Bimodal morphological analyses of native and engineered tissues.

Assessing the morphological features of native and engineered tissues is pivotal to evaluate their degree of development and to identify possible structure-function relationships. Conventional histological or immunohistochemical imaging of stained sections provides limited information about their architecture. Scanning electron microscopy (SEM) yields sub-micrometric resolution images of tissues, but typically cannot be associated with the morphological structures identified by histology. The aim of this study was to establish a technique based on SEM analysis of sections of paraffin-embedded tissues prepared for histological processing (Histo-SEM) for the assessment of morpho-architectural properties of native and engineered tissues. Histo-SEM was performed on sections of cartilaginous, bone and fibrous tissues, native or engineered, in parallel with histological/immunohistochemical staining. Histo-SEM technique allowed evaluating morpho-architectural features typically unreachable by conventional histological staining, like (i) the extent of cartilage maturation based on collagen fibers' diameter and orientation, (ii) the formation of bone tissue/osteoid based on the presence of nanoscale-dense matrix structures, and (iii) tissue integration and vascularization based on collagen fibers' density and average vessel walls thickness of fibrous tissue growing within ectopically implanted porous materials. In conclusion, Histo-SEM allows integrating bimodal morphological assessments of native or engineered tissues and deriving complementary qualitative and quantitative parameters related to their structural organization and level of maturation.