Intermittent Parathyroid Hormone [1-34] Augments Chondrogenesis of the Mandibular Condylar Cartilage of the Temporomandibular Joint

Abstract

Objective: To characterize the long-term effects of intermittent parathyroid hormone (I-PTH) on the mandibular condylar cartilage (MCC) and subchondral bone of the temporomandibular joint, in vivo and in vitro.

Materials and methods: For the in vivo experiments, sixteen 10-week-old mice were divided into 2 groups: (1) I-PTH (n = 8)-subcutaneous daily injection of PTH; (2) control group (n = 8)-subcutaneous daily injection of saline solution. Experiments were carried out for 4 weeks. Mice were injected with calcein, alizarin complexone, and cell proliferation marker before euthanasia. For the in vitro experiments, primary chondrocyte cultures from the MCC of eight 10-week-old mice were treated with I-PTH for 14 days.

Results: There was a significant increase in bone volume, tissue density, mineral deposition, osteoclastic activity, cell proliferation in the cartilage, and cartilage thickness in the I-PTH-treated mice when compared with the control group. In addition, immunohistochemistry in cartilage revealed that I-PTH administration led to an increase in expression of vascular endothelial growth factor and to a decreased expression of sclerostin, matrix metallopeptidase 13, and aggreganase-1 (ADAM-TS4). Quantitative polymerase chain reaction analysis of the I-PTH-treated chondrocytes revealed significantly decreased relative expression of collagen type X (Col10a1), alkaline phosphatase (Alp), and Indian Hedgehog (Ihh) and remarkable increased expression of Sox9, fibroblast growth factor 2 (Fgf2), and proteoglycan 4 (Prg4).

Conclusion: I-PTH administration causes anabolic effects at the subchondral region of the mandibular condyle while triggers anabolic and protective effects at the MCC.

Keywords: mandibular condylar cartilage; parathyroid hormone; temporomandibular joint.

Figure 1.

Increased bone volume and density at the mandibular condyle after long-term intermittent parathyroid hormone (I-PTH) administration. Coronal micro–computed tomography (micro-CT) images of condyles of control (CTRL) and I-PTH-injected mice (A). Quantification of bone parameters: (B) BVF = bone volume fraction, (C) tissue density, (D) trabecular thickness, and (E) trabecular spacing. Histograms (B-E) represent means ± standard deviation (SD) for n = 8 per group. Statistically significant difference between groups: *P < 0.05. Region of interest is illustrated by dotted lines in (A). Scale bar = 500 µm.

Figure 2.

Increased mineralization and bone remodeling at the subchondral region of mandibular condyle after long-term intermittent parathyroid hormone (I-PTH) administration. Sagittal sections of mandibular condyles of control (CTRL) and I-PTH-injected mice illustrating alizarin complexone and calcein labeling (A). Dotted lines in (A) in the alizarin complexone images in CTRL and PTH images represent the tidemark: faint line of alizarin complexone is observed in the PTH image. Quantification of alizarin complexone (red, B) and calcein (green, C) percentage of positive pixels over the subchondral bone area. Sagittal sections of mandibular condyles of control (CTRL) and I-PTH-injected mice stained for tartrate-resistant acid phosphatase (TRAP) (D). Quantification of percentage of TRAP-positive pixels (yellow, E) in the subchondral bone area. Histograms (B, C, and E) represent means ± standard deviation (SD) for n = 5 per group. Statistically significant difference between groups: *P < 0.05. Scale bar = 100 µm (A) and 50 µm (D).

Figure 3.

Increased cartilage thickness and chondrocyte proliferation at the mandibular condylar cartilage (MCC) after long-term intermittent parathyroid hormone (I-PTH) administration. Sagittal sections of mandibular condyles of control (CTRL) and I-PTH-injected mice stained for Safranin O (A). MCC and subchondral bone area (Sub. Bone) are labeled. Quantification of cartilage thickness (B). Sagittal sections stained for 5-ethnyl-2′-deoxyuridine (EdU) (C). Quantification of EdU-positive pixels (yellow), representing the amount of cellular proliferation, over DAPI-positive pixels (blue) at the proliferative zone (D). Histograms (B and D) represent means ± standard deviation (SD) for n = 5 per group. Statistically significant difference between groups: *P < 0.05. Scale bar = 50 µm.

Figure 4.

Decreased expression of sclerostin (SOST), matrix metallopeptidase 13 (MMP13), and aggreganase-1 (ADAM-TS4) and increased expression of vascular endothelial growth factor (VEGF) at the mandibular condyle of intermittent parathyroid hormone (I-PTH)–injected mice. Immunohistochemistry for SOST (A), VEGF (B), MMP13 (C), and ADAM-TS4 (D) in sagittal sections of condyles. Scale bar = 50 µm.

Figure 5.

Gene expression changes in chondrocyte micro mass cultures treated with intermittent parathyroid hormone (I-PTH). Histograms represent relative gene expression tested by quantitative polymerase chain reaction (qPCR) 14 days after I-PTH treatment. The relative expression of Col10a1 (A), Alp (B), and Ihh (E) was markedly decreased, while the expression of Sox9 (C), Fgf2 (D), and Prg4 (F) was significantly increased. Statistically significant difference between groups: *P < 0.05.