Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis

Abstract

Osteoarthritis is a complex disease involving the mechanical breakdown of articular cartilage in the presence of altered joint mechanics and chondrocyte death, but the connection between these factors is not well established. Lubricin, a mucinous glycoprotein encoded by the PRG4 gene, provides boundary lubrication in articular joints. Joint friction is elevated and accompanied by accelerated cartilage damage in humans and mice that have genetic deficiency of lubricin. Here, we investigated the relationship between coefficient of friction and chondrocyte death using ex vivo and in vitro measurements of friction and apoptosis. We observed increases in whole-joint friction and cellular apoptosis in lubricin knockout mice compared with wild-type mice. When we used an in vitro bovine explant cartilage-on-cartilage bearing system, we observed a direct correlation between coefficient of friction and chondrocyte apoptosis in the superficial layers of cartilage. In the bovine explant system, the addition of lubricin as a test lubricant significantly lowered the static coefficient of friction and number of apoptotic chondrocytes. These results demonstrate a direct connection between lubricin, boundary lubrication, and cell survival and suggest that supplementation of synovial fluid with lubricin may be an effective treatment to prevent cartilage deterioration in patients with genetic or acquired deficiency of lubricin.

Fig. 1.

(A) Chondrocyte apoptosis in mice with different Prg4 genotypes. Activated caspase-3 immunodetection in coronal sections of knee joints from 10-wk-old wild-type (Prg4^+/+), heterozygous lubricin knockout (Prg4^+/−), and homozygous lubricin knockout (Prg4^−/−) mice that were not tested using the passive pendulum system. First column, Immunofluoresence detection (red) of active caspase-3. Second column, Same sections fluorescently imaged to reveal DAPI-stained (blue) chondrocyte nuclei. Third column, Merged active caspase-3 and DAPI images. Note the red fluorescence at the cartilage surface represents nonspecific antibody binding to proteinacious deposits that normally accumulate on the cartilage surface in Prg4^−/− mice. In contrast, the punctate red fluorescence observed below the cartilage surface overlaps with DAPI-stained nuclei indicating chondrocytes undergoing apoptosis. In contrast to Prg4^−/− knees, the knees in Prg4^+/+ and Prg4^+/− mice had few chondrocytes with activated caspase-3. Fourth column, TUNEL staining and methyl green counterstaining of cartilage. More apoptotic (brown) chondrocyte nuclei are present in Prg4^−/− cartilage compared with Prg4^+/+ and Prg4^+/− cartilage. (Scale bars, 100 μm.) (Bottom row) Region of interest taken from the image above to show the cells located just below the flattened superficial chondrocytes that are most susceptible to apoptosis. (B) Whole-joint COF in mice with different Prg4 genotypes. Bar graphs depicting mean + 1 SD COF measurements in knee joints from 10-wk-old wild-type (Prg4^+/+), heterozygous lubricin knockout (Prg4^+/−), and homozygous lubricin knockout (Prg4^−/−) mice. Each individual COF measurement is indicated by an open circle. Prg4^−/− knees have significantly higher COF than either Prg4^+/+ or Prg4^+/− knees. *P < 0.01 compared with Prg4^+/+ or Prg4^+/− knees.

Fig. 2.

COF in bovine cartilage bearings lubricated with different test solutions. Bar graphs depicting mean + 1 SD static (unshaded) and kinetic (shaded) COF measurements in bovine cartilage bearings lubricated with either PBS, SF from patients with CACP (CACP-SF), purified human synoviocyte lubricin (HSL), human synovial fluid from normal joints (HSF), and CACP-SF that has been supplemented with HSL (CACP-SF+HSL). Static COF was significantly higher in PBS and CACP-SF lubricated bearings compared with HSL- and HSF-lubricated bearings. Kinetic COF was significantly higher in bearings lubricated with PBS compared with all other groups.

Fig. 3.

Apoptosis in bovine cartilage bearings lubricated with different test solutions. The lower explant cartilage bearings were stained for active caspase-3 (red, left column), and nuclei were counterstained with DAPI (blue, middle column) following friction and wear testing. Images were merged to show colocalization of caspase-3 within cells (right column). We observed many apoptotic cells in the superficial and upper middle zones of PBS and CACP-SF–lubricated samples, whereas few apoptotic cells were observed in the unloaded bearings and in bearings lubricated with HSL, HSF, or CACP-SF + HSL. (Scale bars, 100 μm.)

Fig. 4.

(A) Apoptosis in lower bovine cartilage bearings analyzed by zone. Cartilage explant bearings tested with PBS and CACP-SF had a greater number of cells positive for activated caspase-3 (brown, arrowheads) than bearings lubricated with HSL, HSF, or CACP-SF+HSL and unloaded bearings. Cells negative for activated caspase-3 are stained blue. TUNEL staining (brown) confirmed apoptosis in bearings lubricated with PBS and CACP-SF (arrowheads), but few cells were TUNEL positive in the bearings lubricated with HSL, HSF, or CACP-SF+HSL or in unloaded bearings. Cells negative for TUNEL are stained blue. (Scale bars, 100 μm.) (B) Percentage of activated caspase-3 in lower bovine cartilage bearings. Bearings tested with PBS and CACP-SF had significantly higher percentages of apoptotic cells in the superficial and upper middle zones compared with unloaded bearings and bearings lubricated with HSL and HSF. Error bars indicate SD. (C) Correlation of static COF and activated caspase-3 in zone A. A significant correlation (r² = 0.41) between static COF and activated caspase-3–positive cells in zone A (articular surface, 100 μm) was observed for the mechanically tested bearings, across the different lubricants.