Biomaterials (v.29, #18)
The role of cerium redox state in the SOD mimetic activity of nanoceria
by Eric G. Heckert; Ajay S. Karakoti; Sudipta Seal; William T. Self (pp. 2705-2709).
Cerium oxide nanoparticles (nanoceria) have recently been shown to protect cells against oxidative stress in both cell culture and animal models. Nanoceria has been shown to exhibit superoxide dismutase (SOD) activity using a ferricytochrome C assay, and this mimetic activity that has been postulated to be responsible for cellular protection by nanoceria. The nature of nanoceria's antioxidant properties, specifically what physical characteristics make nanoceria effective at scavenging superoxide anion, is poorly understood. In this study electron paramagnetic resonance (EPR) analysis confirms the reactivity of nanoceria as an SOD mimetic. X-ray photoelectron spectroscopy (XPS) and UV–visible analyses of nanoceria treated with hydrogen peroxide demonstrate that a decrease in the Ce 3+/4+ ratio correlates directly with a loss of SOD mimetic activity. These results strongly suggest that the surface oxidation state of nanoceria plays an integral role in the SOD mimetic activity of nanoceria and that ability of nanoceria to scavenge superoxide is directly related to cerium(III) concentrations at the surface of the particle.
Keywords: Free radical; Nanoparticle; Superoxide; Surface analysis; Cerium oxide
Favourable influence of hydrophobic surfaces on protein structure in porous organically-modified silica glasses
by Bouzid Menaa; Mar Herrero; Vicente Rives; Mayya Lavrenko; Daryl K. Eggers (pp. 2710-2718).
Organically-modified siloxanes were used as host materials to examine the influence of surface chemistry on protein conformation in a crowded environment. The sol–gel materials were prepared from tetramethoxysilane and a series of monosubstituted alkoxysilanes, RSi(OR′)3, featuring alkyl groups of increasing chain length in the R-position. Using circular dichroism spectroscopy in the far-UV region, apomyoglobin was found to transit from an unfolded state to a native-like helical state as the content of the hydrophobic precursor increased from 0 to 15%. At a fixed molar content of 5% RSi(OR′)3, the helical structure of apomyoglobin increased with the chain length of the R-group, i.e. methyln-propyl< n-butyl< n-hexyl. This trend also was observed for the tertiary structure of ribonuclease A, suggesting that protein folding and biological activity are sensitive to the hydrophilic/hydrophobic balance of neighboring surfaces. The observed changes in protein structure did not correlate with total surface area or the average pore size of the modified glasses, but scanning electron microscopy images revealed an interesting relationship between surface morphology and alkyl chain length. The unexpected benefit of incorporating a low content of hydrophobic groups into a hydrophilic surface may lead to materials with improved biocompatibility for use in biosensors and implanted devices.
Keywords: Sol–gel encapsulation; Circular dichroism; Hydrophobicity; Protein stability; Molecular confinement; Surface morphology
The slow resorption with replacement by bone of a hydrothermally synthesized pure calcium-deficient hydroxyapatite
by Takatoshi Okuda; Koji Ioku; Ikuho Yonezawa; Hideyuki Minagi; Yoshinori Gonda; Giichiro Kawachi; Masanobu Kamitakahara; Yasuaki Shibata; Hisashi Murayama; Hisashi Kurosawa; Tohru Ikeda (pp. 2719-2728).
A newly developed calcium-deficient hydroxyapatite composed of rod-shaped particles synthesized by the hydrothermal method (HHA) and stoichiometric hydroxyapatite (SHA) synthesized by the sintering method was used for in vivo implantation and in vitro culture systems to compare these biological responses. In the rabbit femur, implanted HHA was slowly resorbed and about 80% of the implant remained 24 weeks after implantation; however, up to 72 weeks after implantation, most of the implanted HHA was resorbed. The implanted SHA was unresorbed throughout the experimental period, but degradation by the invasion of newly formed bone was seen at 72 weeks after implantation. Bone histomorphometry showed that the volume of newly formed bone and the number of osteoclasts in the implanted region were significantly higher in HHA than in SHA 24 weeks after implantation. In vitro culture of C2C12 cells with the induction of osteoblastic phenotypes using recombinant bone morphogenetic protein-2 showed similar cell density and the induction of alkaline phosphatase activity between the cells on HHA and SHA discs. In vitro osteoclastogenesis of HHA and SHA discs using bone marrow macrophages and recombinant receptor activator of nuclear factor-κB ligand showed higher TRAP activity of osteoclasts cultured on HHA discs. These results showed that slow biodegradability did not always correlate to final replaceability in bone tissue, and suggested that the activity of osteoclasts correlated to the bone-forming activity of osteoblasts.
Keywords: Bone substitute; Biodegradation; Bone graft; Cell culture; Osteoclast
The influence of an in vitro generated bone-like extracellular matrix on osteoblastic gene expression of marrow stromal cells
by Quynh P. Pham; F. Kurtis Kasper; L. Scott Baggett; Robert M. Raphael; John A. Jansen; Antonios G. Mikos (pp. 2729-2739).
The function and development of cells rely heavily on the signaling interactions with the surrounding extracellular matrix (ECM). Therefore, a tissue engineering scaffold should mimic native ECM to recreate the in vivo environment. Previously, we have shown that an in vitro generated ECM secreted by cultured cells enhances the mineralized matrix deposition of marrow stromal cells (MSCs). In this study, MSC expression of 45 bone-related genes using real-time reverse transcriptase polymerase chain reaction (RT-PCR) was determined. Upregulation of osteoblastic markers such as collagen type I, matrix extracellular phosphoglycoprotein with ASARM motif, parathyroid hormone receptor, and osteocalcin, indicated that the MSCs on plain titanium scaffolds differentiated down the osteoblastic lineage and deposited a mineralized matrix on day 12. Significant mineralized matrix deposition was observed as early as day 4 on ECM-containing scaffolds and was associated with the enhancement in expression of a subset of osteoblast-specific genes that included a 2-fold increase in osteopontin expression at day 1 and a 6.5-fold increase in osteocalcin expression at day 4 as well as downregulation of chondrogenic gene markers. These results were attributed to the cellular interactions with growth factors and matrix molecules that are likely present in the in vitro generated ECM since the genes for insulin-like growth factor 1, insulin-like growth factor 2, vascular endothelial growth factor, dentin matrix protein, collagen type IV, cartilage oligomeric protein, and matrix metalloproteinase 13 were significantly upregulated during ECM construct generation. Overall, the data demonstrate that modulation of MSC differentiation occurs at the transcriptional level and gene expression of bone-related proteins is differentially regulated by the ECM. This study presents enormous implications for tissue engineering strategies, as it demonstrates that modification of a biomaterial with an in vitro generated ECM containing cell-generated bioactive signaling molecules can effectively direct gene expression and differentiation of seeded progenitor cell populations.
Keywords: Extracellular matrix; Gene expression; Growth factors; Osteoblast; Scaffold
Influence of cyclic strain and decorin deficiency on 3D cellularized collagen matrices
by Zannatul Ferdous; Luis D. Lazaro; Renato V. Iozzo; Magnus Höök; Kathryn J. Grande-Allen (pp. 2740-2748).
Cyclic strain evokes the expression of the small leucine-rich proteoglycans decorin and biglycan in 2D cultures and native tissues. However, strain-dependent expression of these proteoglycans has not been demonstrated in engineered tissues. We hypothesized that the absence of decorin may compromise the effect of cyclic strain on the development of engineered tissues. Thus, we investigated the contribution of decorin to tissue organization in cyclically strained collagen gels relative to statically cultured controls. Decorin null ( Dcn−/−) and wild-type murine embryonic fibroblasts were seeded within collagen gels and mechanically conditioned using a Flexcell® Tissue Train® culture system. After 8 days, the cyclically strained samples demonstrated greater collagen fibril density, proteoglycan content, and material strength for both cell types. On the other hand, increases in cell density, collagen fibril diameter, and biglycan expression were observed only in the cyclically strained gels seeded with Dcn−/− cells. Although cyclic strain caused an elevation in proteoglycan expression regardless of cell type, the type of proteoglycan differed between groups: the Dcn−/− cell-seeded gels produced an excess of biglycan not found in the wild-type controls. These results suggest that decorin-mediated tissue organization is strongly dependent upon tissue type and mechanical environment.
Keywords: Decorin; Collagen; Tissue engineering; Cyclic strain; Proteoglycan; Biglycan
The formation of pores in the basal lamina of regenerated renal tubules
by Annette Blattmann; Lucia Denk; Raimund Strehl; Hayo Castrop; Will W. Minuth (pp. 2749-2756).
Little information is available concerning the generation of renal tubules, but this information is urgently needed in regenerative medicine for the future treatment of acute and chronic renal failures. Of major interests are the integration of stem/progenitor cells, the cellular development and the tubular growth in a spatial environment. In this regard, we investigated the basal aspect of renal tubules generated at the interphase of an artificial interstitium. Stem/progenitor cells derived from neonatal rabbit kidney were mounted inside a specific tissue holder and covered by layers of polyester fleece. The tissue was then kept in a perfusion culture container for 13 days in chemically defined IMDM containing aldosterone (1×10−7m) as a tubulogenic factor. The spatial development of tubules was registered on whole-mount specimens and on cryo-sections labeled with soybean agglutinin (SBA) and tissue-specific antibodies indicating that collecting duct tubules were developed. Scanning electron microscopy (SEM) revealed that the generated tubules were completely covered by a basal lamina. Most interestingly, the matrix was not consistently composed, but exhibited three categories of pores. The most frequently found pore type had an apparent diameter of 133±26nm followed by a medium-sized pore type of 317±35nm. Another category of pores with a diameter of 605±101nm was rather rarely found. All of the pores were evenly distributed and not restricted to particular sites. The newly detected pores are not related to culture artifacts, since they were also detected in collecting duct tubules of the neonatal rabbit kidney. It remains to be evaluated whether these pores support physiological transport functions or if they indicate the site where extracellular matrix proteins are inserted into newly synthesized basal lamina.
Keywords: Tissue engineering; Perfusion culture; Kidney; Collecting duct; Tubule; Basal lamina
Three-dimensional extracellular matrix-directed cardioprogenitor differentiation: Systematic modulation of a synthetic cell-responsive PEG-hydrogel
by Thomas P. Kraehenbuehl; Prisca Zammaretti; André J. Van der Vlies; Ronald G. Schoenmakers; Matthias P. Lutolf; Marisa E. Jaconi; Jeffrey A. Hubbell (pp. 2757-2766).
We show that synthetic three-dimensional (3D) matrix metalloproteinase (MMP)-sensitive poly(ethylene glycol) (PEG)-based hydrogels can direct differentiation of pluripotent cardioprogenitors, using P19 embryonal carcinoma (EC) cells as a model, along a cardiac lineage in vitro. In order to systematically probe 3D matrix effects on P19 EC differentiation, matrix elasticity, MMP-sensitivity and the concentration of a matrix-bound RGDSP peptide were modulated. Soft matrices ( E=322±64.2Pa, stoichiometric ratio: 0.8), mimicking the elasticity of embryonic cardiac tissue, increased the fraction of cells expressing the early cardiac transcription factor Nkx2.5 around 2-fold compared to embryoid bodies (EB) in suspension. In contrast, stiffer matrices ( E=4036±419.6Pa, stoichiometric ratio: 1.2) decreased the number of Nkx2.5-positive cells significantly. Further indicators of cardiac maturation were promoted by ligation of integrins relevant in early cardiac development (α5β1, αvβ3) by the RGDSP ligand in combination with the MMP-sensitivity of the matrix, with a 6-fold increased amount of myosin heavy chain (MHC)-positive cells as compared to EB in suspension. This precisely controlled 3D culture system thus may serve as a potential alternative to natural matrices for engineering cardiac tissue structures for cell culture and potentially therapeutic applications.
Keywords: Extracellular matrix; Biomimetic hydrogel; Poly(ethylene glycol); Cardiac tissue engineering; P19 embryonal carcinoma cells; Stem cells
Preparation and characterization of pH- and temperature-sensitive pullulan microspheres for controlled release of drugs
by Gheorghe Fundueanu; Marieta Constantin; Paolo Ascenzi (pp. 2767-2775).
Most part of pH- and temperature-sensitive microspheres used for the controlled delivery of drugs are not biodegradable. Therefore, the aim of this work is to prepare pH- and temperature-sensitive microspheres from biodegradable and biocompatible natural polymers. Pullulan microspheres were prepared by suspension cross-linking with epichlorohydrin of an aqueous solution of the polymer. In order to confer them temperature sensitivity, poly( N-isopropylacrylamide- co-acrylamide) was grafted onto pullulan microspheres. Then, the pH-sensitive units (–COOH) were introduced by reaction between the remaining –OH groups of the pullulan with succinic anhydride. The grafted pullulan microspheres are more hydrophilic than pullulan microspheres, their swelling degree as well as water regain increase significantly. The thermo-sensitivity of the carboxylated microspheres depends to the number and the ionization form (–COOH/–COO−) of carboxylic groups. At a low exchange capacity (0.35meq/g), microspheres are thermo-sensitive both in the protonated and deprotonated form of –COOH groups. At a higher exchange capacity (2.25meq/g), microspheres are almost unswellable in the protonated form and swell extensively in the ionized form (up to 28 times than their dried form) loosing in a great extent the thermo-sensitive properties. In isotonic phosphate buffer pH=7.4, both thermo-sensitive and pH/thermo-sensitive microspheres possess a phase transition temperature close to that of the human body temperature. Loading and release profiles of lysozyme, taken as a molecular model system, were investigated.
Keywords: Intelligent microspheres; pH/thermo-responsive copolymers; Pullulan; Lower critical solution temperature; Drug deliveryAbbreviations; AAm; acrylamide; AIBN; N; ,; N; ′-azobisisobutyronitrile; CAN; cerium ammonium nitrate; CP; cloud point; 4-DMAP; 4-dimethylamino pyridine; IPB; isotonic phosphate buffer pH; =; 7.4; LCST; lower critical solution temperature; ms; microspheres; NIPAAm; N; -isopropylacrylamide; PNIPAAm; poly(; N; -isopropylacrylamide); Pul; pullulan; SA; succinic anhydride; SEM; scanning electron microscopy; VPTT; volume phase transition temperature
Glutathione-mediated release of functional plasmid DNA from positively charged quantum dots
by Dan Li; Gaiping Li; Weiwei Guo; Peicai Li; Erkang Wang; Jin Wang (pp. 2776-2782).
DNA was efficiently bound to water-soluble positively charged CdTe quantum dots (QDs) through complementary electrostatic interaction. These QDs–DNA complexes were disrupted and DNA was released by glutathione (GSH) at intracellular concentrations. Interestingly, there was almost no detectable DNA released by extracellular concentration of GSH. The formation of QDs–DNA complexes and GSH-mediated DNA release from the complexes were confirmed by dye displacement assay, electrophoretic mobility shift assay (EMSA), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) experiments. The released DNA retained transcriptional activity and expressed enhanced green fluorescent protein (EGFP) after being transfected into HEK 293 cells. The transfection efficiency measured by flow cytometry (FCM) was comparable with the positive control. The obvious difference of GSH concentration in nature between the intra- and extracellular environments as well as the GSH concentration-dependent triggered release suggests potential applications of these positively QDs in selective unpacking of payload in living cells in a visible manner.
Keywords: QDs; Glutathione; Plasmid DNA; Controlled release
Enhancement of poly(orthoester) microspheres for DNA vaccine delivery by blending with poly(ethylenimine)
by David N. Nguyen; Shyam S. Raghavan; Lauren M. Tashima; Elizabeth C. Lin; Stephen J. Fredette; Robert S. Langer; Chun Wang (pp. 2783-2793).
Poly(orthoester) (POE) microspheres have been previously shown to possess certain advantages for the in vivo delivery of DNA vaccines. In particular, timing of DNA release from POE microspheres in response to acidic phagosomal pH was shown to be an important factor in determining immunogenicity, which was hypothesized to be linked to the natural progression of antigen-presenting cell uptake, transfection, maturation, and antigen presentation. Here we report in vitro characterization of the enhanced efficacy of POE microspheres by blending poly(ethylenimine) (PEI), a well-characterized cationic transfection agent, into the POE matrix. Blending of a tiny amount of PEI (approximately 0.04wt%) with POE caused large alterations in POE microsphere properties. PEI provided greater control over the rate of pH-triggered DNA release by doubling the total release time of plasmid DNA and enhanced gene transfection efficiency of the microspheres up to 50-fold without any significant cytotoxicity. Confocal microscopy results of labeled PEI and DNA plasmids revealed that PEI caused a surface-localizing distribution of DNA and PEI within the POE microsphere as well as focal co-localization of PEI with DNA. We provide evidence that upon degradation, the microspheres of POE–PEI blends released electrostatic complexes of DNA and PEI, which are responsible for the enhanced gene transfection. Furthermore, blending PEI into the POE microsphere induced 50–60% greater phenotypic maturation and activation of bone marrow-derived dendritic cells in vitro, judged by the up-regulation of co-stimulatory markers on the cell surface. Physically blending PEI with POE is a simple approach for modulating the properties of biodegradable microspheres in terms of gene transfection efficiency and DNA release kinetics. Combined with the ability to induce maturation of antigen-presenting cells, POE–PEI blended microspheres may be excellent carriers for DNA vaccines.
Keywords: Polyorthoester; Microsphere; Immune response; Controlled drug release; Gene therapy
A sandwich structured SiO2/cytochrome c/SiO2 on a boron-doped diamond film electrode as an electrochemical nitrite biosensor
by Rong Geng; Guohua Zhao; Meichuan Liu; Mingfang Li (pp. 2794-2801).
A novel sandwich structured SiO2 gel/cytochrome c (Cyt c)/SiO2 gel was designed and constructed on conductive boron-doped diamond (BDD) film substrate. A SiO2 gel membrane was first in situ deposited on the pretreated positive charged H-terminated BDD electrode with a simple and artful surface vapor sol–gel method. Cyt c was subsequently immobilized onto the SiO2 membranes by electrostatic attraction, followed by another SiO2 gel layer in situ depositing on it. The SiO2 interlayer was conceived to play an important role in the resultant sandwich structured SiO2/Cyt c/SiO2/BDD electrode as a selective “semi-open” medium, which guaranteed the immobilized Cyt c to maintain high stability and perform good electrochemistry and biocatalysis responses. The bioactivity of Cyt c was well protected and the immobilized biomolecule even didn't denature at extremely high or low pH condition. More attractively, Cyt c in the sandwich structured electrode could be further oxidized into highly reactive Cyt c π-cation by two-step electrochemical oxidation, which could oxidize NO2− into NO3− in the solution. A sensitive determination approach of nitrite was accordingly built up based on this biocatalytic oxidative interaction for the first time and a possible mechanism of the interaction was herein proposed.
Keywords: Sandwich structured; Protein; Bioelectrocatalysis; Diamond; Nitrite
Three-dimensional polymer scaffolds for high throughput cell-based assay systems
by Ke Cheng; Yinzhi Lai; William S. Kisaalita (pp. 2802-2812).
Many whole cell-based assays in use today rely on flat, two-dimensional (2D) glass or plastic substrates that may not produce results characteristic of in vivo conditions. In this study, a three-dimensional (3D) cell-based assay platform was established by integrating 3D synthetic polymer scaffolds with standard cell culture dishes and multi-well plates. This technology can be used to feasibly modify any traditional 2D cell-based assay vessels for 3D cell-based assay with currently used high throughput screening (HTS) systems. We examined neural stem (NS) cells' growth profile, morphology, cell–matrix interaction, gene expression and voltage gated calcium channel (VGCC) functionality of this novel 3D assay platform. Our results showed that unlike the NS cells cultured on traditional 2D planar surfaces, cells in 3D scaffolds are more physiologically relevant with respect to in vivo characteristics exhibited by in-vivo surrogates such as neural spheres. This new biomimetic cell-based assay platform may provide a broadly applicable 3D cell-based system for use in drug discovery programs and other research fields.
Keywords: Biomimetic material; ECM; Cell culture; Scaffold; Stem cell
A synthetic amyloid lawn system for high-throughput analysis of amyloid toxicity and drug screening
by Koyeli Girigoswami; Sook Hee Ku; Jungki Ryu; Chan Beum Park (pp. 2813-2819).
Amyloid-β (Aβ) is the major constituent of senile plaques in the brains of Alzheimer's disease patients. In order to develop an efficient in vitro system for studying the interaction of cells with Aβ aggregates, we have prepared a synthetic amyloid lawn by immobilizing Aβ peptides over a functionalized glass surface and subsequently incubating the template in a fresh Aβ solution. On the top of different types of amyloid lawns (e.g. monomeric, oligomeric, and fibrillar), we cultivated PC12 cells, creating physical contacts between the cells and the lawns. Results indicated that cell viability was differentially affected when grown atop different Aβ lawns while cells were well adhered onto the surface of these Aβ lawns. The mode of cell death by Aβ lawn was confirmed to be apoptotic rather than necrotic, showing that cells undergo suicide by just contact with Aβ lawn. While conventional ‘solution-based’ methods for testing amyloid toxicity suffer from problems such as lot-to-lot variations, continued fibrillation, and heterogeneous population of aggregates, our ‘surface-based’ lawn system is suitable for high-throughput analysis of amyloid toxicity, which may enable high-throughput screening of potential drug candidates for treating amyloid diseases with the goal of reducing the cell death on the lawn.
Keywords: Self-assembly; Cell viability; Surface modification; Amyloid aggregation
Identification, recombinant production and structural characterization of four silk proteins from the Asiatic honeybee Apis cerana
by Jiahai Shi; Shixiong Lua; Ning Du; Xiangyang Liu; Jianxing Song (pp. 2820-2828).
Unlike silkworm and spider silks assembled from very large and repetitive fibrous proteins, the bee and ant silks were recently demonstrated to consist of four small and non-repetitive coiled-coil proteins. The design principle for this silk family remains largely unknown and so far no structural study is available on them in solution. The present study aimed to identify, express and characterize the Asiatic honeybee silk proteins using DLS, CD and NMR spectroscopy. Consequently, (1) four silk proteins are identified, with ∼6, 10, 9 and 8% variations, respectively, from their European honeybee homologs. Strikingly, their recombinant forms can be produced in Escherichia coil with yields of 10–60mg/l. (2) Despite containing ∼65% coiled-coil sequences, four proteins have very low α-helix (9–27%) but unusually high random coil (45–56%) contents. Surprisingly, β-sheet is also detected in four silk proteins (26–35%), implying the possible presence of β-sheet in the bee and ant silks. (3) Four proteins lacking of the tight tertiary packing appear capable of interacting with each other weakly but this interaction triggers no significant formation of the tight tertiary packing. The study not only implies the promising potential to produce recombinant honeybee silk proteins for the development of various biomaterials; but also provides the first structural insight into the molecular mechanism underlying the formation of the coiled-coil silks.
Keywords: Asiatic honeybee; Apis cerana; Silk protein; Coiled-coil; Recombinant biotechnology; Dynamic light scattering; Circular dichroism