Preventing friction-induced chondrocyte apoptosis: comparison of human synovial fluid and hylan G-F 20

Abstract

Objective: Symptomatic osteoarthritis (OA) is a common painful disease with limited treatment options. A rising number of patients with OA have been treated with intraarticular injections of hyaluronic acid, including the high-molecular-weight hylan G-F 20, which is injected following arthrocentesis. We investigated the effectiveness of hylan G-F 20 to lower coefficient of friction (COF) and prevent chondrocyte apoptosis in vitro.

Methods: A disc-on-disc bovine cartilage bearing was used to measure the static and kinetic COF when lubricated with hylan G-F 20, human synovial fluid (HSF), and phosphate buffered saline (PBS). Following friction testing, we stained paraffin-embedded sections of these cartilage bearings for activated caspase-3, a marker of apoptosis.

Results: Bearings lubricated with hylan G-F 20 had kinetic COF values that were similar to bearings lubricated with PBS, but significantly higher than those lubricated with HSF. There were no significant differences in static COF values in bearings lubricated with hylan G-F 20 as compared to PBS or HSF. However, bearings lubricated with HSF had significantly lower static COF values compared to bearings lubricated with PBS. The mean percentage of caspase-3-positive chondrocytes in the superficial and upper intermediate zones of bearings lubricated with hylan G-F 20 was significantly higher compared to that of bearings lubricated with HSF or unloaded controls, but significantly lower than in those lubricated with PBS.

Conclusion: These findings indicate that joint lubrication may prevent chondrocyte apoptosis by lowering the COF. Further, removal of synovial fluid prior to hylan G-F 20 injection may be detrimental to cartilage health.

Figures

Figure 1. Schematic diagram of friction and wear testing system and a flow chart of experimental protocol

Test lubricant was applied between the bearing surfaces, and the bearings were axially loaded to 18% of the mean total cartilage thickness, and held at that displacement for 8 minutes to allow for fluid depressurization. The large disc was then rotated in torsion +2 revolutions and reset −2 revolutions at an effective velocity of 0.3 mm/s for 12 continuous cycles. Axial load and torque were measured throughout the testing protocol.

Figure 2. COF of bovine osteochondral bearings

Bearings were tested with test lubricants, either PBS, hylan G-F 20 or HSF following the protocol outlined in Figure 1. Lubrication with HSF had the lowest mean static and kinetic COF values. Bearings lubricated with hylan G-F 20 had no significant difference compared to either group in static COF, but had a significantly higher kinetic COF compared to HSF. *p

Figure 3. Activated caspase-3 in superficial and upper intermediate zone chondrocytes

Bearings lubricated with hylan G-F 20 had a significantly higher percentage of cells positive for activated caspase-3 cells in Zone A, Zone B and across all zones compared to unloaded control bearings or bearings lubricated with HSF, indicating that hylan GF-20 is less chondroprotective than HSF. Bearings lubricated with PBS had a significantly higher percentage of apoptotic cells in all zones compared to all other groups. **p

Figure 4. Bovine osteochondral bearings stained for activated caspase-3 (top) and TUNEL (bottom)

Cells positive for activated caspase-3 and TUNEL are brown. Negative cells are stained blue. We observed more apoptotic cells in the PBS and hylan G-F 20 lubricated bearings compared to HSF lubricated bearings and unloaded controls.

Figure 5. Correlation between static COF (left) and kinetic COF (right) and percentage of activated caspase-3 positive cells in Zone A of the superficial zone

We observed a significant correlation between coefficient of friction and percentage of apoptotic cells in Zone A of the cartilage bearings. We observed trends for each lubricant, with the HSF lubricated bearings showing clustering towards the origin, indicating low COF and low percentage of apoptotic cells. In contrast, PBS lubricated bearings clustered in the upper right hand section of the plot, indicating high COF and high percentage of apoptotic cells. Hylan G-F 20 lubricated bearings fell between the two extremes, showing that it has some lubricating and chondroprotective abilities compared to PBS. Note that the x-axis differs between graphs for better visualization of the data points.