Biomaterials (v.30, #17)

The influence of side group modification in polyphosphazenes on hydrolysis and cell adhesion of blends with PLGA by Nicholas R. Krogman; Arlin L. Weikel; Katherine A. Kristhart; Syam P. Nukavarapu; Meng Deng; Lakshmi S. Nair; Cato T. Laurencin; Harry R. Allcock (3035-3041).
Polyphosphazenes have been synthesized with tris(hydroxymethyl)amino methane (THAM) side groups and with co-substituents glycine ethyl ester and alanine ethyl ester. The THAM side group was linked to the polyphosphazene backbone via the amino function. The three pendent hydroxyl functions on each THAM side group were utilized for hydrogen bonding association with poly(glycolic–lactic acid) (PLGA). Co-substitution of the polyphosphazene with both THAM and glycine or alanine ethyl esters was employed to avoid the insolubility of the single-substituent THAM-substituted polyphosphazenes. Both poly[(tris(hydroxymethyl)aminomethane)(ethyl glycinato)phosphazene] and poly[(tris(hydroxymethyl)aminomethane)(ethyl alanato)phosphazene] (1:1 ratio of side groups) were blended with PLGA (50:50) or PLGA (85:15). DSC analysis indicated miscible blend formation, irrespective of the detailed molecular structure of the polyphosphazene or the composition of PLGA in the blend. Hydrolysis studies of the polyphosphazene:PLGA (50:50) blends indicated that the PLGA component hydrolyzed more rapidly than the polyphosphazene. Primary osteoblast cell studies showed good cell adhesion to the polymer blends during 14 days, but subsequent limited cell spreading due to increased surface roughness as the two polymers eroded at different rates.
Keywords: Polyphosphazenes; Amino acid; Bioerodible; THAM; Polymer blends; Osteoblast cells;

Establishment of a comprehensive platform for the assessment of host–biomaterial interaction in vivo is an important issue. Nuclear factor-κB (NF-κB) is an inducible transcription factor that is activated by numerous stimuli. Therefore, NF-κB-dependent luminescent signal in transgenic mice carrying the luciferase genes was used as the guide to monitor the biomaterials-affected organs, and transcriptomic analysis was further applied to evaluate the complex host responses in affected organs in this study. In vivo imaging showed that genipin-cross-linked gelatin conduit (GGC) implantation evoked the strong NF-κB activity at 6 h in the implanted region, and transcriptomic analysis showed that the expressions of interleukin-6 (IL-6), IL-24, and IL-1 family were up-regulated. A strong luminescent signal was observed in spleen on 14 d, suggesting that GGC implantation might elicit the biological events in spleen. Transcriptomic analysis of spleen showed that 13 Kyoto Encyclopedia of Genes and Genomes pathways belonging to cell cycles, immune responses, and metabolism were significantly altered by GGC implants. Connectivity Map analysis suggested that the gene signatures of GGC were similar to those of compounds that affect lipid or glucose metabolism. GeneSetTest analysis further showed that host responses to GGC implants might be related to diseases states, especially the metabolic and cardiovascular diseases. In conclusion, our data provided a concept of molecular imaging-guided transcriptomic platform for the evaluation and the prediction of host–biomaterial interaction in vivo.
Keywords: Bioluminescence imaging; Transcriptomic analysis; Nuclear factor-κB; Genipin-cross-linked gelatin conduit;

Biocompatibility of biodegradable semiconducting melanin films for nerve tissue engineering by Christopher J. Bettinger; Joost P. Bruggeman; Asish Misra; Jeffrey T. Borenstein; Robert Langer (3050-3057).
The advancement of tissue engineering is contingent upon the development and implementation of advanced biomaterials. Conductive polymers have demonstrated potential for use as a medium for electrical stimulation, which has shown to be beneficial in many regenerative medicine strategies including neural and cardiac tissue engineering. Melanins are naturally occurring pigments that have previously been shown to exhibit unique electrical properties. This study evaluates the potential use of melanin films as a semiconducting material for tissue engineering applications. Melanin thin films were produced by solution processing and the physical properties were characterized. Films were molecularly smooth with a roughness (R ms) of 0.341 nm and a conductivity of 7.00 ± 1.10 × 10−5  S cm−1 in the hydrated state. In vitro biocompatibility was evaluated by Schwann cell attachment and growth as well as neurite extension in PC12 cells. In vivo histology was evaluated by examining the biomaterial–tissue response of melanin implants placed in close proximity to peripheral nerve tissue. Melanin thin films enhanced Schwann cell growth and neurite extension compared to collagen films in vitro. Melanin films induced an inflammation response that was comparable to silicone implants in vivo. Furthermore, melanin implants were significantly resorbed after 8 weeks. These results suggest that solution-processed melanin thin films have the potential for use as a biodegradable semiconducting biomaterial for use in tissue engineering applications.
Keywords: Electroactive polymer; Biocompatibility; Nerve tissue engineering;

Optimization strategies for electrospun silk fibroin tissue engineering scaffolds by Anne J. Meinel; Kristopher E. Kubow; Enrico Klotzsch; Marcos Garcia-Fuentes; Michael L. Smith; Viola Vogel; Hans P. Merkle; Lorenz Meinel (3058-3067).
As a contribution to the functionality of scaffolds in tissue engineering, here we report on advanced scaffold design through introduction and evaluation of topographical, mechanical and chemical cues. For scaffolding, we used silk fibroin (SF), a well-established biomaterial. Biomimetic alignment of fibers was achieved as a function of the rotational speed of the cylindrical target during electrospinning of a SF solution blended with polyethylene oxide. Seeding fibrous SF scaffolds with human mesenchymal stem cells (hMSCs) demonstrated that fiber alignment could guide hMSC morphology and orientation demonstrating the impact of scaffold topography on the engineering of oriented tissues. Beyond currently established methodologies to measure bulk properties, we assessed the mechanical properties of the fibers by conducting extension at breakage experiments on the level of single fibers. Chemical modification of the scaffolds was tested using donor/acceptor fluorophore labeled fibronectin. Fluorescence resonance energy transfer imaging allowed to assess the conformation of fibronectin when adsorbed on the SF scaffolds, and demonstrated an intermediate extension level of its subunits. Biological assays based on hMSCs showed enhanced cellular adhesion and spreading as a result of fibronectin adsorbed on the scaffolds. Our studies demonstrate the versatility of SF as a biomaterial to engineer modified fibrous scaffolds and underscore the use of biofunctionally relevant analytical assays to optimize fibrous biomaterial scaffolds.
Keywords: Silk fibroin; Electrospinning; Topography; Mechanical properties; Adsorption; Fibronectin;

Fluorescent microparticles for sensing cell microenvironment oxygen levels within 3D scaffolds by Miguel A. Acosta; Patrick Ymele-Leki; Yordan V. Kostov; Jennie B. Leach (3068-3074).
We present the development and characterization of fluorescent oxygen-sensing microparticles designed for measuring oxygen concentration in microenvironments existing within standard cell culture and transparent three-dimensional (3D) cell scaffolds. The microparticle synthesis employs poly(dimethylsiloxane) to encapsulate silica gel particles bound with an oxygen-sensitive luminophore as well as a reference or normalization fluorophore that is insensitive to oxygen. We developed a rapid, automated and non-invasive sensor analysis method based on fluorescence microscopy to measure oxygen concentration in a hydrogel scaffold. We demonstrate that the microparticles are non-cytotoxic and that their response is comparable to that of a traditional dissolved oxygen meter. Microparticle size (5–40 μm) was selected for microscale-mapping of oxygen concentration to allow measurements local to individual cells. Two methods of calibration were evaluated and revealed that the sensor system enables characterization of a range of hypoxic to hyperoxic conditions relevant to cell and tissue biology (i.e., pO2 10–160 mm Hg). The calibration analysis also revealed that the microparticles have a high fraction of quenched luminophore (0.90 ± 0.02), indicating that the reported approach provides significant advantages for sensor performance. This study thus reports a versatile oxygen-sensing technology that enables future correlations of local oxygen concentration with individual cell response in cultured engineered tissues.
Keywords: Tissue engineering; Hypoxia; Optical sensors; Microspheres; Poly(dimethylsiloxane);

An injectable implant system that immediately forms a film around the injection site of an animal was successfully developed by dissolving microbial polyester poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) in not harmful organic solvents including N-methyl pyrrolidone (NMP), dimethylacetamide (DMAC), 1,4-dioxane (DIOX), dimethyl sulfoxide (DMSO) and 1,4-butanolide (BL), respectively. The formation of the PHBHHx film was the result of contact between aqueous body fluids and the amphiphilic PHBHHx solvents, leading to the controllable precipitation (film formation) of PHBHHx around the contact site. The resultant PHBHHx film assumed the shapes of its surrounding cavities. The resulting porous PHBHHx film was not favorable for attachment of Human Embryo Lung Fibroblast (HELF) cells. As a consequence, the fibroblasts cultured on the PHBHHx film exhibited a spheroid-like morphology. It was found that hydrophilicity of the PHBHHx film prepared using the above technique was significantly reduced compared with the poly(lactic acid) (PLA) film prepared for the same purpose and a PHBHHx film prepared from chloroform casting. This reduced hydrophilicity explains the poor attachment of fibroblast cells to the injectable PHBHHx film, suggesting that the PHBHHx injectable implant system can be developed as a tissue adhesion prevention film for surgical operations.
Keywords: PHB; PHBHHx; Polyhydroxyalkanoates; Implant; Adhesion prevention; Biomaterials;

The role of RGD-tagged helical rosette nanotubes in the induction of inflammation and apoptosis in human lung adenocarcinoma cells through the P38 MAPK pathway by Sarabjeet S. Suri; Felaniaina Rakotondradany; Andrew J. Myles; Hicham Fenniri; Baljit Singh (3084-3090).
The rosette nanotubes (RNTs) are a class of biologically inspired, self-assembling, metal-free, hydrophilic nanotubes, which hold tremendous potential as targeted drug delivery vehicles. We investigated the cell signaling events caused by lysine-functionalized RNTs (K-RNT) co-assembled with Arg-Gly-Asp-Ser-Lys-functionalized RNTs (RGDSK-RNT) for induction of inflammation and apoptosis in human adenocarcinoma (Calu-3) cells. When co-assembled in a ratio of 1:10 μM these composite RNTs (referred to as RGDSK/K-RNTs) rapidly induced phosphorylation of P38 mitogen-activated protein kinase (MAPK) within 2 min. Higher concentrations of RGDSK/K-RNTs (>10:100 μM) resulted in a P38 MAPK-dependent increase in secretion of TNF-α. RGDSK/K-RNTs (1:10–40:400 μM) also caused a concentration- and P38 MAPK-dependent increase in caspase-3 activity and DNA fragmentation in Calu-3 cells at 18 h of exposure. Over-expression of pro-apoptotic genes including caspase-3, BAK1, CIDEB, TP53BP2, FAS, TNF and FASLG supported pro-apoptotic behaviors of these RNTs. We conclude that RGDSK/K-RNTs induce phosphorylation of P38 MAPK, which regulate secretion of TNF-α, activation of caspase-3 and apoptosis in Calu-3 cells. These results suggest that the RNTs could be used as a drug to induce apoptosis in cancer cells or as a versatile platform to deliver a variety of biologically active molecules for cancer therapy.
Keywords: Cell signaling; Apoptosis; P38 MAPK; TNF-α; DNA fragmentation; Caspase-3;

Production, safety and antitumor efficacy of recombinant Oncofetal Antigen/immature laminin receptor protein by Adel L. Barsoum; Bainan Liu; James W. Rohrer; Joseph H. Coggin; J. Allan Tucker; Lewis K. Pannell; Paul O. Schwarzenberger (3091-3099).
We describe here for the first time an efficient high yield production method for clinical grade recombinant human Oncofetal Antigen/immature laminin receptor protein (OFA/iLRP). We also demonstrate significant antitumor activity for this protein when administered in liposomal delivery form in a murine model of syngeneic fibrosarcoma. OFA/iLRP is a therapeutically very promising universal tumor antigen that is expressed in all mammalian solid tumors tested so far. We have cloned the human OFA/iLRP cDNA in a bacterial expression plasmid which incorporates a 6x HIS-tag. Large scale cultures of the plasmid transformed Escherichia coli were performed and the crude HIS-tagged OFA/iLRP was isolated as inclusion bodies and solubilized in guanidine chloride. The protein was then purified by successive passage through three column chromatography steps of immobilized metal affinity, anion exchange, and gel filtration. The resulting protein was 94% pure and practically devoid of endotoxin and host cell protein. The purified OFA/iLRP was tested in mice for safety and efficacy in tumor rejection with satisfactory results. This protein will be used for loading onto autologous dendritic cells in an FDA approved phase I/II human cancer vaccine trial in OFA/iLRP-positive breast cancer patients.
Keywords: Oncofetal Antigen/immature laminin receptor protein; Recombinant protein; Cancer vaccine; Laminin receptor precursor;

In this study, oligopeptide amphiphile containing three blocks of amino acids, Ac-(AF)6-H5-K15-NH2 (FA32), were synthesized and evaluated as carriers for co-delivery of drug and gene. Doxorubicin (DOX), luciferase reporter gene, and p53 gene were used as a model drug and genes. The peptide amphiphile self-assembled into cationic core–shell nanostructures (i.e. micelles), with a CMC value of around 0.042 mg/mL, estimated by fluorescent spectroscopy technique. FA32 nanostructures had an average size of 102 ± 19 nm, and a zeta potential of 22.8 ± 0.2 mV. These nanostructures had a high capacity for DOX encapsulation, with a DOX loading level of up to 22%. In addition, DOX release from the micelles was sustained without obvious initial burst. DOX-loaded micelles were effectively taken up by HepG2 cells, with an IC50 of 1.8 mg/L for DOX-loaded FA32, which was higher than that of free DOX (0.25 mg/L). In addition, FA32 micelles condensed DNA efficiently to form small complexes with net positive charge on the surface. In vitro gene transfection studies showed that FA32 induced comparable gene expression level to polyethylenimine. Co-delivery of drug and gene using FA32 micelles was demonstrated via confocal imaging, luciferase expression in the presence of DOX, and synergy in cytotoxic effect between p53 gene and DOX. It was shown that through simultaneous delivery of both p53 gene and DOX using FA32 micelles, an increase in p53 mRNA expression level as well as end point cytotoxicity towards HepG2 cells was achieved. FA32 micelles, therefore, have a great potential in delivering hydrophobic anticancer drug and gene simultaneously for improved cancer therapy.
Keywords: Amphiphilic oligopeptide; Self-assembly; Cationic micelles; Doxorubicin; p53; Co-delivery;

Despite many successful applications of microelectrode arrays (MEAs), typical two-dimensional in-vitro cultures do not project the full scale of the cell growth environment in the three-dimensional (3D) in-vivo setting. This study aims to on-line monitor in-vitro cell growth in a 3D matrix on the surface-modified MEAs with a dynamic perfusion culture system. A 3D matrix consisting of poly(ethylene glycol) hydrogel supplemented with poly-d-lysine was subsequently synthesized in situ on the self-assembled monolayer modified MEAs. FTIR spectrum analysis revealed a peak at 2100 cm−1 due to the degradation of the structure of the 3D matrix. After 2 wks, microscopic examination revealed that the non-degraded area was around 1500 μm2 and provided enough space for cell growth. Fluorescence microscopy revealed that the degraded 3D matrix was non-cytotoxic allowing the growth of NIH3T3 fibroblasts and cortical neurons in vitro. Time-course changes of total impedance including resistance and reactance were recorded for 8 days to evaluate the cell growth in the 3D matrix on the MEA. A consistent trend reflecting changes of reactance and total impedance was observed. These in-vitro assays demonstrate that our 3D matrix can construct a biomimetic system for cell growth and analysis of cell surface interactions.
Keywords: MEA; Perfusion culture system; Degradation; 3D matrix; Impedance measurement;

Micro and macro rheology of planar tissues by Olga Lokshin; Yoram Lanir (3118-3127).
Tissues are intrinsically non-linear, anisotropic, viscoelastic, and undergo a process of mechanical adaptation (preconditioning). Previous constitutive laws considered one or two of these response aspects, often resulting in inadequate fit to data. Here we developed a general constitutive formulation encompassing the entire set of features. To exemplify this novel approach, constitutive equation for the skin was developed by stochastic incorporation of the fibers' orientation and undulation distributions. Predictions were contrasted with biaxial data of rabbit skin. The significance of each micro-feature was examined by sensitivity analysis. The results show that micro-structure based rheological characterization provides reliable representation under multiple biaxial protocols. Parametric investigation points to the essential roles of the fibers' orientation distributions (elastin and collagen) and waviness (collagen), their respective stress–strain relationship, and their viscoelasticity and preconditioning adaptation. The effect of ground substance is small but significant for model-to-data fit. Although the collagen is two order of magnitude stiffer, the contribution of elastin is predominant at low strains, and still significant (up to 20%) at high strains at which collagen carries the major load. The results are consistent with collagen preconditioning steming from stretch induced increase in the reference length, while in elastin it is the Mullins effect (strain softening). The most important impact of the study is that for the first time the entire scope of multi-axial tissue properties are unified in a single constitutive formulation. The potential implications are on the procedures of tissues characterization and on the design and analysis of artificial tissue scaffolds.
Keywords: Connective tissue; Constitutive modelling; Viscoelasticity; Preconditioning; Micro-structure; In vitro test;