Cartilage boundary lubrication of ovine synovial fluid following anterior cruciate ligament transection: a longitudinal study

Osteoarthritis and Cartilage 23 (2015) 640e647

Cartilage boundary lubrication of ovine synovial fluid following anterior cruciate ligament transection: a longitudinal study M. Atarod y *, T.E. Ludwig z, C.B. Frank x, T.A. Schmidt k, N.G. Shrive ¶ y McCaig Institute for Bone and Joint Health, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada z Biomedical Engineering, University of Calgary, Calgary, Alberta, Canada x Department of Surgery, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada k Faculty of Kinesiology, Biomedical Engineering, University of Calgary, Calgary, Alberta, Canada ¶ Department of Civil Engineering, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada

a r t i c l e i n f o

s u m m a r y

Article history: Received 21 May 2014 Accepted 20 December 2014

Objective: To assess ovine synovial fluid (oSF) from different post-injury time points for (1) proteoglycan4 (PRG4) and hyaluronan (HA) concentration, (2) HA molecular weight (MW) distribution, (3) cartilage boundary lubrication function, and (4) lubricant composition-function relationships. The association between cartilage boundary lubrication and gross cartilage changes after injury was also examined. Methods: oSF was collected 2, 4, 10, and 20 weeks post anterior cruciate ligament (ACL) transection in five skeletally mature sheep. PRG4 and HA concentrations were measured using sandwich enzymelinked immunosorbent assay, and HA MW distribution by agarose gel electrophoresis. Cartilage boundary lubrication of oSF was assessed using a cartilageecartilage friction test. Gross damage to articular cartilage was also quantified at 20 weeks using modified Drez scoring protocol. Results: Early (2e4 weeks) after ACL injury, PRG4 concentrations were significantly higher (P ¼ 0.045, P ¼ 0.037), and HA concentrations were substantially lower (P ¼ 0.005, P ¼ 0.005) compared to 20 weeks. The HA MW distribution also shifted towards lower ranges in the early post-injury stage. The kinetic friction coefficients were significantly higher 2e4 weeks post injury (P ¼ 0.008 and P ¼ 0.049) compared to 20 weeks. Poor cartilage boundary lubricating ability early after injury was associated with cartilage damage at 20 weeks. Conclusion: Altered composition and diminished boundary lubrication of oSF early after ACL transection may pre-dispose the articular cartilage to degenerative changes and initiate osteoarthritis (OA). These observations also provide potential motivation for biotherapeutic interventions at earlier time points post injury. © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Keywords: Synovial fluid ACL injury Cartilage Boundary lubrication Ovine

Introduction Articular cartilage is the lubricious, low-friction, wear resistant, load bearing tissue at the end of long bones in synovial joints1. Severe injuries to the knee joint such as anterior cruciate ligament (ACL) rupture may lead to failure of articular cartilage homeostasis and increased risk of osteoarthritis (OA) development in the long term2,3.

* Address correspondence and reprint requests to: M. Atarod, McCaig Institute for Bone and Joint Health, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 Canada. Tel: 1-403-903-2443; Fax: 1-403-2837742. E-mail addresses: [email protected] (M. Atarod), [email protected] (T.E. Ludwig), [email protected] (C.B. Frank), [email protected] (T.A. Schmidt), [email protected] (N.G. Shrive).

There are a number of biophysical mechanisms that contribute to the lubrication of articulating cartilage surfaces and maintenance of cartilage homeostasis, mainly classified as fluid film and boundary lubrication modes4,5. In the fluid film mechanism, the interstitial fluid within cartilage becomes pressurized, or forced between articular surface asperities, and contributes substantially to the bearing of normal load with little resistance to shear force, leading to a considerably low friction coefficient4. On the other hand, in the boundary mode of lubrication, load is supported by surface-to-surface contact, and the associated frictional properties are determined by lubricant surface molecules4. This mode of lubrication has been suggested to be vital for the protection and maintenance of articular surfaces since the opposing cartilage layers make contact over ~10% of the total area, and this may be where most of the friction occurs4,5.

http://dx.doi.org/10.1016/j.joca.2014.12.017 1063-4584/© 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

M. Atarod et al. / Osteoarthritis and Cartilage 23 (2015) 640e647

Synovial fluid (SF) has been demonstrated to function as an effective friction-lowering boundary lubricant for opposing articular cartilage surfaces in vitro6,7. There are two primary macromolecules that contribute significantly to the boundary lubricating ability of SF: proteoglycan-4 (PRG4), also known as lubricin8 or superficial zone protein (SZP)9, and hyaluronan (HA). These molecules are secreted by chondrocytes in the superficial layer of articular cartilage10, synoviocytes in synovium11 or cells within menisci12, and mediate boundary lubrication of articular cartilage by adsorbing to the articular surfaces and reducing the interaction of opposing tissues4,13. Boundary lubricating ability of SF may be altered in joint disease or acute joint injury, potentially due to alterations in PRG4 and HA concentrations8,14,15. Poor lubrication function of human SF has been reported following inflammatory and traumatic insults including rheumatoid arthritis16 and post-traumatic knee effusions17. Furthermore, animal models of meniscectomy18, posterior cruciate ligament and ACL disruption19, and unilateral ACL transection20 have all indicated altered compositions of PRG4 or HA acutely post injury, associated with articular cartilage damage. Collectively, these studies suggest that normal HA and PRG4 composition is essential for SF lubricating function and normal cartilage homeostasis4,5. A recent study in an ovine injury model suggested that ovine SF (oSF) boundary lubricant composition and function 20 weeks following joint injury were similar to normal levels21. PRG4 concentrations at 20 weeks in sham, ACL/MCL (Medial Collateral Ligament) transection, and meniscectomy oSF (131 ± 26, 225 ± 7, 244 ± 62 mg/ml, respectively) were not significantly different from their respective contralateral controls (193 ± 49, 296 ± 58, 173 ± 54 mg/ml, respectively). Similarly, HA concentrations (0.82 ± 0.11, 0.83 ± 0.29, 1.02 ± 0.08 mg/ml, respectively) were not significantly different from the respective contralateral controls (0.86 ± 0.18, 0.95 ± 0.05, 0.97 ± 0.09 mg/ml, respectively)21. While oSF composition and function at 20 weeks post injury were restored to normal ranges, significant cartilage damage was demonstrated by the ACL/MCL transection and meniscectomy sheep compared to the shams21, suggesting that any potential changes in PRG4 and HA concentration of oSF following injury might have occurred at earlier time points in these ovine models and warrant further investigation. The time course of alteration of lubricant composition and function following joint injury has not been investigated in a large animal model, where gross joint changes could also be evaluated. Thus, the main objective of the present study was to evaluate composition-function alterations in the oSF over time following ACL injury. We also aimed to examine the association between cartilage boundary lubrication changes after joint injury and gross cartilage changes in the longer term. The governing hypothesis was that oSF would exhibit diminished boundary lubricating ability short term (2e4 weeks) post ACL transection, associated with gross cartilage damage at 20 weeks. This objective was tested by the following specific aims: determine (1) PRG4 and HA composition of oSF using enzyme-linked immunosorbent assay (ELISA), (2) HA molecular weight (MW) distribution of oSF using agarose gel electrophoresis, (3) cartilage lubricating ability using a cartilageecartilage in vitro boundary mode friction test of post injury oSF at 2, 4, and 20 weeks, and (4) oSF composition-function relationships. The association between cartilage boundary lubrication and gross cartilage changes post injury was also examined. Materials and methods Sample preparation Five skeletally mature (3-year-old) female Suffolk-cross sheep underwent arthroscopic unilateral ACL transection in their right

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hind stifle joint. oSF samples were aspirated from the operated joint of each subject early post injury (2 and 4 weeks), as well as medium term (10 weeks) and long term (20 weeks) following ACL transection. Prior to SF collection at each time point, the operated limbs underwent 20 cycles of passive flexion-extension, to provide a consistent procedure for SF aspirations. oSF samples were clarified by centrifugation (3000g for 30 min at 4 C) prior to storage at 80 C with protease inhibitors (PIs) and without PIs for HA MW analysis22. The total volume of oSF aspirated at 2, 4, and 20 weeks post injury for each sheep was approximately 1.5 ml, where 90 ml was used for PRG4 ELISA, 1 ml for HA ELISA, 35 ml for HA MW assessments, 1.2 ml for cartilage boundary lubrication tests, and the remaining portion for any further analyses. The amount of SF that could be collected from the joint at the 10 week time point for each sheep was not sufficient (400e600 ml) to allow both the SF composition and the cartilage boundary lubrication tests. Little or no SF could be collected from the pre-injury state or from the contralateral limbs, due to practical difficulties. All procedures were reviewed and approved by the University of Calgary Animal Care Committee and comply with Canadian Council on Animal Care guidelines. PRG4 composition PRG4 concentration in oSF samples was measured using a previously described custom sandwich ELISA22, with anti-PRG4 antibody LPN (Lubricin Polyclonal) (recognizing the C-terminal of PRG423) for capture and peanut agglutinin lectin horse radish peroxidase (PNA-HRP; SigmaeAldrich, St. Louis, MO) for detection. HA composition A commercially available sandwich ELISA (R&D Systems, Minneapolis, MN) was used to measure HA concentration in oSF22. The diluted oSF samples (1:40,000 in 5% Tween 20 in PBS (Phosphate Buffered Saline)) were assessed in triplicate, and a standard HA control curve was used for calculation of HA concentrations22. HA MW distribution The MW distribution of HA in oSF was quantified in duplicate using agarose gel electrophoresis1. The oSF samples, stored without PIs and treated with proteinase K, were diluted to 0.5 mg/ml and Hi-Ladder (0.5e1.5 MDa) and Mega-Ladder (1.5e6.1 MDa; Oklahoma City, OK) MW markers were used as HA MW standards. One blank lane was left between samples for background measurement. After electrophoresis for 3 h at 50 V in a horizontal gel apparatus (Bio-Rad, Mississauga, ON), the gels were stained with 0.005% Stains-All in 50% ethanol and de-stained in 10% ethanol. The migration of HA was evaluated by densitometric analysis with Image J (NIH, Bethesda, MD)22. Cartilage boundary lubricating ability Cartilage boundary lubricating ability of oSF from different postinjury time points (2, 4, and 20 weeks) was evaluated using normal bovine osteochondral cores with a previously described in vitro cartilageecartilage friction test under the boundary lubrication regimen4,21. Briefly, annulus and core-shaped osteochondral samples were prepared from the patellofemoral groove of skeletally mature bovine stifle joints. Samples were nutated vigorously overnight at 4 C in 40 ml of PBS to rid the articular surface of residual SF22. Samples were then stored at 80 C in PBS with PIs until the day before testing, when they were defrosted and again nutated vigorously overnight at 4 C in 40 ml of PBS. Samples were bathed in

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~0.3 ml of the subsequent test lubricant (annulus bathed in ~0.1 ml, core bathed in ~0.2 ml) at 4 C overnight prior to lubrication experiments. The following test sequence was used over 5 consecutive days with n ¼ 4 osteochondral samples for each oSF sample: PBS, 2 weeks oSF, 4 weeks oSF, 20 weeks oSF, and bovine SF (bSF). The amount of oSF collected at the 10 week time point was not sufficient for cartilage boundary lubrication tests and only allowed PRG4 and HA composition assessments. Also, previous studies using this in vitro cartilageecartilage friction test confirmed that up to five sequential tests could be performed on a single osteochondral pair over 5 days, with overnight storage at 4 C between tests, without degradation of the samples22. Cartilage boundary lubrication tests were conducted using a Bose ELF 3200 (Bose EnduraTEC, Minnetonka, MN). Cartilage surfaces were compressed at a constant rate of 0.002 mm/s to 18% of total cartilage thickness, stress relaxed for 40 min to allow for an interstitial fluid depressurization period, then rotated against each other at an effective velocity of 0.3 mm/s with pre-sliding durations (Tps; the duration of time the samples were stationary prior to rotation, also known as dwell time) of 1200, 120, 12, and 1.2 s without removing compression4. The test sequences were then repeated in the opposite direction of rotation. Two friction coefficients were calculated for each lubricant: mstatic,Neq, representing resistance to the onset of motion, and , representing resistance to steady motion, where Neq represents equilibrium axial load and angle brackets indicate that the value is an average4. Gross morphological grading All animals were sacrificed at 20 weeks post injury and their articular cartilage was graded for gross defects at 14 standardized locations spanning the stifle (six tibial plateau, four femoral condyle, two femoral groove, and two patellar locations) by one observer using previously validated modified Drez scoring protocol24, with Grade 0 indicating no damage and Grade 5 showing maximal damage in that region. The “Gross Cartilage Score” was defined as the sum of damage scores in these 14 locations, with a total grade of 70 indicating the worst possible cartilage damage24. Statistical analysis Single-factor analysis of variance (ANOVA) was performed to examine changes in PRG4 and HA composition of oSF, with postsurgical time point as the independent factor. The data set was independent observations and checked for normality using the ShapiroeWilk W test for Normality. A repeated measures ANOVA was also used to assess the effects of lubricant and Tps, as a repeated factor, on mstatic,Neq and . All the analyses were performed in SPSS (IBM SPSS Statistics 19.0, IBM SPSS Inc., Chicago, IL)25. Statistical significance was accepted at P < 0.05. Unless otherwise indicated, data are presented as mean with a 95% confidence interval (CI) (lower limit, upper limit).

Fig. 1. PRG4 and HA concentrations of oSF at different time points post ACL transection for each subject (N ¼ 5). Data are presented as mean ± 95% CI.

HA composition The assumption of data set normality was satisfied. There was also a statistically significant effect of post-surgical time point on HA composition of oSF (P ¼ 0.001). In contrast to PRG4, HA concentrations were significantly lower at 2 weeks (0.156 (0.096, 0.216) mg/ml, P ¼ 0.005) and 4 weeks (0.122 (0.072, 0.172) mg/ml, P ¼ 0.005) compared to 20 weeks (1.052 (0.742, 1.362) mg/ml). HA concentrations at different time points post injury for each subject are demonstrated in Fig. 1. HA MW distribution The MW distribution of HA also demonstrated a shift towards lower forms in the early post-injury stage. The average concentration of high MW HA (3050e6100 kDa) was lowest at 2e4 weeks, but returned to normal ranges at 20 weeks. HA MW distributions at different time points post injury for each subject are depicted in Fig. 2. As observed, significantly higher concentrations of low-MW HA (495e1090 kDa) occur at 2e4 weeks post injury, but a higher concentration of high MW HA (3050e6100 kDa) at 20 weeks.

Results Cartilage boundary lubrication PRG4 composition The assumption of data set normality was satisfied. There was a statistically significant effect of post-surgical time point on PRG4 composition of oSF (P ¼ 0.035). PRG4 concentrations were significantly higher at 2 weeks (527.0 (322.8, 731.2) mg/ml, P ¼ 0.045) and 4 weeks (728.3 (417.9, 1038.7) mg/ml, P ¼ 0.037) post injury compared to 20 weeks (244.0 (212.2, 275.8) mg/ml). PRG4 concentrations at different time points post injury for each subject are demonstrated in Fig. 1.

The cartilage boundary lubricating ability of oSF was significantly diminished early after ACL transection. For static friction, lubricants and Tps were observed to modulate mstatic,Neq. The static friction coefficient significantly increased (P ¼ 0.006) with increasing Tps for all lubricants [Fig. 3(a)]. mstatic,Neq values are compared at Tps ¼ 1200 s between different lubricants. There was also a statistically significant effect of lubricant type on mstatic,Neq (P < 0.001). Values of mstatic,Neq were greatest in PBS (mstatic,Neq ¼ 0.546 (0.486, 0.606)) compared to oSF and bSF. For oSF,

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Fig. 2. HA MW distribution of oSF at different time points post ACL transection for each subject (N ¼ 5).

the 2 week (mstatic,Neq ¼ 0.315 (0.265, 0.365)) and 4 week (mstatic,Neq ¼ 0.251 (0.171, 0.331)) time points had significantly higher (all P < 0.05) friction coefficients compared to 20 weeks (mstatic,Neq ¼ 0.230 (0.130, 0.330)). In all cases, 20 week oSF was not significantly different (P > 0.05) from bSF (mstatic,Neq ¼ 0.249 (0.209, 0.289)) [Fig. 3(a)]. The kinetic friction coefficient was minimally affected by Tps (values at 1.2 s were within 21 ± 3% of those at 1200 s). Therefore, for brevity, and as previously described4, values of are presented only at Tps ¼ 1.2 s. There was a statistically significant effect of lubricant type on (P ¼ 0.022). PBS had the highest friction coefficient ( ¼ 0.205 (0.185, 0.225)) compared to oSF and bSF [Fig. 3(b)]. For oSF, the 2 week ( ¼ 0.049 (0.049, 0.049)) and 4 week ( ¼ 0.042 (0.032, 0.052)) time points had significantly higher (P ¼ 0.008 and P ¼ 0.049, respectively) friction coefficients compared to 20 weeks ( ¼ 0.025 (0.015, 0.035)). In all cases, the 20 week oSF was not significantly different (P ¼ 0.756) from bSF ( ¼ 0.026 (0.026, 0.026)) [Fig. 3(b)]. Composition-function relationships The relationship between kinetic friction coefficient and HA composition of oSF is represented in Fig. 4. Generally, lower concentration of HA early after injury is associated with significantly higher friction coefficients [Fig. 4(a)]. Similarly, higher abundance of high MW HA (3050e6100 kDa) molecules later post injury correspond to lower kinetic friction coefficients [Fig. 4(b)]. Gross cartilage damage In terms of gross morphology, the average (N ¼ 5) “Gross Cartilage Score” was 13.0 ± 6.5 (out of 70). Poor cartilage boundary lubrication early after injury (2e4 weeks) was associated with cartilage damage at 20 weeks (Fig. 5); the higher the frictional changes, the higher the cartilage damage.

Discussion The present study elucidated the time course of alteration of lubricant composition and function following joint injury using a unilateral ACL transection ovine model. The results indicate that oSF composition and cartilage boundary lubricating ability was altered significantly early (2e4 weeks) after ACL injury compared to 20 weeks. Short-term post ACL transection, the boundary lubrication function of oSF was reduced considerably, as indicated by higher than normal friction coefficients. This alteration in lubrication function may be due to the diminished concentration of high MW HA, despite the elevated concentrations of PRG4 in oSF. In the later stage of injury, boundary lubrication function of oSF appears to be almost fully recovered, possibly due to restoration of HA concentration and MW. The decrease in HA concentration early post injury is consistent with previous human and animal models. The normal concentration of HA in human SF ranges from 1.8 to 3.33 mg/ml22 and has been observed to diminish significantly after effusive joint injury26, intra-articular fracture27, and arthritic disease28, or to remain normal in advanced OA29. HA supplementation at early stages of knee OA is a common clinical practice30,31 with the aim of restoring normal SF function and/or delaying articular cartilage degeneration. Other animal models of joint injury have also indicated compromised HA composition of SF in the acute stages of injury32. In the equine model, the HA concentration of SF is reported to decrease from 0.33 to 1.3 mg/ml in normal conditions to 0.18e0.3 mg/ml in post-traumatic arthritis and 0.18e0.74 mg/ml in chronic injury state13. Decreased HA concentration in SF with acute ACL injury has also been reported in rabbit20, canine33, and rat34 models. Although lower HA concentrations following joint injury are evident, one should note the extensive range of HA concentrations reported for normal and diseased SF. The sources of variability may include sample source, species, particular joint and state of health/injury/disease13. Finally, the decrease in oSF HA concentration early after ACL transection, as reported in this study,

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Fig. 4. Composition-function relationships for oSF following ACL transection.

Fig. 3. (A) Average static and (B) average kinetic (at Tps ¼ 1.2 s) friction coefficients for oSF at 2, 4, and 20 weeks post ACL transection (N ¼ 5) (n ¼ 4 osteochondral samples). The amount of oSF collected at the 10 week time point was not sufficient for cartilage boundary lubrication tests and only allowed PRG4 and HA composition assessments. The data are also represented for PBS (negative control) and bovine SF (bSF, positive control). Asterisks (*) indicate significant changes (P < 0.05). Data are presented as mean ± 95% CI. There was very negligible variability in the 2 weeks SF and bSF kinetic friction data compared to other time points.

could be due to changes in the rates of synthesis, secretion, degradation, and/or loss from the joint. The mechanism of these changes remains to be fully elucidated35. The shift towards lower MW HA following ACL transection observed in the present study agrees with findings of previous investigations. High MW of HA is known to provide the viscoelastic component to SF, which in turn affects the cartilage lubricating ability of normal SF in a fluid film mode of lubrication1. Following injury or disease, a decrease in the proportion of high MW HA has been suggested in humans31,36, which corresponds to reduced viscoelasticity of SF and its diminished ability to lubricate the articular cartilage and protect the joint. This observation is also consistent with animal models of injury, where intra-articular administration of high MW HA has been shown to inhibit cartilage degradation in rabbits (meniscectomy37 and ACL/MCL transection38) as well as in a canine model of OA39. Related to cartilage boundary mode lubrication, in an equine osteochondral injury model, the MW distribution of HA in acute-injury SF was shown to shift markedly towards smaller forms, but return to normal state in chronic injury, and this lower HA MW equine SF demonstrated compromised boundary lubrication13. Consistent with the functional outcome associated with the time course alterations in HA

MW observed in this study, high MW HA supplementation to the HA-deficient equine SF restored cartilage boundary lubricating function. Furthermore, a recent study using purified lubricants demonstrated that high MW HA is a key friction-reducing constituent at a cartilageecartilage bio-interface in boundary mode lubrication, along with PRG4, and deficiency in either can result in decreased cartilage boundary lubricating function40. Collectively, these studies suggest high MW HA is an integral part of SF for its normal cartilage boundary lubrication function. The effects of injury on PRG4 concentration in SF appear to be quite variable, both in humans and animals. The concentration of PRG4 in human SF after ACL injury has been shown to be significantly lower compared to the uninjured contralateral joints, returning to normal approximately 12 months post injury41. In later stages of human OA, PRG4 concentrations have been shown to increase42 as well as decrease22. Human knees afflicted with a tibial plateau fracture have demonstrated SF PRG4 concentrations more

Fig. 5. Change in cartilage average kinetic friction (mean 2e4 weeks) early after injury, relative to 20 weeks, plotted against “Gross Cartilage Scores”.

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than two-fold higher compared with the control SF at early stage post injury27. Similar to humans, PRG4 composition of SF following injury in animal models has been reported to increase or decrease. Conversely, in ACL-deficient guinea pig knees14, a significant decrease in PRG4 concentration was observed 9 months following injury, but returned to normal state in late stages of OA. In a rabbit ACL/PCL (Posterior Cruciate Ligament) transection model19, the concentration of PRG4 in SF was indicated to be significantly higher at week 1 post-injury, but decreasing 2e3 weeks after injury. Decreased PRG4 concentration has also been reported early post ACL injury in rats15,34,43e45. In contrast, in an equine osteochondral injury model13, the concentration of PRG4 was demonstrated to increase from 57 mg/ml to 104 mg/ml acutely after injury, and change to 95 mg/ml in the late stages of injury. In the present study, an increase in PRG4 concentrations short term (2e4 weeks) following ACL transection was also observed, in the ovine mode, returning to levels similar observed in normal joints from another study at the same (20 week) time point21. The mechanisms responsible for PRG4 composition changes of SF following acute joint injury remain to be elucidated, but could be due to changes in protein expression/synthesis by synoviocytes and chondrocytes, loss of cells, or an increase in the catabolism of PRG414. The 2e4 fold increase in PRG4 concentration at the early time points following injury is feasible given the altered chemical and mechanical milieu as PRG4 secretion by chondrocytes in explant culture has been shown to be upregulated to that extent, and more, by mechanical46 and chemical stimuli47. These variable PRG4 concentrations may be due to a number of different factors including the type of injury, duration of injury before the SF was aspirated, sample source, and analysis method. Western blotting of SF with LPN and PNA-HRP indicated increased relative high MW immunoreactivity, indicative of full-length PRG4, at early time points (see supplemental figure), qualitatively agreeing with the trends in ELISA values reported here. Diminished cartilage boundary lubricating ability of oSF observed early post ACL transection agrees with previous studies of SF function. SF analysis following transection of rabbit ACL/PCL revealed an association between SF's decreased boundary lubricating ability and damage to articular cartilage matrix19. Similarly, in a rabbit ACL/MCL injury model, a relationship between cartilage damage and SF lubrication function was demonstrated38. Altered SF lubricating ability and associated cartilage degeneration have been reported in other animal models of injury including rats15,34,43e45, ovine18, and canine33. Several human studies have also indicated an association between increased cartilage degradation and decreased joint lubrication following joint injury31,36,41. Collectively, these studies suggest that joint injury may pre-dispose the articular cartilage to an increased risk of wear-induced damage as a consequence of poor boundary lubrication, potentially leading to secondary OA. Our results indicated a similar decrease in cartilage boundary lubrication in the acute stage of injury (2e4 weeks), associated with cartilage damage at 20 weeks. The observed decrease in SF boundary lubrication following ACL injury is likely due to significant decreases in the concentration of high MW HA, despite increased PRG4 concentrations. The data also imply that both molecules being present at physiological levels, and appropriate structural distribution, are important and perhaps necessary for normal cartilage lubrication40. Further investigation is required to fully understand the PRG4-HA interactions in SF, and potential dependences on HA MW and/or PRG4 multimerization23. HA MW bins used for analysis in this study were similar to those reported previously22, which were used for practical reasons (i.e., markers in the ladders used). Alternative binning and/or evaluation of the continuous MW distribution could potentially provide

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further detailed information of changes in HA MW distribution, but would not change the overall conclusions of this study. While small compared to other human/animal models of injury, the N ¼ 5 animals used in this study appeared sufficient to detect functionally significant differences in SF composition and function between early and late stages of injury. Little or no SF could be collected from the contralateral limbs or from the pre-injury state, preventing oSF composition-function comparisons of the experimental limbs to the non-operated controls. However, PRG4 and HA concentrations obtained 20 weeks following ACL transection (212e276 mg/ml and 0.74e1.36 mg/ml, respectively) were similar to the previously reported values for non-operated contralateral limbs in the same ovine model (173e296 mg/ml and 0.86e0.97 mg/ml, respectively)21, which are within normal physiological ranges21,48,49. HA concentration from oSF from the normal knee joint has been previously reported in the range of 0.68e1.65 mg/ml, which is similar to the 0.74e1.36 mg/ml ranges observed in the present work48. There were also no statistically significant differences between cartilage boundary lubrication function of 20 week oSF and healthy bSF. This would indicate that bovine cartilage is an acceptable substrate for measuring the lubricating function of oSF at 20 weeks. For earlier time points, however, the biotribology of pathologic SF on damaged (vs normal) cartilage surfaces is to be evaluated. In conclusion, the present study evaluated the time course of alteration of lubricant composition and function following joint injury and its association with gross cartilage damage. Diminished high MW HA concentration was observed early after ACL injury, which was associated with decreased cartilage boundary lubricating function, despite elevated concentrations of PRG4. In the late stage of injury, oSF composition and function was restored to within normal ranges. Diminished lubrication in the early postinjury stage may pre-dispose the articular cartilage to degenerative changes and initiate premature OA. Future studies focused on elucidation of the link between altered lubricant compositionfunction, including functional interactions of lubricant molecules and potentially dependency on lubricant molecule structure or size, associated cartilage damage, and potential changes in gait post knee injury as well as SF cytokine levels will contribute to further understanding of the biochemical and biomechanical changes associated with OA progression21. These observations also provide potential implications for biotherapeutic interventions (e.g., intraarticular administration of HA with or without PRG4) at earlier time points post injury, to slow or prevent cartilage degradation and OA development. Author contributions We declare that all authors contributed to the conception and design of the original study and approved the final submitted manuscript, in line with Osteoarthritis and Cartilage guidelines. MA and TL were responsible of the data acquisition and analysis. The article was first drafted by MA, and critically reviewed by TL, TS, CF, and NS. NS ([email protected]) and CF ([email protected]) obtained funding for the study, and take full responsibility for the integrity of this work. Role of funding source This work was funded by Natural Sciences and Engineering Research Council of Canada Collaborative Research and Training Experience Program NSERC-CREATE (grant# RT735134), Canadian Institutes of Health Research CIHR (grant# MOP126039) and Alberta Innovates e Health Solutions AIHS (grant# RT10003133). Other than their support, these funding sources had no involvement in the design or execution of the study, in the preparation of the manuscript or the decision to submit this work for publication.

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Conflict of interest The authors have no potential conflicts of interest. Acknowledgments The authors gratefully acknowledge the funding sources: Natural Sciences & Engineering Research Council of Canada Collaborative Research and Training Experience Program (NSERC-CREATE), Canadian Institutes of Health Research (CIHR) and Alberta Innovates Health Solutions (AIHS). We would also like to thank Etienne O'Brien, Bryan Heard, Leslie Jacques, Kristen Barton and Nathan Solbak for performing animal surgeries and collecting SF samples. We also greatly appreciate Suresh Regmi for his assistance with Western Blot experiments. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.joca.2014.12.017. References 1. Kwiecinski JJ, Dorosz SG, Ludwig TE, Abubacker S, Cowman MK, Schmidt TA. The effect of molecular weight on hyaluronan's cartilage boundary lubricating abilityealone and in combination with proteoglycan 4. Osteoarthritis Cartilage 2011;19:1356e62. 2. Beynnon BD, Johnson RJ, Abate JA, Fleming BC, Nichols CE. Treatment of anterior cruciate ligament injuries, part I. Am J Sports Med 2005;33:1579e602. 3. Potter HG, Jain SK, Ma Y, Black BR, Fung S, Lyman S. Cartilage injury after acute, isolated anterior cruciate ligament tear: immediate and longitudinal effect with clinical/MRI follow-up. Am J Sports Med 2012;40:276e85. 4. Schmidt TA, Sah RL. Effect of synovial fluid on boundary lubrication of articular cartilage. Osteoarthritis Cartilage 2007;15:35e47. 5. Schmidt TA, Gastelum NS, Nguyen QT, Schumacher BL, Sah RL. Boundary lubrication of articular cartilage: role of synovial fluid constituents. Arthritis Rheum 2007;56:882e91. 6. Hui AY, McCarty WJ, Masuda K, Firestein GS, Sah RL. A systems biology approach to synovial joint lubrication in health, injury, and disease. Wiley Interdiscip Rev Syst Biol Med 2012;4: 15e37. 7. Katta J, Jin Z, Ingham E, Fisher J. Biotribology of articular cartilageea review of the recent advances. Med Eng Phys 2008;30:1349e63. 8. Jay GD, Torres JR, Rhee DK, Helminen HJ, Hytinnen MM, Cha CJ, et al. Association between friction and wear in diarthrodial joints lacking lubricin. Arthritis Rheum 2007;56:3662e9. 9. Tanimoto K, Kamiya T, Tanne Y, Kunimatsu R, Mitsuyoshi T, Tanaka E, et al. Superficial zone protein affects boundary lubrication on the surface of mandibular condylar cartilage. Cell Tissue Res 2011;344:333e40. 10. DuRaine G, Neu CP, Chan SM, Komvopoulos K, June RK, Reddi AH. Regulation of the friction coefficient of articular cartilage by TGF-beta1 and IL-1beta. J Orthop Res 2009;27: 249e56. 11. Jones AR, Flannery CR. Bioregulation of lubricin expression by growth factors and cytokines. Eur Cell Mater 2007;13:40e5 (discussion 5). 12. Schumacher BL, Schmidt TA, Voegtline MS, Chen AC, Sah RL. Proteoglycan 4 (PRG4) synthesis and immunolocalization in bovine meniscus. J Orthop Res 2005;23:562e8.

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