The results of our study confirm our first hypothesis that intra-articular supplementation with lubricin following ACL injury reduces articular cartilage damage as indicated by radiography and histology. However, we were unable to accept our second and third hypotheses since intra-articular supplementation with HA alone did not have an effect, and the addition of HA combined with lubricin did not further reduce articular cartilage damage compared to lubricin alone. Statistically significant differences in synovial fluid levels of inflammatory mediators and cartilage breakdown products were not seen, possibly due to the multifactorial processes by which post-traumatic cartilage degeneration occurs, the possible lack of a difference at the six week time point, the limited sample size, our ability to reproducibly perform the lavages in such a small knee, or other limitations of the rat model. Given that significant differences were present in the histologic and radiologic scores of cartilage damage, and that the lubricin concentrations in the synovial fluid of the lubricin treated animals remained elevated 1 week after the final injection, intra-articular supplementation with lubricin appears to be a promising approach to minimize the impact of post-traumatic OA in the ACL-deficient knee. However, more study is needed to understand the effects of lubricin and HA treatment on the metabolism of articular cartilage over the long term.
ACL transection has been used previously to induce OA in the rat knee.12,18
Using the modified OARSI scale, Hayami et al demonstrate that histological cartilage damage could be detected within 2 weeks of surgery, which would continued to progress over time.18
Visibly roughened articular surfaces, cartilage defects, and osteophyte formation were present 6 weeks post-surgery the interval selected for this study. Hayami concluded that the rat ACL transection model shared many characteristics (i.e. progressive cartilage damage, subchondral bone sclerosis and osteophyte formation) seen in the human and other animal models of OA.12,18
As with most animal OA models, arthrosis appears to be accelerated in the ACL-deficient rat knee when compared to the human. Although this can be perceived as a limitation, the rat provides a validated model that is small, easy to handle, relatively inexpensive, and that responds to both injury and treatment in a timely manner. It is ideal to provide proof of concept and to then design large animal and clinical studies to translate a novel chondroprotective therapy to clinical practice.
The concentration of lubricin for the injections used in our study, 200 μg/mL, was based upon the level of lubricin measured in normal synovial fluid.24,27,45,54–55
Of note, purified human lubricin was used for injection, rather than native rat lubricin, which may introduce the possibility of immunoreactivity and increased cartilage damage. However, our finding of decreased OA progression in the lubricin supplemented joints suggested that this was not the case. Also, the lubricin molecule is highly conserved across species further minimizing this concern. The concentration of HA and molecular weight of HA for the injection protocol in this study was also based on physiologic concentrations. HA is normally found in high concentrations, 2–4 mg/mL, with an average molecular weight of 1 × 106
Following acute knee injury, synovial fluid levels of lubricin are decreased, likely as a consequence of inflammatory degradation and the subsequent loss of lubricating ability.11,23
It has been shown that patients with Camptodactyly Arthropathy Coxa Vara Pericarditis (CACP) syndrome, who lack the functional gene for lubricin expression, produce synovial fluid which lacks lubricating ability, and hence develop severe precocious arthritis.26
It has also been shown that the lack of the lubricin gene in a mouse model results in increased joint friction and accelerated arthritis progression;45
that increased joint friction results from brief proteolytic degradation of the mucin layer at the surface of intact joints;57
and that in late arthritis after ACL transection, low levels of lubricin are associated with increased whole joint friction.57
These findings lead to the question of whether supplementing the lubricating components of synovial fluid may delay or prevent the progression of post-traumatic OA in the ACL injured patient, the objective of the present study.
Our findings that intra-articular lubricin supplementation reduce radiographic and histologic measures of post-traumatic arthritis are consistent with the data published by Flannery et al., who showed delayed OA progression after intra-articular supplementation using recombinant lubricin.14
In their study, a recombinant form of lubricin, containing a shortened central mucin domain, was injection intra-articularly in the rat knee. OA was induced by sectioning the medial meniscus and medial collateral ligament. The rats received recombinant lubricin injections either 1 or 3 times per week for four weeks after surgery, which were matched with saline injection control animals. The outcome measures in their study showed significant reductions in cartilage degeneration scores, total joint scores, widths of severe lesions, and significant cartilage degeneration widths. Their study did not include radiographic or synovial fluid analyses nor did it use a full length natural lubricin control.14
In this study, the K-L score was selected to evaluate radiographic evidence of cartilage damage.32
Because the radiographs were taken post mortem, weightbearing radiographs were not obtained so joint space narrowing was not included in the K-L assessment. The modified numerical score corresponded to the following descriptors of OA: none, doubtful, minimal, moderate, and severe. When the assessments were being performed, the evaluators were blinded to the animal and treatment groups. The average of the two independent scores (an expert and a novice) were intentionally used to add variability. Despite this, there were significant differences between treatments. Poor reliability between examiners would only undermine the analysis if we did not find differences between groups.
Although HA is thought to contribute to the viscosity of synovial fluid,27,31,39
there has been recent debate about its ability to act as a surface lubricant.50
A recent study evaluating the ability of HA to interact with idealized bearing surfaces has shown that it does not effectively coat the articulating surfaces, as would be required for boundary lubrication.7
In addition, synovial fluid treated with hyaluronidase does not lose its lubricating ability, while synovial fluid treated with proteases, which would remove lubricin, is an ineffective lubricant.22
While HA may not be an effective boundary lubricant, it has been shown that lubricin alters the mechanical properties of HA-containing solutions: HA and lubricin in solution together to lower viscosity and alter diffusion compared to HA-only solutions.27
Based on the results of the current study, the combination of HA plus lubricin does not appear to be synergistic in preventing cartilage damage, though it should be noted that this may be due to the limited sample size which was only 80% powered to detect a 35% between the mean values.
Although significant differences in inflammatory markers (IL-1β, TNF-α) and cartilage breakdown products (CTX-II, sGAG) were not seen in this study, existing data suggest that both HA and lubricin may act as signaling molecules in addition to possessing mechanical lubricating properties. Of interest in this study, HA injection had the greatest effect in lowering IL1- β, while both lubricin- and HA-treated animals had lower levels of TNF- α. The cytokines IL-1β and TNF-α are of importance in cartilage degradation in OA. IL-1β has been found to be spontaneously released by OA cartilage,2
and both IL-β and TNF-α perpetuate cartilage matrix degradation by stimulating the release of stromelysin (MMP-3), collagenases (MMP-1/8/13), gelatinases (MMP-2/9), and by their own action.40,43
It should be noted that the cartilage metabolism biomarker data from this study must be treated with caution due to the relatively small sample size. For these four biomarkers we were only 80% powered to detect a shift of one standard deviation between treatments. Furthermore, lavages were only obtained after euthanasia so we do not know what the concentrations of each marker were during the active course of treatment. Further investigations will need to address the metabolic effects of lubricin and HA treatment on chondrocyte and synoviocyte metabolism, including their effects on inflammation and enzyme expression.
The progression of OA following injury is multifactorial, and a model in which OA progression occurs more slowly, for example after meniscal injury instead of ACL transection, will be studied in the future to further explore the metabolic effects of tribosupplementation. In addition, interval and longer term follow up periods will be included to determine if the observed reduction in OA progression persists after treatment. The delay in OA progression following lubricin treatment shown in this study did not correlate with significant alterations in inflammatory marker expression, but it is also possible that ongoing mechanical damage, which may have been partially prevented by supplementing the lubricating ability of synovial fluid, was the main contributor to OA progression. Further investigations will focus on the effects of supplemental lubricin and HA on the metabolism and mechanical properties of articular cartilage.
What is known about the subject:Lubricin and HA are lubricants of articular cartilage. Although HA is frequently used to treat the arthritic knee, its ability to reduce cartilage damage has not been demonstrated. Lubricin is down regulated following ACL injury, which in turn places the articular cartilage at greater risk for wear.
What this study adds to existing knowledge:This study demonstrates that lubricin provides chondral protection in the ACL injured knee while HA does not in this experimental model.