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In vivo inflammatory response to silicone elastomer particulate debris
In Vivo Inflammatory Response
to Silicone Elastomer Particulate Debris Sanjiv H. Naidu, MD, Hershey, PA, Pedro Beredjiklian, MD, Louis Adler, MD, F. William Bora, Jr, MD, Daniel G. Baker, MD, Philadelphia, PA Silicone elastomer particles (Silastic silicone elastomer, Dow Coming, Midland, MI), polymethylmethacrylate particles, and monosodium urate particles smaller than 10 gm were injected into a rat subcutaneous air pouch lined with synovial membranelike cells. Inflammatory exudate from the air pouch was retrieved at 6 hours, 24 hours, 48 hours, and 72 hours after injection. White blood cell count, tumor necrosis factor, and prostaglandin E2 were measured in the exudate. White blood cell and tumor necrosis factor levels in the exudate were the highest for the silicone group at 6 and 24 hours. Prostaglandin E2 was also significantly higher in the silicone group at 24 hours. We conclude that acute inflammation is particle-type specific and that Silicone elastomer particles are acutely inflammatory. (l Hand Surg 1996;21A:496-500.)
Silicone synovids is a well-known entity. Breakdown of silicone elastomer particles evokes a severe inflammatory response. 1-3 Along with proliferative synovitis, peri-implant bone resorption occurs; the resulting osteolysis leads to severe bone loss and loss of joint function. Several clinical, radiologic, and histologic studies have documented silicone synovitisJ 3 but no study to date has yet quantified the inflammatory response to silicone particles. Observation of implanted materials reveals that the size of particulate material plays an important role in the type of tissue-cellular response. Bulk implantation of metallic or plastic objects into bone or muscle results in a relatively fibrous membrane. In sharp contrast, implantation of the same materials in the form of a powder results in a marked cellular From the Department of Orthopaedic Surgery, Pennsylvania State University, Hershey, PA, the Department of Materials Science and Engineering, University of Pennsylvania, and the Department of Rheumatology, Veterans Administration Hospital, Philadelphia, PA. Partially Funded by the NSF-MRL Program under Grant #])MR 9120668. Received for publication May 23, 1995; accepted in revised form Sept. 2, 1995. Reprint requests: Sanjiv H. Naidu, MD, Hershey Medical Center, RO. Box 850, Hershey, PA 17033.
496
The Journal of Hand Surgery
inflammatory reaction. 4 Goodman et al. noted a significant difference in the interface membrane between bone and cement when two forms of polymethylmethacrylate (PMMA) (bulk vs particles) were compared; the particulate PMMA induced a much thicker and larger membrane. In addition, the number of histiocytes was markedly increased in the case in which the particulate PMMA was used. 4 The exact type of cells that are involved and are thought to be most intimately involved in the inflammatory response are not well elucidated. Phagocytic cells, including macrophages, histiocytes, multinuclear giant cells, and osteoclasts probably all have a role in the loss of integrity at the bone-elastomer implant interface in response to elastomer particulate debris. Monocytes and macrophages have the capacity to produce cytokines such as tumor necrosis factor (TNF); cytokines stimulate bone resorption, activate osteoclasts, and attract polymorphonuclear leukocytes. 5-6 Goldring et al. 7 described the interface membrane surrounding aseptically loosened cemented total hip components as synovial-like and capable of producing prostaglandin E2 (PGE2) and collagenase. Many other pathologic studies have reported similar histologic findings in the membrane between bone
The Journal of Hand Surgery/Vol. 21A No. 3 May 1996 497
and acrylic cement with implants and also have suggested its involvement in osteolysis and joint failure.8-10 We used the rat subcutaneous air pouch model to study the inflammatory response to silicone elastomer particles because it contains cell populations analogous to the membrane surrounding failed prostheses. Edwards et al. u described the formation of a rat air pouch that resembled synovium histologically and could be used to study the inflammatory response to various particulates. Previous studies in this laboratory have demonstrated that the rat air pouch is an excellent system with which to study the role of particulate wear debris in the production of mediators of osteolysis. 12,13 In this article, we report the inflammatory response to three different types of particles, namely Silastic silicone elastomer (Dow Corning, Midland, MI), PMMA (bone cement), and monosodium urate particles (MSU).
Materials and Methods Particle Preparation
Silastic silicone finger joint (Dow Corning, Wright, TN) was intially cut to 3-mm pieces using a razor blade. These elastomer pieces were then put in a high-energy freezer mill with liquid nitrogen as the freezing agent. The silicone elastomer was cooled below its glass transition temperature o f - l l 0 ~ The elastomer was then milled for 45 minutes to 1 hour. The milled elastomer was then suspended and immersed in distilled water for approximately 4 weeks. It was then further mechanically ground using a tissue grinder. The mortar and piston formed a very tight seal; therefore, small silicone elastomer particles were generated by pressurizing the water in the grinding chamber. Polymethylmethacrylate particles were produced by polymerizing Simplex-P cement (Howmedica, Rutherford, NJ; 1 packet powder, 1 vial monomer) and mechanically grinding the cured cement. Particles were produced by grinding cement on cement. Monosodium urate crystals were made by the method of McCarty and Faires. 14 Briefly, 0.45 g NaOH pellets were dissolved in 400 mL distilled water in a glass beaker. The solution was heated until the pellets dissolved; 1.68 g uric acid was then added and dissolved. The solution was allowed to sit overnight in a cool place, and pH was maintained between 7.0 and 7.2. The following morning, the supernatant was decanted, and the MSU crystals were washed in cold saline and dried.
To ensure a clear definition of size ranges, a series of nylon mesh (Spectra mesh; Fisher Scientific, Pittsburgh, PA) of varying sizes, fit to modified plastic Buchner funnels, was used to filter the mechanical wear debris to the appropriate size 15 of under 10 gm. While we were able to generate adequate silicone and MSU particles smaller than 1.2 gm, we were not able to grind PMMA smaller than 10 gm. Particles were filtered using a 0.22-gm filter and then resuspended in sterile saline solution (0.9% NaC1). The silicone suspension was then analyzed by both scanning electron microscopy (JEOL 6006, Tokyo, Japan) and transmission electron microscopy (JEOL 400T, Tokyo, Japan) for characterizing size and morphology. Polymethylmethacrylate and MSU were analyzed by scanning electron microscopy only. The absence of contamination by metal particles from the milling process was confirmed by energy dispersive spectroscopy (Kevex International, Foster City, CA). Particles in saline were counted on a hemocytometer, and an initial pilot dose response mn showed that silicone in the concentration of 106 particles/mL caused a measurable white blood cell (WBC) count increase in the pouch exudate. To elicit a measurable inflammatory response, 107 particles/mL of PMMA and 107 particles/mL of MSU were needed. After preparing samples with the above concentration of particles, the particles were sterilized with gamma irradiation (2.5 mrad). Five milliliters of each of the various suspensions of PMMA, silicone, and MSU were drawn into 10-mL syringes using large-bore (16-gauge) needles in preparation for injection into the rat subcutaneous air pouch. Rat Subcutaneous Air Pouch Preparation
Rat pouches were prepared according to the method of Edwards et al. u Male Sprague-Dawley barrier rats (175-200 g) were anesthetized by ketamine-xylazine injection. The rats were shaved, and alcohol was applied over the dorsal skin. A subcutaneous pouch was created by injecting 20 mL air, using a 25-gauge needle attached to a 0.22-gm filter and a 20-mL syringe. After 4 days, the pouches were reinjected with 10 mL air to keep them inflated. On day 6 (2 days after reinflation), 5 mL particulate suspension was injected. At various times following the injection (6, 24, 48, or 72 hours), the pouch was irrigated with 5 mL sterile saline, and fluid was withdrawn for analysis. A WBC count was performed on fresh aspirates. The remaining exudate was cen-
498 Naidu et al. / Inflammatory Responseto Silicone Elastomer Debris
trifuged, and the resultant supernatant was stored at -70~ for later assays of TNF and PGE2. The rats were placed in a standard CO2 chamber after aspiration of the pouch. A longitudinal incision was made over the dorsum; sharp dissection was performed with iris scissors, and the entire subcutaneous pouch was harvested. The pouch tissue was preserved in formalin for histology (light microscopy). Assays White blood cells were counted with use of a standard counting chamber. The levels of PGE2 were determined by use of a PGE2-specific monoclonal antibody with an enzyme-linked immunosorbent assay and a commercially available kit (Amersham, Cambridge, MA). Tumor necrosis factor activity was measured by the cytotoxic cell assay method of Mosmann et al. 16 Briefly, WEHI fibrosarcoma cells were plated at a concentration of 700,000 cells per well in a 96-microtiter well plate in RPMI (Gibco, Grand Island, NY) cell culture medium containing 1:400 concentration of actinomycin D. Following addition of standard or samples, the cells were incubated for 24 hours at 37~ and 5% CO2. Following this incubation, the live cells were stained and the amount of TNF was then determined using an enzyme-linked immunoabsorbent assay microplate reader. The TNF measured was inversely proportional to the absorbance at 590 nm. The actual TNF quantity was determined from a standard curve. Particles were demonstrated to be free of endotoxin by using an E-Toxate assay kit (Sigma Chemical, St. Louis, MO) after sterilization with gamma irradiation. Experiments
After preparation of the pouches in 60 rats, the animals were randomized to one of three subgroups. Twenty rats were injected with 5 mL Silastic silicone elastomer particles (106 particles/mL), 20 with 5 mL MSU particles (107/mL), and 20 with 5 mL PMMA particles (107/mE). Exudate was retrieved as described previously from five animals in each group at the 6-, 24-, 48-, and 72-hour time points. After the WBC count, the exudate was analyzed for TNF and PGE2 as described previously.
Results Evaluation of Silicone Particles
Silicone particles < 1.2 gm were obtained. Transmission electron microscopy (Fig. 1) showed that the
Figure 1. Transmission electron micrograph of a 75-nmthick section of Silastic silicone elastomer (original magnification, x17,600). Note the dark electron-dense small particles dispersed in the lighter amorphous polymer matrix; these are fumed silica particles used to reinforce polydimethylsiloxane polymer matrix to improve the elastomer tensile properties. Silastic silicone particle is made of two components: the lighter region indicates the cross-linked polydimethylsiloxane polymer; the electron-dense particles dispersed in the polymer matrix represent the fumed silica filler used to augment the mechanical properties of the etastomer. Analysis with energydispersive spectroscopy revealed a single peak consistent with the presence of silicone. Scanning electron microscopy was then used to confirm the sizes of PMMA and MSU particles. Polymethylmethacrylate particles were smaller than 10 gin, and MSU particles were smaller than 1.2 gm. Light microscopy of pouch lining stained by hematoxylin and eosin revealed pseudosynoviaMike lining consistent with the description of Edwards et al. al White Blood Cell Count White blood cell count was the highest in the silicone group at 6 hours (p < .0001) and 24 hours (p < .0001) after injection. There were no significant differences at other time points (Fig. 2A). Tumor Necrosis Factor Tumor necrosis factor levels were the highest in the silicone group at 6 hours (p = .021) and 24 hours (p = .006) after injection. There were no significant differences at other time points (Fig. 2B). Prostaglandin E2 Prostaglandin E2 was also the highest in the silicone group at 24 hours (p = .039) after injection. There were no significant differences at other time points (Fig. 2C).
Discussion Polymethylmethacrylate particles were used as negative controls in this study. Previous studies in
The Journal of Hand Surgery/Vol. 21A No. 3 May 1996
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Figure 2. Temporal profile of inflammatory mediators. (A) The white blood cell counts are significantly higher in the silicone group at 6 hours and 24 hours (p < .0001) after injection. (B) Tumor necrosis is significantly higher in the silicone group at 6 hours (p < .021) and 24 hours (p < .006) after injection. Also, note that the graph is plotted on a logrithmic scale. (C) Prostaglandin E2 is significantly higher in the Silastic silicone group at 24 hours (p < .039) after injection.
the rat model have shown that P M M A is minimally inflammatory. 12 Several conclusions can be made with regard to acute inflammatory response to particulate matter. Not all microparticulate debris elicits a similar degree of inflammation; some, such as silicone, elicit more exuberant acute inflammatory response than others. In our study, particle sizes were equivalent, in that all the particles tested were
499
smaller than 10 ~tm. Silastic silicone particles injected were at one tenth the concentration of either P M M A or MSU; nevertheless, Silastic silicone displayed higher inflammatory mediators than the latter two. Matlaga et al. 17 found that triangular polymer implants were associated with higher acid phosphatase enzyme activity than were round or pentagon-shaped rods. We did not control for particle shapes in our study. Not only particle shape but particulate microstructure may play an important role in the above rat model. Of the tbaee types of particles surveyed above, MSU was distinctly different from the other two particles in that it is purely crystalline. Both P M M A and Sitastic silicone elastomer have a significant amorphous component in their respective polymeric structures. It may be that semicrystalline and amorphous polymers, be they homopolymers or filled elastomer networks, are more inflammatory. Ultrahigh molecular weight polyethylene is a semicrystalline homopolymer. Literature indicates that ultrahigh molecular weight polyethylene particulate wear debris is the main cause for osteolysis in uncemented total joint arthroplasty9; inflammation and correlation with microstructure of particulate debris warrant further investigation at this point. From Figure 1, it is clear that there are actually two components to the Silastic silicone elastomer. The silica particles are too small to be seen by light microscopy. It may be that during the mechanical grinding process, a significant amount of silica particles is dissociated from the elastomer matrix, creating a more concentrated solution of particles than what was actually measured with light microscopy. It may simply be a situation of particle overload leading to exuberant inflammation. Conversely, it may be that the silica filler is actually more inflammatory. By creating small Silastic silicone particles, more silica surface is exposed to the in vivo environment. This increase in surface area of silica in contact with the in vivo environment may also enhance inflammation. However, it is still not clear which component of silicone is inflammatory; it may be that the cross-linked polymer matrix and the silica filler are truly different in inflammatory potential. The lack of significant inflammation in the MSU group merits further discussion. The MSU particles were synthetically prepared by the method of McCarty and Faires. 14 It may be that pure MSU crystals elicit less inflammation than those formed in vivo in gout; MSU crystals formed in vivo may con-
500 Naidu et al. / Inflammatory Response to Silicone Elastomer Debris
tain proinflammatory proteins that may accentuate the inflammatory response. The overall time course of the inflammatory response suggests that there is an initial release of TNF by the pseudosynovial lining cells. The WBC influx into the pouch is concomitant with the increase in TNE Further rise in TNF at 24 hours is probably because of additional TNF release by the WBCs. There is a lag in PGE2 response. Peak PGE2 levels occur at 24 hours; this appears to be temporally related to the influx of WBCs into the pouch tissue. The importance of meticulous particle preparation and analysis of the particles used for studies of biocompatability should not be underestimated. To ensure that the material is tested in an adequate manner, the following criteria must be met: (1) the particles must be free of contamination by bacterial endotoxins; (2) the particle sizes should be equivalent; (3) the particles must be pure and free of other microparticulate contamination. Silicone synovitis is a clinical entity associated with Silastic silicone implant fracture, proliferative synovitis, and diffuse osteolysis. There is a body of literature devoted to clinical and pathologic findings associated with silicone synovitis. This study quantitates acute inflammatory mediator response to silicone elastomer particulate debris. The rat air pouch model is valuable because the inflammatory response to various particulates can be quantitated. It also provides for a convenient materials screening model.
References 1. Worsing RA, Engber WD, Lange TA. Reactive synovitis from particulate Silastic. J Bone Joint Surg 1982;64A: 581-585. 2. Verhaar J, Vermeulen A, Bulstra S, Walenkamp G. Bone reaction to silicone metatarsophalangeal joint hemiprostheses. Clin Orthop 1989;245:228-232. 3. Smith RJ, Atkinson RE, Jupiter JB. Silicone synovitis of the wrist. J Hand Surg 1985;10A:47-60. 4. Goodman SB, Fornasier VL, Kei J. The effects of bulk versus particulate PMMA on bone. Clin Orthop 1988;232: 255-262.
5. Howie DW, Vernon-Roberts B. The synovial response to intraarticular cobalt-chrome wear particles. Clin Orthop 1988;232:244-254. 6. Le J, Vilcek J. Tumor necrosis factor and interleukin-l: cytokines with multiple overlapping biological activities. Lab Invest 1987;56:234-248. 7. Goldring SR, Sciller AL, Roelke M, Rourke CM, O'Neill DA, Harris WH. The synovial like membrane at the bone cement interface in loose total hip replacements and its proposed role in bone lysis. J Bone Joint Surg 1983;65: 575-584. 8. Bullough PG, Dicarlo EF, Hansraj KK, Neves MC. Pathologic studies of total joint replacement. Orthop Clin North Am 1988;19:611-625. 9. Campbell P, Nasser S, Millett D, Amstutz HC. A study of the effects of polyethylene wear debris in cemented and uncemented implants. Trans Orthop Res Soc 1990;15: 441. 10. Eftekhar NS, Nercessian O. Incidence and mechanism of failure of cemented acetabular component in total hip arthroplasty. Orthop Clin North Am 1988;19:557566. 11. Edwards JC, Sedgwick AD, Willoughby DA. The formation of a structure with the features of synovial lining by subcutaneous injection of air: an in vivo tissue culture system. J Pathol 1981;134:147-156. 12. Lazarus M, Cuckler JM, Ducheyne P, Baker D, Schumacher HR. Biocompatability of PMMA with and without barium sulfate in the rat subcutaneous air pouch model. In: St. John KR, ed. Particulate debris from medical implants: mechanisms of formation and biological consequences. Philadelphia: ASTM Special Technical Publication 1144, 1992: 127-134. 13. Nagase M, Baker DG, Schumacher HR. Prolonged inflammatory reactions induced by artificial ceramics in rat air pouch model. J Rheumatol 1988;15:1334-1338. 14. McCarty DJ, Faires JS. Acute arthritis in man and dog after intrasynovial injection of sodium urate crystals. Lancet 1962;2:682-684. 15. Horowitz SM, Doty SB, Lane JM, Burstein AH. Studies of the mechanism by which the mechanical failure of PMMA leads to bone resorption. J Bone Joint Surg 1993;75A: 802-813. 16. Mosmann et al. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. J Immunol Methods 1993;65:5563. 17. Matlaga BF, Yasenchak LR Salthouse TN. Tissue response to implanted polymers: the significance of sample shape. J Biomed Mater Res 1976;10:391-397.
to Silicone Elastomer Particulate Debris Sanjiv H. Naidu, MD, Hershey, PA, Pedro Beredjiklian, MD, Louis Adler, MD, F. William Bora, Jr, MD, Daniel G. Baker, MD, Philadelphia, PA Silicone elastomer particles (Silastic silicone elastomer, Dow Coming, Midland, MI), polymethylmethacrylate particles, and monosodium urate particles smaller than 10 gm were injected into a rat subcutaneous air pouch lined with synovial membranelike cells. Inflammatory exudate from the air pouch was retrieved at 6 hours, 24 hours, 48 hours, and 72 hours after injection. White blood cell count, tumor necrosis factor, and prostaglandin E2 were measured in the exudate. White blood cell and tumor necrosis factor levels in the exudate were the highest for the silicone group at 6 and 24 hours. Prostaglandin E2 was also significantly higher in the silicone group at 24 hours. We conclude that acute inflammation is particle-type specific and that Silicone elastomer particles are acutely inflammatory. (l Hand Surg 1996;21A:496-500.)
Silicone synovids is a well-known entity. Breakdown of silicone elastomer particles evokes a severe inflammatory response. 1-3 Along with proliferative synovitis, peri-implant bone resorption occurs; the resulting osteolysis leads to severe bone loss and loss of joint function. Several clinical, radiologic, and histologic studies have documented silicone synovitisJ 3 but no study to date has yet quantified the inflammatory response to silicone particles. Observation of implanted materials reveals that the size of particulate material plays an important role in the type of tissue-cellular response. Bulk implantation of metallic or plastic objects into bone or muscle results in a relatively fibrous membrane. In sharp contrast, implantation of the same materials in the form of a powder results in a marked cellular From the Department of Orthopaedic Surgery, Pennsylvania State University, Hershey, PA, the Department of Materials Science and Engineering, University of Pennsylvania, and the Department of Rheumatology, Veterans Administration Hospital, Philadelphia, PA. Partially Funded by the NSF-MRL Program under Grant #])MR 9120668. Received for publication May 23, 1995; accepted in revised form Sept. 2, 1995. Reprint requests: Sanjiv H. Naidu, MD, Hershey Medical Center, RO. Box 850, Hershey, PA 17033.
496
The Journal of Hand Surgery
inflammatory reaction. 4 Goodman et al. noted a significant difference in the interface membrane between bone and cement when two forms of polymethylmethacrylate (PMMA) (bulk vs particles) were compared; the particulate PMMA induced a much thicker and larger membrane. In addition, the number of histiocytes was markedly increased in the case in which the particulate PMMA was used. 4 The exact type of cells that are involved and are thought to be most intimately involved in the inflammatory response are not well elucidated. Phagocytic cells, including macrophages, histiocytes, multinuclear giant cells, and osteoclasts probably all have a role in the loss of integrity at the bone-elastomer implant interface in response to elastomer particulate debris. Monocytes and macrophages have the capacity to produce cytokines such as tumor necrosis factor (TNF); cytokines stimulate bone resorption, activate osteoclasts, and attract polymorphonuclear leukocytes. 5-6 Goldring et al. 7 described the interface membrane surrounding aseptically loosened cemented total hip components as synovial-like and capable of producing prostaglandin E2 (PGE2) and collagenase. Many other pathologic studies have reported similar histologic findings in the membrane between bone
The Journal of Hand Surgery/Vol. 21A No. 3 May 1996 497
and acrylic cement with implants and also have suggested its involvement in osteolysis and joint failure.8-10 We used the rat subcutaneous air pouch model to study the inflammatory response to silicone elastomer particles because it contains cell populations analogous to the membrane surrounding failed prostheses. Edwards et al. u described the formation of a rat air pouch that resembled synovium histologically and could be used to study the inflammatory response to various particulates. Previous studies in this laboratory have demonstrated that the rat air pouch is an excellent system with which to study the role of particulate wear debris in the production of mediators of osteolysis. 12,13 In this article, we report the inflammatory response to three different types of particles, namely Silastic silicone elastomer (Dow Corning, Midland, MI), PMMA (bone cement), and monosodium urate particles (MSU).
Materials and Methods Particle Preparation
Silastic silicone finger joint (Dow Corning, Wright, TN) was intially cut to 3-mm pieces using a razor blade. These elastomer pieces were then put in a high-energy freezer mill with liquid nitrogen as the freezing agent. The silicone elastomer was cooled below its glass transition temperature o f - l l 0 ~ The elastomer was then milled for 45 minutes to 1 hour. The milled elastomer was then suspended and immersed in distilled water for approximately 4 weeks. It was then further mechanically ground using a tissue grinder. The mortar and piston formed a very tight seal; therefore, small silicone elastomer particles were generated by pressurizing the water in the grinding chamber. Polymethylmethacrylate particles were produced by polymerizing Simplex-P cement (Howmedica, Rutherford, NJ; 1 packet powder, 1 vial monomer) and mechanically grinding the cured cement. Particles were produced by grinding cement on cement. Monosodium urate crystals were made by the method of McCarty and Faires. 14 Briefly, 0.45 g NaOH pellets were dissolved in 400 mL distilled water in a glass beaker. The solution was heated until the pellets dissolved; 1.68 g uric acid was then added and dissolved. The solution was allowed to sit overnight in a cool place, and pH was maintained between 7.0 and 7.2. The following morning, the supernatant was decanted, and the MSU crystals were washed in cold saline and dried.
To ensure a clear definition of size ranges, a series of nylon mesh (Spectra mesh; Fisher Scientific, Pittsburgh, PA) of varying sizes, fit to modified plastic Buchner funnels, was used to filter the mechanical wear debris to the appropriate size 15 of under 10 gm. While we were able to generate adequate silicone and MSU particles smaller than 1.2 gm, we were not able to grind PMMA smaller than 10 gm. Particles were filtered using a 0.22-gm filter and then resuspended in sterile saline solution (0.9% NaC1). The silicone suspension was then analyzed by both scanning electron microscopy (JEOL 6006, Tokyo, Japan) and transmission electron microscopy (JEOL 400T, Tokyo, Japan) for characterizing size and morphology. Polymethylmethacrylate and MSU were analyzed by scanning electron microscopy only. The absence of contamination by metal particles from the milling process was confirmed by energy dispersive spectroscopy (Kevex International, Foster City, CA). Particles in saline were counted on a hemocytometer, and an initial pilot dose response mn showed that silicone in the concentration of 106 particles/mL caused a measurable white blood cell (WBC) count increase in the pouch exudate. To elicit a measurable inflammatory response, 107 particles/mL of PMMA and 107 particles/mL of MSU were needed. After preparing samples with the above concentration of particles, the particles were sterilized with gamma irradiation (2.5 mrad). Five milliliters of each of the various suspensions of PMMA, silicone, and MSU were drawn into 10-mL syringes using large-bore (16-gauge) needles in preparation for injection into the rat subcutaneous air pouch. Rat Subcutaneous Air Pouch Preparation
Rat pouches were prepared according to the method of Edwards et al. u Male Sprague-Dawley barrier rats (175-200 g) were anesthetized by ketamine-xylazine injection. The rats were shaved, and alcohol was applied over the dorsal skin. A subcutaneous pouch was created by injecting 20 mL air, using a 25-gauge needle attached to a 0.22-gm filter and a 20-mL syringe. After 4 days, the pouches were reinjected with 10 mL air to keep them inflated. On day 6 (2 days after reinflation), 5 mL particulate suspension was injected. At various times following the injection (6, 24, 48, or 72 hours), the pouch was irrigated with 5 mL sterile saline, and fluid was withdrawn for analysis. A WBC count was performed on fresh aspirates. The remaining exudate was cen-
498 Naidu et al. / Inflammatory Responseto Silicone Elastomer Debris
trifuged, and the resultant supernatant was stored at -70~ for later assays of TNF and PGE2. The rats were placed in a standard CO2 chamber after aspiration of the pouch. A longitudinal incision was made over the dorsum; sharp dissection was performed with iris scissors, and the entire subcutaneous pouch was harvested. The pouch tissue was preserved in formalin for histology (light microscopy). Assays White blood cells were counted with use of a standard counting chamber. The levels of PGE2 were determined by use of a PGE2-specific monoclonal antibody with an enzyme-linked immunosorbent assay and a commercially available kit (Amersham, Cambridge, MA). Tumor necrosis factor activity was measured by the cytotoxic cell assay method of Mosmann et al. 16 Briefly, WEHI fibrosarcoma cells were plated at a concentration of 700,000 cells per well in a 96-microtiter well plate in RPMI (Gibco, Grand Island, NY) cell culture medium containing 1:400 concentration of actinomycin D. Following addition of standard or samples, the cells were incubated for 24 hours at 37~ and 5% CO2. Following this incubation, the live cells were stained and the amount of TNF was then determined using an enzyme-linked immunoabsorbent assay microplate reader. The TNF measured was inversely proportional to the absorbance at 590 nm. The actual TNF quantity was determined from a standard curve. Particles were demonstrated to be free of endotoxin by using an E-Toxate assay kit (Sigma Chemical, St. Louis, MO) after sterilization with gamma irradiation. Experiments
After preparation of the pouches in 60 rats, the animals were randomized to one of three subgroups. Twenty rats were injected with 5 mL Silastic silicone elastomer particles (106 particles/mL), 20 with 5 mL MSU particles (107/mL), and 20 with 5 mL PMMA particles (107/mE). Exudate was retrieved as described previously from five animals in each group at the 6-, 24-, 48-, and 72-hour time points. After the WBC count, the exudate was analyzed for TNF and PGE2 as described previously.
Results Evaluation of Silicone Particles
Silicone particles < 1.2 gm were obtained. Transmission electron microscopy (Fig. 1) showed that the
Figure 1. Transmission electron micrograph of a 75-nmthick section of Silastic silicone elastomer (original magnification, x17,600). Note the dark electron-dense small particles dispersed in the lighter amorphous polymer matrix; these are fumed silica particles used to reinforce polydimethylsiloxane polymer matrix to improve the elastomer tensile properties. Silastic silicone particle is made of two components: the lighter region indicates the cross-linked polydimethylsiloxane polymer; the electron-dense particles dispersed in the polymer matrix represent the fumed silica filler used to augment the mechanical properties of the etastomer. Analysis with energydispersive spectroscopy revealed a single peak consistent with the presence of silicone. Scanning electron microscopy was then used to confirm the sizes of PMMA and MSU particles. Polymethylmethacrylate particles were smaller than 10 gin, and MSU particles were smaller than 1.2 gm. Light microscopy of pouch lining stained by hematoxylin and eosin revealed pseudosynoviaMike lining consistent with the description of Edwards et al. al White Blood Cell Count White blood cell count was the highest in the silicone group at 6 hours (p < .0001) and 24 hours (p < .0001) after injection. There were no significant differences at other time points (Fig. 2A). Tumor Necrosis Factor Tumor necrosis factor levels were the highest in the silicone group at 6 hours (p = .021) and 24 hours (p = .006) after injection. There were no significant differences at other time points (Fig. 2B). Prostaglandin E2 Prostaglandin E2 was also the highest in the silicone group at 24 hours (p = .039) after injection. There were no significant differences at other time points (Fig. 2C).
Discussion Polymethylmethacrylate particles were used as negative controls in this study. Previous studies in
The Journal of Hand Surgery/Vol. 21A No. 3 May 1996
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Figure 2. Temporal profile of inflammatory mediators. (A) The white blood cell counts are significantly higher in the silicone group at 6 hours and 24 hours (p < .0001) after injection. (B) Tumor necrosis is significantly higher in the silicone group at 6 hours (p < .021) and 24 hours (p < .006) after injection. Also, note that the graph is plotted on a logrithmic scale. (C) Prostaglandin E2 is significantly higher in the Silastic silicone group at 24 hours (p < .039) after injection.
the rat model have shown that P M M A is minimally inflammatory. 12 Several conclusions can be made with regard to acute inflammatory response to particulate matter. Not all microparticulate debris elicits a similar degree of inflammation; some, such as silicone, elicit more exuberant acute inflammatory response than others. In our study, particle sizes were equivalent, in that all the particles tested were
499
smaller than 10 ~tm. Silastic silicone particles injected were at one tenth the concentration of either P M M A or MSU; nevertheless, Silastic silicone displayed higher inflammatory mediators than the latter two. Matlaga et al. 17 found that triangular polymer implants were associated with higher acid phosphatase enzyme activity than were round or pentagon-shaped rods. We did not control for particle shapes in our study. Not only particle shape but particulate microstructure may play an important role in the above rat model. Of the tbaee types of particles surveyed above, MSU was distinctly different from the other two particles in that it is purely crystalline. Both P M M A and Sitastic silicone elastomer have a significant amorphous component in their respective polymeric structures. It may be that semicrystalline and amorphous polymers, be they homopolymers or filled elastomer networks, are more inflammatory. Ultrahigh molecular weight polyethylene is a semicrystalline homopolymer. Literature indicates that ultrahigh molecular weight polyethylene particulate wear debris is the main cause for osteolysis in uncemented total joint arthroplasty9; inflammation and correlation with microstructure of particulate debris warrant further investigation at this point. From Figure 1, it is clear that there are actually two components to the Silastic silicone elastomer. The silica particles are too small to be seen by light microscopy. It may be that during the mechanical grinding process, a significant amount of silica particles is dissociated from the elastomer matrix, creating a more concentrated solution of particles than what was actually measured with light microscopy. It may simply be a situation of particle overload leading to exuberant inflammation. Conversely, it may be that the silica filler is actually more inflammatory. By creating small Silastic silicone particles, more silica surface is exposed to the in vivo environment. This increase in surface area of silica in contact with the in vivo environment may also enhance inflammation. However, it is still not clear which component of silicone is inflammatory; it may be that the cross-linked polymer matrix and the silica filler are truly different in inflammatory potential. The lack of significant inflammation in the MSU group merits further discussion. The MSU particles were synthetically prepared by the method of McCarty and Faires. 14 It may be that pure MSU crystals elicit less inflammation than those formed in vivo in gout; MSU crystals formed in vivo may con-
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tain proinflammatory proteins that may accentuate the inflammatory response. The overall time course of the inflammatory response suggests that there is an initial release of TNF by the pseudosynovial lining cells. The WBC influx into the pouch is concomitant with the increase in TNE Further rise in TNF at 24 hours is probably because of additional TNF release by the WBCs. There is a lag in PGE2 response. Peak PGE2 levels occur at 24 hours; this appears to be temporally related to the influx of WBCs into the pouch tissue. The importance of meticulous particle preparation and analysis of the particles used for studies of biocompatability should not be underestimated. To ensure that the material is tested in an adequate manner, the following criteria must be met: (1) the particles must be free of contamination by bacterial endotoxins; (2) the particle sizes should be equivalent; (3) the particles must be pure and free of other microparticulate contamination. Silicone synovitis is a clinical entity associated with Silastic silicone implant fracture, proliferative synovitis, and diffuse osteolysis. There is a body of literature devoted to clinical and pathologic findings associated with silicone synovitis. This study quantitates acute inflammatory mediator response to silicone elastomer particulate debris. The rat air pouch model is valuable because the inflammatory response to various particulates can be quantitated. It also provides for a convenient materials screening model.
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5. Howie DW, Vernon-Roberts B. The synovial response to intraarticular cobalt-chrome wear particles. Clin Orthop 1988;232:244-254. 6. Le J, Vilcek J. Tumor necrosis factor and interleukin-l: cytokines with multiple overlapping biological activities. Lab Invest 1987;56:234-248. 7. Goldring SR, Sciller AL, Roelke M, Rourke CM, O'Neill DA, Harris WH. The synovial like membrane at the bone cement interface in loose total hip replacements and its proposed role in bone lysis. J Bone Joint Surg 1983;65: 575-584. 8. Bullough PG, Dicarlo EF, Hansraj KK, Neves MC. Pathologic studies of total joint replacement. Orthop Clin North Am 1988;19:611-625. 9. Campbell P, Nasser S, Millett D, Amstutz HC. A study of the effects of polyethylene wear debris in cemented and uncemented implants. Trans Orthop Res Soc 1990;15: 441. 10. Eftekhar NS, Nercessian O. Incidence and mechanism of failure of cemented acetabular component in total hip arthroplasty. Orthop Clin North Am 1988;19:557566. 11. Edwards JC, Sedgwick AD, Willoughby DA. The formation of a structure with the features of synovial lining by subcutaneous injection of air: an in vivo tissue culture system. J Pathol 1981;134:147-156. 12. Lazarus M, Cuckler JM, Ducheyne P, Baker D, Schumacher HR. Biocompatability of PMMA with and without barium sulfate in the rat subcutaneous air pouch model. In: St. John KR, ed. Particulate debris from medical implants: mechanisms of formation and biological consequences. Philadelphia: ASTM Special Technical Publication 1144, 1992: 127-134. 13. Nagase M, Baker DG, Schumacher HR. Prolonged inflammatory reactions induced by artificial ceramics in rat air pouch model. J Rheumatol 1988;15:1334-1338. 14. McCarty DJ, Faires JS. Acute arthritis in man and dog after intrasynovial injection of sodium urate crystals. Lancet 1962;2:682-684. 15. Horowitz SM, Doty SB, Lane JM, Burstein AH. Studies of the mechanism by which the mechanical failure of PMMA leads to bone resorption. J Bone Joint Surg 1993;75A: 802-813. 16. Mosmann et al. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. J Immunol Methods 1993;65:5563. 17. Matlaga BF, Yasenchak LR Salthouse TN. Tissue response to implanted polymers: the significance of sample shape. J Biomed Mater Res 1976;10:391-397.