The effect of sample preparation technique on determination of structure and nanomechanical properties of human cementum hard tissue

The mechanical properties of a tissue can be evaluated by determining the response of the structure to mechanical loading. This can be accomplished only when the tissue has been prepared with minimum to no artifacts, thus preserving its structure. In this study it was hypothesized that the structure of cementum is inhomogenous, contributing to a significant variation in mechanical properties of cementum. Therefore, the goals of the study were to identify potential artifacts generated by conventional sample preparation techniques such as polishing and ultrasectioning and subsequently characterize the prepared specimens using an atomic force microscope (AFM) and an AFM-nanoindenter. Comparisons between cryofractured, ultrasectioned and polished specimens concluded that ultrasectioned surfaces have significantly lower average surface roughness ‘Ra’ (p<0.05). Microstructure of ultrasectioned specimens characterized using an AFM illustrated Sharpeyʹs fibers (SF) and intrinsic fibers (IF) running perpendicular and parallel to the root surface similar to the observed microstructure of cryofractured cementum. In addition, a 10–50 μm wide cementum dentin junction (CDJ) was distinctly observed in the ultrasectioned specimens but not in polished specimens. The SF and CDJ illustrated relatively higher levels of hydrophilicity under wet conditions. The observed inhomogenous microstructure of the ultrasectioned specimens led to a broader range of nanomechanical properties (modulus: 14.2–25.9 GPa; hardness: 0.48–1.09 GPa). However, masking of the same regions such as SF and CDJ due to smeared cementum in polished specimens resulted in a narrower range of nanomechanical properties (modulus: 18.2–20.8 GPa; hardness: 0.79–0.89 GPa). This effect is most noticeable under wet conditions for ultrasectioned specimens (modulus 2.6–10.9 GPa; hardness 0.05–0.30 GPa) compared to the polished specimens (modulus 12.2–14.5 GPa; hardness 0.33–0.45 GPa). Cementum also was shown to be highly viscoelastic, especially when hydrated. The results suggest ultrasectioning of cementum was superior to polishing preparation technique since it allowed visualization of cementum structures similar to cryofractured specimens while providing a flat surface necessary for AFM-based nanoindentation techniques. Additionally, the structural inhomogeneity observed within ultrasectioned cementum contributed to a broader range of mechanical properties.