The objective of this study was to determine the localization and concentration of SZP in articular cartilage and synovial fluid of patients with advanced OA. The SZP content was further correlated with the articular surface friction coefficient, OA severity, and level of inflammatory cytokines. The main findings of this study can be summarized as follows: (1) OA severity at different regions of human cartilage correlated with the coefficient of friction, (2) SZP expression patterns and friction coefficient showed a dependence on articulating knee joint location and maximum OA score, (3) SZP expression was observed in all samples at the articular surface regardless of OA severity, and (4) high SZP expression was detected in synovial fluid of all subjects with advanced OA.
Regional SZP expression patterns revealed a coupling between biochemical and mechanical function in which physical forces regulate OA severity and joint lubrication through mechanically stimulated secretion of SZP. Particularly for the lateral condyle, for which the maximum OA score was relatively low compared to that of the medial condyle (), a higher SZP expression was observed in the load-bearing region (i.e., SZP expression levels in LA
locations; ). Thus it appears that the physical forces from typical joint contact resulted in increased chondrocyte mechanotransduction, as reported previously (3
) and as confirmed by the immunohistochemistry data for normal human subjects of this study (). It was not possible to confirm this result for the medial condyle of OA samples because the missing cells and tissue of the superficial zone due to the advanced OA precluded a complete analysis.
A surprising finding was the immunolocalization of SZP in the medial condyle despite the high OA score that reflected the loss of superficial zone cells producing the protein (). The fact that SZP was observed in all the samples can be explained by the increased SZP expression in the synovial fluid (with additionally increased intra-articular volumes; ), suggesting that SZP/lubricin in synovial fluid served as a boundary-lubricant reservoir for all surfaces lining the synovial joint. Further, it may be that there are increased binding sites in the damaged tissue structure due to the loss of proteoglycans during OA progression. These interpretations are supported by SZP immunolocalization results representative of severe and minimal OA (). Additionally, the presence of blood in some synovial fluid samples may have been indicative of the harvest procedure and may have underestimated the concentration of SZP overall (). Reduced concentrations of lubricin and markers of joint inflammation have been observed in the synovial fluid of patients within 32 to 364 days from anterior cruciate ligament injury (5
), in contrast to the present study that showed increased SZP expression levels and markers of joint inflammation in some, but not all, late-stage OA patients (). The obtained results suggest an effect of OA stage on SZP/lubricin expression, with a possible rebound and increased SZP expression encountered in the late stage of the disease. However, it is important to consider that the 345 kDa PRG4
gene product was consistently observed using monoclonal antibody S6.79 (), and thus it is unclear how other gene products varied as a function of location and degree of OA severity. Further, because the normal samples were obtained post-mortem, it is unknown if SZP levels precisely reflect concentrations in vivo
SZP (or related biomolecules, such as lubricin) may be ineffective in reducing friction of boundary-lubricated human joints exhibiting advanced OA because other mechanisms may dominate the tribological response. The friction coefficient increased significantly with the maximum OA score, particularly in the case of medial condyles (MA
), despite relatively similar levels of SZP expression with condyles from MP
joint locations (). It is thus possible that either cartilage damage causes an increase in the friction coefficient and/or changes in the friction coefficient lead to cartilage damage. This finding is attributed to other mechanisms that may dominate the tribological behavior (11
). It is well known that the friction coefficient shows a dependence on length scale and operating conditions (e.g., magnitude of contact stresses, apparent contact area, mechanical properties of interacting surfaces, and total sliding distance). For SZP to function properly in the boundary lubrication regime, the adsorbed molecular film must be conformal to the surface topography in a closed-packed arrangement. It is believed that the increased surface roughness and variable tissue stiffness in late-stage OA subjects (13
) intensified the local stresses at asperity contacts, resulting in the rapid removal of the boundary lubricant and preventing its timely replenishment. The higher friction coefficients of OA samples (particularly for load-bearing MA
location) compared to normal subjects indicate that the sliding conditions were not conducive to the maintenance and/or timely replenishment of a continuous and conformal boundary-lubricating layer. This may be attributed to the increased surface roughness of the OA samples (especially those characterized by a high maximum OA score) which promoted the dominance of other friction mechanisms, such as asperity deformation, adhesion, and plowing. These mechanisms are generally characterized by higher energy dissipation (friction) during sliding compared to shearing of a boundary-lubricant layer, which dominated the friction behavior of the smoother normal subjects. Moreover, it has been documented that although synovial fluid from patients with advanced OA lacks superficial zone chondrocytes (in some tissue regions), it maintains normal superficial zone lubricating ability in vitro
, indicating that synovial fibroblasts contribute to joint lubrication through lubricin synthesis (14
). Considering that the SZP/lubricin detected in the present study may possess normal lubricating ability (14
), it is unclear to what extent recombinant lubricin will effectively treat OA (15
), particularly when diagnosed in the late stage.
A strong correlation (r2
> 0.819) of SZP expression and friction coefficient with maximum OA score was found for OA samples () despite the variability inherent to this disease patient population. Meaningful comparisons and tests were conducted because of a sufficiently large sample size (n
= 21); however, such comparisons to normal samples were difficult because of the small sample size (n
= 2). Although the principal objective of this study was to document SZP expression patterns in OA patients, it was also of interest to determine whether regional patterns in young bovine joints (3
) were also present in normal human samples. Patterns similar to those observed in a previous study (3
) were revealed by immunohistochemistry of normal human samples (), where a greater depth of SZP expression in tissue samples was observed in anterior (particularly medial) than posterior locations. Such patterns may be the result of an in vivo
regulation effect of physical forces and joint contact typical of normal physiological activities, a concept reinforced by the OA severity at MA
regions observed in 81% of the OA patients examined. However, ELISA data for normal SZP expression () did not correspond to observed immunohistochemistry patterns. This disparity is attributed to the small sample size (n
= 2) and that repeated testing of only these samples by the ELISA methods yielded highly variable values. Thus, the data of normal SZP expression shown in should be interpreted with caution because further studies are necessary to provide a more comprehensive understanding of regional SZP expression in normal human samples. Additionally, because a human SZP standard was not available, the use of a bovine standard may have altered the magnitude of SZP concentration overall.
Structure-function relationships abound in biological systems, such as Wolff’s law in bone (16
), and the functional significance of single proteins are well documented in nature (17
). The presented results reveal a coupling between biochemical and mechanical function in which physical forces regulate OA severity and joint lubrication through the development of SZP in articular cartilage by mechanotransduction. To our knowledge, this is the first demonstration that the mechanical measurement of the friction coefficient correlates so highly with the histological assessment of cartilage damage in OA patients. The findings of this study suggest that SZP (or similar biomolecules) may be ineffective in reducing friction under boundary lubrication conditions of human joints at advanced stages of OA where other mechanisms may dominate the tribological response. Although the SZP expression may be mediated by TGFβRI signaling (3
), the precise mechanotransduction pathways by which mechanical signals regulate SZP expression and the mechanisms by which SZP lubricates human synovial joints over the lifetime of the organism require further investigation. Such regulatory pathways may provide insight into the progression of cartilage degeneration and regenerative therapies aimed at reconstitution and maintenance of effective boundary lubrication of articular cartilage prior to advanced OA.