The Journal of Bone and Joint Surgery 81:452-61 (1999)
© 1999 The Journal of Bone and Joint Surgery, Inc.
The Effect of Ultra-High Molecular Weight Polyethylene Wear Debris on MG63 Osteosarcoma Cells in Vitro*
D. D. DEAN, PH.D. ,
Z. SCHWARTZ, D.M.D., PH.D ,
Y. LIU, M.D.,
C. R. BLANCHARD, PH.D. ,
C. M. AGRAWAL, PH.D., P.E.,
J. D. MABREY, M.D.,
V. L. SYLVIA, PH.D.,
C. H. LOHMANN, M.D. and
B. D. BOYAN, PH.D., SAN ANTONIO, TEXAS
Investigation performed at the University of Texas Health Science Center at San Antonio, San Antonio
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Abstract
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Background: Focal osteolysis due to ultra-high molecular weight polyethylene wear debris involves effects on both bone resorption and bone formation.
Methods: The response of MG63 osteoblast-like osteosarcoma cells to ultra-high molecular weight polyethylene wear debris isolated by enzymatic digestion of granulomatous tissue obtained from the sites of failed total hip arthroplasties was examined. Scanning electron microscopy, particle-size analysis, and Fourier transform infrared spectroscopy were used to characterize the number, morphology, size distribution, and chemical composition of the particles. Cell response was assessed by adding particles at varying dilutions to confluent cultures and measuring changes in cell proliferation (number of cells and [3H]-thymidine incorporation), osteoblast function (alkaline-phosphatase-specific activity and osteocalcin production), matrix production (collagen production and proteoglycan sulfation), and local cytokine production (prostaglandin-E2 production).
Results: The mean size of the particles was 0.60 micrometer, and 95 percent of the particles had a size of less than 1.5 micrometers. The number of particles per gram of tissue ranged from 1.39 to 3.38 x 109. Three of the four batches of particles were endotoxin-free. Exposure of the cells to particles of wear debris significantly increased the number of cells (p < 0.05) and the [3H]-thymidine incorporation (p < 0.05) in a dose-dependent manner. In contrast, the addition of particles decreased alkaline-phosphatase-specific activity and osteocalcin production. Collagen production and proteoglycan sulfation were also decreased, while prostaglandin-E2 synthesis was increased by the addition of particles.
Conclusions: Ultra-high molecular weight polyethylene particles isolated from human tissue stimulated osteoblast proliferation and prostaglandin-E2 production and inhibited cell differentiation and matrix production. These results indicate that particles of wear debris inhibit cell functions associated with bone formation and that osteoblasts may produce factors in response to wear debris that influence neighboring cells, such as osteoclasts and macrophages.
Clinical Relevance: Particles of wear debris, especially ultra-high molecular weight polyethylene, have been implicated in the loosening of implants and the development of osteolysis. The present study shows that particles of ultra-high molecular weight polyethylene isolated from human tissue inhibit osteoblast functions associated with bone formation. In addition, particles of wear debris induced osteoblasts to secrete factors capable of influencing neighboring cells, such as osteoclasts and macrophages. These results suggest that osteoblasts may play a role in the cascade of events leading to granuloma formation, osteolysis, and failure of orthopaedic implants.
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Introduction
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Little is known about the role of osteoblasts in periprosthetic osteolysis, but it is believed that these cells play a role in the recruitment of osteoclasts and macrophages and their subsequent activation in the presence of wear debris. Goodman et al.12 observed that various concentrations of polyethylene particles in a bone-harvest chamber stimulated not only a marked foreign-body response and fibrosis but also a net reduction in bone formation. Co-culture of osteoblast-like cells and macrophages led to the differentiation of the macrophages into cells with an osteoclast-like phenotype. This phenomenon was not observed in cultures of macrophages alone26,28,32. Moreover, culture of macrophages and calvariae in the presence of wear debris caused an increase in prostaglandin-E2 production compared with that found when macrophages were cultured with particles alone11. In the periprosthetic environment, macrophages are not the only cells that can phagocytose particles. Osteoblasts have been shown to phagocytose collagen fibrils40, the products of the degradation of cobalt-chromium alloy14 or porous polyester-urethane foam31, and small titanium particles41. These findings suggest that these cells may participate in the osteolytic process by responding to particles in specific ways.
A net reduction in bone formation may also be due to decreased osteoblastic activity. MG63 osteoblast-like osteosarcoma cells were found to synthesize less collagen when they were exposed to titanium particles41, indicating that the activity of the cells is down-regulated. In other studies, MG63 cells cultured in the presence of particles of commercial ultra-high molecular weight polyethylene were found to have an increased rate of proliferation and a decreased expression of alkaline phosphatase, a marker of phenotypic expression8,37. Whether this is the case for MG63 cells exposed to ultra-high molecular weight polyethylene particles isolated from human periprosthetic tissue has not been determined, to our knowledge.
As particles of wear debris from the sites of failed total hip arthroplasties, and particularly particles of ultra-high molecular weight polyethylene in granulomatous tissue, have been well characterized5,21,23,38, we tested the hypothesis that particles from these tissues would affect the phenotypic expression of osteoblasts. MG63 cells were incubated with ultra-high molecular weight polyethylene particles isolated by enzymatic digestion of granulomatous tissue from the sites of failed total hip arthroplasties. The effects of these particles were assessed by the measurement of cell proliferation (the number of cells and [3H]-thymidine incorporation), osteoblast function (alkaline-phosphatase-specific activity and osteocalcin production), extracellular matrix synthesis (collagen synthesis and proteoglycan sulfation), and local cytokine production (prostaglandin-E2 production).
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Materials and Methods
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Profile of Patients
Granulomatous tissue was removed from the area behind the loose acetabular cups of four patients (three women and one man) who were being managed with a revision procedure because of aseptic loosening following a total hip arthroplasty. The patients ranged in age from forty-three to seventy-nine years. The time-interval after the primary total hip replacements, which were performed because of osteoarthritis, ranged from eight to twenty-four years. Three of the femoral components (Cases 1, 3, and 4) were fixed without cement, and one (Case 2) was fixed with cement. All of the acetabular components were fixed without cement. The indications for the revisions included pain in the hip and radiographic evidence of loosening as indicated by migration of the femoral component and radiolucency around both components. In three patients, loss of centralization of the femoral head due to severe wear of the polyethylene acetabular cup was observed radiographically.
Control tissue was obtained from a capsule removed during a primary total hip arthroplasty in a sixty-two-year-old man who was being managed for primary osteoarthritis with no evidence of other osseous or autoimmune disease. This study was approved by the Institutional Review Board of the University of Texas Health Science Center at San Antonio, and informed consent was obtained from all patients.
Isolation of Particles of Wear Debris
At the time of resection, the tissue was immediately placed in neutral buffered formalin and stored at 20 degrees Celsius. Particles of wear debris were isolated from the tissue by papain digestion with a modification of the method of Maloney et al.22, as will be described. Two grams of tissue were minced into small (two to three-square-millimeter) pieces and washed four times with ten volumes of ultrapure water over the course of three days to remove the formalin fixative. At the completion of the final wash, the tissue was digested with papain (catalogue number P3125; Sigma Chemical, St. Louis, Missouri). The enzyme was dissolved in 0.05-molar sodium phosphate buffer (pH 6.5) containing two-millimolar N-acetylcysteine at a ratio of three milligrams of papain per ten milliliters of buffer. Five milliliters of this enzyme solution was added for each gram of tissue to be digested. Digestion of the tissue was conducted at 65 degrees Celsius in a shaking water bath for three days. Each day, a fresh aliquot of papain, equivalent to the original amount of enzyme added to digest the tissue, was added. At the end of digestion, the insoluble residue (including particles with a density of greater than one) was removed by centrifugation at 100,000 times gravity for one hour, and the supernatant containing the ultra-high molecular weight polyethylene wear debris was collected. The digest supernatant was then sonicated for ten minutes in a sonicator (model ME2.1; Mettler Electronics, Anaheim, California), and the particles were collected by filtration on a polyester filter with a pore size of 0.2 micrometer (Poretics, Livermore, California).
After the particles in the supernatant had been collected on the filter, they were washed twice with two milliliters of ultrapure water followed by two washes with two milliliters of 70 percent ethanol. The filter containing particles from two grams of tissue was then placed in five milliliters of Dulbecco's modified Eagle medium containing 0.5 percent antibiotics and was stored overnight at 4 degrees Celsius. On the next day, the solution was sonicated again and the filters were removed. The experimental media were prepared by dilution of this stock, which contained 1.35 x 109 particles per milliliter for Case 1, 1.00 x 109 particles per milliliter for Case 2, 1.30 x 109 particles per milliliter for Case 3, and 0.56 x 109 particles per milliliter for Case 4. Each stock solution of particles was mixed with culture medium to yield dilutions of 1:1, 1:10, 1:100, and 1:1000 of the original stock. The stock solution from each patient was tested in two replicate experiments. To simplify presentation of the data, the dilution factors, instead of the numbers of particles, are given in the present report. The control tissue was prepared in exactly the same manner to determine if the digestion protocol produced any residue that affected the behavior of the osteoblast cells.
Characterization of Particles
Morphology
The geometry and surface morphology of the particles were examined with a scanning electron microscope (model 1645; Amray, Bedford, Massachusetts) after collection on the polyester filter, as described, and sputter-coating with gold-palladium. The particles were viewed at magnifications ranging from 1000 to 10,000 times.
Size Distribution
Particles were collected as described for the morphology studies, and photomicrographs were made at magnifications as high as 20,000 times with use of the scanning electron microscope. The equivalent circle diameter of the particles was determined with use of custom software based on the NIH Image program (National Institutes of Health, Bethesda, Maryland)17, and a total of 695 distinct particles were evaluated on representative photomicrographs of specimens from all four patients.
Number of Particles
To determine the number of particles, the particles were collected on the polyester filters as described for the morphology studies, were viewed with scanning electron microscopy at a magnification of 2580 times (Case 1) or 2000 times (Cases 2, 3, and 4), and then were photographed. The particles were counted in ten representative fields, and the average number of particles for all of the fields was determined. These data were then used to determine the number of particles per gram of tissue for each patient by correcting for the area of the filter and the portion of the digest containing particles that were counted.
Chemical Composition
Microscopic Fourier transform infrared spectroscopy was used to determine the chemical composition of the particles. Particles were collected on a polyester filter with a pore size of 0.2 micrometer that had been sputter-coated with gold-palladium. A Nicolet Magna Fourier transform infrared spectroscopy system (Nicolet Biomedical Instruments, Madison, Wisconsin) was used in reflection mode, and spectra were collected with use of thirty-two scan summations at a resolution of sixteen reciprocal centimeters. Spectra were obtained for particles that were clumped together and had an aggregate diameter of more than twenty micrometers. These particles were confirmed to be ultra-high molecular weight polyethylene as peaks at 2923, 2854, 1466, and 718 reciprocal centimeters were observed.
Presence of Endotoxin
The presence of endotoxin in the samples was assessed with use of a commercially available kit (E-TOXATE; Sigma Chemical) according to the manufacturer's directions.
Cell Culture
MG63 osteoblast-like cells, originally isolated from a human osteosarcoma, were obtained from the American Type Culture Collection (Rockville, Maryland). This cell line has been well characterized3,10 and shows numerous osteoblastic traits, including high levels of 1,25-(OH)2D3-responsive alkaline phosphatase, and inhibition of the number of cells after treatment with 1,25-(OH)2D3. Cells were plated at 9300 cells per square centimeter in Dulbecco's modified Eagle medium containing 10 percent fetal bovine serum and 0.5 percent antibiotics (as described), and they were cultured in an atmosphere of 100 percent humidity and 5 percent carbon dioxide at 37 degrees Celsius in twenty-four-well culture plates (Corning, Corning, New York). The media were changed at twenty-four hours and then at seventy-two-hour intervals until the cells reached confluence. At confluence, the media were replaced with experimental media containing dilutions of the particle stock solutions, and the cells were cultured for an additional twenty-four hours unless otherwise noted.
Cell Proliferation
Number of Cells
MG63 cells were released from the culture surface by the addition of 0.25 percent trypsin in Hanks balanced salt solution containing one-millimolar EDTA for ten minutes at 37 degrees Celsius; this was followed by the addition of Dulbecco's modified Eagle medium containing 10 percent fetal bovine serum. The cells were isolated by centrifugation at 500 times gravity for ten minutes. Cell pellets were washed with phosphate-buffered saline solution, resuspended, and counted in a Coulter counter (Coulter Electronics, Hialeah, Florida). Cells obtained in this manner exhibited greater than 95 percent viability as demonstrated by trypan-blue exclusion.
[3H]-Thymidine Incorporation
DNA synthesis was estimated by measurement of [3H]-thymidine incorporation34. Fifty microliters of [3H]-thymidine (from a four-microcurie-per-milliliter stock solution [DuPont NEN Research Products, Boston, Massachusetts]) was added to the cultures. Four hours later, the media were removed and the cultures were washed twice with cold phosphate-buffered saline solution and twice with cold 5 percent trichloroacetic acid and then were treated with ice-cold saturated trichloroacetic acid for thirty minutes. The resulting precipitate was dissolved in 0.3 milliliter of 1 percent sodium dodecyl sulfate at 20 degrees Celsius, and radioactivity was measured with liquid-scintillation spectroscopy.
Osteoblast Function
Alkaline-Phosphatase-Specific Activity
Alkaline-phosphatase-specific activity in isolated cells and in cell and matrix preparations (as will be described) was assayed4 by measurement of the release of p-nitrophenol from p-nitrophenylphosphate at pH 10.2. Protein content was determined with use of micro/macro BCA kits (Pierce Chemical, Rockford, Illinois).
Cells and matrix were isolated with the method described by Hale et al.13 as modified by us2,33. The culture media were decanted, and the cells and matrix were washed twice with phosphate-buffered saline solution and then were removed with a cell-scraper. After centrifugation, the pellet containing the cells and matrix was washed once more with phosphate-buffered saline solution and was resuspended by vortexing in 500 microliters of deionized water containing twenty-five microliters of 1 percent Triton X-100. Isolated cells were resuspended in the same way. Before assay of enzyme activity, both preparations were frozen and thawed three times to ensure that they were completely lysed.
Osteocalcin Production
The production of osteocalcin by the cultures was measured with use of a commercially available radioimmunoassay kit (Human Osteocalcin Kit, Biomedical Technologies, Stoughton, Massachusetts) with rabbit anti-human osteocalcin antibody.
Matrix Production
Collagen Production
Matrix protein synthesis was assessed by measurement of [3H]-proline incorporation into collagenase digestible protein and noncollagenase digestible protein27. At confluence, the media were replaced with 500 microliters (per well) of Dulbecco's modified Eagle medium containing 10 percent fetal bovine serum, antibiotics, fifty micrograms of ß-aminoproprionitrile (Sigma) per milliliter, and five microcuries of L[3H]-proline (New England Nuclear, Boston, Massachusetts) per milliliter. After twenty-four hours, the media were collected. Cells and matrix were removed by scraping and were resuspended in two 0.2-milliliter portions of 0.2-molar NaOH. The media and the cell and matrix fractions were combined, and protein was precipitated with 0.05 milliliter of 100 percent trichloroacetic acid containing 10 percent tannic acid, washed three times with 0.5 milliliter of 10 percent trichloroacetic acid containing 1 percent tannic acid, and then washed twice with ice-cold acetone. The pellets were then dissolved in 500 microliters of 0.05-molar NaOH.
Digestion of the cell-and-matrix pellet was performed with use of highly purified clostridial collagenase, 138 units per milligram of protein, obtained from Calbiochem (San Diego, California). Noncollagenase digestible protein synthesis was calculated after multiplication of the labeled proline in the noncollagenase digestible protein fraction by 5.4 to correct for its relative abundance in collagen29. Percent collagen production was calculated by comparison of the amount of collagenase digestible protein with the total amount of collagenase and noncollagenase digestible protein. The protein content of each fraction was determined with a modification of the method of Lowry et al.20.
[35S]-Sulfate Incorporation
Proteoglycan synthesis was assessed on the basis of [35S]-sulfate incorporation25,36. [35S]-sulfate was added to the media at a final concentration of nine microcuries per milliliter. Four hours later, the media were discarded and the wells were washed once with 500 microliters of phosphate-buffered saline solution. The cells and matrix were collected in two 0.25-milliliter portions of 0.25-molar NaOH. The total volume of the sample was adjusted to 0.7 milliliter by the addition of 0.15-molar NaCl, and the sample was dialyzed against buffer containing 0.15-molar NaCl, twenty-millimolar Na2SO4, and twenty-millimolar Na2HPO4 at pH 7.4 and 4 degrees Celsius until the radioactivity in the dialysate reached background levels. The amount of [35S]-sulfate that was incorporated was determined by liquid scintillation spectroscopy, and the protein content was determined with the method of Lowry et al.20.
Prostaglandin-E2 Production
The amount of prostaglandin E2 that was produced by the cells and released into the media was assessed with use of a commercially available competitive binding radioimmunoassay kit (Dupont NEN Research Products). Previous studies have shown that most of the prostaglandin E2 that is synthesized by MG63 cells is immediately released into the medium rather than being stored in the cells and matrix9,35. For this reason, we determined the prostaglandin-E2 content of the media only.
Statistical Analysis
Data regarding the number of cells and the alkaline-phosphatase-specific activity (Fig. 4) were collected from one of two replicate experiments with use of the same stock solution of particles (that is, from one patient). Data regarding the number of cells (Fig. 3), [3H]-thymidine incorporation (Fig. 5), alkaline-phosphatase-specific activity (Fig. 6), osteocalcin production (Fig. 7), percent collagen production (Fig. 8), [35S]-sulfate incorporation (Fig. 9), and prostaglandin-E2 production (Fig. 10) were collected from duplicate experiments with use of the same particle stock solutions; individual stock solutions were prepared for each of the four patients. The data are presented as treatment:control ratios for the four patients. Each treatment group in each replicate experiment represents the mean and the standard error of the mean for six individual cultures. The data were first tested with analysis of variance, and when statistical differences were observed post hoc testing was performed with the Student t test with Bonferroni's modification. P values of less than 0.05 were considered significant. For analysis of treatment:control ratios, significant differences between treatment groups were determined with use of the Wilcoxon signed-rank test. Again, p values of less than 0.05 were considered significant.
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Fig. 4 Comparison of the effects of ultra-high molecular weight polyethylene particles from granulomatous tissue and the insoluble residue from control tissue on the number of cells (A) and the alkaline-phosphatase-specific activity (B). MG63 cells were treated for twenty-four hours with varying dilutions of the stock solution from Case 2 (109 particles per milliliter) or from the control tissue. The cultures were then digested with trypsin, and the number of cells was determined with use of a Coulter counter or the cells and matrix were assayed for alkaline phosphatase activity. The values are the mean and the standard error of the mean for six cultures. The data are from one of two replicate experiments. An asterisk indicates that the addition of particles significantly affected the number of cells or alkaline-phosphatase-specific activity compared with that in the control (p < 0.05).
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Fig. 3 Effect of ultra-high molecular weight polyethylene particles on the number of MG63 cells. The treatment:control ratios (T/C) for experiments involving particles from four patients are shown; they represent the mean and the standard error of the mean. An asterisk indicates that the addition of particles significantly increased the number of cells compared with that in the control (p < 0.05).
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Fig. 5 Effect of ultra-high molecular weight polyethylene particles on [3H]-thymidine incorporation by MG63 cells. The treatment:control ratios (T/C) for experiments involving particles from four patients are shown; they represent the mean and the standard error of the mean. An asterisk indicates that the addition of particles significantly increased the [3H]-thymidine incorporation compared with that in the control (p < 0.05).
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Fig. 6 Effect of ultra-high molecular weight polyethylene particles on alkaline-phosphatase-specific activity of MG63 cells. The treatment:control ratios (T/C) for experiments involving particles from four patients are shown; they represent the mean and the standard error of the mean. An asterisk indicates that the addition of particles significantly decreased the alkaline-phosphatase-specific activity compared with that in the control (p < 0.05).
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Fig. 7 Effect of ultra-high molecular weight polyethylene particles on osteocalcin production by MG63 cells. The treatment:control ratios (T/C) for experiments involving particles from four patients are shown; they represent the mean and the standard error of the mean. An asterisk indicates that the addition of particles significantly decreased the osteocalcin production compared with that in the control (p < 0.05).
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Fig. 8 Effect of ultra-high molecular weight polyethylene particles on percent collagen production by MG63 cells. The treatment:control ratios (T/C) for experiments involving particles from four patients are shown; they represent the mean and the standard error of the mean. An asterisk indicates that the addition of particles significantly decreased the collagen production compared with that in the control (p < 0.05).
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Fig. 9 Effect of ultra-high molecular weight polyethylene particles on [35S]-sulfate incorporation by MG63 cells. The treatment:control ratios (T/C) for experiments involving particles from four patients are shown; they represent the mean and the standard error of the mean. An asterisk indicates that the addition of particles significantly decreased the [35S]-sulfate incorporation compared with that in the control (p < 0.05).
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Fig. 10 Effect of ultra-high molecular weight polyethylene particles on prostaglandin-E2 production by MG63 cells. The treatment:control ratios (T/C) for experiments involving particles from four patients are shown; they represent the mean and the standard error of the mean. An asterisk indicates that the addition of particles significantly increased the prostaglandin-E2 production compared with that in the control (p < 0.05).
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Results
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Characterization of Particles
The scanning electron micrographs showed three main types of particles (Figs. 1-A and 1-B). The most abundant type of particle was round or slightly oblong with an irregular, grainy surface. Other particles were thin and fibril-like, extending to as long as four micrometers. The least abundant type of particle was longer still and had an oblong or round so-called head at the end of the fibril.
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Figs. 1-A and 1-B: Scanning electron micrographs of ultra-high molecular weight polyethylene particles isolated from tissue obtained at the revision procedures (original magnification x 1000 and bar = 10.0 micrometers for Fig. 1-A; original magnification x 10,000 and bar = 1.0 micrometer for Fig. 1-B).
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Figs. 1-A and 1-B: Scanning electron micrographs of ultra-high molecular weight polyethylene particles isolated from tissue obtained at the revision procedures (original magnification x 1000 and bar = 10.0 micrometers for Fig. 1-A; original magnification x 10,000 and bar = 1.0 micrometer for Fig. 1-B).
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The particles ranged in size from 0.1 to 6.0 micrometers (Fig. 2), and 95 percent of the particles had a size of less than 1.5 micrometers. The mean equivalent circle diameter (and standard error of the mean) of all of the particles that were measured was 0.60 ± 0.45 micrometer. The mean number of particles per field was 667 ± 90 for Case 1, 586 ± 42 for Case 2, 759 ± 97 for Case 3, and 326 ± 13 for Case 4. The number of particles isolated per gram of tissue was 3.38 x 109 for Case 1, 2.50 x 109 for Case 2, 3.24 x 109 for Case 3, and 1.39 x 109 for Case 4.
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Fig. 2 Distribution of the sizes, expressed as equivalent circle diameters in micrometers, of the particles of wear debris isolated from tissue obtained at the revision procedures.
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The particles from three patients were negative for the presence of endotoxin, whereas those from one patient were positive. Nonetheless, as will be described, no significant difference in cell response was observed.
Cell Proliferation
Number of Cells
The addition of wear debris from the granulomatous tissue increased the number of cells in a dose-dependent manner (Fig. 3). Compared with the number of cells in the untreated control, the number of cells was significantly increased by 1.2-fold, 1.4-fold, and 1.5-fold at dilutions of the stock solution of 1:100 (p < 0.05), 1:10 (p < 0.05), and 1:1 (p < 0.001). The increase in the number of cells was consistent among the four donors. In contrast, when control tissue was digested in an identical manner and the insoluble residue was collected on a filter and added to the cultures, there was no effect on the number of cells (Fig. 4, A). This finding indicates that the effect on the cells was due to the ultra-high molecular weight polyethylene particles and was not an artifact of the digestion procedure.
[3H]-Thymidine Incorporation
Ultra-high molecular weight polyethylene particles caused a significant increase in [3H]-thymidine incorporation (Fig. 5). The effect of the particles was found to be concentration-dependent, with significant increases of 1.17-fold, 1.35-fold, and 1.39-fold at dilutions of 1:100 (p < 0.05), 1:10 (p < 0.001), and 1:1 (p < 0.001).
Osteoblast Function
Alkaline-Phosphatase-Specific Activity
Ultra-high molecular weight polyethylene particles caused a decrease in the alkaline-phosphatase-specific activity of MG63 cells (Fig. 6). The effect of the particles was similar regardless of whether the enzyme activity of the cells and matrix or that of the isolated cells was examined. The effect was consistent among the samples from the different patients. There were significant decreases of 23 and 28 percent in alkaline-phosphatase-specific activity at dilutions of 1:10 (p < 0.01) and 1:1 (p < 0.001). In contrast, residue from control tissue had no effect on alkaline-phosphatase-specific activity (Fig. 4, B).
Osteocalcin Production
Particles caused a dose-dependent decrease in osteocalcin production by MG63 cells (Fig. 7). There were significant decreases of 18, 50, and 46 percent at dilutions of 1:100 (p < 0.05), 1:10 (p < 0.001), and 1:1 (p < 0.001), and the effect was consistent among the samples from the four patients.
Matrix Production
Collagen Production
Collagen production by MG63 cells was also decreased by the presence of particles in the culture medium (Fig. 8). Significant decreases of 11, 25, and 27 percent were observed at dilutions of 1:100 (p < 0.05), 1:10 (p < 0.01), and 1:1 (p < 0.001).
[35S]-Sulfate Incorporation
Proteoglycan production by the cells was estimated by [35S]-sulfate incorporation (Fig. 9). There were significant decreases in [35S]-sulfate incorporation of 26 and 38 percent at dilutions of 1:10 (p < 0.01) and 1:1 (p < 0.001).
Prostaglandin-E2 Production
Prostaglandin-E2 production was significantly increased 1.5-fold and 1.7-fold (Fig. 10) in cultures treated with dilutions of 1:10 (p < 0.05) and 1:1 (p < 0.001).
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Discussion
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In the present study, wear debris was isolated, with a new papain digestion protocol, from granulomatous tissue of patients being managed with a revision of a loose total hip prosthesis. The wear debris had a size distribution, morphology, and Fourier transform infrared spectrum typical of ultra-high molecular weight polyethylene particles described previously5,22,23,38. The particle preparations in this study were free of contamination by metal (titanium or titanium alloy) or polymethylmethacrylate particles because the centrifugation step in the isolation procedure sediments these particles on the basis of their densities. The particles were then added to cultures of MG63 cells, and their effect on cell response (proliferation, differentiation, extracellular matrix synthesis, and release of local factors) was examined. We believe that this is the first time that particles of ultra-high molecular weight polyethylene wear debris have been isolated from human tissue and then added to cell cultures to examine their effect on cell response in vitro.
In the current study, the number of ultra-high molecular weight polyethylene particles isolated from the tissue was similar to that found by other investigators22,23. We reported a range of 1.39 to 3.38 x 109 particles per gram wet weight of tissue, whereas Maloney et al.22 reported a range of 0.09 to 4.29 x 109 particles per gram wet weight of tissue and Margevicius et al.23 reported a range of 14 to 141 x 109 particles per gram dry weight of tissue. Because the numbers of particles isolated from each patient in our study were similar, only a limited amount of tissue was available, and we knew of no previous studies of the effect of different concentrations of ultra-high molecular weight polyethylene particles on MG63 cell response, we decided to dilute our particle stock solutions by as much as 1000-fold. Using this approach, we were able to determine the sensitivity and dose-dependent response of the osteoblasts to particles. Interestingly, the number of particles added to our cell cultures at the 1:100 dilution (for example, 107 particles per milliliter in Case 2) was similar to the number of titanium particles (for example, 2.35 x 107 particles per milliliter) necessary to elicit a response from cultures of the same cell type41.
We know that the responses of the cells were due to the wear debris and not to the presence of endotoxin because all of the particle batches elicited comparable effects, even though three batches were endotoxin-free and one contained detectable lipopolysaccharide. It is unlikely that the cell response was due to particles other than ultra-high molecular weight polyethylene because the isolation procedure would have sedimented material with a density of greater than one (for example, polymethylmethacrylate or titanium). The control tissue, when processed in an identical fashion to the tissue obtained during the revision procedures, elicited no response from the osteoblasts during any of the tests. Finally, comparison of treatment:control ratios showed that the observed effects were consistent.
In our study, particles caused a dose-dependent increase in cell proliferation. Both the number of cells and their rate of DNA synthesis were increased. In a recent investigation of the response of MG63 cells to small titanium or polystyrene particles, Yao et al.41 suggested that the size rather than the composition of the particles modulates cell proliferation. In contrast with our findings, Yao et al. observed that small titanium particles had no effect on [3H]-thymidine incorporation. Because the particles in our study were similar in size to the titanium particles in the study by Yao et al., our results indicate that the chemical composition of the particles may in fact play an important role in osteoblast response.
Increased cell proliferation is generally associated with a cessation in the expression of the differentiated phenotype in osteoblasts18. Our results suggest that particles of ultra-high molecular weight polyethylene may inhibit the differentiation of osteoblasts in the periprosthetic environment. MG63 cells exhibited concentration-dependent decreases in alkaline-phosphatase-specific activity as well as osteocalcin production. Increases in alkaline-phosphatase-specific activity occur relatively early as osteoblasts differentiate in culture, whereas osteocalcin production occurs later in the differentiation cascade6,19. The marked decreases noted in our study suggest that the particles affected the cell cycle in some manner, although the mechanism by which this occurs is not clear. Matrix production was also inhibited, indicating that the protein-synthesis processes associated with the G1/S phase were down-regulated39. Yao et al.41 also noted a decrease in collagen mRNA production in MG63 cells exposed to polystyrene and titanium particles.
We found that wear debris affected the amount of alkaline-phosphatase-specific activity in the isolated cells and the cell and matrix fractions similarly, indicating that the cells and the extracellular matrix vesicles were affected. These findings and the observation of reduced proteoglycan and collagen production and suppressed osteocalcin production support the hypothesis that ultra-high molecular weight polyethylene particles inhibit the formation and mineralization of osteoid. Thus, increasing concentrations of these particles at the bone-implant interface may have an adverse effect on the formation of bone at the site of the implant and may ultimately contribute to loosening of the implant.
These data also indicate that exposure to ultra-high molecular weight polyethylene particles can stimulate cytokine release because prostaglandin-E2 production was increased in a concentration-dependent manner. It is not known whether the prostaglandin E2 produced by the MG63 cells was the result of constitutive or inducible cyclooxygenase activity24; thus, it is difficult to determine whether it represents an inflammatory process. It is well known that low levels of prostaglandin E2 are needed for osteoblastic differentiation. At higher levels, this prostanoid stimulates proliferation and differentiation of cells in the monocytic lineage7,30. The fact that the human-derived particles affected the production of prostaglandin-E2 suggests that the production of local factors is modulated in vivo as well.
Inhibition of osteoblastic phenotypic expression in vivo could result in a net loss in bone formation. Although proliferation was enhanced, the phenotype of the new cells is not known. Since we used a cell line, the potential for selecting a subpopulation was reduced compared with that associated with use of a heterogeneous population of bone cells; however, cell lines can respond to stimuli with altered phenotypic expression1,19.
The production of local factors by osteoblasts and osteoclasts, and their effect on each other, in response to exposure to particles has been noted by others. Horowitz and Purdon15 incubated osteoblasts with conditioned medium from macrophages that had been exposed to polymethylmethacrylate. They observed release of prostaglandin E2, granulocyte-macrophage colony-stimulating factor, and interleukin-6 by the osteoblasts, which could potentially result in osteoclast and macrophage recruitment. In periprosthetic tissue, wear debris is in direct contact with osteoblasts, which, by means of increased prostaglandin-E2 production, could stimulate osteoclasts to resorb increased amounts of bone, leading to osteolysis16. The increased production of prostaglandin E2 by osteoblasts in our study was a direct effect of particles of ultra-high molecular weight polyethylene on the cells. Thus, particles of wear debris may even initiate recruitment of macrophages and osteoclasts through dose-dependent, direct effects on osteoblasts.
In summary, in this study MG63 cells were challenged with various concentrations of ultra-high molecular weight polyethylene particles that had been isolated from granulomatous tissue removed at revisions of failed total hip prostheses. The addition of particles to the cultures resulted in a dose-dependent cell response to the particles. The results suggest that particles of ultra-high molecular weight polyethylene can modify phenotypic expression of MG63 cells. As there is direct contact between wear debris and osteoblasts at the bone-implant interface in vivo, the quality of bone as well as the net amount of bone formed are likely to be suppressed by the presence of the particles. Furthermore, increased production of prostaglandin E2 by the osteoblasts may be an important factor in the recruitment and activation of monocytic cells and their further differentiation.
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Footnotes
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*Although none of the authors has received or will receive benefits for personal or professional use from a commercial party related directly or indirectly to the subject of this article, benefits have been or will be received, but are directed solely to a research fund, foundation, educational institution, or other nonprofit organization with which one or more of the authors is associated. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were the Center for the Enhancement of the Biology/Biomaterials Interface at the University of Texas Health Science Center at San Antonio and a grant from the B. Braun Foundation of Germany.
Departments of Orthopaedics (D. D. D., Y. L., C. M. A., J. D. M., V. L. S., C. H. L., and B. D. B.), Periodontics (B. D. B.), and Biochemistry (B. D. B.), University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7774. E-mail address for Dr. Dean: deand@uthscsa.edu. E-mail address for Dr. Boyan: boyanb@uthscsa.edu.
Department of Periodontics, Hebrew University Hadassah Faculty of Dental Medicine, P.O. Box 1172, Jerusalem 91010, Israel.
Southwest Research Institute, 6220 Culebra Road, Building 128, San Antonio, Texas 78238.
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Abstract
Introduction