Hepatoprotective effects of S-adenosylmethionine and silybin on canine hepatocytes in vitro.

Inflammation and oxidative stress are associated with liver injury and development of liver disease. The transcription factors nuclear factor-kappa beta (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2) play critical roles in modulating liver injury and damage. Activation of NF-κB induces production of pro-inflammatory molecules including prostaglandin E2 (PGE2 ), interleukin-8 (IL-8) and macrophage chemotactic protein-1 (MCP-1). Nrf2 regulates genes controlling antioxidants. Our laboratory previously showed that hepatocytes, the primary functional cell type comprising liver tissue, respond to the cytokine interleukin-1 beta (IL-1ß) by increased production of PGE2 , IL-8 and MCP-1. This increase is associated with nuclear translocation of NF-κB. In this study, we evaluated whether primary canine hepatocytes pre-treated with the combination of S-adenosylmethionine (SAMe; 30 and 2000 ng/ml) and silybin (SB; 298 ng/ml), agents with known anti-inflammatory and antioxidant properties, could attenuate IL-1ß-induced inflammation and oxidative stress. The SAMe and SB combination reduced cytokine-induced PGE2 , IL-8 and MCP-1 production while also inhibiting NF-κB nuclear translocation. These changes were accompanied by increased antioxidant enzyme-reduced glutathione (GSH) comparable to control levels. The study shows for the first time that the SAMe and SB combination inhibits both inflammation and oxidative stress through two separate signalling pathways.

Silybin inhibits interleukin-1ß-induced production of pro-inflammatory mediators in canine hepatocyte cultures.

Hepatocytes are highly susceptible to cytokine stimulation and are fundamental to liver function. We established primary canine hepatocyte cultures to study effects of anti-inflammatory agents with hepatoprotective properties. Hepatocyte cultures were incubated with control media alone, silybin (SB), or the more bioavailable silybin-phosphatidylcholine complex (SPC), followed by activation with interleukin-1 beta (IL-1ß; 10 ng/mL). Inflammatory response was measured by prostaglandin E2 (PGE(2) ), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) production and also nuclear factor-kappa B (NF-κB) translocation. Hepatocyte cultures continued production of the phenotypic marker albumin for more than 7 days in culture. IL-1ß exposure increased PGE(2) , IL-8, and MCP-1 production, which was paralleled by NF-κB translocation from the cytoplasm to the nucleus. Pretreatment with SB and SPC significantly inhibited IL-1ß-induced production of pro-inflammatory markers and attenuated NF-κB nuclear translocation. We demonstrate for the first time that primary canine hepatocyte cultures can be maintained in culture without phenotypic loss. The observation that hepatocyte cultures respond to pro-inflammatory IL-1ß activation indicates hepatocytes as primary cellular targets of extrinsic IL-1ß. The ability of SB and SPC to inhibit hepatocyte culture activation by IL-1ß reinforces the notion of their hepatoprotective effects. Our primary canine hepatocyte culture model facilitates identification of hepatoprotective agents and their mechanism of action.

Inhibition of cyclooxygenase-2 expression and prostaglandin E2 production in chondrocytes by avocado soybean unsaponifiables and epigallocatechin gallate.

OBJECTIVE: To evaluate the anti-inflammatory effect of the combination of avocado soybean unsaponifiables (ASU) and epigallocatechin gallate (EGCG) on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production in cytokine-activated equine chondrocytes. METHODS: Production of type II collagen and aggrecan was verified by immunohistochemistry and Western blot. Chondrocytes were incubated with: (1) control media alone, (2) ASU (4 microg/ml; 8.3 microg/ml), (3) EGCG (4, 40, 400 ng/ml), or (4) the combination of ASU and EGCG for 24h. Cells were next incubated with control medium alone or with IL-1beta (10 ng/ml) and TNF-alpha (1 ng/ml). COX-2 gene expression by real-time PCR analysis and NF-kappaB nuclear translocation by immunohistochemistry were performed after 1h of incubation. PGE(2) production was determined by immunoassay after 24h of incubation. RESULTS: Equine chondrocytes responded to cytokine activation by up-regulated gene expression of COX-2 and increased PGE(2) production. Activation was associated with NF-kappaB translocation. Individually, ASU and EGCG marginally inhibited COX-2 expression and PGE(2) production in activated chondrocytes. In contrast, the combination of ASU and EGCG reduced COX-2 expression close to non-activated control levels and significantly inhibited PGE(2) production. These reductions were statistically greater than those of ASU or EGCG alone. The inhibition of COX-2 expression and PGE(2) production was associated with inhibition of NF-kappaB translocation. CONCLUSION: The present study demonstrates that the anti-inflammatory activity of ASU and EGCG is potentiated when used in combination. This combination may offer an attractive supplement or alternative to non-steroidal anti-inflammatory drugs (NSAIDs) in the management of osteoarthritis.

Avocado soybean unsaponifiables (ASU) suppress TNF-alpha, IL-1beta, COX-2, iNOS gene expression, and prostaglandin E2 and nitric oxide production in articular chondrocytes and monocyte/macrophages.

OBJECTIVE: To evaluate the effects of avocado soybean unsaponifiables (ASU) on proinflammatory mediators in chondrocytes and monocyte/macrophage-like cells. DESIGN: To determine the dose response of ASU, chondrocytes (5 x 10(5) cells/well) were incubated at 5% CO(2), 37 degrees C for 72 h with (1) control media alone or (2) ASU at concentrations of 0.3, 0.9, 2.7, 8.3, and 25 microg/ml. Cells were activated with 20 ng/ml lipopolysaccharide (LPS) for 24 h and cell supernatants were analyzed for prostaglandin E(2) (PGE(2)) and nitrite content. Chondrocytes and THP-1 monocyte/macrophages (5 x 10(5) cells/well) were incubated at 5% CO(2), 37 degrees C for 72 h with (1) control media alone or (2) ASU (25 mug/ml). One set of cells was activated for 1 h with LPS (20 ng/ml) for both reverse-transcriptase PCR and real-time PCR analysis of tumor necrosis factor-alpha (TNF-alpha), interleukin-1-beta (IL-1beta), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) expression. One set of cells was activated for 24 h to analyze secreted PGE(2) and nitrite levels in the cellular supernatant. RESULTS: ASU reduced TNF-alpha, IL-1beta, COX-2, and iNOS expression in LPS-activated chondrocytes to levels similar to nonactivated control levels. The suppression of COX-2 and iNOS expression was paralleled by a significant reduction in PGE(2) and nitrite, respectively, in the cellular supernatant. ASU also reduced TNF-alpha and IL-1beta expression in LPS-activated monocyte/macrophage-like cells. CONCLUSION: The present study demonstrates that the anti-inflammatory activity of ASU is not restricted to chondrocytes, but also affects monocyte/macrophage-like cells that serve as a prototype for macrophages in the synovial membrane. These observations provide a scientific rationale for the pain-reducing and anti-inflammatory effects of ASU observed in osteoarthritis patients.

Extraction and characterization of metallic wear debris from total joint arthroplasty.

Wear debris generated from total joint arthroplasty may elicit a granulomatous and inflammatory response and has also been implicated in the development of osteolysis. Technical difficulty in retrieval and isolation of wear material from tissues has hindered the study of their physicochemical properties. The purpose of this study was to retrieve and analyze metallic wear debris from periprosthetic tissue obtained during revision arthroplasty. Tissue from six osteoarthritic patients was obtained during revision arthroplasty. The tissue was minced and then heated in a sodium dodecyl sulfate solution. Undigested tissue was incubated sequentially with papain and pepsin solutions. Metallic wear debris retrieved from the digestion procedure was analyzed by scanning electron microscopy. Wear fragments were seen as irregularly shaped flakes, splinters and polyhedral structures ranging from 1 to 100 microns in size. These structures appeared to be free from non-metallic surface-adherent material. Energy dispersion spectroscopy verified the presence of cobalt, chrome and molybdenum which comprised the implant alloy. Fatigue lines were observed on the surface suggesting brittle wear. Our technique for isolating metallic fragments facilitates the retrieval and preparation of wear debris for analysis of physicochemical properties and how wear debris interacts with cellular elements in surrounding tissue.

Effect of PMMA particles and movement on an implant interface in a canine model.

The pathogenesis of aseptic loosening of total joint prostheses is not clearly understood. Two features are associated with loosened prostheses, namely, particulate debris and movement of the implant. While numerous studies have evaluated the cellular response to particulate biomaterials, few have investigated the influence of movement of the implant on the biological response to particles. Our aim was therefore to test the hypothesis that excessive mechanical stimulation of the periprosthetic tissues induces an inflammatory response and that the addition of particulate biomaterials intensifies this. We allocated 66 adult Beagle dogs to four groups as follows: stable implants with (I) and without (II) particulate polymethylmethacrylate (PMMA) and moving implants with (III) and without (IV) particulate PMMA. They were then evaluated at 2, 4, 6, 12 and 24 weeks. The stable implants were well tolerated and a thin, fibrous membrane of connective tissue was observed. There was evidence of positive staining in some cells for interleukin-6 (IL-6). Addition of particulate PMMA around the stable implants resulted in an increase in the fibroblastic response and positive staining for IL-6 and tumour necrosis factor-alpha (TNF-alpha). By contrast, movement of the implant resulted in an immediate inflammatory response characterised by large numbers of histiocytes and cytokine staining for IL-1beta, TNF-alpha and IL-6. Introduction of particulate PMMA aggravated this response. Animals with particulate PMMA and movement of the implant have an intense inflammatory response associated with accelerated bone loss. Our results indicate that the initiation of the inflammatory response to biomaterial particles was much slower than that to gross mechanical instability. Furthermore, when there was both particulate debris and movement, there was an amplification of the adverse tissue response as evidenced by the presence of osteolysis and increases in the presence of inflammatory cells and their associated cytokines.

Identification of caveolae and detection of caveolin in normal human osteoblasts.

Caveolae, specialised regions of the cell membrane which have been detected in a wide range of mammalian cells, have not been described in bone cells. They are plasmalemmal invaginations, 50 to 100 nm in size, characterised by the presence of the structural protein, caveolin, which exists as three subtypes. Caveolin-1 and caveolin-2 are expressed in a wide range of cell types whereas caveolin-3 is thought to be a muscle-specific subtype. There is little information on the precise function of caveolae, but it has been proposed that they play an important role in signal transduction. As the principal bone-producing cell, the osteoblast has been widely studied in an effort to understand the signalling pathways by which it responds to extracellular stimuli. Our aim in this study was to identify caveolae and their structural protein caveolin in normal human osteoblasts, and to determine which subtypes of caveolin were present. Confocal microscopy showed staining which was associated with the plasma membrane. Transmission electron microscopy revealed the presence of membrane invaginations of 50 to 100 nm, consistent with the appearance of caveolae. Finally, we isolated protein from these osteoblasts, and performed Western blotting using anti-caveolin primary antibodies. This revealed the presence of caveolin-1 and -2, while caveolin-3 was absent. The identification of these structures and their associated protein may provide a significant contribution to our further understanding of signal transduction pathways in osteoblasts.

Beta-1 integrin expression by human nasal chondrocytes in microcarrier spinner culture.

Beta-1 integrin plays a major role in cell attachment and is believed to be involved in mediating the interactions of chondrocytes with their environment. We previously reported that articular chondrocytes propagated in microcarrier spinner culture proliferated and reexpressed their chondrocytic protein. The goal of the present study was to investigate the expression of beta-1 integrin by chondrocytes growing on the surface of microcarriers. Nasal chondrocytes (4 x 10(3)/cm(2)) were seeded on microcarriers and incubated at 37 degrees C, 5% CO(2), 60 rpm. Expression of chondrocyte markers and beta-1 integrin was determined using reverse transcriptase-polymerase chain reaction and immunocytochemical analyses. De novo synthesis of sulfate-containing proteoglycans was studied using 35SO(4) incorporation techniques. Like articular chondrocytes propagated in microcarrier spinner culture, nasal chondrocytes expressed high levels of collagen type II mRNA, whereas collagen type I mRNA levels were low. Aggrecan mRNA was detectable and levels of de novo 35SO(4) incorporation were high. Chondrocytes immunostained intensely for collagen type II and keratan sulfate but did not stain for collagen type I. beta-1 integrin mRNA levels were high, and the protein was immunolocalized to regions of cell-to-cell or cell-to-microcarrier contact. The fact the chondrocytes expressed high levels of beta-1 integrin raises the possibility that this integrin molecule has a role in the maintenance of the chondrocytic phenotype.

Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes.

The search for biocompatible materials that can support the growth and phenotypic expression of osteoblasts and chondrocytes is a major challenge in the application of tissue engineering techniques for the repair of bone and cartilage defects. Chitosan, a copolymer of glucosamine and N-acetylglucosamine, may provide an answer to this search. Chitosan is the deacetylated product of chitin, a ubiquitous biopolymer found in the exoskeleton of insects and marine invertebrates. Little is known about the utility of chitosan in propagating human osteoblasts and chondrocytes. In this study, we test the hypothesis that chitosan promotes the survival and function of osteoblasts and chondrocytes. Chitosan (4%, w/v in 2% HAc) was coated onto plastic coverslips that had been fitted into 24-well plates. Human osteoblasts and articular chondrocytes were seeded on either uncoated or chitosan-coated coverslips at 1 x 10(5)/cells per well. Cultures were incubated at 37 degrees C, 5% CO(2) for a period of 7 days. Cell viability was assessed at that time using a fluorescent molecular probe. The phenotypic expression of osteoblasts and chondrocytes was analyzed by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Osteoblasts and chondrocytes appeared spherical and refractile on chitosan-coated coverslips. In contrast, greater than 90% of cells on plastic coverslips were elongated and spindle shaped after 7 days of culture. Similar to cells propagated on uncoated control wells, greater than 90% of human osteoblasts and chondrocytes propagated on chitosan remained viable. Human osteoblasts propagated on chitosan films continued to express collagen type I whereas chondrocytes expressed collagen type II and aggrecan, as shown by reverse transcriptase-polymerase chain reaction analysis and immunostaining. The present in vitro work demonstrates the biocompatibility of chitosan as a substrate for the growth and continued function of human osteoblasts and chondrocytes. Chitosan may have potential use as a tissue engineering tool for the repair of osseous and chondral defects.

Proinflammatory cytokine expression of IL-1beta and TNF-alpha by human osteoblast-like MG-63 cells upon exposure to silicon nitride in vitro.

This study was designed to determine the effect of Si(3)N(4) disks and particulates on human osteoblast-like MG-63 cells in vitro. The MG-63 (10(5)/mL) cells were plated onto 24-well polystyrene plates fitted with either sintered reaction-bonded (SRBSN) or reaction-bonded (RBSN) 15-mm disks. Controls consisted of wells without Si(3)N(4) disks. Cells propagated at 37 degrees C, 5% CO(2) for 48 h on Si(3)N(4) disks and control polystyrene surfaces exhibited similar proliferative capacities (7000 and 4000 cpm/10(5) cells, respectively, p > 0.05). Cells incubated with 1, 10, or 100 microgram/ml of Si(3)N(4) particles (<1.00 to 5.00 micrometer) for 24 h did not exhibit a decrease in DNA synthetic activity: 12 +/- 1.3 x 10(4), 10.5 +/- 1.5 x 10(4), and 11.0 +/- 1.7 x 10(4) cpm, respectively, compared to 11.6 +/- 2.6 x 10(4) cpm/10(5) for the control cells, as indicated by (3)H-thymidine uptake. Cells propagated on RBSN displayed increased expression of cytokines IL-1beta and TNF-alpha compared to the control cells, as shown by reverse transcriptase-polymerase chain reaction (RT-PCR). In contrast, cells propagated on SRBSN surfaces expressed the same level of IL-1beta and TNF-alpha as that of control cells. Incubation of MG-63 cells with 1-10 microgram/mL of particles did not increase IL-1beta expression. However, at 100 microgram/mL, TNF-alpha expression was greater than that of the control cells. Silicon nitride, evaluated here as disks or as particulates (1-10 microgram/mL), is biocompatible and does not hinder the proliferation or induce proinflammatory cytokine expression of human osteoblast-like MG-63 cells in vitro.

Immunohistochemical evaluation of interface membranes from failed cemented and uncemented acetabular components.

Aseptic loosening of acetabular components is a primary factor compromising the long-term outcome of cemented and cementless total hip replacement. It is unknown whether the pathogenesis of the loosening process is identical for both types of fixation. The specific aim of this study was to determine whether there is a difference in the cellular and cytokine profiles of interface membranes removed from between the implant and the host bone from failed cemented (n = 5) and failed cementless acetabulae (n = 5). Routine histology and immunohistochemical evaluations were completed on each tissue specimen. The monoclonal antibodies used included those specific for cell types (macrophages, fibroblasts, T lymphocytes) and for cytokines (IL-1beta, IL-6, TNF-alpha). The patients were all revised for loosening. The time to revision was significantly longer for the cemented group (16.6 yr; 13-21 range) than for the cementless group (8.9 yr; 4-13 range). In all cases, slides from each group stained positively for each of the cell types and cytokines evaluated. Immunohistochemical analyses indicated a predominance of macrophages and ubiquitous staining for the cytokines IL-1beta and TNF-alpha within the membranes of both patient groups. The intensities of cytokine staining were similar for both patient groups. More regions of fibroblastic connective tissue were observed surrounding failed cementless components as compared to those of the cemented group. The clinical ramification of our findings is that, despite differences in the cellular composition of the periprosthetic membranes, the membranes from failed cemented and cementless implants contain cytokines, which have been shown to be capable of modulating the inflammatory response. These inflammatory mediators are likely to play a significant role in the development of osteolysis and prosthetic loosening.

Effect of prosthetic titanium wear debris on mitogen-induced monocyte and lymphoid activation.

Wear debris generated by joint implant components has been reported to activate inflammatory and immune cells. Particulate debris derived from prosthetic material induces monocytes/macrophages, lymphocytes, synoviocytes, and fibroblasts to secrete cellular products, such as cytokines, which mediate inflammation. It has been speculated that degradation products impair the ability of inflammatory and immune cells to mount a protective response against noxious agents and infectious organisms by interfering with cell activation. Recent in vitro studies suggest that soluble metal ions inhibit T and B cell activation, but it is not known whether insoluble metal particles generated by prosthetic wear in tissue have the same effect. The purpose of the present study was to determine whether titanium wear debris retrieved from periprosthetic tissues surrounding a failed knee prosthesis suppresses activation of human monocytic and lymphoid cells. Peripheral blood monocytes and lymphocytes were incubated with the nonspecific activator pokeweed mitogen (PWM) in the presence or absence of titanium particles. Cell proliferative capacity and production of interleukins IL-1beta and IL-2 were determined as measures of activation. Titanium wear debris induced monocyte secretion of IL-1beta at levels comparable to those induced by PWM alone. In combination with PWM, titanium wear debris stimulated monocytes to secrete higher concentrations of IL-1beta than is stimulated by titanium itself or by PWM alone. Titanium wear debris did not activate lymphocytes, as indicated by marginal changes in DNA synthesis and IL-2 secretion, nor did it suppress the PWM-induced stimulation of DNA synthesis and IL-2 secretion. Our study suggests that nonspecific mitogen activators in spite of exposure to titanium wear debris can stimulate monocytic and lymphoid cells.

Enhanced proliferation and osteocalcin production by human osteoblast-like MG63 cells on silicon nitride ceramic discs.

The biocompatibility of silicon nitride (Si3N4) was assessed in an in vitro model using the human osteoblast-like MG-63 cell line. Cells were propagated on the surface of: reaction-bonded silicon nitride discs, sintered after reaction-bonded silicon nitride discs or control polystyrene surface for 48 h. Compared to cells propagated on polystyrene surface, cells grown on the surface of unpolished silicon nitride discs had significantly lower cell yield and decreased osteocalcin production. In contrast, cells on the surface of polished silicon nitride discs showed similar proliferative capacity to control cells propagated on polystyrene surface. Cells propagated on polished discs also produced higher levels of osteocalcin than cells on unpolished discs. SEM analysis showed cells with well-delineated morphology and cytoplasmic extensions when propagated on polished sintered after reaction-bonded discs. Cells appeared more spherical, when grown on polished reaction-bonded discs. The results of this study suggest that silicon nitride is a non-toxic, biocompatible ceramic surface for the propagation of functional human bone cells in vitro. Its high wear resistance and ability to support bone cell growth and metabolism make silicone nitride an attractive candidate for clinical application. Further studies are needed to explore the feasibility of using silicon nitride clinically as an orthopedic biomaterial.

Effect of particulate bioactive glass on human synoviocyte cultures.

Bioglass is a resorbable glass material that has been shown to induce osteoblast proliferation as well as bone matrix production in vitro. Its physico-chemical properties have been reported to be suitable for use as an implant coating for arthroplasty. However, Bioglass is a ceramic material that can fragment into particulate debris in vivo. The effect of particulate Bioglass on tissue cells has not been defined. In order to determine the biologic response to particulate Bioglass, we tested its effect on human synoviocytes in a cell culture model. At the concentrations of 1.0 and 10, micrograms/mL, particulate Bioglass (sizes ranging from approximately 0.5 to 80 microns) had a low cytotoxic effect. However, these concentrations induced secretion of TNF alpha. The observation that particulate Bioglass elicits release of inflammatory cytokines suggests that the development of this bioceramic implant coating should address techniques that would minimize the generation of particulates.

Human osteoblast-like cells (MG63) proliferate on a bioactive glass surface.

Bioglass, a resorbable glass, previously has been evaluated as a bone graft substitute using cells of animal origin. Limited information is available on its effect on human cells. The objective of this study was to test the hypothesis that Bioglass supports viability and proliferation of human bone cells. As a prototype of human bone cells, the osteoblast cell line MG63 was used and propagated on Bioglass disks. MG63 cells also were seeded onto disks made of titanium (Ti-6Al-4V) and of cobalt chrome (Co-Cr-Mo) alloys. The number of viable cells recovered was similar for Bioglass, titanium, and polystyrene control surfaces. Significantly fewer cells were recovered from CoCr (P < 0.05) compared to Bioglass, Ti-6 Al-4v, and polystyrene surfaces. The proportion of cells undergoing DNA synthesis, estimated by thymidine uptake, was significantly greater on Bioglass and titanium surfaces (P < 0.05) than on the CoCr surface. There were detectable differences in cell morphology on these biomaterials. Functional capacity was tested by assay of osteocalcin production and no differences were detectable among the different biomaterials. This study supports the hypothesis that 45S5 Bioglass provides a favorable environment for human osteoblast proliferation and function. Bioglass may have clinical potential as a bone graft substitute, a bioactive grout, or an implant coating for promoting bony ingrowth in uncemented prostheses.

Glass peek composite promotes proliferation and osteocalcin production of human osteoblastic cells.

An isoelastic intramedullary implant has been developed using a composite of polyetheretherketone and 10% random, chopped E-glass fibers (GPEEK). The effect of this novel material on human bone cells has not been defined. The objective of this study was to test whether GPEEK supported the proliferation of the human bone cell line MG63, which exhibits osteoblastlike characteristics. Cells (1 x 10(5)/mL) were propagated on GPEEK discs with three different surface roughnesses (3, 6, and 9 microns) and on polystyrene plates, for comparison. The reaction of MG63 osteoblastlike cells to the GPEEK polymer composite was analyzed by determination of cell yield, osteocalcin production, and levels of alkaline phosphatase. The viable cells that were retrieved from the GPEEK discs of all three surface roughness had an approximate sixfold increase in number. Osteoblastic function of the cells, indicated by osteocalcin production, was unimpaired after a 5-day culture on the three surfaces of GPEEK. The highest level of osteocalcin was produced by osteoblastic cells propagated on GPEEK with a 9 microns surface roughness. The levels of alkaline phosphatase of these cells were similarly greater for the different degrees of surface roughness. Overall, this study demonstrates that GPEEK supported proliferation of osteoblastlike cells and provided a favorable environment for the continued production of osteocalcin in vitro.

Immunotoxicity of medical devices. Symposium overview.

Determination of the ability of a medical device to interact with the immune system currently involves assessment of the immunogenic potential and biocompatibility of the device or an extract of the device. However, implants are often in the body for extended periods of time and/or are placed by a surgical procedure that in and of itself will generate an acute inflammatory response. This symposium discussed studies that have been performed to evaluate the immunogenicity of various devices consisting of several different compositions (i.e., silicone, metals, and latex) in contact with different anatomical sites, the ability of a device to modulate an inflammatory response generated by a surgical procedure or trauma, and the response of the body to a material left in place for extended periods of time. This symposium brought together scientists from many different disciplines to begin to identify and fill in the gaps in this area.

Silicone does not potentiate development of the scleroderma-like syndrome in tight skin (TSK/+) mice.

Several reports have implicated an association between the development of connective tissue disorders and exposure to silicone in breast implant patients. These connective tissue disorders include local or systemic scleroderma-like syndrome and have been characterized by fibrosis as well as the presence of circulating autoantibodies. Whether silicone does potentiate the development of a scleroderma-like syndrome is still to be defined. The purpose of the present study was to determine the effect of silicone on the tight skin (TSK/+) mouse which develops a scleroderma-like syndrome and on its normal pa/pa TSK/- littermate. Groups of six TSK and five of their normal pa/pa littermates were injected subcutaneously with low molecular weight silicone (LMW-PDMS), high molecular weight silicone gel (HMW-PDMS), incomplete Freunds' adjuvant (IFA) or Hank's balanced salt solution (HBSS) in the dorsal neck area and ventrally in the upper chest region. Serum was obtained prior to and 1 month after injection of silicone, IFA, or control HBSS. Antibody levels to bovine serum albumin (BSA), RNA polymerase (RNAP) and topoisomerase I were determined. Mice were then euthanized and strips of skin from the injection sites as well as samples of kidney and liver were studied histologically. No significant pathological changes were observed in TSK/- mice 1 month following injection with HBSS, LMW-PDMS or HMW-PDMS. Skin samples from TSK/+ mice which received LMW-PDMS showed hyperplasia of the dermis and peri-panniculus carnosus tissue and infiltrates of macrophages containing lipid-like vacuolated materials. Lipid vacuoles were observed throughout the deeper dermis as multiple loculated vacuoles. TSK mice which received HMW-PDMS showed similar thickening of the dermis and the peri-panniculus carnosus connective tissue. There were no significant differences in the histologic characteristics of the silicone-injected TSK/+ mice compared to those that received HBSS or IFA. No detectable changes in the kidney, spleen, or liver samples taken from TSK/+ or TSK/- mice injected with HBSS, IFA or silicone (LMW-PDMS, HMW-PDMS) preparations were noted. Baseline circulating antibody levels to BSA, RNAP and topoisomerase were significantly higher in TSK/+ mice compared to the control TSK/- littermates. Administration of silicone (LMW-PDMS or HMW-PDMS) did not significantly alter circulating antibody levels to BSA, RNAP and topoisomerase in either the TSK/+ or the TSK/- mice. The results of this study indicate that silicone administration does not potentiate the development of the scleroderma-syndrome characterized by skin lesions and presence of circulating antibodies in the TSK/+ model.

Silicone gel and octamethylcyclotetrasiloxane (D4) enhances antibody production to bovine serum albumin in mice.

The purpose of the present study was to determine whether components of silicone breast implants [silicon (polydimethylsiloxane) oil, silicone gel, and octamethylcyclotetrasiloxane (D4)] potentiate the antibody response to bovine serum albumin (BSA) in mice. Seventy A/J mice were divided into seven groups which received the following: group I--phosphate buffer solution (PBS); group II--20 centistoke (cs) silicone oil; group III--50% silicone gel homogenized in silicone oil; group IV--incomplete Freunds' adjuvant (IFA); group V--IFA mixed with an equal volume of silicone oil; group VI--D4; and group VII--IFA mixed with an equal volume of DA. Each 0.1 mL of treatment material(s) was mixed or emulsified with an equal 0.1-mL volume of 250 micrograms/mL BSA in PBS solution. Antibodies to BSA were measured using an enzyme-linked immunosorbent assay. Our study demonstrates for the first time that both D4 and the silicone gel potentiate antibody production to BSA in mice. Histopathologic evaluation of the injection sites reveals granulomas for mice injected with IFA and D4 preparations. Whether D4 or silicone gel acts as an adjuvant against self-antigens has yet to be determined.