Biomaterials (v.26, #34)

Calendar (I).

Endotoxin: The uninvited guest by Maud B. Gorbet; Michael V. Sefton (6811-6817).
In the laboratory environment where biomaterials are synthesized and their biocompatibility assessed, we find that endotoxin contamination is hard to avoid and must not be ignored. In those relatively few cases where endotoxin was known to be present, it has been clearly shown that endotoxin can significantly affect the biological response observed and hence confound any effect of the material. This short review explains what endotoxin is, how to test for it and remove it and what its effect on the biological response to biomaterials is. We advocate routine testing of endotoxin on biomaterials and of reagents used in experimental evaluation of biomaterials and this should be the responsibility of every scientist to ensure the validity of any biomaterial study.
Keywords: Endotoxin; Biomaterials; Cytokines; LPS;

This paper describes the use of surface titration as a more direct probe of the surface chemistry of hydroxyapatite (HA) than zeta-potential measurements. The variation in HA surface charge with pH for two different KCl electrolyte concentrations was determined titrimetrically and the point of zero charge (PZC) found to be at a pH of 7.3±0.1. The curves also demonstrated that HA accumulates positive charge more readily below the PZC than it accumulates negative charge above it. Extended titration data indicated that dissolution occurred more rapidly in increasingly acidic conditions, but was inhibited by increasing electrolyte strength. Similar experiments with 2.5 mM Ca2+ in the electrolyte show that Ca2+ adsorption balances loss of H+ to give a near-neutral surface at any pH above 7 (subject to availability of calcium ions and adsorption sites). The mechanism for adsorption has been shown to be deprotonation of adjacent surface HPO4 2− sites and subsequent adsorption of a calcium ion to the lattice surface site. Furthermore, inhibition of dissolution under alkaline conditions in the presence of Ca2+ suggested that dissolution was driven by desorption of Ca2+. Modelling of the adsorption/desorption processes demonstrated that in both pure water and under physiological conditions phosphate groups will predominate at the HA surface. Furthermore, the (2 0 0) plane was identified as the likely form of the HA surface. These methodologies and findings are particularly relevant to investigation of biological response with respect to modification of surface hydrophobicity and surface energy or charge.
Keywords: Hydroxyapatite; Surface analysis; Surface charge; Interface;

Sol–gel synthesis of a multifunctional, hierarchically porous silica/apatite composite by Jenny Andersson; Sami Areva; Bernd Spliethoff; Mika Lindén (6827-6835).
In this study, a degradable, hierarchically porous silica/apatite composite material is developed from a simple low-temperature synthesis. Mesoporosity is induced in the silica portion by the use of supramolecular templating. The template is further removed by calcination. Firstly, hydroxyapatite is synthesized through a sol–gel method at near room temperature conditions. After the mineralization process, the crystal surface is coated with a mesoporous silica matrix using the templates already present in the bulk solution. The material is characterized by XRD, N2-sorption, FT–IR, SEM/EDS, and TEM. The coating layer is distributed fairly homogeneously over the apatite surface and the coating thickness is easily adjustable and dependent on the amount of added silica precursor. The hybrid material is shown to efficiently induce calcium phosphate formation under in vitro conditions and simultaneously work as a carrier system for drugs.
Keywords: Porous; Apatite; Silica; Osteoconductivity; Drug delivery;

Tuning compliance of nanoscale polyelectrolyte multilayers to modulate cell adhesion by Michael T. Thompson; Michael C. Berg; Irene S. Tobias; Michael F. Rubner; Krystyn J. Van Vliet (6836-6845).
It is well known that mechanical stimuli induce cellular responses ranging from morphological reorganization to mineral secretion, and that mechanical stimulation through modulation of the mechanical properties of cell substrata affects cell function in vitro and in vivo. However, there are few approaches by which the mechanical compliance of the substrata to which cells adhere and grow can be determined quantitatively and varied independent of substrata chemical composition. General methods by which mechanical state can be quantified and modulated at the cell population level are critical to understanding and engineering materials that promote and maintain cell phenotype for applications such as vascular tissue constructs. Here, we apply contact mechanics of nanoindentation to measure the mechanical compliance of weak polyelectrolyte multilayers (PEMs) of nanoscale thickness, and explore the effects of this tunable compliance for cell substrata applications. We show that the nominal elastic moduli E s of these substrata depend directly on the pH at which the PEMs are assembled, and can be varied over several orders of magnitude for given polycation/polyanion pairs. Further, we demonstrate that the attachment and proliferation of human microvascular endothelial cells (MVECs) can be regulated through independent changes in the compliance and terminal polyion layer of these PEM substrata. These data indicate that substrate mechanical compliance is a strong determinant of cell fate, and that PEMs of nanoscale thickness provide a valuable tool to vary the external mechanical environment of cells independently of chemical stimuli.
Keywords: Cell adhesion; Stress analysis; Endothelial cells; Atomic force microscopy; Polyelectrolyte multilayers;

Characteristics of Poly-l-Ornithine-coated alginate microcapsules by Marcus D. Darrabie; William F. Kendall; Emmanuel C. Opara (6846-6852).
Poly-l-Lysine (PLL) is the most widely used biomaterial for providing perm-selectivity in alginate microcapsules for islet transplantation. We had previously reported that Poly-l-Ornithine (PLO) is less immunogenic than PLL, and in the present study, we have compared the physical characteristics of PLO- and PLL-coated hollow alginate microcapsules. Microspheres made with 1.5% alginate were divided into 2 groups that were first coated with either 0.1% PLO or PLL, followed by a second coating with 0.25% alginate. After liquefaction of the inner alginate core with sodium citrate, the microcapsules were washed with saline and used for experiments. Pore size exclusion studies were performed with FITC-labeled lectins incubated with encapsulated pig islets followed by examination for fluorescence activity. Mechanical strength was assessed by an osmotic pressure test and by 36 h of mechanical agitation of microcapsules with inert soda lime beads. The pore size exclusion limit of microcapsules after 20 min of coating was significantly smaller with PLO. While the mean±SEM diameter of PLL-coated microcapsules increased from 718±17 to 821±17 μm ( p < 0.05 ) during 14 days incubation at 37 °C, the PLO group did not change in size. Also, PLL group had a higher percentage of broken capsules (52.7±4.9%) compared to 3.1±2.05% for PLO capsules ( p < 0.0001 , n = 6 ). We conclude that PLO-coated alginate microcapsules are mechanically stronger and provide better perm-selectivity than PLL-coated microcapsules.
Keywords: Alginate; Microencapsulation; Poly-amino acid membrane; Physical properties;

Spontaneously forming hydrogel from water-soluble random- and block-type phospholipid polymers by Mizuna Kimura; Kikuko Fukumoto; Junji Watanabe; Madoka Takai; Kazuhiko Ishihara (6853-6862).
The mixed aqueous solutions of two water-soluble phospholipid polymers, such as poly[2-methacryloyloxyethyl phosphorylcholine(MPC)-co-methacrylic acid(MA)] (rPMA) and poly[MPC-co-n-butyl methacrylate(BMA)] (PMB), spontaneously form a hydrogel at room temperature without any chemical treatment due to hydrogen bonding formation between the carboxyl groups. With the objective of enhancing the hydrogen bonding efficiency, we have focused on the density of the carboxyl groups by controlling the chemical structure and monomer unit sequence. Thus, a random and an ABA-block-type MPC copolymer having carboxylic acids, poly[MPC-co-4-(2-methacryloyloxyethyl) trimellitic acid(MET)] (rPMT) and poly(MA)-poly(MPC)-poly(MA) (bPMA), have been designed. The purpose of this study is to investigate the gelation mechanism and physical properties of a hydrogel composed of rPMA and PMB (ABgel), one of bPMA and PMB (bABgel), and one of rPMT and poly(MPC-co-benzyl methacrylate) (PMBz) (TZgel). The Raman spectroscopic analysis and the rheological study of the dissolution behaviors indicated that the TZgel formation occurred due to inter- and intra-molecular hydrogen bonding formation between the carboxyl groups in the rPMT. The gelation mechanism of the bABgel was investigated by the dynamic light scattering measurement, the scanning electron microscopy observation and the rheological study. The results showed that the bPMA chains aggregate in the aqueous medium and transform into a hydrogel network structure. The bPMA needed much more gelation time than the rPMA due to this transformation. There was no difference between the gelation periods of the ABgel and the TZgel. The compression strengths of the ABgel and the bABgel showed no significant difference, while that of TZgel was lower than ABgel. The reason for this is that the polymer chains and bulky side chains of rPMT inhibit rearranging into a planar conformation and forming hydrogen bondings. These results lead to the conclusion that the properties of these MPC polymer hydrogels can be controlled by not only the chemical structure of the polymer but also the monomer unit sequence containing carboxyl groups.
Keywords: Hydrogel; Gelation; 2-methacryloyloxyethyl phosphorylcholine polymer; Molecular design; Hydrogen bonding;

Solvent and water retention in dental adhesive blends after evaporation by Cynthia K.Y. Yiu; Edna L. Pashley; Noriko Hiraishi; Nigel M. King; Cecilia Goracci; Marco Ferrari; Ricardo M. Carvalho; David H. Pashley; Franklin R. Tay (6863-6872).
This study examined the extent of organic solvent and water retention in comonomer blends with different hydrophilicity (Hoy's solubility parameter for hydrogen bonding, δ h ) after solvent evaporation, and the extent of tracer penetration in polymerised films prepared from these resins. For each comonomer blend, adhesive/solvent mixtures were prepared by addition of (1) 50 wt% acetone, (2) 50 wt% ethanol, (3) 30 wt% acetone and 20 wt% water and (4) 30 wt% ethanol and 20 wt% water. The mixtures were placed in glass wells and evaporated for 30–60 s for acetone-based resins, and 60–120 s for ethanol-based resins. The weight of the comonomer mixtures was measured before and after solvent evaporation. Resin films were prepared for transmission electron microscopy (TEM) after immersion in ammonical silver nitrate. The percentages of solvent and water retained in the comonomer mixtures, and between the acetone and ethanol groups were measured gravimetrically and were statistically compared. In comonomer-organic solvent mixtures, the percentage of solvent retained in acetone and ethanol-based mixtures increased significantly with hydrophilicity of the comonomer blends ( P < 0.05 ). In resin-organic solvent–water mixtures, significantly more solvent and water were retained in the ethanol-based mixtures ( P < 0.0001 ), when compared to acetone-based mixtures after 60 s of air-drying. TEM revealed residual water being trapped as droplets in resin films containing acetone and water. Water-filled channels were seen along the film periphery of all groups and throughout the entire resin films containing ethanol and water. The addition of water to comonomer-ethanol mixtures results in increased retention of both ethanol and water because both solvents can hydrogen bond to the monomers.
Keywords: Acetone; Ethanol; Solubility parameter; Resin; Hydrophilicity;

Interaction between O-carboxymethylchitosan and dipalmitoyl-sn-glycero-3-phosphocholine bilayer by Ai Ping Zhu; Ning Fang; Mary B. Chan-Park; Vincent Chan (6873-6879).
O-carboxylmethylchitosan (OCMCS), a chitosan derivative, has emerged as a strong polymeric biomembrane perturbant. In this study, the interaction between OCMCS and dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was examined with cross-polarization microscopy, differential scanning calorimetry (DSC) and the surface pressure–area isotherms techniques. Cross-polarized light images showed that OCMCS induced the fusion of small DPPC multilamellar vesicles (MLV) to form large lamellar structures. From DSC measurement, the highest degree of fusion was found at the optimum OCMCS concentrations between 0.0625 and 0.2 mg/ml which are orders of magnitude lower than those required for similar reductions with unmodified chitosan as perturbant. At these concentrations, the association of DPPC and OCMCS enhances the fusion of DPPC vesicles. Surface pressure–area isotherms of DPPC monolayer in the presence of OCMCS imply interactions between OCMCS and DPPC in neutral condition. In comparison with chitosan, OCMCS proved to be a more effective membrane perturbant not only in neutral but also in acidic and basic conditions. The physical driving forces for OCMCS-induced perturbation of DPPC bilayer in neutral conditions are mainly hydrogen bonding and hydrophobic interactions. In acidic or basic conditions, the physical driving forces are dominated by the electrostatic interactions. The strong OCMCS–DPPC interaction will potentially increase the effectiveness of OCMCS for gene or drug delivery.
Keywords: O-carboxylmethylchitosan; DPPC; Interaction; Perturbation;

Antimicrobial potency of alkali ion substituted calcium phosphate cements by Uwe Gbureck; Oliver Knappe; Liam M. Grover; Jake E. Barralet (6880-6886).
Potassium and sodium containing nanoapatite cements were produced by the reaction of mechanically activated CaNaPO4 (CSP), CaKPO4 (CPP) and Ca2KNa(PO4)2 (CPCP) with a 2.5% Na2HPO4 solution. The cements exhibited clinically acceptable setting times of approximately 5 min and compressive strengths of 5–10 MPa. The antimicrobial properties of the cements were tested with the agar diffusion test using Streptococcus salvarius, Staphylococcus epidermis and Candida albicans. All types of alkali ion containing cements showed a significantly higher antimicrobial potency with inhibition zones of approx. 4–11 mm than a commercial calcium hydroxide cement which resulted in small inhibition zones around the cement samples of a maximum of 1.5 mm. The antimicrobial properties of all the cements were not found to diminish even after longer incubation times. This behaviour was attributed to the formation of soluble alkaline metal phosphates during setting which increased the pH value in the agar gel around the alkali containing calcium phosphate cement to 8.5–10.7 compared to 6.5–8.0 for the Ca(OH)2 product. The high antimicrobial potency of alkali–calcium phosphate cements may find an application in dentistry as pulp capping agents, root fillers or cavity liners.
Keywords: Calcium phosphate cement; Mechanical activation; Antimicrobial properties; pH value;

Mylar™ and Teflon-AF™ as cell culture substrates for studying endothelial cell adhesion by Charles C. Anamelechi; George A. Truskey; W. Monty Reichert (6887-6896).
The textured and opaque nature of Dacron™ and ePTFE has prevented the use of these fabrics in conventional cell culture techniques normally employed to optimize cell attachment and retention. This lack of optimization has led, in part, to the poor performance of endothelialization strategies for improving vascular graft patency. Here we show that thin, transparent films of Mylar™ and Teflon-AF™ are viable in vitro cell culture mimics of Dacron™ and ePTFE vascular graft materials, particularly for the study of protein mediated endothelial cell (EC) attachment, spreading and adhesion. Glass substrates were used as controls. X-ray photoelectron spectroscopy (XPS) and contact angle analysis showed that Mylar™ and Teflon-AF™ have surface chemistries that closely match Dacron™ and ePTFE. 125I radiolabeling was used to quantify fibronectin (FN) adsorption, and FN and biotinylated-BSA “dual ligand” co-adsorption onto glass, Mylar™ and Teflon-AF™ substrates. Native human umbilical vein endothelial cells (HUVEC) and streptavidin-incubated biotinylated-HUVEC (SA-b-HUVEC) spreading was measured using phase contrast microscopy. Cell retention and adhesion was determined using phase contrast microscopy under laminar flow. All surfaces lacking protein pre-treatment, regardless of surface type, showed the lowest degree of cell spreading and retention. Dual ligand treated Mylar™ films showed significantly greater SA-b-HUVEC spreading up to 2 h, but were similar to HUVEC on FN treated Mylar™ at longer times; whereas SA-b-HUVEC spreading on dual ligand treated Teflon-AF was never significantly different from HUVEC on FN treated Teflon-AF™ at any time point. SA-b-HUVEC retention was significantly greater on dual ligand treated Mylar™ compared to HUVEC on FN treated Mylar™ over the entire range of shear stresses tested (3.54–28.3 dynes/cm2); whereas SA-b-HUVEC retention to dual ligand and HUVEC retention to FN treated Teflon-AF™ gave similar results at each shear stress, with only the mid-range of stresses showing significant difference in cell retention to Teflon-AF™.
Keywords: Teflon-AF; Mylar; Fibronectin; Avidin–biotin; Cell adhesion; Vascular grafts; Endothelialization;

Ligand density characterization of peptide-modified biomaterials by Thomas A. Barber; Gregory M. Harbers; Susan Park; Michele Gilbert; Kevin E. Healy (6897-6905).
A simple fluorescence based characterization method was developed to assess ligand density on peptide-modified biomaterials. The method exploits the exquisite sensitivity of proteolysis for the purpose of liberating a fluorescently labeled probe fragment from an immobilized peptide. The released fragment can then be detected in solution using high-throughput fluorometry. In silico screening tools identified the enzyme chymotrypsin as a promising candidate for releasing a detectable probe fragment from the fluorescently labeled peptide, Ac-CGGNGEPRGDTYRAYK(FITC)GG-NH2. After chymotrypsin digestion of the peptide in solution was first characterized using mass spectrometry and HPLC, a basic enzyme mediated release protocol was developed and implemented to generate peptide-binding isotherms on various peptide-modified biomaterials. The new method is sensitive, has good signal-to-noise ratio (S/N), and is easily standardized. Furthermore, the technique can be applied independent of material chemistry and geometry, making it a suitable alternative to radiolabeling for a wide range of biomaterial applications.
Keywords: Peptide; Surface analysis; Surface modification; IPN; Silane;

Combinatorial screening of cell proliferation on poly(l-lactic acid)/poly(d,l-lactic acid) blends by Carl G. Simon; Naomi Eidelman; Scott B. Kennedy; Amit Sehgal; Chetan A. Khatri; Newell R. Washburn (6906-6915).
We have combined automated fluorescence microscopy with a combinatorial approach for creating polymer blend gradients to yield a rapid screening method for characterizing cell proliferation on polymer blends. A gradient in polymer blend composition of poly(l-lactic acid) (PLLA) and poly(d,l-lactic acid) (PDLLA) was created in the form of a strip-shaped film and was annealed to allow PLLA to crystallize. Fourier transform infrared (FTIR) microspectroscopy was used to determine the composition in the gradients and atomic force microscopy was used to characterize surface topography. Osteoblasts were cultured on the gradients and proliferation was assessed by automated counting of cells using fluorescence microscopy. Surface roughness varied with composition, was smooth on PDLLA-rich regions and was rough on the PLLA-rich regions. Cell adhesion was similar on all regions of the gradients while proliferation was faster on the smooth, PDLLA-rich end of the gradients than on the rough, PLLA-rich end of the gradients. These results demonstrate the feasibility of a new, combinatorial approach for evaluating cell proliferation on polymer blends.
Keywords: Combinatorial methods; Polymer blends; Cell proliferation; Polylactic acid; Microspectroscopic FTIR; Osteoblast;

Biomimetic deposition of apatite coating on surface-modified NiTi alloy by Y.W. Gu; B.Y. Tay; C.S. Lim; M.S. Yong (6916-6923).
TiO2 coatings were prepared on NiTi alloy by heat treatment in air at 300, 400, 600 and 800 °C. The heat-treated NiTi alloy was subsequently immersed in a simulated body fluid for the biomimetic deposition of the apatite layer onto the surface of TiO2 coating. The apatite coatings as well as the surface oxide layer on NiTi alloy were characterized using scanning electron microscopy equipped with energy dispersive spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy and Raman spectroscopy. Results showed the samples heat-treated at 600 °C produced a layer of anatase and rutile TiO2 on the surface of NiTi. No TiO2 was detected on the surface of NiTi after heat treatment at 300 and 400 °C by X-ray diffraction, while rutile was formed on the surface of the 800 °C heat-treated sample. It was found that the 600 °C heat-treated NiTi induced a layer consisted of microcrystalline carbonate containing hydroxyapatite on its surface most effectively, while 300 and 400 °C heat-treated NiTi did not form apatite. This was due to the presence of anatase and/or rutile in the 600 and 800 °C heat-treated NiTi which could provide atomic arrangements in their crystal structures suitable for the epitaxy of apatite crystals, and anatase had better apatite-forming ability than rutile. XPS and Raman results revealed that this apatite layer was a carbonated and non-stoichiometric apatite with Ca/P ratio of 1.53, which was similar to the human bone. The formation of apatite on 600 °C heat-treated NiTi following immersion in SBF for 3 days indicated that the surface modified NiTi possessed excellent bioactivity.
Keywords: Calcium phosphate; Apatite; NiTi; In vitro bioactivity; TiO2; Heat treatment; Simulated body fluid;

Reduced platelet adhesion to titanium metal coated with apatite, albumin–apatite composite or laminin–apatite composite by Masaki Uchida; Atsuo Ito; Katsuko S. Furukawa; Keigo Nakamura; Yuji Onimura; Ayako Oyane; Takashi Ushida; Takashi Yamane; Tamotsu Tamaki; Tetsuya Tateishi (6924-6931).
Titanium metal coated with apatite (HA–Ti), albumin−apatite composite (AA–Ti) or laminin−apatite composite (LA–Ti) was prepared by the immersion of NaOH- and heat-treated titanium metal in a calcium phosphate solution, or one supplemented with albumin or laminin. Platelet adhesion to the obtained materials under flow conditions was investigated in real time using a cone- and plate-type viscometer and fluorescence labeled platelets. Adhesion and activation of the platelets on the HA–Ti, AA–Ti and LA–Ti were definitely suppressed as compared with those on untreated titanium metal with a mirror surface. Furthermore, the numbers of platelets adhered to AA−Ti and LA–Ti are smaller than those adhered to HA–Ti, although the differences were not statistically significant. These findings suggest that HA–Ti, AA–Ti and LA–Ti, especially AA–Ti and LA–Ti, would exhibit thromboresistance that is superior to commercially pure titanium metal in terms of platelet adhesion.
Keywords: Titanium; Apatite; Albumin; Laminin; Platelet adhesion;

The effect of cyclo-DfKRG peptide immobilization on titanium on the adhesion and differentiation of human osteoprogenitor cells by Stéphane Pallu; Chantal Bourget; Reine Bareille; Christine Labrugère; Michel Dard; Andreas Sewing; Alfred Jonczyk; Michel Vernizeau; Marie Christine Durrieu; Joelle Amédée-Vilamitjana (6932-6940).
This study takes place in the field of development of a bioactive surface of titanium alloys. In this paper, titanium was functionalized with cyclo-DfKRG peptide by coating or grafting using different anchors (thiol or phosphonate) as spacers between the surface and the peptide. Cell adhesion, and differentiation of human osteoprogenitor (HOP) cells arising from human bone marrow were investigated.Our results seem to demonstrate that cyclo-DfKRG peptide coating with a phosphonate anchor and grafting procedure contributes to higher cell adhesion and a strong ALP and Cbfa1 mRNA expression, after 10 days of cell seeding. At the contrary, this peptide coated with a thiol anchor stimulates differentiation of HOP within 3 days of culture.
Keywords: Cyclo-DfKRG peptide; Osteoblast; Differentiation; Adhesion;

Effects of bisphosphonates on proliferation and osteoblast differentiation of human bone marrow stromal cells by Fabian von Knoch; Claude Jaquiery; Marc Kowalsky; Stefan Schaeren; Claude Alabre; Ivan Martin; Harry E. Rubash; Arun S. Shanbhag (6941-6949).
Bisphosphonates are well known potent inhibitors of osteoclast activity and are widely used to treat metabolic bone diseases. Recent evidence from in vitro and in vivo studies indicates that bisphosphonates may additionally promote osteoblastic bone formation. In this study, we evaluated the effects of three FDA-approved and clinically utilized bisphosphonates, on the proliferation and osteogenic differentiation of human bone marrow stromal cells (BMSC).BMSC were obtained from patients undergoing primary total hip arthroplasty for end-stage degenerative joint disease. Cells were treated with or without a bisphosphonate (alendronate, risedronate, or zoledronate) and analyzed over 21 days of culture. Cell proliferation was determined by direct cell counting. Osteogenic differentiation of BMSC was assessed with alkaline phosphatase bioassay and gene expression analyses using conventional RT-PCR as well as real-time quantitative RT-PCR.All bisphosphonates tested enhanced the proliferation of BMSC after 7 and 14 days of culture. Steady-state mRNA levels of key genes involved in osteogenic differentiation such as bone morphogenetic protein-2 (BMP-2), bone sialoprotein-II, core-binding factor alpha subunit 1 (cbfa1) and type 1 collagen, were generally increased by bisphosphonate treatment in a type- and time-dependent manner. Gene expression levels varied among the different donors. Enhancement of osteogenic differentiation was most pronounced after 14 days of culture, particularly following zoledronate treatment ( p < 0.05 for BMP-2).In conclusion, using a clinically relevant in vitro model we have demonstrated that bisphosphonates enhance proliferation of BMSC and initiate osteoblastic differentiation. When administered around joint replacements, bisphosphonates may potentially compensate for the deleterious effects of particulate wear debris at the bone–implant interface, by encouraging increased numbers of cells committed to the osteoblastic phenotype, and thus improve the longevity of joint replacements.
Keywords: Alendronate; Risedronate; Zoledronate; Bisphosphonates; Human bone marrow stromal cells; Bone morphogenetic protein; Total joint replacements;

Physicochemical model of alginate–poly-l-lysine microcapsules defined at the micrometric/nanometric scale using ATR-FTIR, XPS, and ToF-SIMS by Susan K. Tam; Julie Dusseault; Stefania Polizu; Martin Ménard; Jean-Pierre Hallé; L’Hocine Yahia (6950-6961).
Alginate–poly-l-lysine–alginate (APA) microcapsules are currently being investigated as a means to immuno-isolate transplanted cells, but their biocompatibility is limited. In this study, we verified the hypothesis that poly-l-lysine (PLL), which is immunogenic when unbound, is exposed at the APA microcapsule surface. To do so, we analysed the microcapsule membrane at the micrometric/nanometric scale using attenuated total reflectance Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry. The results indicate that PLL and alginate molecules interact within the membrane. PLL exists in considerable amounts near the surface, contributing to the majority of the carbon within the outermost 100 Å of the membrane. PLL was also detected at the true surface (the outermost monolayer) of the microcapsules. The exposure of PLL does not appear to result from defects in the outer alginate coating. This physicochemical model of APA microcapsules could explain their immunogenicity and will play an important role in the optimization of the microcapsule design.
Keywords: Microencapsulation; Biocompatibility; Alginate; Poly-l-lysine; Time-of-flight secondary ion mass spectrometry (ToF-SIMS); Islet;

Surface engineering of stainless steel materials by covalent collagen immobilization to improve implant biocompatibility by Rainer Müller; Jochen Abke; Edith Schnell; Frank Macionczyk; Uwe Gbureck; Robert Mehrl; Zbigniev Ruszczak; Richard Kujat; Carsten Englert; Michael Nerlich; Peter Angele (6962-6972).
It was shown recently that the deposition of thin films of tantalum and tantalum oxide enhanced the long-term biocompatibility of stainless steel biomaterials due to an increase in their corrosion resistance. In this study, we used this tantalum oxide coating as a basis for covalent immobilization of a collagen layer, which should result in a further improvement of implant tissue integration. Because of the high degradation rate of natural collagen in vivo, covalent immobilization as well as carbodiimide induced cross-linking of the protein was performed. It was found that the combination of the silane-coupling agent aminopropyl triethoxysilane and the linker molecule N,N′-disulphosuccinimidyl suberate was a very effective system for collagen immobilizing. Mechanical and enzymatic stability testing revealed a higher stability of covalent bound collagen layers compared to physically adsorbed collagen layers. The biological response induced by the surface modifications was evaluated by in vitro cell culture with human mesenchymal stem cells as well as by in vivo subcutaneous implantation into nude mice. The presence of collagen clearly improved the cytocompatibility of the stainless steel implants which, nevertheless, significantly depended on the cross-linking degree of the collagen layer.
Keywords: Surface modification; Steel; Tantalum; Collagen; Coupling agents; Cross-linking; Mesenchymal stem cells;

The enzyme α-chymotrypsin, a model for catalytic proteins, was entrapped in different silicone elastomers that were formed via the condensation-cure room temperature vulcanization (CC-RTV) of silanol terminated poly(dimethylsiloxane) with tetraethyl orthosilicate as a crosslinker, in the presence of different poly(ethylene oxide) oligomers that were functionalized with triethoxysilyl groups. The effects of various chemical factors on both the activity and entrapping efficiency of proteins (leaching) were studied using a 2-level fractional factorial design—a chemometrics approach. The factors studied include the concentration and chain length of poly(ethylene oxide), enzyme content, and crosslinker (TEOS) concentration. The study indicated that poly(ethylene oxide) can stabilize the entrapped α-chymotrypsin in silicone rubber: the specific activity can be maximized by incorporating a relatively high content of short chain, functional PEO. Increased enzyme concentration was found to adversely affect the specific activity. The effect of TEOS was found to be insignificant when PEO was present in the elastomer, however, it does affect the activity positively in the case of simple elastomers.
Keywords: Immobilized enzyme; α-chymotrypsin; Silicone elastomer; Poly(ethylene oxide); Chemometrics;

Inhibition of implant-associated infections via nitric oxide release by Brian J. Nablo; Heather L. Prichard; Renita D. Butler; Bruce Klitzman; Mark H. Schoenfisch (6984-6990).
The in vivo antibacterial activity of nitric oxide (NO)-releasing xerogel coatings was evaluated against an aggressive subcutaneous Staphylococcus aureus infection in a rat model. The NO-releasing implants were created by coating a medical-grade silicone elastomer with a sol–gel-derived (xerogel) film capable of storing NO. Four of the bare or xerogel-coated silicone materials were subcutaneously implanted into male rats. Ten rats were administered 10 μl of a 108  cfu ml−1 S. aureus colony directly into the subcutaneous pocket with the implant prior to wound closure. Infection was quantitatively and qualitatively evaluated after 8 d of implantation with microbiological and histological methods, respectively. A 82% reduction in the number of infected implants was achieved with the NO-releasing coating. Histology revealed that the capsule formation around infected bare silicone rubber controls was immunoactive and that a biofilm may have formed. Capsule formation in response to NO-releasing implants had greater vascularity in comparison with uninoculated or untreated controls. These results suggest that NO-releasing coatings may dramatically reduce the incidence of biomaterial-associated infection.
Keywords: Staphylococcus aureus; Implant infection; Sol–gel; Nitric oxide; In vivo;

Enhanced vascular-related cellular affinity on surface modified copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) by Xiang-Hua Qu; Qiong Wu; Juan Liang; Xue Qu; Shen-Guo Wang; Guo-Qiang Chen (6991-7001).
Random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate, short as PHBHHx, was surface modified by ammonia plasma treatment and/or fibronectin coating, respectively. The improved results were demonstrated by better growth of human umbilical vein endothelial cells (HUVECs) and rabbit aorta smooth muscle cells (SMCs) on the surface of ammonia plasma-treated PHBHHx coated with fibronectin (PFn-PHBHHx), compared with the fibronectin-coated (Fn-PHBHHx) or uncoated PHBHHx, respectively, although XPS analysis and ELISA demonstrated higher fibronectin adsorption on Fn-PHBHHx than on PFn-PHBHHx. Confocal microscopy observation showed that the specific co-localization of fibronectin with F-actin was impaired on PFn-PHBHHx, while it was almost lost on Fn-PHBHHx compared with pristine PHBHHx or plasma-treated PHBHHx (P-PHBHHx). These were attributed to the generation of new nitrogen- and oxygen-containing groups on the PHBHHx surface by the ammonia plasma treatment, which led to increased polar components that enhanced polymer surface energy and hydrophilic properties on P-PHBHHx. The most prominent effect of PFn-PHBHHx was its stimulation of HUVECs proliferation. HUVECs on PFn-PHBHHx formed a confluent monolayer after 3 days of incubation, while SMCs were unable to form a sub-confluent layer. The above evidences revealed that PFn-PHBHHx would benefit endotheliazation rather than SMCs proliferation. We therefore believed that PFn-PHBHHx would be a promising material as a luminal surface of vascular grafts.
Keywords: PHA; PHB; PHBHHx; Surface modification; Plasma treatment; Vascular graft; Fibronectin;

Thermosensitive N -isopropylacrylamide– N –propylacrylamide-vinyl pyrrolidone terpolymers: Synthesis, characterization and preliminary application as embolic agents by Xiaowei Li; Wenguang Liu; Guixiang Ye; Bingqi Zhang; Dunwan Zhu; Kangde Yao; Zuoqin Liu; Xizhong Sheng (7002-7011).
In this article, thermosensitive N-isopropylacrylamide (NIPAAm)–N-propylacrylamide (NPAAm)–vinyl pyrrolidone (VP) terpolymers (PNINAVP) were prepared by varying feed ratios with free radical copolymerization method. The composition ratios and molecular weights of PNINAVP were examined by NMR and GPC. The thermo-responsive behaviors of copolymer solutions in the absence and with addition of Iohexol, a radiopaque agent, were investigated by differential scanning calorimetry (DSC) and rheometer. The sol–gel transition of the copolymer solutions occurred reversibly within 1 min in response to temperature. Incorporation of Iohexol increased the transition time and transition temperature of PNINAVP solutions; the rheological properties were also influenced. It was observed that at body temperature, PNINAVP and Iohexol could form an integrated bulky hydrogel presumably due to the hydrogen bonding between them, which was favorable for the clinical follow-up and reducing toxic side effects. In vitro embolic model experiment indicated that 5 wt% 16:16:1H PNINAVP solution containing Iohexol displayed a satisfactory embolization effect. This solution was injected into the rete mirabiles (RM) of six swines through a microcatheter. The angiographical results obtained immediately after the operation showed a complete occlusion of the RM, and no recanalization was observed at postoperative month 1. The histological examination demonstrated no acute inflammatory reaction inside the RM and surrounding tissue. This work could provide a beneficial guidance for designing a new temperature-sensitive polymer-based embolic agent.
Keywords: N -isopropylacrylamide; N -propylacrylamide; Vinyl pyrrolidone; Thermosensitive; Gelation; Liquid embolic agent;

Chondrocytes isolated from human foetal epiphyseal cartilage were seeded dynamically into polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to produce tissue-engineered cartilage. Several culture techniques with the potential to provide endogenous growth factors and other conditions beneficial for de novo cartilage synthesis were investigated. Osteochondral composite constructs were generated by seeding separate PGA scaffolds with either foetal chondrocytes or foetal osteoblasts then suturing the scaffolds together before bioreactor cultivation. This type of co-culture system provided direct contact between the tissue-engineered cartilage and developing tissue-engineered bone and yielded significant improvements in cartilage quality. In the cartilage section of the composites, the concentrations of glycosaminoglycan (GAG) and total collagen were increased by 55% and 2.5-fold, respectively, compared with control cartilage cultures, while levels of collagen type II were similar to those in the controls. The osteochondral composites were harvested from the bioreactors as single units with good integration between the cartilage and bone tissues. Only the cartilage layer contained GAG while only the bone layer was mineralised. In other experiments, co-culture of tissue-engineered cartilage with pieces of ex-vivo cartilage or ex-vivo bone did not improve the quality of the cartilage relative to control cultures. Addition of 10−6m diacerein to the culture medium also had no effect on the properties of engineered cartilage. This work demonstrates the beneficial effects of generating cartilage tissues in contact with developing bone. It also demonstrates the feasibility of producing composite osteochondral constructs for clinical application using recirculation column bioreactors.
Keywords: Bioreactor; Bone tissue engineering; Cartilage tissue engineering; Co-culture; Osteochondral composite; Polyglycolic acid;

Implantation of preadipocyte-loaded hyaluronic acid-based scaffolds into nude mice to evaluate potential for soft tissue engineering by Karsten Hemmrich; Dennis von Heimburg; Raoul Rendchen; Chiara Di Bartolo; Eva Milella; Norbert Pallua (7025-7037).
The reconstruction of soft tissue defects following extensive deep burns or tumor resections remains an unresolved problem in plastic and reconstructive surgery since adequate implant materials are still not available. Preadipocytes, immature precursor cells found between mature adipocytes in adipose tissue, are a potential material for soft tissue engineering since they can proliferate and differentiate into adipose tissue after transplantation. In previous studies, we identified hyaluronan benzyl ester (HYAFF®11) sponges to be promising carrier matrices. This study now evaluates, in vitro and in vivo, a new sponge architecture with pores of 400 μm either made of plain HYAFF®11 or HYAFF®11 coated with the extracellular matrix glycosaminoglycan hyaluronic acid. Human preadipocytes were isolated, seeded onto carriers and implanted into nude athymic mice. Explants harvested after 3, 8, and 12 weeks were examined for macroscopical appearance, thickness, weight, pore structure, histology, and immunohistochemistry. Compared to previous studies, we found better penetration of cells into both types of scaffolds, with more extensive formation of new vessels throughout the construct but with only minor adipose tissue. Our encouraging results contribute towards a better seeded and vascularised scaffold but also show that the enhancement of adipogenic conversion of preadipocytes remains a major task for further in vivo experiments.
Keywords: Adipose tissue engineering; Hyaluronic acid; Progenitor cell;

Extracellular matrix-enriched polymeric scaffolds as a substrate for hepatocyte cultures: in vitro and in vivo studies by B. Zavan; P. Brun; V. Vindigni; A. Amadori; W. Habeler; P. Pontisso; D. Montemurro; G. Abatangelo; R. Cortivo (7038-7045).
Tissue engineering is a promising approach to developing hepatic tissue suitable for the functional replacement of a failing liver. The aim of the present study was to investigate whether an extracellular cell matrix obtained from fibroblasts-cultured within scaffolds of hyaluronic acid (HYAFF™) could influence the proliferation rate and survival of rat hepatocytes both during long-term culture and after in vivo transplantation. Cultures were evaluated by histological and morphological analysis, a proliferation assay and metabolic activity (albumin secretion). Hepatocytes cultured in extracellular matrix-enriched scaffolds exhibited a round cellular morphology and re-established cell–cell contacts, growing into aggregates of several cells along and/or among fibers in the fabric. Hepatocytes were able to secrete albumin up to 14 days in culture. In vivo results demonstrated the biocompatibility of HYAFF-11TM implanted in nude mice, in which hepatocytes maintained small well-organised aggregates until the 35th day.In conclusion, the presence of a fibroblast-secreted extracellular matrix improved the biological properties of the hyaluronan scaffold, favoring the survival and morphological integrity of hepatocytes in vitro and in vivo.
Keywords: Scaffold; Cell morphology; Hyaluronic acid; Biomaterial; Cell culture; Fibroblast; Hepatocyte;

Mimicking endogenous bone-binding proteins, RGD peptides have been synthesized with polyacidic amino acid domains in order to ionically tether the peptides to bone-like synthetic biomaterials, including hydroxyapatite (HA). However, a direct comparison of unmodified RGD with polyacidic-conjugated RGD has not been performed, and thus a benefit for the acidic domain has not been established. We evaluated the peptide/HA bond of RGD peptides with and without an attached polyglutamate sequence (E7), as well as examined mesenchymal stem cell (MSC) adhesion and morphology as they were affected by the conjugated peptide. We found that significantly more E7RGD was bound to HA than RGD at all coating concentrations tested, and moreover, more E7RGD was retained on the HA surface even after extended washing in serum-free media. Consistent with in vitro results, higher levels of E7RGD than RGD remained on HA that had been implanted in vivo for 24 h, indicating that the polyacidic domain improved peptide-binding efficiency. At several peptide concentrations, E7RGD increased cell adhesion compared to RGD surfaces, establishing a biological benefit for the E7 modification. In addition, HA pre-coated sequentially with low-density E7RGD (1–10 μg/ml) and serum (FBS) stimulated cell adhesion and spreading, compared to either coating alone, suggesting that an ionic linkage allows for the potential adsorption of serum proteins to unoccupied sites, which may be important for bone formation in vivo. Collectively, these results suggest that tethering peptides to HA via a polyglutamate domain is an effective method for improving the peptide/HA bond, as well as for enhancing MSC adhesion.
Keywords: Hydroxyapatite; Mesenchymal stem cell; RGD peptide; Cell adhesion; Cell spreading; Bone;

The behaviour of human mesenchymal stem cells (hMSC) when cultured in contact with a range of silane-modified surfaces was examined to determine if changing the surface chemistry affected the early differentiation potential of mesenchymal stem cells in vitro over a 7-day period. Cells were cultured for 1 and 7 days in direct contact with glass which had been functionalized by surface treatment to provide a range of different surfaces: -CH3, -NH2, -SH, -OH, and -COOH modified surfaces and a clean glass reference (TAAB). Viable cell adhesion was quantified by Lactate Dehydrogenase assay, and morphology and viability was qualitatively evaluated using calcein AM, ethidium homodimer, cytoskeletal (F Actin), extra-cellular matrix (fibronectin and vitronectin) and Hoechst staining (nucleus). The expression of selected differentiation markers, Collagen II (chondrocytes), CBFA1 (bone transcription factor), Collagen I (MSC marker) and TGF-β3 (extra-cellular matrix production) was determined using real time polymerase chain reaction. The expression of ornithine decarboxylase was evaluated as a marker of proliferation. Surfaces of the -NH2 group demonstrated the greatest level of cell adhesion by the 7-day period, and mRNA expression profiles indicated osteogenic differentiation, increased CBFA1 and decreased Collagen II expression. Cells cultured in contact with the -COOH surfaces displayed different cell morphologies, fibronectin and vitronectin spatial distributions compared with the cells in contact with the -NH2 surfaces, in addition to an increase in Collagen II expression, indicative of chondrogenic differentiation. The modifications to the surface chemistry of glass did affect cell behaviour, both in terms of viable cell adhesion, morphology and profiles of mRNA expression, providing the means to alter the differentiation potential of the MSCs.
Keywords: Mesenchymal stem cells; Cell viability; Adhesion; ECM;

A chemically defined surface for the co-culture of melanocytes and keratinocytes by Paula Clare Eves; Alison J. Beck; Alex G. Shard; Sheila Mac Neil (7068-7081).
Patients with stable vitiligo can be helped surgically using transplantation of autologous cultured melanocytes, but there is a need for a culture methodology that is free from xenobiotic agents and for a simple way of delivering cultured melanocytes to the patient to achieve pigmentation with good wound healing. The aim of this study was to develop a chemically defined surface, suitable for the co-culture of melanocytes and keratinocytes which could be used in the future for the treatment vitiligo patients to achieve both restoration of pigmentation and good wound healing. Two keratinocyte growth media and two melanocyte growth media were compared; two of these were serum free. Cells were seeded on a range of chemically defined substrates (produced by plasma polymerisation of acrylic acid, allylamine or a mixture of these monomers) either as mono- or co-cultures. Melanocytes and keratinocytes attached and proliferated on both acid and amine substrates (without significant preferences), and co-cultures of cells proliferated more successfully than individual cultures. One media, M2, which is serum free, supported expansion of melanocytes and to a lesser extent keratinocytes on several plasma polymer substrates. In conclusion, these data indicate that a combination of a chemically defined substrate with M2 media allows serum-free co-culture of melanocytes and keratinocytes.
Keywords: Vitiligo; Plasma polymerisation; Acrylic acid; Allylamine; Keratinocytes; Melanocytes; Skin tissue engineering;

In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells by Yongzhong Wang; Ung-Jin Kim; Dominick J. Blasioli; Hyeon-Joo Kim; David L. Kaplan (7082-7094).
Adult cartilage tissue has limited self-repair capacity, especially in the case of severe damages caused by developmental abnormalities, trauma, or aging-related degeneration like osteoarthritis. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into cells of different lineages including bone, cartilage, and fat. In vitro cartilage tissue engineering using autologous MSCs and three-dimensional (3-D) porous scaffolds has the potential for the successful repair of severe cartilage damage. Ideally, scaffolds designed for cartilage tissue engineering should have optimal structural and mechanical properties, excellent biocompatibility, controlled degradation rate, and good handling characteristics. In the present work, a novel, highly porous silk scaffold was developed by an aqueous process according to these criteria and subsequently combined with MSCs for in vitro cartilage tissue engineering. Chondrogenesis of MSCs in the silk scaffold was evident by real-time RT-PCR analysis for cartilage-specific ECM gene markers, histological and immunohistochemical evaluations of cartilage-specific ECM components. Dexamethasone and TGF-β3 were essential for the survival, proliferation and chondrogenesis of MSCs in the silk scaffolds. The attachment, proliferation, and differentiation of MSCs in the silk scaffold showed unique characteristics. After 3 weeks of cultivation, the spatial cell arrangement and the collagen type-II distribution in the MSCs-silk scaffold constructs resembles those in native articular cartilage tissue, suggesting promise for these novel 3-D degradable silk-based scaffolds in MSC-based cartilage repair. Further in vivo evaluation is necessary to fully recognize the clinical relevance of these observations.
Keywords: Mesenchymal stem cell; MSC; Cartilage tissue engineering; Aqueous-derived silk scaffold;

Delivery of TGF-β1 and chondrocytes via injectable, biodegradable hydrogels for cartilage tissue engineering applications by Hansoo Park; Johnna S. Temenoff; Theresa A. Holland; Yasuhiko Tabata; Antonios G. Mikos (7095-7103).
In this work, novel hydrogel composites, based on the biodegradable polymer, oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) were utilized as injectable cell and growth factor carriers for cartilage tissue engineering applications. Specifically, bovine chondrocytes were embedded in composite hydrogels co-encapsulating gelatin MPs loaded with transforming growth factor-β1 (TGF-β1). Hydrogels with embedded cells co-encapsulating unloaded MPs and those with no MPs served as controls in order to assess the effects of MPs and TGF-β1 on chondrocyte function. Samples were cultured up to 28 days in vitro. By 14 days, cell attachment to embedded gelatin MPs within the constructs was observed via light microscopy. Bioassay results showed that, over the 21 day period, there was a statistically significant increase in cellular proliferation for samples containing gelatin MPs, but no increase was exhibited in samples without MPs over the culture period. The release of TGF-β1 further increased cell construct cellularity. Over the same time period, glycosaminoglycan content per cell remained constant for all formulations, suggesting that the dramatic increase in cell number for samples with TGF-β1-loaded MPs was accompanied by maintenance of the cell phenotype. Overall, these data indicate the potential of OPF hydrogel composites containing embedded chondrocytes and TGF-β1-loaded gelatin MPs as a novel strategy for cartilage tissue engineering.
Keywords: Cartilage tissue engineering; Cell encapsulation; Drug delivery; Injectable hydrogel; Transforming growth factor- β 1;

In vitro evaluation of low-intensity pulsed ultrasound in herniated disc resorption by Sadahiro Iwabuchi; Masaya Ito; Junko Hata; Toshihoro Chikanishi; Yoshiaki Azuma; Hirotaka Haro (7104-7114).
Herniated disc (HD) is often resolved spontaneously without surgical intervention. HD resorption (HDR) is associated with abundant vascularization and infiltration of macrophages (Mφ) into the intervertebral disc (ID), as well as with high levels of matrix metalloproteinases (MMPs). Low-intensity pulsed ultrasound (LIPUS) accelerates bone fracture healing in clinical studies, and angiogenic factors are involved in the mechanism of action. In the present study, we examined the effects of LIPUS on HDR in a rat in vitro HD model. HDR was enhanced by LIPUS as measured by the change in the wet weight of the cultured ID. The secretion of tumor necrosis factor-α (TNF-α) and macrophage chemoattractant protein-1 (MCP-1) from Mφ into the culture medium was stimulated by LIPUS. LIPUS also enhanced matrix metalloproteinase-3 (MMP-3) maturation. Moreover, many apoptotic cell death were observed in the HDR groups with LIPUS exposure. These results suggest that LIPUS enhanced the HDR via MMP-3 activation through TNF-α and MCP-1 pathways. Although animal studies and clinical trial are needed to understand the LIPUS effects on HDR, LIPUS treatment might be an effective treatment for accelerating HDR.
Keywords: In vitro test; Intervertebral disc; Macrophage; Matrix metalloproteinase; Spinal surgery; Low-intensity pulsed ultrasound;

We report a novel method of surface grafting a polymeric phospholipid system containing an acryloyl end group (1stearoyl-2-[12-(acryloyloxy)-dodecanoyl]-sn-glycero-3-phosphocholine) onto medical grade silicone catheters. The surface of silicone catheters was functionalized in a sequence of steps: plasma polymerization of allyl alcohol on the catheter surface, grafting acryloyl moieties and in situ polymerization of the pre-assembled acryloyl terminated phospholipids on the acryloyl functionalized catheter surface. The surface morphological changes analyzed by scanning electron microscopy (SEM) and atomic force microscopy (AFM), a sharp decrease in water contact angle, and appearance of N1s peak in XPS analysis indicated a successful monolayer grafting of the phospholipid. In platelet adhesion tests performed using platelets isolated from rabbit plasma, the phospholipid grafted surface showed fewer adhered platelets, without emerging pseudopodes or aggregation. However, ungrafted catheter surface showed large number of platelets in extensively spread and aggregated states. Thus, this modified phospholipid system and its simple grafting technique was very effective with regard to suppressing in vitro platelet adhesion on the silicon catheter surface.
Keywords: Surface modification; Grafting; Phospholipid; Catheter;

Assessing the in vitro biocompatibility of a novel carbon device for the treatment of sepsis by Susan R. Sandeman; Carol A. Howell; Gary J. Phillips; Andrew W. Lloyd; J. Graham Davies; Sergey V. Mikhalovsky; Steve R. Tennison; Andrew P. Rawlinson; Oleksandr P. Kozynchenko; Hannah L.H. Owen; John D.S. Gaylor; Jennifer J. Rouse; James M. Courtney (7124-7131).
The aim of the present study was to conduct a preliminary investigation into the blood biocompatibility of a novel, uncoated carbon for use in a filtration/adsorption device for the treatment of sepsis. Carbon well prototypes were manufactured from phenol–formaldehyde–aniline-based pyrolysed carbons using monolithic polymer technology. Inflammatory blood cell and plasma protein mediation of the inflammatory response were evaluated using the novel carbon prototypes and compared with dialyser membrane and tissue culture plate controls. Assays determining monocyte and granulocyte adhesion, platelet adhesion and activation, granulocyte activation and complement activation were performed. Preliminary findings suggest an adsorptive but passivating carbon surface. Moderate levels of monocyte and granulocytes adhesion were seen in conjunction with adsorption of plasma proteins to the carbon surface. Activation of granulocyte and adherent platelets was not detected and the complement cascade was not activated by the carbons, indicating a surface compatible with blood contact. The results support the further development of the proposed carbon-based device for the treatment of sepsis.
Keywords: Blood compatibility; Carbon; Cell adhesion/activation; Complement; Sepsis;

Safety of gold in stapes surgery by P. Kwok; M. Schuster; K. Boch; P. Jacob; O. Gleich; J. Strutz (7132-7135).
Gold prostheses in middle ear surgery were found to have a higher extrusion rate than prostheses made from titanium. Incidences of deafness after insertion of a gold piston into the vestibule during stapes surgery have been observed. The aim of this study was to find out to what degree gold cations can diffuse from the prosthesis piston into the perilymph. For this, gold prostheses were incubated in artificial perilymph for four months, after which the gold content of the perilymph was analysed. As gold exhibits a special behaviour in complex fluids like the perilymph, a new analysing method was developed. The results show that gold does leak out of the pistons, that it can be reliably measured and that the amount of gold reaching the perilymph depends on the contact area. As the amount of gold measured in the perilymph stays far below the toxic level, it is very unlikely that the gold cations diffusing from a stapes prosthesis into the perilymph have a toxic effect on the inner ear hair cells. Inflammatory or allergic reactions to gold induced by direct tissue contact, however, cannot be excluded.
Keywords: Gold content; Perilymph; Analysing method; Stapes surgery; Gold stapes prosthesis;

Selectins (E- and P-selectin) and other endothelial expressed leukocyte adhesion molecules (ELAMs) are potential targets for site-specific delivery of therapeutics to the vascular endothelium due to their specific and highly regulated expression in vascular disease. It was recently shown that degradable microspheres coated with antibodies against E-selectin or other ELAMs can target inflammation in vivo. However, targeting ELAMs alone cannot differentiate between normal and diseased state, as a basal level of these LAMs are expressed on endothelium in healthy tissues. Furthermore, leukocytes usually employ two separate adhesion molecules in parallel to home to diseased tissues, and we recently quantified the advantages of a two-receptor display for the targeting of leukocyte mimetics (Eniola AO, Willcox PJ, Hammer DA. Interplay between rolling and firm adhesion elucidated with a cell-free system engineered with two distinct receptor–ligand pairs. Biophys J 2003;85:2720–31). Here, we describe a leukocyte mimetic for targeting therapeutics to the vasculature in inflammatory diseases via two receptors, selectin and intercellular cell adhesion molecule-1 (ICAM-1), where biodegradable, polymer microspheres were co-functionalized with the selectin ligand, sialyl LewisX (sLeX), and an antibody against ICAM-1, anti-ICAM-1 (aICAM-1). These two-receptor targeted particles, at given ratios of sLeX/aICAM-1, firmly adhere to substrate surface in flow only when both targeting ligands can interact with their respective receptors, mimicking the multi-step in vivo leukocyte adhesion in inflammation. Thus, we have faithfully recreated the specificity and extent of leukocyte adhesion in a platform that can allow for local delivery of therapeutics.
Keywords: Selectin; ICAM-1; Leukocyte; Inflammation; Drug targeting; Two-receptor targeting; Microspheres; Poly(lactic-co-glycolic acid) (PLGA);

Chlorhexidine-releasing methacrylate dental composite materials by Danny Leung; David A. Spratt; Jonathan Pratten; Kishor Gulabivala; Nicola J. Mordan; Anne M. Young (7145-7153).
Light curable antibacterial, dental composite restoration materials, consisting of 80 wt% of a strontium fluoroaluminosilicate glass dispersed in methacrylate monomers have been produced. The monomers contained 40–100 wt% of a 10 wt% chlorhexidine diacetate (CHXA) in hydroxyethylmethacrylate (HEMA) solution and 60–0 wt% of a 50/50 mix of urethane dimethacrylate (UDMA) and triethyleneglycol dimethacrylate (TEGDMA). On raising HEMA content, light cure polymerisation rates decreased. Conversely, water sorption induced swelling and rates of diffusion controlled CHXA release from the set materials increased. Experimental composites with 50 and 90 wt% of the CHXA in HEMA solution in the monomer were shown, within a constant depth film fermentor (CDFF), to have slower rates of biofilm growth on their surfaces between 1 and 7 days than the commercial dental composite Z250 or fluoride-releasing dental cements, Fuji II LC and Fuji IX. When an excavated bovine dentine cylinder re-filled with Z250 was placed for 10 weeks in the CDFF, both bacteria and polymers from the artificial saliva penetrated between the material and dentine. With the 50 wt% experimental HEMA/CHXA formulation, this bacterial microleakage was substantially reduced. Polymer leakage, however, still occurred. Both polymer and bacterial microleakage were prevented with a 90 wt% HEMA/CHXA restoration in the bovine dentine due to swelling compensation for polymerisation shrinkage in combination with antibacterial release.
Keywords: Antibacterial; Bacteria; Biofilm; Composite; Controlled drug release; Dental restorative material; Dentine; Fluoride; FTIR; Microbiology; Monomer; PolyHEMA;

Liquid filled nanoparticles as a drug delivery tool for protein therapeutics by Natarajan Venkatesan; Junichiro Yoshimitsu; Yukako Ito; Nobuhito Shibata; Kanji Takada (7154-7163).
In the present study, an attempt was made to study the feasibility of nanoparticulate adsorbents in the presence of an absorption enhancer, as a drug delivery tool for the administration of erythropoietin (EPO) to the small intestine. Liquid filled nano- and micro-particles (LFNPS/LFMPS) were prepared using solid adsorbents such as porous silicon dioxide (Sylysia 550), carbon nanotubes (CNTs), carbon nanohorns, fullerene, charcoal and bamboo charcoal. Surfactants such as a saturated polyglycolysed C8–C18 glyceride (Gelucire 44/14), PEG-8 capryl/caprylic acid glycerides (Labrasol) and polyoxyethylene hydrogenated castor oil derivative (HCO-60) were used as an absorption enhancer at 50 mg/kg along with casein/lactoferrin as enzyme inhibitors. The absorption of EPO was studied by measuring serum EPO levels by an ELISA method after small intestinal administration of EPO-LFNPS preparation to rats at the EPO dose level of 100 IU/kg. Among the adsorbents studied, CNTs showed the highest serum EPO level of 62.7±11.6 mIU/ml. In addition, with the use of casein, EPO absorption was improved, C max 143.1±15.2 mIU/ml. Labrasol showed the highest absorption enhancing effect after intra-jejunum administration than Gelucire 44/14 and HCO-60, 25.6±3.2 and 22.2±3.6 mIU/ml, respectively. Jejunum was found to be the best absorption site for the absorption of EPO from LFNPS. The use of CNTs as LFNPS, improved the bioavailability of EPO to 11.5% following intra-small intestinal administration.
Keywords: Erythropoietin; Carbon nanotubes; Labrasol; Gelucire 44/14; HCO-60; Nanoparticles;

Structure–release rate correlation in collagen gels containing fluorescent drug analog by Silvia H. De Paoli Lacerda; Bruce Ingber; Nitsa Rosenzweig (7164-7172).
The paper examines the release properties of collagen gels that contain covalently bound fluorescent drug analogs. Collagen gels were prepared by fibrilogenesis. The gels were stabilized by cross linking with EDAC/NHS. SEM studies showed that increasing the cross-linking time with EDAC/NHS resulted in decreasing pore size and increasing gel density. Fluorescence spectroscopy measurements showed a clear correlation between decreasing pore size and increasing gel density, and lower release rate from the gels. Additives like chondrotitin-6-sulfate (CS) and amino acids altered the release properties of the cross-linked collagen gels. CS increased the stability of collagen gels to enzymatic degradation and non-enzymatic degradation. This was attributed to increasing gel rigidity due to carbohydrate–protein interactions. The amino acid lysine increased the stability of collagen gels which was attributed to increasing cross-linking level between the collagen fibrils and the primary amine group on the lysine side chain. The amino acid histidine decreased the stability of the gels, particularly to non-enzymatic degradation. These results correlated with increasing pore size following treatment with histidine. Our study shows, for the first time, a clear correlation between structure and release properties of collagen gels. It describes in detail the effect of additives on the structural and release properties of collagen gels. The study focused on gels that were prepared through fibrillogenesis and were therefore similar in structure to native collagen.
Keywords: Collagen; EDAC; Cross linking; Drug delivery; Chondroitin-6-sulfate; Collagenase;

Compatibility of lyotropic liquid crystals with viruses and mammalian cells that support the replication of viruses by Li-Lin Cheng; Yan-Yeung Luk; Christopher J. Murphy; Barbara A. Israel; Nicholas L. Abbott (7173-7182).
We report a study that investigates the biocompatibility of materials that form lyotropic liquid crystals (LCs) with viruses and mammalian cells that support the replication of viruses. This study is focused on aqueous solutions of tetradecyldimethyl-amineoxide (C14AO) and decanol (D), or disodium cromoglycate (DSCG; C23H14O11Na2), which can form optically birefringent, liquid crystalline phases. The influence of these materials on the ability of vesicular stomatitis virus (VSV) to infect human epitheloid cervical carcinoma (HeLa) cells was examined by two approaches. First, VSV was dispersed in aqueous C14AO+D or DSCG, and then HeLa cells were inoculated by contacting the cells with the aqueous C14AO+D or DSCG containing VSV. The infectivity of VSV to the HeLa cells was subsequently determined. Second, VSV was incubated in LC phases of either C14AO+D or DSCG for 4 h, and the concentration (titer) of infectious virus in the LC was determined by dilution into cell culture medium and subsequent inoculation of HeLa cells. Using these approaches, we found that the LC containing C14AO+D caused inactivation of virus as well as cell death. In contrast, we determined that VSV retained its infectivity in the presence of aqueous DSCG, and that greater than 74–82% of the HeLa cells survived contact with aqueous DSCG (depending on concentration of DSCG). Because VSV maintained its function (and we infer structure) in LCs formed from DSCG, we further explored the influence of the virus on the ordering of the LC. Whereas the LC formed from DSCG was uniformly aligned on surfaces prepared from self-assembled monolayers (SAMs) of HS(CH2)11(OCH2CH2)4OH on obliquely deposited films of gold in the absence of VSV, the introduction of 107–108 infectious virus particles per milliliter caused the LC to assume a non-uniform orientation and a colorful appearance that was readily distinguished from the uniformly aligned LCs. Control experiments using cell lysates with equivalent protein concentrations but no virus did not perturb the uniform alignment of the LC.
Keywords: Compatibility; Liquid crystals; Virus; Cells;

Aqueous dispersions of magnetite nanoparticles with NH3 + surfaces for magnetic manipulations of biomolecules and MRI contrast agents by Dar-Bin Shieh; Fong-Yu Cheng; Chia-Hao Su; Chen-Sheng Yeh; Ming-Ting Wu; Ya-Na Wu; Chiau-Yuang Tsai; Chao-Liang Wu; Dong-Hwang Chen; Chen-Hsi Chou (7183-7191).
In the current study, amine surface modified iron-oxide nanoparticles of 6 nm diameter without polymer coating were fabricated in an aqueous solution by organic acid modification as an adherent following chemical coprecipitation. Structure and the superparamagnetic property of magnetite nanoparticles were characterized by selected area electron diffraction (SAED) and superconducting quantum interference measurement device (SQUID). X-ray photoelectron spectrometer (XPS) and zeta potential measurements revealed cationic surface mostly decorated with terminal –NH3 +. This feature enables them to function as a magnetic carrier for nucleotides via electrostatic interaction. In addition, Fe3O4/trypsin conjugates with well-preserved functional activity was demonstrated. The nanoparticles displayed excellent in vitro biocompatibility. The NMR and the in vitro MRI measurements showed significantly reduced water proton relaxation times of both T 1 and T 2. Significantly reduced T 2 and T 2*-weighted signal intensity were observed in a 1.5 T clinical MR imager. In vivo imaging contrast effect showed a fast and prolonged inverse contrast effect in the liver that lasted for more than 1 week. In addition, it was found that the spherical Fe3O4 assembled as rod-like configuration through an aging process in aqueous solution at room temperature. Interestingly, TEM observation of the liver tissue revealed the rod-like shape but not the spherical-type nanoparticles being taken up by the Kupffer cells 120 h after tail vein infusion. Combining these results, we have demonstrated the potential applications of the newly synthesized magnetite nanoparticles in a broad spectrum of biomedical applications.
Keywords: Iron oxides; Nanoparticles; Enzymatic activity; Cytotoxicity; MRI imaging;

Substrate chemistry influences the morphology and biological function of adsorbed extracellular matrix assemblies by Michael J. Sherratt; Daniel V. Bax; Shazia S. Chaudhry; Nigel Hodson; Jian R. Lu; Priya Saravanapavan; Cay M. Kielty (7192-7206).
In addition to mediating cell signalling events, native extracellular matrix (ECM) assemblies interact with other ECM components, act as reservoirs for soluble signalling molecules and perform structural roles. The potential of native ECM assemblies in the manufacture of biomimetic materials has not been fully exploited due, in part, to the effects of substrate interactions on their morphology. We have previously demonstrated that the ECM components, fibrillin and type VI collagen microfibrils, exhibit substrate dependent morphologies on chemically and topographically variable heterogeneous surfaces. Using both cleaning and coating approaches on silicon wafers and glass coverslips we have produced chemically homogeneous, topographically similar substrates which cover a large amphiphilic range. Extremes of substrate amphiphilicity induced morphological changes in periodicity, curvature and lateral spreading which may mask binding sites or disrupt domain structure. Biological functionality, as assayed by the ability to support cell spreading, was significantly reduced for fibrillin microfibrils adsorbed on highly hydrophilic substrates (contact angle 20.7°) compared with less hydrophilic (contact angle 38.3°) and hydrophobic (contact angle 92.8°) substrates. With an appropriate choice of surface chemistry, multifunctional ECM assemblies retain their native morphology and biological functionality.
Keywords: Extracellular matrix (ECM); Atomic force microscopy (AFM); Surface energy; Surface roughness; Protein adsorption; Cell spreading;