A Stepwise Ultrasonic‑Assisted Protocol for Skeletal Preparation from Formalin‑Fixed Cadavers

High‑quality human skeletal specimens remain essential for anatomical education, surgical training, and forensic research; however, preparation of intact skeletons from long‑term formalin‑fixed cadavers remains technically challenging. Formalin‑induced protein cross‑linking renders soft tissues resistant to removal, while conventional boiling or strong‑alkali maceration often results in incomplete cleaning or damage to fragile osseous structures. In this study, we developed and evaluated a stepwise ultrasonic‑assisted protocol for skeletal preparation from formalin‑fixed, post‑dissection cadavers. Thirty adult cadavers fixed in 10% formalin for more than one year were assigned to five processing protocols: a novel experimental protocol integrating weak‑acid pre‑treatment, Ca(OH)₂‑supplemented KOH maceration, and ultrasonic agitation; traditional hot‑water maceration; KOH boiling alone; KOH boiling combined with ultrasound; and KOH boiling combined with calcium hydroxide. Outcomes were assessed based on processing efficiency, completeness of soft‑tissue removal, and preservation of cortical bone and osteological landmarks. Compared with all control methods, the experimental protocol consistently achieved rapid and thorough soft‑tissue removal while maintaining cortical integrity across both robust and fragile bone types. In contrast, control protocols resulted in incomplete tissue removal, cortical damage, or both. Although the method requires operator expertise and access to ultrasonic equipment, it provides a reproducible and scalable approach for high‑fidelity skeletal preparation. This protocol enables effective secondary utilization of formalin‑fixed cadavers and offers a practical solution for anatomical education and research in settings where access to fresh cadavers is limited.