Biomaterials (v.28, #23)

The effect of covalently immobilized rhIL-1ra-ELP fusion protein on the inflammatory profile of LPS-stimulated human monocytes by Dong-Hwan Kim; Jason T. Smith; Ashutosh Chilkoti; William M. Reichert (3369-3377).
The objective of this research was to investigate whether immobilized anti-inflammatory cytokines will signal changes in the inflammatory profile of cultured monocytes. A fusion protein of recombinant human IL-1 receptor antagonist and elastin-like peptide (IL-1ra-ELP) was expressed in Escherichia coli. THP-1 human monocytes were cultured on either carboxyl-terminated self-assembled monolayers (SAMs), or SAMs with either covalently immobilized or soluble IL-1ra-ELP. LPS-stimulated monocytes exposed to either soluble or immobilized IL-1ra-ELP were prevented from cell differentiation, showed attenuated expression of pro-inflammatory cytokines, and had increased production of anti-inflammatory and pro-wound healing cytokines. These results suggest that immobilized anti-inflammatory cytokines have the potential to be immunomodulatory biomaterials.
Keywords: IL-1ra; Monocytes; Cytokines; Inflammation; Wound healing;

Material-based regulation of the myofibroblast phenotype by Melinda C. Cushing; Jo-Tsu Liao; Michael P. Jaeggli; Kristi S. Anseth (3378-3387).
Fibroblast growth factor receptor (FGFR) activation by basic fibroblast growth factor (FGF-2) serves to naturally repress the myofibroblast activation of valvular interstitial cells (VICs). Co-receptors for FGF-2, the heparan sulfate proteoglycans (HSPGs), are key participants in the formation of active FGF-2 signaling complexes. Bioactive environments regulating the myofibroblast phenotype were created by utilizing heparin glycosaminoglycan as a competitive inhibitor of HSPGs. First, soluble heparin was delivered to compete with cell-surface HSPG for the binding of FGF-2. Exogenous soluble heparin prevented serum-dependent activation of the classic mitogen-activated protein kinase (MAPK) and induced myofibroblast alpha smooth muscle actin (αSMA) expression and collagen production. Next, heparin-functionalized hydrogel cell substrates were polymerized from vinyl-modified precursors and rendered adhesive through incorporation of RGDS peptide. Culture of VICs on heparin-modified gels induced αSMA expression and inhibited MAPK activity compared to control gel substrates lacking heparin. Additionally, heparin-functionalized gels continued to induce αSMA expression in serum-free culture conditions, suggesting that bioactivity was independent of exogenous soluble mediators. Biomaterial scaffolds targeting cell surface growth factor receptors are a promising new direction for regulating cell functions in tissue-engineering applications.
Keywords: Cardiac valve tissue engineering; Cell signalling; Fibroblast growth factor receptor; MAPK; Heparin; Hydrogel;

The in vitro development of autologous fibrin-based tissue-engineered heart valves through optimised dynamic conditioning by Thomas C. Flanagan; Christian Cornelissen; Sabine Koch; Beate Tschoeke; Joerg S. Sachweh; Thomas Schmitz-Rode; Stefan Jockenhoevel (3388-3397).
Our group has previously demonstrated the synthesis of a completely autologous fibrin-based heart valve structure using the principles of tissue engineering. The present approach aims to guide more mature tissue development in fibrin-based valves based on in vitro conditioning in a custom-designed bioreactor system. Moulded fibrin-based tissue-engineered heart valves seeded with ovine carotid artery-derived cells were subjected to 12 days of mechanical conditioning in a bioreactor system. The bioreactor pulse rate was increased from 5 to 10 b.p.m. after 6 days, while a pressure difference of 20 mmH2O was maintained over the valve leaflets. Control valves were cultured under stirred conditions in a beaker. Cell phenotype and extracellular matrix (ECM) composition were analysed in all samples and compared to native ovine aortic valve tissue using routine histological and immunohistochemical techniques.Conditioned valve leaflets showed reduced tissue shrinkage compared to stirred controls. Limited ECM synthesis was evident in stirred controls, while the majority of cells were detached from the fibrin scaffold. Dynamic conditioning increased cell attachment/alignment and expression of α-smooth muscle actin, while enhancing the deposition of ECM proteins, including types I and III collagen, fibronectin, laminin and chondroitin sulphate. There was no evidence for elastin synthesis in either stirred controls or conditioned samples. The present study demonstrates that the application of low-pressure conditions and increasing pulsatile flow not only enhances seeded cell attachment and alignment within fibrin-based heart valves, but dramatically changes the manner in which these cells generate ECM proteins and remodel the valve matrix. Optimised dynamic conditioning, therefore, might accelerate the maturation of surgically feasible and implantable autologous fibrin-based tissue-engineered heart valves.
Keywords: Cardiovascular tissue engineering; Heart valve; Extracellular matrix; Fibrin; Bioreactor; Immunochemistry;

Regulation of axon guidance and extension by three-dimensional constraints by Herbert Francisco; Benjamin B. Yellen; Derek S. Halverson; Gary Friedman; Gianluca Gallo (3398-3407).
Axons in vivo are guided by molecular signals acting as attractants and repellents, and possibly by physical constraints encountered in the extracellular environment. We analyzed the ability of primary sensory axons to extend and undergo guidance in three-dimensional (3-D) environments generated using photolithography. Confinement of neurons in fully enclosed square chambers decreased the percentage of neurons establishing axons as a function of chamber width. However, the ability to extend an axon in one or more directions allowed axons to form and extend similarly to those on two-dimensional (2-D) substrata. Live imaging of growth cones interacting with the walls of chambers or corridors revealed that growth cones respond to contact with a 3-D constraint by decreasing surface area, and circumvent constraints by repeated sampling of the constraint until an unobstructed path is encountered. Analysis of the ability of axons to turn around corners in corridors revealed that the angle of the corner and corridor width determined the frequency of turning. Finally, we show that the length of axons can be controlled through the use of 3-D constraints. These data demonstrate that 3-D constraints can be used to guide axons, and control the extent of axon formation and the length of axons.
Keywords: Axon; Growth cone; Myosin; Filopodium; Microfluidics;

Functional histidine/nickel hexacyanoferrate nanotube assembly for biosensor applications by Minghui Yang; Jianhui Jiang; Yashuang Lu; Yan He; Guoli Shen; Ruqin Yu (3408-3417).
By exploring the properties of histidine, functional histidine/nickel hexacyanoferrate nanotube assembly was prepared using nanopore alumina template via a sequential deposition strategy and demonstrated for improved biosensing. The vertically oriented nanotube assembly has a nanotube density ∼8×108  cm−2 and can be stably attached to the glassy carbon electrode surface. The nanotube assembly formed provides an ordered well-defined three-dimensional (3D) structure with good electron transfer efficiency and a large specific surface area with abundant electroactive sites. Gold nanoparticles were then absorbed onto nanotube surfaces through amino group provided by histidine, and were used for further absorption of glucose oxidase into the 3D matrix. The histidine and gold nanoparticles on the nanotube surface provide a favorable microenvironment to keep the bioactivity of the enzymes with a low apparent Michaelis–Menten constant (K Mapp) of 2.15 mm, and the ordered orientation of the nanotube assembly facilitated enzyme–substrate contact. The biosensor was sensitive and selective toward glucose with a linear range covered from 2 μm to 20 mm of glucose. The biosensor was used to determine glucose concentration in real blood samples with satisfactory results.
Keywords: Biosensor; Electrochemistry; Enzyme; Gold nanoparticle; Metal hexacyanoferrate; Nanotube assembly;

Dextran-based in situ cross-linked injectable hydrogels to prevent peritoneal adhesions by Taichi Ito; Yoon Yeo; Christopher B. Highley; Evangelia Bellas; Daniel S. Kohane (3418-3426).
Peritoneal adhesions are serious sequelae of surgery, and can cause significant morbidity and/or mortality due to pain, infertility, and bowel obstruction. We have designed and synthesized novel dextran (DX)-based injectable hydrogels for adhesion prevention, which are formed by mixing hydrazide-modified carboxymethyldextran (CMDX–ADH) with aldehyde-modified DX (DX–CHO) or carboxymethylcellulose (CMC–CHO). At high polymer concentrations, hydrogels formed very quickly upon mixing, e.g. 5% CMDX–ADH with 6% DX–CHO (=CMDX–DX; 1.8 s) and 5% CMDX–ADH with 6% CMC–CHO (=CMDX–CMC; 5.8 s). CMDX–DX shrank after gelling, while CMDX–CMC swelled. CMDX–ADH and CMC–CHO showed minimal to mild cytotoxicity to mesothelial cells and macrophages in vitro, while DX–CHO was very cytotoxic. However, all cross-linked gels had very mild cytotoxicity. When applied in a rabbit sidewall defect-bowel abrasion model of adhesion formation, CMDX–CMC greatly reduced the formation of adhesions while CMDX–DX worsened them.
Keywords: Post-operative adhesion; Dextran; Carboxymethyl dextran; Carboxymethyl cellulose; In situ cross-linking hydrogel; Rabbit sidewall defect-cecum abrasion model;

Protein direct delivery to dendritic cells using nanoparticles based on amphiphilic poly(amino acid) derivatives by Takami Akagi; Xin Wang; Tomofumi Uto; Masanori Baba; Mitsuru Akashi (3427-3436).
Induction of an adaptive immune response by vaccination is possible for a broad range of infectious diseases or cancers. Antigen-loaded polymeric nanoparticles have recently been shown to possess significant potential as vaccine delivery systems and adjuvants. Here we demonstrate the use of nanoparticles composed of amphiphilic poly(amino acid) derivatives as vaccine adjuvants. We prepared protein-loaded, biodegradable nanoparticles composed of hydrophobically modified poly(γ-glutamic acid) (γ-PGA). γ-PGA hydrophobic derivatives (γ-hPGA) formed 200 nm-sized nanoparticles in water. The protein-encapsulated γ-hPGA nanoparticles were efficiently taken up by immature dendritic cells (iDCs). Interestingly, the nanoparticle uptake by iDCs induced DC maturation. The immunization with human immunodeficiency virus (HIV)-1 gp120-encapsulated nanoparticles strongly induced antigen-specific cellular immunity. These results suggest that antigen-loaded γ-hPGA nanoparticles provide a novel delivery tool for vaccination against viral infections or tumors. This system has potential application as a universal delivery system for protein-based vaccines capable of inducing cytotoxic T lymphocyte (CTL).
Keywords: Nanoparticles; Vaccine; Protein delivery; Dendritic cells; Poly(γ-glutamic acid);

Modulation of chondrocyte phenotype via baculovirus-mediated growth factor expression by Li-Yu Sung; Wen-Hsin Lo; Hsin-Yi Chiu; Huang-Chi Chen; Ching-Kuang Chung; Hsiao-Ping Lee; Yu-Chen Hu (3437-3447).
Baculovirus has emerged as a new gene delivery vector thanks to a number of advantages. This study demonstrated that baculovirus conferred efficient gene delivery and mediated expression of growth factors (TGF-β1, IGF-1 and BMP-2) to therapeutic levels in rabbit chondrocytes. Interestingly, the cellular response to growth factor stimulation was dependent on the cell passage. The highly de-differentiated passage 5 (P5) chondrocytes failed to respond to the stimulation by either growth factor. The de-differentiated P3 cells also failed to maintain the chondrocyte phenotype, but baculovirus-mediated BMP-2 expression remarkably reversed the de-differentiation and enhanced the aggrecan and collagen II production in 2D and 3D cultures, as evidenced by cell morphology, histological staining and gene expression analyses. Baculovirus-mediated TGF-β1 expression modestly enhanced the cartilage-specific matrix production, although to a lesser extent. Intriguingly, IGF-1, a well-known chondroinductive protein, failed to stimulate the P3 cells likely due to the loss of IGF-1 receptor expression. In summary, this study proved for the first time the potentials of baculovirus in modulating the differentiation status of chondrocytes in the context of cartilage tissue engineering, but also highlighted the importance of selecting appropriate cell passage and growth factor for genetic manipulation.
Keywords: Baculovirus; Chondrocyte; Gene therapy; Growth factor; Cartilage tissue engineering; De-differentiation;

In this study lactoferrin (Lf) was investigated as a targeting ligand for receptor-mediated gene delivery to human bronchial epithelial cells. A high number of lactoferrin receptors (LfRs) were detected on bronchial epithelial (BEAS-2B), but not on alveolar epithelial (A549) cells by fluorescence microscopy and FACS measurements, suggesting potential targeting selectivity for bronchial epithelial cells. Molecular conjugates with ratios of Lf to branched polyethylenimine 25 kDa (PEI) ranging from 4:1 to 1:40 (mol/mol) were synthesized and analyzed for complexation of plasmid DNA (pDNA), transfection efficiency, and cytotoxicity. Whereas particle size increased with the degree of Lf coupling from 45 to 225 nm, surface charge was not significantly influenced. Transfection studies on BEAS-2B cells revealed that Lf-PEI 1:20 exhibited the highest luciferase gene expression which was 5-fold higher at an N/P ratio (molar ratio of PEI nitrogen to pDNA phosphate) of 4 than PEI and could be inhibited by an excess of free Lf. With A549 cells, no significant enhancement in transfection efficiency between Lf-PEI/pDNA and PEI/pDNA complexes could be observed. Increasing the degree of Lf coupling to PEI resulted in reduced transfection efficiency in both alveolar and bronchial epithelial cells. Cell viability assays resulted in significantly lower cellular toxicity of Lf-PEI/pDNA compared with PEI/pDNA complexes. We suggest that Lf represents a potent targeting ligand for receptor-mediated gene delivery to bronchial epithelial cells and might be a promising candidate for lung gene transfer in vivo.
Keywords: Lactoferrin; Polyethylenimine; Gene delivery; Receptor-mediated; Lung;

Co-polypeptides of 3,4-dihydroxyphenylalanine and l-lysine to mimic marine adhesive protein by Jing Wang; Changsheng Liu; Xin Lu; Min Yin (3456-3468).
Co-polypeptides containing 3,4-dihydroxyphenylalanine (DOPA) and l-lysine were synthesized to mimic the marine adhesive proteins through ring-opening polymerizations. The effects of composition, molecular weight, and curing agents on the adhesive properties, swelling, and degradation behavior of synthetic polypeptide were investigated. Ferric ion was introduced to the system to optimize both adhesive strength and water resistance, simulating the specificity of metal-mediated cross-linking of the marine mussel adhesives. The polypeptide was used to bond on porcine skin and porcine bone in vitro to evaluate the adhesive properties. Furthermore, outcomes of the cell culture of both co-polypeptide and co-polypeptide-modified ethylene vinyl acetate (EVA) demonstrated good cell affinity, which would provide basic data for the application of the co-polypeptide in the biomaterial field.
Keywords: Biomimetic material; Copolymer; Polyamino acid; Tissue adhesive; Swelling;