Biomaterials (v.26, #17)

Calendar (I).

A note from the Editor: Critical 2005 Conferences by David Williams (3377-3378).

Influence of magnesium doping on the phase transformation temperature of β-TCP ceramics examined by Rietveld refinement by R. Enderle; F Götz-Neunhoeffer; M Göbbels; F.A. Müller; P. Greil (3379-3384).
The influence of Mg doping in Ca3(PO4)2 on the transformation temperature of β-tricalcium phosphate (β-TCP) to α-tricalcium phosphate (α-TCP) was examined. A maximum substitution by 14 mol% of Mg2+ on Ca2+ sites in the β-TCP structure was determined for powders calcinated at 1025±10 °C. X-ray powder diffraction analysis (XRPD) in combination with Rietveld method (TOPAS 2.1) was employed for quantitative phase analysis and structural refinement. The synthesized and characterized Mg-doped samples were sintered in air atmosphere in a vertical tube furnace at suitable temperatures ranging from 1460 to 1680 °C and subsequently quenched to room temperature, in order to stabilize α-TCP. The β to α transformation temperatures were determined by differential temperature analysis and XRPD analysis.
Keywords: β-TCP; α-TCP; Transformation temperature; Mg substitution; Rietveld refinement;

In vitro degradation of silk fibroin by Rebecca L. Horan; Kathryn Antle; Adam L. Collette; Yongzhong Wang; Jia Huang; Jodie E. Moreau; Vladimir Volloch; David L. Kaplan; Gregory H. Altman (3385-3393).
A significant need exists for long-term degradable biomaterials which can slowly and predictably transfer a load-bearing burden to developing biological tissue. In this study Bombyx mori silk fibroin yarns were incubated in 1 mg/ml Protease XIV at 37 °C to create an in vitro model system of proteolytic degradation. Samples were harvested at designated time points up to 12 weeks and (1) prepared for scanning electron microscopy (SEM), (2) lyophilized and weighed, (3) mechanical properties determined using a servohydraulic Instron 8511, (4) dissolved and run on a SDS–PAGE gel, and (5) characterized with Fourier transform infrared spectroscopy. Control samples were incubated in phosphate-buffered saline. Fibroin was shown to proteolytically degrade with predictable rates of change in fibroin diameter, failure strength, cycles to failure, and mass. SEM indicated increasing fragmentation of individual fibroin filaments from protease-digested samples with time of exposure to the enzyme; particulate debris was present within 7 days of incubation. Gel electrophoresis indicated a decreasing amount of the silk 25 kDa light chain and a shift in the molecular weight of the heavy chain with increasing incubation time in protease. Results support that silk is a mechanically robust biomaterial with predictable long-term degradation characteristics.
Keywords: Degradation; Silk; Scaffold; Ligament; Mechanical properties;

Modulation of porosity in apatitic cements by the use of α-tricalcium phosphate—calcium sulphate dihydrate mixtures by Enrique Fernández; Maria Daniela Vlad; Maria Montserrat Gel; Jose López; Ricardo Torres; Juan V. Cauich; Marc Bohner (3395-3404).
Calcium phosphate bone cements are injectable biomaterials that are being used in dental and orthopaedic applications through minimally invasive surgery techniques. Nowadays, apatitic bone cements based on α-tricalcium phosphate (α-TCP) are of special interest due to their self-setting behaviour when mixed with an aqueous liquid phase. In this study, a new method to improve osteointegration of α-TCP-based cements is presented. This method consists in the modification of the cement's powder phase with different amounts of calcium sulphate dihydrate (CSD). The resulting hardening properties of the new biphasic cements are a combination between the progressive hardening due to the main α-TCP reactant and the progressive dissolution of the CSD phase, which render a porous material. It was observed that the maximum compressive strength of Biocement-H © (45 MPa) decreased as the amount of CSD increased in the cement powder mixture (≈30 MPa for 25 wt% of CSD). It was also observed that after complete dissolution of the CSD phase a porous apatitic structure appears with a mechanical compressive strength suitable for cancellous bone applications (10 MPa).
Keywords: Cement; Calcium phosphate; Calcium sulphate; Physico-chemical properties;

Fatigue behavior of packable composites by Y. Abe; M.J.A. Braem; Paul Lambrechts; S. Inoue; M. Takeuchi; B. Van Meerbeek (3405-3409).
The aim of this study was to investigate the flexural fatigue behavior of 11 packable composites. Of each material 30 rectangular samples (1.2×5×35 mm) for flexural fatigue test were prepared. The clamped fracture strength and flexural fatigue limit (FFL) of each material were determined using a custom-made fatigue machine, after storage of the samples for one month in water at 37 °C. All data were analyzed using one-way ANOVA and Bonferroni/Dunn's test for multiple comparisons (p<0.05). Regression analysis was used to evaluate the relationship between elastic modulus (Abe et al., 2001), clamped fracture strength or FFL and inorganic filler fraction (vol%). The fracture strengths of all but two materials were in between those of the compact-filled densified composites and the microfine ones. The FFL of the packable composites tested were significantly lower than those of the compact-filled densified composites. Three of the tested materials showed even significantly lower FFL than did the microfine composites. There were statistically significant relationships between both the elastic modulus and the volumetric filler fraction (R 2=0.974, p=1.990×10−7).The great diversity in packable composites makes clinical assessment necessary with regard to a justified use in posterior teeth.
Keywords: Composites; Packable; Condensable; Elastic modulus; Clamped fracture strength; Flexural fatigue limit;

This study examined the engineering and true axial stress–strain behavior of smooth cylindrical and shallow and deep notched cylindrical test specimens, under applied axial tensile loading using non-contacting methods, of both conventional and highly crosslinked ultra-high molecular weight polyethylenes (UHMWPEs). The smooth specimens experienced a uniaxial stress state, while the notched specimens experienced a triaxial stress state in the vicinity of the notch. Materials were all prepared from a single batch of medical grade GUR 1050 resin (Ticona, Bayport, TX). The two conventional UHMWPEs were as-received (virgin) and gamma radiation sterilized at 30 kGy in a nitrogen atmosphere (radiation sterilized). The two highly crosslinked UHMWPEs were each irradiated at 100 kGy and then post-processed with one of either of the two thermal treatments: annealing, which was done below the melt transition temperature (T m), at 110 °C for two hours (110 °C-annealed), and remelting, which was done above T m, at 150 °C (150 °C-remelted). All of the materials showed notch strengthening; that is, a significant elevation of axial yield properties (both engineering and true) for the shallow and deep notched conditions. Axial ultimate properties (engineering and true) were significantly decreased for the notched conditions compared with the smooth condition. Hardening ratios (both true and engineering), which are defined in this work as the ratio of ultimate stress or strain to yield stress or strain, were also found to significantly decrease with notching. The extent of change was dependent on the UHMWPE material. The micromechanism of fracture differed between the smooth and notched conditions. This study suggests that notches inherent in the design of UHMWPE joint replacement components (posts, undercuts, grooves) will have different notch sensitivities depending on the UHMWPE formulation.
Keywords: UHMWPE; Multiaxial; Crosslinking; Notch strengthening; Fracture micromechanisms;

Ultrastructural observation of single-crystal apatite fibres by Mamoru Aizawa; Alexandra E. Porter; Serena M. Best; William Bonfield (3427-3433).
Hydroxyapatite (HAp) has been widely used as a biomaterial for substituting human hard tissues such as bone. By altering the morphology of HAp crystals, novel properties may be produced by controlling the orientation of the crystal planes. Apatite fibres were successfully synthesized by precipitation from aqueous solutions containing Ca(NO3)2, (NH4)2HPO4, urea and HNO3. The products were composed of carbonate-containing apatite fibres with preferred orientation along the { h 0 0 } planes. Examination of individual fibres using transmission electron microscopy showed that the as-synthesized apatite fibres were highly strained single crystals with the c-axis orientation parallel to the long axis of the fibre. The crushed fibres consisted of domains that were preferentially oriented with the c-axis parallel to the long axis of the fibres. When the apatite fibres were heated to 800, 1000 and 1200 °C for 1 h, the domains were removed and grain boundaries, dislocations and voids were formed.
Keywords: Hydroxyapatite; Microstructure; Transmission electron microscopy; Apatite fibre; Single crystal;

Preparation of a chemically anchored phospholipid monolayer on an acrylated polymer substrate by Hyun Kyun Kim; Kwangmeyung Kim; Youngro Byun (3435-3444).
This paper describes a strategy for designing a chemically anchored phospholipid monolayer that could be used as coating materials for biomedical implants. To make a chemically anchored phospholipid monolayer on the polymer substrate, we prepared the mono-acrylated phospholipid (1-palmitoyl-2-[12-(acryloyloxy)-dodecanoyl]-sn-glycero-3-phosphocholine; acryloyl-PC) and the acrylated polymer (poly(octadecylacrylate-co-4-acryloyloxy butylacrylate)), which was synthesized by the acrylation of poly(octadecyl acrylate-co-hydroxybutyl acrylate, poly(OA-co-HA)) with acryloyl chloride. The chemically anchored phospholipid monolayer was prepared by using in situ photopolymerization of a pre-assembled phospholipid monolayer, produced by lipid vesicle fusion, onto the acrylated polymer coated silicon wafer. Optimal condition of vesicle fusion and irradiation time was determined from the degree of hydrophilicity rendered by the polymerized phospholipid surface. The physicochemical properties of polymerized phospholipid monolayer on the substrate were evaluated using water contact angle, field-emission scanning electron micrograph (FE-SEM), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). These results confirmed that the polymerized phospholipid monolayer was chemically anchored on the acrylated polymer substrate. The chemically anchored phospholipid monolayer was stable in aqueous condition for 2 weeks, but the physically adsorbed phospholipid monolayer got removed within 1 day. Moreover, the polymerized phospholipid monolayer also suppressed albumin absorption and platelet adhesion, in vitro. This polymerized phospholipid monolayer provides a new biomimetic system for coating medical devises.
Keywords: Phospholipid monolayer; In situ photopolymerization; Acrylated polymer substrate; Blood compatibility;

Liquid–vapor interfacial tension of blood plasma, serum and purified protein constituents thereof by Anandi Krishnan; Arwen Wilson; Jacqueline Sturgeon; Christopher A. Siedlecki; Erwin A. Vogler (3445-3453).
A systematic study of water–air (liquid–vapor, LV) interfacial tension γ lv of blood plasma and serum derived from four different mammalian species (human, bovine, ovine and equine) reveals nearly identical concentration-dependence ( d γ lv d ln C B ; where C B is plasma/serum dilution expressed in v/v concentration units). Comparison of results to a previously-published survey of purified human-blood proteins further reveals that d γ lv d ln C B of plasma and serum is surprisingly similar to that of purified protein constituents. It is thus concluded that any combination of blood-protein constituents will be substantially similar because d γ lv d ln C B of individual proteins are very similar. Experimental results are further interpreted in terms of a recently-developed theory emphasizing the controlling role of water in protein adsorption. Accordingly, the LV interphase saturates with protein adsorbed from bulk solution at a fixed weight-volume concentration (∼436 mg/mL) independent of protein identity or mixture. As a direct consequence, d γ lv d ln C B of purified proteins closely resembles that of mixed solutions and does not depend on the relative proportions of individual proteins comprising a mixture. Thus variations in the plasma proteome between species are not reflected in d γ lv d ln C B nor is serum different from plasma in this regard, despite being depleted of coagulation proteins (e.g. fibrinogen). A comparison of pendant-drop and Wilhelmy-balance tensiometry as tools for assessing protein γ lv shows that measurement conditions employed in the typical Wilhelmy plate approach fails to achieve the steady-state adsorption state that is accessible to pendant-drop tensiometry.
Keywords: Protein adsorption; Liquid–vapour interfacial tension; Plasma; Serum; Blood proteins;

In vivo evaluation of plasma-sprayed wollastonite coating by Weichang Xue; Xuanyong Liu; XueBin Zheng; Chuanxian Ding (3455-3460).
Wollastonite coatings were prepared by plasma spraying. The bioactivity of wollastonite coatings was investigated in vivo by implanting in dog's muscle, cortical bone and marrow, respectively. The behaviour of bone tissue around wollastonite coatings were examined by histological and SEM observation. After 1 month in the muscle, a bone-like apatite layer was found to form on the surface of the wollastonite coating. When implanted in cortical bone, histological observation demonstrated that bone tissue could extend and grow along the surface of the wollastonite coating. The coating bonded directly to the bone without any fibrous tissue, indicating good biocompatibility and bone conductivity. SEM and EDS analysis revealed that bone did not bond to wollastonite coating directly, but through a Ca/P layer. This suggested that the formation of bone-like apatite layer was very important for bonding to the bone tissue. The amount of bone–implant contact was also measured. Wollastonite coating was shown to stimulate more bone formation on its surface than titanium coating after implantation for 1 month, enhancing the short-term osseointegration properties of implant. The test in marrow indicated that wollastonite coatings could induce new bone formation on their surface showing good bone inductivity property.
Keywords: Wollastonite coating; Plasma spraying; In vivo; Apatite; Bone;

Vascularization and gene regulation of human endothelial cells growing on porous polyethersulfone (PES) hollow fiber membranes by Ronald E. Unger; Kirsten Peters; Quan Huang; Andreas Funk; Dieter Paul; C.J. Kirkpatrick (3461-3469).
Open-cell hollow fibers made of polyethersulfone (PES) manufactured in the absence of solvents with pore diameters smaller than 100 μm were examined for vascularization by human endothelial cells. The goal of this study was to determine whether the 3-D porous character of the PES surface affected human endothelial cell morphology and functions. Freshly isolated human endothelial cells from the skin (HDMEC), from the lung (HPMEC) and from umbilical cords (HUVEC) and two human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS.c1 were added to PES fibers and cell adherence and growth was followed by confocal laser scanning microscopy. Prior coating of PES with gelatin or fibronectin was necessary for adhesion and spreading of cells over the uneven porous surface with time. Confluent cells exhibited typical strong PECAM-1 expression at cell–cell borders. Little expression of the activation markers E-selectin, ICAM-1, and VCAM-1 was observed by RT-PCR of endothelial cells growing on PES. However, after stimulation for 4 h by LPS, activation of these markers was observed and it was shown by immunofluorescent staining that induction occurred in most of the cells, thus confirming an intact functionality. Finally, cells growing as a monolayer on PES migrated to form microvessel-like structures when placed under conditions that stimulated angiogenesis. Thus, human endothelial cells grown on fibronectin-coated PES fibers retain important endothelial-cell specific morphological and functional properties and PES may serve as a useful biomaterial in tissue engineering and biotechnology applications.
Keywords: Polyethersulfone; PES; Vascularization; Endothelial cell; Cell compatibility; Gene regulation;

A new method for processing hydroxyapatite/ultrahigh molecular weight polyethylene (HA/UHMWPE) composite has been developed by combining wet ball milling and swelling. Sintered HA particles were ground in ethanol to ∼50 nm in diameter. The nano-sized HA particles were mechanically mixed with UHMWPE in the ball mill and then compression molded into solid slabs. The slabs were then swollen in a pharmaceutical grade paraffin oil to enhance the UHMWPE chain mobility and HA/UHMWPE interface adhesion before final hot press. The resultant composite exhibits a two-zone network structure formed by a homogeneous HA-rich phase and a UHMWPE-rich phase. This process resulted in a 90% increase in Young's modulus and a 50% increase in the yield strength of HA/UHMWPE composite, comparing with those of unfilled UHMWPE.
Keywords: Hydroxyapatite; UHMWPE; Composite; Swelling; Ball milling;

Thiol groups were attached to polyethylene terephthalate (PET) to promote the transfer of a known platelet inhibitor, nitric oxide (NO), from nitrosated thiols naturally found in the body to PET, followed by the release of NO from PET to prevent platelet adhesion. In order to immobilize the most thiols on the modified polymer, the processing parameters used to attach the following three thiol containing groups were assessed: l-cysteine, 2-iminothiolane, and a cysteine polypeptide. When comparing the immobilized concentrations of thiol groups from each of the optimized processes the amount of immobilized thiol groups increased in order with the following groups: cysteine polypeptide <2-iminothiolane <l-cysteine. The effect of each optimized polymer on platelet adhesion was studied by in vitro experiments utilizing a parallel plate perfusion chamber. Platelets in the following solutions were tested: Tyrode's buffer, 7 μm nitrosated bovine serum albumin in Tyrode's buffer, 50% plasma in Tyrode's buffer, and 50% whole blood in Tyrode's buffer. All of the polymers demonstrated a significant decrease in platelet adhesion compared to controls when exposed to the BSANO, plasma and whole blood solutions. The most significant decrease was for the l-cysteine modified polymer in the plasma solution with a 65% decrease.
Keywords: L-cysteine; 2-Iminothiolane; Cysteine polypeptide; Platelets; Poly(ethylene terephthalate) (PET);

In vitro studies on the effect of delaminated a-C:H film fragments on bone marrow cell cultures by Arie Bruinink; Anouk Schroeder; Gilbert Francz; Roland Hauert (3487-3494).
Amorphous hydrogenated carbon (a-C:H) films have many outstanding properties required for a protective coating material on load bearing medical implants. Recently, titanium doped a-C:H films have been evaluated regarding their effects on bone marrow cell cultures. But many materials that are well-tolerated in bulk form are able to induce toxic reaction if present particulate form. In order to further assess biocompatibility aspects of these two coatings, film delamination has been mimicked in exposure to fluids. In the present study, particles from a-C:H, a-C:H/Ti and a-C:H–a-C:H/Ti bilayer films were added to bone marrow cell cultures in vitro. The results showed that plain a-C:H and to a certain extent a-CH/Ti particles were inert. Both kinds of particles did not significantly stimulate the osteoclast-related enzyme tartrate resistant acid phosphatase (TRAP). A slight increase in cell proliferation and total culture TRAP was found in cultures treated by a-C:H–a-C:H/Ti bilayer films. Latter effect can probably be traced back by the relative high percentage of small particles of a size of around 2 μm. However, if corrected by the cell number also no differences between particle-treated and untreated control cultures could be found, indicating the absence of a toxic effect from delaminated a-C:H coatings.
Keywords: Particles; Bone marrow cells; a-C:H; Titanium; Biocompatibility;

Chemical modification of poly(vinyl chloride) resin using poly(ethylene glycol) to improve blood compatibility by Biji Balakrishnan; D.S. Kumar; Yasuhiko Yoshida; A. Jayakrishnan (3495-3502).
Poly(vinyl chloride) (PVC) was aminated by treating the resin with a concentrated aqueous solution of ethylenediamine. The aminated PVC was then reacted with hexamethylene diisocyanate to incorporate the isocyanate group onto the polymer backbone. The isocyanated PVC was further reacted with poly(ethylene glycol) (PEG) of molecular weight 600 Da. The modified polymer was characterized using infrared and X-ray photoelectron spectroscopy (XPS) and thermal analysis. Infrared and XPS spectra showed the incorporation of PEG onto PVC. The thermal stability of the modified polymer was found to be lowered by the incorporation of PEG. Contact angle measurements on the surface of polymer films cast from a tetrahydrofuran solution of the polymer demonstrated that the modified polymer gave rise to a significantly hydrophilic surface compared to unmodified PVC. The solid/water interfacial free energy of the modified surface was 3.9 ergs/cm2 as opposed to 18.4 ergs/cm2 for bare PVC surface. Static platelet adhesion studies using platelet-rich plasma showed significantly reduced platelet adhesion on the surface of the modified polymer compared to control PVC. The surface hydrophilicity of the films was remarkably retained even in the presence of up to 30 wt% concentration of the plasticizer di-(2-ethylhexyl phthalate). The study showed that bulk modification of PVC with PEG using appropriate chemistry can give rise to a polymer that possesses the anti-fouling property of PEG and such bulk modifications are less cumbersome compared to surface modifications on the finished product to impart anti-fouling properties to the PVC surface.
Keywords: Polyvinyl chloride; Polyethylene oxide; Surface modification; Surface energy; Platelet adhesion; Biocompatibility;

The aim of this in vitro study was to investigate the effect of low-level laser therapy (LLLT) on the attachment, proliferation, differentiation and production of transforming growth factor-ß1 (TGF-β 1) by human osteoblast-like cells (HOB). Cells derived from human mandibular bone were exposed to GaAlAs diode laser at dosages of 1.5 or 3 J/cm2 and then seeded onto titanium discs. Non-irradiated cultures served as controls. After 1, 3 and 24 h, cells were stained and the attached cells were counted under a light microscope. In order to investigate the effect of LLLT on cell proliferation after 48, 72 and 96 h, cells were cultured on titanium specimens for 24 h and then exposed to laser irradiation for three consecutive days. Specific alkaline phosphatase activity and the ability of the cells to synthesize osteocalcin after 10 days were investigated using p-nitrophenylphosphate as a substrate and the ELSA-OST-NAT immunoradiometric kit, respectively. Cellular production of TGF-β 1 was measured by an enzyme-linked immunosorbent assay (ELISA), using commercially available kits. LLLT significantly enhanced cellular attachment ( P < 0.05 ). Greater cell proliferation in the irradiated groups was observed first after 96 h. Osteocalcin synthesis and TGF-β 1 production were significantly greater ( P < 0.05 ) on the samples exposed to 3 J/cm2. However, alkaline phosphatase activity did not differ significantly among the three groups. These results showed that in response to LLLT, HOB cultured on titanium implant material had a tendency towards increased cellular attachment, proliferation, differentiation and production of TGF-β 1, indicating that in vitro LLLT can modulate the activity of cells and tissues surrounding implant material.
Keywords: Low level laser therapy; Titaninum implant; Human osteoblasts; Cell attachment; Differentiation; Proliferation; TGF- β 1 ;

Fabrication and biocompatibility of polypyrrole implants suitable for neural prosthetics by Paul M. George; Alvin W. Lyckman; David A. LaVan; Anita Hegde; Yuika Leung; Rupali Avasare; Chris Testa; Phillip M. Alexander; Robert Langer; Mriganka Sur (3511-3519).
Finding a conductive substrate that promotes neural interactions is an essential step for advancing neural interfaces. The biocompatibility and conductive properties of polypyrrole (PPy) make it an attractive substrate for neural scaffolds, electrodes, and devices. Stand-alone polymer implants also provide the additional advantages of flexibility and biodegradability. To examine PPy biocompatibility, dissociated primary cerebral cortical cells were cultured on PPy samples that had been doped with polystyrene-sulfonate (PSS) or sodium dodecylbenzenesulfonate (NaDBS). Various conditions were used for electrodeposition to produce different surface properties. Neural networks grew on all of the PPy surfaces. PPy implants, consisting of the same dopants and conditions, were surgically implanted in the cerebral cortex of the rat. The results were compared to stab wounds and Teflon implants of the same size. Quantification of the intensity and extent of gliosis at 3- and 6-week time points demonstrated that all versions of PPy were at least as biocompatible as Teflon and in fact performed better in most cases. In all of the PPy implant cases, neurons and glial cells enveloped the implant. In several cases, neural tissue was present in the lumen of the implants, allowing contact of the brain parenchyma through the implants.
Keywords: Biocompatibility; Micropatterning; Electroactive polymer; Neural prosthesis; In vitro test; In vivo test;

In vivo study of the effect of RGD treatment on bone ongrowth on press-fit titanium alloy implants by Brian Elmengaard; Joan E. Bechtold; Kjeld Søballe (3521-3526).
Early bone ongrowth is known to increase primary implant fixation and reduce the risk of early implant failure. Arg–Gly–Asp (RGD) peptide has been identified as playing a key role in osteoblast adhesion and proliferation on various surfaces. The aim for this study is to evaluate the effect of RGD peptide coating on the bony fixation of orthopaedic implants, to justify its further evaluation in clinical applications. Sixteen unloaded cylindrical plasma sprayed Ti6Al4 V implants coated with cyclic RGD peptide were inserted as press-fit in the proximal tibia of 8 mongrel dogs for 4 weeks. Uncoated control implants were inserted in the contralateral tibia. Results were evaluated by histomorphometry and mechanical push-out test. A significant two-fold increase was observed in bone ongrowth for RGD-coated implants. Also, fibrous tissue ongrowth was significantly reduced for RGD-coated implants. Bone volume was significantly increased in a 0–100 μm zone around the implant. The increased bony anchorage resulted in moderate increases in mechanical fixation as apparent shear stiffness was significantly higher for RGD-coated implants. Increases in median ultimate shear strength and energy to failure were also observed. This study demonstrates that cyclic RGD coating increases early bony fixation of unloaded press-fit titanium implants.
Keywords: Integrins; RGD peptide; Implant fixation; Osteoblasts; In vivo;

The healing of confined critical size cancellous defects in the presence of silk fibroin hydrogel by M. Fini; A. Motta; P. Torricelli; G. Giavaresi; N. Nicoli Aldini; M. Tschon; R. Giardino; C. Migliaresi (3527-3536).
In vitro and in vivo behaviour of an injectable silk fibroin (SF) hydrogel was studied through osteoblast cultures and after implantation in critical-size defects of rabbit distal femurs. A commercial synthetic poly(d,l lactide-glycolide) copolymer was used as control material. In vitro biocompatibility was evaluated by measuring LDH release, cell proliferation (WST1), differentiation (ALP, OC), and synthetic activity (collagen I, TGF ß1, IL-6). Bone defect healing rate and quality of the newly formed bone inside the defects were determined in vivo by measuring trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), mineral apposition rate (MAR) and bone formation rate (BFR/B.Pm). In vitro tests indicated that both materials significantly increased cell proliferation in comparison with the negative control. A significant increase in the TGF-β1 level was found for SF hydrogel in comparison with the control material and negative control. Both materials promoted bone healing when used to fill critical size defects in rabbit femurs. The new-formed bone of the SF hydrogel treated defects showed significantly higher BV/TV, Tb.Th, MAR and BFR/B.Pm and lower Tb.Sp values in comparison with the control gel. At 12 weeks the re-grown bone of the SF hydrogel-treated defects appeared more similar to normal bone than that of the control synthetic polymeric material-treated defects, except for the Tb.N value that differed significantly from that of normal bone (p<0.05). MAR and BFR/B.Pm presented significantly (p<0.05) higher values for SF hydrogel-treated defects in comparison with controls treated with a synthetic polymeric material, confirming that SF hydrogel accelerated remodelling processes.
Keywords: Silk; Hydrogel; Osteoblast; Biocompatibility; Animal model;

As a new member of polyhydroxyalkanoate (PHA) family, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) has better mechanical properties compared with poly(3-hydroxybutyrate) (PHB). Still, many properties of PHBHHx and its blend with PHB may be exploited for a wide range of applications, such as tissue engineering and drug delivery. In this study, PHBHHx was found to be completely miscible with PHB in their blends. Crystallization behavior of these polyester blends played an important role in determination of the surface physicochemical properties including surface free energy, chemical states and polarity, and could subsequently govern the cellular responses. Interaction between PHB and PHBHHx influenced the non-dispersion surface free energy component, which governed the total surface free energy. It was shown that the blend with a 1:1 ratio of PHB/PHBHHx possessed the highest surface free energy, which was the most optimal material for chondrocytes adhesion. After 24 h, the amount of chondrocytes adhered on films of PHB/PHBHHx (1:1) was 2.1×104  cells/cm2, 5-times more compared with that on the PHB films (0.4×104  cells/cm2). The polarity of the blends increased with decreasing crystallinity. After 8 days of cultivation, the chondrocytes attached on PHB films were surrounded by both collagen II and collagen X, the amount of extracellular collagen X decreased with increasing polarity contributed by increasing PHBHHx content in the blend, while chondrocytes changed shapes from spherical to flat with increasing polarity. It indicated that endochondral ossification of chondrocytes was remarkably influenced by the crystallinity of the polyesters.
Keywords: Polyhydroxyalkanoates; PHB; PHBHHx; Chondrocytes; Crystallization; Surface free energy; Chemical state; Adhesion; Differentiation;

The effect of simvastatin on polyethylene particle-induced osteolysis by Fabian von Knoch; Anja Heckelei; Christian Wedemeyer; Guido Saxler; Gero Hilken; Franz Henschke; Franz Löer; Marius von Knoch (3549-3555).
This study aimed to investigate the effects of the HMG-CoA reductase inhibitor simvastatin on ultra-high molecular weight polyethylene (UHMWPE) particle-induced osteolysis.The murine calvarial osteolysis model was used in 21 C57BL/J6 mice randomized to three groups. Group I underwent sham surgery only, group II received UHMWPE particles, and group III, particles and simvastatin treatment. After two weeks, calvaria were processed for histomorphometry. Bone resorption was measured as resorption within the midline suture using Giemsa staining. Osteoclast numbers were determined per high-power field using TRAP-staining. Statistical analysis was performed using one-way ANOVA and Student's t-test.Bone resorption in midline suture was 0.094±0.007 mm2 in sham controls (group I), 0.25±0.025 mm2 after particle implantation without further intervention (group II), and 0.131±0.02 mm2 with particle implantation and additional simvastatin treatment (group III) ( p = 0.00003 ). Osteoclast numbers were 15.3±3.6 in group I, 48.7±7.1 in group II and 6.2±3.1 in group III ( p = 0.00002 ).In conclusion, simvastatin treatment markedly decreased UHMWPE particle-induced osteolysis in a murine calvarial model. This finding suggests that simvastatin may have a role for noninvasive prevention and treatment of wear debris-mediated periprosthetic osteolysis after total joint arthroplasty.
Keywords: Osteolysis; Wear debris; Polyethylene; Calvarium; Osteoclast;

In vivo corrosion of four magnesium alloys and the associated bone response by F. Witte; V. Kaese; H. Haferkamp; E. Switzer; A. Meyer-Lindenberg; C.J. Wirth; H. Windhagen (3557-3563).
Degrading metal alloys are a new class of implant materials suitable for bone surgery. The aim of this study was to investigate the degradation mechanism at the bone–implant interface of different degrading magnesium alloys in bone and to determine their effect on the surrounding bone. Sample rods of four different magnesium alloys and a degradable polymer as a control were implanted intramedullary into the femora of guinea pigs. After 6 and 18 weeks, uncalcified sections were generated for histomorphologic analysis. The bone–implant interface was characterized in uncalcified sections by scanning electron microscopy (SEM), element mapping and X-ray diffraction. Results showed that metallic implants made of magnesium alloys degrade in vivo depending on the composition of the alloying elements. While the corrosion layer of all magnesium alloys accumulated with biological calcium phosphates, the corrosion layer was in direct contact with the surrounding bone. The results further showed high mineral apposition rates and an increased bone mass around the magnesium rods, while no bone was induced in the surrounding soft tissue. From the results of this study, there is a strong rationale that in this research model, high magnesium ion concentration could lead to bone cell activation.

3D microenvironment as essential element for osteoinduction by biomaterials by Pamela Habibovic; Huipin Yuan; Chantal M. van der Valk; Gert Meijer; Clemens A. van Blitterswijk; Klaas de Groot (3565-3575).
In order to unravel the mechanism of osteoinduction by biomaterials, in this study we investigated the influence of the specific surface area on osteoinductive properties of two types of calcium phosphate ceramics. Different surface areas of the ceramics were obtained by varying their sintering temperatures.Hydroxyapatite (HA) ceramic was sintered at 1150 and 1250 °C. Biphasic calcium phosphate (BCP) ceramic, consisting of HA and beta-tricalcium phosphate (β-TCP), was sintered at 1100, 1150 and 1200 °C.Changes in sintering temperature did not influence the chemistry of the ceramics; HA remained pure after sintering at different temperatures and the weight ratio of HA and β-TCP in the BCP was independent of the temperature as well. Similarly, macroporosity of the ceramics was unaffected by the changes of the sintering temperature. However, microporosity (pore diameter <10 μm) significantly decreased with increasing sintering temperature. In addition to the decrease of the microporosity, the crystal size increased with increasing sintering temperature. These two effects resulted in a significant decrease of the specific surface area of the ceramics with increasing sintering temperatures.Samples of HA1150, HA1250, BCP1100, BCP1150 and BCP1200 were implanted in the back muscles of Dutch milk goats and harvested at 6 and 12 weeks post implantation. After explantation, histomorphometrical analysis was performed on all implants.All implanted materials except HA1250 induced bone. However, large variations in the amounts of induced bone were observed between different materials and between individual animals.Histomorphometrical results showed that the presence of micropores within macropore walls is necessary to make a material osteoinductive. We postulate that introduction of microporosity within macropores, and consequent increase of the specific surface area, affects the interface dynamics of the ceramic in such a way that relevant cells are triggered to differentiate into the osteogenic lineage.
Keywords: Hydroxyapatite (HA); Biphasic calcium phosphate (BCP); Sintering temperature; Macrostructure; Microstructure; Specific surface area; Osteoinduction;

Engineering of volume-stable adipose tissues by Seung-Woo Cho; Sang-Soo Kim; Jong Won Rhie; Hyun Mi Cho; Cha Yong Choi; Byung-Soo Kim (3577-3585).
Autologous adipose tissues have been clinically used for augmentation of soft tissues lost due to mastectomy or lumpectomy in plastic and reconstructive surgery. However, this therapy has problems of absorption and subsequent volume loss of the implanted adipose tissues. In this study, volume-stable adipose tissues were engineered in vivo using mechanical support structures fabricated from biodegradable synthetic polymers. Dome-shaped mechanical support structures were fabricated by reinforcing poly(glycolic acid) fiber-based matrices with poly(L-lactic acid). The support structures were placed into subcutaneous pockets of athymic mice, and human preadipocytes suspended in fibrin matrix were injected into the space under the support structures (group I). Injection of either fibrin matrix without preadipocytes under the support structures (group II) or fibrin matrix containing preadipocytes into subcutaneous spaces with no support structures (group III) served as controls. Six weeks after implantation, the original implant volume was maintained approximately in groups I and II, whereas, group III showed significant implant shrinkage. The compressive modulus of the mechanical support structures did not change significantly over 6-week incubation in phosphate-buffered saline at 37 °C. Histological analyses of the implants showed regeneration of adipose tissues in group I. In contrast, groups II and III did not show extensive adipose tissue formation. This study demonstrates that volume-stable adipose tissues can be engineered in vivo using mechanical support structures. This technique offers the potential for augmentation of adipose tissues with volume conservation.
Keywords: Volume-stable; Adipose tissue; Mechanical support structure; Preadipocyte; Tissue engineering;

Biodegradable gelatin sponges incorporating various amounts of β-tricalcium phosphate (βTCP) (gelatin-βTCP) were fabricated and the in vitro osteogenic differentiation of mesenchymal stem cells (MSC) isolated from the rat bone marrow in the sponges was investigated. The gelatin sponges incorporating βTCP have an interconnected pore structure with the average size of 180–200 μm, irrespective of the βTCP amount. The stiffness of the sponges became higher with an increase in the amount of βTCP. When seeded into the sponges by an agitated method, MSC were homogeneously distributed throughout the sponge. The morphology of cells attached got more spreaded with the increased βTCP amount. The rate of MSC proliferation depended on the βTCP amount and culture method: the higher the βTCP amount in the stirring culture, the higher the proliferation rate. The deformed extent of gelatin-βTCP sponges was suppressed with the increased amount of βTCP. When measured to evaluate the osteogenic differentiation of MSC, the alkaline phosphatase activity and osteocalcin content became maximum for the sponge with a βTCP amount of 50 wt%, although both the values were significantly high in the stirring culture compared with those in the static culture. We concluded that the attachment, proliferation, and osteogenic differentiation of MSC were influenced by sponge composition of gelatin and βTCP as the cell scaffold.
Keywords: Mesenchymal stem cells; Sponge scaffold; Gelatin; β-tricalcium phosphate; Osteogenic differentiation;

In order to increase the functionality of islets encapsulated in a biohybrid artificial pancreas (BAP), it was proposed that co-encapsulation with insulinotropic agents would improve insulin secretion from islets. To prevent agents from leaking out, conjugation with high-molecular-weight polymers was inevitable. In this study, synthetic glucagon-like peptide-1 (GLP-1) (7–37) was conjugated to a water-soluble polymer, poly(N-vinyl-2-pyrroridone-co-acrylic acid) (5 mol% acrylic acid, M w 445 kDa), via poly(ethylene glycol, M w 3.4 kDa) spacer. The chemical conjugation was confirmed by reverse phase-HPLC and the GLP-1 content in the GLP-1/polymer conjugate (VAPG) was determined by UV spectrophotometry at 280 nm (ca. 29 wt/wt%). In a static insulin secretion test, the VAPG increased insulin secretion up to 200% over a control (no stimulation) at high glucose levels, although the insulinotropic activity of VAPG was slightly lower than that of native GLP-1. The bioactivity of VAPG was prolonged for at least 2 weeks, which was examined by co-encapsulation of the conjugate into islet microcapsules. Dose–response curve revealed that the half-maximal effective dose (ED50) of VAPG was about 55 nm (25 nm for native GLP-1). By N-terminal analysis using aminopeptidase and RP-HPLC, it was confirmed that the lowered bioactivity of VAPG stemmed from the polymer conjugation to N-terminal histidine moieties, which actively participate in binding to GLP-1 receptors, resulting in only 16% of N-terminal histidine remaining intact after the conjugation reaction. Finally, the specific interaction of the VAPG with isolated rat islets was investigated. Total cellular cyclic AMP levels were measured and confocal microscopy was conducted using GLP-1 and VAPG labeled with fluorescent probes. It was found that VAPG effectively increased the cAMP level in islet cells in a glucose concentration-dependent manner. Moreover, the confocal microscopy study showed that the binding of VAPG occurs at the same location where GLP-1 binds but with less affinity than that of native GLP-1. In summary, a GLP-1/polymer conjugate was synthesized for the first time, and its bioactivity was examined, which must result from its specific interaction with isolated islets.
Keywords: Glucagon-like peptide-1 (GLP-1); Polymer; Insulin secretion; Islets of Langerhans; Biohybrid artificial pancreas;

Alginate microcapsules prepared with xyloglucan as a synthetic extracellular matrix for hepatocyte attachment by Seog-Jin Seo; Toshihiro Akaike; Yun-Jaie Choi; Mayumi Shirakawa; Inn-Kyu Kang; Chong-Su Cho (3607-3615).
In this study, xyloglucan (XG) was used as a new synthetic extracellular matrix (ECM) for primary mouse hepatocyte attachment in Ca-alginate (AL) capsules. The rates of hepatocytes adhesion onto collagen type I-, XG-coated and uncoated polystyrene (PS) surface were 89.1%, 91.1% and 25.5%, respectively, at 4 h after incubation at 37 °C. From the inhibition study in a cell adhesion assay, the adhesion rates of freshly isolated hepatocytes and preincubated hepatocytes with 20 mm galactose onto the XG-coated surface were 55.7 and 17.3%, respectively, after 30 min incubation at 37 °C. Flow cytometric analysis showed that the internalization of XG by freshly isolated hepatocytes was stronger than preincubated hepatocytes with 20 mm galactose. The concentration of XG in AL/XG capsules to perform the best liver-specific functions was 0.5 mg/ml, where the highest albumin secretion rates were obtained. The albumin secretion, ammonia elimination rates and cell viability of hepatocytes were slowly decreased with culture time in AL/XG capsules, whereas those were rapidly decreased in AL capsules, indication of the more rapid formation of hepatocyte spheroids in AL/XG capsules than in AL capsules. More than 70% of the seeded hepatocytes in AL/XG capsules participated in spheroid formation after 2 days, whereas most hepatocytes in AL capsules remained as single cells and only a few cells began to form aggregates after 3 days. Intercellular molecule genes, such as connexin (Cx) 32 and E-cadherin, of hepatocyte spheroids in AL or AL/XG capsules were detected by reverse transcriptase–polymerase chain reaction. Cx32 and E-cadherin genes in AL/XG capsules were more rapidly reexpressed and expressed, respectively, than in AL ones. The results suggest that the multicellular spheroid formation of hepatocytes can enhance the liver-specific functions in the three-dimensional space in the presence of XG as a new synthetic ECM owing to the specific interaction between the galactose moieties of XG and asialoglycoprotein receptors of hepatocytes.
Keywords: Hepatocyte; Asialoglycoprotein receptor; Xyloglucan; Alginate; Capsule; Spheroid;

Repair of articular cartilage defects treated by microfracture and a three-dimensional collagen matrix by R. Dorotka; U. Windberger; K. Macfelda; U. Bindreiter; C. Toma; S. Nehrer (3617-3629).
The objective of our study was to evaluate the behavior of ovine chondrocytes and bone marrow stromal cells (BMSC) on a matrix comprising type-I, -II, and -III collagen in vitro, and the healing of chondral defects in an ovine model treated with the matrix, either unseeded or seeded with autologous chondrocytes, combined with microfracture treatment.For in vitro investigation, ovine chondrocytes and BMSC were seeded on the matrix and cultured at different time points. Histological analysis, immunohistochemistry, biochemical assays for glycosaminoglycans, and real-time quantitative PCR for collagens were performed. The animal study described here included 22 chondral defects in 11 sheep, divided into four treatment groups. Group A: microfracture and collagen matrix seeded with chondrocytes; B: microfracture and unseeded matrices; C: microfracture; D: untreated defects. All animals were sacrificed 16 weeks after implantation, and a histomorphometrical and qualitative evaluation of the defects was performed.The in vitro investigation revealed viable cells up to 3 weeks; chondrocytes had a predominantly round morphology, produced glycosaminoglycans, and expressed both collagen markers, whereas BMSC stained positive for antibodies against type-II collagen; however, no mRNA for type-II collagen was amplified. All treatment groups of the animal model showed better defect filling compared to untreated knees. The cell-seeded group had the greatest quantity of repair tissue and the largest quantity of hyaline-like tissue.Although the collagen matrix is an adequate environment for BMSC in vitro, the additionally implanted unseeded collagen matrix did not increase the repair response after microfracture in chondral defects. Only the matrices seeded with autologous cells in combination with microfracture were able to facilitate the regeneration of hyaline-like cartilage.
Keywords: Cartilage tissue engineering; Scaffold; Chondrocyte; Bone marrow;

For the repair of bone defects, a tissue engineering approach would be to combine cells capable of osteogenic (i.e. bone-forming) activity with an appropriate scaffolding material to stimulate bone regeneration and repair. Human mesenchymal stem cells (hMSCs), when combined with hydroxyapatite/β-tricalcium phosphate (HA/TCP) ceramic scaffolds of the composition 60% HA/40% TCP (in weight %), have been shown to induce bone formation in large, long bone defects. However, full repair or function of the long bone could be limited due to the poor remodeling of the HA/TCP material. We conducted a study designed to determine the optimum ratio of HA to TCP that promoted hMSC induced bone formation yet be fully degradable. In a mouse ectopic model, by altering the composition of HA/TCP to 20% HA/80% TCP, hMSC bone induction occurred at the fastest rate in vivo over the other formulations of the more stable 100% HA, HA/TCP (76/24, 63/37, 56/44), and the fully degradable, 100% TCP. In vitro studies also demonstrated that 20/80 HA/TCP stimulated the osteogenic differentiation of hMSCs as determined by the expression of osteocalcin.
Keywords: Calcium phosphate; Mesenchymal stem cell; Bone; Osteogenesis;

Cooperative modulation of neuritogenesis by PC12 cells by topography and nerve growth factor by John D. Foley; Eric W. Grunwald; Paul F. Nealey; Christopher J. Murphy (3639-3644).
Understanding axonal formation and contact guidance are of critical importance for the design of materials that interface with neuronal tissue. Contact guidance of neurites by topographic features is well known, but the role that topography plays in the modulation of neuritogenesis has not been addressed. To test this, we cultured PC12 cells with a range of nerve growth factor (NGF) concentrations on surfaces with ridge widths ranging from 70 to 1900 nm. We find that neuritogenesis by PC12 cells cultured with sub-optimal concentrations of NGF (25 and 5 ng/ml) is modulated by topographic feature size. The threshold for induction of neuritogenesis was markedly reduced when cells were cultured on ridges of 70 and 250 nm. In contrast, contact guidance of neurites was independent of feature size. These results suggest that the scale of topographic features can act cooperatively with NGF signaling to regulate the formation of neurites. These findings may have general relevance to differentiation processes in neurons as well as in other cell types.
Keywords: Nanotopography; Nerve tissue engineering; Neural cell; Nerve growth factor;

Osteogenic differentiation of rat bone marrow stromal cells cultured on Arg–Gly–Asp modified hydrogels without dexamethasone and β-glycerol phosphate by Heungsoo Shin; Johnna S. Temenoff; Gregory C. Bowden; Kyriacos Zygourakis; Mary C. Farach-Carson; Michael J. Yaszemski; Antonios G. Mikos (3645-3654).
In this study, we investigated the effect of signaling peptides incorporated into oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels on in vitro differentiation and mineralization of marrow stromal cells (MSCs) cultured in media without soluble osteogenic supplements (dexamethasone and β-glycerol phosphate). When MSCs were cultured for 16 days on OPF hydrogels modified with Arg–Gly–Asp (RGD) containing peptides, the normalized cell number was dependent on the peptide concentration between days 0 and 5 and reached comparable values at day 10 regardless of the concentration. The alkaline phosphatase (ALP) activity of MSCs on the peptide-modified OPF hydrogels was also concentration-dependent: ALP activity showed peaks on day 10 or day 13 on OPF hydrogels modified with 2.0 and 1.0 μmol peptide/g, which were significantly greater than those on the OPF hydrogels modified with 0.1 μmol peptides/g or no peptide. A characteristic marker of osteoblastic differentiation, osteopontin (OPN), was detected for all the test groups. However, OPN secretion between days 0 and 10 was significantly higher on the peptide modified hydrogels compared to that on tissue culture-treated polystyrene. Taken together, the results indicate that the presence of signaling peptide allows for a favorable microenvironment for MSCs to differentiate into osteoblasts and produce mineralized matrix, although the soluble factors may further enhance calcium deposition. These findings further support the usefulness of OPF hydrogels as scaffolds for guided bone regeneration, and represent an initial step in exploring the complex relationship between soluble and insoluble factors in osteogenic differentiation on biodegradable materials.
Keywords: Osteoblasts; Biomimetic hydrogel; Mineralization; Differentiation; Osteogenic media;

Micropatterning proteins and cells on polylactic acid and poly(lactide-co-glycolide) by Chien-Chi Lin; Carlos C. Co; Chia-Chi Ho (3655-3662).
Techniques for micropatterning proteins and cells on biomaterials are important in tissue engineering applications. Here, we present a method for patterning proteins and cells on poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) substrates that are routinely used as scaffolds in engineering tissues. Poly(oligoethyleneglycol methacrylate) (poly-OEGMA) or poly(oligoethyleneglycol methacrylate-co-methacrylic acid) (poly(OEGMA-co-MA)) was microcontact printed onto substrates to create cell resistant areas. Proteins adsorbed onto the unprinted regions whereas the polymer printed regions effectively repel non-specific protein adsorption. NIH 3T3 fibroblasts remain confined within the patterns on the PLGA and PLA films for up to 2 weeks and aligned their actin cytoskeleton along the line patterns. In comparison to unpatterned cells, fibroblasts confined within line-shaped patterns show fewer actin filaments. This method for controlling the spatial morphology and distribution of cells on synthetic biomaterials could have significant applications in tissue engineering.
Keywords: Micropatterning; Poly(lactide-co-glycolide) (PLGA); Poly(lactide acid) (PLA); Poly(ethylene glycol); Tissue engineering;

Improved cell adhesion and proliferation on synthetic phosphonic acid-containing hydrogels by Jian Tan; Richard A. Gemeinhart; Mandy Ma; W. Mark Saltzman (3663-3671).
Hydrogels with tissue-like mechanical properties are highly attractive scaffolds for tissue engineering. In this study, copolymers containing vinyl phosphonic acid (VPA) and acrylamide (AM) were tested for their swelling, protein uptake in serum supplemented medium, and cell adhesion and proliferation. The swelling of the gels in serum containing culture medium increased with increasing VPA content. The presence of VPA also increased protein uptake of gels in medium; gels polymerized with more than 50% of VPA absorbed as much as 100 μg/cm2 of protein, twice the amount absorbed by gels made with only acrylamide. The adhesion and growth of the three types of cells, NIH 3T3 fibroblast, osteoblast-like MG-63 and Saos-2, were significantly improved on the gels made with 50% or more VPA; the number of adherent Mg-63 cells increased three-fold while the growth rate increased four-fold. Similar results were obtained for Saos-2 and 3T3 cells. The adhesion and growth of the three cell types on gels with sufficient phosphonate content were at least comparable to, or even better than, that on commercially available tissue culture plates. These results suggest great potential of anionic gels in bone tissue engineering.
Keywords: Hydrogel; Osteoblast; Vinyl phosphonic acid; Adhesion; Proliferation; Protein uptake;

Interactions between epidermal–dermal cells via soluble factors provide important signals in regulating the reepithelialization of wounded skin. For example, keratinocytes regulate the expression of keratinocyte growth factor (KGF) in fibroblasts through the release of interleukin-1beta (IL-1β). In this study, a previously developed polyethyleneglycol-based interpenetrating network (IPN) system was utilized as a platform for the delivery of keratinocyte-active factors. The effect of substrate chemistry, culture condition, and the delivery of exogenous keratinocyte-active factors on the keratinocyte behavior and the keratinocyte-fibroblast paracrine relationship was delineated. Adherent keratinocyte density on TCPS and glutaraldehyde-fixed gelatin hydrogels but not on IPN was significantly increased with culture time in the presence of growth supplements independent of the released KGF from the gelatin hydrogel and IPN. In the presence of fibroblasts, adherent keratinocyte density on gelatin hydrogels was higher than that without fibroblasts. This phenomenon was not observed on IPN and polycarbonate membrane. In summary, the delivered exogenous huKGF (i.e., released from a biomaterial matrix) operates in tandem with fibroblasts in regulating keratinocyte activation (i.e., IL-lβ release and adhesion) in a surface-dependent manner. Immunoassay analysis of cell culture keratinocyte-fibroblast paracrine relationship as characterized by IL-1β and KGF could not be established in the presence of IPNs, 0.1% glutaraldehyde-fixed gelatin hydrogels, and polycarbonate membranes.
Keywords: Hydrogels; PEG; Cell adhesion; Fibroblast co-culture; huKGF;

An amperometric biosensor has been developed for the quantitative determination of urea in aqueous solution. The principle is based on the use of pH-sensitive redox active dissolved hematein molecule. The enzyme, urease (Urs), was covalently immobilized on a conducting copolymer poly (N-3-aminopropyl pyrrole-co-pyrrole) film, electrochemically prepared onto an indium-tin-oxide (ITO)-coated glass plate. The covalent linkage of enzyme and porous morphology of the polymer film lead to high enzyme loading and an increased lifetime stability of the enzyme electrode. Amperometric response was measured as a function of concentration of urea, at fixed bias voltage of 0.0 V vs. Ag/AgCl in a phosphate buffer (pH 7.0). The electrode gives a linear response range of 0.16–5.02 mM for urea in aqueous medium. The response time is 40 s reaching to a 95% steady-state current value, and 80% of the enzyme activity is retained for about 2 months.
Keywords: Urea; Polymerization; Biosensor; Covalent immobilization; Peptide linkage;

Factors influencing calcium phosphate cement shelf-life by Uwe Gbureck; Sofia Dembski; Roger Thull; Jake E. Barralet (3691-3697).
Long-term stability during storage (shelf-life) is one major criterion for the use of a material as medical device. This study aimed to investigate the ageing process of β -tricalcium phosphate/monocalcium phosphate cement powders when stored in sealed containers at ambient conditions. This kind of cement type is of interest because it is forming dicalcium phosphate dihydrate (brushite) when set, which is in contrast to hydroxyapatite resorbable in physiological conditions. The stability of cements was checked by either measuring the phase composition of powders as well as the setting time and compressive strength when mixed with sodium citrate as liquid. Critical factors influencing ageing were found to be temperature, humidity and the mixing regime of the powders. Mechanically mixed cement powders which were stored in normal laboratory atmosphere (22 °C, 60% rel. humidity) converted to dicalcium phosphate anhydrous (monetite) within a few days; this could be mechanistically related to a dissolution/precipitation process since humidity condensed on the particles’ surfaces and acted as reaction medium. Various storage conditions were found to be effective in prolonging cement stability which were in order of effectiveness: adding solid citric acid retardant>dry argon atmosphere=gentle mixing (minimal mechanical energy input) ⪢ low temperature.
Keywords: Calcium phosphate cement; Shelf-life; Brushite;

The use of bone cement to treat vertebral compression fractures in a percutaneous manner requires placement of the cement under fluoroscopic image guidance. To enhance visualization of the flow during injection and to monitor and prevent leakage beyond the confines of the vertebral body, the orthopedic community has described increasing the amount of radiopacifier in the bone cement. In this study, static tensile and compressive testing, as well as fully reversed fatigue testing, was performed on three PMMA-based bone cements. Cements tested were Simplex®P with 10% barium sulfate (Stryker Orthopedics, Mahwah, NJ) which served as a control; Simplex®P with 36% barium sulfate prepared according to the clinical recommendation of Theodorou et al.; and KyphX HV-R with 30% barium sulfate (Kyphon Inc., Sunnyvale, CA). Static tensile and compressive testing was performed in accordance with ASTM F451-99a. Fatigue testing was conducted in accordance with ASTM F2118-01a under fully reversed, ±10-, ±15-, and ±20-MPa stress ranges. Survival analysis was performed using three-parameter Weibull modeling techniques. KyphX HV-R was found to have comparable static mechanical properties and significantly greater fatigue life than either of the two control materials evaluated in the present study. The static tensile and compressive strengths for all three PMMA-based bone cements were found to be an order of magnitude greater than the expected stress levels within a treated vertebral body. The static and fatigue testing data collected in this study indicate that bone cement can be designed with barium sulfate levels sufficiently high to permit fluoroscopic visualization while retaining the overall mechanical profile of a conventional bone cement under typical in vivo loading conditions.
Keywords: Polymethylmethacrylate(PMMA); Bone cement; Fatigue; Tension; Compression; Spine; Kyphoplasty; Vertebroplasty; Mechanical behavior; Elastic modulus; Ultimate strength; Vertebrae;

Development of high-viscosity, two-paste bioactive bone cements by S. Deb; L. Aiyathurai; J.A. Roether; Z.B. Luklinska (3713-3718).
Self-curing two-paste bone cements have been developed using methacrylate monomers with a view to formulate cements with low polymerization exotherm, low shrinkage, better mechanical properties, and improved adhesion to bone and implant surfaces. The monomers include bis-phenol A glycidyl dimethacrylate (bis-GMA), urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) as a viscosity modifier. Two-paste systems were formulated containing 60% by weight of a bioactive ceramic, hydroxyapatite. A methacroyloxy silane (A174) was used as a coupling agent due to its higher water stability in comparison to other aminosilanes to silanate the hydroxyapatite particles prior to composite formulation. A comparison of the FT-infrared spectrum of hydroxyapatite and silanated hydroxyapatite showed the presence of the carbonyl groups (∼1720 cm−1), –C=C–(∼1630 cm−1) and Si–O– (1300–1250 cm−1) which indicated the availability of silane groups on the filler surface. Two methods of mixing were effected to form the bone cement: firstly by mixing in an open bowl and secondly by extruding the two pastes by an auto-mixing tip using a gun to dispense the pastes. Both types of cements yielded low polymerization exotherms with good mechanical properties; however, the lower viscosity of UDMA allowed better extrusion and handling properties. A biologically active apatite layer formed on the bone cement surface within a short period after its immersion in simulated body fluid, demonstrating in vitro bioactivity of the composite. This preliminary data thus suggests that UDMA is a viable alternative to bis-GMA as a polymerizable matrix in the formation of bone cements.
Keywords: Orthopaedic bone cements; Hydroxypatite; Composites; Mechanical properties;

Stimulation of bone formation by zoledronic acid in particle-induced osteolysis by Christian Wedemeyer; Fabian von Knoch; Andreas Pingsmann; Gero Hilken; Christoph Sprecher; Guido Saxler; Frank Henschke; Franz Löer; Marius von Knoch (3719-3725).
We investigated the effect of a single subcutaneous dose of zoledronic acid on particle-induced osteolysis and observed excessive regional new bone formation.We utilized the murine calvarial osteolysis model and polyethylene particles in C57BL/J6 mice. Twenty-eight mice were used, seven per group. Specimens were stained with Giemsa dye. The osteoid tissue area was determined. Bone thickness was measured as an indicator of bone growth.Net bone growth was significantly increased in animals with zoledronic acid treatment: 0.02 mm2±0.03 mm2 in animals with particle implantation only (group 2), 0.25 mm2±0.08 mm2 with particle implantation and zoledronic acid treatment directly after surgery (group 3; p = 0.0018 ), and 0.21 mm2±0.11 mm2 with particle implantation and zoledronic acid treatment on the fourth postoperative day (group 4; p = 0.0042 ). The mean bone thickness was 0.2 mm±0.04 mm (range 0.17 mm–0.31 mm) in group 1 (sham controls) and 0.16 mm±0.02 mm (range 0.14 mm–0.19 mm) in group 2, 0.31 mm±0.04 mm (range 0.28 mm–0.39 mm) in group 3, and 0.29 mm±0.02 mm (range 0.28 mm–0.34 mm) in group 4. Student's t-test revealed a statistically significant difference between groups 2 and 3 ( p = 0.00042 ), and groups 2 and 4 ( p = 0.0019 ).In conclusion, our observational study suggests that zoledronic acid may stimulate bone apposition locally in the process of particle-induced osteolysis.
Keywords: Net bone growth; Wear debris; Polyethylene particles; Histomorphometry; Zoledronic acid;

Pharmacologically active microcarriers: a tool for cell therapy by V.M. Tatard; M.C. Venier-Julienne; P. Saulnier; E. Prechter; J.P. Benoit; P. Menei; C.N. Montero-Menei (3727-3737).
To overcome certain problems encountered in cell therapy, particularly cell survival, lack of cell differentiation and integration in the host tissue, we developed pharmacologically active microcarriers (PAM). These biodegradable particles made with poly(d,l-lactic-co-glycolic acid) (PLGA) and coated with adhesion molecules may serve as a support for cell culture and may be used as cell carriers presenting a controlled delivery of active protein. They can thus support the survival and differentiation of the transported cells as well as their microenvironment. To develop this tool, nerve growth factor (NGF)—releasing PAM, conveying PC12 cells, were produced and characterized. Indeed, these cells have the ability to differentiate into sympathetic—like neurons after adhering to a substrate, in the presence of NGF, and can then release large amounts of dopamine. Certain parameters such as the size of the microcarriers, the conditions enabling the coating of the microparticles and the subsequent adhesion of cells were thus studied to produce optimized PAM.
Keywords: Microcarriers; Cell adhesion; Nerve growth factor; PC12; Cell therapy;

Novel PCL-based honeycomb scaffolds as drug delivery systems for rhBMP-2 by B. Rai; S.H. Teoh; D.W. Hutmacher; T. Cao; K.H. Ho (3739-3748).
This study investigated a novel drug delivery system (DDS), consisting of polycaprolactone (PCL) or polycaprolactone 20% tricalcium phosphate (PCL-TCP) biodegradable scaffolds, fibrin Tisseel sealant and recombinant bone morphogenetic protein-2 (rhBMP-2) for bone regeneration. PCL and PCL-TCP-fibrin composites displayed a loading efficiency of 70% and 43%, respectively. Fluorescence and scanning electron microscopy revealed sparse clumps of rhBMP-2 particles, non-uniformly distributed on the rods’ surface of PCL-fibrin composites. In contrast, individual rhBMP-2 particles were evident and uniformly distributed on the rods’ surface of the PCL-TCP-fibrin composites. PCL-fibrin composites loaded with 10 and 20 μg/ml rhBMP-2 demonstrated a triphasic release profile as quantified by an enzyme-linked immunosorbent assay (ELISA). This consisted of burst releases at 2 h, and days 7 and 16. A biphasic release profile was observed for PCL-TCP-fibrin composites loaded with 10 μg/ml rhBMP-2, consisting of burst releases at 2 h and day 14. PCL-TCP-fibrin composites loaded with 20 μg/ml rhBMP-2 showed a tri-phasic release profile, consisting of burst releases at 2 h, and days 10 and 21. We conclude that the addition of TCP caused a delay in rhBMP-2 release. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline phosphatase assay verified the stability and bioactivity of eluted rhBMP-2 at all time points.
Keywords: BMP; Bone tissue engineering; Drug delivery; Fibrin; Polycaprolactone;

Nanoscale anionic macromolecules for selective retention of low-density lipoproteins by Evangelia Chnari; Hamed B. Lari; Lu Tian; Kathryn E. Uhrich; Prabhas V. Moghe (3749-3758).
Synthetically designed anionic nanocarriers that mimic the charge properties of glycosaminoglycans can potentially sequester low-density lipoproteins (LDL) during the treatment of atherosclerosis. In this study, we explore the LDL retentivity of 15–20 nm anionic micelles formed from amphiphilic scorpion-like macromolecules (AScMs) as building blocks. The macromolecules comprise four aliphatic chains attached to mucic acid and a linear polyethylene glycol (PEG) segment to form micellar nanocarriers with a hydrophobic core and hydrophilic corona. Dynamic light scattering and transmission electron microscopy studies indicate that the carboxylate-terminated nanocarriers (20 nm) sequester LDL (22 nm), resulting in complexes with a diameter of 60–90 nm, but neutral ethoxy-terminated nanocarriers do not retain LDL. Further, carboxylate-terminated nanocarriers consistently bound to unoxidized LDL (Relative Electrophoretic Mobility, REM=1.0) and mildly oxidized LDL (REM=1.5), but not highly oxidized LDL (REM=3.6), whereas the neutral nanocarriers displayed no preference/affinity at all, indicating that the nanocarrier-LDL binding is charge-dependent. The binding affinity of unoxidized LDL for differentially charged nanocarriers, formed from varying ratios of carboxylate- and ethoxy-terminated macromolecules, was quantified. The 100% carboxylated nanocarriers elicited the highest binding affinity (K d=567 nm), whereas mixed micelles elicited significantly lower levels of binding affinity. Our results highlight the promise of synthetically designed nanomaterials in lipoprotein retention, a key step in managing the escalation of atherosclerosis.
Keywords: Low-density lipoproteins; Atherosclerosis; Amphiphilic macromolecules; Micelles; Nanotechnology;

Liposome-encapsulated actin–hemoglobin (LEAcHb) artificial blood substitutes by Shuliang Li; Jonathan Nickels; Andre Francis Palmer (3759-3769).
A new approach to enhance the circulation persistence of liposomes has been applied to develop liposome-encapsulated actin–hemoglobin (LEAcHb) dispersions as potential blood substitutes by introducing an actin matrix into the liposome aqueous core. Asymmetric flow field-flow fractionation coupled with multi-angle static light scattering was used to study the shape, size distribution, and encapsulation efficiency of liposome-encapsulated hemoglobin (LEHb) and LEAcHb dispersions. By polymerizing monomeric actin into filamentous actin inside the liposome aqueous core, LEAcHb particles transformed into a disk-like shape. We studied the effect of an encapsulated actin matrix on the size distribution, hemoglobin (Hb) encapsulation efficiency, oxygen affinity, and methemoglobin (MetHb) level of LEAcHb dispersions, and compared them with plain LEHb dispersions (without actin). LEHb, and LEAcHb dispersions extruded through 400 nm membranes were injected into rats and it was observed that LEAcHb dispersions with 1 mg/mL of actin enhanced the circulatory half-life versus LEHb dispersions. The circulatory characteristics of empty PEGylated and non-PEGylated actin–containing liposomes (without Hb) were studied as controls for the LEHb and LEAcHb dispersions in this paper, which displayed maximum circulatory half-lives greater than 72 h. Taken together the results of this study supports our hypothesis that a lipid membrane supported by an underlying actin matrix will extend the circulatory half-life of LEHb dispersions.
Keywords: Liposome; Hemoglobin; Actin; Asymmetric flow field-flow fractionation; Multiangle static light scattering; Blood substitute; Oxygen carrier;