Biomaterials (v.26, #33)

Calendar (I).

Cell sheet engineering: Recreating tissues without biodegradable scaffolds by Joseph Yang; Masayuki Yamato; Chinatsu Kohno; Ayako Nishimoto; Hidekazu Sekine; Fumio Fukai; Teruo Okano (6415-6422).
While tissue engineering has long been thought to possess enormous potential, conventional applications using biodegradable scaffolds have limited the field's progress, demonstrating a need for new methods. We have previously developed cell sheet engineering using temperature-responsive culture dishes in order to avoid traditional tissue engineering approaches, and their related shortcomings. Using temperature-responsive dishes, cultured cells can be harvested as intact sheets by simple temperature changes, thereby avoiding the use of proteolytic enzymes. Cell sheet engineering therefore allows for tissue regeneration by either direct transplantation of cell sheets to host tissues or the creation of three-dimensional structures via the layering of individual cell sheets. By avoiding the use of any additional materials such as carrier substrates or scaffolds, the complications associated with traditional tissue engineering approaches such as host inflammatory responses to implanted polymer materials, can be avoided. Cell sheet engineering thus presents several significant advantages and can overcome many of the problems that have previously restricted tissue engineering with biodegradable scaffolds.
Keywords: Cell sheet; Temperature-responsive culture dish; Biodegradable scaffold; Inflammation;

The first calcium phosphate cements (CPCs) were discovered in the 1980s. Two decades later, the interest for these materials is still rising. The goal of the present document is to review the most recent achievements in the field and to analyze future directions in research and development.
Keywords: Calcium phosphate cement; Vertebroplasty; Delivery; In vivo;

The effect of a large compressive plastic deformation on the melt temperature (T m), lamellar thickness, crystallinity, and density of four UHMWPEs (two conventional and two highly crosslinked) was examined. The materials were prepared from a single batch of medical grade GUR 1050 resin (Ticona, Bayport, TX, USA). 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 two thermal treatments: annealing, which was done below the melt transition temperature (T m), at 110 °C for 2 h (110 °C-annealed); and, remelting, which was done above T m, at 150 °C (150 °C-remelted). Differences in changes upon compression between the materials were examined using ANCOVA analyses. The 150 °C-remelted material showed a significant change in T m and lamellar thickness upon compressive plastic deformation whereas the other three UHMWPE materials did not. However, all of the materials showed significantly decreased crystallinity and density upon compressive deformation. The findings of this study support that microstructural evolution during compressive deformation is a function of UHMWPE formulation, as affected by irradiation and post-irradiation heat treatment.
Keywords: UHMWPE; Crosslinking; Compression; Differential scanning calorimetry; Melt temperature; Lamellar thickness; Crystallinity; Density;

The degree of conversion (DC) of methacrylate dental resins is typically determined by spectroscopically measuring the decrease of the vinyl (C=C) stretching band at 1640 cm−1, ratioed before and after polymerization to an internal standard (aromatic ring quadrant stretching vibration (Ph)) at around 1609 cm−1. While standard methods exist for measuring the intensity of the C=C and Ph peaks from the FTIR or Raman spectrum, these methods either fail under certain circumstances, or lack a physical basis, being purely based on spectral features. In this study, we present a rigorous method (named rotational isomerism method) for determining the intensity of the vinyl and aromatic bands from the FTIR and Raman spectra of dental monomer mixtures, and compare it to a standard baseline method (SBM) and a standard curve fitting method. Model triethyleneglycol dimethacrylate (TEGDMA)/2,2-bis(4-hydroxyphenyl)propane (Bisphenol-A) and TEGDMA/2,2-bis(4-(2-hydroxy-3-methacryloxypropoxy)phenyl)propane (Bis-GMA) mixtures with a series of known C=C/Ph molar ratios were prepared in order to simulate the effect of curing. The accuracy of the new method for measuring the DC was found to be as good as the commonly used SBM. The standard curve fitting method was shown to be inappropriate for measuring C=C/Ph ratios by FTIR spectroscopy due to its inability to realistically simulate the features of the spectra. The insight gained through the use of this new method may be useful for the characterization of other methacrylate biomaterials.
Keywords: Dental restorative material; FTIR; Bis-phenol A derivative; Crosslinking; Raman;

Effects of resin hydrophilicity on water sorption and changes in modulus of elasticity by Shuichi Ito; Masanori Hashimoto; Bakul Wadgaonkar; Nadia Svizero; Ricardo M. Carvalho; Cynthia Yiu; Frederick A. Rueggeberg; Stephen Foulger; Takashi Saito; Yoshihiro Nishitani; Masahiro Yoshiyama; Franklin R. Tay; David H. Pashley (6449-6459).
As acidic monomers of self-etching adhesives are incorporated into dental adhesives at high concentrations, the adhesive becomes more hydrophilic. Water sorption by polymers causes plasticization and lowers mechanical properties. The purpose of this study was to compare the water sorption and modulus of elasticity (E) of five experimental neat resins (EX) of increasing hydrophilicity, as ranked by their Hoy's solubility parameters and five commercial resins.After measuring the initial modulus of all resin disks by biaxial flexure, half the specimens were stored in hexadecane and the rest were stored in water. Repeated measurements of stiffness were made for 3 days. Water sorption and solubility measurements were made in a parallel experiment.None of the specimens stored in oil showed any significant decrease in modulus. All resins stored in water exhibited a time-dependent decrease in modulus that was proportional to their degree of water sorption. Water sorption of EX was proportional to Hoy's solubility parameter for polar forces ( δ p ) with increasing polarity resulting in higher sorption. The least hydrophilic resin absorbed 0.55 wt% water and showed a 15% decrease in modulus after 3 days. The most hydrophilic experimental resin absorbed 12.8 wt% water and showed a 73% modulus decrease during the same period. The commercial resins absorbed between 5% and 12% water that was associated with a 19–42% reduction in modulus over 3 days.
Keywords: Bis-phenol a derivative; Hydrophilicity; Hydroxyethyl methacrylate; Elasticity; Water;

Microtomography assessment of failure in acrylic bone cement by P.E. Sinnett-Jones; M. Browne; W. Ludwig; J.-Y. Buffière; I. Sinclair (6460-6466).
Micromechanical studies of fatigue and fracture processes in acrylic bone cement have been limited to surface examination techniques and indirect signal analysis. Observations may then be mechanically unrepresentative and/or affected by the presence of the free surface. To overcome such limiting factors the present study has utilised synchrotron X-ray microtomography for the observation of internal defects and failure processes that occurred within a commercial bone cement during loading. The high resolution and the edge detection capability (via phase contrast imaging) have enabled clear microstructural imaging of both strongly and weakly absorbing features, with an effective isotropic voxel size of 0.7 μm. Detailed assessment of fatigue damage processes in in vitro fatigue test specimens is also achieved. Present observations confirm a link with macroscopic failure and the presence of larger voids, at which crack initiation may be linked to the mechanical stress concentration set up by adjacent beads at pore surfaces. This study does not particularly support the suggested propensity for failure to occur via the inter-bead matrix; however crack deflections at matrix/bead interfaces and the incidence of crack arrest within beads do imply locally increased resistance to failure and potential improvements in global crack growth resistance via crack tip shielding.
Keywords: PMMA; Bone cement; X-Ray synchrotron tomography; Micromechanical failure;

Effect of processing route and acetone pre-treatment on the biostability of pellethane materials used in medical device applications by Jennifer Elizabeth Taylor; Peter R. Laity; Sharlene Freeburn; Steven S. Wong; Keith Norris; Peck Khunkamchoo; Martin Cable; Geoffrey Andrews; Anthony F. Johnson; Ruth E. Cameron (6467-6476).
Thermoplastic polyurethanes, such as Pellethane 2363 80A™ (Pel80A) and Pellethane 2363 55D™ (Pel55D) are widely used in the medical device industry because of their biological and mechanical properties. However, premature failure in such devices has been observed and attributed to environmental stress cracking (ESC). The current work investigates the possibility of reducing ESC via bulk morphology manipulation. This can be achieved through various processing routes such as solvent-casting (SC) and hot-press quenching (HPQ). The effect of stress on the bulk morphology of Pel55D and Pel80A was evaluated using small-angle X-ray scattering (SAXS) in conjunction with tensile testing. SC samples exhibited greater phase separation compared with HPQ samples. Alignment of hard segment domains became apparent around the point of yield. Onset of ESC with respect to SC and HPQ routines was determined using the Zhao–Stokes glass-wool test with optical (OM) and environment scanning electron microscopy (ESEM). Improvement in biostability of Pel80A was found in HPQ samples compared to those that were SC. A secondary objective of this work was to investigate the effect of acetone pre-treatment on surface morphology. High resolution imaging of acetone treated and untreated SC Pel80A showed significant differences in surface morphology.
Keywords: Polyurethane; Biocompatibility; Haemocompatibility; Oxidation;

A comparative study of calcium phosphate formation on bioceramics in vitro and in vivo by Renlong Xin; Yang Leng; Jiyong Chen; Qiyi Zhang (6477-6486).
Formation of calcium phosphate (Ca-P) on various bioceramic surfaces in simulated body fluid (SBF) and in rabbit muscle sites was investigated. The bioceramics were sintered porous solids, including bioglass, glass-ceramics, hydroxyapatite, α-tricalcium phosphate and β-tricalcium phosphate. The ability of inducing Ca-P formation was compared among the bioceramics. The Ca-P crystal structures were identified using single-crystal diffraction patterns in transmission electron microscopy. The examination results show that ability of inducing Ca-P formation in SBF was similar among bioceramics, but considerably varied among bioceramics in vivo. Sintered β-tricalcium phosphate exhibited a poor ability of inducing Ca-P formation both in vitro and in vivo. Octacalcium phosphate (OCP) formed on the surfaces of bioglass, A-W, hydroxyapatite and α-tricalcium phosphate in vitro and in vivo. Apatite formation in physiological environments cannot be confirmed as a common feature of bioceramics.
Keywords: Calcium phosphate; Bioceramics; Simulated body fluid; Bioactivity; Transmission electron microscopy;

Colloidal gold nanoparticle modified carbon paste interface for studies of tumor cell adhesion and viability by Dan Du; Shengli Liu; Jing Chen; Huangxian Ju; Hongzhen Lian; Jianxin Li (6487-6495).
A non-toxic biomimetic interface for immobilization of living cells and electrochemical exogenous effect study of cell viability was constructed by mixing colloidal gold nanoparticles in carbon paste. A new approach to study the effects of anti-tumor drug and other exogenous factors on cell viability was proposed. The nanoparticles were efficient for preserving the activity of immobilized living cells and preventing their leakage from the electrode surface. The immobilized living AsPC-1 cells (pancreatic adenocarcinoma cells derived from ascites) exhibited an irreversible voltammetric response related to the oxidation of guanine. The presence of guanine was verified by liquid chromatography–mass spectrometry. The contents of guanine in cytoplasm of each AsPC-1 and normal pancreatic cell were detected to be 370 and 22 amol, respectively. The cytotoxic effect of adriamycin resulted in a decrease in peak current of guanine. The optimal exogenous factors that affected cell viability, including pH, temperature and salt concentration of electrolyte, were just consistent with cell growth conditions in culture. This simple and rapid method could be applied for the electrochemical investigation of exogenous effect and characterization of the viability of living cells.
Keywords: Biomimetic interface; Cell immobilization; Exogenous effect; Cyclic voltammetry; AsPC-1 cells; Colloidal gold nanoparticles; Guanine; Anti-tumor drug;

Bioactive bone cements containing nano-sized titania particles for use as bone substitutes by K. Goto; J. Tamura; S. Shinzato; S. Fujibayashi; M. Hashimoto; M. Kawashita; T. Kokubo; T. Nakamura (6496-6505).
Three types of bioactive polymethylmethacrylate (PMMA)-based bone cement containing nano-sized titania (TiO2) particles were prepared, and their mechanical properties and osteoconductivity are evaluated. The three types of bioactive bone cement were T50c, ST50c, and ST60c, which contained 50 wt% TiO2, and 50 and 60 wt% silanized TiO2, respectively. Commercially available PMMA cement (PMMAc) was used as a control. The cements were inserted into rat tibiae and allowed to solidify in situ. After 6 and 12 weeks, tibiae were removed for evaluation of osteoconductivity using scanning electron microscopy (SEM), contact microradiography (CMR), and Giemsa surface staining. SEM revealed that ST60c and ST50c were directly apposed to bone while T50c and PMMAc were not. The osteoconduction of ST60c was significantly better than that of the other cements at each time interval, and the osteoconduction of T50c was no better than that of PMMAc. The compressive strength of ST60c was equivalent to that of PMMAc. These results show that ST60c is a promising material for use as a bone substitute.
Keywords: Titanium oxide; Bioactivity; Osteoconduction; Polymethylmethacrylate;

A new dual acting polymeric coating is described that combines nitric oxide (NO) release with surface-bound active heparin, with the aim of mimicking the nonthrombogenic properties of the endothelial cell (EC) layer that lines the inner wall of healthy blood vessels. A trilayer membrane configuration is employed to create the proposed blood compatible coating. A given polymeric substrate (e.g., the outer surface of a catheter sleeve, etc.) is first coated with a dense polymer layer, followed by a plasticized poly(vinyl chloride) (PVC) or polyurethane (PU) layer doped with a lipophilic N-diazeniumdiolate as the NO donor species. Finally, an outer aminated polymer layer is applied. Porcine heparin is then covalently linked to the outer layer via formation of amide bonds. The surface-bound heparin is shown to possess anti-coagulant activity in the range of 4.80–6.39 mIU/cm2 as determined by a chromogenic anti-Factor Xa assay. Further, the surface NO flux from the underlying polymer layer containing the diazeniumdiolate species can be controlled and maintained at various levels (from 0.5 to 60×10–10  mol cm−2  min−1) for at least 24 h and up to 1 week (depending on the flux level desired) by changing the chemical/polymer composition of the NO release layer. The proposed polymeric coatings are capable of functioning by two complementary anti-thrombotic mechanisms, one based on the potent anti-platelet activity of NO, and the other the result of the ability of immobilized heparin to inhibit Factor Xa and thrombin (Factor IIa). Thus, the proposed polymeric coatings are expected to exhibit greatly enhanced thromboresistivity compared to polymers that utilize either immobilized heparin or NO release alone.
Keywords: Nitric oxide; Heparin; Diazeniumdiolates; Blood compatible polymers; Controlled release; Anti-coagulant; Anti-platelet activity;

The cellular response to transglutaminase-cross-linked collagen by David Y.S. Chau; Russell J. Collighan; Elisabetta A.M. Verderio; Victoria L. Addy; Martin Griffin (6518-6529).
Collagen, type I, is a highly abundant natural protein material which has been cross-linked by a variety of methods including chemical agents, physical heating and UV irradiation with the aim of enhancing its physical characteristics such as mechanical strength, thermal stability, resistance to proteolytic breakdown, thus increasing its overall biocompatibility. However, in view of the toxicity of residual cross-linking agents, or impracticability at large scales, it would be more useful if the collagen could be cross-linked by a milder, efficient and more practical means by using enzymes as biological catalysts.We demonstrate that on treating native collagen type I (from bovine skin) with both tissue transglutaminase (TG2; tTG) and microbial transglutaminase (mTG; Streptoverticillium mobaraense) leads to an enhancement in cell attachment, spreading and proliferation of human osteoblasts (HOB) and human foreskin dermal fibroblasts (HFDF) when compared to culture on native collagen. The transglutaminase-treated collagen substrates also showed a greater resistance to cell-mediated endogenous protease degradation than the native collagen. In addition, the HOB cells were shown to differentiate at a faster rate than on native collagen when assessed by measurement of alkaline phosphatase activity and osteopontin expression.
Keywords: Bioactivity; Biocompatibility; Collagen; Fibroblasts; Osteoblasts; Tissue engineering;

Antibiotic resistance in exopolysaccharide-forming Staphylococcus epidermidis clinical isolates from orthopaedic implant infections by Carla Renata Arciola; Davide Campoccia; Simonetta Gamberini; Maria Elena Donati; Valter Pirini; Livia Visai; Pietro Speziale; Lucio Montanaro (6530-6535).
The opportunistic pathogen Staphylococcus epidermidis is able to produce biofilm and to frequently cause implant infections. In recent years, it has also exhibited an increasing antimicrobial drug resistance. Here, the resistance to a panel of 16 different antibiotics in 342 clinical strains of S. epidermidis from orthopaedic implant infections has been investigated. The isolates were pheno- and genotyped for extracellular polysaccharide production, relevant to staphylococcal biofilm formation, in order to ascertain possible associations with antibiotic resistance.Approximately 10% of the isolates were found to be sensitive to all screened antibiotics. In all, 37–38% were resistant to β-lactams such as oxacillin and imipenem, while the resistance to penicillin, ampicillin, cefazolin, cefamandole, was consistently observed in over 80% of the strains. Erythromycin- and clindamycin- resistant strains were approximately 41% and 16%, respectively. Of the isolates, 10% was resistant to chloramphenicol, 23% to sulfamethoxazole and 26% to ciprofloxacin. Resistance to vancomycin was never observed. Interestingly, exopolysaccharide-producing strains exhibited a significantly higher prevalence in the resistance to the four aminoglycosides (gentamicin, amikacin, netilmicin, tobramycin), to sulfamethoxazole and to ciprofloxacin with respect to non-producing isolates. Moreover, multiple resistance to antibiotics was more frequent among exopolysaccharide-forming strains.
Keywords: Staphylococcus epidermidis; Antibiotic resistance; Methicillin; Aminoglycosides; Biofilm; Implant infections;

Generation of cell adhesive substrates using peptide fluoralkyl surface modifiers by Mark J. Ernsting; Geneviève C. Bonin; Meilin Yang; Rosalind S. Labow; J. Paul Santerre (6536-6546).
Previous studies reported on the delivery of vitamin E to the surface of a polycarbonate polyurethane (PCNU) to produce antioxidant surfaces, using a bioactive fluorinated surface modifer (BFSM). In the current report, a cell adhesive peptide sequence was coupled to the BFSM, and when blended into PCNU, generated a cell adhesive substrate. An NH2-GK*GRGD-CONH2 peptide sequence (referred to as RGD) with a dansyl label (*) on the lysine residue was coupled via the N-terminal to a BFSM precursor molecule. The resulting RGD BFSM was purified and the pmol peptide/mg BFSM value was assayed by amino acid quantification. The migration of the RGD BFSM in a PCNU blend was confirmed by X-ray photoelectron spectroscopy analysis. U937 macrophage-like cells and human monocytes were seeded onto the PCNU and blends of PCNU with non-bioactive fluorinated surface modifier or the RGD BFSM, in order to study the cell response. Both U937 cells and human monocytes adhered in greater numbers to the RGD BFSM substrate when compared to unmodified PCNU or the blend of PCNU with the non-bioactive fluorinated surface modifying macromolecule substrate. The study demonstrated a novel approach for the introduction of peptides onto the surface of polymers by modifying the surface from within the polymer as opposed to the use of cumbersome post-surface modification techniques. The generation of a peptide substrate points to the possibility of producing complex bioactive surfaces using various peptide BFSMs or pharmaceuticals simultaneously to manipulate cell functions.
Keywords: Polyurethane; Surface modification; Fluorinated; RGD peptide; Conjugation; Monocyte; Macrophages; Cell adhesion.;

In vitro hemocompatibility of self-assembled monolayers displaying various functional groups by Claudia Sperling; Rüdiger B. Schweiss; Uwe Streller; Carsten Werner (6547-6557).
Self-assembled monolayers (SAMs) of alkanethiols with various terminating groups (–OH, –CH3, –COOH) and binary mixtures of these alkanethiols were studied with respect to their hemocompatibility in vitro by means of freshly taken human whole blood. The set of smooth monomolecular films with graded surface characteristics was applied to scrutinize hypotheses on the impact of surface chemical–physical properties on distinct blood activation cascades, i.e. to analyze –OH surface groups vs. complement activation, acidic surface sites vs. contact activation/coagulation and surface hydrophobicity vs. thrombogenicity. Blood and model surfaces were analyzed after incubation for the related hemocompatibility parameters. Our results show that the adhesion of leukocytes is abolished on a –CH3 surface and greatly enhanced on surfaces with –OH groups. The opposite was detected for the adhesion of platelets. A strong correlation between the activation of the complement system and the adhesion of leukocytes with the content of –OH groups could be observed. The contact activation for hydrophilic surfaces was found to scale with the amount of acidic surface sites. However, the coagulation and platelet activation did not simply correlate with any surface property and were therefore concluded to be determined by a superposition of contact activation and platelet adhesion.
Keywords: Hemocompatibility; Self-assembled monolayer; Coagulation; Complement; Blood cell adhesion;

Effects of lyophilization on the infectivity of enveloped and non-enveloped viruses in bone tissue by Christine Uhlenhaut; Thomas Dörner; Georg Pauli; Axel Pruss (6558-6564).
Recently reported qualitative experiments proved that retroviral infectivity is not destroyed by lyophilization performed on systemically infected bone and tendon. The now accomplished quantitative determination of residual infectivity for enveloped and non-enveloped viruses allows a validation of the production process regarding viral safety in freeze-dried bone transplants.The lyophilization effect on the infectivity of two non-enveloped viruses (Maus Elberfeld virus, MEV; Porcine parvovirus, PPV) and one enveloped virus (Vesicular Stomatitis virus, VSV) was examined for virus-spiked bone material in comparison to lyophilized viruses, original virus stock, and air-dried viruses. All experiments were carried out with both cell-free and cell-associated virus. Significant differences were observed regarding the reduction of virus titers (TCID50). Infectivity of VSV was reduced by about 3–4 log10 using lyophilization in presence of bone matrix and of MEV by 6–7 log10, while no substantial reduction in virus titers was observed for PPV. Lyophilization of cell-free or cell-associated virus is not sufficient to inactivate viruses completely. However, lyophilization could have an additive effect in line with other production steps used in the manufacturing process.
Keywords: Bone transplants; Lyophilization; Freeze-drying; Virus safety; Tissue bank;

Polyhydoxyalkanoates (PHA) are polyesters produced by microorganisms under unbalanced growth conditions. They are generally biodegradable and thermoprocessable, making them attractive as biomaterials for applications in both conventional medical devices and tissue engineering. Over the past years, PHA, particularly poly 3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly 4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) and poly 3-hydroxyoctanoate (PHO) and its composites have been used to develop devices including sutures, repair devices, repair patches, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, guided tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone marrow scaffolds, and wound dressings. The changing PHA compositions also allow favorable mechanical properties, biocompatibility, and degradation times within desirable time frames under specific physiological conditions. This paper reviews what have been achieved in the PHA tissue engineering area and concluded that the PHA prospective will look very bright in the near future.
Keywords: PHB; Polyhydroxyalkanoates; Tissue engineering; Biodegradability; Biocompatibility; Hemocompatibility;

The gelatin-tricalcium phosphate membranes were cross-linking with low concentration glutaraldehyde solution (GTG). This material has good mechanical property, biocompatibility, and is feasible for surgical manipulation. For axonal regeneration, nerve growth factors (NGF) were immobilized onto the composite (GTG) with carbodiimide. The purpose of this study was to evaluate the release characteristics and bioactivity of NGF after covalent immobilization onto the GTG membranes (GEN). NGF immobilized onto and released from the composite was quantified using ELISA method. PC 12 cells were cultured on the GTG and GEN composites. Cell survival, cytotoxicity, and cellular activity were evaluated by total protein content, LDH activity, and MTT assay respectively. Neurite outgrowth assay was used to evaluate the biological activity of NGF released from GEN composite. From ELISA measurement, the releasing curve for NGF showing two distinctive parts with different slopes indicated that NGF were released from the composite in diffusion-controlled mechanism and degradation-controlled mechanism respectively. While culturing with PC 12 cells, LDH leakage results implied that whether GTG composite cross-linked with NGF or not showed little cytotoxicity. The total protein content and cellular activity of PC 12 cells were lower on GTG and GEN membranes than control group. However, 56%±3.98 of PC 12 cells showed significant neurite outgrowth on GEN membranes which was statistically higher than GTG without NGF immobilization. In addition, sustained release of bioactive NGF for two months had been demonstrated by neurite outgrowth assay. From these experiments, it can be concluded that the technique used in the present study is capable of immobilizing NGF onto GTG membranes covalently and remaining the bioactivity of NGF. Therefore, GEN composite can be materials for sustained release of bioactive NGF and a candidate for future therapeutic application in nerve repair.
Keywords: Biodegradable nerve conduit; Nerve growth factor; PC 12 cell; Neurite outgrowth; NGF release;

The construction of an artificial kidney module by tissue engineering or the application of cell-based therapies for the treatment of renal failure requires exact information regarding the cellbiological mechanisms of parenchyme development in combination with different kinds of biomaterials. To learn more about these processes tissue cultures are frequently used experimental tools. However, apart from experiments with early kidney anlagen there is a lack of suitable in-vitro models regarding the generation and long-term maintenance of renal tubules. In the present paper we like to demonstrate an advanced culture technique, which allows to generate tubular elements derived from renal stem cells. For the growth of tubules it is essential to fine-tune the interface between the embryonic tissue and the dead fluid space within a perfusion culture container by offering a polyester artificial interstitium. Culture was performed in IMDM supplemented with hormones and growth factors but using serum-free conditions over 14 days. Formation of tissue was then analysed by immunohistochemistry and two-dimensional (2D) electrophoresis. Culture in pure IMDM leads to a complete loss of tissue formation. In contrast, application of aldosterone (A) induces the development of numerous polarised tubules. Surprisingly, addition of epidermal growth factor (EGF), a cocktail of insulin, transferrin and selenium (ITS), retinoic acid (RA), cholecalciferol (VitD3) or bovine pituitary extract (BPT) does not further improve development of tubules, but leads to intensive cell clustering and a decrease of tubule formation. 2D Western blots of developing tissue probed with soybean agglutinin (SBA) reveal a unique pattern of newly detected proteins. It is found that growth factors do not support but abolish protein spots upregulated by aldosterone. It remains to be investigated, which cellbiological effect stimulates the embryonic cells to develop tubules in competition to cell clusters at the interphase of an artificial interstitium.
Keywords: Artificial interstitium; Polyester; Kidney; Tubules; Stem cells; Tissue engineering;

The spatial orientation of nerve cells plays a pivotal role in nerve regeneration. Here we report a new method for regulating neuronal cell morphology and guiding neurite extension on standard tissue culture dishes. Random copolymers of oligoethyleneglycol methacrylate and methacrylic acid [poly(OEGMA-co-MA)], microcontact printed on standard tissue culture dishes, resist cell attachment and remain intact in serum-containing medium for up to 2 weeks. Cell viability assay of SH-SY5Y cells demonstrated that poly(OEGMA-co-MA) on the substrate or in solution has no cytotoxic effect. When retinoic acid was added to SH-SY5Y cells, they extended neurites along the line patterns that are significantly longer than cells cultured on non-patterned culture dishes. The ability to guide neurite extension with micrometer precision is valuable for guiding directional growth of neurites and path finding of regenerating nerves.
Keywords: Microfabrication; Neuron; Microcontact printing; SH-SY5Y; Neurite extension;

Engineering of a vascularized scaffold for artificial tissue and organ generation by Heike Mertsching; Thorsten Walles; Michael Hofmann; Johanna Schanz; Wolfram H. Knapp (6610-6617).
Tissue engineering is an emerging field in regenerative medicine to overcome the problem of end-stage organ failure. However, complex tissues and organs need a vascular supply to guaranty graft survival and render bioartificial organ function. Here we developed methods to decellularize porcine small bowl segments and repopulate the remaining venous and arterial tubular structures within these matrices with allogeneic porcine endothelial progenitor cells. Cellular adherence and vitality was characterized by quantitative 2-[18F]-fluoro-2′-desoxy-glucose (FDG) positron emission tomography (PET) and subsequent immunohistological work up. The generated matrices showed insulin-dependent FDG uptake predominantly in the region of the former vascular structures. Stain for vitality and the specific endothelial markers CD31, VE-Cadherin and Flk-1 matched this functional finding. Providing evidence for vitality up to 3 weeks post reconstitution and typical endothelial differentiation, these results indicate that our generated matrix allows the generation of complex bioartificial tissues and organs for experimental and future clinical application.
Keywords: Bioprosthesis; Vascularized scaffold;

Polycaprolactone films doped with poly(lactide-co-glycolide) (65:35) in 0, 10, 20, and 30 (wt%) were prepared and evaluated in terms of morphology, dynamic contact angle analysis, and thermal properties. The unique surface morphology of the doped PCL film resulted, without introducing significant microstructure disruption of PCL aggregation. The doped PCL film registered a lower contact angle and increased hydrophilicity. Osteoblast cells attached to all doped materials, the 10% and 20% doped materials demonstrating the greatest cell growth.
Keywords: Poly (lactide-co-glycolide); Polycaprolactone; Doping; Morphology; Contact angle; Thermal properties; Osteoblast cells;

Effect of isoliquiritigenin on viability and differentiated functions of human hepatocytes maintained on PEEK-WC–polyurethane membranes by Loredana De Bartolo; Sabrina Morelli; Maria Carmela Gallo; Carla Campana; Giancarlo Statti; Maria Rende; Simona Salerno; Enrico Drioli (6625-6634).
In this study, we tested the ability of microporous membranes synthesised from a polymeric blend of modified polyetheretherketone (PEEK-WC) and polyurethane (PU) to support long-term maintenance and differentiation of human liver cells. The effect of isoliquiritigenin (ISL), which is a component of liquorice extract, exhibiting growth stimulatory and antiproliferative dose-dependent effect was investigated by comparing cultures treated with ISL with those untreated.To this purpose, flat-sheet membranes were prepared by a blend of PEEK-WC and PU polymers by phase inverse technique. The morphological and physico-chemical properties were characterised, respectively, by scanning electron microscopy and water contact angle measurements.Human hepatocytes cultured on PEEK-WC–PU membranes were constant up to 1 month albumin production and urea synthesis as well as the synthesis of total proteins. The liver-specific functions were expressed at high levels when cells were cultured on membranes with respect to collagen. Also the biotransformation functions were maintained for all culture periods: the ISL elimination rate increased during the culture time and high values were measured up to 22 days. Thereafter, a decrease was observed. ISL stimulated the proliferation of hepatocytes cultured on both substrata but did not affect their liver-specific functions. Hepatocytes cultured on PEEK-WC–PU membranes responded very well to ISL and expressed high levels of P450 cytochrome.These results demonstrated that long-term maintenance of human liver differentiation can be achieved on PEEK-WC–PU membranes. The incubation with ISL at the investigated concentration could stimulate the proliferation of human hepatocytes in biohybrid systems.
Keywords: Membrane; Human hepatocyte; Liver-specific functions; Biotransformation; Isoliquiritigenin;

Adhesion strength of human tenocytes to extracellular matrix component-modified poly(dl-lactide-co-glycolide) substrates by Ting-Wu Qin; Zhi-Ming Yang; Ze-Zhi Wu; Hui-Qi Xie; Jian Qin; Shao-Xi Cai (6635-6642).
We report a direct measurement of the adhesion strength of human embryonic tenocytes (HETCs) and transformed human embryonic tenocytes (THETCs) to fibronectin (FN)- and type I collagen (CNI)- modified poly(dl-lactide-co-glycolide) (PLGA) substrates with a micropipette aspiration technique. PLGA substrates were first coated with poly-d-lysine (PDL), and then with various concentrations (1 μg/ml, 2 μg/ml, 5 μg/ml, and 10 μg/ml) of FN and CNI in serum-free F12 media. Anti-FN and Anti-CNI antibodies were used to inhibit attachment of tenocytes to FN- and CNI- modified substrates in a dilution range of 1:5000–1:500 and 1:1500–1:250, respectively. The substrates were employed for incubation of HETCs and THETCs for 30 min at 37 °C before the adhesion strength measurements. We found that the adhesion strengths showed a strong dependence on the seeding time and FN or CNI concentrations. Anti-FN and Anti-CNI antibodies significantly compromised adhesion of HETCs and THETCs to the corresponding modified substrates ( P < 0.05 ). These findings show that FN- or CNI-modified polymer substrates offer significant advantages for tissue engineering tendon scaffolds concerning tenocyte adhesion. In addition, HETCs and THETCs bear similar biological behaviors in terms of adhesion, indicating the possibility of using THETCs in place of HETCs in tissue engineering construction of human tendons.
Keywords: Adhesion strength; Tenocyte; Fibronectin; Collagen; Poly(dl-lactide-co-glycolide);

A silanized hydroxypropyl methylcellulose hydrogel for the three-dimensional culture of chondrocytes by C. Vinatier; D. Magne; P. Weiss; C. Trojani; N. Rochet; G.F. Carle; C. Vignes-Colombeix; C. Chadjichristos; P. Galera; G. Daculsi; J. Guicheux (6643-6651).
Articular cartilage has limited intrinsic repair capacity. In order to promote cartilage repair, the amplification and transfer of autologous chondrocytes using three-dimensional scaffolds have been proposed. We have developed an injectable and self-setting hydrogel consisting of hydroxypropyl methylcellulose grafted with silanol groups (Si-HPMC). The aim of the present work is to assess both the in vitro cytocompatibility of this hydrogel and its ability to maintain a chondrocyte-specific phenotype. Primary chondrocytes isolated from rabbit articular cartilage (RAC) and two human chondrocytic cell lines (SW1353 and C28/I2) were cultured into the hydrogel. Methyl tetrazolium salt (MTS) assay and cell counting indicated that Si-HPMC hydrogel did not affect respectively chondrocyte viability and proliferation. Fluorescent microscopic observations of RAC and C28/I2 chondrocytes double-labeled with cell tracker green and ethidium homodimer-1 revealed that chondrocytes proliferated within Si-HPMC. Phenotypic analysis (RT-PCR and Alcian blue staining) indicates that chondrocytes, when three-dimensionnally cultured within Si-HPMC, expressed transcripts encoding type II collagen and aggrecan and produced sulfated glycosaminoglycans. These results show that Si-HPMC allows the growth of differentiated chondrocytes. Si-HPMC therefore appears as a potential scaffold for three-dimensional amplification and transfer of chondrocytes in cartilage tissue engineering.
Keywords: Articular cartilage; Hydrogel; Chondrocytes; Tissue engineering; Biocompatibility;

Fabrication of hydroxyapatite sponges by dextran sulphate/amino acid templating by Rosanna Gonzalez-McQuire; David Green; Dominic Walsh; Simon Hall; Jean-Yves Chane-Ching; Richard O.C. Oreffo; Stephen Mann (6652-6656).
We report a new template-directed method for the fabrication of hydroxyapatite (HAp) sponges by using amino-acid-coated HAp nanoparticles dispersed within a viscous polysaccharide (dextran sulfate) matrix, and describe the use of these materials for the viability and proliferation of human bone marrow stromal cells. The nanoparticles were prepared in the presence of excess amounts of aspartic acid, alanine or arginine, and subsequently organised into macroporous frameworks with typical pore sizes of 100–200 μm during thermal degradation of the dextran matrix. The sponge macrostructure was influenced by changes in the heating rate and sintering time, as well as the use of different amino acids or variations in dextran functional groups. Biocompatibility testing showed retention of cell viability, production of extracellular matrix and alkaline phosphatase expression, suggesting that it should be possible to exploit this novel fabrication method for potential applications in cartilage or soft tissue engineering.

Characterization of irradiated blends of α -tocopherol and UHMWPE by Ebru Oral; Evan S. Greenbaum; Arnaz S. Malhi; William H. Harris; Orhun K. Muratoglu (6657-6663).
Adhesive/abrasive wear in ultra-high molecular weight polyethylene (UHMWPE) has been minimized by radiation cross-linking. Irradiation is followed by melting to eliminate residual free radicals and avoid long-term oxidative embrittlement. However, post-irradiation melting reduces the crystallinity of the polymer and hence its strength and fatigue resistance. We proposed an alternative to post-irradiation melting to be the incorporation of the antioxidant α -tocopherol into UHMWPE prior to consolidation. α -Tocopherol is known to react with oxygen and oxidized lipids, stabilizing them against further oxidative degradation reactions. We blended GUR 1050 UHMWPE resin powder with α -tocopherol at 0.1 and 0.3 wt% and consolidated these blends. Then we gamma-irradiated these blends to 100-kGy. We characterized the effect of α -tocopherol on the cross-linking efficiency, oxidative stability, wear behavior and mechanical properties of the blends. (I) The cross-link density of virgin, 0.1 and 0.3 wt% α -tocopherol blended, 100-kGy irradiated UHMWPEs were 175±19, 146±4 and 93±4 mol/m3, respectively. (II) Maximum oxidation indices for 100-kGy irradiated UHMWPE previously blended with 0, 0.1 and 0.3 wt% α -tocopherol that were subjected to accelerated aging at 80 °C in air for 5 weeks were 3.32, 0.09, and 0.05, respectively. (III) The pin-on-disc wear rates of 100-kGy irradiated UHMWPE previously blended with 0.1 and 0.3 wt% α -tocopherol that were subjected to accelerated aging at 80 °C in air for 5 weeks were 2.10±0.17 and 5.01±0.76 mg/million cycles, respectively. (IV) Both accelerated aged, α -tocopherol-blended 100-kGy irradiated UHMWPEs showed higher ultimate tensile strength, higher yield strength, and lower elastic modulus when compared to 100-kGy irradiated, virgin UHMWPE. These results showed that α -tocopherol-blended 100-kGy irradiated UHMWPEs were not cross-linked to the same extent as the 100-kGy irradiated, virgin UHMWPE.
Keywords: Antioxidant; Polyethylene; Wear mechanism; Lipid; Gamma-irradiation; Hip replacement prosthesis;

Influence of selected wound dressings on PMN elastase in chronic wound fluid and their antioxidative potential in vitro by Ute Schönfelder; Martin Abel; Cornelia Wiegand; Dieter Klemm; Peter Elsner; Uta-Christina Hipler (6664-6673).
Exudates from non-healing wounds contain elevated levels of proteolytic enzymes, like elastase from polymorphonuclear granulocytes (PMN elastase), reactive oxygen species (ROS) and reactive nitrogen species (RNS). The overproduction of proteolytic enzymes leads to reduced concentrations of growth factors and proteinase inhibitors, resulting in an imbalance between degradation and remodelling processes. Thus, the reduction of protein-degrading enzymes and scavenging of ROS and RNS seem to be suitable ways to support the healing process of chronic stagnating wounds. The aim of this study was to test selected wound dressings from different biomaterials (collagen, oxidized regenerated cellulose (ORC) and ORC/collagen mixture), regarding their antioxidative potential in vitro and their influence on the concentration and activity of PMN elastase in chronic wound fluid. Antioxidant capacity of the investigated wound dressing was determined by a pholasin-based chemiluminescent assay. PMN elastase concentration was determined by means of ELISA. Enzyme activities could be measured by a fluorescence assay. As the presented data demonstrates, all tested materials showed antioxidant capacity. In addition, the investigated materials were able to reduce the concentration and activity of PMN elastase. Beside other aspects, such as biocompatibility, biodegradability, fluid absorption and clinical effects (e.g. angiogenesis and microcirculation), the understanding of these properties may help to support the further refinement of wound dressings for improved wound healing.
Keywords: Collagen; Cellulose; Wound dressing; PMN elastase; Free radicals; Wound healing;

Intrinsically radiopaque hydrogels for nucleus pulposus replacement by Erik J.H. Boelen; Catharina S.J. van Hooy-Corstjens; Sjoerd K. Bulstra; André van Ooij; Lodewijk W. van Rhijn; Leo H. Koole (6674-6683).
Degeneration of the intervertebral disc is the most common cause of back pain. In case of early stage degenerative disc disease or traumatic herniations, a suitable treatment may be to replace the nucleus pulposus, preserving the annulus fibrosus. Eight new hydrogel biomaterials were prepared and studied for their potential as a nucleus replacement. The hydrogels were designed according to the following criteria: (i), they should exhibit adequate radiopacity; (ii), they should be non-cytotoxic; (iii), implantation in the dry state and subsequent swelling in situ to fill the entire nucleus cavity; (iv), after swelling they should match the physical–mechanical properties of the native nucleus. The approach was to use copolymers consisting of 2-(4′-iodobenzoyl)-oxo-ethyl methacrylate (4IEMA) and a hydrophilic building block (either N-vinyl-2-pyrrolidinone (NVP) or 2-hydroxyethyl methacrylate (HEMA)); 4 copolymers of NVP/4IEMA and 4 copolymers of HEMA/4IEMA in different compositions (5, 10, 15 and 20 mol% 4IEMA).The study comprised 1H-NMR analysis of the copolymerization reaction NVP+4IEMA. Furthermore, the copolymers were studied with respect to their swelling behavior, mechanical properties, cytotoxicity in vitro and X-ray contrast. Hydrogels with 5 mol% 4IEMA appear to meet all criteria: they are non-cytotoxic, have adequate physical–mechanical properties and feature sufficient radiopacity in a realistic model. The potential implications of these new results with respect to treatment of degenerative disc disease are discussed briefly.
Keywords: Hydrogel; Copolymer; Mechanical properties; Radiopacity; Spinal surgery;

Chitosan as an antibacterial agent and heparin as an anti-adhesive agent were alternatively deposited onto aminolyzed poly(ethylene terephthalate) (PET) films to construct anti-adhesive and antibacterial multilayer films. The contact-angle and UV data verified the progressive buildup of the multilayer film by alternate deposition of the polyelectrolytes. The properties of multilayer films were investigated by contact angle, atomic force microscopy (AFM), lateral force microscopy (LFM) and UV spectra. The results of initial adhesion of Escherichia coli (E. coli) on PET substrates showed that the number of E. coli adhered onto the control PET was in a much greater extent than onto the chitosan/heparin multilayer films, and the number of adhesive bacteria decreased with a decrease in assembly pH. The in vitro antibacterial test indicated that a multilayer of chitosan/heparin could kill the bacteria effectively. The number of viable bacteria decreased by 7% after 7 h in contact with the control PET films, but by 46–68% for the multilayer-modified PET films. Only 3–8% of the cells were viable for the multilayer-modified PET films after 24 h. It is interesting to find the assembly pH has a remarkable effect on the antibacterial property of the multilayer. The number of viable bacteria on the multilayer assembled at pH=3.8, 2.9 and 6.0 decreased by 68%, 58% and 46%, respectively. Such an easy processing and shape-independent method to prepare an anti-adhesive and antibacterial surface may have good potential for surface modification of cardiovascular devices.
Keywords: Antibacterial; Anti-adhesion; Layer-by-layer; pH; PET films;

Peripheral quantitative computed tomography in evaluation of bioactive glass incorporation with bone by Ville-Valtteri Välimäki; Niko Moritz; Jessica J. Yrjans; Michel Dalstra; Hannu T. Aro (6693-6703).
This laboratory study examined the feasibility of non-invasive, in vivo peripheral quantitative computed tomography (pQCT) method in evaluation of bioactive glass incorporation with bone. An intramedullary defect model of the rat tibia was applied. The defect was filled with bioactive glass microspheres (diameter of 250–315 μm) or was left to heal without filling (empty controls). The results of the pQCT analysis were compared with those of histomorphometry. In the control defects, there was a good correlation ( r 2 = 0.776 , p < 0.001 ) between the pQCT density of the intramedullary space and the amount of new bone measured by histomorphometry. In the defects filled with bioactive glass, the use of thresholding techniques of the applied pQCT system (Stratec XCT Research M) failed in separation of new bone formation and bioactive glass particles. However, detailed analysis of the pQCT attenuation profiles showed time-related changes which well matched with the histomorphometric results of new bone formation both in control and bioactive glass filled defects. The biphasic pQCT attenuation profiles of bioactive glass filled defects could be separated into two distinct peaks. In statistical analysis of various variables, the center (i.e. the value of attenuation) of the major attenuation peak was found to be the most significant indicator of the incorporation process. The center of the peak initially decreased (during the first 4 weeks of healing) and thereafter increased. These two phases probably reflect the primary resorption and reactivity of the bioactive glass microspheres in vivo followed by secondary new bone formation on their surfaces. Based on these results, pQCT-method seems to be suitable for in vivo follow-up of the bioactive glass incorporation processes. Although the imaging technique is not able to discriminate the individual microspheres from invading new bone unambiguously, the attenuation profiling seems to give adequate information about the state of the incorporation process. This information may help to establish non-invasive imaging techniques of synthetic bone substitutes for preclinical and clinical testing of their efficacy.
Keywords: pQCT; Bioactive glass; Bone; Bone graft;

Antifungal coating by biofunctionalized polyelectrolyte multilayered films by Olivier Etienne; Claire Gasnier; Corinne Taddei; Jean-Claude Voegel; Dominique Aunis; Pierre Schaaf; Marie-Helène Metz-Boutigue; Anne-Laure Bolcato-Bellemin; Christophe Egles (6704-6712).
The surface of medical devices is a common site of bacterial and fungal adhesion, first step to the constitution of a resistant biofilm leading frequently to chronic infections. In order to prevent such complications, several physical and chemical modifications of the device surface have been proposed. Here, we experiment a new type of topical antifungal coating using the layer-by-layer technique. The nanometric multilayer film obtained by this technique is functionalized by the insertion of a chromogranin A-derived antifungal peptide (CGA 47-66, chromofungin). We show that the embedded peptide keeps its antifungal activity by interacting with the fungal membrane and penetrating into the cell. In vitro studies demonstrate that such an antifungal coating is able to inhibit the growth of yeast Candida albicans by 65% and completely stop the proliferation of filamentous fungus Neurospora crassa. The cytotoxicity of such a coating was also assessed by growing human gingival fibroblasts at its surface. Finally, the antifungal coating of poly(methylmethacrylate), a widely used material for biomedical devices, is successfully tested in an in vivo oral candidiasis rat model. Taken together, these results assessed the functionalized multilayer films containing a new potent antifungal non-toxic peptide, as a novel and promising technique for local antifungal protection.
Keywords: Antimicrobial; Bioactivity; Surface modification; Polyelectrolyte multilayer film; Chromofungin; Chromogranin A;

Mechanism of bone incorporation of β-TCP bone substitute in open wedge tibial osteotomy in patients by Robert D.A. Gaasbeek; Hanneke G. Toonen; Ronald J. van Heerwaarden; Pieter Buma (6713-6719).
A histological study was performed of bone biopsies from 16 patients (17 biopsies) treated with open wedge high tibial osteotomies for medial knee osteoarthritis. The open wedge osteotomies were filled with a wedge of osteoconductive beta tricalcium phosphate (β-TCP) ceramic bone replacement. At the time of removal of the fixation material, core biopsies of the area where the β-TCP was located were taken at different follow-up periods (6–25 months). β-TCP resorption, bone ingrowth and bone remodelling were studied. We hypothesized that the incorporation and remodelling process occurs similarly as in animals.Histology showed a good resorption of the β-TCP with complete incorporation and remodelling into new bone. The different phases as described in animal studies were found. A correlation was found between histological findings and radiological assessment.In conclusion, β-TCP appeared to be a bone replacement material with optimal biocompatibility, resorption characteristics and bone conduction properties for the clinical use.
Keywords: Bone ingrowth; Tricalcium phosphate; Bone remodelling;

The bone response of oxidized bioactive and non-bioactive titanium implants by Young-Taeg Sul; Carina Johansson; Eungsun Byon; Tomas Albrektsson (6720-6730).
A number of experimental and clinical data on so-called oxidized implants have reported promising outcomes. However, little is investigated on the role of the surface oxide properties and osseointegration mechanism of the oxidized implant. Sul [On the Bone Response to Oxidized Titanium Implants: The role of microporous structure and chemical composition of the surface oxide in enhanced osseointegration (thesis). Göteborg: Department of Biomaterials/Handicap Research, University of Göteborg, Sweden; 2002; Biomaterials 2003; 24: 3893–3907] recently proposed two action mechanisms of osseointegration of oxidized implants, i.e. mechanical interlocking through bone growth in pores/other surface irregularities (1) and biochemical bonding (2). The aim of the present study is two-fold: (i) investigating the role of the implant surface chemistry on bone responses; (ii) investigating the validity of the biochemical bonding theory of the oxidized, bioactive bone implants with specific implant surface chemistry. Two groups of oxidized implants were prepared using micro arc oxidation process and were then inserted in rabbit bone. One group consisted of magnesium ion incorporated implants (MgTiO implant), the other consisted of TiO2 stoichiometry implants (TiO implant). Surface oxide properties of the implants were characterized with various surface analytic techniques. After 6 weeks of follow up, the mean peak values of removal torque of Mg implants dominated significantly over TiO implants ( p ⩽ 0.0001 ). Bonding failure generally occurred in the bone away from the bone to implant interface for the MgTiO implant and mainly occurred at the bone to implant interface for the TiO implant that consisted mainly of TiO2 chemistry and significantly rougher surface as compared to the MgTiO implant. Between bone and the Mg- incorporated implant surface, ionic movements and ion concentrations gradient were detected. The current in vivo experimental data may provide positive evidence for the surface chemistry-mediated biochemical bonding theory of oxidized bioactive implants. However, the present study does not rule out potential synergy effects of the oxide thickness, micro-porous structure, crystal structure and surface roughness on improvements of bone responses to oxidized bioactive implants.
Keywords: Oxidized bioactive titanium implants; Surface chemistry; Bone; Biochemical bonding; Osseointegration mechanism;

In vitro studies investigating the influence of electric DC current on bacterial detachment have demonstrated that continuous currents of only 25–125 μA stimulated staphylococcal strains to detach from surgical stainless steel. However, DC currents produce more power that has to be dissipated by the skin as compared to alternating currents. Also, an excess of ions on the steel can cause negative osteogenesis and fixation results. Therefore, it is the aim of this paper to examine whether detachment of Staphylococcus epidermidis from stainless steel surfaces in a parallel plate flow chamber can also be stimulated using electric block currents. Block currents of 15, 60 and 100 μA with different frequencies (0.1–2 Hz) and duty cycles (5–50%) were applied to induce bacterial detachment. Block currents of 100 μA cause detachment of about 76% of adhering staphylococci from stainless steel, whereas in addition the remaining bacteria are less viable, as determined by culturing the remaining bacteria on agar plates. Therewith, block current-induced detachment of adhering bacteria from stainless steel appears to be an equally promising method to prevent infection of orthopaedic fixation pins and screws than application of DC currents.
Keywords: Electric block current; Detachment; Stainless steel; Bactericidal; Staphylococcus epidermidis; Percutaneous implant; Biofilm;

Novel BAB type amphiphilic triblock copolymers consisting of poly (ethylene glycol) (PEG) (B) as hydrophilic segment and poly (ε-caprolactone) (PCL) (A) as hydrophobic block were prepared by coupling reaction using l-lysine methyl ester diisocyanate (LDI) as the chain extender. The triblock copolymers obtained were characterized by FT-IR, 1H NMR, GPC, and DSC. Core-shell type nanoparticles were prepared by nanoprecipitation method and below 100 nm nanoparticles were obtained due to their specific structure. Transmission electron microscopy image demonstrated that these nanoparticles were spherical in shape. Stability of the nanoparticles in biological media was evaluated. Poorly water-soluble anticancer drug 4′-demethyl-epipodophyllotoxin (DMEP) was chosen for controlled drug release because it was easily encapsulated into polymeric nanoparticles via hydrophobic interaction. In vitro release behavior of DMEP from polymeric nanoparticles was investigated, the results showed that the drug release rate can be modulated by the variation of the copolymer composition.
Keywords: Amphiphilic triblock copolymer; Chain extender; Poly (ε-caprolactone); Poly (ethylene glycol); Nanoparticle; Drug release;

Hollow gold nanoparticles encapsulating horseradish peroxidase by Rajiv Kumar; A.N. Maitra; P.K. Patanjali; Parvesh Sharma (6743-6753).
Hollow nanoshells of gold entrapping an enzyme, horseradish peroxidase (HRP), in the cavity of the nanoshell have been prepared in the reverse micelles by leaching out silver chloride (AgCl) from AushellAgClcore nanoparticles with dilute ammonia solution. The particles have been characterised by dynamic laser light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD), and electron diffraction. The particle size is below 100 nm diameter, depending upon the size of the aqueous core of reverse micelles in which these particles have been prepared. This soft-chemical method for the preparation of such particles allows the entrapped enzyme to remain active inside the hollow gold nanoparticles. Small substrate molecules such as o-dianisidine can easily enter through the pores of the nanoshell and can undergo enzymatic oxidation by H2O2. The enzyme kinetics follows Michaelis–Menten mechanism. When the substrate is chemically conjugated with dextran molecule (10 kDa), the enzymatic reaction is practically completely prevented perhaps by the inability of dextran-o-dianisidine conjugate to penetrate the pores of the nanoshells. However, HRP did not show any activity when trapped inside solid gold nanoparticles.
Keywords: Hollow gold nanoparticles; Aushell AgClcore nanoparticles; Enzyme entrapment; Horseradish peroxidase (HRP); Dextran-o-dianisidine;

Polymeric microspheres as stabilizing anchors for oligonucleotide delivery to dendritic cells by Jeffrey R. Kovacs; Ying Zheng; Hongmei Shen; Wilson S. Meng (6754-6761).
The aim of this study is to evaluate a novel microspheric vector for delivery of oligonucleotides (ODN) into dendritic cells (DC). A requirement of decoy-based modulation of transcriptional activities in DC is that the ODN would have to accumulate inside the cell. Using an ex vivo DC culture model, we demonstrate that anionic microspheres (MS) coated with an ornithine/histadine-based cationic peptide (O10H6) is an effective carrier of short ODN. This method does not disrupt the colloidal nature of the microspheric particles. The MS provide stabilizing effect on DNA and O10H6 complexation. Accumulation of ODN in DC is greatly enhanced with the surface modified MS. Taken together, these data demonstrate that the self assembly system of MSO10H6 is an effective delivery vehicle for DNA-based modulation of DC functions.
Keywords: DNA delivery; Dendritic cell; Nanoparticle; Cationic peptide; Oligonucleotide;

Engineered spatial patterns of FGF-2 immobilized on fibrin direct cell organization by Phil G. Campbell; Eric D. Miller; Gregory W. Fisher; Lynn M. Walker; Lee E. Weiss (6762-6770).
The purpose of this study was to initiate the exploration of cell behavioral responses to inkjet printed spatial patterns of hormones biologically immobilized on biomimetic substrates. This approach was investigated using the example of preosteoblastic cell response in vitro to fibroblast growth factor-2 (FGF-2) printed on fibrin films. Concentration modulated patterns of FGF-2, including continuous concentration gradients, were created by overprinting dilute FGF-2 bioinks with a custom inkjet printer. The immobilized FGF-2 was biologically active and the printed patterns persisted up to 10 days under cell culture conditions. Cell numbers increased in register to printed patterns from an initial random uniform cell distribution across the patterned and non-patterned fibrin substrate. Patterned immobilized FGF-2, not cell attachment directed cell organization because the fibrin substrate was homogeneous. The capability to engineer arbitrary and persistent hormone patterns is relevant to basic studies across various fields including developmental biology and tissue regeneration. Furthermore, since this hormone inkjet printing methodology is extensible to create complex three-dimensional structures, this methodology has potential to create therapies for tissue engineering using spatial patterned delivery of exogenous hormones.
Keywords: Tissue engineering; Inkjet technology; Solid freeform fabrication; Growth factor; Printing technology; Biological patterning;

Tuning the sol–gel microenvironment for acetylcholinesterase encapsulation by Sofia Sotiropoulou; Nikos A. Chaniotakis (6771-6779).
The effect of the sol–gel microenvironment on the activity of acetylcholinesterase, an enzyme of high bio-analytical interest, is presented and is correlated to the overall analytical performance of corresponding biosensors. The sol–gel membranes are initially optimized with respect to the catalyst and the TEOS:H2O ratio (r), for mechanical stability, porosity, and hydrophobicity as well as in terms of enzymatic activity. FT-IR and electrochemical impedance spectroscopy (EIS) are used to probe the configuration and rotational mobility of the enzyme within the sol–gel matrices. Overall, it is observed that the rotational mobility of the protein can be correlated with the sensitivity of the biosensor. Optimum biosensor performance is obtained for base-catalyzed sol–gels with r values close to 2. The biosensor has sensitivity of 2.5 μA/mm, a linear range of response between 1 and 3 mm, response time of about 30 s, and sensor-to-sensor reproducibility (RSD) of 3%. These analytical characteristics are far superior to previously reported sol–gel biosensors.
Keywords: Acetylcholinesterase; Sol–gel; Infrared spectroscopy; Electrochemical impedance spectroscopy; Amperometric biosensor;

Characterisation of dentin surfaces processed with KrF excimer laser radiation by Sónia Eugénio; Manickam Sivakumar; Rui Vilar; Ana Maria Rego (6780-6787).
In the present work, the surface microtexture and chemical changes induced in human dentin by laser processing with KrF excimer laser radiation using fluences ranging from 0.5 to 20 J/cm2 were studied by SEM, XPS and FTIR. Two distinct behaviours were observed in the evolution of surface topography. In some samples, the laser-treated surface remained flat, independently of the fluence used. It was covered by a layer formed of redeposited ablation particles, which occluded the dentinal apertures. In other samples the surface topography depended on radiation fluence. When the fluence was lower than 1 J/cm2, preferential removal of intertubular dentin occurs, producing a columnar structure in which the columns are essentially formed of peritubular material. If the fluence exceeded 1 J/cm2the processed surface was flat and covered with resolidified material. Despite these topographic changes, the dentin was not significantly affected by the laser treatment. The observed behaviour can be explained by differences in the constitution of dentin.
Keywords: Dentin; Laser ablation; Surface modification; SEM; XPS; FTIR;

Mathematical modelling of the distribution of newly formed bone in bone tissue engineering by Laurent Pothuaud; Jean-Christophe Fricain; Stephane Pallu; Reine Bareille; Martine Renard; Marie-Christine Durrieu; Michel Dard; Michel Vernizeau; Joelle Amédée (6788-6797).
New bone formation in bone substitutes is usually investigated by histomorphometric global analysis. This study provides a novel mathematical modelling approach of new bone formation in the use of osteoinductive and functionalized biomaterials for bone tissue engineering. We discuss here the repartition and the probability to get new bone formation inside Biphasic Calcium Phosphate (BCP) loaded with autologous osteogenic cells, functionalized with a cyclo RGD peptide, after implantation in rabbits for 2 and 4 weeks. This local analysis allowed us to complement classical global findings and to demonstrate that after 2 weeks of implantation, the probability of new bone formation was significantly higher in RGD-grafted BCP and that new formed bone was largely distributed from the edge to the centre of the implant. While no significant differences were obtained after 4 weeks of implantation between RGD-grafted and non-grafted materials, distribution of new bone formation inside RGD-grafted materials was significantly more homogeneous as demonstrated by our mathematical modelling approach. In conclusion, local analysis of new bone formation inside macroporous substitutes coupled with mathematical modelling constitutes a potential quantitative approach for the evaluation of the osteoconductive and osteoinductive characteristics of such biomaterials.
Keywords: RGD peptide; Animal model; Image analysis; Modelling;

Theoretical prediction of surface bone remodeling in the diaphysis of the long bone under various external loads are made within the framework of adaptive elastic theory. These loads include external lateral pressure, electric and thermal loads. Two solutions are presented for analyzing thermoelectroelastic problems of surface bone remodeling. The analytical solution that gives explicit formulation is capable of modeling homogeneous bone materials, while the semi-analytical solution is suitable for analyzing inhomogeneous cases. Numerical results are presented to verify the proposed formulation and to show the effects of mechanical, thermal and electric loads on surface bone remodeling process.
Keywords: Bone surface remodeling; Piezoelectric; Thermal field;