Key points
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Animal models are critical for understanding disease mechanisms and developing new therapies.
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Species differences in intervertebral disc size, tissue composition, and cellular content associate with distinct biomechanical and biological behaviors.
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Some features of human disc degeneration are mimicked in animal model systems, but the absence of standardized methods for assessing, reporting, and comparing histopathologies of human and animal discs limits our ability to generalize mechanisms from animal models and clinical studies.
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While the discs in large animals can approach the size of a human, the lack of validated pain outcomes creates barriers for solidifying the clinical relevance of animal models of degeneration and regeneration.
Introduction
Animal models are important for understanding disease mechanisms and also serve as a central component of preclinical evaluation of treatments for intervertebral disc (IVD) degeneration-related back pain. Factors, such as physical stress, solute transport, immunity, and tissue spatial relationships undoubtedly influence the manner by which animal IVDs degenerate, either spontaneously with age or after induction with mechanical or chemical insult. Equally important are the phenotypic behaviors of IVD cells that may be species-specific and therefore different from human IVD cells. Therefore, while animal models are an important and necessary bridge between in vitro systems and human clinical studies, they must be applied and interpreted with thoughtful consideration of their limitations and relevance to the particular line of investigation.
A primary obstacle in using animal models for IVD degeneration/regeneration research is whether clinically relevant pathologies exist or can be induced. This is particularly important when testing IVD regenerative therapies, where a primary goal is to determine whether the product (cells, matrices, and/or growth factors) can stimulate a desirable disease-modifying activity within the inhospitable niche of the degenerating human IVD [ ]. If the disease model does not sufficiently mimic the human situation, then animal data may not reasonably forecast future clinical trial outcomes. This fact underscores the importance that the chosen animal model simulates the salient pathological, technical, and clinical features. The larger the discrepancy between the animal model and humans, the greater the risk that the therapy will be ineffectual or even harmful. This difficultly is compounded by the fact that we are still uncovering human IVD pain mechanisms and hence have not fully defined the desired disease-modifying activities for new therapies.
Many animal models have been investigated in attempts to clarify disease mechanisms [ ]. However, these are not necessarily appropriate for judging the quality of a regenerative response. Similarly, there is a diversity of animal models used for preclinical testing of bioactive IVD therapies [ ]. Yet, significant gaps between outcome measures in animals and pain mechanisms in humans muddle decision-making when designing preclinical studies. The purpose of this review is to summarize factors that should be weighed when considering an animal model for degeneration research or therapy development and validation.
Anatomy
Across the various species used for spine research, the IVD comprises the same basic subtissues: nucleus pulposus (NP), annulus fibrosus (AF), and cartilage endplate ( Figs. 4.1 and 4.2 ) (see Chapter 1 ). This is not surprising given the similar developmental origins and functional requirements to facilitate spine flexibility while supporting the load. However, species diversity, principally in size and bipedalism, leads to differences that can become significant in the context of IVD degeneration research and tissue engineering.
Size
There is a tremendous range in IVD size across species. For example, IVD height can range between 0.25 mm for a mouse and up to 11 mm or more for a human ( Fig. 4.1 ; Table 4.1 ). However, when IVD height is normalized by width as a measure of shape, the differences from humans are less extreme and the range from 12% for mouse to 31% for sheep [ ]. Yet, these size differences have important biomechanical and biological consequences. Three centuries ago, Galileo proposed the Square-Cube Law, which states that as a structure’s size increases, its volume (proportional to length cubed) grows faster than its surface area (proportional to length squared). For example, if an animal’s IVD dimension is doubled, then the surface area is increased by a factor of four while the volume is increased by a factor of eight. In this example, the Square-Cube Law indicates that the larger IVD may carry twice as much stress but is required to support nutritionally four times as much tissue. Assuming that the same biomechanical and biological principles apply across species, then as animals become larger, IVD loading and/or the IVD itself needs to be modified for cellular homeostasis to be achieved.
| NP | AF | NP | AF | NP | AF | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Animal | Disc height (mm) | Disc area (mm 2 ) | Normalized compressive stiffness (MPa) | Normalized torsional stiffness (N-m/deg) | GAG per dry weight (μg/mg) | GAG per dry weight (μg/mg) | Water content (%) | Water content (%) | Collagen content per dry weight (μg/mg) | Collagen content per dry weight (μg/mg) |
| Human | 10.91 | 1925 | 9.95 | 0.087 | 466 | 161 | 81 | 72 | 16 | 103 |
| Calf | 6.09 | 1100 | 12.72 | 0.108 | 384 | 66 | 80 | 69 | 60 | 149 |
| Pig | 5.46 | 872 | 15.77 | 0.403 | 379 | 72 | 83 | 59 | 6 | 122 |
| Baboon | 5.97 | 808 | 9.36 | 0.127 | 971 | 333 | 80 | 66 | 19 | 110 |
| Goat | 4.28 | 670 | 7.2 | 0.084 | 335 | 25 | 84 | 66 | 19 | 53 |
| Sheep | 3.4 | 511 | 9.78 | 0.356 | 547 | 133 | 75 | 57 | 20 | 107 |
| Rabbit | 2.4 | 90 | 10.44 | 0.152 | 579 | 160 | 82 | 62 | 78 | |
| Rat lumbar | 0.77 | 11.85 | 5.09 | 0.04 | 384 | 47 | 82 | 65 | ||
| Mouse lumbar | 0.31 | 1.61 | 2.93 | 0.083 | ||||||
| Cow tail | 9.18 | 857 | 8.84 | 0.068 | 548 | 112 | 83 | 69 | 43 | 107 |
| Rat tail | 0.94 | 12.86 | 4.19 | 0.015 | 95 | 20.5 | 75 | 48 | ||
| Mouse tail | 0.44 | 0.095 |
Biochemical composition
Biomechanical properties of spinal IVDs can be linked to their biochemical composition and size [ , ]. Proteoglycans are a major constituent of the IVD NP and, due to their fixed negative charge, provide the capacity to draw in water osmotically, swell, and resist compressive mechanical forces [ ]. Comparative tests indicate that nuclear proteoglycan and water contents are relatively similar between species ( Table 4.1 ), with the exception of rat-tails, which are significantly less, and baboon IVDs, which are significantly more [ ]. Interestingly, despite the range of measured values, proteoglycan and water contents do not correlate with compressive properties across species.
The AF is reinforced with collagen fibers that are oriented to support IVD compression, bending, and axial torsion [ , ]. IVD collagen within the NP is similar between baboon, goat, and sheep IVD, whereas that from the pigs is less than half while that from calves and cows is over three times greater ( Table 4.1 ) [ ]. Similar variations are noted for the AF where goat had 48% less and calf had 45% more than human. Yet, like proteoglycan and water composition, the collagen content of the AF does not correlate strongly with IVD torsional properties.
Biomechanics
Functionally IVD size, shape, and biochemical constituents affect biomechanical properties (see Chapters 2 and 3 ). Axial compressive stiffness varies between 13 N/mm in mice to 2500 N/mm in calf spines [ ]. These differences are less marked if adjusted by the ratio of IVD height to the area, in which case IVD stiffness from calves, cows, pigs, baboons, sheep, rabbits, and rats are not much different from humans [ ]. Similar results have been reported for IVDs tested in axial torsion, where the normalized torsional stiffness (normalized by height divided by the polar moment of inertia) of IVD from goat and mouse (tail and lumbar) was within 10% of human, while calf and bovine IVDs were within 25% [ ]. In this case, however, IVDs from pigs and sheep were approximately three times stiffer, perhaps due to differences in annular material properties [ ]. Taken together, these data indicate that biomechanical properties of animal IVDs, and by extension the regenerative effect of novel therapies assessed in animals, may be scaled to human as an indicator of the potential clinical benefit.
Transport
Because the IVD is avascular, IVD cells rely on diffusion to and from capillaries at the outer AF and vertebral endplate [ ] (see Chapter 10 ). This constraint creates opposing gradients of nutritional factors (e.g., glucose and oxygen) and products of cell metabolism (e.g., lactate [ ]) from the IVD periphery to the IVD center. The Square-Cube Law again makes clear that as IVD size increases, the surface area for transport becomes disproportionately small compared to tissue volume. Another consequence of size differences between species is the IVD tolerance for metabolic demand. Computational studies demonstrate that the kinetics of solute transport into the IVD vary between species in relation to IVD size [ ]. Similarly, diffusion of IVD metabolic products out of the IVD will also have size dependency. These transport phenomena clearly limit the extent to which cellular activity can be sustained within an IVD of a particular size. This concept is beautifully depicted by experimental data for NP cell density versus IVD height across species ( Fig. 4.3 ) [ ]. Importantly, Square-Cube Law scaling also explains why smaller IVDs have a greater healing capacity, as wound-site transport is better able to supply signals and a regenerative milieu, as seen in species-dependent healing of long bone fractures [ ].
Surgical considerations
Variations in IVD size across species have additional practical implications. For example, surgical manipulations in small animals (mice, rats, and to some extent rabbits) may be difficult to execute reproducibly, or may not be meaningful to study therapies that involve implants. It may also be difficult in small IVDs to inject cells reliably that may include viscous carriers (e.g., the mouse NP is approximately 0.13 μL; [ ]). Injecting through small gauge needles can damage cells due to high shear stress, as lower cell viability is associated with small needle diameter and long needle lengths [ ]. Also, working with small volumes can lead to proportionately greater errors in the administered dose due to cell adherence within the syringe.
Size constraints are also a factor when attempting to implant tissues, scaffolds, and devices that are part of a regenerative therapy. Scaling to constructs appropriate for small animals may require significant deviation from the formulation and configuration of implants intended to be used in the clinical situation. For this reason, primates have been a model of choice for more traditional spinal implants and, more recently, IVD arthroplasty devices [ ]. Here, IVD dimensions are more similar to humans and hence more accommodating for implantation. Primates may also be valuable for refining the surgical approach and associated instrumentation.
Another consideration is the availability of imaging (see Chapter 5 ) to facilitate longitudinal studies. The use of magnetic resonance imaging to assess IVD degeneration and healing longitudinally can help increase statistical power and reduce cost by using each animal as its own control. However, the high field strength is needed to achieve a reasonable resolution in small animals (up to 7T) and access to imaging resources may be limited for larger animals.
Bipedalism
In addition to differences in metabolic stress between species, there are also differences in physical loading. While the common notion is that quadrupeds have lower spinal forces than human due to their horizontal spine orientation, research in evolutionary biology indicates that in vivo biomechanical stresses are generally independent of animal size and that tissues across species seem to be operating with a similar factor of safety (ratio of failure load to functional load)—of between 3 and 5 [ ].
All animal spines are loaded by ligament and muscle forces developed during movement and during maintenance of posture against gravitational loading. Even though the spines of quadrupeds are aligned parallel to gravity, significant muscle forces are generated to support the bending and torsional movements required for locomotion [ ]. The observation that vertebral strength [ ] and normalized IVD stiffness (cited above) in animals are comparable to, if not greater than human, also supports the notion that quadruped IVDs are subjected to a significant force in vivo. Direct measurements in vivo have been made: spine forces in the baboon are generally proportionate (by body weight) to humans [ ], while those in sheep are approximately 50% [ ]. Yet the intradiscal pressures in sheep can be significantly higher than in humans depending on the activity [ ]. Similar to the baboon, bovine measurements show that loads and pressures are comparable to humans during static postures, while dynamic pressures tend to be higher [ ].
These studies demonstrate that quadrupeds generate significant spinal forces and pressures that are comparable to that seen in humans. Importantly, they indicate that animals with upright postures (e.g., primates) are not necessarily required to approximate human biomechanical conditions.
Cells
Cells are the heart of IVD tissue homeostasis, and consequently the focus of biological approaches for IVD repair. Disc degeneration can ultimately be linked to undesirable cell behaviors, which include catabolic and proinflammatory activities leading to the deterioration of tissue biomechanical properties. Consequently, therapeutic strategies often target cellular disease-modifying pathways by delivering donor cells, a bioactive agent that stimulates host cells, or a combination of both. Several animal model features can impact the behavior of such strategies, potentially in ways that differ significantly from the human situation.
One main consideration is that NP cells in animals may be notochordal and have behaviors that differ from adult human NP cells. During embryonic development of the spinal column, the NP is formed by notochordal cells (NC) that are sequestered from the developing vertebral bodies and AF [ ]. Rodents, rabbits, and other nonchondrodystrophoid species [ ] maintain a high NC number throughout their lifetime, whereas in other animals such as bovine, goat, and sheep, and human these cells disappear early in life [ ] ( Table 4.2 ).
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