David J. Schurman

Signaling Pathways for Tumor Necrosis Factor-α and Interleukin-6 Expression in Human Macrophages Exposed to Titanium-Alloy Particulate Debris in Vitro*

BACKGROUND Loosening 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. METHODS Macrophages 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-alpha (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-kappaB) 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. RESULTS Exposure of macrophages to titanium-alloy particles in vitro for forty-eight hours resulted in a fortyfold increase in the release of TNF-alpha 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-alpha or IL-6. Thus, phagocytosis of particles was not necessary for induction of the release of TNF-alpha or IL-6 in the cultured macrophages. Ligation of the macrophage CD11b/CD18 receptors by integrin-specific antibodies also increased the release of TNF-alpha 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-alpha and IL-6. Blocking RNA synthesis with actinomycin D or preventing protein synthesis with cycloheximide abolished or decreased particle-induced release of TNF-alpha and IL-6 from the macrophages. Macrophage release of TNF-alpha 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-kappaB 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-kappaB and NF-IL-6. CONCLUSIONS These data suggest that particle induced macrophage release of TNF-alpha and IL-6 does not require phagocytosis but is dependent on tyrosine and serine/threonine kinase activity culminating in activation of

Fatigue life of compact bone—II. Effects of microstructure and density

Fatigue tests to failure of compact bovine bone specimens were conducted at five stress amplitudes (65–108 MN/m2) and four temperature levels (21–45°C). The resulting relationships between fatigue life and stress amplitude, bone temperature, and bone density have been reported (Carter and Hayes, 1975). In the present study, the bone specimens were categorized into four microstructure groups based on the extent of secondary Harversian remodeling. A significant correlation (P < 0·001) between density and microstructure group was shown with primary bone specimens generally being more dense than secondary Haversian specimens. A positive correlation (P < 0·01) between fatigue life and density was revealed within each structural group. In addition, a negative correlation (P < 0·001) between fatigue life and the extent of Haversian remodeling was shown even after appropriate adjustments for density differences. These data suggest that Haversian remodeling of primary bovine bone reduces fatigue resistance not only by decreasing bone density, but also by creating an inherently weaker structure.

Cellular profile and cytokine production at prosthetic interfaces: STUDY OF TISSUES RETRIEVED FROM REVISED HIP AND KNEE REPLACEMENTS

The tissues surrounding 65 cemented and 36 cementless total joint replacements undergoing revision were characterised for cell types by immunohistochemistry and for cytokine expression by in situ hybridisation. We identified three distinct groups of revised implants: loose implants with ballooning radiological osteolysis, loose implants without osteolysis, and well-fixed implants. In the cemented series, osteolysis was associated with increased numbers of macrophages (p = 0.0006), T-lymphocyte subgroups (p = 0.03) and IL-1 (p = 0.02) and IL-6 (p = 0.0001) expression, and in the cementless series with increased numbers of T-lymphocyte subgroups (p = 0.005) and increased TNF alpha expression (p = 0.04). For cemented implants, the histological, histochemical and cytokine profiles of the interface correlated with the clinical and radiological grade of loosening and osteolysis. Our findings suggest that there are different biological mechanisms of loosening and osteolysis for cemented and cementless implants. T-lymphocyte modulation of macrophage function may be an important interaction at prosthetic interfaces.

Mechanoregulation of human articular chondrocyte aggrecan and type II collagen expression by intermittent hydrostatic pressure in vitro

This study addressed the hypothesis that duration and magnitude of applied intermittent hydrostatic pressure (IHP) are critical parameters in regulation of normal human articular chondrocyte aggrecan and type II collagen expression. Articular chondrocytes were isolated from knee cartilage and maintained as primary, high‐density monolayer cultures. IHP was applied at magnitudes of 1, 5 and 10 MPa at 1 Hz for durations of either 4 h per day for one day (4 × 1) or 4 h per day for four days (4 × 4). Total cellular RNA was isolated and analyzed for aggrecan and type II collagen mRNA signal levels using specific primers and reverse transcription polymerase chain reaction (RT‐PCR) nested with beta‐actin primers as internal controls. With a 4 × 1 loading regimen, aggrecan mRNA signal levels increased 1.3‐ and 1.5‐fold at 5 and 10 MPa, respectively, relative to beta‐actin mRNA when compared to unloaded cultures. Changing the duration of loading to a 4 × 4 regimen increased aggrecan mRNA signal levels by 1.4‐, 1.8‐ and 1.9‐fold at loads of 1, 5 and 10 MPa, respectively. In contrast to the effects of IHP on aggrecan, type II collagen mRNA signal levels were only upregulated at loads of 5 and 10 MPa with the 4 × 4 loading regimen. Analysis of cell‐associated protein by western blotting confirmed that IHP increased aggrecan and type II collagen in chondrocyte extracts. These data demonstrate that duration and magnitude of applied IHP differentially alter chondrocyte matrix protein expression. The results show that IHP provides an important stimulus for increasing cartilage matrix anabolism and may contribute to repair and regeneration of damaged or diseased cartilage.

Influences of mechanical stress on prenatal and postnatal skeletal development.

A new theory is introduced to describe some of the influences of mechanical stresses on chondroosseoubiology. It is proposed that degeneration and ossification is a normal process for all cartilage in the appendicular skeleton, which is (1) accelerated by intermittently applied shear stresses (or strain energy), and (2) inhibited or prevented by intermittently applied hydrostatic pressure. These concepts were applied using finite element computer models in an effort to predict the ossification pattern of the prenatal and postnatal femoral anlage. The theoretical calculations successfully predicted the key features of skeletal morphogenesis including the development of (1) the primary ossification site, (2) a tubular diaphysis and marrow cavity, (3) meta-phys-al and epiphyseal trabecular bone, (4) the location and geometry of the growth plate, (5) the appearance and location of the secondary ossific nucleus, and (6) the existence and thickness distribution of articular cartilage. The results suggest that degenerative joint disease in immobilized or non-load-bearing mature joints may be a manifestation of the final stage in the ossification of the anlage. In nonfunctional joints, the absence or reduction of intermittent hydrostatic pressure in the articular cartilage permits cartilage degeneration and the progressive advance of the ossification front toward the joint surface until the articular cartilage has been ossified.

The function of the primary ligaments of the knee in varus-valgus and axial rotation

Abstract Four in vitro human knee specimens have been loaded, two with varus-valgus femoral rotations and two with internal and external axial tibial rotations. Each specimen has been tested in full extension and in 30° of flexion. All orthogonal components of applied force and moment required to cause the rotations were measured as were all resultant orthogonal components of load on the femur. Curves fit to the data were studied to establish the portions of the applied load transmitted by each ligament.

Fibroblast response to metallic debris in vitro. Enzyme induction cell proliferation, and toxicity.

Bovine synovial fibroblasts in primary monolayer culture were exposed to particulate metallic debris. The effects of the metallic particles on the synthesis and secretion of proteolytic enzymes and on cell proliferation and viability were examined. Uniform suspensions of titanium, titanium-aluminum, cobalt, and chromium particles, ranging in size from approximately 0.1 to ten micrometers (average, one to three micrometers), were prepared; the particle concentrations (the volume of particles divided by the total volume of the suspension) ranged from 0.0005 to 5 per cent. Aliquots of the particle suspensions were added to the synovial fibroblast cultures. The final particle concentrations in the media ranged from 0.0000083 to 0.83 per cent. After seventy-two hours of exposure, each medium was harvested and was assayed for proteolytic and collagenolytic activity and for hexosaminidase levels. Neutral metalloproteases, quantified by collagenolytic and caseinolytic (proteolytic) activity, represent enzymes, secreted by cells, that are capable of degrading extracellular matrix. Hexosaminidase is a marker for lysosomal enzyme activity that can include more than thirty enzymes, such as proteases, lipases, nucleases, and phosphatases. Cell proliferation was quantified by uptake of 3H-thymidine. Cell morphology was examined by scanning electron microscopy. Titanium, titanium-aluminum, and chromium significantly stimulated 3H-thymidine uptake at low particle concentrations (p < 0.01, p < 0.002, and p < 0.002, respectively). Exposure to cobalt, even at the lowest particle concentration, resulted in a significant decrease in thymidine uptake (p = 0.027). At the highest particle concentrations, all particles were toxic, as evidenced by the absence of thymidine uptake. At high particle concentrations, all of the metals caused a decrease in caseinolytic (proteolytic) and collagenolytic activity in the culture media. Titanium elevated the lysosomal enzyme marker, hexosaminidase, except at high concentrations. Chromium and titanium-aluminum had no significant effect on hexosaminidase at any particle concentration, while cobalt decreased all enzyme markers at mid-particle to high-particle concentrations. Scanning electron microscopy demonstrated that the morphological response of fibroblasts to titanium included membrane-ruffling and extension of filopodia, typical of active fibroblasts. In contrast, exposure to cobalt at the same concentration resulted in cell crenation, indicative of cell death.

Septic arthritis in postoperative anterior cruciate ligament reconstruction.

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.

The function of the primary ligaments of the knee in anterior-posterior and medial-lateral motions

Abstract The functions of the primary ligaments of the human knee have been investigated during anterior, posterior, medial and lateral tibial displacements. The ligaments of in vitro knee specimens have been loaded to large load levels and all three resultant orthogonal components of force and of moment acting on the femur as a result of the applied tibial displacements have been measured. Least squares cubic spline curves fit to the data have been analyzed to determine what percentage of a given applied load is transmitted by each ligament. These curves can also provide data for a computer model of the human leg.

Range of motion studies for total hip replacements. A comparative study with a new experimental apparatus.

Significant differences in ROM exist between different THR prosthesis designs: several of the prosthesis designs tested are marginal in flexion; several millimeters of socket wear will decrease the ROM. The results also emphasize the importance of proper component orientation at surgery. The surgeon has less latitude in orienting the components of a THR with limited ROM. Subluxation and dislocation due to rim contact can be minimized with most prosthetic units by instructing the patients to abduct and/or externally rotate their hips during acute flexion. Analyses suggest that impingement of prosthesis neck and socket rim may lead to increased risk of dislocation and increased rim wear. Prostheses with adequate ROM for everyday activities should provide stability, less frequent neck and socket contact with decreased rim wear, less force transmission to acrylic-bone interface, and less diminution of ROM with wear of the socket wall.