Received: 16 July 2020 Revised: 25 May 2021 Accepted: 26 May 2021

DOI: 10.1002/jsp2.1162

SPECIAL ISSUE ARTICLE

A comprehensive tool box for large animal studies of

intervertebral disc degeneration

Naomi N. Lee1 | Elias Salzer2 | Frances C. Bach3 | Andres F. Bonilla4 |

James L. Cook1 | Zulma Gazit5 | Sibylle Grad6 | Keita Ito2 |
Lachlan J. Smith7 | Andrea Vernengo6,8 | Hans-Joachim Wilke9 |
Julie B. Engiles10 | Marianna A. Tryfonidou3
1
Thompson Laboratory for Regenerative Orthopaedics, University of Missouri, Columbia, Missouri
2
Orthopaedic Biomechanics, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands
3
Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
4
Preclinical Surgical Research Laboratory, Department of Clinical Sciences, Colorado State University, Colorado
5
Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California
6
AO Research Institute Davos, Davos, Switzerland
7
Departments of Neurosurgery and Orthopaedic Surgery, University of Pennsylvania, Philadelphia, Pennsylvania
8
Department of Chemical Engineering, Rowan University, Glassboro, New Jersey
9
Institute of Orthopaedic Research and Biomechanics, University Hospital Ulm, Ulm, Germany
10
Department of Pathobiology, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania

Correspondence
Marianna A. Tryfonidou, Faculty of Veterinary Abstract
Medicine, Department of Clinical Sciences, Preclinical studies involving large animal models aim to recapitulate the clinical situa-
Utrecht University, Yalelaan 108, 3584 CM
Utrecht, The Netherlands. tion as much as possible and bridge the gap from benchtop to bedside. To date, stud-
Email: [email protected] ies investigating intervertebral disc (IVD) degeneration and regeneration in large
Julie B. Engiles, New Bolton Center, Murphy
animal models have utilized a wide spectrum of methodologies for outcome evalua-
Laboratory, University of Pennsylvania,
382 West Street Road, Kennett Square, tion. This paper aims to consolidate available knowledge, expertise, and experience in
Pennsylvania, PA 19348, USA.
large animal preclinical models of IVD degeneration to create a comprehensive tool
Email: [email protected]
box of anatomical and functional outcomes. Herein, we present a Large Animal IVD
Funding information
Scoring Algorithm based on three scales: macroscopic (gross morphology, imaging,
AO Foundation, Grant/Award Number:
AO-03-W16; Foundation for the National and biomechanics), microscopic (histological, biochemical, and biomolecular analyses),
Institutes of Health, Grant/Award Numbers:
and clinical (neurologic state, mobility, and pain). The proposed algorithm encom-
R01AR066517, R01AR077435, T32 grant
OD011126; Fulbright Association, Grant/ passes a stepwise evaluation on all three scales, including spinal pain assessment, and
Award Number: ICETEX program; German
relevant structural and functional components of IVD health and disease. This com-
Research Council, Grant/Award Number: DFG
Wi 1352/14-3; H2020 Societal Challenges, prehensive tool box was designed for four commonly used preclinical large animal
Grant/Award Number: GAP # 825925;
models (dog, pig, goat, and sheep) in order to facilitate standardization and applicabil-
ReumaNederland, Grant/Award Number:
LLP22; US Department of Veteran's Affairs, ity. Furthermore, it is intended to facilitate comparison across studies while discern-
Grant/Award Number: I01RX001321
ing relevant differences between species within the context of outcomes with the

Naomi N. Lee and Elias Salzer, contributed equally (shared first authorship); Julie B. Engiles and Marianna A. Tryfonidou contributed equally (shared last authorship)

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2021 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.

JOR Spine. 2021;4:e1162. jorspine.com 1 of 36

https://doi.org/10.1002/jsp2.1162
2 of 36 LEE ET AL.

goal to enhance veterinary clinical relevance as well. Current major challenges in pre-
clinical large animal models for IVD regeneration are highlighted and insights into
future directions that may improve the understanding of the underlying pathologies
are discussed. As such, the IVD research community can deepen its exploration of
the molecular, cellular, structural, and biomechanical changes that occur with IVD
degeneration and regeneration, paving the path for clinically relevant therapeutic
strategies.

KEYWORDS
biomechanical testing, clinical assessment, disc disease, dog, goat, histopathology, intervertebral
disc, low back pain, neck pain, pig, sheep, spine disorders, spine research

1 | I N T RO DU CT I O N access to specialty equipment, and other technical expertise. For

example, availability of biomedical research quality-specific pathogen-
Chronic low back pain has been reported as the leading cause of years free animal sources is dependent on geographical location and
1,2
lost to disability for the past three decades. Intervertebral disc (IVD) methods in inducing disc degeneration in large animal models are
degeneration is recognized in at least 40% of cases of symptomatic dependent on individual institutions or research groups. There are
back pain.3,4 Much effort has been aimed toward the development of four species that predominate: dog (canine), goat (caprine), pig (por-
more effective diagnostic, preventative, and therapeutic strategies for cine), and sheep (ovine), in alphabetic order. Regardless of the model,
IVD degeneration and preclinical studies involving large animal models the current literature reports a wide variety of experimental method-
are still considered a critical translational tool. Advantages of large ani- ologies, with three or fewer distinct methodologies employed in most
mal models include the use of whole-organ/whole-body systems, rela- studies (Figure 1; Supporting Information, Appendix S1). This observa-
tive vertebra-disc geometry,5biomechanics,6,7 similar pathology, and tion was discussed during a breakout session during the ORS PSRS
8
clinically relevant outcome measures (reviewed by Thorpe et al ). fifth International Spine Research Symposium on large animal models.
Most large animal models require an experimentally induced There was a strong consensus that consolidating these methodologies
insult or injury to the IVD in order to create and study IVD degenera- and their specific application in large animal studies of IVD degenera-
tion, with each method for induction having specific advantages and tion and regeneration would represent an invaluable resource for the
9–12
disadvantages. It is beyond the scope of this article to provide a spine research community. Therefore, by consolidating knowledge
detailed review of large animal IVD models, their strengths and weak- from literature as well as expertise, and experiences from the IVD
nesses, and the extent to which these models reproduce all the char- research community, a basic set of tools was compiled allowing for
acteristics of pathological human IVDs. Instead, the reader is referred complementary functional and anatomical evaluations in large animal
9–12
to previous reviews on this topic. Furthermore, there are species- models. Here, a workflow is presented based on general consider-
specific biological considerations, including overall size, age of skeletal ations pertaining to study design and tissue processing of spinal speci-
maturation, spinal anatomy, and cellular composition in the nucleus mens, and considerations and suggestions for three scales of read
pulposus (NP) (Table 1), and IVD biomechanics, and from a clinical per- outs: macroscopic (gross morphology, imaging, and biomechanics),
spective, differences in pain-related behaviors. Within the particular microscopic (histological, biochemical, and biomolecular analyses), and
species employed, confounding factors including sex (hormones), clinical (neurologic state, mobility, and pain).
genetics, and susceptibility to developing spontaneous IVD conditions
all need to be considered. Preclinical findings from experimental ani-
mal models can be supplemented with clinical studies involving client- 1.1 | General considerations for study designs
owned companion animals, for example, canine patients. Dogs
develop spontaneous degenerative IVD diseases and manifest with The ARRIVE (Animal Research: Reporting of In Vivo Experiments)
well-recognized clinical presentations. The diagnostics and treatment guidelines14,15 were developed to increase reproducibility and scien-
13
are closely comparable to those for humans (reviewed by Lee et al ). tific rigor by providing a checklist of information to include when
Although these studies are emerging, as yet their implementation is reporting animal studies. Many publishers require manuscripts to
limited. closely follow the reporting guidelines and thereby support the 3Rs
The large animal model and spinal segments (eg, cervical, lumbar) principles (Replacement, Reduction, and Refinement). To further
selected for a particular study are highly dependent on the specific reduce the number of animals, IVD degeneration can be induced at
aims of that study. Also, institutions are highly variable in terms of multiple levels employing nonadjacent levels to limit interference
infrastructure supporting animal procurement, housing, biosecurity, among the IVDs studied. In such a multilevel approach, within each
 LEE ET AL. 3 of 36

animal/lumbar spine, the following control conditions may be, at a

NCD, non-chondrodystrophic laboratory dogs (eg, Mongrel Hounds); CD, chondrodystrophic laboratory dogs (eg, Beagles). In laboratory animal sciences, the terms “Mongrel dogs” or “laboratory hounds” have
become interchangeable in a sense that they refer to non-Beagle (CD) laboratory dogs. They represent the NCD dog population. These animals are purpose-bred in USDA Class A dealer facilities which certify
Goats exhibit distinct behaviors from sheep
minimum, included for proper comparisons: a nonoperated intact

The age at skeletal maturity may range depending on the specific breed being utilized.193,194 Furthermore, body weight and size of animal can widely vary depending on breed and breeding practices; this is

clear pedigree and specific pathogen-free. The benefits of using these animals include avoidance of unclear pedigree and health status which can often be seen in livestock obtained for biomedical research
and they tend to be much more active,
(nondegenerated) IVD, an experimentally degenerated, but
untreated IVD, and a sham operated IVD (eg, surgical exposure and

curious, and orally investigative.

vehicle only). The inclusion of these controls may facilitate assess-
ment of the intrinsic repair capacity of the large animal model
<90 kg by 36 mo of age
being employed.
The multilevel approach may complicate understanding the caus-
ative relationship between the clinical scale read out parameters (such
as painful symptoms) and IVD degeneration or the intervention being
15-18 y

studied. In order to remove a confounding factor of having more than

2-3 y
Goat

one degenerative IVD causing pain and disability, these research ques-
tions may require models that involve inducing IVD degeneration at a
Young males are often castrated by

Calm and docile with relative ease

Ram: <135 kg by 36 mo of age192

single level per animal in contrast to the abovementioned multilevel

approach.
Longitudinal imaging follows IVDs from their native state to
induced degeneration and subsequent experimental therapeutic inter-
ventions, thereby allowing determination of changes from baseline
1 mo of age.

measurements. Although the IVDs in each region (lumbar thoracic and

of handling
Ewe: <90 kg

cervical) are of relatively similar size, the lumbosacral IVD is larger

10-12 y
Sheep

and subjected to different biomechanical conditions compared to the

2-3 y

other lumbar IVDs.16Thus, since it is a level where clinical pathologies

particularly evident in sheep that are bread either for meat or wool (eg, sheep bred for wool are often <50 kg body weight).

are commonly found and treated, in both humans and canines, in the
authors' opinion the lumbosacral IVD is preferably separated from
recommended intact boars be separated.
result in welfare concerns. It is especially
Aggression and fighting within groups may
Domestic: 115-130 kg at sexual maturity

the others in the experimental design of large animal studies and not
Miniature: 30-50 kg at sexual maturity

pooled or randomized with other levels.

Overview of experimental domesticated large animal models used in IVD studies

1.2 | General considerations for processing spinal

Social hierarchy exists.

specimens

The standard directional anatomical nomenclature for quadrupeds is

(5-6 mo)
18 mo-4 y
15-20 y

shown in Figure 2A. By following the process described in

Figure 2B, the macro- and microscopic parameters described here
Pig

can be applied to each investigated IVD. As such, gross morphologi-

cal grading can be combined with histopathological schemes that
among intact dogs) may result in welfare
Social species with the ability to establish,

Conflicts within group housing (especially

evaluate cellular and matrix microstructure and molecular composi-

tional information. For all three scales, previously published, well-
maintain, or change a relationship.

validated schemes to evaluate these parameters can be applied and

possible drawbacks and opportunities are discussed in the future
Max. body weight varies strongly depends on breed.

perspectives section.
Immediately after euthanasia, imaging (eg, plain radiography, com-
CD: less than 15 kg
NCD: 10-11 mo191

puter tomography (CT) and magnetic resonance imaging (MRI)) can be

NCD: 20-30 kg

purposes from a sale barn or open herd.

CD: 8-9 mo191

performed in situ, prior to recovering the vertebral column from the

concerns.

animal and extracting the functional spinal units. A functional spinal

10–15 y
Dogb

unit (FSU, also referred to as spinal motion segment [SMS]) comprises

one IVD between the adjacent vertebral bodies, intact interbody liga-
ments, and facet joints, but with paraspinal muscle tissue removed.
Average expected

considerations

The pedicles and posterior column elements are typically removed to

Age at skeletal

Bodyweightc
maturitya

isolate the ventral column unit (VCU, equivalent to anterior column

Behavioral
lifespan
TABLE 1

unit (ACU) in humans; Figure 2B). Isolated VCUs can be further

divided transversely (axially) or sagittally for macroscopic evaluation
and further processing. Transversely cutting the IVD will result in a
b
a

c
4 of 36 LEE ET AL.

F I G U R E 1 Variety of experimental methodologies reported for outcome evaluation. A sample of recent peer reviewed manuscripts
employing four common large animal models (ie, canine, caprine, ovine, porcine; n = 10 per species) to study IVD degeneration or therapeutic
strategies in the past two decades. The number of six main outcome measures concomitantly used (macroscopic, histologic, radiologic,
biochemical, biomechanical, pain), A, and the detailed use of each type of outcome, B, were registered and demonstrated the majority of the
studies employed 3-4 outcome measures; with limited concomitant biomechanical analysis and absent pain assessment. C, When IVD
degeneration was induced (“Deg. Induction”; 92% of all studies), healthy and degenerate controls (“Deg. Control” may be either induced or
naturally occurring disc degeneration) were regularly, but not consistently, reported. Spontaneous degeneration was only reported for canine
species. Adherence to the ARRIVE guidelines was mentioned in one study. D, Available scoring systems in histological and radiological outcomes
(“evaluation” refers to yes/no evaluation and “scoring” specifies within those studies whether or not a scoring scheme was employed), including
quantitative MR imaging were seldom employed. The studies included are provided in the Supporting Information, Appendix S1

cross-section consisting of the AF and NP. This cut is acceptable for (ECM) components during subsequent tissue processing.17 Instead, en
studies that focus on basic analyses of AF and NP; however, evalua- bloc fixation of the intact VCU can prevent these artifacts.
tion of the cartilaginous endplate (CEP) is not possible. Sagittal sec- Combining multiple types of characterizations of tissue samples
tioning, which preserves the anatomic orientation and context provides an in-depth understanding of IVD regeneration. Proper plan-
between components of the FSU, is suggested for macroscopic and ning is necessary so that nondestructive procedures, like imaging and
microscopical evaluation and grading of AF, NP, and CEP as shown in biomechanics, can be carried out prior to destructive tests such as
Figure 2B. The workflow proposed by the authors in Figure 2B and gross morphological evaluation, or those that alter the physical prop-
the Supporting Information (Appendix S1) allows for the complete erties of the tissue, such as fixation for histology. If storage is
evaluation of gross morphological analysis in combination with imag- unavoidable, a single freeze-thaw cycle is generally considered accept-
ing, histology, biochemical, and biomechanical analysis (outlined in able prior to imaging or biomechanics, with the caveat that this may
more detail in the Supplementary flowchart, Appendix S1). Transverse partially compromise planned macro- and microscale outcome param-
sectioning prior to fixation is not recommended as without constraint eters. Preparation of tissue for RNA or protein assays is preferably
of the IVD by the vertebral bodies, the NP tissue may swell, distorting done using freshly isolated, unfixed, and undecalcified specimens. For
tissue architecture and allowing leaching out of extracellular matrix biochemical and biomolecular analysis, tissue is usually snap frozen in
LEE ET AL. 5 of 36

FIGURE 2 Legend on next page.

6 of 36 LEE ET AL.

liquid nitrogen and stored at 80 C.18 For biomechanical testing, the original scheme are implemented, additional assessment of inter-
use of fresh specimens is preferred, but if this is not feasible, speci- and intraoperator variability may be warranted.
mens can be properly stored in controlled conditions until testing as In reflecting whether to employ gross morphological grading,
described in the respective section. the following can be considered: (a) the ease of integrating macro-
scopic evaluation into the overall experiment workflow;
(b) available access to at least two independent evaluators
1.3 | Macroscopic scale (experts or trainable personnel) to perform accurate and consis-
tent evaluations of IVD morphology; and (c) the relative advan-
1.3.1 | Gross morphological grading tages and disadvantages that macroscopic evaluation provides
over other types of assessments, if all cannot be performed. With
Gross morphological grading refers to the scoring of an IVD's patho- respect to this final consideration, advantages of gross morpho-
logical state through inspection of internal macrostructure, either logical grading include its low cost, the requirement for minimal
directly or using photographs. Direct physical inspection, while pos- specialized equipment or technical expertise, and its ability to pro-
ing some practical limitations, has advantages over digital imaging, vide a higher resolution assessment of IVD and vertebral bone
as tactile assessment of the exposed tissue enables more effective macrostructure compared to radiologic/MRI evaluations. Unlike
localization of defects such as AF delamination and circumferential histology, there is minimal pre-analytical variability that could
tears, rim lesions, and NP tissue granulation. Nevertheless, high- impact results (eg, tissue microtomy artifacts due to tissue shrink-
quality photographs are most frequently used for macroscopic ing or expansion, damage from sub-sectioning, or variations in
evaluation. staining). Accurate and consistent histologic evaluation typically
The most commonly adopted gross morphological grading requires specialized training so that tissue artifacts listed above
scheme for IVDs was published by Thompson et al.19 This scheme is are not misinterpreted as actual lesions (eg, tears in the AF matrix
based on a refinement of the comprehensive descriptions of human degeneration). The major disadvantages of macroscopic evalua-
20
IVD pathology by Vernon-Roberts, and includes assessments of tions include acquiring subsections that result in tissue destruc-
the major substructures of the IVD (NP, AF, and CEPs) in addition to tion, and unlike imaging, it cannot be applied longitudinally in vivo.
the adjacent vertebral body margins. In developing and validating Macroscopic evaluation schemes are also inherently subjective,
this scheme, Thompson et al applied it to 68 IVDs from 15 human with grades potentially subject to ambiguity and high inter-
spines obtained postmortem from individuals aged 16-89 years. observer variability even when performed by experts.
Mid-sagittal VCU sections (including the osseous endplates) were
graded by three experienced and blinded individuals, and the
authors undertook extensive validation to ascertain intra- and 1.3.2 | Implementation
interoperator variability, which were found to be 85%-87%, and
61%-88%, respectively. The parameters described herein for gross morphological grading
While this scheme was originally designed for application to of large animal IVDs are drawn in large part from the original study
human IVDs (giving it high clinical relevance), it has frequently of Thompson et al19 (to which readers are encouraged to refer).
been adapted for large animal IVDs. A literature search revealed IVDs should be bisected using a single, smooth cut using a straight-
approximately 60 studies that cite the Thompson grading scheme edged trimming knife or similar implement, in order to minimize
with application to dog, 21 sheep, 22 pig, 23 or goat IVDs 24 (select cutting artifact that could confound assessments. Following the
examples cited with examples for each species shown in scheme of Thompson et al,19 IVDs are assigned a grade from 1 to
Figure 3). Of note, the Thompson grading scheme has been 5, where 1 is healthy and 5 is most degenerate. Grading criteria for
adapted and applied to IVDs cut in the transverse plane by some each IVD substructure (NP, AF, CEP, and vertebral bodies) are
researchers 25 ; however, this presents the significant limitation shown in Table 2 (example images in Figure 3). Both left and right
that the CEPs, BEPs, and vertebral bodies cannot be assessed. In hemi-IVDs should be graded and averaged for each of the
instances where this, and other significant deviations from the assessors.

F I G U R E 2 A, Comparative Anatomy of biped and quadruped spines. B, Flowchart of possible port-mortem procedures for evaluation of all
read out parameters from each functional spinal unit (FSU). A, Anatomic analogy between humans and quadrupeds. B, Once the spinal column is
extracted, the spinal cord can be removed en bloc (a). Transverse processes can be removed by trimming the lateral aspects of the spine (b). To
isolate FSUs, cuts are made transversely through the mid-vertebrae (c). Dorsal aspects can be removed by cutting sagittally through the spinal
canal (d) resulting in individual ventral column units (VCUs) (e). The isolated VCU can then be transversely transected into two identical parts (e).
Thereafter, both parts can be used to take digital photographs for macroscopic evaluation and subsequently, one part may be fixed for
histopathology (f). From the second part, the nucleus pulposus (NP) and annulus fibrosus (AF) tissue can be isolated from the cartilaginous end
plates (CEPs) and vertebra with a surgical blade for biochemical analysis (g-i). Note that prior to the described post-mortem procedures (advanced)
imaging and non-destructive biomechanical analysis can be conducted as described in this manuscript
LEE ET AL. 7 of 36

F I G U R E 3 Gross Morphology. Exemplary gross morphological grading images for discs from goats (caprine), sheep (ovine), pigs (porcine) and dogs
(canine). Human IVDs are included for comparison. Grades (1–5) have been assigned according to the criteria outlined by Thompson et al,19 where Grade
1 corresponds to healthy IVDs and Grade 5 the most severely degenerated. Noteworthy characteristics specific to each image series include: the
translucent, notochordal NP region present in healthy dog and pig IVDs, compared to the more cartilaginous NP in healthy sheep, goat and human IVDs;
and the presence of growth plates in sexual and skeletal maturity goat and sheep and in the immature pigs. Gross morphological grading includes
assessments of the major substructures of the IVD (NP, AF, and CEPs) in addition to the adjacent vertebral body margins and as such, the gross changes
may differ depending on the method of disc degeneration and the therapeutic approach tested. Examples in this figure are from induced disc degeneration
models via chondroitinase ABC (goat) and partial nuclectomy (ovine) and from naturally occurring disc degeneration (canine). The worst grade of the
different substructures is used to define the final score. Animal IVDs are oriented with the ventral side facing down. Human IVDs are oriented with the
anterior side facing to the right and are from naturally occurring disc degeneration. n.a.: not available; Relative size differences need to be considered
between the different species; grading is independent of the relative IVD size. Goat, dog, and pig images were kindly provided by Prof. Dr. Theo Smit,
Dr. Niklas Bergknut, Prof. Dr. Hans-Joachim Wilke respectively. Images of human IVDs were adapted from Wilke et al198 and Galbusera et al199

1.4 | Imaging the research approach, radiographs can provide information on mor-
phology and density of vertebral bodies, IVD degenerative changes
Imaging is frequently employed for the evaluation of IVD degenera- including IVD height loss, spondylolisthesis,26 osteophyte formation,
tion/regeneration in experimental large animal models, noninvasive or bridging via spondylosis formation in late stages of IVD degenera-
longitudinal assessments during in vivo studies. Imaging is also com- tion. Additionally, information on possible adverse effects of the ther-
monly performed on ex vivo samples at study end points. The most apeutic approach can be indicated by radiographs, for example,
common imaging modalities applied are plain radiography and MRI, extradiscal bone formation following intradiscal injections due to leak-
while other advanced imaging modalities may be available in specific age of the therapeutic agent. Compared with other imaging tech-
research infrastructures, including computed tomography (CT) and niques, some advantages of plain radiographs are the low cost and
ultra-high field MRI, providing additional spatial information with high availability across institutions.
improved resolution. Depending on the infrastructure and equipment, radiographs can
be obtained either in the standing position (goat, sheep) or conducted
under deep sedation or anesthesia (dog, pig, sheep, goat) to ensure
1.4.1 | Plain radiography proper muscle relaxation, and to allow for positioning and minimizing
distress to the animal according to “Refinement,” one of the 3Rs prin-
Plain radiography is a widely used and accessible tool to evaluate the ciples. Positioning of the animals for radiographic evaluation depends
boney structures of the spine. Depending on the model and on the spinal segment of interest; ideally the region of interest is
8 of 36 LEE ET AL.

centered in the radiograph as parallax errors increase at the borders

Early chondrophytes or osteophytes at

of the image. Furthermore, it is important to maintain the vertebral
column parallel and as close as possible to the x-ray cassette for a bet-
ter evaluation of IVD spaces. General aspects to further consider are
tight collimation to enhance detail. Next to the most common lateral
view, ancillary views such as ventrodorsal, flexed, and extended views

Osteophytes <2 mm

Osteophytes >2 mm
Rounded margins can be acquired when evaluating cervical IVDs.27
Vertebral bodies

Pointed margins
Disc height index (DHI) quantifies changes in IVD height

margins
(Figure 4) and is considered a more accurate method than absolute
IVD height measurement as it corrects for positioning and animals of
differing sizes. The DHI is calculated by taking the average of two-
dimensional measurements obtained from the dorsal, middle, and
irregularity and focal sclerosis in subchondral bone ventral portions of the IVD and dividing those by the average of the
Fibrocartilage extending from subchondral bone;

adjacent vertebral body heights.28 The DHI was proposed by Sasaki

et al29 and thoroughly studied and advanced by Masuda et al.30 It is
essential to maintain a consistent level of sedation (or no sedation)
during radiography of each animal and at each time point, in order to
obtain a similar degree of muscle relaxation and minimize confounding
Hyaline; uniform thickness

effects on IVD space narrowing. These measurements are most fre-

Focal defects in cartilage
Cartilaginous end plates

quently obtained using plain radiographs; although the resolution and

three-dimensionality afforded by MRI may allow for more accurate
Irregular thickness

Diffuse sclerosis

measurements.

1.4.2 | Magnetic resonance imaging

Currently, MRI is considered the most definitive noninvasive

Mucinous material between lamellae in inner AF
Extensive mucinous infiltration; loss of annular-

imaging tool in the clinical assessment of IVD degeneration, and in

nuclear demarcation, in-folding of inner AF

large animal models is always conducted under anesthesia to prevent

The macroscopic grading scheme adapted from Thompson et al19

Discrete fibrous lamellae

Annulus fibrosus (AF)

Focal disruptions
lamellae
Horizontal (vertical) clefts parallel to

Clefts extend through NP and AF

White fibrous tissue peripherally
Consolidated fibrous tissue
Nucleus pulposus (NP)

end plate
Bulging gel
TABLE 2

F I G U R E 4 The disc height index. It is suggested to quantify

Grade

changes in IVD height on plain radiographs or high field MR images.

1
2
3

According to Masuda et al28

LEE ET AL. 9 of 36

motion artifacts. Within this context, little is known on how the level induction of IVD degeneration and once degeneration is established
of relaxation in large animal models may affect outcomes, likewise prior to the administration of therapies. This not only allows determi-
diurnal variations on signal intensity and IVD height reported in nation of baseline measurements, but also enables registration of con-
humans.31 In terms of grading degeneration, MRI provides valuable founders that may affect interpretation of the study results, such as
information on signal intensity (related to hydration state), IVD height variability in the level of IVD degeneration and pathologic changes,
and the structure and differentiation of its components. Complica- like the less documented vertebral body and endplate changes termed
tions such as extradiscal bone formation due to leakage of stem cells “Modic changes” at the vertebral body level.43
32,33 34
or growth factors, or other lesions including Schmorls nodes,
can also be detected by MRI.
In 2001, a grading system using MRI was developed by Pfirrmann 1.5 | Biomechanical assessment
35
et al, for the semiquantitative assessment of the human lumbar IVD
degeneration condition. T2-weighted-MRI sequences without fat sat- The IVDs of large quadrupedal animals are often assumed to be under
uration were used for this purpose as the signal loss of the IVD on different loads than those of humans because of the difference in hor-
these sequences correlates with progressive degenerative changes. izontal vs vertical spine orientation, along with other anatomical dif-
The Pfirrmann scale ranges from 1 to 5 and considers: the structure ferences. However, to keep the spine of quadrupeds in a horizontal
and signal intensity of the NP, the height of the IVD, as well as the position, substantial contractile forces arising from combinations of
distinction of the NP and AF (Table 3, Figure 5). This grading system is muscular contraction and reactive forces in the ligaments are neces-
still the most accepted and commonly used MRI grading system for sary, as evidenced by the density and orientation of their vertebral
36,37
the evaluation of IVD degeneration. The Pfirrmann grading sys- trabecular structure.10,44,45 As such, the basic biomechanics of large
tem was proposed using 1 T MRI images almost 20 years ago, and the animal spines are not much different to those of humans, and there-
quality of images of high and low magnetic fields in MRI have fore, changes in biomechanical properties evaluated in these models
improved since then. As such, an updated MRI disc degeneration have translational applicability to human IVD health and disease. Nev-
38
grading system may be considered. For this purpose, the present ertheless, the degeneration model employed may interfere with the
manuscript provides representative images for the four commonly evaluation of the biomechanical effects of the treatment strategy,
used large animal species in the field (Figure 5). such as cavity formation by enzymatic digestion.46 A basic description
Various quantitative MR sequences have been shown to reliably of IVD biomechanics and load displacement curves (LDC) is provided
indicate biochemical changes in the IVD, including hydration status, in the Supporting Information (Appendix S1) and the normal, healthy
proteoglycan content, and IVD degeneration as reported for goats,39 lumbar range of motion (ROM) for the common experimental large
sheep,40 pigs,41 and dogs.42 Relying on signal intensity alone (eg, from animal species in comparison to those of humans is shown in Figure 6.
single T2-weighted images) for quantitative assessments is problem- The most common spine sample type for biomechanical evalua-
atic, as signal intensity will vary based on position in the magnet. In tion is the FSU, as it is the basic functional repeating unit of the spine;
the absence of performing truly quantitative MR sequences, normaliz- however, two or more coupled FSUs are also sometimes evaluated.
ing IVD signal intensity to that of adjacent structures such as cerebral To investigate the biomechanical properties of only the IVD, VCUs
spinal fluid or bone marrow (which are expected to remain constant) are often employed. Although their biomechanical behaviors are
is one option to address this. Considering the advantages of qualita- related, different levels of FSUs or VCUs are ideally compared individ-
tive and quantitative MRI, researchers can plan longitudinal follow up ually.47,48 If the research question and the infrastructure allow, nonde-
during in vivo studies. Ideally, MRI analysis is conducted prior to structive biomechanical testing on freshly isolated specimens which

TABLE 3 The MRI-based grading scheme adapted from Pfirrmann et al35

Grade Structure Distinction between NP Signal intensity Height of IVD

and AF
1 Homogeneous and bright white Clear Hyperintense and isointense Normal
to CSF
2 Nonhomogeneous with or without Clear Hyperintense and isointense Normal
horizontal bands to CSF
3 Nonhomogeneous and gray Unclear Intermediate Normal to slightly
decreased
4 Nonhomogeneous and gray to black Lost Intermediate to hypointense Normal to moderately
decreased
5 Nonhomogeneous and black Lost Hypointense Collapsed disc space

Notes: Pfirrmann grading is performed using T2-weighted sequences with 1.5 Tesla or higher. From the high-quality images, four factors are scored: overall
structure, the distinction of specific components, signal intensity, and the height of the IVD.
Abbreviations: AF, Annulus fibrosus; CSF, cerebrospinal fluid; NP, Nucleus pulposus.
10 of 36 LEE ET AL.

F I G U R E 5 Healthy and degenerated IVDs (Pfirrmann graded) in large animal species and human. Typical examples of T2-weighted MR
images from human (3 T); goat, sheep and pig with experimentally induced IVD degeneration (3 T), and dog with naturally occurring IVD
degeneration (1.5 T). Note that the intensity of the IVD signal is compared to the intensity of the cerebrospinal fluid (Table 6) Grade 1: The
healthy IVD shows a homogenous structure with a hyperintense signal intensity and normal IVD height. Grade 2: The structure of the IVD is no
longer homogeneous and the signal is still intense. Horizontal gray bands may be present in the IVD that is related to a beginning unclear
distinction between NP and AF. Grade 3: Signal intensity is intermediate and the height of the IVD is slightly but visibly decreased with unclear
distinction between NP and AF. Grade 4: Signal intensity is hypointense and there is no longer a distinction between NP and AF, the IVD height
is moderately decreased. Grade 5: Inhomogeneous structure of the IVD with a hypointense signal intensity and a collapsed IVD space. Note that
in naturally occurring IVD disease at these stages spondylosis occurs eventually, potentially fusing the segment with progression (eg, dog, Grade
5). NP: nucleus pulposus, AF: annulus fibrosus. Human and sheep MRIs were kindly provided by Frank Niemeyer169 and Marion Fussilier,178
respectively

can be further processed for complementary readouts is ideal negative impact.50 For a longer duration, specimens may be frozen, at
(Figure 2; Supporting Information, Appendix S1). 20 C or lower for best sample preservation based on the available
literature body which is summarized in detail in the Supporting Infor-
mation (Appendix S1). For the retrieval and storage of IVD tissues
1.6 | Ex vivo biomechanical testing (CEP, NP, and AF), we suggest storing the entire intact IVD and
harvesting the tissue directly before testing, if the tissue cannot be
1.6.1 | Specimen preparation and storage tested fresh.51

Whenever possible, fresh specimens are preferred for biomechanical

testing. If specimens need to be stored, a 0.15 M PBS-soaked gauze 1.6.2 | Test preparations
can be placed around the unit and subsequently covered by at least
two plastic bags/cling foils49 to prevent dehydration. Generally, Prior to testing, the tissue or IVD specimen can be thawed while
mainly cartilaginous specimens (ie, IVDs not containing NP noto- sealed to prevent dehydration. If samples need to be tested multiple
chordal cells) may be stored at 4 C for at most overnight without a times over consecutive days, freeze-thawing can be avoided by
LEE ET AL. 11 of 36

storing at 4 C and sealing to prevent dehydration.52 The loading his- height varies during day and night (diurnal changes) due to water
tory of the sample influences the reproducibility of the biomechanical flux caused by the changing average load magnitude. This affects,
53–55
evaluations, for example, due to variations in hydration. The IVD among other parameters, the intradiscal pressure.56 There may be
water content variations from storage conditions, which are ideally
balanced out prior to testing. This can be addressed by applying
axial compression load (“pre-load”) or displacement, or allow passive
hydration without load with the goal to equilibrate to a physiologi-
cally realistic situation.57
During testing, the specimens are frequently kept in a tem-
perature and humidity-controlled test environment that best
mimics the in vivo situation. Accordingly, temperature is kept
constant at 37 C, or alternatively room temperature at a relative
humidity of ca. 100% at all times. Humidity can be controlled,
for example, by spraying 0.15 M PBS, in an environmental cham-
ber, wrapping in soaked gauze or by creating a reservoir.48,58 Of
note, swelling will occur in a saline bath without axial loading.59
At 37 C, catabolic enzymes are active, and degeneration is
enhanced. Therefore, specimens cannot be tested for a longer
duration than several hours without the use of protease inhibi-
tors and antifungal agents. Biomechanical experiments are fre-
quently carried out at room temperature, however, the
viscoelastic properties of the IVD and adjacent structures are
temperature dependent. 60,61
Specimens are frequently “preconditioned,” usually for at least
three cycles to achieve a consistent response in creep,62 stress relaxa-
tion, and in load-displacement curves.47 Furthermore, the strain rate,
preload, and follower load can be optimized in preliminary studies, as
they are, for example, dependent on animal, IVD size and level, age,
biomechanical test, or degeneration state.

1.6.3 | Testing of FSU or VCU specimens

For multiple degrees-of-freedom (see Supporting Information,

Appendix S1) FSU-testing and analysis recommendations, we refer to
the review by Wilke et al.47 In addition, individual VCU specimens
may be tested in unconfined compression63 to measure axial, time-
dependent characteristics, for example, creep-recovery.49,64

F I G U R E 6 The range of motion (ROM) of healthy lumbar IVDs.

The ROM of individual healthy lumbar IVD levels of sheep200 and
pig201 as well as the average ROM of dog (average from L4-L5 to
L6-L7, unpublished preliminary data from Prof. Dr Hans-Joachim
Wilke) and goat202 (average from T13-L1 to L5-L6) compared to
human203 specimens (unpublished ROM data, described in Kettler
et al203). Note that the sheep spine contains 6 or 7, the dog 7, the
goat and the pig 6 lumbar vertebrae compared to 5 in human. Values
are mean (SD) ROM in  for each species; the moment and number of
IVDs studied for the large animal models is given in the figure caption.
Pig: 15 spines from 6-month-old cross of Pie'train boar with hybrid
pig. Sheep: 14 spines from 3 to 4-year-old female merino sheep. Goat:
8 spine from 3- to 4-year-old female Dutch milk goats. Dog: 2 spines
from 2 years old non-chondrodystrophic dogs. Human: 111 adult
donors
12 of 36 LEE ET AL.

Biomechanical parameters of healthy IVDs are IVD-level dependent type or pathology is to be examined, the direction of sectioning
and as such should not be averaged on a spine region but evaluated should be adapted accordingly. For example, if histologic evaluation of
65
individually. the endplate is not needed, transverse sections can be used to provide
more surface area of NP and AF to evaluate (personal communication,
Dr Chantelle Bozynski, Missouri Orthopedic Institute). A properly pre-
1.6.4 | Testing the AF, NP, and CEP pared section is essential to accurately observe the fine cellular and
tissue component structures. Here, we describe common fixation
The AF lamellae experience high circumferential and longitudinal ten- and processing procedures, and scoring systems for microscale ana-
sile, shear, and radial compression forces. The orientation during test- lyses, providing technical and analytical considerations, and potential
ing is relevant as the tissue is anisotropic.66 The AF can be tested in drawbacks with respect to their application. A more detailed version
uni- or biaxial tension, in shear, or under unconfined or confined com- of this histopathology section is provided in the Supporting Informa-
pression (ie, with more controlled boundary conditions). For each test, tion (Appendix S1).
single or multiple lamellae may be used.48,67,68
Due to a high fixed charge density, the NP can imbibe large
amounts of water. If the glycosaminoglycans (GAGs) responsible for 1.7.2 | Tissue processing for histopathology
this swelling and the water content are altered (because GAGs leach
out or water is imbibed in unloaded conditions69), the time-dependent Fixation
mechanical properties change drastically.70–72 Furthermore, the NP Fixation and decalcification procedures are very important for proper
experiences shear stresses, for example, during bending or axial rota- histological analysis of IVDs. Although cryo-compound fixation and
tion. As the NP is in vivo confined by the bony and cartilaginous cryo-sectioning can be performed for IVD analysis, in the authors'
endplates (BEP and CEP) and the AF, compression and relaxation tests experience, stained sections of paraffin embedded decalcified speci-
are frequently performed in confined conditions.73,74 Unconfined mens provide superior morphology for evaluation of cellular and ECM
compression is not physiological, but may be used to determine NP components. However, histologic tissue artifacts that might
material parameters, for example, Poisson's ratio.75 During axial load- unintentionally be generated during fixation, decalcification, dehydra-
ing, the intradiscal pressure increases in the confined NP, which can tion, or sectioning procedures should not be confused with authentic
be measured.76,77 Reitmaier et al have published a method for in vivo degenerative features in IVD tissues (see Table 4). For most applica-
78
measurements of intradiscal pressures in large animals and have tions, IVD tissues are fixed to preserve the cells and ECM and prevent
compared this to human intradiscal pressures.79 autolysis. To minimize the required fixation time and possible con-
Compared to the NP and AF, little attention has been given to founding effects of inappropriate fixation (eg, leaching out of ECM
the CEP, even though it plays an important role in IVD degenera- molecules17), the sample can be trimmed to the required size before
80
tion. Inhibition of endplate nutritional pathways leads to IVD degen- fixation (Figure 2B). To prevent unconstrained swelling of the NP tis-
eration81 and the CEP it is also responsible for limiting the loss of NP sue during fixation, the CEP needs to be maintained. Frequently used
matrix proteins. Thus, CEP properties should ideally be considered fixatives for IVD research purposes are 10% neutral buffered formalin
when studying IVD regeneration, especially because solute transport or 4% paraformaldehyde. Cryo-compound fixation of samples immedi-
might be affected, for example, due to CEP degradation caused by ately after removal of the CEPs can allow for fast processing with
enzymatically induced IVD degeneration. Biomechanical evaluation of postfixing after sectioning.86–88 For immunohistochemistry, ethanol
82 83
the CEP can include confined compression or uniaxial tension, or fixation can avoid some of the antigen retrieval steps.
permeability testing84,85testing.
Decalcification
In IVD tissues, the CEP and flanking bony portions of the vertebral
1.7 | Microscopic scale bodies are ideally maintained for complete histological scoring of all
IVD substructures. Decalcification prior to paraffin embedding is
The overall goal of microscale evaluation is to provide characterization therefore essential for microtome sectioning. The decalcification
of the microscopic architecture and composition of the IVD, including method chosen can lead to a compromise between time requirements
cellular and ECM components at the histological, biochemical, and and preservation of tissue morphology, as the main difference
biomolecular level. between decalcification solutions is the speed of decalcification.
Ethylenediaminetetraacetic acid (0.5 M EDTA) as a chelating agent is
the method of choice for gentle decalcification, and generally the best
1.7.1 | Histopathology at preserving tissue morphology46,89 and is most compatible with
immunohistochemistry. However, from weeks to months may be
For histological grading, it is standard practice to evaluate the micro- needed for EDTA decalcification, especially for large animal speci-
structure of the NP, AF, and CEP on mid-sagittal tissue sections; how- mens. Alternative decal solutions and techniques are discussed in the
ever, if a region of interest of the IVD relevant to a specific treatment Supporting Information (Appendix S1).
LEE ET AL. 13 of 36

TABLE 4 Histological grading scheme of disc degeneration compiled based on validated schemes40,96–100

of notochordal cells (NCs)a in the NP (H & E; dog and pig model only)—Figure 9
Abundantly present (>90% of total cells) within the entire NP 0
Present in moderate amounts (50%-90% of total cells) 1
Present in low amounts (<50% of total cells) 2
Absent 3
Nucleus pulposus cell (NPC)b clusters (H&E)—Figure 10 and 11
No clusters 0
Scattered small cell clusters (ie, 2-7 nuclei per cluster) 1
Frequent large cell clusters (ie, >8 nuclei per cluster) 2
Presence of huge cell clusters (ie, >15 nuclei per cluster) 3
NP cell loss (karyolysis) and cell necrosis (pyknosis or karyorrhexis) (H&E)—Figure 11 and Figure 12
None—lacunae all contain viable nuclei 0
Rarely present. Few (<25%) lacunae demonstrate karyolysis, pyknosis, or karyorrhexis 1
Frequently present. 25%-50% of lacunae demonstrate karyolysis, pyknosis, or karyorrhexis 2
Karyolysis, pyknosis, or karyorrhexis predominates (>50%) 3
NP Matrix staining (AB-PSR)—Figures 7 and 10
Blue proteoglycan ECM stain dominates 0
Reduction in blue proteoglycan ECM staining (ie, fading) 1
Reduction in blue proteoglycan ECM staining with presence of collagen staining (up to 50% of area) 2
Loss of blue proteoglycan ECM staining and/or dominance of collagen staining (>50% of area) 3
AF morphology (AB-PSR)—Figure 13
Well-organized, well-defined, uniform collagen lamellae form concentric half-ring arcs throughout entire AF 0
Mild disorganization/delamination of collagen fiber lamellae with some disruption or loss of concentric layers (<25%) 1
Moderately disorganization/delamination of collagen fiber lamellae with progressive disruption or loss of concentric layer (25-75%) 2
Complete disorganization/delamination/collapse of AF; almost all concentric collagen lamellae are severely disrupted or lost (>75%) 3
AF Cellular (H&E) and ECM metaplasiac (AB-PSR)/distinction between AF and NP—Figure 14 and Figure 15
Clear distinction between AF and NP tissue with intense blue proteoglycan ECM staining in NP: 0
spindle-shaped fibrocytic nuclei populate AF with no or rare individual metaplastic cells resembling NPCs in the AF
Distinction less clear: loss of annular-nuclear demarcation. Proliferation of cells resembling NPC's is focally restricted (ie, perilesional) or 1
mild proliferation with >75% lacunae containing single fibrocytic nuclei (ie, rare to few small NPC-like clusters)
Distinction less clear: loss of annular-nuclear demarcation. Regionally extensive perilesional proliferation of cells resembling NPC's or 2
moderate proliferation involving 25%-50% AF lacunae that contain more than one cell nucleus (ie, frequent NPC-like clusters).
Poorly or no discernable annular-nuclear demarcation. Regionally extensive perilesional proliferation of cells resembling NPC's or 3
moderate proliferation involving >50% AF lacunae that contain more than one cell nucleus (ie, frequent NPC-like clusters).
Tears and cleft formationd in the AF/NP (H&E, AB-PSR)—Figure 13 and Figure 15; Figure 18
Absent 0
Rarely present. Few small concentric-oriented clefts confined within a single region of the AF (ie, inner, mid or outer layer), 1
without formation of transverse clefts
Larger and more numerous clefts or formation of transverse clefts that bridge an adjacent AF region (ie, inner-to-mid, or outer-to-mid) 2
Abundantly present, large clefts; formation of transverse clefts that bridge more than one layer of the AF; or 3
presence of cellular infiltration/neovascularization
CEP morphology (Saf O/FG, AB-PSR)—Figures 16, 17, and 18
Uniformly thickness and contour without disruption 0
Focal to multifocal thickness and/or contour irregularities without disruption 1
Focal endplate disruptions (thin clefts, fissures or chondroid ECM extrusion up to 10% total area 2
with or without irregularities in thickness/contour
Frequent or large areas of endplate disruption with chondroid ECM extrusion >10% total area 3
with or without formation of Schmorl's nodes
Bone modeling—at the external AF/bone interface (H&E)—Figures 7, 8, and 19
Absent 0
Limited, bone remodeling that forms pointed EP contours or small nodular exostoses that to not impinge on AF 1

(Continues)
14 of 36 LEE ET AL.

TABLE 4 (Continued)

Bone modeling—at the external AF/bone interface (H&E)—Figures 7, 8, and 19

Larger vertebral exostoses that protrude cranio-caudally and impinge on outer layers of the AF 2
Abundant new bone formation with partial to complete bridging spondylosis 3

Notes: All categories apply to all species reviewed herein; however, modifications of the scheme or analysis protocols (eg, additional histochemical or
immunohistochemical stains) may be warranted, depending on the induction method employed to create a lesion (eg, surgical AF disruption vs chemical-
induced nucleotomy) or the research question (eg, for processes concerning angiogenesis, nerve ingrowth and inflammation, repair strategy employed).
The reader is referred to Figures 7-18 for representative bright field microscopy examples of the different scoring categories. It remains to be determined
when reactive degenerative changes included in the scoring may reflect a regenerative response in the context of a regenerative therapy.
Abbreviations: AB-PSR, Alcain blue/picrosirius red stain; AF, annulus fibrosus; CEP, cartilaginous endplate; ECM, Extracellular matrix; H&E, hematoxylin &
eosin stain; NP, nucleus pulposus; NPC, NP cells; Saf O/FG, safranin O/fast green stain.
a
Notochordal cells are large vacuolated cells present within embryonic development in the notochord and once the IVD has developed, reside within the
NP. There are slight differences in the morphology of the NCs between species (pigs, non-chondrodystrophic dogs; Figure S5), whereas some species (sheep, goat)
do not have vacuolated NCs at the ages they are used in animal experiments therefore this component of the grading scheme should not be applied to those
models. The non-vacuolated smaller NP cells residing within the core of the IVD are termed NP cells (NPCs).
b
Note that NPC clusters as presented in this article are analogous to “chondrocyte proliferation” as presented in other articles (eg, Bergknut et al97 and
Boos et al94).
c
Note that cellular and ECM metaplasia presented in this article as NPC-like is analogous to the terminology “chondroid metaplasia” or “mucous
degeneration” as presented in other references (eg, Bergknut et al97 and Boos et al94).
d
Tears and clefts needs to be distinguished from processing artifacts. Nonartifactual tears and clefts commonly occur within the context of a degenerative
milieu characterized by cellular and ECM metaplasia (eg, AF cells obtain an NPC morphology with increased AB staining of the surrounding ECM and/or
presence of NPC clusters, cell loss or cell necrosis). Artifactual clefts tend to have sharp, jagged margins within regions of healthy cells and ECM. Scoring of
tears and clefts may have to be considered within the context of method of lesion induction (ie, outer annulus surgical defect vs chemical nucleotomy).

Mounting and staining can be evaluated by individuals with red-green color vision deficiency,
Thin paraffin-embedded sections (5-10 μm) should be mounted on the most common type of color-blindness. Because the alcian blue-
positively charged glass slides (Supporting Information, Appendix S1). picrosirius red, safranin O or toluidine blue-fast green staining intensity
Due NP tissue's tendency to swell during the rehydration steps, which are relative and can vary between individuals of the same species, ide-
can cause wrinkles and reduce its adherence to the slide resulting in ally staining should be conducted in a batched fashion, where all sam-
either poor quality sections (eg, folds) or even loss of sections, adher- ples within a set are stained simultaneously under the same conditions
ence to the slide can be enhanced by drying the sections overnight at (eg, concentration, timing, and temperature), and compared with a
37 C prior to further processing, or precoating the slides with species-matched internal control.91 For protocols and specific examples
gelatin.90 of these histochemical stains, see Supporting Information, Appendix S1.
The standard histological staining for the general assessment of
tissue structure and cell morphology is hematoxylin/eosin (H&E) that Histological grading scheme
stains cell nuclei blue/purple (hematoxylin) and the collagenous ECM Several histological grading schemes have been described in the litera-
shades of pink (eosin) (Figures 7 and 8). Areas with high proteoglycan ture for the different species (Supporting Information, Appendix S1),
91
content (eg, NP) stain blue-gray. Suggested histochemical stains for but to date there is no consensus on a histological grading scheme
the evaluation of the IVD ECM are Alcian blue/picrosirius red that is appropriate for all IVD large animal models. Therefore, to pro-
(AB-PSR)92 and safranin O/fast green.91 Alcian blue binds with nega- mote standardization and facilitate cross-species comparisons of
tively charged macromolecules in the ECM through electrostatic results, the authors have surveyed and reviewed the human and vet-
forces, thus stains sulfated and unsulfated GAGs blue at a pH of erinary scientific literature to scrutinize all available schemes (includ-
2.5.92 Picrosirius red is an elongated birefringent molecule that com- ing the human Boos94 and Rutges95) to synthesize a single
plexes with collagen fibers and exploits the anisotropic properties of comprehensive histological grading scheme that can be applied to
collagen fibers, staining fibers orange to red under standard bright commonly used large animal models (dog, pig, sheep, and goat;
field illumination, and enhancing their birefringence under polarized Table 4). The authors sought within the individually published
light, thus allowing for clear identification of collagen fiber structural schemes40,96–100 comparable features of IVD degeneration shared
91,93
organization or depletion. Safranin O is a cationic dye that binds across all species and incorporated scoring methods that provide uni-
to the carboxyl and sulfate groups (negative charged) of GAGs.91 Fast formity as well as simplicity so that tissues could be evaluated by
green serves as a counterstain that stains GAG-depleted areas green. researchers without specialized pathology training, degrees, or board-
Safranin O/fast green stain is commonly employed in cartilage pathol- certifications.
ogy and as such could be employed to further study CEP/BEP histo- The development of a semiquantitative four-point grading
pathology. Toluidine blue, a cationic dye that stains GAGs with more scheme from 0 to 3 (Table 4; Figure 7-18) separates scores into a
intense staining due to its higher sulfur affinity than safranin O,91 scheme that corresponds to none, mild, moderate, and severe. Pro-
counter-stained with fast-green is an alternative staining method. Tolui- vided images are accompanied by different histological subscores to
dine blue-fast green staining provides an additional advantage in that it demonstrate histologic changes that are not necessarily dependent on
LEE ET AL. 15 of 36

F I G U R E 7 Composite depicting progressive stages of induced IVD degeneration in goat (left images, H&E) and sheep (right images bright-field
microscopy AB/PSR); mid-sagittal lumbar spine sections, dorsal aspects of discs oriented to the left). IVD degeneration was enzymatically induced by
chondroitinase-ABC† disc injection in 3-year-old, skeletally mature goats 12-14 weeks prior to harvest. Nuclectomy was performed in 2-4 year-old,
skeletally mature sheep 4-5 months prior to harvest. †Amsbio, Cambridge, MA. Grade 0 (normal, healthy controls) IVDs have a distinct nucleus
pulposus (NP) that stains blue on H&E and deep turquoise on AB/PSR with a well-defined NP-annulus fibrosus (AF) interface. Hemi-concentric well-
defined lamellae of the AF stain eosinophilic with H&E. The cartilage endplates (CEP) are thin uniform contiguous bands. The bony endplates (BEPs;
stained pink on H&E and dark red on AB-PSR) are comprised of uniform, regularly spaced arrays of bone trabeculae that are often flanked by
persistent cartilage growth plates of the vertebral bodies (arrowheads). Section artifacts include clefts (short arrows) within the NP, AF or CEP
interface that have sharp margins with abrupt transition to clear space devoid of degenerative matrix or cells (see Figure S2). Grade 1 (mild disc
degeneration) IVDs show loss of basophilic/turquoise staining and reduced definition between the NP and AF that corresponds to reduced NP
glycosaminoglycan (GAG) content and chondroid metaplasia of the AF, respectively. Inner to mid lamellae of the AF in this region (arrows) contain fine
clefts spanned by proteoglycan-rich fibrillated collagen corresponding to microtears (black frame, see Figure S2). CEPs retain their discrete, uniform
contour but the trabecular bone of flanking BEPs has compacted (ie, endplate sclerosis). Grade 2 (moderate disc degeneration) changes show almost
complete loss of NP basophilia (H&E) with poor definition of NP-AF interface. Inner to mid lamellae of the AF in this region (arrows) show larger,
more extensive clefts in the AF (black frames, see Figure S2) and progressive compaction of flanking trabecular bone. In the sheep, although
proteoglycan staining of NP persists, there is loss of the inner to mid AF layers with NP protrusion into this region (arrows) that coincides to
narrowing of the disc space and regional endplate thickening. Grade 3 (severe disc degeneration) changes include complete loss of NP basophilia
(H&E) and more severely depleted NP GAG staining (AB-PSR) with loss of NP architecture and poor discernment of NP-AF interface. There is a
collapse of IVD space and clefts within the distorted, degenerate NP (black frame, see Figure S2) and extrusion of degenerate chondroid IVD material
beyond the CEPs that extends to the flanking cartilage growth plates of the vertebral bodies (arrows). The chondroid material (white arrow) stains
dark blue on AB/PSR (see Figure S2). A triangular cleft at the dorsal aspect of the endplate (asterisk) is an artifact of sectioning. In the sheep, there is
herniation of the ventral annulus with osteophytes that span cranial and caudal vertebral bodies (arrowheads). †Amsbio, Cambridge, MA
16 of 36 LEE ET AL.

F I G U R E 8 Low magnification composite of naturally occurring IVD degeneration. H&E and AB-PSR stained bright-field photomicrographs of
mid-sagittal sections of dog IVDs in various stages of naturally occurring degeneration. Mild shows degeneration and loss of glycosaminoglycan
(GAG) staining within the nucleus pulposus (NP; asterisks) with increased GAG staining (chondroid metaplasia) of the annulus fibrosus (AF) and
loss of definition. Moderate shows progressive NP and AF matrix degeneration with the production of small nodular exostoses (ie,
syndesmophytes) at the dorsal margins of the AF (arrows); the ventral aspect of the H&E panel contains section artifact (arrowheads) and cannot
be evaluated. Severe shows collapse of the IVD with partial dorsal and ventral extrusion of degenerate NP and AF matrix (arrowheads) and
ventral bridging exostoses (arrows) compatible with intervertebral ankylosis (eg, self-fusion). End-stage IVDs show more complete extrusion of
degenerate IVD matrix dorsally (arrowheads) and ventrally (asterisks) with complete collapse of IVD space and foci of bone-to-bone contact;
ventrally, a large exostosis (arrows) surrounds the extruded IVD material (arrows). Reprinted with permission from Spine152 and further modified
to serve the needs of demonstrating naturally occurring IVD degeneration changes. Note that these are representative images from a naturally
occurring disc degeneration model and not from a large animal model where disc degeneration is induced either chemically or surgically. In the
canine species the growth plates close and are as such absent in these sections indicating that they are from skeletally mature dogs

or restricted to any given species, breed, age, method of induction, or species, including human, would need to be considered for further
time frame. Since there is not yet a consensus on which parameter improvement of the scoring scheme in the future. Consultation with a
might contribute more or less to the overall degenerative process or highly experienced IVD expert or veterinary pathologist can provide
clinical outcome, in this newly developed scheme, all categories have important insights and assistance in modifying or developing an
a score of 0-3 so that all parameters contribute equally to the final appropriate scheme to best fit the model. For example, in models that
score. The authors recognize that histologic scoring system without develop significant inflammation, given the limited inflammatory reac-
the use of quantitative image analysis is inherently subjective, and tion typically present within IVD degeneration where ECM extrusion
therefore grading schemes that showed good consistency and repeat- is not present, inflammatory infiltrates may indicate either postsurgical
ability for intra- and interobserver scores97 were included. The infection or a type of hypersensitivity reaction specific to the surgical
authors emphasize that this uniform grading scheme has benefits in modifications or implant devices/materials.101 In this case, the authors
evaluating components of the IVD (NP, AF, and CEP) as separate enti- suggest that researchers develop an additional component to the
ties with respect to their architecture and cellular/ECM components, grading scheme that encompasses the spectra of inflammatory
and can be used to evaluate changes associated with different experi- changes specific102 to the model. Moreover, note that new bone for-
mental models (ie, lesion induction method). The authors recognize mation and subchondral bone sclerosis are best determined and/or
that grading schemes may not apply, in part or in total, to certain quantified via imaging (plain radiography and μCT), since for histologi-
experimental models or research questions and that modifications of cal analysis only one specific location is analyzed, and therefore the
published grading schemes are common and often appropriate. New severity or extent of vertebral osteophytosis and sclerosis can be mis-
insights on IVD histopathology and comparative analysis across interpreted. Other aspects of IVD pathology not included in this
LEE ET AL. 17 of 36

F I G U R E 9 Nucleus pulposus
notochordal cell depletion. H&E
stained photomicrographs of
progressive notochordal cell
(NC) depletion within the nucleus
pulposus (NP) in the dog IVD.
Grade 0 shows that >90% of cells
within the NP comprise a mixture
of individualized and clusters of
small and large vacuolated NCs
(arrows). Grade 1 shows 30%-
40% NC depletion replaced by a
population of nonvacuolated cells
having large, eccentrically
located, spindle-shaped nuclei
(arrow). Grade 2 shows
degeneration and loss of nearly
all vacuolated notochordal cells
with remaining cells having
centralized round hyperchromatic
nuclei within lacunae (arrows).
Grade 3 shows the complete loss
of vacuolated NCs replaced by
similar cells (arrows) as described
for Grade 2

uniform scheme include, in addition to inflammation, analysis and/or biomolecular analysis. For gene expression analysis,
neovascularization, and nerve invasion/fiber characterization. These RNAlater solution is suitable to protect cellular RNA if the tissue
parameters will need to be evaluated on a case-by-case basis and will cannot immediately be processed or snap frozen. If other bioactive
depend on the research question posed. components need to be measured from tissue extracts, the tissues
can be collected in a purpose-specialized lysis buffer that extracts
Immunostaining and protects the proteins to be measured, homogenized, and stored
Immunohistochemistry (IHC) and immunofluorescence are applied to at 80 C until biochemical analysis to extract growth factors/cyto-
identify specific cellular or extracellular proteins in the tissue. Immu- kines for measurements. Prior to tissue digestion, samples are
nostaining is particularly challenging for large animal studies, as most weighed and then dried (eg, lyophilization or oven) in order to nor-
commercially available antibodies are developed to react with anti- malize biochemical data for wet weight. Thereafter, the supernatant
gens from human and rodent proteins. Cross-reactivity with the par- and pellet can be enzymatically digested overnight, and the DNA
ticular species, including appropriate positive and negative controls, and GAG content can be measured.18,104 A hydroxyproline assay can
needs to be confirmed to ensure specific immunostaining. Further- be used to determine the digested samples hydroxyproline/collagen
more, decalcification and antigen retrieval present additional chal- content.105 Note that these are merely quantitative assays that do
lenges for obtaining high-quality and consistent immunostaining. not provide information on the ECM quality in terms of GAG or col-
Within this context, immunostaining procedures need to be optimized lagen integrity. As alternatives to wet or dry weight for normaliza-
from the first step to achieve satisfactory and reproducible results. tion, results may also be normalized to total protein, cell number, or
The spine researcher undertaking immunostaining is referred to DNA content.
detailed information provided in the (Supporting Information, For gene expression profiling, the specific challenges of
Appendix S1) and to Binch et al reporting on these protocols for cartilaginous tissues like the NP include low cellularity, difficult
human samples.103 homogenization, and ECM rich in glycosaminoglycans that are
copurified during extraction and interfere with the RT-qPCR reac-
tion.106 Tissue homogenization can be achieved with variable
1.8 | Biochemical, gene, and protein analysis methods utilizing either mincing, cryo-sectioning, or cryo-pulveriza-
tion. RNA extraction techniques can be optimized to ensure satisfac-
For this analysis, the NP and AF tissue (from 1/4 IVD; Figure 2B) is tory RNA yield and quality from GAG-rich tissues, for example, via
carefully separated, cut into small pieces, snap frozen in dry ice or additional enzymatic digestion steps upon collection of the tissue or
liquid nitrogen, and stored at 80 C until required for biochemical high salt coprecipitation of GAGs during the RNA extraction
18 of 36 LEE ET AL.

F I G U R E 1 0 Nucleus
pulposus cell clusters and matrix
staining. H&E-stained (left) and
AB-PSR (right, bright-field
microscopy) stained
photomicrographs of grades
corresponding to progressively
increasing nucleus pulposus
(NP) cell clusters (arrows) and loss
of proteoglycan matrix staining in
the goat and sheep IVD. Grade
0 shows individual NP cells
(arrows) evenly dispersed in a
homogeneously blue-gray (H&E,
sheep) and dark turquoise
(AB-PSR, goat) extracellular
matrix. Grade 1 shows small,
scattered NP cell clusters of
2 nuclei (arrows) embedded in a
light gray-pink extracellular matrix
(H&E, sheep) and heterogenous
light-dark blue matrix staining
(AB-PSR, goat). Grade 2 shows
more frequent NP cell clusters of
>8 nuclei (arrows) and increased
heterogeneity of matrix staining
that is hypereosinophilic (H&E,
sheep) to dark red (AB-PSR, goat)
between 25-50% of the total NP
area. Grade 3 show presence of
huge (>15 nuclei) NP cell clusters
(arrows) that contain pyknotic and
viable nuclei (H&E, goat) and
predominance of dark red matrix
staining (AB-PSR, goat)

process.107–109 In the case of limited starting material, unbiased pre- correct normalization of these profiles is essential. To date, while sets
amplification of the target sequences may provide an alternative.18,110 of stable reference genes have been studied for some human, dog and
Although the authors anticipate that these techniques may also be goat IVD tissues,18,106,111 they remain to be determined for most spe-
suitable for other species, age groups and IVD levels than those cies and tissues. As gene expression levels are not always correlated
employed in the reported work, the RNA isolation method would with protein levels, parallel quantification of cytokine, chemokines,
need to be validated to make final conclusions. To normalize target ECM molecules, and other proteins of interest in the tissue extracts
gene expressions, it is suggested to use more than one stably employing Western blots and/or commercially available testing kits,
expressed reference gene. Gene expression profiling is a tool that is ELISAs or multiplex immunoassays is, in the authors opinion,
often used to address the findings from a mechanistic perspective and advantageous.
LEE ET AL. 19 of 36

F I G U R E 1 1 Nucleus pulposus cell clusters with cell necrosis. H&E stained photomicrographs of cell clusters within the nucleus pulposus in
the dog IVD. Grade 0 shows individualized vacuolated notochordal cells. Grade 1 shows conversion of vacuolated notochordal cells into
nonvacuolated cells resembling chondrocytes within lacunae with occasional two-cell clusters (arrows). Grade 2 shows presence of larger cell
clusters (double arrows) having >8 nuclei per cluster in addition to scattered cell necrosis (arrowheads) characterized by nuclear fading
(karyolysis), pyknosis, and fragmentation (karyolysis). Grade 3 shows the presence of huge cell clusters (double arrows) having >15 nuclei per
cluster along with cell necrosis (arrowhead). Typical cell loss and necrosis is further illustrated in Figure 12. Of note, cell clusters are typically
considered a hallmark of degeneration but have also been related to cellular proliferation. As such, within the context of therapeutic strategies
studied, cell clustering may reflect an attempt for regeneration rather than a degenerative reactive response180

1.9 | Clinical scale models and have not yet been properly validated specifically for back
pain related to IVD degeneration.
At present, there are no methods established solely for the purpose of Recognizing pain and assessing its intensity and duration are
assessing or quantifying spinal pain in large animal models. However, essential to ethical hypothesis-driven research as clinically relevant
behavioral and physiologic parameters can be used to evaluate the outcome measures, as a potential source of experimental error, and as
degree of pain and responses to analgesia or therapies.112 Behavioral or a key component of animal welfare.114 In addition to recognition of
physiologic parameters that can produce quantifiable outcome measure- pain, pain management with effective analgesia and humane end-
ments can be included a priori during study design. In this article, we pro- points for uncontrollable pain during an experimental study are
vide an overview of behaviors and clinical parameters that may be required components of the 3R principle (Replacement, Reduction, and
included to evaluate research outcomes and instruct future recommen- Refinement). Pain is the result of complex combinations of physiologi-
dations for relevant preclinical outcome measures in large animal models. cal, immunological, cognitive, and behavioral parameters. As such, pain
may be generated by causes other than known interventions, includ-
ing husbandry and aging related factors that could skew experimental
1.9.1 | General pain assessment of large animal data. Therefore, behavioral indices (Table 5) and careful extrapolation
models from the clinical presentations should be considered when assessing
pain in animals.114,115
To date, there is a relative paucity of data regarding assessment of Behavioral indicators of pain are often markedly different for lab-
pain in preclinical large animal models (Figure 1) and even in animal oratory animals when compared to domesticated animals of the same
models in general8 despite the fact that pain is the most clinically rele- species. As such species-specific pain indicators are required to suc-
vant parameter for patients suffering from spinal conditions. While cessfully incorporate behavioral and/or nociceptive indicators as mea-
small animal pain models are available113 based on a single IVD level surable data. Perception of pain, nociceptive response generators, and
intervention, they remain largely qualitative for preclinical large animal clinical presentations of pain can be species dependent. There are no
20 of 36 LEE ET AL.

F I G U R E 1 2 Nucleus
pulposus cell death characterized
by karyolysis (empty lacunae) and
pyknosis (shrunken nuclei). H&E-
stained photomicrographs in
goats showing progressive cell
loss (arrowheads) and necrosis
(arrows) for Grade 0 (none),
Grade 1 (<25% cells), Grade
2 (25%-50% cells), and Grade
3 (>50% cells). Karyolysis: High
(40) magnification H&E stained
photomicrographs feature
karyolysis characterized by
lacunae devoid of nuclei (white
arrowhead) in contrast to the
adjacent viable cell (white arrow)
containing a distinct round
nuclear membrane with single
nucleolus. Pyknosis is
characterized by cells having
shrunken hyperchromatic nuclei
(white arrowheads) in contrast to
viable cells (white arrows) having
open nuclei containing discrete
nucleoli; karyorrhexis (ie, nuclear
fragmentation) another
morphologic manifestation of cell
death is not shown

generally accepted methods or objective criteria for assessing pain in nociceptive mechanical stimulation with incrementing levels of force,
large animal models employed for IVD regeneration. However, a where upon the withdrawal threshold is determined. Depending on
detailed neurologic examination by a veterinary expert can help local- severity, duration, and degree of neurologic deficit, Von Frey thresh-
1
ize spinal pain. Due to the highly specific nature of research investi- olds may increase or decrease.
gations, the optimal pain scale for a given experiment should ideally Gait analysis can be another quantitative tool for lameness120
be as objective, standardized, and repeatable as is possible based on caused by IVD-related back pain. There are several different
species, methods, and resources as determined a priori using generally methods to perform gait analysis including kinematic gait analysis,
accepted or validated methods. kinetic gait analysis (force plate), and temporospatial gait analysis
(pressure sensing walkways). Pain can be highly variable among
species, strain/breeds, and even individuals in the same
1.9.2 | Quantitative measures of pain cohort.121,122 It can also vary greatly based on study-specific
interventions. As such, animals are acclimatized to the environ-
While limited, there are analyses that can produce objective quantita- ment and the measurement method and trained for consistent
tive data assessing pain and/or functional deficits in large animal gait analysis; baseline measurements based on at least three-day
models. One suggested measure is the Von Frey test, which has been recordings prior to the intervention can correct for individual vari-
used widely and validated in several species including dog, pig, sheep, ances. Objective gait analysis specifically for low back pain has
young goats, and humans116 to assess the mechanical nociceptive not been applied in large animal models and is in its infancy even
threshold.117–119 Von Frey filaments are applied to the skin to deliver in the veterinary clinical field.
LEE ET AL. 21 of 36

F I G U R E 1 3 Annulus fibrosus (AF) morphology and tears and cleft formation in the AF/nucleus pulposus (NP). AB/PSR stained low
magnification bright-field photomicrographs of progressively severe AF tears in sagittally sectioned goat IVDs from the same model featured in
Figure 7. Outer AF of the ventral aspect of the disc is oriented to the left and inner AF/NP to the right. Grade 0 AF fibrous lamellae are intact,
uniformly aligned and stained; dark blue linear streaks (arrow) are staining artifacts. Grade 1 AF tears form irregular clefts within the inner annulus
in areas of mild matrix degeneration (arrows). Grade 2 AF tears show clefts extending from the outer NP through inner (black arrow) to mid (white
arrow) annulus. Grade 3 AF tears comprise large irregularly divergent clefts (asterisk) within a large region of degenerate matrix that extends from
inner to outer lamellae. One cleft extends along the interface with the outer endplate (EP) and is partially filled with proteoglycan-rich fibrillated
matrix (white arrow). High (10) magnification inset of H&E stained AF from the framed area show remnants of AF lamellae (arrowheads)
separated by a fibrous stroma containing many vascular profiles (arrows); scale bar = 100 μm

Lastly, general activity can be monitored using commercially avail- Therefore, pain assessment categories need to be developed based on
able activity monitoring devices,123 external/implantable telemetry,124 individual projects with considerations of the Pig Grimace Scale and other
and/or video analysis.125 These methods provide ways to quantify general physiological and behavior cues127 for pain in pigs.
general activity level which is expected to decrease with increased Even though their behavior is different, general signs of pain in labo-
pain perception in animals. Especially, external/implantable telemetry ratory sheep and goats are similar. Sheep have been reported to tolerate
has been used widely in large animals in various research settings as severe injury without overt signs of pain or distress.114 Pain can lead to
some telemetry devices have capabilities to record and store detailed the cessation of rumination, eating, and drinking. Curling of the lips has
and continuous physiologic parameters such as heart rate, ECG, respi- been observed as a reliable indicator of pain. Goats are more likely to
ration, and glucose levels along with activity level. There are advan- vocalize with pain and may grind their teeth. Increased agitation (foot
tages and disadvantages of each activity monitoring method that stomping) and frequent changes in posture could indicate pain. Especially
pertain to the purpose of study. in dairy breeds of goat and sheep, rapid decrease in milk production can
be observed along with body weight loss and decreased body condition.
The Sheep Grimace Scale (SGS) has been established for use in laboratory
1.9.3 | Species-specific recognition and assessment sheep following orthopedic procedures (unilateral tibia osteotomy)128
of pain and pain generated by hoof abnormalities.129

Assessment of pain in laboratory pigs can be challenging as mature pigs

do not tend to show markedly altered behavioral indicators. Pain assess- 1.9.4 | Recognition and assessment of pain in dogs
ment is better established for piglets as they are employed extensively in and clinical grading
the production and research settings. The Pig Grimace Scale (PGS) has
been developed for use in piglets.126 To date, there are no published Dogs in pain115 often appear stiff or are unwilling to move when
behavior indices specifically for orthopedic experimental models in swine. aroused. They may stop greeting research or husbandry staff members
22 of 36 LEE ET AL.

F I G U R E 1 4 Cellular (H&E) and matrix metaplasia (AB-PSR, bright-field microscopy) of the annulus fibrosus (AF)/distinction between annulus
fibrosus and nucleus pulposus (NP). Alcian Blue-Picrosirius Red (AB-PSR)-stained goat IVD low magnification photomicrographs taken at similar
locations of the disc demonstrate progressive loss of AF lamellar organization and definition of AF-NP interface. Corresponding high
magnification H&E stained photomicrographs showing fibrous to chondroid cell metaplasia. Note that scores for AF lamellar architecture vs
AF-NP matrix definition/cell metaplasia may segregate independently. Grade 0 shows well-defined red collagen lamellae with a sharp transition
to turquoise glycosaminoglycan (GAG) staining at the AF-NP interface; AF fibrocytes are uniformly spindle to stellate shaped. Grade 1 shows
some loss of definition of collagen lamellae with less clear staining distinction at the AF-NP interface; AF cells demonstrate more rounded
profiles, but retain single nuclei within lacunae. Grade 2 shows distortion of AF collagen lamellae with turquoise GAG staining extending into
outer lamellae; 25%-50% AF cells contain 2 nuclei within lacunae. Grade 3 shows complete disorganization with collapse of the AF and disc space
(arrowheads) demarcate remnant lamellae of outer annulus and arrows demarcate markedly distorted endplate margins). The mid to inner AF
lamellae have been replaced by collagen-rich fibrovascular tissue admixed with islands of turquoise GAG-rich material containing cells that form
multinucleate clusters within lacunae (H&E, arrows), compatible with chondroid cellular and matrix metaplasia
LEE ET AL. 23 of 36

F I G U R E 1 5 Cellular and matrix metaplasia of the annulus fibrosus (AF) with tears and cleft formation in naturally occurring IVD disease. Low
and high magnification AB-PSR-stained bright-field photomicrographs of AF degeneration and tearing within a spontaneous IVD disease model of
skeletally mature non-chondrodystrophic dogs. The top left panel (Grade 0/1) shows an intact AF without tears (Grade 0), although there are
small scattered cleft-like artifacts of processing (asterisks); there is mild matrix degeneration based on the increased AB staining of the mid-to-
outer annulus layers (Grade 1 cellular and matrix metaplasia). Higher magnification of Grade 0 cellular and matrix metaplasia shows predominant
fibrillar collagen matrix containing single spindle-shaped cells (arrows). The top right panel (Grade 3/3) shows large tears extending through
multiple layers of the AF (Grade 3 tears) surrounded by degenerate (ie, dark blue, GAG-rich) matrix with strong AB staining in the outer AF layers
(arrowheads) and occasional bridging of the clefts by frayed collagen fibers; the majority of cells have nucleus-pulposus cell (NPC) morphology
with nuclear clusters (Grade 3 cellular and matrix metaplasia). Higher magnification (Grade 3) demonstrates the loss of fibrillar collagen replaced
by proteoglycan-rich matrix with predominant NPC-like cells within large round lacunae and frequent clusters with >4 nuclei (yellow arrows;
Grade 3 cellular and matrix degeneration)

and may consistently retreat to the extents or protected areas of their simultaneous benefit to pets and pet owners. Both
housing. Dogs can also exhibit pain by being restless, agitated, and chondrodystrophic (CD) and nonchondrodystrophic (NCD) breeds
hypersensitive. Some dogs may attempt to bury or hide food when pain- spontaneously develop IVD disease. The onset and anatomic loca-
ful. Dogs in pain may bite when painful areas are palpated, which can be tion of IVD disease are different and this provides opportunities to
mistaken for aggressive behavior. Vocalization can be evident; however, model different IVD disease phenotypes. A brief overview of the
excessive barking is unlikely in the presence of pain. Growling, whimper- most common clinical entities in canine veterinary patients is out-
ing, or howling are indicators of pain in dogs; however, neither lack of lined in Table 6. Researchers are encouraged to discuss with veteri-
vocalization nor excessive vocalization is a reliable indicator of pain. nary specialists in identifying the most appropriate clinical entity to
There is no published or validated grimace scale for laboratory dogs to be employed in their translational endeavor once the intended
the authors' knowledge. However, there are several validated pain approach has been proven to be safe and have sufficient efficacy.
assessment methods for use in client-owned dogs including The Mel- In addition to the pain assessment methods, in clinical patients,
bourne Pain Scale, MPS; Colorado State University Canine Acute Pain gait/posture analysis can be performed to monitor changes due to
Scale; The Canine Brief Pain Inventory, CBPI; Helsinki Chronic Pain IVD perturbations. Pain associated with cervical and
Index, HCPI; Canine Orthopedic Index, COI; Glasgow Composite Pain thoracolumbar IVD disease (spontaneous or induced) can be
Scale, GCPS),130–133 which may have utility for application to laboratory assessed via neurologic grading of IVD disease. Table 7 shows the
dogs when the differences are considered. most commonly used clinical scales when assessing IVD disease in
Within the context of large animal models, spontaneously dogs. As the clinical grade increases, clinical signs become worse
occurring disorders of the spine in client-owned dogs are common with decreasing normal functions. Also, higher grades indicate
and can serve as effective models for translational research with severity of impacts on PNS and/or CNS and worse prognosis for
24 of 36 LEE ET AL.

F I G U R E 1 6 Sheep-cartilage
end-plate (CEP) morphology
(AB-PSR). AB-PSR stained bright-
field photomicrographs of
progressive CEP disruption in the
goat IVD. Grade 0 shows intact
CEP of uniform contour and
thickness. Grade 1 shows regional
thinning or the CEP (arrow).
Grade 2 shows multifocal
disruption of the CEP <10% of
the total area (arrows) with
limited extrusion of IVD matrix
into the bony endplate. Grade
3 shows a focal small disruption
(arrowhead) adjacent to a large
regional disruption of the CEP
involving >30% of total area with
extrusion of IVD matrix deep
within the bony endplate (arrows).
Note the Grade 3 image was
obtained at half magnification of
Grades 0-2 in order to
demonstrate the extent of the
endplate disruption

return to normal functions. This particular scoring scheme can be As such, where possible involvement of both sexes in studies will
extrapolated to the other large animal models. allow discernment of this effect. In small animal models, strains of dif-
ferent genetic backgrounds are beginning to be employed for a better
understanding of IVD aging and pathology135,136 and genetic back-
1.10 | Perspective—future outlook ground has been shown to play a role in rodent models137; however,
these remain relatively unexplored in large animal models. There is
The current manuscript presents a comprehensive tool box for con- the well described role of the FGFR4 retrogene in the canine
ducting in vivo large animal studies of IVD treatment with the focus species,138 and livestock animal models typically involve genetic poly-
on methodology and complementary outcome parameters (key points morphisms or mutations affecting skeletal developments. The IVD
summarized in Table 8). Moving forward, key areas for refinement and may potentially show similar genetic traits, but only their effects on
expansion with specific focus on the comprehensive tool box are dis- bone phenotype have been described so far. An example is the quan-
cussed below to facilitate comprehensive analysis and a better under- titative trait locus mapping to the growth differentiation factor-8
standing of the underlying pathology and model-specific differences. (GDF8 or myostatin) gene reported in Texel sheep139,140 and in cross-
The most significant species-specific confounders affecting out- breeds like the Swifters, with GDF8 shown to inhibit chondrogenesis
comes and their utilization include sex and breed-dependent genetic by suppressing SOX9 and collagen type II expression.141 Within this
background. IVD (patho)physiology is potentially influenced by sex context, there are reports of spontaneous NP18,142–146 and AF147,148
hormone differences and as well as related to the species-dependent repair in large animal models. While those may partly relate to IVD
reproduction cycle, particularly considering the well described effects size and as such to nutritional status of the IVD,149 to date there is
134
of sex on bone (patho)physiology within the field of osteoporosis. limited understanding of the differences in intrinsic repair capacity
LEE ET AL. 25 of 36

F I G U R E 1 7 Cartilaginous endplate (CEP) and bony endplate (BEP) morphology (Safranin O/Fast Green). Safranin-O/Fast Green stained low
magnification photomicrographs of progressive CEP disruption in the sheep model where a surgically induced annulus fibrosus (AF) defect was
implanted with a composite tissue engineering repair device (asterisks) and harvested at 3 months postsurgery. Grade 0 shows the intact CEP of
uniform contour and thickness. The vertebral growth plate (black arrowheads) flanks the bony endplate, which in goats and sheep tends to persist
past the age of sexual and skeletal maturity. Grade 1 shows multifocal irregularities in the thickness and contour of the CEP (yellow arrowheads).
Grade 2 shows focal disruption of the CEP <10% of the total area (black arrows). Grade 3 shows regional CEP disruption >30% of total area with
extrusion of blue-green IVD matrix deep within the bony endplate (black arrows)

across large animal species and how these translate into changes in Furthermore, macroscopic grading is ideally complemented by more
the outcomes of the three scales, that is, macroscopic, microscopic, detailed scoring and analysis of the different degenerative processes
and clinical, displayed in this manuscript. at the microscopic or biochemical level. The degenerative processes
With the exception of dogs, IVD geometries have been reported of the different components of the IVD (NP, AF, cartilage, and bony
for goats, pigs and sheep5–7,150 and summarized by Fusellier et al.151 EPs) do not necessarily go together for all the studied parameters and
Furthermore, varying measurement methodologies (eg, application of may also progress differently depending on the model used to induce
axial load) makes comparison between the available studies difficult. IVD degeneration.
Although basic geometries have been documented, anatomical details In this respect, CEP, BEP, and the vertebral body changes are to
are still insufficient. For example, CEP-thickness for dog,152 pig,153 and date relatively understudied while they have been related to IVD
human154 were reported, but not for goat and sheep, and these may be pathobiology,157,158 and prolonged low back pain and disability.43
important when selecting models for investigation of IVD nutrient trans- Macroscopic and microscopic outcome parameters can contribute to
port. Furthermore, potential damage to the CEP (eg, due to freezing or improving the comprehension of BEP/CEP and vertebral body pathol-
enzymes155,156) and its role in degeneration needs to be further investi- ogy and need to be further improved and expanded based on the new
gated. For future studies, one might also consider freezing specimens scoring scheme of human IVD degeneration.159 Within the basic set
51
comparable to cryopreservation for total IVD transplantation. of parameters, macroscopic grading of mid-sagittal planes, even
Macroscopic grading based on gross morphology and MRI imag- though more cumbersome in its processing due to challenges in the
ing are simplified and accepted as five-point scale systems enabling decalcification processes and subsequent paraffin sectioning can
overall scoring and subsequent reporting of IVD degeneration across quantify changes of the CEP and vertebral bone. Furthermore, MRI
large animal models and humans. However, because of their coarse imaging produces indispensable data by allowing longitudinal investiga-
nature, to preserve their efficacy and enable cross-study comparisons, tion of experimental manipulations in large animal models. It enables
liberal adaptations of these schemes are preferably avoided. evaluation of the diffusion capacity of the EP and bone marrow lesions
26 of 36 LEE ET AL.

F I G U R E 1 8 Annulus fibrosus (AF) tears and cleft formation with endplate changes in the canine model with spontaneous IVD degeneration
(AB-PSR stain). High magnification AB-PSR stained bright-field photomicrographs highlighting spontaneous degenerative changes and tearing at the
interface between the annulus fibrosus (AF) and bony endplate (BEP) in the dog IVD. Normal (healthy) shows a proteoglycan-rich segment of the
nucleus pulposus and inner AF without tears and an intact cartilage endplate (CEP), although there are small scattered cleft-like artifacts of processing
(asterisks). Moderate changes include linear clefts arising within a region of reduced proteoglycan matrix staining, compatible with matrix
degeneration, that extend from the AF through the CEP (white arrow). Severe changes in the left lower panel shows longitudinal tears forming wide
clefts (double-headed arrows) within the outer fibrous portion of the AF centered within a focus of proteoglycan-rich degenerative matrix with
irregular thinning of the CEP. Severe changes in the lower right panel show numerous coalescing linear fissures within the AF that form free
fragments (arrowheads) within a region of inconsistent proteoglycan matrix staining; fissures extend from the AF into the BEP (yellow arrows)

of the vertebral bodies associated with degenerated IVDs, known as developed to overcome these limitations in human IVD degeneration
“Modic changes” described in human literature. Modic changes have and have thus far only been limited used in large animal studies. Those
been commonly associated with neck160 and low back pain,161 and with include advanced MR imaging intended to evaluate and establish rela-
IVD degeneration43,162–165; however, identification and classification tions of IVD degeneration with water content, the collagenous ECM
can depend on the MRI field strength,157 and to date they remain rela- structure, or proteoglycan content, including T2 mapping,170T1rho-
18,166,167
tively unexplored in large animal models. These advanced imag- weighted,171 and sodium imaging,172 delayed gadolinium-enhanced
ing parameters could be further explored as they may shed more magnetic resonance imaging (dGEMRIC)173 and the ultrashort time-
information on the therapeutic potency of the employed experimental to-echo (UTE) MRI technique174 to assess the cartilaginous endplate,
strategies in regressing Modic changes. and chemical exchange saturation transfer imaging (CEST)-based
In addition to Modic changes, MR imaging, especially advanced imaging to determine GAGs41 and pH175 changes within the IVD tis-
techniques, assists in improving the in-depth knowledge of IVD sue. Although these techniques still have the limitation of requiring
degeneration while facilitating the 3Rs principles. Furthermore, con- complex equipment and training, it is anticipated that they will play an
sidering that the degenerative changes scored on MRI are a contin- increasingly important role in defining imaging biomarkers that relate
uum rather than the predefined five grades, the subjectivity of a five- to lower back pain and thereby complementing the outcomes of
grade scoring system could be further improved with the aid of artifi- future large animal studies.
cial intelligence and machine learning currently available for human Bony changes can be studied in a qualitative and quantitative
applications.168,169 Sensitivity of standard signal intensity-based MRI manner with plain radiography, and postmortem via μCT or high-
is limited in early grades of degeneration and may lead to a gap in the resolution peripheral quantitative computed tomography (HR-pQCT)
recognition of early tissue alterations occurring in large animal models. thereby providing a more complete representation of bone pathology
To this end, several biochemically sensitive MRI techniques have been of the entire IVD segment studied (eg, focal vs diffuse endplate
LEE ET AL. 27 of 36

T A B L E 5 General assessments and behavioral/physiological correlation may provide an inaccurate representation, especially if
indicators of pain that apply to large animal models no changes are detected in the examined section(s). While guide-
General considerations when conducting assessments of pain in large lines for assessing the bone microstructure have been defined for
animal models rodents,176 these need to be fine-tuned for the purpose of assessing
• The safety of laboratory personnel and animals should always be bone microstructure and the BEP in large animal models. Correla-
maintained as a high priority. tion of μCT with other parameters like histological measurements of
• The same laboratory personnel should conduct assessments of the
the BEP has been reported in the pig177 and goat,100 showing this
animals for the duration of the study. Specific training needs to be
provided so that scoring can be consistent. imaging technique can provide valuable information regarding the
• Baseline assessments of pain should be determined after adequate IVD degeneration process. In the future, morphometrical measure-
acclimation to the experimental housing, environment, and ments provided by μCT could be used to define and evaluate the
conditions.195
overall architecture of the spine segment including IVD height,179
• Observe the animal's behavior without disturbing it first (see
vertebral bone mineral density and architecture, and changes of the
behavioral indices).
• Consider that the animal's behavior can change drastically in the BEP and vertebrae.100,178
presence of a new observer or characteristics (ie, observer's sex, The presented histological grading scheme with consensus for all
perfume, glasses, hat, color of clothes, etc.). four considered large animal IVD model species was based on individ-
• Examine the entire animal prior to focusing on areas of anticipated
ual, validated schemes and helps to compare inter-study results
or perceived pain.
• Initially assess for pain by gently palpating or handling presumed regardless of the experimental model employed (ie, large animal spe-
painful areas before performing any approved provocative cies or method of lesion induction). This newly developed scheme can
maneuvers. be used by researchers without specialized pathology training. More-
• Monitor accurate weights and food consumption for each animal.
over, all categories have a score of 0-3 (healthy, mild, moderate,
Also, note any changes in micturition or defecation.
• Determine and communicate definitive criteria regarding severe degeneration) so that all parameters contribute equally to the
definitions, measures, and levels of pain requiring intervention final score. The authors propose this scheme as a starting point based
along with specific actions in response prior to study initiation. on the available literature on large animal models, although new
Behavioral/Physiological indices insights in IVD histopathology and comparative analysis across spe-
General behaviors cies, including man, should be considered for further improvement of
• Agitation, restlessness the scoring scheme. In the authors' opinion, until more information
• Recumbency and immobility
becomes available or a consensus evolves that correlate specific histo-
• Isolation, social desynchronization
• Aggressiveness logic parameters with clinical disease or outcomes, that the newly
• Loss of appetite (weight loss, decreased urinary/fecal output) developed scheme improves objectivity compared with previous his-
Physiological changes tologic grading schemes which contained unequal categories,40,96,97
• Heart rate enabling certain categories to outweigh the others. When studying
• Respiratory rate
regeneration in large models of disc degeneration, it remains to be
• Blood pressure
determined which changes, currently interpreted as degenerative
• Internal temperature
reactive changes, may be species-dependent relating to a differential
Calls/Vocalization
• Number and duration intrinsic repair capacity. Within this context, specific histological fea-
• Intensity tures, like NP cell clustering, could be the result of a regenerative res-
• Spectral composition ponse.180For histological and immunohistochemical evaluation, the
Postures/gait possibility of automation is increasingly implemented to provide more
• Posture changes (e.g., hunched)
objective assessment compared to the conventional grading that may
• Tonic immobility, stiffness
depend on the individual observer. Nevertheless, automated methods
• Locomotion/Lameness/Gait changes
need to be precisely described and validated, as they are susceptible
Altered behavior/mutilation
• Trembling, Spasm to artifacts. With emerging artificial intelligence and machine learning
• Scratching, licking, biting with localized pain technologies, misinterpretations by automated evaluations will be fur-
• Withdrawal reflex ther diminished.
• Shaking
Employing consistent methodology is imperative for proper
comparative meta-analysis of the generated data to evaluate trans-
latability of therapeutic. However, the relationship between struc-
sclerosis, extent of osteophytosis, or structural abnormalities in the tural tissue changes, function of the IVD, and clinical disease
adjacent vertebral bodies associated with altered remodeling), as remains a topic of debate. With respect to characterizing structural
compared to histology, which is limited to one thin section. Within changes of degenerated and repaired IVD tissues, GAG and collagen
this context, in the histopathological grading scheme, scoring for composition are reported based on the basic set of measurements
bony endplate sclerosis is no longer integrated and scoring for new (ie, stains and quantification by the DMMB and HYP assays). How-
bone formation (eg, exostosis or osteophytosis) without radiologic ever, these methods do not give insight into quality of the ECM that
28 of 36 LEE ET AL.

TABLE 7 Neurologic Grading of IVD disease

Notes: Dog breeds can be categorized into chondrodystrophic (CD) or non-chondrodystrophic (NCD) breeds. CD breeds have distinctive conformational differences that are characterized by short limbs and long
torso length compared to limb strengths. The clinical signs relate to the fact that the diseased IVD is affecting the peripheral nervous system (PNS) or central nervous system (CNS). Localization is done based on
function that will cause urinary bladder to be flaccid, eventually leading to urinary incontinence and
Clinical signs are related to the dynamic movement and compression of spinalcord and nerve roots by
Clinical

Chronic pain with acute episodes, progressive hind limb weakness and muscle wasting. May lead to
progressive sensory and motor deficits in the hind limbs. Nerve root signature (pain apparent on
protruded cervical IVD. Nerve root signature (pain apparent on palpation or traction of the limb)
grade Representation

Cervical-chronic protrusion—C5-C7; Wobbler Syndrome caused by cervical vertebral instability

palpation or traction of the limb). Damage to the lower motor neurons will influence bladder
0 Normal gait
1 Normal gait, cervical or thoracolumbar pain,
hyperesthesia
2 Ambulatory paraparesis with decreased proprioception
3 Non-ambulatory paraparesis (unable to walk or stand
unassisted) with absent proprioception
4 Paralysis (not able to stand or walk), +/ Urinary
incontinence, conscious deep pain perception present
5 Paralysis, conscious deep pain perception absent,
Cauda Equina Syndrome—L6-L7-S1, chronic protrusion197

urinary, and fecal incontinence

Notes: As the clinical grade increases, clinical signs become more severe
and prognosis for regaining affected functions worsens. While this scheme
has been specifically reported for scoring of the neurologic grade in canine
species that suffer from clinical disc-disease, the representation can be
extrapolated to other large animal models involving the species-specific
pain indicators.

is reflected in the type of these macromolecules and lengths of the

fecal incontinence.

proteoglycan and glycosaminoglycan chains present. Therefore,

mass spectrometry, high-performance liquid chromatography, gel
chromatography, sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE), agarose gels, and Western blots, for exam-
ple, help with thorough characterization.181 Alterations in the
NCD

polydispersity of aggrecan182 and fragmentation of small leucine-

rich proteoglycans, biglycan, and fibromodulin183,184 have been
Acute pain that may be accompanied by motor deficits in the hindquarters depending on the level
Clinical signs may include hyperesthesia of the neck and forelimbs, muscle spasms and pain in the

of spinal cord trauma. Damage to the upper motor neuron causes the urinary bladder to be full
and tense which leads to difficulty with expression of the urinary bladder eventually leading to

reported with degeneration. Likewise, hydroxyproline assays can

Spontaneous IVD diseases and common clinical signs in client-owned dogs

potentially be coupled with qualitative analysis (eg, immunostaining)

to determine total collagen content and distinguish among the dif-
ferent types of collagen present. Furthermore, similar analytical
thorough clinical and neurological examination complemented by imaging modalities.

methods apply to de novo engineered tissue to better understand

the full spectrum of the regenerative strategy on proteoglycans, col-
lagens, and other noncollagenous proteins. Finally, even though the
nucleus-endplate integration as well as the interlamellar cohesion
and elastic network were described,185–187 they are seldom studied
in degeneration and regeneration studies.
While it is generally accepted that in progressive IVD degenera-
cervical musculature, paresis/paralysis of all limbs196

tion structural changes compromise IVD biomechanical properties

resulting in the long-term in pain, biomechanical tests are rarely per-
formed when investigating regenerative strategies. This is likely due
Thoracolumbar-Acute herniation—T11-L3

to limited resources or technical support. With the proposed algo-

rithm, nondestructive mechanical evaluation is feasible—with com-
Cervical-Acute herniation—C2-C5

plexity depending on the available equipment. However, comparative

studies of large animals are still needed to deepen the existing knowl-
edge and increase translatability. For example, IVDs with notochordal
urinary incontinence.

cell-rich NPs (pigs, NCD dogs) differ significantly regarding spine bio-
mechanical properties, which are proposed to be in relation with the
different cell types present in the NP and inherent differences in ECM
TABLE 6

composition.188 Nonetheless, it has also been suggested that their

AFs do not differ substantially from the other large animal species.10
CD

However, it has yet to be determined exactly what contributes to

LEE ET AL. 29 of 36

TABLE 8 Highlights of the comprehensive tool box for large animal studies in IVD degeneration and regeneration

Macroscopic scale
• Gross morphological grading is relatively low cost, easy to implement, and provides overall information on IVD macrostructure.
• Imaging via radiography is a rapid and inexpensive modality
• Radiography can be complemented by MRI to obtain the Disc Height Index and detailed information on degenerative changes in vivo.
• T2-weighed images are species-dependent as notochordal cell-rich IVDs have a higher signal intensity.
• Non-destructive biomechanical tests allow functional evaluation of the IVD.
Microscopic scale
• A new four-point histological grading scheme with consensus for all large animal IVD models has been compiled, based on validated schemes for
individual species.
• On mid-sagittal sections all relevant IVD components: the nucleus pulposus, the annulus fibrosus, and the cartilage endplates can be evaluated
• H&E for cell morphology and extracellular matrix strains enable microscopic IVD grading.
Clinical scale
• Following the ARRIVE guidelines and consultancy with veterinary and animal welfare experts helps determine
• pain-related outcome measures, analgesia rescue protocols, and humane endpoints.
• Client-owned dog patients represent different clinical entities that could contribute to translational research and lead to the development of
therapeutic strategies.
• Post-operative evaluations for neurologic state, pain, and other clinical parameters may be species-specific.
• It is beneficial to have a standard assessment scheme that will decrease inter-evaluator variability.

F I G U R E 1 9 Bone modeling at external annulus

fibrosus (AF)-bone interface. Low magnification
bright-field photomicrographs of AB-PSR stained
hemisections of intervertebral IVDs from goat
(induced IVD degeneration) and dog specimens
(naturally occurring IVD degeneration). Grade
0 shows a smooth bony contour (white arrows)
between the AF-bone interface without peripheral
nodular exostoses. Grade 1 shows a focal nodular
exostosis (black arrows) at the periphery of the AF-
bone interface. Grade 2 shows regionally extensive
nodular exostosis that protrudes from the bony
endplate into the AF (white arrows). Grade 3 shows
a large nodular exostosis surrounding the extruded
IVD. Similar histopathological changes have also
been extensively described in the annular lesion
ovine model of IVD degeneration,204 including bony
remodeling of the annular rim205 and are also
depicted in Figure 7, right panel
30 of 36 LEE ET AL.

differences in clinical presentation and lesion progression supported by AO Foundation and AOSpine International. The authors
between CD and NCD dogs. Further exploration of the correlation would like to thank (a) Stacy Turpin Cheavens, MS, CMI for the creation
between ECM composition, IVD biomechanics, and lesion morphology of graphical abstract and Figures 2 and 4 and Chantelle Bozynski, DVM,
between these two spontaneous dog models could help elucidate the DACVP of the University of Missouri Department of Orthopedic Sur-
biomechanical effects of IVD lesions and ECM composition in humans. gery for consultation on the histopathology grading; (b) Nora
Changes at the macroscopic level have been reported to be more Goudsouzian and Dirk Nehrbass, AO Research Institute Davos, for con-
189
prevalent in patients suffering from back pain but they are also pre- sultation on the histopathology section; (c) Theo Smit, PhD, Department
sent in asymptomatic patients.190 As such, in the authors opinion, of Orthopedic Surgery, Amsterdam University Medical Center, the
study designs addressing only the macroscopic and microscopic Netherlands for contribution to Figure 3 (goats); (Niklas Bergknut, DVM,
parameters of IVD degeneration/regeneration do not necessarily PhD, Dipl. ECVN for contribution to Figure 3 (dogs); (d) Marion Fusellier,
determine the clinical relevance of the therapeutic strategy being 
DVM, PhD, of Ecole Nationale Vétérinaire, Agroalimentaire et de l'Ali-
studied. Herein, behavioral and/or pain outcome measures are reliable mentation Nantes-Atlantique, Nantes (ONIRIS) for contribution to
indicators of how effective candidate therapeutic interventions may Figure 5 (sheep); (e) Guy Grinwis, DVM, PhD, Faculty of Veterinary
be. There are better tools becoming available with the advancement Medicine, Utrecht University, the Netherlands for contribution to
of technology such as motion analysis and telemetry, which will per- Figure 8; (f) Sarah Gullbrand, PhD and Robert Mauck, PhD from the
mit access to highly detailed and quantifiable data without interfering McKay Orthopaedic Research Laboratory, University of Pennsylvania,
with the wellbeing of the laboratory animals. Furthermore, collabora- Philadelphia, PA, USA for their contributions to Figures 7, 10, 12, 13,
tion with veterinary experts to establish clinical parameters relevant 14, and 19. The content is solely the responsibility of the authors and
to the spinal pathology being studied, for example, acute does not necessarily represent the official views of the National Insti-
thoracolumbar IVD herniation in the CD dog or progressive lumbosa- tutes of Health.
cral IVD protrusion in the large breed working dog, that are relevant
to the individual studies is valuable. CONFLIC T OF INT ER E ST
In conclusion, this comprehensive tool box for large animal IVD The authors have the following declarations: Naomi N. Lee; Elias
models comprised of macroscopic, microscopic and clinical outcome Salzer; Andres F. Bonilla; Frances C. Bach; Julie B. Engiles; Andrea
parameters can assist in generating a comprehensive data set to Vernengo: No conflicts to declare. James L. Cook: Artelon: Paid con-
address the full spectrum of changes associated with IVD degenera- sultant Arthrex, Inc: IP royalties; Paid consultant; Paid presenter or
tion and regeneration in studies with large animal models. Within this speaker; Research support AthleteIQ: IP royalties ConforMIS:
context, there are opportunities for further refinement and expansion, Research support CONMED Linvatec: Paid consultant Coulter Foun-
including the implementation of the emerging innovative techniques dation: Research support DePuy Synthes, A Johnson & Johnson Com-
we have touched on throughout this article. By employing such an pany: Research support Eli Lilly: Paid consultant; Research support
algorithm, metadata analysis will be less complicated, even across spe- Journal of Knee Surgery: Editorial or governing board Merial: Research
cies, and provide insight into model-dependent differences and how support Midwest Transplant Network: Board or committee member
they could translate to understanding human pathology, improving Musculoskeletal Transplant Foundation: Board or committee member;
translatability and clinical relevance which will ultimately serve IP royalties; Research support National Institutes of Health (NIAMS &
patients suffering from pain and disability due to IVD degeneration. NICHD): Research support Purina: Research support Schwartz Bio-
medical: Paid consultant Thieme: Publishing royalties, financial or
ACKNOWLEDGMENTS material support Trupanion: Paid consultant U.S. Department of
Research and salary support for NNL is provided through NIH T32 grant Defense: Research support Zimmer-Biomet: Research support. Sibylle
OD011126. Elias Salzer, Frances C. Bach, Sibylle Grad, Andrea Grad; Zulma Gazit: JOR Spine—scientific advisory board. Keita Ito: NC
Vernengo, Keita Ito, Hans-Joachim Wilke, and Marianna A. Tryfonidou Biomatrix: Paid consultant and shareholder; Global Spine Journal: Dep-
received funding from the European Union's Horizon 2020 research and uty editor: Biomechanics and Modeling in Mechanobiology: Editorial
innovation program iPSpine under the grant agreement #825925 (www. board. Lachlan J. Smith: JOR Spine—scientific advisory board; PLOS
ipspine.eu). Research and salary support for Andres F. Bonilla is provided One—academic editorial board member; Connective Tissue
through the Fulbright—ICETEX program, and the Preclinical Surgical Research—Associate Editor; National MPS Society—Scientific Advi-
Research Laboratory of CSU. Marianna A. Tryfonidou receives funding sory Board; ORS Spine Section—committee member. Hans-Joachim
from the Dutch Arthritis Society (LLP22). Zulma Gazit was supported by Wilke: Grammer AG: Paid consultant; European Spine Journal: Deputy
the National Institute of Arthritis and Musculoskeletal and Skin Diseases Editor. German Spine Foundation: Vice President. Marianna A.
of the National Institutes of Health under Awards Number Tryfonidou: JOR Spine—scientific advisory board; SentryX—scientific
R01AR066517. Lachlan J. Smith received support from the National advisor.
Institutes of Health (R01AR077435) and the US Department of Vet-
eran's Affairs (I01RX001321). Hans-Joachim Wilke was supported by AUT HOR S' CONT R IBUT IONS
the German Research Council (DFG Wi 1352/14-3) and AO Spine All authors: conceived set up, drafted in working groups parts of the
Foundation AO-03-W16; Sibylle Grad and Andrea Vernengo are manuscript (macro scale: Elias Salzer, Andres F. Bonilla, Zulma Gazit,
LEE ET AL. 31 of 36

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