David G. Castner

Biomedical surface science: Foundations to frontiers

Surfaces play a vial role in biology and medicine with most biological reactions occurring at surfaces and interfaces. The foundations, evolution, and impact of biomedical surface science are discussed. In the 19th century, the first observations were made that surfaces control biological reactions. The advancements in surface science instrumentation that have occurred in the past quarter of a century have significantly increased our ability to characterize the surface composition and molecular structure of biomaterials. Similar advancements have occurred in material science and molecular biology. The combination of these advances have allowed the development of the biological model for surface science, where the ultimate goal is to gain a detailed understanding of how the surface properties of a material control the biological reactivity of a cell interacting with that surface. Numerous examples show that the surface properties of a material are directly related to in vitro biological performance such as protein adsorption and cell growth. The challenge is to fully develop the biological model for surface science in the highly complex and interactive in vivo biological environment. Examples of state-of-the-art biomedical surface science studies on surface chemical state imaging, molecular recognition surfaces, adsorbed protein films, and hydrated surfaces are presented. Future directions and opportunities for surface scientists working in biomedical research include exploiting biological knowledge, biomimetics, precision immobilization, self-assembly, nanofabrication, smart surfaces, and control of non-specific reactions.

Time-of-flight secondary ion mass spectrometry: techniques and applications for the characterization of biomaterial surfaces.

The surface of a biomaterial plays a critical role in the success of an implant. Much effort is currently being focused on controlling the chemistry at biomaterial surfaces to ensure favorable results in vivo. The successful tailoring of the surface chemistry will require a detailed surface characterization to verify that the desired changes have been made. This will include the ability to determine the composition, structure, orientation, and spatial distribution, of the molecules and chemical structures on the surface. TOF-SIMS is a powerful surface characterization technique that is able to address these requirements through both spectral analysis and direct chemical state imaging. The flexibility of the TOF-SIMS technique, and the wealth of data produced have generated much interest in its use for biomaterial characterization. This review discusses the strengths, weaknesses, and challenges of static TOF-SIMS for biomaterial surface characterization. First the basic principles of TOF-SIMS are introduced, giving an overview of the technique. Next, sample type, and other sample considerations are discussed. Then data interpretation is overviewed using examples from both spectral and imaging data. Finally, quantitative SIMS analysis is discussed and an outlook for TOF-SIMS analysis of biomaterials will be given.

PEO-like plasma polymerized tetraglyme surface interactions with leukocytes and proteins: in vitro and in vivo studies

Polyethylene oxide (PEO) surfaces reduce non-specific protein and cell interactions with implanted biomaterials and may improve their biocompatibility. PEO-like polymerized tetraglyme surfaces were made by glow discharge plasma deposition onto fluorinated ethylene propylene copolymer (FEP) substrates and were shown to adsorb less than 10 ng/cm2 of fibrinogen in vitro. The ability of the polymerized tetraglyme surfaces to resist leukocyte adhesion was studied in vitro and in vivo. Polymerized tetraglyme and FEP were implanted subcutaneously in mice and removed after 1 day or 4 weeks. Histological analysis showed a similar degree of fibrous encapsulation around all of the 4-week implants. Darkly stained wells were present in the fibrous tissues at the tissue-material interface of both FEP and tetraglyme. Scanning electron micrographs showed that in vivo macrophage adhesion to polymerized tetraglyme was much higher than to FEP. After 2-hour contact with heparinized whole blood, polymorphonuclear leukocyte (PMN) adhesion to polymerized tetraglyme was much higher than to FEP, while platelet adhesion to polymerized tetraglyme was lower than to FEP. When PMNs isolated from blood were suspended in 10% autologous plasma, cell adhesion to polymerized tetraglyme was higher than to FEP; however when the cells were suspended in heat inactivated serum, cell adhesion to FEP was higher than to polymerized tetraglyme. The surface chemistry of polymerized tetraglyme did not change after 2-hour blood contact, but displayed nitrogen functional groups after 1-day implantation and became slightly degraded after 4-week implantation. The surface chemistry of FEP did not change significantly after blood contact or implantation. Loosely bound proteins such as fibrinogen on polymerized tetraglyme may contribute to the adhesion of PMNs and macrophages and ultimately to fibrous encapsulation (the foreign body response) around the implants.

Application of Surface Chemical Analysis Tools for Characterization of Nanoparticles

AbstractThe important role that surface chemical analysis methods can and should play in the characterization of nanoparticles is described. The types of information that can be obtained from analysis of nanoparticles using Auger electron spectroscopy (AES), X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (TOF-SIMS), low-energy ion scattering (LEIS), and scanning-probe microscopy (SPM), including scanning tunneling microscopy (STM) and atomic force microscopy (AFM), are briefly summarized. Examples describing the characterization of engineered nanoparticles are provided. Specific analysis considerations and issues associated with using surface-analysis methods for the characterization of nanoparticles are discussed and summarized, with the impact that shape instability, environmentally induced changes, deliberate and accidental coating, etc., have on nanoparticle properties. FigureAtomic force microscopy image of Cu2O nanodots formed on a SrTiO3 substrate.

Low temperature plasma treatment of asymmetric polysulfone membranes for permanent hydrophilic surface modification

A plasma treatment that renders asymmetric polysulfone membranes permanently hydrophilic is reported. Our modification strategy entails treating these membranes downstream from an inductively coupled rf plasma source. Contact angle measurements confirm that the membranes are completely wettable with water as a result of H2O plasma treatment. More importantly, the hydrophilic modification is permanent as plasma-treated membranes remain wettable for more than 16 months after plasma treatment. This treatment achieves the desired change in wettability for microporous as well as ultrafiltration polysulfone membranes, illustrating the universality of this method. XPS analysis of treated membranes demonstrates this dramatic change in wettability is a result of chemical changes in the membrane induced by plasma treatment. Moreover, the membrane modification is complete as the plasma penetrates the thickness of the membrane, thereby modifying the entire membrane cross-section.

Characterization of adsorbed protein films by time of flight secondary ion mass spectrometry

Time of flight secondary ion mass spectrometry (ToF-SIMS) is a useful technique in the study of adsorbed protein films because of its high surface sensitivity and chemical selectivity. However, the protein mass spectra generated by ToF-SIMS are complex fragmentation patterns of a polymer consisting of 20 different monomers (i.e., amino acids). Principal component analysis (PCA) was implemented to classify several reference positive ion protein spectra according to protein and substrate type. Furthermore, the positive ion 74/102 and 120/130 SIMS intensity ratios, radiolabeled experiments, and PCA were used to track the relative surface concentrations of bovine serum albumin and bovine fibronectin in a binary adsorption experiment. In all cases, the combination of ToF-SIMS and PCA proved capable in classifying proteins by their type (in the case of pure protein spectra) and relative surface concentration (in the case of the binary protein spectra).

Surface Characterization of Extracellular Matrix Scaffolds

Extracellular matrix (ECM) scaffolds prepared from different tissue sources or using different methods have been demonstrated to have distinctive effects upon cell adhesion patterns and the ability to support and maintain differentiated phenotypes. It is unknown whether the molecular composition or the ultrastructure of the ECM plays a greater role in determining the phenotype of the cells with which it comes into contact. However, when implanted, the topology and ligand landscape of the material will determine the host molecules that bind and the type and behavior of cells that mediate the host response. Therefore, a comprehensive understanding of surface characteristics is essential in the design of scaffolds for specific clinical applications. The surface characteristics of ECM scaffolds derived from porcine urinary bladder, small intestine, and liver as well as the effects of two commonly used methods of chemical cross-linking upon UBM were investigated. Electron microscopy and time of flight secondary ion mass spectroscopy were used to examine the surface characteristics of the scaffolds. The results show that ECM scaffolds have unique morphologic and structural properties which are dependant on the organ or tissue from which the scaffold is harvested. Furthermore, the results show that the surface characteristics of an ECM scaffold are changed through chemical cross-linking.

Limits of detection for time of flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS): detection of low amounts of adsorbed protein

Characterization of biomaterial surfaces requires analytical techniques that are capable of detecting a wide concentration range of adsorbed protein. This range includes detection of low amounts of adsorbed protein (<10 ng/cm2) that may be present on non-fouling biomaterials. X-ray Photoelectron Spectroscopy (XPS) and Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) are surface sensitive techniques capable of detecting adsorbed proteins. We have investigated the lower limits of detection of both XPS and ToF-SIMS on four model substrates each presenting unique challenges for analysis by XPS and ToF-SIMS: mica, poly(tetrafluoroethylene), allyl amine plasma polymer and heptyl amine plasma polymer. The detection limit for XPS ranged from 10 ng/cm2 of fibrinogen (on mica) to 200 ng/cm2 (on allyl amine plasma polymers). The detection limit for ToF-SIMS ranged from 0.1 ng/cm2 of fibrinogen to 100 ng/cm2, depending on the substrate and data analysis. Optimal conditions provided detection limits between 0.1 ng/cm2 and 15 ng/cm2 on all of the substrates used in this study. While both techniques were shown to be effective in detecting protein, the sensitivity of both XPS and ToF-SIMS was shown to be dependent on substrate surface chemistry and the organization of the adsorbed protein film. This study specifically highlights the applicability of ToF-SIMS in the characterization of low level protein adsorption.

Ultrasensitive probing of the protein resistance of PEG surfaces by secondary ion mass spectrometry

The highly sensitive surface analytical techniques X-ray photoelectron spectroscopy (XPS) and time-of-flight static secondary ion mass spectrometry (ToF-SIMS) were used to test the resistance of poly(ethylene glycol) (PEG) coatings towards adsorption of lysozyme (LYS) and fibronectin (FN). PEG coatings were prepared by grafting methoxy-terminated aldehyde-PEG (MW 5000 Da) onto two amino-functionalised surfaces with different amine group densities, generated by radio frequency glow discharge polymerisation of n-heptylamine and allylamine. Grafting was performed at the lower critical solution temperature to maximise the graft density of the PEG chains. XPS showed that the grafted density of PEG chains was slightly higher on the allylamine surface. XPS detected no adsorption of either protein on either PEG coating. ToF-SIMS analysis, on the other hand, found, in the positive ion spectra, minute but statistically significant signals assignable to amino acid fragment ions from both proteins adsorbed to the lower density PEG coating and from LYS but not FN on the higher density PEG coating. Negative ion spectra contained relatively more intense protein fragment ion signals for the lower density PEG coating but no changes assignable to adsorbed proteins on the higher density PEG coating. These results demonstrate the importance of utilising highly sensitive techniques to study protein adsorption on surfaces intended to be protein resistant, and that both positive and negative ion ToF-SIMS spectra should be acquired to probe for possible very low levels of protein adsorption.

Probing the orientation and conformation of alpha-helix and beta-strand model peptides on self-assembled monolayers using sum frequency generation and NEXAFS spectroscopy.

The structure and orientation of amphiphilic alpha-helix and beta-strand model peptide films on self-assembled monolayers (SAMs) have been studied with sum frequency generation (SFG) vibrational spectroscopy and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. The alpha-helix peptide is a 14-mer, and the beta-strand is a 15-mer of hydrophilic lysine and hydrophobic leucine residues with hydrophobic periodicities of 3.5 and 2, respectively. These periodicities result in the leucine side chains located on one side of the peptides and the lysine side chains on the other side. The SAMs were prepared from the assembly of either carboxylic acid- or methyl-terminated alkyl thiols onto gold surfaces. For SFG studies, the deuterated analog of the methyl SAM was used. SFG vibrational spectra in the C-H region of air-dried peptides films on both SAMs exhibit strong peaks near 2965, 2940, and 2875 cm(-1) related to ordered leucine side chains. The orientation of the leucine side chains was determined from the phase of these features relative to the nonresonant gold background. The relative phase for both the alpha-helix and beta-strand peptides showed that the leucine side chains were oriented away from the carboxylic acid SAM surface and oriented toward the methyl SAM surface. Amide I peaks observed near 1656 cm(-1) for the alpha-helix peptide confirm that the secondary structure is preserved on both SAMs. Strong linear dichroism related to the amide pi* orbital at 400.8 eV was observed in the nitrogen K-edge NEXAFS spectra for the adsorbed beta-strand peptides, suggesting that the peptide backbones are oriented parallel to the SAM surface with the side chains pointing toward or away from the interface. For the alpha-helix the dichroism of the amide pi* is significantly weaker, probably because of the broad distribution of amide bond orientations in the alpha-helix secondary structure.