Biomaterials (v.27, #31)

Calendar (I).

Finalizing the properties of porous scaffolds of aliphatic polyesters through radiation sterilization by Peter Plikk; Karin Odelius; Minna Hakkarainen; A.C. Albertsson (5335-5347).
Porous scaffolds made of various l,l-lactide (LLA), 1,5-dioxepane-2-one (DXO) and ε-caprolactone (CL) copolymers were sterilized by EB- and γ-irradiation. Differences in the comonomers, composition and the microstructure of the starting materials were used to influence the degradation mechanism and susceptibility towards irradiation and by this means to achieve sterilized scaffolds with predicted end-properties. The chemical changes and the formation of low-molecular-weight products were determined by SEC, 1H nuclear magnetic resonance (NMR), 13C NMR and gas chromatography-mass spectrometry (GC-MS). The degradation mechanism changed from random chain scission to cross-linking depending on the choice of monomers, the copolymer composition and the monomer sequences. Copolymerization of LLA with small amounts of CL or DXO increased the stability compared to that of the LLA homopolymer. Changing DXO to CL in a LLA copolymer also increased the stability. The type of radiation and the microstructure of the copolymer chains determined which of the monomer sequences were more prone to degrade. The most abundant low-molecular-weight product identified after sterilization was DXO monomer. Traces of LLA and CL monomers were also identified. Modification of the copolyester microstructure changed the degradation mechanism and the susceptibility towards irradiation. This allows the use of radiation sterilization to finalize the scaffold properties.
Keywords: Electron beam; Gamma irradiation; Scaffold; Copolymer; Polycaprolactone; Polylactic acid;

Ni(II) activates the Nrf2 signaling pathway in human monocytic cells by Jill B. Lewis; Regina L. Messer; Veronica V. McCloud; Petra E. Lockwood; Stephen D. Hsu; John C. Wataha (5348-5356).
Nickel is a component of biomedical alloys that is released during corrosion and causes inflammation in tissues by as yet unknown mechanisms. Recent data show that Ni(II) at concentrations of 10–50 μm amplifies lipopolysaccharide-triggered, NFκB-mediated cytokine secretion from monocytes. In the current study, we tested the hypothesis that Ni(II) amplifies cytokine secretion by activating the Nrf2 antioxidant pathway rather than by enhancing activity of the NFκB signaling pathway. Human THP1 monocytes were exposed to Ni(II) concentrations of 10–30 μm for 6–72 h, then immunoblots of whole-cell lysates or cytosolic and nuclear proteins were used to detect changes in Nrf2 or NFκB signaling. Our results show that Ni(II) increased (by 1–2 fold) whole-cell Nrf2 levels and nuclear translocation of Nrf2, and amplified lipopolysaccharide (LPS)-induction of Nrf2 (by 3–5 fold), but had no detectable effect on the initial activation or nuclear translocation of NFκB. Because Nrf2 target gene products are known regulators of NFκB nuclear activity, our results suggest that Ni(II) may affect cytokine secretion indirectly via modulation of the Nrf2 pathway.
Keywords: Dental alloy; Inflammation; Monocyte; Nickel; Biocompatibility;

Fibrinogen adsorption, platelet adhesion and activation on mixed hydroxyl-/methyl-terminated self-assembled monolayers by Sofia N. Rodrigues; Inês C. Gonçalves; M.C.L. Martins; Mário A. Barbosa; Buddy D. Ratner (5357-5367).
The effect of surface wettability on fibrinogen adsorption, platelet adhesion and platelet activation was investigated using self-assembled monolayers (SAMs) containing different ratios of longer chain methyl- and shorter chain hydroxyl-terminated alkanethiols (C15CH3 vs. C11OH) on gold.Protein adsorption studies were performed using radiolabeled human fibrinogen (HFG). Platelet adhesion and activation studies with and without pre-adsorbed fibrinogen, albumin and plasma were assessed using scanning electron microscopy (SEM) and a glutaraldehyde-induced fluorescence technique (GIFT). Results demonstrated a linear decrease of HFG adsorption with the increase of OH groups on the monolayer (increase of the hydrophilicity). Platelet adhesion and activation also decrease with increase of hydrophilicity of surface. Concerning SAMs pre-immersed in proteins, fibrinogen adsorption was related with high platelet adhesion and activation. The passivant effect of albumin on platelet adhesion and activation was only demonstrated on SAMs contained C11OH. When all the blood proteins are present (plasma) platelet adhesion was almost absent on SAMs with 65% and 100% C11OH. This could be explained by the higher albumin affinity of the SAMs with 65% C11OH and the lower total protein adsorption associated with SAMs with 100% C11OH.
Keywords: Platelet activation; Platelet adhesion; Fibrinogen; Protein adsorption; SEM; Self assembled;

In vitro model of glial scarring around neuroelectrodes chronically implanted in the CNS by Vadim S. Polikov; Michelle L. Block; Jean-Marc Fellous; Jau-Shyong Hong; W. Monty Reichert (5368-5376).
A novel in vitro model of glial scarring was developed by adapting a primary cell-based system previously used for studying neuroinflammatory processes in neurodegenerative disease. Midbrains from embryonic day 14 Fischer 344 rats were mechanically dissociated and grown on poly-d-lysine coated 24 well plates to a confluent layer of neurons, astrocytes, and microglia. The culture was injured with either a mechanical scrape or foreign-body placement (segments of 50 μm diameter stainless steel microwire), fixed at time points from 6 h to 10 days, and assessed by immunocytochemistry. Microglia invaded the scraped wound area at early time points and hypertrophied activated astrocytes repopulated the wound after 7 days. The chronic presence of microwire resulted in a glial scar forming at 10 days, with microglia forming an inner layer of cells coating the microwire, while astrocytes surrounded the microglial core with a network of cellular processes containing upregulated GFAP. Vimentin expressing cells and processes were present in the scrape at early times and within the astrocyte processes forming the glial scar. Neurons within the culture did not repopulate the scrape wound and did not respond to the microwire, although they were determined to be electrically active through patch clamp recording. The time course and relative positions of the glia in response to the different injury paradigms correlated well with stereotypical in vivo responses and warrant further work in the development of a functional in vitro test bed.
Keywords: Neural prosthesis; Foreign-body response; In vitro test; Cell culture; Brain; Biocompatibility;

Blood compatibility of novel water soluble hyperbranched polyglycerol-based multivalent cationic polymers and their interaction with DNA by Rajesh Kumar Kainthan; Muthiah Gnanamani; Munia Ganguli; Tanay Ghosh; Donald E. Brooks; Souvik Maiti; Jayachandran N. Kizhakkedathu (5377-5390).
A novel class of hyperbranched polymers based on polyglycerol (PG) and poly(ethylene glycol) (PEG) are synthesized by multibranching anionic ring opening polymerization. Multivalent cationic sites are added to these polymers by a post-amination and quarternization reactions. Blood compatibility studies using these polymers at different concentrations showed insignificant effects on complement activation, platelet activation, coagulation, erythrocyte aggregation and hemolysis compared to branched cationic polyethyleneimine (PEI). The degree of quarternization does not have large influence on the blood compatibility of the new polymers. Cytotoxicity of these polymers is significantly lower than that of PEI and is a function of quarternized nitrogen present in the polymer. Also, these polymers bind DNA in the nanomolar range and are able to condense DNA to highly compact, stable, water soluble nanoparticles in the range of 60–80 nm. Gel electrophoresis studies showed that they form electroneutral complexes with DNA around N/P ratio 1 irrespective of the percentage of quarternization under the conditions studied.
Keywords: Hyperbranched polymers; Cationic polymers; Blood-compatibility; Drug delivery; DNA; AFM;

Micromolding of shape-controlled, harvestable cell-laden hydrogels by Judy Yeh; Yibo Ling; Jeffrey M. Karp; Jay Gantz; Akash Chandawarkar; George Eng; James Blumling III; Robert Langer; Ali Khademhosseini (5391-5398).
Encapsulation of mammalian cells within hydrogels has great utility for a variety of applications ranging from tissue engineering to cell-based assays. In this work, we present a technique to encapsulate live cells in three-dimensional (3D) microscale hydrogels (microgels) of controlled shapes and sizes in the form of harvestable free standing units. Cells were suspended in methacrylated hyaluronic acid (MeHA) or poly(ethylene glycol) diacrylate (PEGDA) hydrogel precursor solution containing photoinitiator, micromolded using a hydrophilic poly(dimethylsiloxane) (PDMS) stamp, and crosslinked using ultraviolet (UV) radiation. By controlling the features on the PDMS stamp, the size and shape of the molded hydrogels were controlled. Cells within microgels were well distributed and remained viable. These shape-specific microgels could be easily retrieved, cultured and potentially assembled to generate structures with controlled spatial distribution of multiple cell types. Further development of this technique may lead to applications in 3D co-cultures for tissue/organ regeneration and cell-based assays in which it is important to mimic the architectural intricacies of physiological cell–cell interactions.
Keywords: Microgels; Tissue engineering; Micromolding; Biomaterials; Cell encapsulation;

One challenge of particular importance in tissue engineering is to improve vascularization of larger size defects, which would then facilitate a sufficient supply with oxygen and nutrients to the central regions of a larger tissue-engineered construct or in highly vascularized tissues. In this study, we show that outgrowth endothelial cells (OECs) derived from human peripheral blood can serve as a source of human autologous endothelial cells and can be used in combination with fibroin silk fiber meshes for applications in tissue engineering. OEC reveal a highly differentiated endothelial phenotype as well as a high phenotypic stability during their expansion. Furthermore, OEC showed very promising results in the endothelialization of fibroin silk fiber meshes, maintaining their endothelial characteristics and functions. On the fibroin fiber meshes OECs formed differentiated endothelial cell layers covering the single fibers as shown by data from scanning electron microscopy, immunofluorescence, and gene expression analysis. After embedding in a wound-healing matrix, mimicked by fibrin gels, OEC migrated from the fibroin scaffolds into the fibrin and formed a microvessel-like network. Thus, we conclude that OEC could serve as a valuable source of autologous endothelial cells, supporting pro-angiogenic therapies in combination with silk fibroin-based scaffolding materials in the field of tissue engineering and regenerative medicine.
Keywords: Angiogenesis; Endothelial cells; Autologous cells; Stem cells; 3-D-scaffolds; Silk fibroin;

Porous acellular bovine pericardia seeded with mesenchymal stem cells as a patch to repair a myocardial defect in a syngeneic rat model by Hao-Ji Wei; Sung-Ching Chen; Yen Chang; Shiaw-Min Hwang; Wei-Wen Lin; Po-Hong Lai; Huihua Kenny Chiang; Lee-Feng Hsu; Hang-Hsing Yang; Hsing-Wen Sung (5409-5419).
A patch is often mandatory to repair myocardial defects; however, currently available patches lack the possibility of regeneration. To overcome this limitation, a porous acellular bovine pericardium seeded with BrdU-labeled mesenchymal stem cells (MSCs) was prepared (the MSC patch) to repair a surgically created myocardial defect in the right ventricle of a syngeneic rat model. The bovine pericardium before cell extraction was used as a control (the Control patch). The implanted samples were retrieved at 4- and 12-week postoperatively ( n = 5 per group at each time point). At retrieval, no aneurysmal dilation of the implanted patches was seen for both studied groups. No apparent tissue adhesion was observed for the MSC patch throughout the entire course of the study, while for the Control patch, two out of the five studied animals at 12-week postoperatively had a filmy adhesion to the chest wall. On the inner (endocardial) surface, intimal thickening was observed for both studied groups; however, no thrombus formation was found. Intact layers of endothelial and mesothelial cells were identified on the inner and outer (epicardial) surfaces of the MSC patch. Smooth muscle cells together with neo-muscle fibers, neo-glycosaminoglycans and neo-capillaries were observed within the pores of the MSC patch. Some cardiomyocytes, which stained positively for BrdU and α-sacromeric actin, were observed in the MSC patch, indicating that the implanted MSCs can engraft and differentiate into cardiomyocytes. Additionally, a normality of the local electrograms on the epicardial surface of the MSC patch was observed. In contrast, no apparent tissue regeneration was observed for the Control patch throughout the entire course of the study, while only abnormal electrogram signals were seen on its epicardial surface. In conclusion, the MSC patch may preserve the structure of the ventricular wall while providing the potential for myocardial tissue regeneration.
Keywords: Mesenchymal stem cells; Porous acellular bovine pericardium; Cardiomyocytes; Myocardial tissue regeneration; Angiogenesis;

A tris(2,2′-bipyridyl)cobalt(III)-bovine serum albumin composite membrane for biosensors by Ying Zhuo; Ruo Yuan; Yaqin Chai; Aili Sun; Ying Zhang; Jiuzhi Yang (5420-5429).
A concept based on a novel redox-biocompatible composite protein membrane fabrication, double enzyme membrane modification technique and antibody immobilization, was exploited to develop a highly sensitive amperometric enzyme immunosensor for detection of carcinoembryonic antigen (CEA). In this concept, a solution of bovine serum albumin (BSA) containing horseradish peroxidase (HRP) is coated on the gold electrode in such a way that the first enzyme membrane is achieved. Then tris(2,2′-bipyridyl) cobalt(III) (Co(bpy)3 3+), as a mediator, was embedded in BSA–HRP composite membrane vis the electrostatic force and hydrophobe functions. Later a self-assembled conductive nano-Au monolayer was constructed onto the resultant electrode surface by electrostatic interaction between the negatively charged nano-Au and positively charged Co(bpy)3 3+. Protein A is used as a binding material to achieve an adjusted (but not random) orientation of the antibodies surface for efficient combination of antigens. Finally, the HRP, was employed to block the possible remaining active sites and avoid the non-specific adsorption, which acts not only as a blocking reagent instead of the commonly used BSA but also as the conventional enzyme-labeling to amplify the response of the antigen–antibody reaction. The immunosensor constructed with the double layer biocatalytic HRP membranes and the desirable Co(bpy)3 3+/BSA redox-biocompatible composite membrane performed high sensitivity and a wide linear response to CEA in the range of 0.50–80.00 ng/mL with a limit of detection of 0.14 ng/mL, as well as good stability and long-term life.
Keywords: Amperometric enzyme immunosensor; Bovine serum albumin (BSA); Co(bpy)3 3+; Horseradish peroxidase (HRP); Carcinoembryonic antigen (CEA);

Micropatterning with aerosols: Application for biomaterials by Louis Gagné; Gerardo Rivera; Gaetan Laroche (5430-5439).
Adhesion and proliferation behaviors of bovine aortic endothelial cells (BAECs) were investigated on surfaces micropatterned with peptides using a novel approach. This micropatterning technique allows modification of macroscopic three-dimensional (3D) biomaterials surfaces and exploits the semi-random properties of aerosols and the principles of liquid atomization. The possibility to control cell behaviors on polytetrafluoroethylene (PTFE) surfaces tailored with this micropatterning approach was evaluated. CGRGDS and CWQPPRARI peptides were selected for their adhesive, migration and spreading properties. Culture of BAECs on patterned PTFE showed the possibility of modulating cell behaviors. The study showed that CGRGDS spots with a diameter of 10±2 μm over a background of CWQPPRARI peptides was the most effective combination to enhance endothelialization of PTFE. This micropatterning technique is innovative, easily adaptable, simple, and rapid for covering large 3D areas.
Keywords: Cell adhesion; Cell proliferation; Cell spreading; Endothelialization; Micropatterning; RGD peptide;

Controlled drug release devices of pH-sensitive, complexing poly(acrylic acid-g-ethylene glycol) (P(AA-g-EG)) hydrogels were prepared by free radical solution UV polymerization. The effects of hydrogel composition, polymerization conditions and surrounding environment on theophylline release kinetics and drug transport mechanisms were evaluated in these P(AA-g-EG) polymer networks. Release studies indicated a dependence of the theophylline release kinetics and diffusion coefficients on the hydrogel structure, polymerization conditions and pH of the environment. The theophylline transport mechanism was studied by fitting experimental data to five different model equations and calculating the corresponding parameters. The Akaike information criterion was also considered to elucidate the best-fit equation. Results indicated that in most release cases, the drug release mechanism was anomalous (non-Fickian). This indicates that such systems may, under certain conditions, provide release characteristics approaching zero-order release. The pH of the dissolution medium appeared to have a strong effect on the drug transport mechanism. At more basic pH values, Case II transport was observed, indicating a drug release mechanism highly influenced by macromolecular chain relaxation. The results obtained in this research work lead us to the conclusion that P(AA-g-EG) hydrogels can be successfully used as drug delivery systems. Their versatility to be designed with specifically tuned release properties renders these biomaterials promising pharmaceutical carriers for therapeutic agents.
Keywords: Hydrogel; Controlled drug release; Copolymer; Poly(acrylic acid); Poly(ethylene glycol);

Surface immobilization of active vascular endothelial growth factor via a cysteine-containing tag by Marina V. Backer; Vimal Patel; Brian T. Jehning; Kevin P. Claffey; Joseph M. Backer (5452-5458).
Developing tissue engineering scaffolds with immobilized growth factors requires facile and reliable methods for the covalent attachment of functionally active proteins. We describe here a new approach to immobilize recombinant proteins based on expression of the protein of interest with a 15-aa long fusion tag (Cys-tag), which avails a free sulfhydryl group for site-specific conjugation. To validate this approach, we conjugated a single-chain vascular endothelial growth factor expressed with an N-terminal Cys-tag (scVEGF) to fibronectin (FN) using a common thiol-directed bi-functional cross-linking agent. We found that the FN-scVEGF conjugate retains VEGF activity similar to that of free scVEGF when used as a soluble ligand. Cells expressing VEGF receptor VEGFR-2 grown on plates coated with FN-scVEGF displayed morphological phenotypes similar to those observed for cells grown on FN in the presence of equivalent amounts of free scVEGF. In addition, 293/KDR cell growth stimulation was observed in the same concentration range with either immobilized or free scVEGF. The effects of immobilized scVEGF, and soluble scVEGF were blocked by NVP-AAD777-NX, a VEGF receptor tyrosine kinase inhibitor. These data indicate that site-specific immobilization via Cys-tag provides a facile and reliable method for permanent deposition of functionally active growth factors on synthetic or protein scaffolds with applications for advanced tissue engineering.
Keywords: Site-specific conjugation; Immobilization; Growth factors; VEGF; Tissue engineering;

Biomimetic strategies focusing on presenting short bioadhesive oligopeptides, including the arginine–glycine–aspartic acid (RGD) motif present in numerous adhesive proteins, on a non-fouling support have emerged as promising approaches to improve cellular activities and healing responses. Nevertheless, these bio-inspired strategies are limited by low activity of the oligopeptides compared to the native ligand due to the absence of complementary or modulatory domains. In the present analysis, we generated well-defined biointerfaces presenting RGD-based ligands of increasing complexity to directly compare their biological activities in terms of cell adhesion strength, integrin binding and signaling. Mixed self-assembled monolayers of alkanethiols on gold were optimized to engineer robust supports that present anchoring groups for ligand tethering within a non-fouling, protein adsorption-resistant background. Controlled bioadhesive interfaces were generated by tethering adhesive ligands via standard peptide chemistry. On a molar basis, biointerfaces functionalized with the FNIII7–10 recombinant fragment presenting the RGD and PHSRN adhesive motifs in the correct structural context exhibited significantly higher adhesion strength, FAK activation, and cell proliferation rate than supports presenting RGD ligand or RGD–PHSRN, an oligopeptide presenting these two sites separated by a polyglycine linker. Moreover, FNIII7–10-functionalized surfaces displayed specificity for α 5 β 1 integrin, while cell adhesion to supports presenting RGD or RGD–PHSRN was primarily mediated by α v β 3 integrin. These results are significant to the rational engineering of bioactive materials that convey integrin binding specificity for directed cellular and tissue responses in biomedical and biotechnological applications.
Keywords: Biomimetic; Integrins; Cell adhesion; Focal adhesion; FAK; Self-assembled monolayers;