Ovine models of intervertebral disc degeneration

Introduction

Animal models have played an important role in the attempt to clarify how intervertebral disc degeneration (IVDD) evolves over time. They have also been vital in helping determine how structural, environmental, or biomechanical risk factors may initiate, promote, or otherwise regulate these degenerative changes (1). Attempting to investigate these degenerative changes has led to the development of a large number of animal models. In most studies these animal models have been either small or large animal. While mice, rats and rabbits represent common choices among small animal models, large animal models such as dogs, pigs, goats and sheep have frequently been employed (2). Nevertheless, given the complexity of human IVDD, the perfect animal model does not exist (3) (Table 1). Selection of an appropriate animal model depends on the research question, but also must take into consideration size, intervertebral disc (IVD) anatomy, cellularity characteristics, biomechanical forces, and ethical considerations (6).

When considering all of these factors, the ovine spine has emerged as a prominent model for studying IVDD. This is due to the sheep’s similar cellular, biomechanical, and anatomical similarities to the human spine and IVD (7-9). In addition, the ovine species aligns with humans relative to body weight, bone mineral composition, bone and disc metabolism and bone healing (10,11). Despite its quadrupedal posture, the ovine spine demonstrates notable biomechanical similarities to the human spine, particularly in intradiscal pressure patterns within the lumbar region during activity and rest (12,13). Additionally, like humans, the ovine IVD undergoes chondroid metaplasia with skeletal maturation, with the consequently differentiation/loss of the notochordal cell remnants (14). Beyond these physiological similarities, ethical considerations further endorse the use of ovine models over other models such as non-human primates or companion animals. In this literature review, we seek to describe the key features of IVDD shared between sheep and humans, elucidate current ovine models of IVDD, and underscore the relevance of these models in advancing our understanding of disease mechanisms, diagnosis, prognosis, prevention, and treatment of IVDD.

Advantages of the ovine model

Anatomical and physiological similarities

Sheep share key anatomical and physiological similarities with humans, including comparable body weight and bone mineral composition (10). Notably, adult sheep typically weigh between 60 and 100 kg, closely aligning with human body mass. This body size, combined with the presence of long bones of sufficient dimensions, makes sheep well-suited for the implantation of human-sized orthopedic spine implants and prostheses—a feature not achievable in smaller animal models (10,15). Furthermore, studies evaluating bone ingrowth into porous implants placed in the distal femur of sheep, a weight-bearing model, have demonstrated bone healing patterns remarkably similar to those observed in humans (16-18). These findings highlight the translational relevance of sheep, particularly given the anatomical parallels between ovine and human spines, and support their use as a valuable preclinical model for advancing research in spinal anatomy, biomechanics, and surgical interventions.

The ovine IVD closely resembles the structural and cellular phenotype of the human disc, making it an ideal model compared to other animal models. Macroscopically, the ovine IVD exhibits a structure like the human IVD, featuring an organized and well-defined annulus fibrosus (AF), a discernible transition zone between the AF and a clearer, gelatinous nucleus pulposus (NP) (Figure 1). The most pronounced parallels in the principal dimensions of the sheep and human spine are predominantly evident in the thoracic and lumbar regions (20). However, the sheep cervical spine has been deemed a suitable model for cervical spine research, particularly supported by quantitative data, with the motion segment C3–C4 demonstrating the most reliability as a model for its human counterpart (21).

Figure 1 Representative images illustrate comparative axial gross views of healthy lumbar intervertebral discs from human (left image), and ovine (right image) specimens, showcasing remarkable anatomical similarities. These visual comparisons accentuate shared structural features, enhancing our comprehension of the inter-species parallels in disc anatomy. The figure was reused from an open access article from Stolworthy et al. (19) under the terms of the Creative Commons Attribution 4.0 International License.

Despite its quadrupedal position, sheep experience mechanical stresses such range of motion, neutral zone, and stiffness parameters similar to those of the human spine (12). Biomechanical assessments and intradiscal pressure evaluation have demonstrated favorable comparability between human and ovine lumbar spines in terms of overall disc shape and most biomechanical parameters, including range of motion, neutral zone, and load distribution between the IVD (22). Similar hydration patterns between human and ovine IVD over time were observed, shedding light on the importance of disc hydration and its impact on stiffness under various loading conditions; findings reinforcing the utility of the ovine model for disc research—old sheep (23). In addition, when testing the biomechanical range of motion of sheep and human spines with implanted devices, the qualitative effects of the devices have been shown to be very similar (24). These biomechanical changes are also age-related comparable. For instance, research indicates that the intra-lamellar matrix within individual AF lamellae of sheep is weaker and more compliant in middle-aged and elderly ovine IVDs compared to young IVDs, mirroring observations in humans (22). Considering the relevance of the ovine model to understand spine conditions even cutting-edge technological advancements such as computational finite element models have been developed to validate both sheep cervical and lumbar spine models. These studies indicate that despite geometric variations, the human and ovine IVDs are functionally adapted to generate similar internal stresses (22,25).

Notochordal cells are specialized cells that form the notochord, a temporary rod-like structure present in the embryos of vertebrate animals (26). These cells are initially present in the NP, the inner core of the IVD (14); but beyond their developmental features, notochord cells are crucial for promoting extracellular matrix (ECM) synthesis and regulating cellular activity in the NP, thus playing a key part in maintaining healthy IVDs homeostasis (27,28). The persistent existence of notochordal cells is crucial, as they significantly influence processes into the IVD, including proteoglycan metabolism, hyaluronan production, and potential contributions to progenitor cell function (2). In some species, including humans, these notochordal cells in the IVDs undergo a process of cellular differentiation as part of the natural aging and development process. This results in the eventual replacement in the NP of the notochord cells by a chondrocyte phenotype cells (29-32). Studies have demonstrated that the loss of notochordal cells is a critical trigger in the onset of IVDD, underscoring their essential role in maintaining disc health (33,34). Specifically, evidence suggest that the reduction of these notochordal cells leads to an imbalance in homeostasis of the IVDs and the subsequent degradation of the ECM (35,36), both of which are critical factors contributing to IVDD (35,37). Therefore, whether notochordal cells are present or not in IVDs is an important factor to consider in the selection of the appropriate animal model (35,38).

Sheep are among the few large animal models that can differentiate their notochordal cells rapidly following birth (27,38). Similar to humans during adolescence, sheep undergo a differentiation process of their notochord cells linked to skeletal maturation (14,39). These similarities in the process of differentiation of notochordal cells in ovine models presents a clear advantage, closely mirroring clinical conditions observed in human patients with IVDD (14). This similarity enhances the relevance of the ovine model, particularly in evaluating potential cellular therapeutic interventions for disc-related conditions (Figure 2).

Figure 2 Representative images present comparative sagittal views of lumbar intervertebral discs from human (first row) and ovine (second row) specimens, showcasing notable anatomical similarities. Grades 1–5 have been assigned according to the Thompson grading scale for gross disc changes (40), and their translation to the ovine model. These visual comparisons emphasize shared structural features, enhancing our understanding of interspecies parallels in gross disc anatomy. The figure was reused from an open access article from Lee et al. (41) under the terms of the Creative Commons Attribution 4.0 International License.

Ethical considerations

Ovine models are generally more widely accepted from an ethical standpoint than non-human primate models such as baboons or macaques, which raise substantial ethical and practical concerns, including high costs and specialized housing requirements (42). The growing utilization of sheep as animal models for orthopedic research is partly due to ethical concerns and public apprehension regarding the use of companion animals for medical research. Furthermore, their availability as animals for consumption makes sheep less ethically objectionable compared to companion animals, contributing to their growing popularity in orthopedic research. Unlike companion animals or non-human primates, sheep have less emotional attachment from owners or caregivers, further facilitating their use as research subjects (10,22).

Pathological changes associated with IVDD in sheep

Following induced degeneration in sheep IVDs, several histological, biochemical, and biomechanical changes are observed (Figure 3). These changes closely mimic human disc degeneration, confirming the similarities between species and highlighting the sheep a valuable model for studying IVDD pathophysiology and evaluating potential treatments. Among the histological changes, the most relevant is the decreased cell density of NP, with an increased cell clustering, and presence of cell death (41). The NP also loses its gelatinous consistency and becomes more fibrous. For the AF, the clearest change is the disorganization of collagen fibers, presence of clefts, and fissures (43). Additionally, an increased vascular infiltration and inflammatory cell presence in the AF is observed. In the case of the endplates, an increased calcification and irregularities, with reduced cell viability has been shown (44).

Figure 3 Diagram illustrating the differences between healthy (“naïve”) ovine lumbar intervertebral discs (left) and those with physically induced degeneration (right). The top images depict the gross morphology of an intact disc compared to a disc 32 weeks post-partial annulotomy, showing evident structural deterioration. The bottom images present T2-weighted MRI scans of the same discs, where the healthy disc (left) exhibits high signal intensity in the nucleus pulposus, reflecting normal hydration, while the degenerated disc (right) shows a markedly reduced signal, consistent with loss of hydration and disc degeneration. MRI, magnetic resonance imaging.

Among the most common biochemical changes observed in IVDD in sheep are the alterations in proteoglycan, collagen, matrix metalloproteinases (MMPs), and inflammatory mediators. A hallmark of disc degeneration is the significant reduction in proteoglycan content, particularly aggrecan, which compromises the disc’s ability to retain water, leading to dehydration and loss of disc height (45,46). Collagen composition also shifts, with an increase in type I collagen and a decrease in type II collagen within the NP, reflecting fibrotic transformation. Additionally, the AF exhibits altered collagen ratios, further compromising its structural integrity (47). Another change is the elevated levels of MMPs and other degradative enzymes, contributing to ECM breakdown (48). Similarly, increased production of pro-inflammatory cytokines such as IL-1β, TNF-α, and other inflammatory markers is noted (49,50). These biochemical signatures not only drive the degenerative cascade but also reinforce the value of the ovine model in mimicking key molecular features of human disc degeneration for translational research.

IVDD in sheep models exhibits biomechanical changes that closely parallel those observed in humans, reinforcing the translational relevance of ovine studies. A primary alteration is the significant reduction in proteoglycan content, particularly aggrecan, leading to decreased water retention within the NP. This dehydration compromises the disc’s hydrostatic pressure, resulting in diminished disc height and turgor. Consequently, the disc’s stiffness increases while its elasticity decreases, impairing its capacity to absorb and distribute mechanical loads effectively (12). These changes alter load transmission across the disc and adjacent vertebrae, potentially escalating stress on surrounding spinal structures and contributing to further degenerative processes.

Methods for inducing IVDD in ovine models

Numerous methods have been devised to assess and induce IVDD in sheep, positioned as a one of the most common large animal models to gauge clinical conditions, potential targets, and therapeutic approaches. It is crucial to emphasize that while these methods aim to mimic human IVDD by instigating a degenerative sequence through acute injuries, none of these approaches can perfectly replicate a condition identical to human disorders. These in vivo approaches can be broadly categorized into three groups: spontaneous, damage-induced, and mechanical models. Additionally, in vitro methods have proven valuable in advancing our understanding of the pathophysiology of IVDD. Each model contributes uniquely to our understanding of IVDD in sheep and other large animal models, offering valuable insights for translational research and therapeutic advancements in the context of human degenerative disc diseases.

Spontaneous models

Aging is considered the most relevant factor associated with the occurrence of IVDD in humans, and this is not different in the ovine specie (Figure 4) (52). Spontaneous age-related changes indicative of IVDD has been observed in sheep as early as 2 years of age through various imaging modalities such as X-ray, computed tomography (CT), and magnetic resonance imaging (MRI) (53). The alignment of these imaging findings with histologic evaluations further confirm that skeletally mature sheep exhibit signs of IVDD compared to their younger counterparts (54). Although imaging and histological studies demonstrate the presence of age-related degenerative changes, there are no reports describing associated clinical musculoskeletal or neurological signs in sheep. A rare case report described IVDD with disc prolapse and Schmorl’s node formation associated with clinical signs such as stiffness and reluctance to move (55); however, such cases are exceptional and do not establish a consistent pattern of symptomatic disc degeneration in this species. Sheep rarely develop clinically symptomatic IVD disease under normal husbandry conditions, likely because they do not live long enough for advanced degeneration to become clinically evident (56). Other studies have similarly revealed that measurements of bone volume, osteoid volume, and mineral apposition rate in older sheep are comparable to those observed in men and post-menopausal women in their 6–7th decade of life (57). This parallel, together with the fact that most animal models require experimentally or injury-induced trauma to initiate IVDD, underscores the value of sheep as a model in which spontaneous, age-related degeneration closely mirrors the natural progression of human IVDD, without the need for additional intervention or trauma.

Figure 4 Cross-sections of a human (left) and ovine (right) intervertebral disc, both exhibiting degenerative changes. The human disc shows a dehydrated, discolored nucleus pulposus (arrow) surrounded by intact annulus fibrosus rings. The ovine disc demonstrates similar spontaneous, age-related degeneration from a 9-year-old (1). Attempting to investigate these degenerative changes has led to the development of a large number of animal models. In most studies these animal models have been either small or large old sheep. This comparison underscores the morphological parallels in disc degeneration between the two species. Ovine disc is an original image from the authors. Human disc image modified and used with permission of Ruel et al. (51).

Damage-induced models

Needle puncture

The needle puncture model involves puncturing the AF, the outer structure of the IVDs, using various types and sizes of needles. While this model is predominantly used in small animal models, it has also been established in large animal models, including sheep (58). Induction of IVDD by needle puncture model is relatively straightforward, entailing the insertion of a needle into the AF, with or without disrupting the NP, leading to depressurization and/or AF damage, depending primarily on the needle size (59). Studies have shown that when sheep discs are punctured with a 27-gauge needle, which accounts for approximately 10% of the disc height in the ovine lumbar disc, no significant resultant differences in axial properties compared to intact discs, except for a roughly 15% change in the torque range, occurred (60). Researchers have observed that NP migration can occur through an 18-gauge needle puncture, suggesting that the likelihood of acute herniation increases with the needle diameter (61). However, other studies have reported that needle puncture, regardless of needle size, has minimal or no significant impact on inducing IVDD (62).

The insertion depth of the needle can be precisely determined through radiographic imaging or by observing the length of the emerged needle. This method, typically approached surgically, is commonly attempted through a lateral or postero-lateral approach under fluoroscopic guidance to ensure accurate needle placement within the IVD space (63,64). CT-guide needle puncture has been demonstrated in sheep cadavers as a straightforward procedure, allowing for precise positioning of the needle into the lumbo-sacral disc (65,66). While this approach offers valuable training opportunities for less experienced surgeons or radiologists performing IVD injections, it is associated with increased radiation exposure. Based on our experience from numerous preclinical studies, a primary limitation of needle puncture models is that the NP does not necessarily herniate following AF disruption, due to the viscous consistency of the NP and the AF’s rapid healing response. In fact, we have consistently observed that a complete window opening of the AF is often necessary to effectively displace the NP. Nevertheless, the continued development of less invasive techniques aims to minimize surgical trauma, reduce healing time, and limit the inflammatory response induced by both the procedure and the disc injury itself (67). Overall, optimizing these approaches is crucial to improving the reproducibility and translational relevance of disc degeneration models.

Induced chemical

Chemonucleolysis, involving the chemical digestion of the NP, originally served as a non-surgical treatment to digest the herniated NP tissue (68). At the present time this method is being used to induce IVDD in several animal models such as rabbits, sheep and goats (69). Numerous chemical agents have been investigated as potential initiators of the pathophysiological progression of IVDD, with some of the most commonly studied agents including chymopapain, chondroitinase ABC (C-ABC), and 5-bromodeoxyuridine (70-72).

The use of chymopapain, was first reported clinically as a treatment for disc protrusion (70). This proteolytic enzyme derived from the papaya latex, digests disc glycosaminoglycan chains within the disc and therefore induced IVDD through the loss of disc height and alteration of its biomechanical properties (38). This enzyme degrades IVD’s proteoglycans in a manner that is dependent on the dosage, with higher doses even inducing destruction of the AF in small animal models (73,74). In sheep, the first reported use dates back to 1986, when it was shown that chymopapain had a bactericidal effect (75). Subsequent studies provided further evidence regarding the ease of removing the NP following the administration of chymopapain to ovine IVDs (76).

Chondroitinase ABC is a different enzyme that digests chondroitin sulfate isomers. Injecting C-ABC induces degradation of the polysaccharide chains within the proteoglycan, causing disc height loss and alteration of biomechanical stability (69). Similar to chymopapain, studies using C-ABC have demonstrated a dose-related reduction in intradiscal pressure when C-ABC is administered (72,77). Another agent used to induce chemonucleolysis is 5-bromodeoxyuridine (BrdU). BrdU is known for consistently triggering genome instability, leading to a senescence-like phenomenon in mammalian cells, irrespective of cell types or species (71). It is believed that employing an animal model that integrates age-related alterations in disc cells presents advantages compared to more invasive degenerative models that directly damage the matrix of disc tissue. In sheep, injection of BrdU leads to the loss of T2-weighted signal intensity on MRI, decrease in disc height, and loss of the normal architecture and cell density after 14 weeks (78).

As mentioned earlier, the injection of these enzymes results mostly in the loss of proteoglycan, a crucial element in the clinically observed pathophysiological process of IVDD (79). However, the use of chemical agents to induce IVDD in animal models remains controversial, as the mere insertion of the needle, required to deliver the agent may itself cause degeneration through mechanical puncture. It has been demonstrated that techniques employing even a small-gauge needle to puncture the IVD led to an expedited progression of IVDD, herniation, diminished disc height and signal, along with the emergence of reactive endplate changes (61). Therefore, prudent evaluation of the risks and benefits is essential when considering procedures involving any type of disc injection.

Induced physical

The deliberate surgical injury to the IVD is a well-established approach for inducing IVDD. This injury can be directed towards the endplate, the AF, or the AF and NP.

Endplate damage

Studies have shown that the vertebral endplate serves as the primary pathway for intravascular solute transport into the NP of IVDs (80). Therefore, impeding endplate perfusion can result in restricted solute transport into the intranuclear tissue of the IVD and lead to IVDD. After nucleotomy through the transpedicular approach across the endplates, it is observed that mechanical nucleotomy facilitates the formation of a cavity within IVD, preserving the integrity of the AF (81). This preservation has allowed the injection of different volumes of hydrogels, scaffolds, and different tissue engineering constructs, facilitating preclinical testing in the ovine model (58,81). However, in addition to inducing damage to the endplates, this transpedicular approach can lead to neurological impairment and leakage of injected materials into the systemic circulation (82). Consideration of these adverse effects is imperative before proceeding with this surgical approach.

Annular defect/annulotomy

Annular lesions have been demonstrated to cause immediate changes to the mechanics of the ovine IVD (83). Among the most common type of lesions are the needle puncture, stabs or slid incisions, (also known as cruciate and box/window defects). These type of lesions to AF have been compared in mature sheep showing the in vivo and ex vivo advantages and limitations for each method (43,83). For instance, cruciate-style AF defects with removal of NP material have been used to evaluated whether experimental, injectable, and bioactive biomaterials (84). Induced damage to the AF in ovine discs has also been produced by creating concentric tears using injection of saline solution into the AF. These concentric tears of the AF induce mechanical changes by decreasing the stiffness of the IVD (85). Interestingly, studies inducing annulotomy have shown that while the outer AF displayed some healing capacity, the initial defect caused deformation and bulging of collagen bundles, ultimately resulting in tear extension towards the inner AF and complete failure (71). In our experience, AF defects trend towards natural healing and then a longer inflammatory process causes a fibrotic scar that could lead to bone formation causing spondylosis of the disc level.

Overall, these defects induced to the exterior lateral layers of the IVD aim to recreate a model of disc herniation. However, one important consideration is that even aggressive removal of AF does not result in disc herniation (43). According to the definition of disc herniation, a displacement of at least 25% of the disc circumference is required to classify it as a herniation (86). Unfortunately, this level of NP displacement has not been demonstrated in sheep. Therefore, careful consideration needs to be addressed in the selection of the sheep as a IVD herniation model.

Nucleotomy

Nucleotomy, whether partial or total depending on the research question or therapeutic intervention being tested, involves the surgical removal or partial excision of the NP (87). In the ovine model, a combination of methods, such as the transpedicular approach and chemonucleolysis, is often utilized to achieve complete nucleotomy (88,89). By creating a focal defect within the IVD, nucleotomy disrupts the structural integrity, resulting in altered biomechanics, tissue remodeling, and degenerative changes reminiscent of human IVDD. Studies employing nucleotomy in ovine models have yielded valuable insights into the pathophysiology of disc degeneration, shedding light on the role of mechanical loading, biochemical changes, and inflammatory responses in disease progression. Notably, the establishment of a model with an intact AF has revealed that mechanical nucleotomy results in a more reproducible and less destructive cavity within the NP (81). This controlled defect facilitates the reliable injection of biomaterials such as hydrogels or tissue engineering constructs, making it an advantageous platform for testing regenerative therapies (89). Overall, nucleotomy-based ovine models represent a robust and translationally relevant approach for studying disc degeneration and evaluating novel therapeutic interventions.

Mechanical models

Mechanical alteration of spinal kinematics is a well-established approach for inducing IVDD in sheep models. The most commonly employed method involves the surgical stabilization of spinal motion segments to disrupt normal biomechanical loading (90,91). Alternatively, controlled spinal compression has also been used, leading to a decrease in IVD height and range of motion (92). Another approach involves mechanical destabilization through AF incision, which produces characteristic degenerative changes, including a reduction in disc height, an increase in the neutral zone during biomechanical testing, and significant depletion of proteoglycans and collagen. This method is further associated with molecular changes, including upregulation of collagen types I and II, aggrecan, versican, perlecan, MMPs (MMP-1 and MMP-13), and ADAMTS-5, mirroring key aspects of human IVDD (48).

Mechanical immobilization of the ovine lumbar spine can be achieved through pedicle screw and rod implantation while preserving the integrity of the AF and endplates. Studies report a significant reduction in disc height after both 6 and 26 weeks of immobilization, with marked degenerative changes observed compared to non-immobilized controls. Notably, the absence of a significant difference between the 6- and 26-week timepoints suggests that the degeneration model develops reliably within just 6 weeks, although progressive degeneration of adjacent facet joints continues over time (48). Collectively, these kinematic alteration models offer reliable and reproducible methods for inducing IVDD in sheep, providing valuable platforms for studying IVDD pathophysiology and evaluating therapeutic interventions.

In vitro models

The organ culture system for ovine IVDs, including the vertebral endplates, has been well established and provides a valuable model for investigating the effects of nutrition and mechanical loading on intact disc explants (93). This culture system has been shown to maintain disc cells in their native three-dimensional environment under uniaxial diurnal loading for up to seven days (93). Studies have demonstrated that during this period, cell viability and glycosaminoglycan synthesis rates remain unchanged. However, the expression of catabolic genes is significantly upregulated, while the expression of anabolic genes tends to be downregulated (93). Another study showed in vitro the regulation of MMP-2 in ovine NP cells. These results also indicated that transforming growth factor-beta 1 (TGF-beta 1) and insulin-like growth factor-1 (IGF-I) decreased active MMP-2 levels. These findings shed light on the poor healing potential of dense, avascular tissues like the IVD (94).

IVD degeneration under various loading conditions have been extensively used in vitro with the ovine IVDs to explore the mechanisms of structural failure. For instance, an in vitro evaluation of healthy mature ovine lumbar motion segments subjected to flexion and vibration loading (1,300±500 N) was conducted to simulate moderately severe physiological exposure. Microstructural analysis of the damaged discs revealed delamination and disruption of the inner and mid-AF layers, along with limited diffuse tracking of NP material (95).

Further in vitro studies using ovine lumbar spinal segments in an IVD loading simulator have investigated the impact of five different loading combinations on disc integrity. These studies demonstrated that specific loading patterns can lead to failures at the endplate junction and AF failures. Notably, similar to what was mentioned in in vivo models, herniation was not observed in any of the segments subjected to these loading combinations (96). In parallel with mechanical loading investigations, MRI has been employed to assess relaxation times and provide detailed anatomical and functional information about the ovine lumbar IVD under uniaxial compression. This advanced imaging modality offers comprehensive insights into disc structure and function under mechanical load, contributing significantly to our understanding of disc biomechanics (97). Similarly, MRI T2 relaxation time analysis has been utilized to evaluate the effects of advanced glycation end-products (AGEs) on IVD hydration. AGEs, which accumulate in the disc with aging and degeneration, were shown to reduce water content in a dose-dependent manner without significantly affecting proteoglycan and collagen composition (98,99). Furthermore, an in vitro study demonstrated that ovine IVDs can be maintained in culture for up to 21 days under simulated physiological loading, both under normal and restricted nutritional conditions. However, when nutrition was limited, cell viability rapidly declined to 50–60% within days and remained at that level throughout the three-week period (100). Together, these in vitro studies highlight the ovine IVD’s ability to replicate key mechanical and biochemical responses observed in human discs, reinforcing its relevance as a translational model for investigating the pathophysiology of disc degeneration and testing novel therapeutic strategies.

Limitations of the ovine model

A recent scientific consensus concluded that all models appeared to lack some aspect of translatability and therefore the justification for careful selection of one model over another (3). The fact that many models exist for studying IVDD further supports the fact that no single model is better than another at this time. Therefore, careful selection of a particular model should be considered based on the research question to be solved (101). In that sense, sheep may not be the most appropriate model for evaluating factors such as IVDD induced behavioral or pain responses. Although is possible to monitor neurological changes in dorsal root ganglion (DRG) of animals with IVDD, sheep are very stoic animals who can hide any signs of pain (102). This makes difficult to make any type of clinical or even mechanical evaluation to determinate pain sensations.

In addition, when a large animal model may be desired, husbandry may be prohibitive, particularly when a large number of animals are required for a well powered sample study. Availability is also important, making small animals such as rodents an attractive option. This is due to a number of factors, such as ease of breeding and thus their improved attainability, study reproducibility, relatively quick maturation and aging and lower maintenance costs. Funding considerations can also influence model choice and is an important aspect when planning a study that features a preclinical animal model (103).

Ovine models of IVDD may be more acceptable to animal ethics committees compared to companion animals and are readily available, reasonably outbred, and less expensive to purchase than other large species. Additionally, sheep are easy to manage and handle during surgical procedures (104). Moreover, sheep do not require environmental enrichment as they thrive in their natural pastures. Clinically relevant technologies like MRI, CT, and positron emission tomography (PET) scans, as well as clinical equipment such as anesthesia, physiological monitoring, and surgical instrumentation, can be used with sheep. Disadvantages of the sheep model includes higher maintenance costs and the need for larger facilities.

Future directions of ovine models of IVDD

Recent advancements in the sheep models of disc degeneration hold promise for furthering our understanding of this complex condition and facilitating the development of novel therapeutic strategies. One promising area of research involves the application of genetic engineering technologies in sheep, allowing researchers to investigate specific genetic factors involved in disc formation and degeneration (105). By introducing targeted genetic modifications, such as gene knockouts or overexpression, researchers can elucidate the role of individual genes or pathways in the pathogenesis of disc degeneration, paving the way for targeted interventions. For example, employing genetic modifications using CRISPR/Cas9 technology (106), could enable the spontaneous simulation of disc degeneration by activating senescence pathways. Looking ahead, the introduction of inducible Cas9 in the IVDs of sheep may establish programmable translational models for testing therapeutic interventions.

Adjacent segment degeneration (ASD) following spinal fusion represents a major clinical challenge in human patients, characterized by accelerated degeneration, altered biomechanics, and pain in motion segments adjacent to the fused area (107-109). Given the anatomical and loading similarities between ovine and human spines, the ovine model is well suited to investigate ASD mechanisms and mitigation strategies. Studies in sheep have already shown that fusion combined with sagittal malalignment can alter biomechanics at neighboring levels and produce degenerative changes in adjacent facet joints, supporting the face validity of ASD-like pathology in this species (110). Established ovine posterolateral and interbody fusion models further enable controlled, longitudinal assessment of adjacent segment kinematics, histology, and molecular remodeling (111-113). Building on these foundations, future studies should pair instrumented fusion with longitudinal imaging, quantitative gait/behavioral readouts, and targeted molecular profiling to define the cascade of adjacent-level alterations and pain-related biomarkers, ultimately informing fusion strategies or motion-preserving technologies that reduce ASD risk in humans.

One of the major challenges with the ovine model of IVDD is establishing a reliable assessment of pain. The stoic nature of sheep makes it particularly difficult to critically asses their level of pain. However, recent efforts have focused on refining behavioral assessments of pain in sheep. The Ovine Grimace Scale (OGS), adapted from similar systems in other species, has shown potential for detecting pain based on subtle changes in facial expressions following a variety of orthopedic procedures (114,115). Preliminary work from our group is underway to develop gait analysis protocols in sheep, similar to those used in rodents, employing treadmills, Tekscan technology and force plates to quantify locomotor changes (116,117). In parallel, artificial intelligence tools are being applied to evaluate sheep behavior, including gait patterns and facial expressions, before and after spine surgical procedures with the goal of objectively identifying pain-related behaviors. Another promising avenue is the investigation of pain-related neuroanatomical structures, particularly the DRG. The DRG contains the sensory neurons responsible for transmitting pain signals to the central nervous system. Recent studies have functionally characterized the ovine DRG in the context of peripheral sensitization after osteochondral defects (102), suggesting its potential as a biomarker and mechanistic target for pain assessment in disc degeneration. Expanding this research into DRG responses associated with IVDD could offer critical insights into the neurobiological underpinnings of discogenic pain in sheep models.

While inflammatory and immune processes are well documented in human IVDD, large animal models such as sheep remain comparatively underexplored in this regard. In vitro studies using ovine NP cells have demonstrated that stimulation with IL-1β upregulates the expression of catabolic enzymes such as MMP-2 and MMP-3, indicating that disc cells in sheep are capable of mounting inflammatory responses similar to those observed in human degenerative discs (50). In vivo ovine disc injury models have also described vascular invasion and focal lymphocytic infiltration near AF defects, suggesting a partial loss of immune privilege during degeneration (43). However, comprehensive immune profiling, remains largely absent in ovine studies. Preliminary proteomic work from our group has evaluated molecular changes across different stages of disc degeneration in sheep, revealing patterns comparable to those described in human IVDD and identifying potential therapeutic targets associated with inflammation, matrix remodeling, and cellular stress responses (118). These findings reinforce the translational relevance of the ovine model and emphasize the need for further investigation into the immunological and molecular mechanisms underlying disc degeneration.

The refinement of surgical techniques and integration of advanced imaging technologies have greatly enhanced the development and translational relevance of ovine models for spinal research. The sheep spine’s anatomical dimensions and biomechanical characteristics make it particularly compatible with intraoperative imaging and computer-assisted surgical systems used in human spine surgery. These technologies, including intraoperative CT, fluoroscopy, and surgical navigation, enable real-time three-dimensional visualization and precise targeting of IVDs or vertebral structures (Figure 5). Their application reduces procedural variability, improves accuracy in creating standardized disc injuries or performing fusion procedures, and allows immediate verification of implant or instrument placement.

Figure 5 Application of intraoperative imaging and surgical navigation for spine surgery in an ovine model of intervertebral disc degeneration. (A) Intraoperative photograph demonstrating the use of an O-arm integrated with and Stealthstation S8 (Medtronic, USA) for real-time surgical navigation. The image shows the planned trajectory to access the T12 vertebra in a 6-month-old sheep using a minimally invasive approach. (B) Intraoperative CT image confirming accurate pedicle screw placement at T12 was achieved. CT, computed tomography.

The adoption of these approaches in ovine models mirrors current human surgical practices, thereby strengthening their clinical translatability. Importantly, minimally invasive and image-guided techniques reduce surgical trauma and recovery time, improving animal welfare and experimental consistency (119). Collectively, these advancements have positioned the ovine model as a critical platform for the preclinical evaluation of novel spinal surgical procedures, biomaterials, and regenerative therapies (119).

Conclusions

The ovine model for IVDD exhibits several favorable traits as an outstanding IVD model, including the absence of notochordal cells, comparable body mass to humans, and exposure to similar biomechanical forces affecting the IVD. A significant potential critique of this model relates to the quadrupedal nature of sheep, as previous biomechanical studies have indicated substantial comparability in many biomechanical properties between ovine and human lumbar spines, despite the quadrupedal/bipedal distinction (12).

When choosing a preclinical model for spine research, it is critical to consider that biologic and biomechanical components of the healthy IVD and IVDD are inextricably linked. Furthermore, it is important to acknowledge and address limitations of ovine models including subtle differences in biomechanics, genetics, physiology, and lifestyles. As such, comprehensive outcome assessments with correlations among metrics are important for validity and translatability. Taken together, preclinical studies using the spontaneous aging, induced in vivo, and in vitro ovine models of ovine models can guide targeted research toward developing valid and effective tools for early diagnosis, prevention, and treatments both for veterinary and human patients.

Acknowledgments

The authors would like to thank the Preclinical Surgical Research Laboratory at Colorado State University for providing the resources, collaborative environment, and technical expertise that made this work possible. Special recognition is given to the Fulbright Program, particularly Pasaporte a la Ciencia – Fulbright Colombia, for supporting A.F.B.’s PhD studies.

Footnote

Peer Review File: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-136/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-25-136/coif). V.V.P. reports ongoing institutional research support, consulting, and advisory, or other collaborative relationships with Globus, SI-Bone, Mainstay Medical, Simplify Medical, Medical Metrics, Inc., Cerapedics, Zygofix, SpineWelding, Orthobond Corporation, Johnson & Johnson, Ecential Robotics, Pfizer, Performat, and Orthofix. All relationships are paid to the institution. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This article is a narrative review and does not include any original studies involving human participants or animals. No Institutional Review Board (IRB) or Institutional Animal Care and Use Committee (IACUC) approval was required.

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