Changes in physical properties of decellularized bone after long-term treatment in phosphate-buffered saline

Phosphate-buffered saline (PBS) is used as the washing solution, diluent, and stock solution to prepare decellularized bone. However, limited information exists regarding the effects of PBS alone on changes in bone structure and mechanical properties. This study examined the effects of prolonged PBS treatment (up to 14 days) on the bone matrix. Histological staining showed a reduction in cellular components (partial decellularization) after extended treatment, while collagen fibers were retained. Although PBS-treated bone showed remarkably decreased bone strength, the loss of inorganic materials was limited to the bone surface of the tibia. Collagen fibers in the PBS-treated bones showed marked irregularity and an increase in their diameter. These results suggest that prolonged PBS treatment affects the structure and network of collagen fibers, similar to inorganic materials, in the bone matrix. Our results provide basic insights into the preparation of functional decellularized bone for dental applications.