SEARCH

SEARCH BY CITATION

Keywords:

  • articular cartilage;
  • bovine patella model;
  • calcified cartilage;
  • early osteoarthritis;
  • grading systems;
  • histology

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author contributions
  9. Funding
  10. Conflict of interest
  11. References

The bovine patella model has been used extensively for studying important structure–function aspects of articular cartilage, including its degeneration. However, the degeneration seen in this model has, to our knowledge, never been adequately compared with human osteoarthritis (OA). In this study, bovine patellae displaying normal to severely degenerate states were compared with human tissue displaying intact cartilage to severe OA. Comparisons of normal and OA features were made with histological scoring, morphometric measurements, and qualitative observations. Differential interference contrast microscopy was used to image early OA changes in the articular cartilage matrix and to investigate whether this method provided comparable quality of visualisation of key structural features with standard histology. The intact bovine cartilage was found to be similar to healthy human cartilage and the degenerate bovine cartilage resembled the human OA tissues with regard to structural disruption, cellularity changes, and staining loss. The extent of degeneration in the bovine tissues matched the mild to moderate range of human OA tissues; however, no bovine samples exhibited late-stage OA. Additionally, in both bovine and human tissues, cartilage degeneration was accompanied by calcified cartilage thickening, tidemark duplication, and the advancement of the cement line by protrusions of bony spicules into the calcified cartilage. This comparison of degeneration in the bovine and human tissues suggests a common pathway for the progression of OA and thus the bovine patella is proposed to be an appropriate model for investigating the structural changes associated with early OA.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author contributions
  9. Funding
  10. Conflict of interest
  11. References

In diagnosing osteoarthritis (OA), clinicians rely largely on patient history, physical examination, and standard imaging techniques (Lories & Luyten, 2010; Bijlsma et al. 2011). Cartilage loss can be detected using weight-bearing radiographic imaging of joint space narrowing, which yields an indirect measure of cartilage thickness (Kellgren & Lawrence, 1957; Felson & Neogi, 2004), and MRI technology to quantitatively measure cartilage volume and thickness (Hunter & Guermazi, 2012). However, these diagnostic tools are normally only used when the patient complains of pain and the joint has already progressed to a mid-to-late stage of the disease (Satku et al. 2003; Hunter & Guermazi, 2012).

Detecting earlier changes in OA, before cartilage loss and the onset of pain-related symptoms, is essential for the validation of new interventions that might halt or slow the rate of OA advancement (Bijlsma et al. 2011). Recently, MRI using T2 and T1ρ mapping of collagen and proteoglycan (PG) contents, respectively, has been investigated as a potential method for detecting early OA (Eckstein et al. 2006), although such measurements may not be specific to these tissue constituents (Menezes et al. 2004). Detection of gross mechanical property deficiencies (Knecht et al. 2006) and mild or subtle structural alterations in the osteochondral tissues (Thambyah & Broom, 2009) can provide additional insights into the health of cartilage and its progression to OA.

Although techniques used to characterise OA include genetic, proteomic, and structural approaches (Buckwalter & Martin, 2006; Lories & Luyten, 2010), histological grading (Mankin et al. 1971; Hacker et al. 1997; Pritzker et al. 2006; Hunziker, 2007; Pastoureau et al. 2010; Moody et al. 2012) is, arguably, the most widely used method to assess the severity of OA in human tissue biopsies, in tissue removed during joint replacement, and in animal models to allow for comparison across species. The Osteoarthritis Research Society International's Cartilage Histopathology Assessment System (OOCHAS) (Pritzker et al. 2006) and Mankin's Histologic/Histochemical Grading System (abbreviated to Mankin) (Mankin et al. 1971) are both commonly used, but they have different goals.

The Osteoarthritis Research Society International's Cartilage Histopathology Assessment System gives a composite grade useful for quick analysis, whereas the Mankin system provides independent scores of osteoarthritic features which may assist in distinguishing between different patterns of OA development (Moody et al. 2012). Both grading systems have their limitations. The Mankin system poorly assesses mild and moderate OA (Custers et al. 2007), reducing its utility for the detection of the early disease. OOCHAS evaluates animal models of OA more reliably than Mankin (Custers et al. 2007) and includes, in addition to the grade, a staging component that can be used either separately or in combination with the grade. However, OOCHAS disregards any changes in the calcified cartilage and bone until advanced stages are reached, in which the cartilage becomes eroded.

Structural features of OA include articular cartilage (AC) swelling and increased thickness in early stages of the disease (de Bri et al. 1995; Broom et al. 2001; Thambyah et al. 2012), AC fraying and decrease in thickness as the disease progresses (Outerbridge & Outerbridge, 2001; Thambyah & Broom, 2007), and eventual exposure of bone. Chondrocytes become hypertrophic, multiply into clusters, and undergo apoptosis (Radin et al. 1991; Brandt et al. 2003; McGlashan et al. 2008). The PG content of the cartilage matrix decreases (Pritzker et al. 2006). Calcification advances episodically into the AC, resulting in tidemark duplication (Lemperg, 1971; Oegema & Thompson, 1990, 1992; Thambyah & Broom, 2007). The zone of calcified cartilage (ZCC) is invaded by cone-shaped bony spicules (Thambyah & Broom, 2007), each of which contains a vascular channel (Burr & Radin, 2003). The ZCC has been shown to increase in thickness with advancing calcification (Goldring & Goldring, 2010; Thambyah et al. 2012) in the early stages of degeneration or decrease with an advancing cement line (Thambyah & Broom, 2009). However, calcified cartilage and subchondral bone changes are not well correlated with OA in the published literature.

Animal models used to study OA include the dog, guinea pig, horse, mouse, rabbit, rat, rhesus monkey, sheep, and goat (Pritzker, 1994; Aigner et al. 2010). Various methods have been applied to induce degeneration, including impact loading, surgical intervention, immobilisation, and degradative agents (e.g. trypsin or hyaluronidase) (Smith & Little, 2007). However, such methods may not simulate any naturally occurring event associated with the initiation and progression of OA. The current models of spontaneous OA include mouse, rat, guinea pig, dog, and macaque (Smith & Little, 2007), yet these models have significantly smaller joints than humans and significantly thinner cartilage. These differences in size, structure, and load bearing limit the biomechanical relevance of these models to humans (Smith & Little, 2007).

Given the limitations of the above noted models, our laboratory has made extensive use of the bovine patella (Broom et al. 1996; Flachsmann et al. 2000, 2005; Broom & Flachsmann, 2003; Thambyah & Broom, 2007, 2009; Bevill et al. 2010; Thambyah et al. 2012). This large animal model displays a range of naturally occurring OA-like changes in the osteochondral tissues (Bartels, 1975), making it suitable for investigating the progression of the degenerative process, beginning with the entirely normal joint tissues through to full bone eburnation. Further, previous studies have described the changes seen in the bovine patella as OA-like, including thickening, softening, fissuring of the AC, and osteophyte formation (Bartels, 1975; Thambyah & Broom, 2009). Degeneration in this model tends to be localised to the distal-lateral quadrant, which, conveniently, provides a near flat surface and is thus well-suited to mechanical testing (Mente & Lewis, 1994).

Thus, the bovine patella has proved invaluable for joint tissue studies; however, to our knowledge it has never been validated as a model of early OA. Using the OOCHAS and Mankin histological grading systems in combination with differential interference microscopy (DIC), this study investigated whether mild to moderate degenerative changes occurring in bovine patellae are representative of the early stages of human OA.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author contributions
  9. Funding
  10. Conflict of interest
  11. References

Tissue

Thirty-three bovine patellae from mature females were obtained immediately following slaughter and stored at −20 °C. Following thawing in cold running water, the articular surface of each patella was stained with India ink to assess the extent of macroscopically visible surface disruption (Fig. 1). Sixteen showed no macroscopic surface disruption, henceforth referred to as ‘intact’, and 17 showed macroscopic disruptions < 1.5 cm in diameter and were thus denoted as ‘mildly degenerate.’ The percentage of the patella surface that displayed India ink retention was recorded and used to assess the OOCHAS stage component (Pritzker et al. 2006).

image

Figure 1. Macroscopic view of bovine patellae with India ink staining. (A) Intact, OOCHAS Grade 0, right patella. (B) Mildly degenerate, OOCHAS Grade 4, left patellae. Box indicates the block cut from the distal-lateral region where degeneration is common. Samples were then cut into two sections for histology and DIC microscopy.

Download figure to PowerPoint

A cartilage-on-bone block was sawn from the distal-lateral region of each patella (the site where degeneration is commonly observed; Thambyah & Broom, 2007) and sliced into two neighbouring sections of 2.5 mm width (see Fig. 1) using a saline-irrigated diamond bone saw (Buehler Isomet) at a blade rotational speed of 500 rpm. These two samples from each patella were then processed, one for histological grading and the other for DIC microscopy, as described below.

Human femoral condyle tissues were obtained with consent from seven patients undergoing partial or total knee arthroplasty at Auckland City Hospital (Ethics: NTX/08/12/117). Within 1 h of surgery, cartilage-on-bone slices from both grossly intact and diseased regions, approximately 30 × 10 × 2 mm, were obtained. The OOCHAS stage component was not measured in the human samples because only fragments of the joint surface were available from joint replacement surgery.

Histology

The bovine patella tissues were fixed in 10% formalin for a minimum of 2 days, decalcified in 10% formic acid for at least 4 weeks, dehydrated in graded concentrations of ethanol, embedded with paraffin wax, surface decalcified with H2O2, and then microtomed to 5-μm-thick sections. These sections were then stained for PGs with Safranin-O and Fast Green. The human femoral condyle tissues were fixed in 10% neutral buffered formalin for 24 h, then decalcified in Gooding and Stewart's solution containing 5% formaldehyde/25% formic acid for 4 weeks. They were then dehydrated in graded ethanol prior to embedding in paraffin wax. Sections 5 μm thick were obtained and similarly stained for PGs with Safranin-O and Fast Green.

Bovine and human histological sections were imaged using bright field transmission microscopy at appropriate magnifications and graded by the principal researcher (E.H.-T.) using both the Mankin and OOCHAS cartilage grading systems (Mankin et al. 1971; Pritzker et al. 2006). Scores were counterchecked by the other authors.

The intact bovine patella tissues were compared with the grossly intact human condylar tissue from joint replacement surgery and with published healthy cartilage histology (Hunziker et al. 2002). The intact human samples may, in fact, represent an early OA state as they originate from an otherwise osteoarthritic joint (Thambyah & Broom, 2009). However, they allow for comparison of features such as staining, cellularity, and morphometry between intact bovine and human cartilage. The degenerate bovine cartilage was compared with both the human OA condylar tissue and with histological images of femoral head, femoral condyle, tibial plateau, and patella tissues in the osteoarthritis scoring atlases developed from the Mankin and OOCHAS grading systems (Pritzker et al. 2006; Pauli et al. 2012).

DIC optical imaging

Bovine cartilage-bone tissues were fixed in 10% formalin for a minimum of 2 days, decalcified in 10% formic acid for 3 days, snap-frozen in liquid nitrogen, and then cryo-sectioned to obtain 30-μm-thick sections. Sections were then wet-mounted in saline, cover-slipped onto slides, and imaged using DIC microscopy (Thambyah & Broom, 2009).

Morphometry

To quantitatively compare intact and degenerate bovine tissues, morphometry was performed on images obtained from both DIC and histological sections. The aim was to investigate whether the two very different modes of sample preparation and imaging provided comparable quality of visualisation of key structural features.

For analysis, 6–12 vertical lines were drawn on each image according to the systematic random sampling method of Hunziker (2007) using imagej (Rasband, 1997–2012). The following measurements were performed at each vertical line, namely, total cartilage thickness (including both AC and ZCC), superficial layer thickness (depth over which chondrocytes are arranged parallel to the articular surface), ZCC thickness, number of duplicate tidemarks, and subchondral bone plate thickness (i.e. distance between cement line and the first distal occurrence of a marrow space (Muraoka et al. 2007; de Bri et al. 1995). The frequency of bony spicule protrusions was calculated over the length of the sample.

Reflected light microscopy

Bovine cartilage-on-bone blocks were fine-ground wet on a flat surface using graded carborundum papers from P120 to P1200 and imaged dry using reflected light microscopy.

Statistical analysis

All data were analysed for statistically significant differences (P < 0.05) between groups using non-parametric Mann–Whitney U-tests. Correlations were tested with Spearman's rank correlation coefficient.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author contributions
  9. Funding
  10. Conflict of interest
  11. References

Representative examples of intact and mildly degenerate bovine patellae are shown in Fig. 1 together with the location on the distal-lateral quadrant from which the test samples were taken. Each sample region showed a range of degeneration as shown in Fig. 2. Both the Mankin and OOCHAS grading systems assess the most severe degeneration present on the joint surface (Pritzker et al. 2006) and thus the maximum scores for both the bovine and human tissues are shown in Table 1. Results of the six different morphometric measurements (see 'Methods') from the bovine tissues using both DIC and histological images are shown in Table 2.

Table 1. Histology scoring of bovine and human tissues using the Mankin and OOCHAS systems.
 MankinOOCHAS
Structure (0–6)Cellularity (0–3)Safranin-O staining (0–4)Tidemark integrity (0–1)Grade (0–6)Stage (0–4)% Joint surface showing India ink
  1. Mean ± standard error in the mean.

  2. a

    Statistical significance (P < 0.05) between bovine intact and bovine mildly degenerate tissues.

Bovine intact patella (n = 16)1.0 ± 0.2a0.6 ± 0.2a0.6 ± 0.2a0.6 ± 0.10.9 ± 0.2a1.1 ± 0.2a7.5 ± 1.3a
Bovine mildly degenerate patella (n = 17)3.4 ± 0.2a1.3 ± 0.1a1.8 ± 0.1a0.2 ± 0.13.7 ± 0.1a3.0 ± 0.1a38.2 ± 2.6a
Human (n = 7)3.7 ± 1.32.3 ± 0.52.1 ± 0.90.6 ± 0.54.4 ± 0.9N/AN/A
Table 2. Bovine morphometrics from DIC microscopy and histology.
 Total cartilage thickness (mm)Superficial layer thickness (μm)Calcified cartilage (ZCC) thickness (μm)Subchondral bone thickness (μm)Number of duplicate tidemarksBony spicule frequency (per mm)
  1. Mean ± standard error in the mean. Statistical significance (P < 0.05) *between intact and mildly degenerate and between DIC and histology measurements. Note that superficial layer thickness and bony spicule frequency were not recalculated from histology sections. Two histology samples were damaged in processing and could not be measured.

Intact DIC (n = 16)2.4 ± 0.1*,76.3 ± 6.9*194 ± 12*582 ± 491.3 ± 0.4*,2.8 ± 0.2
Mildly degenerate DIC (n = 17)2.7 ± 0.2*,11.3 ± 4.9*248 ± 12*603 ± 606.3 ± 0.4*,2.4 ± 0.2
Intact histology (n = 15)2.1 ± 0.1*,N/A191 ± 11*594 ± 441.0 ± 0.45*,N/A
Mildly degenerate histology (n = 16)2.5 ± 0.1*,N/A263 ± 14*565 ± 594.3 ± 0.54*,N/A
image

Figure 2. Range of degeneration visible in each sample as graded by the OOCHAS system (0–6). Bars locate each individual sample within the continuum of osteoarthritic degeneration with zero being normal and six being the most severe. The bovine samples represented here are from the distal-lateral quadrant of the patella and the human samples are from the femoral condyles.

Download figure to PowerPoint

Histological comparison of intact bovine and intact human tissues

The intact bovine patella cartilage was found to be histologically similar to both the intact human cartilage sampled from the non-lesion sites and that reported for healthy human cartilage elsewhere (Eckstein et al. 1992; Hunziker et al. 2002). The average thickness of the bovine cartilage was 2.1 mm and compares favourably with that from the intact human femoral condyles (average 2.2 mm) and from other previously reported human medial femoral condyle cartilage (average 2.5 mm) (Hunziker et al. 2002). Further, Eckstein et al. (1992) found that the total cartilage thickness in the intact human patella ranged from 3.0 to 5.5 mm.

Relative to our human condylar tissue, the bovine cartilage and bone stained more strongly with Safranin-O and Fast Green, respectively (Fig. 3), but stained with similar intensities to the body of published human cartilage histology (Pritzker et al. 2006; Pauli et al. 2012).

image

Figure 3. Typical histological sections of bovine and human cartilage-on-bone tissue. Articular and calcified cartilage are orange/red. The superficial layer and subchondral bone are blue. The samples shown are (A) bovine intact (OOCHAS grade 1), (B) bovine degenerate (OOCHAS grade 4), (C) human intact (OOCHAS grade 1) age 60 male, (D) human degenerate (OOCHAS grade 4), age 62 male. Scale bar: 1 mm.

Download figure to PowerPoint

The zonality of the bovine cartilage was similar to the human, with approximately 20% of the articular cartilage comprising the superficial and transitional zones and the remaining 80% being radial. The superficial zone was slightly thinner than the transitional zones with 7.5 and 12% of the total cartilage thickness, respectively. The average thickness of the bovine ZCC was 0.19 mm, representing 6–10% of the total cartilage thickness, which closely follows that of intact human cartilage measured to be ~ 10% in our samples and ~ 5% in the literature (Hunziker et al. 2002). Intact bovine subchondral bone thickness averaged 0.6 mm, which is greater than the human subchondral bone plate with values in the literature quoted from as little as 10 μm up to 3 mm thick (Clark & Huber, 1990), with Hunziker et al. (2002) giving an average of 0.19 mm and Frisbie et al. (2006) giving an average of 0.4 mm.

The bovine cartilage cellularity resembled that in the human tissue. Superficial zone chondrocytes were elliptical and formed single-cell chondrons. The transition zone contained rounded, single-cell chondrons. The radial zone contained clusters of chondrocytes in vertical chondrons.

Histological comparison of degenerate bovine and human OA tissues

Histologically, the degenerate bovine cartilage resembled the human OA tissues when structural disruptions, changes in cellularity, staining loss, and tidemark integrity were assessed using the Mankin scale. Figure 2 shows the ranges of degeneration present in the lesion region of both bovine and human tissues. The tissues of both species display a similar range of degeneration with human showing some more advanced stages of degeneration.

Figure 4 illustrates the progression of structural disruption in the bovine tissues. The most severe form presented with a Mankin structure score of 4.5 of 6 and had matrix erosion into the mid-to-deep zone but not into the calcified cartilage or bone (Fig. 4F).

image

Figure 4. Bovine cartilage structure atlas using representative images with varying severities of cartilage structural degeneration as graded by the Mankin structure score. (A) Intact cartilage with smooth surface and normal appearance (grade 0). (B) Cartilage with uneven surface, grade 1. (C) Cartilage with surface discontinuity, grade 2. (D) Cartilage with vertical fissures extending into the mid-zone, grade 3. (E) Cartilage surface delamination and matrix loss, grade 4. (F) Cartilage matrix excavation, grade 4.5, maximum degeneration observed. Objective lens ×2. Scale bar: 1 mm.

Download figure to PowerPoint

Cellularity changes in the bovine degeneration (Fig. 5) included diffuse hypercellularity, cloning into large chondrocyte clusters, and hypocellularity. The Mankin cellularity scores ranged from 0 to 3 of 3. The bovine chondrocyte clusters (Fig. 5D) strongly resembled the human OA clusters (Pauli et al. 2012).

image

Figure 5. Bovine cartilage cellularity atlas using representative images with varying severities of cellularity changes as graded by the Mankin cellularity score. (A) Normal (one to two cells per chondron), grade 0. (B) Diffuse hypercellularity, grade 1. (C) Diffuse hypercellularity, grade 1.5. (D) Chondrocyte cloning, grade 2. (E) Hypocellularity, grade 3. Objective lens ×40. Scale bar: 100 μm.

Download figure to PowerPoint

Stain depletion in the bovine was similar to that observed in human OA tissues and ranged from normal to moderate reduction (0–2 of 4 in the Mankin staining score) with no complete loss of articular cartilage staining (Fig. 6). It should be noted that all bovine tissues, both intact and degenerate, showed greater staining intensity than the human tissues (cf. Fig. 3A vs. 3C); however, those human and bovine tissues with a similar severity of AC disruption exhibited similar intensities of Safranin-O staining loss (cf. Fig. 3B vs. 3D).

image

Figure 6. Bovine cartilage staining atlas using representative images with varying degrees of Safranin-O stain loss as graded by the Mankin staining score. (A) Normal staining, grade 0. (B) Slight reduction in staining, particularly in the superficial zone, grade 1. (C) Moderate reduction of staining extending down to the mid zone, grade 2. Objective lens ×2. Scale bar: 1 mm.

Download figure to PowerPoint

Whereas the human atlas of OA cartilage (Pauli et al. 2012) shows blood vessels crossing the tidemark (Mankin tidemark integrity score = 1), the majority of blood vessels in both human and bovine tissue were contained within bony spicules, shown in Fig. 7. In both tissues, most of the spicules did not reach the uppermost tidemark (Fig. 7A1–A5, B1–B4; Mankin tidemark integrity score = 0). These bony spicules infrequently reached the uppermost tidemark but never progressed further into the non-calcified cartilage (Fig. 7A6, B5; Mankin tidemark integrity score = 1). Whether the spicules reached the uppermost tidemark did not correlate with the level of degeneration in the tissue. Additionally, the heavy decalcification involved in histology preparation may obscure the tidemarks and thus a spicule that appears to cross the uppermost tidemark may actually be crossing a duplicate tidemark that is not uppermost.

image

Figure 7. Representative images of the ZCC comparing bovine and human bony spicules with their central canals. Series A are bovine spicules and series B are human. The human spicules closely resemble those in the bovine tissue. A1–A5 show bovine spicules that do not reach the uppermost tidemark and thus are grade 0 on the Mankin tidemark integrity score. A6 shows a spicule graded 1 because it contacts the uppermost tidemark. B1–B4 show human spicules that do not reach the uppermost tidemark, grade 0. B5 shows a spicule graded 1. Objective lens ×20. Scale bar: 200 μm. Arrows indicate uppermost tidemarks.

Download figure to PowerPoint

Comparing intact and degenerate bovine tissues

Quantitative comparisons of intact and mildly degenerate bovine tissue were performed to evaluate the type of degeneration occurring and allow comparison with human OA.

Table 1 compares the Mankin and OOCHAS osteoarthritis scoring between intact and mildly degenerate bovine tissue groups. With respect to structure, the majority of sections from the macroscopically intact bovine tissues (i.e. exhibiting no visible microscopic India ink staining) showed, to varying degrees, minor surface irregularities and were graded from 0 to 2.5 on the OOCHAS scale. The mildly degenerate tissues (showing macroscopic lesions < 1.5 cm diameter) were graded significantly higher than the intact tissues with the OOCHAS grade. The OOCHAS stage component (assessed macroscopically) was also greater in the mildly degenerate tissues, with an average of 40% of the patella surface showing Indian ink retention vs. only 7% retention in the intact patella surfaces.

The Mankin structure, cellularity, and Safranin-O staining scores were significantly greater in the mildly degenerate than in the intact tissues. These three criteria were also positively correlated with the OOCHAS grade for all intact and mildly degenerate bovine tissues (correlation coefficients 0.88, 0.68, and 0.79, respectively). The Mankin tidemark integrity score was not significantly different between the intact and mildly degenerate bovine tissues and showed no correlation with the OOCHAS grade.

Table 2 summarises the morphometric differences between the intact and mildly degenerate bovine tissues. Morphometric measurements were performed on both DIC and histological sections to allow comparison of the two techniques (see later). Relative to the intact tissues, the mildly degenerate tissues exhibited significantly greater total cartilage thickness, ZCC thickness, and number of duplicate tidemarks. In the intact tissues, the mean thickness of the superficial layer was significantly greater than that of the mildly degenerate samples. There were no significant differences between the intact and mildly degenerate tissues in subchondral bone thickness. OOCHAS grade correlated with superficial cartilage thickness (r = −0.82), number of duplicate tidemarks (r = 0.77), and ZCC thickness (r = 0.49). Total cartilage thickness (TCT) correlated with OOCHAS grade with a quadratic relationship (constant = 1.7, b1 = 0.71, b2 = −0.14) (R2 = 0.38). This relationship shows that cartilage thickness increases as OOCHAS score increases from grade 0 to grade 3, but decreases from grade 3 onwards.

Bony spicules were present in the ZCC of all bovine samples from both the intact and the mildly degenerate groups (Fig. 7). They showed no clear differences in morphometry or frequency (see Table 2). Also, when imaged at higher resolution with DIC microscopy, the AC mid-zone matrix was consistently different between the groups (Fig. 8). In the intact tissues, the matrix texture appeared smooth (Fig. 8, Series A), whereas in the mildly degenerate tissues there were varying degrees of fibrosity, ranging from a pronounced crimp (Fig. 8, Series B), suggesting an in-phase collapse and aggregation of the previously spatially discrete fibrillar architecture characteristic of the normal matrix (Broom et al. 2001; Nickien et al. 2013), to severe matrix destructuring (Fig. 8, Series C).

image

Figure 8. High resolution images of cartilage mid-zone matrix ‘texture’ showing a range of destructuring of the fibrillar network. Series 1: bovine tissue in DIC microscopy. Series 2: corresponding bovine tissue in histology. Series 3: human tissue with similar fibrosity in histology. Within each series, images labelled A–C represent (A) smooth texture in intact tissue, (B) in-phase crimping in mildly degenerate tissues, and (C) pronounced fibrous texture in moderately degenerate tissues. Objective lens ×40. Scale bar: 100 μm. Radial direction is vertical in all images.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author contributions
  9. Funding
  10. Conflict of interest
  11. References

In conducting this study we recognised the potential or actual limitations of using the bovine animal model. Clinically relevant details such as animal-specific age or history of joint effusion and lameness were not available to us. However, all samples were derived from a broad population of New Zealand dairy cattle whose primary habitat was relatively flat, grassy plains with a regular though small fraction of their time spent on concrete platforms. There was no certainty that any collected pair of patellae originated from the same animal and this necessarily limited the validity of any statistical method requiring independent observations (Mann–Whitney U-test and Spearman's rank correlation coefficient). Also, the patellae only infrequently displayed degeneration as advanced as late-stage human OA, seldom presenting typically end-stage features of cartilage denudation, joint deformation, complete Safranin-O stain loss, or hypocellularity – most probably because, in the dairy industry, the animals are generally slaughtered for economic reasons at an age well before that at which end-stage joint problems develop.

Despite the above limitations, the many thousands of bovine patellae that our laboratory has collected and classified over several decades have been shown to exhibit consistent site-specific morphological changes and these changes are represented in the 33 patellae forming the basis of the present study.

The morphometric and histological features found in the bovine tissues were generally consistent with those seen across the spectrum from healthy to moderate OA as defined by both the OOCHAS and Mankin grading systems. These ‘standardized histological scoring systems are needed to assess the severity of degradation in human tissues and experimental models’ (Pauli et al. 2012). Thus, the significance of the present study is its provision of an appropriate validation of the bovine patellae model for OA in relation to the vast number of past studies that have used these same scoring systems to define the extent of OA progression.

Also important is how the characterisation of bovine patellae OA in the present study fits with related, earlier published works. It has previously been shown that bovine patella tissues, representative of those used in the present study and exhibiting only mild or subtle structural changes, do indeed display significantly different mechanical responses from their healthy controls (Broom & Flachsmann, 2003; Broom et al. 2005; Thambyah & Broom, 2007; Thambyah et al. 2012). That such subtle structural changes, in the present study verified as manifestations of early OA in a clinically relevant manner using the Mankin and OOCHAS scoring systems, are indeed mechanically significant is an important starting point in considering the mechanical and structural determinants of OA initiation.

The present study showed that the intact bovine patella and human femoral condyle tissues had almost identical average cartilage thicknesses. This similarity of anatomical scale allows for both comparable mechanical testing and ease of correlation with structural responses, which contrasts with the experimental difficulties associated with smaller animal models. The bovine patella does display a thicker subchondral plate than that in the human condyle (Hunziker et al. 2002). However, the definition of subchondral bone plate thickness is often unclear (Frisbie et al. 2006) and its measurement is highly variable (Hunziker et al. 2002; Frisbie et al. 2006), so any potential relevance of this parameter for studying the progression of OA will always depend on the accuracy of its measurement.

Although the bovine cartilage stained more intensely for PGs than the human cartilage, both tissues showed a similar level of stain loss for an equivalent degree of structural disruption. This suggests a similar loss of PGs in bovine and human cartilage degeneration.

Two recognised OA grading systems were used in this study to compensate for their individual methodological and analytical limitations. Both grading systems neglect early or subtle changes in the ZCC and subchondral bone, changes which have been shown to occur in the development of OA (Radin & Rose, 1986; Oegema & Thompson, 1992; Oegema et al. 1997). The OOCHAS system assesses changes in the tissues only at more severe levels of degeneration, whereas the Mankin system focuses solely on whether vascularity crosses the tidemark. Our bovine tissues did not strictly follow the tidemark integrity changes associated with OA according to the Mankin system; however, tidemark integrity changes have been shown to correlate poorly with spontaneous OA (Mapp et al. 2008). Neither system discusses the multiplication of tidemarks in the advancement of calcification or the appearance of bony spicules as a sign of subchondral bone remodelling, although they are known to be features of human OA (Goldring, 2009).

Additionally, using the OOCHAS system proved challenging for both the human and bovine tissue when choosing an appropriate grade for features supposedly occurring simultaneously; for example, a grade 4 is assigned when cartilage matrix loss is present with cyst formation, and yet there were cases where cysts were not present with such matrix loss in both species. In general, the progression of OA features was as consistent in the bovine tissue as it was in the human.

The articular cartilage thickening in the mildly degenerate bovine tissue (see Fig. 4 and Table 2) is known to be associated with a general destructuring of the collagen fibrillar network (McIlwraith et al. 2010; Nickien et al. 2013). Although not noted in the Mankin and OOCHAS grading systems, this fibril-level destructuring of the cartilage matrix results in swelling, which is a recognised feature of human OA tissue (Maroudas & Venn, 1977). However, as degeneration progressed beyond an OOCHAS grade of 3, the combination of surface irregularities, radial fissures, and upper matrix loss, results in the observed decrease in bovine cartilage thickness.

Bony spicules were observed in both the bovine and human tissues. Although any association between spicule formation and OA remains unclear, their presence does suggest subchondral bone remodelling, a process that has been found to occur in OA tissues (Burr, 2004). Considering that spicules were present in both the intact and degenerate bovine and human tissues (Fig. 7), if they are in fact associated with the development of OA, this suggests that they are a very early occurrence that persists with degeneration. Even in the ostensibly healthy tissue there may be subtle changes at the microscopic level that represent the initial part of a continuum leading eventually to fully developed OA. This raises the intriguing question of whether the intact (i.e. macroscopically normal) tissues reflect a genuinely healthy state or, alternatively, a ‘pre-OA’ state in which microscopic changes have already commenced.

Both DIC microscopy of fully hydrated, mildly decalcified sections and histological assessment of dehydrated, heavily decalcified sections provide a means of analysing changes occurring with degeneration; however, each method is suited to different purposes. In the current study, histological staining proved useful for viewing PG loss (Fig. 6). Alternatively, DIC microscopy provided high resolution images of the AC matrix structure with a level of clarity not possible with histology. In comparing the normal and mildly degenerate bovine tissues, with DIC optical imaging the smooth/amorphous appearance of the healthy matrix (Fig. 8A) has been shown to reflect a dense, pseudo-random fibrillar network (Broom & Flachsmann, 2003; Nickien et al. 2013). With increasing severity of degeneration this fibrillar network destructures and the fibrils aggregate into a microscopically visible fibrosity (Fig. 8B,C).

DIC microscopy imaged the ZCC tidemarks more clearly than the histological sections (cf. Fig. 9B,C) and allowed a greater number to be observed (see Table 2), probably because less decalcification was needed for the thicker sections imaged under DIC. These same DIC sections also showed cell clusters in the mid-to-late stage OA tissues more clearly than the histological sections (Fig. 10). Interestingly, while comparing the two imaging methods using sections from the same samples there were obvious artefacts in the histological sections that were not seen in the corresponding DIC sections. These included section folding (visible in all images of Fig. 4), tearing, and an uneven articular surface (Fig. 4B). Thus, when using histological grading systems such as Mankin and OOCHAS for cartilage degeneration it is important to keep in mind that an uneven articular surface may be due to artefact and may falsely be classified as grade 1 degeneration. Total cartilage thicknesses obtained from the histological sections averaged 260 μm less than those from the corresponding DIC sections (see Table 2). Histological methods may therefore mask subtle cartilage swelling and thickness changes that are early features of OA. As DIC microscopy is performed using fully hydrated tissue sections and requires minimal preparation time, we recommend it as an important imaging tool in joint tissue research for both its ease of application and the additional structural insights that it provides.

image

Figure 9. Imaging the zone of calcified cartilage (ZCC) of mildly degenerate bovine patellae. Uppermost tidemark shown by black arrow, cement line shown with white arrow. (A) Reflected light microscopy shows indistinct tidemarks and bony spicules (dotted lines). (B) Safranin-O and Fast Green stained histology sections show multiple tidemarks as dark lines. (C) DIC microscopy of hydrated sections without staining shows more clearly the multiple tidemarks. Scale bars: 100 μm.

Download figure to PowerPoint

image

Figure 10. High resolution DIC microscopy image of cartilage showing chondrocyte cloning (grade 2 Mankin cellularity score). Image from 30-μm hydrated section with no staining. Objective lens ×40. Scale bar: 100 μm.

Download figure to PowerPoint

The data from the present study can now be used to refine and advance our earlier schematic model of progression to OA (Thambyah & Broom, 2009). Both this earlier version and the new schematic model (Fig. 11) illustrate changes occurring in the ZCC and subchondral bone. The uppermost tidemark forms a singular boundary in the healthy state (Fig. 11A) which, in the early degenerative stage, progresses to tidemark duplication and ZCC thickening (Fig. 11C–D). Along with this thickening, bony spicules are also shown to project from the cement line into the calcified cartilage where they may reach as far as the uppermost tidemark (Fig. 11B). Further progression of OA is depicted as the advancement of the cement line into the ZCC space via in-filling around the bony spicules. This progression described in the earlier model was then suggested to form the context for the structural demise of the joint with severe loss of the overlying cartilage in end-stage OA (Thambyah & Broom, 2009).

image

Figure 11. Schematic of the proposed progression of OA through (A) healthy, (B) intact ‘pre-OA’, (C) early OA, (D) mid-stage OA, and (E) late-stage OA. The bovine samples from the present study fall largely within A–D, whereas the human tissues from joint replacement fall largely within D and E. The schematic incorporates structural changes in the cartilage, calcified cartilage, and subchondral bone as well as proteoglycan loss shown with histological staining. The insets indicate the collagen fibril-level changes.

Download figure to PowerPoint

Included in the new schematic model (Fig. 11) is the histological staining from the present study to indicate the spectrum of relative PG loss occurring with OA (Fig. 6). Additionally, the collagen fibril-level destructuring of the cartilage matrix is included in the schematic; the intact state is represented as a pseudo-random fibrillar network (Fig. 11A inset) and as OA progresses, network destructuring and fibril aggregation occurs (Fig. 11C inset).

Finally, the schematic model illustrates the progression from a healthy state to a still intact ‘pre-OA’ state and then through to early-, mid-, and late-stage OA (A–E in Fig. 11). The bovine tissues demonstrate primarily stages A–D, with a few instances of more severe degeneration (D–E). By contrast, the human tissue from patients with fully developed OA would primarily be categorised as D–E. Thus, by combining the structural and histological changes in the bovine and human tissues, we propose a model for OA progression that spans the full spectrum from healthy to severely degenerate.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author contributions
  9. Funding
  10. Conflict of interest
  11. References

We show that the bovine patella model represents a suitable large animal model of early OA. This model more closely mimics the thickness, morphometry, and zonality of human cartilage than all other models including rabbit, sheep, dog, goat, and equine tissues (Frisbie et al. 2006; Smith & Little, 2007). Further, the large, relatively flat surface of the bovine patella provides an ideal site for mechanical studies. We have shown that this model demonstrates common features of OA: (i) swelling, fibrillation, and loss of the cartilage matrix, (ii) PG loss, (iii) calcified cartilage thickening, and (iv) tidemark duplication. We consider the development of OA in this model to be more akin to primary, spontaneous OA as it requires no artificial inducement. The collection method employed provides a large number of samples inexpensively. Additionally, the model demonstrates the full continuum of conditions from intact to severely degenerate, with a high frequency of early and mid-stages of the disease, a phase that in humans is still poorly understood.

Author contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author contributions
  9. Funding
  10. Conflict of interest
  11. References

Acquisition of data and statistical analysis: Emily Hargrave-Thomas. Drafting the article: Emily Hargrave-Thomas, Ashvin Thambyah, Neil Broom. Study conception and design, revision and approval of the article: Emily Hargrave-Thomas, Ashvin Thambyah, Neil Broom, and Susan McGlashan.

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author contributions
  9. Funding
  10. Conflict of interest
  11. References

The authors gratefully acknowledge funding provided by the University of Auckland (Doctoral Scholarship awarded to E.H.-T.) and the Marsden Fund (Royal Society of New Zealand). Neither sponsor had any involvement in the study.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author contributions
  9. Funding
  10. Conflict of interest
  11. References