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Techniques for analyzing biomaterial surface structure, morphology and topography
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Techniques for analyzing biomaterial surface structure, morphology and topography N . S . M U R T H Y , Rutgers ± The State University of New Jersey, USA
Abstract: This is a brief review of the key techniques that are most useful in characterizing the surface structure, morphology, and topography of biomaterials. The emphasis is on the use of these techniques for the evaluation of materials used in prostheses, biomedical implants and tissue scaffolds. The first part describes the techniques using light, electrons and scanning probes to examine the surface morphology. Profilometry that provides quantitative measure of the surface roughness is also presented. Next, techniques that characterize the surface structures at the molecular level, x-ray scattering and Raman spectroscopy, are presented. The enhancements that make these classical techniques surface sensitive are discussed. Finally, contact-angle measurements are discussed in the context of the effect of surface structure and topography on the energetics of the interaction between the substrate and the adsorbing molecules. Principles of each of the techniques are described and illustrated with examples relevant to biomedical applications. Key words: surface structure, microscopy, x-ray scattering, Raman spectroscopy, contact-angle.
9.1
Introduction
Besides surface chemistry, other surface properties such as stiffness and roughness are known to dictate the protein adsorption and the cell response and thus determine the viability of a biomedical device [1]. Surface chemistry provides signals for the cells to attach, proliferate and differentiate. Substrate stiffness affects the cell motility and thus affects the cell behavior. The significance of nanometer to micrometer size topographical features is not well understood, and is still an active area of research. Surface roughness, in particular, influences the wetting behavior and the biocompatibility properties of solid substrates. This in turn affects the performance of biomaterials in various biomedical applications including, sutures, bone pins, implants, and in general, tissue scaffolds. The influence of substrate geometry on cell response has been recognized since the early 1900s. But it is only since the early 1990s that there has been a systematic effort to study the effect of texture on cellular attachment,
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proliferation and motility [2, 3]. Protein adsorption, cellular response and eventually bacterial growth have been shown to be directly affected by surface roughness. These studies have shown that substrate topography in the micrometer range can be used to modify and control the response of the cells and biocompatibility when implanted into tissue. This chapter deals with the characterization of surface topography and structure of materials used in prostheses, biomedical implants and tissue scaffolds. The most common techniques for the physical characterization of surfaces are listed in Table 9.1 [4]. In contrast to the extensive reviews of these techniques that have been published [4, 5], this chapter will present the utility of the most commonly used techniques for examining the surfaces of biomaterials as it relates to protein adsorption and cellular response in biological environments. The goal is not to be exhaustive, but to present recent developments in optical, electron and scanning probe microscopies, x-ray scattering, Raman imaging and surface energy measurements. Principles and the general overview of the different techniques will be presented, and the methods illustrated with examples.
9.2
Surface morphology and topography
Surfaces of biomaterials invariably have features that span length scales from sub-nm to m, and are highly dependent on processing and thermal history. Subnm features are typically due to phase separation and crystallization, and the m-size features are due to steep gradients and pores of different sizes. These surface structures can be discussed in terms of morphology (e.g., domain structure) and topography (e.g., surface roughness due to holes, peaks, ridges and valleys). These features have a large influence on the bulk properties of the material such as strength, but more importantly on the interactions with the surrounding tissue and fluid in a biological environment. It is also possible that surface mechanical properties such as hardness and stiffness influence the surface-protein/cell interactions by themselves, independently of the morphology and surface chemistry. A classical technique for surface hardness measurement is the one based on diamond stylus. Young's modulus can also be calculated from load-displacement measurements. A 2D surface stiffness image can be created by scanning the surface with the stylus. Nanoindentation measurements are routinely performed using scanning force microscopes [6, 7]. These mechanical properties will not be discussed here. Instead, this section will deal with the direct imaging of the surface features on length scales of 1 to 10 m. There are many microscopic techniques that provide surface morphology and topographic data at different length scales. The technique to be chosen depends on the information that is being sought. Some of the factors that need to be considered are the lateral resolution (nm to m), the dimensions of the surface to be examined (m to mm), the depth of the surface (nm to m), and the sample
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Table 9.1 Essential features of the various techniques used for characterizing surface structure and morphology [24]
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Technique
Probe
Depth analyzed (m)
Lateral resolution (m) (magnification)
Information
Comments
Optical microscopy, OM
Light
0.1
0.3 (2 to 2000)
Surface roughness, structure
Many possibilities, good height resolution with interference techniques
Scanning electron microscopy, SEM
Electrons
2
0.1 (20 to 1 105 )
Surface topography
Vacuum technique
Scanning force microscopy, SFM
Cantilever
0.001
0.0005 (1000 to 2 106 )
Surface topography, composition, toughness
Atomic resolution, many different modes
Profilometry
Light/ cantilever
0.01
5
Surface texture
Quantitative measure of surface roughness
X-ray reflectometry, XR Grazing incidence x-ray small-angle scattering, GISAXS
X-rays
0.5
1
Surface roughness, thin surface layers, lateral structure
Flat surfaces required
Neutron reflectometry, NR
Neutrons
0.5
±
Surface roughness, enrichment layer
Deuterated compound needed
Micro-indentation, MI
Cantilever
100
200
Surface hardness, modulus
Quantitative interpretation difficult
Confocal Raman imaging
Light
50
2
Chemical species
Raman active bands required
Pendent drop
Liquid
0.2
Surface tension/ contact angle, ST
Liquid drop
0.1
1000
Interface tension
Indirect technique
Surface energy
Easy to use, molecular information difficult
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environment (vacuum, ambient, metal coated or immersed in water). Optical microscopy is the most versatile of the methods for examining the surface morphology [8]. However, the lateral resolution is limited to ~ 300 nm. Scanning near field techniques (SNOM) and interference techniques enhance the utility of the technique [9]. For higher resolution, the most established techniques are the scanning and transmission electron microscopy, scanning tunneling microscopy and atomic force microscopy.
9.2.1
Confocal microscopy
One of the main drawbacks of a conventional light microscope is that the object planes outside of the focal plane contribute equally to the image as those points at the focal plane, thus producing a blurred image. Confocal laser scanning microscopy (CLSM) [10, 11], also called laser scanning confocal microscopy (LSM), provides a blur-free image by eliminating the out-of-focus light or glare and can be used to collect serial optical sections from thick specimens. In a conventional wide-field microscope, the entire specimen is illuminated from a suitable lamp and the image formed by a series of lenses is viewed directly by eye or captured on a detector. In contrast, in confocal microscopy, the surface is scanned by one or more focused beams of light, usually a laser, and the image is reconstructed on a computer (Fig. 9.1). Laser light from the illuminating aperture passes through an excitation filter (not shown), reflected
9.1 Schematic of the confocal principle in epifluorescence scanning mode.
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by the dichroic mirror and is brought into focus by the objective lens to a diffraction limited spot at the focal plane within the specimen. The light from the specimen is passed through a pinhole that provides the crucial spatial filtering, and is detected by a photomultiplier tube (PMT). Although unstained specimens can be viewed using light reflected from the specimen, the samples are usually stained or labeled with one or more fluorescent probes. In this fluorescence mode, fluorescence emissions excited both within the illuminated in-focus voxel (volume picture element) and within the illuminated cones above and below it, are collected by the objective and pass through the dichroic mirror and the emission filter (not shown). In both of these modes, only the light from the in-focus voxel is able to pass unimpeded through the imaging aperture to be detected by the PMT. The emissions from regions below the focal plane and from above it have different primary image plane foci and are thus severely attenuated by the imaging aperture, contributing little to the final confocal image. The output from the PMT is built into a 2D image. Image resolution is typically 0.2 m in the transverse plane (xy-plane) and ~ 0.5 m in the z-plane. The greatest advantage is the possibility of making a 3D image of the surface of the sample within a depth of 100±200 m. Application Confocal microscopy is widely used to examine surface and near-surface features in biomaterials such as bone [12], dentin and enamel [13], imaging cells on substrates, topography of substrates, and porous structures in scaffolds. Confocal images are typically obtained using the light reflected from the sample (epitransmission or reflectance) or by capturing the fluorescent light that is excited in the sample by the incident beam (epifluorescence). In one example, CLSM was used to study the bimodal porous structure in cancellous bone. The images shown in Fig. 9.2 were obtained in reflectance mode from a single location on defatted and deproteinized cancellous canine bone specimen [12]. Both the surface and near-surface features can be seen in the Z-stack overlay image shown in the figure. The many distinct macropores have micropores along their walls (indicated with arrows). The macropores are interconnected through the micropores in the intertrabecular space. Such features are difficult to see with other imaging techniques. Confocal microscopy is an effective tool to nondestructively image the surface and near-surface structures in a way that is not possible with other techniques such as microcomputed tomography and magnetic resonance imaging.
9.2.2
Scanning electron microscopy (SEM)
SEM reveals the surface features at nm lateral resolutions, about two orders of magnitude better than optical microscopes, and is often preferred over optical
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9.2 Z-stack overlay of the confocal image of a cancellous bone obtained in reflection mode from a Zeiss 210 CLSM (Carl-Zeiss, Jena, Germany) using an Ar 488 nm laser. The figure shows macropores and interconnecting micropores (indicated with arrows) [12]. Reprinted with permission from `Confocal Laser Scanning Microscopy as a Tool for Imaging Cancellous Bone' by I.O. Smith et al., published in Biomed. Mater. Res. Part B: Appl. Biomater. (2006) 79B: 185± 192, ß 2006, Wiley Periodicals, Inc.
microscopy because of the increased depth of field at low resolutions. However, at resolutions approaching sub-m (3±6 nm, best case), SEM is limited by its depth of field (<<1 m at high magnification), and it cannot image subsurface features [12]. SEM uses a beam of electrons that interact with the specimen in a vacuum environment (Fig. 9.3). Various interactions occur between the electron beam and the sample surface including transmission of the primary electrons, secon-
9.3 Schematic diagram of a scanning electron microscope.
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dary electron emission, back scattering, absorption and emission of light and xrays. In transmission electron microscopy (TEM), the transmitted electrons are used to produce an image, the contrast arising from the difference in electron absorption and scattering in different parts of the sample. In SEM, the primary beam is rastered over the sample, and the secondary electrons that are emitted from the surface of the material are collected and composed to provide an electron micrograph. The contrast arises from either electron absorption or emission. Application SEM is widely used because of the fairly simple sample preparation and the ease of using the equipment. It is used to study scaffolds, fiber mats, surfaces of bones and joints. One common application is the use of SEM to examine the surface roughness of implants that can often be related to its performance in vivo. There are several studies in which titanium surfaces have been examined after different types of surface treatments such as after sand-blasting with Al2O3 particles, plasma spraying and elecrolytically coating with hydroxyapatite [14]. Another example of surface treatment is laser ablation. Laser ablation provides a means to modify the surface and thus control the tissue interactions. Figure 9.4 shows an example of how SEM was useful in showing the changes in the surface texture upon ablation of poly(ethylene terephthalate) (PET) surface [15]. PET is widely used in biomedical applications including implantable sutures, surgical mesh, vascular grafts and heart valves [16]. The montage in the figure shows the progressive change in the morphology with the increase in the laser fluence, from an unirradiated area to the left to the ablated area on the right. The figure shows the texture changes from ripple into densely packed cones, before these cones are ablated and removed at higher beam fluence. The figure is most illustrative in that m resolution can be achieved while examining a relatively large area ~ 0.1 mm, and also the large depth of field that can be obtained. Sample size is limited to about 10 mm in diameter. If the sample is not electrically conductive, then a thin (20±30 m) metallic coating, typically gold, platinum or god/palladium alloy is sputter coated onto the sample to prevent electrical charging of the specimen. In addition to imaging the surface features, SEM is often combined with energy dispersive x-ray analysis (EDX) to obtain a map of the elemental distribution.
9.2.3
Scanning force or atomic force microscopy (SFM or AFM)
This is one in a family of scanning probe techniques that have been used successfully in high resolution imaging and molecular imaging of biological
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9.4 A montage of the images from an SEM showing the laser-ablated regions on the surface of PET injection molded plaque. The fluence of the laser beam increases from left to right [15]. Reprinted with permission from: `Self-assembled and etched cones on laser-ablated polymer surfaces' by N.S. Murthy et al., J. Appl. Phys. (2006) 100: 023538, 1±12, ß 2006, American Institute of Physics.
ß Woodhead Publishing Limited, 2011 9.5 (a) Illustration of the principle of AFM. (b±d): A schematic of the three common AFM operation modes. (b) Contact mode. (c) Tapping mode. (d) Phase imaging mode [17]. Reprinted with permission from `Atomic force microscopy of biomaterials surfaces and interfaces' by K.D. Jandt, Surface Science (2001) 491: 303±332, ß 2001, Elsevier.
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macromolecules since their invention in the 1980s [17, 18]. In SFM, the force between a probe and the sample surface is used to map the surface morphology and to study the interaction forces in biological systems down to the picoNewton range, typical of single ligand-receptor interaction. Other techniques, not discussed here, use tunneling current, chemical affinity and other phenomena to probe the surface characteristics. In SFM, a 3D image of the surface is created by scanning a micron-size cantilever across the surface (Fig. 9.5). The cantilever is typically made of silicon or silicon nitride, with a tip whose radius of curvature is ~ 10 nm. The forces between the tip and the surface, which are ~ nN and include contributions from Van der Waals interactions, chemical bonding, electrostatic forces and solvation forces, deflect the cantilever. The deflection of the cantilever due to these forces is measured using a laser spot reflected from the top surface of the cantilever into an array of photodiodes. This signal is used maintain a constant force via a feedback loop, or to maintain a constant height, depending on the scan mode that is chosen. The sample is moved across the tip by piezo crystals. The AFM can be operated in a number of modes depending on the application, and three of these are presented in Figs 9.5(b±d). In the static mode, also called the contact mode, the force between the tip and the surface is kept constant during scanning, and the change in the cantilever deflection required to maintain this constant force is used to obtain an image of the sample's surface topography. This mode is suitable for hard surfaces but not for biomaterials which are soft and whose surface may have weakly adsorbed molecules. In the dynamic mode, also called the tapping mode, the cantilever is vibrated close to its resonant frequency, and the changes in amplitude and phase are monitored. The reduction in amplitude is monitored and used to map the surface topographical features. When the AFM is operated in the tapping mode, it is possible to take advantage of the difference in the phase of the cantilever oscillation and the signal sent to the cantilever. This phase difference depends on the sample's hardness, elasticity and adhesion. This phase lag is monitored and a 2D phase image can be produced to qualitatively map the surface viscoelastic properties of the surface such as to map the distribution of soft biomolecules on a stiff substrate. Application Scanning probe microscopes are useful in characterizing surfaces at atomic resolutions in a variety of environments from ultrahigh vacuum to aqueous solutions. It is also used to study time-dependent phenomena such as conformational change of the molecules and the shapes of the whole cells adsorbed onto a surface, hydration induced changes, crystallization and corrosion processes. A typical contact mode AFM image is shown in Fig. 9.6a, and is compared with a corresponding SEM image of a similar sample in Fig. 9.6b. These images
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9.6 Comparison of (a) AFM (deflection in contact mode) and (b) SEM (secondary electron) imaging technique on similar, dry titanium/titanium oxide surfaces (2 2 m 2 ). Oxide domes are visible in both the images [19]. Reprinted with permission from `Direct observation of hydration of TiO2 on Ti using electrochemical AFM: freely corroding versus potentiostatically held' by J.P. Bearinger, C.A. Orme and J.L. Gilbert, Surface Science (2001) 491: 370± 387, ß 2001, Elsevier.
were a part of a study to characterize the morphology of the titanium surfaces in saline-based oxide hydration events in situ under varying biomedically relevant electrochemical conditions in the hydrated state [19]. The figure shows the surface in air prior to immersion. The oxide domes are more clearly visible in AFM than in SEM. Average dome height in the AFM image is 4:3 1:7 nm and the average dome width is 91 16 nm. Phase imaging is widely used to image soft particles embedded in stiffer matrices. A biologically relevant example of the use of the phase image is the observation of the adsorption of biomolecules in the presence of roughness that is inherent in biomaterial surfaces. AFM images of a National Heart Lung and Blood Institute (NHLBI) reference polydimethylsiloxane (PDMS) with and without adsorbed fibrinogen are shown in Fig. 9.7 [20]. Fibrinogen is a plasma protein important for blood coagulation and platelet aggregation, and PDMS is a commonly used substrate for protein adsorption and cell studies. The images were obtained in the tapping mode. The topographic data is presented in Fig. 9.7a and the phase data in Fig. 9.7b. Proteins were adsorbed at a concentration of 500 ng/ml and the surface concentration is ~ 10 molecules/m2. Although isolated fibrinogen molecules adsorbed onto the surface can be observed in the height image, they are more clearly visible in the phase image in which the topography of the PDSM substrate does not appear because of phase imaging. Highest resolution images are obtained when the AFM is operated in the contact mode. However, because the tip is in constant contact with the sample, the shear forces applied to the sample during scanning can potentially damage
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9.7 AFM tapping mode data from PDMS with and without adsorbed fibrinogen [20]. (a) Height image. (b) Phase image. Reprinted with permission from `Individual plasma proteins detected on rough biomaterials by phase imaging AFM' by N.B. Holland and R.E. Marchant, J. Biomed. Mater. Res. (2000) 51: 307±315, ß 2000, Wiley Periodicals, Inc.
the surface, especially the weakly bound protein molecules adsorbed onto the biomaterials. In the tapping mode, the tip is not in contact with the sample during scanning. Thus the forces applied to the sample surface are negligible. Furthermore, in the tapping mode, phase imaging can be used to map surface inhomogeneities that give rise to variations in viscoelasticity of the material surface but do not alter the physical appearance or the topography of the surface.
9.2.4
Profilometry
The microscopic techniques discussed in Sections 9.2.1±9.2.3 provide an image of the surface texture, but not the quantitative measure of the surface roughness. This can be done by profilometry, in which a probe, mechanical (contact) or optical (noncontact), is passed across the surface [21]. The probe follows the contours at each point on the surface, and the height of the probe at each point is recorded and the resulting 1D scan or a 2D map is analyzed. A mechanical stylus captures the features over a large area (~ 100 mm), with a x-y resolution of 5 m and a z-resolution of 0.01 m. An optical probe such as a confocal laser scanning microscope has a range of ~ 2 mm, x-y resolution of 5 m and the z resolution of 0.01 m. AFM covers a much smaller area (100 m) but provides much higher resolutions (0.2 m in x-y and 1.5 pm in z). The force applied with a mechanical stylus is 5 mN compared to 1 N in AFM. Parameters such as arithmetic average of the absolute values of all points of the profile (Ra), root means square values of all the heights around the mean (Rq) are often used to quantify the roughness. These, however, do not reflect the distance between the features and their shape.
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Application Profilometry is frequently used in the evaluation of surfaces of metallic surgical implants. These implants are treated in various ways to bring about chemical and topographic modifications of the surface to improve the osseointegration process and the biomechanical performance of implants. In one study profilometry was used to measure the surface roughness in three different materials, poly-L-lactic acid (PLLA), poly-DL-lactic acid (PDLA), and sodium alginate hydrogel (AGA-100) used in clinical practice, and correlate this to the osteoblast adhesion. These were coated onto mechanically and chemically treated (sand-blasted and acid-etched) Ti substrates. Figure 9.8 shows how the changes in the surface roughness due to different coatings can be monitored using contact profilometry images. These results were used to identify a specific roughness parameter (peak density) which mainly controls the amount of osteoblast adhesion a crucial factor in rapid `osteointegration' [22]. The average roughness was 4.31 in the control (sand-blasted and acid-etched) sample, and was 2.39, 1.39 and 2.78 in AGA-100, PLLA and PDLA coated samples,
9.8 Contact profilometry of Ti surfaces [22]. (a) The initial sand-blasted and acid-etched surface prior to coating. (b) Coated with PLLA. (c) Coated with PDLA. (d) Coated with sodium alginate hydrogel AGA-100. Reprinted with permission from `Contact profilometry and correspondence analysis to correlate surface properties and cell adhesion in vitro of uncoated and coated Ti and Ti6Al4V disks' by A. Bagno et al., Biomaterials (2004) 25: 2437±2445, ß 2001, Elsevier.
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respectively, reflecting the modification of the surface roughness to different degrees depending on the coating material. Only AGA-100 was able to preserve, to some extent, the roughness in the starting surface. Only the starting and AGA100 coated samples allowed the osteoblasts to adhere, and the other two seemed to hinder the adherence. Further evidence suggests that it is indeed this surface roughness and not the chemical composition that is responsible for the differences in adhesion. Profilometers generate an image of the surface height. Size of the area measured and the size of the probe set the upper and lower limits on the size of the features that can be characterized. The nature of the probe and the force exerted by the probe on the surface limit the range of surfaces that can be investigated by these techniques. Although optical techniques are more appropriate for relatively soft materials, stylus technique is more appropriate for examining large areas with substantial slopes within the surface structure [23]. The advantages of this method are that it is direct, less expensive and reproducible.
9.3
Surface structure and spatial distribution
The techniques described in the previous section are useful in examining the morphology, texture and surface topography. At the other extreme, there are surface spectroscopic techniques such as Auger electron spectroscopy, x-ray photoelectron spectroscopy and secondary ion mass spectrometry that provide the chemical makeup or the elemental composition at or near the surface. These techniques are discussed in Chapter 8. Between these two extremes, there is another category of techniques for examining the structure, the molecular organization, surface orientation that change as a result of surface treatment. These structures near the surface can be studied using methods that are surfaceenhanced versions of the classic x-ray scattering, IR and Raman spectroscopic techniques. These classical techniques have been adapted so that x-ray, IR or visible light penetrate only the surface layers of the material, and provide information about the surface structure. This section will discuss the use of x-ray scattering and Raman spectroscopic methods for analyzing the surface structures that arise as a result of surface orientation, phase separation, crystallization and degradation.
9.3.1
X-ray reflectivity and scattering
When x-rays are incident on a surface at very shallow angles, two events can occur. First, just like light, x-rays are also reflected from surfaces. But, because the refractive index of solids for x-rays is < 1, this specular reflection (reflected angle equal to incident angle) occurs for incidence angles less than the critical angle, ~ 0.1ë (Fig. 9.9a). In this configuration, known as reflectometry, the profile of the reflected beam is analyzed to characterize roughness as well as investigate phenomena such as interdifussion, blending, surface-induced order,
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adsorption or surface enrichment of components [24]. X-ray reflectometry is an ideal tool to quantify the surface roughness in the nm length scale. If the interface between the sample and air is not perfectly sharp and smooth then the reflected intensity will deviate from that predicted by the simple Fresnel equations. These deviations are analyzed to obtain the density profile of the interface normal to the surface. Such reflectivity measurements provide surface roughness [25] and the structure of deposited membranes [26]. In a related second technique, the x-rays are incident at a very shallow angle ( , Fig. 9.9b) such that the path within the sample is considerably long so as to attenuate the beam within a small depth from the surface. This depth depends on the absorption coefficient of the substrate, and typically a few m for polymers. In this technique, called grazing-incidence scattering, the intensity measured function of the scattering angle 2 will reveal the structure near the surface of
9.9 (a) Schematic of the sample geometry used in x-ray reflectivity measurements. (b) Geometry of the sample, incident and scattered beam used in the data collection in grazing-incidence scattering. (c) Measured x-ray reflectivity plotted as R/RFresnel versus Qz, where RFresnel is the reflectivity of an infinitely sharp, step-like interface [26]. Reprinted with permission from `Characterization of biological thin films at the solid-liquid interface' by C.E. Miller et al., Phys. Rev. Lett. (2005) 94: 238104, 1±4, ß 2005, American Institute of Physics. (d) Arrangement of the head groups and the hydrocarbon tail region modeled based on the data in (c).
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the sample. By varying the angle of incidence, one can probe layers of increasing thickness and analyze the structure and chemical composition by means of diffraction or fluorescence experiments. Grazing incidence small-angle x-ray scattering (GISAXS) provides surface sensitive information at length scales of 1±100 nm. Such measurements have been used to tailor the island density, shape and size distributions. Applications The example shown in Fig. 9.9 illustrates the use of x-ray reflectivity to characterize the phospholipids bilayer membranes at the solid-water interface. The x-ray reflectivity curve typically obtained is shown in Fig. 9.9c. This curve is modeled using a model, in this case, a 4-slab model made of outer head groups, hydrocarbon tails, inner head groups, and water cushion (Fig. 9.9d). The lipid used in this case was 1,2-Dioleoyl-Sn-Glycero-3-phosphocholine (DOPC). Ê hydrocarbon tail region, outer head The data can be explained in terms of 23.2 A Ê Ê group region thickness of 10 A with a 6 A roughness, and the inner head group Ê with 3.8 A Ê roughness. Finally, the model also suggests a thin thickness of 8 A Ê 4 A layer of water between the lipid and the quartz substrate. Such x-ray reflectivity measurements have been used to model the surface roughness as well as the structure near the solid-solution interface, and in the case just discussed, that of the lipid bilayer. The technique requires that the surfaces be flat, extending to a few centimeters. The measurements are performed with x-rays illuminating the surface at grazing incidence, typically a few milliradians close to the critical angle for total reflection at the material-air interface. Because of the reduced intensity due to tight collimation, such measurements are typically performed at synchrotron sources.
9.3.2
Confocal Raman spectroscopy
Raman spectroscopy, like infrared spectroscopy, is highly sensitive to molecular species present in the material. Combining this with the confocal principle described in Section 9.1.2, makes it a powerful tool for obtaining the structural information near the surface. Unlike infrared spectroscopy, Raman spectroscopy is relatively insensitive to water, and hence is potentially more useful in the characterization of the microstructure of biomaterials where water is invariably present. Recent advances in laser sources, optical elements and detector technology have enabled mapping of the surfaces based on the identity and concentration of the selected species. It can be used to nondestructively image the surface to a depth of ~ 50 m, and has been used, for instance, to analyze the surface and assess the residual stress fields in artificial hip joints [27, 28], in addition to studying the oxidation state, crystallization behavior and phase fractions.
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In typical analytical measurements, the Raman spectral intensities are directly proportional to the concentration of the corresponding species within the relatively small deviation in Raman response. Concentrations can be measured from the intensities of an interference-free peak after calibration using a reference material. The most common way to Raman map a surface is to find a characteristic strong band in the species of interest, a band that is free from interference from other species, and map the intensity of this band. However, when strongly overlapping bands are present, as in most biomaterials, chemometrics methods such as multivariate curve resolution are used to obtain the Raman images [29]. Spatial resolutions of 2 m in the x±y plane and 3 m to a depth of 50 m in the z-plane are achievable. Application Confocal Raman imaging is commonly used to examine the changes that occur near the surface due to adsorption of molecules, hydration (with D2O labeled samples), and degradation. By combining the micrometric lateral resolution of a laser beam focused on the polymer surface with a proper selection of Raman bands, it is possible to obtain a highly resolved 2D map of the conformational population patterns, including crystalline and amorphous phase fractions, and oxidation states. In one example, confocal spectroscopic techniques were used to study the microscopic features due to wear in acetabular cups made of ultra-high molecular weight polyethylene (UHMWPE) before and after implantation in vivo (Plate IV between pages 208 and 209) [30]. The figure shows significant oxidation gradients along the subsurface of the long-term implanted retrieval, especially in the main wear zone. A clear relationship is found between orthorhombic crystalline fraction and degree of oxidation along the subsurface, and this was attributed to partial crystallization of polyethylene on oxidation. It is important to note that these confocal experiments show relatively high crystallinity in the immediate subsurface of the cup during implantation lifetime in vivo. This could be related to the formation of polyethylene debris on wear contact. Thus, near-surface UHMWPE gets oxidized and crystallizes during aging in vivo, which could induce significant embrittlement of the polymeric subsurface.
9.4
Energetics
Surface energy is the work required to increase unit surface area at constant temperature, pressure and composition. Surface tension, which is related to surface energy, determines the wettability of a surface and as such plays an important role in determining the biocompatibility, adsorption processes and adhesion in biomaterials. It reflects surface roughness, and is sensitive to both
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surface segregation of components and surface contamination. Surface tension also reflects the strength of bonding within the bulk materials. Hard solids with covalent, ionic or metallic bonds have high surface energy surfaces (surface tension 500±5000 mJ/m2). Most polymers are soft solids in which van der Waals and hydrogen bonding forces play a major role and which have low energy surfaces (< 100 mJ/m2).
9.4.1
Contact angle measurements
The surface tension is most often determined by measuring the contact angle of a drop of liquid positioned on the surface. The contact angle is the angle at which a liquid-vapor interface meets the solid surface. This angle is determined by the interactions across the three interfaces. In Fig. 9.10, SL, SV and LV are the interfacial energy (surface tension) at the solid-liquid, solid-vapor and liquid-vapor interfaces, respectively, and the Young's relation given in the figure relates the contact angle to these three forces. There are several methods of determining the surface tensions including measuring the direct force by Wilhelmy balance, contact angles using sessile drop or captive air bubble method, and capillary penetration into porous systems [31]. Contact angle using sessile drop is the most widely used and will be illustrated here. A drop of fluid, water for most biomaterials, is placed on the surface of interest. The contact angle is determined from the tangent to the liquid drop at contact where the liquid and the solid intersect. Surface roughness or heterogeneity can be evaluated by measuring the contact-angle hysteresis, i.e. by measuring the advancing and receding contact angles [32]. As the volume of drop is increased, and the drop advances over the surface, the edge of the liquid is pinned at the boundaries of the wetting and non-wetting interfaces (if the surface is heterogeneous) or the sharp edge of the surface boundary (if the surface is rough). Thus, the contact angle will be higher than the equilibrium value. Similarly, because of the same pinning the contact angle will be lower than the equilibrium value when the volume of the droplet decreases as the liquid is withdrawn from the droplet. The resulting hysteresis can be used to characterize the surface roughness or heterogeneity.
9.10 Illustration of the Young's force balance giving the equilibrium contact angle.
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Application The use of contact-angle measurement will be illustrated with studies of dynamic contact-angle measurements and adsorption of -globulin made as a function of Wenzel ratio (roughness factor calculated as the ratio of the actual and the projected surface areas) [33]. Contact-angle hysteresis measurements were made on well-defined nanostructured surfaces that were fabricated by growing germanium nanopyramids on Si(001) surfaces. AFM images shown in Fig. 9.11 indicate the different surface morphologies. These surfaces are made of nanopyramids and the density of these pyramids determines the surface roughness It has been reported that surface roughness modifies the contact angles and the contact-angle hysteresis of wetting. There is no consensus as to whether the contact angle increases with roughness and on the effect of roughness on the hysteresis behavior [33]. The results show that the advancing contact angle of water monotonically increases by 20ë from the flat substrates to substrates with maximum pyramid density whereas the receding contact angle remains constant. The biocompatibility of materials is determined at the most fundamental level by protein adsorption. The amount of adsorbed proteins bovine -globulin (BGG) increases significantly with the density of nanopyramids on the substrate, i.e., with the roughness of the surface. Increase in the effective surface by 7% causes a 2- to 4-fold increase in the adsorbed protein. However, the activity of the adsorbed BGG was found to decrease with pyramid density. These results demonstrate that wetting behavior and biocompatibility are both strongly correlated with the surface nanoarchitecture, and thus illustrate the importance of careful surface topographic measurements in understanding the surface-induced biological processes.
9.5
Future trends
With great advances in surface modification discussed elsewhere in this book, the need for rapid characterization of the surface features inexpensively and reproducibly will continue to grow. Optical microscopy will continue to be a most versatile tool to examine surface morphology. Moving beyond the confocal microscopes, near-field scanning optical microscopy can deliver resolutions normally associated with electron microscopy [34]. It uses tapered glass optical elements and techniques of scanned probe imaging (e.g., AFM) to provide images at 50 nm resolution, far below the Rayleigh resolution limit of half the wavelength of the imaging radiation (~ 250 nm). A major limitation of SEM is that the samples that are imaged are typically in a high vacuum environment. Innovative sample chambers are now being used that permit the samples to be in different environments such as moisture. One trend in SEM has been to work at a low incident beam energy (< 5 keV) in order to minimize beam penetration,
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9.11 (a±d) AFM images indicating the increase in surface roughness with increasing nanopyramid density. (e) Advancing (open circles) and receding (filled circles) contact angle hysteresis for water in surfaces with different nanopyramid density. (f) Protein adsorption of BCG and anti BCG vs. roughness factor [34]. Reprinted with permission from `Impact of nanometerscale roughness on contact-angle hysteresis and globulin adsorption' by B. Muller, et al., J. Vac. Sci. Technol. (2001) 19: 1715±1720, ß 2001, AVS: Science & Technology.
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9.11 Continued
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beam spreading, and sample charging and thus improve surface image resolution. Alternatively, high energy beams are being used obtain complementary information such as subsurface features that can be combined with the data obtained at low energies. Scanning probe techniques continue to evolve with the development of new probes to map different aspects of the surface. Availability of high-intensity x-ray synchrotron sources makes it much easier to obtain reflectivity and grazing incidence measurements for the detailed evaluation of surface structures. In more traditional techniques such as contact angle measurement, automation of the measurement and analysis enables highly reliable data to be obtained even by those who may lack specialized skills. In all these techniques, technology is used to move towards the ultimate goal of viewing features at high resolutions in a large dynamic system at high imaging speeds.
9.6
References
1. Wong, J.Y., J.B. Leach and X.Q. Brown, Balance of chemistry, topography, and mechanics at the cell±biomaterial interface: issues and challenges for assessing the role of substrate mechanics on cell response. Surface Science, 2004. 570: 119±133. 2. Chen, C.S. et al., Geometric control of cell life and death. Science, 1997. 276: 1425± 1428. 3. Huang, S. and D.E. Ingber, Shape-dependent control of cell growth, differentiation, and apoptosis: switching between attractors in cell regulatory networks. Exp. Cell. Res., 2000. 261: 91±103. 4. Stamm, M., ed. Polymer surfaces and interfaces: characterization, modification and applications, 2008. Berlin: Springer. 5. Merrett, K. et al., Surface analysis methods for characterizing polymeric biomaterials. J. Biomater. Sci. Polymer Edn, 2002. 13(6): 593±621. 6. Haque, F., Application of nanoindentation to development of biomedical materials. Surface Engineering, 2003. 19(4): 253±268. 7. Bedoui, F., F. Sansoz and N.S. Murthy, Incidence of nanoscale heterogeneity on the nanoindentation of a semicrystalline polymer: experiments and modeling. Acta Materialia, 2008. 56: 2296±2306. 8. Sawyer, L.C. and D.T. Grubb, Polymer Microscopy. 3rd edn, 2008. Springer, 540. 9. Ohtsu, M. and H. Hori, Near-field nano-optics ± From basic principles to nanofabrication and nanophotonics, 1999. New York: Kluwer Academic/Plenum. 10. Semwogerere, D. and E.R. Weeks, 'Confocal microscopy', in Encyclopedia of Biomaterials and Biomedical Engineering, 2005. pp. 1±10. 11. Shotton, D., Confocal scanning optical microscopy and its applications for biological specimens. Journal of Cell Science, 1989. 94: 175±206. 12. Smith, I.O. et al., Confocal laser scanning microscopy as a tool for imaging cancelous bone. J. Biomed. Mat. Res. Part B: Appl. Biomat., 2006. 79B: 185±192. 13. Curtis, R.V. and T.F. Watson, eds. Dental biomaterials: Imaging, testing and modelling, 2009. Cambridge: Woodhead Publishing. 14. Prado da Silva, M.H. et al., Surface analysis of titanium dental implants with different topographies. Mat. Res., 2000. 3(3): 61±67. 15. Murthy, N.S. et al., Self-assembled and etched cones on laser-ablated polymer surfaces. J. Appl. Phys., 2006. 100: 1±12.
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16. von Recum, A.F., ed. Handbook of Biomaterials Evaluation: Scientific, Technical and Clinical Testing of Implant Materials, 2nd edn, 1998. Boca Raton, FL: CRC Press. 17. Jandt, K.D., Atomic force microscopy of biomaterials surfaces and interfaces. Surface Science, 2001. 491(3): 303±332. 18. Siedlecki, C.A. and R.E. Marchant, Atomic force microscopy for characterization of the biomaterials interface. Biomaterials, 1998. 19(4±5): 441±454. 19. Bearinger, J.P., C.A. Orme and J.L. Gilbert, Direct observation of hydration of TiO2 on Ti using electrochemical AFM: freely corroding versus potentiostatically held. Surface Science, 2001. 491: 370±387. 20. Holland, N.B. and R.E. Marchant, Individual plasma proteins detected on rough biomaterials by phase imaging AFM. J. Biomed. Mater. Res., 2000. 51: 307±315. 21. Assender, H., V. Bliznyuk and K. Porfyrakis, How surface topography relates to materials' properties. Science, 2002. 297: 973±976. 22. Bagno, A. et al., Contact profilometry and correspondence analysis to correlate surface properties and cell adhesion in vitro of uncoated and coated Ti and Ti6Al4V disks. Biomaterials, 2004. 25: 2437±2445. 23. Wennerberg, A. et al., Characterizing three-dimensional topography of engineering and biomaterial surfaces by confocal laser scanning and stylus techniques. Med. Eng. Phys., 1996. 18: 548±556. 24. Stamm, M., Polymer surfaces, interfaces and thin films studied by x-ray and neutron reflectometry, in Scattering in polymeric and colloidal systems, W. Brown and K. Mortensen, eds, 2000. Amsterdam: Gordon and Breach Science Publishers, pp. 495±534. 25. Stone, V.W. et al., Roughness of free surfaces of bulk amorphous polymers as studied by x-ray surface scattering and atomic force microscopy. Phys. Rev. B, 1999. 60: 5883±5894. 26. Miller, C.E. et al., Characterization of biological thin films at the solid-liquid interface by X-ray reflectivity. Phys. Rev. Lett., 2005. 94: 238104 (1-4). 27. Pezzotti, G., Stress microscopy and confocal Raman imaging of load-bearing surfaces in artificial hip joints. Exp. Rev. Med. Dev., 2007. 4(2): 165±189. 28. Pezzotti, G. et al., Confocal Raman spectroscopic analysis of ceramic hip joints. Key Engineering Materials, 2006. 309±311: 1211±1214. 29. Andrew, J.J. and T.M. Hancewicz, Rapid analysis of Raman image data using twoway multivariate curve resolution. Appl. Spectro., 1998. 52: 797±807. 30. Pezzotti, G. et al., Confocal Raman spectroscopic analysis of cross-linked ultra-high molecular weight polyethylene for application in artificial hip joints. J. Biomed. Opt., 2007. 12(1): 0141011 (1±14). 31. Grundke, K., Characterization of polymer surfaces by wetting and electrokinetic measurements ± contact angle, interfacial tension and zeta potential, in Polymer surfaces and interfaces, M. Stamm, ed., 2008. Berlin: Springer. 32. Extrand, C.W., in Encyclopedia of surface and colloid science, P. Somasundaran, ed., 2006. Boca Raton, FL: CRC Press, pp. 2876±2891. 33. Muller, B. et al., Impact of nanometer-scale roughness on contact-angle hysteresis and globulin adsorption. J. Vac. Sci. Technol., 2001. 19(5): 1715±1720. 34. Doose, S., Trends in Biological Optical Microscopy. Chem. Phys. Chem., 2008. 9: 523±528.
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Plate IV (a) Photograph of a long-term retrieved cup. (b±e) Maps of wear index (related to oxidation index) collected at increasing depths of focal plane z0 of the confocal probe on the long-term retrieved UHMWPE acetabular cup shown in (a). (b and d): Nonwear zone. (c and e): Wear zone. Reprinted with permission from Pezzotti G et al., `Confocal Raman spectroscopic analysis of cross-linked ultra-high molecular weight polyethylene for application in artificial hip joints', J. Biomed. Opt. (2007) 12: 014011 1±14, 2007, SPIE.
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Techniques for analyzing biomaterial surface structure, morphology and topography N . S . M U R T H Y , Rutgers ± The State University of New Jersey, USA
Abstract: This is a brief review of the key techniques that are most useful in characterizing the surface structure, morphology, and topography of biomaterials. The emphasis is on the use of these techniques for the evaluation of materials used in prostheses, biomedical implants and tissue scaffolds. The first part describes the techniques using light, electrons and scanning probes to examine the surface morphology. Profilometry that provides quantitative measure of the surface roughness is also presented. Next, techniques that characterize the surface structures at the molecular level, x-ray scattering and Raman spectroscopy, are presented. The enhancements that make these classical techniques surface sensitive are discussed. Finally, contact-angle measurements are discussed in the context of the effect of surface structure and topography on the energetics of the interaction between the substrate and the adsorbing molecules. Principles of each of the techniques are described and illustrated with examples relevant to biomedical applications. Key words: surface structure, microscopy, x-ray scattering, Raman spectroscopy, contact-angle.
9.1
Introduction
Besides surface chemistry, other surface properties such as stiffness and roughness are known to dictate the protein adsorption and the cell response and thus determine the viability of a biomedical device [1]. Surface chemistry provides signals for the cells to attach, proliferate and differentiate. Substrate stiffness affects the cell motility and thus affects the cell behavior. The significance of nanometer to micrometer size topographical features is not well understood, and is still an active area of research. Surface roughness, in particular, influences the wetting behavior and the biocompatibility properties of solid substrates. This in turn affects the performance of biomaterials in various biomedical applications including, sutures, bone pins, implants, and in general, tissue scaffolds. The influence of substrate geometry on cell response has been recognized since the early 1900s. But it is only since the early 1990s that there has been a systematic effort to study the effect of texture on cellular attachment,
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proliferation and motility [2, 3]. Protein adsorption, cellular response and eventually bacterial growth have been shown to be directly affected by surface roughness. These studies have shown that substrate topography in the micrometer range can be used to modify and control the response of the cells and biocompatibility when implanted into tissue. This chapter deals with the characterization of surface topography and structure of materials used in prostheses, biomedical implants and tissue scaffolds. The most common techniques for the physical characterization of surfaces are listed in Table 9.1 [4]. In contrast to the extensive reviews of these techniques that have been published [4, 5], this chapter will present the utility of the most commonly used techniques for examining the surfaces of biomaterials as it relates to protein adsorption and cellular response in biological environments. The goal is not to be exhaustive, but to present recent developments in optical, electron and scanning probe microscopies, x-ray scattering, Raman imaging and surface energy measurements. Principles and the general overview of the different techniques will be presented, and the methods illustrated with examples.
9.2
Surface morphology and topography
Surfaces of biomaterials invariably have features that span length scales from sub-nm to m, and are highly dependent on processing and thermal history. Subnm features are typically due to phase separation and crystallization, and the m-size features are due to steep gradients and pores of different sizes. These surface structures can be discussed in terms of morphology (e.g., domain structure) and topography (e.g., surface roughness due to holes, peaks, ridges and valleys). These features have a large influence on the bulk properties of the material such as strength, but more importantly on the interactions with the surrounding tissue and fluid in a biological environment. It is also possible that surface mechanical properties such as hardness and stiffness influence the surface-protein/cell interactions by themselves, independently of the morphology and surface chemistry. A classical technique for surface hardness measurement is the one based on diamond stylus. Young's modulus can also be calculated from load-displacement measurements. A 2D surface stiffness image can be created by scanning the surface with the stylus. Nanoindentation measurements are routinely performed using scanning force microscopes [6, 7]. These mechanical properties will not be discussed here. Instead, this section will deal with the direct imaging of the surface features on length scales of 1 to 10 m. There are many microscopic techniques that provide surface morphology and topographic data at different length scales. The technique to be chosen depends on the information that is being sought. Some of the factors that need to be considered are the lateral resolution (nm to m), the dimensions of the surface to be examined (m to mm), the depth of the surface (nm to m), and the sample
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Table 9.1 Essential features of the various techniques used for characterizing surface structure and morphology [24]
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Technique
Probe
Depth analyzed (m)
Lateral resolution (m) (magnification)
Information
Comments
Optical microscopy, OM
Light
0.1
0.3 (2 to 2000)
Surface roughness, structure
Many possibilities, good height resolution with interference techniques
Scanning electron microscopy, SEM
Electrons
2
0.1 (20 to 1 105 )
Surface topography
Vacuum technique
Scanning force microscopy, SFM
Cantilever
0.001
0.0005 (1000 to 2 106 )
Surface topography, composition, toughness
Atomic resolution, many different modes
Profilometry
Light/ cantilever
0.01
5
Surface texture
Quantitative measure of surface roughness
X-ray reflectometry, XR Grazing incidence x-ray small-angle scattering, GISAXS
X-rays
0.5
1
Surface roughness, thin surface layers, lateral structure
Flat surfaces required
Neutron reflectometry, NR
Neutrons
0.5
±
Surface roughness, enrichment layer
Deuterated compound needed
Micro-indentation, MI
Cantilever
100
200
Surface hardness, modulus
Quantitative interpretation difficult
Confocal Raman imaging
Light
50
2
Chemical species
Raman active bands required
Pendent drop
Liquid
0.2
Surface tension/ contact angle, ST
Liquid drop
0.1
1000
Interface tension
Indirect technique
Surface energy
Easy to use, molecular information difficult
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environment (vacuum, ambient, metal coated or immersed in water). Optical microscopy is the most versatile of the methods for examining the surface morphology [8]. However, the lateral resolution is limited to ~ 300 nm. Scanning near field techniques (SNOM) and interference techniques enhance the utility of the technique [9]. For higher resolution, the most established techniques are the scanning and transmission electron microscopy, scanning tunneling microscopy and atomic force microscopy.
9.2.1
Confocal microscopy
One of the main drawbacks of a conventional light microscope is that the object planes outside of the focal plane contribute equally to the image as those points at the focal plane, thus producing a blurred image. Confocal laser scanning microscopy (CLSM) [10, 11], also called laser scanning confocal microscopy (LSM), provides a blur-free image by eliminating the out-of-focus light or glare and can be used to collect serial optical sections from thick specimens. In a conventional wide-field microscope, the entire specimen is illuminated from a suitable lamp and the image formed by a series of lenses is viewed directly by eye or captured on a detector. In contrast, in confocal microscopy, the surface is scanned by one or more focused beams of light, usually a laser, and the image is reconstructed on a computer (Fig. 9.1). Laser light from the illuminating aperture passes through an excitation filter (not shown), reflected
9.1 Schematic of the confocal principle in epifluorescence scanning mode.
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by the dichroic mirror and is brought into focus by the objective lens to a diffraction limited spot at the focal plane within the specimen. The light from the specimen is passed through a pinhole that provides the crucial spatial filtering, and is detected by a photomultiplier tube (PMT). Although unstained specimens can be viewed using light reflected from the specimen, the samples are usually stained or labeled with one or more fluorescent probes. In this fluorescence mode, fluorescence emissions excited both within the illuminated in-focus voxel (volume picture element) and within the illuminated cones above and below it, are collected by the objective and pass through the dichroic mirror and the emission filter (not shown). In both of these modes, only the light from the in-focus voxel is able to pass unimpeded through the imaging aperture to be detected by the PMT. The emissions from regions below the focal plane and from above it have different primary image plane foci and are thus severely attenuated by the imaging aperture, contributing little to the final confocal image. The output from the PMT is built into a 2D image. Image resolution is typically 0.2 m in the transverse plane (xy-plane) and ~ 0.5 m in the z-plane. The greatest advantage is the possibility of making a 3D image of the surface of the sample within a depth of 100±200 m. Application Confocal microscopy is widely used to examine surface and near-surface features in biomaterials such as bone [12], dentin and enamel [13], imaging cells on substrates, topography of substrates, and porous structures in scaffolds. Confocal images are typically obtained using the light reflected from the sample (epitransmission or reflectance) or by capturing the fluorescent light that is excited in the sample by the incident beam (epifluorescence). In one example, CLSM was used to study the bimodal porous structure in cancellous bone. The images shown in Fig. 9.2 were obtained in reflectance mode from a single location on defatted and deproteinized cancellous canine bone specimen [12]. Both the surface and near-surface features can be seen in the Z-stack overlay image shown in the figure. The many distinct macropores have micropores along their walls (indicated with arrows). The macropores are interconnected through the micropores in the intertrabecular space. Such features are difficult to see with other imaging techniques. Confocal microscopy is an effective tool to nondestructively image the surface and near-surface structures in a way that is not possible with other techniques such as microcomputed tomography and magnetic resonance imaging.
9.2.2
Scanning electron microscopy (SEM)
SEM reveals the surface features at nm lateral resolutions, about two orders of magnitude better than optical microscopes, and is often preferred over optical
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9.2 Z-stack overlay of the confocal image of a cancellous bone obtained in reflection mode from a Zeiss 210 CLSM (Carl-Zeiss, Jena, Germany) using an Ar 488 nm laser. The figure shows macropores and interconnecting micropores (indicated with arrows) [12]. Reprinted with permission from `Confocal Laser Scanning Microscopy as a Tool for Imaging Cancellous Bone' by I.O. Smith et al., published in Biomed. Mater. Res. Part B: Appl. Biomater. (2006) 79B: 185± 192, ß 2006, Wiley Periodicals, Inc.
microscopy because of the increased depth of field at low resolutions. However, at resolutions approaching sub-m (3±6 nm, best case), SEM is limited by its depth of field (<<1 m at high magnification), and it cannot image subsurface features [12]. SEM uses a beam of electrons that interact with the specimen in a vacuum environment (Fig. 9.3). Various interactions occur between the electron beam and the sample surface including transmission of the primary electrons, secon-
9.3 Schematic diagram of a scanning electron microscope.
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dary electron emission, back scattering, absorption and emission of light and xrays. In transmission electron microscopy (TEM), the transmitted electrons are used to produce an image, the contrast arising from the difference in electron absorption and scattering in different parts of the sample. In SEM, the primary beam is rastered over the sample, and the secondary electrons that are emitted from the surface of the material are collected and composed to provide an electron micrograph. The contrast arises from either electron absorption or emission. Application SEM is widely used because of the fairly simple sample preparation and the ease of using the equipment. It is used to study scaffolds, fiber mats, surfaces of bones and joints. One common application is the use of SEM to examine the surface roughness of implants that can often be related to its performance in vivo. There are several studies in which titanium surfaces have been examined after different types of surface treatments such as after sand-blasting with Al2O3 particles, plasma spraying and elecrolytically coating with hydroxyapatite [14]. Another example of surface treatment is laser ablation. Laser ablation provides a means to modify the surface and thus control the tissue interactions. Figure 9.4 shows an example of how SEM was useful in showing the changes in the surface texture upon ablation of poly(ethylene terephthalate) (PET) surface [15]. PET is widely used in biomedical applications including implantable sutures, surgical mesh, vascular grafts and heart valves [16]. The montage in the figure shows the progressive change in the morphology with the increase in the laser fluence, from an unirradiated area to the left to the ablated area on the right. The figure shows the texture changes from ripple into densely packed cones, before these cones are ablated and removed at higher beam fluence. The figure is most illustrative in that m resolution can be achieved while examining a relatively large area ~ 0.1 mm, and also the large depth of field that can be obtained. Sample size is limited to about 10 mm in diameter. If the sample is not electrically conductive, then a thin (20±30 m) metallic coating, typically gold, platinum or god/palladium alloy is sputter coated onto the sample to prevent electrical charging of the specimen. In addition to imaging the surface features, SEM is often combined with energy dispersive x-ray analysis (EDX) to obtain a map of the elemental distribution.
9.2.3
Scanning force or atomic force microscopy (SFM or AFM)
This is one in a family of scanning probe techniques that have been used successfully in high resolution imaging and molecular imaging of biological
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9.4 A montage of the images from an SEM showing the laser-ablated regions on the surface of PET injection molded plaque. The fluence of the laser beam increases from left to right [15]. Reprinted with permission from: `Self-assembled and etched cones on laser-ablated polymer surfaces' by N.S. Murthy et al., J. Appl. Phys. (2006) 100: 023538, 1±12, ß 2006, American Institute of Physics.
ß Woodhead Publishing Limited, 2011 9.5 (a) Illustration of the principle of AFM. (b±d): A schematic of the three common AFM operation modes. (b) Contact mode. (c) Tapping mode. (d) Phase imaging mode [17]. Reprinted with permission from `Atomic force microscopy of biomaterials surfaces and interfaces' by K.D. Jandt, Surface Science (2001) 491: 303±332, ß 2001, Elsevier.
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macromolecules since their invention in the 1980s [17, 18]. In SFM, the force between a probe and the sample surface is used to map the surface morphology and to study the interaction forces in biological systems down to the picoNewton range, typical of single ligand-receptor interaction. Other techniques, not discussed here, use tunneling current, chemical affinity and other phenomena to probe the surface characteristics. In SFM, a 3D image of the surface is created by scanning a micron-size cantilever across the surface (Fig. 9.5). The cantilever is typically made of silicon or silicon nitride, with a tip whose radius of curvature is ~ 10 nm. The forces between the tip and the surface, which are ~ nN and include contributions from Van der Waals interactions, chemical bonding, electrostatic forces and solvation forces, deflect the cantilever. The deflection of the cantilever due to these forces is measured using a laser spot reflected from the top surface of the cantilever into an array of photodiodes. This signal is used maintain a constant force via a feedback loop, or to maintain a constant height, depending on the scan mode that is chosen. The sample is moved across the tip by piezo crystals. The AFM can be operated in a number of modes depending on the application, and three of these are presented in Figs 9.5(b±d). In the static mode, also called the contact mode, the force between the tip and the surface is kept constant during scanning, and the change in the cantilever deflection required to maintain this constant force is used to obtain an image of the sample's surface topography. This mode is suitable for hard surfaces but not for biomaterials which are soft and whose surface may have weakly adsorbed molecules. In the dynamic mode, also called the tapping mode, the cantilever is vibrated close to its resonant frequency, and the changes in amplitude and phase are monitored. The reduction in amplitude is monitored and used to map the surface topographical features. When the AFM is operated in the tapping mode, it is possible to take advantage of the difference in the phase of the cantilever oscillation and the signal sent to the cantilever. This phase difference depends on the sample's hardness, elasticity and adhesion. This phase lag is monitored and a 2D phase image can be produced to qualitatively map the surface viscoelastic properties of the surface such as to map the distribution of soft biomolecules on a stiff substrate. Application Scanning probe microscopes are useful in characterizing surfaces at atomic resolutions in a variety of environments from ultrahigh vacuum to aqueous solutions. It is also used to study time-dependent phenomena such as conformational change of the molecules and the shapes of the whole cells adsorbed onto a surface, hydration induced changes, crystallization and corrosion processes. A typical contact mode AFM image is shown in Fig. 9.6a, and is compared with a corresponding SEM image of a similar sample in Fig. 9.6b. These images
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9.6 Comparison of (a) AFM (deflection in contact mode) and (b) SEM (secondary electron) imaging technique on similar, dry titanium/titanium oxide surfaces (2 2 m 2 ). Oxide domes are visible in both the images [19]. Reprinted with permission from `Direct observation of hydration of TiO2 on Ti using electrochemical AFM: freely corroding versus potentiostatically held' by J.P. Bearinger, C.A. Orme and J.L. Gilbert, Surface Science (2001) 491: 370± 387, ß 2001, Elsevier.
were a part of a study to characterize the morphology of the titanium surfaces in saline-based oxide hydration events in situ under varying biomedically relevant electrochemical conditions in the hydrated state [19]. The figure shows the surface in air prior to immersion. The oxide domes are more clearly visible in AFM than in SEM. Average dome height in the AFM image is 4:3 1:7 nm and the average dome width is 91 16 nm. Phase imaging is widely used to image soft particles embedded in stiffer matrices. A biologically relevant example of the use of the phase image is the observation of the adsorption of biomolecules in the presence of roughness that is inherent in biomaterial surfaces. AFM images of a National Heart Lung and Blood Institute (NHLBI) reference polydimethylsiloxane (PDMS) with and without adsorbed fibrinogen are shown in Fig. 9.7 [20]. Fibrinogen is a plasma protein important for blood coagulation and platelet aggregation, and PDMS is a commonly used substrate for protein adsorption and cell studies. The images were obtained in the tapping mode. The topographic data is presented in Fig. 9.7a and the phase data in Fig. 9.7b. Proteins were adsorbed at a concentration of 500 ng/ml and the surface concentration is ~ 10 molecules/m2. Although isolated fibrinogen molecules adsorbed onto the surface can be observed in the height image, they are more clearly visible in the phase image in which the topography of the PDSM substrate does not appear because of phase imaging. Highest resolution images are obtained when the AFM is operated in the contact mode. However, because the tip is in constant contact with the sample, the shear forces applied to the sample during scanning can potentially damage
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9.7 AFM tapping mode data from PDMS with and without adsorbed fibrinogen [20]. (a) Height image. (b) Phase image. Reprinted with permission from `Individual plasma proteins detected on rough biomaterials by phase imaging AFM' by N.B. Holland and R.E. Marchant, J. Biomed. Mater. Res. (2000) 51: 307±315, ß 2000, Wiley Periodicals, Inc.
the surface, especially the weakly bound protein molecules adsorbed onto the biomaterials. In the tapping mode, the tip is not in contact with the sample during scanning. Thus the forces applied to the sample surface are negligible. Furthermore, in the tapping mode, phase imaging can be used to map surface inhomogeneities that give rise to variations in viscoelasticity of the material surface but do not alter the physical appearance or the topography of the surface.
9.2.4
Profilometry
The microscopic techniques discussed in Sections 9.2.1±9.2.3 provide an image of the surface texture, but not the quantitative measure of the surface roughness. This can be done by profilometry, in which a probe, mechanical (contact) or optical (noncontact), is passed across the surface [21]. The probe follows the contours at each point on the surface, and the height of the probe at each point is recorded and the resulting 1D scan or a 2D map is analyzed. A mechanical stylus captures the features over a large area (~ 100 mm), with a x-y resolution of 5 m and a z-resolution of 0.01 m. An optical probe such as a confocal laser scanning microscope has a range of ~ 2 mm, x-y resolution of 5 m and the z resolution of 0.01 m. AFM covers a much smaller area (100 m) but provides much higher resolutions (0.2 m in x-y and 1.5 pm in z). The force applied with a mechanical stylus is 5 mN compared to 1 N in AFM. Parameters such as arithmetic average of the absolute values of all points of the profile (Ra), root means square values of all the heights around the mean (Rq) are often used to quantify the roughness. These, however, do not reflect the distance between the features and their shape.
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Application Profilometry is frequently used in the evaluation of surfaces of metallic surgical implants. These implants are treated in various ways to bring about chemical and topographic modifications of the surface to improve the osseointegration process and the biomechanical performance of implants. In one study profilometry was used to measure the surface roughness in three different materials, poly-L-lactic acid (PLLA), poly-DL-lactic acid (PDLA), and sodium alginate hydrogel (AGA-100) used in clinical practice, and correlate this to the osteoblast adhesion. These were coated onto mechanically and chemically treated (sand-blasted and acid-etched) Ti substrates. Figure 9.8 shows how the changes in the surface roughness due to different coatings can be monitored using contact profilometry images. These results were used to identify a specific roughness parameter (peak density) which mainly controls the amount of osteoblast adhesion a crucial factor in rapid `osteointegration' [22]. The average roughness was 4.31 in the control (sand-blasted and acid-etched) sample, and was 2.39, 1.39 and 2.78 in AGA-100, PLLA and PDLA coated samples,
9.8 Contact profilometry of Ti surfaces [22]. (a) The initial sand-blasted and acid-etched surface prior to coating. (b) Coated with PLLA. (c) Coated with PDLA. (d) Coated with sodium alginate hydrogel AGA-100. Reprinted with permission from `Contact profilometry and correspondence analysis to correlate surface properties and cell adhesion in vitro of uncoated and coated Ti and Ti6Al4V disks' by A. Bagno et al., Biomaterials (2004) 25: 2437±2445, ß 2001, Elsevier.
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respectively, reflecting the modification of the surface roughness to different degrees depending on the coating material. Only AGA-100 was able to preserve, to some extent, the roughness in the starting surface. Only the starting and AGA100 coated samples allowed the osteoblasts to adhere, and the other two seemed to hinder the adherence. Further evidence suggests that it is indeed this surface roughness and not the chemical composition that is responsible for the differences in adhesion. Profilometers generate an image of the surface height. Size of the area measured and the size of the probe set the upper and lower limits on the size of the features that can be characterized. The nature of the probe and the force exerted by the probe on the surface limit the range of surfaces that can be investigated by these techniques. Although optical techniques are more appropriate for relatively soft materials, stylus technique is more appropriate for examining large areas with substantial slopes within the surface structure [23]. The advantages of this method are that it is direct, less expensive and reproducible.
9.3
Surface structure and spatial distribution
The techniques described in the previous section are useful in examining the morphology, texture and surface topography. At the other extreme, there are surface spectroscopic techniques such as Auger electron spectroscopy, x-ray photoelectron spectroscopy and secondary ion mass spectrometry that provide the chemical makeup or the elemental composition at or near the surface. These techniques are discussed in Chapter 8. Between these two extremes, there is another category of techniques for examining the structure, the molecular organization, surface orientation that change as a result of surface treatment. These structures near the surface can be studied using methods that are surfaceenhanced versions of the classic x-ray scattering, IR and Raman spectroscopic techniques. These classical techniques have been adapted so that x-ray, IR or visible light penetrate only the surface layers of the material, and provide information about the surface structure. This section will discuss the use of x-ray scattering and Raman spectroscopic methods for analyzing the surface structures that arise as a result of surface orientation, phase separation, crystallization and degradation.
9.3.1
X-ray reflectivity and scattering
When x-rays are incident on a surface at very shallow angles, two events can occur. First, just like light, x-rays are also reflected from surfaces. But, because the refractive index of solids for x-rays is < 1, this specular reflection (reflected angle equal to incident angle) occurs for incidence angles less than the critical angle, ~ 0.1ë (Fig. 9.9a). In this configuration, known as reflectometry, the profile of the reflected beam is analyzed to characterize roughness as well as investigate phenomena such as interdifussion, blending, surface-induced order,
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adsorption or surface enrichment of components [24]. X-ray reflectometry is an ideal tool to quantify the surface roughness in the nm length scale. If the interface between the sample and air is not perfectly sharp and smooth then the reflected intensity will deviate from that predicted by the simple Fresnel equations. These deviations are analyzed to obtain the density profile of the interface normal to the surface. Such reflectivity measurements provide surface roughness [25] and the structure of deposited membranes [26]. In a related second technique, the x-rays are incident at a very shallow angle ( , Fig. 9.9b) such that the path within the sample is considerably long so as to attenuate the beam within a small depth from the surface. This depth depends on the absorption coefficient of the substrate, and typically a few m for polymers. In this technique, called grazing-incidence scattering, the intensity measured function of the scattering angle 2 will reveal the structure near the surface of
9.9 (a) Schematic of the sample geometry used in x-ray reflectivity measurements. (b) Geometry of the sample, incident and scattered beam used in the data collection in grazing-incidence scattering. (c) Measured x-ray reflectivity plotted as R/RFresnel versus Qz, where RFresnel is the reflectivity of an infinitely sharp, step-like interface [26]. Reprinted with permission from `Characterization of biological thin films at the solid-liquid interface' by C.E. Miller et al., Phys. Rev. Lett. (2005) 94: 238104, 1±4, ß 2005, American Institute of Physics. (d) Arrangement of the head groups and the hydrocarbon tail region modeled based on the data in (c).
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the sample. By varying the angle of incidence, one can probe layers of increasing thickness and analyze the structure and chemical composition by means of diffraction or fluorescence experiments. Grazing incidence small-angle x-ray scattering (GISAXS) provides surface sensitive information at length scales of 1±100 nm. Such measurements have been used to tailor the island density, shape and size distributions. Applications The example shown in Fig. 9.9 illustrates the use of x-ray reflectivity to characterize the phospholipids bilayer membranes at the solid-water interface. The x-ray reflectivity curve typically obtained is shown in Fig. 9.9c. This curve is modeled using a model, in this case, a 4-slab model made of outer head groups, hydrocarbon tails, inner head groups, and water cushion (Fig. 9.9d). The lipid used in this case was 1,2-Dioleoyl-Sn-Glycero-3-phosphocholine (DOPC). Ê hydrocarbon tail region, outer head The data can be explained in terms of 23.2 A Ê Ê group region thickness of 10 A with a 6 A roughness, and the inner head group Ê with 3.8 A Ê roughness. Finally, the model also suggests a thin thickness of 8 A Ê 4 A layer of water between the lipid and the quartz substrate. Such x-ray reflectivity measurements have been used to model the surface roughness as well as the structure near the solid-solution interface, and in the case just discussed, that of the lipid bilayer. The technique requires that the surfaces be flat, extending to a few centimeters. The measurements are performed with x-rays illuminating the surface at grazing incidence, typically a few milliradians close to the critical angle for total reflection at the material-air interface. Because of the reduced intensity due to tight collimation, such measurements are typically performed at synchrotron sources.
9.3.2
Confocal Raman spectroscopy
Raman spectroscopy, like infrared spectroscopy, is highly sensitive to molecular species present in the material. Combining this with the confocal principle described in Section 9.1.2, makes it a powerful tool for obtaining the structural information near the surface. Unlike infrared spectroscopy, Raman spectroscopy is relatively insensitive to water, and hence is potentially more useful in the characterization of the microstructure of biomaterials where water is invariably present. Recent advances in laser sources, optical elements and detector technology have enabled mapping of the surfaces based on the identity and concentration of the selected species. It can be used to nondestructively image the surface to a depth of ~ 50 m, and has been used, for instance, to analyze the surface and assess the residual stress fields in artificial hip joints [27, 28], in addition to studying the oxidation state, crystallization behavior and phase fractions.
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In typical analytical measurements, the Raman spectral intensities are directly proportional to the concentration of the corresponding species within the relatively small deviation in Raman response. Concentrations can be measured from the intensities of an interference-free peak after calibration using a reference material. The most common way to Raman map a surface is to find a characteristic strong band in the species of interest, a band that is free from interference from other species, and map the intensity of this band. However, when strongly overlapping bands are present, as in most biomaterials, chemometrics methods such as multivariate curve resolution are used to obtain the Raman images [29]. Spatial resolutions of 2 m in the x±y plane and 3 m to a depth of 50 m in the z-plane are achievable. Application Confocal Raman imaging is commonly used to examine the changes that occur near the surface due to adsorption of molecules, hydration (with D2O labeled samples), and degradation. By combining the micrometric lateral resolution of a laser beam focused on the polymer surface with a proper selection of Raman bands, it is possible to obtain a highly resolved 2D map of the conformational population patterns, including crystalline and amorphous phase fractions, and oxidation states. In one example, confocal spectroscopic techniques were used to study the microscopic features due to wear in acetabular cups made of ultra-high molecular weight polyethylene (UHMWPE) before and after implantation in vivo (Plate IV between pages 208 and 209) [30]. The figure shows significant oxidation gradients along the subsurface of the long-term implanted retrieval, especially in the main wear zone. A clear relationship is found between orthorhombic crystalline fraction and degree of oxidation along the subsurface, and this was attributed to partial crystallization of polyethylene on oxidation. It is important to note that these confocal experiments show relatively high crystallinity in the immediate subsurface of the cup during implantation lifetime in vivo. This could be related to the formation of polyethylene debris on wear contact. Thus, near-surface UHMWPE gets oxidized and crystallizes during aging in vivo, which could induce significant embrittlement of the polymeric subsurface.
9.4
Energetics
Surface energy is the work required to increase unit surface area at constant temperature, pressure and composition. Surface tension, which is related to surface energy, determines the wettability of a surface and as such plays an important role in determining the biocompatibility, adsorption processes and adhesion in biomaterials. It reflects surface roughness, and is sensitive to both
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surface segregation of components and surface contamination. Surface tension also reflects the strength of bonding within the bulk materials. Hard solids with covalent, ionic or metallic bonds have high surface energy surfaces (surface tension 500±5000 mJ/m2). Most polymers are soft solids in which van der Waals and hydrogen bonding forces play a major role and which have low energy surfaces (< 100 mJ/m2).
9.4.1
Contact angle measurements
The surface tension is most often determined by measuring the contact angle of a drop of liquid positioned on the surface. The contact angle is the angle at which a liquid-vapor interface meets the solid surface. This angle is determined by the interactions across the three interfaces. In Fig. 9.10, SL, SV and LV are the interfacial energy (surface tension) at the solid-liquid, solid-vapor and liquid-vapor interfaces, respectively, and the Young's relation given in the figure relates the contact angle to these three forces. There are several methods of determining the surface tensions including measuring the direct force by Wilhelmy balance, contact angles using sessile drop or captive air bubble method, and capillary penetration into porous systems [31]. Contact angle using sessile drop is the most widely used and will be illustrated here. A drop of fluid, water for most biomaterials, is placed on the surface of interest. The contact angle is determined from the tangent to the liquid drop at contact where the liquid and the solid intersect. Surface roughness or heterogeneity can be evaluated by measuring the contact-angle hysteresis, i.e. by measuring the advancing and receding contact angles [32]. As the volume of drop is increased, and the drop advances over the surface, the edge of the liquid is pinned at the boundaries of the wetting and non-wetting interfaces (if the surface is heterogeneous) or the sharp edge of the surface boundary (if the surface is rough). Thus, the contact angle will be higher than the equilibrium value. Similarly, because of the same pinning the contact angle will be lower than the equilibrium value when the volume of the droplet decreases as the liquid is withdrawn from the droplet. The resulting hysteresis can be used to characterize the surface roughness or heterogeneity.
9.10 Illustration of the Young's force balance giving the equilibrium contact angle.
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Application The use of contact-angle measurement will be illustrated with studies of dynamic contact-angle measurements and adsorption of -globulin made as a function of Wenzel ratio (roughness factor calculated as the ratio of the actual and the projected surface areas) [33]. Contact-angle hysteresis measurements were made on well-defined nanostructured surfaces that were fabricated by growing germanium nanopyramids on Si(001) surfaces. AFM images shown in Fig. 9.11 indicate the different surface morphologies. These surfaces are made of nanopyramids and the density of these pyramids determines the surface roughness It has been reported that surface roughness modifies the contact angles and the contact-angle hysteresis of wetting. There is no consensus as to whether the contact angle increases with roughness and on the effect of roughness on the hysteresis behavior [33]. The results show that the advancing contact angle of water monotonically increases by 20ë from the flat substrates to substrates with maximum pyramid density whereas the receding contact angle remains constant. The biocompatibility of materials is determined at the most fundamental level by protein adsorption. The amount of adsorbed proteins bovine -globulin (BGG) increases significantly with the density of nanopyramids on the substrate, i.e., with the roughness of the surface. Increase in the effective surface by 7% causes a 2- to 4-fold increase in the adsorbed protein. However, the activity of the adsorbed BGG was found to decrease with pyramid density. These results demonstrate that wetting behavior and biocompatibility are both strongly correlated with the surface nanoarchitecture, and thus illustrate the importance of careful surface topographic measurements in understanding the surface-induced biological processes.
9.5
Future trends
With great advances in surface modification discussed elsewhere in this book, the need for rapid characterization of the surface features inexpensively and reproducibly will continue to grow. Optical microscopy will continue to be a most versatile tool to examine surface morphology. Moving beyond the confocal microscopes, near-field scanning optical microscopy can deliver resolutions normally associated with electron microscopy [34]. It uses tapered glass optical elements and techniques of scanned probe imaging (e.g., AFM) to provide images at 50 nm resolution, far below the Rayleigh resolution limit of half the wavelength of the imaging radiation (~ 250 nm). A major limitation of SEM is that the samples that are imaged are typically in a high vacuum environment. Innovative sample chambers are now being used that permit the samples to be in different environments such as moisture. One trend in SEM has been to work at a low incident beam energy (< 5 keV) in order to minimize beam penetration,
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9.11 (a±d) AFM images indicating the increase in surface roughness with increasing nanopyramid density. (e) Advancing (open circles) and receding (filled circles) contact angle hysteresis for water in surfaces with different nanopyramid density. (f) Protein adsorption of BCG and anti BCG vs. roughness factor [34]. Reprinted with permission from `Impact of nanometerscale roughness on contact-angle hysteresis and globulin adsorption' by B. Muller, et al., J. Vac. Sci. Technol. (2001) 19: 1715±1720, ß 2001, AVS: Science & Technology.
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9.11 Continued
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beam spreading, and sample charging and thus improve surface image resolution. Alternatively, high energy beams are being used obtain complementary information such as subsurface features that can be combined with the data obtained at low energies. Scanning probe techniques continue to evolve with the development of new probes to map different aspects of the surface. Availability of high-intensity x-ray synchrotron sources makes it much easier to obtain reflectivity and grazing incidence measurements for the detailed evaluation of surface structures. In more traditional techniques such as contact angle measurement, automation of the measurement and analysis enables highly reliable data to be obtained even by those who may lack specialized skills. In all these techniques, technology is used to move towards the ultimate goal of viewing features at high resolutions in a large dynamic system at high imaging speeds.
9.6
References
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16. von Recum, A.F., ed. Handbook of Biomaterials Evaluation: Scientific, Technical and Clinical Testing of Implant Materials, 2nd edn, 1998. Boca Raton, FL: CRC Press. 17. Jandt, K.D., Atomic force microscopy of biomaterials surfaces and interfaces. Surface Science, 2001. 491(3): 303±332. 18. Siedlecki, C.A. and R.E. Marchant, Atomic force microscopy for characterization of the biomaterials interface. Biomaterials, 1998. 19(4±5): 441±454. 19. Bearinger, J.P., C.A. Orme and J.L. Gilbert, Direct observation of hydration of TiO2 on Ti using electrochemical AFM: freely corroding versus potentiostatically held. Surface Science, 2001. 491: 370±387. 20. Holland, N.B. and R.E. Marchant, Individual plasma proteins detected on rough biomaterials by phase imaging AFM. J. Biomed. Mater. Res., 2000. 51: 307±315. 21. Assender, H., V. Bliznyuk and K. Porfyrakis, How surface topography relates to materials' properties. Science, 2002. 297: 973±976. 22. Bagno, A. et al., Contact profilometry and correspondence analysis to correlate surface properties and cell adhesion in vitro of uncoated and coated Ti and Ti6Al4V disks. Biomaterials, 2004. 25: 2437±2445. 23. Wennerberg, A. et al., Characterizing three-dimensional topography of engineering and biomaterial surfaces by confocal laser scanning and stylus techniques. Med. Eng. Phys., 1996. 18: 548±556. 24. Stamm, M., Polymer surfaces, interfaces and thin films studied by x-ray and neutron reflectometry, in Scattering in polymeric and colloidal systems, W. Brown and K. Mortensen, eds, 2000. Amsterdam: Gordon and Breach Science Publishers, pp. 495±534. 25. Stone, V.W. et al., Roughness of free surfaces of bulk amorphous polymers as studied by x-ray surface scattering and atomic force microscopy. Phys. Rev. B, 1999. 60: 5883±5894. 26. Miller, C.E. et al., Characterization of biological thin films at the solid-liquid interface by X-ray reflectivity. Phys. Rev. Lett., 2005. 94: 238104 (1-4). 27. Pezzotti, G., Stress microscopy and confocal Raman imaging of load-bearing surfaces in artificial hip joints. Exp. Rev. Med. Dev., 2007. 4(2): 165±189. 28. Pezzotti, G. et al., Confocal Raman spectroscopic analysis of ceramic hip joints. Key Engineering Materials, 2006. 309±311: 1211±1214. 29. Andrew, J.J. and T.M. Hancewicz, Rapid analysis of Raman image data using twoway multivariate curve resolution. Appl. Spectro., 1998. 52: 797±807. 30. Pezzotti, G. et al., Confocal Raman spectroscopic analysis of cross-linked ultra-high molecular weight polyethylene for application in artificial hip joints. J. Biomed. Opt., 2007. 12(1): 0141011 (1±14). 31. Grundke, K., Characterization of polymer surfaces by wetting and electrokinetic measurements ± contact angle, interfacial tension and zeta potential, in Polymer surfaces and interfaces, M. Stamm, ed., 2008. Berlin: Springer. 32. Extrand, C.W., in Encyclopedia of surface and colloid science, P. Somasundaran, ed., 2006. Boca Raton, FL: CRC Press, pp. 2876±2891. 33. Muller, B. et al., Impact of nanometer-scale roughness on contact-angle hysteresis and globulin adsorption. J. Vac. Sci. Technol., 2001. 19(5): 1715±1720. 34. Doose, S., Trends in Biological Optical Microscopy. Chem. Phys. Chem., 2008. 9: 523±528.
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Plate IV (a) Photograph of a long-term retrieved cup. (b±e) Maps of wear index (related to oxidation index) collected at increasing depths of focal plane z0 of the confocal probe on the long-term retrieved UHMWPE acetabular cup shown in (a). (b and d): Nonwear zone. (c and e): Wear zone. Reprinted with permission from Pezzotti G et al., `Confocal Raman spectroscopic analysis of cross-linked ultra-high molecular weight polyethylene for application in artificial hip joints', J. Biomed. Opt. (2007) 12: 014011 1±14, 2007, SPIE.
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