FIBROBLAST RESPONSE TO METALLIC DEBRIS IN-VITRO - ENZYME-INDUCTION, CELL-PROLIFERATION, AND TOXICITY

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 0.000083 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 H-3-thymidine. Cell morphology was examined by scanning electron microscopy. Titanium, titanium-aluminum, and chromium significantly stimulated H-3-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. CLINICAL RELEVANCE: Osteolysis has emerged as a serious problem in patients who have had a total joint arthroplasty without cement. The role of metallic debris in the osteolysis and loosening processes remains unknown. The data presented here show that in vitro fibroblasts exhibit a proliferative response after direct exposure to low concentrations of titanium, titanium-aluminum, and chromium particles. Thus, metallic debris may be instrumental in the formation of a fibrous membrane around implants that have been inserted without cement. Formation of such a membrane may, in turn, act as a conduit for polyethylene debris generated at the articular surface, providing access to the space between the implant and the bone. Thus, metallic and polymeric wear debris may play a synergistic role in the development of osteolysis and the loosening of the implants in patients who have had a total joint arthroplasty.