Development of a Rat Caudal Interbody Fusion Model: A Pilot Feasibility Study

Summary

This study demonstrated the technical feasibility of a pilot rat caudal interbody fusion model using posterior cage and plate fixation. Histomorphometric analysis at 12 weeks confirmed continuous bone bridging and distinct intra-cage bone maturation patterns. This technical model serves as a preliminary foundation for future large-scale studies.

Abstract

Rat caudal vertebrae are widely used in bone regeneration and spinal fusion research as convenient sites for testing bone graft materials and implants. Prior rat tail fusion models have achieved intervertebral fusion through disc removal and bone grafting, or with external fixation devices. Nonetheless, no such small-animal model has been developed that uses an interbody fusion cage analogous to that used clinically. It was hypothesized that an evaluable bone fusion environment could be established without neurological complications by performing a posterior cage insertion mimicking clinical procedures, supplemented with plate fixation, in the rat caudal vertebrae. This preliminary pilot study (n = 1) was designed to evaluate technical feasibility. A surgical approach was developed in which a miniaturized titanium cage was precisely inserted into the resected disc space between the adjacent caudal vertebrae, closely mirroring the human surgical procedure. Titanium was selected for the cage owing to its widespread clinical use and biocompatibility. The initial results demonstrated the technical feasibility of this approach. The implanted cage maintained disc height and spinal alignment. Progressive bone ingrowth through the cage was observed, culminating in confirmed continuous bone bridging across the intervertebral space by 12 weeks postoperatively. The presence of continuous trabecular structure and the absence of intervening fibrous tissue were confirmed radiologically and histologically. Overall, this cage-based rat tail fusion model provides a promising platform for evaluating novel biomaterials and investigating mechanisms of intervertebral bone fusion. However, the lack of a control group and statistical analysis, due to the single-subject design, limits the generalizability of these findings and necessitates further validation.

Introduction

Interbody fusion is an established surgical treatment for various spinal disorders, including instability, spondylolisthesis, and degenerative diseases; notably, it has evolved over approximately 70 years as a standard procedure^1,2. The procedure involves removing the intervertebral disc and inserting a cage or bone graft to promote solid fusion between adjacent vertebrae^3,4. Moreover, adding posterior instrumentation with cages reduces pseudarthrosis rates and expands its clinical indications⁵. Nevertheless, complications such as pseudarthrosis and cage subsidence remain significant clinical challenges, warranting reliable preclinical model development to evaluate new materials and fusion-promoting factors^6,7.

Rats have been widely used in spinal fusion research owing to their economic feasibility, rapid reproduction, rapid healing, and handling ease^8,9. Nonetheless, the existing rat interbody fusion models exhibit significant limitations. Specifically, the anterior lumbar interbody fusion (ALIF) approach in rats requires major abdominal vessel and organ manipulation, resulting in high surgical invasiveness and substantial complication risks^10,11. The posterior lumbar interbody fusion (PLIF) approach involves an unavoidable risk of neurological injury due to the extremely small spinal canals and neural elements, making neural retraction nearly impossible¹². Consequently, most rat studies have been limited to posterolateral fusion (PLF), which structurally fails to investigate the mechanisms of bone fusion within the cage, a critical aspect in clinical settings¹³. Furthermore, while caudal models exist, they lack an evaluation system that integrates a clinical-like interbody cage with posterior plate fixation.

The caudal spine of rats offers unique interbody fusion research advantages. Caudal discs demonstrate strong geometric, compositional, and mechanical similarities to the human lumbar discs, making them increasingly popular in disc research^14,15. Additionally, caudal spine surgery provides easy surgical access, cost-effectiveness, and rapid postoperative recovery compared with lumbar approaches¹⁶. However, to date, no studies have reported successful posterior cage insertion in the rat caudal spine with a detailed histological evaluation of bone formation within and around the cage.

Histological assessment, particularly of undecalcified bone histomorphometry, remains the gold standard for evaluating osseointegration and bony fusion^17,18. Specifically, it enables quantitative analysis of critical bone formation parameters, including bone volume/tissue volume (BV/TV), trabecular number, and trabecular thickness, while allowing bone maturity qualitative assessment by distinguishing woven from lamellar bone^19,20. Such a detailed histomorphometric evaluation can reveal the spatial and temporal osteoconduction patterns within the cage and at the cage-vertebra interfaces^21,22. Nonetheless, a comprehensive histomorphometric evaluation of cage osteoconduction in rat models is lacking.

This study aimed to develop a pilot rat caudal interbody fusion model using posterior cage insertion and supplemental plate fixation to demonstrate its technical feasibility. This study presents a detailed surgical technique and a comprehensive histological evaluation of bone formation within the cage and at the vertebral endplate interfaces. It was hypothesized that an evaluable bone fusion environment could be established without neurological complications by performing a posterior cage insertion mimicking clinical procedures, supplemented with plate fixation, in the rat caudal vertebrae. This model provides an efficient platform for evaluating interbody fusion materials and, in the existing literature, represents an initial technical exploration of successful posterior cage placement using histomorphometric osteoconduction assessment in a rat model.

Protocol

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. The titanium cage was sterilized by gamma irradiation at 25 kGy prior to implantation.

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. Ophthalmic ointment was applied to both eyes to prevent corneal desiccation, and the animal was placed on a thermostatically controlled heating pad maintaining body temperature at approximately 37 °C throughout the procedure. 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). The fur over the tail base and caudal dorsum was clipped over an area extending at least 150% of the planned incision length (approximately 1.3 cm proximal to the tail base, encompassing the entire area to be exposed by the drape), and loose hair was removed. The clipped area and the entire tail were then 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. Lidocaine (2 mg/kg, 1%) was infiltrated subcutaneously along the planned incision site as a skin line block, and 5 min were allowed to elapse before incision. 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

A self-retaining retractor was placed to displace the skin and soft tissues away from the site to be flattened, ensuring direct visualization of the bone surface and preventing nonspecific soft-tissue injury from the rotating burr. 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 preloaded into the holes in 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). Before closure, loose and ragged tissue ends created during blunt dissection were sharply excised and removed from the surgical site to promote clean wound healing. 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 (5 mg/kg) was administered subcutaneously immediately after surgery and at 12, 24, and 48 h postoperatively, for a total of four doses. Skin sutures were removed 10–14 days postoperatively. 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. For each in vivo imaging session, the animals were anesthetized with the same three-drug combination and doses of medetomidine, midazolam, and butorphanol used for surgery. After scanning, anesthesia was reversed with atipamezole (0.75 mg/kg, subcutaneously). 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.

Results

Body weight (Table 1)

Body weight was measured up to 12 weeks postoperatively, demonstrating a steady increase (from 221.8 g to 346.8 g), indicating physiological homeostasis and indirectly supporting the absence of severe chronic distress. This was corroborated by daily multimodal assessments confirming normal activity and uncomplicated wound healing.

Wound (Figure 3)

Discussion

Here, a novel interbody fusion model of the rat caudal spine was established. This model involves inserting a titanium cage via a posterior approach augmented with plate-and-screw fixation. Subsequently, intracage bone formation was evaluated using detailed histomorphometric analysis. The distinct features of this model are threefold: 1) it closely mimics clinical posterior lumbar interbody fusion or transforaminal lumbar interbody fusion procedures by combining an interbody cage with posterior instrumentation; 2) it uti...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-0 nylon sutures	Natsume	ER-25-30N2
Bright-field microscope	Keyence. (Osaka, Japan)	BZ-X800
Butorphanol	FUJIFILM	021-19001
Calcein	FUJIFILM	76082
Carprofen	FUJIFILM	037-19761
Cefalexin	FUJIFILM	036-11051
Computerized image analyzer	System Supply Co. (Nagano, Japan)	Histometry RT CSS-840
Confocal laser-scanning microscope for fluorescence merging	Carl Zeiss. (Jena, Germany)	LSM 980
Curette	Natsume	E-8-00
Diamond saw microtome	Leica. (Wetzlar, Germany)	SP1600
Electric drill	TAMIYA	N/A	https://www.amazon.co.jp /%E3%82%BF%E3%83% 9F%E3%83%A4-%E3% 82%AF%E3%83%A9% E3%83%95%E3%83% 88%E3%83%84%E3% 83%BC%E3%83%AB% E3%82%B7%E3%83% AA%E3%83%BC%E3% 82%BA-No-42-%E9%9B %BB%E5%8B%95%E3% 83%8F%E3%83%B3%E3 %83%87%E3%82%A3% E3%83%AA%E3%83%A5 %E3%83%BC%E3%82% BF%E3%83%BC-%E3% 83%97%E3%83%A9%E3 %83%A2%E3%83%87%E3 %83%AB%E7%94%A8%E5 %B7%A5%E5%85%B7/dp/B 01LZ05WWY/ref=sr_1_2?ad grpid=54785347873&dib=eyJ 2IjoiMSJ9.f61YHQv80N0nxws VOdlrIFtTsQoJICvSb4t4rVt97 FEBWkOo4ilX5O2lZH-5vADU HT9CRXO308QpVFqdBNli0C osd4iBSfSzEcKxP9HMX4DhiI S-Qko5ix33W7FjPQqpgTWbj3 CV_vBytnvfqvSyoGN9IB8445o 5Kc0pjeKPG-SRR3Ixpqr2-FT_ ZgUyWstoyQVFTcv_Rq_K4-E OWnuT41BBYtBh757EOAoBe z2lkCd6Psz99wZxfUuvzRUAH Ecq9QV6EmiT9LAY8dYYq08k aLt7Klqx2k-MdV8XbWRxgKk. 7TWzXBGA1PeNmB4me_Cz4 mMFAKBAv5H3YXAw_HPuLd E&dib_tag=se&hvadid=651687 588880&hvdev=c&hvexpln=0& hvlocphy=1009309&hvnetw=g& hvocijid=5538300633798619650 --&hvqmt=e&hvrand=55383006 33798619650&hvtargid=kwd-33 4267191534&hydadcr=4289_13 319726&jp-ad-ap=0&keywords= %E3%82%BF%E3%83%9F%E3 %83%A4+%E9%9B%BB%E5% 8B%95%E3%83%8F%E3%83% B3%E3%83%87%E3%82%A3% E3%83%89%E3%83%AA%E3% 83%AB&mcid=6b8ff8c984ba3d45 be477b407ef2d1ba&qid=1767395 580&sr=8-2
hand drill	VEAGIA	N/A	https://www.amazon.co.jp/dp/B0D2 9B4W18/ref=sspa_dk_detail_5?pd _rd_i=B0D29B4W18&pd_rd_w=xe kNz&content-id=amzn1.sym.f293b e60-50b7-49bc-95e8-931faf86ed1e &pf_rd_p=f293be60-50b7-49bc-95e8 -931faf86ed1e&pf_rd_r=HPW6B367R QC0VPB7WTTS&pd_rd_wg=E8mAS &pd_rd_r=dbeb0aa7-1bea-4f40-99dd -38bcb05c8234&sp_csd=d2lkZ2V0T mFtZT1zcF9kZXRhaWw&th=1
Medetomidine	FUJIFILM	135-17473
Midazolam	FUJIFILM	135-13791
Pin vise	ANEX	N/A	https://www.amazon.co.jp/%E3%82% A2%E3%83%8D%E3%83%83%E3% 82%AF%E3%82%B9-ANEX-%E3% 83%A9%E3%83%90%E3%83%BC %E3%82%B0%E3%83%AA%E3% 83%83%E3%83%97-%E3%83%94 %E3%83%B3%E3%83%90%E3% 82%A4%E3%82%B9%E5%8F% 8E%E7%B4%8D%E5%BC%8F -No-98-R/dp/B000T4D0XQ/ ref=pd_bxgy_thbs_d_sccl_1/3 55-1353338-1511720?pd_rd_w =pnGb2&content-id=amzn1.sy m.fb3df7da-4601-4c73-b94b-e2 75cd0eb364&pf_rd_p=fb3df7da -4601-4c73-b94b-e275cd0eb364 &pf_rd_r=KBMW265N2ZK5TTH 76ZW7&pd_rd_wg=ApIxl&pd_rd_ r=e53648bf-e253-43a7-add1-966f f5070c9a&pd_rd_i=B000T4D0XQ &th=1
Plastic internal fixation material	Matec Co. Ltd. (Hiroshima, Japan)	N/A	Polyoxymethylene, 3 mm wide × 18 mm long × 1 mm high, custom-fabricated
Polarized light microscope	Olympus. (Tokyo, Japan)	BX-53
Small forceps	Natsume	A-31
Stainless steel screw	Matsumoto Sangyo Co., Ltd. (Chiba, Japan)	TD-00224	#0 special truss head [1805]+, 1.2 × 14, passivated
Titanium cage	Stryker. (MI, USA)	N/A	A Cascadia TL titanium cage (Stryker, MI, USA) was machined to a width of 2 mm, depth of 2 mm, and height of 2 mm by Fujiflex Co., Ltd. (Nara, Japan)
Wire cutter	Natsume	B-16