Biomaterials (v.27, #12)

Calendar (I).

Photolithographic patterning of polyethylene glycol hydrogels by Mariah S. Hahn; Lakeshia J. Taite; James J. Moon; Maude C. Rowland; Katie A. Ruffino; Jennifer L. West (2519-2524).
A simple, inexpensive photolithographic method for surface patterning deformable, solvated substrates is demonstrated using photoactive poly(ethylene glycol) (PEG)-diacrylate hydrogels as model substrates. Photolithographic masks were prepared by printing the desired patterns onto transparencies using a laser jet printer. Precursor solutions containing monoacryloyl-PEG-peptide and photoinitiator were layered onto hydrogel surfaces. The acrylated moieties in the precursor solution were then conjugated in monolayers to specific hydrogel regions by exposure to UV light through the transparency mask. The effects of UV irradiation time and precursor solution concentration on the levels of immobilized peptide were characterized, demonstrating that bound peptide concentration can be controlled by tuning these parameters. Multiple peptides can be immobilized to a single hydrogel surface in distinct patterns by sequential application of this technique, opening up its potential use in co-cultures. In addition, 3D structures can be generated by incorporating PEG-diacrylate into the precursor solution. To evaluate the feasibility of using these patterned surfaces for guiding cell behavior, human dermal fibroblast adhesion on hydrogel surfaces patterned with acryloyl–PEG–RGDS was investigated. This patterning method may find use in tissue engineering, the elucidation of fundamental structure–function relationships, and the formation of immobilized cell and protein arrays for biotechnology.
Keywords: Photolithography; Biomimetic; Patterning; Polyethylene glycol;

Heparin incorporating liposomes as a delivery system of heparin from PET-covered metallic stents: Effect on haemocompatibility by Georgia Koromila; George P.A. Michanetzis; Yannis F. Missirlis; Sophia G. Antimisiaris (2525-2533).
We investigate the possibility of coating polymer-covered stents with heparin-encapsulating liposomes for improving their haemocompatibility. Thin-film hydration (for multilamellar vesicles, MLV), and the dehydration–rehydration vesicle (DRV) methods are used for preparation of low-molecular weight heparin (LMWH)-encapsulating liposomes with varying lipid compositions. Liposomes are characterized for LMWH encapsulation and retention. For measurement of LMWH, a chromogenic technique is adjusted. For evaluation of heparin release from vesicles in platelet poor plasma (PPP) coagulation time is measured in presence of liposomal samples. Results reveal that LMWH encapsulation in liposomes is higher in DRV, however compositions with high encapsulation are leaky during buffer incubation. Most liposomes release LMWH slowly during plasma incubation (retention after 24 h ranges between 74% and 95%). Concerning the haemocompatibility of polyethylene terephthlate-covered stents after coating with LMWH-encapsulating liposomes, there is a marked increase (higher for DRV-coated stents compared to MLV) in plasma recalcification time compared to the control (plain blood) and reference (non-coated stent), which increases with blood–material contact time. This is probably due to LMWH release, demonstrating that encapsulated LMWH retains its biological functionality. Interestingly, the DRV-coated stents retained a high plasma recalcification time and a large number of liposomes on the stents (as proven by SEM studies) even after extensive washing (high shear conditions), proving that this method may be functional under high flow applying in vivo conditions.
Keywords: Heparin; Liposome; Haemocompatibility; Blood; Stability; Endoprosthesis;

Pattern stability under cell culture conditions—A comparative study of patterning methods based on PLL-g-PEG background passivation by Jost W. Lussi; Didier Falconnet; Jeffrey A. Hubbell; Marcus Textor; Gabor Csucs (2534-2541).
Despite the rapidly increasing number of publications on the fabrication and use of micro-patterns for cell studies, comparatively little is know about the long-term stability of such patterns under cell culture conditions. Here, we report on the long-term stability of cellular patterns created by three different patterning techniques: selective molecular assembly patterning, micro-contact printing and molecular assembly patterning by lift-off. We demonstrate that although all three techniques were combined with the same background passivation chemistry based on assembly of a PEG-graft copolymer, there are considerable differences in the long-term stability between the three different pattern types under cell culture conditions. Our results suggest that these differences are not cell-dependent but are due to different (substrate-dependent) interactions between the patterned substrate, the passivating molecule and the serum containing cellular medium.
Keywords: Selective molecular assembly patterning; SMAP; Microcontact printing; Molecular assembly patterning by lift-off; MAPL; Pattern degradation;

Stem cell-coated titanium implants for the partial joint resurfacing of the knee by Karl-Heinz Frosch; Anja Drengk; Petra Krause; Volker Viereck; Nicolai Miosge; Carola Werner; Detlev Schild; Ewa K. Stürmer; Klaus M. Stürmer (2542-2549).
The goal of the present study was to evaluate the partial surface replacement of the knee with stem cell-coated titanium implants and to provide a basis for a successful treatment of large osteochondral defects.Mesenchymal stem cells (MSCs) were isolated from bone marrow aspirates of adult sheep. Round titanium implants with a diameter of 2×7.3 mm were seeded with autologous MSC and inserted into an osteochondral defect in the medial femoral condyle. As controls, defects received either an uncoated implant or were left untreated. Nine animals with 18 defects were sacrificed after 6 months. Histological evaluation was performed by intravital polychrome fluorescent labelling, intravital perfusion with Indian ink, microradiographs and differential staining with toluidine blue. The quality of regenerated cartilage was assessed by in situ hybridization of collagen type II and immunohistochemistry of collagen types I and II.In 50% of the cases, defects treated with MSC-coated implants showed a complete regeneration of the subchondral bone layer. In these cases collagen type II and only traces of collagen type I were detected. A high level of collagen type II mRNA expression compared to articular cartilage indicates regenerating hyaline-like cartilage. A total of 50% of MSC-coated and uncoated implants failed to osseointegrate and formation of fibrocartilage was observed. Untreated defects as well as defects treated with uncoated implants demonstrated incomplete healing of subchondral bone and formation of fibrous cartilage. A modified histological score according to Wakitani significantly demonstrated better results for cell-coated implants (8.8±6.4) than for uncoated implants (5.5±3.9) and for untreated defects (2.8±2.5).Our results demonstrate that, in a significant number of cases, a partial joint resurfacing of the knee with stem cell-coated titanium implants occur. A slow bone and cartilage regeneration and an incomplete healing in half of the MSC-coated implants are limitations of the presented method. To improve our approach and optimize the experimental parameters, further investigations are needed prior to clinical application.
Keywords: Tissue engineering; Osteochondral defects; Stem cells; Titanium implants;

Fabrication of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) microstructures using soft lithography for scaffold applications by Zheyao Wang; Huan Hu; Yu Wang; Yawu Wang; Qiong Wu; Litian Liu; Guoqiang Chen (2550-2557).
This paper reports two soft lithographic methods, micromolding and hot embossing, to produce biodegradable poly (3-hydroxybutyrate-co-3-ftydroxyhexanoate) (PHBHHx) arrays of microstructures for hosting and culturing cells in a local microenvironment by controlled shape. Silicon masters with high-aspect-ratio microfeatures were fabricated using KOH and DRIE anisotropic etching. These silicon masters were used as molds to construct PHBHHx microstructures using micromolding and hot embossing. Using silicon rather than conventional PDMS as molds allowed microstructures with feature size of 20 μm and height of 100 μm to be realized. PHBHHx microstructures with different configurations including circles, rectangles, and octagons were fabricated to investigate the effects of topography on cell culture. Mouse fibroblast cell lines L929 were cultured on PHBHHx microstructures in vitro to investigate the biocompatibility. This study demonstrates the feasibility of microfabrication of PHBHHx structures with micro-scale feature size using soft lithography, and the results show that PHBHHx microstructures can be created to mimic cellular microenvironment for cell culture, providing a convenient means to investigate relationships of microstructures and cell functions.
Keywords: Cell culture; PHBHHx; Scaffold; Soft lithography;

Microfabrication of poly (glycerol–sebacate) for contact guidance applications by Christopher J. Bettinger; Brian Orrick; Asish Misra; Robert Langer; Jeffrey T. Borenstein (2558-2565).
Controlling cell orientation and morphology through topographical patterning is a phenomenon that is applicable to a wide variety of medical applications such as implants and tissue engineering scaffolds. Previous work in this field, termed contact guidance, has demonstrated the application of this cellular response on a wide variety of material substrates such as silicon, quartz, glass, and poly(di-methyl siloxane) typically using ridge–groove geometries with sharp feature edges. One limitation of these studies in terms of biomedical applications is the choice of material. Therefore, demonstrating contact guidance and topography in a biodegradable material platform is a promising strategy for controlling cellular arrangements in tissue engineering scaffolds. This study investigates several strategies to advance contact guidance strategies and technology to more practical applications. Flexible biodegradable substrates with rounded features were fabricated by replica-molding poly(glycerol–sebacate) on sucrose-coated microfabricated silicon. Bovine aortic endothelial cells were cultured on substrates with microstructures between 2 and 5 μm in wavelength and with constant feature depth of 0.45 μm. Cells cultured on substrates with smaller pitches exhibited a substantially higher frequency of cell alignment and smaller circularity index. This work documents the first known use of using a flexible, biodegradable substrate with rounded features for use in contact guidance applications. The replica-molding technique described here is a general process that can be used to fabricate topographically patterned substrates with rounded features for many biomaterials. Furthermore, these results may lead to further elucidation of the mechanism of cell alignment and contact guidance on microfabricated substrates.
Keywords: Microstructure; Biodegradation; Biocompatibility; Nanotopography;

A simple and effective method of biomacromolecule immobilization on biomaterial surface for direct tuning of biophysical parameters such as the initial cell deformation rate, degree of cell spreading and adhesion kinetics is important for tissue engineering. The photochemical immobilization of azide-chitosan (Az-CS) on poly (lactide-co-glycolide) acid (PLGA) is applied here. Chitosan immobilization on PLGA through the photoactive azide group further facilitates subsequent grafting of other biocompatible biomacromolecules like gelatin (Gel) through the active amine groups on CS. This study quantitatively compares the 3T3 fibroblast adhesion dynamics on three PLGA surfaces (Gel-CS-PLGA, CS-PLGA and unmodified PLGA surfaces) using Confocal-Reflectance Interference Contrast Microscopy (C-RICM) together with phase contrast imaging. CS-PLGA and Gel-CS-PLGA surfaces developed were confirmed by X-ray photoelectron spectroscopy, atomic force microscopy and water contact angle and cell adhesion contact dynamics measurements. The cell adhesion was strongest on the Gel-CS-PLGA surface and lowest on unmodified PLGA. The steady state adhesion energy attained by the cells on gelatin modified PLGA surface is determined as 4.0×10−8  J/m2, which is about 400 times higher than that on PLGA surface (1.1×10−10  J/m2). Significantly increased cell adhesion with Gel-CS-PLGA is postulated to result in increased cell spreading. Our integrated biophysical method can quantify the transient contact dynamics and is sufficiently accurate to discriminate even between Gel and CS modified surfaces.
Keywords: Adhesion contact dynamics; Poly (lactide-co-glycolide acid) (PLGA); Azide-chitosan; Surface modification;

Biochemical consequences of alginate encapsulation: A NMR study of insulin-secreting cells by Nicholas E. Simpson; Samuel C. Grant; Lenita Gustavsson; Vilje-Mia Peltonen; Stephen J. Blackband; Ioannis Constantinidis (2577-2586).
In this study we explore the biochemical consequences of alginate encapsulation on βTC3 cells. 13C NMR spectroscopy and isotopomer analysis were used to investigate the effects of encapsulation on several enzymatic processes associated with the TCA cycle. Our data show statistically significant differences in various enzymatic fluxes related to the TCA cycle and insulin secretion between monolayer and alginate-encapsulated cultures. The principal cause for these effects was the process of trypsinization. Embedding the trypsinized cells in alginate beads did not have a compounded effect on the enzymatic fluxes of entrapped cells. However, an additional small but statistically significant decrease in insulin secretion was measured in encapsulated cells. Finally, differences in either enzymatic fluxes or glucose consumption as a function of bead diameter were not observed. However, differences in T 2, assessed by 1H NMR microimaging, were observed as a function of bead diameter, suggesting that smaller beads became more organized with time in culture, while larger beads displayed a looser organization.
Keywords: Alginate; Metabolism; 13C NMR; Isotopomer;

Protein interactions with subcutaneously implanted biosensors by Raeann Gifford; Joseph J. Kehoe; Sandra L. Barnes; Boris A. Kornilayev; Michail A. Alterman; George S. Wilson (2587-2598).
Biofouling of in vivo glucose sensors has been indicated as the primary reason for sensitivity losses observed during the first 24 h after implant [Wisniewski N, Moussy F, Reichert WM. Characterization of implantable biosensor membrane biofouling. Fresen J Anal Chem 2000; 366(6–7): 611–621]. Identification of the biomolecules that contribute to these sensitivity perturbations is the primary objective of the research presented. Active needle-type glucose sensors were implanted in Sprague–Dawley rats for 24 h, and then a proteomics approach was used to identify the substances absorbed to the sensors. MALDI–TOF mass spectrometry was the primary tool utilized to identify the biomolecules in sensor leachate samples and species absorbed directly on sensor membranes excised from explanted in vivo sensors. Not surprisingly serum albumin was identified as the primary biomolecule present, however, predominantly as endogenous fragments of the protein. In addition, several other biomolecule fragments, mainly less than 15 kD, were identified. Based on these findings, it is concluded that fragments of larger biomolecules infiltrate the sensor membranes causing diminished glucose diffusivity, thus decreasing in vivo sensitivity.
Keywords: MALDI–TOF mass spectrometry; Glucose sensor biofouling; Inflammatory response; Protein identification; In vivo; Sensitivity loss;

pH triggered release of protective poly(ethylene glycol)-b-polycation copolymers from liposomes by Debra T. Auguste; Steven P. Armes; Krystyna R. Brzezinska; Timothy J. Deming; Joachim Kohn; Robert K. Prud’homme (2599-2608).
Triggered release of adsorbed polymers from liposomes enables protection against immune recognition during circulation and subsequent intracellular delivery of DNA. Polycationic blocks, poly[2-(dimethylamino) ethyl methacrylate] (DMAEMA) (0.8, 3.1, 4.9, or 9.8 kg/mol) or polylysine (K) (3 kg/mol), act as anchors for poly(ethylene glycol) (PEG) (2 or 5 kg/mol) protective blocks. In addition, a copolymer with 15 strictly alternating blocks of PEG (2 kg/mol) and cationic amine sites was evaluated as a protective coating. Incorporation of 1,2-dioleoyl-3-dimethylammonium-propane, a titratable lipid with a pKa of ∼6.7, allows the liposome's net charge to increase as the pH shifts from 7.4 in the bloodstream to 5.5 in the endosome. The increased net liposome cationicity results in decreased cationic polymer adsorption. The EMPEG113–DMAEMA31 and EMPEG113–DMAEMA62 association constants decrease from 3.1 and 6.2 (mg/m2)/(mg/ml) at pH 7.4 to 1.7 and 3.2 (mg/m2)/(mg/ml) at pH 5.5, respectively. However, EMPEG45–DMAEMA5, EMPEG45–DMAEMA20, and EMPEG45–N–DP15 did not show a strong response to changes in pH. Cationic polymer adsorption exceeds calculated values for liposome neutralization, resulting in adsorption profiles in the brush regime.
Keywords: Adsorption; Drug delivery; Controlled drug release; Gene therapy; Polyethylene oxide; Liposomes;

Sustained delivery of growth factors from methylidene malonate 2.1.2-based polymers by Laurent Désiré; Evgueni Mysiakine; David Bonnafous; Patrick Couvreur; Serge Sagodira; Pascal Breton; Elias Fattal (2609-2620).
The incorporation of growth factors into new methylidene malonate 2.1.2-based biocompatible polymeric blends of oligomers and polymers to improve their stability and controlled release was investigated. Five growth factors were used in this study: FGF2, PDGF, TGF-β, NGF and GM-CSF. Formulation in poly(methylidene malonate 2.1.2) blends was achieved by a four-step optimized process, using different oligomers/polymers ratios. Once dried, formulations could be subsequently stored at 4 or 20 °C or immediately subjected to degradation in conditioned cell culture medium. Toxicity of blends and their degradation products were evaluated in several cell lines with MTT. Bioactivity and biospecificity of the formulated growth factors were investigated using MTT and immunohistochemical staining. Combined ELISA and crystal violet colorimetric assays were performed to analyze growth factors release. Limited toxicities were observed for unloaded poly(methylidene malonate 2.1.2) blends. Once optimized, growth factors formulations did not reveal lower bioactivities or loss of biospecificity. Moreover, a sustained release over a 21-day period with more than 90% of preserved bioactivity was reached. To conclude, dual growth factor delivery was made possible by the mean of poly(methylidene malonate 2.1.2) blends. These studies demonstrate the ability of methylidene malonate 2.1.2-based polymeric blends for the delivery of growth factors.
Keywords: Growth factors; Controlled drug release; Peptide; Methylidene malonate 2.1.2;

A new, facile method to prepare the heparin-functionalized PLGA nanoparticle (HEP-PLGA NP) for the controlled release of growth factors is developed. This system is composed of PLGA as a hydrophobic core, Pluronic F-127 as a hydrophilic surface layer, and heparin as the functional moiety. HEP-PLGA NPs were prepared by a solvent-diffusion method without chemical modification of the components. The entrapment of heparin molecules was confirmed by a negatively increased zeta potential value and the specific binding affinity to antithrombin III. The average diameter and the surface charge of the nanoparticles were ranged from 139±2 to 188±4 nm and from –26.0±1.1 to –44.1±1.3 mV by increasing the amount of heparin during the nanoparticle preparation. Accordingly, the amount of heparin on the nanoparticle increased from 0% to 4.7%. As a model in vitro release experiment, lysozyme was loaded into HEP-PLGA NPs, and a sustained release profile over 2 weeks was obtained with maintaining its bioactivity. The release of rhVEGF, one of the heparin-binding growth factors, showed a more sustained and prolonged profile than that of lysozyme over one month.
Keywords: Heparin; Pluronic; PLGA; Nanoparticle; Controlled release; Growth factor;

Fabrication and characterization of heparin functionalized membrane-mimetic assemblies by Po-Yuan Tseng; Shyam M. Rele; Xue-Long Sun; Elliot L. Chaikof (2627-2636).
A membrane-mimetic assembly incorporating surface bound heparin was fabricated as a system to improve the hemocompatibility of blood-contacting devices. As a model system, heparin was chemically modified by end-point conjugation to biotin and immobilized onto membrane-mimetic thin films via biotin–streptavidin interactions. Heparin surface density, determined by radiochemical titration, confirmed that surface density was directly related to the molar concentration of biotinylated lipid within the assembled membrane-mimetic film. The capacity of surface bound heparin to promote ATIII-mediated thrombin inactivation was investigated in a parallel plate flow chamber under simulated venous and arterial wall shear rates of 50 and 500 s−1, respectively. Significantly, we observed that the rate of thrombin inactivation approached a maximum at a heparin surface concentration greater than 4.4 pmol/cm2 (61 ng/cm2). In the process, mass transport limited regimes were identified for heparin potentiated thrombin inactivation under both simulated venous and arterial conditions.
Keywords: Anticoagulant; Biomimetics; Heparin; Antithrombogenic; Membrane-mimetic;

Membrane–mimetic thin films containing thrombomodulin (TM) and/or heparin were produced and their capacity to inhibit thrombin generation evaluated in a continuous flow system. Tissue factor (TF) along with TM and heparin were immobilized in spatially restricted zones as components of a membrane–mimetic film. Specifically, TF was positioned as an upstream trigger for thrombin generation and TM and/or heparin positioned over the remaining downstream portion of test films. Peak and steady-state levels of thrombin were decreased by antithrombin III (ATIII), as well as by surface bound heparin and TM. Although physiologic concentrations of ATIII have the capacity to significantly inhibit thrombin activity, surface bound TM and heparin nearly abolished steady-state thrombin responses. In particular, surface bound TM appears to be superior to heparin in reducing local thrombin concentrations. These studies are the first to demonstrate the additive effect of surface bound heparin and TM as a combined interactive strategy to limit TF-induced thrombin formation.
Keywords: Membrane–mimetic; Self-assembly; Heparin; Thrombomodulin; Blood-contacting material;