Biomaterials (v.27, #22)

Calendar (I).

Influence of surface pretreatment of titanium- and cobalt-based biomaterials on covalent immobilization of fibrillar collagen by Rainer Müller; Jochen Abke; Edith Schnell; Dieter Scharnweber; Richard Kujat; Carsten Englert; Darius Taheri; Michael Nerlich; Peter Angele (4059-4068).
Collagen type-I is a major component of the extracellular matrix of most tissues and it is increasingly utilized for surface engineering of biomaterials to accelerate receptor-mediated cell adhesion. In the present study, coatings with layers of fibrillar type-I collagen were prepared on titanium, titanium alloy, and cobalt alloy to improve initial osteoblast adhesion and implant–tissue integration. To suppress the quick in vivo degradation rate of collagen the deposited layers were covalently immobilized at the metal surfaces as well as chemically cross-linked. The application of different oxidation techniques to the metallic substrates resulted in surfaces with varying hydroxyl group contents, which directly influenced the amount of immobilized silane coupling agents. It was found that a high density of surface-bound coupling agents increased the stability of the covalently linked collagen layers. After coating of metallic biomaterials with a cross-linked collagen layer, an improved cellular response of human osteoblast-like cells (MG-63) in vitro could be recognized.
Keywords: Surface modification; Titanium alloys; Cobalt alloys; Collagen; Coupling agents; Cross-linking;

The differentiation of bone marrow mesenchymal stem cells into chondrocyte-like cells on poly-l-lactic acid (PLLA) scaffolds by Stephen M. Richardson; Judith M. Curran; Rui Chen; Anne Vaughan-Thomas; John A. Hunt; Anthony J. Freemont; Judith Alison Hoyland (4069-4078).
While intervertebral disc (IVD) degeneration is associated with the majority of cases of low back pain, current treatments are symptomatic rather than curative. Tissue engineering offers a treatment that both cures the problem of disc degeneration and restores normal disc function. One of the major problems for any tissue engineering strategy, however, is ensuring that both the cells and matrices used are suitable for the target tissue. In this study, we have developed and studied a potential system for tissue engineering of the nucleus pulposus (NP) of the severely degenerate IVD. While cells from degenerate discs are not suitable for tissue engineering, bone-marrow-derived mesenchymal stem cells, which are capable of differentiating into chondrocyte-like cells such as those found within the NP of the disc, offer a potential source of cells. We have used transfection with adenoviral SOX-9, a transcription factor involved in differentiation of MSCs along the chondrogenic lineage, combined with culture in a specialised medium, to differentiate monolayer MSCs to NP-like (chondrocyte-like) cells, as shown by real-time quantitative polymerase chain reaction for NP-marker genes. We have also replicated these findings on porous, biodegradable three-dimensional (3D) poly-l-lactic acid scaffolds and shown expression and deposition of NP matrix markers such as type II collagen and aggrecan. We are therefore proposing predifferentiation of human MSCs and seeding on porous, biodegradable 3D synthetic polymer scaffolds as a realistic tissue engineering strategy for regeneration of the degenerate human IVD.
Keywords: Intervertebral disc; Mesenchymal stem cell; Polylactic acid; Confocal microscopy; PCR; TGF;

Osteogenic differentiation of mesenchymal stem cells in self-assembled peptide-amphiphile nanofibers by Hossein Hosseinkhani; Mohsen Hosseinkhani; Furong Tian; Hisatoshi Kobayashi; Yasuhiko Tabata (4079-4086).
The proliferation and differentiation of mesenchymal stem cells (MSC) was investigated in a three dimensional (3-D) network of nanofibers formed by self-assembly of peptide-amphiphile (PA) molecules. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. The sequence of arginine-glycine-aspartic acid (RGD) was included in peptide design as well. A 3-D network of nonofibers was formed by mixing cell suspensions in media with dilute aqueous solution of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. When rat MSC were seeded into the PA nanofibers with or without RGD, larger number of cells attached was observed in the PA nanofibers including RGD. When measured to evaluate the osteogenic differentiation of MSC, the alkaline phosphatase (ALP) activity and osteocalcin content became maximum for the PA nanofibers including RGD compared with those without RGD, although both the values were significantly higher compared with those in the static tissue culture plate (2-D culture). We concluded that the attachment, proliferation, and osteogenic differentiation of MSC were influenced by PA nanofibers as the cell scaffold.
Keywords: Scaffold; Mesenchymal stem cells; Peptide amphiphile; Nanofibers; Osteogenic differentiation;

Extensive cell–cell or cell–matrix interaction in three-dimensional (3D) culture is important for the maintenance of adult hepatocyte function and the maturation of hepatic progenitors. However, although there is significant interest in inducing the transdifferentiation of adult stem cells into the hepatic lineage, very few studies have been conducted in a 3D culture configuration. The aim of this study is to investigate the differentiation of mesenchymal stem cells (MSC) into hepatocytes in a pellet configuration, with or without the presence of small intestinal submucosa (SIS). After 4 weeks of differentiation with growth factors bFGF, HGF, and OsM, we obtained hepatocyte-like cells that expressed a subset of hepatic genes, secreted albumin and urea, stored glycogen, and showed inducible CYP3A4 mRNA levels. When these cells were implanted into livers of hepatectomized rats, they secreted human albumin into the bloodstream. The hepatic differentiation of MSC was faster in cell pellets without SIS. The plausible explanations for this finding may be related to the mass transport issues of the two different pellets and the role of cell–cell contact over cell–matrix interactions. The findings of this study should help in the design of optimal culture configurations for efficient hepatic differentiation of adult stem cells.
Keywords: Stem cell; Hepatocyte; Cell adhesion; ECM;

Directed growth and selective differentiation of neural progenitor cells on micropatterned polymer substrates by Jennifer B. Recknor; Donald S. Sakaguchi; Surya K. Mallapragada (4098-4108).
Directional growth and differentiation of adult rat hippocampal progenitor cells (AHPCs) were investigated on micropatterned polymer substrates in vitro. Astrocytes or AHPCs cultured on micropatterned polystyrene substrates chemically modified with laminin exhibited over 75% alignment in the groove direction. AHPCs co-cultured with astrocytes preferentially acquired neuronal morphology, with nearly double the percentage of cells expressing class III β-tubulin on the micropatterned half of the substrate, as opposed to the planar half of the substrate, or compared to those growing in the absence of astrocytes. This indicates that substrate three-dimensional topography, in synergy with chemical (laminin) and biological (astrocytes) guidance cues, facilitates neuronal differentiation of the AHPCs. Through multi-dimensional cell–cell interactions, this environment provides spatial control selectively enhancing neuronal differentiation and neurite alignment on topographically different regions of the same substrate. Integrating these cues is important in understanding and controlling neural stem cell differentiation and designing scaffolds for guided nerve regeneration.
Keywords: Neural stem/progenitor cell; Astrocyte; Co-culture; Micropatterning; Nerve tissue engineering;

Guidance of liver and kidney organotypic cultures inside rectangular silicone microchannels by Eric Leclerc; Anne Corlu; Laurent Griscom; Regis Baudoin; Cécile Legallais (4109-4119).
We have studied the effect of rectangular polydimethylsiloxane (PDMS) microchannels on the behavior of embryonic liver and kidney explants maintained in contact with these microchannels. The microchannel widths were varied from 35 to 300 μm and depth from 45 to 135 μm. The growth of these tissue types were compared to the development on flat silicone and plastic control material. At seeding, due to the viscoelastic properties of both organs, “capillary-like filling” was observed inside the narrowest microchannels. In those cases, the tissues grew to a confluent layer joining the microchannels with no cell migration and proliferation inside the microchannels. In the largest microchannels, only a weak migration was observed and the cellular behavior appears quite similar to that of PDMS flat culture conditions. In intermediate geometries, we observed different tissue growth properties inside the microchannels when compared to other sizes. The liver tissues progressed inside those microchannels with an average growth velocity of up to 72 μm/dayresulting to form a dense three-dimensional multicellular ‘liver-like tissue’. Scanning electron microscopy (SEM) observations demonstrated that the tissue was organized like an epithelial layer with round cells embedded in an extracellular matrix. Liver cell mobility may result primarily from the activity of the marginal cells, whereas the submarginal cells appeared passively dragged. Parenchymal organization demonstrating differentiated states was also observed. Kidney grew mainly on the microchannel walls and the tissues never appeared dense and organized as the liver ones.
Keywords: Rectangular microchannels; PDMS; Organotypic cultures; Liver tissue engineering;

Repair of osteochondral defects with biphasic cartilage-calcium polyphosphate constructs in a Sheep model by R.A. Kandel; M. Grynpas; R. Pilliar; J. Lee; J. Wang; S. Waldman; P. Zalzal; M. Hurtig (4120-4131).
There has been interest in developing novel biological treatments to repair focal cartilage defects. We have developed a method of forming biphasic constructs (“osteochondral”-type plug) in vitro consisting of cartilaginous tissue, formed on and anchored to the intended articulation surface of a porous ceramic substrate. The purpose of this study was to evaluate the biochemical and biomechanical properties and morphology of in vitro-formed biphasic constructs 3 and 9 months after implantation into 4 mm diameter full thickness osteochondral defects in the trochlear groove of sheep stifles. The implants withstood loading in vivo up to 9 months with evidence of fusion to adjacent native cartilage and fixation by bone ingrowth into the ceramic substrate. The cartilage layer was eroded from those implants that were proud to the joint surface. Control implants (ceramic only) had fibrous tissue on the articulating surface after implantation for 3–4 months. Neither the cellularity nor proteoglycan content of the implanted cartilage, when it remained, changed significantly between 3 and 9 months and the collagen content increased slightly. The elastic equilibrium modulus of the cartilage improved with time with the greatest improvement (10-fold) occurring early during the first 3–4 months after implantation. This study suggests that biphasic constructs may be suitable to repair joint defects as the implants were maintained up to 9 months in sheep. Importantly the mechanical properties of the implanted cartilage improved significantly after implantation suggesting that cartilage can mature in vivo after implantation. The results indicate that further study of this treatment approach is warranted to attempt to overcome the technical surgical difficulties identified in this study.
Keywords: Articular cartilage; Transplantation; Tisssue engineering;

A materials design of a new supramolecular hydrogel self-assembled between α-cyclodextrin and a biodegradable poly(ethylene oxide)–poly[( r )-3-hydroxybutyrate]–poly(ethylene oxide) (PEO–PHB–PEO) triblock copolymer was demonstrated. The cooperation effect of complexation of PEO segments with α-cyclodextrin and the hydrophobic interaction between PHB blocks resulted in the formation of the supramolecular hydrogel with a strong macromolecular network. The in vitro release kinetics studies of fluorescein isothiocyanate labeled dextran (dextran-FITC) model drug from the hydrogel showed that the hydrogel was suitable for relatively long-term sustained controlled release of macromolecular drugs, which many simple triblock copolymer hydrogel systems could not achieve. The hydrogel was found to be thixotropic and reversible, and can be applied as a promising injectable drug delivery system.
Keywords: Self-assembly; Supramolecule; Cyclodextrin; Triblock copolymer; Injectable hydrogel; Drug delivery;

Degradation behavior of dextran hydrogels composed of positively and negatively charged microspheres by Sophie R. Van Tomme; Cornelus F. van Nostrum; Stefaan C. de Smedt; Wim E. Hennink (4141-4148).
This paper reports on the degradation behavior of in situ gelling hydrogel matrices composed of positively and negatively charged dextran microspheres. Rheological analysis showed that, once the individual microspheres started to degrade, the hydrogel changed from a mainly elastic to a viscoelastic network. It was shown with gels composed of equal amounts of cationic and anionic microspheres, that both a higher crosslink density of the particles and a decrease in water content of the hydrogels resulted in a slower degradation, ranging from 65 to 140 days. Dispersions containing cationic, neutral or anionic microspheres completely degraded within 30, 55 or 120 days, respectively. The microspheres were loaded with rhodamine-B-dextran and degradation was studied with confocal microscopy and fluorescence spectroscopy. After a lag time of 3 days rhodamine-B-dextran started to release from the positive microspheres with a 50% release after 16 days. In contrast, release of rhodamine-B-dextran from the negative microspheres started after 10 days with a 50% release after 36 days.The faster degradation of the positively charged microspheres as compared to the negatively charged microspheres is attributed to stabilization of the transition state in the hydrolysis process by the protonated tertiary amine groups present in the cationic microspheres. On the other hand, the presence of negatively charged groups causes repulsion of hydroxyl anions resulting in a slower degradation. Combining the oppositely charged microspheres in different ratios makes it possible to tailor the network properties and the degradation behavior of these hydrogels, making them suitable for various applications in drug delivery and tissue engineering.
Keywords: Dextran microspheres; Self-gelling; Injectable hydrogel; Degradation behavior; Release;

Control of drug accessibility on functional polyelectrolyte multilayer films by Constant Vodouhê; Erell Le Guen; Juan Mendez Garza; Gregory Francius; Christophe Déjugnat; Joëlle Ogier; Pierre Schaaf; Jean-Claude Voegel; Philippe Lavalle (4149-4156).
A surface coating based on polylysine/hyaluronic acid multilayers was designed and acted as a reservoir for an antiproliferative agent, paclitaxel (Taxol). Absolutely no chemical modification of polyelectrolytes or of the drug was needed and the final architecture was obtained in an extremely simple way using the layer-by-layer method. The paclitaxel dose available for human colonic adenocarcinoma cells HT29 seeded on the films could be finely tuned. Moreover, the accessibility of the drugs was controlled by adding on the top of the drug reservoir a capping made of synthetic polyelectrolyte multilayers. This capping was also required to allow adhesion of HT29 cells. Paclitaxel activity was maintained after embedding in the polyelectrolyte multilayers and cellular viability could be reduced by about 80% 96 h after seeding. The strategy described in this paper could be valuable for various other drug/cell systems.
Keywords: Paclitaxel; HT29 cells; Polyelectrolyte multilayers; Bioactive films;

Use of chitosan bandage to prevent fatal infections developing from highly contaminated wounds in mice by Marina Burkatovskaya; George P Tegos; Emilia Swietlik; Tatiana N Demidova; Ana P Castano; Michael R. Hamblin (4157-4164).
HemCon® bandage is an engineered chitosan acetate preparation used as a hemostatic control dressing, and its chemical structure suggests that it should also be antimicrobial. We tested its ability to rapidly kill bacteria in vitro and in mouse models of infected wounds. We used the Gram-negative species Pseudomonas aeruginosa and Proteus mirabilis and the Gram-positive Staphylococcus aureus that had all been stably transduced with the entire bacterial lux operon to allow in vivo bioluminescence imaging. An excisional wound in Balb/c mice was inoculated with 50–250 million cells followed after 30 min by application of HemCon bandage, alginate sponge bandage, silver sulfadiazine cream or no treatment. HemCon was more adhesive to the wound and conformed well to the injury compared to alginate. Animal survival was followed over 15 days with observations of bioluminescence emission and animal activity daily. Chitosan acetate treated mice infected with P. aeruginosa and P. mirabilis all survived while those receiving no treatment, alginate and silver sulfadiazine demonstrated 25–100% mortality. Chitosan acetate was much more effective than other treatments in rapidly reducing bioluminescence in the wound consistent with its rapid bactericidal activity in vitro as well as its light-scattering properties. S. aureus formed only non-lethal localized infections after temporary immunosuppression of the mice but HemCon was again more effective in reducing bioluminescence. The data suggest that chitosan acetate rapidly kills bacteria in the wound before systemic invasion can take place, and is superior to alginate bandage and silver sulfadiazine that may both encourage bacterial growth in the short term.
Keywords: Chitin/chitosan; Alginate; Infection; Wound dressing; Antimicrobial; Haemostasis;

Molecularly imprinted polymers for tobacco mosaic virus recognition by Linden D. Bolisay; James N. Culver; Peter Kofinas (4165-4168).
Molecular imprinted Polymers (MIP) targeted for Tobacco mosaic virus (TMV) have been synthesized. Batch equilibrium studies using imprinted and non-imprinted polymer hydrogels in TMV and TNV solutions were conducted to determine virus-binding capacities. TMV-imprinted hydrogels showed increased binding to TMV (8.8 mg TMV/g polymer) compared to non-imprinted hydrogels (4.2 mg TMV/g polymer). Furthermore, TMV-imprinted hydrogels exhibited increased binding to TMV compared to TNV, while non-imprinted hydrogels bound similar amounts of TMV or TNV. This research has demonstrated that molecular imprinting of viruses can be used to selectively induce binding of target viruses based on shape differences of their virions.
Keywords: Hydrogel; Molecular imprinting; Virus;

Nanofibrous poly(acrylonitrile-co-maleic acid) membranes functionalized with gelatin and chitosan for lipase immobilization by Peng Ye; Zhi-Kang Xu; Jian Wu; Christophe Innocent; Patrick Seta (4169-4176).
Nanofibrous membranes with an average diameter of 100 and 180 nm were fabricated from poly(acrylonitrile-co-maleic acid) (PANCMA) by the electrospinning process. These nanofibrous membranes contain reactive groups which can be used to covalently immobilize biomacromolecules. Two natural macromolecules, chitosan and gelatin, were tethered on these nanofibrous membranes to fabricate dual-layer biomimetic supports for enzyme immobilization in the presence of 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxyl succinimide (NHS). Lipase from Candida rugosa was then immobilized on these dual-layer biomimetic supports using glutaraldehyde (GA), and on the nascent PANCMA fibrous membrane using EDC/NHS as coupling agent, respectively. The properties of the immobilized lipases were assayed. It was found that there is an increase of the activity retention of the immobilized lipase on the chitosan-modified nanofibrous membrane (45.6±1.8%) and on the gelatin-modified one (49.7±1.8%), compared to that on the nascent one (37.6±1.8%). The kinetic parameters of the free and immobilized lipases, K m and V max, were also assayed. In comparison with the immobilized lipase on the nascent nanofibrous membrane, there is an increase of the V max value for the immobilized lipases on the chitosan- and gelatin-modified nanofibrous membranes. Results also indicate that the pH and thermal stabilities of lipases increase upon immobilization. The residual activities of the immobilized lipases are 55% on the chitosan-modified nanofibrous membrane and 60% on the gelatin-modified one, after 10 uses.
Keywords: Electrospinning; Nanofibrous membranes; Biomacromolecules; Enzyme immobilization; Lipase;

Detection of a specific DNA sequence by electrophoresis through a molecularly imprinted polymer by Masayo Ogiso; Norihiko Minoura; Toshio Shinbo; Toshimi Shimizu (4177-4182).
To develop a simple and inexpensive DNA detection method, we prepared a molecularly imprinted polymer (MIP) gel for recognizing a specific double-stranded DNA (dsDNA) target sequence in MIP gel electrophoresis (MIPGE). During MIPGE, migration of the target sequence of dsDNA should be hindered by the capture effect of the binding sites in the MIP gel. This migration hindrance of target dsDNA was determined by plotting the relationship between the migration distance in the MIP gel and that in polyacrylamide gel, commonly used in gel electrophoresis. Using this plot, detection of a target dsDNA from a mixture of different-sized dsDNA fragments was achieved. Moreover, we found the detection method successfully distinguished between a target and its base-pair substitutes. These results suggest that MIPGE could be employed for detection of a target dsDNA sequence.
Keywords: Biomimetic material; Biosensor; DNA; Hydrogel; Polymerization;