R. Lane Smith

A framework for the in vivo pathomechanics of osteoarthritis at the knee

The in vivo pathomechanics of osteoarthritis (OA) at the knee is described in a framework that is based on an analysis of studies describing assays of biomarkers, cartilage morphology, and human function (gait analysis). The framework is divided into an Initiation Phase and a Progression Phase. The Initiation Phase is associated with kinematic changes that shift load bearing to infrequently loaded regions of the cartilage that cannot accommodate the loads. The Progression Phase is defined following cartilage breakdown. During the Progression Phase, the disease progresses more rapidly with increased load. While this framework was developed from an analysis of in vivopathomechanics, it also explains how the convergence of biological, morphological, and neuromuscular changes to the musculoskeletal system during aging or during menopause lead to the increased rate of idiopathic OA with aging. Understanding the in vivo response of articular cartilage to its physical environment requires an integrated view of the problem that considers functional, anatomical, and biological interactions. The integrated in vivoframework presented here will be helpful for the interpretation of laboratory experiments as well as for the development of new methods for the evaluation of OA at the knee.

Periprosthetic Osteolysis in Total Hip Arthroplasty: the Role of Particulate Wear Debris

Much of the basic science research regarding total joint arthroplasty currently is focused on the biological mechanisms of osteolysis and aseptic loosening. However, the clinical problem of osteolysis is not new; in fact. osteolysis was one of the first problems associated with hip replacement surgery. Charnley’s experiment with Teflon acetabular components’ resulted in accelerated wear and particle generation: at that time, the destruction of hone associated with this problem was thought to represent culture-negative infection. Harris et al.2’ first described localized bone resorption in the femur in association with loose cemented total hip replacements. In that study. four hips having extensive resorption of bone that appeared radiographically as an infection or tumor were reported. However, the pattern of bone lysis (expansile lytic lesions) was distinctly different from that commonly noted in association with aseptic loosening. which is usually characterized by a linear resorption pattern. Histological analysis demonstrated sheets of macrophages with interspersed giant cells and birefningent material. which could not be identified further at that time. Subsequently, Jasty et al.” and Maloney et al.47 described focal femoral osteolysis in patients who had a stable femoral component that had been inserted with cement (Fig. 1-A). Histological analysis of the soft-tissue membrane revealed findings that were similar to those reported in association with aseptic loosening. The phenomenon of hone loss in association with cemented implants became known as cement disease35. This so-called cement disease was one of the reasons for the development of implants that were designed to he inserted without cement. It is now clear, however. that the term cement disease was a misnomer: osteolysis has been identified with increasing frequency in associ-

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

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.

Effect of size, concentration, surface area, and volume of polymethylmethacrylate particles on human macrophages in vitro

This study investigated effects of different sizes, concentrations, volumes, and surface areas of polymethylmethacrylate (PMMA) particles on human macrophages. Adherent peripheral blood monocytes isolated from five healthy individuals were exposed for 48 h to phagocytosable (0.325 micron and 5.5 microns) and nonphagocytosable (200 microns) spherical particles. Each particle size was tested over a range of concentrations (10(4)-10(11) particles per milliliter [0.325 micron], 10(2)-10(7) particles per milliliter [5.5 microns], 10(1)-10(4) particles per milliliter [200 microns]) to provide overlap in number, volume, and surface area. Primary human monocyte/macrophages were cultured in macrophage serum-free medium and 5% fetal calf serum. Macrophage viability was assessed by 3H-thymidine uptake and activation was quantified by release of interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, prostaglandin E2 (PGE2), and the lysosomal enzyme hexosaminidase. Medium alone served as a negative control; lipopolysaccharide (10 micrograms/mL) was also tested. PMMA particles were not toxic to human macrophages at any concentration tested. The smallest phagocytosable particles (0.325 micron) stimulated the release of interleukin-1 beta, interleukin-6, prostaglandin E2, and hexosaminidase at concentrations of 10(10)-10(11) particles/mL. The release of cytokines, PGE2, and hexosaminidase depended on the size, concentration, surface area, and volume of the phagocytosable particles. This study demonstrates that PMMA particle load Mi.e., the concentration of phagocytosable particles per tissue volume, characterized by size, surface area, and volume, rather than simply particle number-determines the degree of macrophage activation.

COX-2 selective NSAID decreases bone ingrowth in vivo.

Whether non‐steroidal anti‐inflammatory drug (NSAID)—induced suppression of bone ingrowth is due to cyclooxygenase‐1 (COX‐1) inhibition, cyclooxygenase‐2 (COX‐2) inhibition, or through a yet unidentified pathway is unknown. In this study, the effects of a non‐specific COX‐1 and COX‐2 inhibitor, versus a specific COX‐2 inhibitor on bone ingrowth and tissue differentiation are examined in vivo. Harvest chambers were implanted unilaterally in the tibiae of eight mature, New Zealand white rabbits. After a 6‐week period for osseointegration of the chamber, the following oral treatments were given for 4 weeks each, followed by a harvest in each case: drinking water with no NSAID (control 1), Naproxen sodium–a COX‐1 and COX‐2 non‐specific inhibitor at a dose of 110 mg/kg/day in the drinking water, drinking water with no NSAID (control 2), and Rofecoxib—a COX‐2 inhibitor at a dose of 12.5 mg/day inserted directly into the rabbits mouth. Harvested specimens were snap frozen, cut into serial 6 μm sections and stained with hematoxylin and eosin for general morphological characterization, and alkaline phosphatase (osteoblast marker). Sections were also processed for immunoperoxidase staining using monoclonal antibodies to identify cells expressing the vitronectin receptor (osteoclast‐like cells). With drinking water alone, the percentage of bone ingrowth averaged 24.8 ± 2.9% and 29.9 ± 4.5% respectively. Naproxen sodium in the drinking water and oral Rofecoxib decreased bone ingrowth significantly (15.9 ± 3.3%, p = 0.031 and 18.5 ± 2.4%, p = 0.035 compared to drinking water respectively). Both Naproxen sodium (p = 0.026) and Rofecoxib (p = 0.02) decreased the number of CD51 positive osteoclast‐like cells per section compared with drinking water alone. Rofecoxib decreased the area of osteoblasts per section area (p = 0.014) compared to controls, although the value for Naproxen sodium did not reach statistical significance. The results of the present study suggest that bone formation is suppressed by oral administration of an NSAID which contains a COX‐2 inhibitor. COX‐2 inhibitors currently taken for arthritis and other conditions may potentially delay fracture healing and bone ingrowth.

Quantitation of glycosaminoglycan hexosamine using 3-methyl-2-benzothiazolone hydrazone hydrochloride

Abstract This procedure allows analysis of hexosamine present in glycosaminoglycans under conditions of mild acid treatment. It utilizes the color complex formed by interaction of 3-methyl-2-benzothiazolone hydrazone hydrochloride (MBTH) and the 2,5-anhydrohexoses produced by deamination of hexosamines. Both glucosamine and galactosamine behave equivalently in this assay.

Revision joint replacement, wear particles, and macrophage polarization.

Currently, younger, more active patients are being offered total joint replacement (TJR) for end-stage arthritic disorders. Despite improved durability of TJRs, particle-associated wear of the bearing surfaces continues to be associated with particulate debris, which can activate monocyte/macrophages. Activated macrophages then produce pro-inflammatory factors and cytokines that induce an inflammatory reaction that activates osteoclasts leading to bone breakdown and aseptic loosening. We hypothesized that activated macrophages in tissues harvested from revised joint replacements predominantly express an M1 pro-inflammatory phenotype due to wear-particle-associated cell activation, rather than an M2 anti-inflammatory phenotype. We further questioned whether it is possible to convert uncommitted monocyte/macrophages to an M2 phenotype by the addition of interleukin-4 (IL-4), or whether it is necessary to first pass through an M1 intermediate stage. Retrieved periprosthetic tissues demonstrated increased M1/M2 macrophage ratios compared to non-operated osteoarthritic synovial tissues, using immunohistochemical staining and Western blotting. Uncommitted monocyte/macrophages with/without polymethyl-methacrylate particles were transformed to an M2 phenotype by IL-4 more efficiently when the cells were first passed through an M1 phenotype by exposure to endotoxin. Wear particles induce a pro-inflammatory microenvironment that facilitates osteolysis; these events may potentially be modulated favorably by exposure to IL-4.

Modulating osteogenesis of mesenchymal stem cells by modifying growth factor availability

Growth factors control the proliferation and differentiation of osteoprogenitor cells. This study explores the effects of modulating growth factors (VEGF, IGF-1, FGF-2 and BMP-2) on osteogenesis of mesenchymal stem cells (MSCs) in vitro. Constant and profiled delivery protocols, in accordance with protein expression in vitro, were applied to deliver or neutralize growth factors. Cell number, alkaline phosphatase (ALP-2) and osteocalcin (OC) expression, and mineralization were measured as outcome variables. Profiled addition of VEGF increased MSC proliferation. Constant and profiled application of FGF-2 and neutralization of IGF-1 and BMP-2 decreased ALP-2 levels. Profiled addition of BMP-2 vastly increased OC release from MSCs, but constant addition of IGF-1, constant and profiled neutralization of IGF-1 and FGF-2 reduced OC levels. Constant addition of IGF-1 and FGF-2, as well as profiled loading of FGF-2 decreased mineralization of MSCs. This study indicated that endogenous IGF-1 and FGF-2 are essential to osteogenesis; excess IGF-1 and FGF-2 were inhibitory to bone formation. Selective, temporally specific addition of growth factors, such as BMP-2 and VEGF appears to be an important strategy to enhance osteogenesis.

Effects of shear stress on nitric oxide and matrix protein gene expression in human osteoarthritic chondrocytes in vitro.

Mechanical loading alters articular cartilage metabolism. However, mechanisms underlying intracellular signaling and communication between cells in response to mechanical stresses remain enigmatic. This study tested the hypothesis that shear stress‐induced nitric oxide (NO) production participates in the regulation of matrix protein gene expression. The data presented here demonstrate that exposure of human osteoarthritic chondrocytes to a continuously applied shear stress (1.64 Pa) upregulated NO synthase gene expression and increased NO release by 1.8‐, 2.4‐, and 3.5‐fold at 2, 6, and 24 h, respectively. Exposure of chondrocytes to a short duration of shear stress for 2 h resulted in the release of accumulation of NO in the culture medium. Exposure of chondrocytes to shear stress for 2, 6, and 24 h inhibited type II collagen mRNA signal levels by 27%, 18% and 20% after a constant post‐shear incubation period of 24 h. Aggrecan mRNA signal levels were inhibited by 30%, 32% and 41% under identical conditions. Addition of an NO antagonist increased type II collagen mRNA signal levels by an average of 1.8‐fold (137% of the un‐sheared control) and reestablished the aggrecan mRNA signal levels by an average of 1.4‐fold after shear stress (92% of the un‐sheared control) (ANOVA p < 0.05). These data support the hypothesis that shear stress‐induced NO release may influence the development of degenerative joint diseases by inhibiting matrix macromolecule synthesis.