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Stem cells hold great promise for treating cartilage degenerative diseases such as osteoarthritis (OA). The efficacy of stem cell-based therapy for cartilage repair is highly dependent on their interactions with local cells in the joint. This study aims at evaluating the interactions between osteoarthritic chondrocytes (OACs) and adipose-derived stem cells (ADSCs) using three dimensional (3D) biomimetic hydrogels. To examine the effects of cell distribution on such interactions, ADSCs and OACs were co-cultured in 3D using three co-culture models: conditioned medium (CM), bi-layered, and mixed co-culture with varying cell ratios. Furthermore, the effect of transforming growth factor (TGF)-β3 supplementation on ADSC–OAC interactions and the resulting cartilage formation was examined. Outcomes were analyzed using quantitative gene expression, cell proliferation, cartilage matrix production, and histology. TGF-β3 supplementation led to a substantial increase in cartilage matrix depositions in all groups, but had differential effects on OAC–ADSC interactions in different co-culture models. In the absence of TGF-β3, CM or bi-layered co-culture had negligible effects on gene expression or cartilage formation. With TGF-β3 supplementation, CM and bi-layered co-culture inhibited cartilage formation by both ADSCs and OACs. In contrast, a mixed co-culture with moderate OAC ratios (25% and 50%) resulted in synergistic interactions with enhanced cartilage matrix deposition and reduced catabolic marker expression. Our results suggested that the interaction between OACs and ADSCs is highly dependent on cell distribution in 3D and soluble factors, which should be taken into consideration when designing stem cell-based therapy for treating OA patients.

Objective This study assessed T1ρ relaxation dispersion, measured by magnetic resonance imaging (MRI), as a tool to noninvasively evaluate cartilage material and biochemical properties. The specific objective was to answer two questions: (1) does cartilage initial elastic modulus ( E0) correlate with T1ρ dispersion effects and (2) does collagen or proteoglycan content correlate with T1ρ dispersion effects? Design Cadaveric patellae with and without visible cartilage damage on conventional MR were included. T2 and T1ρ relaxation times at 500 and 1000 Hz spin-lock field amplitudes were measured. We estimated T1ρ dispersion effects by measuring T1ρ relaxation time at 500 and 1000 Hz and T2 relaxation time and using a new tool, the ratio T1ρ/T2. Cartilage initial elastic modulus, E0, was measured from initial response of mechanical indentation creep tests. Collagen and proteoglycan contents were measured at the indentation test sites; proteoglycan content was measured by their covalently linked sulfated glycosaminoglycans (sGAG). Pearson correlation coefficients were determined, taking into account the clustering of multiple samples within a single patella specimen. Results Cartilage initial elastic modulus, E0, increased with decreasing values of T1ρ/T2 measurements at both 500 Hz ( P = 0.034) and 1000 Hz ( P = 0.022). 1/T1ρ relaxation time (500 Hz) increased with increasing sGAG content ( P = 0.041). Conclusions T1ρ/T2 ratio, a new tool, and cartilage initial elastic modulus are both measures of water–protein interactions, are dependent on the cartilage structure, and were correlated in this study.

WHERE ARE WE NOW?: Biological treatments, defined as any nonsurgical intervention whose primary mechanism of action is reducing the host response to wear and/or corrosion products, have long been postulated as solutions for osteolysis and aseptic loosening of total joint arthroplasties. Despite extensive research on drugs that target the inflammatory, osteoclastic, and osteogenic responses to wear debris, no biological treatment has emerged as an approved therapy. We review the extensive preclinical research and modest clinical research to date, which has led to the central conclusion that the osteoclast is the primary target. We also allude to the significant changes in health care, unabated safety concerns about chronic immunosuppressive/antiinflammatory therapies, industry's complete lack of interest in developing an intervention for this condition, and the practical issues that have narrowly focused the possibilities for a biologic treatment for wear debris-induced osteolysis. WHERE DO WE NEED TO GO?: Based on the conclusions from research, and the economic, regulatory, and practical issues that limit the future directions toward the development of a biologic treatment, there are a few rational approaches that warrant investigation. These largely focus on FDA-approved osteoporosis therapies that target the osteoclast (bisphosphonates and anti-RANK ligand) and recombinant parathyroid hormone (teriparatide) prophylactic treatment to increase osseous integration of the prosthesis to overcome high-risk susceptibility to aseptic loosening. The other roadblock that must be overcome if there is to be an approved biologic therapy to prevent the progression of periprosthetic osteolysis and aseptic loosening is the development of radiological measures that can quantify a significant drug effect in a randomized, placebo-controlled clinical trial. We review the progress of volumetric quantification of osteolysis in animal studies and clinical pilots. HOW DO WE GET THERE?: Accepting the aforementioned rigid boundaries, we describe the emergence of repurposing FDA-approved drugs for new indications and public (National Institutes of Health, FDA, Centers for Disease Control and Prevention) and private (universities and drug and device manufactures) partnerships as the future roadmap for clinical translation. In the case of biologic treatments for wear debris-induced osteolysis, this will involve combined federal and industry funding of multicenter clinical trials that will be run by thought leaders at large medical centers.

Background: An intra-articular injection of local anesthetics is a common procedure for diagnostic and therapeutic purposes. It has been shown that these agents are toxic to articular cartilage and synovial tissue in a dose- and time-dependent fashion, and in some cases, they may lead to postarthroscopic glenohumeral chondrolysis (PAGCL). However, the role of apoptosis in cell death is still unclear, and the potential role of apoptosis inhibition in minimizing chondrocyte and synovial cell death has not been reported. Purpose: (1) To quantify the degree of apoptotic cell death in chondrocytes and synovial cells exposed to local anesthetics, and (2) to determine whether caspase inhibition could reduce cell death. Study Design: Controlled laboratory study. Methods: Human chondrocytes and synovial cells were expanded in vitro and exposed to normal saline, 0.5% bupivacaine, 0.5% ropivacaine, 1% lidocaine, or 1:1000 epinephrine for 90 minutes. Apoptosis was then detected at 1, 3, 5, and 7 days after exposure using terminal deoxynucleotidyl transferase (TdT)–mediated dUTP nick-end labeling (TUNEL) and immunohistochemistry. Apoptosis was then inhibited using the pan-caspase inhibitor z-vad- fmk. Results were normalized to normal saline controls and analyzed by generalized regression models and pairwise confidence intervals. Results: Analysis of cumulative chondrocyte apoptosis relative to controls after anesthetic exposure demonstrated more than 60% cell death with 0.5% bupivacaine and 1:1000 epinephrine. The greatest chondroprotective effect of caspase inhibition occurred with 0.5% ropivacaine. Similarly, in synovial cells, epinephrine was also very cytotoxic; however, 1% lidocaine induced the most apoptosis. Synovial cells exposed to 0.5% ropivacaine were again most sensitive to protective caspase inhibition. Conclusion: Local anesthetics induce chondrocyte and synovial cell apoptosis in a time-dependent fashion, with peak apoptosis occurring 5 days after exposure. Both chondrocytes and synovial cells are most sensitive to caspase inhibition after exposure to 0.5% ropivacaine. Clinical Relevance: Apoptosis inhibition may be an effective strategy in minimizing chondrocyte and synovial cell death after exposure to anesthetics. Further investigation is clinically warranted.

Wear particles generated with use of total joint replacements incite a chronic macrophage-mediated inflammatory reaction, which leads to implant failure. Macrophage activation may be polarized into two states, with an M1 proinflammatory state dominating an alternatively activated M2 anti-inflammatory state. We hypothesized that IL-4, an activator of M2 macrophages, could modulate polyethylene (PE) particle-induced osteolysis in an experimental murine model. Four animal groups included (a) calvarial saline injection with harvest at 14 days (b) single calvarial injection of PE particles subcutaneously (SC) without IL-4 (c) PE particles placed as in (b), then IL-4 given SC for 14 consecutive days and (d) PE particles as in (b) then IL-4 beginning 7 days after particle injection for 7 days. The calvarial bone volume to total tissue volume was measured using microCT and histomorphometry. Calvaria were cultured for 24 h to assess release of RANKL, OPG, TNF-α, and IL-1ra and isolation and identification of M1 and M2 specific proteins. MicroCT and histomorphometric analysis showed that bone loss was significantly decreased following IL-4 administration to PE treated calvaria for both 7 and 14 days. Western blot analysis showed an increased M1/M2 ratio in the PE treated calvaria, which decreased with addition of IL-4. Cytokine analysis showed that the RANKL/OPG ratio and TNF-α/IL-1ra ratio decreased in PE-treated calvaria following IL-4 addition for 14 days. IL-4 delivery mitigated PE particle-induced osteolysis through macrophage polarization. Modulation of macrophage polarization is a potential treatment strategy for wear particle induced periprosthetic osteolysis.

Purpose Exogenously administered mesenchymal stem cells and bioactive molecules are known to enhance tendon healing. Biomolecules have been successfully delivered using sutures that elute growth factors over time. We sought to evaluate the histologic and biomechanical effect of delivering both cells and bioactive substrates on a suture delivery vehicle in comparison with sutures coated with bioactive substrates alone. Methods Bone marrow–derived stem cells were harvested from Sprague-Dawley rat femurs. Experimental cell and substrate-coated, coated suture (CS) group sutures were precoated with intercellular cell adhesion molecule 1 and poly-L-lysine and seeded with labeled bone marrow–derived stem cells. Control (substrate-only [SO] coated) group sutures were coated with intercellular cell adhesion molecule 1 and poly-L-lysine only. Using a matched-paired design, bilateral Sprague-Dawley rat Achilles tendons (n = 105 rats) were transected and randomized to CS or SO repairs. Tendons were harvested at 4, 7, 10, 14, and 28 days and subjected to histologic and mechanical assessment. Results Labeled cells were present at repair sites at all time points. The CS suture repairs displayed statistically greater strength compared to SO repairs at 7 days (12.6 ± 5.0 N vs 8.6 ± 3.7 N, respectively) and 10 days (21.2 ± 4.9 N vs 16.4 ± 4.8 N, respectively). There was no significant difference between the strength of CS suture repairs compared with SO repairs at 4 days (8.1 ± 5.1 N vs 6.6 ± 2.3 N, respectively), 14 days (22.8 ± 7.3 N vs 25.1 ± 9.7 N, respectively), and 28 days (40.9 ± 12.4 N vs 34.6 ± 15.0 N, respectively). Conclusions Bioactive CS sutures enhanced repair strength at 7 to 10 days. There was no significant effect at later stages. Clinical relevance The strength nadir of a tendon repair occurs in the first 2 weeks after surgery. Bioactive suture repair might provide a clinical advantage by jump-starting the repair process during this strength nadir. Improved early strength might, in turn allow earlier unprotected mobilization.

We evaluated the fate of pluripotential stem cells adherent to a suture carrier after being passed through tendon tissue in vitro.FiberWire suture segments were coated with poly-L-lysine (PLL) and a 2 × 10(6) C3H10T1/2 (a mouse embryo pluripotential cell line) cell suspension. The sutures were incubated for 7 days, passed through two 1-cm segments of acellularized rabbit Achilles tendons and tied (horizontal mattress). The repairs were frozen and sectioned (6 μm). The sections were stained with 4',6-diamidino-2-phenylindole and a live/dead viability/cytotoxicity (calcein/ethidium homodimer) kit and examined with fluorescent microscopy to evaluate cell presence and viability. Alamar Blue was used in parallel to assess metabolic activity.PLL-coated sutures showed a 3-fold increase in fluorescence when compared with the phosphate-buffered saline-coated controls. At day 3, fluorescence was 2.2 times greater. At day 5, a 2-fold increase was found, and at day 8 there was no significant difference in values. Furthermore, after delivery of the cells into tendon, fluorescence readings for the samples (n = 19) showed 9450 compared with the positive control at 21,218. At 96 hours the mean was 27,609 compared with 34,850 for the positive control. The difference in fluorescence means at 48 hours and 96 hours were significant (p.001). Live-dead and DAPI staining confirmed the presence of live cells at the tendon repair site.Sutures seeded with pluripotential embryonic cells deliver cells to a tendon repair site. The cells deposited at the repair site survive the trauma of passage and remain metabolically active, as seen in staining and metabolic assay studies. Use of bioactive sutures leads to repopulation of the acellular zone surrounding sutures within the tendon.

Periprosthetic osteolysis of joint replacements caused by wear debris is a significant complication of joint replacements. Polymethylmethacrylate (PMMA) particles have been shown to inhibit osteogenic differentiation, but the molecular mechanism has not been previously determined. In this study, we exposed differentiating MC3T3-E1 preostoblast cells to PMMA particles and determined the changes that occurred with respect to p38 mitogen-activated protein kinase (MAPK) activity and the transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMP) signaling pathways. In the absence of particles, MC3T3-E1 cells demonstrate activation of p38 MAPK on day 8 of differentiation; however, when treated with PMMA particles, differentiating MC3T3-E1 cells demonstrate the suppression of p38 activity on day 8 and show activation of p38 on days 1 and 4. On day 4 of particle exposure, the differentiating MC3T3-E1 cells show significant downregulation of TGF-beta1 expression, which is involved in osteoblast differentiation, and a significant upregulation of the expression of BMP3 and Sclerostin (SOST), which are negative regulators of osteoblast differentiation. By day 8 of particle exposure, the changes in TGF-beta1, BMP3, and SOST expression are opposite of those seen on day 4. This study has demonstrated the distinct changes in the molecular profile of MC3T3-E1 cells during particle-induced inhibition of osteoblast differentiation. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

The aim of this study was to test the hypothesis that a change in serum cartilage oligomeric matrix protein (COMP) concentration is related to joint load during a 30-min walking exercise in patients with medial compartment knee osteoarthritis (OA) and in age-matched control subjects. Blood samples were drawn from 42 patients with medial compartment knee OA and from 41 healthy age-matched control subjects immediately before, immediately after, and 0.5, 1.5, 3.5, and 5.5 h after a 30-min walking exercise on a level outdoor walking track at self-selected normal speed. Serum COMP concentrations were determined using a commercial ELISA. Basic time-distance gait variables were recorded using an activity monitor. Joint loads were measured using gait analysis. Serum COMP concentrations increased immediately after the walking exercise (+6.3% and +5.6%; p < 0.001) and decreased over 5.5 h after the exercise (-11.1% and -14.6%; p < 0.040 and p = 0.001) in patients and control subjects, respectively. The magnitude of increase in COMP concentration did not differ between groups (p = 0.902) and did not correlate with any variables describing ambulatory loads at the joints of the lower extremity. These results, taken together with a previous study of a younger healthy population, suggest the possibility that the influence of ambulatory loads on cartilage turnover is dependent on age.

Continued production of wear debris affects both initial osseointegration and subsequent bone remodeling of total joint replacements (TJRs). However, continuous delivery of clinically relevant particles using a viable, cost effective, quantitative animal model to simulate the scenario in humans has been a challenge for orthopedic researchers. In this study, we successfully infused blue-dyed polystyrene particles, similar in size to wear debris in humans, to the intramedullary space of the mouse femur for 4 weeks using an osmotic pump. Approximately 40% of the original particle load (85 microL) was delivered into the intramedullary space, an estimate of 3 x 10(9) particles. The visible blue dye carried by the particles confirmed the delivery. This model demonstrated that continuous infusion of particles to the murine bone-implant interface is possible. In vivo biological processes associated using wear debris particles can be studied using this new animal model.

Orthopedic wear debris has been implicated as a significant inhibitory factor of osteoblast differentiation. Polymethylmethacrylate (PMMA) particles have been previously shown to inhibit the differentiation of osteoprogenitors in heterogeneous murine marrow stromal cell cultures, but the effect of PMMA particles on pure osteoprogenitor populations remains unknown. In this study, we challenged murine MC3T3-E1 osteoprogenitor cells with PMMA particles during their initial differentiation in osteogenic medium. MC3T3-E1 cultures challenged with PMMA particles showed a gradual dose-dependent decrease in mineralization, cell number, and alkaline phosphatase activity at low particle doses (0.038–0.150% v/v) and complete reduction of these outcome parameters at high particle doses (≥0.300% v/v). MC3T3-E1 cultures challenged with a high particle dose (0.300% v/v) showed no rise in these outcome parameters over time, whereas cultures challenged with a low particle dose (0.075% v/v) showed a normal or reduced rate of increase compared to controls. Osteocalcin production was not significantly affected by particles at all doses tested. MC3T3-E1 cells grown in conditioned medium from particle-treated MC3T3-E1 cultures showed a significant reduction in mineralization only. These results indicate that direct exposure of MC3T3-E1 osteoprogenitors to PMMA particles results in suppression of osteogenic proliferation and differentiation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:932–936, 2008

This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-beta1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-beta1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.

Aseptic loosening of implants following total joint arthroplasty remains a major cause of implant failure. Particulate debris generated primarily from wear results in inflammatory mediated periprosthetic osteolysis. Titanium is a commonly utilized metal in joint arthroplasty and titanium debris induces the production of the pro-inflammatory cytokine IL-1. To further elucidate the role of IL-1, this study examined the response of murine femora to the presence of titanium particles following implantation of an intramedullary rod in mice lacking the receptor for IL-1. We hypothesized that the inflammatory effects of wear debris on bone would be mitigated in IL-1R1 deficient mice with a resultant decrease in resorption. Femora receiving titanium particles demonstrated a marked inflammatory response in wild-type mice with increased endocortical resorption, periprosthetic membrane formation, and significant histomorphometric changes. Femora exposed to titanium particles in the knockout mice also demonstrated osteolysis with irregular deposition of trabecular bone and increased cortical porosity. The persistence of inflammation and osteolysis, despite the lack of functional IL-1R1, suggests a multi-factorial role for IL-1 in the proinflammatory cascade resulting from wear debris. This intramedullary murine model provides the ability to evaluate and quantify the proinflammatory cascade in an in vivo model approximating prosthesis failure.

In this study, we delineate the sequential expression of selected growth factors associated with bone formation in vitro. Mineralization, osteocalcin, and alkaline phosphatase (ALP-2) were measured...

Periprosthetic bone loss induced by implant wear debris may be a combined effect of osteolysis and reduced bone formation resulting from particle-induced suppression of osteoprogenitor differentiation. This study investigated the time-dependent effects of polymethylmethacrylate (PMMA) particles on the osteogenic capability of bone marrow osteoprogenitor cells during the early phase of differentiation. Murine bone marrow cells were challenged with PMMA particles (0.30% v/v) on the first day of growth in osteogenic medium. Particles were removed from culture after 1, 3, and 5 days, respectively, after which cell growth in osteogenic medium was continued until the 15th day. Bone marrow osteoprogenitor cells exposed to particles during the first 5 days of differentiation showed complete, irreversible inhibition of proliferation, alkaline phosphatase expression, and mineralization. Osteoprogenitors exposed to particles for more than 5 days showed the same degree of inhibition, while those exposed to particles for less than 5 days showed a diminished inhibitory response. Conditioned medium from particle-treated cells did not suppress osteogenic development, demonstrating that suppression of osteogenesis was not due to secreted inhibitory factors. This study has shown that the early phase of osteoprogenitor differentiation is a crucial time period during which exposure to PMMA particles causes irreversible inhibition of osteogenesis. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:450–457, 2007

Wear particles from total joint arthroplasties are constantly being generated throughout the lifetime of an implant. Since mesenchymal stem cells and osteoprogenitors from the bone marrow are the precursors of osteoblasts, the reaction of these cells to orthopaedic wear particles is critical to both initial osseointegration of implants and ongoing regeneration of the periprosthetic bed. Particles less than 5 microm can undergo phagocytosis by mature osteoblasts, with potential adverse effects on cellular viability, proliferation and function. The specific effects are dependent on particle composition and dose. Metal and polymer particles in non-toxic doses stimulate pro-inflammatory factor release more than ceramic particles of a similar size. The released factors inhibit markers of bone formation and are capable of stimulating osteoclast-mediated bone resorption. Mesenchymal stem cells and osteoprogenitors are also profoundly affected by wear particles. Titanium and polymethylmethacrylate particles inhibit bone cell viability and proliferation, and downregulate markers of bone formation in a dose- and time-dependent manner. Future studies should delineate the molecular mechanisms by which particles adversely affect mesenchymal stems cells and the bone cell lineage and provide strategies to modulate these effects.

Aseptic implant loosening of total joint replacements often results from particle-mediated bone loss, which may be a combined effect of osteolysis and suppressed bone formation. Bone regeneration in the prosthetic bed depends on the activity of osteoblasts and their differentiation from osteoprogenitors in the bone marrow. This study investigated the effects of polymethylmethacrylate (PMMA) particles on the ability of bone marrow osteoprogenitors to differentiate into osteoblasts in vitro. Murine bone marrow cells challenged with PMMA particles on the first day of differentiation in osteogenic medium showed a dose-dependent decrease in osteoprogenitor proliferation, alkaline phosphatase expression, and mineralization. Undifferentiated bone marrow cells pretreated with PMMA particles in nonosteogenic medium for 5 days also showed a dose-dependent loss in osteogenic potential, which was sustained throughout subsequent growth in particle-free, osteogenic medium. Bone marrow cells challenged with PMMA particles after the fifth day of differentiation in osteogenic medium showed significant reductions in cellular proliferation, but not alkaline phosphatase expression and mineralization, indicating that bone marrow cells were most sensitive to particle treatment during the first 5 days of differentiation. This study demonstrated that PMMA particles inhibit osteoblastic differentiation of bone marrow osteoprogenitor cells, which may contribute to periprosthetic bone loss and implant failure.

The effects of a short course of a COX-2 inhibitor on bone healing when the drug is discontinued are unknown. We examined the effects of rofecoxib on bone ingrowth over a 6-week period using a well-defined animal model. The Bone Harvest Chamber was implanted bilaterally in mature rabbits. After osseointegration of the chamber, the following treatments were given for 6 weeks each, followed by a harvest in each case: control-no drug; oral rofecoxib (12.5 mg/day) for the first 2 of 6 weeks; washout period-no drug; oral rofecoxib for the last 2 of 6 weeks; washout period-no drug; rofecoxib given continuously for all 6 weeks. Harvested specimens were snap-frozen, cut into serial 6-μm sections, and stained with hematoxylin and eosin and alkaline phosphatase (osteoblast marker), and processed using immunohistochemistry to identify the vitronectin receptor (osteoclast-like cells). Rofecoxib given continuously for 6 weeks yielded statistically less bone ingrowth compared to the control treatment. Rofecoxib given during the initial or final 2 weeks of a 6-week treatment did not appear to interfere with bone ingrowth. This suggests that the effects of COX-2 inhibitors on bone are less profound when the drug is administered for a short period of time. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res 72A: 279–287, 2005

Background We examined the effects of ultra-high molecular weight polyethylene (UHMWPE) particles on mononuclear cell proinflammatory gene expression in a novel murine model. We hypothesized that m...

Wear debris from total joint replacement prostheses is implicated in periprosthetic osteolysis and implant loosening. The pathophysiology of this biological process remains unclear. Animal models of particle-induced osteolysis have proven useful in the study of specific tissue responses to wear debris. However, existing in vivo murine models of particle-mediated inflammation do not permit analysis of cortical bone degradation. This study describes a murine model of particle disease using an intramedullary rod in the mouse femur to parallel the clinical situation. The model consists of placing a 10-mm-long Kirschner wire retrograde in both femurs of C57b1/6 male mice via a medial parapatellar arthrotomy. Phagocytosable titanium particles were also implanted unilaterally to replicate generation of wear debris. Mice were sacrificed at 2, 10, and 26 weeks and whole femurs were cultured for 72 h. Levels of interleukin-6, monocyte chemotactic protein-1, and macrophage colony stimulating factor were assayed by ELISA. Transverse histological sections, at the level of the implant, were taken and stained with hematoxylin and eosin (H&E). Results demonstrated increased expression of proinflammatory mediators at 2 weeks in femora with rod and particles compared to femora with rods alone. Destruction of the endosteum was evident at 2, 10, and 26 weeks in the femora with titanium. This novel murine model of particle-induced intramedullary inflammation may facilitate cost-effective genetic studies and offers investigators a simple, clinically relevant intramedullary model to readily examine the pathogenesis of particle-mediated periprosthetic osteolysis.

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.

Objective This study tested the hypothesis that intermittent hydrostatic pressure applied to human osteoarthritic chondrocytes modulates matrix metalloproteinase and pro-inflammatory mediator release in vitro . Design Human osteoarthritic articular chondrocytes were isolated and cultured as primary high-density monolayers. For testing, chondrocyte cultures were transferred to serum-free medium and maintained without loading or with exposure to intermittent hydrostatic pressure (IHP) at 10MPa at a frequency of 1Hz for periods of 6, 12 and 24h. Levels of matrix metalloproteinase-2, -9 (MMP-2, -9), tissue inhibitor of metalloproteinase-1 (TIMP-1), and the pro-inflammatory mediators, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), released into the culture medium were assessed by ELISA. Matrix metalloproteinase activity was confirmed by zymographic analysis. Results In the absence of IHP, levels of MMP-2, TIMP-1, IL-6, and MCP-1 in the chondrocyte culture medium increased in a time-dependent manner. Application of IHP decreased MMP-2 levels at all time periods tested, relative to unloaded control cultures maintained for the same time periods. Although 84/82kDa bands were faintly detectable by zymography, MMP-9 levels were not quantifiable in medium from loaded or unloaded cultures by ELISA. TIMP-1 levels were not altered in response to IHP at any time period tested. IL-6 and MCP-1 levels decreased in cultures exposed to IHP at 12 and 24h, relative to unloaded control cultures maintained for the same time periods. Conclusion IHP decreased release of MMP-2, IL-6 and MCP-1 by osteoarthritic chondrocytes in vitro suggesting that pressure influences cartilage stability by modulating chondrocyte expression of these degradative and pro-inflammatory proteins in vivo .

Although interleukin-1 (IL-1) has been implicated in the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are largely unknown. Recently, a cytokine-driven, stromal cell-free mouse osteoclastogenesis model was established. A combination of macrophage colony stimulating factor (M-CSF) and receptor activator of NFkappaB ligand (RANKL) was proven to be sufficient in inducing differentiation of bone marrow hematopoietic precursor cells to bone-resorbing osteoclasts in the absence of stromal cells or osteoblasts. This study utilizes this model to examine the impact of human IL-1beta on in vitro osteoclastogenesis of bone marrow progenitor cells. We found that osteoclast precursor cells failed to undergo osteoclastogenesis when treated with IL-1 alone. In contrast, IL-1 dramatically up-regulated osteoclastogenesis by 2.5- to 4-folds in the presence of RANKL and M-CSF. The effect can be significantly blocked by IL-1 receptor antagonist (p < 0.01). Tumor necrosis factor-alpha (TNF-alpha) was undetectable in the culture medium of differentiating osteoclasts induced by IL-1. Adding exogenous TNF-alpha neutralizing antibody had no influence on the IL-1-induced effect as well. These results show that in the absence of stromal cells, IL-1 exacerbates osteoclastogenesis by cooperating with RANKL and M-CSF, while TNF-alpha is not involved in this IL-1-stimulated osteoclast differentiation pathway.

This study tests the hypothesis that transcription factor activation by exposure of macrophages to titanium particles can be modulated by the addition of the antiinflammatory cytokine, interleukin 10 (IL-10). The experiments were carried out with primary human monocyte/macrophages that were treated in the presence or absence of IL-10 with and without exposure to titanium particles. The time course for experiments varied from 1 h-5 h for analysis of nuclear protein and up to 48 h for analysis of cytokine release. Transcription factor translocation to the nucleus was analyzed using electrophoretic gel shift assays and cytokine release was quantified by enzyme-linked immunosorbent assay. Addition of titanium particles increased release of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-1 beta (IL-1 beta). In addition, titantium particle induced translocation of the transcription factors, NF-kappa B and NF-IL6, in the nucleus within 1 h. Treatment of macrophages with IL-10 prior to exposure to titanium particles decreased translocation of NF-IL6 but did not significantly alter nuclear levels of NF-kappa B. In addition, pretreatment of the cells with IL-10 decreased particle-induced cytokine release. These data show that antiinflammatory cytokines may provide a mechanism by which particle-induced inflammatory response may be modulated in vivo.

Osseointegration of porous-coated implants during revision arthroplasty procedures is often impeded due to the presence of residual granuloma, particulate debris, and a sclerotic, dysvascular bone bed. We hypothesized that local infusion of recombinant fibroblast growth factor (FGF-2) would increase bone ingrowth in an in vivo model of tissue differentiation in the rabbit tibia in the presence of phagocytosable polyethylene particles. A drug test chamber (DTC) was implanted in the proximal medial tibial metaphysis of mature rabbits unilaterally. The chamber contained a 1× 1 × 5-mm tunnel for tissue ingrowth, and was connected to an osmotic diffusion pump. FGF-2 was infused at dosages of 0, 0.5, 5, 50, or 500 ng/day for a 3-week period, with subsequent harvesting of the ingrown tissue after each 3-week treatment. The effects of ultrahigh molecular weight polyethylene particles (0.5-μm diameter) on tissue ingrowth were determined by adding particles to the chamber at concentrations of 5.8 × 1011 (low dose) or 1.7 × 1012 (high dose) particles/mL, with and without infusion of 50 ng/day of FGF for 3 weeks. The tissue forming in the chamber was harvested after each treatment for histologic processing and morphometric analysis of bone ingrowth. Statistical analysis was performed using parametric tests (ANOVA), nonparametric tests (Kruskal–Wallis test) and post hoc tests. In the absence of particles, infusion of 50 ng FGF-2 per day yielded the greatest amount of bone ingrowth. The high dose of particles suppressed bone ingrowth into the chamber, but the low dose particles did not (p = 0.0002, 95% confidence limits = 9.19–18.80). Infusion of 50 ng FGF-2 per day significantly increased net bone formation in the presence of high-dose UHMWPE particles (p = 0.039, 95% confidence limits = 1.41–6.79). There was a trend for decreased numbers of vitronectin-receptor positive (osteoclast-like) cells with the addition of FGF-2, compared to particles alone (p = 0.08). Local delivery of FGF-2 may prove useful in mitigating the adverse effects of wear debris (e.g., in treating early osteolytic lesions), and facilitating osseointegration of revision total joint replacements in situations where the bone bed is suboptimal and residual particles and granulomatous tissue are present. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 65A: 454–461, 2003

Wear particles generated after total joint arthroplasty activate monocyte/macrophages and incite formation of a granulomatous periprosthetic tissue associated with bone loss and implant loosening. This study tested the hypothesis that selective opsonization of orthopedic implant biomaterial wear particles by human serum proteins influences monocyte/macrophage activation. Serum protein binding to metallic, polymeric, and ceramic particles was determined by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Individual proteins bound to particles were subsequently identified using two-dimensional SDS-PAGE, microsequencing techniques, and SWISS-PROT analysis. Effects of selective protein opsonization on particle-induced monocyte/macrophage activation were assessed by quantification of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha release. Results from one-dimensional gel analyses revealed distinct serum protein-binding patterns specific for each material tested. Two-dimensional gel analysis together with amino acid sequencing of the prominent protein species confirmed the presence of albumin and alpha-1-antitrypsin bound to all particles tested. In contrast to the metallic particles, apolipoprotein was a major species associated with polymeric particles. Opsonization of PMMA particles with purified preparations of each of the identified proteins showed that albumin significantly enhanced particle-induced monocyte/macrophage activation. These data confirm orthopedic biomaterial specific binding of human serum proteins and demonstrate that albumin exacerbates particle-induced monocyte/macrophage activation. Alterations in the chemical and surface properties of orthopedic biomaterials to modulate protein interactions may improve implant longevity.

This study examined the relationship between acetabular cartilage properties after hemiarthroplasty surgery and surgical variables including femoral head size and position. Nineteen sheep received unilateral hip arthroplasties and were euthanized one year post-operatively to harvest the femora and acetabula. Cartilage histology, biochemistry and material properties were determined from samples located in the superior load-bearing region. Femoral head size mismatch, leg length difference, anterior-posterior and medial lateral offset and anteversion were measured. In the acetabulum. substantial cartilage degradation occurred with widespread librillation and significant changes in the biochemical and material properties compared to the intact contralateral joint. Regression analyses on the surgical variables explained 75-80% of the changes in tissue biochemistry but did not explain the material changes. Head size mismatch and leg length difference were the most significant contributors of the five variables examined and therefore may be critical to successful outcome in hemiarthroplasty.

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.

C-C chemokines are soluble mediators that occur in a periprosthetic granuloma and influence recruitment, localization and activation of inflammatory cells. This study tested effects of titanium and polymethylmethacrylate (PMMA) particles on expression of selected C-C chemokines in cultured human fibroblasts. The C-C chemokines analyzed included monocyte chemoattractant protein-1. 2 (MCP-1. 2), monocyte inflammatory protein-1 alpha (MIP-1 alpha), and regulated on activation, normal T-cell expressed and secreted protein (RANTES). Interleukin-1 beta (IL-1 beta) served as a known stimulator of chemokine release while interleukin-6 (IL-6) expression served as a marker for fibroblast activation. Protein and mRNA signal levels were determined by ELISA and RT-PCR, respectively. The results demonstrated that exposure of fibroblasts to titanium and PMMA particles resulted in increased release of MCP-1 in a dose- and time-dependent manner. After 24 h, titanium particles maximally upregulated MCP-1 release 7-fold while PMMA particles increased MCP-1 levels 2-fold, when compared to unchallenged fibroblasts. MCP-2, MIP-1 alpha and RANTES levels remained unchanged following exposure of fibroblasts to titanium or PMMA particles at any concentration or time point tested. However, IL-1 beta stimulated release of MCP-1, MCP-2, and RANTES, but not MIP-1 alpha from the fibroblasts. IL-1 beta, not particles, exhibited the most prominent effect on MCP-1 mRNA levels. Increased release of MCP-1 from fibroblasts exposed to titanium and PMMA particles coincided with increased release of IL-6. This study suggests that release of chemoattractant factors from fibroblasts localized in periprosthetic membranes enhances the chronic inflammatory process leading to bone resorption and implant loosening.

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-alpha 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-alpha 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.

The local delivery of exogenous growth factors may help achieve a stable, long-lasting prosthetic interface around primary and revision joint replacements. This study examines the effects of local infusion of transforming growth factor beta (TGFbeta) in an in vivo model of tissue differentiation within bone. The Drug Test Chamber was implanted in the proximal medial tibial metaphysis of 8 mature rabbits unilaterally. The chamber contained a 1 x 1 x 5 mm canal for tissue ingrowth. The chamber was connected to an osmotic diffusion pump via polyvinyl tubing. 3.5 microg of recombinant TGFbeta1 was infused for 1 day or 1 week with subsequent harvesting of the ingrown tissue after 3 weeks. Each TGFbeta treatment was followed by two, 3-week infusions of carrier alone and harvesting of the ingrown tissue. TGFbeta for 1 day increased, and TGFbeta for 1 week decreased the percentage of bone in the chamber, compared to the initial control harvest after carrier alone. These changes, however, did not reach statistical significance. The number of vitronectin receptor positive cells in total, adjacent to bone and away from bone was higher after treatment with TGFbeta for 1 day, compared to 1 week. In an "unperturbed" bone ingrowth system (i.e., if bone ingrowth is not initially suppressed by other stimuli), this dose of TGFbeta did not enhance bone ingrowth using the DTC model.

Periprosthetic membranes commonly observed at sites of total joint implant loosening exhibit abundant macrophages and particulate debris. Macrophages phagocytose orthopedic debris and release the pro-inflammatory mediators interleukin-1, interleukin-6, tumor necrosis factor-alpha, and prostaglandin E2. In addition, other immunologic agents, such as interferon-gamma, are present in tissues harvested from the bone-implant interface of failed orthopedic implants. The present study examined the effects of interferon-gamma on polymethylmethacrylate (PMMA) particle-challenged monocyte/macrophages in vitro. The effects of interferon-gamma were determined by measuring interleukin-6 and tumor necrosis factor-alpha release by primary human monocyte/macrophages following exposure to PMMA particles. Exposure of the monocyte/macrophages to PMMA particles resulted in a dose-dependent release of interleukin-6 and tumor necrosis factor-alpha at 48 h. The interleukin-6 release in response to PMMA particle challenge was stimulated by 76% and 127% in the presence of 1.0 and 10.0 ng/mL of interferon-gamma, respectively. Interferon-gamma challenge alone did not alter interleukin-6 release relative to controls. In contrast to interleukin-6, interferon-gamma challenge stimulated tumor necrosis factor-alpha release in a dose-dependent manner. In the presence of particles, addition of 1.0 and 10.0 ng/mL of interferon-gamma resulted in 17% and 171% increases in the levels of tumor necrosis factor-alpha release, respectively, relative to cultures challenged solely with particles. Blocking antibody to IFN-gamma inhibited the effect of IFN-gamma on particle-induced interleukin-6 and tumor necrosis factor-alpha release. The data presented in this study demonstrate that the immunologic modulator interferon-gamma exacerbates monocyte/macrophage release of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in response to PMMA particle challenge in vitro.

This study reports on the effects of phagocytosable particles on proinflammatory mediator release in an animal model. Bone harvest chambers (BHCs) were implanted bilaterally into mature rabbits; phagocytosable ultrahigh molecular weight polyethylene (UHMWPE) and polystyrene (PS) particles, and the carrier sodium hyaluronate (HE) were tested for their ability to stimulate proinflammatory mediator release. Tissues were harvested after 3, 4, or 6 weeks. Retrieved tissues were placed into culture medium. The release of the proinflammatory mediators interleukin-6 (IL-6), interleukin-1beta (IL-1beta), and tumor necrosis factor alpha (TNF-alpha) into the culture medium was assessed using bioassays. DNA content and dry weights were also measured. The maximal biological response to the PE particles with respect to TNF-alpha and IL-1beta was observed at three weeks with 11- and fivefold stimulations over controls, respectively. The maximal response to PE particles with respect to IL-6 was observed at 4 weeks with a twofold stimulation over controls. Similar patterns were seen with PS particles; however, PE particles stimulated higher cytokine release. PE particles stimulated the expression of IL-1beta, IL-6, and TNF-alpha in the BHC model. Cell culture and human retrieval studies also implicate these proinflammatory mediators in loosening and osteolysis of total joint replacements. Thus, the BHC is a useful in vivo model to document the effects of particles on the evolution of biological responses to particulate debris.

Histomorphometry is a useful tool for assessing the dynamics of bone in orthopaedic research. We compared a new fluorescence imaging method with a commonly used computer-interactive manual method using hematoxylin and eosin staining to quantify the total bone area in 6-μm thick frozen undecalcified sections processed from a bone chamber. Eighteen sections were analyzed by both methods, and the correlation coefficient was 0.92. The advantages of the fluorescence imaging are that it is simple, objective, and correlates strongly with traditional more tedious methods for histomorphometric analysis of bone. (The J Histotechnol 31:15, 2008)Submitted July 24, 2007; accepted with revisions January 9, 2008

This study tested whether macrophages respond differently to retrieved titanium-alloy particles than they do to machined titanium-alloy particles and assessed whether pretreatment of machined titanium-alloy particles with human serum would influence macrophage activation and cytokine release in vitro. Human monocyte/macrophages were isolated from normal healthy donors and exposed to increasing concentrations of machined and retrieved titanium-alloy particles. The profile of cytokine release was determined by commercially available ELISA kits. Machined titanium-alloy particles were opsonized with human serum and added to macrophage cultures. Serum protein binding was confirmed by SDS-PAGE analysis. The results showed that machined titanium-alloy particles and retrieved titanium-alloy particles stimulate a similar level of cytokine release when tested at comparable concentrations. Opsonization of the machined particles with human serum increased the macrophage release of cytokines in the first 12 h after exposure compared to nonopsonized particles. At 24 h, the opsonized particles stimulated significantly higher levels of cytokine release, but only at the greatest particle concentrations. This study demonstrates that machined titanium alloy induces a metabolic response in macrophages similar to that of titanium-alloy particles retrieved from failed total hip arthroplasty. In addition, these data show that serum protein binding to orthopedic biomaterial debris alters the macrophage reaction to the particles.

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.

Abstract: Aminopterin and its N10-methyl analog; methotrexate, were synthesized in the late 1940s and methotrexate was subsequently incorporated into clinical use as an antitumor agent in the mid-1950s. In more recent years methotrexate has also become accepted as an effective agent for treatment of rheumatoid arthritis. Structure-activity studies have been ongoing over the past 40 years in an effort to develop a safer and more effective version of this powerful antifolate. Early work featured replacement of nitrogen atoms at various positions in the pterin ring, substitution about the phenyl ring in the side chain and alteration in the glutamic acid subunit. These studies shed considerable light on structural requirements for antitumor activity. However, new agents with serious potential for clinical introduction were not encountered until structural modification of the region bridging the pterin and benzoyl glutamate side chain took place. A series of 10-substituted-10-deazaaminopterin analogs was investigated and found to have activity superior to methotrexate in several tumor models in vivo. There was in vitro evidence for facilitated transport into tumor cells versus sensitive host tissue cells and increased polyglutamylation within the tumor cells such that a selective accumulation was achieved in tumor tissue. 10-Ethyl-10-deazaaminopterin (Edatrexate) was found to be clinically effective against lung and breast solid tumors. Our laboratories have continued to pursue structural modifications of 10-deaza, 5,10-dideaza and 8,10-dideazaaminopterins in an effort to improve upon the efficacy of edatrexate. Additional variation has been made in the "bridge region" and various heterocyclic rings have been introduced in place of the side chain phenyl ring. These modifications have resulted in additional new antitumor agents and compounds with activity exceeding that of methotrexate in rheumatoid arthritis.

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.

The purpose of this study was to examine the effects of particles, derived from metals commonly used in joint prostheses, on chondrocyte proliferation, metabolism, and morphology in vitro. Chondrocyte viability was influenced by the type and concentration of metal particle added. Cobalt was toxic to chondrocytes at all particle concentrations (0.83–0.000083%, v/v), whereas the chromium, titanium and titanium-aluminum particles only effected chondrocyte viability at high concentrations. The metabolic response of chondrocytes to particulate debris as assessed by caseinase, collagenase, and hexosaminidase activities were variable at low concentrations but were always reduced at high concentrations (0.83% v/v). Prostaglandin E2 levels in the medium showed a steady increase when particle load increased, except in the medium of chondrocytes exposed to titanium-aluminum. Scanning electron microscopy of chondrocytes exposed to titanium showed ruffled cell borders and frequent membrane blebbings. This was in contrast to chondrocytes exposed to cobalt, where the crenated appearance indicated cell death, and titanium-aluminum, where the cells appeared quiescent. These findings show that metal particles alter chondrocyte viability and metabolism and suggest that particulate debris may influence the integrity and stability of articular cartilage following hemiarthroplasty. © 1994 John Wiley & Sons, Inc.

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.

Wear particles generated from total joint arthroplasty (TJA) stimulate macrophages to release chemokines. The role of chemokines released from wear particle-stimulated macrophages on the migration of macrophages and osteoprogenitor cells in vitro has not been elucidated. In this study, we challenged murine macrophages (RAW 264.7) with clinically relevant polymethyl methacrylate (PMMA, 1-10 microm) and ultra high molecular weight polyethylene (UHMWPE, 2-3 microm) particles. The chemotactic effects of the conditioned media (CM) were tested in vitro using human macrophages (THP-1) and human mesenchymal stem cells (MSCs) as the migrating cells. CM collected from both particle types had a chemotactic effect on human macrophages, which could be eliminated by monocyte chemotactic protein-1 (MCP-1) neutralizing antibody. Blocking the CCR1 receptor eliminated the chemotactic effect, while CCR2 antibody only partially decreased THP-1 cell migration. CM from PMMA but not UHMWPE-exposed macrophages led to chemotaxis of MSCs; this effect could be eliminated by macrophage inflammatory protein-1 alpha (MIP-1alpha) neutralizing antibody. Neither CCR1 nor CCR2 blocking antibodies showed an effect on the migration of MSCs. Chemokines released by macrophages stimulated by wear particles can have an effect on the migration of macrophages and MSCs. This effect seems to be dependent on the particle type, and may be modulated by MCP-1 and MIP-1alpha, however, more than one chemokine may be necessary for chemotaxis.

Polymethylmethacrylate (PMMA) particles have been shown to inhibit the differentiation of osteoprogenitor cells, but the mechanism of this inhibitory effect has not been investigated. We hypothesize that the inhibitory effects of PMMA particles involve impairment of osteoprogenitor viability and direct inhibition of transcription factors that regulate osteogenesis. We challenged MC3T3-E1 osteoprogenitors with PMMA particles and examined the effects of these materials on osteoprogenitor viability and expression of transcription factors Runx2, osterix, Dlx5, and Msx2. MC3T3-E1 cells treated with PMMA particles over a 72-h period showed a significant reduction in cell viability and proliferation as indicated by a dose- and time-dependent increase in supernatant levels of lactate dehydrogenase, an intracellular enzyme released from dead cells, a dose-dependent decrease in cell number and BrdU uptake, and the presence of large numbers of positively labeled Annexin V-stained cells. The absence of apoptotic cells on TUNEL assay indicated that cell death occurred by necrosis, not apoptosis. MC3T3-E1 cells challenged with PMMA particles during the first 6 days of differentiation in osteogenic medium showed a significant dose-dependent decrease in the RNA expression of Runx2, osterix, and Dlx5 on all days of measurement, while the RNA expression of Msx2, an antagonist of Dlx5-induced osteogenesis, remained relatively unaffected. These results indicate that PMMA particles impair osteoprogenitor viability and inhibit the expression of transcription factors that promote osteoprogenitor differentiation.

In order to optimize and modulate bone formation it is essential to understand the expression patterns of key bone-specific growth factors, as osteoprogenitor cells undergo the processes of proliferation, differentiation and maturation. This study reports the sequential expression of bone-related growth and transcription factors when bone marrow-derived osteoprogenitor cells from C57BL mice were cultured on allograft bone discs. Mineralization and osteocalcin protein levels were used to track osteogenic differentiation and maturation. Bone-related growth factors, such as Bmp-2, Bmp-7, Ctnnb-1, Fgf-2, Igf-1, Vegf-a and Tgf-β1, and transcription factors, such as Runx-2 and osteocalcin, were examined by enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Total density of mineralized bone was significantly increased 7.6 ± 0.7% in allografts cultured with cells, compared with a 0.5 ± 2.0% increase in the controls without cells (p < 0.01). Osteocalcin protein levels peaked at day 4. Protein expression showed peaks of BMP-2 and TGF-β1 on day 2, with VEGF peaking on day 8, and IGF-1 decreasing on day 2. mRNA for Pdgf-a peaked on day 2; Bmp-2 on days 4 and 16; Ctnnb-1 on days 8 and 20; Vegf-a, Fgf-2, Runx-2 and Igf-1 on day 12; Tgf-β1 on day 16; and Pdgf-b on day 20. Osteogenic growth factors correlated with Runx-2 and Ctnnb-1, whereas a predominant vascular growth factor, Vegf-a, did not follow this pattern. Specific bone-related genes and proteins were expressed in a time-dependent manner when osteoprogenitor cells were cultured on cortico-cancellous bone discs in vitro.

In this article, cross-relaxation imaging is applied to human ex vivo knee cartilage, and correlations of the cross-relaxation imaging parameters with macromolecular content in articular cartilage are reported. We show that, unlike the more commonly used magnetization transfer ratio, the bound pool fraction, the cross-relaxation rate (k) and the longitudinal relaxation time (T(1)) vary with depth and can therefore provide insight into the differences between the top and bottom layers of articular cartilage. Our cross-relaxation imaging model is more sensitive to macromolecular content in the top layers of cartilage, with bound pool fraction showing moderate correlations with proteoglycan content, and k and T(1) exhibiting moderate correlations with collagen.

The aim of this study was to test the hypothesis that a change in serum cartilage oligomeric matrix protein (COMP) concentration is related to joint load during a 30-min walking exercise in patients with medial compartment knee osteoarthritis (OA) and in age-matched control subjects. Blood samples were drawn from 42 patients with medial compartment knee OA and from 41 healthy age-matched control subjects immediately before, immediately after, and 0.5, 1.5, 3.5, and 5.5 h after a 30-min walking exercise on a level outdoor walking track at self-selected normal speed. Serum COMP concentrations were determined using a commercial ELISA. Basic time-distance gait variables were recorded using an activity monitor. Joint loads were measured using gait analysis. Serum COMP concentrations increased immediately after the walking exercise (+6.3% and +5.6%; p0.001) and decreased over 5.5 h after the exercise (-11.1% and -14.6%; p0.040 and p = 0.001) in patients and control subjects, respectively. The magnitude of increase in COMP concentration did not differ between groups (p = 0.902) and did not correlate with any variables describing ambulatory loads at the joints of the lower extremity. These results, taken together with a previous study of a younger healthy population, suggest the possibility that the influence of ambulatory loads on cartilage turnover is dependent on age.

Successful bone engineering requires an understanding of the effects of mechanical stress on osteoblast differentiation. Therefore, we examined the effects of varying magnitude and duration of fluid shear stress on factors associated with osteoblastic differentiation.Using a cone viscometer, primary neonatal rat calvarial osteoblasts were exposed to continuous fluid shear stress at varying doses: 0.21, 0.43, and 0.85 Pa for varying time periods. Gene expression was analyzed using Northern blots and nitric oxide production was quantified with the colorimetric Griess reaction.Fluid shear stress stimulated comparable transient increases in TGF-beta1 and TGF-beta3 expression by 3 hours. TGF-beta1 expression returned to baseline by 12 hours at all shear doses. In contrast, TGF-beta3 expression decreased by 22 percent and 47 percent at 12 hours in response to 0.43 Pa and 0.85 Pa, respectively. Osteopontin and Msx-2 expression patterns were consistent with a more differentiated phenotype at all shear levels. The maximum level of shear stress increased nitric oxide production 2.5-fold at 12 hours and 6.0-fold at 24 hours.These data demonstrate differential regulation of TGF-beta1 and TGF-beta3 isoforms with fluid shear stress. Furthermore, because osteopontin and Msx-2 changes were consistent with progressive differentiation at all levels of shear stress, dosage appears to be less important than the presence of an effective physical stimulus. Lastly, nitric oxide does not appear to be the primary regulator of early transcriptional changes found in this study.

In vitro models are important investigative tools in understanding the biological processes involved in wear-particle-induced chronic inflammation and periprosthetic osteolysis. In the clinical scenario, particles are produced and delivered continuously over extended periods of time. Previously, we quantified the delivery of both polystyrene and polyethylene particles over 2- and 4-week time periods using osmotic pumps and collection tubes. In the present study, we used explanted mice femora in organ culture and showed that continuous intramedullary delivery of submicron-sized polymer particles using osmotic pumps is feasible. Furthermore, infusion of 2.60 x 10(11) particles per mL (intermediate concentration) of ultrahigh molecular weight polyethylene (UHMWPE) for 2 weeks and 8.06 x 10(11) particles per mL (high concentration) UHMWPE for 4 weeks both yielded significantly higher scores for bone loss when compared with controls in which only mouse serum was infused.

Orthopedic wear debris has been implicated as a significant inhibitory factor of osteoblast differentiation. Polymethylmethacrylate (PMMA) particles have been previously shown to inhibit the differentiation of osteoprogenitors in heterogeneous murine marrow stromal cell cultures, but the effect of PMMA particles on pure osteoprogenitor populations remains unknown. In this study, we challenged murine MC3T3-E1 osteoprogenitor cells with PMMA particles during their initial differentiation in osteogenic medium. MC3T3-E1 cultures challenged with PMMA particles showed a gradual dose-dependent decrease in mineralization, cell number, and alkaline phosphatase activity at low particle doses (0.038-0.150% v/v) and complete reduction of these outcome parameters at high particle doses (or =0.300% v/v). MC3T3-E1 cultures challenged with a high particle dose (0.300% v/v) showed no rise in these outcome parameters over time, whereas cultures challenged with a low particle dose (0.075% v/v) showed a normal or reduced rate of increase compared to controls. Osteocalcin production was not significantly affected by particles at all doses tested. MC3T3-E1 cells grown in conditioned medium from particle-treated MC3T3-E1 cultures showed a significant reduction in mineralization only. These results indicate that direct exposure of MC3T3-E1 osteoprogenitors to PMMA particles results in suppression of osteogenic proliferation and differentiation.

Polyethylene wear debris induces progressive osteolysis by increasing bone degradation and suppressing bone formation. Polyethylene particles inhibit the function of mature osteoblasts, but whether polyethylene particles also interfere with the proliferation and differentiation of osteoprogenitor cells is unknown. In this study, we investigated the effects of ultrahigh molecular weight polyethylene (UHMWPE) particles on the osteogenic activity of primary murine bone marrow osteoprogenitors and MC3T3-E1 preosteoblastic cells in vitro. Submicron-sized UHMWPE particles generated from wear simulator tests were isolated from serum-containing solution by density gradient centrifugation. The particles were coated onto the surface of culture wells at concentrations of 0.038, 0.075, 0.150, 0.300, and 0.600% v/v in a layer of type I collagen matrix. Primary murine bone marrow cells and MC3T3-E1 preosteoblasts were seeded onto the particle-collagen matrix and induced to differentiate in osteogenic medium for 20 days. Exposure of both cell populations to UHMWPE particles resulted in a dose-dependent decrease in mineralization, proliferation, alkaline phosphatase activity, and osteocalcin production when compared with control cells cultured on collagen matrix without particles. Complete suppression of osteogenesis was observed at particle concentrations > or =0.150% v/v. This study demonstrated that UHMWPE particles inhibit the osteogenic activity of osteoprogenitor cells, which may result in reduced periprosthetic bone regeneration and repair.

This paper reports the effects of adding partially purified bovine thyroid calcitonin, thyrocalcitonin, to adult bovine articular cartilage cells. Thyrocalcitonin stimulated chondrocyte proliferation fourfold under low serum (0.5%) culture conditions. In serum-free medium, thyrocalcitonin stimulated cell proliferation more than twofold. With high-density cultures in serum-free medium, chondrocyte glycosaminoglycan (GAG) synthesis was stimulated 60% by thyrocalcitonin. Cell-associated radioactivity was increased twofold. In contrast to thyrocalcitonin, addition of human and salmon calcitonin peptides as well as the thyroid hormones T3 and T4 had no effect on adult cartilage cell proliferation or GAG synthesis. The data reported here suggest the existence of a thyroid-derived factor, independent of calcitonin peptides or thyroid hormones, which stimulates adult articular chondrocyte metabolism.