SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Mechanical loading through a mechano-adaptive response modifies articular cartilage structure and contributes to osteoarthritis (OA). However, the specific mechanical stimuli involved in joint health and disease remain poorly defined, partly due to a lack of in vivo models of controlled loading. The present study was undertaken to develop and characterize a novel nonsurgical murine model in which applied loads to the knee joint are highly adjustable.

Methods

Animals experienced normal locomotion, except during loading. Loads were applied to the right knees of 8-week-old CBA mice, 3 times a week for 2 weeks (and assessed immediately or after 3 weeks of nonloading), or for 5 weeks, or just once (and assessed immediately or after 2 weeks of nonloading). Histologic features of loaded and control contralateral joints, including articular cartilage lesions, osteophyte formation, and pathologic features, were examined. Ex vivo visualization during loading was performed by microfocal computed tomography (micro-CT).

Results

Two weeks of loading produced articular cartilage lesions only at sites of maximal contact as exhibited by micro-CT; after 3 weeks without further loading, joints in another group of mice identically loaded revealed significant increases in mean lesion severity to levels seen following 5 weeks of loading. Single application of load also induced lesions, but in this case, 2 weeks of solely habitual use did not lead to further deterioration. Only repetitive loading induced loss of Safranin O staining. Loading also led to osteophyte formation, meniscal ossification, synovial hyperplasia and fibrosis, and cruciate ligament pathology, with a severity that was dependent upon the loading regimen utilized.

Conclusion

We describe for the first time a noninvasive model of murine knee joint loading. This will further the study of mechanical and genetic interactions in joint health and in OA initiation and progression.

Mechanical loading is known to modify articular cartilage structure through a mechano-adaptive homeostatic response (1, 2) and to contribute to osteoarthritis (OA) (3–5). Despite these fundamental roles, the specific mechanical stimuli that are detrimental or beneficial to joint health remain undefined. This lack of understanding is at least partly due to a paucity of in vivo models in which specific components of mechanical loading can be controlled noninvasively.

Most studies examining the effects of mechanical loading on articular cartilage and chondrocytes have been performed in vitro. Previous investigators have applied specific forces to chondrocyte monolayers or, for example, 3-dimensional scaffolds (6–8). However, as with most in vitro models, these disrupt crucial interactions between cells and their extracellular matrix (ECM) in intact tissues. While the use of articular cartilage explants diminishes these concerns (9–11), explant models fail to retain relationships between distinct joint tissues and are complicated significantly by dissection and culture.

In vivo models preserve these chondrocyte–ECM interactions and those of intact cartilage with other tissues. The most extensively used in vivo models modifying mechanical loads involve surgical joint destabilization. Initial studies used canine anterior cruciate ligament transection in the Pond-Nuki model (12) or rabbit knee meniscectomy (13). These produced major advances and highlighted a need for similar models in smaller, genetically modifiable species, such as mice.

The development of surgical murine models now allows induction of lesions with varying severity (14); several models are considered representative of human OA (15, 16). However, these are severely disadvantaged by the need for surgery, which increases infection risk, causes trauma, and directly affects joint cell metabolism. The intransience of the modified loading that current models utilize also means that the models are not useful for studying responses to specific episodes of loading or discrete components of the mechanical milieu. Availability of a model that, once characterized, allows application of adjustable noninvasive loading is obviously an attractive prospect.

Responses to differing loading regimens were first examined in rabbits by Radin et al (17). In those studies, magnitude and frequency of applied loads were changed, and joints later analyzed (18). Damage was induced in other rabbit models using a single adjustable impact (19, 20). Development and characterization of similar nonsurgical models in mouse knee joints would offer greater advantages, as mice are often the animal of choice in such studies and their use allows examination of responses in mutant and transgenic animals.

The advantage of nonsurgical joint loading is also inherent in exercise and disuse models (21, 22). However, these are hampered by reliance on a relatively uncontrolled mechanical input as the stimulus. There is, therefore, a need for a nonsurgical murine model that allows for adjustment of specific components of the applied load, including its magnitude, frequency, and loading cycle shape.

In this report we describe a newly developed nonsurgical model for loading of mouse knee joints. The loading is applied through natural points of articulation, with the contralateral joint experiencing normal gait between loading episodes, providing an internal control. The model has allowed us to demonstrate induction of focal articular cartilage lesions. Another attractive characteristic is its controllability, which enabled us to distinguish between the short- and long-term effects of single- or multiple-loading episodes on articular cartilage integrity. Additionally, we describe histologic changes in various other joint tissues, induced by adjusting the number of exposures to a specific loading pattern. With its versatility and modifiability, this model may allow discrimination between joint responses to physiologic and pathologic loading, as well as a more thorough investigation of the interaction between genetic and mechanical influences in OA initiation and progression.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Animals.

Male CBA mice (Charles River) were kept in polypropylene cages at 21°C (±2°C), subjected to 12-hour light and dark cycles, and fed a standard RM1 maintenance diet ad libitum (Rat and Mouse No.1; Special Diet Services). All procedures were in compliance with the Animals (Scientific Procedures) Act 1986 and were approved by the local ethics committee.

In vivo loading.

The right knee joints of 8-week-old CBA mice were loaded using a model described for use in examining tibial bone responses to loading (23). Mice were anesthetized with isoflurane throughout the procedure and the right tibia positioned vertically between custom-made cups, with knee and ankle joints in deep flexion (Figure 1A). Using a servo-hydraulic materials testing machine (model HC10; Dartec), axial compressive loads were applied through the knee joint via the upper cup that was attached to the actuator, while the lower cup was linked to the load cell, in order to monitor the loads applied.

thumbnail image

Figure 1. Diagrammatic representation of the loading model, loading application pattern, and loading regimens. A, Estimated position of the hind limb and loading direction when placed in the loading apparatus. B, Diagram of a single cycle of applied load, showing hold and peak load magnitudes, rate of load application, and intervening peak and baseline hold times (pattern used in groups 1–5). C, Diagrammatic representation of the 5 different loading regimens (groups 1–5); 1 episode was composed of 40 cycles. Except where indicated otherwise, the regimen involved the application of repeated loading episodes on 3 occasions each week.

Download figure to PowerPoint

Loading regimen.

All studies used a single loading pattern (Figure 1B) in which peak loads of 9N were applied for 0.05 seconds, with a rise and fall time each of 0.025 seconds and a baseline hold time of 9.9 seconds. Baseline 2N loads maintained the tibia in position between periods of peak loading. Forty cycles of this pattern were applied to the right knee in each loading episode, and the left knee was used as a nonloaded control. The application of 9N loads using this particular regimen was previously shown to be insufficient in magnitude to induce osteotrophic effects at various sites along the tibial diaphysis (23).

Right knees in 5 groups of mice received differing numbers of loading episodes or were examined at varying lengths of time after the final loading episode. Mouse joints in group 1 (n = 7) were loaded on alternate days, 3 times each week for 2 weeks, and examined 2 days after the final loading episode; joints in group 2 (n = 7) were loaded 3 times each week for 2 weeks, but were examined after an additional 3 weeks without further loading; joints in group 3 (n = 9) were loaded 3 times each week for 5 weeks, and examined 2 days after the final loading episode; joints in group 4 (n = 6) were loaded for a single episode, and examined 2 hours later; and joints in group 5 (n = 6) were also only loaded once, but examined after an additional 2 weeks without further loading (Figure 1C). The rationale behind these 5 regimens was that comparing their outcomes would allow us to distinguish between the effects of single and multiple loading episodes, as well as their immediate and longer-term impact on joint integrity.

Histologic analysis.

After cervical dislocation, right (loaded) and left (nonloaded) joints were dissected, fixed for 24 hours in neutral buffered formalin, decalcified for 7 days in Immunocal (Quartett), and processed for standard paraffin embedding. Coronal 6-μm sections from individual knees were cut across the entire joint, and a quarter of the entire set from regular intervals were stained with toluidine blue (0.1% in 0.1M solution of acetate buffer [pH 5.6]).

Lesion severity in articular cartilage in these joints was graded using the system described by Chambers et al (24), with some modification. Grade 0 corresponded to normal articular cartilage; grade 1, rough articular cartilage surface or lesions in the superficial zone; grade 2, lesions reaching the intermediate articular cartilage zone; grade 3, lesions reaching the tidemark or loss of articular cartilage; grades 4 and 5, loss of articular cartilage across 20–50% or 50–80% of the condylar surface, respectively; and grade 6, loss of articular cartilage with exposed subchondral bone. Assigning grades to each of the 4 compartments (lateral and medial tibia and femur) in sections across each entire joint allowed for determination of a maximum lesion grade (i.e., most severe lesion) per joint and per compartment. In addition, we slightly modified this grading system to produce a mean score for each joint and each compartment. By grading lesion severity in each compartment in multiple slides (each containing 5 sections), taken at 120-μm intervals across the entire joint, a mean grade per joint (±SEM) and per compartment was also obtained. These mean grades provide a measure of the extent (representing the relative volume) of the articular cartilage lesion in each joint and compartment.

Osteophyte severity and location were recorded in sections stained with toluidine blue and graded as follows (25): 0 = no osteophytes; 1 = predominantly cartilaginous; 2 = mixed cartilage and bone with chondrocyte hypertrophy and new bone formation; 3 = predominantly bone with marrow spaces. Histologic evaluation was also used to assess pathophysiologic changes in synovial lining, menisci, and ligaments. Selected serial sections were stained with 0.1% Safranin O and with 0.02% fast green to evaluate articular cartilage proteoglycan (PG) content.

Knee joint visualization by micro-CT during applied loading.

A limited number of cadaveric specimens (2 knee joints from 8-week-old mice) were examined by micro-CT to determine whether the location of articular cartilage lesions (induced by in vivo loading) coincided with likely points of articular cartilage “contact” between the tibia and femur. The relative positions of the tibia and femur were visualized in mouse limbs positioned into x-ray–translucent replica cups in a test rig that was identical to that used for in vivo loading. An initial preload (<0.5N) was applied to locate the knee joint securely, and the test rig was then placed in the x-ray tomography machine (V|TOME|X; General Electric) with a nanotomography head and scanned. After the completion of this preload scan, the load was subsequently increased to 9N, allowed to stabilize, and the joints rescanned (0.5° rotation, 100 kV, 70 μA, isotropic voxel size 19.5 μm). Filters were applied to the images prior to reconstruction to remove artifacts, including beam-hardening and ring artifacts.

Statistical analysis.

Statistical analyses comparing loaded and contralateral joints were performed using a paired Wilcoxon's signed rank test, and comparison between different loading regimens were compared by Kruskal-Wallis test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Application of multiple loading episodes induces reproducible focal lesions in the articular cartilage of mouse knee joints.

Previous investigations have shown that the specific loading pattern used in this study (Figure 1A) applied at peak loads of 9N or lower to 8-week-old mouse tibiae 3 times per week for 2 weeks induced minimal or no diaphyseal osteogenesis (8). Since these loads failed to exert discernible osteotrophic effects, we hypothesized that 9N loads are predominantly borne by cartilage. Therefore, we first examined effects on articular cartilage integrity (24, 26) at 9N peak loads using an identical loading pattern. We found that lesions were induced by 6 loading episodes (over 2 weeks), with significant increases in both the maximum (from a mean ± SEM of 0.11 ± 0.07 in contralateral control knees to 3.64 ± 0.12; P = 0.022) and mean (from 0.0075 ± 0.0072 to 0.66 ± 0.09; P = 0.015) grade across the entire joint.

Destabilization models promote articular cartilage lesions and PG loss in specific compartments (27, 28). Assessment performed to establish whether our model showed such compartmentalization indicated that articular cartilage integrity was adversely affected by loading only on the lateral femur, with significant increases in both lesion severity (maximum) and extent (mean score) (Figure 2). In contrast, neither the lateral tibial compartment, the medial tibial compartment, nor the medial femur showed any histologic lesions; articular cartilage in these compartments remained perfectly normal. Closer examination of lateral femur lesions showed a loss of articular cartilage, sufficient to expose the tidemark (Figure 2); loss of Safranin O staining demonstrated reduced PG content in the uncalcified cartilage that was directly adjacent to the lesions. This indicates that repeated application of loading induces reproducible focal articular cartilage lesions that are accompanied by local loss of structural integrity.

thumbnail image

Figure 2. Restricted location of load-induced lesions in the articular cartilage of the lateral femur compartment of the femorotibial joint. A and B, Sections of joints from 8-week-old CBA control mice (A) or group 1 CBA mice (loading applied 3 times each week for 2 weeks) (B) were stained with either toluidine blue (upper panels) or Safranin O (lower panels). Low-power images of the entire joint (left panels) and high-power images of the lateral (middle panels) and medial (right panels) compartments are shown. Control joints exhibited regular articular cartilage surface in all 4 compartments (upper panels) and regular distribution of proteoglycan (lower panels); loaded joints exhibited localized lesions restricted to the lateral femur (upper panels) accompanied by loss of proteoglycan (lower panels). In all panels, the femur and tibia are shown at the top and bottom of the photomicrographs, respectively. Original magnification × 5 in left panels; × 20 in middle and right panels. C and D, Mean ± SEM lesion severity scores in each compartment of control joints (open bars) and loaded joints (shaded bars). Mean (C) and maximum (D) lesion severity scores are shown. Statistically significant load-induced lesions were restricted to the lateral femur. ∗∗∗ = P < 0.001 versus control. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002(ISSN)1529-0131.

Download figure to PowerPoint

To investigate why these lesions were prevalent on the lateral femur, we visualized similarly loaded joints in cadaveric specimens by micro-CT during load application. The results indicated that the articulating femoral and tibial aspects became more closely opposed, with decreased joint space predominantly within the lateral compartment (versus the medial compartment) upon 9N load application (Figures 3B and E). This supports the idea that the location of load-induced compression in the lateral compartment colocalized with the position of reproducible focal lesions and loss of cartilage structural integrity observed by histologic analysis.

thumbnail image

Figure 3. Direct visualization of the knee joint during application of peak 9N loads. A, B, D, and E, Microfocal computed tomography images of the lateral (A and B) and medial (D and E) compartments of a nonloaded joint (A and D) and a loaded joint (B and E) shown from a medial view. Joint space “narrowing” between the femur (top bone) and the tibia (bottom bone) was more marked in the lateral compartment (B) than in the medial compartment (E). C and F, Photomicrographs of sagittal plane sections. Image in F is a higher-magnification view of the boxed area in C. Histologically defined lesions are evident in lateral compartment locations, which coincide with most marked load-induced “narrowing” between the femur and tibia (seen in A, B, D, and E). Original magnification × 2.5 in C; × 10 in F. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002(ISSN)1529-0131.

Download figure to PowerPoint

Single loading episodes induce nonprogressing articular cartilage lesions, and repeated episodes promote their spontaneous subsequent progression.

The versatility of our model allowed us to modify the number and scope of loading episodes to examine changes that may occur following their cessation, after a return to solely habitual use (without the application of loads). This was exploited to determine whether lesions induced by 6 loading episodes (over 2 weeks) showed evidence of spontaneous progression, and to determine the outcome of application of a single loading episode. We observed that articular cartilage lesions were consistently confined to the lateral femur, and contralateral joints in all groups showed normal cartilage without lesions (data not shown). By comparing joints loaded for 6 episodes over 2 weeks (group 1) with those also loaded for 6 episodes but allowed an additional 3 weeks of solely habitual use (group 2), we found a significantly greater mean grade of OA-like lesions after the period of habitual use (Figure 4A). In contrast, maximum severity of lesions showed no further deterioration (Figure 4B). Diminished Safranin O staining was observed around the lesions in both groups (Figure 4C). These data suggest that the 2-week regimen with 3 further weeks of habitual use is sufficient to induce lesions that worsen in their extent, but not in their maximum severity.

thumbnail image

Figure 4. Nonprogressing articular cartilage lesions induced by single-load regimens and spontaneous subsequent articular cartilage lesion progression promoted by multiple-load regimens. Right joints were loaded using 5 different regimens (groups 1–5). A, Mean lesion severity induced by application of loading increased within all groups, except group 5. Compared with 2 weeks of applied loading in group 1, mean lesion severity also increased in joints loaded for 2 weeks and examined after 3 weeks without additional loading (group 2) and in joints loaded for 5 weeks (group 3); joints loaded only once showed no spontaneous lesion progression (group 4 versus group 5). B, Maximum lesion severity induced by application of loading increased within all groups, except group 5. Compared with 2 weeks of applied loading in group 1, the maximum incidence of lesions did not increase in joints loaded for 2 weeks and examined after 3 weeks (group 2), but increased in joints loaded for 5 weeks (group 3); joints loaded only once showed no spontaneous lesion progression (group 4 versus group 5). Values in A and B are the mean ± SEM. ∗ = P < 0.05; ∗∗ = P < 0.01, versus nonloaded joints (open bars); & = P < 0.05. NS = not significant. C, Toluidine blue staining (upper panels) showed evidence of articular cartilage lesions in only the lateral femur compartment of loaded joints, and loss of Safranin O staining (lower panels) from uncalcified cartilage regions was seen in joints from groups 1–3. In contrast, a single loading episode resulted in lesions without loss of Safranin O staining (groups 4 and 5). Original magnification × 20.

Download figure to PowerPoint

To determine whether deterioration (between weeks 2 and 5) occurs as rapidly as the expected load-induced promotion of lesions, we studied mice that were loaded for 15 episodes over 5 weeks (group 3). Analysis of this group showed that the maximum severity and mean extent of lesions were significantly greater than in joints loaded for only 2 weeks (Figures 4A and B), but showed no significant increase when compared with joints loaded for 2 weeks, followed by 3 weeks of normal use (Figures 4A and B). Corresponding decreases in cartilage PG content were also found around the lesions (Figure 4C).

Examination of joints immediately after a single loading episode (group 4) showed that this was also sufficient to induce marked articular cartilage lesions, with a severity somewhat similar to that induced by 2 weeks of loading, but less severe than that induced by 5 weeks of loading (Figures 4A and B). However, joints loaded only once did not show any notable decreases in Safranin O staining (Figure 4C). Examination of joints after 2 weeks of habitual use following a single loading episode did not reveal significant deterioration or reduction in Safranin O staining. This indicates that single loading is sufficient to induce nonprogressive lesions, but with repeated loading episodes, a “threshold” promoting subsequent articular cartilage lesion progression without additional applied loading is exceeded.

Differing loading regimens induce specific responses in distinct joint tissues.

Many articular tissues show important changes in response to mechanical stimuli and OA (29–31). Therefore, we also examined various articular tissue components for any load-induced changes in groups 1–5. Osteophyte formation (32) was graded and location and incidence noted (Table 1). We found that 2 weeks of loading (group 1) led to grade 1 osteophyte generation on the lateral aspect of the medial femoral condyle near the cruciate ligament insertion, in 4 of 7 mice examined (57%). These osteophytes were at an early chondrogenic stage, with pronounced toluidine blue staining of the ECM, and resident chondrocyte-like cells (Figure 5). Three weeks without additional loading (group 2) did not further modify their severity or incidence (Table 1). In contrast, loading of joints 15 times over 5 weeks (group 3) led to an increased incidence of osteophytes but no change in the severity of osteophytes on the medial femur in 8 of 9 mice examined, as well as formation of additional osteophytes on the lateral aspect of the lateral femoral condyle in these joints (Table 1). Not surprisingly, no osteophyte formation was seen in joints that were examined immediately after a single load (group 4); however, grade 1 osteophytes were observed after 2 weeks of further habitual use (group 5; 2 of 6 [33%] and 1 of 6 [16%] on medial and lateral femoral compartments, respectively) (Table 1).

Table 1. Responses induced by different loading regimens in various joint tissues*
 Group
Control12345
  • *

    Incidence (animals affected/animals per group) of pathologic changes in joints loaded using different regimens, as determined histologically. As expected, no pathologic changes were found in nonloaded left joints (control). MF = medial femur; LF = lateral femur; MC = meniscal chondroplasia; MO = meniscal ossification; ST = synovial thickening; SF = synovial fibrosis.

  • Mouse joints in group 1 were loaded on alternate days 3 times each week for 2 weeks and examined 2 days after the final loading episode; joints in group 2 were loaded 3 times each week for 2 weeks and were examined after an additional 3 weeks without further loading; joints in group 3 were loaded 3 times each week for 5 weeks and examined 2 days after the final loading episode; joints in group 4 were loaded for a single episode and examined 2 hours later; and joints in group 5 were also only loaded once but examined after an additional 2 weeks without further loading.

Osteophytes      
 MF0/74/74/78/90/62/6
 LF0/70/70/77/90/61/6
Meniscus      
 MC0/77/77/79/90/65/6
 MO0/71/77/79/90/60/6
Synovium      
 ST0/77/74/79/90/92/6
 SF0/77/72/79/90/60/6
Cruciate ligament0/77/77/79/90/66/6
thumbnail image

Figure 5. Specific responses induced by different applications of loading in distinct joint tissues. A, Schematic diagram of a knee joint showing locations of histologic load-induced changes. This schematic highlights specific affected regions of the joint. LF = lateral femur; MF = medial femur; LT = lateral tibia; MT = medial tibia. Toluidine blue–stained sections corresponding to the areas labeled B–G in A are shown in B–G. Left panels in B–F and upper panel in G show normal histologic features in control joints; right panels in B–F and lower panel in G show loaded joints. B and C, Early stages of osteophyte formation on the lateral aspects of the lateral femur (B) and medial femur (C). D, Changes in the lateral aspect of the lateral meniscus, with chondroplasia (upper panels) and ossification (lower panels) in the fibrocartilage area. E and F, Synovial fibrosis between the lateral tibia and meniscus and synovial lining cell hyperplasia in the area lining the fat pad and ligaments close to the lateral meniscus. G, Cruciate ligament changes, including cell clustering and hypocellular, matrix-rich areas. H, Higher-magnification views of the boxed areas in the lower right panel of D (upper panel) and the lower panel in G (lower panel). Original magnification × 20 in B–G; × 40 in H. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002(ISSN)1529-0131.

Download figure to PowerPoint

Normal murine menisci become ossified (33), containing marrow cavities and outer compartments comprising fibrocartilage. We found that the lateral meniscus showed significant changes in response to applied loading. In all groups that experienced repeated loading (for 2 weeks, 2 weeks followed by 3 weeks of habitual use, and 5 weeks [groups 1, 2, and 3, respectively]), menisci showed chondroplasia (lateral aspect of lateral meniscus) (Figure 5); cells lost their fibroblast-like shape and became round and large, resembling hypertrophic chondrocytes, and toluidine blue staining became more intense, consistent with increased PG deposition (Figure 5). Analysis immediately after just 1 loading episode revealed no overt changes, but meniscal chondroplasia was apparent after 2 further weeks of solely habitual use (83%) (Table 1). Incidence of this phenotype reached 100% in all groups exposed to repetitive loading (groups 1–3). Ossification was also seen in the lateral meniscus in joints that had been loaded for 2 weeks (17% of group 1), and in those that had been loaded for 2 weeks and examined after a further 3 weeks (group 2) (100%), and those loaded for 5 weeks (group 3) (100%); none of the animals undergoing a single loading episode (groups 4 and 5) showed meniscal ossification.

The synovial lining in control joints was thin, with a 1–2-cell-thick intimal surface. Loading led to increases in subintimal fibrosis and to lining cell hyperplasia (thickening). These synovial changes were evident in all joints loaded for 2 weeks (group 1), and most of them were marked laterally, between the tibia and meniscus, along the fat pad close to the lateral meniscus and the medial edge of the lateral tibia (Figure 5); the incidence of synovial change decreased after 3 weeks of further habitual use (group 2) (Table 1). All mice loaded for 5 weeks (group 3) showed fibrotic and hyperplastic synovium, but this was not seen in joints loaded once (group 4), although there was a slight increase in synovial hyperplasia (in 2 of 6 mice) following a further 2 weeks of habitual use (group 5).

Finally, anterior cruciate ligaments showed changes near their tibial insertion. In control joints, there was a smooth transition from cartilage to ligament, with cells aligned and spindle shaped. Applied loads led to development of a rounded, hypertrophic appearance with proliferation to clusters of 4 or more cells, localized decreases in cell content, and increased PG-rich matrix staining (Figure 5). These were consistent features of joints loaded for either 5 weeks or 2 weeks (groups 1–3), but were not observed in joints that had been subjected to a single load episode (group 4). However, the prevalence of these features increased markedly after a further 2 weeks without loading (group 5).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

The in vivo model described herein offers opportunities to noninvasively apply specific mechanical loading elements without surgery, and to study their effects in the intact murine knee joint. The advantages of our model are the adjustability and reproducibility of the episodes of applied loading. We have shown that articular cartilage injury can be induced after 1 loading episode, thereby offering a new model of localized articular cartilage damage. In addition, we found that the application of multiple loading episodes induces lesions that resemble those observed in OA; these lesions increase in severity without further specific applied loading. This provides an opportunity to investigate articular cartilage responses to lesion induction and the mechanisms involved in the progression of OA-like lesions.

Using a specific regimen of loading episodes, articular cartilage lesions were reproducibly restricted to the lateral femur. The likelihood that these were a direct product of localized loading is increased when the supporting results of micro-CT imaging performed during loading application are considered. These showed close apposition across the lateral compartment, with increases from 2N (holding load) to 9N (peak loads) in episodes of compressive loading, indicating that load-related “contact” likely coincides with articular cartilage lesions in the lateral femur. This predominant lateral damage is probably due to the design of the apparatus, in which the knee is held in a valgus position by the custom-made cup during loading.

Our studies also displayed a relative protection of the directly opposed tibial articular cartilage, which is likely to share a virtually identical mechanical challenge. There are many possible explanations for this. The tibial articular cartilage might have distinct structural properties that make it inherently less prone to trauma. Additionally, the tibia may be relatively protected because of the applied loading direction; micro-CT imaging results support the idea that our model probably loads the tibia in a direction that is relatively in accordance with normal use. In contrast, the femoral epiphysis likely receives nonaxial, nonphysiologic compressive loads. This restricted localization of femoral lesions is potentially advantageous from a research perspective. This compartment is not commonly affected by either spontaneous or surgically induced OA (14, 33), and our model may provide a new means for exploring relationships between genetic and load-induced susceptibility to cartilage damage. Though protection against overt lesions in other compartments may be considered advantageous, a lack of histologic lesions might not necessarily signify a lack of biochemical changes, and the use of opposing tibiae as internal controls should be undertaken with caution. The femoral compartment in contralateral, nonloaded joints is a suitable control.

Our model also allows the number of loading episodes to be varied. This profoundly influenced the character of articular cartilage lesions; they increased only in mean lesion grade, but not maximum, following repetitive loading with only habitual use. Lesions induced by the application of multiple loading episodes progressed by becoming more extensive (anteroposterially) without an apparent need for further loading, but not more severe. In contrast, lesions induced by a single application of loading were severe, but not susceptible to such progression. This leads us to hypothesize that a threshold or “point of no return” is reached after 2 weeks of repeated loading, instigating lesion progression independently of additional applied loading. This new model may allow the nature of this threshold to be defined and its underlying mechanisms to be identified. It may also allow adjustment of joint loading to further optimize the induction of cartilage lesions.

Our data suggest that PG loss identifies articular cartilage that is vulnerable to progression resembling that seen in OA (27, 34, 35). By using transgenic and mutant mice, our model could enable elucidation of important processes, as well as new targets for limiting articular cartilage lesion induction and, vitally, for slowing OA progression. Other models are limited by permanent alteration of joint mechanics, whereas the controllability, adjustability, and temporary nature of the applied loading make our model very versatile.

OA influences the whole joint; osteophyte formation and synovial inflammation and fibrosis are late OA hallmarks. Our observations revealed localized medial osteophyte formation, near the cruciate ligament insertion into the femoral groove. This resembled changes that follow instability induced by collagenase injection (36). More prolonged loading (5 weeks) promotes additional osteophyte formation on the lateral femur. To our knowledge, few studies have considered the location of murine osteophytes. Blaney-Davidson et al (36) found that the location was highly dependent upon the model. Future studies may help to define the exact mechanical and molecular cues that control osteophyte formation.

OA is also often associated with synovial lining cell hyperplasia and fibrosis (37–39). However, these features were seen only in joints that received continued loading, and since synovium appeared to rapidly return to normal upon loading cessation, this suggests that synovium has acute sensitivity to mechanical stimuli. Mechanical loads control both synovial fluid and lining cell behavior in vivo (40, 41), and this model provides a new tool for understanding synovial pathophysiology.

Damage or transection of cruciate ligaments usually induces OA. In our model, loading caused marked changes in the cruciate ligament within 2 weeks. The changes resembled those found in OA joints (42), where they are speculated to be the product of high mechanical stress. It is possible that these changes influence OA progression, and our model may make it possible to test such hypotheses. The response of murine menisci to excessive loading resembles secondary ossification. In loaded joints, meniscal chondrocytes become hypertrophied and blood vessels invade (after 2 weeks) before a marrow cavity surrounded by ossified matrix is formed (5 weeks) (43–45). Together with osteophyte formation, synovial hyperplasia and fibrosis, and cruciate ligament changes, this load-induced meniscal ossification indicates the diversity of the specific end points (OA hallmarks) that might be selected for measurement in our model. Their relevance to OA, combined with the likelihood that this model can be modified to accentuate specific desired characteristics, strengthen its potential for use in studying the joint as an organ. The rapid appearance of these OA hallmarks strongly implicates mechanical influences in their development. This model may therefore help identify specific features of the joint loading environment that drive the appearance of these late changes in human OA.

Translating these findings to human OA should be undertaken with great caution, as mouse models have many shortcomings. These include those inherent to all animal models and those specific to mice, such as limitations associated with size, weight, and gait. Further limitations specific to mice include distinct epiphyseal and metaphyseal trabecular bone architecture and mass, and lack of intracortical bone remodeling, which will affect the mechanical environment of articular cartilage during episodes of loading. Mouse articular cartilage composition and organization also hinder direct comparison with human OA. These and many other additional limitations are, however, inevitable consequences of the use of animal models.

In conclusion, we have developed a model for mechanical loading in mouse knee joints, which offers opportunities to study the effects of various loading magnitudes and regimens on joint health and disease. Because the mouse has ready possibilities for genetic manipulation, the interaction between loading and genetics can be more fully investigated. Another attractive feature in this model is its controllability. We have found that identification of short- and long-term effects of single or multiple loading episodes on articular cartilage integrity enables discrimination between spontaneously progressing and nonprogressing lesions, and this model can also be used to regulate the effects of loading on various other joint tissues. The versatility of this model should allow discrimination between joint responses to physiologic and pathologic loading and will, over a longer period of time, enable better elucidation of the interactions between age and genetic and mechanical influences in OA initiation and progression.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Pitsillides had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Poulet, Pitsillides.

Acquisition of data. Poulet, Hamilton, Shefelbine.

Analysis and interpretation of data. Poulet, Pitsillides.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We are indebted to Professor Gordon Blunn, Royal National Orthopaedic Hospital (London, UK), for his help in constructing the cups used in our loading device, and to Rachel Murray, Animal Care Trust at the Royal Veterinary Hospital for the initial studies she completed.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES