Bovine synovial fibroblasts in primary monolayer culture were exposed to
particulate metallic debris. The effects of the metallic particles on the
synthesis and secretion of proteolytic enzymes and on cell proliferation
and viability were examined. Uniform suspensions of titanium,
titanium-aluminum, cobalt, and chromium particles, ranging in size from
approximately 0.1 to ten micrometers (average, one to three micrometers),
were prepared; the particle concentrations (the volume of particles divided
by the total volume of the suspension) ranged from 0.0005 to 5 per cent.
Aliquots of the particle suspensions were added to the synovial fibroblast
cultures. The final particle concentrations in the media ranged from
0.0000083 to 0.83 per cent. After seventy-two hours of exposure, each
medium was harvested and was assayed for proteolytic and collagenolytic
activity and for hexosaminidase levels. Neutral metalloproteases,
quantified by collagenolytic and caseinolytic (proteolytic) activity,
represent enzymes, secreted by cells, that are capable of degrading
extracellular matrix. Hexosaminidase is a marker for lysosomal enzyme
activity that can include more than thirty enzymes, such as proteases,
lipases, nucleases, and phosphatases. Cell proliferation was quantified by
uptake of 3H-thymidine. Cell morphology was examined by scanning electron
microscopy. Titanium, titanium-aluminum, and chromium significantly
stimulated 3H-thymidine uptake at low particle concentrations (p < 0.01,
p < 0.002, and p < 0.002, respectively). Exposure to cobalt, even at
the lowest particle concentration, resulted in a significant decrease in
thymidine uptake (p = 0.027). At the highest particle concentrations, all
particles were toxic, as evidenced by the absence of thymidine uptake. At
high particle concentrations, all of the metals caused a decrease in
caseinolytic (proteolytic) and collagenolytic activity in the culture
media. Titanium elevated the lysosomal enzyme marker, hexosaminidase,
except at high concentrations. Chromium and titanium-aluminum had no
significant effect on hexosaminidase at any particle concentration, while
cobalt decreased all enzyme markers at mid-particle to high-particle
concentrations. Scanning electron microscopy demonstrated that the
morphological response of fibroblasts to titanium included
membrane-ruffling and extension of filopodia, typical of active
fibroblasts. In contrast, exposure to cobalt at the same concentration
resulted in cell crenation, indicative of cell death.