Bio

Academic Appointments


Research & Scholarship

Current Research and Scholarly Interests


Our group is interested in the molecular and cell biology underlying bone and cartilage metabolism in health and disease. Normal daily activities are linked to the ability of the articular cartilage to withstand normal joint forces that may reach 5-7 times body weight and bone homeostasis depends on daily mechanical loading histories. The phenotypic stability of cartilage and bone depends on a complex interplay between stimuli influencing cell metabolism, physical forces, cytokines, hormones and growth factors, and the genetic expression determining the material properties of the tissue. Our lab applies modern biochemical techniques to analyze:

1. Mechanisms of cartilage degradation in inflammation and sepsis;
2. Stimulation of cartilage growth and repair by growth factors and hormones in serum-free culture;
3. Effects of adherence and deposition of glycocalyx on bacterial resistance to antibiotic treatment;
4. Effects of mechanical stresses and strains on cartilage and bone cell gene expression and matrix
syntheses;
5. Analysis of metal particles on bone resorption and prosthetic loosening in total joint arthroplasty.

The experimental techniques include development of primary cultures of human chondrocytes, quantification of proteoglycan and collagen synthesis and degradation, zymogen and kinetic analysis of neutral metalloproteinases, western analysis of protein expression, northern and slot blot analysis of mRNA levels and cloning of connective tissue and bacterial genes.

Teaching

2013-14 Courses


Publications

Journal Articles


  • Local effect of IL-4 delivery on polyethylene particle induced osteolysis in the murine calvarium. Journal of biomedical materials research. Part A Rao, A. J., Nich, C., Dhulipala, L. S., Gibon, E., Valladares, R., Zwingenberger, S., Smith, R. L., Goodman, S. B. 2013; 101 (7): 1926-1934

    Abstract

    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-?, 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-?/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. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

    View details for DOI 10.1002/jbm.a.34486

    View details for PubMedID 23225668

  • Local effect of IL-4 delivery on polyethylene particle induced osteolysis in the murine calvarium JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Rao, A. J., Nich, C., Dhulipala, L. S., Gibon, E., Valladares, R., Zwingenberger, S., Smith, R. L., Goodman, S. B. 2013; 101A (7): 1925-1934
  • Functional assessment of the acute local and distal transplantation of human neural stem cells after spinal cord injury SPINE JOURNAL Cheng, I., Mayle, R. E., Cox, C. A., Park, D. Y., Smith, R. L., Corcoran-Schwartz, I., Ponnusamy, K. E., Oshtory, R., Smuck, M. W., Mitra, R., Kharazi, A. I., Carragee, E. J. 2012; 12 (11): 1040-1044

    Abstract

    Spinal cord injury can lead to severe functional impairments secondary to axonal damage, neuronal loss, and demyelination. The injured spinal cord has limited regrowth of damaged axons. Treatment remains controversial, given inconsistent functional improvement. Previous studies demonstrated functional recovery of rats with spinal cord contusion after transplantation of rat fetal neural stem cells.We hypothesized that acute transplantation of human fetal neural stem cells (hNSCs) both locally at the injury site as well as distally via intrathecal injection would lead to improved functional recovery compared with controls.Twenty-four adult female Long-Evans hooded rats were randomized into four groups with six animals in each group: two experimental and two control. Functional assessment was measured after injury and then weekly for 6 weeks using the Basso, Beattie, and Bresnahan Locomotor Rating Score. Data were analyzed using two-sample t test and linear mixed-effects model analysis.Posterior exposure and laminectomy at T10 level was used. Moderate spinal cord contusion was induced by the Multicenter Animal Spinal Cord Injury Study Impactor with 10-g weight dropped from a height of 25 mm. Experimental subjects received either a subdural injection of hNSCs locally at the injury site or intrathecal injection of hNSCs through a separate distal laminotomy. Controls received control media injection either locally or distally.Statistically significant functional improvement was observed in local or distal hNSCs subjects versus controls (p=.034 and 0.016, respectively). No significant difference was seen between local or distal hNSC subjects (p=.66).Acute local and distal transplantation of hNSCs into the contused spinal cord led to significant functional recovery in the rat model. No statistical difference was found between the two techniques.

    View details for DOI 10.1016/j.spinee.2012.09.005

    View details for Web of Science ID 000311807300013

    View details for PubMedID 23063425

  • A relationship between mechanically-induced changes in serum cartilage oligomeric matrix protein (COMP) and changes in cartilage thickness after 5 years OSTEOARTHRITIS AND CARTILAGE Erhart-Hledik, J. C., Favre, J., Asay, J. L., Smith, R. L., Giori, N. J., Muendermann, A., Andriacchi, T. P. 2012; 20 (11): 1309-1315

    Abstract

    To evaluate the hypothesis that a mechanical stimulus (30-min walk) will produce a change in serum concentrations of cartilage oligomeric matrix protein (COMP) that is associated with cartilage thickness changes on magnetic resonance imaging (MRI).Serum COMP concentrations were measured by enzyme-linked immunosorbent assay in 17 patients (11 females, age: 59.0±9.2 years) with medial compartment knee osteoarthritis (OA) at study entry immediately before, immediately after, 3.5 h, and 5.5 h after a 30-min walking activity. Cartilage thickness changes in the medial femur and medial tibia were determined from MR images taken at study entry and at 5-year follow-up. Relationships between changes in cartilage thickness and COMP levels, with post-activity concentrations expressed as a percentage of pre-activity levels, were assessed by the calculation of Pearson correlation coefficients and by multiple linear regression analysis, with adjustments for age, sex, and body mass index (BMI).Changes in COMP levels 3.5 h and 5.5 h post-activity were correlated with changes in cartilage thickness in the medial femur and tibia at the 5-year follow-up. The results were strengthened after analyses were adjusted for age, sex, and BMI. Neither baseline pre-activity COMP levels nor changes in COMP levels immediately post-activity were correlated with cartilage thickness changes.The results of this study support the hypothesis that a change in COMP concentration induced by a mechanical stimulus is associated with cartilage thinning at 5 years. Mechanically-induced changes in mechano-sensitive biomarkers should be further explored in the context of stimulus-response models to improve the ability to assess OA progression.

    View details for DOI 10.1016/j.joca.2012.07.018

    View details for Web of Science ID 000309853400013

  • The effect of suture coated with mesenchymal stem cells and bioactive substrate on tendon repair strength in a rat model. journal of hand surgery Yao, J., Woon, C. Y., Behn, A., Korotkova, T., Park, D., Gajendran, V., Smith, R. L. 2012; 37 (8): 1639-1645

    Abstract

    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.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.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).Bioactive CS sutures enhanced repair strength at 7 to 10 days. There was no significant effect at later stages.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.

    View details for DOI 10.1016/j.jhsa.2012.04.038

    View details for PubMedID 22727924

  • Effect of a CCR1 receptor antagonist on systemic trafficking of MSCs and polyethylene particle-associated bone loss BIOMATERIALS Gibon, E., Yao, Z., Rao, A. J., Zwingenberger, S., Batke, B., Valladares, R., Smith, R. L., Biswal, S., Gambhir, S. S., Goodman, S. B. 2012; 33 (14): 3632-3638

    Abstract

    Particle-associated periprosthetic osteolysis remains a major issue in joint replacement. Ongoing bone loss resulting from wear particle-induced inflammation is accompanied by continued attempts at bone repair. Previously we showed that mesenchymal stem cells (MSCs) are recruited systemically to bone exposed to continuous infusion of ultra high molecular weight polyethylene (UHMWPE) particles. The chemokine-receptor axis that mediates this process is unknown. We tested two hypotheses: (1) the CCR1 receptor mediates the systemic recruitment of MSCs to UHMWPE particles and (2) recruited MSCs are able to differentiate into functional mature osteoblasts and decrease particle-associated bone loss. Nude mice were allocated randomly to four groups. UHMWPE particles were continuously infused into the femoral shaft using a micro-pump. Genetically modified murine wild type reporter MSCs were injected systemically via the left ventricle. Non-invasive imaging was used to assay MSC migration and bone mineral density. Bioluminescence and immunohistochemistry confirmed the chemotaxis of reporter cells and their differentiation into mature osteoblasts in the presence of infused particles. Injection of a CCR1 antagonist decreased reporter cell recruitment to the UHMWPE particle infusion site and increased osteolysis. CCR1 appears to be a critical receptor for chemotaxis of MSCs in the presence of UHMWPE particles. Interference with CCR1 exacerbates particle-induced bone loss.

    View details for DOI 10.1016/j.biomaterials.2012.02.003

    View details for Web of Science ID 000302425400003

    View details for PubMedID 22364730

  • Cross-relaxation Imaging of Human Articular Cartilage MAGNETIC RESONANCE IN MEDICINE Stikov, N., Keenan, K. E., Pauly, J. M., Smith, R. L., Dougherty, R. F., Gold, G. E. 2011; 66 (3): 725-734

    Abstract

    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.

    View details for DOI 10.1002/mrm.22865

    View details for Web of Science ID 000293988000013

    View details for PubMedID 21416504

  • Role of the Toll-like receptor pathway in the recognition of orthopedic implant wear-debris particles BIOMATERIALS Pearl, J. I., Ma, T., Irani, A. R., Huang, Z., Robinson, W. H., Smith, R. L., Goodman, S. B. 2011; 32 (24): 5535-5542

    Abstract

    The inflammatory response to prosthetic implant-derived wear particles is the primary cause of bone loss and aseptic loosening of implants, but the mechanisms by which macrophages recognize and respond to particles remain unknown. Studies of innate immunity demonstrate that Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPS). All TLRs signal through myeloid differentiation factor 88 (MyD88), except TLR3 which signals through TIR domain containing adapter inducing interferon-beta (TRIF), and TLR4 which signals through both MyD88 and TRIF. We hypothesized that wear-debris particles may act as PAMPs/DAMPs and activate macrophages via TLRs. To test this hypothesis, we first demonstrated that inhibition of MyD88 decreases polymethylmethacrylate (PMMA) particle-induced production of TNF-? in RAW 264.7 macrophages. Next we compared particle-induced production of TNF-? among MyD88 knockout (MyD88(-/-)), TRIF knockout (TRIF(-/-)), and wild type (WT) murine macrophages. Relative to WT, disruption of MyD88 signaling diminished, and disruption of TRIF amplified the particle-induced production of TNF-?. Gene expression data indicated that this latter increase in TNF-? was due to a compensatory increase in expression of MyD88 associated components of the TLR pathway. Finally, using an in vivo model, MyD88(-/-) mice developed less particle-induced osteolysis than WT mice. These results indicate that the response to PMMA particles is partly dependent on MyD88, presumably as part of TLR signaling; MyD88 may represent a therapeutic target for prevention of wear debris-induced periprosthetic osteolysis.

    View details for DOI 10.1016/j.biomaterials.2011.04.046

    View details for Web of Science ID 000292431100001

    View details for PubMedID 21592562

  • Effects of Intermittent Hydrostatic Pressure and BMP-2 on Osteoarthritic Human Chondrocyte Metabolism In Vitro JOURNAL OF ORTHOPAEDIC RESEARCH Smith, R. L., Lindsey, D. P., Dhulipala, L., Harris, A. H., Goodman, S. B., Maloney, W. J. 2011; 29 (3): 361-368

    Abstract

    This study examined effects of intermittent hydrostatic pressure (IHP) and a chondrogenic growth factor, bone morphogenetic protein-2 (BMP-2), on anabolic, catabolic, and other metabolic markers in human osteoarthritic (OA) chondrocytes in vitro.Articular chondrocytes, isolated from femoral OA cartilage and maintained in high-density monolayer culture, were examined for effects of BMP-2 and IHP on gene expression of matrix-associated proteins (aggrecan, type II collagen, and SOX9) and catabolic matrix metalloproteinases (MMP-2 and MMP-3) and culture medium levels of the metabolic markers MMP-2, nitric oxide (NO), and glycosaminoglycan (GAG). The results were analyzed using a mixed linear regression model to investigate the effects of load and growth factor concentration.IHP and BMP-2 modulated OA chondrocyte metabolism in accordance with growth factor concentration independently, without evidence of synergism or antagonism. Each type of stimulus acted independently on anabolic matrix gene expression. Type II collagen and SOX9 gene expression were stimulated by both IHP and BMP-2 whereas aggrecan was increased only by BMP-2. IHP exhibited a trend to decrease MMP-2 gene expression as a catabolic marker whereas BMP-2 did not. NO production was increased by addition of BMP-2 and IHP exhibited a trend for increased levels. GAG production was increased by BMP-2.This study confirmed the hypothesis that human OA chondrocytes respond to a specific type of mechanical load, IHP, through enhanced articular cartilage macromolecule gene expression and that IHP, in combination with a chondrogenic growth factor BMP-2, additively enhanced matrix gene expression without interactive effects.

    View details for DOI 10.1002/jor.21250

    View details for Web of Science ID 000287173500009

    View details for PubMedID 20882590

  • Prediction of glycosaminoglycan content in human cartilage by age, T1 rho and T2 MRI OSTEOARTHRITIS AND CARTILAGE Keenan, K. E., Besier, T. F., Pauly, J. M., Han, E., Rosenberg, J., Smith, R. L., Delp, S. L., Beaupre, G. S., Gold, G. E. 2011; 19 (2): 171-179

    Abstract

    A relationship between T1? relaxation time and glycosaminoglycan (GAG) content has been demonstrated in chemically degraded bovine cartilage, but has not been demonstrated with quantitative biochemistry in human cartilage. A relationship has also been established between T2 relaxation time in cartilage and osteoarthritis (OA) severity. We hypothesized that T1? relaxation time would be associated with GAG content in human cartilage with normal T2 relaxation times.T2 relaxation time, T1? relaxation time, and glycosaminoglycan as a percentage of wet weight (sGAG) were measured for top and bottom regions at 7 anatomical locations in 21 human cadaver patellae. For our analysis, T2 relaxation time was classified as normal or elevated based on a threshold defined by the mean plus one standard deviation of the T2 relaxation time for all samples.In the normal T2 relaxation time subset, T1? relaxation time correlated with sGAG content in the full-thickness and bottom regions, but only marginally in the top region alone. sGAG content decreased significantly with age in all regions.In the subset of cartilage specimens with normal T2 relaxation time, T1? relaxation time was inversely associated with sGAG content, as hypothesized. A predictive model, which accounts for T2 relaxation time and the effects of age, might be able to determine longitudinal trends in GAG content in the same person based on T1? relaxation time maps.

    View details for DOI 10.1016/j.joca.2010.11.009

    View details for Web of Science ID 000287470600005

    View details for PubMedID 21112409

  • Viability and proliferation of pluripotential cells delivered to tendon repair sites using bioactive sutures--an in vitro study. journal of hand surgery Yao, J., Korotkova, T., Smith, R. L. 2011; 36 (2): 252-258

    Abstract

    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 ?m). 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.

    View details for DOI 10.1016/j.jhsa.2010.10.004

    View details for PubMedID 21186083

  • Effects of orthopedic polymer particles on chemotaxis of macrophages and mesenchymal stem cells. Journal of biomedical materials research. Part A Huang, Z., Ma, T., Ren, P., Smith, R. L., Goodman, S. B. 2010; 94 (4): 1264-1269

    Abstract

    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.

    View details for DOI 10.1002/jbm.a.32803

    View details for PubMedID 20694994

  • Modulating osteogenesis of mesenchymal stem cells by modifying growth factor availability CYTOKINE Huang, Z., Ren, P., Ma, T., Smith, R. L., Goodman, S. B. 2010; 51 (3): 305-310

    Abstract

    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.

    View details for DOI 10.1016/j.cyto.2010.06.002

    View details for Web of Science ID 000281108200016

    View details for PubMedID 20580248

  • Surveillance of systemic trafficking of macrophages induced by UHMWPE particles in nude mice by noninvasive imaging. Journal of biomedical materials research. Part A Ren, P., Huang, Z., Ma, T., Biswal, S., Smith, R. L., Goodman, S. B. 2010; 94 (3): 706-711

    Abstract

    Macrophages constitute a major part of the cell response to wear particles produced at articulating and nonarticulating interfaces of joint replacements. This foreign body reaction can result in periprosthetic osteolysis and implant loosening. We demonstrate that ultra-high molecular weight polyethylene (UHMWPE) particles induce systemic trafficking of macrophages by noninvasive in vivo imaging and immunohistochemistry. The distal femora of nude mice were injected with 60 mg/mL UHMWPE suspension or saline alone. Reporter RAW264.7 macrophages that stably expressed the bioluminescent reporter gene and the fluorescence reporter gene were injected intravenously. Bioluminescence imaging was performed using an in vivo imaging system immediately after macrophage injection and at 2-day intervals. Compared with the nonoperated contralateral femora, at day 4, 6, and 8, the bioluminescent signal of femora containing UHMWPE suspension increased 1.30 +/- 0.09-, 2.36 +/- 0.92-, and 10.32 +/- 7.61-fold, respectively. The values at same time points for saline-injected control group were 1.08 +/- 0.07-, 1.14 +/- 0.27-, and 1.14 +/- 0.35-fold, respectively. The relative bioluminescence of the UHMWPE group was higher at all postinjection days and significantly greater than the saline group at day 8 (p < 0.05). Histological analysis confirmed the presence of reporter macrophages within the medullary canal of mice with implanted UHMWPE particles. The presence of UHMWPE particles induced enhanced bone remodeling activity. Clinically relevant UHMWPE particles stimulated the systemic recruitment of macrophages during an early time course using the murine femoral implant model. Interference with systemic macrophage trafficking may potentially mitigate UHMWPE particle-induced periprosthetic osteolysis.

    View details for DOI 10.1002/jbm.a.32744

    View details for PubMedID 20213815

  • Effects of Tensile Strain and Fluid Flow on Osteoarthritic Human Chondrocyte Metabolism In Vitro JOURNAL OF ORTHOPAEDIC RESEARCH Mawatari, T., Lindsey, D. P., Harris, A. H., Goodman, S. B., Maloney, W. J., Smith, R. L. 2010; 28 (7): 907-913

    Abstract

    This study examined the hypothesis that tensile strain and fluid flow differentially influence osteoarthritic human chondrocyte metabolism. Primary high-density monolayer chondrocytes cultures were exposed to varying magnitudes of tensile strain and fluid-flow using a four-point bending system. Metabolic changes were quantified by real-time PCR measurement of aggrecan, IL-6, SOX-9, and type II collagen gene expression, and by determination of nitric oxide levels in the culture medium. A linear regression model was used to investigate the roles of strain, fluid flow, and their interaction on metabolic activity. Aggrecan, type II collagen, and SOX9 mRNA expression were negatively correlated to increases in applied strain and fluid flow. An effect of the strain on the induction of nitric oxide release and IL-6 gene expression varied by level of fluid flow (and visa versa). This interaction between strain and fluid flow was negative for nitric oxide and positive for IL-6. These results confirm that articular chondrocyte metabolism is responsive to tensile strain and fluid flow under in vitro loading conditions. Although the articular chondrocytes reacted to the mechanically applied stress, it was notable that there was a differential effect of tensile strain and fluid flow on anabolic and catabolic markers.

    View details for DOI 10.1002/jor.21085

    View details for Web of Science ID 000278654500011

    View details for PubMedID 20063382

  • Polymethylmethacrylate particle exposure causes changes in p38 MAPK and TGF-beta signaling in differentiating MC3T3-E1 cells. Journal of biomedical materials research. Part A Ma, G. K., Chiu, R., Huang, Z., Pearl, J., Ma, T., Smith, R. L., Goodman, S. B. 2010; 94 (1): 234-240

    Abstract

    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.

    View details for DOI 10.1002/jbm.a.32686

    View details for PubMedID 20166219

  • Flexor Tendon Tissue Engineering: Bioreactor Cyclic Strain Increases Construct Strength TISSUE ENGINEERING PART A Saber, S., Zhang, A. Y., Ki, S. H., Lindsey, D. P., Smith, R. L., Riboh, J., Pham, H., Chang, J. 2010; 16 (6): 2085-2090

    Abstract

    Mutilating injuries of the hand and upper extremity result in tendon losses too great to be replaced by autologous grafts. The goal of this study was to use tissue engineering techniques to produce additional tendon material. We used a custom bioreactor to apply cyclic mechanical loading onto tissue-engineered tendon constructs to study ultimate tensile stress (UTS) and elastic modulus (E). Constructs used were acellularized rabbit hindpaw flexor digitorum profundus equivalents reseeded with tenocytes or left unseeded. Tendon constructs were subjected to a stretch force of 1.25 N over a 5-day course. Seeded tendon constructs that were exposed to bioreactor loading had a significantly increased UTS (71.17 +/- 14.15 N) compared to nonloaded controls (35.69 +/- 5.62 N) (p = 0.001). Similarly, seeded constructs exposed to bioreactor loading also had a significantly higher E (1091 +/- 169 MPa) compared to nonloaded controls (632 +/- 86 MPa) (p = 0.001). This study shows that cyclic loading of tendon constructs increases the UTS and elastic modulus of seeded constructs. The use of the bioreactor may therefore accelerate the in vitro production of strong, nonimmunogenic tendon material that can potentially be used clinically to reconstruct significant tendon losses.

    View details for DOI 10.1089/ten.tea.2010.0032

    View details for Web of Science ID 000278164800026

    View details for PubMedID 20109062

  • Polymethylmethacrylate Particles Impair Osteoprogenitor Viability and Expression of Osteogenic Transcription Factors Runx2, Osterix, and Dlx5 JOURNAL OF ORTHOPAEDIC RESEARCH Chiu, R., Smith, K. E., Ma, G. K., Ma, T., Smith, R. L., Goodman, S. B. 2010; 28 (5): 571-577

    Abstract

    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.

    View details for DOI 10.1002/jor.21035

    View details for Web of Science ID 000277311700003

    View details for PubMedID 20014320

  • Biocompatibility of poly(ethylene glycol)/poly(acrylic acid) interpenetrating polymer network hydrogel particles in RAW 264.7 macrophage and MG-63 osteoblast cell lines. Journal of biomedical materials research. Part A Yim, E. S., Zhao, B., Myung, D., Kourtis, L. C., Frank, C. W., Carter, D., Smith, R. L., Goodman, S. B. 2009; 91 (3): 894-902

    Abstract

    Hydrogel polymers comprise a novel category of synthetic materials being investigated for use in cartilage replacement. One candidate compound, a poly(ethylene glycol)/poly(acrylic acid) (PEG/PAA) interpenetrating polymer network (IPN), was developed for use in corneal prostheses and was recently engineered for potential orthopedic use. The current study examined the effects of particles of this compound on two cell lines (MG-63 osteoblast-like cells and RAW 264.7 macrophages) over a 48-h time course. To mimic the effects of wear debris, particles of the compound were generated and introduced to the cells. In the MG-63 cell line, the particles had no significant effect on cell viability measured by PicoGreen assay and trypan blue exclusion. In contrast, a significant decrease in cell viability was detected in the Raw 264.7 macrophage cells at the final timepoint with the highest concentration of hydrogel (3.0% v:v). A concentration- and time-dependent increase in TNF-alpha release characteristic of other known biocompatible materials was also detected in RAW 264.7 cells, but nitric oxide and interleukin (IL)-1beta showed no response. In addition, the MG-63 cell line demonstrated no IL-6 response. Particles of the PEG/PAA IPN thus seem to stimulate biological responses similar to those in other biocompatible materials.

    View details for DOI 10.1002/jbm.a.32311

    View details for PubMedID 19072924

  • Change in Serum COMP Concentration Due to Ambulatory Load Is Not Related to Knee OA Status JOURNAL OF ORTHOPAEDIC RESEARCH Muendermann, A., King, K. B., Smith, L., Andriacchi, T. P. 2009; 27 (11): 1408-1413

    Abstract

    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.

    View details for DOI 10.1002/jor.20908

    View details for Web of Science ID 000271310700002

    View details for PubMedID 19422040

  • Flexor Tendon Tissue Engineering: Acellularized and Reseeded Tendon Constructs PLASTIC AND RECONSTRUCTIVE SURGERY Chong, A. K., Riboh, J., Smith, R. L., Lindsey, D. P., Pham, H. M., Chang, J. 2009; 123 (6): 1759-1766

    Abstract

    Tissue engineering of flexor tendons requires scaffolds with adequate strength and biocompatibility. The biomechanical properties of acellularized and reseeded flexor tendon scaffolds are unknown. Acellularized tendons and reseeded constructs were tested to determine whether the treatment process had altered their biomechanical properties.Rabbit flexor tendons were acellularized using a freeze-thaw cycle followed by trypsin and Triton-X treatment. Complete acellularization of the tendon samples was confirmed by histology and by attempting to obtain viable cells by trypsin treatment of acellularized tendon. Reseeded constructs were obtained by incubating acellularized tendons in a tenocyte suspension. Tensile testing was performed to compare the ultimate tensile stress and elastic modulus of acellularized tendons and reseeded flexor tendon constructs to control flexor tendons.The treatment protocol successfully acellularized flexor tendons. No cells were seen within the tendon on histologic assessment, and no viable cells could be obtained from acellularized tendon. Acellularized tendon was successfully reseeded with tenocytes, although cell adhesion was limited to the surface of the tendon scaffold. Tensile testing showed that acellularized tendon had the same ultimate stress and elastic modulus as normal tendons. Reseeded tendons had the same elastic modulus as normal tendons, but hind-paw tendon constructs showed a decrease in ultimate stress compared with normal tendons (50.09 MPa versus 66.01 MPa, p = 0.026).Acellularized flexor tendons are a potential high-strength scaffold for flexor tendon tissue engineering. This approach of acellularization and reseeding of flexor tendons may provide additional intrasynovial graft material for hand reconstruction.

    View details for DOI 10.1097/PRS.0b013e3181a65ae7

    View details for Web of Science ID 000266674600018

    View details for PubMedID 19483576

  • Ultrahigh molecular weight polyethylene wear debris inhibits osteoprogenitor proliferation and differentiation in vitro JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Chiu, R., Ma, T., Smith, R. L., Goodman, S. B. 2009; 89A (1): 242-247

    Abstract

    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.

    View details for DOI 10.1002/jbm.a.32001

    View details for Web of Science ID 000263981300024

    View details for PubMedID 18442106

  • Efficacy of a p38 mitogen activated protein kinase inhibitor in mitigating an established inflammatory reaction to polyethylene particles in vivo JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Ma, T., Ren, P., Larsen, D. M., Suenaga, E., Zilber, S., Genovese, M., Smith, R. L., Goodman, S. B. 2009; 89A (1): 117-123

    Abstract

    The inhibitor of p38 mitogen-activated protein kinase (MAPK) is of interest in the nonoperative treatment of periprosthetic osteolysis due to wear particles. Previous studies demonstrated that an oral p38 MAPK inhibitor did not suppress bone formation when given during the initial phase of tissue differentiation. However, the oral p38 MAPK inhibitor also did not curtail the foreign body and chronic inflammatory response to particles when given simultaneously. The purpose of the current study was to examine the efficacy of a p38 MAPK inhibitor, SCIO-323, on mitigating an established inflammatory reaction that parallels the clinical situation more closely. The Bone Harvest Chamber was implanted in rabbits and submicron polyethylene particles were placed in the chamber for 6 weeks. The contents of the chambers were harvested every 6 weeks. Oral treatment with the SCIO-323 included delivery for 3 weeks and stopping for 3 weeks, delivery for 3 weeks after an initial 3-week delay, and delivery for 6 weeks continuously. Administration of the SCIO-323 continuously for 6 weeks with/without the presence of particles, or for the initial 3 of 6 weeks had minor effects on bone ingrowth. After establishing a particle-induced chronic inflammatory reaction for 3 weeks, administration of SCIO-323 for a subsequent 3 weeks suppressed net bone formation. The activity of osteoclast-like cells remained low among all treatments when compared with the first control. Using the present model, the oral p38 MAPK inhibitor was ineffective in improving bone ingrowth in the presence of polyethylene particles.

    View details for DOI 10.1002/jbm.a.31957

    View details for Web of Science ID 000263981300011

    View details for PubMedID 18431764

  • In vivo murine model of continuous intramedullary infusion of particles--a preliminary study. Journal of biomedical materials research. Part B, Applied biomaterials Ma, T., Ortiz, S. G., Huang, Z., Ren, P., Smith, R. L., Goodman, S. B. 2009; 88 (1): 250-253

    Abstract

    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.

    View details for DOI 10.1002/jbm.b.31175

    View details for PubMedID 18777575

  • New Bone Formation by Murine Osteoprogenitor Cells Cultured on Corticocancellous Allograft Bone JOURNAL OF ORTHOPAEDIC RESEARCH Nelson, E. R., Huang, Z., Ma, T., Lindsey, D., Jacobs, C., Smith, R. L., Goodman, S. B. 2008; 26 (12): 1660-1664

    Abstract

    The gold standard for bone grafting in orthopedics is autograft, however autograft has a limited supply and is associated with significant morbidity at the harvest site. One alternative, allograft bone, provides an osteoconductive scaffold, is in less limited supply, and it does not require a harvest from the patient. However, allograft lacks both osteogenic cells and osteoinductive proteins that make autograft bone so advantageous. This study provides a model to investigate strategies for augmentation of corticocancellous allograft bone discs with bone marrow-derived osteoprogenitor cells (OPCs) plus exogenous growth factors in vitro. In this model, allograft bone discs were created by cutting 1-mm thick slices from the distal femur and proximal tibia of euthanized mice. The allografts were sterilized and scanned by micro-computed tomography (microCT) to provide the pre-culture graft volume and trabecular characteristics. The discs were then seeded with OPCs harvested from murine bone marrow. The seeded grafts were placed in organ culture until harvest, after which they were re-scanned by microCT and the data compared to the corresponding pre-culture data. In addition, bone morphogenetic protein-7 (BMP-7, also know as osteogenic protein-1 or OP-1), basic fibroblast growth factor (bFGF), and OP-1 combined with bFGF were added on a daily basis to the cultures. After final microCT scanning, all grafts were sectioned and evaluated histologically after hematoxylin and eosin (H&E) staining. microCT scans of cultured allografts with cells at 3, 5, and 9 weeks showed a time-dependent, statistically significant increase in bone volume. The trabecular thickness (Tb.Th.) of grafts, from both groups that were augmented with OP-1, showed a statistically significant increase in trabecular thickness of allografts with OPCs. These data suggest that bone marrow-derived OPCs adhere to, and produce, new bone on corticocancellous allograft in vitro. When exogenous OP-1 is added to this model, an increase in the production of bone onto the corticocancellous allograft bone disc is seen. This model allows for the investigation of the effects of multiple growth factors, and other interventions, on OPCs seeded onto allograft bone in vitro.

    View details for DOI 10.1002/jor.20676

    View details for Web of Science ID 000260934700017

    View details for PubMedID 18524004

  • Continuous Intramedullary Polymer Particle Infusion Using a Murine Femoral Explant Model JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS Ortiz, S. G., Ma, T., Regula, D., Smith, R. L., Goodman, S. B. 2008; 87B (2): 440-446

    Abstract

    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.

    View details for DOI 10.1002/jbm.b.31122

    View details for Web of Science ID 000260355000017

  • Bioactive Sutures for Tendon Repair: Assessment of a Method of Delivering Pluripotential Embryonic Cells JOURNAL OF HAND SURGERY-AMERICAN VOLUME Yao, J., Korotkova, T., Riboh, J., Chong, A., Chang, J., Smith, R. L. 2008; 33A (9): 1558-1564

    Abstract

    Pluripotential embryonic cells may be seeded onto sutures intended for tendon repair. These cells may be influenced to adhere to suture material using adhesion substrates, and furthermore, these cells may remain in culture attached to those sutures. These cell-impregnated sutures may be useful for promoting healing of tendon repairs.Ten-centimeter segments of 4-0 sutures (FiberWire) were coated overnight with 10 microg/mL fibronectin, 10 microg/mL poly-l-lysine, or phosphate-buffered saline. The sutures were placed in dishes and covered with a suspension of C3H10T1/2 cells at concentrations of 1 x 10(6), 2 x 10(6), or 4 x 10(6) cells for 24 hours. The sutures were then placed into low adhesion polypropylene tubes with Dulbecco's modified Eagle's medium and 10% fetal bovine serum for 7 days. The presence of viable cells on these sutures was assessed by the colorimetric Alamar blue cell proliferation assay. Spectrophotometry was used to quantify the relative amount of cell proliferation across the experimental groups. The sutures were also visually inspected using phase-contrast light microscopy.Our results show that at all seeding densities (1 x 10(6), 2 x 10(6), and 4 x 10(6) cells), the suture segments coated with poly-l-lysine and fibronectin showed a significant increase in C3H10T1/2 cell adhesion. Coating the suture with poly-l-lysine increased the adherent cell number to 17% of the initial seeding concentration compared with 2% for the control. Fibronectin coating increased the number of adherent viable cells present to 6.6%.Pluripotential embryonic cells may be seeded onto sutures, adhere, and survive in culture. Coating sutures with poly-l-lysine and fibronectin offers significant improvement in retention of viable cells. This technique may be a useful adjunct for future tendon healing studies.

    View details for DOI 10.1016/j.jhsa.2008.06.010

    View details for Web of Science ID 000260725900015

  • An in vivo murine model of continuous intramedullary infusion of polyethylene particles BIOMATERIALS Ma, T., Huang, Z., Ren, P., McCally, R., Lindsey, D., Smith, R. L., Goodman, S. B. 2008; 29 (27): 3738-3742

    Abstract

    Wear debris affects both initial osseointegration and subsequent bone remodeling of total joint replacements (TJRs). To study the complex cascade associated with the continuous generation of particles, a robust animal model is essential. To date, an animal model that incorporates continuously delivered particles to an intramedullary orthopaedic implant has not been available. In this study, we successfully infused clinically relevant ultra high molecular weight polyethylene particles, previously isolated from joint simulator tests, to the intramedullary space of the mouse femur for 4 weeks using a subcutaneous osmotic pump. Reduction of bone volume following the 4-week infusion of UHMWPE was detected by microCT. UHMWPE particles also changed the level of Alkaline Phosphatase expression in the infused femurs. Continuous infusion of particles to the murine bone-implant interface simulated the clinical scenario of local polymer wear particle generation and delivery in humans and can be used to further study the biological processes associated with wear debris particles.

    View details for DOI 10.1016/j.biomaterials.2008.05.031

    View details for Web of Science ID 000258439300014

    View details for PubMedID 18561997

  • Fluid shear stress magnitude, duration, and total applied load regulate gene expression and nitric oxide production in primary calvarial osteoblast cultures PLASTIC AND RECONSTRUCTIVE SURGERY Gonzalez, O., Fong, K. D., Trindade, M. C., Warren, S. M., Longaker, M. T., Smith, R. L. 2008; 122 (2): 419-428

    Abstract

    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.

    View details for DOI 10.1097/PRS.0b013e31817d5ff1

    View details for Web of Science ID 000258136900011

    View details for PubMedID 18626357

  • Polymethylmethacrylate particles inhibit osteoblastic differentiation of MC3T3-E1 osteoprogenitor cells JOURNAL OF ORTHOPAEDIC RESEARCH Chiu, R., Ma, T., Smith, R. L., Goodman, S. B. 2008; 26 (7): 932-936

    Abstract

    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.

    View details for DOI 10.1002/jor.20618

    View details for Web of Science ID 000256744600006

    View details for PubMedID 18302244

  • Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium ANNALS OF BIOMEDICAL ENGINEERING Wagner, D. R., Lindsey, D. P., Li, K. W., Tummala, P., Chandran, S. E., Smith, R. L., Longaker, M. T., Carter, D. R., Beaupre, G. S. 2008; 36 (5): 813-820

    Abstract

    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.

    View details for DOI 10.1007/s10439-008-9448-5

    View details for Web of Science ID 000254755800013

    View details for PubMedID 18266109

  • Validation and quantification of an in vitro model of continuous infusion of submicron-sized particles JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS Ortiz, S. G., Ma, T., Epstein, N. J., Smith, R. L., Goodman, S. B. 2008; 84B (2): 328-333

    Abstract

    Wear particles produced from total joint replacements have been shown to stimulate a foreign body and chronic inflammatory reaction that results in periprosthetic osteolysis. Most animal models that simulate these events have used a single injection of particles, which is not representative of the clinical scenario, in which particles are continuously generated. The goal of this study was to evaluate the feasibility of an osmotic pump for the continuous delivery of clinically relevant submicron-sized particles over an extended period of time. Blue-dyed polystyrene particles and retrieved ultra-high molecular weight polyethylene (UHMWPE) particles, both suspended in mouse serum, were loaded into an Alzet mini-osmotic pump. Pumps were attached to vinyl tubing that ended with hollow titanium rods, simulating a metal implant, which was suspended in a collection vessel. The number of particles collected was evaluated over 2- and 4-week time periods. Delivery of both the polystyrene and UHMWPE particles was feasible over pump concentrations of 10(9) to 10(11) particles per pump. Furthermore, delivery efficiency of polystyrene particles decreased with increasing initial particle concentration, whereas delivery efficiency of UHMWPE particles increased slightly with increasing initial particle concentration. For UHMWPE, approximately one-third of the particles in the pump were collected at 4 weeks. This in vitro study has quantified the efficiency of a unique particle pumping system that may be used in future in vivo investigations to develop a murine model of continuous particle infusion.

    View details for DOI 10.1002/jbm.b.30875

    View details for Web of Science ID 000252472900003

    View details for PubMedID 17595028

  • Histomorphometric Analysis of the intramedullary bone response to titanium particles in wild-type and IL-1R1 knowk-out mice: A preliminary study JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS Bragg, B., Epstein, N., Ma, T., Goodman, S., Smith, R. L. 2008; 84B (2): 559-570

    Abstract

    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.

    View details for DOI 10.1002/jbm.b.30904

    View details for Web of Science ID 000252472900032

    View details for PubMedID 17618512

  • The sequential expression profiles of growth factors from osteroprogenitors to osteoblasts In vitro TISSUE ENGINEERING Huang, Z., Nelson, E. R., Smith, R. L., Goodman, S. B. 2007; 13 (9): 2311-2320

    Abstract

    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 to monitor the differentiation and maturation of osteoprogenitor cells collected from C57BL mice. Bone-related growth factors, including transforming growth factor beta (TGF-beta), fibroblast growth factor 2 (FGF-2), platelet-derived growth factor (PDGF), insulinlike growth factor (IGF)-1, vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP)-2, and BMP-7, were selected. Enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR) were used to measure growth factors at the protein and messenger ribonucleic acid (mRNA) level, respectively. The results found that ALP-2 expression increased progressively over time, whereas mineralization and osteocalcin did not become evident until culture day 14. VEGF and IGF-1 were upregulated early during proliferation. PDGF and TGF-beta mRNA expression was bimodal. FGF-2 and BMP-2 mRNAs were expressed only later in differentiation. FGF-2 mRNA signal levels were highest at day 14 and remained prominent through day 28 of culture. BMP-2 showed a similar profile as FGF-2. BMP-7 was not detectable using RT-PCR or ELISA. Strong correlations existed for the expression patterns between several early-response growth factors (VEGF, TGF-beta, and IGF-1) and were also evident for several late-response growth factors (BMP-2, PDGF, and FGF-2). Differential expression for grouped sets of growth factors occurs during the temporal acquisition of bone-specific markers as osteoprogenitor cell maturation proceeds in vitro.

    View details for DOI 10.1089/ten.2006.0423

    View details for Web of Science ID 000249391900016

    View details for PubMedID 17523879

  • Modulation of allograft incorporation by growth factors over a prolonged continuous infusion of duration in vivo BONE Ma, T., Gutnick, J., Salazar, B., Larsen, M. D., Suenaga, E., Zilber, S., Huang, Z., Huddleston, J., Smith, R. L., Goodman, S. 2007; 41 (3): 386-392

    Abstract

    Morselized cancellous allograft bone is frequently used in the reconstruction of bone defects in cases of revision total joint replacement, trauma, spine fusion and treated infection. However, the initial lack of viable bone cells in morselized allograft bone significantly slows the process of graft incorporation compared to autograft bone. This study examined the effects of prolonged local infusion of the growth factors bone morphogenic protein-7 (BMP-7 or OP-1) and fibroblast growth factor-2 (FGF-2 or basic FGF) in the process of allograft incorporation using a rabbit tibial chamber model. New bone formation was evaluated by two indices, the activity of alkaline phosphatase and the level of birefringence. The markers of osteoclast-like cells were also measured. Without the infusion of the growth factors, lower levels of new bone formation were observed in the allograft group, compared to the autograft group. Infusion of growth factors FGF-2 and OP-1, singly or in combination, for 4 weeks, diminished this difference. The numbers of osteoclast-like cells were much higher in the allograft group before the growth factors were delivered. The infusion of FGF, singly, diminished this difference. However, the infusion of OP-1 or the combination of FGF and OP-1 did not decrease the number of osteoclast-like cells to a level comparable to autograft only. Local infusion of growth factors appears to be a useful adjunct to promote the incorporation of allograft bone in vivo.

    View details for DOI 10.1016/j.bone.2007.05.015

    View details for Web of Science ID 000248898600012

    View details for PubMedID 17613298

  • Effects of a p38 MAP kinase inhibitor on bone ingrowth and tissue differentiation in rabbit chambers JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Goodman, S. B., Ma, T., Spanogle, J., Chiu, R., Miyanishi, K., Oh, K., Plouhar, P., Wadsworth, S., Smith, R. L. 2007; 81A (2): 310-316

    Abstract

    The effects of an oral p38 mitogen-activated protein kinase (MAPK) inhibitor and polyethylene particles separately and together on tissue differentiation in the bone harvest chamber (BHC) in rabbits over a 3-week treatment period were investigated. The harvested tissue was analyzed histomorphometrically for markers of bone formation (percentage of bone area), osteoblasts (alkaline phosphatase staining), and osteoclasts (CD51, the alpha chain of the vitronectin receptor). Polyethylene particles decreased the percentage of bone ingrowth and staining for alkaline phosphatase. The p38 MAPK inhibitor alone decreased alkaline phosphatase staining. When the oral p38 MAPK inhibitor was given and the chamber contained polyethylene particles, there was a suppression of bone ingrowth and alkaline phosphatase staining. In contrast to oral non-steroidal anti-inflammatory drugs (NSAIDs) and local Interleukin-1 receptor antagonist (IL-1ra) administration, the oral p38 MAPK inhibitor alone did not suppress bone formation when given during the initial phase of tissue differentiation. Particle-induced inflammation and the foreign body reaction were not curtailed when the p38 MAPK inhibitor was given simultaneously with particles. Additional experiments are needed to establish the efficacy of p38 MAPK inhibitor administration on mitigating an established inflammatory and foreign body reaction that parallels the clinical situation more closely.

    View details for DOI 10.1002/jbm.a.30983

    View details for Web of Science ID 000245688500006

    View details for PubMedID 17120215

  • Kinetics of polymethylmethacrylate particle-induced inhibition of osteoprogenitor differentiation and proliferation JOURNAL OF ORTHOPAEDIC RESEARCH Chiu, R., Ma, T., Smith, R. L., Goodman, S. B. 2007; 25 (4): 450-457

    Abstract

    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.

    View details for DOI 10.1002/jor.20328

    View details for Web of Science ID 000245063900004

    View details for PubMedID 17205559

  • Effects of orthopaedic wear particles on osteoprogenitor cells BIOMATERIALS Goodman, S. B., Ma, T., Chiu, R., Ramachandran, R., Smith, R. L. 2006; 27 (36): 6096-6101

    Abstract

    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.

    View details for DOI 10.1016/j.biomaterials.2006.08.023

    View details for Web of Science ID 000242121900013

    View details for PubMedID 16949151

  • Effects of local infusion of OP-1 on particle-induced and NSAID-induced inhibition of bone ingrowth in vivo JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Ma, T., Nelson, E. R., Mawatari, T., Oh, K. J., Larsen, D. M., Smith, R. L., Goodman, S. B. 2006; 79A (3): 740-746

    Abstract

    Excessive polyethylene wear particles from joint replacements may lead to periprosthetic osteolysis and loosening. Nonsteroidal anti-inflammatory drugs (NSAIDs) decrease fracture healing and bone ingrowth. We hypothesized that continuous local infusion of OP-1 (BMP-7) would increase local bone formation in the presence of two different adverse stimuli, polyethylene particles, and an oral NSAID. The Drug Test Chamber (DTC) was implanted in the proximal tibia of mature rabbits. The tissue growing into the chamber was exposed to OP-1 solution (110 ng/day), which was infused via an osmotic pump. Infusion of OP-1 alone for 6 weeks enhanced local bone formation in the chamber by 80% (p < 0.05) over infusion of carrier alone. In the presence of polyethylene particles, infusion of OP-1 increased local bone formation by 38% (p < 0.05) over treatment with particles and carrier. Oral administration of NSAID reduced local bone formation by 58% (p < 0.05); this suppressive effect caused by NSAIDS was completely reversed by the infusion of OP-1 (p < 0.05). These findings underline a potential role for local treatment with OP-1 to increase bone formation in the presence of potentially adverse stimuli such as polyethylene wear particles or NSAID use.

    View details for DOI 10.1002/jbm.a.30949

    View details for Web of Science ID 000242134700034

    View details for PubMedID 16988970

  • Dose- and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta 3-induced chondrogenesis by adult human mesenchymal stem cells in vitro TISSUE ENGINEERING Miyanishi, K., Trindade, M. C., Lindsey, D. P., Beaupre, G. S., Carter, D. R., Goodman, S. B., Schurman, D. J., Smith, R. L. 2006; 12 (8): 2253-2262

    Abstract

    This study examined effects of varying magnitudes of intermittent hydrostatic pressure (IHP) applied for different times on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro. hMSCs were exposed to 0.1, 1, and 10 MPa of IHP at a frequency of 1 Hz for 4 h/day for 3, 7, and 14 days in the presence of transforming growth factor (TGF-beta3). Chondrogenesis was characterized by gene expression, macromolecule production, and extracellular matrix deposition. Exposure of hMSCs to 0.1 MPa of IHP increased SOX9 and aggrecan mRNA expression by 2.2- and 5.6-fold, respectively, whereas type II collagen mRNA expression responded maximally at 10 MPa. Production of sulfated glycosaminoglycan responded to IHP of 1 MPa and 10 MPa, whereas collagen levels increased only at 10 MPa. Morphologically, matrix condensation occurred with increased IHP, concomitant with collagen expression. This study demonstrated that different levels of IHP differentially modulate hMSC chondrogenesis in the presence of TGF-beta3. The data suggest that tissue engineering of articular cartilage through application or recruitment of hMSCs can be facilitated by mechanical stimulation.

    View details for Web of Science ID 000240345800019

    View details for PubMedID 16968165

  • Comparison of VEGF-producing cells in periprosthetic osteolysis BIOMATERIALS Spanogle, J. P., Miyanishi, K., Ma, T., Epstein, N. J., Smith, R. L., Goodman, S. B. 2006; 27 (21): 3882-3887

    Abstract

    The pro-angiogenic cytokine vascular endothelial growth factor (VEGF) has been implicated in periprosthetic osteolysis and subsequent aseptic loosening of implants following total hip arthroplasty (THA). The goal of this study was to investigate whether increased VEGF at the bone-implant interface is secondary to a greater number of VEGF-producing cells or to increased VEGF production by individual cells. Real time polymerase chain reaction (RT-PCR) techniques were used to assess the expression of VEGF mRNA (isoforms 121, 165, 189) in periprosthetic tissues from revision THAs. Immunofluorescence was used to determine both differences in overall cellularity and in VEGF-producing cell type (macrophages, fibroblasts, endothelial cells) between patients with periprosthetic osteolysis (OL) and a control group undergoing primary THA for osteoarthritis (OA). Quantitative analysis of VEGF release in periprosthetic membranes via RT-PCR demonstrated no significant difference in the per-cell mRNA production of VEGF isoforms 121 165, or 189 between OL and OA patient groups. Immunofluorescence showed both higher cellularity and higher overall VEGF expression in the OL group. Immunofluorescence also showed a significant increase in macrophages in the OL group, but no significant difference in the proportion of fibroblasts or endothelial cells between the OL and OA groups. Co-localization of CD68+ and CD11b+ macrophage fluorescent signals with VEGF signal was greater in the OL group than in the OA group. Our results demonstrate that increased VEGF in OL periprosthetic tissue compared to OA synovium is correlated to increased numbers of VEGF-producing CD68+ and CD11b+ macrophages. Impact statement: Aseptic loosening, caused in large part by OL, remains the major cause of failed THAs leading to revision surgery. At the bone-implant interface, we found increased numbers of macrophages-cellular mediators of OL-and increased VEGF expression. VEGF may be a possible target for therapeutic intervention in mitigating OL.

    View details for DOI 10.1016/j.biomaterials.2006.02.035

    View details for Web of Science ID 000237467200002

    View details for PubMedID 16540164

  • Polymethylmethacrylate particles inhibit osteoblastic differentiation of bone marrow osteoprogenitor cells JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Chiu, R., Ma, T., Smith, R. L., Goodman, S. B. 2006; 77A (4): 850-856

    Abstract

    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.

    View details for DOI 10.1002/jbm.a.30697

    View details for Web of Science ID 000237792300022

    View details for PubMedID 16596588

  • The effects of titanium and polymethylmethacrylate particles on osteoblast phenotypic stability JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Ramachandran, R., Goodman, S. B., Smith, R. L. 2006; 77A (3): 512-517

    Abstract

    Wear particles generated following total joint arthroplasty interact with cells at the periprosthetic margin and induce an inflammatory response that contributes to osteolysis, aseptic loosening, and implant failure. This study examined the long-term effects of particles from two commonly implanted materials, titanium (Ti) and polymethylmethacrylate (PMMA), on cell viability and metabolism over a 21-day time course, using the human osteoblast-like cell line MG-63. Addition of particles was not associated with increased cell death or nitric oxide production at the particle concentration chosen. Collagen production was increased with exposure to titanium particles, whereas alkaline phosphatase and osteocalcin expression remained unchanged following exposure to both types of particles. The data show that titanium but not PMMA particles shifts bone cell metabolism to preferentially produce fibrous tissue rather than bone.

    View details for DOI 10.1002/jbm.a.30649

    View details for Web of Science ID 000237431300009

    View details for PubMedID 16482550

  • Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro TISSUE ENGINEERING Miyanishi, K., Trindade, M. C., Lindsey, D. P., Beaupre, G. S., Carter, D. R., Goodman, S. B., Schurman, D. J., Smith, R. L. 2006; 12 (6): 1419-1428

    Abstract

    This study examined the effects of intermittent hydrostatic pressure (IHP) and transforming growth factor-beta 3 on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro. Chondrogenic gene expression was determined by quantifying mRNA signal levels for SOX9, a transcription factor critical for cartilage development and the cartilage matrix proteins, aggrecan and type II collagen. Extracellular matrix production was determined by weight and histology. IHP was applied to hMSCs in pellet culture at a level of 10 MPa and a frequency of 1 Hz for 4 h per day for periods of 3, 7, and 14 days. hMSCs responded to addition of TGF-beta 3 (10 ng/mL) with a greater than 10-fold increase (p < 0.01) in mRNA levels for each, SOX9, type II collagen, and aggrecan during a 14-day culture period. Applying IHP in the presence of TGF-beta 3 further increased the mRNA levels for these proteins by 1.9-, 3.3-, and 1.6-fold, respectively, by day 14. Chondrogenic mRNA levels were increased with just exposure to IHP. Extracellular matrix deposition of type II collagen and aggrecan increased in the pellets as a function of treatment conditions and time of culture. This study demonstrated adjunctive effects of IHP on TGF-beta 3-induced chondrogenesis and suggests that mechanical loading can facilitate articular cartilage tissue engineering.

    View details for Web of Science ID 000239570400004

    View details for PubMedID 16846340

  • Microarray analysis of mechanical shear effects on flexor tendon cells PLASTIC AND RECONSTRUCTIVE SURGERY Fong, K. D., Trindade, M. C., Wang, Z., Nacamuli, R. P., Pham, H., Fang, T. D., Song, H. J., Smith, L., Longaker, M. T., Chang, J. 2005; 116 (5): 1393-1404

    Abstract

    Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. The authors hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing.To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis.Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, the authors also found up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with the authors' microarray data.The authors demonstrate an overall antifibrotic expression pattern in response to shear stress in tendon cells that may provide insight into the mechanisms by which early mobilization decreases adhesion formation without impaired tendon healing.

    View details for DOI 10.1097/01.prs.0000182345.86453.4f

    View details for Web of Science ID 000232421100029

    View details for PubMedID 16217485

  • UHMWPE wear debris upregulates mononuclear cell proinflammatory gene expression in a novel murine model of intramedullary particle disease ACTA ORTHOPAEDICA Epstein, N. J., Bragg, W. E., Ma, T., Spanogle, J., Smith, R. L., Goodman, S. B. 2005; 76 (3): 412-420

    Abstract

    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 mononuclear cell gene transcription of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), interleukin-6 (IL-6) and macrophage chemoattractant protein-1 (MCP-1) would be upregulated by the addition of polyethylene particles in this murine intramedullary rod model.The model involved a stainless steel Kirschner wire inserted retrograde with a line-to-line fit in bilateral femora of C57bl/6 mice. Additionally, the right femora were injected with 3 x 10(9) UHMWPE particles. Mononuclear marrow cells were isolated by bone marrow aspiration and Ficoll-Paque centrifugation at 2, 4 and 10 weeks post-surgery. Total RNA was isolated and real-time RT-PCR was performed to quantify gene expression. Histological specimens of explanted femora were also analyzed to track the changes in periprosthetic tissue.UHMWPE particles stimulated gene transcription in mononuclear cells when examined at 2, 4 and 10 weeks post-surgery, compared to the rod-only group. Relative levels of IL-1beta and MCP-1 mRNA increased in a linear fashion over the 10-week time-course. IL-6 mRNA showed increased expression which peaked at 4 weeks. TNF-alpha mRNA expression was variable and reached a minimum at 4 weeks. The addition of UHMWPE particles stimulated ingress of macrophages and multinuclear cells of macrophage origin to the bone-implant interface.In this model, a single bolus of UHMWPE particles had a long-term effect on gene transcription in mononuclear cells which perpetuated a chronic inflammatory state. This murine model of intramedullary particle-induced inflammation simulates periprosthetic events associated with implant wear in humans, and may contribute to a more mechanistic understanding of wear-debris associated prosthesis failure.

    View details for DOI 10.1080/17453670510041321

    View details for Web of Science ID 000231005300021

    View details for PubMedID 16156472

  • Interleukin-1 modulates periprosthetic tissue formation in an intramedullary model of particle-induced inflammation JOURNAL OF ORTHOPAEDIC RESEARCH Epstein, N. J., Warme, B. A., Spanogle, J., Ma, T., Bragg, B., Smith, R. L., Goodman, S. B. 2005; 23 (3): 501-510

    Abstract

    Interleukin-1 (IL-1) is a proinflammatory cytokine that has been implicated in wear-debris associated total joint replacement failure. We hypothesized that the absence of the IL-1 type-1 receptor would mitigate the inflammatory response to titanium particles and decrease periprosthetic inflammatory tissue in a murine intramedullary rod model.An intramedullary rod with and without commercially pure titanium particles was placed in the femora of 24 wild type mice (WT) and 24 mice lacking a functional type-1 receptor to IL-1. Femora were analyzed histologically and by ELISA of organ culture explant supernatants.The presence of titanium particles in WT mice stimulated increased expression of interleukin-6 (IL-6) and macrophage chemoattractant protein-1 (MCP-1) relative to rod only controls. In contrast, IL-6 and MCP-1 expression were diminished in IL-1r1-KO mice exposed to titanium particles. Additionally, the formation of a periprosthetic fibro-inflammatory membrane in IL-1r1-KO mice was blunted at 2 weeks when compared to that in wild-type mice. Inflammatory changes and the quality of periprosthetic bone of IL-1r1-KO mice was similar to WT mice in response to titanium particles.These results implicate IL-1 as an important modulator in the local inflammatory response to intramedullary titanium particles. MCP-1 appears to be significantly modulated in IL-1r1-KO mice in response to titanium particles. This may be responsible, in part, for the diminished periprosthetic membrane observed in IL-1r1-KO mice at 2 weeks. Expansion of this murine model of intramedullary particle-induced inflammation to other gene targets may contribute to a more mechanistic understanding of wear-debris associated prosthesis failure.

    View details for DOI 10.1016/j.orthres.2004.10.004

    View details for Web of Science ID 000229375000001

    View details for PubMedID 15885468

  • Temporal effects of a COX-2-selective NSAID on bone ingrowth JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Goodman, S. B., Ma, T., Mitsunaga, L., Miyanishi, K., Genovese, M. C., Smith, R. L. 2005; 72A (3): 279-287

    Abstract

    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-microm 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.

    View details for DOI 10.1002/jbm.a.30231

    View details for Web of Science ID 000227223200005

    View details for PubMedID 15666361

  • Pharmacologic modulation of periprosthetic osteolysis CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Goodman, S. B., Trindade, M., Ma, T., Genovese, M., Smith, R. L. 2005: 39-45

    Abstract

    Wear and periprosthetic osteolysis of total joint replacements continue to be the most important problems in arthroplasty surgery. Despite the introduction of improved technologies including alternative bearing surfaces for TJRs, wear is inevitable because of relative movement at different interfaces and processes such as electrolysis and material degradation. Worn, clinically failing implants need to be followed closely and revised when appropriate. However, early wear and minor osteolysis do not result necessarily in progressive failure of the prosthesis. Indeed such cases may be followed up clinically and radiographically to establish the functional and biologic sequelae of wear and the timeline of these events. This scenario provides an opportunity to modulate the adverse biologic reaction associated with wear particles that includes chronic inflammation, the foreign body response, and periprosthetic bone destruction. Currently, immunological events associated with wear particles are becoming understood more clearly. Strategies to mitigate adverse processes associated with wear debris include local or systemic administration of immune modulators, signaling molecules, anti-inflammatory agents and growth factors, and altering osteoclast function. Ultimately, prevention of accelerated wear and periprosthetic osteolysis will be achieved with improved bearing surfaces and prosthetic designs.

    View details for DOI 10.1097/01/blo.0000149998.88218.05

    View details for Web of Science ID 000226145500005

    View details for PubMedID 15662302

  • Proinflammatory mediator expression in a novel murine model of titanium-particle-induced intramedullary inflammation JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS Warme, B. A., Epstein, N. J., Trindade, M. C., Miyanishi, K., Ma, T., Saket, R. R., Regula, D., Goodman, S. B., Smith, R. L. 2004; 71B (2): 360-366

    Abstract

    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.

    View details for DOI 10.1002/jbm.b.30120

    View details for Web of Science ID 000224846700018

  • Osteoarthritis - Current treatment and future prospects for surgical, medical, and biologic intervention CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Schurman, D. J., Smith, R. L. 2004: S183-S189

    Abstract

    The treatment of osteoarthritis includes a wide spectrum of approaches. This article reviews current practices in medical, pharmaceutical and surgical treatment with a perspective toward the immediate, distant and far distant future. At present, with the exception of surgery, all other treatments are palliative. That is to say that many of these treatments relieve pain and increase function. However, on the basis of medical evidence, these treatments do not change the course of the disease. Surgical interventions, including joint replacement and osteotomy, reverse the progress of osteoarthritis and provide long-term improved function and pain relief for specific joints. The goal of treating osteoarthritis is to arrest and reverse its progress regionally or globally through biologic methodology. Meaningful progress for biologic intervention accumulates annually. Pluripotent mesenchymal cells can be coaxed into chondrocytes or stem cells. Cytokines, growth factors, chemokines, protease inhibitors, kinases, apoptosis, mechanics, and genetics are increasingly recognized to play key roles in the control of the articular cartilage behavior. Knowledge of their roles and relationships advance toward solutions related to osteoarthritis. In the future, biologic control may be harnessed to regrow joints or limbs, as currently occurs naturally in newts and salamanders. Fortunately, until then we have ever improving joint replacement.

    View details for DOI 10.1097/01.blo.0000143555.33848.c4

    View details for Web of Science ID 000224524400031

    View details for PubMedID 15480065

  • The mechanobiology of articular cartilage development and degeneration CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Carter, D. R., Beaupre, G. S., Wong, M., Smith, R. L., Andriacchi, T. P., Schurman, D. J. 2004: S69-S77

    Abstract

    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.

    View details for DOI 10.1097/01.blo.0000144970.05107.7e

    View details for Web of Science ID 000224524400014

    View details for PubMedID 15480079

  • Pressure and shear differentially alter human articular chondrocyte metabolism - A review CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Smith, R. L., Carter, D. R., Schurman, D. J. 2004: S89-S95

    Abstract

    Homeostasis of articular cartilage depends in part on mechanical loads generated during daily activity whereas inappropriate joint loads result in focal degeneration of cartilage, as occurs in osteoarthritis. We will review results of a series of questions regarding the effects of two types of mechanical loads-intermittent hydrostatic pressure and shear stress-on adult human articular chondrocytes in high-density monolayer culture. Intermittent hydrostatic pressure increased aggrecan and Type II collagen gene expression in normal chondrocytes and induced changes in the cell-associated proteins of normal and osteoarthritic chondrocytes. Hydrostatic pressure also counteracted inhibitory effects of bacterial lipopolysaccharide on matrix protein expression by cultured chondrocytes. Application of shear stress to osteoarthritic chondrocytes increased the release of the proinflammatory mediator, nitric oxide, decreased aggrecan and Type II collagen expression, and induced molecular changes associated with apoptosis whereas hydrostatic pressure increased matrix macromolecule expression. The findings show that the types of load comprising the mechanical loading environment of articular cartilage considerably alter chondrocyte metabolism and suggest that mechanical stimulation may be used for in vitro or in vivo approaches for cartilage engineering.

    View details for DOI 10.1097/01.blo.0000143938.30681.9d

    View details for Web of Science ID 000224524400016

    View details for PubMedID 15480081

  • Intermittent hydrostatic pressure inhibits matrix metalloproteinase and pro-inflammatory mediator release from human osteoarthritic chondrocytes in vitro OSTEOARTHRITIS AND CARTILAGE Trindade, M. C., Shida, J., Ikenoue, T., Lee, M. S., Lin, E. Y., Yaszay, B., Yerby, S., Goodman, S. B., Schurman, D. J., Smith, R. L. 2004; 12 (9): 729-735

    Abstract

    This study tested the hypothesis that intermittent hydrostatic pressure applied to human osteoarthritic chondrocytes modulates matrix metalloproteinase and pro-inflammatory mediator release in vitro.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 10 MPa at a frequency of 1 Hz for periods of 6, 12 and 24 h. 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.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 84/82 kDa 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 24 h, relative to unloaded control cultures maintained for the same time periods.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.

    View details for DOI 10.1016/j.joca.2004.05.008

    View details for Web of Science ID 000224046200007

    View details for PubMedID 15325639

  • Human interleukin-1-induced murine osteoclastogenesis is dependent on RANKL, but independent of TNF-alpha CYTOKINE Ma, T., Miyanishi, K., Suen, A., Epstein, N. J., Tomita, T., Smith, R. L., Goodman, S. B. 2004; 26 (3): 138-144

    Abstract

    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.

    View details for DOI 10.1016/j.cyto.2004.02.001

    View details for Web of Science ID 000221898200007

    View details for PubMedID 15135808

  • Effects of interleukin-10 on titanium particle-induced macrophage transcription factor activation and cytokine expression in vitro JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Wong, N., Trindade, M. C., Patel, R., Yaszay, B., Goodman, S. B., Smith, R. L. 2004; 69A (1): 40-46

    Abstract

    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.

    View details for DOI 10.1002/jbm.a.20097

    View details for Web of Science ID 000220224600005

  • A framework for the in vivo pathomechanics of osteoarthritis at the knee ANNALS OF BIOMEDICAL ENGINEERING Andriacchi, T. P., Mundermann, A., Smith, R. L., Alexander, E. J., Dyrby, C. O., Koo, S. 2004; 32 (3): 447-457

    Abstract

    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 vivo pathomechanics, 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 vivo framework 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.

    View details for Web of Science ID 000222465100016

    View details for PubMedID 15095819

  • Interleukin 1 receptor antagonist inhibits localized bone formation in vivo JOURNAL OF RHEUMATOLOGY Ma, T., Miyanishi, K., Trindade, M. C., Genovese, M., Regula, D., Smith, R. L., Goodman, S. B. 2003; 30 (12): 2547-2552

    Abstract

    To test the in vivo effects of interleukin 1 receptor antagonist (IL-1ra) on bone formation and tissue ingrowth using an implantable bone ingrowth chamber that can be infused with test solutions.The bone ingrowth chamber was implanted in the proximal tibia of 10 mature NZW rabbits unilaterally. After an initial osseointegration period, the chambers were emptied of tissue and infused with either 0.05% bovine serum albumin (BSA) in phosphate buffered saline (PBS) or an IL-1ra solution for 4-week periods, which were separated by 4-week periods of no infusion. Tissue samples harvested from each chamber were snap-frozen and examined by histology and immunohistochemistry.The chambers were filled with longitudinally-oriented woven bone in a fibrovascular stroma during periods of infusion of 0.05% BSA in PBS or during periods without infusion. In contrast, infusion of IL-1ra for 4 weeks prevented tissue ingrowth in 4 of 6 chambers, and in 2 chambers exhibiting tissue ingrowth, bone formation was decreased. Bone formation remained at a lower level during the subsequent two 4-week periods without infusion after IL-1ra was discontinued, compared to samples prior to the IL-1ra treatment.The results showed that tissue ingrowth and bone formation were suppressed in an in vivo model by continuous infusion of IL-1ra at an early phase of tissue regeneration and differentiation.

    View details for Web of Science ID 000187441800008

    View details for PubMedID 14719192

  • Expression of nitric oxide, peroxynitrite, and apoptosis in loose total hip replacements. Journal of biomedical materials research. Part A Puskas, B. L., Menke, N. E., Huie, P., Song, Y., Ecklund, K., Trindade, M. C., Smith, R. L., Goodman, S. B. 2003; 66 (3): 541-549

    Abstract

    Nitric oxide (NO) is an effector molecule associated with inflammation, immune function, bone metabolism, and the induction of apoptosis. This study examined the role of NO, peroxynitrite (ONOO(-)), and apoptosis in cases of revision total hip replacements (THRs). We hypothesized that apoptosis and excess production of NO contribute to the inflammatory reaction to orthopedic biomaterial wear debris that is associated with loosening and osteolysis. Periprosthetic membranous specimens were collected from revised cemented acetabular components with simple loosening and ballooning osteolysis. Synovial samples from patients undergoing primary THR were used as controls. The presence of macrophages (CD68(+)) and levels of inducible nitric oxide synthase (INOS), endothelial nitric oxide synthase (EcNOS), ONOO(-) (Nitro, assayed by the amount of nitrated tyrosine residues), and apoptosis (TUNEL staining) were examined using immunohistochemistry. Increased expression for INOS, EcNOS, and ONOO(-) in both the loose/osteolytic and the loose/non-osteolytic groups was observed when compared to the synovium group. There were no significant differences between the loose/osteolytic group and loose/non-osteolytic group for these biologic markers. TUNEL staining showed a significant increase in apoptosis in the loose/osteolytic group compared to the loose/non-osteolytic group and synovial tissues. These findings suggest that NO and NO-derived molecules, such as ONOO(-), may be involved in sustaining the foreign-body reaction to wear debris. NO and ONOO(-) may prove to be useful markers of prosthetic loosening whereas apoptosis may be a marker distinguishing ballooning from simple osteolysis.

    View details for PubMedID 12918037

  • Periprosthetic osteolysis: Induction of vascular endothelial growth factor from human monocyte/macrophages by orthopedic biomaterial particles JOURNAL OF BONE AND MINERAL RESEARCH Miyanishi, K., Trindade, M. C., Ma, T., Goodman, S. B., Schurman, D. J., Smith, R. L. 2003; 18 (9): 1573-1583

    Abstract

    VEGF and VEGF receptor, Flt-1, expression was observed in periprosthetic tissues surrounding loosened total joint implants. Exposure of monocyte/macrophages to titanium particles resulted in increased VEGF expression, p44/42 MAPK activation, and VEGF-dependent macrophage chemotaxis. Increased levels of angiogenic factors, such as VEGF, may be critically important in wear debris-induced implant loosening after total joint arthroplasty.Periprosthetic osteolysis after total hip arthroplasty occurs in association with formation of a vascularized granulomatous tissue in response to particulate debris.This study examined expression of vascular endothelial growth factor (VEGF) and the VEGF receptor in 10 periprosthetic tissues from loosened prostheses and quantified effects of titanium particles on VEGF release, intracellular signaling, and VEGF-dependent chemotaxis in primary cultures of human monocyte/macrophages.Double immunofluorescent staining showed that VEGF and Flt-1 co-localized with cells positive for the macrophage marker, CD11b, in the periprosthetic tissues. Monocyte/macrophages challenged with titanium particles showed a dose- and time-dependent release of VEGF ranging from 2.8- to 3.1-fold and exhibited increased expression of VEGF121 and VEGF165 mRNAs, reaching levels up to 5.0- and 8.6-fold, respectively, by 48 h (p < 0.01). Exposure of monocyte/macrophages to titanium particles upregulated phosphorylated-p44/42 mitogen-activated protein kinase (MAPK) within 30 minutes. Particle-induced activation of p44/42 MAPK and release of VEGF were dose-dependently suppressed by pretreatment of cells with PD98059, a specific inhibitor of p44/42 MAPK. Monocyte/macrophages challenged with titanium particles also showed a time-dependent activation of AP-1, a transcription factor associated with VEGF expression (p < 0.01). Supernatants from particle-challenged monocyte/macrophages increased macrophage chemotactic activity by 30%, which was significantly inhibited by anti-VEGF neutralizing antibody (p < 0.01).This study suggests that induction of VEGF release from monocyte/macrophages in response to orthopaedic biomaterial wear debris may contribute to periprosthetic osteolysis and implant loosening.

    View details for Web of Science ID 000184943900003

    View details for PubMedID 12968666

  • Regulation of nitric oxide and bcl-2 expression by shear stress in human osteoarthritic Chondrocytes in vitro JOURNAL OF CELLULAR BIOCHEMISTRY Lee, M. S., Trindade, M. C., Ikenoue, T., Goodman, S. B., Schurman, D. J., Smith, R. L. 2003; 90 (1): 80-86

    Abstract

    Onset and progression of cartilage degeneration is associated with shear stress occurring in diarthrodial joints subjected to inappropriate loading. This study tested the hypothesis that shear stress induced nitric oxide is associated with altered expression of regulatory onco-proteins, bcl-2, and Fas (APO-1/CD95) and apoptosis in primary human osteoarthritic chondrocyte cultures. Shear stress induced membrane phosphatidylserine and nucleosomal degradation were taken as evidence of chondrocyte apoptosis. Application of shear stress upregulated nitric oxide in a dose-dependent manner and was associated with increases in membrane phosphatidylserine and nucleosomal degradation. Increasing levels of shear stress decreased expression of the anti-apoptotic factor, bcl-2, from 44 to 10 U/ml. Addition of the nitric oxide antagonists, L-N(5)-(1-iminoethyl) ornithine and Nomega-nitro-L-arginine methyl ester (L-NAME), reduced shear stress induced nucleosomal degradation by 62% and 74%, respectively. Inhibition of shear stress induced nitric oxide release by L-NAME coincided with a 2.7-fold increase of bcl-2, when compared to chondrocytes exposed to shear stress in the absence of L-NAME. These data suggest that shear stress induced nitric oxide is associated with changes in apoptotic regulatory factors that alter chondrocyte metabolism and may contribute to joint degeneration.

    View details for DOI 10.1002/jcb.10611

    View details for Web of Science ID 000185103600009

    View details for PubMedID 12938158

  • Local infusion of FGF-2 enhances bone ingrowth in rabbit chambers in the presence of polyethylene particles. Journal of biomedical materials research. Part A Goodman, S. B., Song, Y., Yoo, J. Y., Fox, N., Trindade, M. C., Kajiyama, G., Ma, T., Regula, D., Brown, J., Smith, R. L. 2003; 65 (4): 454-461

    Abstract

    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 1x 1 x 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-microm diameter) on tissue ingrowth were determined by adding particles to the chamber at concentrations of 5.8 x 10(11) (low dose) or 1.7 x 10(12) (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.

    View details for PubMedID 12761835

  • Human serum opsonization of orthopedic biomaterial particles: Protein-binding and monocyte/macrophage activation in vitro JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Sun, D. H., Trindade, M. C., Nakashima, Y., Maloney, W. J., Goodman, S. B., Schurman, D. J., Smith, R. L. 2003; 65A (2): 290-298

    Abstract

    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.

    View details for DOI 10.1002/jbm.a.10477

    View details for Web of Science ID 000182627600022

  • Modulation of bone ingrowth and tissue differentiation by local infusion of interleukin-10 in the presence of ultra-high molecular weight polyethylene (UHMWPE) wear particles. Journal of biomedical materials research. Part A Goodman, S., Trindade, M., Ma, T., Lee, M., Wang, N., Ikenou, T., Matsuura, I., Miyanishi, K., Fox, N., Regula, D., Genovese, M., Klein, J., Bloch, D., Smith, R. L. 2003; 65 (1): 43-50

    Abstract

    Interleukin-10 (IL-10) is a cytokine that plays a major role in suppressing the inflammatory response, particularly cell-mediated immunity that is characteristic of the TH1 response. The purpose of this study was to determine whether local infusion of IL-10 could mitigate the suppression of bone ingrowth associated with polyethylene wear particles. Drug test chambers were implanted in the proximal tibia of 20 mature New Zealand White rabbits. The DTC provided a continuous 1 x 1 x 5-mm canal for tissue ingrowth. After a 6-week period for osseointegration, the DTC was then connected to an osmotic diffusion pump. IL-10 at doses of 0.1-100 ng/mL (0.25 microL/h) was infused with or without ultra-high molecular weight polyethylene particles (0.5 +/- 0.2 microm diameter, 10(12) particles/mL) present in the chamber for a 3- or 6-week period. The tissue in the chamber was harvested after each treatment; sections were stained with hematoxylin and eosin for morphometric analysis. Osteoclast-like cells were identified by immunohistochemical staining using a monoclonal antibody directed against the alpha chain of the vitronectin receptor, CD51. Osteoblasts were identified using alkaline phosphatase staining. In dose-response studies, infusion of 1 ng/mL IL-10 yielded the greatest bone ingrowth in the presence of particles. The addition of polyethylene particles evoked a marked foreign body reaction and fibrosis; bone ingrowth was significantly suppressed (p = 0.0003). Bone ingrowth was increased by over 48% with infusion of IL-10 for the final 3 weeks of a 6-week ultra-high molecular weight polyethylene particle exposure compared with particles alone (p = 0.027). IL-10 is a cytokine that plays a major role in suppressing the inflammatory response, especially cell-mediated immunity that is characteristic of the TH1 response. Local infusion of immune-modulating cytokines such as IL-10 may prove to be useful in abating particle-induced periprosthetic osteolysis.

    View details for PubMedID 12635153

  • The role of the TH1 and TH2 immune responses in loosening and osteolysis of cemented total hip replacements. Journal of biomedical materials research. Part A Arora, A., Song, Y., Chun, L., Huie, P., Trindade, M., Smith, R. L., Goodman, S. 2003; 64 (4): 693-697

    Abstract

    The mechanisms underlying the development of osteolysis and aseptic loosening have an impact on the longevity of total hip replacements (THRs). This study examines the specific roles of lymphocytes in the TH1 and TH2 subsets in osteolysis and aseptic loosening of THR. Tissue from periprosthetic regions from patients with loose, cemented acetabular components were used to determine the TH1 and TH2 cytokine profile. Twelve tissue specimens from patients with radiographic signs of osteolysis, and nine tissue specimens from patients with no signs of osteolysis were harvested during revision surgery. Immunohistochemistry using primary antibodies against CD3, interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and IL-10 was performed on frozen sections to determine the percentage of positive cells for each of the sections. No statistically significant differences in the percentage of positive cells expressing cytokines characteristic of the TH1 pathway (IFN-gamma, IL-2) or TH2 pathway (IL-4, IL-10) were found when comparing osteolytic and non-osteolytic tissues. However, significant numbers of T cells (averaging about 10% of the total cells) and TH1 and TH2 immune cytokines (averaging 3-5% of cells) implicate a possible role for immune processes at the prosthetic interface.

    View details for PubMedID 12601781

  • Intermittent hydrostatic pressure inhibits shear stress-induced nitric oxide release in human osteoarthritic chondrocytes in vitro JOURNAL OF RHEUMATOLOGY Lee, M. S., Trindade, M. C., Ikenoue, T., Schurman, D. J., Goodman, S. B., Smith, R. L. 2003; 30 (2): 326-328

    Abstract

    To test the effects of intermittent hydrostatic pressure (IHP) on nitric oxide (NO) release induced by shear stress and matrix macromolecule gene expression in human osteoarthritic chondrocytes in vitro.Chondrocytes isolated from cartilage samples from 9 patients with osteoarthritis were cultured and exposed to either shear stress or an NO donor. Nitrite concentration was measured using the Griess reaction. Matrix macromolecule mRNA signal levels were determined using reverse-transcriptase polymerase chain reaction and quantified by imaging analysis software.Exposure to shear stress upregulated NO release in a dose and time-dependent manner. Application of IHP inhibited shear stress induced NO release but did not alter NO release from chondrocytes not exposed to shear stress. Shear stress induced NO or addition of an NO donor (sodium nitroprusside) was associated with decreased mRNA signal levels for the cartilage matrix proteins, aggrecan, and type II collagen. Intermittent hydrostatic pressure blocked the inhibitory effects of sodium nitroprusside but did not alter the inhibitory effects of shear stress on cartilage macromolecule gene expression.Our data show that shear stress and IHP differentially alter chondrocyte metabolism and suggest that a balance of effects between different loading forces preserve cartilage extracellular matrix in vivo.

    View details for Web of Science ID 000180709700022

    View details for PubMedID 12563690

  • COX-2 selective NSAID decreases bone ingrowth in vivo JOURNAL OF ORTHOPAEDIC RESEARCH Goodman, S., Ma, T., Trindade, M., Ikenoue, T., Matsuura, I., Wong, N., Fox, N., Genovese, M., Regula, D., Smith, R. L. 2002; 20 (6): 1164-1169

    Abstract

    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 rabbit's mouth. Harvested specimens were snap frozen, cut into serial 6 microm 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.

    View details for Web of Science ID 000179400800005

    View details for PubMedID 12472224

  • Fibroblast expression of C-C chemokines in response to orthopaedic biomaterial particle challenge in vitro JOURNAL OF ORTHOPAEDIC RESEARCH Yaszay, B., Trindade, M. C., Lind, M., Goodman, S. B., Smith, R. L. 2001; 19 (5): 970-976

    Abstract

    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.

    View details for Web of Science ID 000170771100032

    View details for PubMedID 11562149

  • Interleukin-10 inhibits polymethylmethacrylate particle induced interleukin-6 and tumor necrosis factor-alpha release by human monocyte/macrophages in vitro BIOMATERIALS Trindade, M. C., Lind, M., Nakashima, Y., Sun, D. H., Goodman, S. B., Schurman, D. J., Smith, R. L. 2001; 22 (15): 2067-2073

    Abstract

    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. Populations of activated lymphocytes are often seen in periprosthetic membranes. These lymphocytes may modulate the monocyte/macrophage response to particulate debris and influence aseptic loosening. In addition, other immunologic agents, such as interleukin-10, are present in tissues harvested from the bone-implant interface of failed total joint arthroplasties. The present study examined the effects of interleukin-10 on polymethylmethacrylate (PMMA) particle challenged human monocyte/macrophages in vitro. Human monocyte/macrophages isolated from buffy coats of five healthy individuals were exposed to 1-10 microm PMMA particles. Interleukin-10 was added to the monocyte/macrophages with and without the addition of PMMA particles. Interleukin-10-induced alterations in monocyte/macrophage metabolism were determined measuring interleukin-6 and tumor necrosis factor-alpha release by the cells 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. Interleukin-10 reduced the levels of interleukin-6 and tumor necrosis factor-alpha release by macrophages in response to PMMA particles in a dose-dependent manner. At 48 h, particle-induced interleukin-6 release was inhibited by 60 and 90% with 1.0 and 10.0 ng/ml treatments of interleukin-10, respectively. At 48 h, particle-induced tumor necrosis factor-alpha release was inhibited by 58 and 88% with 1.0 and 10.0 ng/ml treatments of interleukin-10, respectively. Interleukin-10 challenge alone did not significantly alter basal interleukin-6 or tumor necrosis factor-alpha release relative to control cultures. The data presented in this study demonstrate that the anti-inflammatory cytokine, interleukin-10, inhibits monocyte/macrophage release of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in response to PMMA particle challenge in vitro.

    View details for Web of Science ID 000169365300001

    View details for PubMedID 11432585

  • In vitro reaction to orthopaedic biomaterials by macrophages and lymphocytes isolated from patients undergoing revision surgery BIOMATERIALS Trindade, M. C., Lind, M., Sun, D., Schurman, D. J., Goodman, S. B., Smith, R. L. 2001; 22 (3): 253-259

    Abstract

    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.

    View details for Web of Science ID 000166303300008

    View details for PubMedID 11197500

  • Effects of local infusion of TGF beta on bone ingrowth in rabbit chambers JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Goodman, S., Song, Y., Chun, L., Aspenberg, P., Plouhar, P., Glancy, T., Regula, D., Smith, R. L. 2000; 53 (5): 475-479

    Abstract

    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.

    View details for Web of Science ID 000089469300005

    View details for PubMedID 10984694

  • G-protein activity requirement for polymethylmethacrylate and titanium particle-induced fibroblast interleukin-6 and monocyte chemoattractant protein-1 release in vitro JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Trindade, M. C., Schurman, D. J., Maloney, W. J., Goodman, S. B., Smith, R. L. 2000; 51 (3): 360-368

    Abstract

    Periprosthetic granulomatous membranes consisting of fibroblasts, macrophages, lymphocytes, foreign body giant cells, and abundant particulate debris occur at sites of implant loosening. Previous studies demonstrate that fibroblasts respond to particulate debris through the release of interleukin-6 (IL-6), prostaglandin E(2), and matrix metalloproteinases in vitro. C-C chemokines are observed in granulomatous tissue surrounding loosened prosthetic implants and are released by macrophages and fibroblasts in response to particle challenge in vitro. This study tested the hypothesis that G protein activity is required for fibroblast activation by titanium and polymethylmethacrylate (PMMA) particles, and that inhibition of G protein activity would alter IL-6 and and monocyte chemoattractant protein-1 (MCP-1) release from activated fibroblasts. The specific inhibitor of G protein activity, pertussis toxin, was added to the fibroblasts to examine the effects of G protein activity with respect to the production of IL-6 and MCP-1 by orthopedic biomaterial-challenged fibroblasts in vitro. Interleukin-1beta (IL-1beta), a proven activator of MCP-1 and interleukin-6, was used as a positive control. Exposure of fibroblasts to titanium and polymethylmethacrylate (PMMA) particles resulted in a dose-dependent release of MCP-1 and IL-6. Challenge with PMMA particles at doses of 0.150%, 0.300%, and 0.600% vol/vol increased the release of interleukin-6 by 7-, 19-, and 22-fold, respectively, compared to fibroblasts exposed to serum-free culture medium alone at 24 h. Challenge with PMMA particles at doses of 0.075%, 0.150%, 0.300%, and 0.600% vol/vol increased the release of MCP-1-6 by 2.5-, 3.6-, 4. 3-, and 4.5-fold, respectively, compared to fibroblasts exposed to serum-free culture medium alone. Challenge with titanium particles at concentrations of 0.075%, 0.150%, 0.300%, and 0.600% vol/vol increased the release of interleukin-6 by 2.6-, 6.4-, 9.6-, and 10. 0-fold, respectively, compared to fibroblasts exposed to serum-free culture medium alone at 24 h. Challenge with titanium particles at concentrations of 0.038%, 0.075%, 0.150%, 0.300%, and 0.600% vol/vol increased the release of MCP-1 by 2.9-, 3.1-, 5.8-, 5.4-, and 5. 8-fold, respectively, compared to fibroblasts exposed to serum-free culture medium alone. Pretreatment of fibroblasts with pertussis toxin inhibited the release of interleukin-6 and MCP-1 from PMMA and titanium particle challenged fibroblasts in a dose-dependent manner. PMMA particle induced fibroblast IL-6 release was inhibited by 23.6% and 35.3% with 20- and 200-ng/mL doses of pertussis toxin, respectively. Titanium particle induced fibroblast IL-6 release was inhibited by 48.2% and 56.3% with 20- and 200-ng/mL doses of pertussis toxin, respectively. PMMA particle-induced fibroblast MCP-1 release was inhibited by 36.0%, 50.4%, and 60.1% with 2-, 20- and 200-ng/mL doses of pertussis toxin, respectively. Titanium particle-induced fibroblast MCP-1 release was inhibited by 15.5%, 53.2%, and 64.6% with 2-, 20-, and 200-ng/mL doses of pertussis toxin, respectively. This study suggests that fibroblasts localized in periprosthetic membranes are a source of macrophage chemoattractant factors and proinflammatory mediators that may influence granuloma formation and lead to periprosthetic bone resorption. Furthermore, this study shows that G proteins are involved in particle-induced fibroblast activation, as evidenced by decrease levels of particle induced IL-6 and MCP-1 release following pertussis toxin treatment. (c) 2000 John Wiley & Sons, Inc.

    View details for Web of Science ID 000087914500009

    View details for PubMedID 10880077

  • Induction of interleukin-6 release in human osteoblast-like cells exposed to titanium particles in vitro CALCIFIED TISSUE INTERNATIONAL Shida, J., Trindade, M. C., Goodman, S. B., Schurman, D. J., Smith, R. L. 2000; 67 (2): 151-155

    Abstract

    Orthopaedic wear debris induces release of bone-resorbing factors from macrophages and fibroblasts. However, the extent to which elemental metallic particles induce bone cells to express factors contributing to implant loosening remains unclear. This study showed that exposure of MG-63 osteoblast-like cells to titanium particles at a concentration of 0.30% v/v resulted in a 15-fold increase in IL-6 release into the culture medium after 24 hours, when compared with cells without particles. Northern blots revealed that exposure of MG-63 cells to titanium particles at a concentration of 0.30% v/v for 24 hours increased IL-6 mRNA signal levels by 9.6-fold, when compared with control cultures. Pretreatment of MG-63 cells with cytochalasin B prevented the particle-induced increase of IL-6 expression but did not alter the basal level of IL-6 release from cells cultured in the absence of particles. The protein kinase C inhibitor, H7, and the serine/threonine kinase inhibitor, genistein, abolished the particle-induced increase in IL-6 release at a concentration of 100 microM for each compound. In contrast, an inhibitor of protein kinase A, HA1004, had no effect on the particle-induced increase in IL-6 release. The transcription factors, nuclear factor IL-6 and nuclear factor kappa B, translocated into the nucleus within 1 hour of particle exposure. This study showed that osteoblast-like cells respond to titanium particles through increased expression of the proinflammatory cytokine, IL-6, in a process requiring phagocytosis and intracellular signaling pathways. These results suggest that osteoblasts play a direct role in implant loosening because of localized release of soluble mediators such as interleukin-6.

    View details for Web of Science ID 000088387400010

    View details for PubMedID 10920220

  • Degradative enzymes in osteoarthritis. Frontiers in bioscience Smith, R. L. 1999; 4: D704-12

    Abstract

    A central feature of the osteoarthritic disease process involves erosive destruction of the articular cartilage extracellular matrix (ECM) on the surfaces of diarthrotic joints. The resultant loss of joint function makes studies on mechanisms underlying ECM degradation critical for treatment of the disease and prevention of disability. Candidate pathways to account for the loss of cartilage involve expression of a combination of proteases that degrade the major cartilage matrix macromolecules, aggrecan and type II collagen. The specific types of enzymatic activities associated with the progressive removal of ECM and severity of joint disease include the matrix metalloproteinases, collagenase, gelatinase and aggrecanase(s). The degradative enzymes originate in synovial cells, cartilage cells, the chondrocytes, distributed within the ECM and leukocytes that actively invade the joint space. Specific enzymes arising from each of these tissues exhibit selective ECM degrading properties; the different categories of these tissue-derived enzymes will be discussed in this chapter. A perspective on the efficacy of existing agents and the potential for development of novel therapeutic agents is also included. While the degradative enzymes serve as a focal point for therapeutic intervention, a fundamental understanding of the mechanisms underlying degradative enzyme expression in osteoarthritis remains an important goal for prevention of disease.

    View details for PubMedID 10525483

  • Interferon-gamma exacerbates polymethylmethacrylate particle-induced interleukin-6 release by human monocyte/macrophages in vitro JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Trindade, M. C., Lind, M., Goodman, S. B., Maloney, W. J., Schurman, D. J., Smith, R. L. 1999; 47 (1): 1-7

    Abstract

    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.

    View details for Web of Science ID 000081599800001

    View details for PubMedID 10400874

  • Proinflammatory mediator release in response to particle challenge: Studies using the bone harvest chamber JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Trindade, M. C., Song, Y., Aspenberg, P., Smith, R. L., Goodman, S. B. 1999; 48 (4): 434-439

    Abstract

    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.

    View details for Web of Science ID 000081440800006

    View details for PubMedID 10421684

  • Signaling pathways for tumor necrosis factor-alpha and interleukin-6 expression in human macrophages exposed to titanium-alloy particulate debris in vitro. journal of bone and joint surgery. American volume Nakashima, Y., Sun, D. H., Trindade, M. C., Maloney, W. J., Goodman, S. B., Schurman, D. J., Smith, R. L. 1999; 81 (5): 603-615

    Abstract

    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.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.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.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

    View details for PubMedID 10360689

  • Expression of inflammatory mediators by human macrophages in response to particulate debris in vitro JOINT ARTHROPLASTY Nakashima, Y., Sun, D. H., Trindade, M., Maloney, W. J., Goodman, S. B., Schurman, D. J., Smith, R. L., Ushijima, M., Iwamoto, Y. 1999: 65-75
  • Induction of macrophage C-C chemokine expression by titanium alloy and bone cement particles. journal of bone and joint surgery. British volume Nakashima, Y., Sun, D. H., Trindade, M. C., Chun, L. E., Song, Y., Goodman, S. B., Schurman, D. J., Maloney, W. J., Smith, R. L. 1999; 81 (1): 155-162

    Abstract

    Particulate wear debris is associated with periprosthetic inflammation and loosening in total joint arthroplasty. We tested the effects of titanium alloy (Ti-alloy) and PMMA particles on monocyte/macrophage expression of the C-C chemokines, monocyte chemoattractant protein-1 (MCP-1), monocyte inflammatory protein-1 alpha (MIP-1alpha), and regulated upon activation normal T expressed and secreted protein (RANTES). Periprosthetic granulomatous tissue was analysed for expression of macrophage chemokines by immunohistochemistry. Chemokine expression in human monocytes/macrophages exposed to Ti-alloy and PMMA particles in vitro was determined by RT-PCR, ELISA and monocyte migration. We observed MCP-1 and MIP-1alpha expression in all tissue samples from failed arthroplasties. Ti-alloy and PMMA particles increased expression of MCP-1 and MIP-1alpha in macrophages in vitro in a dose- and time-dependent manner whereas RANTES was not detected. mRNA signal levels for MCP-1 and MIP-1alpha were also observed in cells after exposure to particles. Monocyte migration was stimulated by culture medium collected from macrophages exposed to Ti-alloy and PMMA particles. Antibodies to MCP-1 and MIP-1alpha inhibited chemotactic activity of the culture medium samples. Release of C-C chemokines by macrophages in response to wear particles may contribute to chronic inflammation at the bone-implant interface in total joint arthroplasty.

    View details for PubMedID 10068024

  • Effects of serum protein opsonization on cytokine release by titanium-alloy particles JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Maloney, W. J., Sun, D. H., Nakashima, Y., James, R., Smith, R. L. 1998; 41 (3): 371-376

    Abstract

    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.

    View details for Web of Science ID 000074523300005

    View details for PubMedID 9659605

  • Effects of particulate debris on macrophage-dependent fibroblast stimulation in coculture. journal of bone and joint surgery. British volume Lind, M., Trindade, M. C., Yaszay, B., Goodman, S. B., Smith, R. L. 1998; 80 (5): 924-930

    Abstract

    The interactions between the different cell types in periprosthetic tissue are still unclear. We used a non-contact coculture model to investigate the effects of polymethylmethacrylate (PMMA) particles and human macrophage-derived soluble mediators on fibroblast activation. Macrophages were either exposed or not exposed to phagocytosable PMMA particles, but fibroblasts were not. Increasing numbers of macrophages were tested in cocultures in which the fibroblast cell number was held constant and cultures of macrophages alone were used for comparison of cytokine release. We used the release of interleukin-1 beta (IL-1beta), interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-alpha), lysosomal enzyme and metalloproteinase activity to assess the cultivation of macrophages and fibroblasts. In cocultures, IL-6 release was increased 100-fold for both unchallenged and particle-challenged cultures when compared with macrophage cultures alone. Furthermore, particle-challenged cocultures had threefold higher IL-6 levels than unchallenged cocultures. Release of TNF-alpha was similar in cocultures and in macrophage cultures. IL-1beta release in cocultures was independent of the macrophage-fibroblast ratio. Lysosomal enzyme activity and metalloproteinase activity were increased in cocultures. Our data show that macrophages and fibroblasts in coculture significantly increase the release of IL-6 and to a less degree other inflammatory mediators; particle exposure accentuates this effect. This suggests that macrophage accumulation in fibrous tissue may lead to elevated IL-6 levels that are much higher than those caused by particle activation of macrophages alone. This macrophage-fibroblast interaction represents a novel concept for the initiation and maintenance of the inflammatory process in periprosthetic membranes.

    View details for PubMedID 9768911

  • In vitro, in vivo, and tissue retrieval studies on particulate debris CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Goodman, S. B., Lind, M., Song, Y., Smith, R. L. 1998: 25-34

    Abstract

    The biologic effects of wear debris are an important factor limiting the longevity of total joint replacements. In vivo, in vitro, and tissue retrieval studies have underlined a central role for the macrophage in the etiology of loosening and periprosthetic osteolysis. Wear particles from the materials used for total joint replacement activate macrophages to secrete proinflammatory factors. Complex interactions between macrophages and other cells stimulate bone resorption and suppress bone formation at the prosthetic interface. To improve the long term outcome of joint replacements, future research must find innovative approaches to minimize the production and biologic effects of wear debris.

    View details for Web of Science ID 000074714400005

    View details for PubMedID 9678030

  • Induction of matrix metalloproteinase expression in human macrophages by orthopaedic particulate debris in vitro. journal of bone and joint surgery. British volume Nakashima, Y., Sun, D. H., Maloney, W. J., Goodman, S. B., Schurman, D. J., Smith, R. L. 1998; 80 (4): 694-700

    Abstract

    We exposed human macrophages isolated from the peripheral blood of healthy donors to metal and bone-cement particles from 0.2 to 10 microm in size. Zymography showed that macrophages exposed to titanium alloy and polymethylmethacrylate (PMMA) particles released a 92- and 72-kDa gelatinase in a dose- and time-dependent manner. Western immunoblotting confirmed that the 92- and 72-kDa gelatinolytic activities corresponded to matrix metalloproteinase-9 and matrix metalloproteinase-2 (MMP-9, MMP-2), respectively. Western immunoblotting also indicated that titanium alloy and PMMA particles increased the release of MMP-1. Northern blotting showed elevated mRNA signal levels for MMP-1, MMP-2, and MMP-9 after exposure to both types of particle. Collagenolytic activity also increased in the macrophage culture medium in response to both types of particle. Our findings support the hypothesis that macrophages release MMPs in proportion to the amount of particulate debris within periprosthetic tissues.

    View details for PubMedID 9699840

  • Effects of polyethylene particles on tissue surrounding knee arthroplasties in rabbits JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Sacomen, D., Smith, R. L., Song, Y., Fornasier, V., Goodman, S. B. 1998; 43 (2): 123-130

    Abstract

    Clinical studies suggest a role for polyethylene (PE) wear debris in the pathogenesis of osteolysis and loosening of total joint replacements. In this study, submicron particles of ultrahigh molecular weight PE (UHMWPE) were placed around pressfit tibial hemiarthroplasties in rabbits to determine the biological reaction. After 6 months the periprosthetic tissue was harvested and characterized biochemically by measuring the extracellular matrix macromolecules, collagen, and glycosaminoglycan (GAG) and quantifying the expression of inflammatory/osteolytic mediators [prostaglandin E2 (PGE2), hexosaminidase, transforming growth factor beta (TGF beta), and interleukins-6 and -1 (IL-6, IL-1)]. Particle exposure resulted in a decrease in levels of total extracellular matrix molecules including a 53% decrease in total GAG (p < 0.05) and a 74% decrease in total collagen (p < 0.005). Collagen content remained significantly decreased when normalized for cellularity (DNA content). Total TGF beta release exhibited a downward trend (p = 0.06) in the particle exposed group. Hexosaminidase and PGE2 levels did not show a difference between groups; however, when normalized for cellularity, PGE2 values exhibited an upward trend in the particle exposed group (p = 0.1). IL-6 was undetected by bioassay and ELISA. Previous studies emphasized that PE debris enhances the degradation of bone. The data from this in vivo model suggest that submicron UHMWPE particles may also act to inhibit biosynthetic pathways of bone and mesenchymal tissue. Decreased levels of collagen, GAG, and TGF beta expression may indicate suppression of bone formation, possibly through a downregulation of osteoblast activity.

    View details for Web of Science ID 000073961000006

    View details for PubMedID 9619430

  • Joint infection ACTA ORTHOPAEDICA SCANDINAVICA Schurman, D. J., Smith, R. L. 1998; 69: 14-16
  • Joint infection. Acta orthopaedica Scandinavica. Supplementum Schurman, D. J., Smith, R. L. 1998; 281: 14-16

    View details for PubMedID 9771535

  • Staphylococcal septic arthritis: Antibiotic and nonsteroidal anti-inflammatory drug treatment in a rabbit model JOURNAL OF ORTHOPAEDIC RESEARCH Smith, R. L., KAJIYAMA, G., Schurman, D. J. 1997; 15 (6): 919-926

    Abstract

    This study evaluated the effects of combining antibiotic therapy with the application of a nonsteroidal anti-inflammatory drug on the degradation of articular cartilage for an animal model of Staphylococcal septic arthritis. Rabbits were infected intra-articularly with Staphylococcus aureus. Antibiotic treatment started 18 hours after infection and continued for 7 days. Treatment with the nonsteroidal anti-inflammatory drug naproxen sodium started 24 hours before infection and continued for either 3 or 7 weeks. The cartilage matrix of uninfected and infected knees was quantified by analysis of glycosaminoglycan and collagen content. Three weeks after infection, the combined treatment of the nonsteroidal anti-inflammatory drug and antibiotics reduced the loss of glycosaminoglycan and collagen from the cartilage of the infected knee by 15 and 30%, respectively, compared with antibiotic treatment alone. Continuing treatment with naproxen sodium for 7 weeks reduced the loss of collagen by 50% when compared with antibiotic treatment alone. The longer period of treatment with naproxen sodium showed little further effect on the loss of glycosaminoglycan than that observed for the 3-week treatment. Treatment with this drug and antibiotics reduced swelling of the knee and levels of prostaglandin E2 in the synovial fluid. The data support the hypothesis that decreasing post-infectious inflammation by adding the drug to a standard antibiotic regimen reduces cartilage damage from Staphylococcal septic arthritis.

    View details for Web of Science ID 000072261300018

    View details for PubMedID 9497819

  • Stabilized analogs of thymopentin .3. Evaluation of ketomethylene pseudopeptides for antiarthritic properties JOURNAL OF MEDICINAL CHEMISTRY Smith, R. L., Dousman, L., Waud, W. R., DeGraw, J. I. 1997; 40 (15): 2407-2411

    Abstract

    This study analyzed the role of ketomethylene pseudopeptides of thymopentin as potential agents for the treatment of arthritis. The analogs were tested in vivo using assessment of inflammation and antibody production in the mouse type II collagen arthritis model and the rat adjuvant arthritis model. The compounds were also tested for immune-potentiating activity in vitro using induction of the lymphocyte marker, Thy-1.2, in mouse spleen cells and stimulation of T-cell proliferation. The results show that certain of the compounds exhibit disease-remitting properties for arthritis as evidenced by reduction of paw swelling in the mouse and rat models and decreased incidence of disease in the mouse model. The active compounds were dose specific and represented a range in efficacy. In spite of effects on arthritis, type II collagen antibody levels were not altered in the mouse model. Selected compounds also exhibited immune potentiating properties as evidenced by induction of Thy-1.2 expression and stimulation of T-cell proliferation. The absence of effect of the compounds on type II collagen antibody production suggests that the antiarthritic activity of the effective compounds results from alteration of cell-mediated immunity.

    View details for Web of Science ID A1997XL78700013

    View details for PubMedID 9240355

  • Analogues of methotrexate in rheumatoid arthritis .2. Effects of 5-deazaaminopterin, 5,10-dideazaaminopterin, and analogues on type II collagen-induced arthritis in mice JOURNAL OF MEDICINAL CHEMISTRY Piper, J. R., DeGraw, J. I., Colwell, W. T., Johnson, C. A., Smith, R. L., Waud, W. R., Sirotnak, F. M. 1997; 40 (3): 377-384

    Abstract

    Twenty-six compounds derived from the 5-deaza- and 5,10-dideazaaminopterin series of aminopterin analogues were evaluated for antiarthritic activity in the mouse type II collagen model. New compounds in the 5-deaza series were prepared by alkylation of an appropriate N-substituted (4-aminobenzoyl)-L-glutamic acid dialkyl ester or N-(5-amino-2-thenoyl)-L-glutamate diester with a 2,4-diamino-5-alkyl-6-(bromomethyl)-5-deazapteridine. The resultant 5-deazaaminopterin diesters were saponified to provide the target 5-deaza analogues. 5,10-Dideazaaminopterins were synthesized by similar alkylation of the carbanions of appropriate 4-carboxyphenylacetic, (5-carboxy-2-thienyl)acetic, or (5-carboxy-2-pyridyl)acetic acid dimethyl esters. The diesters of the 2,4-diamino-4-deoxy-10-carboxy-5,10-dideazapteroic acid types so obtained were saponified and then readily decarboxylated by heating in Me2SO solution to provide the 2,4-diamino-5,10-dideazapteroic acid-type intermediates. Peptide coupling with diethyl L-glutamate followed by ester hydrolysis at room temperature afforded the new 5,10-dideazaaminopterin analogues. 5-Deazaaminopterins bearing an alkyl substituent at the 5-position were generally quite effective as antiinflammatory agents. Thus 5-propyl-5-deazaaminopterin, 5-methyl-10-propargyl-5-deazaaminopterin, 5-methyl-10-allyl-5-deazaaminopterin, 5-ethyl-5-deazamethotrexate, and 2,5-disubstituted thiophene analogue of 5-methyl-5-deazaaminopterin showed potencies greater than methotrexate by intraperitoneal or oral administration and were active over a considerably broader dose range. Useful activity in the 5,10-dideaza series was only observed for 5,10-dideazaaminopterin and its 10-methyl analogue. Alkyl substitution at C-5 or C-10 was generally detrimental to antiinflammatory activity in this series.

    View details for Web of Science ID A1997WF08600014

    View details for PubMedID 9022805

  • Analogues of methotrexate in rheumatoid arthritis .1. Effects of 10-deazaaminopterin analogues on type II collagen-induced arthritis in mice JOURNAL OF MEDICINAL CHEMISTRY DeGraw, J. I., Colwell, W. T., Crase, J., Smith, R. L., Piper, J. R., Waud, W. R., Sirotnak, F. M. 1997; 40 (3): 370-376

    Abstract

    Carbonation of the dianions (LDA) of 5-methylthiophene-2-carboxylic, 2-methylpyridine-5-carboxylic, and 3-methylpyridine-6-carboxylic acids provided the respective carboxy heteroarylacetic acids. The crude diacids were directly esterified in MeOH-HCl to afford the diesters. Alkylation of the sodio anions with ethyl iodide yielded the appropriate alpha-ethyl diesters. The anions of the various diester substrates were then alkylated by 2,4-diamino-6-(bromomethyl)-pteridine followed by ester saponification at room temperature to afford the respective 2,4-diamino-4-deoxy-10-carboxy-10-deazapteroic acids. The 10-carboxyl group was readily decarboxylated by heating in DMSO at temperatures of 110-135 degrees C to give the diamino 10-deaza heteropteroic acid intermediates. Coupling with diethyl L-glutamate followed by ester hydrolysis afforded the target aminopterins. The analogues were evaluated for antiinflammatory effect in the mouse type II collagen model. The thiophene analogue of 10-ethyl-10-deazaaminopterin was found to be an effective inhibitor in terms of reduced visual evidence of inflammation and swelling as determined by caliper measurement.

    View details for Web of Science ID A1997WF08600013

    View details for PubMedID 9022804

  • Analogs of methotrexate in rheumatoid arthritis CHEMISTRY AND BIOLOGY OF PTERIDINES AND FOLATES 1997 DeGraw, J. I., Colwell, W. T., Piper, J. R., Waud, W. R., Sirotnak, F. M., Smith, R. L. 1997: 127-132
  • Anti-lipid A antibodies in childhood arthritis: Methods of immobilization affect quantitation and crossreactivity measured by ELISA JOURNAL OF RHEUMATOLOGY Miller, J. J., Zhu, S. L., Smith, R. L. 1996; 23 (12): 2125-2131

    Abstract

    To find an optimal method to study antibodies reactive with monophosphoryl lipid A characteristic of oligoarticular arthritis in children.ELISA using 3 different methods of immobilization were compared, in (1) HCO3 buffer, pH 9.6; (2) HCl, pH 2.0; and (3) methanol. Competitive inhibition studies were carried out to quantitate relative avidity of cross reactions with suspected autoantigens.Sera from healthy children reacted significantly more strongly with monophosphoryl lipid A after immobilization in acid or in methanol than in a basic buffer. Sera from children with oligoarticular arthritis reacted more strongly than normal sera with the basic buffer method and even more strongly with the methanol method, but were not distinguishable from normal sera with the acid method. Results with individual oligoarticular sera correlated from method to method, but results with normal sera did not. Collagen types I and II, cardiolipin, and denatured DNA can block the anti-monophosphoryl lipid A reactivity to varying degrees on plates prepared with basic buffer, but only collagen type I and DNA block reactivity on plates prepared with methanol.The epitope on monophosphoryl lipid A recognized by oligoarticular sera is differentially affected by the method of immobilization. The crossreactivity of the anti-monophosphoryl lipid A antibody in this disease is confirmed.

    View details for Web of Science ID A1996VX84300021

    View details for PubMedID 8970051

  • RT-PCR analysis of MMP-9 expression in human articular cartilage chondrocytes and synovial fluid cells BIOTECHNIC & HISTOCHEMISTRY Tsuchiya, K., Maloney, W. J., Vu, T., Hoffman, A. R., Schurman, D. J., Smith, R. L. 1996; 71 (4): 208-213

    Abstract

    This report presents a method for analyzing matrix metalloproteinase-9 (92 kD gelatinase) MMP-9 mRNA expression using reverse transcription polymerase chain reaction (RT-PCR). With this method, chondrocytes isolated from small samples of osteoarthritic cartilage showed significantly elevated signal for MMP-9 mRNA compared to normal cartilage. In addition, cells of synovial fluid samples aspirated from osteoarthritic joints also exhibited MMP-9 expression using this technique. RT-PCR proved to be a sensitive method for assessing MMP-9 regulation in osteoarthritic and normal cartilage, and may provide a useful index of arthritic cartilage and synovial fluid cell metabolism when limited tissue is available.

    View details for Web of Science ID A1996VE64300007

    View details for PubMedID 8874859

  • In vitro activation of human fibroblasts by retrieved titanium alloy wear debris JOURNAL OF ORTHOPAEDIC RESEARCH Manlapaz, M., Maloney, W. J., Smith, R. L. 1996; 14 (3): 465-472

    Abstract

    Titanium-aluminum-vanadium wear particles isolated from the soft-issue membrane of a failed total hip arthroplasty were added to human fibroblasts in cell culture. The cellular response to particle challenge was determined by assaying for levels of interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, prostaglandin E2, basic fibroblast growth factor, platelet-derived growth factor-AB, and transforming growth factor-beta. Collagenase and gelatinase activities were analyzed by zymography and [3H]collagen degradation. Cell viability was assessed by measuring the uptake of [3H]thymidine. Over the range of particle concentrations tested, cell viability, as demonstrated by [3H]thymidine uptake, remained unaffected. Fibroblasts exhibited a dose-dependent release of interleukin-6 in response to exposure to titanium-aluminum-vanadium particles. At 6 and 48 hours, the highest concentration of titanium alloy particles (0.189% [vol/vol]) resulted in 7-fold and 16-fold increases in interleukin-6 release, respectively, when compared with negative controls. Neither interleukin-1 beta nor tumor necrosis factor-alpha was detected in the culture medium at any particle concentration tested for both dermal and foreskin fibroblasts. The pattern of prostaglandin E2 release by fibroblasts mirrored the pattern of interleukin-6 release. Fibroblasts exposed to the highest concentration of titanium alloy particles showed an increase in collagenase activity, starting at 12 hours. When medium samples were treated with amino phenylmercuric acetate to activate latent enzymes, a statistically significant increase in collagenase activity was observed as early as 6 hours (p < 0.001). Substrate gel analysis of medium from fibroblasts stimulated by high particle concentrations also showed an increase in gelatinolytic activity when compared with unstimulated controls. Analysis of medium samples for growth factors showed an increase in basic fibroblast growth factor at low particle concentrations, beginning at 12 hours. Levels of platelet-derived growth factor-AB and transforming growth factor-beta were not detectable in the controls or at any particle concentration tested. The results of this study showed that fibroblasts exposed to titanium alloy wear particles become activated and release proinflammatory mediators that influence bone metabolism. These data support the hypothesis that direct activation of fibroblasts by particulate wear may play a role in particle-mediated osteolysis. Fibroblast activation coupled with the biologic response of macrophages to wear debris in the loosening membrane may have a synergistic effect on pathologic bone resorption.

    View details for Web of Science ID A1996UT65500016

    View details for PubMedID 8676260

  • Effect of size, concentration, surface area, and volume of polymethylmethacrylate particles on human macrophages in vitro JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Gonzalez, O., Smith, R. L., Goodman, S. B. 1996; 30 (4): 463-473

    Abstract

    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.

    View details for Web of Science ID A1996UD18300006

    View details for PubMedID 8847354

  • Chondrocytes from osteoarthritic cartilage have increased expression of insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) and -5, but not IGF-II or IGFBP-4 JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Olney, R. C., Tsuchiya, K., Wilson, D. M., Mohtai, M., Maloney, W. J., Schurman, D. J., Smith, R. L. 1996; 81 (3): 1096-1103

    Abstract

    Osteoarthritis is a disease in which articular cartilage metabolism is altered, leading to cartilage destruction. As insulin-like growth factor-I (IGF-I) is the major anabolic mediator for articular cartilage, and the IGF-binding proteins (IGFBPs) are an integral part of the IGF axis, they may play a role in the pathophysiology of osteoarthritis. Chondrocytes isolated from fibrillated and normal appearing areas of osteoarthritic human cartilage and from normal cartilage were studied for IGF and IGFBP expression. IGF and IGFBP messenger ribonucleic acids were analyzed by a RT-quantitative PCR technique and Northern blotting. In osteoarthritic chondrocytes, IGF-I message was increased 3.5-fold, IGFBP-3 was increased 24-fold, and IGFBP-5 was increased 16-fold over normal chondrocytes. Chondrocytes from normal appearing areas of cartilage from osteoarthritic joints had intermediate levels. Message levels for beta-actin, IGF-II, and IGFBP-4 were unchanged between the cartilage types. IGF and IGFBP production were analyzed by Western ligand blots and RIAs of conditioned medium from cartilage cultured in serum-free conditions. IGF-I was undetectable in conditioned medium from normal cartilage and increased in that from osteoarthritic cartilage. Osteoarthritic cartilage samples produced IGFBP-2, -3, and -4; glycosylated IGFBP-4; and IGFBP-5. IGFBP-2, -3, and -5 production was increased in osteoarthritic cartilage. Proteases with activity against IGFBP-3 and -5 were also produced by osteoarthritic cartilage. The observation that IGFBP-3 and -5 expression and production are elevated in osteoarthritic cartilage suggests that they may be acting as a competitor for IGF-I in osteoarthritic cartilage, thus reducing the anabolic stimulation of this tissue and contributing to the net loss of cartilage in this disease.

    View details for Web of Science ID A1996TZ90600042

    View details for PubMedID 8772582

  • Human macrophage response to retrieved titanium alloy particles in vitro CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Maloney, W. J., James, R. E., Smith, R. L. 1996: 268-278

    Abstract

    Titanium alloy particles were isolated from membranes obtained at revision arthroplasty. Addition of these retrieved particles to human monocytes/macrophages in cell culture resulted in morphologic change and metabolic activation. Cells exposed to these particles actively phagocytized the metallic debris, resulting in an increase in cytoplasm and a polarization of ingested metal. The metabolic response of the macrophages included increased release of prostaglandin E2, interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha, and increased hexosaminidase activity. Increased release of interleukin-1 beta was maximal 6 to 12 hours after particle exposure. These data show that retrieved titanium alloy particles activate macrophages in vitro in an analogous fashion to that observed around failed arthroplasties.

    View details for Web of Science ID A1996TP57400032

    View details for PubMedID 8542704

  • In vitro stimulation of articular chondrocyte mRNA and extracellular matrix synthesis by hydrostatic pressure JOURNAL OF ORTHOPAEDIC RESEARCH Smith, R. L., RUSK, S. F., Ellison, B. E., Wessells, P., Tsuchiya, K., Carter, D. R., CALER, W. E., Sandell, L. J., Schurman, D. J. 1996; 14 (1): 53-60

    Abstract

    This study tested the effects of hydrostatic pressure (10 MPa) on adult articular chondrocyte mRNA and extracellular matrix synthesis in vitro. High density primary cultures of bovine chondrocytes were exposed to hydrostatic pressure applied intermittently at 1 Hz or constantly for 4 hours in serum-free medium or in medium containing 1% fetal bovine serum. mRNAs for aggrecan, types I and II collagen, and beta-actin were analyzed by Northern blots and quantified by slot blots. Proteoglycan synthesis was quantified by 35SO4 uptake into cetylpyridinium chloride-precipitable glycosaminoglycans, and cell-associated aggrecan and type-II collagen were detected by immunohistochemical techniques. In serum-free medium, intermittent pressure increased aggrecan mRNA signal by 14% and constant pressure decreased type-II collagen mRNA signal by 16% (p < 0.05). In the presence of 1% fetal bovine serum, intermittent pressure increased aggrecan and type-II collagen mRNA signals by 31% (p < 0.01) and 36% (p < 0.001), respectively, whereas constant pressure had no effect on either mRNA. Intermittent and constant pressure stimulated glycosaminoglycan synthesis 65% (p < 0.001) and 32% (p < 0.05), respectively. Immunohistochemical detection of cell-associated aggrecan and type-II collagen was increased in response to both intermittent and constant pressure. These data support the hypothesis that physiologic hydrostatic pressure directly influences the extracellular matrix metabolism of articular chondrocytes.

    View details for Web of Science ID A1996UA58800009

    View details for PubMedID 8618166

  • ISOLATION AND CHARACTERIZATION OF WEAR PARTICLES GENERATED IN PATIENTS WHO HAVE HAD FAILURE OF A HIP-ARTHROPLASTY WITHOUT CEMENT JOURNAL OF BONE AND JOINT SURGERY-AMERICAN VOLUME Maloney, W. J., Smith, R. L., Schmalzried, T. P., Chiba, J., HUENE, D., Rubash, H. 1995; 77 (9): 1301-1310

    Abstract

    Wear particles from thirty-five membranes obtained during revision hip-replacement operations were studied after digestion of the soft tissue with papain. The particles were isolated and were characterized with use of light and scanning electron microscopic techniques, x-ray microanalysis, and an automated particle analyzer. The mean size of the polyethylene particles was 0.5 micrometer, and the metal particles were a mean of 0.7 micrometer, as determined with scanning electron microscopy. The automated particle analyzer revealed a mean particle diameter of 0.63 micrometer (more than 90 per cent of all particles were less than 0.95 micrometer) and a mean of 1.7 billion particles per gram of tissue, compared with only 143 million per gram of tissue for the control samples. X-ray microanalysis revealed metal debris in sixteen (46 per cent) of the thirty-five membranes after digestion. Thirteen (50 per cent) of the twenty-six membranes surrounding a titanium-alloy stem contained metal particles, compared with three of the nine membranes surrounding a chromium-cobalt stem. Metal debris was present in only one of the twelve membranes surrounding a titanium-alloy stem without a porous coating, compared with twelve of the fourteen membranes surrounding a titanium-alloy stem with a porous coating. This tenfold difference in prevalence was significant (p < 0.005). On the average, the total number of particles (expressed in millions per gram of tissue) associated with the bipolar acetabular components was twice that associated with the fixed acetabular components. In addition, there was a trend toward a larger mean size of the polyethylene particles in association with the bipolar cups. Our data indicate that particulate prosthetic debris in the tissues around failed femoral components that have been inserted without cement constitutes a class of particles that are predominantly less than one micrometer in size and are present in amounts of more than one billion particles per gram of tissue. Routine histological methods did not detect this class of wear debris and led to a gross underestimation of the amount of debris in these membranes.

    View details for Web of Science ID A1995RV56800002

    View details for PubMedID 7673277

  • NEW ANALOGS OF METHOTREXATE IN CANCER AND ARTHRITIS CURRENT MEDICINAL CHEMISTRY DeGraw, J. I., Colwell, W. T., Piper, J. R., Sirotnak, F. M., Smith, R. L. 1995; 2 (2): 630-653
  • INTERLEUKIN-1 AND TUMOR-NECROSIS-FACTOR-ALPHA INCREASE INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-3 (IGFBP-3) PRODUCTION AND IGFBP-3 PROTEASE ACTIVITY IN HUMAN ARTICULAR CHONDROCYTES JOURNAL OF ENDOCRINOLOGY Olney, R. C., Wilson, D. M., Mohtai, M., Fielder, P. J., Smith, R. L. 1995; 146 (2): 279-286

    Abstract

    IGF-I is the major anabolic factor for cartilage matrix production. Chondrocytes and cartilage treated with interleukin-1 alpha (IL-1 alpha), and chondrocytes from several models of inflammatory joint disease, exhibit reduced responsiveness to IGF-I. Since the IGF-binding proteins (IGFBPs) modulate the effects of IGF-I, we examined the effect of IL-1 alpha and tumor necrosis factor-alpha (TNF-alpha) on IGFBP production by normal human articular chondrocytes in primary culture. Western ligand blots and immunoprecipitation of conditioned medium samples showed that articular chondrocytes produced IGFBPs-2, -3 and -4 and glycosylated IGFBP-4. Both IL-1 alpha and TNF-alpha increased chondrocyte production of IGFBP-3, but did not alter IGFBP-4 production. The activity of a neutral metalloprotease with the ability to cleave IGFBP-3 was also increased by IL-1 alpha. These data suggest that the cytokines IL-1 alpha and TNF-alpha may act to reduce IGF-I access to chondrocytes by increasing production of IGFBP-3. This may be a factor in the decreased matrix production in the inflammatory arthritides.

    View details for Web of Science ID A1995RM22600013

    View details for PubMedID 7561640

  • NONSTEROIDAL ANTIINFLAMMATORY DRUGS - EFFECTS ON NORMAL AND INTERLEUKIN-1 TREATED HUMAN ARTICULAR CHONDROCYTE METABOLISM IN-VITRO JOURNAL OF RHEUMATOLOGY Smith, R. L., KAJIYAMA, G., Lane, N. E. 1995; 22 (6): 1130-1137

    Abstract

    To test the effects of nonsteroidal antiinflammatory drugs (NSAID), naproxen, ibuprofen and diclofenac, and salicylates on normal and interleukin 1 (IL-1) treated human articular chondrocyte metabolism in vitro.Normal adult human articular chondrocytes were isolated and cultured as primary monolayers; the cells were treated with NSAID and salicylates at low and high plating density for assessing effects on proliferation and matrix synthesis and IL-1 modulated cell metabolism, respectively.Cell proliferation was inhibited by ibuprofen and high doses of salicylates. Glycosaminoglycan (GAG) synthesis was stimulated by ibuprofen at 10 micrograms/ml but was not changed by any other drugs at similarly low concentrations; at medium to high concentrations, only the salicylates inhibited GAG synthesis. Collagen synthesis was unaffected by any drug at the concentrations tested. IL-1 induced prostaglandin E2 release was completely inhibited by the NSAID and partially inhibited by the salicylates. IL-1 induced IL-6 release was inhibited by ibuprofen and the salicylates where as IL-1 induced APMA-activated collagenase was only inhibited by the salicylates.Normal human chondrocytes respond differentially to naproxen, ibuprofen, dicolfenac, and the salicylates; these observations suggest that each drug may exhibit unique attributes with respect to longterm efficacy on cartilage metabolism.

    View details for Web of Science ID A1995RD29700024

    View details for PubMedID 7674242

  • Lysosomal enzyme production at the interface surrounding loose and well-fixed cemented tibial hemiarthroplasties in the rabbit knee. Journal of investigative surgery Goodman, S. B., Kang, T., Smith, R. L. 1993; 6 (5): 413-418

    Abstract

    Fourteen mature New Zealand white female rabbits had a right, cemented, tibial hemiarthroplasty using a stemmed, fluted, titanium alloy, condylar-type prosthesis. In one group (seven rabbits), polymethyl methacrylate (PMMA) was used to cement the prosthesis firmly. In a second group (seven rabbits), the prosthesis was treated with cement ex vivo; the prosthesis and cured cement were then implanted, and rotated once within the bone to ensure that the prosthesis was loose fitting. Roentgenograms performed postoperatively and at 3 months were graded for new (i.e., not present on the immediate postoperative radiograph) radiolucent lines. At 3 months, the tissue adjacent to the implant was harvested sterilely and cultured over a 3-day period; the tissues and culture supernatants were then assayed for total protein, DNA content, and lysosomal enzyme activity (N-acetyl-beta-D-glucosaminidase and beta-glucuronidase). The mean cumulative grading of new lucent lines was 0.4 +/- 0.3 (mean +/- standard error) for the well-fixed prosthetic group and 2.0 +/- 0.6 for the loose prosthetic group. The tissue surrounding loose prostheses contained more DNA and total protein, and produced greater amounts of lysosomal enzymes compared to well-fixed prostheses. The control left sides were not statistically different for any parameter analyzed. The increased DNA content demonstrates an increase in cellularity of the tissue surrounding loose prostheses. Normalization of the relative amount of enzyme released as a function of cellularity (DNA) suggests that the influx of cells into the area surrounding loose prostheses may be more important to the overall increase in lysosomal enzyme release than increased production of lysosomal enzymes by individual cells.

    View details for PubMedID 8292569

  • THE EFFECTS OF BONE-CEMENT POWDER ON HUMAN ADHERENT MONOCYTES MACROPHAGES IN-VITRO JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Davis, R. G., Goodman, S. B., Smith, R. L., Lerman, J. A., Williams, R. J. 1993; 27 (8): 1039-1046

    Abstract

    This study reports the effects of Simplex bone cement powder (BC) on the proliferation and production of bone resorbing factors in vitro by human adherent monocytes/macrophages. Adherent peripheral blood cells were isolated from seven healthy individuals and exposed to a dispersion of BC powder (1 mg/mL), phytohemagglutinin (PHA, 40 micrograms/mL), or medium alone at different periods of cell incubation (days 0-2, 0-7, 5-7, or 10-12). Cell proliferation was quantified by incorporation of 3H-thymidine uptake. Culture supernatants were evaluated for levels of interleukin 1-like activity (IL-1) by murine thymocyte proliferation assay, prostaglandin E2 (PGE2) by radioimmunoassay, lysosomal enzyme activity (N-acetyl-beta-D-glucosaminidase and beta-glucuronidase using fluorometry, and collagen and casein degrading activity using radioactive substrates. Human adherent peripheral blood cells showed a proliferative response to PHA that coincided with cell maturation; BC did not inhibit PHA-induced cell proliferation of either adherent or nonadherent blood cells, indicating the non-toxic nature of these particles at the concentrations tested. BC stimulated increased release of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase; the levels of PGE2, IL-1, collagenase, and caseinase were unchanged.

    View details for Web of Science ID A1993LM05200008

    View details for PubMedID 8408116

  • PRODUCTION AND HORMONAL-REGULATION OF INSULIN-LIKE GROWTH-FACTOR BINDING-PROTEINS IN BOVINE CHONDROCYTES ENDOCRINOLOGY Olney, R. C., Smith, R. L., Kee, Y., Wilson, D. M. 1993; 133 (2): 563-570

    Abstract

    Linear growth results from proliferation and differentiation of chondrocytes within the growth plates and is regulated, in part, by the insulin-like growth factors (IGFs). IGF binding proteins (IGFBPs) also appear to play a significant, but yet unclear, role. To examine IGFBP production by chondrocytes, we isolated bovine chondrocytes from adult articular, fetal articular, and fetal growth plate cartilage, and maintained them in primary culture as high-density monolayers or encapsulated in alginate beads. Cells were cultured in serum-free conditions with human GH (hGH), insulin, hIGF-I, or hIGF-II. Human IGF-I resulted in higher DNA content in all three of the chondrocyte types. Conditioned medium samples were analyzed for IGFBPs by Western ligand blotting. Chondrocytes released IGFBPs of 24, 29, 33, 39, and 43 kilodaltons (kDa). Deglycosylation and immunoblotting identified the 39/43-kDa doublet as IGFBP-3 and the 33-kDa band as IGFBP-2. All chondrocyte types released 29- and 24-kDa IGFBP bands constitutively. Adult articular chondrocytes increased production all IGFBPs in response to IGF-I, but particularly the 29-kDa band (17-fold increase). Fetal articular chondrocytes showed a similar pattern, but with less of an increase when treated with IGF-I. Fetal growth plate chondrocytes primarily showed increases in IGFBP-3 and the 24-kDa form (4.7- and 2.7-fold, respectively) in response to IGF-I. Although the role of IGFBPs in IGF mediation of articular and growth plate chondrocyte metabolism requires further research, we show here that bovine chondrocytes produce IGFBPs, and the IGFs regulate this production.

    View details for Web of Science ID A1993LQ84600021

    View details for PubMedID 7688290

  • EXPRESSION OF 92-KD TYPE-IV COLLAGENASE GELATINASE (GELATINASE-B) IN OSTEOARTHRITIC CARTILAGE AND ITS INDUCTION IN NORMAL HUMAN ARTICULAR-CARTILAGE BY INTERLEUKIN-1 JOURNAL OF CLINICAL INVESTIGATION Mohtai, M., Smith, R. L., Schurman, D. J., Tsuji, Y., Torti, F. M., Hutchinson, N. I., STETLERSTEVENSON, W. G., Goldberg, G. I. 1993; 92 (1): 179-185

    Abstract

    We report here that a 92-kD gelatinolytic metalloproteinase is expressed as protein and mRNA in human osteoarthritic cartilage, but not in normal adult articular cartilage. Western immunoblotting demonstrated that the 92-kD gelatinolytic activity corresponded to 92-kD type IV collagenase/gelatinase (gelatinase B); mRNA for gelatinase B was identified by Northern blotting. Chondrocytes from normal cartilage also exhibited mRNA for 72-kD type IV collagenase/gelatinase (gelatinase A), tissue collagenase, and stromelysin-1, and these mRNAs were increased in osteoarthritic cartilage. Regional analysis of osteoarthritic cartilage samples from four individuals revealed that gelatinase B mRNA was expressed in grossly fibrillated areas; two of four nonfibrillated cartilage samples failed to exhibit the mRNA, but did have increased levels of mRNA for other neutral metalloproteinases. IL-1 alpha treatment of normal human cartilage explants or isolated chondrocytes induced increased levels of gelatinase B and increased mRNA for tissue collagenase and stromelysin-1. Under identical conditions, mRNA levels for gelatinase A were not increased indicating that regulation of this enzyme in human articular chondrocytes is distinct from that of other metalloproteinases. Our data showing expression of gelatinase B in fibrillated cartilage suggest that it is a marker of progressive articular cartilage degradation in osteoarthritis.

    View details for Web of Science ID A1993LL77300024

    View details for PubMedID 8325982

  • Fibroblast response to metallic debris in vitro. Enzyme induction cell proliferation, and toxicity. journal of bone and joint surgery. American volume Maloney, W. J., Smith, R. L., CASTRO, F., Schurman, D. J. 1993; 75 (6): 835-844

    Abstract

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

    View details for PubMedID 8314824

  • RABBIT KNEE IMMOBILIZATION - BONE REMODELING PRECEDES CARTILAGE DEGRADATION JOURNAL OF ORTHOPAEDIC RESEARCH Smith, R. L., Thomas, K. D., Schurman, D. J., Carter, D. R., Wong, M., VANDERMEULEN, M. C. 1992; 10 (1): 88-95

    Abstract

    This study analyzed processes underlying osteoporosis and osteoarthrosis after short-term immobilization of the right hind limb of postadolescent (2.8 kg) and mature (4.0 kg) rabbits. After 3 weeks, the lateral posterior aspect of the lateral tibial plateau and the lateral femoral condyle of the immobilized limb exhibited prominent subchondral vascular eruptions. Femoral metaphyseal bone density decreased 27 and 18% in the immobilized limbs of postadolescent and mature rabbits, respectively. Calcein green fluorescence increased 1.9-fold (p less than 0.001) in the metaphyseal trabeculae of immobilized femurs. With immobilization, sulfate incorporation into femoral cartilage glycosaminoglycan increased, although total cartilage glycosaminoglycan and hydroxyproline levels were unchanged. Thymidine incorporation into DNA increased four- to fivefold in tibial and femoral cartilage of the immobilized limb. In this study, bone loss and remodeling preceded erosive cartilage degradation.

    View details for Web of Science ID A1992GX48200010

    View details for PubMedID 1370179

  • INHIBITION OF INTERLEUKIN-1 INDUCED CHONDROCYTE PROTEASE ACTIVITY BY A CORTICOSTEROID AND A NONSTEROIDAL ANTIINFLAMMATORY DRUG JOURNAL OF RHEUMATOLOGY Lane, N. E., Williams, R. J., Schurman, D. J., Smith, R. L. 1992; 19 (1): 135-139

    Abstract

    We report that administration of the corticosteroid, methylprednisolone (PRED) inhibited interleukin 1 (IL-1) induction of chondrocyte caseinolytic activity (25-55%) and collagenolytic activity (15-24%). The nonsteroidal antiinflammatory drug (NSAID), naproxen (NAP) had no effect on either enzyme activity over a therapeutic range (7-30 micrograms/ml) but at 120 micrograms/ml inhibited IL-1 induced caseinolytic and collagenolytic activity by 17 and 19%, respectively. However, PRED (2 micrograms/ml) in combination with NAP (30 micrograms/ml) significantly increased the inhibition of caseinolytic activity (p less than 0.001) compared to that observed with PRED (2 micrograms/ml) alone. The suppression of IL-1 induced collagenolytic activity noted with PRED in combination with NAP did not exceed that observed with PRED alone.

    View details for Web of Science ID A1992HF28900027

    View details for PubMedID 1556675

  • PURIFIED STAPHYLOCOCCAL CULTURE-MEDIUM STIMULATES NEUTRAL METALLOPROTEASE SECRETION FROM HUMAN ARTICULAR-CARTILAGE JOURNAL OF ORTHOPAEDIC RESEARCH Williams, R. J., Smith, R. L., Schurman, D. J. 1991; 9 (2): 258-265

    Abstract

    Human articular cartilage released significantly increased levels of metal-dependent enzymes capable of degrading collagen, casein, and gelatin at a neutral pH following exposure to a sterile, purified fraction of Staphylococcus aureus culture medium. Neutral metalloprotease activity was determined by radiolabeled substrate assays and substrate gel analysis. The enzymes were activated with 4-aminophenylmercuric acetate and were inhibited by 1,10-phenanthroline and ethylenediamine tetraacetic acid. Protein immunoblots demonstrated that type I collagenase and stromelysin (matrix metalloproteinase III) secretion was increased following staphylococcal medium challenge. The profile of enzymatic activity induced by staphylococcal medium was directly comparable to that observed with interleukin-1, which was used as a positive control. The staphylococcal medium had no inherent proteolytic activity. Increased production of the neutral metalloproteases collagenase and stromelysin may significantly contribute to the extensive cartilage destruction noted in staphylococcal septic arthritis.

    View details for Web of Science ID A1991EW65600013

    View details for PubMedID 1846914

  • Mechanical overload of a single compartment induces early degenerative changes in the rabbit knee: a preliminary study. Journal of investigative surgery Goodman, S. B., Lee, J., Smith, R. L., Csongradi, J. C., Fornasier, V. L. 1991; 4 (2): 161-170

    Abstract

    The purpose of this experiment was to determine whether mechanical overload of a single compartment of the knee in rabbits via proximal tibial osteotomy could produce early changes consistent with degenerative arthritis. Ten New Zealand white female rabbits were allocated into two groups. Group 1 (five animals) underwent a right 20 degrees valgus proximal tibial osteotomy to overload the lateral compartment of the knee. Group 2 (five animals) underwent a tibial osteotomy without malalignment (sham controls). The osteotomies were stabilized with a mini AO/ASIF plate and screws, allowing early mobilization. The left leg in each animal was left intact and served as a control. Animals were sacrificed after 3 months. Histological grading of the cartilage was performed according to Mankin et al. The mean histological gradings for the right minus the left knee were the same for the lateral and medial compartments in the 0 degrees sham osteotomy group. However, the mean histological grading of the "overloaded" lateral compartment was 2.4 times greater than the medial compartment in the 20 degrees valgus osteotomy group. These findings suggest that histological evidence of degenerative changes can be surgically induced in the rabbit knee by creating a biomechanical overload of one compartment.

    View details for PubMedID 2069926

  • SOLUBLE MEDIATORS OF ARTICULAR-CARTILAGE DEGRADATION IN JUVENILE RHEUMATOID-ARTHRITIS CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Smith, R. L. 1990: 31-37

    Abstract

    Juvenile rheumatoid arthritis (JRA) involves a wide range of joint tissues. Tissue changes include proliferation of synovial cells, alterations in synovial fluid, and degradation of articular cartilage. Synovial cell proliferation results in increased numbers of fibroblasts and lymphocytes. Changes in the synovial fluid include increased content of antibodies, altered complement ratios, increased levels of factors stimulating cartilage-mediated proteoglycan degradation, and decreased levels of insulinlike growth factor I. Levels of cytokine such as interleukin-1 and interleukin-2 vary with cell culture and assay technique. Cartilage degradation is apparent from increased quantities of proteoglycan and glycosaminoglycan in serum and synovial fluid. Type II collagen peptide antibodies are also prevalent in JRA patients. Cartilage degradation appears linked to factors in JRA synovial fluid. Conditioned medium of peripheral blood mononuclear cells from patients with JRA also stimulates increased release of cartilage proteoglycan. Thus, the outcome in JRA likely reflects activities of interacting soluble factors that directly influence cartilage homeostasis.

    View details for Web of Science ID A1990EA94400006

    View details for PubMedID 2208870

  • GLUCOSAMINIDASE, GALACTOSAMINIDASE, AND GLUCURONIDASE IN THE GROWTH PLATE JOURNAL OF ORTHOPAEDIC RESEARCH Gamble, J. G., HAIMSON, R., Smith, R. L. 1990; 8 (5): 764-768

    Abstract

    The activities of glucosaminidase, galactosaminidase, and glucuronidase were determined in fractions of bovine growth plate cartilage. Glucosaminidase and galactosaminidase activities were lowest in the area corresponding to the reserve cartilage and increased from the upper to the lower portions of the hypertrophic zones of the growth plate, reaching a maximum in the calcifying cartilage. Glucuronidase activity showed a distinct spike of activity in the calcifying cartilage. The spatial distribution of these activities suggests a role in calcification and in the dissolution of the extracellular matrix at the chondro-osseous junction of the growth plate.

    View details for Web of Science ID A1990DU81900017

    View details for PubMedID 2388115

  • QUANTITATION AND RELATIVE DISTRIBUTION OF EXTRACELLULAR-MATRIX IN STAPHYLOCOCCUS-EPIDERMIDIS BIOFILM JOURNAL OF ORTHOPAEDIC RESEARCH VANPETT, K., Schurman, D. J., Smith, R. L. 1990; 8 (3): 321-327

    Abstract

    The relationship between adherence of bacteria to foreign bodies and their deposition of extracellular matrix was examined on glass and suture material. To quantitate bacterial adherence, uptake of [3H]thymidine into bacterial DNA was analyzed. Corresponding amounts of extracellular matrix were measured by a new technique using [14C]glucose incorporation. This study shows that [14C]glucose preferentially labeled bacterial strains in proportion to biofilm production. The ratio of 3H14C in high biofilm producers was 0.9 and in low producers it was 3.7. Radioactive identification of organisms as high and low producers was confirmed by electron microscopy. The results presented here show that production and accumulation of biofilm over time is a stable characteristic in different strains of S. epidermidis. The use of ratios reflecting radiolabeling of bacteria and biofilm by [3H]thymidine and [14C]glucose, respectively, is a quantitative yet simple technique to assess extracellular matrix of different strains of S. epidermidis.

    View details for Web of Science ID A1990CZ95500002

    View details for PubMedID 2324850

  • SEPTIC ARTHRITIS - STAPHYLOCOCCAL INDUCTION OF CHONDROCYTE PROTEOLYTIC ACTIVITY ARTHRITIS AND RHEUMATISM Williams, R. J., Smith, R. L., Schurman, D. J. 1990; 33 (4): 533-541

    Abstract

    We report herein that cartilage proteolytic activity increased in bovine and rabbit articular cartilage after treatment with a purified staphylococcal culture medium or intraarticular infection with Staphylococcus aureus. Staphylococcal culture medium increased the release of gelatinolytic, collagenolytic, and caseinolytic activity into the medium of isolated chondrocytes or cartilage organ culture. The proteolytic activities were determined in assays using radiolabeled substrate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staphylococcal culture medium was proteolytically inactive by both assay techniques. RNA synthesis of isolated chondrocytes was unaffected by staphylococcal culture medium, whereas overall protein synthesis was inhibited by 84%. An analysis of extracts of Staphylococcus aureus-infected rabbit knee cartilage by substrate gels showed increased gelatinolytic and caseinolytic activity compared with extracts of uninfected knee cartilage. Our data suggest that the rapid loss of proteoglycan and persistent degradation of cartilage in staphylococcal septic arthritis is due to the production and activation of chondrocyte proteases.

    View details for Web of Science ID A1990CZ97400011

    View details for PubMedID 1691643

  • STIMULATION OF ADULT CHONDROCYTE METABOLISM BY A THYROID-DERIVED FACTOR JOURNAL OF ORTHOPAEDIC RESEARCH Jones, D. G., Smith, R. L. 1990; 8 (2): 227-233

    Abstract

    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.

    View details for Web of Science ID A1990CN61700010

    View details for PubMedID 2303955

  • INFLAMMATION AND TISSUE-REPAIR SPORTS-INDUCED INFLAMMATION : CLINICAL AND BASIC SCIENCE CONCEPTS Schurman, D. J., Goodman, S. B., Smith, R. L. 1990: 277-284
  • BIOCHEMICAL-MECHANISMS UNDERLYING CARTILAGE DESTRUCTION IN INFECTIOUS ARTHRITIS ARTICULAR CARTILAGE AND KNEE JOINT FUNCTION Smith, R. L., Schurman, D. J. 1990: 197-211
  • GROWTH-HORMONE STIMULATES INSULIN-LIKE GROWTH FACTOR-I ACTIONS ON ADULT ARTICULAR CHONDROCYTES JOURNAL OF ORTHOPAEDIC RESEARCH Smith, R. L., PALATHUMPAT, M. V., Ku, C. W., Hintz, R. L. 1989; 7 (2): 198-207

    Abstract

    We report effects of adding insulin-like growth factor I (IGF-I) and methionyl human growth hormone (GH), alone or in combination, to adult bovine articular chondrocytes plated at high density. Purified human and synthetic IGF-I stimulated chondrocyte DNA and proteoglycan synthesis. GH had no effect on either process. However, GH added in combination with IGF-I increased proteoglycan, cell-associated proteoglycan, and keratan sulfate synthesis over levels observed with IGF-I alone. IGF-I and GH did not alter the hydrodynamic size of proteoglycans or synthesis of collagen. Our results show that GH and IGF-I act together to stimulate adult chondrocyte extracellular matrix synthesis.

    View details for Web of Science ID A1989T300200006

    View details for PubMedID 2918420

  • INDUCTION OF ARTICULAR-CARTILAGE DEGRADATION BY RECOMBINANT INTERLEUKIN 1-ALPHA AND 1-BETA CONNECTIVE TISSUE RESEARCH Smith, R. L., Allison, A. C., Schurman, D. J. 1989; 18 (4): 307-316

    Abstract

    The purpose of this study was to compare the effects of human recombinant interleukin 1, alpha and beta, on articular cartilage. The effects of rIL-1 alpha and rIL-1 beta on proteoglycan degradation and synthesis following treatment of bovine articular cartilage in serum-free organ culture were quantified. Purified human IL-1 and both rIL-1 alpha and rIL-1 beta induced a two-fold or greater increase in glycosaminoglycan (GAG) release from cultured articular cartilage. Levels or rIL-1 alpha as low as 15 pM induced increased proteoglycan degradation whereas identical levels of rIL-1 beta did not. Killing of the cartilage cells abolished induced GAG release by all forms of IL-1. Analysis of proteoglycan size following IL-1 treatment showed limited degradation of material released into the culture medium or remaining within cartilage. Both forms of recombinant IL-1 inhibited GAG synthesis when continually present in the culture medium. Actinomycin D and cycloheximide inhibited IL-1 dependent cartilage destruction whereas indomethacin did not.

    View details for Web of Science ID A1989T696500007

    View details for PubMedID 2787228

  • QUANTITATION OF GLYCOCALYX PRODUCTION IN COAGULASE-NEGATIVE STAPHYLOCOCCUS JOURNAL OF ORTHOPAEDIC RESEARCH Tsai, C. L., Schurman, D. J., Smith, R. L. 1988; 6 (5): 666-670

    Abstract

    The purpose of this study was to develop sensitive and accurate methods of quantitating bacterial glycocalyx. Coagulase-negative staphylococci were cultured in trypticase soy broth. Quantitation of slime production was evaluated using various methods of fixation and staining. The amount of dye associated with bacterial slime was assessed spectrophotometrically following solubilization of the dye-biofilm complex by 0.2 M NaOH at 85 degrees C for 1 h. Carnoy's solution was optimal for fixation of the slime, and toluidine blue staining was most reproducible. Fifteen strains of Staphylococcus epidermidis showed a gradation in biofilm production ranging from high, medium, to low that was strain stable. Irrespective of the technique used, high, medium, and low producers of bacterial slime remained in the same category and always showed significantly different optical density readings (p less than 0.05). In our experiments, solubilization of a toluidine blue-bacterial biofilm complex was a direct, simple, and efficient method for reproducibily quantitating glycocalyx. This method provides a useful means of rapid quantitation of biofilm production to assess its role in the infection process and in the response to antibiotic therapy.

    View details for Web of Science ID A1988P849300006

    View details for PubMedID 3404323

  • TIME-VARYING MAGNETIC-FIELDS - EFFECTS OF ORIENTATION ON CHONDROCYTE PROLIFERATION JOURNAL OF ORTHOPAEDIC RESEARCH Elliott, J. P., Smith, R. L., Block, C. A. 1988; 6 (2): 259-264

    Abstract

    The purpose of this study was to determine the effect of orientation of pulsed electromagnetic fields (PEMFs) on cellular proliferation and extracellular matrix synthesis. Bovine articular chondrocytes were cultured in PEMFs (repetitive pulse at 72 Hz) generated using Helmholtz coils oriented either parallel (horizontal) or perpendicular (vertical) to the plane of cell adhesion. Dissipation of signal energy in the form of heat increased the temperature of the PEMF coils by 2 degrees C and the tissue culture medium by 1 degree C. Therefore, control coils, which emitted no PEMFs, were heated to the temperature of PEMF coils by circulating water. Chondrocytes were cultured in 16-mm-well culture plates, and the data for individual wells were pooled as triplicates. Although not observed by microscopic examination of individual wells, positionally dependent electric field effects may be minimized by this approach. PEMFs generated by coils oriented vertically significantly decreased chondrocyte proliferation. The effect was dependent on the concentration of serum in the culture media. At 3% serum concentration, the total cell number attained after 10 days of culture was reduced by 50% in stimulated cultures when compared with controls. At 5% serum concentration, there was no effect. PEMFs applied by coils oriented horizontally did not alter proliferation of articular chondrocytes. PEMFs had no effect on synthesis of extracellular matrix by chondrocytes plated at high density, irrespective of orientation.

    View details for Web of Science ID A1988M409700012

    View details for PubMedID 3343631

  • BIOCHEMISTRY OF FUSION MASS CONSOLIDATION IN THE SHEEP SPINE JOURNAL OF ORTHOPAEDIC RESEARCH Slater, R., Nagel, D., Smith, R. L. 1988; 6 (1): 138-144

    Abstract

    We report here the biochemistry of fusion mass consolidation in sheep spines during a 1-year period following autogenous cortical-cancellous bone grafting and stabilization with Harrington distraction rods. Biochemical analysis of vertebral fusion mass included determination of wet weight and dry weight and quantification of glycosaminoglycan, collagen, calcium, and phosphate following extraction with neutral EDTA and proteolytic hydrolysis with papain. Our results showed that at 1 week after surgery, the fusion mass consisted of original cortical and cancellous bone graft material. The cortical bone graft was partially resistant to EDTA-papain treatment, resulting in a residue containing hydroxyproline and mineral. At 12 weeks after surgery, the fusion mass had become a homogeneous material, which, like cancellous bone graft, was completely susceptible to treatment by EDTA-papain. Collagen content of consolidating fusion mass was highest at 16 weeks after surgery when normalized to dry weight; glycosaminoglycan content was highest within 6 weeks after surgery. Mineral content was lowest at the 6-week stage but by 12 weeks after surgery, it was comparable with original bone grafting material. At 24 and 52 weeks after surgery, fusion mass consolidation was characterized by an increase in the proportion of organic and mineral components resistant to EDTA-papain. The appearance of the EDTA-papain-resistant material in the fusion mass coincided with formation of lamellar bone and successful consolidation.

    View details for Web of Science ID A1988L307200017

    View details for PubMedID 3275430

  • The effect of antibiotics on the destruction of cartilage in experimental infectious arthritis. journal of bone and joint surgery. American volume Smith, R. L., Schurman, D. J., KAJIYAMA, G., Mell, M., Gilkerson, E. 1987; 69 (7): 1063-1068

    Abstract

    In joints with bacterial arthritis, continuing prolonged destruction of cartilage may occur in spite of prompt, effective antibiotic therapy. We measured the extent to which early antibiotic therapy with ceforanide altered the degradation of the cartilage after arthritis due to Staphylococcus aureus had been produced in the knee joint in rabbits. Degradation of the cartilage was quantified by analyses for glycosaminoglycan and collagen. Three weeks after the infection was produced, the cartilage had lost more than half of its glycosaminoglycan whether the antibiotic therapy had been started at one, two, or seven days after infection. Beginning the antibiotic treatment one day after infection reduced over-all loss of collagen by 37 per cent and decreased the area of erosion of the infected articular surfaces. When antibiotic treatment was begun at four, eight, or twelve hours after infection, the loss of glycosaminoglycan averaged 18 per cent. Prophylaxis with antibiotics completely prevented any degradation of the cartilage. Clinical relevance: The findings reported here show how rapidly cartilage loses glycosaminoglycan when it is involved by arthritis caused by staphylococci and how early treatment of the infection reduces the loss of collagen. There is less protection against loss of glycosaminoglycan. The results emphasize the need for early diagnosis and treatment of infectious synovitis and support the rationale for early administration of antibiotics without waiting for identification of the responsible bacteria.

    View details for PubMedID 3654698

  • Surgical approach to the management of septic arthritis. Orthopaedic review Schurman, D. J., Smith, R. L. 1987; 16 (4): 241-245

    View details for PubMedID 3454937

  • BIOCHEMISTRY AND ANTIGENICITY OF OSTEOARTHRITIC AND RHEUMATOID CARTILAGE JOURNAL OF ORTHOPAEDIC RESEARCH Schurman, D. J., PALATHUMPAT, M. V., DeSilva, A., KAJIYAMA, G., Smith, R. L. 1986; 4 (3): 255-262

    Abstract

    The purpose of this study was to test whether cartilage serves as the source or repository of antigenic components active in the stimulation of inflammation in rheumatoid arthritis through an analysis of peripheral blood lymphocyte proliferation. Articular cartilage samples were obtained from patients with osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis undergoing joint replacement surgery. Each sample was homogenized and characterized biochemically with respect to the content of proteoglycan, collagen, and immunoglobulin. Proteoglycan content of rheumatoid cartilage was reduced by 71% when compared to osteoarthritic cartilage; the proteoglycan content of ankylosing spondylitis cartilage was reduced by 40% when compared to osteoarthritic cartilage. Immunoglobulins were detectable in all cartilage samples when analyzed by ELISA or end-plate titration. Lymphocyte proliferation, quantified by uptake of 3H-thymidine, was unaltered by addition of cartilage fragments, low (saline) and high salt extracts (2.0 M CaCl2), or cartilage residues. Both autologous and heterologous lymphocytes were tested against the cartilage samples with no difference in reactivity. Purified bovine articular proteoglycans and Type II collagen were also inactive. Although tetanus toxoid and phytohemagglutinin were effective stimulants of proliferation, lymphocytes from arthritis patients were suppressed relative to those of normal individuals. Analysis of arthritic articular cartilage by these techniques failed to demonstrate the presence of antigen(s) stimulating proliferation of peripheral blood lymphocytes.

    View details for Web of Science ID A1986D614100001

    View details for PubMedID 3734934

Conference Proceedings


  • POLYMETHYLMETHACRYLATE PARTICLES INHIBIT HUMAN MESENCHYMAL STEM CELL DIFFERENTIATION INTO OSTEOBLASTS Chiu, R., Smith, R. L., Goodman, S. LIPPINCOTT WILLIAMS & WILKINS. 2010: 109-109
  • POLYMETHYLMETHACRYLATE PARTICLES INHIBIT HUMAN MESENCHYMAL STEM CELL DIFFERENTIATION INTO OSTEOBLASTS Chiu, R., Smith, R. L., Goodman, S. LIPPINCOTT WILLIAMS & WILKINS. 2010: 150-150
  • POLYMETHYLMETHACRYLATE PARTICLE-INDUCED SUPPRESSION OF OSTEOPROGENITOR DIFFERENTIATION INVOLVES INHIBITION OF RUNX2, OSTERIX, DLX5, AND beta-CATENIN EXPRESSION Chiu, R., Smith, R. L., Goodman, S. LIPPINCOTT WILLIAMS & WILKINS. 2009: 228-228
  • POLYMETHYLMETHACRYLATE PARTICLE-INDUCED SUPPRESSION OF OSTEOPROGENITOR DIFFERENTIATION INVOLVES IMPAIRMENT OF OSTEOPROGENITOR VIABILITY BY NECROSIS Chiu, R., Smith, R. L., Goodman, S. LIPPINCOTT WILLIAMS & WILKINS. 2009: 140-140
  • Polymethylmethacrylate bone cement particles inhibit MC3T3-E1 osteoprogenitor differentiation in vitro Chiu, R., Smith, R. L., Goodman, S. B. LIPPINCOTT WILLIAMS & WILKINS. 2008: 242-243
  • Ultrahigh molecular weight polyethylene wear debris suppresses osteoblasttc differentiation of bone marrow osteoprogenitors and MC3T3-E1 preosteoblasts in vitro Chiu, R., Smith, R. L., Goodman, S. B. LIPPINCOTT WILLIAMS & WILKINS. 2008: 243-243
  • Inhibition of marrow stromal cell osteogenesis by polymethylmethacrylate wear particles and soluble factors released from polymethylmethacrylate particle-activated macrophages. Chiu, R., Smith, R. L., Goodman, S. B. LIPPINCOTT WILLIAMS & WILKINS. 2007: S155-S155
  • Polymethylmethacrylate particles inhibit osteoblastic differentiation of bone marrow osteoprogenitor cells in vitro. Chiu, R., Ma, T., Smith, R. L., Goodman, S. B. LIPPINCOTT WILLIAMS & WILKINS. 2006: S169-S169
  • Effects of shear stress on articular chondrocyte metabolism Smith, R. L., Trindade, M. C., Ikenoue, T., Mohtai, M., Das, P., Carter, D. R., Goodman, S. B., Schurman, D. J. IOS PRESS. 2000: 95-107

    Abstract

    The articular cartilage of diarthrodial joints experiences a variety of stresses, strains and pressures that result from normal activities of daily living. In normal cartilage, the extracellular matrix exists as a highly organized composite of specialized macromolecules that distributes loads at the bony ends. The chondrocyte response to mechanical loading is recognized as an integral component in the maintenance of articular cartilage matrix homeostasis. With inappropriate mechanical loading of the joint, as occurs with traumatic injury, ligament instability, bony malalignment or excessive weight bearing, the cartilage exhibits manifestations characteristic of osteoarthritis. Breakdown of cartilage in osteoarthritis involves degradation of the extracellular matrix macromolecules and decreased expression of chondrocyte proteins necessary for normal joint function. Osteoarthritic cartilage often exhibits increased amounts of type I collagen and synthesis of proteoglycans characteristic of immature cartilage. The shift in cartilage phenotype in response to altered load yields a matrix that fails to support normal joint function. Mathematical modeling and experimental studies in animal models confirm an association between altered loading of diarthrotic joints and arthritic changes. Both types of studies implicate shear forces as a critical component in the destructive profile. The severity of cartilage destruction in response to altered loads appears linked to expression of biological factors influencing matrix integrity and cellular metabolism. Determining how shear stress alters chondrocyte metabolism is fundamental to understanding how to limit matrix destruction and stimulate cartilage repair and regeneration. At present, the precise biochemical and molecular mechanisms by which shear forces alter chondrocyte metabolism from a normal to a degenerative phenotype remain unclear. The results presented here address the hypothesis that articular chondrocyte metabolism is modulated by direct effects of shear forces that act on the cell through mechanotransduction processes. The purpose of this work is to develop critical knowledge regarding the basic mechanisms by which mechanical loading modulates cartilage metabolism in health and disease. This presentation will describe the effects of using fluid induced shear stress as a model system for stimulation of articular chondrocytes in vitro. The fluid induced shear stress was applied using a cone viscometer system to stimulate all the cells uniformly under conditions of minimal turbulence. The experiments were carried using high-density primary monolayer cultures of normal and osteoarthritic human and normal bovine articular chondrocytes. The analysis of the cellular response included quantification of cytokine release, matrix metalloproteinase expression and activation of intracellular signaling pathways. The data presented here show that articular chondrocytes exhibit a dose- and time-dependent response to shear stress that results in the release of soluble mediators and extracellular matrix macromolecules. The data suggest that the chondrocyte response to mechanical stimulation contributes to the maintenance of articular cartilage homeostasis in vivo.

    View details for Web of Science ID 000088104400010

    View details for PubMedID 10912182

  • Interleukin-4 inhibits granulocyte-macrophage colony-stimulating factor, interleukin-6, and tumor necrosis factor-alpha expression by human monocytes in response to polymethylmethacrylate particle challenge in vitro Trindade, M. C., Nakashima, Y., Lind, M., Sun, D. H., Goodman, S. B., Maloney, W. J., Schurman, D. J., Smith, R. L. JOHN WILEY & SONS INC. 1999: 797-802

    Abstract

    The outcome of total joint arthroplasty is determined by biological events at the bone-implant interface. Macrophages phagocytose implant or wear debris at the interface and release proinflammatory mediators such as interleukins 1 and 6, tumor necrosis factor-alpha, and prostaglandin E2. These mediators are thought to contribute to the resorption of periprosthetic bone. Previous studies of tissues harvested from the bone-implant interface of failed orthopaedic implants demonstrated a possible role for two other cytokines, granulocyte-macrophage colony-stimulating factor and interleukin-4. The present study examined the effects of in vitro challenge with polymethylmethacrylate particles on the expression of granulocyte-macrophage colony-stimulating factor by primary human monocytes/macrophages and the role of interleukin-4 in regulating this expression. The polymethylmethacrylate particles caused a dose-dependent release of granulocyte-macrophage colony-stimulating factor at 48 hours. This release was accompanied by increased expression of interleukins 6 and 1beta and tumor necrosis factor-alpha. Release of the lysosomal enzyme hexosaminidase also increased in response to the particles. Interleukin-4 inhibited the expression of granulocyte-macrophage colony-stimulating factor, interleukin-6, and tumor necrosis factor-alpha at 48 hours in a dose-dependent manner. The data presented in this study confirm the hypothesis that interleukin-4 downregulates particle-induced activation of macrophages, as demonstrated by the decreased release of proinflammatory mediators.

    View details for Web of Science ID 000084603300001

    View details for PubMedID 10632444

  • Osteoarthritis: Differential expression of matrix metalloproteinase-9 mRNA in nonfibrillated and fibrillated cartilage Tsuchiya, K., Maloney, W. J., Vu, T., Hoffman, A. R., Huie, P., Sibley, R., Schurman, D. J., Smith, R. L. WILEY-BLACKWELL. 1997: 94-100

    Abstract

    Expression of matrix metalloproteinase-9 mRNA in osteoarthritic and normal cartilage was analyzed using reverse transcription-polymerase chain reaction and in situ hybridization. Fifty-four osteoarthritic cartilage samples were obtained from 24 patients undergoing total knee arthroplasty. Sixteen normal cartilage samples were obtained from non-osteoarthritic knees of four autopsy cases. With normal cartilage, reverse transcription-polymerase chain reaction analysis for matrix metalloproteinase-9 mRNA showed that chondrocytes exhibited only a trace signal. In analysis of osteoarthritic cartilage, chondrocytes of moderately and severely fibrillated cartilage exhibited a 73-fold and 110-fold increase in matrix metalloproteinase-9 mRNA signal, respectively, relative to normal cartilage. Chondrocytes of nonfibrillated osteoarthritic cartilage exhibited a 6-fold increase (p < 0.02) in matrix metalloproteinase-9 mRNA signal relative to normal cartilage. Analysis of matrix metalloproteinase-9 mRNA expression in fresh-frozen sections of normal and osteoarthritic cartilage by in situ hybridization confirmed these results. This study showed that reverse transcription-polymerase chain reaction provides a sensitive index of mRNA levels in normal and osteoarthritic cartilage samples and suggests that increased expression of matrix metalloproteinase-9 precedes fibrillation of cartilage in the development of osteoarthritis.

    View details for Web of Science ID A1997WL95000013

    View details for PubMedID 9066532

  • Nitric oxide and G proteins mediate the response of bovine articular chondrocytes to fluid-induced shear Das, P., Schurman, D. J., Smith, R. L. WILEY-BLACKWELL. 1997: 87-93

    Abstract

    Mechanical loading alters the metabolism of articular cartilage, possibly due to effects of shear stress on chondrocytes. In cultured chondrocytes, glycosaminoglycan synthesis increases in response to fluid-induced shear. This study tested the hypothesis that shear stress increases nitric oxide production in chondrocytes, and nitric oxide then influences glycosaminoglycan metabolism. Inhibitors of nitric oxide synthase, G proteins, phospholipase C, potassium channels, and calcium channels were also analyzed for effects on nitric oxide release and glycosaminoglycan synthesis. Fluid-induced shear was applied to primary high-density monolayer cultures of adult bovine articular chondrocytes using a cone viscometer. Nitric oxide release in chondrocytes increased in response to the duration and the magnitude of the fluid-induced shear. Shear-induced nitric oxide production was reduced in the presence of nitric oxide synthase inhibitors-but was unaffected by pertussis toxin, neomycin, tetraethyl ammonium chloride, or verapamil. The increase in glycosaminoglycan synthesis in response to shear stress was blocked by nitric oxide synthase inhibitors, pertussis toxin, and neomycin but not by tetraethyl ammonium chloride or verapamil. The phospholipase C inhibitor, neomycin, also decreased glycosaminoglycan synthesis in the absence of flow-induced shear. As studied here, shear stress increased nitric oxide production by chondrocytes, and the shear-induced change in matrix macromolecule metabolism was influenced by nitric oxide synthesis, G protein activation, and phospholipase C activation.

    View details for Web of Science ID A1997WL95000012

    View details for PubMedID 9066531

  • Osteoblast adhesion to orthopaedic implant alloys: Effects of cell adhesion molecules and diamond-like carbon coating Kornu, R., Maloney, W. J., Kelly, M. A., Smith, R. L. JOHN WILEY & SONS INC. 1996: 871-877

    Abstract

    In total joint arthroplasty, long-term outcomes depend in part on the biocompatibility of implant alloys. This study analyzed effects of surface finish and diamond-like carbon coating on osteoblast cell adhesion to polished titanium-aluminum-vanadium and polished or grit-blasted cobalt-chromium-molybdenum alloys. Osteoblast binding was tested in the presence and absence of the cell adhesion proteins fibronectin, laminin, fibrinogen, and vitronectin and was quantified by measurement of DNA content. Although adherence occurred in serum-free medium, maximal osteoblast binding required serum and was similar for titanium and cobalt alloys at 2 and 12 hours. With the grit-blasted cobalt alloy, cell binding was reduced 48% (p < 0.05) by 24 hours. Coating the alloys with diamond-like carbon did not alter osteoblast adhesion, whereas fibronectin pretreatment increased cell binding 2.6-fold (p < 0.05). In contrast, fibrinogen, vitronectin, and laminin did not enhance cell adhesion. These results support the hypothesis that cell adhesion proteins can modify cell binding to orthopaedic alloys. Although osteoblast binding was not affected by the presence of diamond-like carbon, this coating substance may influence other longer term processes, such as bone formation, and deserves further study.

    View details for Web of Science ID A1996WC77400004

    View details for PubMedID 8982128

  • Periprosthetic osteolysis in total hip arthroplasty: The role of particulate wear debris Maloney, W. J., Smith, R. L. AMER ACAD ORTHOPAEDIC SURGEONS. 1996: 171-182

    View details for Web of Science ID A1996BH44V00020

    View details for PubMedID 8727736

  • HOLMIUM - YAG LASER EFFECTS ON ARTICULAR-CARTILAGE METABOLISM IN-VITRO Smith, R. L., Montgomery, L., Fanton, G., Dillingham, M., Schurman, D. J. SPIE - INT SOC OPTICAL ENGINEERING. 1994: 149-153
  • PRODUCTION AND HORMONAL-REGULATION OF INSULIN-LIKE GROWTH-FACTOR BINDING-PROTEINS IN BOVINE CHONDROCYTES Olney, R. C., Smith, R. L., Kee, Y., KAJIYAMA, G., Wilson, D. M. SLACK INC. 1992: A81-A81
  • CHARACTERIZATION OF SPECIFIC INSULIN-LIKE GROWTH FACTOR-L SOMATOMEDIN-C RECEPTORS ON HIGH-DENSITY, PRIMARY MONOLAYER-CULTURES OF BOVINE ARTICULAR CHONDROCYTES Watanabe, N., DOLLAR, L. A., Hintz, R. L., Smith, R. L., Rosenfeld, R. G. SLACK INC. 1984: A54-A54

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