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. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: Arthritis Rheumatol. 2014 May;66(5):1256–1265. doi: 10.1002/art.38375

Early Response of Mouse Joint Tissues to Noninvasive Knee Injury Suggests Treatment Targets

P Wu 1,5, N Holguin 1, M J Silva 1,4, M Fu 5, W Liao 5, L J Sandell 1,2,3
PMCID: PMC4310559  NIHMSID: NIHMS630811  PMID: 24470303

Abstract

Objective

Joint trauma can lead to a spectrum of acute lesions, including cartilage degradation, ligament or meniscus tears, and synovitis, all potentially associated with osteoarthritis. The goal of this study was to generate and validate a murine model of knee joint trauma following non-invasive controlled injurious compression in vivo and to investigate early molecular events.

Methods

The right knees of 8-week old mice were placed in a hyperflexed position and subjected to compressive joint loading at one of three peak forces (3, 6, 9 N) for 60 cycles in a single loading period and harvested at 5, 9 and 14 days post loading (n=3–5 mice for each time point and for each loading). The left knees were not loaded and served as the contralateral controls. Histological, immunohistochemical and ELISA analyses were performed to evaluate acute pathologic features in chondrocyte viability, cartilage matrix metabolism, synovial reaction, and serum COMP levels.

Results

Acute joint pathology was associated with increased injurious loads. All loading regimens induced chondrocyte apoptosis, cartilage matrix degradation, disruption of cartilage collagen fibril arrangement, and increased levels of serum COMP. We also observed that 6N loading induced mild synovitis by day 5 whereas at 9 N, with tearing of the anterior cruciate ligament, severe posttraumatic synovitis and ectopic cartilage formation were observed.

Conclusion

We have established and analyzed some early events in a murine model of knee joint trauma with different degrees of over-loading in vivo. These results suggest that immediate therapies particularly targeted to apoptosis and synovial cell proliferation could affect the acute posttraumatic reaction to potentially limit chronic consequences and osteoarthritis.

Introduction

Osteoarthritis is a frequent cause of disability following trauma to weight-bearing joints. It is estimated that at least 12% of symptomatic OA has a post-traumatic etiology (1). In the young population of recent war veterans, joint injuries resulted in OA in 60% of all injuries and 100% of knee injuries (2), causing unprecedented disability. The majority of joint injuries do not involve articular fractures; rather, they consist of damage at the level of the cells and matrices often associated with ligamentous, meniscal, or joint capsule damage. Joint trauma is usually associated with several pathological events, including cell death, altered cartilage structure, and release of blood into the joint space in the absence of articular stabilization (3). Injuries severe enough to result in ACL rupture or meniscus tears, eventually involve cartilage destruction and osteophyte formation (4), thus the magnitude of load and subsequent articular injury severity may play a fundamental role in the pathologic progression of OA and also serve as important prognostic indicators for clinical outcomes. The mechanisms by which the extent of injury severity may lead to increased cartilage degeneration and synovial response are unclear, although cartilage and synovial pathology are believed to be critical to the development of OA. The early response resulting from articular injury may initiate events important to the progression of OA, but those early disease events remain incompletely characterized.

In order to gain insight into early response of joint tissue following increasing severity of knee injuries, we adapted a noninvasive murine model of compressive load to the knee joint that results in injuries to the articular cartilage and other joint tissues (5). Currently, invasive mouse knee injury models are major tools for OA research, including injecting collagenase into the joint (6, 7), using a needle to induce cruciate transection in the closed knee (8), or using surgical techniques to transect or injure the ligaments of the knee or the medial meniscus (9, 10). However, these models do not faithfully mimic clinically-relevant injury conditions, and the use of invasive surgical procedures may introduce confounding factors associated with the surgery itself, which may mask the native biologic response to injury. Consequently, the noninvasive murine model used here is especially attractive for studies of early pathological events in OA.

The objective of this investigation was to examine the effect of a loading method that allows for precise control of the loading history and where the joint injury is created during a single loading session. We utilized this model to determine the time course of articular cartilage and synovial changes following knee injury in mice with increasing magnitude of peak force. We hypothesize that the determination of the time course and dose dependence of pathologic changes in tissues in the joint after traumatic injury will help establish the targets for treatments and eventually the window of opportunity for therapies aimed at ultimately limiting chronic consequences and potentially OA.

Methods

Loading procedure

Eight-week-old male C57BL/6 mice (Jackson laboratory, Bar Harbor, ME) were used in the studies approved by the Animal Studies Committee of Washington University. While under anesthesia, right tibiae (n = 54) were positioned vertically, with the ankle upward and the knee downward in deep flexion, between custom-made cups (Figure 1A). Using a materials testing machine (Instron ElectroPulse E1000, Norwood, MA), axial compressive loads were applied through the knee joint via the upper loading cup (attached to the actuator) and the lower fixed cup (linked to the load cell) (11). Three different loads were applied: group 1 (n=13) was subjected to peak force of 3 N, group 2 (n=13) was subjected to peak force of 6 N and group 3 (n=13) was subjected to peak force of 9 N. At the start of the loading session, a 0.5 N compressive preload was applied. Sixty cycles of loading were then applied. Each cycle consisted of a triangle wave with a rise time to peak force of 0.17 × and a fall time of 0.17 s followed by a 10 s rest period (holding at 0.5 N) (Figure 1B) (5, 12). For the 9 N loading group, a discontinuity in the force-displacement curve was seen during the first cycle. Based on pilot studies this corresponds to ACL rupture. Knee joints in 3 groups were examined at 5 days (n=5), 9 days (n=3) and 14 days (n=5) after the loading. The left knees served as the contralateral controls. Three age-matched mice were used as uninjured controls. Animals had access ad libitum to standard mouse chow and water.

Figure 1.

Figure 1

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Noninvasive mouse knee loading model induces cartilage damage in lateral femoral condyle. (A) the right tibia of each test mouse was positioned vertically with the ankle upward and the knee downward in deep flexion, between custom-made cups; (B) Diagram of an example cycles of applied load, showing hold and peak load magnitudes, rate of load application, and interval time between peaks (C) Serial sagittal 10-μm sections from individual knees were cut through the entire lateral femoral condyle. (D) From the lateral side to the medial side of knee joint, serial sections were stained with Safranin-O fast green (to evaluate articular cartilage proteoglycan content), or Picrosirius Red, and a set of sections next to those shown was left unstained for immunohistochemical staining. Bar=200 μm.

Histological and immunohistochemical analyses of cartilage and synovium

Knee joints were dissected, fixed, decalcified, and paraffin embedded. Serial sagittal 10-μm sections from individual knees were cut through the entire lateral femoral condyle as reported elsewhere (5, 13) (Figure 1C, D). Selected sections were stained with Safranin-O fast green (to evaluate articular cartilage proteoglycan content), or Picrosirius Red (to evaluate collagen content) (data not shown), according to standard protocols. From the serial Safranin-O stained sections in each sample, we selected one section with the widest cartilage lesion that featured focal loss of Safranin-O staining, minor fissuring of articular cartilage, and atrophy of articular chondrocytes. According to these histological changes, it is possible to identify the demarcation between the normal and injured cartilage tissue, and the length of the cartilage lesion range was measured.

Synovial pathology was evaluated based on four Safranin-O fast green stained sections per knee joint using a modified form of an established synovitis score for changes in synovial lining thickness (14) with the scale, 0, normal thickness of synovial lining (1–2 cells); 1, thickness 2–4 cells; 2, thickness 4–9 cells; and 3, thickness ≥ 10 cells.

TUNEL assay for apoptosis detection

Detection of apoptotic cells was performed with the terminal deoxnucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay.

Immunofluorescence staining for ECM molecules

Sections were deparaffinized, rehydrated and digested with proteinase K (10 μg/ml for 20 min at 37°C) to enhance antibody penetration and then blocked with 10% serum in PBS for 1 h at room temperature. Next, the slides were incubated with the IIF antibody against the type II collagen triple helical domain (15) (1:100) at 4°C overnight in the blocking solution, washed and incubated with a second primary antibody against either aggrecan (LEC7, G3 domain of aggrecan, a gift from Dr. Kurt Doege, Louisiana State University, (16) (1:100), type I collagen (Abcam, USA) (1:200), or COMP (gift from Dr. Dick Heinegard, University of Lund, Sweden) (1:100). Fluorescent detection of each protein was achieved using either secondary goat Alexa Fluor 488-conjugated anti-rabbit IgG (1:250) or goat Alexa Fluor 594-conjugated anti-rat IgG antibodies (1:250) (Invitrogen, USA), and counterstained with DAPI (1:1000)(Vector Laboratories, Burlingame, CA). The images were captured using a 20× objective on an Eclipse E800 microscope (Nikon), Q Imaging Retiga 2000R Fast 1394 camera and MetaMorph software v 7.7 (Molecular Devices, Sunnyvale, CA).

Serum levels of COMP

Blood was collected by cardiac puncture at days 5, 9 and 14 in injured mice and uninjured mice to collect serum. Serum levels of COMP were measured using a commercially available animal assay (MD Bioproducts, St Paul MN) at 1:10 dilution, as per the manufacturer’s protocol.

Statistical analysis

Statistics were performed with SPSS software (SPSS Inc., Chicago IL). Each data point represents the mean and standard deviation. Histological scores and ELISA results for injured and uninjured knees were compared by Student t-tests or one-way ANOVA followed by Tukey’s post hoc test with statistical significance set at p=0.05.

Results

Acute joint pathology is associated with increased injurious load in the mouse knee

The injury protocol, sectioning and loading regimen are shown in Figure 1. In the joints loaded at 3 N and 6 N, the alignment of fibers in the ligament indicated normal tension in anterior cruciate ligament. In the 9 N joints, ruptured ACL led to lax fiber alignment in the ligament, increased joint gap, and anterior translation of tibia (Figure 2A). In these animals, each 9 N peak load resulted in rupture of the ligament, whereas the ligament was intact in the 3 N and 6 N peak loads. Therefore, the outcome varies with the dose, with cartilage damage sustained at all peak loads and ACL rupture at 9 N peak load thus additionally resulting in instability of the entire joint.

Figure 2.

Figure 2

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Acute joint pathology is associated with increased injurious load in the mouse knee. (A) F indicates the femur; T indicates the tibia; the asterisk indicates anterior cruciate ligament (ACL); the hash sign indicates joint gap; and dotted lines indicate the joint alignment. Only the 9 N load induced ACL rupture indicated by disrupted alignment of the ligament fibrils, increased joint gap, and the forward position of the tibia in relation to the femur; (B) indication of the portion of cartilage and synovium we selected for evaluation; (C, D) comparison of the length of the cartilage lesion range (based on Safranin O staining) and the synovitis score among groups, ** indicated significant difference from the other groups, p<0.01; (E) Safranin O stained sections of injured cartilage, dotted lines indicate the range of loss of staining; (F) TUNEL staining, dotted curve lines indicate the cartilage surface; (G) enlargement of the synovial lining cell layer was assessed from Safranin O stained sections of mouse knee joints, the red squares indicate regional synovial lining cells.

The location of the injured cartilage as well as the region sampled for synovial proliferation are shown in Figure 2B. Based on the sections with most severe loss of Safranin-O staining per knee, the extent of articular cartilage lesions (e.g. the length of PG loss region) was measured (Figure 2C). Loss of Safranin-O staining can be seen in all the injured limbs (Figure 2E). The more severe cartilage injuries were associated with higher compressive load: the average lesion range in each group was 0.29 ±0.03mm for 9 N, 0.26±0.05mm for 6 N (p=0.085 compare to 9 N group), and 0.17±0.03 mm for 3 N (p<0.001 compare to 6 N or 9 N group). There was a trend toward increase of the lesion range from day 9 to day 14 in 6 N and 9 N loading groups (data not shown), but the results did not reach statistical significance across these three time points post-injury within each loading group. This was possibly due to the greater variation in the pathological changes in articular cartilage by 2 weeks of habitual use following a single loading episode (Figure 2C, E).

Apoptosis was observed within the lesion domain by the TUNEL reaction with each loading intensity indicative of DNA fragmentation, fluorescing green (Figure 2F). With the highest load at 9 and 14 days, fewer apoptotic cells were observed possibly due to the removal of cell debris.

To evaluate posttraumatic synovial response in all groups, we used a modified form of an established synovitis score for changes in synovial lining thickness (17). Synovitis scores for 9 N loading were significantly higher than that of 6 N group at all time points (p<0.01), while in 3 N group, there was no significant difference with the control group. The applied loading magnitude was correlated with the synovitis score (Figure 2D, G). Within the cartilage lesions, the range of Safranin O staining loss (Figure 2E) was coincident with the range of chondrocyte apoptosis (Figure 2F) indicating pathological changes in extracellular matrix structural integrity. Cell death was evident at all loads at the site of femoral-tibial contact, however 9 N impacts resulted in cell death in the deeper zone cartilage compared to 3 N and 6 N impacts with loss of DAPI staining in more superficial zones (Figure 2F).

Chondrocyte ECM

To better characterize the composition and distribution of the ECM, we focused on four major matrix constituents, type II collagen, aggrecan, COMP, and type I collagen, and compared their distribution patterns at the injured site to the adjacent uninjured cartilage. The immunofluorescence signal of type II collagen in the injured area was not significantly different from that of the uninjured area. In the uninjured cartilage region, aggrecan presented a smooth and intense pericellular distribution pattern. Note that the antibody for aggrecan used in this study was raised against the G3 domain of aggrecan: this domain is often removed from the aggrecan in the interterritorial region (16). Type II collagen was uniformly present in all cartilage zones, although the intensity of fluorescence appears higher in the calcified cartilage. A large reduction in pericellular aggrecan thickness and intensity in extracellular distribution was evident around the cells in the injured region with an increased presence of aggrecan inside the cells (Figure 3A): this was coincident with loss of proteoglycan content in injured site as observed by Safranin-O staining (Figure 2E).

Figure 3.

Figure 3

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Double immunofluorescence stained sections showing cartilage matrix degradation at 5 days and 14 days after injurious load. Dotted parallel lines indicate the range of injured cartilage region, and white arrows indicate abnormal distribution pattern of matrix proteins. (A) ACAN (green) and type II collagen (red); (B) COMP (green) and type II collagen (red); (C) type I collagen (green) and type II collagen (red) in injured joints. Nuclei are counterstained with Dapi (blue). Bar=100 μm.

In contrast to aggrecan, COMP distribution varied with the depth of the cartilage. It was distributed around chondrocytes in the uninjured area in the superficial zone of the cartilage; while in the injured area, COMP-positive cells extended to the middle zone in all groups (Figure 3B). The pericellular distribution of COMP was less well organized in the injured area compared to the normal region (Figure 3B). More cells became COMP-positive from 5 to 14 days post injury, with increasing cell staining for COMP with increased compression. For type I collagen, characteristic of bone, we observed traces of type I collagen in the injured region of cartilage on day 14 in all groups and on day 5 in 9 N group, while it was absent in the uninjured cartilage (Figure 3C).

Aggrecan is known to be internalized after degradation likely mediated by CD44, the hyaluronan receptor (18, 19). Nagase and colleagues (20) recently demonstrated that the enzyme that cleaves aggrecan, ADAMTS-5, is more active when cells are dead than alive due to inefficient inhibition, in dead or dying cells, of the enzyme by the cell surface receptor, LRP1. To investigate whether the cells with internalized aggrecan fragments (Figure 3A) were dying, we stained these cells using the TUNEL technique to identify cells undergoing DNA fragmentation indicative of apoptosis. We found that cells with clearly defined pericellular aggrecan were TUNEL negative and had intact nuclei staining with DAPI, therefore alive. The cells lacking pericellular aggrecan and with internalized aggrecan were TUNEL positive (Figure 4), indicating that the aggrecan internalization coincided with cells dying by apoptosis.

Figure 4.

Figure 4

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Knee joint cartilage injured by 3 N compressive load showing a reduction in pericellular aggrecan thickness and intensity in extracellular distribution that was evident around the cells in the impact area. (A) Loss of Safranin-O staining (S.O.) was observed in the impact area. (B) Representative images of TUNEL assay combined with immunofluorescence staining for aggrecan (ACAN). Inferior ACAN encapsulation and thickness around apoptotic chondrocytes (nuclei are stained in green) in the injured area, compared to clear pericellular aggrecan in TUNEL negative cells (nuclei are stained in blue). Bar=20 μm.

Temporal synovial reaction and early ectopic cartilage formation

The synovial lining in control joints and 3N loading group was thin, with a 1–2 cell thick intimal surface. Both 6 N and 9 N loading led to synovial cell proliferation and lining cell hyperplasia in a dose-dependent manner (Figure 2D). Safranin O stained neo-cartilage tissue was observed in regions of synovium and meniscus, and in the anterior aspect of the lateral femoral condyle, potentially the origin of chondro-osteophytes (Figure 5A). No similar ectopic neo-cartilage tissue formation was seen in joints in the 3 N and 6 N loading groups (data not shown). In the 9 N loading group, significant synoviocyte proliferation was observed at 5 days after injury (Figure 5B). On day 9, several cells with rounded, enlarged nuclei formed clusters in subintimal zone; there was localized decrease in cell density, and increase in PG-rich matrix staining. Aggrecan and COMP were extensively deposited while very little type II collagen was present in the synovia. On day 14, however, the chondrogenic cell clusters showed increased ECM production and localized type II collagen co-staining with aggrecan and COMP (Figure 5B).

Figure 5.

Figure 5

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Temporal synovial reaction and early ectopic cartilage formation were observed in 9 N group. (A) Neo-cartilage tissue was observed in specific regions in the injured joints; (B) images of temporal reaction in the regional synovia indicated with a black square in (A) at day 5, 9 and 14. Consecutive sections were stained with Safranin O, or double immunofluorescence with ACAN (green) and type II collagen (red) or COMP (green) and type II collagen (red); S indicates synovium; M indicated meniscus; F indicates femoral condyle. White arrows indicated new formed cartilage tissue. (C) Temporal changes in concentrations of COMP fragments in serum after injury. ***: significantly different (p<0.01) from all groups; ** on day 5, 3 N group significantly different from 6N group (p<0.05) and 9 N group(p<0.01); * on day 9, 9 N group significantly different (p<0.05) from the others.

Posttraumatic serum COMP levels increased

With the significant presence of COMP in the proliferated synovial cells, we measured COMP levels in serum. COMP was significantly increased by load within 5 days, especially in the 9 N loading (Figure 5C). On day 5, the serum COMP level in 3 N group was 0.27 U/dL, which was significant lower than that of the 6 N or 9 N groups (0.30 or 0.31 U/dL respectively, p<0.01); on day 9, serum COMP level in 6 N group was decreased to 0.26 U/dL, which was similar to the 3 N group. It remained significant higher in the 9 N group on day 9 (0.29 U/dL, p<0.05), while on day 14, there was no significant difference in serum COMP levels among these three groups, although all were elevated when compared to control values.

Discussion

The identification of early events leading to OA is a primary goal of the OA research field and will likely offer testable targets for therapies. In this study we used a non-invasive method for inducing localized articular cartilage injury by 3 N, 6 N or 9 N compressive loads in mouse knees in vivo and identified specific cellular responses to injury. These rapid and substantial early changes in different joint tissues with intricately linked biological relationships indicate that immediate therapies that are specifically targeted to mechanical load-induced cell apoptosis and synoviocyte proliferation could affect the acute posttraumatic reaction to ultimately limit chronic consequences and potentially OA.

By comparison with other similar non-invasive murine models, different loading regimens induced different pathologic sequelae. Multiple loading produced cartilage and bone changes in older mice (21). Poulet and colleagues (5) reported that a single loading episode induced only nonprogressing articular cartilage lesions within 2 weeks, and repeated episodes promoted their spontaneous subsequent progression. In their loading regimen, peak loads of 9 N were applied for 0.05 seconds, with a rise and fall time each of 0.025 seconds and 40 cycles in a single session (5). In our model, peak loads were applied for 0.33 seconds, with a rise and fall time each of 0.17 seconds and 60 cycles in a single session. We observed a trend towards increase in the lesion range from day 9 to day 14 in 9 N loading group, but the results did not reach statistical significance across these three time points post-injury. Our results support the idea that cycling mechanical stimuli in a single loading session could lead to local cartilage damage in lateral femoral compartment; however the cartilage injury did not progress to degeneration within 2 weeks post-injury. It is likely that degeneration would develop after a longer interval, given the early changes we observed and based on results from others. In particular, Christiansen and colleagues reported that one-cycle loading of 12 N induce ACL rupture and resulted in a mild OA by 56 days, with the cartilage lesions localized in all the compartments of the knee joint (13). It is possible that the ACL rupture induced by this one-cycle load released energy that might reduce the direct damage to the cartilage. Accordingly, this one-cycle load/ACL rupture approach provides a condition of joint instability rather than direct cartilage injury. Thus, cartilage degeneration that was observed in all the knee joint compartments by 56 days was likely induced by habitual use following ACL rupture. In the studies reported here, using 60 cycles, at 9 N we induced ACL rupture plus local cartilage damage due to the 60 cycles of loading (i.e. 1 cycle to rupture the ACL followed by 59 cycles at progressively increasing displacement).

Cartilage Response

All three levels of mechanical load caused cell and matrix damage. The impact zone was easily and reproducibly observed by loss of Safranin-O staining in the femur by 5 days after injury and the size of the impact zone was dose-dependent. The loss of Safranin-O staining was accompanied by apoptosis of the cells in the superficial zone, at 3N or 6N, and in the deeper zone, at 9 N peak force. When we investigated the damage to matrix molecules, the response was found to be very specific. The two matrix proteins investigated, COMP and aggrecan, exhibited differential responses. Aggrecan appeared to be fragmented around cells where Safranin-O staining had been lost and appeared to be inside the cell rather than in the ECM. While this had not been observed in vivo, Embry and Knudson (18) and Fosang and colleagues (19) had found that upon cleavage of aggrecan with ADAMTS-5, the fragments of aggrecan are internalized into the chondrocyte by the cell surface receptor for hyaluronic acid, CD44. Recently, Nagase and colleagues reported that ADAMTS-5 is bound in an inactive state by the cell surface receptor LRP-1 and activated when cells die (20). Our results are consistent with these previous reports, however, we cannot totally rule out that the reduced pericellular staining is solely due to decreased synthesis of aggrecan. In light of these findings, we co-stained the sections with TUNEL and aggrecan that allowed us to reveal a striking difference between the impact zone and the surrounding cartilage: only dying cells appeared to be internalizing their aggrecan; live cells clearly had a substantial pericellular complement of aggrecan. Whether aggrecan is fragmented or intact cannot be directly determine by these studies. These results along with those cited above, demonstrate how chondrocytes respond to injurious compression and the specific molecular events that lead to rapid matrix alterations – even at the lowest force used in this study. The specific time course of events in aggrecan breakdown and apoptosis remains to be studied.

The COMP distribution was independent from aggrecan distribution. Pericellular COMP is primarily observed in the superficial cartilage zone with very little being seen in the middle and deep zones. Initially, the superficial zone COMP appeared to be internalized by the chondrocytes. In the lower zone, over time and with increasing impact, COMP synthesis appeared to increase, demonstrating more pericellular COMP in all zones at higher impacts over time. Thus, the chondrocyte response to mechanical stress includes synthesis of COMP. By assaying mRNA levels and protein levels, we have recently reported the same COMP response to compression in three dimensional cultures (22). According to in vitro studies from other laboratories, mechanical load stimuli could increase COMP mRNA expression levels and the synthesis of COMP (23), indicating that cartilage tissue can remodel its ECM in response to altered mechanical environment.

Other studies have documented some of these early events. Carames and colleagues (2012) showed that cell death and loss of sGAG in articular cartilage subjected to mechanical injury was a time-dependent process (24). The pharmacologic inhibition of mTORC-1 prevented cell apoptosis by partly enhancing autophagy, and consequently reducing sGAG loss after mechanical injury (24). In bovine cartilage explant studies, Seol and colleagues (25) proposed that HMGB-1 released during apoptosis induced migration and proliferation of chondroprogenitor cells. However, the efficacy of these cells was called into question because they produced relatively high levels of pro-inflammatory molecules and matrix metalloproteinases considered to be detrimental to the cartilage.

Synovial Response

Synovial proliferation was correlated with loading intensity, however, at 9 N with rupture of the ACL, synovial proliferation increased substantially. The rapid and considerable synovial responses following 9 N load may be due to several contributing factors including ACL rupture, destabilization of the joint, induced inflammatory cytokines and hemarthrosis. The extent of synovial proliferation remained the same up to two weeks, however, the sequelae of events within the synovia continued to develop. By five days, the synovium had reached maximum proliferation with some matrix synthesis; by nine days, the cells were synthesizing cartilaginous ECM molecules throughout the tissue. And by 14 days, cartilaginous nodules appeared in the tissue.

Based on systematic review by de Lange-Brokaar and colleagues, synovitis is a common feature of OA and the proinflammatory cytokines tumor necrosis factor TNF-α and interleukin IL-1β were most frequently detected in OA synovial tissue (26). Synovitis is usually characterized by the presence of infiltrating immune cells, such as macrophages, T cells and mesenchymal stem cells (17). Kurth and colleagues reported the existence of resident MSCs in the knee joint synovium undergoing proliferation and chondrogenic differentiation following injury in vivo (27). Our study indicated an important role of synovial cells in the process of neo-cartilage metaplasia that was observed primarily at the transition zone where the synovium connects to the meniscus or articular cartilage, mimicking the osteophyte observed in OA. A similar observation of ectopic chondrogenesis is from Watson and colleagues who reported that gene delivery of TGF-β1 induced massive proliferation of synovial fibroblasts and chondrometaplasia of synovium in vivo (28).

Serum COMP

The extracellular matrix protein COMP is secreted by chondrocytes, synoviocytes, fibroblasts, and even osteoblasts (29, 30). Not only with cartilage damage, but physical exercise such as running, cycling and swimming can also increase COMP serum levels (31). Our results showed higher serum COMP levels after injury in all groups within the first 5 days, and all groups had a downward trend with a slow decline, however, the differences between different loading groups were small. We expected to see more COMP in the sera of mice subjected to 9N loading due to the larger number of cells in the joint producing COMP. However, our results are consistent with those of Christiansen et al. (13) who observed a similar level of COMP from 1 day to 56 days post injury. It is possible that there is additional COMP coming from other tissues after injury and the amount contributed by the cartilage and synovium is relatively small. However, both our studies and those of Christiansen agree in that the increased COMP could be a biomarker to detect alterations in metabolism of articular cartilage and joint tissue.

Ectopic Chondrogenesis

Ectopic bone is a significant problem in injured joint tissues. By 14 days post injury, focal regions of chondrogenesis were seen in the meniscus, synovium and periosteum identified by enlargement of the cells, intense rather than generalized staining with Safranin-O, and reaction with aggrecan and type II collagen antibodies. Depending on the location of these regions of chondrogenesis, they will result in synovial chondrocalcinosis, chondro-osteophyte or ectopic bone. The presence of bone was confirmed by (13) by micro CT at day 56 after injury.

Tochigi and Buckwalter showed that human intra-articular fractures resulted in acute chondrocyte death that predominated along fracture lines and spontaneously progressed in the forty-eight hours following injury (32). Lotz and colleagues reported that mechanical injury lead to cell death which was because of injury induced autophagy suppression. In these studies, pharmacologic enhancement of autophagy prevented cell and matrix damage, suggesting a novel approach for chondrocyte protection (33). A recent study by Olson et al, found that compared to C57BL/6 mice, MRL/MpJ mice had less severe intraarticular and systemic inflammation following joint injury, which may halt the progression of post-traumatic arthritis (34).

Translating these results to a human traumatic joint injury should be undertaken with great caution, as there are many shortcomings inherent to murine model, such as mouse articular cartilage cell density and arrangement, matrix structure and composition and organization that are different from human articular cartilage. We used 8 week old mice which are not skeletally mature, and the corresponding human age may be younger than the target population for knee injuries and treatment. Additionally, limitations associated with animal gait and knee joint mechanics in habitual activities were also different from human knee joint.

CONCLUSIONS

In conclusion, the findings of the current study indicate that various magnitudes of injurious loading cause acute joint pathological alterations including localized cell death, matrix damage, and synovitis to different extents. Only the 9 N loading resulted in ACL rupture and consequent cartilage metaplasia in synovium. Future studies will investigate the role of injury-induced cytokines or other events that may be vital in establishing the window of opportunity for therapeutic interventions aimed at inhibiting posttraumatic synovial hyperplasia and cartilage metaplasia in synovium. Treatment of these events could affect the acute posttraumatic reaction to ultimately limit chronic consequences and potentially OA. These results provide basis for novel therapeutic approaches that would target specific acute events, help to determine which events are most important in the development of OA and test targeted treatments at the early time of injury or later.

Acknowledgments

This work was supported by a Grand Opportunity Grant funded by the American Recovery and Reinvestment Act and awarded by the NIH (RC2-AR-058978 to Drs. Jim Cheverud and Linda Sandell), RO1AR050847 and RO1AR045550 (LJS). The histologic analysis was performed and supported in part by the NIH grant P30-AR057235.

References

  • 1.Brown TD, Johnston RC, Saltzman CL, Marsh JL, Buckwalter JA. Posttraumatic osteoarthritis: a first estimate of incidence, prevalence, and burden of disease. Journal of orthopaedic trauma. 2006;20(10):739–44. doi: 10.1097/01.bot.0000246468.80635.ef. [DOI] [PubMed] [Google Scholar]
  • 2.Rivera J, Wenke J, Buckwalter JA, Ficke J, Johnson A. Posttraumatic Osteoarthritis Caused by Battlefield Injuries: The Primary Source of Disability in Warriors. Journal American Academy of Orthopaedic Surgeons. 2012;20(1):S64–S9. doi: 10.5435/JAAOS-20-08-S64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Martin JA, Buckwalter JA. Post-traumatic osteoarthritis: the role of stress induced chondrocyte damage. Biorheology. 2006;43(3–4):517–21. [PubMed] [Google Scholar]
  • 4.von Porat A, Roos EM, Roos H. High prevalence of osteoarthritis 14 years after an anterior cruciate ligament tear in male soccer players: a study of radiographic and patient relevant outcomes. Ann Rheum Dis. 2004;63(3):269–73. doi: 10.1136/ard.2003.008136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Poulet B, Hamilton RW, Shefelbine S, Pitsillides AA. Characterizing a novel and adjustable noninvasive murine joint loading model. Arthritis Rheum. 2011;63(1):137–47. doi: 10.1002/art.27765. [DOI] [PubMed] [Google Scholar]
  • 6.van Osch GJ, van der Kraan PM, Blankevoort L, Huiskes R, van den Berg WB. Relation of ligament damage with site specific cartilage loss and osteophyte formation in collagenase induced osteoarthritis in mice. J Rheumatol. 1996;23(7):1227–32. [PubMed] [Google Scholar]
  • 7.Joosten LA, Smeets RL, Koenders MI, van den Bersselaar LA, Helsen MM, Oppers-Walgreen B, et al. Interleukin-18 promotes joint inflammation and induces interleukin-1-driven cartilage destruction. Am J Pathol. 2004;165(3):959–67. doi: 10.1016/S0002-9440(10)63357-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Hanashi D, Koshino T, Uesugi M, Saito T. Effect of femoral nerve resection on progression of cartilage degeneration induced by anterior cruciate ligament transection in rabbits. J Orthop Sci. 2002;7(6):672–6. doi: 10.1007/s007760200119. [DOI] [PubMed] [Google Scholar]
  • 9.Kamekura S, Hoshi K, Shimoaka T, Chung U, Chikuda H, Yamada T, et al. Osteoarthritis development in novel experimental mouse models induced by knee joint instability. Osteoarthritis Cartilage. 2005;13(7):632–41. doi: 10.1016/j.joca.2005.03.004. [DOI] [PubMed] [Google Scholar]
  • 10.Glasson SS, Blanchet TJ, Morris EA. The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthritis Cartilage. 2007;15(9):1061–9. doi: 10.1016/j.joca.2007.03.006. [DOI] [PubMed] [Google Scholar]
  • 11.Silva MJ, Brodt MD, Lynch MA, Stephens AL, Wood DJ, Civitelli R. Tibial loading increases osteogenic gene expression and cortical bone volume in mature and middle-aged mice. PLoS One. 2012;7(4):e34980. doi: 10.1371/journal.pone.0034980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Brodt MD, Silva MJ. Aged mice have enhanced endocortical response and normal periosteal response compared with young-adult mice following 1 week of axial tibial compression. Journal Bone Mineral Research. 2010;25(9):2006–15. doi: 10.1002/jbmr.96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Christiansen BA, Anderson MJ, Lee CA, Williams JC, Yik JH, Haudenschild DR. Musculoskeletal changes following non-invasive knee injury using a novel mouse model of post-traumatic osteoarthritis. Osteoarthritis Cartilage. 2012;20(7):773–82. doi: 10.1016/j.joca.2012.04.014. [DOI] [PubMed] [Google Scholar]
  • 14.Lewis JS, Hembree WC, Furman BD, Tippets L, Cattel D, Huebner JL, et al. Acute joint pathology and synovial inflammation is associated with increased intra-articular fracture severity in the mouse knee. Osteoarthritis Cartilage. 2011;19(7):864–73. doi: 10.1016/j.joca.2011.04.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Oganesian A, Zhu Y, Sandell LJ. Type IIA procollagen amino propeptide is localized in human embryonic tissues. The journal of histochemistry and cytochemistry: official journal of the Histochemistry Society. 1997;45(11):1469–80. doi: 10.1177/002215549704501104. [DOI] [PubMed] [Google Scholar]
  • 16.Sandy JD, Thompson V, Doege K, Verscharen C. The intermediates of aggrecanse-dependent cleavage of aggrecan in rat chondrosarcoma cells treated with interleukin-1. Biochem J. 2000;351:161–66. doi: 10.1042/0264-6021:3510161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Krenn V, Morawietz L, Burmester GR, Kinne RW, Mueller-Ladner U, Muller B, et al. Synovitis score: discrimination between chronic low-grade and high-grade synovitis. Histopathology. 2006;49(4):358–64. doi: 10.1111/j.1365-2559.2006.02508.x. [DOI] [PubMed] [Google Scholar]
  • 18.Embry JJ, Knudson W. G1 domain of aggrecan cointernalizes with hyaluronan via a CD44-mediated mechanism in bovine articular chondrocytes. Arthritis Rheum. 2003;48(12):3431–41. doi: 10.1002/art.11323. [DOI] [PubMed] [Google Scholar]
  • 19.Fosang AJ, Last K, Stanton H, Weeks DB, Campbell IK, Hardingham TE, et al. Generation and novel distribution of matrix metalloproteinase-derived aggrecan fragments in porcine cartilage explants. Journal of Biological Chemistry. 2000;275(42):33027–37. doi: 10.1074/jbc.M910207199. [DOI] [PubMed] [Google Scholar]
  • 20.Yamamoto K, Troeberg L, Scilabra SD, Pelosi M, Murphy CL, Strickland DK, et al. LRP-1-mediated endocytosis regulates extracellular activity of ADAMTS-5 in articular cartilage. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2012;2:511–21. doi: 10.1096/fj.12-216671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Ko FC, Dragomir C, Plumb DA, Goldring SR, Wright TM, Goldring MB, et al. In vivo cyclic compression causes cartilage degeneration and subchondral bone changes in mouse tibiae. Arthritis Rheum. 2013;65(6):1569–78. doi: 10.1002/art.37906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Wu P, DeLassus E, Patra D, Liao W, Sandell LJ. Effects of serum and compressive loading on the cartilage matrix synthesis and spatiotemporal deposition around chondrocytes in 3D culture. Tissue Eng Part A. 2013;19(9–10):1199–208. doi: 10.1089/ten.tea.2012.0559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Wong M, Siegrist M, Cao X. Cyclic compression of articular cartilage explants is associated with progressive consolidation and altered expression pattern of extracellular matrix proteins. Matrix Biol. 1999;18(4):391–9. doi: 10.1016/s0945-053x(99)00029-3. [DOI] [PubMed] [Google Scholar]
  • 24.Carames B, Taniguchi N, Otsuki S, Blanco FJ, Lotz M. Autophagy is a protective mechanism in normal cartilage, and its aging-related loss is linked with cell death and osteoarthritis. Arthritis Rheum. 2010;62(3):791–801. doi: 10.1002/art.27305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Seol D, McCabe DJ, Choe H, Zheng H, Yu Y, Jang K, et al. Chondrogenic progenitor cells respond to cartilage injury. Arthritis Rheum. 2012;64(11):3626–37. doi: 10.1002/art.34613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.de Lange-Brokaar BJ, Ioan-Facsinay A, van Osch GJ, Zuurmond AM, Schoones J, Toes RE, et al. Synovial inflammation, immune cells and their cytokines in osteoarthritis: a review. Osteoarthritis Cartilage. 2012;20(12):1484–99. doi: 10.1016/j.joca.2012.08.027. [DOI] [PubMed] [Google Scholar]
  • 27.Kurth TB, Dell’accio F, Crouch V, Augello A, Sharpe PT, De Bari C. Functional mesenchymal stem cell niches in adult mouse knee joint synovium in vivo. Arthritis Rheum. 2011;63(5):1289–300. doi: 10.1002/art.30234. [DOI] [PubMed] [Google Scholar]
  • 28.Watson RS, Gouze E, Levings PP, Bush ML, Kay JD, Jorgensen MS, et al. Gene delivery of TGF-beta1 induces arthrofibrosis and chondrometaplasia of synovium in vivo. Lab Invest. 2010;90(11):1615–27. doi: 10.1038/labinvest.2010.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Recklies AD, Baillargeon L, White C. Regulation of cartilage oligomeric matrix protein synthesis in human synovial cells and articular chondrocytes. Arthritis Rheum. 1998;41(6):997–1006. doi: 10.1002/1529-0131(199806)41:6<997::AID-ART6>3.0.CO;2-G. [DOI] [PubMed] [Google Scholar]
  • 30.Di Cesare PE, Fang C, Leslie MP, Tulli H, Perris R, Carlson CS. Expression of cartilage oligomeric matrix protein (COMP) by embryonic and adult osteoblasts. J Orthop Res. 2000;18(5):713–20. doi: 10.1002/jor.1100180506. [DOI] [PubMed] [Google Scholar]
  • 31.Celik O, Salci Y, Ak E, Kalaci A, Korkusuz F. Serum cartilage oligomeric matrix protein accumulation decreases significantly after 12weeks of running but not swimming and cycling training - A randomised controlled trial. Knee. 2013;20(1):19–25. doi: 10.1016/j.knee.2012.06.001. [DOI] [PubMed] [Google Scholar]
  • 32.Tochigi Y, Buckwalter JA, Martin JA, Hillis SL, Zhang P, Vaseenon T, et al. Distribution and progression of chondrocyte damage in a whole-organ model of human ankle intra-articular fracture. J Bone Joint Surg Am. 2011;93(6):533–9. doi: 10.2106/JBJS.I.01777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Carames B, Taniguchi N, Seino D, Blanco FJ, D’Lima D, Lotz M. Mechanical injury suppresses autophagy regulators and pharmacologic activation of autophagy results in chondroprotection. Arthritis Rheum. 2012;64(4):1182–92. doi: 10.1002/art.33444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Lewis JS, Jr, Furman BD, Zeitler E, Huebner JL, Kraus VB, Guilak F, et al. Genetic and cellular evidence of decreased inflammation associated with reduced incidence of posttraumatic arthritis in MRL/MpJ mice. Arthritis and rheumatism. 2013;65(3):660–70. doi: 10.1002/art.37796. [DOI] [PMC free article] [PubMed] [Google Scholar]

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