Biomaterials (v.30, #11)
Biomimetic strategies based on viruses and bacteria for the development of immune evasive biomaterials
by Matthew T. Novak; James D. Bryers; William M. Reichert (pp. 1989-2005).
The field of biomaterials has begun to focus upon materials strategies for modulating the immune response. While certain approaches appear promising, they are currently limited to isolated facets of inflammation process. It is well documented that both bacteria and viruses have highly developed methods for evading the immune system, providing inspiration for a more biomimetic approach to materials design. This review presents the immune evasive tactics employed by viruses and bacteria, and offers suggestions for future directions that apply these principles to design of immune evasive biomaterials.
Keywords: Inflammation; Wound healing; Immunomodulation; Bacteria; Biomimetic material
TEGDMA-induced oxidative DNA damage and activation of ATM and MAP kinases
by Alexander Eckhardt; Nicol Gerstmayr; Karl-Anton Hiller; Carola Bolay; Claudia Waha; Gianrico Spagnuolo; Carlos Camargo; Gottfried Schmalz; Helmut Schweikl (pp. 2006-2014).
The development of strategies for the protection of oral tissues against the adverse effects of resin monomers is primarily based on the elucidation of underlying molecular mechanisms. The generation of reactive oxygen species beyond the capacity of a balanced redox regulation in cells is probably a cause of cell damage. This study was designed to investigate oxidative DNA damage, the activation of ATM, a reporter of DNA damage, and redox-sensitive signal transduction through mitogen-activated protein kinases (MAPKs) by the monomer triethylene glycol dimethacrylate (TEGDMA). TEGDMA concentrations as high as 3–5mm decreased THP-1 cell viability after a 24h and 48h exposure, and levels of 8-oxoguanine (8-oxoG) increased about 3- to 5-fold. The cells were partially protected from toxicity in the presence of N-acetylcysteine (NAC). TEGDMA also induced a delay in the cell cycle. The number of THP-1 cells increased about 2-fold in G1 phase and 5-fold in G2 phase in cultures treated with 3–5mm TEGDMA. ATM was activated in THP-1 cells by TEGDMA. Likewise, the amounts of phospho-p38 were increased about 3-fold by 3mm TEGDMA compared to untreated controls after a 24h and 48h exposure period, and phospho-ERK1/2 was induced in a very similar way. The activation of both MAPKs was inhibited by NAC. Our findings suggest that the activation of various signal transduction pathways is related to oxidative stress caused by a resin monomer. Signaling through ATM indicates oxidative DNA damage and the activation of MAPK pathways indicates oxidative stress-induced regulation of cell survival and apoptosis.
Keywords: Dental resin; TEGDMA; DNA oxidation; ATM; p38; ERK1/2
The use of combinatorial topographical libraries for the screening of enhanced osteogenic expression and mineralization
by Jette Lovmand; Jeannette Justesen; Morten Foss; Rune Hoff Lauridsen; Michael Lovmand; Charlotte Modin; Flemming Besenbacher; Finn Skou Pedersen; Mogens Duch (pp. 2015-2022).
Nano- and microstructured surfaces are known to impact on the binding and differentiation of cells, but the detailed basic understanding of the underlying regulatory mechanisms is still scarce, which impedes the rational design of smart biomaterials. Towards a comprehensive analysis of the interplay between topographical parameters such as feature design and lateral and vertical dimensions we here report on a combinatorial screening approach, BioSurface Structure Array (BSSA) of test squares each with a distinct topography. Using such BSSA libraries of 504 topographically distinct surface structures, we have identified combinations of size, gap and height of structures which enhance mineralization as well as the expression of osteogenic markers of a preosteoblastic murine cell line. This generic BSSA screening platform is a versatile technology for the systematic identification of surfaces with specific biological properties, and it may for example be useful for optimizing the design of biomaterials for regulating cellular behaviour.
Keywords: Biocompatibility; Cell culture; Microstructure; Surface topography
The temporal and spatial dynamics of microscale collagen scaffold remodeling by smooth muscle cells
by Yonggang Pang; Areck A. Ucuzian; Akie Matsumura; Eric M. Brey; Andrew A. Gassman; Vicki A. Husak; Howard P. Greisler (pp. 2023-2031).
Smooth muscle cells (SMCs) and collagen scaffolds are widely used in vascular tissue engineering but their interactions in remodeling at the microscale level remained unclear. We characterized microscale morphologic alterations of collagen remodeled by SMCs in six dimensions: three spatial, time, multichannel and multi-position dimensions. In live imaging assays, computer-assisted cell tracking showed locomotion characteristics of SMCs; reflection and fluorescent confocal microscopy and spatial reconstruction images of each time point showed detailed morphologic changes of collagen fibers and spatial collagen–SMC interactions. The density of the collagen around the SMCs was changed dynamically by the leading edges of the cells. The density of the collagen following 24h of cell-induced remodeling increased 51.61±9.73% compared to unremodeled collagen containing cells for 1h ( P<0.0001, n=40) (NS vs. collagen without cells). Fast Fourier transform analysis showed that the collagen fibers' orientation changed from random (alignment index=0.047±0.029, n=40) after 1h into concordant with that of the SMCs (alignment index=0.379±0.098, P<0.0001, n=40) after 24h. Mosaic imaging extended the visual field from a single cell to a group of cells in one image without loss of optical resolution. Direct visualization of alignment of actin fibers and collagen fibers showed the molecular machinery of the process of scaffold remodeling. This is a new approach to better understanding the mechanism of scaffold remodeling and our techniques represent effective tools to investigate the interactions between cells and scaffold in detail at the microscale level.
Keywords: Hydrogel; Collagen; Smooth muscle cell; Arterial tissue engineering; Scaffold; Confocal microscopy
The effect of an rhBMP-2 absorbable collagen sponge-targeted system on bone formation in vivo
by Rick Visser; Pilar M. Arrabal; Jose Becerra; Ursula Rinas; Manuel Cifuentes (pp. 2032-2037).
Reparation of bone defects remains a major clinical and economic concern, with more than 3 million bone grafts performed annually only in the United States and the EU. The search for alternatives to autologous bone grafting led to the approval by the FDA of an absorbable collagen carrier combined with rhBMP-2 for the treatment of certain bone diseases and fractures. The present work is focused on the production of a collagen-targeted rhBMP-2 based system to improve bone formation. We produced a modified rhBMP-2 with only an additional collagen-binding decapeptide derived from the von Willebrand factor and tested its affinity to collagen and its ability to induce ectopic bone formation in vivo when implanted in combination with absorbable collagen sponges or hydroxyapatite. The results showed not only that the rhBMP2–CBD had an increased affinity to collagen, but also that this binding was very stable during a prolonged period of time. In vivo experiments demonstrated that this rhBMP2–CBD maintained its osteoinductive activity, being capable of inducing new bone formation even at lower concentrations than native rhBMP-2. These results indicate that the combination of the fusion protein with absorbable collagen may be a suitable and safer alternative to rhBMP-2 for bone repair purposes.
Keywords: BMP (bone morphogenetic protein); Collagen; Bone tissue engineering; Osteogenesis; Recombinant protein
The stimulation of myoblast differentiation by electrically conductive sub-micron fibers
by Indong Jun; Sungin Jeong; Heungsoo Shin (pp. 2038-2047).
Myotubes assemble with bundles of myofibers to form the structural units in skeletal muscle. Therefore, myotube formation plays an important role in restoring muscular functions, and substrates to promote the differentiation of myoblasts to myotubes need to be developed for muscle tissue engineering. In this study, we developed electrically conductive composite fibers of poly(l-lactide-co-ɛ-caprolactone) (PLCL) blended with polyaniline (PANi) using an electrospinning method, and then investigated the effect of these composite fibers on the differentiation of myoblasts. The prepared PLCL/PANi fibers showed no significant difference in fiber diameter or contact angle, regardless of the incorporation of PANi. The fibers containing 30% PANi (PLCL/PANi-30) maintained elastic properties of maximum elongation at break (160±14.4%). The composite fibers were cytocompatible, as the DNA content on each fiber was similar for up to 8 days of C2C12 myoblast culture. After 4 days of culture, the number of cells positive for sarcomeric myosin was 3.6-times greater on the electrically conductive fibers (21±1 and 19±2 for PLCL/PANi-15 and -30 fibers, respectively) than on the PLCL/PANi-0 fibers (6±2). Furthermore, the level of myogenin expression detected on day 8 of culture on PLCL/PANi-15 was approximately 1.6-fold greater than the PLCL/PANi-0 fibers. Similar results were observed for the expression of other genes including troponin T (2-fold greater) and the myosin heavy chain gene (3-fold greater). These results indicate that electrically conductive substrates can modulate the induction of myoblasts into myotube formation without additional electrical stimulation, suggesting that these fibers may have potential as a temporary substrate for skeletal tissue engineering.
Keywords: Electrospinning; Electrically conductive fibers; Myoblasts; Proliferation; Differentiation
Guided growth of neurons and glia using microfabricated patterns of parylene-C on a SiO2 background
by Evangelos Delivopoulos; Alan F. Murray; Nikki K. MacLeod; John C. Curtis (pp. 2048-2058).
This paper describes a simple technique for the patterning of glia and neurons. The integration of neuronal patterning to Multi-Electrode Arrays (MEAs), planar patch clamp and silicon based ‘lab on a chip’ technologies necessitates the development of a microfabrication-compatible method, which will be reliable and easy to implement. In this study a highly consistent, straightforward and cost effective cell patterning scheme has been developed. It is based on two common ingredients: the polymer parylene-C and horse serum. Parylene-C is deposited and photo-lithographically patterned on silicon oxide (SiO2) surfaces. Subsequently, the patterns are activated via immersion in horse serum. Compared to non-activated controls, cells on the treated samples exhibited a significantly higher conformity to underlying parylene stripes. The immersion time of the patterns was reduced from 24 to 3h without compromising the technique. X-ray photoelectron spectroscopy (XPS) analysis of parylene and SiO2 surfaces before and after immersion in horse serum and gel based eluant analysis suggests that the quantity and conformation of proteins on the parylene and SiO2 substrates might be responsible for inducing glial and neuronal patterning.
Keywords: Cell adhesion; Fibronectin; Neural network; Patterning; Protein adsorption; XPS
The development of photochemically crosslinked native fibrinogen as a rapidly formed and mechanically strong surgical tissue sealant
by Christopher M. Elvin; Alan G. Brownlee; Mickey G. Huson; Tracy A. Tebb; Misook Kim; Russell E. Lyons; Tony Vuocolo; Nancy E. Liyou; Timothy C. Hughes; John A.M. Ramshaw; Jerome A. Werkmeister (pp. 2059-2065).
We recently reported the generation of a highly elastic, crosslinked protein biomaterial via a rapid photochemical process using visible light illumination. In light of these findings, we predicted that other unmodified, tyrosine-rich, self-associating proteins might also be susceptible to this covalent crosslinking method. Here we show that unmodified native fibrinogen can also be photochemically crosslinked into an elastic hydrogel biomaterial through the rapid formation of intermolecular dityrosine. Photochemically crosslinked fibrinogen forms tissue sealant bonds at least 5-fold stronger than commercial fibrin glue and is capable of producing maximum bond strength within 20s. In vitro studies showed that components of the photochemical crosslinking reaction are non-toxic to cells. This material will find useful application in various surgical procedures where rapid curing for high strength tissue sealing is required.
Keywords: Fibrinogen; Tissue sealant; Photochemical crosslinking; Dityrosine; Ruthenium; Biomaterial
The toxicokinetics and distribution of 2-hydroxyethyl methacrylate in mice
by J. Durner; H. Kreppel; J. Zaspel; H. Schweikl; R. Hickel; Franz X. Reichl (pp. 2066-2071).
The cytotoxicity of dental composites has been attributed to the release of residual monomers from polymerized resin-based composites due to the degradation processes or the incomplete polymerisation of materials. 2-Hydroxyethyl methacrylate (HEMA) is one of the major components released from dental resin-based composites. It was shown in vitro that HEMA was released into the adjacent biophase from such materials during the first days after placement. In this study uptake, distribution, and excretion of 14C-HEMA applied via gastric tube or subcutaneous administration at dose levels well above those encountered in dental care were examined in mice to test the hypothesis that HEMA can reach cytotoxic levels in mammalian tissues. 14C-HEMA was taken up rapidly from the stomach and intestines after gastric administration and was widely distributed in the body following administration by each route. Most 14C was excreted within one day as14CO2. Two metabolic pathways of 14C-HEMA can be described. The peak HEMA levels in all tissues examined after 24h were lower than known toxic levels. Therefore the study did not support the hypothesis.
Keywords: Dental restorative material; Biocompatibility; Biodegradation; Composite
The adsorption and lubrication behavior of synovial fluid proteins and glycoproteins on the bearing-surface materials of hip replacements
by Marcella Roba; Marco Naka; Emanuel Gautier; Nicholas D. Spencer; Rowena Crockett (pp. 2072-2078).
The selectivity of synovial fluid protein adsorption onto ultra-high molecular weight polyethylene (UHMWPE) and alumina (Al2O3), and in particular the ability of glycoproteins to adsorb in the presence of all the other synovial fluid proteins, was investigated by means of fluorescence microscopy and gel electrophoresis (SDS-PAGE). The non-specific nature of protein adsorption from synovial fluid indicated that the lubrication of artificial hip-joint materials may not be attributable to a single protein as has been frequently suggested. The friction behavior of polyethylene (PE) sliding against Al2O3 in solutions of bovine serum albumin (BSA), alpha-1-acid glycoprotein (AGP) and alpha-1-antitrypsin (A1AT) was investigated by means of colloidal probe atomic force microscopy. BSA was shown to be a poorer boundary lubricant than the phosphate buffered saline used as a control. This was attributed to denaturation of the BSA upon adsorption, which provided a high-shear-strength layer at the interface, impairing the lubrication. Interestingly, both the glycoproteins AGP and A1AT, despite their low concentrations, improved lubrication. The lubricating properties of AGP and A1AT were attributed to adsorption via the hydrophobic backbone, allowing the hydrophilic carbohydrate moieties to be exposed to the aqueous solution, thus providing a low-shear-strength fluid film that lubricated the system. The amount of glycoprotein adsorbed on hydrophobic surfaces was determined by means of optical waveguide lightmode spectroscopy (OWLS), allowing conclusions to be drawn about the conformation of the glycan residues following adsorption.
Keywords: Synovial fluid; Hip implants; Lubrication; Glycoproteins; Adsorption; Alumina
Periprosthetic tissue reactions observed at revision of total intervertebral disc arthroplasty
by Ilona M. Punt; Jack P.M. Cleutjens; Thorvald de Bruin; Paul C. Willems; Steven M. Kurtz; Lodewijk W. van Rhijn; Geert Willem H. Schurink; André van Ooij (pp. 2079-2084).
Wear, wear particle induced inflammation, and osteolysis following total disc arthroplasty were, until recently, not thought to be present due to limited intervertebral motion and the lack of a synovial membrane between the lower lumbar vertebrae. The purpose of this study was to evaluate the periprosthetic tissue reactions associated with total disc arthroplasty revision surgery. Periprosthetic samples of fibrous tissue were collected in all patients during revision surgery of SB Charité III disc prostheses. Revision was indicated for intractable pain after an average of 8 years. Histological evaluation was performed in tissue samples of 16 patients using light microscopy and polarized light microscopy with a magnification of 100×. Polyethylene particles were detected in 15 of 16 patients. The smallest particles were the most numerate. A positive correlation was present between the number of particles per mm2 and the extent of the chronic inflammatory reaction in the periprosthetic fibrous tissue. Osteolysis was observed in one patient. In the tissue samples containing polyethylene particles, TNF-α and IL-6 were determined by immunohistochemistry. TNF-α and IL-6 were co-expressed as a subset of mononuclear macrophages and giant cells.
Keywords: Total disc arthroplasty; Charité; Lumbar spine; Polyethylene wear; Inflammation
Nanostructured biocomposite substrates by electrospinning and electrospraying for the mineralization of osteoblasts
by Deepika Gupta; J. Venugopal; S. Mitra; V.R. Giri Dev; S. Ramakrishna (pp. 2085-2094).
Nanotechnology has enabled the engineering of nanostructured materials to meet current challenges in bone replacement therapies. Biocomposite nanofibrous scaffolds of poly(l-lactic acid)- co-poly( ɛ-caprolactone), gelatin and hydroxyapatite (HA) were fabricated by combining the electrospinning and electrospraying techniques in order to create a better osteophilic environment for the growth and mineralization of osteoblasts. Electrospraying of HA nanoparticles on electrospun nanofibers helped to attain rough surface morphology ideal for cell attachment and proliferation and also achieve improved mechanical properties than HA blended nanofibers. Nanofibrous scaffolds showed high pore size and porosity up to 90% with fiber diameter in the range of 200–700nm. Nanofibrous scaffolds were characterized for their functional groups and chemical structure by FTIR and XRD analysis. Studies on cell–scaffold interaction were carried out by culturing human fetal osteoblast cells (hFOB) on both HA blended and sprayed PLACL/Gel scaffolds and assessing their growth, proliferation, mineralization and enzyme activity. The results of MTS, ALP, SEM and ARS studies confirmed, not only did HA sprayed biocomposite scaffolds showed better cell proliferation but also enhanced mineralization and alkaline phosphatase activity (ALP) proving that electrospraying in combination with electrospinning produced superior and more suitable biocomposite nanofibrous scaffolds for bone tissue regeneration.
Keywords: Electrospinning; Electrospraying; Biocomposite; Hydroxyapatite; Mineralization; Bone tissue engineering
The use of electron beam lithographic graft-polymerization on thermoresponsive polymers for regulating the directionality of cell attachment and detachment
by Naokazu Idota; Takahiko Tsukahara; Kae Sato; Teruo Okano; Takehiko Kitamori (pp. 2095-2101).
A simple process for nano-patterned cell culture substrates by direct graft-polymerization has been developed using an electron beam (EB) lithography system requiring no photo-masks or EB-sensitive resists. The compound N-isopropylacrylamide (IPAAm) was locally polymerized and grafted directly by EB lithographic exposure onto hydrophilic polyacrylamide (PAAm)-grafted glass surfaces. The size of the surface grafted polymers was controlled by varying the area of EB dose, and a minimal stripe pattern with a 200nm line-width could be fabricated onto the surface. On the stripe-patterned surfaces, above the lower critical solution temperature (LCST), the cells initially adhered and spread with an orientation along the pattern direction. The magnitude of the spreading angle and elongation of adhered cells depended on the pattern intervals of the grafted PIPAAm. When culture temperature was lower than the LCST, cultured cells detached from the surfaces with strong shrinkage along the pattern direction, and sometimes folded and became parallel with the stripe pattern. This patterned cell recovery technique may be useful for the construction of muscle cell sheets with efficient shrinkage/relaxation in a specific direction and spheroidal 3D cell structures, with application to tissue engineering and microfluidic cellular devices.
Keywords: Electron beam; Polymerization; Thermally responsive material; Cell patterning; Cell morphology
A nanoscale drug-entrapment strategy for hydrogel-based systems for the delivery of poorly soluble drugs
by Mei-Chin Chen; Hung-Wen Tsai; Chin-Tang Liu; Shu-Fen Peng; Wei-Yun Lai; Shiang-Jiuun Chen; Yen Chang; Hsing-Wen Sung (pp. 2102-2111).
The hydrophilic nature of hydrogel matrices makes them disadvantageous to entrap poorly soluble therapeutic agents and greatly restricts their applications as drug-delivery systems. In this study, we demonstrated that sustained delivery of lipophilic drugs in hydrogel-based devices can be readily achieved by enhancing retention of drugs within micelles. This nanoscale drug-entrapment strategy was applied to develop a polymeric drug-eluting stent. Sirolimus, a lipophilic anti-proliferative/immunosuppressive drug, was entrapped into the hydrophobic core of Pluronic L121 micelles and then blended in a chitosan-based strip and crosslinked by an epoxy compound to fabricate test stents. It was found that the use of such a nanoscale drug-entrapment strategy was able to significantly increase the loading efficiency of lipophilic drugs, prevent the drug from aggregation and beneficially reduce its initial burst release; thus, the duration of drug release was extended considerably. When implanting the stent in rabbit infrarenal abdominal aortas, in-stent restenosis was markedly reduced and less inflammatory reaction was observed, while unfavorable effects such as delayed endothelial healing caused by the overdose of sirolimus could be significantly evaded.
Keywords: Hydrogel; Drug-eluting stent; Micelle; Drug delivery; Chitosan
Poly(amidoamine) dendrimer–drug conjugates with disulfide linkages for intracellular drug delivery
by Yunus E. Kurtoglu; Raghavendra S. Navath; Bing Wang; Sujatha Kannan; Robert Romero; Rangaramanujam M. Kannan (pp. 2112-2121).
Understanding and improving drug release kinetics from dendrimer–drug conjugates are key steps to improve their in vivo efficacy. N-Acetyl cysteine (NAC) is an anti-inflammatory agent with significant potential for clinical use in the treatment of neuroinflammation, stroke and cerebral palsy. There is a need for delivery of NAC which can enhance its efficacy, reduce dosage and prevent it from binding plasma proteins. For this purpose, a poly(amidoamine) dendrimer–NAC conjugate that contains a disulfide linkage was synthesized and evaluated for its release kinetics in the presence of glutathione (GSH), cysteine (Cys), and bovine serum albumin (BSA) at both physiological and lysosomal pH. The results indicate that the prepared conjugate can deliver ∼60% of its NAC payload within 1h at intracellular GSH concentrations at physiological pH, whereas the conjugate did not release any drug at plasma GSH levels. The stability of the conjugate in the presence of bovine serum albumin at plasma concentrations was also demonstrated. The efficacy of the dendrimer–NAC conjugate was measured in activated microglial cells (target cells in vivo) using the reactive oxygen species (ROS) assay. The conjugates showed an order of magnitude increase in antioxidant activity compared to free drug. When combined with intrinsic and ligand-based targeting with dendrimers, these types of GSH sensitive nanodevices can lead to improved drug release profiles and in vivo efficacy.
Keywords: Dendrimers; PAMAM dendrimers; Neuroinflammation; N; -Acetyl cysteine; Intracellular drug delivery; Glutathione-sensitive release
The influence of the sequential delivery of angiogenic factors from affinity-binding alginate scaffolds on vascularization
by Inbar Freeman; Smadar Cohen (pp. 2122-2131).
This study describes the features of tissue-engineering scaffold capable of sequentially delivering three angiogenic factors. The scaffold consists of alginate-sulfate/alginate, wherein vascular endothelial growth factor (VEGF) platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) are bound to alginate-sulfate with an affinity similar to that realized upon their binding to heparin. Factor release rate from the scaffold was correlated with the equilibrium binding constants of the factors to the matrix, thus enabling the sequential delivery of VEGF, PDGF-BB and TGF-β1. In alginate scaffolds lacking alginate-sulfate, release of the adsorbed proteins was instantaneous. After subcutaneous implantation for 1 and 3 months in rats, the blood vessel density and percentage of mature vessels were 3-fold greater in the triple factor-bound scaffolds than in the factor-adsorbed or untreated scaffolds. Moreover, vascularization within the triple factor-bound scaffolds was superior to that found in scaffolds delivering only basic fibroblast growth factor. Application of this novel scaffold may be extended to the combined delivery of additional heparin-binding angiogenic factors or combinations of growth factors active in different tissue regeneration processes.
Keywords: Alginate-sulfate; Alginate scaffolds; Angiogenic factors; Sequential delivery; Tissue engineering; Vascularization
Conducting polymers for electrochemical DNA sensing
by Hui Peng; Lijuan Zhang; Christian Soeller; Jadranka Travas-Sejdic (pp. 2132-2148).
Conducting polymers (CPs) are a class of polymeric materials that have attracted considerable interest because of their unique electronic, chemical and biochemical properties, making them suitable for numerous applications such as energy storage, memory devices, chemical sensors, and in electrocatalysis. Conducting polymer-based electrochemical DNA sensors have shown applicability in a number of areas related to human health such as diagnosis of infectious diseases, genetic mutations, drug discovery, forensics and food technology due to their simplicity and high sensitivity. This review paper summarizes the advances in electrochemical DNA sensing based on conducting polymers as active substrates. The various conducting polymers used for DNA detection, along with different DNA immobilization and detection methodologies are presented. Current trends in this field and newly developed applications due to advances in nanotechnology are also discussed.
Keywords: Conducting polymers; Electrochemical DNA sensor; Electropolymerization
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