37,38 Although not used for the study of bioceramics, Giannuzzi et al. have demonstrated the ability to use FIB sequential milling and imaging to produce three dimensional reconstructions of biomaterial-bone interfaces.39 Such three dimensional interfacial information was useful for determining the extent of bone ingrowth selleck chemicals KPT-330 into implanted titanium samples, and could easily be applied to the bioceramic interface in much the same way. Cryo-preparation The use of cryogenic-based techniques is quite common in the study of biological tissues. Cryofracturing has been used by Steflik et al. to ensure interfacial tissues remain intact on tissue blocks when removing implants from apposing tissue.
40 Of late, advancements in cryo-FIBSEM and cryo-TEM instrumentation have enabled the complete preparation, transfer and investigation of biological specimens from start to finish under cryogenic conditions, eliminating the need for rigorous tissue processing.41 This may in fact be the best route for preparing and examining biological structures in their native state. Resolving the Interface: The Techniques Light microscopy (LM) Ground sections must be produced, as described earlier, for light microscopic evaluation of the intact interface between non-demineralized tissue and implants, so that features such as bone-implant contact and bone area can be quantified. It has been shown that the thickness of the ground sections are of importance for quantification, as thicker samples include more overlapping information and often result in an over-estimation of bone implant contact.
42 Furthermore, the cutting direction may influence the quality of sections, thereby also interfering with quantification.43 Different staining protocols enable the identification of features for qualitative histology, such as discrimination of woven bone tissue, mature bone tissue, as well as cellular activity. Additionally, with the injection of calcium binding dyes during healing, the mineralization front can be tracked in the ground-section, contributing information regarding, e.g., the origin of bone tissue growth. Linear or circular polarized light microscopy, covered comprehensively elsewhere,44 may also be employed to identify the orientation of collagen in bone, enabling identification of regions of woven or lamellar bone in contact with calcium phosphates.
45 Scanning electron microscopy (SEM) The scanning electron microscope is a valuable characterization tool for bone-implant interfacial analysis. Of the variety of electron-matter interactions that occur in the SEM (Fig. 1), the detection of backscattered electrons is the Cilengitide most useful in the study of calcium phosphates and bone. Backscattered electrons are highly Z-dependent and therefore create Z-contrast images. This is crucial for studying calcium phosphates in contact with bone, since their chemical similarities make them difficult to distinguish otherwise.