Biomaterials (v.28, #13)

Biodegradable magnesium–hydroxyapatite metal matrix composites by Frank Witte; Frank Feyerabend; Petra Maier; Jens Fischer; Michael Störmer; Carsten Blawert; Wolfgang Dietzel; Norbert Hort (2163-2174).
Recent studies indicate that there is a high demand to design magnesium alloys with adjustable corrosion rates and suitable mechanical properties. An approach to this challenge might be the application of metal matrix composite (MMC) based on magnesium alloys. In this study, a MMC made of magnesium alloy AZ91D as a matrix and hydroxyapatite (HA) particles as reinforcements have been investigated in vitro for mechanical, corrosive and cytocompatible properties. The mechanical properties of the MMC-HA were adjustable by the choice of HA particle size and distribution. Corrosion tests revealed that HA particles stabilised the corrosion rate and exhibited more uniform corrosion attack in artificial sea water and cell solutions. The phase identification showed that all samples contained hcp-Mg, Mg17Al12, and HA before and after immersion. After immersion in artificial sea water CaCO3 was found on MMC-HA surfaces, while no formation of CaCO3 was found after immersion in cell solutions with and without proteins. Co-cultivation of MMC-HA with human bone derived cells (HBDC), cells of an osteoblasts lineage (MG-63) and cells of a macrophage lineage (RAW264.7) revealed that RAW264.7, MG-63 and HBDC adhere, proliferate and survive on the corroding surfaces of MMC-HA. In summary, biodegradable MMC-HA are cytocompatible biomaterials with adjustable mechanical and corrosive properties.
Keywords: Magnesium; Hydroxyapatite; Metal matrix composite; Corrosion; Mechanical properties; Cytotoxicity;

Systematic study of osteoblast and fibroblast response to roughness by means of surface-morphology gradients by Tobias P. Kunzler; Tanja Drobek; Martin Schuler; Nicholas D. Spencer (2175-2182).
The surface roughness of a medical implant is of great importance since the surface is in direct contact with the host tissue (e.g. bone, fibrous tissue). The response of cells to roughness is different depending on the cell type. However, the influence of roughness on cell behavior has only rarely been systematically studied. We have developed a surface-modification process to produce roughness gradients that cover a wide range of roughness values on one substratum. Such gradients allow for systematic investigations of roughness on cell behavior. Gradients were fabricated using a two-step roughening and smoothening process, involving sandblasting and a subsequent chemical polishing step. In order to produce a set of identical surfaces we applied a replica technique. Cell experiments were carried out with rat calvarial osteoblasts (RCO) and human gingival fibroblasts (HGF). RCOs showed a significantly increased proliferation rate with increasing surface roughness. The footprint of osteoblasts varied in size at different positions on the gradient, remaining small on the rough end of the gradient and increasing considerably as the roughness decreased. HGF showed the opposite proliferation behavior, proliferation decreasing with increasing roughness. The fibroblast morphology was found to be similar to that seen for osteoblasts.
Keywords: Osteoblast; Fibroblast; Cell proliferation; Cell morphology; Surface roughness; Surface modification;

We report on a culture method for the rapid production of a strong and thick natural matrix by human cells for tissue engineering applications. Dermal fibroblasts were cultured for three weeks at high density on porous substrates in serum-containing or chemically defined media. The mechanical and biochemical properties of the resulting cell-derived matrix (CDM) were compared to those of standard fibroblast-populated collagen and fibrin gels and native human skin. We found that the ultimate tensile strength of CDM cultured in our chemically defined media (313±8.7 kPa) is significantly greater than for collagen gels (168±39.3 kPa), fibrin gels (133±8.0 kPa) and CDM cultured with serum (223±9.0 kPa), but less than native skin (713±55.2 kPa). In addition to the biomechanics, this *CDM is also biochemically more similar to native skin than the collagen and fibrin gels in terms of all parameters measured. As *CDM is produced by human cells in a chemically defined culture medium and is mechanically robust, it may be a viable living tissue equivalent for many connective tissue replacement applications requiring initial mechanical stability yet a high degree of biocompatibility.
Keywords: Extracellular matrix (ECM); Soft tissue biomechanics; Mechanical properties; Connective tissue; Wound healing;

Spatial cues for the enhancement of retinal pigment epithelial cell function in potential transplants by Christina J. Lee; Harvey A. Fishman; Stacey F. Bent (2192-2201).
Retinal pigment epithelial (RPE) cellular morphology and function are vital to the health of the retina. In age-related macular degeneration, RPE dysfunction and changes in Bruch's membrane occur. Thus, a potential cure is a dual-layer biomimetic transplant consisting of a layer of healthy RPE cells cultured on a support membrane. In this study, we investigated human anterior lens capsule as a replacement for Bruch's membrane and also explored different seeding methods as ways of inducing the desired cellular morphology and function. Using in vitro assays, we demonstrated that RPE cells cultured on lens capsule exhibited epithelial characteristics, such as the presence of actin belts and the formation of tight junctions in the monolayer. Bovine photoreceptor outer segments were also incubated with the RPE cells in order to quantify the binding and ingestion activity of the RPE cells. With these assays, we determined that cells seeded by centrifugation appeared to possess the most epithelial-like morphology, with the shortest overall length and the smallest elongation. They also exhibited enhanced metabolic activity, with a 1.5-fold increase over conventional gravity seeding. Thus, the spatial cues provided by centrifugation may assist cells in assuming native RPE function. Therefore, a dual-layer transplant, with RPE cells organized by centrifugation onto lens capsule, appears promising in achieving native retinal function.
Keywords: Cell morphology; Epithelial cell; Membrane; Micropatterning; Ophthalmology; Retina;

This paper analyzes the alignment and differentiation of myoblast cells adherent to surfaces having model chemistries and microtopographical patterns. The patterns strongly influenced cellular alignment but did not modulate expression of differentiation marker proteins in either primary or C2C12 myoblasts. Topographic patterns consisted of embossed ridges and grooves or arrays of holes, with feature sizes ranging from 5–75 μm. The topographic surfaces were prepared with a uniform self-assembled monolayer that presented CH3 molecules for fibronectin adsorption. The myoblast cell models were cultured in differentiation conditions on the substrates. For both cell models, cells aligned to grooves, with groove width modulating orientation, and preferentially orientated parallel to rows of holes. None of the patterns significantly modulated cell density or differentiation as examined through sarcomeric myosin and acetylcholine receptor expression. The results indicate that for the specific configuration examined, microscale topography modulates myoblast alignment, but does not have significant impact on cell density or differentiation.
Keywords: Surface topography; Surface chemistry; Differentiation; Myoblast; Hot embossing; Sarcomeric myosin;

Electrospun aliphatic polycarbonates as tailored tissue scaffold materials by Alexander Welle; Mario Kröger; Manfred Döring; Kerstin Niederer; Elvira Pindel; Ioannis S. Chronakis (2211-2219).
Two different aliphatic polycarbonates were synthesised from CO2 and the respective epoxides. Poly(propyl carbonate) (PPC) was prepared by heterogeneous catalysis with zinc glutarate. Poly(cyclohexyl carbonate) (PCHC) was prepared via living copolymerisation homogeneously catalysed by a 3-amino-2-cyanoimidoacrylate zinc acetate complex and subjected to electrospinning. The obtained nanofibres had a well-defined morphology free of beads along the fibres and with slightly porous structures on their surface. Subsequently, low-power deep UV irradiations, previously applied for photochemical surface modifications of two-dimensional and three-dimensional scaffolds from biostable polymers, were performed. Here, an effect on surface and bulk properties of PPC nanofibres was observed. Surface modifications of both polymers affected plasma protein adsorption. Photochemical bulk modifications observed for the first time on PPC nanofibres are indicating the possibility of spatial control of biodegradation rates, hence allow for control of the progression of host/implant interactions in vivo. In particular PPC was used for cell culture of L929 fibroblasts and primary rat hepatocytes. Even delicate primary cells showed good adhesion to the scaffolds and high viability.
Keywords: Aliphatic polycarbonate; Electrospinning; Biodegradable nanofibres; Non-woven fabric; Deep UV irradiation;

A new thermosensitive hydrogel was designed and prepared by simply mixing N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) and poly(ethylene glycol) (PEG) with a small amount of α-β-glycerophosphate (α-β-GP). The optimum preparative condition was investigated, and the obtained formulation underwent thermal transition from solution below or at room temperature to non-flowing hydrogel around 37 °C in several minutes. As a new formulation, its potential use as nasal drug delivery system was studied. It can be dropped or sprayed easily into nasal cavity and spread on the nasal mucosa in solution state. After being administered into nasal cavity, the solution transformed into viscous hydrogel at body temperature, which decreased nasal mucociliary clearance rate and released drug slowly. Morever, quaternized chitosan as absorption enhancer has been studied extensively in several reports and proved its non-toxicity, mucoadhesivity and the capacity to open the tight junctions between epithelial cells. Therefore, in this study insulin as a model drug was entrapped in this formulation and its release behavior in vitro was also investigated. The enhancement of absorption of fluorescein isothiocyanate (FITC)-labeled insulin in rat nasal cavity by this formulation was proved by confocal laser scanning microscopy (CLSM). The cytoxicity and the change of the blood glucose concentration after nasal administration of this hydrogel were also investigated. The hydrogel formulation decreased the blood glucose concentration apparently (40–50% of initial blood glucose concentration) for at least 4–5 h after administration, and no apparent cytoxicity was found after application. These results showed that HTCC–PEG–GP formulation can be used as nasal drug delivery system to improve the absorption of hydrophilic macromolecular drugs.
Keywords: N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride; Poly(ethylene glycol); Hydrogel; Nasal drug delivery system; Absorption enhancer;

Mucoadhesion mechanism of chitosan and thiolated chitosan-poly(isobutyl cyanoacrylate) core-shell nanoparticles by Irene Bravo-Osuna; Christine Vauthier; Alessandra Farabollini; Giovanni Filippo Palmieri; Gilles Ponchel (2233-2243).
The study is focused on the evaluation of the potential bioadhesive behaviour of chitosan and thiolated chitosan (chitosan-TBA)-coated poly(isobutyl cyanoacrylates) (PIBCA) nanoparticles. Nanoparticles were obtained by radical emulsion polymerisation with chitosan of different molecular weight and with different proportions of chitosan/chitosan-TBA. Mucoadhesion was ex vivo evaluated under static conditions by applying nanoparticle suspensions on rat intestinal mucosal surfaces and evaluating the amount of nanoparticles remaining attached to the mucosa after incubation. The analysis of the results obtained demonstrated that the presence of either chitosan or thiolated chitosan on the PIBCA nanoparticle surface clearly enhanced the mucoadhesion behaviour thanks to non-covalent interactions (ionic interaction and hydrogen bonds) with mucus chains. Both, the molecular weight of chitosan and the proportion of chitosan-TBA in the formulation influenced the nanoparticle hydrodynamic diameter and hence their transport through the mucus layer. Improved interpenetration ability with the mucus chain during the attachment process was suggested for the chitosan of high molecular weight, enhancing the bioadhesiveness of the system. The presence of thiol groups on the nanoparticle surface at high concentration (200×10−6  μmol SH/cm2) increased the mucoadhesion capacity of nanoparticles by forming covalent bonds with the cysteine residues of the mucus glycoproteins.
Keywords: Chitosan; Thiolated chitosan; Poly(isobutyl cyanoacrylate); Mucoadhesion; Core-shell nanoparticles;

Retroviral microarray-based platform on nanostructured TiO2 for functional genomics and drug discovery by Roberta Carbone; Luca Giorgetti; Andrea Zanardi; Ida Marangi; Elisabetta Chierici; Gero Bongiorno; Francesca Fiorentini; Mario Faretta; Paolo Piseri; Pier Giuseppe Pelicci; Paolo Milani (2244-2253).
Living-cell microarrays are powerful tools for functional genomics and drug discovery. However, despite several attempts to improve this technology, it is still a challenge to obtain microarrays of cells efficiently overexpressing or downregulating specific genes to address complex phenotypes. Here, we present a cell-based microarray for phenotype screening on primary and cancer cells based on the localized reverse infection by retroviruses. Viral vectors are immobilized on a nanostructured titanium dioxide (ns-TiO2) film obtained by depositing a supersonic beam of titania clusters on a glass substrate. We validated the retroviral cell array by overexpression of GFP reporter genes in primary and cancer cells, and by RNA interference of p53 in primary cells by analyzing effects in cell growth. We demonstrate that ns-TiO2 retroviral arrays are an enabling tool for the study of gene function of families of genes for complex phenotypes and for the identification of novel drug targets.
Keywords: Nanostructured titanium oxide; Cell-based microarrays;