Development and Validation of a Modified Ovine Model for Anterior Cervical Discectomy and Fusion (ACDF) Studies

Development and Validation of a Modified Ovine Model for Anterior Cervical Discectomy and Fusion (ACDF) Studies | 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 Development and Validation of a Modified Ovine Model for Anterior Cervical Discectomy and Fusion (ACDF) Studies Guang-Shen Li, Gang Wu, Yu-Li Ma, Wei-Xing Zhou, Lei Yang, Chun-Mao Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7280766/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 Background Sheep cervical spines resemble human spines in size and mechanics. However, anatomical differences prevent unmodified use of human fusion devices. Endplate preparation variations may affect results. To develop and validate a low-cost, accurate, and simple ovine cervical spine model system for intervertebral fusion mimicking the human spine. Methods We obtained CT scans from 10 male sheep cervical specimens. Measurements included intervertebral space height; anterior-posterior diameter; left-right diameter; maximum cage depth. We designed an anatomically matched fusion device. Two male sheep underwent ACDF using this cage. Postoperative imaging assessed cage position. After 10 weeks, we explanted fused segments. Bone integration was assessed via micro-CT and histological analysis. Results We successfully fabricated the model system using metal 3D printing. The system underwent size, shape, and mechanical validation. No device displacement occurred postoperatively. Micro-CT and histology demonstrated significant bone ingrowth, with stable osseointegration confirmed. Conclusion. This stable, anatomically accurate sheep model provides a reliable system for intervertebral fusion experiments. It serves as a valuable reference for large animal studies. Anterior cervical discectomy and fusion (ACDF) experimental animal model sheep cervical cage Micro-CT compressive strength elastic modulus intervertebral parameters 3D printing bone ingrowth Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Anterior cervical discectomy and fusion (ACDF) remains the gold standard for treating degenerative cervical disc disorders. This procedure removes pathological discs, inserts an interbody cage, and stabilizes the spine with an anterior plate. ACDF effectively decompresses neural structures, restores intervertebral height, and reestablishes cervical alignment. While proven safe, surgical outcomes increasingly depend on cage design and biomechanical performance. Polyetheretherketone (PEEK) cages face significant limitations. High complication rates include subsidence, pseudoarthrosis, cage loosening, and migration(1). These failures drive research into improved cage designs, particularly through animal studies. Most studies, however, implant human-designed cages without anatomical adaptation—a critical oversight. Primates offer optimal anatomical similarity but impose prohibitive costs and ethical constraints. Sheep provide a validated alternative. Their cervical spine biomechanics, disc morphology, and upright posture during standing closely mimic humans. Goats share similar advantages, but pigs exhibit horizontal cervical alignment unsuitable for human translation. Small rodents (rabbits, rats, mice) lack adequate intervertebral space for cage testing. To address species-specific anatomy, we integrated metal 3D printing (Ti6Al4V alloy) to fabricate sheep-adapted cages. This technology enables rapid prototyping of complex geometries, ensures compressive strength exceeding physiological demands, and leverages titanium’s proven biocompatibility. We designed cage surfaces to match sheep intervertebral endplate contours and optimized fixation systems for vertebral anchorage. This study establishes a functionally accurate ACDF sheep model using anatomically tailored 3D-printed titanium cages. Our model demonstrates high surgical feasibility, reduces experimental complexity, and provides reliable assessment of fusion dynamics—advancing translational research in cervical spine reconstruction. Methods This study included 10 male sheep cervical spine specimens, one male sheep carcass, and two live male sheep, all provided by the Large Animal Experimental Center of China Medical City. The animals weighed 40–55 kg and were 12 months old. CT scans were obtained from the 10 specimens. Six were used to measure intervertebral space dimensions and determine cage sizing. Two were used to design the cage's contact surfaces and fixation approach based on the anatomical features of the sheep cervical spine. The remaining two were used for virtual assembly simulation. The cage was then implanted into one sheep carcass to establish a feasible ACDF model. The system was further validated in vivo using two live sheep, followed by functional and imaging evaluations. All live animals were housed individually in controlled environments. The protocol was approved by the Institutional Animal Care and Use Committee of the Large Animal Experimental Center, China Medical City (Approval No. 2021112801) and complied with the "Guidelines for the Care and Use of Laboratory Animals." CT Imaging and Parameter Measurement All ten spine specimens were scanned using a 64-slice CT scanner (SIEMENS, SOMATOM Definition AS+, Germany). Six specimens were used to measure intervertebral space dimensions and determine cage sizing. Two specimens were used to model the cage contact surfaces and fixation approach. The remaining 8 specimens were used for virtual assembly simulation. CT images were imported into 3D Slicer (5.9.0-2025-05-27, Slicer Community, www.Slicer.org) for analysis. For each intervertebral level (e.g., C2/3), the adjusted sagittal plane (ASP), adjusted coronal plane (ACP), and adjusted axial plane (ATP) were manually aligned, as shown in Figure 1 A. Disc height and vertebral endplate dimensions were measured on the aligned images, as shown in Figure 1 B-D. Parameter definitions included: Anterior disc height: vertical distance from the anterior C2 inferior endplate to the C3 superior endplate in the ACP (C2/3A). Middle disc height: vertical distance from the middle of the C2 inferior endplate to the C3 superior endplate in the ACP(C2/3M). Posterior disc heights: vertical distance from the posterior of the C2 inferior endplate to the C3 superior endplate in the ACP (C2/3P). Maximum cage depth (MCD): distance from the anterior margin of the C2 inferior endplate to the posterior margin of the C3 superior endplate (in the ASP). Anterior-posterior diameter (APD): distance between the posterior C2 inferior endplate and the anterior C3 superior endplate. Left-right diameter (LRD): width of the C3 superior endplate in the coronal view. MCD and the anterior angle (MAA): the angle between the MCD and the anterior vertebral edge (Supplementary Figure 1). Each measurement was repeated three times and averaged. Cage Design and Validation Three-dimensional models of the cervical vertebrae were reconstructed from the CT data of two randomly selected specimens. The C2/3 and C4/5 intervertebral spaces were segmented, and the anterior endplate surfaces were extracted. A generalized interbody fusion cage shape was modeled based on these surfaces. The morphological design of the cage was refined through virtual assembly based on CT scans from eight additional ovine cervical spines, and its feasibility was subsequently confirmed by cadaveric implantation in one animal specimen. In the ASP, the cage-to-plate angle (CPA) is designed based on the size of the MAA. The plate length was standardized to 6 mm to accommodate screw placement. A representative CPA was selected based on measurements from multiple segments. Comparative Analysis of Interbody Cage Dimensions in ACDF Patients and Sheep Cervical Spine Models To evaluate the suitability of the sheep cervical spine for the cage, dimensional comparisons were performed between human and sheep models. Preoperative cervical CT scans from randomly selected ACDF patients were used to measure APD and LRD of the intervertebral space ( Figure 1 E–H). The cage area (APD × LRD) and the intervertebral space area were calculated, and their ratio was obtained. Sheep APD and LRD values were derived from previously collected data. Ratios of cage-to-disc space area were compared across species. Human cages measured 12 mm (APD) and 14.5 mm (LRD); sheep cage dimensions followed the current design. Similar ratios indicated geometric compatibility, supporting the relevance of the sheep model for ACDF research. Biomechanical Testing Specimens were loaded in a universal testing machine (SENS, CMT7505, China), as shown in Figure 2 A. A preload (<40 N) ensured full contact, and axial compression was applied at 0.15 mm/s until failure. Compressive strength (σ = P/A) (2) was calculated as the peak force divided by the contact area (cylindrical indenter, 1 cm diameter). The elastic modulus(E = σ/ε) (3) was derived from the slope of the linear portion of the stress–strain curve ( Figure 2 B). Tests were conducted at room temperature, and specimens were kept moist with saline-soaked gauze to prevent dehydration. Cage Fabrication Interbody fusion cages were fabricated by selective laser melting (SLM) using Ti6Al4V alloy. The porous lattice consisted of rhombic dodecahedral units (cell size 2.5 mm, porosity 70%, strut diameter 0.5 mm). Lateral pores (0.5 mm) connected the internal lattice. After printing, excess titanium powder was removed with high-pressure air and the surfaces were sandblasted. Cages were rinsed with water, ultrasonically cleaned, and autoclaved for sterilization. To facilitate mechanical testing of the intervertebral fusion device, we employed cubic specimens with identical porous architecture ( Figure 2 C). Surgical Procedure A midline longitudinal incision was made in the anterior neck. Muscles and fascia were separated layer by layer using a combination of electrocautery and blunt dissection. A retractor was used to displace the trachea and esophagus to the left and the vascular sheath to the right, exposing the anterior surfaces of the C2 and C3 vertebral bodies and the corresponding intervertebral disc. A Caspar retractor distracted the C2–C3 intervertebral space. The intervertebral disc and cartilage endplates were removed. The custom interbody cage was implanted into the C2/3 disc space and secured with an anterior plate and screws. The wound was irrigated and closed in layers. Postoperative care included ceftiofur (5 mg/kg, twice daily) for infection prevention and analgesics. Postoperative Evaluation Neurological function was assessed at recovery and at 2, 5, and 10 weeks postoperatively using the Tarlov scoring system(4). Blood samples (5 mL) were collected on the day of surgery (fasting). Red blood cell count (RBC), hemoglobin (HGB), white blood cell count (WBC), and neutrophil count (Neu) were measured using an automated hematology analyzer. Abnormal results were confirmed by blood smear. Reference ranges were: RBC 5.50–14.2 × 10^9/L; HGB 63–132 g/L; WBC 5.10–15.8 × 10^9/L; Neu 1.32–8.96 × 10^9/L. Cage position was monitored intraoperatively by fluoroscopy (SIEMENS Artis One, Germany). Lateral radiographs were taken immediately postoperatively and at 2, 5, and 10 weeks to assess cage placement. Imaging and Histological Analysis At 10 weeks, sheep were euthanized and the C2–C5 segments were harvested. Specimens were fixed in formalin and scanned by micro-CT (Skyscan 1076, Bruker, Belgium) at 12.6 μm resolution (70 kV, 141 μA). Images were reconstructed in 3D Slicer. After adjusting the views, the Segment Editor and Boolean operations were used to define the regions of interest (ROI). The intervertebral space was divided into four ROIs: anterior, central, peripheral, and posterior, corresponding to the orange, red, cyan, and green regions in Figure 5 G. The designed cage featured a porous structure with uniform lattice units. Bone ingrowth into the porous implant was quantified by the average grayscale within the cage lattice units. For histology, intervertebral disc tissues trimmed to 3×3×3 mm³ were fixed in 4% paraformaldehyde for 48 hours, dehydrated, and embedded in methyl methacrylate. Polymerized blocks were sectioned and ground to 5–10 μm using an EXAKT system. Sections were stained with 1% toluidine blue, Goldner’s trichrome, and Masson’s trichrome for evaluation. Statistical Analysis Data are presented as mean ± SD. One-way ANOVA followed by Tukey’s post hoc test (SPSS 17.0) was used for comparisons. A p-value < 0.05 was considered statistically significant. Results Anatomical feasibility and morphology The C2–C6 cervical disc spaces in sheep can accommodate interbody cages. We selected the C2/3 level for testing. Sheep vertebral bodies are taller and taper centrally, with sharp anterior edges and “bamboo-like” anterior protrusions; human vertebrae are more cylindrical with flatter anterior surfaces( Figure 1 A, E, F, I). Three-dimensional reconstructions showed the sheep C2/3 disc slopes downward from front to back (spoon-shaped) and is thicker at the sides ( Figure 3 A-C). By contrast, human C6/7 discs are nearly vertical with concave endplates ( Figure 1 J). Despite these differences, the vertical orientation of the sheep spine under load is similar to humans(5), and the relatively flat anterior disc surface provides a stable platform for cage placement. Sagittal disc height (CT analysis) Twenty-three intervertebral spaces from six sheep were analyzed by CT. Mean sagittal heights were 4.48 ± 1.05 mm anteriorly, 2.83 ± 0.42 mm in the middle, and 2.01 ± 0.41 mm posteriorly ( Table 1 and Figure 4 A-C). Disc height dropped significantly from anterior to posterior (mean difference 2.47 mm, P<0.001), about 1.65 mm from anterior to middle and 0.82 mm from middle to posterior. In each space, the anterior height was greatest and the posterior height smallest. From C2/3 down to C5/6 levels, both anterior and middle heights tended to decrease, while posterior height increased slightly. Cage design optimization A consistent anterior platform (2.83 ± 0.42 mm thick) was identified across sheep specimens (3.01 mm at C2/3, 2.67 mm at C4/5), as shown in Figure 3 B-E. After discectomy, the disc height increased, so we prepared cages of four heights (3, 4, 5, 6 mm) to accommodate variation. The cage body was porous, composed of rhombic dodecahedral unit cells (2.5 mm units, 70% porosity, 0.5 mm struts, 1.4 mm pores). An anterior solid plate (6×6×1.5 mm with a 4 mm screw hole) was integrated at the cage’s front. The CPA (150°) was chosen to match the MAA. The final design is shown in Figure 3 F and G, with cage dimensions of 12 × 12 mm. The simulated and actual implantation of the interbody fusion cage using 3D Slicer and cadaveric specimens is shown in Figure 3 H–K. Vertebral bone strength Ten sheep cervical vertebrae (C3–C6) were compression-tested. Strength ranged from 12.0 to 52.5 MPa (median 21.5 MPa) and the mean elastic modulus was 581.4 ± 235.1 MPa ( Figure 4 K–L). Thus, an interbody cage must withstand at least 52.5 MPa . Human–sheep intervertebral comparison Data from 10 human ACDF patients (40 levels) were compared with 6 sheep (23 levels) as shown in Table 2 . Human mean cage-to- intervertebral (C/I) ratios were 40.98 ± 4.56% overall (38.89 ± 2.61% at surgical levels). Sheep mean C/I ratios was 42.82 ± 5.21% overall (38.64 ± 4.31% at surgical levels), as shown in Figure 4 J. These ratios were statistically similar (P=0.14 overall, P=0.88 at surgical levels), supporting the use of similarly sized implants. Porous cage mechanics Three 10×10×10 mm cubes (same architecture as the cage) were tested. They showed compressive strength 139.3 ± 8.6 MPa and elastic modulus 2.43 ± 0.01 GPa ( Figure 4 K–L), slightly above vertebral values. This indicates the cage structure has sufficient stiffness and strength to match the bone. Postoperative safety and fusion ACDF was performed on two sheep at C2/3 (two segments total). Both animals recovered uneventfully, with no paralysis, normal limb function, and no infection. Neurological function (Tarlov grade) remained intact at 4/4 ( Figure 5 A). Sequential angiography (immediately after surgery and at 2, 5, and 10 weeks) showed no cage displacement ( Figure 5 E). Routine blood tests (pre-op through 2 weeks post-op) remained within normal ranges (RBC, WBC, neutrophils, hemoglobin; Figure 5 B–C). Bone ingrowth Ten weeks after implantation, micro-CT showed extensive new bone within the porous cage and continuous callus formation ( Figure 5 F). Grayscale analysis revealed uniform bone density across the cage lattice, indicating homogeneous integration ( Figure 5 D). Histology with toluidine blue and Masson’s trichrome staining confirmed marked bone tissue infiltration throughout the porous structure ( Figure 6 ). Discussion Animal model relevance Preclinical validation of cervical cages requires appropriate animal models. Non-human primates closely mimic human anatomy but are costly and limited (6). Alternative large animals are needed for economical and practical fusion studies. Advantages of sheep Sheep (and similarly sized goats) offer several advantages. Their larger size and well-defined neck anatomy (clear separation of esophagus and major vessels) simplify anterior cervical surgery(5). The sheep spine is held vertically in stance like humans, and even has greater motion than typical post-ACDF patients, providing a demanding test for implants. Compared to pigs or calves, sheep cervical biomechanics are more human-like(7). These features, along with accessibility and lower cost, make sheep an ideal large-animal model for cervical fusion research. Model and cage design A consistent anterior disc platform (2.83 ± 0.42 mm high at C2/3) was found for cage anchoring ( Figure 3 D–E). Despite sheep vertebrae having a convex anterior profile, the C/I matched human levels, justifying similar implant sizing. However, sheep vertebrae are taller and narrower, and their disc spaces tilt upward, preventing use of standard human ACDF plates. Simple bilateral plate designs were unstable in trials. To address this, an integrated cage-plate device was developed: the cage attaches at 150° to an anterior plate that conforms to the lower vertebral edge. The cage fits tightly between the superior and inferior endplates, and its porous surface adds friction against migration. No osteotomy is required. The design’s increased cage height further resists migration. This streamlined system also reduces surgical exposure and steps, improving stability without extra hardware. Anatomical challenges Sheep discs differ anatomically from humans: the superior endplate is convex and the inferior is concave (spoon-shaped), and the space slopes upward anteriorly(8). This geometry lowers intrinsic stability and raises risk of anterior cage migration. Moreover, the posterior disc region is very shallow (2.0 ± 0.41mm) and angled, making it unsafe to work near the spinal cord ( Figure 1 A). Therefore, we confined discectomy and cage placement to the anterior two-thirds of the disc, as recommended by prior studies. The MCD (~14.5 mm overall, 12.3 mm at C2/3) was chosen to fit this region. Using human-designed cages would require reshaping the curved endplates, potentially increasing bleeding and affecting bone healing. Our sheep-specific cage fits the natural endplates without osteotomy, maintaining consistent conditions for fusion assessment. Imaging and biomechanics Metal implants typically cause CT artifacts. Our 3D-printed titanium cage did produce artifacts on standard CT, but high-resolution micro-CT effectively visualized bone ingrowth. The porous design allowed direct micro-CT assessment of osseointegration, supplemented by histology. Additionally, sheep size permits standard biomechanical tests: specimens spanning one level above and below the fusion can be loaded to measure range-of-motion and assess fusion directly. Limitations The high cost of large-animal experiments limited our sample size. Although we analyzed bone ingrowth even at the scale of individual lattice units, biological variability and differences between discs cannot be fully eliminated in a small cohort. Larger studies with longer follow-up are needed to confirm these findings. Conclusion We established a sheep cervical fusion model and a custom interbody system that mimic human anterior fusion surgery. The integrated cage-plate design fits sheep anatomy and provides stable fixation. The 3D-printed porous cage supports robust bone ingrowth and allows fusion to be quantified by micro-CT, histology, and mechanical testing. This platform is a versatile tool for preclinical evaluation of new cage designs, materials, or drug-delivery strategies to enhance spinal fusion. Abbreviations ACDF：Anterior cervical discectomy and fusion PEEK: Polyetheretherketone ASP :Adjusted sagittal plane ACP: Adjusted coronal plane ATP: Adjusted axial plane MCD: Maximum cage depth APD: Anterior-posterior diameter LRD: Left-right diameter MAA: MCD and the anterior angle CPA: Cage-to-plate angle SLM: selective laser melting RBC: Red blood cell HGB: Hemoglobin WBC: White blood cell Neu: Neutrophil ROI: Regions of interest Declarations Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding Science and Technology Plan Project of Taizhou (TS202415), Chen Chunmao Doctoral Research Grant of The Affiliated Taizhou People's Hospital of Nanjing Medical University. Contributions GSL prepared writing—review and editing methodology, conducted data curation and writing—original draft. GW prepared writing—original draft, and investigation. LY performed data curation and formal analysis. MYL and WXZ provided software and project administration. HJL and CMC analyzed supervision and funding acquisition. CZ did supervision and funding acquisition. Corresponding author Correspondence to Chun-Mao Chen and Hai-Jun Li. Ethics declarations Ethics approval and consent to participate The sheep cervical spine specimens, sheep carcass, and live male sheep used in our experiment was purchased at the Jiangsu MEDPHOENIX Medical Technology Co., Ltd. This study was approved by Jiangsu MEDPHOENIX Medical Technology Co., Ltd. Medical Laboratory Animal Ethics Committee, IACUC No. 2021112801. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements Not applicable References Li G, Yang L, Wu G, Qian Z, Li H. An update of interbody cages for spine fusion surgeries: from shape design to materials. Expert Rev Med Devices. 2022 Dec 2;19(12):977–89. Wilga C, Ferry L, Dumont E. The effect of jaw suspension on cartilage strength in elasmobranchs. J Morphol. 2024 July;285(7):e21745. Baleani M, Erani P, Acciaioli A, Schileo E. Tensile Yield Strain of Human Cortical Bone from the Femoral Diaphysis Is Constant among Healthy Adults and across the Anatomical Quadrants. Bioengineering (Basel). 2024 Apr 19;11(4):395. Chen Y, Yang H, Xie N, Zhang S, Zou X, Deng C, et al. Could extended laminectomy effectively prevent spinal cord injury due to spinal shortening after 3-column osteotomy? BMC Musculoskelet Disord. 2023 Aug 17;24:658. Yang H, Wang J, Xu H, Zhang F, Lyu F. Technical Notes for Establishing a Cervical Fusion Model in Goats Based on Distinctive Anatomy. Tissue Engineering Part C [Internet]. 2022 Aug 5 [cited 2022 Sept 13]; Available from: http://www-liebertpub-com-s.webvpn.njmu.edu.cn:8118/doi/full/10.1089/ten.tec.2022.0133 Liu J, Yang Z, Wu X, Huang Z, Huang Z, Chen X, et al. Comparison of the anatomical morphology of cervical vertebrae between humans and macaques: related to a spinal cord injury model. Exp Anim. 2021 Feb 6;70(1):108–18. Sh Y, Fr X, Dm L, Ck W, Fy T. A Dynamic Interbody Cage Improves Bone Formation in Anterior Cervical Surgery: A Porcine Biomechanical Study. Clinical orthopaedics and related research [Internet]. 2021 Jan 11 [cited 2021 Nov 12];479(11). Available from: http://pubmed-ncbi-nlm-nih-gov-s.webvpn.njmu.edu.cn:8118/34343157/ Agazzi S, van Loveren HR, Trahan CJ, Johnson WM. Refinement of interbody implant testing in goats: a surgical and morphometric rationale for selection of a cervical level. Laboratory investigation. J Neurosurg Spine. 2007 Nov;7(5):549–53. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7280766","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":499720839,"identity":"a52a0568-bace-4034-85a9-e67c4c3108e5","order_by":0,"name":"Guang-Shen Li","email":"","orcid":"","institution":"The Affiliated Taizhou People's Hospital of Nanjing Medical University, Taizhou School of Clinical Medicine, Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guang-Shen","middleName":"","lastName":"Li","suffix":""},{"id":499720840,"identity":"36fee8db-6aab-4c5b-98ac-1d88275b0259","order_by":1,"name":"Gang Wu","email":"","orcid":"","institution":"The Affiliated Taizhou People's Hospital of Nanjing Medical University, Taizhou School of Clinical Medicine, Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Wu","suffix":""},{"id":499720841,"identity":"893031c0-3bb0-4468-9e3a-3b5ba06c6af2","order_by":2,"name":"Yu-Li Ma","email":"","orcid":"","institution":"Shanghai Sanyou Medical Co.,Ltd","correspondingAuthor":false,"prefix":"","firstName":"Yu-Li","middleName":"","lastName":"Ma","suffix":""},{"id":499720844,"identity":"13968a8a-d2e5-44be-8788-ec245d752b48","order_by":3,"name":"Wei-Xing Zhou","email":"","orcid":"","institution":"Shanghai Sanyou Medical Co.,Ltd","correspondingAuthor":false,"prefix":"","firstName":"Wei-Xing","middleName":"","lastName":"Zhou","suffix":""},{"id":499720845,"identity":"c9c16952-917f-4350-bc22-637ae3b30131","order_by":4,"name":"Lei Yang","email":"","orcid":"","institution":"The Affiliated Taizhou People's Hospital of Nanjing Medical University, Taizhou School of Clinical Medicine, Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Yang","suffix":""},{"id":499720848,"identity":"61342d73-8955-41e9-93be-335b95387893","order_by":5,"name":"Chun-Mao Chen","email":"","orcid":"","institution":"The Affiliated Taizhou People's Hospital of Nanjing Medical University, Taizhou School of Clinical Medicine, Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chun-Mao","middleName":"","lastName":"Chen","suffix":""},{"id":499720850,"identity":"55449c17-c6aa-495a-8c89-94b4cbebe503","order_by":6,"name":"Hai-Jun Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYBACNv7mAwcSeGrk7NvbDz5I+GFDWAufxLHEBw9kjhkb8JxJNnjYk0ZYixxDjrHhAxvmRAMJBzPJB2yHiXAYwxkziYQctgRzCYa0igSewwz87d0J+LUwt5VJJJyRybOc3XjsRoJFOoPEmbMbCNhyeJtEYg9bMcOdA2k3EnisGQwkcglpSTCTSPzHnNhwI8GsIIGNmRgtKcYGCTzMiRuAWhgS2JyJ0AIK5ASeY8aSPWeSgS5M4yHoF/n+5gMHfwCjkp+9/eDHHz9s5Pjbe/FrwQA8pCkfBaNgFIyCUYAVAAB0S0vB9+N6fQAAAABJRU5ErkJggg==","orcid":"","institution":"The Affiliated Taizhou People's Hospital of Nanjing Medical University, Taizhou School of Clinical Medicine, Nanjing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Hai-Jun","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-08-03 01:53:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7280766/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7280766/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89273546,"identity":"829a3aa7-6252-4af9-a7ab-4275bab9b25b","added_by":"auto","created_at":"2025-08-18 09:09:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5070346,"visible":true,"origin":"","legend":"\u003cp\u003eMeasurement methods for intervertebral spaces in humans and sheep. (A) Views of the sheep cervical spine in the ASP, ACP and ATP. The ATP (red axis) aligns with the anterior platform of C2’s lower endplate. The yellow axis represents the ASP, and the green axis the ACP. (B, C) Magnified ASP view of the sheep cervical spine. (D) Magnified ACP view of the sheep cervical spine. (E) ACP view of the human cervical spine based on the long axis of the adjacent vertebrae. (F) ACP view of the human cervical spine, with the axial plane positioned at the tip of the uncus. (G) ATP view of the human cervical spine based on the spinal canal and spinous process, measuring the left-right diameter (LRD) of the intervertebral space. (H) ATP view of the human cervical spine based on the spinal canal and spinous process, measuring the anterior-posterior diameter (APD) of the intervertebral space. (I) 3D reconstruction view of the C3/4 intervertebral space. (J) Frontal, top, and side views of the human cervical intervertebral space. ASP: Adjusted sagittal plane (yellow axis). ACP: Adjusted coronal plane (green axis). ATP: Adjusted transverse plane (red axis). C2: Second cervical vertebra (axis vertebra). C3: Third cervical vertebra. C4: Fourth cervical vertebra. C2/3A: Anterior height of the C2/3 intervertebral space. C2/3M: Middle height of the C2/3 intervertebral space. C2/3P: Posterior height of the C2/3 intervertebral space. APD: Anterior-posterior diameter. LRD: Left-right diameter. MCD: Maximum cage depth. White circle: Spinal canal. Yellow arrow: Spinous process.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7280766/v1/7ce9ec28a22335909d7336cc.jpg"},{"id":89273543,"identity":"6e841b60-d843-4881-8775-f40cd961c2b5","added_by":"auto","created_at":"2025-08-18 09:09:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6370992,"visible":true,"origin":"","legend":"\u003cp\u003eMechanical testing and 3D printing. (A) Universal testing machine and enlarged view of vertebral body fixation. (B) Stress-strain curve of the sheep cervical vertebra, with a compressive strength of 15.1 MPa. (C) Cubic 3D-printed porous structure used for calculating the compressive strength of the intervertebral fusion device. (D) 3D-printed intervertebral fusion device. (E) Micro-CT scan results of the 3D-printed intervertebral fusion device. Black arrow: Compression test fixture. VB: Vertebral body.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7280766/v1/9d44de0a48a9a8b19e29e206.jpg"},{"id":89273545,"identity":"9a90e810-6290-4f6f-923e-7dbdd821ca40","added_by":"auto","created_at":"2025-08-18 09:09:54","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5694349,"visible":true,"origin":"","legend":"\u003cp\u003eDesign steps of the sheep cervical intervertebral fusion device and simulation of implantation. (A) Reconstructed 3D image of sheep cervical vertebrae C2 and C3 with an enlarged view of the intervertebral space. (B, C) Extraction of the C2/3 intervertebral space. (D, E) Identification of the appropriate location for placing the intervertebral fusion device through C2/3 intervertebral space extraction. (F, G) Design of the intervertebral fusion device shape based on the suitable placement site. (H) Simulation of intervertebral fusion device implantation in the ASP, ACP and ATP. (I) Three-dimensional view of the simulated implantation of the intervertebral fusion device. (J, K) Implantation of the intervertebral fusion device and DR in the sheep cadaver specimen. ASP: Adjusted sagittal plane. ACP: Adjusted coronal plane. ATP: Adjusted transverse plane. C2: Second cervical vertebra (axis). C3: Third cervical vertebra. White circle: Spinal canal. Green arrow: Intervertebral fusion device. Red arrow: Longus colli muscle. Black arrow: Casper retractor screw.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7280766/v1/129a319c19d2325327d232f2.jpg"},{"id":89274296,"identity":"59342436-f550-4eb6-b4f3-bbb21b88a098","added_by":"auto","created_at":"2025-08-18 09:17:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3744812,"visible":true,"origin":"","legend":"\u003cp\u003eAnatomical parameters of sheep and human cervical vertebrae and mechanical test results of the intervertebral fusion device. (A) Anterior intervertebral space height from C2 to C6. (B) Middle intervertebral space height from C2 to C6. (C) Posterior intervertebral space height from C2 to C6. (D) Maximum cage depth (MCD), defined as the distance from the anterior platform of the upper vertebral endplate to the posterior platform of the lower vertebral endplate along the ATP. (E) Left-right diameter (LRD) of the sheep cervical intervertebral space. (F) Anterior-posterior diameter (APD) of the sheep cervical intervertebral space. (G) Left-right diameter (LRD) of the human cervical intervertebral space. (H) Anterior-posterior diameter (APD) of the human cervical intervertebral space. (I) Angle between the MCD and the vertebral anterior edge in the sheep cervical spine's ASP (MAA). (J) The ratio of the product of the anterior-posterior diameter (APD) and lateral diameter (LRD) of the intervertebral fusion device to that of the intervertebral space in both the surgical segment and all segments of the sheep and human cervical spines. (K) Compressive strength (σ) of the sheep cervical spine and the intervertebral fusion device. (L) Elastic modulus (E) of the sheep cervical spine and the intervertebral fusion device.\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7280766/v1/1589b09143310ff3bfdacd48.jpg"},{"id":89273547,"identity":"609f5d2a-6512-4b6c-aa99-2aff1a0aca04","added_by":"auto","created_at":"2025-08-18 09:09:54","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8291612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eX-ray and Follow-up Results\u003c/strong\u003e.(A) Preoperative and postoperative neurological function scores.(B) Preoperative and postoperative RBC, WBC, and Neu levels in sheep.(C) Preoperative and postoperative HGB levels in sheep.( E) Lateral X-ray follow-up results at immediate post-surgery, 2 weeks, 5 weeks, and 10 weeks in two sheep.( F) Micro-CT scan results of the sheep’s cervical spine at 10 weeks post-surgery, after fixation in formalin.(G) ROI analysis from the Micro-CT results: orange, red, cyan, and green ROIs are located in the anterior, middle, peripheral, and posterior regions of the intervertebral space.C2: Second cervical vertebra (axis).C3: Third cervical vertebra. Green arrow: Intervertebral fusion device. ASP: Adjusted sagittal plane (yellow axis). ACP: Adjusted coronal plane (green axis). ATP: Adjusted transverse plane (red axis).\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7280766/v1/8fb41101120a48fef7048980.jpg"},{"id":89273548,"identity":"15c3d17f-b429-43ad-99d2-578a0f1794e8","added_by":"auto","created_at":"2025-08-18 09:09:55","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":30929820,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative histological images of sheep cervical intervertebral disc space.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A–B) Toluidine blue staining under low (A) and high (B) magnification.\u003c/p\u003e\n\u003cp\u003e(C–D) Goldner’s trichrome staining under low (C) and high (D) magnification.\u003c/p\u003e\n\u003cp\u003e(E–F) Masson’s trichrome staining under low (E) and high (F) magnification.\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7280766/v1/cdbf5a902c9926dfc0a8ffd6.jpg"},{"id":89273542,"identity":"8407bd99-96f8-446d-b3a6-bed948871e1b","added_by":"auto","created_at":"2025-08-18 09:09:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCT Views of Anterior Vertebral Margin and Fixation Methods\u003c/strong\u003e.(A) Sagittal plane CT image of the sheep cervical spine from C2 to C5.(B) Transverse plane CT image corresponding to the red line in (A).(C) Simulated placement of a conventional anterior cervical plate.(D) Simulated assembly of the integrated ACDF animal model system designed in this study. SP: Sagittal plane; TP: Transverse plane;C2: Second cervical vertebra (axis); C3: Third cervical vertebra; C4: Fourth cervical vertebra; C5: Fifth cervical vertebra; White circle: Spinal canal; Yellow arrow: Spinous process; Blue arrow: Inferior anterior edge of vertebral body; Black arrow: Early anterior plate prototype from this study; Green arrow: ACDF animal model system; Red triangle: Line perpendicular to the anterior vertebral surface.\u003c/p\u003e","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-7280766/v1/e9c9d8df693b95de20be43ff.png"},{"id":94825779,"identity":"5e999adc-e076-4ebd-9c32-190d775441cd","added_by":"auto","created_at":"2025-10-31 06:50:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":60812582,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7280766/v1/9c4bdd7b-d018-49f4-abf8-f649bb2f1587.pdf"},{"id":89273549,"identity":"bc873ee2-c65b-4110-91a4-f607d29a3b0a","added_by":"auto","created_at":"2025-08-18 09:09:55","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":49369220,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7280766/v1/e5e8025f549b8c8e1dcaa280.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and Validation of a Modified Ovine Model for Anterior Cervical Discectomy and Fusion (ACDF) Studies","fulltext":[{"header":"Background","content":"\u003cp\u003eAnterior cervical discectomy and fusion (ACDF) remains the gold standard for treating degenerative cervical disc disorders. This procedure removes pathological discs, inserts an interbody cage, and stabilizes the spine with an anterior plate. ACDF effectively decompresses neural structures, restores intervertebral height, and reestablishes cervical alignment. While proven safe, surgical outcomes increasingly depend on cage design and biomechanical performance.\u003c/p\u003e\n\u003cp\u003ePolyetheretherketone (PEEK) cages face significant limitations. High complication rates include subsidence, pseudoarthrosis, cage loosening, and migration(1). These failures drive research into improved cage designs, particularly through animal studies. Most studies, however, implant human-designed cages without anatomical adaptation\u0026mdash;a critical oversight.\u003c/p\u003e\n\u003cp\u003ePrimates offer optimal anatomical similarity but impose prohibitive costs and ethical constraints. Sheep provide a validated alternative. Their cervical spine biomechanics, disc morphology, and upright posture during standing closely mimic humans. Goats share similar advantages, but pigs exhibit horizontal cervical alignment unsuitable for human translation. Small rodents (rabbits, rats, mice) lack adequate intervertebral space for cage testing.\u003c/p\u003e\n\u003cp\u003eTo address species-specific anatomy, we integrated metal 3D printing (Ti6Al4V alloy) to fabricate sheep-adapted cages. This technology enables rapid prototyping of complex geometries, ensures compressive strength exceeding physiological demands, and leverages titanium\u0026rsquo;s proven biocompatibility. We designed cage surfaces to match sheep intervertebral endplate contours and optimized fixation systems for vertebral anchorage.\u003c/p\u003e\n\u003cp\u003eThis study establishes a functionally accurate ACDF sheep model using anatomically tailored 3D-printed titanium cages. Our model demonstrates high surgical feasibility, reduces experimental complexity, and provides reliable assessment of fusion dynamics\u0026mdash;advancing translational research in cervical spine reconstruction.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis study included 10 male sheep cervical spine specimens, one male sheep carcass, and two live male sheep, all provided by the Large Animal Experimental Center of China Medical City. The animals weighed 40\u0026ndash;55 kg and were 12 months old.\u003c/p\u003e\n\u003cp\u003eCT scans were obtained from the 10 specimens. Six were used to measure intervertebral space dimensions and determine cage sizing. Two were used to design the cage\u0026apos;s contact surfaces and fixation approach based on the anatomical features of the sheep cervical spine. The remaining two were used for virtual assembly simulation.\u003c/p\u003e\n\u003cp\u003eThe cage was then implanted into one sheep carcass to establish a feasible ACDF model. The system was further validated in vivo using two live sheep, followed by functional and imaging evaluations.\u003c/p\u003e\n\u003cp\u003eAll live animals were housed individually in controlled environments. The protocol was approved by the Institutional Animal Care and Use Committee of the Large Animal Experimental Center, China Medical City (Approval No. 2021112801) and complied with the \u0026quot;Guidelines for the Care and Use of Laboratory Animals.\u0026quot;\u003c/p\u003e\n\u003cp\u003eCT Imaging and Parameter Measurement\u003c/p\u003e\n\u003cp\u003eAll ten spine specimens were scanned using a 64-slice CT scanner (SIEMENS, SOMATOM Definition AS+, Germany). Six specimens were used to measure intervertebral space dimensions and determine cage sizing. Two specimens were used to model the cage contact surfaces and fixation approach. The remaining 8 specimens were used for virtual assembly simulation.\u003c/p\u003e\n\u003cp\u003eCT images were imported into 3D Slicer (5.9.0-2025-05-27, Slicer Community, www.Slicer.org) for analysis. For each intervertebral level (e.g., C2/3), the adjusted sagittal plane (ASP), adjusted coronal plane (ACP), and adjusted axial plane (ATP) were manually aligned, as shown in \u003cstrong\u003eFigure 1\u003c/strong\u003e A. Disc height and vertebral endplate dimensions were measured on the aligned images, as shown in\u0026nbsp;\u003cstrong\u003eFigure 1\u003c/strong\u003eB-D. Parameter definitions included:\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eAnterior disc height:\u003c/strong\u003e vertical distance from the anterior C2 inferior endplate to the C3 superior endplate in the ACP (C2/3A).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eMiddle disc height:\u003c/strong\u003e vertical distance from the middle of the C2 inferior endplate to the C3 superior endplate in the ACP(C2/3M).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003ePosterior disc heights:\u0026nbsp;\u003c/strong\u003evertical distance from the posterior of the C2 inferior endplate to the C3 superior endplate in the ACP (C2/3P).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eMaximum cage depth (MCD):\u003c/strong\u003e distance from the anterior margin of the C2 inferior endplate to the posterior margin of the C3 superior endplate (in the ASP).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eAnterior-posterior diameter (APD):\u003c/strong\u003e distance between the posterior C2 inferior endplate and the anterior C3 superior endplate.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eLeft-right diameter (LRD):\u003c/strong\u003e width of the C3 superior endplate in the coronal view.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eMCD and the anterior angle (MAA):\u003c/strong\u003e the angle between the MCD and the anterior vertebral edge (Supplementary Figure 1).\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eEach measurement was repeated three times and averaged.\u003c/p\u003e\n\u003cp\u003eCage Design and Validation\u003c/p\u003e\n\u003cp\u003eThree-dimensional models of the cervical vertebrae were reconstructed from the CT data of two randomly selected specimens. The C2/3 and C4/5 intervertebral spaces were segmented, and the anterior endplate surfaces were extracted. A generalized interbody fusion cage shape was modeled based on these surfaces. The morphological design of the cage was refined through virtual assembly based on CT scans from eight additional ovine cervical spines, and its feasibility was subsequently confirmed by cadaveric implantation in one animal specimen.\u003c/p\u003e\n\u003cp\u003eIn the ASP, the cage-to-plate angle (CPA) is designed based on the size of the MAA. The plate length was standardized to 6 mm to accommodate screw placement. A representative CPA was selected based on measurements from multiple segments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative Analysis of Interbody Cage Dimensions in ACDF Patients and Sheep Cervical Spine Models\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;To evaluate the suitability of the sheep cervical spine for the cage, dimensional comparisons were performed between human and sheep models. Preoperative cervical CT scans from randomly selected ACDF patients were used to measure APD and LRD of the intervertebral space (\u003cstrong\u003eFigure 1\u003c/strong\u003eE\u0026ndash;H). The cage area (APD \u0026times; LRD) and the intervertebral space area were calculated, and their ratio was obtained. Sheep APD and LRD values were derived from previously collected data. Ratios of cage-to-disc space area were compared across species. Human cages measured 12 mm (APD) and 14.5 mm (LRD); sheep cage dimensions followed the current design. Similar ratios indicated geometric compatibility, supporting the relevance of the sheep model for ACDF research.\u003c/p\u003e\n\u003cp\u003eBiomechanical Testing\u003c/p\u003e\n\u003cp\u003eSpecimens were loaded in a universal testing machine (SENS, CMT7505, China), as shown in \u003cstrong\u003eFigure 2\u003c/strong\u003eA. A preload (\u0026lt;40 N) ensured full contact, and axial compression was applied at 0.15 mm/s until failure. Compressive strength (\u0026sigma;\u0026nbsp;= P/A)\u0026nbsp;(2)\u0026nbsp;was calculated as the peak force divided by the contact area (cylindrical indenter, 1 cm diameter). The elastic modulus(E =\u0026nbsp;\u0026sigma;/\u0026epsilon;)\u0026nbsp;(3)\u0026nbsp;was derived from the slope of the linear portion of the stress\u0026ndash;strain curve (\u003cstrong\u003eFigure 2\u003c/strong\u003eB). Tests were conducted at room temperature, and specimens were kept moist with saline-soaked gauze to prevent dehydration.\u003c/p\u003e\n\u003cp\u003eCage Fabrication\u003c/p\u003e\n\u003cp\u003eInterbody fusion cages were fabricated by selective laser melting (SLM) using Ti6Al4V alloy. The porous lattice consisted of rhombic dodecahedral units (cell size 2.5 mm, porosity 70%, strut diameter 0.5 mm). Lateral pores (0.5 mm) connected the internal lattice. After printing, excess titanium powder was removed with high-pressure air and the surfaces were sandblasted. Cages were rinsed with water, ultrasonically cleaned, and autoclaved for sterilization.\u003c/p\u003e\n\u003cp\u003eTo facilitate mechanical testing of the intervertebral fusion device, we employed cubic specimens with identical porous architecture (\u003cstrong\u003eFigure 2\u003c/strong\u003eC).\u003c/p\u003e\n\u003cp\u003eSurgical Procedure\u003c/p\u003e\n\u003cp\u003eA midline longitudinal incision was made in the anterior neck.\u0026nbsp;Muscles and fascia were separated layer by layer using a combination of electrocautery and blunt dissection. A retractor was used to displace the trachea and esophagus to the left and the vascular sheath to the right, exposing the anterior surfaces of the C2 and C3 vertebral bodies and the corresponding intervertebral disc.\u0026nbsp;A Caspar retractor distracted the C2\u0026ndash;C3 intervertebral space. The intervertebral disc and cartilage endplates were removed. The custom interbody cage was implanted into the C2/3 disc space and secured with an anterior plate and screws. The wound was irrigated and closed in layers. Postoperative care included ceftiofur (5 mg/kg, twice daily) for infection prevention and analgesics.\u003c/p\u003e\n\u003cp\u003ePostoperative Evaluation\u003c/p\u003e\n\u003cp\u003eNeurological function was assessed at recovery and at 2, 5, and 10 weeks postoperatively using the Tarlov scoring system(4). Blood samples (5 mL) were collected on the day of surgery (fasting). Red blood cell count (RBC), hemoglobin (HGB), white blood cell count (WBC), and neutrophil \u0026nbsp; count (Neu) were measured using an automated hematology analyzer. Abnormal results were confirmed by blood smear. Reference ranges were: RBC 5.50\u0026ndash;14.2 \u0026times; 10^9/L; HGB 63\u0026ndash;132 g/L; WBC 5.10\u0026ndash;15.8 \u0026times; 10^9/L; Neu 1.32\u0026ndash;8.96 \u0026times; 10^9/L.\u003c/p\u003e\n\u003cp\u003eCage position was monitored intraoperatively by fluoroscopy (SIEMENS Artis One, Germany). Lateral radiographs were taken immediately postoperatively and at 2, 5, and 10 weeks to assess cage placement.\u003c/p\u003e\n\u003cp\u003eImaging and Histological Analysis\u003c/p\u003e\n\u003cp\u003eAt 10 weeks, sheep were euthanized and the C2\u0026ndash;C5 segments were harvested. Specimens were fixed in formalin and scanned by micro-CT (Skyscan 1076, Bruker, Belgium) at 12.6 \u0026mu;m resolution (70 kV, 141 \u0026mu;A). Images were reconstructed in 3D Slicer.\u0026nbsp;After adjusting the views, the Segment Editor and Boolean operations were used to define the regions of interest (ROI). The intervertebral space was divided into four ROIs: anterior, central, peripheral, and posterior, corresponding to the orange, red, cyan, and green regions in \u003cstrong\u003eFigure 5\u003c/strong\u003eG. The designed cage featured a porous structure with uniform lattice units.\u0026nbsp;Bone ingrowth into the porous implant was quantified by the average grayscale within the cage lattice units.\u003c/p\u003e\n\u003cp\u003eFor histology, intervertebral disc tissues trimmed to 3\u0026times;3\u0026times;3 mm\u0026sup3;\u0026nbsp;were fixed in 4% paraformaldehyde for 48 hours, dehydrated, and embedded in methyl methacrylate. Polymerized blocks were sectioned and ground to 5\u0026ndash;10 \u0026mu;m using an EXAKT system. Sections were stained with 1% toluidine blue, Goldner\u0026rsquo;s trichrome, and Masson\u0026rsquo;s trichrome for evaluation.\u003c/p\u003e\n\u003cp\u003eStatistical Analysis\u003c/p\u003e\n\u003cp\u003eData are presented as mean \u0026plusmn; SD. One-way ANOVA followed by Tukey\u0026rsquo;s post hoc test (SPSS 17.0) was used for comparisons. A p-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAnatomical feasibility and morphology\u003c/p\u003e\n\u003cp\u003eThe C2\u0026ndash;C6 cervical disc spaces in sheep can accommodate interbody cages. We selected the C2/3 level for testing. Sheep vertebral bodies are taller and taper centrally, with sharp anterior edges and \u0026ldquo;bamboo-like\u0026rdquo; anterior protrusions; human vertebrae are more cylindrical with flatter anterior surfaces(\u003cstrong\u003eFigure 1\u003c/strong\u003eA, E, F, I). Three-dimensional reconstructions showed the sheep C2/3 disc slopes downward from front to back (spoon-shaped) and is thicker at the sides (\u003cstrong\u003eFigure 3\u003c/strong\u003eA-C). By contrast, human C6/7 discs are nearly vertical with concave endplates (\u003cstrong\u003eFigure 1\u003c/strong\u003eJ). Despite these differences, the vertical orientation of the sheep spine under load is similar to humans(5), and the relatively flat anterior disc surface provides a stable platform for cage placement.\u003c/p\u003e\n\u003cp\u003eSagittal disc height (CT analysis)\u003c/p\u003e\n\u003cp\u003eTwenty-three intervertebral spaces from six sheep were analyzed by CT. Mean sagittal heights were 4.48 \u0026plusmn; 1.05 mm anteriorly, 2.83 \u0026plusmn; 0.42 mm in the middle, and 2.01 \u0026plusmn; 0.41 mm posteriorly (\u003cstrong\u003eTable 1\u003c/strong\u003e and\u0026nbsp;\u003cstrong\u003eFigure 4\u003c/strong\u003eA-C). Disc height dropped significantly from\u0026nbsp;anterior to posterior\u0026nbsp;(mean difference 2.47 mm, P\u0026lt;0.001), about 1.65 mm from anterior to middle and 0.82 mm from middle to posterior. In each space, the anterior height was greatest and the posterior height smallest. From C2/3 down to C5/6 levels, both anterior and middle heights tended to decrease, while posterior height increased slightly.\u003c/p\u003e\n\u003cp\u003eCage design optimization\u003c/p\u003e\n\u003cp\u003eA consistent anterior platform (2.83 \u0026plusmn; 0.42 mm thick) was identified across sheep specimens (3.01 mm at C2/3, 2.67 mm at C4/5), as shown in\u0026nbsp;\u003cstrong\u003eFigure 3\u003c/strong\u003eB-E. After discectomy, the disc height increased, so we prepared cages of four heights (3, 4, 5, 6 mm) to accommodate variation. The cage body was porous, composed of rhombic dodecahedral unit cells (2.5 mm units, 70% porosity, 0.5 mm struts, 1.4 mm pores). An anterior solid plate (6\u0026times;6\u0026times;1.5 mm with a 4 mm screw hole) was integrated at the cage\u0026rsquo;s front. The CPA (150\u0026deg;) was chosen to match the MAA. The final design is shown in\u0026nbsp;\u003cstrong\u003eFigure 3\u003c/strong\u003eF and G, with cage dimensions of 12 \u0026times; 12 mm. The simulated and actual implantation of the interbody fusion cage using 3D Slicer and cadaveric specimens is shown in\u0026nbsp;\u003cstrong\u003eFigure 3\u003c/strong\u003eH\u0026ndash;K.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVertebral bone strength\u003c/p\u003e\n\u003cp\u003eTen sheep cervical vertebrae (C3\u0026ndash;C6) were compression-tested. Strength ranged from 12.0 to 52.5 MPa (median 21.5 MPa) and the mean elastic modulus was 581.4 \u0026plusmn; 235.1 MPa (\u003cstrong\u003eFigure 4\u003c/strong\u003eK\u0026ndash;L). Thus, an interbody cage must withstand at least 52.5 MPa .\u003c/p\u003e\n\u003cp\u003eHuman\u0026ndash;sheep intervertebral comparison\u003c/p\u003e\n\u003cp\u003eData from 10 human ACDF patients (40 levels) were compared with 6 sheep (23 levels) as shown in\u0026nbsp;\u003cstrong\u003eTable 2\u003c/strong\u003e. Human mean cage-to-\u0026nbsp;intervertebral\u0026nbsp;(C/I) ratios were 40.98 \u0026plusmn; 4.56% overall (38.89 \u0026plusmn; 2.61% at surgical levels). Sheep mean C/I ratios was 42.82 \u0026plusmn; 5.21% overall (38.64 \u0026plusmn; 4.31% at surgical levels), as shown in\u0026nbsp;\u003cstrong\u003eFigure 4\u003c/strong\u003eJ. These ratios were statistically similar (P=0.14 overall, P=0.88 at surgical levels), supporting the use of similarly sized implants.\u003c/p\u003e\n\u003cp\u003ePorous cage mechanics\u003c/p\u003e\n\u003cp\u003eThree 10\u0026times;10\u0026times;10 mm cubes (same architecture as the cage) were tested. They showed compressive strength 139.3 \u0026plusmn; 8.6 MPa and elastic modulus 2.43 \u0026plusmn; 0.01 GPa (\u003cstrong\u003eFigure 4\u003c/strong\u003eK\u0026ndash;L), slightly above vertebral values. This indicates the cage structure has sufficient stiffness and strength to match the bone.\u003c/p\u003e\n\u003cp\u003ePostoperative safety and fusion\u003c/p\u003e\n\u003cp\u003eACDF was performed on two sheep at C2/3 (two segments total). Both animals recovered uneventfully, with no paralysis, normal limb function, and no infection. Neurological function (Tarlov grade) remained intact at 4/4 (\u003cstrong\u003eFigure 5\u003c/strong\u003eA). Sequential angiography (immediately after surgery and at 2, 5, and 10 weeks) showed no cage displacement (\u003cstrong\u003eFigure 5\u003c/strong\u003eE). Routine blood tests (pre-op through 2 weeks post-op) remained within normal ranges (RBC, WBC, neutrophils, hemoglobin;\u0026nbsp;\u003cstrong\u003eFigure 5\u003c/strong\u003eB\u0026ndash;C).\u003c/p\u003e\n\u003cp\u003eBone ingrowth\u003c/p\u003e\n\u003cp\u003eTen weeks after implantation, micro-CT showed extensive new bone within the porous cage and continuous callus formation (\u003cstrong\u003eFigure 5\u003c/strong\u003eF). Grayscale analysis revealed uniform bone density across the cage lattice, indicating homogeneous integration (\u003cstrong\u003eFigure 5\u003c/strong\u003eD). Histology with toluidine blue and Masson\u0026rsquo;s trichrome staining confirmed marked bone tissue infiltration throughout the porous structure (\u003cstrong\u003eFigure\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;6\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAnimal model relevance\u003c/p\u003e\n\u003cp\u003ePreclinical validation of cervical cages requires appropriate animal models. Non-human primates closely mimic human anatomy but are costly and limited\u0026nbsp;(6). Alternative large animals are needed for economical and practical fusion studies.\u003c/p\u003e\n\u003cp\u003eAdvantages of sheep\u003c/p\u003e\n\u003cp\u003eSheep (and similarly sized goats) offer several advantages. Their larger size and well-defined neck anatomy (clear separation of esophagus and major vessels) simplify anterior cervical surgery(5). The sheep spine is held vertically in stance like humans, and even has greater motion than typical post-ACDF patients, providing a demanding test for implants. Compared to pigs or calves, sheep cervical biomechanics are more human-like(7). These features, along with accessibility and lower cost, make sheep an ideal large-animal model for cervical fusion research.\u003c/p\u003e\n\u003cp\u003eModel and cage design\u003c/p\u003e\n\u003cp\u003eA consistent anterior disc platform (2.83\u0026nbsp;\u0026plusmn; 0.42\u0026nbsp;mm high at C2/3) was found for cage anchoring (\u003cstrong\u003eFigure 3\u003c/strong\u003eD\u0026ndash;E). Despite sheep vertebrae having a convex anterior profile, the C/I matched human levels, justifying similar implant sizing. However, sheep vertebrae are taller and narrower, and their disc spaces tilt upward, preventing use of standard human ACDF plates. Simple bilateral plate designs were unstable in trials. To address this, an integrated cage-plate device was developed: the cage attaches at 150\u0026deg; to an anterior plate that conforms to the lower vertebral edge. The cage fits tightly between the superior and inferior endplates, and its porous surface adds friction against migration. No osteotomy is required. The design\u0026rsquo;s increased cage height further resists migration. This streamlined system also reduces surgical exposure and steps, improving stability without extra hardware.\u003c/p\u003e\n\u003cp\u003eAnatomical challenges\u003c/p\u003e\n\u003cp\u003eSheep discs differ anatomically from humans: the superior endplate is convex and the inferior is concave (spoon-shaped), and the space slopes upward anteriorly(8). This geometry lowers intrinsic stability and raises risk of anterior cage migration. Moreover, the posterior disc region is very shallow (2.0\u0026nbsp;\u0026nbsp;\u0026plusmn; 0.41mm) and angled, making it unsafe to work near the spinal cord (\u003cstrong\u003eFigure 1\u003c/strong\u003eA). Therefore, we confined discectomy and cage placement to the anterior two-thirds of the disc, as recommended by prior studies. The MCD (~14.5 mm overall, 12.3 mm at C2/3) was chosen to fit this region. Using human-designed cages would require reshaping the curved endplates, potentially increasing bleeding and affecting bone healing. Our sheep-specific cage fits the natural endplates without osteotomy, maintaining consistent conditions for fusion assessment.\u003c/p\u003e\n\u003cp\u003eImaging and biomechanics\u003c/p\u003e\n\u003cp\u003eMetal implants typically cause CT artifacts. Our 3D-printed titanium cage did produce artifacts on standard CT, but high-resolution micro-CT effectively visualized bone ingrowth. The porous design allowed direct micro-CT assessment of osseointegration, supplemented by histology. Additionally, sheep size permits standard biomechanical tests: specimens spanning one level above and below the fusion can be loaded to measure range-of-motion and assess fusion directly.\u003c/p\u003e\n\u003cp\u003eLimitations\u003c/p\u003e\n\u003cp\u003eThe high cost of large-animal experiments limited our sample size. Although we analyzed bone ingrowth even at the scale of individual lattice units, biological variability and differences between discs cannot be fully eliminated in a small cohort. Larger studies with longer follow-up are needed to confirm these findings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe established a sheep cervical fusion model and a custom interbody system that mimic human anterior fusion surgery. The integrated cage-plate design fits sheep anatomy and provides stable fixation. The 3D-printed porous cage supports robust bone ingrowth and allows fusion to be quantified by micro-CT, histology, and mechanical testing. This platform is a versatile tool for preclinical evaluation of new cage designs, materials, or drug-delivery strategies to enhance spinal fusion.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACDF：Anterior cervical discectomy and fusion\u003c/p\u003e\n\u003cp\u003ePEEK: Polyetheretherketone\u003c/p\u003e\n\u003cp\u003eASP :Adjusted sagittal plane\u003c/p\u003e\n\u003cp\u003eACP: Adjusted coronal plane\u003c/p\u003e\n\u003cp\u003eATP: Adjusted axial plane\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMCD:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMaximum cage depth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAPD: Anterior-posterior diameter\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLRD: Left-right diameter\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAA: MCD and the anterior angle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCPA: Cage-to-plate angle\u003c/p\u003e\n\u003cp\u003eSLM: selective laser melting\u003c/p\u003e\n\u003cp\u003eRBC: Red blood cell\u003c/p\u003e\n\u003cp\u003eHGB: Hemoglobin\u003c/p\u003e\n\u003cp\u003eWBC: White blood cell\u003c/p\u003e\n\u003cp\u003eNeu: Neutrophil\u003c/p\u003e\n\u003cp\u003eROI: Regions of interest\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eScience and Technology Plan Project of Taizhou (TS202415), Chen Chunmao Doctoral Research Grant of\u0026nbsp;The Affiliated Taizhou People\u0026apos;s Hospital of Nanjing Medical University.\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eGSL prepared writing\u0026mdash;review and editing methodology, conducted data curation and writing\u0026mdash;original draft. GW prepared writing\u0026mdash;original draft, and investigation. LY performed data curation and formal analysis. MYL and WXZ provided software and project administration. HJL and CMC analyzed supervision and funding acquisition. CZ did supervision and funding acquisition.\u003c/p\u003e\n\u003cp\u003eCorresponding author\u003c/p\u003e\n\u003cp\u003eCorrespondence to Chun-Mao Chen and Hai-Jun Li.\u003c/p\u003e\n\u003cp\u003eEthics declarations\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;sheep cervical spine specimens, sheep carcass, and live male sheep\u0026nbsp;used in our experiment was purchased at the\u0026nbsp;Jiangsu MEDPHOENIX Medical Technology Co., Ltd. This study was approved by\u0026nbsp;Jiangsu MEDPHOENIX Medical Technology Co., Ltd. Medical Laboratory Animal Ethics Committee,\u0026nbsp;IACUC No. 2021112801.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eAdditional information\u003c/p\u003e\n\u003cp\u003ePublisher\u0026apos;s Note\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi G, Yang L, Wu G, Qian Z, Li H. An update of interbody cages for spine fusion surgeries: from shape design to materials. Expert Rev Med Devices. 2022 Dec 2;19(12):977\u0026ndash;89. \u003c/li\u003e\n\u003cli\u003eWilga C, Ferry L, Dumont E. The effect of jaw suspension on cartilage strength in elasmobranchs. J Morphol. 2024 July;285(7):e21745. \u003c/li\u003e\n\u003cli\u003eBaleani M, Erani P, Acciaioli A, Schileo E. Tensile Yield Strain of Human Cortical Bone from the Femoral Diaphysis Is Constant among Healthy Adults and across the Anatomical Quadrants. Bioengineering (Basel). 2024 Apr 19;11(4):395. \u003c/li\u003e\n\u003cli\u003eChen Y, Yang H, Xie N, Zhang S, Zou X, Deng C, et al. Could extended laminectomy effectively prevent spinal cord injury due to spinal shortening after 3-column osteotomy? BMC Musculoskelet Disord. 2023 Aug 17;24:658. \u003c/li\u003e\n\u003cli\u003eYang H, Wang J, Xu H, Zhang F, Lyu F. Technical Notes for Establishing a Cervical Fusion Model in Goats Based on Distinctive Anatomy. Tissue Engineering Part C [Internet]. 2022 Aug 5 [cited 2022 Sept 13]; Available from: http://www-liebertpub-com-s.webvpn.njmu.edu.cn:8118/doi/full/10.1089/ten.tec.2022.0133\u003c/li\u003e\n\u003cli\u003eLiu J, Yang Z, Wu X, Huang Z, Huang Z, Chen X, et al. Comparison of the anatomical morphology of cervical vertebrae between humans and macaques: related to a spinal cord injury model. Exp Anim. 2021 Feb 6;70(1):108\u0026ndash;18. \u003c/li\u003e\n\u003cli\u003eSh Y, Fr X, Dm L, Ck W, Fy T. A Dynamic Interbody Cage Improves Bone Formation in Anterior Cervical Surgery: A Porcine Biomechanical Study. Clinical orthopaedics and related research [Internet]. 2021 Jan 11 [cited 2021 Nov 12];479(11). Available from: http://pubmed-ncbi-nlm-nih-gov-s.webvpn.njmu.edu.cn:8118/34343157/\u003c/li\u003e\n\u003cli\u003eAgazzi S, van Loveren HR, Trahan CJ, Johnson WM. Refinement of interbody implant testing in goats: a surgical and morphometric rationale for selection of a cervical level. Laboratory investigation. J Neurosurg Spine. 2007 Nov;7(5):549\u0026ndash;53. \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":"Anterior cervical discectomy and fusion (ACDF), experimental animal model, sheep cervical, cage, Micro-CT, compressive strength, elastic modulus, intervertebral parameters, 3D printing, bone ingrowth","lastPublishedDoi":"10.21203/rs.3.rs-7280766/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7280766/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSheep cervical spines resemble human spines in size and mechanics. However, anatomical differences prevent unmodified use of human fusion devices. Endplate preparation variations may affect results. To develop and validate a low-cost, accurate, and simple ovine cervical spine model system for intervertebral fusion mimicking the human spine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe obtained CT scans from 10 male sheep cervical specimens. Measurements included intervertebral space height; anterior-posterior diameter; left-right diameter; maximum cage depth. We designed an anatomically matched fusion device. Two male sheep underwent ACDF using this cage. Postoperative imaging assessed cage position. After 10 weeks, we explanted fused segments. Bone integration was assessed via micro-CT and histological analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe successfully fabricated the model system using metal 3D printing. The system underwent size, shape, and mechanical validation. No device displacement occurred postoperatively. Micro-CT and histology demonstrated significant bone ingrowth, with stable osseointegration confirmed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis stable, anatomically accurate sheep model provides a reliable system for intervertebral fusion experiments. It serves as a valuable reference for large animal studies.\u003c/p\u003e","manuscriptTitle":"Development and Validation of a Modified Ovine Model for Anterior Cervical Discectomy and Fusion (ACDF) Studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-18 09:09:49","doi":"10.21203/rs.3.rs-7280766/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":"1ecd7062-c9ec-4aed-a248-39b88d34d52a","owner":[],"postedDate":"August 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-30T17:08:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-18 09:09:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7280766","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7280766","identity":"rs-7280766","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}