Animal studies

Sixty 7-month-old female Sprague-Dawley rats were randomly divided into five groups: OVX with vehicle treatment ( n = 12), OVX with an early intra-articular alendronate injection (on the day of OVX) ( n = 12), OVX with a late alendronate treatment at 4 weeks after OVX ( n = 12), sham-operated ( n = 12), and normal controls ( n = 12). All animal procedures were approved by the institutional animal care and use committee in accordance with the guidelines of the National Institutes of Health.

Bilateral OVX were performed under general anaesthesia as described previously. For local administration of alendronate (Novartis Pharma Stein AG, Switzerland), a small incision was made in the skin between the eye and ear and the location of the articular capsules of the TMJ. A needle was then inserted from a posterosuperior direction into the inferior joint cavity, so that the disc was avoided. Alendronate was injected on the day of OVX for the early alendronate treatment group or at week 4 after OVX for the late alendronate treatment group. Subsequent injections were administered weekly for 4 weeks at a dosage of 0.1 mg alendronate in 0.02 ml saline solution (5 mg/ml). We chose week 4 after ovariectomy to define early vs. late intervention, because previous studies have shown significant osteoporosis-like changes in the condyle by this time-point. The dose of alendronate was chosen based on the local effective concentration established in a previous in vivo preliminary study, in which local delivery of 1 mg/ml alendronate gel significantly improved bone fill compared with placebo gel in patients with perodontitis. In the present study, we set the concentration at 5 mg/ml, five times higher than the local effective concentration, because of the potential drug dilution within the articular cavity. The rats in the sham-operated and the OVX/vehicle groups were injected with an equivalent volume of saline. Six animals per group were sacrificed at week 10 and week 16 after OVX, respectively ( Fig. 1 ).

Experimental design. Early alendronate treatment was started on the day of ovariectomy, while late treatment was started at 4 weeks after the ovariectomy. Six animals in each group were killed at 10 and 16 weeks after ovariectomy, respectively.

Micro-computed tomography (micro-CT)

The condyles were scanned using a micro-CT system (μ-CT 80 Scanner; Scanco Medical, Brüttisellen, Switzerland). The system was set to 55 kV, 145 mA, and 500 ms integration time. The binary images obtained with a resolution of 1024 × 1024 pixels and isotropic voxel size of 18 μm were reconstructed to three dimensions for qualitative and quantitative evaluations (threshold 250–700). Two regions were defined as the sampling sites: (1) a ‘subchondral region’, comprising the region connected to cartilage, and (2) a ‘central region’, comprising the region beneath the former ( Fig. 2 A). The following parameters were calculated to describe the bone structure: bone volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp).

The effects of intra-articular injection of alendronate (ALN) on the subchondral bone of the TMJ. Determination of the sampling site for subchondral bone (A). The sampling site is shown as a square. Point A is the posterior protruding point of the condylar cartilage, point B is the anterior protruding point of the condylar cartilage, and point C is the anteroposterior midpoint of the sampling site; this point was used as the reference point to obtain the sampling site. The subchondral region is the region connected to cartilage; the central region is the region beneath the subchondral region. Example micro-CT images of the subchondral bone from the normal control rats (B and C), the OVX rats with early ALN treatment (D–G), the OVX rats with late ALN treatment (H–K), and the OVX rats with vehicle treatment (L–O). Bar represents 1 mm. Measurements of the parameters from the subchondral bone at 10 weeks (P) and 16 weeks (Q). *Group significantly different from the early ALN treatment rats ( P < 0.05); ^# group significantly different from OVX rats treated with late ALN and vehicle ( P < 0.05).

Histology and tartrate-resistant acid phosphatase (TRAP) staining

The condyles were paraffin-embedded and every fifth slide was stained with haematoxylin and eosin (H&E) or TRAP (Sigma–Aldrich, St. Louis, MO, USA). The thickness of the cartilaginous layer of the condyle was measured ( Fig. 3 A and B ). TRAP-positive cells were counted in the subchondral bone region or the central bone region of three serial sections per sample.

Effects of intra-articular injection of alendronate (ALN) on the cartilage of the TMJ. The thickness of the cartilaginous layer was measured perpendicular to the articular surface at the anterior, central, and posterior regions along the circumference of the condyle, as indicated by the red line (A and B). The articular cartilage of the condyle from the OVX rats in the early ALN treatment group (C and D), the late ALN treatment group (E and F), and the vehicle treatment group (G and H). Bar represents 500 μm. The thickness of the cartilaginous layer at 10 weeks (I) and 16 weeks (J) after OVX. ^# Group significantly different from OVX rats treated with late ALN and vehicle ( P < 0.05).

Immunohistochemistry

Primary antibody for matrix metalloproteinase (MMP)-13 was purchased from Biovision (Milpitas, CA, USA). Immunohistochemical staining was performed according to the manufacturer’s instructions. Briefly, tissue sections were washed twice with phosphate-buffered saline (PBS) containing 0.3% Tween 20 for 1 h and then incubated with anti-MMP-13 polyclonal antibodies, followed by horseradish peroxidase-labelled secondary antibody (Dako, Carpinteria, CA, USA); the colour (brown) was developed using 0.5 mg/ml 3,3′-diaminobenzidine tetrahydrochloride. The same procedures were carried out without the primary antibody for negative controls.

Real-time quantitative polymerase chain reaction (QRT-PCR)

Total RNA was isolated from the subchondral bone using Trizol reagents (Invitrogen Life Technologies, Carlsbad, CA, USA). Reverse transcription was performed using the PrimeScript RT Reagent Kit (Takara Bio, Shiga, Japan) and the first-strand cDNA was synthesized using oligo(dT)15 primers (Takara Bio, Shiga, Japan). Primer pairs were 5′-GAAGATCAGCCCAGACGAGATT-3′ and 5′-TGCTCGCTGGGTTTGCA-3′ for osteoprotegerin (OPG), 5′-AGCGCTTCTCAGGAGTTCCA-3′ and 5′-GCCGGGCCACATCGA-3′ for receptor activator of nuclear factor-κB ligand (RANKL), and 5′-TATGACTCTACCC ACGGCAAGT-3′ and 5′-ATACTCAGCACCAGCATCACC-3′ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gene expression levels were normalized to GAPDH mRNA.

Statistical analysis

All data were expressed as the mean ± standard error of the mean. Statistical analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). The statistical significance of differences between groups was analyzed using one-way analysis of variance and Fisher’s protected least significant difference test. Any P -value < 0.05 was considered statistically significant.