tooth is mainly affected by the seal of the restorative material on the dental structures.Although leakage through the dentin–DBS interface has been widely reported, 3D investigation technique and accurate non-destructive measurements of leakage as functions of mechanical cycling have never been provided. To address these issues, the properties of the material interface are analysed using micro-tensile static and dynamic tests, assisted by the finite element modelling and by the X-ray computed micro-tomography. The dual energy absorption technique, with the synchrotron beam light, has been developed to investigate, in a non-destructive manner, the effect of mechanical cycling on leakage of a silver nitrate staining solution at the dentin–DBS interface.The effect of the pulpal roof on the stress distribution in the coronal dentin–DBS–composite interface has been investigated and the level at which the state of stress can be assumed to be uniform within acceptable limits has been defined.The tensile static and dynamic results suggest that the adhesive strength for the multi-step DBS resulted significantly higher than the other investigated DBS.Imaging results indicate that 3D leakage occurs radially at the dentin–adhesive interface through the interface itself rather than through the unconditioned dentin bulk; moreover, the dynamic tensile loading allows a more diffuse staining penetration.
Keywords: Dentine; Polymer; Interface; Dual energy X-ray absorption;

Nanoscale variation in surface charge of synthetic hydroxyapatite detected by chemically and spatially specific high-resolution force spectroscopy by Jennifer Vandiver; Delphine Dean; Nelesh Patel; William Bonfield; Christine Ortiz (271-283).
The normal intersurface forces between nanosized probe tips functionalized with COO- and NH3 +-terminated alkanethiol self-assembling monolayers and dense polycrystalline phase pure synthetic hydroxyapatite (HA) were measured via a powerful nanomechanical technique called chemically specific high-resolution force spectroscopy. The data taken on approach of the probe tip to the HA surface was compared to the nonlinear Poisson–Boltzmann-based electrostatic double layer theory to predict the surface charge per unit area of the HA, σ HA (C/m2), as a function of ionic strength, position within a variety of grains, and across grain boundaries. The average σ HA was found to be ∼−0.02 C/m2 and to vary from −0.0037 to −0.072 C/m2 with nanoscale position in relation to grain boundaries and crystal planes up to −0.19 C/m2/μm. Positional measurement of nanoscale surface properties holds great promise in elucidating the molecular origins of physicochemical processes occurring at the biomaterial interface.
Keywords: Hydroxyapatite; Surface analysis; Bioactivity; Atomic force microscopy; Bone repair; Biocompatibility;

The effect of biomimetic apatite structure on osteoblast viability, proliferation, and gene expression by Yu-Fen Chou; Weibiao Huang; James C.Y Dunn; Timothy A Miller; Benjamin M Wu (285-295).
The conventional biomimetic apatite coating process can be accelerated by immersing substrates into concentrated simulated body fluid (5× SBF) at 37°C to form an initial coating of apatite precursor spheres, and transform the precursors into plate-like apatite structures. Depending on processing parameters, different apatite structures can be created over the same substrate. The purpose of this study is to investigate the effects of the different apatite microenvironment on cell spreading, viability, proliferation, and gene expression. MC3T3-E1 preosteoblasts were cultured on five surfaces: conventional apatite (CA), precursor apatite spheres (PreA), large plate-like apatites (LgA), small plate-like apatites (SmA), and tissue culture grade polystyrene (TCPS). PreA induced significantly higher cell death during the first two weeks. TCPS supported more uniform spreading (1 day) and higher proliferation (2 weeks) than CA, LgA, and SmA. Apatites restricted spreading and promoted the extension of cellular projections along the textured surfaces under confocal microscopy observation. By 3 weeks, LgA induced highest expression of mature osteogenic markers osteocalcin (OCN) and bone sialoprotein (BSP) in both regular and osteogenic culture media based on quantitative real-time RT-PCR. The results of this study suggest differential cell responses to subtle changes in apatite microenvironment.
Keywords: Apatite structure; Bone tissue engineering; Biomimetic material; Osteoblast; Gene expression; mRNA;

Rapid fabrication of keratin–hydroxyapatite hybrid sponges toward osteoblast cultivation and differentiation by Akira Tachibana; Sumika Kaneko; Toshizumi Tanabe; Kiyoshi Yamauchi (297-302).
Wool keratin sponges were reported to be useful scaffolds for long-term and high-density cell cultivation (J. Biotechnol. 93 (2002) 165). The hybrid of the keratin sponges with calcium phosphate materials gave the additional function. Two rapid fabrication methods for calcium phosphate hybrid biomaterials were described. Firstly, the CaP-precipitated sponges were obtained by only the immersion of the carboxyl-sponges, chemically introduced with high amount of carboxyl groups on the sponges, in calcium and phosphate ions containing buffers such as PBS(+) for only 1–3 days. Neither sponge, introduced with amino or amido groups or non-treated, gave significant calcium phosphate precipitation. The carboxyl-sponges were mimics of matrix gamma-carboxyglutamic acid protein, which is responsible for osteoblast calcification. Secondly, the hydroxyapatite particle suspension was added onto carboxyl-sponges to fabricate trapped sponge. The trapped hydroxyapatite particles might interact with keratin protein of the sponge walls. Preliminary experiments measuring the expression of alkaline phosphatase, early osteoblast differentiation marker, suggested that both hybrid sponges, CaP-precipitated and trapped sponges, alter the differentiation pattern of preosteoblasts, MC3T3-E1.
Keywords: Wool keratin sponge; Hydroxyapatite hybrid; Osteoblast scaffolds; Osteoblast differentiation;

Effects of a bone-like mineral film on phenotype of adult human mesenchymal stem cells in vitro by William L Murphy; Susan Hsiong; Thomas P Richardson; Craig A Simmons; David J Mooney (303-310).
Multipotent cell types are rapidly becoming key components in a variety of tissue engineering schemes, and mesenchymal stem cells (MSCs) are emerging as an important tool in bone tissue regeneration. Although several soluble signals influencing osteogenic differentiation of MSCs in vitro are well-characterized, relatively little is known about the influence of substrate signals. This study was aimed at elucidating the effects of a bone-like mineral (BLM), which is vital in the process of bone bonding to orthopedic implant materials, on the osteogenic differentiation of human MSCs in vitro. Growth of a BLM film (carbonate apatite, Ca/P=1.55) on poly(lactide-co-glycolide) (PLG) substrates was achieved via surface hydrolysis and subsequent incubation in a modified simulated body fluid. The BLM film demonstrated significantly increased adsorption of fibronectin, and supported enhanced proliferation of human mesenchymal stem cells (hMSCs) relative to PLG substrates. In the absence of osteogenic supplements hMSCs did not display a high expression of osteogenic markers on BLM or PLG. In the presence of osteogenic supplements hMSCs exhibited greater expression of osteogenic markers on PLG substrates than on BLM substrates, as measured by alkaline phosphatase activity and osteocalcin production. Taken together, these data support the concept that substrate signals significantly influence MSC growth and differentiation, highlighting the importance of carrier material composition in stem cell-based tissue engineering schemes.
Keywords: Biomineralization; Bone; Poly(lactide-co-glycolide); Tissue engineering; Biomimetic;

Tissue engineering of bone: effects of mechanical strain on osteoblastic cells in type I collagen matrices by A Ignatius; H Blessing; A Liedert; C Schmidt; C Neidlinger-Wilke; D Kaspar; B Friemert; L Claes (311-318).
The aim of the present study was to investigate the effect of cyclic uniaxial mechanical strain on a human osteoblastic precursor cell line (hFOB 1.19) in three-dimensional type I collagen matrices. Cell seeded collagen constructs were mechanically stretched by a daily application of cyclic uniaxial strain using a special motor-driven apparatus and compared to unstretched controls. Expression of genes involved in cell proliferation and osteoblastic differentiation as well as matrix production were investigated by analyzing the mRNA of histone H4, core binding factor 1, alkaline phosphatase, osteopontin, osteocalcin, and collagen type I (Col I) up to a cultivation period of 3 weeks using real-time PCR. Cyclic stretching of cell seeded Col I matrices at a magnitude occurring in healing bone increased cell proliferation and slightly elevated the expression of nearly all investigated genes over unstrained controls at various time points. It was concluded that mechanical load promotes the proliferation and differentiation of osteoblastic precursor cells in a Col I matrix and that the application of mechanical stimuli may have a beneficial effect on in vitro tissue formation.
Keywords: Bone tissue engineering; Collagen type I matrices; Three-dimensional cell culture; Mechanical strain; Osteoblastic differentation;

Implantation of bone marrow mononuclear cells using injectable fibrin matrix enhances neovascularization in infarcted myocardium by Ju Hee Ryu; Il-Kwon Kim; Seung-Woo Cho; Myeong-Chan Cho; Kyung-Kuk Hwang; Hainan Piao; Shuguang Piao; Sang Hyun Lim; Yoo Sun Hong; Cha Yong Choi; Kyung Jong Yoo; Byung-Soo Kim (319-326).
Neovascularization may improve cardiac function and prevent further scar tissue formation in infarcted myocardium. A number of studies have demonstrated that bone marrow-derived cells have the potential to induce neovascularization in ischemic tissues. In this study, we hypothesized that implantation of bone marrow mononuclear cells (BMMNCs) using injectable fibrin matrix further enhances neovascularization in infarcted myocardium compared to BMMNC implantation without matrix. To test this hypothesis, infarction was induced in rat myocardium by cryoinjury. Three weeks later, rat BMMNCs were mixed with fibrin matrix and injected into the infarcted myocardium. Injection of either BMMNCs or medium alone into infarcted myocardium served as controls. Eight weeks after the treatments, histological analyses indicated that implantation of BMMNCs using fibrin matrix resulted in more extensive tissue regeneration in the infarcted myocardium compared to BMMNC implantation without matrix. Examination with fluorescence microscopy revealed that cells labeled with a fluorescent dye prior to implantation survived in the infarcted myocardium at 8 weeks of implantation. Importantly, implantation of BMMNCs using fibrin matrix resulted in much more extensive neovascularization in infarcted myocardium than BMMNC implantation without matrix. The microvessel density in infarcted myocardium was significantly higher (p<0.05) when BMMNCs were implanted using fibrin matrix (350±22 microvessels/mm2) compared to BMMNC implantation without matrix (262±13 microvessels/mm2) and medium injection (76±9 microvessels/mm2). In addition, average internal diameter of microvessels was significantly larger (p<0.05) in BMMNC implantation with fibrin matrix group (14.6±1.2 μm) than BMMNC implantation without matrix group (10.2±0.7 μm) and medium injection group (7.3±0.5 μm). These results suggest that fibrin matrix could serve as a cell implantation matrix that enhances neovascularization efficacy for myocardial infarction treatment.
Keywords: Fibrin marix; Bone marrow mononuclear cells; Myocardial infarction; Neovascularization;

New promising techniques for depositing hydroxyapatite (HA) and calcium phosphate (CaP) coatings on medical devices are continuously being investigated. Given the vast number of experimental deposition process currently available, this review will focus only on CaP and/or HA coatings produced using the sputtering process. This review will discuss the characterization of sputtered CaP coatings before and after post-deposition treatments and tissue responses to some of the characterized coating surfaces. From the studies observed in the literature, current research on sputtered CaP coatings has shown some promises that may eliminate some of the problems associated with the plasma-spraying process. It has been generally accepted that sputtered HA and CaP coatings improve bone strength and initial osseointegration rate. However, optimal coating properties required to achieve maximal bone response are yet to be reported. As such, the use of well-characterized sputtered CaP and/or HA surfaces in the evaluation of biological responses should be well documented to avoid controversial results. In addition, future investigations of the sputtering process should include clinical trials, to continue the understanding of bone responses to coated-implant surfaces of different properties, and the possibility of coupling sputtered HA and CaP coatings with growth factors.
Keywords: Sputter coatings; Calcium phosphate; Hydroxyapatite; Biological responses; Materials characterization;

Periodontal ligament and gingival fibroblast adhesion to dentin-like textured surfaces by Corinna Bruckmann; X.Frank Walboomers; Kenichi Matsuzaka; John A Jansen (339-346).
It is known that the (micro-) structure of a substrate surface is of major influence on the growth behaviour of adherent cells. In the current study, we aimed to produce a surface that exactly mimics the structure of natural dentin, and to describe the effect of this surface on the growth behaviour of primary periodontal ligament fibroblasts (PDLF) or gingival fibroblasts (GF). First, we used scanning electron microscopy (SEM) and morphometric techniques to analyse the porous dentin structure. Then, using a template made by photolithographic techniques, cell culture dishes with similar surface structure were made. On these dishes, and on smooth controls, primary PDLF and GF were seeded and assayed up to 14 days for proliferation, alkaline phosphatase (ALP) activity, and collagen content. Also, cell morphology was observed with SEM and transmission electron microscopy (TEM). Results showed that GF showed significantly less ALP activity than PDLF. Abundant collagen fibres were only formed by GF grown on the textured surfaces. SEM assessment showed equal spreading of both cell types on smooth and textured surfaces. TEM showed a preferential deposition of ECM material in the texture porosity. From our study we can conclude that dentin-like surfaces have no negative effect on either cell type, and could be used to enhance extracellular matrix deposition in GF formation. However, considerable differences were observed between primary cells from different animals. Therefore, final efficacy of the surfaces remains to be proven in implantation experiments.
Keywords: Cell adhesion; Dentin; Dental implant; ECM (extracellular matrix); Fibroblast; Micropatterning; Surface topography; Biomimetic material;

Erratum to “Oxidation of orthopaedic UHMWPE” by L Costa; K Jacobson; P Bracco; E.M.Brach del Prever (347-348).