Material and methods

All procedures were approved by the Institutional Animal Care and Use Committee of the University of Washington. While under isoflurane anesthesia, 50 female New Zealand rabbits, 5 months old at acquisition, received unilateral injections of either 10 units of BoNT/A (n = 31; Botox; Allergan, Irvine, Calif) or an equivalent volume of 0.9% saline solution (n = 19) to the inferior portion of the superficial masseter muscle (split among 3 injection points ); the contralateral uninjected side served as an intra-animal control. Most of the animals were derived from the previous study. These animals were killed at 4 weeks (atrophy) or 12 weeks (recovery) after injection, therefore establishing 4 test groups: BoNT/A 4 week, BoNT/A 12 week, saline solution 4 week, and saline solution 12 week. Sample sizes for analysis ( Tables I-IV ) varied from 7 to 13 per group because of sectioning or staining errors. Three additional rabbits were untreated controls and were killed at 1 to 2 weeks after acquisition.

One week before they were killed, each animal was anesthetized and injected intravenously with bromodeoxyuridine (BrdU), a thymidine analog that labels cells in the S-phase of replication (40 mg/kg as a 10 mg/mL solution in phosphate-buffered saline solution). Anesthetized animals were killed by perfusion with 0.9% phosphate buffered saline solution followed by 4% paraformaldehyde. The mandibular condyles of each rabbit were removed from the mandibular ramus, decalcified in Immunocal (Decal Chemical, Tallman, NY), embedded in paraffin, and sectioned in the coronal plane at 7 to 10 μm. The observers (T.M. for condylar measurements; H.A.H.D. for the BrdU labeled cell counts and subjective obervations) were blinded to the treatment of the specimens.

For the condylar observations and morphometrics, 3 coronal sections through the widest portion of each condyle were stained with hematoxylin and eosin. Damaged sections were discarded, resulting in a few cases in which only 1 or 2 sections were used. Our original intent was to follow the standard staining and BrdU reaction with immunohistochemical stains for osteoblastic and osteoclastic markers. However, because of problems with decalcification and infiltration leading to the necessity of reembedding many specimens, the cellular contents of many marrow cavities (where osteoblastic and osteoclastic cells would have been found) were disconnected from bone surfaces; this caused distortion and in some cases missing marrow contents. Thus, we could not reliably identify either osteoblasts or osteoclasts in the marrow cavities. Instead, each section was qualitatively examined for signs of atypical morphology (unusual shape, surface defects, and so on) and resorption (multinucleated osteoclasts in Howship’s lacunae or scalloped surface). Then the section was aligned along the long axis of the condylar process and imaged at 10 times magnification (E400 microscope; Nikon, Tokyo, Japan) using MetaVue (Molecular Devices, Sunnyvale, Calif). Measurements were made with MetaMorph (Molecular Devices) and averaged for the 3 sections per condyle.

The measurements are illustrated in Figure 1 . The thickness of the condylar cartilage was measured at the most apical portion of the articular surface and included all zones (fibrous, proliferative, maturational, and hypertrophic), since separate measurements of each zone were not adequately repeatable. The percent of trabecular bone area (areal density) was assessed by superimposing a standardized grid over the chosen region and counting the numbers of intersections that fell on bone; this number was divided by the total number of grid intersections to yield a proportion. The 2 chosen regions were subchondral and midcondylar ( Fig 1 ). Cortical thickness was measured on the medial and lateral sides of the condylar neck perpendicular to the long axis of the condyle at a standard distance from the midcondylar region.

Coronal section of a rabbit condyle showing measurements. A, Morphometrics. Condylar cartilage thickness (fibrous plus chondrocytic layers, CTh ) was measured at the apex of the condyle. For replicating cell counts, the cartilage was divided into medial, central, and lateral thirds (defined by radial lines at 60° angles from a line passing through the condylar poles). The percent of trabecular bone area was calculated from the subchondral ( SC ) and midcondylar ( MC ) regions ( shaded ). The subchondral region was defined by shifting the outline of the superior curvature of the condylar bone inferiorly by 750 μm; the resulting subchondral area was always 750 μm in maximum height but varied in the other dimensions with individual condyle size and shape. The midcondylar region was defined by a 750 × 750 μm area directly inferior to the subchondral region and in line with the apex. Cortical thickness was measured on the medial ( MTh ) and lateral ( LTh ) sides of the condylar neck perpendicular to the long axis of the condyle and 1500 μm from the most inferior aspect of the midcondylar regions. B, A 50 × 50 grid, 5.3 × 5.3 mm, was superimposed on the image. The percent of trabecular bone area was calculated as the number of intersections overlying bone ( black dots ) divided by the total number of intersections in each region.

For cell replication, an additional 5 sections were chosen from the central region of each condyle; 1 slide was used as a negative control, and 4 were reacted for BrdU using a kit and following the manufacturer’s instructions (Becton Dickinson, Franklin Lakes, NJ). The sections were lightly counterstained with methyl green. A brown nucleus was the defining feature for a BrdU-positive (replicating) cell. The cartilage was divided into thirds (medial, central, lateral) as shown in Figure 1 . Three superficial-to-deep zones were identified for counting BrdU-labeled cells: dense fibrous connective tissue, designated the “fibrous zone”; small, flat cells underlying and parallel to the fibrous zone, designated “proliferative zone”; and an area grading from slightly larger ovoid cells to large spherical hypertrophic chondrocytes, designated “maturing and hypertrophic zone” ( Fig 2 ). However, the boundaries of these zones were not distinct. In addition to the condylar cartilage counts, positive cells in the periostea of the condylar poles and neck and in the lining of marrow cavities were noted. Counts from the 4 sections per condyle were averaged.

Zones of the condylar cartilage. The central third of a condyle (BoNT/A-injected side, 4-week end point) is shown in a BrdU-reacted section ( methyl green counterstain ). Labeled cells were rare in the fibrous zone ( Fib Z ) but common at the lower part of the proliferative zone ( Pro Z ) and its junction with the mature plus hypertrophic zones ( M&H Z ), as well as at the mineralizing bone front.

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