Foam Cells

PU foams, of three different densities, were used in this study. Closed cell PU foam of density 0.16 g.cm^-3 and 0.32 g.cm^-3 (American Society for Testing and Materials, ASTM, Grade 10 and Grade 20) [1 ] was used to model low and medium density cancellous bone respectively. Open cell rigid foam of density 0.09 g.cm^-3 was used to model very low density cancellous bone. All PU foams were purchased in block form, with dimensions 130 × 180 × 40 mm, from Sawbones^® Europe AB, Malmö, Sweden. The foam densities were supplied by Sawbones^® Europe AB.
Using a sharpened tube, six cylindrical cores of 9 mm diameter were drilled from each of the three different density PU foam blocks. The cores were taken using the method described by Li and Aspden [14 (link)], in which the cylindrical axis of the core was roughly perpendicular to the surface of the PU block (this is the preferred orientation of the "trabeculae"). The exact diameter of the PU cylinders was determined as an average of four measurements; this was necessary to account for the inhomogeneity of the 0.09 g.cm^-3 open cell PU foam in particular.
For this study, two different cylinder lengths were chosen to test for any buckling or shape effects. A cylinder, of length of 7.7 ± 0.2 mm, was chosen so that results could be compared with those from a published study of human OP cancellous bone [14 (link)]. In order to investigate the effect of specimen dimensions, a cylinder, of length 3.9 ± 0.1 mm, was also investigated. This length was obtained from a standard for testing rubbers [16 ]. The reason for choosing this standard was to ensure that the specimens did not bulge during compression; rubbers have a Poisson's value of about 0.5 and so maintain an almost constant volume during compression; as a result, they bulge more than most other materials [17 (link),18 (link)]. Dimensions were measured with digital vernier callipers (Fisher Scientific UK Ltd., Leicestershire).
Six cylinders were prepared for each cylinder length and each density of PU foam block. The required cylinder length was achieved by either using a small pair of scissors, for the 0.09 g.cm^-3 PU foam, or by rubbing the PU foam cylinder on a sheet of sandpaper (medium grade M2, SupaDec, RS Components Ltd., Northamptonshire, UK), for the 0.16 g.cm^-3 and 0.32 g.cm^-3 PU foams.

Frozen sections were cut at a thickness of 5 μm and mounted onto microscope slides. Sequential sections were used for a single antibody or for two antibodies (co-expression- as specified in Tables 1, 2) including for the negative controls. Samples were fixed with acetone for 15 min. at 4°C and washed with PBS. Then, incubated with the primary antibodies diluted at 1:100 in blocking buffer of 10% normal goat serum in RPMI-1640 medium, overnight at 4°C (2 (link)). The following cellular markers were used to identify and quantify cell populations in the carotid plaques; PMNs were identified by primary antibodies for CD66b, NE, and MPO and macrophages were identified by CD163. Double staining of CD66b(mono)/CD163(poly) was performed to identify potential co-expression. Additional markers included the scavenger receptors CD36 and CD68 for foam cells, the oxidative stress marker 3-NT, hypoxia inducible factor 1α (HIF-1α), VEGF, CD31 – for vessel identification by the presence of endothelial cells, and smooth muscle cell actin (SMC-actin), a marker of arterial wall remodeling.
After overnight incubation with primary antibodues the slides were washed and incubated with 1/400 secondary antibodies in blocking buffer, at room temperature, for 40 min. Secondary antibodies included Cy2 (CF 488A)-conjugated goat anti-rabbit IgG and/or Cy5 (CF 647)-conjugated goat anti-mouse IgG (Biotium, Hayward, CA). Isotype controls included: purified mouse IgG1 (clone MOPC-21, BioLegend, San Diego, CA), and normal rabbit IgG (sc-2027, Santa Cruz Biotechnologies, Santa Cruz, CA). After 40 min. incubation, slides were washed and mounted with mounting medium containing 4’, 6-diamidino-2-phenylindole (DAPI) for nuclear staining (Vectashield H-1000, Vector lab. Inc. Burlingame, CA).

Frozen sections were cut at a 5 μm thickness and mounted on microscope slides. The 5-μm-thick sections were stained with hematoxylin and eosin (H&E), and lipid deposits in the plaques were visualized by Oil Red O staining as previously described (3 (link), 15 (link)). Primary antibodies against CD66b, CD163, and CD68 cellular markets were diluted to 1:100, and a Histostain-Plus Kit AEC, Broad Spectrum (Invitrogen) was used for their detection. The sections were incubated with the primary antibodies for 2 hrs. at 37°C. Then, the sections were incubated with secondary antibody from the Histostain-Plus kit, for 30 min at 37°C. The 3-amino-9-ethylcarbazole (AEC) was used as a chromogen to detect the antibodies according to manufacturer’s instructions. Polymorphonuclear neutrophils (PMNs) were identified by the expression of CD66b, macrophages were identified by the expression of CD163 and foam cells were identified by the expression of CD68 scavenger receptors. Isotype controls were used as specified in the list of antibodies. Lipid deposits were stained with Oil Red O. Sequential sections were stained each for an antibody including for negative controls. At least 3 different sections were cut from the center of each plaque for each CD marker and in each section at least 5 different fields were analyzed.
Collagen and non-collagen proteins were detected by differential staining in tissue sections with two dyes - Sirius Red for all collagens and Fast Green for non-collagen proteins.

Carotid plaques were removed by standard surgical techniques and minimal manipulation to the specimens. Immediately after the surgery, plaques were stored in phosphate buffered saline (PBS) at 4°C. The specimens were embedded into an optimum cutting temperature (OCT) compound (LEICA, 020108926) and stored at -80°C for further analysis. Samples were analyzed by immunohistology, immunohistochemistry, and immunofluorescence using confocal microscopy. For immunohistochemistry plaque samples were analyzed for various CD cellular markers including CD66b, CD163, CD68, and lipids. Additional cellular markers were used for confocal microscopy. Quantitative analyses of the expression of various markers were performed as previously described (2 (link), 15 (link)) and as specified below. Mouse monoclonal primary antibodies were used to identify neutrophils (anti-CD66b), macrophages-foam cells (anti-CD163) and anti-3-nitrotyrosine for oxidative-nitrosative stress. Rabbit primary polyclonal antibodies were used for double-labeling the cells with additional markers including scavenger receptors anti-CD68 and anti-CD36, anti-NE, anti-MPO, anti-Vascular Endothelial Growth Factor (VEGF), anti- CD31, and anti- smooth muscle actin (SMC-actin). Polyclonal anti-CD163 was used for double-labeling with anti-CD66b. Intra/extra cellular lipids and lipid crystals were determined by immunohistochemistry with Oil Red O staining (15 (link)).