Biomaterials (v.27, #4)

Calendar (I).

What future for zirconia as a biomaterial? by Jérôme Chevalier (535-543).
The failure events of Prozyr® femoral heads in 2001–2002 have opened a strong, controversial issue on the future of zirconia as a biomaterial. The aim of this paper is to review and analyze the current knowledge on ageing process and on its effect on the long term performance of implants in order to distinguish between scientific facts and speculation. Current state of the art shows the strong variability of zirconia to in vivo degradation, as a consequence of the strong influence of processing on ageing process. As different zirconia from different vendors have different process related microstructure, there is a need to assess their ageing sensitivity with advanced and accurate techniques, and ISO standards should be modified, especially to gain confidence from clinicians. There is a trend today to develop alumina–zirconia composites as an alternative to monolithic alumina and zirconia: the issue of ageing is also discussed for these composites.
Keywords: Zirconia; Ageing; Explant analysis; Orthopedics;

The degradation of P(DLA X -ran-CL Y )-b-PEG-b-P(DLA X -ran-CL Y )s ( P(DLA X -ran-CL Y ): Poly(d,l-lactide-ran-ε-caprolactone), PEG: Poly(ethylene glycol), X: d,l-lactyl unit fraction, Y: ε-caproyl unit fraction) is investigated. The fraction of DLA in the both end blocks is varied while the overall molecular weight and molecular weight of PEG are kept constant. DSC, XRD and GPC are employed to track the degradation process up to 200 days. Also the change in the surface and cross-sectional morphology is provided by SEM micro-photographs. The result of water absorption and weight loss characterization reveals that the incorporation of DLA in the polyester block could be an effective tool to facilitate degradation as well as water absorption. By tracking the change of molecular weight and polydispersity, chain scission and transport or removal of degraded product from the specimen were found to play a complex role in overall degradation.
Keywords: Poly(d,l-lactide-ran-ε-caprolactone)-b-poly(ethylene glycol)-b-poly(d,l-lactide-ran-ε-caprolactone); Triblock copolymer; Degradation; Crystallinity; Morphology;

Physico-chemical and biological evaluation of excimer laser irradiated polyethylene terephthalate (pet) surfaces by G. Mayer; N. Blanchemain; C. Dupas-Bruzek; V. Miri; M. Traisnel; L. Gengembre; D. Derozier; H.F. Hildebrand (553-566).
The aim of this work was to investigate the consequences of excimer laser irradiation on the physico-chemical and biological properties of polyethylene terephthalate (PET) films, currently used for medical devices. Three PET films from different origins were studied in the present work, chosen with respect to their chemical and physical properties, which are of high importance for ulterior medical application as vascular prostheses. Multiple assays were carried out to characterize the physical and chemical effects of the laser irradiation: surface morphology tests (light microscopy, Dektak profilometer and confocal laser scanning microscopy) showed the strong transformation of the surface with the laser treatment. Contact angle measurements revealed a significant increase of the surface energy for each PET depending on the applied fluency. Finally XPS characterization of the surface demonstrated the appearance of new chemical species favorable for cell attachment. This aspect had to be strongly considered regarding to the multiple biological effects of laser irradiated surfaces on living cells. Different cell culture experiments were carried out with L132 human epithelial cells after 6-days culture: proliferation and vitality rate, cell adhesion and cell morphology. Results clearly revealed that laser treatment improved cell proliferation (up to 140% with respect to controls), vitality (10% higher than controls), morphology and adhesion kinetics (more than 16% of control). A significant correlation (R 2=0.906) was also established on one PET between the fluencies of laser treatment and the cellular response. These results emphasized high importance of the choice of the PET material for a medical application: only one of the three considered PET films showed really improved cellular response.
Keywords: Laser; Polyethylene terephthalate; Surface analysis; Cytocompatibility; Cell adhesion; Cell activation;

The effect of charged groups on protein interactions with poly(HEMA) hydrogels by Megan S. Lord; Martina H. Stenzel; Anne Simmons; Bruce K. Milthorpe (567-575).
Proteins, lipids and other biomolecules interact strongly with the acrylic-based biomaterials used for contact lenses. Although hydrogels are nominally resistant to protein fouling, many studies have reported considerable amounts of protein bound to poly(2-hydroxyethylmethacrylate) (PHEMA) lenses. This study examined the binding of a series of biomolecules (tear protein analogues, mucin and cholesterol) to poly(methylmethacrylate) (PMMA) and three HEMA-based hydrogels (PHEMA, HEMA plus methacrylic acid (P(HEMA–MAA)), HEMA plus methacrylic acid plus N-vinylpyrrolidone (P(HEMA–MAA–NVP))) by use of a quartz crystal microbalance with dissipation (QCM-D) monitoring. The QCM-D estimates changes in the mass and viscous constant for the adsorbed layer through measurements of frequency and dissipation.Protein interaction with each of the test materials caused a net increase in mass of the material indicating protein binding except for lysozyme interacting with P(HEMA–MAA). A net decrease in mass was observed for lysozyme interacting with P(HEMA–MAA) which may be ascribed to lysozyme collapsing the hydrogel by expelling water. A net mass decrease was observed for cholesterol interacting with each of the hydrogel materials, while a mass increase was observed on PMMA.
Keywords: Hydrogels; Protein adsorption; Quartz crystal microbalance; Contact lens material;

Galactose-carrying polymers as extracellular matrices for liver tissue engineering by C.S. Cho; S.J. Seo; I.K. Park; S.H. Kim; T.H. Kim; T. Hoshiba; I. Harada; T. Akaike (576-585).
Extracellular matrix (ECM) plays important roles in tissue engineering because cellular growth and differentiation, in the two-dimensional cell culture as well as in the three-dimensional space of the developing organism, require ECM with which the cells can interact. Especially, the bioartificial liver-assist device or regeneration of the liver-tissue substitutes for liver tissue engineering requires a suitable ECM for hepatocyte culture because hepatocytes are anchorage-dependent cells and are highly sensitive to the ECM milieu for the maintenance of their viability and differentiated functions. Galactose-carrying synthetic ECMs derived from synthetic polymers and natural polymers bind hepatocytes through a receptor-mediated mechanism, resulting in enhanced hepatocyte functions. Attachment and functions of hepatocytes were affected by physico-chemical properties including ECM geometry as well as the type, density and orientation of galactose. Also, cellular environment, medium composition and dynamic culture system influenced liver-specific functions of hepatocytes beside ECM.
Keywords: Galactose; Extracellular matrix; Hepatocyte; Tissue engineering; Asialoglycoprotein receptors;

Study of osteoblastic cells in a microfluidic environment by E. Leclerc; B. David; L. Griscom; B. Lepioufle; T. Fujii; P. Layrolle; C. Legallaisa (586-595).
Bone tissue engineering consists of culturing osteoblastic cells onto synthetic three-dimensional (3D) porous scaffolds. The organization of bone cells into 3D scaffolds is crucial for ex vivo tissue formation. Diffusional rates of nutrients could be greatly improved by perfusing media through the 3D microporous scaffolds. However, bone cells cultured in vitro are responsive to a variety of different mechanical signals including fluid flow and shear stresses. In this work, we attempt to study osteoblastic cells behaviour cultured within microdevices allowing continuous and homogenous feeding of cells. We have fabricated polydimethylsiloxane PDMS microdevices with a 3D microstructured channel network. Mouse calvarial osteoblastic cells MC3T3-E1 were seeded at 2×106  cells/ml and cultured into the microdevices under flow rates of 0, 5, 35 μl/min. Cells attached and proliferated well in the designed microdevices. Cell viability was found around 85% up to 1 to 2 weeks for shear stress value under 5 mPa. The alkaline phosphatase (ALP) activity was enhanced 3- and 7.5-fold inside the microdevices under static and dynamic flow of 5 μl/min as compared to flat static cultures in PDMS coated Petri dishes. Therefore, osteoblastic cells could be successfully cultured inside the microdevices under dynamic conditions and their ALP activity was enhanced. These results are promising for bone cell growth and differentiation as well as future tissue regeneration using larger 3D microfluidic microdevices.
Keywords: Microdevices; Dynamic cell cultures; Osteoblast; Bone tissue engineering;

Effect of fiber diameter on spreading, proliferation, and differentiation of osteoblastic cells on electrospun poly(lactic acid) substrates by Anand S. Badami; Michelle R. Kreke; M. Shane Thompson; Judy S. Riffle; Aaron S. Goldstein (596-606).
Electrospinning is a promising method to construct fused-fiber biomaterial scaffolds for tissue engineering applications, but the efficacy of this approach depends on how substrate topography affects cell function. Previously, it has been shown that linear, parallel raised features with length scales of 0.5–2 μm direct cell orientation through the phenomenon of contact guidance, and enhance phenotypic markers of osteoblastic differentiation. To determine how the linear, random raised features produced by electrospinning affect proliferation and differentiation of osteoprogenitor cells, poly(lactic acid) and poly(ethylene glycol)-poly(lactic acid) diblock copolymers were electrospun with mean fiber diameters of 0.14–2.1 μm onto rigid supports. MC3T3-E1 osteoprogenitor cells cultured on fiber surfaces in the absence of osteogenic factors exhibited a lower cell density after 7 and 14 days of culture than cells cultured on spin-coated surfaces, but cell density increased with fiber diameter. However, in the presence of osteogenic factors (2 mm β-glycerophosphate, 0.13 mm l-ascorbate-2-phosphate), cell density after 7 and 14 days of culture on fiber surfaces was comparable to or exceeded spin-coated controls, and alkaline phosphatase activity after 14 days was comparable. Examination of cell morphology revealed that cells grown on fibers had smaller projected areas than those on planar surfaces. However, cells attached to electrospun substrates of 2.1 μm diameter fibers exhibited a higher cell aspect ratio than cells on smooth surfaces. These studies show that topographical factors designed into biomaterial scaffolds can regulate spreading, orientation, and proliferation of osteoblastic cells.
Keywords: Polylactide; Poly(ethylene glycol); Electrospinning; Cell proliferation; Tissue engineering;

Structural characterization of bioengineered human corneal endothelial cell sheets fabricated on temperature-responsive culture dishes by Takeshi Ide; Kohji Nishida; Masayuki Yamato; Taizo Sumide; Mika Utsumi; Takayuki Nozaki; Akihiko Kikuchi; Teruo Okano; Yasuo Tano (607-614).
For the purpose of corneal regenerative medicine, we fabricated human corneal endothelial cell sheets on temperature-responsive dishes, which could be non-invasively harvested as intact, transplantable sheets by simply reducing the culture temperature. Cells demonstrated hexagonal cell shape with numerous microvilli and cilia, and also exhibited abundant cytoplasmic organelles similar to these cells in vivo. Immunofluorescence for type IV collagen and fibronectin revealed that abundant extracellular matrix (ECM) was deposited on the basal surface throughout culture, and the deposited ECM was harvested along with the cell sheets by reducing culture temperature to 20 °C. Faint ECM remnants were observed on the dish surfaces after cell sheet detachment. Immunofluorescence for ZO-1 showed that tight junctions were established between cells, and immunoblotting indicated that intact ZO-1 was maintained during cell sheet harvest, while conventional proteolytic cell harvest methods resulted in the degradation of ZO-1. These results suggest that these transplantable corneal endothelial cell sheets can be applied to treat patients with damaged corneas.
Keywords: Cell culture; Cornea; Extracellular matrix; Endothelial cell; Ophthalmology; Thermally responsive material;

Mouse embryonic stem cell colonisation of carbonated apatite surfaces by Amanda J. Melville; Janine Harrison; Kārlis A. Gross; John S. Forsythe; Alan O. Trounson; Richard Mollard (615-622).
Apatites play a crucial role in the body and have been used extensively in biomedical implants. The influence on stem cell behaviour is not known and so this study will explore whether sintered carbonated apatites are favourable for propagation of stem cells. Different weight substitutions of carbonated apatite, specifically 2.5 wt% (2.5 wt%CAP) and 5 wt% (5 wt%CAP), were sintered and characterised prior to the investigation of their potential as a matrix for the support of mouse embryonic stem (ES) cells. Characterisation of the apatites included elemental analysis, X-ray diffraction, surface roughness, specific surface area, density, and solubility. The ability of carbonated apatite to support mouse ES cell colonisation and maintenance in the presence of leukaemia inhibitory factor was determined by an enumeration of live versus dead cells within a population, and immunoreactivity to Oct4, a transcription factor and stem cell marker, following growth on each matrix. It was found that while both compositions allowed for the colonisation of mouse ES cells, the cells were not maintained in an undifferentiated state, as evidenced by a reduction in the number of cells staining positive for Oct4 expression. This study shows that an increase in carbonate content within sintered apatites leads to a higher cell number, a desired aspect for stem cells to populate scaffolds intended for tissue engineering. This study presents carbonated apatites as a suitable matrix for the initial colonisation and differentiation of ES cells for tissue engineering applications.
Keywords: Mouse embryonic stem cells; Morphology; Differentiation; Proliferation; Carbonated apatite; Tissue engineering;

The effect of sodium ascorbate on the mechanical properties of hyaluronan-based vascular constructs by Chiara Arrigoni; Davide Camozzi; Barbara Imberti; Sara Mantero; Andrea Remuzzi (623-630).
Esterified hyaluronic acid (HYAFF) is routinely used for clinical tissue engineering applications such as skin and cartilage. In a previous study we developed a technique for in vitro generation of cylindrical constructs from cellularized HYAFF flat sheets. In the present investigation we studied the possibility to improve mechanical properties of this vascular construct by the addition of sodium ascorbate (SA). Non-woven HYAFF flat sheets were seeded with porcine aortic smooth muscle cells (SMCs) and cultured for 14 or 28 days with standard medium or medium additioned with SA. In selected experiments HYAFF sheets seeded with SMCs were wrapped to obtain cylindrical shape and then cultured in control medium or SA additioned medium for up to 28 days. We estimated cell viability for flat sheets, and performed histological examination, analysis of extracellular matrix (ECM) deposition and mechanical tests on tubular constructs. The number of viable cells and ECM deposition increased with time in constructs cultured in the presence of SA, as compared to control group. Moreover, SA improved mechanical properties of the vascular construct lowering material stiffness and increasing tensile strength as compared to untreated controls. The addition of SA to the medium improved cell proliferation and ECM synthesis on this biodegradable material, which leads to the formation of well organized, mechanical resistant tissue-engineered structure.
Keywords: Tissue engineering; Hyaluronic acid; Smooth muscle cell; Sodium ascorbate; Mechanical properties;

Upon implantation, calcium phosphate (Ca–P) surfaces form on materials that are bone bioactive. In this study, the evolving surface characteristics associated with calcium phosphate precipitation are modeled using self-assembled monolayers (SAMs), in a one-step nucleation process. SAMs were used to create amine (–NH2), carboxyl (–COOH) and hydroxyl (–OH) functionalized surfaces by grafting 3-aminopropyltriethoxysilane, 3-triethoxysilylpropyl succinic anhydride and glycidoxypropyl tri-methoxysilane, respectively, onto oxidized silicon wafers. The SAM surfaces were characterized using ellipsometry to establish the presence of grafted molecules. On the surfaces incubated in simulated physiological fluids for 7 days, the thickness of Ca–P layer grew slowly over the first few hours, increasing strongly between 1 and 5 days and then slowed down again. FTIR showed the dependence of calcium phosphate morphology on the type of surface groups, with stronger P–O bands seen on the OH-terminated surface. SEM analysis showed dispersed Ca–P precipitates on the –COOH and –OH terminated surfaces after 1 day immersion. After 7 days, all SAM surfaces were covered with uniformly dispersed and denser Ca–P precipitates. The underlying Ca–P layer showed cracks on the –NH2-terminated surface. Rutherford backscattering spectrometry (RBS) data analysis confirmed that Ca/P ratio is in excellent agreement with the theoretical value of 1.67 for hydroxyapatite. X-ray diffraction (XRD) analysis also showed evidence of apatite formation on all the surfaces, with stronger evidence on the –OH-terminated surface. Highly porous Ca–P precipitates were observed on the SAM surfaces portrayed by the AFM scans with nanoscale RMS roughness. Thus, using highly controlled surface chemistry, under physiological conditions, in vitro, this study demonstrates that a hydroxylated surface enhances Ca–P nucleation and growth relative to other surfaces, thereby supporting the concept of its beneficial effect on bone tissue formation and growth.
Keywords: Self-assembled monolayers; Calcium phosphate; Apatite; Bone; Precipitation;

Reduced thrombogenicity of nitinol stents—In vitro evaluation of different surface modifications and coatings by Gunnar Tepe; Joerg Schmehl; Hans P Wendel; Sivio Schaffner; Stephan Heller; Marc Gianotti; Claus D Claussen; Stephan H Duda (643-650).
The material and the surface patterns of intravascular stents play a pivotal role in activating platelets and triggering adherence of inflammatory cells that consecutively leads to renarrowing caused by neointimal hyperplasia. To improve these features, besides mechanical and chemical modifications, ways of masking the stent by covering have been developed. In addition, polymer-coated stents are used as vehicle for local drug delivery. But as substances used for this application are described to possess an inflammatory potential, this aspect has to be evaluated.In the present study we compared different approaches to surface alterations applied to a nitinol stent design. Besides commonly used techniques like passivation and electropolishing, we evaluated coatings with heparin, aluminium and a polyurethane polymer regarding their thrombogenic and inflammatory characteristics. By weaving thin elastomer fibres a graft was generated. The previously described Chandler loop was used to simulate arterial flow conditions ex vivo using rotating PVC tubings filled with human blood. All stents received 120 min of blood contact. To determine thrombocyte activation and inflammatory reaction, the platelet count and levels of β-TG, TAT and PMN-elastase were assesed. Scanning electron microscopy was used to visualize the reactions.Mechanical polishing and passivation did not improve the stent surface characteristics while sandblasting, electropolishing and aluminium covering decreased activation of the coagulation cascade. In terms of thrombogenicity, the heparin coating had no beneficial effect. The lowest thrombogenic potential was found in the Polyurethane-coated stent group. All stents showed similar levels of polymorph nuclear granulocyte elastase except for the membrane design.While mechanical and chemical modifications are able to reduce thrombogenicity, coating with this particular polyurethane polymer seems to be superior to these approaches regarding the parameters assessed in this experimental setting. The Chandler loop is a valuable tool to test polymeric coatings ex vivo since these modifications may reduce drug performance by inducing inflammatory reaction themselves.
Keywords: Nitinol; Stent; Thrombogenicity; Polyurethane; Surface modification;

Number-concentration of nanoparticles in liposomal and polymeric multiparticulate preparations: Empirical and calculation methods by Hila Epstein; Eyal Afergan; Tamar Moise; Yoram Richter; Yinon Rudich; Gershon Golomb (651-659).
The actual number of particles in formulations of nanoparticles (NP) is of importance for quality assurance, comprehensive physicochemical characterization, and pharmacodynamics. Some calculation methods that have been previously employed are limited because they rely on several assumptions and are not applicable for certain preparations. Currently there are no validated experimental methods for determining the particle number-concentration (N c) of liposomal and polymeric nanoparticulate preparations (<500 nm). This study examines a new empirical method for counting the number of particles in nanoparticulate formulations including drug-containing liposomes and polymeric NP. In the new method, suspended NP are nebulized to form aerosol droplets which are dried and counted using a scanning mobility particle sizer (SMPS). Experiments were conducted with three different preparations, empty liposomes (200 and 400 nm), drug-loaded liposomes (200 nm), and polymeric NP (150 nm). It was verified that no detrimental morphological or structural changes of the formulations have been induced by the SMPS technique, and that the obtained N c values represent the original particles. It is concluded that nano-formulations with concentrations of up to 107 particles per 1 cm3 air, corresponding to approximately 1012 particles per 1 ml solution, can be directly counted within the size range of 30–900 nm. The measured values are compared to newly developed theoretical calculations to assess the viability of these calculations.
Keywords: Particles; Number concentration; Nanoparticles; Liposomes;

Preparation, cellular transport, and activity of polyamidoamine-based dendritic nanodevices with a high drug payload by Parag Kolhe; Jayant Khandare; Omathanu Pillai; Sujatha Kannan; Mary Lieh-Lai; Rangaramanujam M. Kannan (660-669).
Dendrimers are emerging as a relatively new class of polymeric biomaterials with applications in drug delivery, and imaging. Achieving a high drug payload in dendrimers, and understanding the therapeutic effect of the dendrimer–drug conjugates are receiving increasing attention. A high drug payload nanodevice was obtained by covalent conjugation of ibuprofen to a polyamidoamine (PAMAM-G4-OH) dendrimer. Using DCC as a coupling agent, 58 molecules of ibuprofen were covalently conjugated to one molecule of generation 4 PAMAM-OH dendrimer. Cellular entry of the fluoroisothiocynate (FITC)-labeled dendrimer–drug conjugate was evaluated in vitro by using human lung epithelial carcinoma A549 cells by flow cytometry, confocal microscopy and UV/Visible spectroscopy. The pharmacological activity of the dendrimer–ibuprofen conjugate was compared to pure ibuprofen at various time points by measuring the suppression of prostaglandin E2. Significant amounts of the conjugate entered the cells rapidly within 15 min. Suppression of prostaglandin was noted within 30 min for the dendrimer–drug conjugates versus 1 h for the free ibuprofen. The results suggest that dendrimers with high drug payload improve the drug's efficacy by enhanced cellular delivery, and may produce a rapid pharmacological response. These dendrimer–drug conjugates can potentially be further modified by attaching antibodies and ligands for targeted drug delivery.
Keywords: PAMAM dendrimers; Dendrimer–drug conjugates; Drug delivery; Ibuprofen; Cellular transport;

Erratum to “Osteoconductive modifications of Ti-implants in a goat defect model: Characterization of bone growth with SR μCT and histology” by Ricardo Bernhardt; Juliette van den Dolder; Sussane Bierbaum; Rene Beutner; Dieter Scharnweber; John Jansen; Felix Beckmann; Hartmut Worch (670).