Reduction of friction by recombinant human proteoglycan 4 in IL-1α stimulated bovine cartilage explants

Abstract

A boundary lubricant attaches and protects sliding bearing surfaces by preventing interlocking asperity-asperity contact. Proteoglycan-4 (PRG4) is a boundary lubricant found in the synovial fluid that provides chondroprotection to articular surfaces. Inflammation of the diarthrodial joint modulates local PRG4 concentration. Thus, we measured the effects of inflammation, with Interleukin-1α (IL-1α) incubation, upon boundary lubrication and PRG4 expression in bovine cartilage explants. We further aimed to determine whether the addition of exogenous human recombinant PRG4 (rhPRG4) could mitigate the effects of inflammation on boundary lubrication and PRG4 expression in vitro. Cartilage explants, following a 7 day incubation with IL-1α, were tested in a disc-on-disc configuration using either rhPRG4 or saline (PBS control) as a lubricant. Following mechanical testing, explants were studied immunohistochemically or underwent RNA extraction for real-time polymerase chain reaction (RT-PCR). We found that static coefficient of friction (COF) significantly decreased to 0.14 ± 0.065 from 0.21 ± 0.059 (p = 0.014) in IL-1α stimulated explants lubricated with rhPRG4, as compared to PBS. PRG4 expression was significantly up regulated from 30.8 ± 19 copies in control explants lubricated with PBS to 3330 ± 1760 copies in control explants lubricated with rhPRG4 (p < 0.001). Explants stimulated with IL-1α displayed no increase in PRG4 expression upon lubrication with rhPRG4, but with PBS as the lubricant, IL-1α stimulation significantly increased PRG4 expression compared to the control condition from 30.8 ± 19 copies to 401 ± 340 copies (p = 0.015). Overall, these data suggest that exogenous rhPRG4 may provide a therapeutic option for reducing friction in transient inflammatory conditions and increasing PRG4 expression. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:580-589, 2017.

Keywords: Interleukin-1; PRG4; articular cartilage; biomechanics; lubricin; post-traumatic osteoarthritis; tribology, friction.

Figure 1

Diagram demonstrating methods of explant harvest, preparation, and mechanical stimulation of bovine cartilage explant pairs. Panel A displays the medial femoral condylar region of the bovine knee from which 12 and 6mm explant pairs were harvested from the anterior, mid, and posterior regions. Panel B displays the two groups of explant pairs incubated in culture medium supplemented with or without IL-1α for 7-days following explant pair harvest. Panel C shows the explant pairs with their intervening test lubricants. Panel D displays the mechanical stimulation set up including explant pair mounting to the machine components using ethyl cyanoacrylate glue, the magnitude and direction of the load applied to the explant pairs, and the platform rotation from which torque values were obtained for COF calculation.

Figure 2

PRG4 Immunohistochemistry. Immunohistological detection of PRG4 by 9g3 (green) and cell nuclei via DAPI (blue) in non-mechanically stimulated (−Mechanical) and mechanically stimulated (+Mechanical) bovine cartilage explants cultured with IL-1α (+IL-1α, IL) or IL-1α free media (−IL-1α, Control) is shown in six representative images. Either phosphate buffered saline (PBS) or recombinant PRG4 (rhPRG4) was used as a lubricant for mechanically stimulated explants. IL-1α treated, non-mechanically stimulated explants (n=20) displayed decreased PRG4 compared to control treated, non-mechanically stimulated explants (Control, n=20). IL-PBS explants (n=13) displayed no change in PRG4 while IL-rhPRG4 explants (n=16) displayed increased PRG4 on the surface comparable to Control explants and Control-rhPRG4 explants (n=19). Control-PBS explants (n=14) displayed marked decrease in PRG4 compared to Control and Control-rhPRG4 explants. Scale bar is 50 microns.

Figure 3

(A) Total COF Across Cartilage Explant Groups. Dynamic (right) and static (left) coefficient of friction (COF) data for mechanically stimulated, IL-1α supplemented (IL, gray) or IL-1α free (Control, black) explants lubricated with either phosphate buffered saline (PBS, left) or recombinant PRG4 (rhPRG4, right) are illustrated above. IL-PBS explants (n=13) displayed significantly higher static COF compared to Control-PBS explants (n=14, *p=0.02) IL-PBS explants also displayed significantly higher static COF compared to IL-rhPRG4 explants (n=16, *p=0.014). Values represented as mean ± standard deviation. (B) COF by Cartilage Region. Dynamic (right) and static (left) COF of the anterior, mid and posterior areas of the medial femoral condyle is shown. No significant difference was found for static and dynamic COF by cartilage region.

Figure 4

Immunohistochemistry of PRG4 in the Presence of Exogenous Rhodamine Labeled rhPRG4. Representative images showing exogenous rhPRG4 (red) and a combination of exogenous and endogenous PRG4 for IL-1α treated (+IL-1α) and control (−IL-1α) explants with (+rhPRG4) or without (−rhPRG4) rhodamine labeled rhPRG4. Green indicates detection of all PRG4 present by 9g3(whether endogenous or exogenous) and red indicates exogenous, rhodamine-labeled rhPRG4. Counter staining of cell nuclei with DAPI is blue. Samples lubricated with rhPRG4 were either mechanically stimulated (+Mechanical) or not mechanically stimulated (−Mechanical). IL-1α stimulated explants without rhPRG4 (n=5) exhibited little to no endogenous PRG4, while IL-1α stimulated samples with rhPRG4 (n=5) displayed greater amounts of exogenous rhPRG4 penetrating into the cartilage superficial zone. IL-1α stimulated samples appear to exhibit more exogenous rhPRG4 attachment than their IL-1α free counterparts (n=5). This suggests that IL-1α stimulation diminishes endogenous PRG4 while allowing for increased exogenous rhPRG4 attachment and penetration into the cartilage tissue. Scale bar is 100 microns.

Figure 5

PRG4 mRNA Expression Across Cartilage Explant Groups PRG4 mRNA for mechanically stimulated bovine cartilage explants cultured with IL-1α supplemented medium (IL, gray) or IL-1α free medium (Control, black) is shown above. Either phosphate buffered saline (PBS) or recombinant PRG4 (rhPRG4) was used as a lubricant during mechanical stimulation. PRG4 expression was significantly up regulated from 30.8 ± 19 copies in control explants lubricated with PBS to 3330 ± 1760 copies in control explants lubricated with rhPRG4 (n=3, p<0.001). PRG4 expression significantly increased from 30.8 ± 19 copies in IL-1α free explants lubricated with PBS to 401 ± 340 copies in IL-1α stimulated explants lubricated with PBS (n=3, p=0.015). Explants stimulated with IL-1α and lubricated with rhPRG4 trended toward significantly decreased PRG4 expression from 3330 ± 1760 copies in IL-1α free explants lubricated with rhPRG4 to 525 ± 41.2 copies (n=3, p=0.057). Values represented as geometric mean ± geometric standard error.