Intervertebral fusion models are predominantly performed on the cervical and lumbar spine in large animals, but in the rat interbody fusion model, an intertransverse process fusion model is more popular than the coccygeal interbody fusion model[19–22]. Xi Liang et al. used a goat cervical fusion model and found that the porous n-HA/PA66 strut offered the potential for cervical reconstruction after corpectomy[19]. Lovorka Grgurevic et al. constructed a sheep spine fusion model via anterior lumbar interbody fusion (ALIF) and posterolateral lumbar fusion (PLF)[20]. Jason R. Kang et al. used a rat intertransverse process fusion model to prove that varenicline had no adverse effect on spinal fusion[21]. Yu Cheng Yeh et al. characterized a coccygeal interbody fusion model and suggested it is an efficient model for future material and mechanical testing[22]. For research using animal models, larger animal models incur higher experimental costs. However, the existing rat model cannot fully simulate clinical surgery for interbody fusion. Therefore, we constructed a novel rat intervertebral fusion model based on anterior lumbar corpectomy and fusion. In this study, we developed an internal fixation for rat spinal fusion on account of directly measured L5 geometry. Bony fusion was observed at both four and eight weeks post-surgery, with improved fusion rates at eight weeks.
To our knowledge, there is no rat spinal fusion model constructed by anterior lumbar interbody fusion (ALIF). The surgical method is critical to this study and has a theoretical foundation in the research of Marc Antoine Rousseau et al., who reported the ventral approach to the lumbar spine of SD Rats [17]. The main points of this technique include: 1) The intestines and organs should be carefully protected during exposure by covering with wet gauze until fixation is complete; 2) The surgeon should be familiar with the anatomical structure of SD rats, particularly anatomical markers such as the posterior vena cava and ilio-lumbar vessels, which are key to locating the ventral aspect of the spine; 3) Due to the neurovascular plexus at L4 and the shielding effect of the pelvis at L6, the L5 vertebrae was selected to undergo corpectomy. A metal marker should be used for locating the L5 vertebrae via X-ray before corpectomy; 4) When revealing the ventral aspect of L5, the vertebral vessel across the middle of the L5 body can be seen (Fig. 3b). Thermocoagulation of the vertebral vessel ensures a clear surgical field. 5) Stringent aseptic measures should be adopted during model construction. To keep the incision clean and closed, skin interrupted sutures should be performed more intensive and every rat should be isolated in individual cages.
Micro-CT is an emerging non-destructive imaging method that has become an essential assessment tool in animal spinal fusion research[23]. Spatial changes in bony architecture can be observed, and the amount of newly formed bone tissue can be calculated by Micro-CT, which enables comparisons between different interventions. Micro-CT has detected increases in bone mineral density (BMD), percent bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and decreases in trabecular separation (Tb.Sp) in a series of studies promoting spinal fusion [24–27]. In this study, BV/TV and Tb.N were greater in group B compared to group A, which is in line with previous research[24]. However, Tb.Th was lower in group B. This observation might be related to the early shaping processes of regenerated osseous tissue, which still needs further study. According to the Micro-CT images, we found that bone imaging within the titanium cage was more obvious in group B and that the forming trabecular architecture at the interfaces in group B (Fig. 5a). Overall, this study revealed that intervertebral fusion became more apparent as time progressed.
Histological examination is considered the gold standard method for assessing bone microstructure[28]. In this process, a section of bone tissue is stained with hematoxylin and eosin (H&E), Masson's trichrome (MT), and methylene blue-acid fuchsin (MB/AF). Staining with H&E can easily identify calcium deposits, but cannot clearly differentiate osteoid and osseous tissues[29]. As a special staining technique, MT stain has been routinely used, although scanty bone tissue might be lost in the background of collagen[30]. It has been reported that MB/AF stain is a better way to differentiate bone and stromal tissues[31]. In this study, bone sections were stained with MB/AF. Four weeks post-surgery, the contents in the titanium cage was predominantly fibrous connective tissue. Newly formed bone tissue could be seen at both rostral and caudal ends of the cage, accompanied by a small cartilage formation. Eight weeks post-surgery, there was a mass of newly formed bone tissue in the titanium cage with less cartilage and fibrous connective tissue compared to the four-week post-surgery images. Quantitative analysis suggested the area ratio of newly formed bone of group B (eight weeks) was significantly greater than in group A (four weeks). As such, the histological results confirmed that intervertebral fusion occurred in this animal model.
As trabecular bone grows along the direction of mechanical loading[32], a limitation of this study is the different spinal mechanics between rats and humans. Rats are quadrupedal animals with the spine parallel to the ground, while humans are bipedal with the spine perpendicular to the ground. Therefore, the human spinal mechanical environment cannot be directly simulated in the rat model. Future research could explore the effect of placing water and food at height to induce the rats to rise on their hind legs to obtain a similar mechanical stimulus.