Periprosthetic soft tissue obtained at the time of hip revision surgery was fixed without decalcification. Histologic evaluation of formalin-fixed, paraffin embedded tissues was performed. Polarized light microscopy was utilized to identified birefringent polyethylene (UHMWPE) particles. B cell marker: CD20 monoclonal mouse anti-human (mAb) (clone L26; dilution 1:3000) (Dako Corp., Carpinteria, CA.), T cell marker: CD3 polyclonal rabbit anti-human (#A0452; dilution 1:200) (Dako Corp., Carpinteria, CA.), macrophage marker CD68 mAb (clone PGM-1; dilution 1:25) (Dako Corp., Carpinteria, CA.) were utilized for immunohistochemical studies (DakoCytomation Signet Acuity kit, Dako DAB +, and DakoCytomation Autostainer Universal Staining system). Formalin-fixed, paraffin embedded tissues were cut on a microtome at a thickness of 4um on Superfrost-plus microscope slides (Cardinal Health Care, Dublin, OH). Tissue slides were dried over night at room temperature, deparaffinize with xylene, cleared with graded ETOH (100% ×2, 95%, 70%), and rinsed with ddH2O. Antigen retrieval was performed by microwave (Biogenex E-Z Retriever microwave) HIER using Citrate buffer pH6.0 at 97° for 10 minutes. Slides were allowed to cool for 20 minutes, then rinsed in ddH2O and placed into a TBS buffer with Tween-20 (2.5 ml to 5 liters of TBS). Slides were incubated with the primary antibodies for 30 minutes, rinsed, and secondary antibodies with labeled polymer were applied for 25 minutes. Antigen-antibody reaction was visualized using diaminobenzidine chromogen (DAB) applied for 7 minutes. Sections were counterstained in Hematoxylin for 30 seconds, cleared in 2% Glacial acetic acid for 30 seconds, rinsed in hot water then in 0.2% Ammonia Water for 10 seconds, rinsed in water, then dehydrated in ETOH and in Xylene before manual cover slipping.
To determine surface expression of various cell surface markers, cells were washed with cold PBS, and labeled for 30 min on ice with saturating amounts of primary mAb in staining buffer (PBS/0.1 % BSA/ 0.01% NaN3). The following anti human mAbs were used; HLA-DR (clone TU36), CD 40 (clone 5C3), B7-1/CD80 (clone L307.4) , B7-2 /CD86 (clone 2231 ), CD51/61 (clone 23C3), CD14 (M5E2), CD11c (clone BLy6), CD16 (clone 3G8), CD19 (H1B19), CD83 (clone HB15e) and TCR (clone R73) from BD Biosciences, Pharmingen, San Diego, CA. Appropriate isotype controls were included in the experiment.
Fluorescence assisted cell sorting of the appropriately stained samples was performed at our in house facility using the FACSCalibur flow cytometer and cellquest software program (BD Biosciences Mountain View, CA, USA) was utilized for the acquisition of the histogram. Histogram analysis was performed by Flo Jo 7.2 and the mean fluorescence was calculated.
Peripheral blood was obtained from the New York Blood Bank. The monocyte population was separated using CD11C or CD14 conjugated MicroBeads (Miltenyi Biotec). Purified CD11C+ cells were cultured in GM-CSF/IL4 (30 ng/ml plus 10 ng/ml) (RD Systems) for dendritic cells, M-CSF (30 ng/ml) for macrophages or RANK-L (30 ng/ml) and M-CSF (30 ng/ml) for osteoclasts for 5 to 6 days in DMEM (GIBCO, Grand Island, NY, USA), supplemented with 50 U/ml penicillin, 50 g/ml streptomycin, 2 mM l-glutamine, 0.1 mM nonessential amino acids, 1 mM pyruvate, and 10 mM Hepes. CD14+ monocytes were used without further differentiation.
To the assess damage to endosomal compartments we utilized transmission electron microscopy. Patient tissue samples as well as cultured Monocytes, DC, MØ (treated or untreated with UHMWPE) were fixed with a mixture of 2% paraformaldehyde and 2.5 % glutaraldehyde in cacodylate buffer 0.1 M, pH 7.4 at 4°C Fixed cells were infiltrated in sequentially increasing concentrations of LX112-Araldite to 100%, embedded in BEEM capsules, and placed in a 60 °C oven for 4 days. Using a Dupont Sorvall Porter-Blum ultra microtome MT-1, semi-thin sections (0.5um) were stained with 1:1 mixture of 1.0% Methylene blue and 1.0% Azure B, observed with a light microscope, and subsequently selected regions were thin-sectioned and collected on 300 mesh copper grids. Tissue sections were stained with uranyl acetate followed by lead citrate, and viewed with a Jeol JEM-1200EX electron microscope at 80 kV.
Scanning electron microscopy was performed to view the morphology and particle size of patient sample purified particles. The samples were mounted on a stub but not sputter coated and viewed at 300X magnification.
Primary dendritic cells were grown on culture slides with GMCSF for 6 days with and without UHMWPE. The cells were treated either with LysoTracker Green DND-26 (Molecular probes cat no L7526; excitation/emission 504/511 nm) at 75 nM concentration for 30 mins individually or in combination with cathepsin specific activity probe, GB123 (excitation/emission 646/666 nm) (15) washed, counterstained with DAPI (Molecular probes cat no D1306; excitation/emission 358/461 nm) and fixed in 4% paraformaldehyde.
The stained slides were finally mounted and examined under high-resolution laser scanning confocal microscope (Leica AOBS system). Images were captured at 450 nm excitation and emission at 510 nm at 63X under oil emersion objective. All images were collected under identical PMT detector settings. The images were exported into Adobe Photoshop for final processing.
Analysis of ß-hexosaminidase release from human monocyte derived DC was determined by addition of 100ul of incubation buffer from UHMWPE treated or untreated cells to 100ul of reaction mixture (5ml 0.4M Sodium acetate pH 4.4, 5ml of 8mM 4-methylumbelliferyl-N-acetyl-B-D glucopyranoside in water, 0.250ml Triton X-100, 9.750 ml water) in a 96 well plate incubated at 370C for 30 minutes in an hybridization oven. The reaction will be stopped by addition of 75ul of 2M Na2CO3, and the fluorescence measured in a spectrofluorimeter (FluorStar Optima, BMG Labtechnologies Ltd, Durham, NC) at excitation 350nm/emission 450nm.
Western Blot Analysis of cathepsin S
Equal volumes (100 uL) from the supernatants of primary DCs untreated (control) or treated with different sized UHMWPE at 72 hours were separated on a 10% SDS-PAGE, blotted on nitrocellulose membrane (0.4 um), blocked for nonspecific binding with PBS/Tween 20 (5%) for one hour at room temperature, and further processed for incubation with anti-cathepsin S antibodies (Sc-6503 Santa Cruz). The incubation with anti-cathepsin S (goat polyclonal, 1/200 dilution from stock) in PBS/Tween 20 (0.5%) was performed over night at 4°C, further incubated with secondary antibody (anti-goat coupled with horseradish peroxidase (HRP)) for 1 hour at room temperature and developed with enhanced chemi-luminescent kit from Pierce.
Western Blot Analysis to determine inflammasome activation
Jaws cells untreated and treated 24 hours with UHMWPE in presence and absence of selective inhibitors mix of cathepsins, such as calpain inhibitor II (ALLM) and Pepstatin A were cultured. Western analysis of the total cell lysate was then probed with antibodies specific to Cathepsin B, Caspase 1 p10 fragment, IL-1β, II-18 proteins to evaluate the inflammasome activation. Cell lysates from human monocytes cultured with or without particles for 6 hours in the presence or absence of TLR stimulation were likewise immunoblotted to detect expression of Pro-IL1β.
Gene Chip Assay
Gene expression analysis was performed on control and 24 hour m-PE treated human DC. RNA was extracted using a Qiagen kit. Five micrograms of total RNA were hybridized on the Human Toll-like Receptor Oligo GEarray (SuperArray Biosciences Corporation). Data are reported as average hybridization numbers for each gene subtracted for background.
The HEK 293/ TLR 1/2 clones (Invivogen) were used to determine TLR1/2, activation by UHMWPE. The clone was transfected with the NF-Kβ cis- reporter enhancer (pNF- b-LUC, Stratagene) and an independent GFP containing plasmid using Fugene 6 transfection reagent (Roche). The expression of GFP was measured by FACS. Forty-eight hours post transfection the cells were treated with UHMWPE, as well as a positive control like PGN (peptidoglycan) and Zymosan for TLR 1/2 . The luciferase readout was measured at different time points using the standard Luciferase reporter assay kit (Promega).
In vitro processing of Collagen I by cathepsins
Collagen I processing was performed in vitro using human recombinant cathepsins (B, D, L and S) all purchased from Biomol. Briefly, native Collagen-I (Sigma) (5-10 ug/assay) was incubated with different units (0.2-0.5) of each enzyme in the buffer for each cathepsin assay: cathepsin B (120 mM sodium acetate, with 1mM DTT, pH 5.0); cathepsin D (100 mM sodium acetate, pH 3.5); cathepsin L (400 mM sodium acetate, with 8 mM DTT, pH 5.5); cathepsin S (50 mM sodium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5). The reaction mixtures were incubated at 37°C and at 6 hours. 20 ul of the reaction mix were quenched with equal volume of SDS/beta-mercaptoethanol denaturing sample buffer for SDS polyacrylamide electrophoresis (PAGE). The products of the enzymatic digestion of collagen-I (different length fragments) were monitored using 7.5% SDS-PAGE. The gels were stained using standard procedures with colloidal blue or for higher sensitivity with silver stain. In order to identify fragments generated only from cathepsin processing of collagen-I, all enzymatic reactions were performed in parallel using selective inhibitors of cathepsins, such as calpain inhibitor II (ALLM) and Pepstatin A.
The cytokine assay was performed with the control and PE treated (48hrs) dendritic cells using the Bio-rad human 17-plex panel (cat no#171-A11171) reagent kit. To the pre-wet wells the appropriate beads were added and washed adequately. Next the standards and the samples are added and incubated for 30 minutes. The wells are then washed three times and the appropriate detection antibody is added and incubation is continued for another 30 minutes. The wells were again washed and streptavidin-PE was added and after 10 mins washed again and re-suspended in 150 ul of PBS and the 96 well micro-plate is finally analyzed for fluorescence in a Luminex 200 system. IL1β production from human monocytes was determined using an OptEIA ELISA kit (BD Biosciences).
UHMWPE and cement particles
Ultra high molecular weight Polyethylene was purchased from Sigma (cat no #434272). The UHMWPE was surface modified with average size of 53-75 micron according to manufacturers datasheet. UHMWPE can only be dissolved at elevated temperatures in aromatic hydrocarbons, such as toluene or xylene. For our studies we resuspended 20 mg/ml of UHMWPE in PBS ( Phosphate buffered saline) to form a suspension solution. UHMWPE was always vortexed strongly just before addition and was added at a ratio of 20 ul of the vortexed suspension per ml of culture media. The particles of various sizes were obtained from Biomet Orthopedics Inc (Warsaw IN-46581) at a number of 1 × 105 particles in 1ml of distilled autoclaved water in flame sealed amber vials. Particles of Simplex Bone Cement (Howmedica Osteonics, Allendale, NJ) were prepared by filing using a Grobet Warding File 4 Cut: #4 Pattern:Swiss (MSC Industrial Supply Company). After filing, particles were soaked at 4°C in 70% EtOH overnight, then washed twice and stored in sterile PBS solution, pH 7.2. Endotoxin contamination of particles was excluded by limulus assay (E-Toxate; Sigma Chemical, St. Louis, Missouri).