$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
All animal experiments were conducted in accordance with the protocols approved by the Jikei University Animal Care and Use Committee (approval number 2023–034C1). This study was designed as a pilot feasibility study. To assess the technical viability of this highly delicate and novel posterior cage insertion in the rat caudal spine prior to large-scale validation, a single animal (n = 1) was used, per ethical guidelines, to minimize animal use during the initial technical evaluation. Male Sprague-Dawley (SD) rats were selected as the standard strain for spinal fusion research. The inclusion criteria were healthy males aged 8–10 weeks, weighing between 200–250 g, to ensure that the intervertebral disc height (1.8–2.2 mm) is compatible with the 2 mm cage. Animals exhibiting > 10% weight loss during the acclimatization period, decreased activity, or visible tail deformities were excluded. For this study, a male Sprague–Dawley rat (8 weeks old, weighing 221.8 g; Japan SLC, Shizuoka, Japan) was utilized. The animal was housed in a controlled environment at 25°C and a relative humidity of 50% (allowable range 30–70%) with a 12 h light/dark cycle and was provided ad libitum access to water and a standard rodent diet. Rats were housed in solid-bottom cages provided with nesting materials and chew sticks for environmental enrichment. A 3-day acclimatization period was implemented prior to the surgical procedure. To prevent cage mates from gnawing on the tail implants, the rat was maintained under single-housing conditions throughout the study, which was a critical factor in this experimental design.
Preparation of screws and plates
Internal fixation materials were prepared prior to the surgical procedure. Commercially available stainless-steel screws (original diameter: 1.2 mm, length: 14 mm) were cut to a precise length of 6 mm using a wire cutter (Figure 1A). These screws were sterilized via standard steam autoclaving at 121 °C for 20 min. In a polyoxymethylene (POM) internal fixation plate (18 mm length, 3 mm width, 1 mm thickness), screw holes with a diameter of 1.2 mm were drilled at 12 mm intervals using a manual drill (Figure 1B–D). Subsequently, the corners of the POM plates were rounded using a wire cutter to minimize irritation to the surrounding soft tissue (Figure 1E). The plates were sterilized using ethylene oxide (EtO) gas or hydrogen peroxide gas plasma, given the polymer's thermal sensitivity. The titanium cage features an interconnected lattice structure with longitudinal channels measuring 300–500 µm in diameter. Its surface was prepared using Lamellar 3D Titanium Technology to achieve a micro-surface roughness of 3–5 µm, which facilitates direct bony ongrowth.
Anesthesia and surgical procedure
No preoperative fasting was performed to avoid unnecessary metabolic stress. Anesthesia was induced via a triple anesthetic mixture consisting of medetomidine (0.15 mg/kg), midazolam (2 mg/kg), and butorphanol (2.5 mg/kg), which was administered via subcutaneous injection into the dorsum using a 25-gauge needle23. Adequate surgical plane of anesthesia was assessed every 15 min by confirming the loss of the pedal withdrawal reflex (toe pinch), maintaining pink mucous membranes, and monitoring the respiratory rate (70–110 breaths/min). The surgical procedure for interbody fusion of the caudal vertebrae was subsequently performed. The animal was placed in the prone position (Figure 2A), and the entire tail was disinfected following a three-round alternating scrub protocol (70% ethanol, povidone-iodine scrub, and 70% ethanol) to ensure a sterile field. To identify the target vertebrae, manual palpation was performed starting from the sacrum distally. The first mobile joint was identified as the sacrum-Co1 junction, and the segment immediately distal was designated as Co2, defining the Co2–Co3 target site. A longitudinal incision of approximately 2 cm was made on the dorsal aspect, spanning from the second to the third caudal vertebrae (Co2–Co3) (Figure 2B), to expose the posterior surface of each vertebra. During the exposure, care was taken to identify and avoid damage to the dorsal caudal vein; blunt dissection and bipolar electrocautery were used to manage minor bleeders. To prepare the fusion site, the annulus fibrosus between the Co2 and Co3 vertebrae was incised using a scalpel (Figure 2C). The annulus fibrosus and nucleus pulposus were then meticulously removed via curettage using a curette and small forceps (Figure 2D). This step was performed under high-power surgical magnification (≥3.5x). Completeness of removal was objectively defined by the visual confirmation of punctate bleeding (the "bloody endplate" sign) and the total absence of residual soft tissue. Unacceptable endplate damage was defined as deep erosions involving > 25% of the total area or penetration into the cancellous bone > 0.5 mm. A titanium cage (2 × 2 mm cross-section, 2 mm height) (Figure 2E) was subsequently inserted deep into the intervertebral space until a physical stop was reached and full-surface contact with both endplates was visually confirmed, creating a firm interference fit (Figure 2F, 2G).
Fixation and wound closure
To facilitate plate fixation, the bone surface around the designated hole sites in the Co2 and Co3 vertebrae was flattened using an electric handheld router operating at 7,800 RPM equipped with a 2.0 mm spherical file-type burr (Figure 2H). Bone holes were individually drilled at intervals corresponding to the screw holes of the plastic internal fixation plate using a hand drill equipped with a 1.2 mm diameter drill bit (Figure 2I). Two 6 mm stainless steel screws were then pre-loaded into the holes. of the plastic plate (Figure 2J). Screws were inserted at a 90° angle to a standardized depth of 5 mm with a 12 mm center-to-center spacing (Figure 2K). Finally, the skin incision was closed with 4-0 nylon sutures, and the tail was disinfected once again with 70% ethanol to complete the procedure.
Postoperative management and euthanasia
During the immediate recovery phase (Day 0), the animal was monitored every 15 min, and rectal temperature was supported until it achieved full sternal recumbency and purposeful ambulation. For long-term assessment (Weeks 1–12), a daily scoring system based on Body Condition Score (BCS), grooming status, and general activity was implemented, alongside daily surgical site monitoring for infection or dehiscence. Moist chow was provided on the cage floor during the first 48 h. Body weight was measured weekly. Humane endpoints for early euthanasia were strictly defined as: weight loss ≥20%, BCS ≤2.0, persistent inability to reach food/water > 12 h, signs of severe pain/dyspnea, or severe surgical site complications (e.g., necrosis). Animals meeting these criteria were humanely euthanized within 24 h. Postoperative care included the administration of cefalexin (15 mg/kg) intramuscularly immediately after surgery and at 24 h postoperatively to prevent infection. For analgesia, carprofen (2 mg/kg) was administered subcutaneously immediately after surgery and at 12, 24, and 48 h postoperatively, for a total of four doses. At 12 weeks post-implantation, the rat was euthanized via an isoflurane overdose. The segment from the second to the third caudal vertebra was then harvested en bloc and fixed in 70% ethanol.
Training recommendations, troubleshooting, and sample size. To facilitate the successful adoption of this demanding protocol, new operators must perform 3–5 practice procedures on rat cadavers, achieving proficiency when three consecutive placements show no radiographic malpositioning. Common troubleshooting includes pre-measuring disc height via X-ray to optimize cage size selection and re-verifying dorsal bone flatness to prevent cage migration. If the screw purchase is insufficient, utilizing lower drill speeds is recommended. For future validation studies, a minimum sample size of n = 6 to 10 per group is recommended (with an additional 10–20% for attrition) to achieve sufficient statistical power. Furthermore, future studies must incorporate control groups (e.g., empty cage or autograft) to validate the biomaterial’s performance.
Radiographic and histological analysis
Micro-computed tomography (CT) scanning and digital radiography were performed using a micro-CT scanning system with a tube voltage of 80 kV and a current of 0.1 mA. A 1.0 mm-thick aluminum filter was utilized during acquisition to minimize beam-hardening artifacts caused by the titanium implant. For longitudinal assessment, in vivo imaging was conducted at 1 and 4 weeks postoperatively with a 120 µm voxel size. Ex vivo imaging was conducted at 12 weeks postoperatively using a 24 mm specimen holder with a 24 µm voxel size. CT data and radiographic images were reconstructed using a standard filtered back-projection algorithm and analyzed using a DICOM viewer. Quantitative criteria were defined to assess bone union. Radiologically, successful fusion was confirmed when a continuous trabecular structure bridging the cranial and caudal vertebral endplates was observed across at least three consecutive slices on micro-CT. A global threshold of 700 Hounsfield Units (HU) was applied during image segmentation. Histomorphometrically, a BV/TV ratio of 70% or greater within the cage was considered the threshold for physical spatial fulfillment and fusion. Histologically, bone union was defined by complete osseous bridging from the cranial to caudal sides without the interposition of fibrous tissue. For histological analysis, sequential fluorochrome labeling was performed by subcutaneous injection of calcein (8 mg/kg) 2 days before euthanasia to identify the spatial distribution of active mineralization at the terminal 12-week time point. The excised caudal vertebrae were fixed in 70% ethanol for 7 days and stained en bloc with Villanueva bone stain. Specimens were dehydrated through ascending grades of ethanol, embedded in methyl methacrylate resin, and mid-sagittally sectioned at the precise geometric center of the cage, as determined by pre-sectioning Micro-CT guidance, using a diamond saw microtome. Undecalcified ground specimens (5 µm thickness) were prepared at the Ito Bone Histomorphometry Institute (Niigata, Japan). Images were acquired using a bright-field microscope and a confocal laser-scanning microscope for fluorescence merging. Further examination was performed under a polarized light microscope, and histomorphometric analysis was conducted using a computerized image analyzer. The Region of Interest (ROI) was defined as the total internal volume of the cage, excluding the metal struts, and was subdivided into Cranial and Caudal Intracage ROIs. Bone tissue was identified by its purple/magenta color under Villanueva staining. Lamellar bone (Lm.V) was defined by its highly organized, parallel-lamellar structure under polarized light, whereas woven bone (Wo.V) displayed disorganized fiber patterns.
