Materials for Biomedical Engineering. Mohamed N. Rahaman

surface roughness, and on the type of cell as well. Consequently, an understanding of the interaction between surface topography and cell response is essential in creating implants with the desired surface topography. The scale of roughness that has the greatest effect on cell response is approximately equal to the cell dimension or smaller (Figure 5.19). This is because a surface will be perceived as approximately smooth by cells if the scale of roughness is larger than the cell diameter. Additionally, cells can respond differently to a “spiky” surface when compared to a smoothly undulating surface even though the peak to valley distance of the roughness is the same (Xu et al. 2017).

Figure 5.19 Schematic illustrating surface roughness parameters that can have a strong influence on cell response: scale of roughness spacing relative to the cell dimension and spikiness of roughness feature.

      5.5.1 Surface Roughness Parameters

      Surface roughness is commonly quantified in terms of the vertical height of the surface relative to a mean or reference line, defined such that the area between the roughness profile and the mean line is the same above and below the line. While a variety of parameters are used to quantitate the topography of materials, the average roughness R_a and the root mean square roughness R_q are most often used. R_a is the arithmetic mean of the average values of the vertical deviation from the mean line of the roughness profile whereas R_q is the square root of the arithmetic mean of the square of the vertical deviation from the mean line. As these two parameters are primarily concerned with the relative departure of the roughness profile in the vertical direction only, they do not provide information about the slopes, shapes, and sizes of the surface asperities or about their spacing. It is possible for surfaces of widely different profiles to have approximately the same R_a or R_q values. In general, these two parameters are more useful for characterizing surfaces of the same type that are produced by the same method.

      5.5.2 Characterization of Surface Topography

      Characterization of surface topography commonly starts with examining the surface of one or more representative specimens of the material in a scanning electron microscope (SEM) (Figure 5.18). Qualitative information about topographical features such as surface roughness at a microscale or nanoscale, the waviness and spacing of the surface roughness, and the presence of surface flaws can be obtained using this technique. Thereafter, other techniques, classified into two broad categories, depending on whether a component of the measuring instrument makes contact with the specimen surface (contact type) or not (noncontact type), can be used to obtain topographical data such as surface roughness parameters. As a contact‐type technique has the potential for damaging a soft surface, applied loads on the contacting component (for example, a stylus) should not exceed the hardness of the surface. The major techniques used for biomaterials are profilometry and atomic force microscopy (AFM), both of which can be used in a contact or noncontact mode.

      Scanning Electron Microscopy (SEM)

SEM is a widely used and versatile technique for characterizing the microstructure and topography of materials based on its simplicity, type of information obtained and its scale of resolution (down to a few nanometers). It relies on scanning a finely focused electron beam over the surface of a specimen and analyzing the interaction of the beam with the material. In general, an incident electron beam can produce a variety of emissions from a material, a few of which are used in the SEM (Figure 5.20). Depending on the thickness of the specimen and the energy of the incident electron beam, a certain fraction of the incident electrons will be scattered in the forward direction, used in transmission electron microscopy (TEM), a fraction will be absorbed and the remaining fraction will be scattered in the backward direction. As the capacity of atoms in the specimen to scatter the incident electrons is a function of their atomic number, these backscattered electrons are used in the SEM to provide compositional contrast between different (solid) phases at the surface.

Figure 5.20 Emissions produced by the interaction of an electron beam with a solid specimen.

      The incident electron beam can also generate secondary effects. One type of effect is that the incident electrons can knock electrons out of their orbits around the atom. These electrons may have enough energy to escape from the specimen and become what are called secondary electrons if they are near the surface (within ~20–100 nm). These secondary electrons are used to produce an image of the microstructure (Figure 3.23) and topography (Figure 5.18) of the material. Another type of secondary effect occurs when an electron undergoes a transition from one energy level to fill a vacant site in a lower energy level of an excited atom, generating radiation in the form of X‐rays or light. As the X‐rays from each element will have a different energy (or wavelength), we can detect which element emitted them by measuring their energy in a spectrometer. This is the basis of energy‐dispersive X‐ray (EDS) analysis in the SEM.

EDS analysis provides rapid qualitative and quantitative information about the elemental composition of the specimen volume that is being analyzed by the electron beam, which, in the SEM is commonly an area of approximately 1 μm by 1 μm and a depth of ~1 μm. For qualitative analysis, the presence of an element is determined from a comparison of the energy values of the peaks in the spectrum with the energy values of known elements (Figure 5.21). Quantitative analysis, such as the percentage of each element in the specimen, is determined from the relative intensity of the peaks present in the spectrum. Semiquantitative analysis without standards, with an accuracy of approximately ±5%, can be quickly performed using software, but much greater accuracy is achieved using calibration standards of known compositions that contain the elements present in the specimen.

Figure 5.21 EDS spectrum of a borosilicate glass examined in the SEM, showing the elements (in addition to B) present in a surface layer of thickness ~1 μm.

      In common with most electron‐beam techniques, conventional SEMs require the use of a high vacuum environment that is shared by the electron beam column and the specimen chamber. This necessitates the use of clean, dry specimens that often have a different surface chemistry from the actual biomaterials implanted in vivo. Electrically insulating materials such as ceramics and polymers must also be sputter‐coated with a thin layer of a conducting material, typically a gold–palladium alloy or carbon, to reduce static charging at the specimen surface. In view of these limitations, the environmental scanning electron microscope (ESEM) has been developed which allows the examination of specimens under a variety of conditions more relevant to the use of biomaterials. The ESEM uses pressure‐limiting apertures that separate the electron beam column from the specimen chamber, allowing the use of a variety of humidity levels, pressure, temperature, and ambient gas or