In vitro interaction between silicone gel and human monocyte-macrophages.

Controversy remains regarding the ability of silicone materials to induce a specific immune reaction versus a nonspecific inflammatory response. Histopathological analysis of the tissue around failed breast implants reveals chronic inflammation with silicone gel droplets either surrounded by giant cells or engulfed by macrophages, areas of fibrosis, and necrosis. Macrophages are the key cells engulfing or forming foreign body giant cells. To address the mechanisms of silicone-induced inflammation a model of human monocyte-derived macrophages (MDMs) was developed. After sonication of silicone gel, the silicone droplets were embedded in Type I collagen and used to coat glass coverslips; human MDMs were subsequently seeded on the coverslips and maintained in culture for up to 7 days. The advantage of the model was that human macrophages could be studied histologically, and cytochemically as they interacted with well-characterized silicone materials. Initial analysis of the human macrophages shows phagocytosis of the silicone gel within hours of exposure to the material. Analysis for pro-inflammatory cytokines reveals significant transient secretion of IL-1 (p < 0.01) over controls by human macrophages upon exposure to silicone gel at 24 h.

The effect of polyethylene particle phagocytosis on the viability of mature human macrophages.

Macrophages are the major cell type observed in the inflammatory membrane retrieved at implant revision surgery. In this study, mature human monocyte-derived macrophages (MDM) were adapted to a previously established in vitro model to examine the influence of high-density polyethylene (HDPE) particulate (4-10 microm) on MDM viability. HDPE particles were suspended in soluble type I collagen, which subsequently was solidified on glass coverslips. Mature human macrophages, derived from differentiating peripheral blood monocytes on polystyrene for 10 days, were incubated in culture media on collagen controls and collagen-particle substrata for 31 days. Histologic analysis demonstrated that MDMs were in contact with the particles at 2 h. The majority of the particles were associated with the cells within 24 h. Based on electron microscopy, those cells associated with the particles appeared to be morphologically activated rather than necrotic or apoptotic. Assessment of cell viability revealed no differences among the groups at 24 h, but at 31 days significantly more viable cells and higher DNA values were found associated with the particle groups versus the collagen controls. The histologic results validate human mature MDMs as a clinically relevant cell type for study of the role of polyethylene particulate in aseptic loosening. The cell viability results indicate that phagocytosis of HDPE is not toxic to MDMs but in fact prolongs MDM survival. The long-lived MDMs may play a role in perpetuating chronic inflammation surrounding implants.

Changes in macrophage morphology and prolonged cell viability following exposure to polyethylene particulate in vitro.

The interaction of macrophages and ultra-high molecular weight polyethylene (PE) wear plays an important role in perpetuating chronic inflammation at the bone implant interface, leading to peri-implant osteolysis and mechanical failure of the implant. A model to study the interaction of human mature macrophages with orthopaedic biomaterial wear has been previously developed. With the use of the model, in this study, the mature human monocyte-derived macrophages (MDMs) were observed with light, fluorescent, and scanning electron microscopy (SEM), as well as transmission electron microscopy (TEM). The cell viability was investigated using calcein and ethidium staining. Following exposure to PE particulate, the morphology of the human MDMs was heterogeneous: rounded, flattened, and elongated. There was no morphological evidence of cytotoxicity or apoptosis. The MDM viability was not influenced by phagocytosis of PE particulate in a negative fashion. In fact, more prolonged cell viability was observed in the human MDMs exposed to PE particulate when compared to controls.

Differential response to chemically altered polyethylene by activated mature human monocyte-derived macrophages.

Macrophages and polyethylene (PE) particulate are currently recognized as being the two common denominators in the development of chronic inflammation, periprosthetic osteolysis, and subsequent implant failure. In this study, the effect of PE particulate surface chemistry on mature human monocyte-derived macrophage (MDM) function was investigated. Virgin high-density PE (HDPE: 4-10 microm) and HDPE oxidized by irradiation, thermal and chemical treatment were characterized by FT-IR and suspended in soluble type I collagen, which was subsequently solidified on glass coverslips. Human MDMs, derived from differentiating monocytes on polystyrene for 14 days, were trypsinized and cultured on collagen-particle substrata and collagen controls for 31 days. Analysis of conditioned media collected at 24h incubation showed a significantly higher level of IL-1beta secretion in virgin HDPE over oxidized HDPE or collagen controls, and a significant inhibition of IL-6 secretion in both virgin and oxidized samples. Esterase activity was increased in the medium at a significantly higher level in the virgin HDPE versus controls with the highest activity observed in oxidized HDPE at 31 days. These results illustrate the effect of PE particle surface chemistry (oxidation) on MDM cytokine secretion and esterase activity, and highlight the need to further investigate the potential of PE surface chemistry on modulating MDM function.

The effect of polyethylene particle chemistry on human monocyte-macrophage function in vitro.

Osteolysis remains the most important problem in orthopedic implant failure. Wear debris from the implant contains polyethylene (PE) particulate which has been shown to activate monocyte-derived macrophages (MDM). Although the response of MDM has been shown to be influenced by the size, shape, and chemical type of PE, the effect of chemically altered PE on MDM has not been studied. In this study, human MDM were seeded onto glass coverslips coated with virgin high density (HD)PE and chemically modified HDPE (impregnated with ppm levels of CoCl(2) and oxidized by heat) mixed with type I collagen and cultured for 96 h. Light microscopic evaluation demonstrated consistent phagocytosis of the HDPE particulate that was confirmed by scanning electron and transmission electron microscopy with little evidence of cytotoxicity. Evaluation of pro-inflammatory mediator secretion by MDMs in response to the virgin and chemically modified HDPE revealed significant differences in interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and IL-6 secretion. A significant elevation of IL-1 secretion was observed after initial exposure to virgin HDPE particles compared with controls (p = 0.001). IL-1 secretion was also elevated in the low oxidized particle groups (p = 0.001), whereas the highly oxidized particles were not different than controls. Secretion of both IL-6 (p = 0.03) and TNF-alpha (p = 0.007) were significantly elevated by the low oxidized HDPE particles whereas the virgin and highly oxidized groups showed no difference. The different effects on MDM activation when HDPE surface chemistry was altered, highlight the importance of defining the particle properties when studying the role of MDM activation in in vitro systems and extrapolating these observations to the in vivo situation.

The role of the macrophage in periprosthetic bone loss.

Aseptic loosening after total joint replacement remains the most common reason for long-term implant failure. Macrophages activated by submicron wear particles of the polyethylene liner used in joint replacement have been shown to be the source of periprosthetic bone loss. Understanding the role of material chemistry in macrophage activation and the subsequent effects that macrophage-derived enzymes play in the degradation of implanted biomaterials is key to developing methods for prolonging the lifespan of implantable materials.

Intradermal injection of autologous dermal fibroblasts improves wound healing in irradiated skin.

BACKGROUND: Despite its well-recognized benefits in the management of several solid tumors, the use of radiotherapy prior to surgery is associated with a high incidence of significant surgical wound healing complications. Radiation-induced damage to dermal fibroblasts has been proposed as an important cause. We hypothesized that the introduction of normal, unirradiated fibroblasts into previously irradiated skin would enhance healing of the subsequent surgical wound. MATERIALS AND METHODS: Four groups of wounds were examined in female Wistar rats: (1) unirradiated skin (n = 10), (2) irradiated skin injected with tissue culture medium alone (n = 17), (3) irradiated skin injected with autologous dermal fibroblasts (n = 17), and (4) irradiated skin injected with irradiated autologous dermal fibroblasts (n = 7). Wounds were evaluated biomechanically and histologically. RESULTS: The biomechanical values of breaking load, ultimate tensile strength, elastic modulus, and toughness were significantly greater in the irradiated wounds injected with fibroblasts than those injected with medium only. These cell-injected wounds did not perform as well biomechanically as those in unirradiated skin. Irradiating the cells prior to injection resulted in biomechanical results no better than those in medium-injected wounds. CONCLUSIONS: These results demonstrate that injection of normal, unirradiated fibroblasts significantly improves healing of the irradiated surgical wound. These cells are likely better able to respond to the proliferative, migratory, and synthetic demands of the wound healing environment, as injection of irradiated cells has an equivalent effect on healing as injection of medium alone.

Effect of interleukin-6 secreted by engineered human stromal cells on osteoclasts in human bone.

The effect of elevated human IL-6 (hIL-6) production by human bone marrow (Hu-BM) stromal cells on osteoclasts in human bone was examined. Human bone was implanted into nonobese diabetic mice with severe combined immunodeficiency (Hu-Bone-NOD/SCID mice). Immunohistochemistry of bone implants and mouse spleens (at 20 weeks), showed human CD45+ cells, B cells, and macrophages in both tissues. Thus, Hu-BM cells survive human bone transplantation and infiltrate mouse tissue. Bone implants had 75 +/- 12% (mean +/- SD) human CD45+ cells, and 9 +/- 4% mouse hematopoietic cells. A retrovirus vector containing the human IL-6 gene was used to transduce Hu-BM stromal cells (IL-6/stromal) and the PA317 cell line (IL-6/PA317). IL-6/ stromal cells (secreting, on average, 17 microg of hIL-6/10(6) cells per 24 h) were injected directly into human bone implants in Hu-Bone-NOD/SCID mice. IL-6/PA317 cells (secreting 16 microg/mL of hIL-6/10(6) cells per 24 h) were injected intraperitoneally into Hu-Bone-NOD/SCID mice. Analyses of sera from both groups of animals showed elevated levels of IL-6. However, only bone implants engrafted with IL-6/stromal cells had a statistically significant increase in osteoclast-lined mineralized trabecular bone surface (BS). Thus, a high concentration of serum hIL-6 in Hu-Bone-NOD/SCID mice alone does not increase osteoclast-lined BS in bone implants. Most importantly, it is the type of human BM cell that secretes the high levels of hIL-6 that is most critical.

The role of T cells in polyethylene particulate induced inflammation.

OBJECTIVE: To investigate the role of T lymphocytes in ultra-high molecular weight polyethylene (UHMWPE) induced inflammation in joint arthroplasty. METHOD: We address the role of T cells in wear induced inflammation by injecting the knee joints of both immune competent rats and mice and severe combined immunodeficient (SCID) mice with UHMWPE. Histological and immunohistochemical analysis of the synovial tissues was compared. Interaction between human T cells and UHMWPE particles was examined in vitro using T cell activation assays. RESULTS: Histological and immunohistochemical analysis of the knees of the immune competent animals showed significant UHMWPE induced inflammation. In contrast, the tissue in the SCID mice knee joints showed very little inflammatory response to UHMWPE despite phagocytosis of the particulate. Since the SCID mice have no functional T or B lymphocytes, it is highly likely that the lack of inflammation in knee joints may be due to the absence of mouse T cells, as the infiltration of T cells into the joint tissue may enhance the inflammatory response to UHMWPE particles. T cell activation assays showed that T cells were not directly activated by UHMWPE particles and the nature of the interaction was not revealed from these experiments. CONCLUSIONS: Although T cells are not directly involved in UHMWPE particle induced inflammation, as shown by the T cell activation assays, the histological data from the mice studies clearly show differences in the amplitude of inflammation from animals with and without functional T cells. Our studies suggest that the T cells may enhance the inflammatory response due to a bystander effect. Since the macrophages upon ingestion of UHMWPE particles release several cytokines including tumor necrosis factor-alpha, interleukin 1, and IL-6, it is possible that T cells in the vicinity of these macrophages may become attracted to the knee joint and activated due to cytokine release.

A biomechanical assessment of ligaments preventing dorsoradial subluxation of the trapeziometacarpal joint.

Degenerative arthritis of the trapeziometacarpal joint is commonly associated with ligament laxity and joint subluxation. Specifically, key pinch in an affected joint often results in dorsoradial joint subluxation. This study examined the role the 4 ligaments of the trapeziometacarpal joint play in preventing dorsoradial subluxation. Six fresh-frozen cadaver hands were dissected of all soft tissue to expose the joint capsule and ligaments of the trapeziometacarpal joint. Serial random sectioning of the intermetacarpal ligament, anterior oblique ligament, palmar oblique ligament, and dorsoradial collateral ligament (RCL) was performed. Dorsoradial displacement of the metacarpal shaft in relation to the trapezium was measured using a linear variable dimension transformer. In all 6 specimens, sectioning of the RCL resulted in the greatest dorsoradial subluxation of the metacarpal. The mean displacement due to sectioning of the RCL was 1.4 mm, compared with 0.08 mm for the intermetacarpal ligament, 0.06 mm for the anterior oblique ligament, and 0.2 mm for the palmar oblique ligament. The importance of the RCL in preventing dorsoradial subluxation may have clinical significance. This study suggests that repairing or reconstructing the RCL during ligament reconstruction of the trapeziometacarpal joint should be considered.

Macrophage phagocytosis of polyethylene particulate in vitro.

In this study, an in vitro model has been developed to examine the interactions of macrophages with ultrahigh molecular-weight polyethylene (UHMWPE) and high-density polyethylene (HDPE) particles. Polyethylene particles are the major constituent of the material debris formed as a result of orthopedic implant wear. However, the study of polyethylene particle interactions with cells has been limited. UHMWPE (18-20 microns) and HDPE (4-10 microns) were suspended in soluble collagen type I and subsequently solidified on glass coverslips. The particle chemistry was characterized by Fourier transform infra-red spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). Mouse cell line macrophages (IC-21) were established on the collagen-particle substrata and maintained for up to 24 h. The response of the cells to the particles was examined by light and transmission electron microscopy (LM and TEM), as well as by scanning electron microscopy (SEM), and compared to cells on control collagen surfaces without particles. Histological analysis of the samples revealed that the macrophages surrounded larger particles (18-20 microns) and the cells appeared to be attached to the surface of the particles, and the smaller particles (4-10 microns) had been phagocytosed within 2 h. Inflammatory cytokines (TNF-alpha, IL-1 alpha, IL-1 beta, and IL-6), lysosomal enzymes (beta-galactosidase and hexosaminidase), and prostaglandin E2 were released into the medium, and IL-1 alpha, IL-1 beta, PGE2, beta-galactosidase, and hexosaminidase levels were significantly increased over collagen control values. The results demonstrate active phagochemotaxis by macrophages for wear particulates and validate this model as a means of studying the specific in vitro interactions of polyethylene with cells.

Human hematopoiesis in SCID mice implanted with human adult cancellous bone.

The persistence of hematopoietic cells from human adult cancellous bone fragments implanted subcutaneously into CB-17 scid/scid mice was studied. Recipient mice received either no pretreatment (control group) or pretreatment with 3 Gy total-body irradiation and anti-asialo GM1 sera (ASGM1; pretreated group) before implantation. Pretreated severe combined immunodeficient (SCID) mice implanted with human bone were subsequently given ASGM1 every 7 days for the duration of the experiments. At 12 weeks postimplantation, flow cytometry of cells from pretreated and control animal tissues detected human CD45+ cells in the mouse spleen (mean, 7.8% and 3.4% positive cells, pretreated and control animals, respectively), bone marrow (BM; mean, 16.5% and 4.8% positive cells, respectively), and blood (mean, 5.5% and < 2% positive cells, respectively), and in the implanted human bone (73% and 8.9% positive cells, respectively). At 12 weeks, pretreated mice had human granulocyte-macrophage colony-forming cells (GM-CFC) and burst-forming units-erythrocyte (BFU-E) in the implanted human bone in the murine BM and in some of the spleens. The spleens also had extensive infiltration of human B cells and macrophages. Mean serum levels of human IgG in pretreated animals were 14 micrograms/mL during weeks 6 to 12, compared with trace levels (< 1 microgram/mL) in control mice. Bone from patients with acute myeloblastic leukemia (AML) was also implanted in pretreated SCID mice, and retrieved at 8 weeks for analysis. Comparison of preimplantation and implanted samples showed that the original histology was maintained, and massive infiltration of human CD68+ cells was observed in the mice spleens and BM. Implantation of AML bone in SCID mice facilitates analysis of in situ AML cell interaction with stromal cells in the leukemic state, and therapies against AML can be tested in this system, especially the selective killing of AML cells in the presence of other BM cells. Furthermore, this model requires no exogenous administration of cytokines to maintain human hematopoiesis with both normal or AML bone. Because the structure and function of both normal and diseased human adult bone is maintained, this animal model should facilitate investigation of both normal human hematopoiesis and hematopoietic malignancies.

The inflammatory response to particulate wear debris in total hip arthroplasty.

OBJECTIVE: To identify the cell types and patterns of cell distribution associated with particulate wear debris in the membranes of patients who undergo revision of hip arthroplasty. DESIGN: Case series. SETTING: Three university-affiliated hospitals. PATIENTS: Twenty-nine patients who underwent revision of a hip arthroplasty for reasons other than infection. Eight patients were excluded because loosening of the prosthesis was found to be associated with infection, leaving 21 patients, 18 with aseptic loosening and 3 with recurrent dislocation. INTERVENTION: Retrieval of inflammatory membranes from loosened total hip prostheses. MAIN OUTCOME MEASURES: Results of aerobic and anaerobic tissue cultures, number of macrophages, T cells and B cells, and presence and histologic pattern of particulate debris in the membranes. RESULTS: None of the tissue cultures grew any organisms. All membranes contained T cells and macrophages, but B cells were present in only three patients at a low level. Particulate debris, surrounded by macrophages and foreign-body giant cells, was present in all membranes. Three histologic patterns were noted: type I--mostly fibrous tissue with cells and macrophages widely scattered through membrane (3 membranes); type II--fibrous membrane with a significant number of T cells, macrophages and foreign-body giant cells and well-defined foreign-body granulomas (7 membranes); type III--diffusely scattered T cells, ill-defined granulomas, "foamy" macrophages and fewer foreign-body giant cells than in type II (11 membranes). CONCLUSIONS: These findings document the key cellular elements involved in cell-mediated immunity in association with particulate wear debris in aseptically loosened total hip arthroplasty membranes. T-cell amplification of the innate macrophage inflammatory reaction to particulate debris may play a role in the mechanism of particle clearance.

Distal femoral varus osteotomy for valgus deformity of the knee.

Twenty-four patients who had degenerative arthritis of the lateral compartment of the knee that was associated with a valgus deformity and a superolateral tilt of the joint line were treated by distal femoral varus osteotomy. A surgical technique that was designed to produce a horizontal joint line and a tibiofemoral angle of zero degrees is described. At an average length of follow-up of four years, twenty-two of twenty-four patients had a successful result as judged by our protocol for evaluation. One osteotomy had to be revised for failure of fixation, and one patient had a pulmonary embolism that had a satisfactory outcome after the administration of anticoagulants. One patient required manipulation of the knee at six months. The simple surgical technique that we used was effective in realigning the femoral with the tibial axis in patients who had a valgus deformity of the knee, a superolateral tilt of the joint line, and osteoarthritis of the lateral compartment.