Biomedical subjects

Buddy D Ratner

Publications and source records attributed to Buddy D Ratner.

At least 37 records · Page 2Linked to original sources

PubMed2005

Preparative soft and reactive landing of multiply charged protein ions on a plasma-treated metal surface.

Soft landing on a plasma-treated metal surface of multiply protonated protein ions from the gas phase results in a substantial retention of protein function, as demonstrated for trypsin and streptavidin. The majority of trypsin ions soft-landed at hyperthermal kinetic energies are undamaged and retain 72-98% of enzymatic activity after being washed into solution. A small fraction of trypsin ions that were landed at nominal kinetic energies of 130-200 eV remain tethered to the surface and show approximately 50% enzymatic activity. The streptavidin tetramer is found to dissociate to monomer units upon multiple charging in electrospray. The majority of soft-landed monomers can be washed into solution where they show affinity to biotin. The layer of streptavidin monomer that is immobilized on the surface can be detected if fluorescence-tagged and retains the ability to reversibly bind biotin. A mechanism is proposed to explain nondestructive protein ion discharge on the surface that considers proton migration from the soft-landed cations to the metal oxide layer and metal ion reduction by electron transfer from the bulk metal.

Biotin↗

PubMed2005

Controlling osteopontin orientation on surfaces to modulate endothelial cell adhesion.

Osteopontin (OPN) is an important extracellular matrix protein that has been shown to impact wound healing, inflammation, and the foreign body reaction, and has been identified as a potential surface component for engineered biomaterials. OPN contains the arginine-glycine-aspartic acid (RGD) moiety that has been shown to mediate cell adhesion through interactions with integrins. In its preferred orientation and conformation on a surface, the functional domains of OPN will be presented to cells to the greatest extent. However, control of protein orientation and conformation is still challenging. In this work, we investigated OPN adsorption and cell adhesion to the OPN layer on self-assembled monolayers (SAMs) of alkanethiols terminated with various functional groups and on a gold surface. The four SAM terminal groups studied were --CH3, --OH, --NH2, and --COOH, representing hydrophobic, hydrophilic but neutral, positively charged, and negatively charged surfaces, respectively. Surface plasmon resonance biosensor and atomic force microscopy were used to characterize the adsorption of OPN on these surfaces. An in vitro cell adhesion assay of bovine aortic endothelial cells was performed to test the functionality of OPN on various SAMs. Surface plasmon resonance results showed that the amount of protein adsorbed on the --NH2 surface is close to a monolayer and similar to that on the --COOH surface, consistent with the atomic force microscopy results. However, based on cell adhesion experiments, both cell count and average cell spreading area on the --NH2 surface are much higher than those on the --COOH surface. From these results, it is suggested that the orientation and conformation of OPN on a positively charged --NH2 surface is more favorable for cell adhesion and spreading than on a negatively charged --COOH surface. The surface coverage of bovine aortic endothelial cells on the surfaces studied decreased in the following order: --NH2 > Au > --CH3 > --COOH > --OH whereas the mean cell spreading area decreased in the following order: --NH2 > Au > --CH3 > --COOH. Our studies show that surface properties will alter OPN behavior on surfaces, thus influencing cell interactions.

Adsorption↗

PubMed2005

The role of osteopontin in foreign body giant cell formation.

Foreign body giant cells (FBGCs) are a hallmark of the foreign body reaction caused by biomaterial implantation and are thought to contribute to biomaterial degradation and the duration of the response. Osteopontin (OPN) is a secreted, acidic matricellular protein with multiple phosphorylation sites that is highly expressed at sites of inflammation. OPN wildtype and knockout mice were implanted with poly(vinyl alcohol) sponges and explanted at 14 days. OPN knockout mice had more foreign body giant cells but fewer macrophages surrounding the implants than their wildtype counterparts. In an in vitro human FBGC assay, addition of soluble OPN was found to reduce macrophage fusion to giant cells. These are the first studies to show a direct inhibitory role of OPN in FBGC formation in response to implantation.

Animals↗

PubMed2005

Surface characterization of the extracellular matrix remaining after cell detachment from a thermoresponsive polymer.

The temperature-responsive behavior of poly(N-isopropyl acrylamide) (pNIPAM) directly affects the attachment and detachment of cells cultured on these surfaces. At culture temperatures, cells behave similarly to those on tissue culture polystyrene (TCPS), while at room temperature, cells cultured on pNIPAM spontaneously detach as a confluent sheet. In comparison, cells grown on TCPS remain attached indefinitely after the temperature drop, requiring enzymatic or mechanical removal. In this work, we present an examination of the response of bovine aortic endothelial cells (BAECs) and extracellular matrix (ECM) proteins to plasma polymerized NIPAM (ppNIPAM) surfaces using X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and immunostaining. Immunoassay results reveal that, although fibronectin, laminin, and collagen closely associate with the cell sheet, some collagen may be associated with the surface, as well. Our XPS results indicate that ppNIPAM surfaces after cell liftoff differ from their blank counterparts, the primary distinction being the presence of amide and alcohol species on ppNIPAM surfaces used for cell culture, possibly owing to the presence of a proteinaceous film. Finally, a comparison between ppNIPAM-treated surfaces used for cell culture versus control surfaces by principal component analysis of the ToF-SIMS data confirms that the surfaces differ; the presence of molecular ion fragments from amino acids (e.g., alanine, glycine, and proline) is the chief reason for this difference. Therefore, from our surface characterization of ppNIPAM-coated TCPS after cell liftoff, we conclude that although low-temperature liftoff of the BAEC monolayer is accompanied by the majority of the components of the ECM, some of the ECM proteins still remain at the surface.

Acrylic Resins↗

PubMed2005

Protein adsorption on oligo(ethylene glycol)-terminated alkanethiolate self-assembled monolayers: The molecular basis for nonfouling behavior.

A study of protein resistance of oligo(ethylene glycol) (OEG), HS(CH2)11(OCH2CH2)nOH (n = 2, 4, and 6), self-assembled monolayers (SAMs) on Au(111) surfaces is presented here. Hydroxyl-terminated OEG-SAMs are chosen to avoid the hydrophobic effect observed with methyl-terminated OEG-SAMs, particularly at high packing densities. The structure of the OEG-SAM surfaces is controlled by adjusting the assembly solvent. These SAMs were characterized by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Protein adsorption on these surfaces was investigated by surface plasmon resonance (SPR). OEG-SAMs assembled from mixed ethanol and water solutions show higher packing density on gold than those from pure ethanol solution. For EG2OH- and EG4OH-SAMs, proteins (i.e., fibrinogen and lysozyme) adsorb more on the densely packed SAMs prepared from mixed ethanol and water solutions, while EG6OH-SAMs generally resist protein adsorption regardless of the assembly solvent used.

Adsorption↗

PubMed2005

Plasma deposition and surface characterization of oligoglyme, dioxane, and crown ether nonfouling films.

Plasma-deposited PEG-like films are emerging as promising materials for preventing protein and bacterial attachment to surfaces. To date, there has not been a detailed surface analysis to examine the chemistry and molecular structure of these films as a function of both precursor size and structure. In this paper, we describe radio-frequency plasma deposition of a series of short-chain oligoglymes, dioxane, and crown ethers onto glass cover slips to create poly(ethylene glycol)-like coatings. The resultant films were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (TOF-SIMS), dynamic contact angle goniometry, and radiolabeled fibrinogen adsorption. Detailed analysis of the high-mass (120-300 m/z) TOF-SIMS oligoglyme film spectra revealed six classes of significant fragments. Two new models are proposed to describe how these fragments could be formed by distinct film-building processes: incorporation of intact and fragmented precursor molecules. The models also provide for the incorporation of hydrocarbon--a species that is not present in the precursors but is evidenced in XPS C(1s) spectra of these films. Two additional models describe the effects of incorporating intact and fragmented cyclic precursors.

Anti-Infective Agents↗

PubMed2005

Fibro-porous meshes made from polyurethane micro-fibers: effects of surface charge on tissue response.

The purpose of this research was to evaluate the influence of surface charge on fibrous encapsulation, cell nuclei density, and vessel ingrowth into small-fiber, fibro-porous, biomaterial meshes. Meshes electrospun from polyurethane with mean fiber diameters of 5.8 microm and mean fiber spacing of 64.9 microm were plasma coated with films of different relative surface charge: Hexafluoropropylene (HF) (neutral), N,N-dimethylaminoethyl methacrylate (NN) (positive charge), and methacrylic acid (MA) (negative charge). Samples were implanted in rat subcutaneous dorsum for 5 weeks then fibrous capsule presence around the implants, cell nuclei density, and vessel number were assessed. Results showed that within the resolution of the histological analysis methods used, no implant experienced fibrous encapsulation. There was no significant difference between cell nuclei density and coating for the four groups: uncoated, HF-coated, NN-coated, and MA-coated. HF-coated and NN-coated samples had lower vessel numbers than uncoated samples (p = 0.055 and 0.032, respectively). MA-coated samples had vessel numbers not significantly different from uncoated polyurethane (slightly negatively charged) samples (p = 0.879). The results suggest that negatively charged surfaces may facilitate vessel ingrowth into fibro-porous mesh biomaterials.

Animals↗

PubMed2005

Protein adsorption on 18-alkyl chains immobilized on hydroxyl-terminated self-assembled monolayers.

Surfaces of devices that contact blood accumulate adsorbed and denatured proteins perhaps triggering activation of the coagulation system. A renewable layer of albumin would biologically "passivate" the surface and prevent thrombus formation. Based on the approach of selectively binding albumin to fatty acids, different percentages of a compound with 18 carbons (C18) were immobilized on OH-terminated self-assembled monolayers (SAMs). Fourier transform infrared reflection absorption spectroscopy (IRAS), ellipsometry, contact angle (and surface free energy) and X-ray photoelectron spectroscopy (XPS) measurements were used to characterize these surfaces and proved that there is an efficient immobilization of C18. There is an increase of the thickness and hydrophobicity of SAMs with an increasing percentage of C18. Adsorption of human serum albumin (HSA) was evaluated using radiolabelled (125)I-HSA and IRAS. This study showed a gradual increase of HSA adsorption with the increase of surface hydrophobicity. Regarding competitive binding and exchangeability of albumin towards fibrinogen, it was proved, by radiolabelling, that SAMs prepared from solutions with 2.5% C18 presented considerable adsorption in a selective and reversible way.

Adsorption↗

PubMed2005

Esophageal epithelial cell interaction with synthetic and natural scaffolds for tissue engineering.

As an initial step towards a tissue-engineered esophagus, rat esophageal epithelial cells (REEC) were isolated and characterized for epithelial identity, adhesion protein preference, and in vitro interaction with natural and synthetic scaffolds. The scaffolds consisted of AlloDerm (LifeCell Corporation, Branchburg, NJ), poly(L-lactic acid) (PLLA), poly(lactic-co-glycolic) acid (75:25) (PLGA75), poly(lactic-co-glycolic) acid (50:50) (PLGA50), and polycaprolactone/poly(L-lactic acid) (50:50) (PCL/PLLA). Various factors-including calcium concentration, scaffold composition, and pore size--were evaluated for their influence on epithelial growth and differentiation. By day 18, keratinocytes seeded on AlloDerm cultured under high Ca(++) (1.5mm) conditions showed a proliferating basal cell layer, epithelial stratification (5--6 layers) and a thick keratin layer. The synthetic scaffolds (PLGA, PLLA, PCL/PLLA) also showed complete surface coverage, regions of proliferating basal cells, and evidence of stratification (2--3 layers) and keratinization. The highly porous nature of the synthetic scaffolds, however, limited the formation of a continuous epithelial layer and resulted in a lack of overall spatially-defined differentiation. In conclusion, rat esophageal epithelial cells were successfully isolated and characterized, with cells seeded on AlloDerm showing superior epithelial organization and stratification compared to synthetic scaffolds. Modification of the synthetic scaffold's surface properties and pore size may be necessary to mimic epithelial behavior on natural scaffolds.

Animals↗

PubMed2005

Synthesis of segmented poly(ether urethane)s and poly(ether urethane urea)s incorporating various side-chain or backbone functionalities.

A series of linear and branched functionalized high-molecular-weight segmented poly (ether urethane)s and poly(ether urethane urea)s were prepared by chain-extending isocyanate pre-polymers based on poly(tetramethylene oxide) with molecular weight 1000 and 4,4'-diphenylmethane diisocyanate. Different functional groups were incorporated within the polymer backbone or on side-chains by using several chain extenders during the synthesis: glycerol, 2,2-bis (hydroxymethyl) propionic acid, 1H, 1H,2H,3H,3H-perfluoroundecane-1,2-diol, 1H,1H,8H,8H-dodecanefluoro-1,8-octanediol, 1,3-diamino-2-hydroxypropane and 3,5-diaminobenzoic acid, using the method of gradual approach to stoichiometry. In some cases, pendant functional groups were used as reactive sites for the further attachment of side groups. Polymers were characterized using 1H-NMR and FT-IR spectroscopies and GPC in conjunction with chemical structural confirmation by a model compound comparison study of 4,4'-diphenylmethane diisocyanate or trifluoro-p-tolyl isocyanate reacted with 1,3-diamino-2-hydroxypropane and 1,4-butanediol.

Chromatography, Gel↗

PubMed2005

Poly(ether urethane)s incorporating long alkyl side-chains with terminal carboxyl groups as fatty acid mimics: synthesis, structural characterization and protein adsorption.

The object of this work was to produce polyurethanes with greater affinity for albumin (Alb) and improved hemocompatibility by introduction of carboxyl-terminated alkyl side-chains that better mimic fatty acids, in contrast to methyl terminated alkyl side-chains used previously. Synthesis of poly(ether urethane)s (PEUs) with long alkyl side-chains via a multi-step solution addition polymerization is described. The synthesis is based upon the polymerization of a diisocyanate pre-polymer with various chain extenders and reaction with Br-terminated compound in the final stage. The side-chains had terminal methyl or carboxylic groups, and were attached either directly to the polymer backbone or to an oligo(ethylene glycol) spacer. The bulk structure of the PEUs was confirmed by 1H-NMR and the surface polymer structure was characterized by ToF-SIMS. The influence of the incorporated C16-alkyl, C16-carboxyalkyl and oxyethylene-C16-carboxyalkyl side-chains attached to the polymer backbone on fibrinogen (Fg) and Alb adsorption from blood plasma, and Fg adsorption from buffer solutions and binary mixtures with Alb was measured. Incorporation of C16-alkyl or C16-carboxyalkyl side-chains into PEUs caused relatively small changes in Fg and Alb adsorption. PEUs with oxyethylene-C16-carboxyalkyl side-chains exhibited the lowest Fg adsorption and the highest Alb adsorption among all the tested polymers.

Adsorption↗

PubMed2004

Novel cell patterning using microheater-controlled thermoresponsive plasma films.

A novel approach is reported for cell patterning based on addressable microheaters and a poly(N-isopropyl acrylamide) (pNIPAM) themoresponsive coating. This thermoresponsive coating is created by a radio frequency NIPAM plasma and is denoted as plasma polymerized NIPAM (ppNIPAM). Films of ppNIPAM with a good retention of monomer side-chain functionality are produced using low-power continuous plasma deposition. Cell adhesion and cell detachment tests indicate that the surface switches between adhesive and nonadhesive behaviors as a function of temperature. The use of a photolithographically fabricated microheater array allows the ppNIPAM transition to occur spatially under the control of individual heaters. This localized change in the surface adhesive behavior is used to direct site-specific cell attachment. Patterned adhesion of two types of cells has been visualized on the array through fluorescent markers. Applications for diagnostic devices, cell-based sensors, tissue engineering, and cell transfection are envisioned.

Acrylic Resins↗

PubMed2004

Enhancing the biological activity of immobilized osteopontin using a type-1 collagen affinity coating.

The covalent attachment of biomolecules onto surfaces represents a step toward the improvement of biomaterial properties by providing relevant biological signals of interest to the cell culture or tissue environment. The chemistries involved, however, often attach proteins to the surface in a random fashion, rather than the conformation or orientation most easily recognized by cells and other proteins both in vitro and in vivo. An alternative approach is to take advantage of natural interactions to both bind and orient a biomolecule "naturally," thereby enhancing its biological activity. Type 1 collagen has been shown to bind to osteopontin (OPN), a protein implicated in processes such as wound healing, endothelial cell survival, and angiogenesis. This study seeks to characterize, quantify, and exploit this interaction in order to present a more naturally recognized form of OPN to the environment surrounding a biomaterial. Binding of OPN to type 1 collagen was confirmed using Surface Plasmon Resonance (SPR). Radio-iodination of OPN showed that binding to collagen was dose-dependent and maximal in basic conditions. Principal component analysis of Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) data identified differences in OPN immobilized via different techniques. Adhesion of bovine aortic endothelial cells on OPN immobilized using the affinity coating was also significantly enhanced compared to controls. Investigation into the in vivo relevance of this immobilization method is currently underway.

Animals↗

PubMed2004

Methods for generating protein molecular ions in ToF-SIMS.

One of the greatest challenges in mass spectrometry lies in the generation and detection of molecular ions that can be used to directly identify the protein from the molecular weight of the molecular ion. Typically, proteins are large (MW > 1000), nonvolatile, and/or thermally labile, but the vaporization process produced by many mass spectrometry techniques including time-of-flight secondary ion mass spectrometry (ToF-SIMS) is inherently limited to generating ions from smaller compounds or fragments of the parent molecule, making the identification of proteins complex. The application of specific molecules to aid in the generation of high molecular weight ions in ToF-SIMS has been recognized for some time. In this study we have developed a matrix-SAM substrate preparation technique based on the self-assembly of a matrix-like molecule, mercaptonicotinic acid (MNA), on gold. We then compare this substrate with two existing ToF-SIMS sample preparation techniques, cationized alkane thiol and matrix-enhanced SIMS (MESIMS). The results of this study illustrate that while there is a range of methods that can be used to improve the molecular ion yield of proteins in ToF-SIMS, their efficacy and reproducibility vary considerably and crucially are linked to the sample preparation and/or protein application methods used. Critically, the MNA modified substrate was able to simultaneously induce molecular ions for each protein present in a multicomponent solution, suggesting that this sample preparation technique may have future application in proteomics and DNA analysis.

Gold↗

PubMed2004

Probing the orientation of surface-immobilized immunoglobulin G by time-of-flight secondary ion mass spectrometry.

Static time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful surface analysis technique for the characterization of protein films because of its chemical selectivity and surface sensitivity. In this study, static ToF-SIMS and principal component analysis (PCA), a multivariate data analysis method, were combined to probe the orientation of surface-immobilized immunoglobulin G (IgG). IgG orientation can enhance its ability to detect its antigen in immunoassay techniques. The IgG used in this work is the mouse monoclonal anti-human chorionic gonadotropin (anti-hCG). Anti-hCG films on different well-defined substrates have been studied using its F(ab')2 and Fc fragments as references. Atomic force microscopy was used to characterize these protein films before static ToF-SIMS analysis. The results from PCA of ToF-SIMS spectra were related to the antibody primary amino acid composition and its three-dimensional structure.

Amino Acid Sequence↗

PubMed2004

Generation of porous microcellular 85/15 poly (DL-lactide-co-glycolide) foams for biomedical applications.

Porous 85/15 poly (DL-lactide-co-glycolide) or PLGA foams were produced by the pressure quench method using supercritical CO2 as the blowing agent. The rate of CO2 uptake and CO2 equilibrium concentration in PLGA at different processing conditions were studied by performing sorption experiments. The effects of saturation pressure and temperature on average cell size and relative density of the resulting foams were also studied. The time required to approach equilibrium exhibited a minimum with increasing saturation pressure. The diffusion coefficient and equilibrium concentration of CO2 in PLGA increased with an increasing pressure in an approximately linear relationship. Porous PLGA foams were generated with relative densities ranging from 0.107 to 0.232. Foams showed evidence of interconnected cells with porosities as high as 89%. The pore size ranged from 30 to 100 microm.

Biocompatible Materials↗

PubMed2004

Biomaterials: where we have been and where we are going.

Since its inception just over a half century ago, the field of biomaterials has seen a consistent growth with a steady introduction of new ideas and productive branches. This review describes where we have been, the state of the art today, and where we might be in 10 or 20 years. Herein, we highlight some of the latest advancements in biomaterials that aim to control biological responses and ultimately heal. This new generation of biomaterials includes surface modification of materials to overcome nonspecific protein adsorption in vivo, precision immobilization of signaling groups on surfaces, development of synthetic materials with controlled properties for drug and cell carriers, biologically inspired materials that mimic natural processes, and design of sophisticated three-dimensional (3-D) architectures to produce well-defined patterns for diagnostics, e.g., biological microelectromechanical systems (bioMEMs), and tissue engineering.

Adsorption↗

PubMed2003

Characterization and analysis of osteopontin-immobilized poly(2-hydroxyethyl methacrylate) surfaces.

A wide array of technologies exist for the characterization and quantification of molecules present at surfaces. We have used several of these experimental and instrumental techniques for the analysis of a novel biomaterial surface. Osteopontin, an extracellular matrix molecule involved in wound-healing processes, has been chosen as a relevant model protein to immobilize onto poly(2-hydroxyethyl methacrylate) [poly(HEMA)]. Electron spectroscopy for chemical analysis and time-of-flight secondary ion mass spectrometry were used to verify the surface chemistry and the presence of protein. Iodination of osteopontin yielded quantitative data supportive of dose-dependent immobilization. Enzyme-linked immunosorbent assay was also used to investigate the presence of osteopontin on poly(HEMA). Finally, the cell adhesive properties of immobilized osteopontin were confirmed by using a bovine aortic endothelial cell adhesion assay. The use of multiple tools to characterize the many facets of a biomaterial surface will undoubtedly improve our understanding of the surface and facilitate the amelioration of in vivo performance.

Animals↗