Biomaterials (v.27, #11)

Calendar (I).

The significance of infection related to orthopedic devices and issues of antibiotic resistance by Davide Campoccia; Lucio Montanaro; Carla Renata Arciola (2331-2339).
Over the last 15 years, with the advent of modern standards in the control of sterility within the operating room environment and adequate protocols of peri-operative antibiotic prophylaxis, the incidence of infections associated to orthopedic implants has become very low. Nevertheless, the event of infection still represents one of the most serious and devastating complications which may involve prosthetic devices. It leads to complex revision procedures and, often, to the failure of the implant and the need for its complete removal. In orthopedics, for the enormous number of surgical procedures involving invasive implant materials, even if nowadays rare, infections have a huge impact in terms of morbidity, mortality, and medical costs.The difficult battle to prevent and fight bacterial infections associated to prosthetic materials must be played on different grounds. A winning strategy requires a clear view of the pathogenesis and the epidemiology of implant-related infections, with a special attention on the alarming phenomenon of antibiotic resistance. In this regard staphylococci are the prevalent and most important causative pathogens involved in orthopedic implant-related infections, and, thus, the main enemy to defeat. In this paper, we offer an overview of the complexity of this battleground and of the current and new, in our opinion most promising, strategies in the field of biomaterials to reduce the risks and counteract the establishment of implant infections.
Keywords: Orthopedic surgery; Microbial infections; Implant materials; Staphylococcus; Antibiotic resistance;

Surface study of collagen/poloxamine hydrogels by a ‘deep freezing’ ToF-SIMS approach by Alejandro Sosnik; Rana N.S. Sodhi; Peter M. Brodersen; Michael V. Sefton (2340-2348).
In order to determine the presence of collagen molecules at the surface of a collagen-modified poloxamine hydrogel (a semi-interpenetrating network), the surface composition was studied using Time-of-Flight Secondary Ion Mass Spectra (ToF-SIMS). Collagen was added to the poloxamine hydrogel (poloxamine is a commercially available four-arm poly(ethylene oxide)/poly(propylene oxide) block copolymer, PEO/PPO) to promote the attachment of endothelial or liver cells. X-ray photoelectron spectroscopy (XPS) of dry samples showed a sharp increase in the N content from 0.6% in a pure poloxamine hydrogel to 8.8% in the collagen-containing material. Afterwards, the surface was studied by a ‘deep freezing’ ToF-SIMS approach under progressive heating from −120 to −60 °C. The positive spectrum of collagen/poloxamine at −65 °C displayed distinct signals corresponding to different amino acid fragments such as CH4N+ (30  m / z , Gly), C3HN2 + (43  m / z , Arg), C2H6N+ (44  m / z , Ala) and C4H5N2 + (81  m / z , His) and others corresponding to the PEO and PPO blocks of poloxamine. In addition, the negative spectrum showed peaks at 26  m / z (CN), 32  m / z (S) and 42  m / z (CNO) characteristic of fragments of the collagen molecule. Imaging experiments indicated the homogeneous distribution of the collagen on the surface. These results supported the use of ToF-SIMS for the surface characterization of hydrated hydrogels and confirmed the collagen presence as the means whereby cells attach to the modified poloxamine matrix.
Keywords: Surface analysis; ‘Deep freezing’ ToF-SIMS; Hydrogels; Collagen; Poloxamine;

Strong mechanical properties and controllable biodegradability, together with biocompatibility, are the important requirement for the development of medical implant materials. In this study, an ultraviolet (UV) radiation method was developed to achieve controlled degradation for bacterial biopolyester poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) which has a low biodegradation rate that limits its application for many implant applications required quick degradation. When UV radiation was applied directly to PHBHHx powder, significant molecular weight (Mw) losses were observed with the powder, Mw reduction depended on the UV radiation time. At the same time, a broad PHBHHx Mw distribution was the result of inhomogeneous radiation. Interestingly, this inhomogeneous radiation helped maintain the mechanical properties of films made of the UV-radiated powder. In comparison, the PHBHHx films subjected to direct UV radiation became very brittle although their degradation was faster than that of the PHBHHx powders subjected to direct UV radiation. After 15 weeks of degradation in simulated body fluid (SBF), films prepared from 8 and 16 h UV-treated PHBHHx powders maintained 92% and 87% of their original weights, respectively, while the untreated PHBHHx films lost only 1% of its weight. Significant increases in growth of fibroblast L929 were observed on films prepared from UV-radiated powders. This improved biocompatibility could be attributed to increasing hydrophilic functional groups generated by increasing polar groups C–O and C=O. In general, UV-treated PHBHHx powder had a broad Mw distribution, which contributed to fast degradation due to dissolution of low Mw polymer fragments, and strong mechanical property due to high Mw polymer chains. Combined with its improved biocompatibility, PHBHHx is one more step close to become a biomedical implant material.
Keywords: UV radiation; Biodegradation; PHBHHx; PHB; Mechanical property;

Increased osteoblast functions on undoped and yttrium-doped nanocrystalline hydroxyapatite coatings on titanium by Michiko Sato; Marisa A. Sambito; Arash Aslani; Nader M. Kalkhoran; Elliott B. Slamovich; Thomas Jay Webster (2358-2369).
In order to improve orthopedic implant performance, the objective of this in vitro study was to synthesize nanocrystalline hydroxyapatite (HA) powders to coat titanium. HA was synthesized through a wet chemical process. The precipitated powders were either sintered at 1100 °C for 1 h in order to produce UltraCap HA (or microcrystalline size HA) or were treated hydrothermally at 200 °C for 20 h to produce nanocrystalline HA. Some of the UltraCap and nanocrystalline HA powders were doped with yttrium (Y) since previous studies demonstrated that Y-doped HA in bulk improved osteoblast (or bone-forming cell) function over undoped HA. The original HA particles were characterized using X-ray diffraction (XRD), inductively coupled plasma–atomic emission spectroscopy (ICP–AES), BET, a laser particle size analyzer, and scanning electron microscopy (SEM). These powders were then deposited onto titanium by a novel room-temperature process, called IonTite™. The properties of the resulting HA-coatings on titanium were compared to respective properties of the original HA powders. The results showed that the chemical and physical properties of the original HA powders were retained when coated on titanium by IonTite™, as determined by XRD, SEM, and atomic force microscopy (AFM) analysis. More importantly, results showed increased osteoblast adhesion on the nanocrystalline HA IonTite™ coatings compared to traditionally used plasma-sprayed HA coatings. Results also demonstrated greater amounts of calcium deposition by osteoblasts cultured on Y-doped nanocrystalline HA coatings compared to either UltraCap IonTite™ coatings or plasma-sprayed HA coatings. These results encourage further studies on nanocrystalline IonTite™ HA coatings on titanium for improved orthopedic applications.
Keywords: Nanocrystalline; Y-doped hydroxyapatite; Plasma-spray; Osteoblast; Orthopedic; Nanotechnology;

Strategies for spinal cord injury repair are limited, in part, by poor drug delivery techniques. A novel drug delivery system (DDS) is being developed in our laboratory that can provide localized release of growth factors from an injectable gel. The gel must be fast-gelling, non-cell adhesive, degradable, and biocompatible as an injectable intrathecal DDS. A gel that meets these design criteria is a blend of hyaluronan and methylcellulose (HAMC). Unlike other injectable gels, HAMC is already at the gelation point prior to injection. It is injectable due to its shear-thinning property, and its gel strength increases with temperature. In vivo rat studies show that HAMC is biocompatible within the intrathecal space for 1 month, and may provide therapeutic benefit, in terms of behavior, as measured by the Basso, Beattie and Bresnahan (BBB) locomotor scale, and inflammation. These data suggest that HAMC is a promising gel for localized delivery of therapeutic agents to the injured spinal cord.
Keywords: Biocompatibility; Injectable; Spinal cord injury; Shear-thinning; Thermally responsive material; Intrathecal;

Physical characterization of vascular grafts cultured in a bioreactor by Laura Buttafoco; Paula Engbers-Buijtenhuijs; Andre A. Poot; Piet J. Dijkstra; Istvan Vermes; Jan Feijen (2380-2389).
Tubular scaffolds of collagen and elastin (weight ratio 1:1) with interconnected pores were prepared by freeze drying and crosslinked with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in the presence or absence of a Jeffamine spacer (poly(propylene glycol)-bis-(2-aminopropyl ether), J230). The crosslinked and uncrosslinked matrices had porosities of 90% and average pore sizes of 131–151 μm. Smooth muscle cells (SMC) were cultured in the crosslinked and uncrosslinked tubular scaffolds under pulsatile flow conditions (mean flow rate 9.6 ml/min, 120 beats/min, pressure 80–120 mmHg). All the constructs could withstand cyclic mechanical strain in the absence of any mechanical support without cracking or suffering permanent deformation. After 7 d, SMC were homogeneously distributed throughout the uncrosslinked and EDC/NHS crosslinked constructs, whereas hardly any cell was observed on the luminal side of J230/EDC/NHS crosslinked matrices. Considering the better mechanical performance of EDC/NHS crosslinked matrices compared to non-crosslinked constructs after 7 d of culture, SMC were dynamically cultured in the former scaffolds for 14 d. During this period, the high strain stiffness of the constructs increased more than two-fold to 38±2 kPa, whereas the low strain stiffness doubled to 8±2 kPa. The yield stress and yield strain were 30±10 kPa and 120±20%, respectively. SMC were homogeneously distributed throughout the EDC/NHS crosslinked collagen/elastin constructs and collagen fibres tended to orient in the circumferential direction.
Keywords: Collagen; Crosslinking; Elastin; Smooth muscle cells; Vascular grafts;

Biological characterisation of vascular grafts cultured in a bioreactor by Paula Engbers-Buijtenhuijs; Laura Buttafoco; Andre A. Poot; Piet J. Dijkstra; Rob A.I. de Vos; Lotus M.Th. Sterk; Rob H. Geelkerken; Istvan Vermes; Jan Feijen (2390-2397).
In this study, the development is described of a tissue-engineered construct mimicking the structure of a natural blood vessel. Smooth muscle cells (SMC) were cultured under pulsatile flow conditions in porous tubular scaffolds composed of crosslinked type I insoluble collagen and insoluble elastin. Under these dynamic culture conditions, average wall shear rate, systolic and diastolic pressures and pressure wave-forms comparable to conditions in the human carotid artery were obtained. Culturing of SMC in tubular scaffolds under dynamic conditions resulted in enhanced tissue formation compared to static conditions. Higher SMC numbers, a more homogeneous distribution of SMC throughout the scaffolds and higher collagen mRNA expression levels were found when cells were cultured under dynamic compared to static conditions. mRNA expression levels of markers of proliferation and apoptosis showed that the higher cell numbers in the scaffolds cultured under dynamic conditions can be explained by increased cell proliferation but not by decreased apoptosis. Glucose consumption and lactate formation by the cells showed that cell metabolism was more aerobic under dynamic compared to static conditions. Lining of the dynamically cultured constructs with a luminal monolayer of endothelial cells might result in vessels suitable for in vivo applications.
Keywords: Vascular tissue engineering; Smooth muscle cells; Collagen; Elastin; Cell proliferation; Apoptosis;

The role of MMP-I up-regulation in the increased compliance in muscle-derived stem cell-seeded small intestinal submucosa by Rebecca A. Long; Jiro Nagatomi; Michael B. Chancellor; Michael S. Sacks (2398-2404).
We have previously observed that muscle-derived stem cells (MDSC) seeded onto porcine small intestinal submucosa (SIS) increase the mechanical compliance of the engineered tissue construct [Lu SH, Sacks MS, Chung SY, Gloeckner DC, Pruchnic R, Huard J, et al. Biaxial mechanical properties of muscle-derived cell seeded small intestinal submucosa for bladder wall reconstitution. Biomaterials 2005;26(4):443–9]. To date, however, the initial remodeling events which occur when MDSC are seeded onto SIS have yet to be elucidated. One potential mechanism responsible for the observed increase in mechanical compliance is the release of matrix metalloproteinase-I (MMP-I). To investigate this finding, MDSC (∼1×106) were cultured on single-layer SIS cell culture inserts (4.7 cm2) for 1–10 days. MDSC MMP-I activity on SIS in the supernatant at 1, 3, 5, 7, and 10 days was determined using a collagenase assay kit. MMP-I activity of the MDSC/SIS was significantly higher (p<0.0025) after one day in culture compared to specimens collected from subsequent time points and the unseeded control. To further study the initial remodeling events, the impact of MMP-I on mechanical compliance was examined. SIS was incubated with 0.16 U/mL collagenase-I for 3, 4.5, 5, and 24 h, then biaxial mechanical testing was performed. After 5 h of digestion with collagenase-I, mechanical compliance under 1 MPa peak stress was increased by 7% in the circumferential direction, compared to control SIS. These findings suggest that the release of MMP-I in response to initial seeding on SIS and subsequent breakdown of collagen fibers is the mechanism responsible for an increase in mechanical compliance.
Keywords: Muscle-derived stem; SIS; Biaxial mechanical testing; Bladder wall; Tissue engineering;

Polypyrrole doped with 2 peptide sequences from laminin by William R. Stauffer; Xinyan T. Cui (2405-2413).
In the field of neural tissue engineering, electrically conducting, biocompatible surfaces are of great interest. Over the past several decades conducting polymers have been studied as candidate surfaces because they fit these criteria. Several attempts have been made to combine the conductivity and biocompatibility of conducting polymers with biomolecules that could promote specific cell attachment and growth. In this report the laminin fragments CDPGYIGSR (p31) and RNIAEIIKDI (p20) are used as dopants in electropolymerization of the conducting polymer polypyrrole (PPy). The electrical properties of the resulting films are analyzed by impedance spectroscopy and cyclic voltammetry and compared to gold. PPy/p20 surfaces consistently demonstrate the lowest impedance and largest charge capacity for a given deposition charge. Next, in vitro studies using primary neurons cultured in a defined media and primary astrocytes in a serum containing media were performed; neuron density and neurite length, as well as astrocyte density, were quantified. Surfaces doped with a combination of the two peptides (PPy/p20-p31) consistently supported the highest neuronal density. It is shown that surfaces doped with the laminin fragment p20 had significantly longer primary neurites than either the p31 doped or poly(styrenesulfonate) doped PPy surfaces. Finally, the astrocyte studies demonstrate that PPy surfaces have significantly less astrocyte adhesion in culture than the common electrode material, gold.
Keywords: Biocompatibility; Neural cell; Neural prosthesis; Laminin; Cell adhesion; Brain; Electroactive polymer; Electrochemistry;

45S5 Bioglass®-derived glass–ceramic scaffolds for bone tissue engineering by Qizhi Z. Chen; Ian D. Thompson; Aldo R. Boccaccini (2414-2425).
Three-dimensional (3D), highly porous, mechanically competent, bioactive and biodegradable scaffolds have been fabricated for the first time by the replication technique using 45S5 Bioglass® powder. Under an optimum sintering condition (1000 °C/1 h), nearly full densification of the foam struts occurred and fine crystals of Na2Ca2Si3O9 formed, which conferred the scaffolds the highest possible compressive and flexural strength for this foam structure. Important findings are that the mechanically strong crystalline phase Na2Ca2Si3O9 can transform into an amorphous calcium phosphate phase after immersion in simulated body fluid for 28 days, and that the transformation kinetics can be tailored through controlling the crystallinity of the sintered 45S5 Bioglass®. Therefore, the goal of an ideal scaffold that provides good mechanical support temporarily while maintaining bioactivity, and that can biodegrade at later stages at a tailorable rate is achievable with the developed Bioglass®-based scaffolds.
Keywords: Scaffolds; Bone tissue engineering; Mechanical properties; Bioactivity; Biodegradation; Replication technique;

Demineralized bone matrix gelatin as scaffold for osteochondral tissue engineering by Xudong Li; Li Jin; Gary Balian; Cato T. Laurencin; D. Greg Anderson (2426-2433).
To develop a single-unit osteochondral tissue with demineralized bone matrix gelatin (BMG), rabbit chondrocytes were cultured on demineralized bone matrix gelatin for 6 weeks. The engineered osteochondral tissue was characterized with histology, immunolocalization, TEM, SEM, biochemical assay, and gene expression analysis. About 1.3 mm viable neo-cartilage was produced on demineralized BMG. RT-PCR, immunohistochemistry, TEM, biochemical assay, and histology revealed hyaline-like cartilage with zonal layers, intense type II collagen expression, and abundant proteoglycan content formed upon BMG compared with normal cartilage. But hydroxyproline content and type I collagen gene and protein expressions were significantly lower. We consider engineering cartilage tissue with chondrocytes cultured on allogenic demineralized BMG is a good approach for osteochondral tissue engineering.
Keywords: Cartilage tissue engineering; Chondrocyte; Bone graft; Cell culture; Collagen;

Migration stability of α-tocopherol in irradiated UHMWPE by Ebru Oral; Keith K. Wannomae; Shannon L. Rowell; Orhun Kamil Muratoglu (2434-2439).
The oxidation resistance of irradiated ultra-high molecular weight polyethylene (UHMWPE) components used in total joint arthroplasty can be improved by adding α-tocopherol (vitamin E) through diffusion. To ensure long-term oxidative stability, a minimum α-tocopherol concentration needs to be maintained throughout these components. Migration of α-tocopherol out of the components is one mechanism that could compromise long-term oxidative stability. We hypothesized that α-tocopherol could elute out during standard implant fabrication steps such as cleaning as well as during in vivo use. We doped 85 kGy irradiated UHMWPE with α-tocopherol at 120 °C and homogenized at 120 °C. We determined the extent of elution of α-tocopherol or its effect on oxidative stability following cleaning in isopropyl alcohol (IPA) and following 5 million cycles (MC) of simulated normal gait in bovine serum. There was no significant elution of α-tocopherol in repeated and prolonged cleaning in IPA as measured by average surface and bulk α-tocopherol concentrations. There was no change in the oxidative stability following 5 MC of hip simulator testing, indicating minimal elution during simulated normal gait.
Keywords: Antioxidant; Vitamin E; Highly cross-linked polyethylene; Joint replacement; Degradation; Oxidation;

Although total joint replacement has become commonplace in recent years, bacterial infection remains a significant complication following this procedure. One approach to reduce the incidence of joint replacement infection is to add antimicrobial agents to the bone cement used to fix the implant. In this in vitro study, we investigated the use of chitosan nanoparticles (CS NP) and quaternary ammonium chitosan derivative nanoparticles (QCS NP) as bactericidal agents in poly(methyl methacrylate) (PMMA) bone cement with and without gentamicin. The antibacterial activity was tested against Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). A 103-fold reduction in the number of viable bacterial cells upon contact with the surface was achievable using QCS NP at a nanoparticle/bone cement weight ratio of 15%. The inhibition of S. aureus and S. epidermidis growth on the surface of the CS NP and QCS NP-loaded bone cements was clearly shown using the LIVE/DEAD Baclight bacterial viability kits and fluorescence microscopy. The CS NP and QCS NP also provided a significant additional bactericidal effect to gentamicin-loaded bone cement. The antibacterial effectiveness remained high even after the modified bone cements had been immersed for 3 weeks in an aqueous medium. No cytotoxic effect of the CS NP- and QCS NP-loaded cements was shown in a mouse fibroblast MTT cytotoxicity assay. Mechanical tests indicated that the addition of the CS and QCS in nanoparticulate form allowed the retention of a significant degree of the bone cement's strength. These results indicate a new promising strategy for combating joint implant infection.
Keywords: Antibacterial; PMMA bone cement; Chitosan; Nanoparticles; Mechanical properties;

Combination devices—those comprising drug releasing components together with functional prosthetic implants—represent a versatile, emerging clinical technology promising to provide functional improvements to implant devices in several classes. Landmark antimicrobial catheters and the drug-eluting stent have heralded the entrance, and significantly, routes to FDA approval, for these devices into clinical practice. This review describes recent strategies creating implantable combination devices. Most prominent are new combination devices representing current orthopedic and cardiovascular implants with new added capabilities from on-board or directly associated drug delivery systems are now under development. Wound coverings and implantable sensors will also benefit from this combination enhancement. Infection mitigation, a common problem with implantable devices, is a current primary focus. On-going progress in cell-based therapeutics, progenitor cell exploitation, growth factor delivery and advanced formulation strategies will provide a more general and versatile basis for advanced combination device strategies. These seek to improve tissue–device integration and functional tissue regeneration. Future combination devices might best be completely re-designed de novo to deliver multiple bioactive agents over several spatial and temporal scales to enhance prosthetic device function, instead of the current ‘add-on’ approach to existing implant device designs never originally intending to function in tandem with drug delivery systems.
Keywords: Combination devices; Drug delivery; Stents; Bone cement; Growth factors; Antibiotics;

Delivery of basic fibroblast growth factor (bFGF) from photoresponsive hydrogel scaffolds by Fotios M. Andreopoulos; Indushekhar Persaud (2468-2476).
Exogenous growth factor therapy has shown a notable promise in accelerating the healing of acute and chronic wounds. However, their susceptibility to enzymatic degradation and short contact time with the wound bed warrant the use of sophisticated delivery vehicles that stabilize the encapsulated peptides and control their rate of release. Herein, we describe the synthesis of a nitrocinnamate-derived polyethylene glycol (PEG-NC) hydrogel system and study the release kinetics of basic fibroblast growth factor (bFGF) as a function of hydrogel properties. Long-wave ultraviolet irradiation (365 nm) was used to alter the physical properties of the gel scaffold (i.e. degree of swelling) and consequently control the release rates of the encapsulated bFGF. The degree of swelling (DS) decreased from 10.7 to 8 as the length of irradiation increased from 5 to 30 min. Similarly, the DS decreased from 17.5 to 11.5 by increasing the initial PEG-NC concentration from 10 to 30 w/v% while keeping the crosslinking irradiation at 10 min. Radiolabeled I125 studies were used to monitor the release of bFGF from PEG-NC hydrogels with variable swellabilities. By increasing the length of irradiation from 2 to 10 min the rate of bFGF release from PEG-NC gel scaffolds was decreased by 29% due to the enhanced crosslinking density. The bFGF-releasing PEG-NC hydrogels were not cytotoxic to human neonatal fibroblast cells and the released growth factor maintained its activity and induced fibroblast proliferation and collagen production in vitro. The addition of heparin within the gel scaffolds further increased the growth factor's activity.
Keywords: bFGF; Hydrogels; PEG; Growth factor delivery; Photosensitivity;

Nerve growth factor expression by PLG-mediated lipofection by Kevin J. Whittlesey; Lonnie D. Shea (2477-2486).
Biomaterials capable of efficient gene delivery provide a fundamental tool for basic and applied research models, such as promoting neural regeneration. We developed a system for the encapsulation and sustained release of plasmid DNA complexed with a cationic lipid and investigated their efficacy using in vitro models of neurite outgrowth. Sustained lipoplex release was obtained for up to 50 days, with rates controlled by the fabrication conditions. Released lipoplexes retained their activity, transfecting 48.2±8.3% of NIH3T3 cells with luciferase activity of 3.97×107  RLU/mg. Expression of nerve growth factor (NGF) was employed in two models of neurite outgrowth: PC12 and primary dorsal root ganglia (DRG) co-culture. Polymer-mediated lipofection of PC12 produced bioactive NGF, eliciting robust neurite outgrowth. An EGFP/NGF dual-expression vector identified transfected cells (GFP-positive) while neurite outgrowth verified NGF secretion. A co-culture model examined the ability of NGF secretion by an accessory cell population to stimulate DRG neurite outgrowth. Polymer-mediated transfection of HEK293T with an NGF-encoding plasmid induced outgrowth by DRG neurons. This system could be fabricated as implants or nerve guidance conduits to support cellular and tissue regeneration. Combining this physical support with the ability to locally express neurotrophic factors will potentiate regeneration in nerve injury and disease models.
Keywords: Gene transfer; PLG; Nerve growth factor; Nerve regeneration; Nerve guide;

Combined microscale mechanical topography and chemical patterns on polymer cell culture substrates by Joseph L. Charest; Marcus T. Eliason; Andrés J. García; William P. King (2487-2494).
This paper presents a technique to independently form mechanical topography and surface chemical patterns on polymer cell substrates, and studies the response of osteoblast cells to these surface patterns. The patterns were formed in two separate steps: hot embossing imprint lithography formed the mechanical topography and microcontact printing created the chemical pattern. The resulting substrate had surface features consisting of embossed grooves 4 μm deep and 8 μm wide spaced by 16 μm wide mesas and microcontact printed adhesive lanes 10 μm wide with spacings that ranged from 10 to 100 μm. When presented with either mechanical topography or chemical patterns alone, the cells significantly aligned to the pattern presented. When presented with mechanical topography overlaid with an orthogonal chemical pattern, the cells aligned to the mechanical topography. As the chemical pattern spacing was increased, osteoblasts remained aligned to the mechanical topography. Unlike traditional microfabrication approaches based on photolithography and wet chemistry, the patterning technique presented is compatible with a large number of biomaterials, could form patterns with features much smaller than 1 μm, and is highly scalable to large substrates.
Keywords: Micropatterning; Surface topography; Chemical patterns; Alignment; Adhesion;

N-halamine/quat siloxane copolymers for use in biocidal coatings by J. Liang; Y. Chen; K. Barnes; R. Wu; S.D. Worley; T.-S. Huang (2495-2501).
A series of copolymers incorporating N-halamine siloxane and quaternary ammonium salt siloxane units has been prepared. The primary function of the quat units was to render the siloxane copolymers soluble in water. The copolymers have been coated onto cotton swatches and evaluated for biocidal efficacy against Staphylococcus aureus and Escherichia coli O157:H7. It was determined that both N-halamine and quat functional groups were effective against S. aureus, but only the N-halamine units were effective against Escherichia coli O157:H7. The copolymers should be useful for applications for which aqueous media is preferred over organic solvents to be used during coating procedures.
Keywords: Bacteria; Bioactivity; Siloxane;

Photoimmobilized array of panel cells for assay of antibodies by Yoshihiro Ito; Tetsuya Yamauchi; Makoto Uchikawa; Yoshihide Ishikawa (2502-2506).
Antibodies in blood are checked with panel blood cells before blood transfusion. In this investigation, for the first time, a panel cell-microarray was prepared by using a photoimmobilization method. Different types of red blood cells were microarrayed on a plate. A water-soluble photoreactive polymer as a matrix was synthesized by the coupling reaction of azidoaniline with poly(2-methacryloyloxyethylphosphorylcholine-co-methacrylic acid). The polymer was mixed with cells and the mixtures were microspotted on substrate and photoirradiated after drying in air. For the antibody assay, monoclonal antibodies or human serum was added to the cell-arrayed plate and adsorbed antibodies were detected by horseradish peroxidase-labeled secondary antibody, which recognized the adsorbed antibodies. Antibodies specifically adsorbed on the immobilized cells as expected. The aggregation method has been available for this type of assay, but extensive experience was needed to apply it correctly. The method using a cell array will be useful for antibody detection.
Keywords: Adsorption; Cross-linking; Phosphorylcholine; Protein adsorption;

Age, dehydration and fatigue crack growth in dentin by Devendra Bajaj; Naryana Sundaram; Ahmad Nazari; D. Arola (2507-2517).
A preliminary study of the effects from age and dehydration on fatigue crack growth in human dentin was conducted. Compact tension (CT) fatigue specimens of coronal dentin were prepared from extracted molars and subjected to high cycle fatigue (105<N<106) under Mode I loading. Young hydrated dentin (mean age=25±7 years), old hydrated dentin (mean age=55±14 years) and young dehydrated dentin (mean age=20±2 years) were examined. Fatigue crack growth rates were quantified according to the Paris Law in terms of the crack growth exponent (m) and coefficient (C). The average fatigue crack growth exponent for the young hydrated dentin ( m = 1 3.3 ± 1.1 ) was significantly less than that for the hydrated old ( m = 2 1.6 ± 5.2 ; p < 0.0 0 3 ) and dehydrated young dentin ( m = 1 8.8 ± 2.8 ; p < 0.0 1 ). Fatigue cracks in the old dentin underwent initiation at a lower stress intensity range than in young dentin and propagated at as significantly faster rate (over 100×). Differences in the microscopic features of the fracture surfaces from the old and young dentin suggested that particular mechanisms contributing to energy dissipation and crack growth resistance in the young hydrated dentin were not present in the old dentin. Based on results of this study, the fatigue crack growth resistance of human dentin decreases with both age of the tissue and dehydration.
Keywords: Age; Dentin; Fatigue; Fracture toughness;