In vitro characterization of biomineralization
The analysis of biomaterials must begin at an in vitro level to better understand material characteristics, how they will behave in an in vivo-like environment, and minimize the use of animals. Often these techniques are employed as end-point analyses. For this reason, many in vitro methods for assessing biomineralization are destructive. A brief overview of these techniques has been provided in the following section. It should be noted that these techniques can all be applied to ex vivo samples also. The techniques are presented in order of increasing spatial resolution. The actual spatial resolution for each technique is not quoted, as this will vary with the configuration of a particular instrument, e.g., lens magnification, detector pixel density, as well as the refractive index of the media with which it interacts.
Histology is one of the oldest and most popular means of assessing biomineralization and a perfect example of a qualitative but destructive in vitro technique for the identification of biomineralized tissue. Extensive sample manipulation through chemical fixation and processing (from embedding through to sectioning) before staining and imaging is required (Carleton et al., 1980), which can be time-consuming. Although sample preparation is extensive and laborious, requiring some level of experience, it is cheap and therefore popular among researchers. The dyes used in histology are either acidic or basic, and their use depends on the chemistry of the target tissue. Thus they can be used to highlight specific regions within tissues, i.e., intra- or extracellular components. Once dyed, high magnification images are taken using traditional bright-field microscopy techniques.
Specifically for biomineralization a further limitation of histology is that, depending on the stain used, a demineralizing step may also be required which defeats the purpose of staining for mineral content (Carleton et al., 1980). The most common biomineralization stains are Von Kossa, Alizarin Red, Safranin O, Picrosirius Red, Aniline Blue, Masson’s Trichrome, and Sanderson’s Bone Stain (Horobin and Kiernan, 2002).