Histopathology
Though no one particular standard offers exceptional correlation to OA, histopathology is currently the gold standard for assessing of OA in animal models [
189]. The histology samples, in conjunction with immunohistochemical staining, can be used to classify and measure the degree of degeneration in the joint. One of the first techniques that were used to grade OA was reported by Collins et al. [
190] and Curran et al. [
191]. Collins and co-workers [
192‐
194] in a series of articles reported the variations in the uptake of
35S and subsequent chondroitin-sulfate synthesis by cartilage cells in the costal and articular cartilages of the patella in humans with different stages of OA. Their observation on articular cartilage tissues obtained from human cadaver was that sulfate utilization was higher and commensurate with the degree of damage to articular cartilage [
190]. They further showed that contrary to the popular belief, damage to the articular cartilage is not caused by loss of chondrocytes [
193,
195]. In fact, increased activity of sulfate utilization by chondrocytes in damaged cartilage pointed to active chondrocytes in those tissues. To further enhance the applicability of this technique, Collins et al. and several other research teams [
194,
195] used new visualization technique (auto-radiography) and quantification technique (radiochemistry). Collins and co-workers [
193], in addition, developed a scoring system based on histological data to classify the knee based on the level of damage to the cartilage. The extent of damage in the knee was classified into four groups: grades 0, I, II, and III and IV, respectively. The first group, i.e., grade 0, had smooth cartilage surface with no defects; the second group, grade I, however, exhibited limited damage to the superficial zones but did not extend deeply into the bone. The third group, grade II, illustrated fibrillations extending into the deep zones, and in the last group (III and IV), significant loss of cartilage along with deep exposure of the bare bones. A major drawback of this system was the specimens were obtained from either surgical removal of the patella or from necropsies. Hence, neither the pathogenesis of the disease nor the progression of OA can be studied by this model.
A point-based grading system was subsequently developed by Mankin et al. [
196,
197]. Here, surgically removed human femoral heads were histopathologically correlated with biochemical changes in DNA and carbohydrate synthesis. The DNA and carbohydrate content were studied by the incorporation of
3H-thymidine and
35SO
4, respectively
. Higher carbohydrate content correlated with lower disease progression, even though the same could not be concluded for DNA. From the experimental observation, a new 14-point grading system based on cellular, histochemical, and biomechanical changes was created [
198]. This system is known as the Mankin score system or more commonly known as Histologic/Histochemical Grading System (HHGS) [
196,
199,
200].
Although the Mankin score and previous grading systems were extensively used in animal models to study OA, they present challenges while investigating early or intermittent stages of OA. Several modified grading systems such as modified-Mankin or modified-HHGS have therefore been developed to address the poor reproducibility and intra and inter-observer variations of Mankin scoring system [
198]. At the same time, Mankin scale can be successfully used to study sodium monoiodoacetate induced OA due to the rapid progression of the disease to form terminal OA. Other scoring systems commonly used in animal models include O’Driscoll, International Cartilage Repair Society (ICRS and ICRSII), and modified O’Driscol scores [
189,
201,
202]. A recent study comparing the various histological scoring systems for OA showed that the ICRSII, O’Driscoll, and modified O’Driscoll scores had higher reliability than other histopathological scores, including the Mankin score [
203].
To enhance reproducibility, decrease intra- and inter-observer variations, and standardize the assessment and reporting techniques across animal models, the OARSI formed a working group in 2010 to develop a standard OA grading system [
54]. The five cardinal principles the working committee used to determine ideal OA histopathological system were simplicity, utility, scalability, extendibility, and comparability [
204]. The OARSI working group’s recommendation aimed to address some of the deficiencies observed in preclinical studies such as lack of defining clear distinction of OA subsets, established clinical trial endpoints, evaluation of biomarkers, histopathology, and exclusion of other arthritis types.
Some of the remarkable progress made by this committee were established clinical trial end points, defined subsets of OA and guidelines to evaluate new features of OA (apart from cartilage) and evaluate histopathology in animal models. Based on the severity of OA, the working group classified OA into seven grades with grade 0 being uninvolved or intact cartilage and grade 6 involving deformation of articular contour. Unlike the older scoring techniques, the OARSI technique specifically relied on the depth of progression into the cartilage to grade OA. By borrowing concepts from cancer pathology, efforts were also made to designate the severity of OA lesions by stages [
16]. The OARSI working group provides this information through a released set of guidelines for each animal used in animal models [
51,
54,
205‐
211].
Imaging modalities
Imaging modalities frequently used to investigate OA in humans include x-rays, MRI, μ-CT scans, and ultrasound. Traditionally, OA is evaluated with radiographs in the clinic to demonstrate joint space width (JSW) and the formation of osteophytes [
212]. Radiographs also permit the visualization of subchondral sclerosis and subchondral cysts [
213]. Various animal models with rats [
214], rabbits [
215], and dogs [
216] have been studied using radiography including the most famous Pond-Nuki model (dogs) [
217]. In rats and rabbits, radiography has been used to study subchondral bone remodeling and joint space narrowing. Recent research, however, suggests cartilage loss alone is not the sole contributor to OA, but changes in the morphology of menisci also play an equally responsible role [
218‐
221]. Unfortunately, radiography, which is the current gold standard for imaging OA, lacks sensitivity to visualize such variations [
222]. Moreover, changes in the flexed position used in the follow-up imaging also might lead to conflicting conclusions, which severely restricts the application of radiography in OA [
223]. In addition, radiography allows only late stage visualization of OA and does not allow direct visualization of cartilage itself. To some degree, utilizing computer tomography (CT) arthroscopy circumvents this problem. Unfortunately, this technique is invasive [
224]. Despite these disadvantages, radiography is still widely used in the clinical setting. Various grading schemes such as Kellegren-Lawrence, OARSI classification scores, WOMAC, Knee Injury and Osteoarthritis Outcome Score (KOOS), and VAS have been developed over the years and are widely used [
225‐
228].
Magnetic resonance imaging (MRI), unlike radiography, is capable of visualizing not only the cartilage but also the menisci, ligaments, synovium, and biochemical markers pertaining to OA [
229]. By virtue of its ability to phase contrast tissues, it can distinguish and study individual tissues. Despite its high cost, due to its potential and capabilities, MRI is a fast advancing tool replacing radiography in characterizing and detecting early stages of OA [
33,
230,
231]. For high resolution imaging, a minimum of 1 Tesla (T) scanners are typically required. Currently, the most widely used models in clinics are the 1.5-T scanners. But recently, the 3-T model has been introduced and is fast becoming the choice for imaging [
232]. Higher field strength scanners (7 T) are currently under development [
233] and are expected to result in higher signal to noise ratios, albeit with minor issues such as chemical shifts.
Application of utilizing these MRI techniques in animal models is summarized in Table
5. With significant advancements in instruments and hardware and with its superior capability, MRI, unlike radiography, is expected to take a leading role in future animal model experiments to study various aspects of OA [
234]. The difficulty in utilizing radiology has prompted the development of these alternate techniques to study OA in animals. Till date, MRI has been utilized to study various animal models, small and large, including rat, rabbit, guinea pig, dog, and non-human primates (rhesus macaque) [
234‐
240]. For example, in rat osteoarthritis models, several osteoarthritic changes can be monitored in vivo with the use of MRI [
241‐
243]. In rabbit models, cartilage thinning and swelling, decrease in proteoglycan content, and mild subchondral changes can be observed which are typically difficult to visualize using radiography [
244]. MRI has also been used to acquire 3D images of cartilage volume loss in a naturally occurring OA caused by obesity in the guinea pig model [
245]. Some surgical models which induce OA and have used MRI to study changes include ACLT and Medial Meniscus Tear [
244,
246]. In much smaller animal models such as mice, standard MRI measurements are not possible; however, micro-MRI has been utilized to study ACLT induced OA [
247] and in
Brtl mouse models [
248].
Table 5
Examples of various MRI techniques used in OA animal models
T1-rho | Rabbit-ACLT | Cartilage degeneration [ 326] |
| Rat-meniscectomy | Decrease in cartilage thickness and loss of cartilage [ 327] |
| Rat-ACLT | Loss of proteoglycans, collagens and hydration changes [ 328] |
| Canine-stifle model | Osteophytosis and synovial thickening [ 329] |
| Guinea pig model | Cartilage thickness to study age related OA [ 330] |
| Rabbit model | Proteoglycan loss, disruption of collagen network [ 239] |
T2-mapping | Rabbit-antigen induced OA | Synovitis, macrophages [ 331] |
| Goat knee-papain induced OA | |
| Guinea pigs-aging | Cysts, osteophytes, sclerosis, cartilage degeneration [ 333] |
| Rabbit-papain induced | Cartilage thickness, loss of proteoglycan [ 334] |
| Rabbit-medial meniscectomy | |
dGEMRIC | Goat-osteochondral defect | Glycosaminoglycan content [ 336] |
2D spin echo and 3D gradient echo | Canine model | OA bone abnormalities, intraosseous cysts [ 337] |
| Rabbit-ACLT | Articular cartilage degradation, osteophyte formation, subchondral bone changes [ 338] |
| Rabbit-ACLT and meniscectomy | Synovial effusion, meniscus and ACL lesions, and osteophytes [ 339] |
| Rat-ACLT | Cartilage volume/thickness [ 242] |
| Rat-meniscectomy | Cartilage degeneration, subchondral bone defects, and osteophytes [ 235] |
| Goat-osteochondral defect | Osteochondral repair and bone lesions [ 340] |
| Mouse (C57BL/6) | Articular synovial space, subchondral bone [ 317] |
Sodium MRI | Porcine (intra-articular injection (IL-1beta) | Proteoglycan content [ 341] |
Magnetization transfer | Rat model (antigen induced) | Macrophage infiltration, changes in water content [ 342] |
| Goat knee-papain | Collagen concentration, proteoglycan depletion [ 332] |
| Rabbit-medial meniscectomy | Collagen framework, proteoglycan loss [ 239] |
Cartilage is essentially composed of collagen, proteoglycans, and water [
26]. All three components play a complex role in the functioning of the tissue. Any change in their composition causes debilitating effect on the tissue and ultimately leads to OA. That is another reason why radiography ultimately fails in its ability to study OA. Site-specific studies can be fortunately performed, unlike in radiography, by MRI using various techniques such as gradient recalled echo (GRE), spin echo (SE), fast SE, and 3D SE, which have profound impact in studying the morphological changes of the cartilage during OA [
249]. To enhance the physiological imaging, techniques such as T1 and T2 relaxometry [
250], chemical exchange saturation transfer (CEST) [
251], magnetization transfer (MT) [
252], sodium MRI [
253], diffusion-weighted imaging (DWI) [
254], digital tensor imaging (DTI) [
255], and, more recently, delayed gadolinium-enhanced magnetic resonance (dGEMRIC) [
256] imaging of cartilage have been used to visually observe the glycosaminoglycan (GAG) component of cartilage (Table
5).
For instance, T1 in the rotating frame (T1-rho) works by measuring the spin-lattice relaxation in the rotating frame, and any loss of aggrecan can be measured indirectly by observing the motion of water molecules [
257]. T1-rho has been reported to be used for studying cartilage degeneration, decrease in cartilage thickness, loss of proteoglycans, and changes in synovium (Table
5). On the other hand, in T2 mapping, an increase in relaxation times indicates the inefficiency of water molecules to exchange the energy inside the matrix [
258]. Some of the features of OA that are typically studied, as summarized in Table
5, using T2 mapping include synovitis, macrophages, collagen order, sclerosis, and proteoglycan loss. Combining one of the techniques with dGEMRIC ensures GAG content can also be estimated. An added advantage with this technique is that it is reproducible, and statistical difference in specimen can be observed in as little as 10 weeks [
259].
Typically, the most imaging modalities for OA involve characterizing proteoglycans, but some techniques such as DWI and DTI work by studying the orientation as well as the flow of water molecules through the cartilage. In DWI when diffusion sensitizing agents are applied, water molecules possess a random directionality with a uniform signal intensity. However, when it encounters a diffusion, it undergoes a signal drop, which indicates unhealthy cartilage [
260]. DTI, which is an advanced imaging technique, is capable of measuring not only diffusion of water but also the direction of the flow which aids in mapping the cartilage tissue [
261]. MRI, similar to nuclear magnetic resonance (NMR) spectroscopy, works based on the fact that any atom with odd number of protons with non-zero spin would exhibit magnetic resonance phenomenon [
262]. In that aspect,
23Na can also be used instead of conventionally used
1H to image cartilage and other relevant tissues. When
23Na atoms bind with the negatively charged GAG chains in the cartilage, any loss of GAG results in diminished Na ions, which indicates loss of cartilage due to OA [
263]. Despite its high potential to study the cartilage, using
23Na requires specialized coils which inhibit their clinical use. Their far lower Larmor frequency and concentration at resonance frequency (signal strength) compared with
1H further dampens its case to be used for MRI imaging [
264]. But with significant improvements in instrument hardware, it can be envisaged that
23Na would be a tool of interest in the near future to detect early stages of cartilage changes with OA. Study of loss of proteoglycan is typically studied using this MRI imaging technique (Table
5).
Apart from the loss of proteoglycans as described by Collins et al., it has been reported that synovitis, the inflammation to the synovial fluid, also plays a key role in the early stages of OA [
31]. Plain radiography is incapable of imaging synovial fluid and is thus not used for this purpose. Ultrasound and MRI are the most commonly used modalities to image synovitis. Non-contrast-enhanced (CE) and gadolinium (Gd)-based CE-MRI are two techniques commonly used to observe synovitis [
265,
266]. In addition, 2D spin echo and 3D gradient echo are the other two techniques employed to study synovitis. Aside from synovitis, these techniques can detect intraosseous cysts; lesions in the meniscus, bone, and ACL; and subchondral bone defects and can also map articular synovial space. Ultrasound has found some success in animals and humans to detect other early osteoarthritic changes [
33,
267]. The ultrasound serves as a quicker and cost effective method to study outcomes in animals (Table
5).
The OARSI currently recommends MRI for morphologic evaluation in humans and also for use in preclinical trials [
16,
33,
230]. An added advantage in using MRI is its simplicity in developing a grading system which facilitates uniformity, comparability, and reproducibility across various models. Since MRI is fast emerging as a tool for imaging OA in humans, it is expected to play a key role in studying OA in animal models. Some of the grading systems that are commonly used with MRI include Whole-Organ Magnetic Resonance Imaging Score (WORMS), Boston-Leeds OA Knee Score (BLOKS), and MRI OA Knee Score (MOAKS), with BLOKS and MOAKS being the most widely used scoring systems in MRI based modalities [
268‐
271].
μ-CT is another powerful technique utilized to study 3D structures reconstructed from slices of 2D images [
212]. It is widely used to study bone formation, healing, and remodeling. However, as with radiography, CT even with multisource spiral CT scanners is yet to find any significant application in visualizing OA (knee), especially in its initial stages [
272]. With that said, although its application might be restricted for knee OA, it has huge potential for hip and TMJ OA [
273]. However, as mentioned before, it could be an excellent tool to visualize changes in the bone joints, and MRI with its significant advantages can easily replace CT for knee OA. A more invasive version of CT, optical coherence tomography, is frequently used to study the diseased state of cartilage by affixing with an arthroscope. Also, by combining with other techniques such as MRI and positron emission tomography, CT is expected to make significant contribution in studying early stages of OA [
274]. In addition, by utilizing contrast agents, contrast resolution of the cartilage images can be enhanced. Recently, μ-CT has been utilized to image subchondral changes and thus follow progression of OA in rats and mice [
275]. In rat and mice models, for instance, collagenase-induced subchondral changes and cortical bone loss have been reported using μ-CT technology [
276,
277].
Positron emission tomography (PET) is a unique technique used primarily in oncology, cardiology, and neuroscience [
278]. It allows measurement of functioning of tissues by using compounds that are short-lived positron emitting nuclides [
279]. A widely used positron emission (PE) nuclide is fluorine-18 fluorodeoxyglucose (
18F-FDG) [
280,
281]. Typically, it is used to detect glucose uptake by cells, and fortunately, it can also be utilized for OA as glucose uptake take place in cartilage by proteoglycans. Apart from OA, PET has potential to investigate chondrosarcomas and tumors in the bone [
282,
283]. Recently,
18F-FDG based PET was utilized in a rat model to investigate the early stages of OA. This study indicated its significant potential to detect OA within 2 weeks of induction, while, in histology, a minimum of 8 weeks was required [
284]. Even though PET was not extensively used for OA evaluation previously, it is rapidly finding niches in investigating OA in conjunction with other techniques such as CT and MRI.
In addition to the currently used imagining studies, FRI has shown success in non-invasive mouse models to quantify the biological responses and time course in OA [
141]. In a recent study, bioluminescence has also shown promise in mouse models of osteoarthritis to measure cartilage changes [
285]. For this study, chondrocyte mutation in the CreER
T2 protein, which is activated by tamoxifen injection, was successfully applied to mice undergoing joint destabilization studies and treadmill exercises. The technique might well prove useful as a non-invasive imaging modality for future studies of cartilage degeneration.
Biomarkers
Biomarkers of cell degeneration and inflammation can serve as a measure of disease progress or treatment outcomes in clinical osteoarthritis. These molecules are precursors or products of metabolism released in the serum, urine, and synovial fluid, and their levels correlate with osteoarthritic changes in the joint. The Burden of Disease, Investigative, Prognostic, Efficacy of Intervention and Diagnostic (BIPED) classification [
286] has been applied to these biomarkers to develop and analyze their effectiveness in OA research. Several biomarkers are commercially available for use in clinical trials [
6,
33,
287,
288]. Well-published biomarkers are urinary C-telopeptide of type II collagen (CTX-II) and serum cartilage oligomeric matrix protein (COMP) [
289,
290]. Other clinical biomarkers include serum hyaluronic acid (HA), serum and urine Coll2-1 (a peptide of the alpha-helical region of type II collagen) and its nitrated form Coll2-1 NO
2, and YKL-40 (also known as chitinase 3-like 1, CHI3L1, or cartilage glycoprotein-39) [
291‐
294]. Despite their availability, further investigation into the applicability of these markers in clinical research is needed due to the lack of consistency in results of its application [
288]. Research is ongoing to evaluate new biomarkers for preclinical and clinical studies. In animal models of osteoarthritis, this research also assesses the usefulness of biomarkers in studying early osteoarthritic changes and the effect of treatment.
In an
STR/ort mouse model of primary OA, MMP-3 was found to be a sensitive biomarker to detect early OA changes [
295]. A novel COMP enzyme-linked immunosorbent assay (ELISA) was used to study COMP fragments as a biomarker of OA in the serum of induced mice models. This was found to correlate with results in humans using this assay [
296]. Serum xylosyltransferase 1 (
Xylt1) is increased in mice models of OA under a background of mice with high bone forming potential. This study suggests an application of this marker in studying OA risk in young adults [
297]. There have also been promising results in the application of biomarker research in other small animal models. In rats, this was accomplished using immunohistochemical staining of histological sections [
298]. The MIA model has been utilized in rats to develop an aggrecanse model of cartilage degradation, using aggrecan neoepitope release in synovial fluid to follow these changes [
299]. The rat MIA model has also been used to test meloxicam as a treatment for OA and the ability of the drug to reduce the biomarker CTX-II [
300]. CTX-II has been associated with cartilage changes in conjunction with differences in animal age in a rabbit-ACLT model [
301,
302]. Rabbit models of ACLT have shown a similar correlation of the biomarkers HA and chondroitin-sulfate 846 epitope, with the severity of OA in the joint [
303]. Guinea pigs have been assessed to determine the usefulness of biomarkers in spontaneous models [
97].
In recent years, several biomarker research studies have involved the use of dog models. Dogs share the same MMPs as humans and biomarker research can be translated better to clinical studies [
304]. In a canine model of ACLT, serum levels of CTX-II were elevated indicating that this model is sensitive and specific for early articular changes in OA [
305]. Serum levels of fetuin B and complement C3 were also elevated in this surgical model in another study [
306]. Garner et al. on a surgically induced canine model showed an increase in monocyte chemoattractant protein-1 (MCP-1) and IL-8 in the synovial fluid [
307]. Another study by Alam et al. utilizing a surgical canine model showed a correlation between disease progression and the serum or synovial fluid levels of tartrate resistant acid phosphatase (TRAP), matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) [
308]. These substances could serve as possible biomarkers to study early OA changes in other animal models and humans. Tenascin-C is another biomarker found in both canine and human synovial fluid during osteoarthritic changes, and this substance could play an additional role in increasing joint degradation [
309]. Finally, Coll2-1 and Coll2-1 NO
2 as biomarkers were also found to correlate with OA changes in the canine ACLT model [
310].
Regrettably, no gold standard exists in the literature for animal studies and translation from in vitro to in vivo studies, then clinical studies, has met with difficulties [
311]. In animal studies, biomarkers are most useful when taken directly from the joint synovial fluid [
16]. Yet, this is not always feasible in the smaller joints of small animal models such as mice; aspirated samples from these studies would be insufficient. Although biomarkers could be measured from other sources, such as urine samples, their levels are influenced by other diseases or metabolic conditions just as in clinical studies. Therefore, more biomarkers have been developed for animals with larger joints such as guinea pigs and dogs [
97,
307]. Other animals utilizing biomarkers are sheep and horses [
312,
313]. Used in conjunction with imaging studies, biomarkers can give a greater characterization of the disease process in both large animal models and humans [
33,
312,
314].