BackgroundLoosening of the implant after total joint arthroplasty remains a serious problem. The activation of macrophages by wear debris from implants, mediated by the release of cytokines that ...elicit bone resorption, may lead to loosening. The purpose of the present study was to elucidate the mechanisms of macrophage activation by titanium particles from the components of implants and to identify the signaling pathways involved in particle-mediated release of cytokines. MethodsMacrophages were isolated from mononuclear leukocytes obtained from healthy human donors and were exposed to titanium-alloy particles that had been obtained from periprosthetic membranes collected at revision total joint arthroplasties and then enzymatically prepared. The experimental protocols included examination of the effects of the inhibition of phagocytosis and the binding of antibodies to macrophage complement receptors on particle-induced macrophage activation. The release of the proinflammatory cytokines TNF-α (tumor necrosis factor-alpha) and IL-6 (interleukin-6) was used to assess macrophage activation. The signaling pathways involved in the induction of cytokine release were analyzed by identification of phosphorylated proteins with use of the Western blot technique and by translocation of the transcription factors nuclear factor-kappa B (NF-κB) and nuclear factor-interleukin-6 (NF-IL-6) into the nuclear protein fraction with use of electrophoretic mobility shift assays. The role of serine/threonine and tyrosine kinase pathways in the activation of nuclear factors and the release of cytokines was examined with use of selective pharmacological agents. ResultsExposure of macrophages to titanium-alloy particles in vitro for forty-eight hours resulted in a fortyfold increase in the release of TNF-α and a sevenfold increase in the release of IL-6 (p < 0.01). Phagocytosis of particles occurred in approximately 73 percent of the macrophages within one hour of exposure. Pretreatment of the macrophages with cytochalasin B reduced phagocytosis by 95 percent but did not reduce the release of TNF-α or IL-6. Thus, phagocytosis of particles was not necessary for induction of the release of TNF-α or IL-6 in the cultured macrophages. Ligation of the macrophage CD11b/CD18 receptors by integrin-specific antibodies also increased the release of TNF-α and IL-6. Antibodies to CD11b/CD18 receptors (macrophage Mac-1 receptors) reduced phagocytosis of particles by 50 percent (p < 0.05). (The CD11b/CD18 macrophage receptor is the macrophage receptor for the complement component CR3bi. The CD11b/CD18 macrophage receptor can also bind to ICAM-1 and ICAM-2. CD is the abbreviation for cluster of differentiation, and ICAM is the abbreviation for intercellular adhesion molecule.) Inhibition of phagocytosis was not accompanied by a decrease in the release of TNF-α and IL-6. Blocking RNA synthesis with actinomycin D or preventing protein synthesis with cycloheximide abolished or decreased particle-induced release of TNF-α and IL-6 from the macrophages. Macrophage release of TNF-α and IL-6 in response to particles coincided with increased tyrosine phosphorylation and mitogen-activated protein kinase activation. Inhibition of tyrosine and serine/threonine kinase activity decreased the particle-induced release of cytokines. Exposure of macrophages to either titanium-alloy particles or to antibodies to the receptor proteins CD11b and CD18 for thirty minutes activated the transcription factors NF-κB and NF-IL-6. Inhibition of particle phagocytosis did not block activation of the transcription factors. However, inhibition of tyrosine and serine/threonine kinase activity decreased the activation of NF-κB and NF-IL-6. ConclusionsThese data suggest that particle-induced macrophage release of TNF-α and IL-6 does not require phagocytosis but is dependent on tyrosine and serine/threonine kinase activity culminating in activation of the transcription factors NF-κB and NF-IL-6. Clinical RelevanceRetrieval studies have documented numerous macrophages in association with particulate debris in granulomatous tissue surrounding failed total joint replacements. However, the molecular basis on which wear particles induce macrophage expression of proinflammatory cytokines and bone-resorbing factors remains unclear. This in vitro study showed that particles incite the release of proinflammatory cytokines from macrophages in the absence of phagocytosis. These results imply that contact of wear particles with macrophage cell-surface membrane proteins, such as the complement receptor CD11b/CD18, is sufficient signal for release of proinflammatory cytokines. The data further suggest that release of proinflammatory cytokines follows transmission of a membrane recognition event through intracellular signaling pathways that effect gene activation and protein synthesis. Therefore, these data indicate that a reduction in the formation of wear particles can be expected to improve the outcome after total joint arthroplasty by decreasing macrophage activation.
The development, maintenance, and destruction of cartilage are regulated by mechanical factors throughout life. Mechanical cues in the cartilage fetal endoskeleton influence the expression of genes ...that guide the processes of growth, vascular invasion, and ossification. Intermittent fluid pressure maintains the cartilage phenotype whereas mild tension (or shear) promotes growth and ossification. The articular cartilage thickness is determined by the position at which the subchondral growth front stabilizes. In mature joints, cartilage is thickest and healthiest where the contact pressure and cartilage fluid pressure are greatest. The depth-dependent histomorphology reflects the local fluid pressure, tensile strain, and fluid exudation. Osteoarthritis represents the final demise and loss of cartilage in the skeletal elements. The initiation and progression of osteoarthritis can follow many pathways and can be promoted by mechanical factors including: (1) reduced loading, which activates the subchondral growth front by reducing fluid pressure; (2) blunt impact, causing microdamage and activation of the subchondral growth front by local shear stress; (3) mechanical abnormalities that increase wear at the articulating surface; and (4) other mechanically related factors. Research should be directed at integrating our mechanical understanding of osteoarthritis pathogenesis and progression within the framework of cellular and molecular events throughout ontogeny.
BackgroundPrimary total hip replacement performed through an incision that is ≤10 cm in length has been advocated as a minimally invasive technique. Proponents have claimed that mini-incision ...techniques reduce blood loss, transfusion requirements, postoperative pain, and the length of the hospital stay compared with standard techniques through a longer incision. However, we are aware of no well-designed comparison study that supports these claims. The purpose of the present study was to compare the short-term results of a mini-incision with a standard incision technique for total hip replacement.MethodsA consecutive series of patients who underwent 135 primary unilateral total hip replacements (fifty with use of a mini-incision ≤10 cm and eighty-five with use of a standard incision) by three surgeons at one hospital were studied. Each surgeon selected patients to have a mini-incision procedure and performed a standard approach in the remaining patients. A posterior approach was used for all procedures. In-hospital data were collected retrospectively, and the initial postoperative radiographs were analyzed. Because of the selection process, the patients who had a mini-incision had both a significantly lower average body-mass index (p = 0.008) and a lower average score on the American Society of Anesthesiologists rating (p = 0.006), indicating that they were thinner and healthier than the patients who had a standard incision.ResultsWith the numbers of patients available, no significant differences were found between the groups with respect to the average surgical time, intraoperative blood loss, in-hospital transfusion rate, length of hospital stay, or the patientsʼ disposition after discharge. The mini-incision group was found to have a significantly higher risk of a wound complication (p = 0.02), a higher percentage of acetabular component malposition (p = 0.04), and poor fit and fill of femoral components inserted without cement (p = 0.0036).ConclusionsThere was no evidence that the mini-incision technique resulted in less bleeding or less trauma to the soft tissues of the hip, factors that would have produced a quicker recovery and a shorter hospital stay, than did the standard technique. The present study, which was based on the authorsʼ initial experience with the mini-incision technique, failed to confirm the positive clinical outcomes reported by previous uncontrolled cohort studies, and the findings suggest that further analysis of this new technique is needed before it can be recommended for general use.Level of EvidenceTherapeutic study, Level III-2 (retrospective cohort study). See Instructions to Authors for a complete description of levels of evidence.
Homeostasis of articular cartilage depends in part on mechanical loads generated during daily activity whereas inappropriate joint loads result in focal degeneration of cartilage, as occurs in ...osteoarthritis. We will review results of a series of questions regarding the effects of two types of mechanical loads-intermittent hydrostatic pressure and shear stress-on adult human articular chondrocytes in high-density monolayer culture. Intermittent hydrostatic pressure increased aggrecan and Type II collagen gene expression in normal chondrocytes and induced changes in the cell-associated proteins of normal and osteoarthritic chondrocytes. Hydrostatic pressure also counteracted inhibitory effects of bacterial lipopolysaccharide on matrix protein expression by cultured chondrocytes. Application of shear stress to osteoarthritic chondrocytes increased the release of the proinflammatory mediator, nitric oxide, decreased aggrecan and Type II collagen expression, and induced molecular changes associated with apoptosis whereas hydrostatic pressure increased matrix macromolecule expression. The findings show that the types of load comprising the mechanical loading environment of articular cartilage considerably alter chondrocyte metabolism and suggest that mechanical stimulation may be used for in vitro or in vivo approaches for cartilage engineering.
The normal loading of joints during daily activities causes the articular cartilage to be exposed to high levels of intermittent hydrostatic pressure. This study quantified effects of intermittent ...hydrostatic pressure on expression of mRNA for important extracellular matrix constituents. Normal adult bovine articular chondrocytes were isolated and tested in primary culture, either as high-density monolayers or formed aggregates. Loaded cells were exposed to 10 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for periods of 2, 4, 8, 12, and 24 hrs. Other cells were intermittently loaded for a period of 4 hrs per day for 4 days. Semiquantitative reverse transcription polymerase chain reaction assays were used to assess mRNA signal levels for collagen types II and I and aggrecan. The results showed that type II collagen mRNA signal levels exhibited a biphasic pattern, with an initial increase of approximately five-fold at 4 and 8 hrs that subsequently decreased by 24 hrs. In contrast, aggrecan mRNA signal increased progressively up to three-fold throughout the loading period. Changing the loading profile to 4 hrs per day for 4 days increased the mRNA signal levels for type II collagen nine-fold and for aggrecan twenty-fold when compared to unloaded cultures. These data suggest that specific mechanical loading protocols may be required to optimally promote repair and regeneration of diseased joints.
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Dostopno za:
DOBA, FSPLJ, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
Periprosthetic tissues observed at sites of loose total joint implants exhibit abundant macrophages, lymphocytes, fibroblasts and particulate debris. Macrophages phagocytose orthopaedic debris and ...release proinflammatory cytokines, chemokines, matrix metalloproteinases and other substances. In addition, other cell types present in tissues harvested from the bone–implant interface are thought to influence periprosthetic bone resorption. The present study examined the effects of polymethylmethacrylate (PMMA), cobalt chrome molybdenum alloy (CoCr), and titanium-alloy particle challenge on macrophages co-cultured with lymphocytes in vitro. Potential synergistic effects of lymphocytes on macrophage activation were determined by measuring interleukin-6 and tumor necrosis factor-
α release following exposure to orthopaedic biomaterial particles. Exposure of macrophages or macrophages co-cultured with lymphocytes to all three types of particles resulted in increased release of interleukin-6 and tumor necrosis factor-
α at 48
h, when compared to macrophages or macrophages co-cultured with lymphocytes, respectively, cultured in the absence of particles. Lymphocytes isolated from periprosthetic tissues secreted increased basal levels of cytokines relative to peripheral blood lymphocytes. Higher doses of PMMA and titanium-alloy particles stimulated increased levels of cytokine release in the macrophage and macrophage/lymphocyte groups. In contrast, a higher dose of CoCr particles (0.075% v/v) was not as effective as the 0.015% v/v dose, indicating probable CoCr toxicity. The macrophage/lymphocyte co-culture did not show synergism between the two types of cells with respect to cytokine release. T-cells at the bone-implant interface may alter the biological response to particulate debris.
This study tested the effects of hydrostatic pressure (10 MPa) on adult articular chondrocyte mRNA and extracellular matrix synthesis in vitro. High density primary cultures of bovine chondrocytes ...were exposed to hydrostatic pressure applied intermittently at 1 Hz or constantly for 4 hours in serum-free medium or in medium containing 1% fetal bovine serum. mRNAs for aggrecan, types I and II collagen, and beta-actin were analyzed by Northern blots and quantified by slot blots. Proteoglycan synthesis was quantified by 35SO4 uptake into cetylpyridinium chloride-precipitable glycosaminoglycans, and cell-associated aggrecan and type-II collagen were detected by immunohistochemical techniques. In serum-free medium, intermittent pressure increased aggrecan mRNA signal by 14% and constant pressure decreased type-II collagen mRNA signal by 16% (p < 0.05). In the presence of 1% fetal bovine serum, intermittent pressure increased aggrecan and type-II collagen mRNA signals by 31% (p < 0.01) and 36% (p < 0.001), respectively, whereas constant pressure had no effect on either mRNA. Intermittent and constant pressure stimulated glycosaminoglycan synthesis 65% (p < 0.001) and 32% (p < 0.05), respectively. Immunohistochemical detection of cell-associated aggrecan and type-II collagen was increased in response to both intermittent and constant pressure. These data support the hypothesis that physiologic hydrostatic pressure directly influences the extracellular matrix metabolism of articular chondrocytes.
A review of postoperative infected anterior cruciate ligament reconstructions was done on 3500 consecutive arthroscopic procedures. The purpose was to assess incidence, diagnosis, treatment, and ...outcome factors. Six postoperative intraarticular infections were detected. Average followup was 3 years (range, 2-8 years). The rate of infection was 0.14%. Five men and one woman with a median age of 32.5 years (range, 20-51 years) comprised the study group. The average interval from the onset of symptoms to the initial arthroscopic intervention was 7.5 days (range, 2-20 days). Staphylococcus aureus was present in three knees, Staphylococcus epidermidis in two, and Streptococcus nonhemolytic in one. All patients had initial arthroscopic debridement and lavage followed by 6 weeks of intravenous antibiotics. Two grafts were removed: one patient had delayed ligament reconstruction and the other had total knee arthroplasty. The remaining patients had full range of motion. In the group with the best result, two patients had Staphylococcus epidermidis and one had Staphylococcus aureus, which was treated 2 days after clinical symptoms began. The other two patients infected with Staphylococcus aureus had unsatisfactory results. Anterior cruciate ligament infection is rare, but diagnosable. When treated early with appropriate antibiotic therapy and arthroscopic debridement, four of six grafts were retained. If the infection does not respond rapidly to early therapy, then graft removal is an option.
This study tested the effects of fluid-induced shear on high density monolayer cultures of adult articular chondrocytes. Fluid-induced shear (1.6 Pa) was applied by cone viscometer to normal human ...and bovine articular chondrocytes for periods of 24, 48, and 72 hours. At 48 and 72 hours, fluid-induced shear caused individual chondrocytes to elongate and align tangential to the direction of cone rotation. Fluid-induced shear stimulated glycosaminoglycan synthesis by 2-fold (p < 0.05) and increased the length of newly synthesized chains in human and bovine chondrocytes. In human chondrocytes, the hydrodynamic size of newly synthesized proteoglycans also was increased. After 48 hours of fluid-induced shear, the release of prostaglandin E2 from the chondrocytes was increased 10 to 20-fold. In human chondrocytes, mRNA signal levels for tissue inhibitor of metalloproteinase increased 9-fold in response to shear compared with the controls. In contrast, mRNA signal levels for the neutral metalloproteinases, collagenase, stromelysin, and 72 kD gelatinase, did not show such major changes. This study demonstrated that articular chondrocyte metabolism responds directly to physical stimulation in vitro and suggests that mechanical loading may directly influence cartilage homeostasis in vivo.
VEGF and VEGF receptor, Flt‐1, expression was observed in periprosthetic tissues surrounding loosened total joint implants. Exposure of monocyte/macrophages to titanium particles resulted in ...increased VEGF expression, p44/42 MAPK activation, and VEGF‐dependent macrophage chemotaxis. Increased levels of angiogenic factors, such as VEGF, may be critically important in wear debris‐induced implant loosening after total joint arthroplasty.
Introduction: Periprosthetic osteolysis after total hip arthroplasty occurs in association with formation of a vascularized granulomatous tissue in response to particulate debris.
Materials and Methods: This study examined expression of vascular endothelial growth factor (VEGF) and the VEGF receptor in 10 periprosthetic tissues from loosened prostheses and quantified effects of titanium particles on VEGF release, intracellular signaling, and VEGF‐dependent chemotaxis in primary cultures of human monocyte/macrophages.
Results: Double immunofluorescent staining showed that VEGF and Flt‐1 co‐localized with cells positive for the macrophage marker, CD11b, in the periprosthetic tissues. Monocyte/macrophages challenged with titanium particles showed a dose‐ and time‐dependent release of VEGF ranging from 2.8‐ to 3.1‐fold and exhibited increased expression of VEGF121 and VEGF165 mRNAs, reaching levels up to 5.0‐ and 8.6‐fold, respectively, by 48 h (p < 0.01). Exposure of monocyte/macrophages to titanium particles upregulated phosphorylated‐p44/42 mitogen‐activated protein kinase (MAPK) within 30 minutes. Particle‐induced activation of p44/42 MAPK and release of VEGF were dose‐dependently suppressed by pretreatment of cells with PD98059, a specific inhibitor of p44/42 MAPK. Monocyte/macrophages challenged with titanium particles also showed a time‐dependent activation of AP‐1, a transcription factor associated with VEGF expression (p < 0.01). Supernatants from particle‐challenged monocyte/macrophages increased macrophage chemotactic activity by 30%, which was significantly inhibited by anti‐VEGF neutralizing antibody (p < 0.01).
Conclusions: This study suggests that induction of VEGF release from monocyte/macrophages in response to orthopaedic biomaterial wear debris may contribute to periprosthetic osteolysis and implant loosening.
