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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Orthop Res. Author manuscript; available in PMC Jul 1, 2011.
Published in final edited form as:
PMCID: PMC2875364
NIHMSID: NIHMS162481

A Comparison of Differential Biomarkers of Osteoarthritis with and without Post-traumatic Injury in the Hartley Guinea Pig Model

Abstract

The objective was to compare biomarkers of articular cartilage metabolism in synovial fluid from Hartley guinea pig knees with and without anterior cruciate ligament transection (ACLT) to establish whether there are detectable differences in biomarker levels between primary and secondary OA. Synovial fluid lavages and knees were obtained from 3 months (control group) and 12 months (primary OA group) animals. Another group of animals (post-traumatic OA group) underwent unilateral ACLT at 3 months and samples were obtained 9 months post-surgery. Synovial fluid concentrations of stromal cell-derived-factor (SDF-1), collagen fragments (C2C), proteoglycan (GAG), lubricin, matrix metalloproteinase-13 (MMP-13), and Interleukin-1 (IL-1β) were evaluated. Cartilage damage was assessed via histology. The highest concentrations of C2C and SDF-1 in synovial fluid were found in the post-traumatic OA group, moderate concentrations were found in the primary OA group, and low concentrations in the control group. GAG release in synovial fluid was similar to C2C and SDF-1. The lubricin concentrations were significantly lower in ACLT joints than either the control or 12-month primary OA groups, but not between the control and primary OA groups. Higher levels of MMP-13 and IL-1β were detected in the joints of the post-traumatic OA group as compared to the control or primary OA groups. Histology revealed greatest OA damage in the post-traumatic OA group, followed by moderate and minimal damage in primary OA and control groups, respectively. This study indicates that the biomarkers and progression of OA may differ in the Hartley guinea pig models with and without post-traumatic OA.

Keywords: Cartilage, osteoarthritis, trauma, ACL, biomarker, guinea pig

Introduction

Primary OA has been modeled using the Hartley guinea pig,1-6 where the extent of cartilage damage has been correlated with age, body weight, and mechanical loading.1,6,7 Surgically induced animal models of secondary OA, including anterior cruciate ligament transection (ACLT),8-10 are frequently utilized to study mechanisms and therapies for primary OA. However, it remains unknown whether there are pathological differences between primary and secondary OA that could be detected using biochemical and morphometric measurements.

Because lubricin provides chondroprotection to the knee, 11-13 it may be an important biomarker for post-traumatic OA. Lubricin is a multifaceted glycoprotein present in synovial fluid, which binds to the lamina spendens, and provides boundary lubrication.11-13 ACL injury has been shown to decrease lubricin levels following injury in animal models10,11 and patients.14 It has been hypothesized that the loss of lubricin may be a factor placing the ACL injured patient at risk for post-traumatic OA.

In addition to lubricin, this study also tracked several other biomarkers that may be important for tracking primary and post-traumatic OA: SDF-1, MMP-13, C2C, GAG, BC-3, and IL-1β. Stromal cell-derived-factor 1 (SDF-1) is a chemokine originally isolated from a bone stromal cell line.18 The concentration of SDF-1 has been shown to be related to arthritis development.19 SDF-1 upregulates matrix metalloproteinase-13 (MMP-13),20 a proteolytic enzyme secreted by chondrocytes, that has been shown to cleave type II collagen and proteoglycans.21 C2C and glycosaminoglycans (GAG) are markers of collagen type II fragmentation21 and aggrecan release,22 respectively. BC-3, which recognizes an aggrecanase (ADAMTS-1, -4 & -5)-generated N-terminal neoepitope after cleavage within the interglobular domain, is another biomarker of aggrecan turnover23. Likewise, Interleukin-1β (IL-1β) is an inflammatory cytokine that is involved in lubricin catabolism and cartilage degeneration,15-17 and may play a role in both primary and post-traumatic OA.

The objective of this study was to compare synovial fluid biomarker concentrations (SDF-1, lubricin, C2C, GAG, BC-3, MMP-13, and IL-1β) between Hartley guinea pig knees with (secondary OA) and without ACLT (primary OA) to that of younger controls (no OA). We hypothesized that biomarkers of cartilage metabolism may be different with and without ACLT; in particular that the SDF-1 concentrations will increase in primary OA, and lubricin levels will decrease in secondary OA.

Methods

Specimens

Fifteen male Hartley guinea pigs, 3-months of age, were obtained from Charles River Laboratories (Wilmington, MA). The animals were a subset of those used for a previous study.10 Five animals were euthanized immediately and formed the baseline control (Group 1; no OA); four animals were maintained under standard housing conditions until 12 months old and then euthanized (Group 2; primary OA group); and six 3-month old guinea pigs underwent surgical ACLT in the right leg and were euthanized nine months post-operatively (Group 3; ACLT; secondary OA group). Synovial fluid lavages were performed and the hind limbs harvested immediately after euthanasia. A sample of synovium from the capsule was also harvested. IACUC approval was obtained prior to initiating the study.

ACL Transection Procedure

ACLT was surgically performed in the right knee of the Group 3 animals as previously described.10 Once anesthetized, an incision was made through the capsule just lateral to the patellar tendon. The ACL was cut with a scalpel. Manual laxity testing verified its functional loss. The joint capsule, fascia, and skin were closed in layers. Post-operatively the animals were allowed to bear weight on limbs as tolerated.

Synovial Fluid Collection and Analyses

Synovial fluid lavages were collected from the knees after euthanasia.10 100μL of isotonic saline solution was measured with a pipette, and was injected intra-articularly using a 30 gauge insulin syringe inserted through the inferior patellar tendon. With injection, the joint capsule was visibly distended. The knee was then manually cycled through flexion and extension ten times to distribute the fluid within the joint before collection via joint aspiration. It was noted that approximately half of the fluid that was injected was recovered. The synovial fluid was centrifuged at 2,000 g for 10 minutes to remove cells and debris and frozen at −80°C until analysis. Once thawed, seven biochemical markers of articular cartilage metabolism were measured in the synovial fluid lavages: SDF-1, lubricin, C2C, sulfated-glycosaminoglycans (GAG), BC-3, MMP-13, and IL-1β.

SDF-1 concentrations were quantified using a double-antibody sandwich ELISA (DSA00, Human CXCL12/SDF-1 α, R&D, Minneapolis, MN).19,20 100 μL of the standard or the sample (1:20 dilution) were added per well and incubated for 2 hours at room temperature (RT). Each well was washed three times. 200 μL of SDF-1α conjugate was added to each well and incubated for 2 hours. After wash, 200 μL of substrate solution were added to each well for 30 minutes at RT. 50 μL of stop solution were added to each well and the density was determined within 30 minutes using a microplate reader set to 450 nm.

Lubricin levels were assayed via a sandwich ELISA using peanut agglutinin (PNA) and anti-lubricin mouse monoclonal antibody S6.7924 as previously reported.10 96 well plates were coated overnight with PNA at 4°C in 50 mM sodium bicarbonate buffer, pH 9.5 at a final concentration of 10 μg/ml. The following day, serial dilutions of purified human lubricin and aspirated synovial fluid (1:20 dilution) were incubated on the PNA-coated plates for 60 minutes at RT. After wash with PBS, S6.79 was added at a 1:10,000 dilution and incubated for 60 minutes at RT, followed by washing with PBS. Goat anti-mouse IgG-alkaline phosphatase was added at 1:1,000 dilution to the plate and incubated for 60 minutes at RT. Finally, 4-methylumbelliferyl phosphate (4-MUP) was added and the fluorescence was measured using 465 nm and 550 nm as emission and excitation wavelengths.

C2C concentrations were quantified by ELISA (60-1001-001, IBEX Technologies Montreal, Quebec).25 Plates were coated with 100 μL/well of C2C coating conjugate overnight at 4°C. 50 μL of standards and samples (1:20 dilution) were added to the polypropylene mixing plate. Then 50 μL of C2C antibody diluted in assay buffer was added per well. The plates were pre-incubated for 30 minutes at RT. After washing six times, 80 μL of antigen-antibody mixture was transferred from each well to the corresponding wells of the ELISA plate, and the ELISA plates were incubated for 1 hour at RT. After wash, 100 μL /well GAM-HRP conjugate was added into the ELISA plate and incubated for 30 minutes at RT. 100 μL of tetramethylbenzidine (TMB) added into each well after wash and incubated for 30 minutes at RT. 100 μL of stop solution was added into each well and the plate was read at 450 nm.

The concentrations of sulfated glycosaminoglycans (GAG) were quantified spectrophotometrically with dimethylmethylene blue dye (DMMB) using bovine chondroitin sulfate as standard controls.22 The assay is based on the formation of an insoluble blue colored complex between GAG and DMMB that was quantified at a 525 nm absorbance wavelength.

BC-3, MMP-13, and IL-1β were detected by Western blot using group pooled samples (Group 1: n=3; Group 2: n=3; Group 3: n=4). Total protein was quantified as described in the BAC Protein Assay Reagent Kit (Pierce, Rockford. IL). 10 μg of total protein was electrophoresed in 10% SDS PAGE under reducing conditions. After electrophoresis, proteins were transferred onto Immobilon-PVDF membrane (NJ0HYB0010, Fisher Scientific) and probed with a monoclonal antibody against aggrecan fragments (BC3-ab3773, Abcam Inc, Cambridge MA), MMP-13 (mAB171; R&D systems Inc., Minneapolis MN),23,26, and IL-1β (D-17; Santa Cruz Biotechnology, Inc, Santa Cruz CA). The antibodies were diluted 1:1,000 in PBS-T containing 1% bovine serum albumin. Horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) or goat anti-rabbit IgG (H+L) (Bio-Rad Laboratories, Richmond, CA) was diluted 1:3000 in PBS-T and used as the secondary antibody. Visualization of immunoreactive proteins was achieved by using the ECL Western blotting detection reagents (Amersham, Arlington Heights, IL) and by subsequently exposing the membrane to Kodak X-Omat AR film.

Histology

The proximal tibia were removed from the harvested joints and immersed in 10% formalin for 72 hours. The specimens were decalcified in Richman-Gelfand-Hill solution and bisected in the sagittal 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 then taken along the sagittal plane at the midline of each compartment of the tibial plateau. The slices were then cut into 6 μm sections, mounted on slides, and stained with safranin-O/fast green. The severity of cartilage damage of each joint was then assessed using the modified Mankin grading system.27 Three independent and blinded observers scored each section and the scores for the medial and lateral tibial condyles were averaged within each joint.

Statistics

Mixed linear models were used to compare the 3 groups on the concentrations of SDF-1, lubricin, C2C, GAG, and the histological measurements of cartilage damage. Groups were modeled with heterogeneous variances. Residual estimates of maximum likelihood were used to fit the models to provide unbiased estimates for missing data due to the limited availability of synovial fluid samples. Post-hoc paired comparisons between the 3 experimental groups were carried out with orthogonal contrasts using the Holm test to maintain alpha at 0.05. Adjusted p-values are reported to account for the multiple comparisons. All data are presented as means ± standard deviations. The relationships between the Mankin score versus the SDF-1, lubricin, C2C. and GAG concentrations in synovial fluid were assessed with regression analysis.

Results

Histology

Safranin O staining revealed the greatest OA lesions in the 12-month ACLT joints (Group 3), followed by moderate lesions in the 12-month primary OA joints (Group 2), and virtually no damage in the 3-month control joints (Group 1) (Fig. 1). All group comparisons between the mean Mankin scores were significant (p<0.001). We also observed changes in the synovial membrane. There was only one layer of synovial membrane in the 3-month-old control animals. Two or three layers of synovial membrane were found in 12-month-old primary OA animals (Group 2) and a thicker membrane was observed in 12-month-old ACLT animals (Group 3) (Fig. 2).

Fig. 1
(A) Safranin O staining and (B) the modified Mankin score showed a marked increase in cartilage damage for the 12 month ACLT knees (Group 3; n=7) as compared to the 12-month joints (Group 2; n=6) and 3-month control joints (Group 1; n=7). Loss of proteoglycan ...
Fig. 2
The ACLT joints (Group 3) had thicker synovial membranes compared to the 12 month joints (Group 2) and the 3-month controls (Group 1).

SDF-1

The concentrations of SDF-1 were dependent on the animal group (p<0.0001) (Fig. 3A). Only a relatively small amount of SDF-1 was found in the knee of the control animals (Group 1). The mean concentration of SDF-1 increased by 90% in the primary OA group (Group 2) as compared to the control group (Group 1) (p=0.002). The mean concentration increased by 166% in the secondary OA group (Group 3) relative to the control group (p<0.0001). The concentration of SDF-1 in the secondary OA group (Group 3) was significantly greater than that of the primary OA group (Group 2) (p=0.005). There was a significant correlation (p<0.0001) between the SDF-1 concentration in synovial fluid and the Mankin score (r2 = 0.58; n=20) (Fig 3B). An increase in SDF-1 release corresponded to an increase in Mankin score.

Fig. 3
(A) The SDF-1 concentrations of the 12 month primary OA (Group 2; n=6), the 12 month ACLT (Group 3; n=7), and the 3 month control animals (Group 1; n=7) were significantly different. (B) There was a significant correlation (p<0.001; n=20) between ...

Lubricin

The concentrations of lubricin were dependent on the animal group (p<0.0001) (Fig. 4A). Relatively high levels of lubricin were detected in the control animals (Group 1). The mean concentration of lubricin decreased by 6% in the primary OA group (Group 2) as compared to the control group (Group 1), though this was not significantly different (p=0.460). The mean concentration of lubricin in the secondary OA group (Group 3) decreased by 75% relative to the control group (Group 1) (p<0.0001). The concentration of lubricin in the secondary OA group (Group 3) was significantly less than that of the primary OA group (Group 2) (p<0.0001). There was a significant inverse correlation (p<0.0001) between the lubricin concentration and the Mankin score (r2 = 0.83; n=20) where a decrease in lubricin corresponded to an increase in Mankin grade (Fig 4B).

Fig. 4
(A) Mean lubricin concentrations were significantly different between the 12 month (Group 2; n=6) and the 12 month ACLT (Group 3; n=7) but there was no significant difference between the 3 month (Group 1; n=7) and 12 month (Group 2) animals. (B) There ...

C2C

The concentration of C2C was dependent on the animal group (p<0.0001) (Fig. 5). Only a relatively small amount of C2C was found in the knee of the control animals (Group 1). The mean concentration of C2C increased by 443% in the primary OA group (Group 2) relative to the control group (Group 1) (p<0.0001). The mean concentration increased significantly by 1359% in the secondary OA group (Group 3) relative to the control group (p=0.014). The mean concentration of C2C in the secondary OA group (Group 3) was significantly greater than that of the primary OA group (Group 2) (p=0.044). There was a significant (p<0.0001) correlation between the C2C concentration in synovial fluid and the Mankin score (r2=0.898; n=11). An increase in C2C release corresponded to an increase in Mankin grade.

Fig. 5
Mean concentrations of C2C in the synovial fluid lavages were significantly different between the 12 month (Group 2; n=3) and the 12 month ACLT (Group 3; n=5) animals, and the 3 month controls (Group 1; n=4).

GAG

The concentration of GAG was dependent on the animal group (p<0.0001) (Fig. 6A). Relatively small amounts of GAG were found in the knees of the control animals (Group 1). The mean concentration of GAG significantly increased by 130% in the primary OA group (Group 2) relative to the control group (Group 1) (p<0.0001). The mean concentration of GAG significantly increased by 229% in the secondary OA group (Group 3) relative to the control group (p<0.0001). The mean concentration of GAG in the secondary OA group (Group 3) was significantly greater than that of the primary OA group (Group 2) (p=0.019). There was a significant correlation (p=0.0001; n=17) between the GAG concentration in synovial fluid and the Mankin score (r2=0.658). An increase in GAG release corresponded to an increase in Mankin grade.

Fig. 6
(A) Mean concentrations of GAG were significantly different between the 12 month (Group 2; n=6) and the 12 month ACLT (Group 3; n=7) animals and the 3 month controls (Group 1; n=6). (B) Western blot analysis of BC-3 qualitatively supported these findings ...

Western blot analysis of aggrecan fragments (BC3) also showed that the amount of proteoglycan fragments in the lavage was much higher in ACLT animals compared to 12-month-old primary OA (Fig. 6B).26 There were almost no aggrecan fragments detected in the control animals (Fig. 6B).

MMP-13 and IL-1β

Western blot analyses showed that the ACLT guinea pig joints had higher expression levels of MMP-13 and IL-1β than those of the 12-month-old primary OA joints (Fig. 7). Only weak expressions of MMP-13 and IL-1β were detected in 3-month-old control animals (Fig. 7).

Fig. 7
Western blot analysis showed that the increased levels of MMP-13 (pooled sample; Group 1 n=3; Group 2 n=3, Group 3 n=4) and IL-1β (pooled sample; Group 1 n=3; Group 2 n=3, Group 3 n=4) release were found in the ACLT group compared to the 12-month ...

Discussion

The data produced in this study suggest that the mechanism of OA progression with and without post-traumatic injury may be different. The highest levels of SDF-1 and MMP-13 were found in the post-traumatic ACLT animals (Group 3), which also had more severe histological cartilage damage in comparison to those with primary OA (Group 2). C2C and GAG release into synovial fluid were significantly increased, which also highlights differences between the two degenerative pathways. Considering the high level of pro-inflammatory cytokines in the ACLT (Group 3) animals, it is possible that these molecules may play a more significant role in secondary OA when compared to primary OA.

In addition, SDF-1 has also been implicated in inflammation-induced hypoxia and murine collagen-induced arthritis by attracting leukocytes to the inflamed joints.28 Although SDF-1 plays a multifaceted role in joint disease, our data suggests that SDF-1 may be an important marker of cartilage degeneration. Furthermore, recent studies have shown that reducing SDF-1 levels by synovectomy or blocking CXCR4, a specific receptor for SDF-1 by CXCR4 antibody, is associated with a decrease in MMP-13 expression in human OA.20 Therefore, increased inflammation in the ACLT model may be associated with increased SDF-1 and MMP-13 levels.19,20

Coincidentally, Western blot analysis revealed a uniquely strong presence of IL-1β that may be associated with the down-regulation of lubricin. IL-1β's role is not clear in that IL-1β knockout mice have greater cartilage degeneration compared to wild types;29 however, previous research also suggests that IL-1β involves increasing collagen and aggrecan breakdown in primary OA joints.15,30 A recent study by Huebner and Kraus demonstrated that IL-1β levels were not different between Hartley guinea pigs with idiopathic OA when compared to Strain 13 guinea pigs, which do not exhibit idiopathic OA, suggesting that IL-1β may not be a marker for primary OA.31

Lubricin was found in lower concentrations in the ACLT joints (Group 3) compared to the age-matched primary OA joints (Group 2). This finding suggests that increased inflammatory cytokines following injury may be correlated with a decrease in lubricin synovial fluid concentrations.11,14,32 Surprisingly, there is no significant difference in lubricin concentration between control (Group 1) and primary OA (Group 2) animals even though the latter exhibits higher degrees of cartilage degeneration. This indicates that the concentration of lubricin in synovial lavages may not be a sensitive biomarker of primary OA in the Hartley guinea pig model. A recent study suggests that lubricin concentrations may fluctuate due to changes in catabolism of lubricin by inflammatory cytokines and alterations in synoviocyte or superficial zone chondrocyte activities that produce the lubricin.10 The impact of lubricin loss following traumatic knee injury is worth noting because a decrease in lubricin has been shown to increase whole joint coefficient of friction making it more susceptible to wear.10,33

One limitation of the study is the possible interplay of the primary and secondary OA variables between the animal groups. Previous studies have reported that Hartley guinea pigs begin to develop mild OA by 9 months of age, and uniformly develop OA three months later through natural aging.7 In this study, the secondary OA model was superimposed on the primary OA model by performing the ACLT at 3 months. However, we do not feel this is a major concern, given the magnitude of the differences between the biomarker concentrations in Groups 2 and 3 after such a short duration. Furthermore, the extent of cartilage damage between the 12 month animals with primary OA (Group 2) and the 12 month animals with ACLT (Group 3) is very different.

Synovial fluid samples were collected from guinea pig knees after an injection of 100μL of saline. Lavage was required to get the synovial fluid out of the small joint cavity. An established protocol was followed to ensure that the synovial fluid and saline were mixed by cycling the knee before aspiration. It was noted that approximately half (~50μL) of the fluid injected into the joint was aspirated from the knee of each animal, that this volume appeared to be consistent across all three animal groups, and that the biomarker concentrations were similar between each knee within each animal. Furthermore, there was no evidence of joint swelling before the lavages. In addition, the total protein content in each synovial fluid sample was quantified for Western blot analysis. The Western blots show an increasing content of proteoglycan fragments, MMP-13, and IL-1β in the synovial samples of the older (Group 2) and ACLT animals (Group 3) than those from the younger ACL-intact animals (Group 3) with the same total protein content. Nonetheless, it is important to note this data analysis does not preclude other variables such as changes in synovial vascular permeability of protein content with OA onset.

Another potential limitation is that surgical ACLT may not be as traumatic as an ACL injury sustained during physical activity. Bone bruises and chondral lesions frequently occur and these concomitant injuries may also play a role in the development of secondary OA. Nonetheless, the animal ACLT model has been frequently used to study OA and it has been shown previously to mimic human OA both macroscopically and biochemically.34,35 ACLT, whether surgical or injury induced, renders the knee mechanically unstable, altering the joint loading conditions and biomechanical properties of the tissues inducing both acute inflammation and low grade, intra-articular inflammation in the joint. Thus, it appears to be a reasonable model for traumatic joint injury.

In summary, this study suggests that the pathological progression of OA, and biomarkers thereof, may be different in the Hartley guinea pig OA models with and without ACLT. Because cartilage degeneration in the ACL deficient model is accelerated in comparison to primary OA, future studies are necessary to identify whether the differences in these biomarkers are due to severity of OA or different pathologic mechanisms between primary and secondary OA.

Acknowledgments

The project was supported by National Institutes of Health (R03-AR052479, RO1-AG017021, P20-RR024484; RO1-AR049199) and the Aircast Foundation.

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