Mandible protraction alters Type I collagen, osteocalcin and osteonectin gene expression in adult mice condyle

Abstract

Mandible condyle remodeling is a great challenge on craniofacial growth studies. The great majority of the reports deals with growing period. However, there is a great necessity to clarify the importance of functional stimulation on adult mandible condyle remodeling. By using an adult mouse model, we investigated the influence of mandible forwarding on condyle remodeling and gene expression by bone forming cells. Tomographic and scintigraphic evaluations showed sagittal growth and cell activity enhancement. RT-PCR showed that Type I collagen, osteocalcin and osteonectin expression level can be altered. We showed that functional stimulation is necessary to maintain the regular gene expression by condyle bone forming cells in adult mice. It opens new frame for further investigations aiming new clinical approaches to temporomandibular joint problems treatment, as well as mandible retrusion treatment.

Keywords: CT; bone biology; condyle growth; gene expression; growth evaluation; molecular biology.

Figure 1 a, b

Mandibular advancement. (a) To generate a physiological mandibular advancement, that would lead to stimulation on condyle region, every 3 days during 21 days, the lower incisors were cut by 1 mm to induce protrusion when the animal was feeding. In left panel we can see the control animal with regular distance between inferior and superior incisor, and on right panel we can see the experimental animal immediately after cutting, showing an increase on the overjet; (b) the weight gaining of the animals indicated that on the first days the experimental group was not used to the increase of overjet and had weight loss, but after 9 days they started to eat normally and recover the weight gaining (first point of the graphic is 3 and last point of the graphic is 18, because we checked the weight on the cutting day).

Figure 2 a–c

Tomography. (a) After anesthesia, the animals were positioned on the equipment table as showed on the picture; (b) The software used allows the user to measure linear distances in 3 dimensions. After anesthesia, the animals were analyzed in coronal (right panel) and transversal (left upper panel) plane to localize the condyle. The measurement was done on sagittal plane (left lower panel). Measurements were done separately in left and right condyle; (c) After 21 days, the experimental group showed an increase on sagittal dimension of the condyle. Results reflects means ± SD of 3 different experiments (p< 0.05).

Figure 3 a, b

Bone Scintigraphy. (a) Representative scintigraphic image for the definition of the region of interest (ROI). Red arrow is reference point to show one ROI at the condyle region. The ROI (circle) was established and the radio isotope count [accumulation count of 99mtechnetium-methylene-diphosphonate (99mTc-MDP)] was measured. As inferred by the color bar scale, red shows a high accumulation of 99mTc-MDP and blue shows a low accumulation of 99mTc-MDP; (b) Uptake ratio of 99mTc-MDP at the condyle region after protrusion of the mandible. The vertical axis shows the uptake ratio. The count rates of 99mTc-MDP in the protruded mandible condyle were significantly higher than control after 21 days. Results express mean ± SD of 2 different experiments for each period (p< 0,05).

Figure 4

Immunofluorescence for osteopontin. Confocal images obtained from the condyle macerate. A great number of cells was positive for osteopontin, indicating that they were bone forming cells. Green indicates osteopontin staining and red are nucleus stained by propidium iodide. Transmission image included in merged image.

Figure 5

Real-time PCR. Diferential expression of: (a) Collagen (Col1), (b) Osteocalcin (Bglap) and (c) Osteonectin (Sparc) evaluated at 7 days and 21 days. All of them showed lower expression on experimental group at 7 days, compared to control (p<0.05). On 21 days no statistical difference on gene expression was observed (p<0.05), indicating that the experimental group showed an enhance on gene expression during the period from 7 to 21 days; (d) Differential expression of Type I Collagen, Osteocalcin and Osteonectin by experimental group analyzed in terms of control % (p<0.05).