Six 6-month-old male GOHI/SPF guinea pigs (400–500 g) were purchased from RCC Ltd (Itingen, Switzerland) and six 6-month-old DH guinea pigs (750–800 g) from Charles River Laboratories (Sulzfeld, Germany). The animals were housed singly, had free access to food and water, and were exposed to a 12-hour-light/12-hour-dark cycle. They were sacrificed at 11 months of age by an intracardial injection of T-61 euthanasia solution (0.3 mL/kg of body weight: Schering-Plough, Quebec, Canada). At the time of sacrifice, the mean weights of the GOHI/SPF and DH animals were 785 g and 1020 g, respectively.
The 24 hindlimbs were amputated, and the bones were freed of soft tissues. The proximal tibias (bearing the medial and lateral menisci), which were destined for histomorphometry, were chemically fixed for 2 to 4 days at ambient temperature in a solution containing 2.5% glutaraldehyde (Merck and Co, Inc, Whitehouse Station, NJ) and 2.5% formaldehyde, which was buffered with 0.1 mol/L sodium cacodylate (pH 7.4) (Merck). They were then dehydrated in ethanol and embedded in methylmethacrylate. Using a diamond band saw (Exact Medical Instruments, Oklahoma City, OK), and with a random start at the left-hand margin of the specimen, five 200-μm-thick coronal sections, 1.2 mm apart, were prepared from each tibia. The sections were glued to Plexiglas® holders, ground to a thickness of approximately 100 to 150 μm with a Polycut™ E microtome (Reichert-Jung, Heidelberg, Germany), polished, and surface-stained with McNeal’s tetrachrome, toluidine blue O, and basic fuchsin in preparation for examination in a Vanox AH-2 light microscope (Olympus, Tokyo, Japan). The 24 distal femurs destined for micro-CT were chemically fixed in 10% formaldehyde at ambient temperature. They were not further processed.
Five coronal sections through each tibia were photographed in a Vanox AH-2 light microscope at a final magnification of ×170 (Fig. ). In accordance with Cavalieri’s principle [12
], the total volume of tibial-plateau cartilage was determined stereologically from the photomicrographs using the point-counting technique [11
]. Cartilage tissue was deemed to have undergone degeneration if proteoglycans had been lost from the extracellular matrix (no reactivity after staining with toluidine blue) or if less than 50% of the chondrocytes was viable (as gauged by their structural integrity). Damage to the cartilage surface was considered to have occurred if its structural continuity had been interrupted in any way, such as by fibrillations or by more yawning apertures. The volume of damaged cartilage tissue was likewise determined using the point-counting technique and Cavalieri’s principle. This volume was then expressed relative to the total volume of tibial-plateau cartilage.
Light micrographs of the five coronal sections through the medial and lateral tibial plateau (Stain, McNeal’s tetrachrome, toluidine blue O, and basic fuchsin; original magnification, ×2.5).
Light micrographs (×170) of the third (middle) section through the medial tibial plateau were used to measure the thicknesses of the articular cartilage layer, calcified cartilage layer, and subchondral bone plate. Ten to 12 vertical lines, 20 μm apart, were drawn from the cartilage surface down to the lower end of the subchondral bone plate. The height of each layer was measured along these lines using a ruler [9
]. The border between the hyaline cartilage layer and zone of calcified cartilage was defined by the position of the tidemark. The border between the calcified cartilage layer and subchondral bone plate was identified by the abrupt change in the staining properties of the two tissue types. The lower border of the subchondral bone plate was demarcated by a bone marrow space (Fig. ). If a vertical test line coincided with a region in which the subchondral bone plate merged with the cancellous bone, no measurement was made. The measurements made along the 10 to 12 lines were averaged.
Fig. 2A–C (A) Low- and (B, C) high-magnification light micrographs of the medial tibial plateau in an 11-month-old guinea pig (DH strain) are shown (Stain, McNeal’s tetrachrome, toluidine blue O, and basic fuchsin; original magnification, ×2.5 [A] (more ...)
The 24 formalin-fixed femurs were imaged using a micro-CT scanner (Explore Locus SP micro-CT Scanner; GE Healthcare, Waukesha, WI). For each femur, 499 projections were produced using an isotropic voxel size of 16 μm. To avoid dehydration during scanning, each femur was immersed in a water-filled specimen tube; it was secured with its long axis parallel to the z-axis of the image coordination system to minimize beam-hardening effects [37
]. After image acquisition, each scan was corrected for variations in temperature and xray attenuation.
Reconstructed images of the femoral epiphysis were divided into four rectangular prisms (1.5 × 1.5 × 0.5 mm): anterior medial, anterior lateral, posterior medial, and posterior lateral (Fig. ), which were analyzed with MicroView 2.0 software (GE Healthcare, London, ON, Canada). The following parameters were determined directly from the images [15
]: total volume of tissue, total volume of trabecular bone, total surface area of trabecular bone, thickness of the trabecular bone layer, number of trabeculae, and volumetric bone mineral density. From these measurements, we calculated the trabecular separation and connectivity, the volume fraction of bone, and the structure model index (SMI). The latter parameter quantifies the plate-like (SMI = 0) or rod-like (SMI = 3) characteristics of the cancellous bone [16
Fig. 3 For the 3D analysis, reconstructed micro-CT images of the femoral condyle were divided into four rectangular prisms (1.5 × 1.5 × 0.5 mm): anterior medial, anterior lateral, posterior medial, and posterior lateral. The boxed area (more ...)
A 3D isosurface image of each distal femur was obtained using the volume-rendering function (Fig. ). On volume-rendered 3D images, osteophytic tissue (newly formed bone of low density) was identified and the implicated zone marked in color on the corresponding 2D projection. The total volume of the osteophytic tissue was then calculated by relating the measured value to the known voxel volume.
An isosurface image of a distal femur shows the osteophytic tissue, which has been colored.
Numerical data are expressed as means together with standard error of the mean. For the statistical analysis of differences between two groups, means were compared using an independent-samples t-test. Some of the histologic data did not follow a normal distribution. Hence, all histologic data were subjected to a two-tailed Mann-Whitney U-test. For the statistical analysis of differences among the four bony sites (anterior medial, anterior lateral, posterior medial, and posterior lateral), means were compared using a one-way ANOVA and by applying a post-hoc test and Fisher’s least significance. The data were analyzed using the SPSS® 14.0 for Windows® statistical package (SPSS Inc, Chicago, IL).