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Protein engineering approaches to biomaterials design
Maskarinec, S. A. and D. A. Tirrell (2005), Curr Opin Biotechnol 16(4): 422-6.
Abstract: Biomaterials play crucial roles in reconstructive surgery, tissue engineering and regenerative medicine. Protein engineering offers powerful solutions to the challenges posed by the creation of well-defined, multifunctional materials that guide cell and tissue behavior. Especially challenging is the complex interplay between mechanical and biological properties in determining the success or failure of biomaterials designed for clinical use.

Protein kinase A gating of a pseudopodial-located RhoA/ROCK/p38/NHE1 signal module regulates invasion in breast cancer cell lines
Cardone, R. A., A. Bagorda, et al. (2005), Mol Biol Cell 16(7): 3117-27.
Abstract: Metastasis results from a sequence of selective events often involving interactions with elements of the tumor-specific physiological microenvironment. The low-serum component of this microenvironment confers increased motility and invasion in breast cancer cells by activating the Na+/H+ exchanger isoform 1 (NHE1). The present study was undertaken to characterize the signal transduction mechanisms underlying this serum deprivation-dependent activation of both the NHE1 and the concomitant invasive characteristics such as leading edge pseudopodia development and penetration of matrigel in breast cancer cell lines representing different stages of metastatic progression. Using pharmacological and genetic manipulation together with transport and kinase activity assays, we observe that the activation of the NHE1 and subsequent invasion by serum deprivation in metastatic human breast cells is coordinated by a sequential RhoA/p160ROCK/p38MAPK signaling pathway gated by direct protein kinase A phosphorylation and inhibition of RhoA. Fluorescence resonance energy transfer imaging of RhoA activity and immunofluorescence analysis of phospho-RhoA and NHE1 show that serum deprivation dynamically remodels the cell, forming long, leading edge pseudopodia and that this signal module is preferentially compartmentalized in these leading edge pseudopodia, suggesting a tight topographic relation of the signaling module to an invasion-specific cell structure.

Protein layer coating method on metal surface by electrochemical process through genetical introduced tag
Haruyama, T., T. Sakai, et al. (2005), Biomaterials 26(24): 4944-7.
Abstract: The technology of mercaptide self-assembly has previously been used to coat proteins onto metals, but is not practical for many surfaces. In this study, a short peptidic tag was employed for the electrochemical immobilization of proteins on metals (ECtag). A 6-mer histidine (alpha-amino-1H-imidazole-4-propionic acid) homopeptide was employed as an ECtag ligand, which forms coordinate bonds with Ni2+ and other divalent metal ions. Protein A was chosen as a model protein for the immobilization, and was genetically tagged with an ECtag for immobilization onto a Pt electrode surface through the reduction of ECtag:Ni2+ to ECtag:Ni by the electrode potential.

Protein structural deformation induced lifetime shortening of photosynthetic bacteria light-harvesting complex LH2 excited state
Chen, X. H., L. Zhang, et al. (2005), Biophys J 88(6): 4262-73.
Abstract: Photosynthetic bacterial light-harvesting antenna complex LH2 was immobilized on the surface of TiO(2) nanoparticles in the colloidal solution. The LH2/TiO(2) assembly was investigated by the time-resolved spectroscopic methods. The excited-state lifetimes for carotenoid-containing and carotenoidless LH2 have been measured, showing a decrease in the excited-state lifetime of B850 when LH2 was immobilized on TiO(2). The possibility that the decrease of the LH2 excited-state lifetime being caused by an interfacial electron transfer reaction between B850 and the TiO(2) nanoparticle was precluded experimentally. We proposed that the observed change in the photophysical properties of LH2 when assembled onto TiO(2) nanoparticles is arising from the interfacial-interaction-induced structural deformation of the LH2 complex deviating from an ellipse of less eccentric to a more eccentric ellipse, and the observed phenomenon can be accounted by an elliptical exciton model. Experiment by using photoinactive SiO(2) nanoparticle in place of TiO(2) and core complex LH1 instead of LH2 provide further evidence to the proposed mechanism.

Protein-acrylamide copolymer hydrogels for array-based detection of tyrosine kinase activity from cell lysates
Brueggemeier, S. B., D. Wu, et al. (2005), Biomacromolecules 6(5): 2765-75.
Abstract: We describe the development of an array-based assay for the molecular level detection of tyrosine kinase activity directly from cellular extracts. Glutathione S-transferase-Crkl (GST-Crkl) fusion proteins are covalently immobilized into polyacrylamide gel pads via copolymerization of acrylic monomer and acrylic-functionalized GST-Crkl protein constructs on a polyacrylamide surface. The resulting hydrogels resist nonspecific protein adsorption, permitting quantitative and reproducible determination of Abl tyrosine kinase activity and inhibition, even in the presence of a complex cell lysate mixture. Half-maximal inhibition (IC50) values for imatinib mesylate inhibition of GST-Crkl (SH3) phosphorylation by v-Abl in a purified system and Bcr-Abl within a K562 cell lysate were determined to be 1.5 and 20 microM, respectively. Additionally, the protein-acrylamide copolymer arrays detected CML cell levels as low as 15% in a background of Bcr-Abl- leukemic cells and provided the framework for the parallel evaluation of six tyrosine kinase inhibitors. Such a system may have direct application to the detection and treatment of cancers resulting from upregulated tyrosine kinase activity, such as chronic myeloid leukemia (CML). These findings also establish a basis for screening tyrosine kinase inhibitors and provide a framework on which protein-protein interactions in other complex systems can be studied.

Protein-calcium carbonate coprecipitation: a tool for protein encapsulation
Petrov, A. I., D. V. Volodkin, et al. (2005), Biotechnol Prog 21(3): 918-25.
Abstract: A new approach of encapsulation of proteins in polyelectrolyte microcapsules has been developed using porous calcium carbonate microparticles as microsupports for layer-by-layer (LbL) polyelectrolyte assembling. Two different ways were used to prepare protein-loaded CaCO3 microparticles: (i) physical adsorption--adsorption of proteins from the solutions onto preformed CaCO3 microparticles, and (ii) coprecipitation--protein capture by CaCO3 microparticles in the process of growth from the mixture of aqueous solutions of CaCl2 and Na2CO3. The latter was found to be about five times more effective than the former (approximately 100 vs approximately 20 mug of captured protein per 1 mg of CaCO3). The procedure is rather mild; the revealed enzymatic activity of alpha-chymotrypsin captured initially by CaCO3 particles during their growth and then recovered after particle dissolution in EDTA was found to be about 85% compared to the native enzyme. Core decomposition and removal after assembly of the required number of polyelectrolyte layers resulted in release of protein into the interior of polyelectrolyte microcapsules (PAH/PSS)5 thus excluding the encapsulated material from direct contact with the surrounding. The advantage of the suggested approach is the possibility to control easily the concentration of protein inside the microcapsules and to minimize the protein immobilization within the capsule walls. Moreover, it is rather universal and may be used for encapsulation of a wide range of macromolecular compounds and bioactive species.

Protein-like molecular architecture: biomaterial applications for inducing cellular receptor binding and signal transduction
Fields, G. B., J. L. Lauer, et al. (1998), Biopolymers 47(2): 143-51.
Abstract: The development of biomaterials with desirable biocompatibility has presented a difficult challenge for tissue engineering researchers. First and foremost, materials themselves tend to be hydrophobic and/or thrombogenic in nature, and face compatibility problems upon implantation. To mediate this problem, researchers have attempted to graft protein fragments onto biomaterial surfaces to promote endothelial cell attachment and minimize thrombosis. We envisioned a novel approach, based on the capability of biomolecules to self-assemble into well-defined and intricate structures, for creating biomimetic biomaterials that promote cell adhesion and proliferation. One of the most intriguing self-assembly processes is the folding of peptide chains into native protein structures. We have developed a method for building protein-like structural motifs that incorporate sequences of biological interest. A lipophilic moiety is attached onto a N alpha-amino group of peptide chain, resulting in a "peptide-amphiphile." The alignment of amphiphilic compounds at the lipid-solvent interface is used to facilitate peptide alignment and structure initiation and propagation, while the lipophilic region absorbs to hydrophobic surfaces. Peptide-amphiphiles containing potentially triple-helical or alpha-helical structural motifs have been synthesized. The resultant head group structures have been characterized by CD spectroscopy and found to be thermally stable over physiological temperature ranges. Triple-helical peptide-amphiphiles have been applied to studies of surface modification and cell receptor binding. Cell adhesion and spreading was promoted by triple-helical peptide-amphiphiles. Cellular interaction with the type IV collagen sequence alpha 1(IV) 1263-1277 increased signal transduction, with both the time and level of induction dependent upon triple-helical conformation. Collectively, these results suggest that peptide-amphiphiles may be used to form stable molecular structure on biomaterial surfaces that promote cellular activities and improve biocompatibility.

Protein-mediated macrophage adhesion and activation on biomaterials: a model for modulating cell behavior
Kao, W. J., J. A. Hubbell, et al. (1999), J Mater Sci Mater Med 10(10/11): 601-5.
Abstract: The elucidation of proteins involved in biomaterial-modulated macrophage behavior is critical for the improvement of material performance and the initial exploration of material design capable of manipulating macrophage function for tissue engineering. In this paper, several in vitro and in vivo techniques are presented to demonstrate means of delineating a part of the complex molecular mechanisms involved in the interaction between biomaterial and macrophage adhesion and phenotypic development. The following conclusions were reached: (1) using radioimmunoassay, complement component C3 was found to be critical in mediating human macrophage adhesion on polyurethanes. (2) The presence of a diphenolic antioxidant additive in polyurethanes increased the propensity for complement upregulation but did not affect adherent macrophage density. (3) The subcutaneous cage-implant system was utilized to delineate interleukin-4 participation in the fusion of adherent macrophages to form foreign body giant cells in vivo in mice. The injection of purified interleukin-4 neutralizing antibody into the implanted cages significantly decreased the giant cell density; conversely, the giant cell density was significantly increased by the injection of recombinant interleukin-4 when compared with the controls. (4) The RGD and PHSRN amino acid sequences of the central cell binding domain and the PRRARV sequence of the C-terminal heparin binding domain of human plasma fibronectin were utilized to study the structure-functional relationship of protein in mediating macrophage behavior. Polyethyleneglycol-based networks grafted with the RGD-containing peptide supported higher adherent human macrophage density than surfaces grafted with other peptides. The formation of foreign body giant cell was highly dependent on the relative orientation between PHSRN and RGD domains located in a single peptide.

Proteomic analysis of protein adsorption: serum amyloid P adsorbs to materials and promotes leukocyte adhesion
Kim, J. K., E. A. Scott, et al. (2005), J Biomed Mater Res A 75(1): 199-209.
Abstract: Serum and plasma protein adsorption on materials was analyzed using gel electrophoresis and ion trap mass spectrometry. Following incubation of polypropylene, polyethylene terephthalate (PET), or polydimethylsiloxane (PDMS) with 5% serum for longer than 4 h, we found unexpectedly high amounts of the pentraxin serum amyloid P. It was previously shown that serum amyloid P is constitutively expressed in humans, functions as an opsonin, and interacts with the Fcgamma receptors on leukocytes. We demonstrate that serum amyloid P adsorbed to tissue culture polystyrene, PDMS, and PET promotes the adhesion of granulocytes and monocytes in the presence of calcium. The methods developed for these studies may be useful for the large-scale study of protein adsorption and do not rely on radiolabeling or the availability of antibodies.

Proteomic profiling of erythrocyte proteins by proteolytic digestion chip and identification using two-dimensional electrospray ionization tandem mass spectrometry
Tyan, Y. C., S. B. Jong, et al. (2005), J Proteome Res 4(3): 748-57.
Abstract: Self-assembled monolayers (SAMs) on coinage metal provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition, and other interfacial phenomena. The bonding of enzyme to SAMs of alkanethiols onto gold surfaces is exploited to produce an enzyme chip. In this work, the attachment of trypsin to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water soluble N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as coupling agent. A two-dimensional liquid-phase separation scheme coupled with mass spectrometry is presented for proteomic analysis of erythrocyte proteins. The application of proteomics, particularly with reference to analysis of proteins, will be described. Surface analyses have revealed that the X-ray Photoelectron Spectroscopy (XPS) C1s and N1s core levels illustrate the immobilization of trypsin. These data are also in good agreement with Fourier Transformed Infrared Reflection-Attenuated Total Reflection (FTIR-ATR) spectra for the peaks at Amide I and Amide II. Using two-dimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nano-HPLC-ESI-MS/MS) system observations, analytical results have demonstrated the erythrocyte proteins digestion of the immobilized trypsin on the functionalized SAMs surface. For such surfaces, it also shows the enzyme digestion ability of the immobilized trypsin. The experiment results revealed the identification of 272 proteins from erythrocyte protein sample. The terminal groups of the SAMs structure can be further functionalized with biomolecules or antibodies to develop surface-base diagnostics, biosensors, or biomaterials.

Pseudo 3D single-walled carbon nanotube film for BSA-free protein chips
Byon, H. R., B. J. Hong, et al. (2005), Chembiochem 6(8): 1331-4.

Pseudomonas fluorescens 134 as a biological control agent (BCA) model in cell immobilization technology
Russo, A., M. Basaglia, et al. (2005), Biotechnol Prog 21(1): 309-14.
Abstract: Antifungal activity against Rhizoctonia solani was achieved in vivo through the application of Pseudomonas fluorescens strain 134 encapsulated in sodium alginate beads of different sizes (0.5, 1, and 2 mm). The activity was compared to that obtainable with chemical treatments and bead-derived liquid formulations. The latter was obtained by dissolving alginate beads of 1 and 0.5 mm in 1% Na-citrate solution before application, without any significant (P < 0.05) reduction of bacterial numbers during the dissolution process. The dry bead formulations were applied next to the seeds in plant inoculation experiments, resulting in a reduction of disease symptoms, which were markedly reduced when the liquid formulation was applied. Moreover, the rate of disease symptoms related to liquid formulations from both 1 and 0.5 mm beads was comparable (near to 10%) to that of chemical treatment. Pseudomonas fluorescens strain 134 delivered as both dry and liquid formulations was able to colonize cotton root at a population density of about 10(8) CFU/g fresh root, 15 days after sowing.

Pulmonary emboli from blood-biomaterial interaction
Coleman, J. E., K. Ramberg, et al. (1988), ASAIO Trans 34(3): 882-6.
Abstract: The problem of surface thrombosis and subsequent embolization remains entrenched as a yet incompletely surmounted barrier to the development of truly satisfactory intravascular prosthetic devices. A baboon ex vivo shunt was used to determine the interaction of Indium-111 platelets and potential biomaterials. The uptake of Indium-111 platelets was monitored continuously by gamma camera scanning. Several of the materials tested demonstrated a saw-toothed pattern of platelet activity, with accumulation followed by rapid decline. Neither PTFE nor Dacron exhibited this pattern. Post shunt scans of the animals' chests showed discrete foci of platelet activity in the lungs, corresponding to each embolic event noted on the material's scan. In conclusion, the search for a smooth surface as a blood material interface may produce a material which accumulates and then sloughs significant platelet aggregates. It is crucial that these materials be subjected to vigorous testing to determine their safety prior to initiation of clinical trials.

Pulmonary inflammation caused by chitosan microparticles
Huang, Y. C., A. Vieira, et al. (2005), J Biomed Mater Res A 75(2): 283-7.
Abstract: Chitosan is a cationic biopolymer derived from chitin with potential therapeutic applications such as controlled drug delivery to mucosal-epithelial surfaces in the body. Inhaled chitosan microparticles (CM), for example, are of potential interest in pulmonary pharmacotherapy. In this context, we examine some basic reactions of lung tissue to CM. Inhaled CM (2-10 mg/kg of particles) induce dose-dependent proinflammatory effects in rat lungs; these effects are documented in increases in bronchoalveolar lavage fluid protein (BALF-P) and lactate dehydrogenase activity (BALF-LDH) and increases in lung tissue myeloperoxidase (MPO) activity and leukocyte migration. Overall, the biochemical parameters (i.e., average of BALF-P, BALF-DH, and MPO) indicate that the inflammation response is 1.8-fold greater than controls without CM; the same inflammation parameters, however, are 1.9-fold lower with CM compared with the proinflammatory effects of lipopolysaccharide (LPS). Cytological examination of BALF shows a large infiltration of polymorphonuclear neutrophils to lung tissue: more than a sixfold increase in this population of inflammatory cells, after inhalation of CM relative to air inhalation controls. Thus, the results indicate that inhaled CM can have significant proinflammatory effects on lung tissues; these effects are mild relative to LPS but need to be considered in the context of therapeutic applications via pulmonary delivery if such concentrations of CM are used.

Pulpal responses following direct pulp capping of healthy dog teeth with dentine adhesive systems
Koliniotou-Koumpia, E. and D. Tziafas (2005), J Dent 33(8): 639-47.
Abstract: OBJECTIVE: The aim of the present study was to evaluate the pulpal responses following direct pulp capping of mechanically exposed teeth with new dentine adhesive systems, in the preclinical model of dog teeth. METHODS: Class V cavities (approximately 2.50 mm wide, 3.00 mm long, 1.5-2.0 mm deep) were prepared on the buccal surface of permanent maxillary and mandibulary molars, two rooted premolars, canines and third incisors. The cavities were assigned to five experimental groups, representing one control group treated with a Ca(OH)2-based material and four experimental groups where the adhesive systems Clearfil SE Bond, Prompt-L-Pop, Etch & Prime 3.0 and Single Bond were tested. The pulpal tissue responses to dentine adhesives were assessed at post-operative periods of 7, 21, 65 days. RESULTS: Variable responses were recorded, which were characterized by moderate to severe inflammatory reactions, progressive extension of tissue necrosis with time and total absence of continuous hard tissue bridge formation after pulp capping with each of the four adhesive systems. Application of a Ca(OH)2-based material was characterized by inflammatory cell infiltration, limited tissue necrosis as well as partial to complete hard tissue bridging. CONCLUSIONS: It seems evident that application of dentine adhesive systems in direct contact with the mechanically exposed pulp of healthy dog teeth cannot lead to acceptable repair of the dentine-pulp complex e.g. wound healing with tertiary dentine bridge formation.

Pure and filler free silicone rubber biomaterial; a new concept in preparing silicone rubber
Chawla, A. S. (1976), J Bioeng 1(1): 51-3.

Purification of PA700, the 19S regulatory complex of the 26S proteasome
DeMartino, G. N. (2005), Methods Enzymol 398: 295-306.
Abstract: The 26S proteasome is a 2,400,000-Da protease complex that selectively degrades proteins modified by polyubiquitin chains. The 26S proteasome is composed of two 700,000-Da multisubunit complexes: the 20S proteasome, which serves as the proteolytic core of the complex, and PA700, an ATPase regulatory complex responsible for the binding, modification, and delivery of substrates to the proteolytic chamber. Thus, PA700 mediates multiple functions essential for ubiquitin-dependent proteolysis by the 26S proteasome. This chapter reviews briefly the structure and function of PA700, details the methodology for its large-scale purification from mammalian tissues, and describes a simple functional PA700 assay based on the stimulation of proteasome activity.

Purification of polymeric biomaterials
Wandrey, C. and D. S. Vidal (2001), Ann N Y Acad Sci 944: 187-98.
Abstract: Employing a combined filtration and precipitation method, the endotoxin concentration in sodium alginate (SA) and sodium cellulose sulfate (SCS) was reduced to a value of 200 EU/g polymer. This is one tenth of the regulatory threshold calculated, for example, for an appropriate bioartificial pancreas that consists of approximately 420,000 encapsulated islets of Langerhans. The low endotoxin (ET) levels were maintained below this threshold during a six-month storage period. The purification procedure of the polymers did not negatively influence the final microcapsule properties. The mechanical stability of microcapsules from purified material is even slightly higher than that of microcapsules from the original polymers. A second approach to avoid endotoxin release from the device is its direct complexation during the bead or capsule formation process. The durability of endotoxin binding in binary, ternary, and quaternary complexes could be demonstrated for storage in culture medium and saline. Very low total endotoxin release from the complexes was detected after three months in culture medium and five months in saline. This complexation is primarily based on electrostatic interactions with the participating cationic components and provides additional security for the final bioartificial organ or delivery device.

Pyogenic granuloma following silicone punctal plugs: report of two cases
Musadiq, M., S. Mukherji, et al. (2005), Orbit 24(2): 149-51.
Abstract: PURPOSE: To report two cases of pyogenic granuloma following silicone punctal plugs. METHODS: Interventional case reports. RESULTS: The authors report two cases of pyogenic granuloma secondary to silicone punctal plugs and their lacrimal sequelae following surgical removal in one case and conservative treatment with removal of the plug alone in another. The former case developed no lacrimal sequelae while the second case developed punctal scarring. Spontaneous loss of plugs was common to both the cases. CONCLUSIONS: Patients with punctal plugs should be informed about plug-related problems and encouraged to report them.

QCM-D studies of attachment and differential spreading of pre-osteoblastic cells on Ta and Cr surfaces
Modin, C., A. L. Stranne, et al. (2006), Biomaterials 27(8): 1346-54.
Abstract: The quartz crystal microbalance with dissipation (QCM-D) technique was employed to characterize initial cell adhesion in terms of attachment and spreading of pre-osteoblastic MC3T3-E1 cells on Ta and Cr surfaces. Evaluation of initial cell adhesion established a correlation between input cell number and the shifts in frequency (f) and dissipation (D). The f-shift was found to be much larger in serum-free medium as compared to a medium including serum; hence, initial cell adhesion was subsequently evaluated in serum-free medium. During the first hour of adhesion, we found a positive correlation between the QCM-D f-shift and the average area of the spread cells, as measured by cryo-scanning electron microscopy (cryo-SEM). Finally, the QCM-D technique was used to study cell adhesion on different metal oxide surfaces. Initial cell adhesion on Ta was found to induce a larger f-shift as compared to Cr, indicating larger spreading of cells on Ta. Cryo-SEM data confirmed that spreading of cells on Cr was on average only two-thirds the spreading on Ta. Our results demonstrate that the QCM-D technique is a versatile technique to quickly distinguish initial cell-surface interactions on different biomaterials.

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Last Modified: 8 February 2006