A methodology to evaluate different histological preparations of soft tissues: Intervertebral disc tissues study

A tissue preparation method will inevitably alter the tissue content. This study aims to evaluate how different common sample preparation methods will affect the tissue morphology, biomechanical properties, and chemical composition of samples. The study focuses on intervertebral disc (IVD) tissue; however, it can be applied to other soft tissues. Raman spectroscopy synchronized with nanoindentation instrumentation was employed to investigate the compositional changes of IVD, specifically, nucleus pulposus (NP) and annulus fibrosus (AF), together with their biomechanical properties of IVD. These properties were examined through the following histological specimen types: fresh cryosection (control), fixed cryosection, and paraffin-embedded. The IVD tissue could be located using an optical microscope under three different preparation methods. Paraffin-embedded samples showed the most explicit details where the lamellae structure of AF could be identified. In terms of biomechanical properties, there was no significant difference between the fresh and fixed cryosection (p > 0.05). In contrast, the fresh cryosection and paraffin-embedded samples showed a significant difference (p < 0.05). It was also found that the tissue preparations affected the chemical content of the tissues and structure of the tissue, which are expected to contribute to biomechanical properties changes. Fresh cryosection and fixed cryosection samples are more promising to work with for biomechanical assessment in histological tissues. The findings fill essential gaps in the literature by providing valuable insight into the characteristics of IVD at the microscale. This study can also become a reference for a better approach to assessing the mechanical properties and chemical content of soft tissues at the microscale.