Material and methods

Forty-eight 21-day-old male CD-1 mice (Saitama Experimental Animals Supply, Saitama, Japan) were used. The mice were randomly divided into 6 groups to receive (ad libitum) diets of varying hardness and durations: control (3 animals), HD/1 week (9 animals), HD/4 weeks (9 animals), SD/1 week (9 animals), SD/4 weeks (9 animals), and HSD/4 weeks (9 animals). The 3 control mice were used to generate Figure 1 , whereas 3 sets of 3 mice from each of the remaining 5 groups were used to generate Figures 2 to 5 . At the end of the experimental period, the mice were killed by cervical dislocation after inhalation anesthesia with diethyl ether (Funakoshi, Tokyo, Japan), which was applied to cotton wool and placed with the mouse in an enclosed space. All animal procedures, including care and handling, conformed to the guidelines of our Institutional Animal Care and Use Committee, and the study protocol was approved by the Animal Research Committee of Showa University.

Gene expression analysis in mandibular condylar chondrocytes and tibial cartilage chondrocytes using real-time PCR (n=3): A, P75; B, Wnt1; C, Musashi1; and D, Nestin. Gene expression is normalized against the expression of the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene. ^∗ P <0.05.

Histologic analysis of condylar cartilage: A and B, histologic sections from the superior regions of the condyles of mice at 4 weeks ( W ) and C-E, 7 weeks of age (n = 3). Mice were fed the HD or SD for 1 or 4 weeks, or the HSD for 2 weeks each, respectively, over a total of 4 weeks. APC , Articular, proliferative, and chondroblastic zones; H , hypertrophic zone. Scale bars, 20 μm.

Immunostaining and proliferation analysis of condylar chondrocytes (n = 3): A and B, histologic sections from the superior region of mouse condyles at 4 weeks ( W ) and C-E, 7 weeks. Scale bars, 100 μm. The mice were fed the HD or SD for 1 or 4 weeks, or the HSD for 2 weeks each, with measurements taken at 4 or 7 weeks of age, respectively. Proliferation was measured by the number of cells positively stained for proliferating cell nuclear antigen. ^∗∗ P <0.01.

Immunostaining and quantitative gene expression of A and B, osteopontin, and C and D, type X collagen in mandibular condylar chondrocytes (n = 3). The mice were fed the HD or SD for 1 or 4 weeks ( W ), or the HSD for 2 weeks each, respectively, over a total of 4 weeks. Scale bars, 20 μm.

Immunostaining of Musashi1 in mandibular condylar chondrocytes (n = 3). The mice were fed the HD or SD for 1 week ( W ). Histologic sections from the superior region of the condyles at 4 weeks of age are shown: A and B, entire chondrocytic zone. The stained area was 100 × 100 μm. Scale bars, 20 μm. C, Graphic representation of the number of Musashi1-positive cells in mice fed the HD or SD for 1 week.

Three mice were killed at 21 days of age. The remaining mice were randomly divided into 5 groups of 9 when weaned: 18 mice were fed the SD, with 9 killed at 4 weeks of age and 9 killed at 7 weeks of age; 18 mice were fed the HD, with 9 killed at 4 weeks of age and 9 killed at 7 weeks of age; and 9 mice were fed the HSD every other week, and then killed at 7 weeks of age. The HD group received an ordinary laboratory diet for mice in a hard pellet form. The SD group received the ordinary diet after it was ground and mixed with water in standardized proportions (2 parts food to 5 parts water). No significant differences in weight were identified among the mice in the any of the groups either at randomization or when they were killed.

Tissue processing for hematoxylin and eosin and immunohistochemical staining were carried out. The dissected cartilage specimens from the mandibular condyles were fixed in 4% paraformaldehyde solution (pH 7.4) overnight at 4°C and then decalcified in 10% Na ₂ EDTA (pH 7.2) for 2 weeks. Ten specimens from each group were embedded in paraffin and sectioned (7-μm thickness). The slices were coronal sections, which were carefully selected at the center of the mandibular condylar cartilage.

Five condylar sections from each group were stained with hematoxylin and eosin. The condylar cartilage was divided into 2 parts, comprising the articular, proliferative, and chondroblastic zones in one part, and the hypertrophic zone in another. The representative thicknesses were composed of the mean measurements of these zones.

Immune complexes were formed by mixing a mouse antibody against proliferating cell nuclear antigen (1:200 dilution; Oncogene, Boston, Mass) and EnVisionHRP antimouse detection reagent (Dako, Copenhagen, Denmark) for 1 hour at room temperature. After 60 minutes at room temperature, normal mouse serum (Dako code X0910) was added to the immune complex.

Five sections from each group were incubated with polymer immune complexes for 1 hour at room temperature. Deparaffinized paraffin sections were incubated with HRP-labeled primary antibodies using diaminobenzidine as the substrate. Equivalent dilutions and concentrations of mouse IgG2a (1:200 X0943; Dako) were used as negative controls.

Five sections from each group were incubated for 2.5 hours at room temperature with an antitype-X collagen antibody (1:100 dilution LSL-LB-0092 whole rabbit serum; LSL, Tokyo, Japan), which was detected using an Alexa Fluor 568-conjugated goat antirabbit polyclonal IgG (H+L) (Molecular Probes, Eugene, Ore) as a secondary antibody. The sections were observed with a fluorescence microscope (Eclipse TE300; Nikon, Tokyo, Japan) and an imaging system (AQUA COSMOS; Nikon).

Equivalent dilutions and concentrations of rabbit immunoglobulin fraction (1:100 X0903; Dako) were used as the negative controls.

We performed immunohistochemistry for osteopontin. Five sections from each group were incubated for 3 hours at room temperature with an antiosteopontin antibody (1:100 dilution LSL-LB-4225 whole rabbit serum; LSL), which was detected by incubation with EnVision + HRP antirabbit detection reagent (Dako) for 1 hour at room temperature. To prevent nonspecific staining, we incubated the sections with a blocking reagent containing normal mouse serum for an additional 1 hour at room temperature. Deparaffinized paraffin sections were reacted with HRP-labeled primary antibodies using diaminobenzidine as the substrate.

Equivalent dilutions and concentrations of rabbit immunoglobulin fraction (1:100 X0903; Dako) were used as negative controls.

Five sections from each group were incubated for 1 hour at room temperature with an anti-Musashi1 antibody (1:10 dilution 1877-1 rabbit Monoclonal IgG; Epitomics, Burlingame, Calif), which was detected by incubation with EnVision + HRP antirabbit detection reagent for 1 hour at room temperature. To prevent nonspecific staining, we incubated the sections with a blocking reagent containing normal mouse serum for an additional 1 hour at room temperature. Deparaffinized paraffin sections were reacted with HRP-labeled primary antibodies using diaminobenzidine as the substrate. In all experiments, negative controls were performed in which the primary antibody was omitted. Equivalent dilutions or concentrations of rabbit immunoglobulin fraction (1:10 X0903; Dako) were used as negative controls.

For the reverse transcription and real-time PCR, mandibular condylar cartilage was collected by dissection under a light microscope and snap-frozen in liquid nitrogen. Total RNA from 3 mice (nonpooled) was extracted from the total cartilage layer of the mandibular condyle and the tibial cartilage using an RNeasy Protect Mini Kit (Qiagen, Hilden, Germany) with RNase-free DNaseI treatment (Qiagen). Following the manufacturer’s protocol, we were able to extract sufficient quantities from 1 mouse. In addition, we confirmed by absorption spectrometer that the mRNA obtained was adequate and of good quality. First-strand cDNA was synthesized from 5 μg of total RNA using Super Script II RT (Gibco, Grand Island, NY) and random hexamers (Gibco). For real-time PCR, we used an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Darmstadt, Germany) with TaqMan probes. The final mixture (20 μL) contained 4 μL of cDNA sample, 10 μL of TaqMan Universal PCR Master Mix (Perkin-Elmer Applied Biosciences, Foster City, Calif), and 1 μL of primers for osteopontin, type X collagen, P75, Wnt-1, Musashi1, and Nestin (Mm00436767-m1Spp1, lot 455039; Mm00487041-m1, lot 479307; Mm00446296-m1, lot 958101; Mm01300555-g1, lot 812039; Mm00485224-m1, lot 649865; and Mm00450205-m1, lot 74463). Gene expression levels were normalized by the corresponding expression level of glyceraldehyde-3-phosphate dehydrogenase as a housekeeping gene. The PCR conditions were 2 minutes at 50°C, 10 minutes at 95°C, and 40 cycles of 15 seconds at 95°C and 1 minute at 60°C.