Lubricin is a surface-active mucin glycoprotein which physiosorbs to surfaces providing chondroprotective and anti-adhesive properties. Lubricin protects the cartilage surface, inhibits synoviocyte overgrowth (17
) and prevents wear of articular cartilage. Camptodactyly-arthropathy-coxa-vara pericarditis (CACP) syndrome is an autosomal recessive disorder characterized by non-inflammatory arthropathy and synovial hyperplasia attributable to absent lubricin expression (18
). SF boundary lubrication provided by lubricin studied in vitro
, was decreased in a patient population with traumatic joint synovitis and RA (6
). In contrast, SF from patients with OA appears to provide near normal lubricating ability in vitro
). The observation of compromised joint lubrication also extends to patients with an acute ACL injury.
The significant decrease in SF lubricin levels of the ACL deficient joint was marked within the early days post-injury. As inflammatory cytokines diminished, SF lubricin levels in injured joints approached that of the contralateral control over the course of a year (), though we can not predict how the lubricin concentrations would continue to change after 12 months. The decrease in SF lubricin following ACL injury has been corroborated in animal models. In the guinea pig, transection of the ACL significantly reduced SF lubricin levels 9-months after surgery, and this corresponded to an increase in the coefficient of friction of the articulating surfaces (19
). Following rabbit ACL and PCL transection, SF lubricin concentrations in lavage progressively decreased, lacked full-length lubricin on Western blot and appeared associated with increased cartilage damage (20
As expected, there were higher SF concentrations of IL-1β, TNF-α and IL-6 at an early stage following the injury. Other investigators have reported that the levels of these cytokines were highest within 24 hours after injury and that they decreased there after (21
). In our study, IL-6 and TNF-α remained detectable for almost 6 months following injury, while IL-1β SF levels quickly declined at 3 months following the injury (). The injured SF contained higher levels of IL-6 and TNF-α compared to IL-1β when the injury was 60 days old or less. Our findings also demonstrate that these cytokines were undetected in the contralateral joint. Similar findings were reported in a study of the natural history of ACL-deficient knees at different stages of injury acuity (22
There were many cytokine assays for which concentration estimates either fell below the manufacturer’s recommended minimum detectable limit, or the individual assay’s estimate fell below that of the zero concentration control estimate. Both these scenarios are likely generated by “truly” low or zero concentrations. This is supported by the finding that the probability of a detectable cytokine concentration was significantly higher for injured than contralateral knees for all three cytokines (60% vs. zero for TNF-a, 50% vs. zero for IL1-B, and 97% vs. zero for IL-6; Fisher’s exact p<.0001 for each). When conservatively treated as missing data, this non-random effect may bias results, particularly in terms of our primary hypothesis; cytokines catabolize lubricin, and therefore their concentrations are inversely related to lubricin concentrations. To evaluate the impact, three approaches were performed when handling “truly low” values when analyzing cytokines relative to lubricin concentrations: 1) considered them missing, 2) imputed as 3, .01 or 0.04 pg/ml (the minimum detection limit for IL-1β, TNF-α, and IL-6, respectively), and 3) imputed as the minimum value detected. Neither, the first approach nor the latter two suggested contradictory results but rather reaffirmed the positive findings or tempered the negative findings (i.e. lower p-values, marginally significant for IL-1β using minimum detectable, but not minimum observed), so the results of the first method were reported here.
The cytokines IL-1β, TNF-α and IL-6 upregulate proteolytic enzymes, that may degrade lubricin and lead to the loss of SF chondroprotection. IL-1β and TNF-α can stimulate the secretion of latent cysteine proteases e.g. cathepsin B from cultured synovial fibroblasts from patients with OA and RA (23
). In an antigen-induced arthritis model, a decrease in synovial lubricin expression was found to be associated with an increase expression of cathepsin B and IL-1β (24
). Inhibition of cathepsin B activity in pooled SF from patients with RA inhibited the proteolytic degradation of re-introduced human lubricin in these fluids (25
). In the present study, procathepsin B levels were elevated in SF from injured joints while remaining undetectable in the contralateral joint SF. Procathepsin B is the precursor to cathepsin B. SF cathepsin B activity was not measured in this study, but it seems plausible to assume that an increase in SF procathepsin B levels would be associated with increased SF cathepsin B activity (26
). Similarly, NE was detected in SF from the injured joints and not the contralateral joints. NE is a serine protease that was shown to completely degrade lubricin (4
) and increase cartilage friction, and hence wear, in vivo
Changes in SF sGAG levels were evident at an early stage following ACL injury. The significant increase in SF sGAG levels following the injury maybe indicative of articular cartilage damage. Within 3 months of the injury, there was overlap of SF sGAG levels between the injured and contralateral joints, indicating that proteoglycan catabolism may be contained. This could be consistent with a superficial injury to cartilage characterized by larger differences in SF lubricin, than SF GAG, between injured and contralateral joints. Following ACL and PCL transection in the rabbit model, low lubricin levels and the appearance of collagen type II peptides appeared to precede the appearance of sGAG in the SF (20
). This highlights an important difference between lubricin and sGAG metabolism following injury that might connect an ACL injury to a nidus leading to secondary OA.
In the present study we used sGAG as a biomarker for proteoglycan turnover, and we found that the sGAG concentrations were elevated in the injured knee when compared to the contralateral knee. Various markers of cartilage matrix turnover have been previously evaluated following ACL injury (27
). Lohmander et al reported a marked increase in SF proteoglycan fragments within 3–4 weeks of ACL injury that remained high in many patients out to 4 years after injury (29
). Likewise, the proteoglycan turnover epitope 846 has also been shown to be elevated following ACL injury, and this was correlated with collagen turnover markers including cartilage oligomeric matrix protein (COMP) and C-propeptide of type II collagen (CPII) (30
). In addition, procollagen type II C-propeptide (PIICP), a marker of collagen type II synthesis, and the release of cross-linked collagen type II into SF have been shown to increase following ACL injury (28
). These studies support the finding that cartilage metabolism is altered following injury and may be indicators for OA in this patient population. In the current study, the pattern of lubricin SF changes following ACL injury shows an early decrease in SF lubricin levels, which appear to recover within 1 year after injury, however the metabolic changes appear to persist. It is interesting to note that the SF sGAG concentrations were variable when the lubricin concentrations were high in the contralateral knee (). However, in the injured knee, low lubricin levels were consistently associated with high sGAG concentrations (). The implications of this remain to be proven but suggest that proteoglycan turnover is increased in the presence of low lubricin values. Thus, it is our hypothesis that the pro-inflammatory cytokines initiate a cascade of events that lead to a decrease in joint lubrication and an increase in cartilage wear. Although the lubricin levels eventually return to normal, ongoing damage may have been initiated.
In this study, the patient population was relatively young with no history of degenerative joint disease. Each patient served as his own control by aspirating undiluted SF from the contralateral joint allowing for meaningful comparison between the injured and the contralateral joint. We attempted to limit confounding factors by the study inclusion criteria. Nonetheless, other factors such as bone bruising, minor meniscal damage, and unseen trauma could also influence the results. Other limitations of this study include the cross-sectional nature of the study, and the possible dilution of lubricin and other markers in the injured joint due to transudate. To prevent the dilution effect, lubricin levels were also compared after normalizing them to SF total protein levels without changing the results. It is also unknown what happens to the lubricin concentrations after the 12 month time period. The true concentration must eventually asymptote at a biologically plausible value. Unfortunately we do not have lubricin concentration data from patients more than one year from injury since the focus of the study was on the acute injury state. Future long-term longitudinal studies will be directed at measuring the temporal changes in lubricin levels following ACL injury and its treatment.
In addition, alterations in joint kinematics in the contralateral joint may occur, and it is not completely known how it may affect lubricin metabolism. It was assumed that the contralateral knee was not affected, an assumption supported by the finding that the lubricin concentration did not significantly change with time (). The dynamic shear of cartilage appears important in expression of lubricin by superficial zone chondrocytes (32
). Relieving load could lead to lower expression of articular lubricin by cartilage. This could mean that normal lubricin levels would have been higher than we observed in the contralateral joints. Finally, the relationship between lubricin in SF and lubricin bound to the articular cartilage is not completely known. However, there is evidence that equilibrium may exist between SF and surface-associated lubricin (34
In this study, we have examined the effects of an ACL injury on SF lubricin and its relationship to markers of inflammation and cartilage damage. SF lubricin level was significantly decreased following the injury and subsequently increased as the inflammation subsided. The decrease in SF lubricin in the early stages places the joint at elevated risk of wear-induced damage. Blocking the effects of TNF-α, IL-1β, and IL-6 after injury may prove valuable in preventing the decrease in SF lubricin, which in turn could possibly preserve chondroprotection. The results of this study also suggest that lubricin levels can be used as a marker of cartilage health, especially in the early stages of an acute disease process.