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

A Stepwise Ultrasonic‑Assisted Protocol for Skeletal Preparation from Formalin‑Fixed Cadavers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Stepwise Ultrasonic‑Assisted Protocol for Skeletal Preparation from Formalin‑Fixed Cadavers Jianhua Liu, XuYi Hu, LiangJie Deng, Zhenfu Zhao, Baohua Luo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8805669/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. bone specimen preparation alkaline maceration ultrasonic cleaning formalin-fixed cadaver Figures Figure 1 Figure 2 1. Introduction Human bones, despite the widespread use of anatomical models(Myles et al., 2025 , Gnanasegaram et al., 2020 ) and 3D software(Cortese and Falletta, 2025 , Smith et al., 2022 ), remain unmatched in their ability to provide a precise and authentic three-dimensional understanding in anatomical education, surgical training, and forensic science. Human bone preparation for teaching purposes primarily involves dissecting at bony articulation sites, separating the bones, removing soft tissue, and bleaching the bones(Savitri et al., 2023 ). The needed equipment and time period may vary depending on the method used, the size of the cadaver body, and other environmental factors. Currently, the key to preparing skeletal specimens from animal or human cadavers lies in thoroughly and non-destructively removing soft tissues—such as muscle, tendon, and ligament—adhering to the bone surface. Conventional processing approaches mainly include physical boiling (hot-water maceration) and chemical maceration methods(Topp et al., 2012 , Savitri et al., 2023 ). For cadavers that have been fixed in formalin for prolonged periods, proteins within soft tissues undergo denaturation and cross-linking, rendering the tissues exceptionally tough; consequently, simple prolonged boiling is almost ineffective for stripping soft tissue(Pal et al., 2022 ). According to our past experience, chemical maceration methods—particularly heated treatment with strong alkali solutions such as potassium hydroxide (KOH) and sodium hydroxide (NaOH)—can decompose soft tissues, but this process has several limitations. On the one hand, while strong alkalis corrode soft tissue, they can also dissolve and damage hydroxyapatite, the principal inorganic component of bone(Domagala et al., 2021 ). This is especially likely to injure structurally fragile regions, such as vertebral processes and the thin lamina of the scapula, resulting in specimens with incomplete morphology. On the other hand, during alkaline maceration, the degraded soft tissue forms a viscous layer on the bone surface; this mucilaginous film severely impedes contact between fresh alkali solution and the underlying tissue, markedly reducing processing efficiency and making the overall procedure time-consuming and inconsistent in outcome. Therefore, developing an efficient method to remove soft tissue from bone while maximizing preservation of skeletal structural integrity has become an urgent technical challenge. In this study, we present a novel stepwise protocol specifically developed for formalin-fixed, post-dissection cadavers. We evaluated its performance against four conventional methods, with emphasis on preservation of structural integrity as well as reproducibility, scalability, and sustainability. 2. Materials and Methods 2.1. Ethical Considerations All procedures adhered to institutional guidelines for cadaveric use in teaching and research. Informed consent was obtained from donors prior to death, in accordance with national regulations. 2.2. Cadaver Selection 30 adult cadavers (13 males, 17 females; age range 40–89 years), previously fixed in 10% formalin for >1 year and used for undergraduate dissection courses, were included in this study. All had undergone complete evisceration and superficial and deep dissection as part of the regional anatomy curriculum. 2.3. Stepwise Protocol Step 1: Disarticulation and Selective Soft-Tissue Removal Each cadaver was repositioned supine. Major muscle masses, viscera, and neurovascular bundles were removed using standard dissection tools. Bones were then systematically separated: Vertebrae from the cervical (C1–C7), thoracic (T1–T12), and lumbar (L1–L5) regions were individually detached. Intervertebral discs, ligaments, and the spinal cord were excised until cortical bone was exposed. Transverse processes were protected by retaining 2–3 mm of surrounding muscle. Sacrum: Anterior and posterior sacral foramina were cleared of nerve roots; sacral canal contents removed. Iliac surfaces preserved until later stages. Hip Bones: Separated at sacroiliac joints with care to avoid tearing. Acetabular cartilage removed; iliac fossa protected by leaving 5 mm muscle layer. Long Bones: Femur, tibia, fibula, humerus, radius, ulna — periosteum and tendons removed at insertions; intermuscular septa retained. Hands/Feet: Metacarpals/metatarsals and phalanges separated at joints; collected in stainless steel mesh baskets (2–3 mm pore). Shoulder Girdle: Scapulae processed with 5 mm muscle coverage over thin dorsal/anterior surfaces. Thorax: Sternum cleaned gently; rib heads and tubercles smoothed; rib bodies left intact with intercostal muscles. Note: All cuts followed anatomical planes to preserve osteological landmarks. Step 2: Acid Priming Each bone group was immersed briefly (5 min) in 1% HCl to facilitate penetration of subsequent alkaline solution and neutralize residual formaldehyde. Step 3: Alkaline Maceration with Ultrasonics Bone groups were placed in separate 54-L 304 stainless‑steel tanks equipped with bottom‑mounted ultrasonic transducers (model: SB-1000DT, Ningbo Scientz Biotechnology Co., Ltd, China), operating at a fixed frequency of 40 kHz with a nominal ultrasonic power output of 500 W and a working volume of 54 L. The ultrasonic transducers were integrated into the base of the stainless‑steel tank to ensure uniform energy distribution. Solutions consisted of: 30% KOH (w/v). Protectant: Saturated Ca (OH)₂ added just before boiling. Temperature was maintained at 100°C, and exposure times were adjusted according to bone type, ranging from 8 min for ribs to 20 min for long bones, based on thickness and structural fragility. The use of 30% (w/v) KOH in the present study was intentionally higher than the concentrations typically reported for fresh or minimally fixed specimens, which are commonly in the range of 5–10%. Formalin fixation induces extensive protein cross‑linking, markedly increasing the resistance of soft tissues to alkaline degradation. In preliminary trials using 5–15% KOH, soft‑tissue removal from long‑term formalin‑fixed cadavers was incomplete even after prolonged boiling (>60 min), while higher concentrations (>35%) resulted in unacceptable cortical damage (based on preliminary optimization experiments). A concentration of 30% KOH was therefore selected as a practical compromise to ensure rapid soft‑tissue dissolution, with bone mineral protection achieved through the addition of calcium hydroxide and strict time control. Step 4: Neutralization and Rinsing Immediately after alkaline treatment, bone specimens were transferred to running tap water and rinsed continuously for 10 minutes. This process was repeated once per hour for 3–5 days, with fresh water each time, until the surface pH stabilized at neutral. A final 30‑minute rinse in distilled water was performed prior to dehydration. Step 5: Dehydration and Bleaching Bones were fully submerged in acetone (≥5 cm above the bone surface) for a total of 16 weeks. Acetone was replaced every 4 weeks (four cycles in total). Following dehydration, specimens were immersed in 5–10% hydrogen peroxide for 2–3 days for whitening. Step 6: Air-Drying Final drying on wire racks in a ventilated area for 1–2 weeks. 2.4 Experimental groups The study evaluated five processing protocols (see Table 1): Experimental group (novel protocol): The complete stepwise method developed in this study. Control group 1: Traditional hot‑water maceration. Control group 2: Boiling in 30% KOH only, with no ultrasound, no calcium hydroxide, and no acid pre‑treatment. Control group 3: Boiling in 30% KOH with ultrasound, but without calcium hydroxide and without acid pre‑treatment. Control group 4: Boiling in 30% KOH with calcium hydroxide added, but without ultrasound and without acid pre‑treatment. Table 1. Experimental Groups and Corresponding Processing Conditions Method Sample Size Acid Pre‑treatment KOH (30%) Ultrasound Calcium Hydroxide Description Experimental group 6 Yes Yes Yes Yes Novel stepwise protocol Control group 1 6 No No (water only) No No Traditional hot‑water maceration Control group 2 6 No Yes No No KOH boiling only Control group 3 6 No Yes Yes No KOH boiling + ultrasound Control group 4 6 No Yes No Yes KOH boiling + calcium hydroxide 2.5 Chemical Mechanisms 2.5.1. Tissue Dissolution via KOH KOH induces two primary reactions: Saponification: Breaks ester bonds in lipids → glycerol + potassium fatty acid salts (soap). Triglyceride + 3 KOH → Glycerol + 3 RCOO⁻K⁺ Peptide Hydrolysis: Cleaves amide bonds in proteins → amino acid potassium salts. Protein + H₂O + KOH → Peptides/Amino Acids + K⁺ salts These reactions dissolve cellular membranes, collagen fibers, and connective tissues, facilitating separation from bone. 2.5.2. Bone Protection via Ca(OH)₂ Hydroxyapatite [Ca₅(PO₄)₃OH] dissolution equilibrium: Ca₅(PO₄)₃OH ⇌ 5Ca²⁺ + 3PO₄³⁻ + OH⁻ Addition of Ca(OH)₂ increases [Ca²⁺] and [OH⁻], shifting the equilibrium leftward via Le Chatelier’s Principle, thereby suppressing demineralization, and the ultrasonic waves disrupt viscous biofilm layers formed during corrosion, enhancing mass transfer and reducing localized over-corrosion. 2.6 Evaluation Criteria Specimen quality was evaluated based on three outcome measures: (1) completeness of soft‑tissue removal (classified as complete, partial, or incomplete based on visual inspection), (2) cortical bone integrity (presence of cortical lifting, whitening, or cancellous exposure), and (3) preservation of osteological landmarks in fragile regions. Assessments were performed independently by two experienced anatomists, and observations were recorded photographically and summarized descriptively. 3. Results Five bone‑processing protocols were compared (see Table 1 ). Control 1 was applied to all types of bones mentioned above and required 24–48 hours, yielding no effective soft-tissue removal — tissue remained firmly adhered (see Table 2 ). The experimental protocol—which integrates weak‑acid pre‑soaking, Ca(OH)₂‑supplemented KOH treatment, and 40 kHz ultrasonic agitation—consistently produced clean, smooth bone surfaces with no residual soft tissue and preserved cortical integrity across all bone types (see Fig. 1 ). In contrast, Control 2 (KOH alone) left a viscous soft-tissue layer and caused cortical damage (see Table 2 ). Control 3 (KOH + ultrasound) improved surface cleanliness but still damaged thin bone regions (see Table 2 ). Control 4 (KOH + Ca(OH)₂) preserved bone integrity but left residual soft tissue due to the lack of ultrasound(see Table 2 ). Only the experimental protocol achieved both rapid, thorough soft-tissue removal and complete preservation of structural integrity — a critical advantage for high-fidelity skeletal preparation. Table 2 Comparative Outcomes of Experimental and Control Protocols Group Processing method Soft‑tissue removal Cortical integrity Typical defects Experimental group Acid pre‑treatment + 30% KOH + Ca(OH)₂ + ultrasound (40 kHz) Complete Preserved No residual soft tissue; cortical bone intact; osteological landmarks well preserved Control group 1 Hot‑water maceration (100°C) Incomplete Preserved Soft tissue contracted and discolored but remained firmly adherent to bone Control group 2 30% KOH boiling only Partial Damaged Viscous soft‑tissue residue; cortical lifting and whitening in thin or fragile regions Control group 3 30% KOH boiling + ultrasound Complete Damaged Cortical erosion and exposure of cancellous bone, especially in thin structures Control group 4 30% KOH boiling + Ca(OH)₂ Partial Preserved Residual deep soft tissue after mucus removal; bone morphology largely intact Notes: Comparisons among all groups were performed using the anterior surface of the scapula as a representative example. Green arrows indicate soft tissues intentionally preserved for protective purposes. Yellow arrows indicate residual soft tissues that were not completely removed. Red arrows indicate areas of cortical bone damage. The experimental group demonstrated clean and smooth bone surfaces with clearly visible cortical bone and preservation of only intentionally retained soft tissues. Control Group 1 showed color changes and contraction of soft tissues following boiling, with muscles and fascia remaining tightly adherent to the bone. Control Group 2 exhibited surface mucus and residual deep soft tissues after cleaning, along with cortical bone damage in thinner areas. Control Group 3 showed complete soft‑tissue removal and clear cortical bone but with cortical damage in fragile regions. Control Group 4 retained some residual soft tissue after mucus removal but showed intact cortical bone without obvious damage. 4. Discussion We present a novel, stepwise protocol specifically designed for formalin-fixed, post-dissection cadavers. This method combines controlled alkaline maceration with ultrasonic agitation and bone-specific soft-tissue preservation strategies to protect structurally vulnerable bone regions. In addition, incorporation of calcium hydroxide mitigates mineral loss during alkali exposure, thereby preserving long-term skeletal integrity. Collectively, these features improve specimen quality while enhancing reproducibility, scalability, and sustainability. The preparation of high-quality human skeletal specimens is fundamental to anatomical education. Traditionally, bone specimens are derived from fresh, unfixed cadavers via prolonged boiling or maceration to remove soft tissues(Lander et al., 2014 ). However, these approaches suffer from poor reproducibility, vague procedural instructions, and frequent damage to cortical bone — particularly affecting small, thin-walled, or irregularly shaped bones such as vertebrae(García-Donas et al., 2017 ). In China, modern anatomical education continues to rely heavily on formalin-fixed cadavers due to biosafety regulations and ethical considerations(Pan et al., 2020 ). These specimens, once dissected, are typically cremated, representing a significant waste of scarce educational resources(Signorelli et al., 2025 , Mégevand et al., 2017 ). Given the declining rates of body donation worldwide (Ahmed et al.), there is an urgent need to repurpose dissected cadavers for secondary educational and research applications, including skeletal preparation, within existing regulatory frameworks(Habicht et al., 2018 , Jenkin and Keay, 2025 ). The principal technical challenge in obtaining high-quality bone specimens from formalin-fixed cadavers lies in the profound effects of formalin on soft tissues. Formalin induces protein denaturation and extensive cross-linking, rendering soft tissues exceptionally tough and resistant to removal (Mason and O'Leary, 1991 ); consequently, conventional boiling methods are largely ineffective. Although standard strong-alkali maceration can degrade such tissues, the process is slow, difficult to control, and frequently causes irreversible damage to bone—particularly in thin or fragile anatomical regions—thereby compromising specimen integrity and overall quality. Moreover, when strong alkali is used alone, degraded soft tissues form a viscous film on the bone surface, which impedes further alkali penetration and markedly reduces processing efficiency. The technical strategy employed in the present study directly addresses these limitations through a combination of complementary mechanisms. First, the introduction of ultrasound markedly accelerates alkali-mediated soft-tissue digestion by continuously disrupting and removing the viscous degradation layer that otherwise blocks further reaction. Second, the addition of calcium hydroxide suppresses dissolution of bone mineral salts via a common-ion effect, thereby protecting the hydroxyapatite matrix and preserving osseous structural integrity during alkaline treatment. Third, weak-acid pre-soaking neutralizes alkali that may infiltrate the internal microarchitecture of the bone, providing an additional layer of “inside–out” protection beyond surface preservation. Together, these elements substantially shorten processing time while yielding skeletal specimens with clean surfaces and intact morphology, achieving concurrent improvements in both processing efficiency and specimen quality. When preparing bone specimens, marked regional differences in bone thickness and mechanical strength impose additional constraints on processing conditions. Thin and fragile structures—such as vertebral transverse processes, the iliac fossa, and the costal and dorsal surfaces of the scapula—are particularly susceptible to damage under aggressive alkaline maceration. To mitigate this risk, we implemented an anatomy-guided trimming strategy in which a controlled layer of residual muscle was deliberately retained over vulnerable regions prior to chemical treatment. During alkaline maceration, this retained tissue functions as a temporary sacrificial barrier, preferentially reacting with and buffering the alkali, thereby reducing direct chemical attack on the underlying bone. This region-specific modification proved critical for preserving delicate skeletal structures and was a key factor in achieving intact, high-quality whole-body skeletal specimens. Comparative analysis of the experimental and control groups (see Table 2 ) further underscores the necessity of integrating multiple protective and accelerative mechanisms within a single protocol. Traditional hot-water maceration (Control 1) was ineffective for formalin-fixed specimens, requiring prolonged processing while failing to detach soft tissue. Strong alkali treatment alone (Control 2) improved tissue degradation but resulted in inefficient removal due to viscous residue formation and caused substantial cortical damage, particularly in thin or fragile bones. The addition of ultrasound (Control 3) effectively eliminated residual soft tissue by disrupting the degradation layer, yet this benefit came at the cost of increased bone damage in vulnerable regions. Conversely, supplementation with calcium hydroxide alone (Control 4) preserved bone integrity through mineral protection but failed to achieve complete soft-tissue removal in the absence of ultrasonic agitation. Only the experimental protocol—combining weak-acid pre-soaking, calcium hydroxide–enhanced alkaline treatment, and ultrasonic assistance—simultaneously achieved rapid, thorough soft-tissue removal and consistent preservation of skeletal morphology across all bone types. Conclusion In summary, the protocol described in this study provides an effective solution to the long-standing challenges associated with preparing skeletal specimens from formalin-fixed cadavers. By integrating ultrasonic-assisted alkaline maceration, calcium hydroxide–mediated mineral protection, weak-acid pre-soaking, and anatomy-guided soft-tissue preservation, the method enables efficient soft-tissue removal while maintaining structural integrity across both robust and fragile skeletal regions. Limitations Nevertheless, the protocol requires careful operator judgment in trimming and timing, particularly for anatomically delicate structures, and the use of ultrasonic equipment may limit accessibility in resource-constrained settings. Despite these considerations, the method substantially improves specimen quality, processing efficiency, and sustainability, and offers a practical framework for the secondary utilization of dissected cadavers in anatomical education and research. Declarations Acknowledgements The authors sincerely thank those who donated their bodies to science so that anatomical research could be performed. Results from such research can potentially increase mankind's overall knowledge that can then improve patient care. Therefore, these donors and their families deserve our highest gratitude. Author contribution Conceptualization, J.L. and A.R.; Methodology, J.L., X.H., L.D., B.L., and A.R.; Investigation, X.H. and L.D.; Data curation, J.L.; Visualization, J.L.; Writing – original draft, A.R. and J.L.; Writing – review & editing, A.R.; Resources, J.L.; Project administration, A.R.; Supervision, Z.Z. Funding: Shenzhen University Laboratory and Equipment Management Research Fund (No. 2024018). Construction and Application of a Safety Management System for Human Morphology Teaching Laboratories. Consent to Participate: All body donors involved in this study provided informed consent during their lifetime to participate in anatomy‑related research and teaching prior to death. Ethics Approval: This study was reviewed and approved by the Ethics Committee of the Body Donation Receiving Station, School of Medicine, Shenzhen University. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the Declaration of Helsinki. Ethics declaration: not applicable. Conflict of interest: The authors declare no conflict of interest. Consent to publish : The authors confirm that this manuscript has not been published elsewhere and is not under consideration by another journal. All authors have read and approved the submitted version. References AHMED, S. A., HEGAZY, N. N., ABDEL MALAK, H. W., CLIFF KAYSER, W., 3RD, ELRAFIE, N. M., HASSANIEN, M., AL-HAYANI, A. A., EL SAADANY, S. A., AI-YOUBI, A. O. & SHEHATA, M. H. 2020. Model for utilizing distance learning post COVID-19 using (PACT)™ a cross sectional qualitative study. BMC Med Educ, 20 , 400. CORTESE, K. & FALLETTA, P. 2025. Anatomy at the threshold: Teaching the human body in a hybrid age. Anat Sci Educ, 18 , 1301-1309. DOMAGALA, Z., DOMAŃSKI, J., SMYCZEK, N. & GALK, C. 2021. 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Effects of formaldehyde fixation on protein secondary structure: A calorimetric and infrared spectroscopic investigation. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 39 , 225-9. MéGEVAND, P., WOODTLI, A., YULZARI, A., COSGROVE, G. R., MOMJIAN, S., STIMEC, B. V., CORNIOLA, M. V. & FASEL, J. H. D. 2017. Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model. J Vis Exp . MYLES, C., GORMAN, L. & JONES, J. F. X. 2025. 3D printing variation: Teaching and assessing hepatobiliary variants in human anatomy. Anat Sci Educ, 18 , 885-896. PAL, A., BHANARKAR, U. & RAY, B. 2022. Embalming with Formalin – Benefits and Pitfalls. Scholars International Journal of Anatomy and Physiology, 5 , 70-77. PAN, S. Q., CHAN, L. K., YAN, Y. & YANG, X. 2020. Survey of Gross Anatomy Education in China: The Past and the Present. Anat Sci Educ, 13 , 390-400. SAVITRI, YAKKUNDI, R. C., MATAPATHI, N. & TALAWAR, V. 2023. A review of Bone Preparation Techniques for Anatomical Studies. Journal of Ayurveda and Integrated Medical Sciences, 8 , 112-117. SIGNORELLI, F., RASTEGAR, V., PALERMO, M., LAINO, D., ZEOLI, F. & VISOCCHI, M. 2025. Long-Term Preservation of Human Head and Neck Specimens for Neurosurgical Training: A Technical Note. Brain Sci, 15. SMITH, C. F., FREEMAN, S. K., HEYLINGS, D., FINN, G. M. & DAVIES, D. C. 2022. Anatomy education for medical students in the United Kingdom and Republic of Ireland in 2019: A 20-year follow-up. Anat Sci Educ, 15 , 993-1006. TOPP, T., MüLLER, T., HUSS, S., KANN, P. H., WEIHE, E., RUCHHOLTZ, S. & ZETTL, R. P. 2012. Embalmed and fresh frozen human bones in orthopedic cadaveric studies: which bone is authentic and feasible? Acta Orthop, 83 , 543-7. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigureS1.StepbyStepWorkflowoftheUltrasonicAssistedSkeletalPreparationProtocol.rar Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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10:24:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8805669/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8805669/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102948056,"identity":"8968ef17-86df-4302-9084-b59ba664f584","added_by":"auto","created_at":"2026-02-18 19:29:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":403098,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFinal Bone Specimens Prepared Using the above Experimental Protocol\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8805669/v1/7fd0e0a51f60aed4db4d1632.png"},{"id":102948055,"identity":"723c7a96-25a8-4b62-a34e-02b973b9ee32","added_by":"auto","created_at":"2026-02-18 19:29:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":737261,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVisual Assessment of Bone Surface Integrity and Soft Tissue Removal in Experimental and Control Groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNotes：Comparisons among all groups were performed using the anterior surface of the scapula as a representative example. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eGreen arrows\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e indicate soft tissues intentionally preserved for protective purposes. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eYellow arrows\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e indicate residual soft tissues that were not completely removed.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e Red arrows\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e indicate areas of cortical bone damage. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eThe experimental group\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e demonstrated clean and smooth bone surfaces with clearly visible cortical bone and preservation of only intentionally retained soft tissues. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eControl Group 1\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e showed color changes and contraction of soft tissues following boiling, with muscles and fascia remaining tightly adherent to the bone. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eControl Group 2\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e exhibited surface mucus and residual deep soft tissues after cleaning, along with cortical bone damage in thinner areas. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eControl Group 3\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e showed complete soft‑tissue removal and clear cortical bone but with cortical damage in fragile regions. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eControl Group 4\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e retained some residual soft tissue after mucus removal but showed intact cortical bone without obvious damage.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8805669/v1/834a02f0b033e0ea3033c3b1.png"},{"id":103049798,"identity":"d5ad68fa-44c9-413e-ab85-15da96d938b8","added_by":"auto","created_at":"2026-02-20 07:46:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2242617,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8805669/v1/5d2ccdfe-e9db-479a-8ad4-9563e56af22f.pdf"},{"id":102948057,"identity":"1545e7c2-3f62-41d4-88e8-137bb924602f","added_by":"auto","created_at":"2026-02-18 19:29:42","extension":"rar","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":58333377,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS1.StepbyStepWorkflowoftheUltrasonicAssistedSkeletalPreparationProtocol.rar","url":"https://assets-eu.researchsquare.com/files/rs-8805669/v1/6e22e02068c84c526c3f4802.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Stepwise Ultrasonic‑Assisted Protocol for Skeletal Preparation from Formalin‑Fixed Cadavers","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHuman bones, despite the widespread use of anatomical models(Myles et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, Gnanasegaram et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and 3D software(Cortese and Falletta, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, Smith et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), remain unmatched in their ability to provide a precise and authentic three-dimensional understanding in anatomical education, surgical training, and forensic science. Human bone preparation for teaching purposes primarily involves dissecting at bony articulation sites, separating the bones, removing soft tissue, and bleaching the bones(Savitri et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The needed equipment and time period may vary depending on the method used, the size of the cadaver body, and other environmental factors. Currently, the key to preparing skeletal specimens from animal or human cadavers lies in thoroughly and non-destructively removing soft tissues\u0026mdash;such as muscle, tendon, and ligament\u0026mdash;adhering to the bone surface. Conventional processing approaches mainly include physical boiling (hot-water maceration) and chemical maceration methods(Topp et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Savitri et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor cadavers that have been fixed in formalin for prolonged periods, proteins within soft tissues undergo denaturation and cross-linking, rendering the tissues exceptionally tough; consequently, simple prolonged boiling is almost ineffective for stripping soft tissue(Pal et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). According to our past experience, chemical maceration methods\u0026mdash;particularly heated treatment with strong alkali solutions such as potassium hydroxide (KOH) and sodium hydroxide (NaOH)\u0026mdash;can decompose soft tissues, but this process has several limitations. On the one hand, while strong alkalis corrode soft tissue, they can also dissolve and damage hydroxyapatite, the principal inorganic component of bone(Domagala et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This is especially likely to injure structurally fragile regions, such as vertebral processes and the thin lamina of the scapula, resulting in specimens with incomplete morphology. On the other hand, during alkaline maceration, the degraded soft tissue forms a viscous layer on the bone surface; this mucilaginous film severely impedes contact between fresh alkali solution and the underlying tissue, markedly reducing processing efficiency and making the overall procedure time-consuming and inconsistent in outcome.\u003c/p\u003e \u003cp\u003eTherefore, developing an efficient method to remove soft tissue from bone while maximizing preservation of skeletal structural integrity has become an urgent technical challenge.\u003c/p\u003e \u003cp\u003eIn this study, we present a novel stepwise protocol specifically developed for formalin-fixed, post-dissection cadavers. We evaluated its performance against four conventional methods, with emphasis on preservation of structural integrity as well as reproducibility, scalability, and sustainability.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e2.1. Ethical Considerations\u003c/p\u003e\n\u003cp\u003eAll procedures adhered to institutional guidelines for cadaveric use in teaching and research. Informed consent was obtained from donors prior to death, in accordance with national regulations.\u003c/p\u003e\n\u003cp\u003e2.2. Cadaver Selection\u003c/p\u003e\n\u003cp\u003e30 adult cadavers (13 males, 17 females; age range 40\u0026ndash;89 years), previously fixed in 10% formalin for \u0026gt;1 year and used for undergraduate dissection courses, were included in this study. All had undergone complete evisceration and superficial and deep dissection as part of the regional anatomy curriculum.\u003c/p\u003e\n\u003cp\u003e2.3. Stepwise Protocol\u003c/p\u003e\n\u003cp\u003eStep 1: Disarticulation and Selective Soft-Tissue Removal\u003c/p\u003e\n\u003cp\u003eEach cadaver was repositioned supine. Major muscle masses, viscera, and neurovascular bundles were removed using standard dissection tools. Bones were then systematically separated:\u003c/p\u003e\n\u003cp\u003eVertebrae from the cervical (C1\u0026ndash;C7), thoracic (T1\u0026ndash;T12), and lumbar (L1\u0026ndash;L5) regions were individually detached. Intervertebral discs, ligaments, and the spinal cord were excised until cortical bone was exposed.\u003c/p\u003e\n\u003cp\u003eTransverse processes were protected by retaining 2\u0026ndash;3 mm of surrounding muscle.\u003c/p\u003e\n\u003cp\u003eSacrum: Anterior and posterior sacral foramina were cleared of nerve roots; sacral canal contents removed. Iliac surfaces preserved until later stages.\u003c/p\u003e\n\u003cp\u003eHip Bones: Separated at sacroiliac joints with care to avoid tearing. Acetabular cartilage removed; iliac fossa protected by leaving 5 mm muscle layer.\u003c/p\u003e\n\u003cp\u003eLong Bones: Femur, tibia, fibula, humerus, radius, ulna \u0026mdash; periosteum and tendons removed at insertions; intermuscular septa retained.\u003c/p\u003e\n\u003cp\u003eHands/Feet: Metacarpals/metatarsals and phalanges separated at joints; collected in stainless steel mesh baskets (2\u0026ndash;3 mm pore).\u003c/p\u003e\n\u003cp\u003eShoulder Girdle: Scapulae processed with 5 mm muscle coverage over thin dorsal/anterior surfaces.\u003c/p\u003e\n\u003cp\u003eThorax: Sternum cleaned gently; rib heads and tubercles smoothed; rib bodies left intact with intercostal muscles.\u003c/p\u003e\n\u003cp\u003eNote: All cuts followed anatomical planes to preserve osteological landmarks.\u003c/p\u003e\n\u003cp\u003eStep 2: Acid Priming\u003c/p\u003e\n\u003cp\u003eEach bone group was immersed briefly (5 min) in 1% HCl to facilitate penetration of subsequent alkaline solution and neutralize residual formaldehyde.\u003c/p\u003e\n\u003cp\u003eStep 3: Alkaline Maceration with Ultrasonics\u003c/p\u003e\n\u003cp\u003eBone groups were placed in separate 54-L 304 stainless‑steel tanks equipped with bottom‑mounted ultrasonic transducers (model: SB-1000DT, Ningbo Scientz Biotechnology Co., Ltd, China), operating at a fixed frequency of 40 kHz with a nominal ultrasonic power output of 500 W and a working volume of 54 L. The ultrasonic transducers were integrated into the base of the stainless‑steel tank to ensure uniform energy distribution. Solutions consisted of: 30% KOH (w/v). Protectant: Saturated Ca (OH)₂ added just before boiling. Temperature was maintained at 100\u0026deg;C, and exposure times were adjusted according to bone type, ranging from 8 min for ribs to 20 min for long bones, based on thickness and structural fragility.\u003c/p\u003e\n\u003cp\u003eThe use of 30% (w/v) KOH in the present study was intentionally higher than the concentrations typically reported for fresh or minimally fixed specimens, which are commonly in the range of 5\u0026ndash;10%. Formalin fixation induces extensive protein cross‑linking, markedly increasing the resistance of soft tissues to alkaline degradation. In preliminary trials using 5\u0026ndash;15% KOH, soft‑tissue removal from long‑term formalin‑fixed cadavers was incomplete even after prolonged boiling (\u0026gt;60 min), while higher concentrations (\u0026gt;35%) resulted in unacceptable cortical damage (based on preliminary optimization experiments). A concentration of 30% KOH was therefore selected as a practical compromise to ensure rapid soft‑tissue dissolution, with bone mineral protection achieved through the addition of calcium hydroxide and strict time control.\u003c/p\u003e\n\u003cp\u003eStep 4: Neutralization and Rinsing\u003c/p\u003e\n\u003cp\u003eImmediately after alkaline treatment, bone specimens were transferred to running tap water and rinsed continuously for 10 minutes. This process was repeated once per hour for 3\u0026ndash;5 days, with fresh water each time, until the surface pH stabilized at neutral. A final 30‑minute rinse in distilled water was performed prior to dehydration.\u003c/p\u003e\n\u003cp\u003eStep 5: Dehydration and Bleaching\u003c/p\u003e\n\u003cp\u003eBones were fully submerged in acetone (\u0026ge;5 cm above the bone surface) for a total of 16 weeks. Acetone was replaced every 4 weeks (four cycles in total). Following dehydration, specimens were immersed in 5\u0026ndash;10% hydrogen peroxide for 2\u0026ndash;3 days for whitening.\u003c/p\u003e\n\u003cp\u003eStep 6: Air-Drying\u003c/p\u003e\n\u003cp\u003eFinal drying on wire racks in a ventilated area for 1\u0026ndash;2 weeks.\u003c/p\u003e\n\u003cp\u003e2.4 Experimental groups\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study evaluated five processing protocols (see Table 1):\u003c/p\u003e\n\u003cp\u003eExperimental group (novel protocol): The complete stepwise method developed in this study.\u003c/p\u003e\n\u003cp\u003eControl group 1: Traditional hot‑water maceration.\u003c/p\u003e\n\u003cp\u003eControl group 2: Boiling in 30% KOH only, with no ultrasound, no calcium hydroxide, and no acid pre‑treatment.\u003c/p\u003e\n\u003cp\u003eControl group 3: Boiling in 30% KOH with ultrasound, but without calcium hydroxide and without acid pre‑treatment.\u003c/p\u003e\n\u003cp\u003eControl group 4: Boiling in 30% KOH with calcium hydroxide added, but without ultrasound and without acid pre‑treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. \u0026nbsp;Experimental Groups and Corresponding Processing Conditions\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eMethod\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003eSample Size\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eAcid Pre‑treatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eKOH (30%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eUltrasound\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eCalcium Hydroxide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eExperimental group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eNovel stepwise protocol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eControl group 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eNo (water only)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eTraditional hot‑water maceration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eControl group 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eKOH boiling only\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eControl group 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eKOH boiling + ultrasound\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eControl group 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eKOH boiling + calcium hydroxide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.5 Chemical Mechanisms\u003c/p\u003e\n\u003cp\u003e2.5.1. Tissue Dissolution via KOH\u003c/p\u003e\n\u003cp\u003eKOH induces two primary reactions:\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eSaponification: Breaks ester bonds in lipids\u0026nbsp;\u0026rarr;\u0026nbsp;glycerol + potassium fatty acid salts (soap).\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eTriglyceride + 3 KOH\u0026nbsp;\u0026rarr;\u0026nbsp;Glycerol + 3 RCOO⁻K⁺\u003c/p\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003ePeptide Hydrolysis: Cleaves amide bonds in proteins\u0026nbsp;\u0026rarr;\u0026nbsp;amino acid potassium salts.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eProtein + H₂O + KOH\u0026nbsp;\u0026rarr;\u0026nbsp;Peptides/Amino Acids + K⁺ salts\u003c/p\u003e\n\u003cp\u003eThese reactions dissolve cellular membranes, collagen fibers, and connective tissues, facilitating separation from bone.\u003c/p\u003e\n\u003cp\u003e2.5.2. Bone Protection via Ca(OH)₂\u003c/p\u003e\n\u003cp\u003eHydroxyapatite [Ca₅(PO₄)₃OH] dissolution equilibrium:\u003c/p\u003e\n\u003cp\u003eCa₅(PO₄)₃OH\u0026nbsp;⇌\u0026nbsp;5Ca\u0026sup2;⁺ + 3PO₄\u0026sup3;⁻ + OH⁻\u003c/p\u003e\n\u003cp\u003eAddition of Ca(OH)₂ increases [Ca\u0026sup2;⁺] and [OH⁻], shifting the equilibrium leftward via Le Chatelier\u0026rsquo;s Principle, thereby suppressing demineralization, and the ultrasonic waves disrupt viscous biofilm layers formed during corrosion, enhancing mass transfer and reducing localized over-corrosion.\u003c/p\u003e\n\u003cp\u003e2.6 Evaluation Criteria\u003c/p\u003e\n\u003cp\u003eSpecimen quality was evaluated based on three outcome measures: (1) completeness of soft‑tissue removal (classified as complete, partial, or incomplete based on visual inspection), (2) cortical bone integrity (presence of cortical lifting, whitening, or cancellous exposure), and (3) preservation of osteological landmarks in fragile regions. Assessments were performed independently by two experienced anatomists, and observations were recorded photographically and summarized descriptively.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003eFive bone‑processing protocols were compared (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Control 1 was applied to all types of bones mentioned above and required 24\u0026ndash;48 hours, yielding no effective soft-tissue removal \u0026mdash; tissue remained firmly adhered (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe experimental protocol\u0026mdash;which integrates weak‑acid pre‑soaking, Ca(OH)₂‑supplemented KOH treatment, and 40 kHz ultrasonic agitation\u0026mdash;consistently produced clean, smooth bone surfaces with no residual soft tissue and preserved cortical integrity across all bone types (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, Control 2 (KOH alone) left a viscous soft-tissue layer and caused cortical damage (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Control 3 (KOH\u0026thinsp;+\u0026thinsp;ultrasound) improved surface cleanliness but still damaged thin bone regions (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Control 4 (KOH\u0026thinsp;+\u0026thinsp;Ca(OH)₂) preserved bone integrity but left residual soft tissue due to the lack of ultrasound(see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Only the experimental protocol achieved both rapid, thorough soft-tissue removal and complete preservation of structural integrity \u0026mdash; a critical advantage for high-fidelity skeletal preparation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparative Outcomes of Experimental and Control Protocols\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProcessing method\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSoft‑tissue removal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCortical integrity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTypical defects\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcid pre‑treatment\u0026thinsp;+\u0026thinsp;30% KOH\u0026thinsp;+\u0026thinsp;Ca(OH)₂ + ultrasound (40 kHz)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eComplete\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePreserved\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo residual soft tissue; cortical bone intact; osteological landmarks well preserved\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl group 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHot‑water maceration (100\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIncomplete\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePreserved\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSoft tissue contracted and discolored but remained firmly adherent to bone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl group 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30% KOH boiling only\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDamaged\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eViscous soft‑tissue residue; cortical lifting and whitening in thin or fragile regions\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl group 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30% KOH boiling\u0026thinsp;+\u0026thinsp;ultrasound\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eComplete\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDamaged\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCortical erosion and exposure of cancellous bone, especially in thin structures\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl group 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30% KOH boiling\u0026thinsp;+\u0026thinsp;Ca(OH)₂\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePreserved\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eResidual deep soft tissue after mucus removal; bone morphology largely intact\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eNotes: Comparisons among all groups were performed using the anterior surface of the scapula as a representative example.\u003c/em\u003e \u003cb\u003eGreen arrows\u003c/b\u003e \u003cem\u003eindicate soft tissues intentionally preserved for protective purposes.\u003c/em\u003e \u003cb\u003eYellow arrows\u003c/b\u003e \u003cem\u003eindicate residual soft tissues that were not completely removed.\u003c/em\u003e \u003cb\u003eRed arrows\u003c/b\u003e \u003cem\u003eindicate areas of cortical bone damage.\u003c/em\u003e \u003cb\u003eThe experimental group\u003c/b\u003e \u003cem\u003edemonstrated clean and smooth bone surfaces with clearly visible cortical bone and preservation of only intentionally retained soft tissues.\u003c/em\u003e \u003cb\u003eControl Group 1\u003c/b\u003e \u003cem\u003eshowed color changes and contraction of soft tissues following boiling, with muscles and fascia remaining tightly adherent to the bone.\u003c/em\u003e \u003cb\u003eControl Group 2\u003c/b\u003e \u003cem\u003eexhibited surface mucus and residual deep soft tissues after cleaning, along with cortical bone damage in thinner areas.\u003c/em\u003e \u003cb\u003eControl Group 3\u003c/b\u003e \u003cem\u003eshowed complete soft‑tissue removal and clear cortical bone but with cortical damage in fragile regions.\u003c/em\u003e \u003cb\u003eControl Group 4\u003c/b\u003e \u003cem\u003eretained some residual soft tissue after mucus removal but showed intact cortical bone without obvious damage.\u003c/em\u003e\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eWe present a novel, stepwise protocol specifically designed for formalin-fixed, post-dissection cadavers. This method combines controlled alkaline maceration with ultrasonic agitation and bone-specific soft-tissue preservation strategies to protect structurally vulnerable bone regions. In addition, incorporation of calcium hydroxide mitigates mineral loss during alkali exposure, thereby preserving long-term skeletal integrity. Collectively, these features improve specimen quality while enhancing reproducibility, scalability, and sustainability.\u003c/p\u003e \u003cp\u003eThe preparation of high-quality human skeletal specimens is fundamental to anatomical education. Traditionally, bone specimens are derived from fresh, unfixed cadavers via prolonged boiling or maceration to remove soft tissues(Lander et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, these approaches suffer from poor reproducibility, vague procedural instructions, and frequent damage to cortical bone \u0026mdash; particularly affecting small, thin-walled, or irregularly shaped bones such as vertebrae(Garc\u0026iacute;a-Donas et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In China, modern anatomical education continues to rely heavily on formalin-fixed cadavers due to biosafety regulations and ethical considerations(Pan et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These specimens, once dissected, are typically cremated, representing a significant waste of scarce educational resources(Signorelli et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, M\u0026eacute;gevand et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Given the declining rates of body donation worldwide (Ahmed et al.), there is an urgent need to repurpose dissected cadavers for secondary educational and research applications, including skeletal preparation, within existing regulatory frameworks(Habicht et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Jenkin and Keay, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe principal technical challenge in obtaining high-quality bone specimens from formalin-fixed cadavers lies in the profound effects of formalin on soft tissues. Formalin induces protein denaturation and extensive cross-linking, rendering soft tissues exceptionally tough and resistant to removal (Mason and O'Leary, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1991\u003c/span\u003e); consequently, conventional boiling methods are largely ineffective. Although standard strong-alkali maceration can degrade such tissues, the process is slow, difficult to control, and frequently causes irreversible damage to bone\u0026mdash;particularly in thin or fragile anatomical regions\u0026mdash;thereby compromising specimen integrity and overall quality. Moreover, when strong alkali is used alone, degraded soft tissues form a viscous film on the bone surface, which impedes further alkali penetration and markedly reduces processing efficiency. The technical strategy employed in the present study directly addresses these limitations through a combination of complementary mechanisms. First, the introduction of ultrasound markedly accelerates alkali-mediated soft-tissue digestion by continuously disrupting and removing the viscous degradation layer that otherwise blocks further reaction. Second, the addition of calcium hydroxide suppresses dissolution of bone mineral salts via a common-ion effect, thereby protecting the hydroxyapatite matrix and preserving osseous structural integrity during alkaline treatment. Third, weak-acid pre-soaking neutralizes alkali that may infiltrate the internal microarchitecture of the bone, providing an additional layer of \u0026ldquo;inside\u0026ndash;out\u0026rdquo; protection beyond surface preservation. Together, these elements substantially shorten processing time while yielding skeletal specimens with clean surfaces and intact morphology, achieving concurrent improvements in both processing efficiency and specimen quality.\u003c/p\u003e \u003cp\u003eWhen preparing bone specimens, marked regional differences in bone thickness and mechanical strength impose additional constraints on processing conditions. Thin and fragile structures\u0026mdash;such as vertebral transverse processes, the iliac fossa, and the costal and dorsal surfaces of the scapula\u0026mdash;are particularly susceptible to damage under aggressive alkaline maceration. To mitigate this risk, we implemented an anatomy-guided trimming strategy in which a controlled layer of residual muscle was deliberately retained over vulnerable regions prior to chemical treatment. During alkaline maceration, this retained tissue functions as a temporary sacrificial barrier, preferentially reacting with and buffering the alkali, thereby reducing direct chemical attack on the underlying bone. This region-specific modification proved critical for preserving delicate skeletal structures and was a key factor in achieving intact, high-quality whole-body skeletal specimens.\u003c/p\u003e \u003cp\u003eComparative analysis of the experimental and control groups (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) further underscores the necessity of integrating multiple protective and accelerative mechanisms within a single protocol. Traditional hot-water maceration (Control 1) was ineffective for formalin-fixed specimens, requiring prolonged processing while failing to detach soft tissue. Strong alkali treatment alone (Control 2) improved tissue degradation but resulted in inefficient removal due to viscous residue formation and caused substantial cortical damage, particularly in thin or fragile bones. The addition of ultrasound (Control 3) effectively eliminated residual soft tissue by disrupting the degradation layer, yet this benefit came at the cost of increased bone damage in vulnerable regions. Conversely, supplementation with calcium hydroxide alone (Control 4) preserved bone integrity through mineral protection but failed to achieve complete soft-tissue removal in the absence of ultrasonic agitation. Only the experimental protocol\u0026mdash;combining weak-acid pre-soaking, calcium hydroxide\u0026ndash;enhanced alkaline treatment, and ultrasonic assistance\u0026mdash;simultaneously achieved rapid, thorough soft-tissue removal and consistent preservation of skeletal morphology across all bone types.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, the protocol described in this study provides an effective solution to the long-standing challenges associated with preparing skeletal specimens from formalin-fixed cadavers. By integrating ultrasonic-assisted alkaline maceration, calcium hydroxide\u0026ndash;mediated mineral protection, weak-acid pre-soaking, and anatomy-guided soft-tissue preservation, the method enables efficient soft-tissue removal while maintaining structural integrity across both robust and fragile skeletal regions.\u003c/p\u003e\n\u003ch3\u003eLimitations\u003c/h3\u003e\n\u003cp\u003eNevertheless, the protocol requires careful operator judgment in trimming and timing, particularly for anatomically delicate structures, and the use of ultrasonic equipment may limit accessibility in resource-constrained settings. Despite these considerations, the method substantially improves specimen quality, processing efficiency, and sustainability, and offers a practical framework for the secondary utilization of dissected cadavers in anatomical education and research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors sincerely thank those who donated their bodies to science so that anatomical research could be performed. Results from such research can potentially increase mankind's overall knowledge that can then improve patient care. Therefore, these donors and their families deserve our highest gratitude.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, J.L. and A.R.; Methodology, J.L., X.H., L.D., B.L., and A.R.; Investigation, X.H. and L.D.; Data curation, J.L.; Visualization, J.L.; Writing – original draft, A.R. and J.L.; Writing – review \u0026amp; editing, A.R.; Resources, J.L.; Project administration, A.R.; Supervision, Z.Z.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eShenzhen University Laboratory and Equipment Management Research Fund (No. 2024018). Construction and Application of a Safety Management System for Human Morphology Teaching Laboratories.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u0026nbsp;\u003c/strong\u003eAll body donors involved in this study provided informed consent during their lifetime to participate in anatomy‑related research and teaching prior to death.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval:\u0026nbsp;\u003c/strong\u003eThis study was reviewed and approved by the Ethics Committee of the Body Donation Receiving Station, School of Medicine, Shenzhen University. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration:\u0026nbsp;\u003c/strong\u003enot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e: The authors confirm that this manuscript has not been published elsewhere and is not under consideration by another journal. All authors have read and approved the submitted version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAHMED, S. A., HEGAZY, N. N., ABDEL MALAK, H. W., CLIFF KAYSER, W., 3RD, ELRAFIE, N. M., HASSANIEN, M., AL-HAYANI, A. A., EL SAADANY, S. A., AI-YOUBI, A. O. \u0026amp; SHEHATA, M. H. 2020. 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Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model. \u003cem\u003eJ Vis Exp\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eMYLES, C., GORMAN, L. \u0026amp; JONES, J. F. X. 2025. 3D printing variation: Teaching and assessing hepatobiliary variants in human anatomy. \u003cem\u003eAnat Sci Educ,\u003c/em\u003e 18\u003cstrong\u003e,\u003c/strong\u003e 885-896.\u003c/li\u003e\n\u003cli\u003ePAL, A., BHANARKAR, U. \u0026amp; RAY, B. 2022. Embalming with Formalin \u0026ndash; Benefits and Pitfalls. \u003cem\u003eScholars International Journal of Anatomy and Physiology,\u003c/em\u003e 5\u003cstrong\u003e,\u003c/strong\u003e 70-77.\u003c/li\u003e\n\u003cli\u003ePAN, S. Q., CHAN, L. K., YAN, Y. \u0026amp; YANG, X. 2020. Survey of Gross Anatomy Education in China: The Past and the Present. \u003cem\u003eAnat Sci Educ,\u003c/em\u003e 13\u003cstrong\u003e,\u003c/strong\u003e 390-400.\u003c/li\u003e\n\u003cli\u003eSAVITRI, YAKKUNDI, R. C., MATAPATHI, N. \u0026amp; TALAWAR, V. 2023. A review of Bone Preparation Techniques for Anatomical Studies. \u003cem\u003eJournal of Ayurveda and Integrated Medical Sciences,\u003c/em\u003e 8\u003cstrong\u003e,\u003c/strong\u003e 112-117.\u003c/li\u003e\n\u003cli\u003eSIGNORELLI, F., RASTEGAR, V., PALERMO, M., LAINO, D., ZEOLI, F. \u0026amp; VISOCCHI, M. 2025. Long-Term Preservation of Human Head and Neck Specimens for Neurosurgical Training: A Technical Note. \u003cem\u003eBrain Sci,\u003c/em\u003e 15.\u003c/li\u003e\n\u003cli\u003eSMITH, C. F., FREEMAN, S. K., HEYLINGS, D., FINN, G. M. \u0026amp; DAVIES, D. C. 2022. Anatomy education for medical students in the United Kingdom and Republic of Ireland in 2019: A 20-year follow-up. \u003cem\u003eAnat Sci Educ,\u003c/em\u003e 15\u003cstrong\u003e,\u003c/strong\u003e 993-1006.\u003c/li\u003e\n\u003cli\u003eTOPP, T., M\u0026uuml;LLER, T., HUSS, S., KANN, P. H., WEIHE, E., RUCHHOLTZ, S. \u0026amp; ZETTL, R. P. 2012. Embalmed and fresh frozen human bones in orthopedic cadaveric studies: which bone is authentic and feasible? \u003cem\u003eActa Orthop,\u003c/em\u003e 83\u003cstrong\u003e,\u003c/strong\u003e 543-7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"bone specimen preparation, alkaline maceration, ultrasonic cleaning, formalin-fixed cadaver","lastPublishedDoi":"10.21203/rs.3.rs-8805669/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8805669/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHigh‑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.\u003c/p\u003e","manuscriptTitle":"A Stepwise Ultrasonic‑Assisted Protocol for Skeletal Preparation from Formalin‑Fixed Cadavers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-18 19:29:34","doi":"10.21203/rs.3.rs-8805669/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b15e3843-afb6-4b96-b070-cc771ccc9a5d","owner":[],"postedDate":"February 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-02T02:49:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-18 19:29:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8805669","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8805669","identity":"rs-8805669","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}