Background
Although many studies utilize the guinea pig model to study both idiopathic and posttraumatic Osteoarthritis, it is unknown if the pathological mechanisms are the same. Even though OA is primarily classified as a non-inflammatory arthritis, biomechanical stresses affecting the articular cartilage and subchondral bone and biochemical changes in the articular cartilage and synovial membrane are important in its pathogenesis and may be linked to low grade inflammation [
1]. Mounting research suggests that synovium mediated inflammatory cytokines may mechanistically differentiate idiopathic and post-traumatic OA, in addition to accelerating disease progression in the post-traumatic situation.
In a previous study evaluating biomarker concentrations between idiopathic and posttraumatic OA in Hartley guinea pigs, Wei et al. reported that 12-month-old ACL transected Hartley guinea pigs, those with PTOA, have accelerated articular cartilage damage when compared to age-matched ACL-intact guinea pigs with idiopathic OA [
2]. The greater cartilage damage observed in the ACL-transected knees may be due to either an acceleration of idiopathic OA progression or differences in the pathogenesisincluding the associated inflammatory factors.
It was also found that same-age ACL transected animals presented thicker synovial membranes than that of the idiopathic OA animals. Synovial hyperplasia has been associated with cartilage damage in both animal models and human patients. Synoviocytes have been shown to increase synthesis of SDF-1 and different cytokines such as IL-1β and TNFα during inflammation [
3‐
7]. These signaling molecules cause an up-regulation of aggrecanase and matrix metalloproteinase synthesis in chondrocytes and synoviocytes, which digest surrounding cartilage [
8]. Therapeutic agents that target the inflammatory cytokines IL-1β and TNF-α have been successful in treating rheumatoid arthritis (RA) and related diseases [
9]. Other chemokines, such as SDF-1, may also be involved in OA pathology [
10]. Thus, destabilization of the joint via ACL transection may induce greater synovial membrane proliferation compared to a morphologically equivalent idiopathic OA model, which in turn could alter cytokine profiles, and perpetuate the pathogenesis of post-traumatic OA.
SDF-1 has also been found in higher concentrations in ACL transected Hartley guinea pig joints compared to age-matched idiopathic OA joints [
2]. Briefly, SDF-1 is an 8KDa chemokine originally isolated from a bone stromal cell line [
11]. Through its interaction with CXCR4, it has been shown to stimulate movement of stem cells out of the bone marrow and into circulating blood [
12‐
14]. SDF-1/CXCR interaction also activates calcium, Erk, and p38 MAP kinase signaling pathways in chondrocytes, thereby inducing the release of MMPs, and other proteins required for chemotaxis and other biological processes [
15,
16]. Specifically, SDF-1 has been shown to increase the concentration of MMP-3, −9, and −13, thus increasing of the destruction of the ECM proteins [
17]. Studies have shown that blocking SDF-1’s receptor, CXCR4 decreases MMP-13 expression in in vitro human chondrocytes [
16]. Moreover, it has been shown that synovial cells significantly increase synthesis of SDF-1 during inflammation-induced hypoxia, and that SDF-1 plays a central role in the pathogenesis of murine collagen-induced arthritis by attracting leukocytes to the inflamed joints [
18,
19]. Thus, SDF-1 may be an important component in differentiating between idiopathic and post-traumatic OA.
In this study, we tested the hypotheses that post-traumatic (ACL transected) and idiopathic (ACL intact) OA in the Hartley Guinea pig model lead to similar changes in cartilage morphology, but progress via different pathological mechanisms. We assessed pathological mechanisms by evaluating the synovial fluid SDF-1 and MMP-13 concentrations; in addition to quantifying synovial membrane morphology between anatomically similar idiopathic and post-traumatic joints.
Methods
Animals
Thirty male Hartley guinea pigs (Elm Labs; Chelmsford, MA) were randomly divided into 5 groups of six animals. Group 1 (
n = 6) represented the baseline ACL-intact control group (no OA), which were euthanized via CO
2 at 3-months of age. Group 2 (
n = 6) represented the idiopathic OA group, which were maintained until 12 months of age. Group 3 (
n = 6) underwent anterior cruciate ligament transection (ACLT) on the right knee at 3 months and were euthanized at 5.5-months, Group 4 (n = 6) consisted of ACLT animals sacrificed at 6.5-months, and Group 5 (n = 6) consisted of ACLT animals sacrificed at 7.5-month-old. After completion of the first phase of the study, a sham group, group 6 (n = 6), underwent a 1.5 cm sagittal joint capsule incision and was sacrificed at an experimentally determined equivalency point (5.5-months). The sham sacrifice time point was selected to match the time at which the ACLT joints showed similar cartilage damage to the idiopathic OA joints. The contralateral (ACL-intact) knees of the ACL-transected animals served as an additional control (Groups 3, 4, 5; Left knees). The right knee of the sham group (Group 6) was used as a control to account for the biomechanical instability created by the ACLT procedure while the left knee of the sham animals served as a control to gauge the development of post-traumatic OA of the ACLT animals. Three months was determined as an appropriate age to initiate the experiment as guinea pigs have reached skeletal maturity without exhibiting evidence of cartilage damage [
20]. All animals were individually housed under standard conditions.
Surgery
The surgical animals were anesthetized with an intraperitoneal injection of ketamine (40 mg/Kg) and medetomidine (0.5 mg/Kg). The right knee was shaved and prepped with betadine. Animals were placed in the prone position, and a 2 cm midline incision was made over the anterior knee. The skin was mobilized to expose the patellar tendon. A 1.5 cm incision through the joint capsule was made immediately lateral to the patellar tendon. The patella was then everted, and the ACL was incised with the knee in a flexed position. Manual testing of anterior laxity confirmed complete sectioning of the ACL. In sham animals, only a 1.5 cm incision was made penetrating the joint capsule. For all operated groups, the joint capsule and fascia were closed in layers using interrupted 4-0 Vicryl sutures, and skin using 4-0 Nylon sutures. Post-operative analgesia was maintained using buprenorphine hydrochloride (.05 mg/Kg SQ for 3 days). Sutures were removed 10 days post-surgery. Animals were allowed to bear weight on limbs as tolerated, and equal load bearing was noted between limbs within 3 weeks of surgery.
Macroscopic analysis
The proximal tibias were amputated and immersed in 10% (
v/v) formalin for at least 72 h. Before histological processing, gross morphological cartilage lesions on tibial condyles were visualized and qualitatively analyzed via India ink staining, in which the cartilage surface is painted, for 15 s, with a 20% (v/v) dilution of blue India ink (Parker, Quink) in phosphate buffered saline containing protease inhibitors [
21]. Cartilage samples were qualitatively analyzed for lesion severity and extent.
Histology
The specimens were then decalcified in 10% EDTA solution and bisected in the coronal plane. They were processed in a Tissue-Tek VIP 1000 tissue processor (Model#4617, Miles, Elkhart, IN) and embedded in a single block of Paraplast X-tra (Fisher, Santa Clara, CA). Blocks were trimmed to expose tissue using a rotary microtome (Model#2030, Reichart-Jung, Austria). Slices were taken from the mid-coronal plane within each condyle and cut into 6 μm thick sections, taken 0 μm, 100 μm, and 200 μm, from the midcoronal line. Sections were then mounted on slides and stained with safranin-O/fast green. The severity of cartilage damage of each joint was assessed using the modified Mankin grading system [
20]. Three independent observers scored both the medial and tibial condyle compartments using a digital imaging camera (Nikon microscope E 800). All scoring was done in a random order and in a blinded fashion. The worst scores for each medial and lateral tibial compartment were statistically analyzed. Medial, lateral, and infrapatellar compartments of synovial membrane specimens were dissected, fixed, embedded, cut and stained as described above. Using the Pelletier grading system, the worst synovial scores from each joint were analyzed [
22]. All histological imaging utilized the SPOT digital imaging camera (Diagnostic Instruments, Sterling Heights, Michigan).
Synovial fluid (SF) collection and analysis
Before dissecting the joints, 100 μL of isotonic saline solution was injected intra-articularly, the knee was manually cycled through flexion and extension ten times and then the joint was aspirated. The lavaged synovial fluid was centrifuged at 2000 g for 10 min to remove cells and debris, and then frozen at −80 °C until analysis.
Prior to the SDF-1 and MMP-13 analysis, 20 μL aliquots were treated with 15 U/ml of bovine testicular hyaluronidase for 10 min at 37 °C to reduce viscosity. SDF-1 and MMP-13 concentrations in the SF samples were measured in duplicate. Commercially available double-antibody sandwich enzyme-linked immunosorbent assays (ELISA) were used to detect, SDF-1α (DSA00, R&D, Minneapolis, MN) and MMP-13 (F13 M00, R&D Systems, Minneapolis, MN) in the synovial lavages following the manufacturer’s instructions.
Statistics
Analyses of variance (ANOVA) for repeated measures were used to compare the histological measurements of cartilage damage and synovial membrane inflammation between the six groups. Similar analyses were performed to evaluate the concentrations of SDF-1 and MMP-13. The different weights of the guinea pigs between groups were considered using analysis of covariance (ANCOVA). A two-way mixed absolute interclass reliability coefficient (ICC) was calculated to evaluate the reliability of both the Mankin and Pelletier scoring systems. Follow-up pair-wise comparisons between multiple experimental groups were carried out with orthogonal contrasts using the Tukey’s test (α = .05) and test of homogeneity. Adjusted p-values were reported to account for the multiple comparisons. Prior to analysis, normality was confirmed with the Shapiro-Wilk test. Statistics were performed on SPSS software (SPSS Inc., Chicago, Illinois).
Discussion
Differentiating the pathophysiology of idiopathic and post-traumatic OA may provide important data regarding the development of therapeutic strategies for different types of OA. The data obtained in this study suggest that the mechanism of disease progression with and without traumatic injury may be different. We found that synovial inflammation, which was independent of SDF-1 and MMP-13, was markedly different between idiopathic and post-traumatic OA. We determined that the cartilage damage seen at 12 months with idiopathic OA was histologically equivalent to that seen at 5.5 months in ACLT animals with post-traumatic OA as indicated by the modified Mankin scores. These findings were also supported by the gross and histological morphology assessments (Figs.
1 and
2).
As hypothesized, histological differences were noted for synovitis in all experimental groups (Fig.
4). The association between OA and synovitis is not fully understood, though previous studies suggest that synovitis is involved. Fernandez-Madrid et al. reported that 73% of OA patients have synovitis, and that the presence of synovitis in early OA may predict the need for joint replacement surgery [
23]. It has been hypothesized that synovitis can trigger bone catabolism through the activation of Toll-like receptors (TLRs) and complement cascades [
24‐
27]. Furthermore, synovectomy is an effective surgical method for preventing cartilage destruction and relieving pain in both OA and RA patients [
28].
Synovitis has been well characterized in rheumatoid arthritis studies regarding inflammatory cell adhesion and activation, the production of mediators (such as cytokines, chemokines, and growth factors), angiogenesis, joint destruction, fibrosis, and bone resorption [
29]. Accordingly, synovitis associated changes were observed microscopically with synovial and villous hyperplasia, surface fibrin deposition, and subintimal fibrosis (Fig.
4) present in both sham left and right, 5.5-ACLT unoperated, 12-month primary OA, and ACLT joints.
In our study, angiogenesis, fibrosis, and leukocyte infiltration were unique to the ACLT joints, suggesting that synovitis is an important mechanism in accelerating cartilage damage. Potential causes of this inflammation could be due to capsulotomy and altered joint biomechanics which in turn may alter cytokine profiles [
30]. The combination of both is more likely as the age-matched ACLT joints in our study had both more severe synovitis compared to all other groups and higher modified Mankin scores compared to the sham operated knee. Furthermore, due to the exposure of the joint during capulotomy, the ACL transected animals would be expected to have greater inflammation and synoviocyte activation compared to the sham treated animals.
The idiopathic OA model of this study showed a moderate level of synovitis with notable synovial hyperplasia and fibrosis despite being significantly less severe than the ACLT groups. This result suggests that synovitis may be a part of idiopathic OA pathology but to a lesser degree than in post-traumatic OA following ACL transection. The cause of synovitis in idiopathic OA is unknown, but studies have highlighted potential mechanisms. Because of the lack of a basement membrane and a highly vascularized subintimal layer in the synovial membrane, it is likely that age-related mediators, among others, may induce synovitis [
31]. Huebner et al. demonstrated that Hartley guinea pigs, which exhibit idiopathic OA with aging, had greater levels of IL-6 compared with age-matched Strain 13 guinea pigs, which do not exhibit OA [
32]. This finding implicates this cytokine in synovitis with idiopathic OA [
33]. Synovitis in idiopathic OA may elicit the SDF-1 and MMP-13 signaling pathways, among others, similar to those activated with post-traumatic OA and in combination with age-related changes in proteoglycan turnover, collagen, and vascular changes [
34‐
38].
Further exploration is needed to determine the interactions between the inflammatory mediators and synovitis. Although elevated in relatively-accelerated cartilage damage in our previous study, SDF-1’s induction of cartilage degradation via MMP-13 may not be involved in pathologically differentiating idiopathic and post-traumatic OA, as no statistical significance was observed between the 12-month primary OA guinea pig knees and the 5.5-ACLT knees. There was also a moderate correlation between increases in SDF-1 and modified Mankin scores for all treatment groups (r
2 = .592), suggesting that SDF-1 may provide a downstream mechanism for both idiopathic and post-traumatic OA. Unfortunately, no other studies have compared SDF-1’s involvement in idiopathic or post-traumatic OA models. Because SDF-1 and MMP-13 do not seem to play a greater role in either idiopathic or post-traumatic OA in guinea pig models, other inflammatory factors may be involved. Elsaid et al. has proposed a possible mechanism of secondary OA pathogenesis via synovial upregulation of IL-1β, TNFα, procathepsin B, and neutrophil elastase, which are capable of being synthesized by the synovium and downregulate lubricin, a chondroprotective and lubricating protein of articular cartilage [
39].These cytokines may also regulate chondrogenic production of inflammatory factors, synovial cell overgrowth, and collagenase [
40].
Surprisingly, the contralateral unoperated knee in ACL transected animals presented with moderate OA. Compensatory biomechanical processes may cause a shift in knee kinematics, sensitization of chondrocytes, followed by subsequent reorganization and degeneration favoring catabolic over anabolic cellular processes [
31]. Cartilage damage in the contralateral joint may also be further exacerbated by systemic inflammatory effects related to OA in the operated knee.
There were some limitations to the study. First, we were only able to obtain a mean volume of 120 μL from each animal, and this amount reduced our ability to run additional ELISAs and other experiments. Many previous studies have used GAG, C2C, and COMP to quantify the extent of cartilage damage. However, our limited amounts of synovial fluid did not allow us to test these markers. Serum concentration assays were possible, but may not have been a completely valid option as Kojima et al. found that synovial fluid proteoglycan concentrations did not reflect reductions of proteoglycan in articular cartilage [
41]. Nonetheless, the modified Mankin score has proven to be reliable in characterizing OA progression [
42]. Second, we did not record the precise location of synovitis in the knee, which may provide important data regarding the mechanism of synovitis [
43]. Third, the PTOA model in this study may have also been exacerbated given underlying idiopathic OA pathology. Given that the study did not include an idiopathic-osteoarthritis-free ACLT group, some of the observed changes may have occurred as a result of natural OA progression in the Guinea pig. To this regard, studies have only identified modest joint disruption at the 5.5-month time point, with the onset of moderate OA associated changes at 12-months of age [
44,
45].
Although not detailed in this work, previous mechanistic work has shown that SDF-1 is produced from the synovium to interact, through paracrine signaing, with its receptor CXCR4 on the articular surface. Kanbe et al. showed that SDF-1 is secreted primarily by synovial fibroblasts in models of both osteoarthritis and rheumatoid arthritis at a high level [
18]. Further looking at the production of SDF-1, it was shown that in surgical models where the synovium was excised, the levels of SDF-1 in both the synovial fluid and serum dropped precipitously, suggesting that the responsible cells were localized to the synovium [
29]. A potential limitation, SDF-1 has also been shown to be produced in subchondral bone marrow [
18,
46]. Further research is needed to explore the relative contributions of synovium-derived versus marrow-derived SDF-1, but previous work showing large reductions of SDF-1 concentration after synovectomy without global pharmacologic therapy supports the thesis that the critical volume of SDF-1 originates from the synovium. Furthermore, immunohistochemical methods have been used to localize SDF-1 to the synovial membrane, where SDF-1 positivity was localized in cells lining synovial tissue and in perisynovial epithelial and lymphocytic cells and found to be absent on the articular surface [
18,
29].
In summary, this study indicates that the pathogenesis of post-traumatic OA may be synovium mediated and its pathology does not progress uniquely through an SDF-1 related pathway. Future studies are needed to assess other inflammatory pathways related to synovitis that may initiate and maintain post-traumatic OA, and to help differentiate its pathological mechanism from idiopathic OA.