Abstract
Unilateral fracture of the condylar neck in immature subjects might lead to mandible asymmetry and condyle remodelling. A rat model was used to investigate mandibular deviation and condylar remodelling associated with condyle fracture. 72 4-week-old male rats were randomly divided into three groups: an experimental group (unilateral transverse condylar fracture induced surgically), a sham operation group (surgical exposure but no fracture), and a non-operative control group (no operation). The rats were killed at intervals up to 9 weeks after surgery, and outcomes were assessed using various measures of mandible deviation, histological and X-ray observation, and immunohistochemical measures of expression levels of connective tissue growth factor (CTGF) and type II collagen (Col II). The fracture led to the degeneration of mandibular size, associated with atrophy of fractured condylar process. Progressive remodelling of cartilage and increasing expression levels of CTGF and Col II were found. The authors conclude that condylar fracture can lead to asymmetries in mandible and condyle remodelling and expression of CTGF and Col II in condylar cartilage on both the ipsilateral and the contralateral sides.
Of the facial bones, the mandible is the most frequently fractured. The condylar neck is a primary site of mandibular fracture, accounting for 16.1–41% of all fractures; the incidence in children and adolescents is higher than in adults. Open and closed reduction surgeries are the most commonly used, and effective, treatments for repairing condylar neck fractures, but there is controversy over the advantages and disadvantages of both methods. Studies have described favourable results for condylar fracture when closed methods are used in children, and these conclusions are supported by animal experiments. Remodelling following condylar fractures, following treatment by conservative procedures has been demonstrated by radiography and histology. Some evidence suggests that unilateral fracture of the condylar neck in immature subjects leads to skeletal changes, culminating in mandible asymmetry.
The effects of unilateral condylar fracture, with or without treatment, on the subsequent changes of condyle and mandible are required to promote a better understanding of condylar remodelling and mandibular growth, which is the purpose of the present study, employing growing rats.
Materials and methods
72 4-week-old male Sprague-Dawley rats were fed an ordinary diet of rodent feed (Laboratory Animal Center of Kunming Medical College, Yunnan, China). Beginning 2 days after the operation, powdered feed alone was offered, then mixed feed containing conventional feed and powdered feed was offered for another 2 days, and, thereafter, conventional feed alone was used. The animals were randomly divided into three equal groups of 24 animals: experimental group (unilateral transverse condylar process fracture induced surgically on right side); sham operation group (surgical access only performed on the right side), and control group (no operation). 6 animals were killed at each of four time points.
Surgical procedures
The rats were anaesthetized by intramuscular injection of ketamine hydrochloride and underwent a longitudinal, 1 cm pre-auricular incision on the right side. Blunt dissection through the masseter muscle was performed and the condylar process was exposed just below the zygomatic arch. The right condylar neck was cut with eye scissors by the exterior joint capsule at the level of the sigmoid notch. The right side is designated the ipsilateral side and the left side the contralateral side. For the sham operation group, the condylar neck was exposed but not fractured. The non-operative control group received no treatment. The rats were killed at 1, 3, 5 and 9 weeks after the operation, and their heads were isolated and fixed in 4% paraformaldehyde. The choice of time of death was intended to encompass both growth responses and morphological changes due to the normal ageing process.
Image analysis and mandible measurements
Heads were divided along the median sagittal plane. X-ray images were taken of the right side for both the sham operation group and the control group and of both sides for the experimental group using a standard dental X-ray machine (Oralix AC, Gendex, Italy, 65 kV, 7.5 mA). The cut surface was oriented horizontally, with the X-ray tube aimed at the temporomandibular joint (TMJ) area perpendicular to the sagittal plane of the specimen. The exposure time was 0.16 s.
The specimens were trimmed, the skull removed, and the soft tissue cleaned to expose each anatomical landmark fully. Measurements were taken using a divider calliper and a Vernier calliper (0.02 mm resolution) with reference to the following anatomic landmarks ( Fig. 1 ): Id-Ap, the distance from the infradentale (Id) to the angular process (Ap); TM-AN, the distance from the distal surface of the mandibular third molar to the antegonial notch (AN); and Cds-Ap, the distance from the most superior point of the condylar head (Cds) to the angular process (Ap).
Histology and immunohistochemical staining
The trimmed specimens were decalcified, embedded in paraffin and sectioned into transcondylar slices of 4 μm. Slices were stained with haematoxylin and eosin (H–E), then underwent two-step immunohistochemical staining using rabbit anti-rat connective tissue growth factor (CTGF) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-rat collagen type II (Col II) monoclonal antibody (NeoMarkers, Fremont, California, USA). Visualization of bound antibodies utilized a two-step anti-rabbit/mouse universal immunohistochemical detection kit (Shanghai Gene Company, Shanghai, China).
Images were captured with a digital camera microscope (NIKON 50i, Kanagawa, Japan) at a magnification of 50×, and analysed with an HPIAS-1000 high-resolution colour graphic analysis system (Championimage, Wuhan, China). The thicknesses of the fibrous zone (FZ), proliferative zone (PZ), chondroblast zone (CZ), hypertrophic zone (HZ), and the total thickness of the four zones were determined in the median sagittal sections of the middle part of the condyle.
Qualitatively, brown staining is a positive immunohistochemical signal indicating protein expression. All sections were viewed at the same magnification (200×) and light intensity, and photographs were taken in six randomly selected microscopic fields. In order to quantify the expression, the photographs were analysed with the HPIAS-1000 high-resolution graphic system to determine the mean greyscale values (0–255) of each field. Lower mean greyscale values represent stronger expression because the extent and intensity of brown staining cause a decrease in white-light transmission.
SPSS statistical software (SPSS Inc., Chicago, IL, USA) was used for an analysis of variance of mandible measurements date and greyscale values from the various experimental groups. The data are presented as mean ± standard deviation (SD), and significance was taken as P < 0.05.
Results
Condylar imaging and mandibular measurements
At the different time points, the TMJ X-ray images of both sides for the experimental group and the right side for the sham operation group and the control group were compared. General observations ( Fig. 2 ) showed that on the day of surgery, the fracture line was clearly seen on the ipsilateral side. 1 week after the operation, the condylar structure was not clear on the ipsilateral side. 3 weeks after the operation, the condylar depression was seen at the condylar crest on the ipsilateral side, the condyle was smaller than on the contralateral side, and the condylar neck showed discontinuous, low density. 5 weeks after the operation, the condyle on the ipsilateral side was significantly smaller in the experimental group than in the other groups, and the condyle showed irregular morphology. 9 weeks after the operation, the condyle on the ipsilateral side was smaller in the experimental group than in the other groups, with a blunt and round condylar crest.
Quantitative measures reinforced the general observations. The Cds-Ap dimension was significantly smaller on the ipsilateral side than on the contralateral side throughout the experiment ( P < 0.05). The Id-Ap and TM-AN dimensions had not changed 1 week after the operation. At 3, 5 and 9 weeks after surgery, the authors obtained significantly smaller Id-Ap, TM-AN, and Cds-Ap measurements on the ipsilateral side of the experimental group compared with either the contralateral side or with the non-operative control group ( P < 0.05). No significant differences for Id-Ap, TM-AN and Cds-Ap were found between the contralateral sides of the sham operation group and the non-operative control group ( P > 0.05) ( Table 1 ).
| 1 week post-op | 3 weeks post-op | 5 weeks post-op | 9 weeks post-op | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Id-Ap | TM-AN | Cds-Ap | Id-Ap | TM-AN | Cds-Ap | Id-Ap | TM-AN | Cds-Ap | Id-Ap | TM-AN | Cds-Ap | |
| R | 17.57 ± 5.93 | 4.58 ± 0.23 | 6.56 ± 0.41 # | 18.97 ± 0.81 # , * | 4.75 ± 0.13 # , * | 6.60 ± 0.36 # , * | 21.64 ± 0.72 # , * | 5.18 ± 0.28 # , * | 7.40 ± 0.24 # , * | 21.73 ± 0.64 # , * | 5.55 ± 0.25 # , * | 7.68 ± 0.70 # , * |
| L | 17.84 ± 4.65 | 4.68 ± 0.19 | 6.96 ± 0.34 | 19.51 ± 0.42 | 4.97 ± 0.19 | 7.31 ± 0.27 | 22.38 ± 0.54 | 5.43 ± 0.26 | 7.82 ± 0.57 | 22.98 ± 0.75 | 5.73 ± 0.41 | 8.39 ± 0.38 |
| S | 17.30 ± 3.82 | 4.71 ± 0.15 | 6.67 ± 0.21 | 19.81 ± 0.28 | 5.26 ± 0.26 | 6.82 ± 0.14 | 24.18 ± 2.10 | 5.58 ± 0.15 | 7.61 ± 0.06 | 22.22 ± 0.42 | 5.63 ± 0.29 | 7.80 ± 1.00 |
| B | 17.69 ± 2.76 | 4.89 ± 0.08 | 6.57 ± 0.20 | 20.14 ± 0.43 | 5.16 ± 0.13 | 7.15 ± 0.10 | 23.14 ± 1.24 | 5.64 ± 0.23 | 7.81 ± 0.01 | 22.55 ± 0.21 | 5.93 ± 0.19 | 8.22 ± 0.60 |
# P < 0.05 the ipsilateral side vs . the contralateral side.
* P < 0.05 the ipsilateral side, the contralateral side and the right side of sham operation group vs . the right side of the non-operative control group.
Histological observation and measurements
On the contralateral side, the condylar surface was smooth, and the FZ, PZ, CZ and HZ had clear structures and boundaries with good continuity. The subchondral bone trabeculae were finger-shaped and oriented perpendicular to the joint surface. With increasing age, the condylar cartilage became gradually thinner. On the ipsilateral side, the condylar surface retained good integrity, but the thickness of the condylar cartilage had decreased significantly. The subchondral trabecular space had increased in size, and the trabecular bone had become thinner and disarranged ( Fig. 3 ).
The condylar neck fractures affected the growth of each measurement zone ( Figs 4 and 5 ). 1 week after the operation, there were no significant differences between the thicknesses of the zones for each group. 3 weeks after surgery, morphologic changes of the ipsilateral condyle were seen in the experimental group, where the FZ and PZ had thickened. The CZ and HZ had become thinner, and the total thicknesses were diminished in the experimental group compared with the other groups. There were no significant differences between the other groups. 5 weeks after the surgery, the FZ on the ipsilateral side was thicker than that seen in the non-operative control group. The PZ on the ipsilateral side of the experimental group was still larger than that of the other groups, and the CZ, HZ and total thickness were smaller on the ipsilateral side than the corresponding thicknesses in other groups. 9 weeks after surgery, the FZ and PZ on the ipsilateral side were thicker in the experimental group than in other groups, while the CZ, HZ and total thickness were all smaller.
In each group, the FZ was found to have significantly thickened 3 weeks after the operation (compared to 1 week after the operation), but it then appeared to thin out again, so that, at 9 weeks post-operation it was significantly thinner than its status at 5 weeks. In each group, the growth pattern of the PZ also consisted generally of a thickening followed by thinning, but the pattern is complicated with respect to time and anatomical symmetry. Specifically, the PZ became thicker on both sides for 3 weeks after the operation, but then began to diminish on the contralateral sides of all three groups, even in the non-operative controls. On the ipsilateral side, the authors observed significant thinning only by 9 weeks in the experimental and sham operated groups. The CZ had become thinner 3 and 5 weeks after the operation compared to the previous time points in all groups. The HZ behaved differently on the two sides: on the ipsilateral side of the experimental group it had thinned 3 weeks after the operation compared to 1 week after operation, while on the contralateral side, in both experimental and sham operation groups, it had thickened over the same time period. The total thickness was reduced, and this reduction was significant on the ipsilateral side; there were no significant differences between other groups ( Fig. 5 ).
Immunohistochemical staining
CTGF expression was positive (indicated by brown colour) by immunohistochemical staining in the cytoplasm of the CZ and HZ of the condylar cartilage ( Fig. 6 ). Using greyscale quantitation ( Table 2 ; where intensity values varies inversely with the degree of expression) to compare protein expression among groups, the authors observed no significant differences in CTGF expression among the two control groups and the contralateral side of the operated group at 1, 5 and 9 weeks after surgery. At 3 weeks after surgery, CTGF expression on the ipsilateral side of the experimental group showed slightly more intense staining than that seen in the other three groups. When the authors track expression of CTGF ( Table 2 ) on the ipsilateral side with time, they observe an increase at 3 weeks, persisting at 5 weeks, and decreasing at 9 weeks after the operation. The expression of CTGF after surgery was elevated by 5 weeks (compared with 3 weeks) on the contralateral side of the experimental group, as well as in the sham operation group and the non-operative control group ( Table 2 ).
| Time point | Ipsilateral side | Contralateral side | Sham operation (right side) | Blank control (right side) |
|---|---|---|---|---|
| 1 w post-op | 165.66 ± 2.90 | 165.60 ± 3.96 | 166.83 ± 1.36 | 164.93 ± 4.92 |
| 3 w post-op | 155.63 ± 1.88 # , T , * | 166.26 ± 1.84 | 165.46 ± 3.78 | 166.26 ± 4.27 |
| 5 w post-op | 152.26 ± 1.69 | 157.60 ± 6.55 # | 158.30 ± 2.33 # | 158.06 ± 4.08 # |
| 9 w post-op | 163.66 ± 1.75 # | 162.20 ± 2.65 | 162.26 ± 0.75 | 161.66 ± 2.36 |
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