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Biomaterials (v.29, #5)

Editorial board (pp. co2).

Molecular simulation of protein adsorption and desorption on hydroxyapatite surfaces by J.-W. Jia-Wei Shen; Tao Wu; Qi Wang; H.-H. Hai-Hua Pan (pp. 513-532).
Protein adsorption and desorption on material surfaces play a key role in the biocompatibility of medical implants, biomineralization and protein separation. In this report, the adsorption and desorption behavior of the 10th type III module of fibronectin (FN-III10) with different orientations on hydroxyapatite (HAP) (001) surface were systematically studied by molecular dynamics (MD) and steered MD simulations. These studies show that the electrostatic energy plays a dominant role in the interaction between the model protein and the HAP surface. The values of the interaction energy not only relates to the number of adsorbed sites but also the type. The charged –COO and –NH3+ are the strongest groups that interact with the surface, while other groups like charged guanido group, neutral amino and hydroxyl groups have considerable interactions with the surface. The effects of these groups on interaction energy were quantitatively investigated.

Keywords: Fibronectin; Hydroxyapatite; Protein adsorption and desorption; Cell adhesion; RGD peptide


The rheological properties of silated hydroxypropylmethylcellulose tissue engineering matrices by Ahmed Fatimi; Francois Tassin Jean François Tassin; Sophie Quillard; Monique A.V. Axelos; Pierre Weiss (pp. 533-543).
This paper describes the rheological properties of silated hydroxypropylmethylcellulose (HPMC-Si) used in biomaterials domain as a three-dimensional synthetic matrix for tissue engineering. The HPMC-Si is an HPMC grafted with 3-glycidoxypropyltrimethoxysilane (GPTMS). HPMC and HPMC-Si were studied. It is shown that although silanization reduces the hydrodynamic volume in dilute solution, it does not affect significantly the rheological behavior of the concentrated solutions. The HPMC-Si viscous solution (pH 12.8) cross-links by decreasing the pH using an acid buffer, since HPMC-Si solution transforms into an elastic state. The kinetics of cross-linking and final elastic properties is influenced by several parameters such as polymer concentration, pH and temperature. pH and temperature play an important role in the silanol condensation, mainly responsible for network formation. The study of the gelation process revealed the dependence of the final concentration of HPMC-Si hydrogel on cross-linking kinetics and viscoelastic properties. The percolation theory was applied to determine gel point and to discuss the dependence of storage ( G′) and loss ( G″) moduli on frequency. Results showed that both G′ and G″ exhibit a power-law behavior with an exponent (0.68) which extends over the entire frequency range. This method is the only one to characterize the time where a liquid viscous phase shifts to hydrogel with elastic properties. In this case it was about 23min for a final pH of 7.4.

Keywords: Biomaterials; Tissue engineering; Cellulosic derivative; Silanol; Rheological properties; Gelation


Silicon-induced DNA damage pathway and its modulation by titanium plasma immersion ion implantation by Jiang Jiang; Kaifu Huo; Zhengwei Wu; Shaopeng Chen; Shihao Pu; Zengliang Yu; Xuanyong Liu; Paul K. Chu (pp. 544-550).
Micronuclei tests (MNT) using the Chinese Hamster Ovary (CHO) cells and Chinese Hamster Ovary Mutant cells (XRS5) have been conducted to evaluate the biocompatibility of silicon and titanium plasma-implanted silicon. Pure Si induces high MN ratios of the two cell lines and thus has poor biocompatibility. The MN ratio of CHO cells is higher than background by about 44% and the MN ratio of XRS5 cells is even higher by about 180%, suggesting that most of the cellular DNA damages on the Si wafer are DNA double-strand breaks (DSB) and are efficiently repaired by the nonhomologous end-joining (NHEJ) pathway. The surface biocompatibility of Si can be enhanced by Ti plasma immersion ion implantation (PIII). The altered oxidized species on the Ti plasma-implanted surface block cellular DSB repaired by the NHEJ pathway and decrease the MN ratio of XRS5 cells. By increasing the Ti implantation time and consequently the Ti implant fluence, the oxygen binding energy shifts toward a lower energy and the intensity of the Si peaks corresponding to SiO2 continually diminishes and even disappear. At the same time, the MN ratios of the two cell lines decrease. Our results suggest that the rest of the DNA damages which cannot be repaired by the NHEJ pathway may be blocked because the surface bonding changes from predominantly Si–O on the 10min Ti-implanted Si to Ti–O on the 120min Ti-implanted Si. Our results also suggest that the genotoxicity of cell assay such as MNT and DSB is a valid method to investigate biocompatibility.

Keywords: Si wafers; Titanium; Plasma immersion ion implantation; Micronuclei; DNA-double strand breaks; Biocompatibility


Complement activation on surfaces modified with ethylene glycol units by Yusuke Arima; Mitsuaki Toda; Hiroo Iwata (pp. 551-560).
Poly(ethylene glycol) (PEG) has been widely used for the preparation of biomedical devices and drug carriers to reduce the interaction of proteins with artificial materials. However, unanticipated body reactions such as hypersensitivity reactions caused by PEG-modified surfaces have been reported. Body reactions to PEG-modified materials still remain unclear. In this study, complement activation on surfaces modified with ethylene glycol units was examined. Two surfaces modified with tri(ethylene glycol)-terminated alkanethiol (HS-TEGOH) and methoxy-terminated PEG-thiol (HS-mPEG) were employed as model surfaces. Complement activation was assessed by the binding of an antibody against complement C3b after exposure to diluted human serum using a surface plasmon resonance (SPR) apparatus. Strong complement activation was observed on HS-TEGOH but not on HS-mPEG surfaces. The terminal hydroxyl group of HS-TEGOH is involved in complement activation. Although the HS-mPEG surface just after preparation did not induce complement activation, it became a stronger activator with storage period under room light at room temperature. Complement activation on the HS-mPEG surface was further accelerated by irradiation with UV light. These results suggest that functional groups which can activate the complement system are introduced onto the HS-mPEG surface by oxidation during long-term storage and UV irradiation.

Keywords: Complement; Polyethylene oxide; Protein adsorption; Surface modification; Surface analysis


Functionality of endothelial cells on silk fibroin nets: Comparative study of micro- and nanometric fibre size by Borys Bondar; Sabine Fuchs; Antonella Motta; Claudio Migliaresi; Charles J. Kirkpatrick (pp. 561-572).
Biomimetic material design, such as mimicking nanostructured components of the extracellular matrix, is an actual challenge for biomaterial research with a high impact on tissue engineering and regenerative medicine. Thus, understanding the cellular response at the cell biological and molecular level and the consequences of various chemically or physically modified biomaterials is highly important. In the present study we assessed the response of human umbilical vein endothelial cells (HUVEC) and outgrowth endothelial cells (OEC) from endothelial progenitor cells to different variants of nanofibrous silk fibroin nets in comparison to microfibrous silk fibroin scaffolds with regard to cellular morphology, proliferation, formation of intercellular contacts as well as integrin-dependent adhesion. Endothelial cells (ECs) grown on nanometric nets formed a differentiated and interconnected endothelial monolayer with no significant changes in the expression of intercellular contact molecules or proliferation rates compared with cells grown on micrometric nets. Nevertheless, quantitative real-time PCR revealed a higher expression level of integrin- β1 in ECs grown on nanofibrous fibroin nets compared to the microfibrous samples. In addition, single nano-fibres were recognised by the integrin-receptor mechanism supporting the formation of focal adhesion at the interface of ECs and nanometric nets. These findings indicate that the nanometric silk fibroin scaffolds did not interfere with the formation of a differentiated and interconnected EC layer. On the contrary, nanofibre variation of the fibroin net architecture induced changes in ECs at the molecular level in terms of the increased expression of adhesion molecules such as integrin- β1.

Keywords: Nanotopography; Endothelial cells; Adhesion molecule; Stem cell; Integrins; Biomimetic material


Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro by Aditya V. Vashi; Efthimia Keramidaris; Keren M. Abberton; Wayne A. Morrison; Jeremy L. Wilson; Andrea J. O’Connor; Justin J. Cooper-White; Erik W. Thompson (pp. 573-579).
Due to increasing clinical demand for adipose tissue, a suitable scaffold for engineering adipose tissue constructs is needed. In this study, we have developed a three-dimensional (3-D) culture system using bone marrow-derived mesenchymal stem cells (BM-MSC) and a Pluronic F-127 hydrogel scaffold as a step towards the in vitro tissue engineering of fat. BM-MSC were dispersed into a Pluronic F-127 hydrogel with or without type I collagen added. The adipogenic differentiation of the BM-MSC was assessed by cellular morphology and further confirmed by Oil Red O staining. The BM-MSC differentiated into adipocytes in Pluronic F-127 in the presence of adipogenic stimuli over a period of 2 weeks, with some differentiation present even in absence of such stimuli. The addition of type I collagen to the Pluronic F-127 caused the BM-MSC to aggregate into clumps, thereby generating an uneven adipogenic response, which was not desirable.

Keywords: Adipose tissue engineering; Thermally responsive material; Copolymer; Cell culture; Lipid; In vitro; test


Strong biofilm production, antibiotic multi-resistance and high gelE expression in epidemic clones of Enterococcus faecalis from orthopaedic implant infections by Carla Renata Arciola; Lucilla Baldassarri; Davide Campoccia; Roberta Creti; Valter Pirini; Johannes Huebner; Lucio Montanaro (pp. 580-586).
Enterococcus faecalis is an opportunistic pathogen, which today represents one of the leading aetiologic agents of nosocomial infections and, increasingly, of implant infections. Here, in a collection of 43 E. faecalis isolated from implant orthopaedic infections, virulence-related phenotypes (biofilm and gelatinase production) and genotypes ( gelE and esp) were studied to characterize epidemic clones identified and grouped by ribotyping. The presence of the esp gene and a marked and steady biofilm formation ability appeared to be the features associated with the clonal spreading, as well as a conspicuous gelatinase production, whereas the simple presence of gelE appeared non-specific of the epidemic clones. Antibiotic multi-resistance and strong biofilm production abilities together with a high phenotypic expression of gelatinase are an important equipment of E. faecalis to colonize peri-prosthesis tissues and to spread out as causative agents of implant orthopaedic infections.

Keywords: Enterococcus faecalis; Biofilm; esp; gelE; Antibiotic resistance; Ribotyping


In vivo wound healing of diabetic ulcers using electrospun nanofibers immobilized with human epidermal growth factor (EGF) by Ji Suk Choi; Kam W. Leong; Hyuk Sang Yoo (pp. 587-596).
Biodegradable polymers were electrospun and recombinant human epidermal growth factor (EGF) was immobilized on the electrospun nanofibers for the purpose of treating diabetic ulcers. Amine-terminated block copolymers composed of poly( ε-caprolactone) [PCL] and poly(ethyleneglycol) [PEG] and PCL were electrospun to biocompatible nanofibers with functional amine groups on the surface via PEG linkers. EGF was chemically conjugated to the surface of the nanofibers. The conjugation amount of EGF on the nanofibers was quantitated by X-ray photoelectron scattering. Human primary keratinocytes were cultivated on EGF-conjugated nanofibers in order to investigate the effect of EGF nanofibers on the differentiation of keratinocytes. Wound healing effects of the EGF nanofibers were confirmed in diabetic animals with dorsal wounds. The expression of keratinocyte-specific genes significantly increased with application of EGF-conjugated nanofibers. The EGF-nanofibers exerted superior in vivo wound healing activities compared to control groups or EGF solutions. Furthermore, immunohistochemical-staining results showed that EGF-receptor (EGFR) was highly expressed in the EGF nanofiber group. This study showed that EGF-conjugated nanofiber could potentially be employed as a novel wound healing material by increasing proliferation and phenotypic expression of keratinocytes.

Keywords: Wound healing; Nanofibers; EGF; Diabetic ulcers; Electrospinning


A stimulus-responsive contrast agent for ultrasound molecular imaging by Mark A. Borden; Hua Zhang; Robert J. Gillies; Paul A. Dayton; Katherine W. Ferrara (pp. 597-606).
Complement activation by targeting ligands is an important issue that governs the fate of targeted colloidal contrast agents for molecular imaging. Here, we extend previous work on a stimulus-responsive microbubble construct, in which the ligand is normally buried by a polymeric overbrush and transiently revealed by ultrasound radiation force, to show reduced complement activation and focused adhesion to cells using a physiological peptide ligand. Attachment of C3/C3b in vitro and production of soluble C3a anaphylotoxin in vitro and in vivo decreased significantly for the buried-ligand architecture vs. the conventional exposed-ligand motif and no-ligand control. Additionally, the buried-ligand architecture prevented adhesion of Arg–Gly–Tyr (RGD)-bearing microbubbles to integrin-expressing human umbilical vein endothelial cells (HUVEC) when driven by buoyancy in a static chamber, but it did not affect adhesion efficiency when activated by ultrasound radiation force pulses. These results show, for the first time, the molecular mechanism for reduced immunogenicity for the buried-ligand architecture and feasibility of targeting with this motif using a physiological ligand–receptor pair.

Keywords: Molecular imaging; RGD peptide; Angiogenesis; Complement; Immune response


Dual-role self-assembling nanoplexes for efficient gene transfection and sustained gene delivery by Ankit Agarwal; Robert C. Unfer; Surya K. Mallapragada (pp. 607-617).
Novel cationic pentablock copolymers with poly(diethylamino ethyl methacrylate) blocks covalently attached to parent triblock Pluronic copolymers have been designed and developed as sustained release non-viral gene delivery vectors. These copolymers electrostatically condense plasmid DNA into nanostructures (nanoplexes) and further self-assemble above critical concentration to form thermoreversible hydrogels at physiological temperatures. Unlike other sustained gene delivery systems of non-ionic copolymers that release naked DNA, hydrogels of pentablock copolymer/DNA nanoplexes dissolve in excess buffers to release DNA compacted inside the nanoplexes. These hydrogels permit aqueous pharmaceutical formulations that do not involve organic solvents and are non-invasively injectable with syringes into localized tissues where they instantly form hydrogels in situ. The hydrogels were found to have better mechanical strength than Pluronic gels. Hydrogels of nanoplexes containing 15wt% copolymer dissolved to release nanoplexes up to 5 days in vitro, compared to rapid release of up to 90% entrapped naked DNA from only Pluronic gels by day 1. The release profile of the nanoplexes from the hydrogels could be modulated by changing the concentration of copolymer or plasmid DNA in the hydrogel formulation. Since DNA is electrostatically bound to copolymer molecules, it does not freely diffuse out of the polymeric network, preventing initial release bursts observed with other such controlled release gels/matrices/microspheres. The released nanoplexes were colloidally stable, preserved the integrity of supercoiled plasmid DNA, and gave good transfection efficiencies in vitro upon dissolution. These novel copolymers, thus, act as both nanoscale gene delivery vectors and macroscale sustained delivery agents, and make a clinically viable long-term sustained gene delivery system.

Keywords: Gene delivery; Controlled release; Non-viral vectors; Hydrogels; Polymer


Relevance of bi-functionalized polyelectrolyte multilayers for cell transfection by Florent Meyer; Maria Dimitrova; Justyna Jedrzejenska; Youri Arntz; Pierre Schaaf; Benoit Frisch; J.-C. Jean-Claude Voegel; Joelle Ogier (pp. 618-624).
In an effort to develop new biomaterial coatings, it was shown that polyelectrolyte multilayers constitute a very powerful tool to render surfaces biologically active. The challenge is to multi-functionalize surfaces in a controlled way. We show here, for the first time, that it is possible to functionalize multilayer films simultaneously with two molecules acting in totally different ways on cells, namely plasmid DNA (pDNA), pre-complexed with poly(ethyleneimine) (PEI), and a peptide molecule, NDPMSH. This peptide, grafted to poly(l-glutamic acid) (PGA) was used as a signal molecule for melamona cells B16-F1 and for its ability to enhance gene delivery in a receptor-independent manner. The PGA–NDPMSH chains are embedded in poly-(allylamine hydrochloride)/poly-(sodium 4-styrene sulfonate) multilayers and the pDNA–PEI complexes are deposited on top of the films. It is shown that melanoma cells (B16-F1) are efficiently transfected after 24h of contact with functionalized films. When brought in contact with Huh-7 cells that do not express the peptide receptors, these films trigger significantly the transfection rate, showing that it is possible to enhance the transfection process by incorporating specific peptides into multilayer films. Moreover, transfected cells sorted by flow cytometry produce melanin, demonstrating both activation via the peptide signaling pathway and cell transfection.

Keywords: Polyelectrolyte multilayers; Transfection; Polyplexes; α; -MSH; Melanocytes

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