Simultaneous Evaluation of Stiffness and Histology in Orthopedic Diseases Using Scanning Acoustic Microscope

Organs with different levels of stiffness support the musculoskeletal system. Light microscopy cannot evaluate organ stiffness, whereas scanning acoustic microscopy (SAM) discriminates stiffness based on speed-of-sound (SOS) because sound waves pass faster in stiffer tissues. This study aimed to evaluate SOS imaging for orthopedic diseases using formalin-fixed paraffin-embedded sections. SOS imaging in SAM uses unstained light microscopic (LM) sections to prevent the bias of staining variation. Digital SOS values are comparable in different organ components and diseases. Mouse organs with the lowest mean SOS values included the adipose tissue, bone marrow, calcified cartilage, and nucleus pulposus; those with intermediate values included hyaline cartilages, osteoid, skeletal muscles, cortical and trabecular bones, and ligaments; and those with the highest values comprised fibrocartilages of the vertebral disc and meniscus. Water contents and delipidating procedures decreased SOS values. Collagenous density and arrangement affected higher SOS values. The trabecular bones of mice were thinner and showed significantly lower values of SOS than those of humans. Various orthopedic diseases and disorders displayed the characteristic SOS images. In osteoporosis, the trabecular bone becomes thin with lower SOS, indicating lesser stiffness to cause fractures. Comparison of woven and lamellar bones revealed that woven bones with lower SOS had lesser stiffness to fracture. Changes in SOS values indicated intramembranous bone formation. The trabecular bone develops from the connective tissues with an abrupt increase in SOS values. The regenerating process of bone fractures was monitored using SOS images, in which the granulation tissues transformed into calli in the osteoid to grow a new mineralized bone. The stiffness increased in phases, which appeared in SOS values. Although several methods have been used to visualize the stiffness of biological tissues, SAM only needs 10-µm unstained slides and can simultaneously compare mechanical stiffness and histology. SOS images provide informative mechanical alterations of the bone, cartilage, and connective tissues to assess the status and diagnose a disorder.