Abstract
To examine the hypothesis that conservative treatment is applicable to younger patients with bilateral mandibular condylar fractures, we studied the effect of ageing on the healing of bilateral mandibular condylar fractures in a rat model. Male Sprague–Dawley rats aged 3, 6, and 36 weeks ( n = 25/cohort, total n = 75) were divided into a fracture group ( n = 12) and a sham control group ( n = 12); one rat from each cohort was used as a normal unoperated control. Cell proliferation was evaluated using the bromodeoxyuridine (BrdU) labelling index (LI). Osteochondrogenesis was assessed by the expression of Indian hedgehog (Ihh), type X collagen, and osteocalcin in the condylar head. Condylar fracture healing was found to be delayed by ageing. BrdU LI values in the fracture groups were higher in younger rats than in older rats at 8 weeks after fracture. The number of Ihh-positive cells in the fracture groups increased significantly up to 2 weeks after fracture, and then gradually decreased until 8 weeks after fracture. The findings of this study support the clinical concept of conservative treatment of bilateral condylar fractures in younger patients, but functional issues regarding ramus height and its consequences on occlusion have not been tested in this study.
The mandibular condyle is one of the most frequent sites of maxillofacial fracture. In the treatment of condylar as well as other maxillofacial fractures, treatment goals include the restoration of occlusion, facial symmetry, and jaw function. The most important aspect in managing bilateral condylar fractures with no contact between the segments is to restore mandibular ramus height by an open reduction. Chen et al. reported that open reduction and rigid fixation provided satisfactory functional outcomes. Newman reported better clinical outcomes over the long term following open surgery for bilateral condylar fractures. However, open reduction has always been challenging because of the complexities involved in achieving stable osteosynthesis and safely approaching the temporomandibular joint (TMJ). Controversy therefore remains about the type of treatment to use. In children in particular, conservative treatment is usually advocated because surgical treatment of condylar fractures tends to cause growth disturbances rather than eliminate them. Conservative treatment may also be indicated in elderly patients with severe systemic disease, since surgical intervention poses a greater risk in this patient population. Indeed, in a study of patients older than 60 years suffering from craniomaxillofacial trauma, it was reported that 50% did not receive any surgical correction.
In a previous study in rats, we demonstrated the effect of ageing on the recovery of cellular proliferative ability in unilateral mandibular condylar fracture using bromodeoxyuridine (BrdU) immunohistochemistry. The findings suggested that conservative treatment may be applicable in younger patients. On the other hand, clinically, children with bilateral condylar fractures can show functional adaptability and osseous remodelling of the condyles. The results of a study involving computed tomography imaging before and after non-surgical treatment, suggested that the reestablishment of function did not occur as a result of anatomic restoration of condylar height. However, in a study of closed reduction of bilateral condylar fractures, age was determined to be an important factor in assessing the potential for mandibular functional impairment.
Healing and remodelling of condylar fractures following conservative treatment has been demonstrated in radiographic studies, and there have been a number of histomorphological studies on the healing of experimental fractures. However, there have been no detailed experimental studies on the healing of bilateral condylar fractures. According to Fuentes et al. , a lateral functional shift in the mandible induces increases in mandibular condylar cartilage thickness and proliferative activity in the protruded side of the condyle.
Indian hedgehog (Ihh), which is produced by pre-hypertrophic chondrocytes in the growth plate, regulates chondrocyte proliferation and the rate of chondrocyte maturation, together with parathyroid hormone-related protein (PTHrP). Osteocalcin is a vitamin K-dependent non-collagenous extracellular matrix protein that is limited to only a few specialized cell types, including osteoblasts, chondrocytes, and odontoblasts, which have calcium-binding properties and participate in osteogenesis through mineralization and bone remodeling. Expression of osteocalcin by chondrocytes correlates with advanced maturity. In an in vitro study, osteocalcin was expressed in chondrocytes at the late stage of differentiation and throughout endochondral ossification, which suggests that cartilage cells develop phenotypic markers associated with the mineralizing matrix.
In an effort to explore the feasibility of conservative treatment of dislocated bilateral condylar fractures in a younger clinical population, this study aimed to elucidate the effect of ageing on condylar remodelling after experimental bilateral condylar fractures in rats. We evaluated the expression of BrdU, Ihh, type X collagen, and osteocalcin to reveal the status of cell proliferation and osteochondrogenesis.
Methods
The animal protocol was approved by the local animal ethics committee in 2008. Male Sprague–Dawley rats aged 3, 6, and 36 weeks ( n = 25/cohort, total n = 75) were divided into a fracture group ( n = 12) and a sham control group ( n = 12), with one rat from each cohorts used as a normal unoperated control.
Surgical procedures
Surgical procedures and tissue preparation were performed following our previously described method, with some modifications. In the fracture group, a 1-cm incision was made over the mandibular angle parallel to the inferior border of the mandible on each side under general anaesthesia (intraperitoneal injection of 50 mg/kg pentobarbital sodium). The masseter and periosteum were elevated, and the condylar neck was exposed. A horizontal osteotomy of the condylar neck was performed at the lowest level of the sigmoid notch using a No. 700 fissure burr, with saline irrigation ( Fig. 1 ). The periosteum, muscle sling, and skin were closed with a 4-0 absorbable polyglycolic acid suture. In the sham control group, the condylar neck was exposed in the same manner and the wound closed.
No mechanical splinting or drug therapy was used. The rats were fed freely on a standard rat diet and water. Body weight was measured every day postoperatively.
Tissue preparation
The control rats were killed immediately, while the fracture and sham control groups were prepared for microscopic evaluation at 1, 2, 4, and 8 weeks ( n = 3/week) after fracture. All rats were injected intraperitoneally with a dose of 40 mg/kg BrdU (Sigma Chemical Co., St Louis, MO, USA), diluted in 10 mg/ml of phosphate-buffered saline (PBS). BrdU was administered to each animal at noon each day to eliminate diurnal variation in the labelling index (LI). One hour later, immediately after injection of an intraperitoneal overdose of pentobarbital sodium, the thoracic cavity was exposed and pericardiac perfusion with 0.5% heparinized saline (500 ml) was performed. This was followed by perfusion of 10% neutral phosphate-buffered formalin (pH 7.4; 1 l).
After fixation, a decalcifying fluid (10% ethylenediaminetetraacetic acid 2-Na solution in 0.1 mol/l Tris buffer, pH 7.3) was perfused (26.4 ml/min) for 4–7 days through a microtube pump (EYELA MP-2, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). Approximately 3 mm-thick sections were cut in the frontal plane from the nasal apex to the TMJ region with a microtome blade (Feather Co., Gifu, Japan).
All sections were dehydrated in a graded series of ethanol and processed for routine paraffin embedding. Four micron-thick, coronal, midcondylar sections were then cut, mounted on slides, and placed on a hot plate at 37 °C overnight to promote adhesion. Routine haematoxylin–eosin (HE) staining was done on the first section, and the second section was stained with Azan.
Immunohistochemistry
BrdU immunohistochemical staining was done following the procedure in our previous study. After deparaffinization with xylene, the slides were rehydrated through a graded series of ethanol, incubated for 30 min in 0.3% hydrogen peroxide in methanol to quench peroxidase activity, and then treated with 0.1% trypsin (Sigma Chemical Co.) in 0.05 mol/l Tris buffer solution (pH 7.6) at 4 °C for 10 min. For Ihh staining, sections were baked overnight at 55 °C. For type X collagen staining, sections were digested with testicular hyaluronidase (25 mg/ml; Sigma Chemical Co.) in PBS for 1 h at 37 °C. For osteocalcin staining, sections were digested with proteinase K (0.4 mg/ml; Takara Bio Inc., Shiga, Japan) for 5 min at room temperature.
Sections for BrdU immunohistochemical staining were further immersed in 4 N HCl for 30 min and in 0.1 M boric acid buffer for 10 min. All sections for immunohistochemistry were sequentially incubated with diluted 1.5% horse blocking serum to block non-specific reactions, then treated with primary antibodies: mouse monoclonal BrdU antibody (diluted 1:1000, for 30 min at room temperature; Becton Dickinson, Franklin Lakes, NJ, USA), goat polyclonal Ihh antibody (diluted 1:500, for 1 h at 37 °C; Santa Cruz, CA, USA), rabbit polyclonal type X collagen antibody (diluted 1:500, for 30 min at room temperature; LSL Co., Tokyo, Japan), and mouse monoclonal osteocalcin antibody (diluted 1:200, for 1 h at room temperature; Takara Bio Inc.). Subsequent immunohistochemical procedures were performed by applying avidin–biotin complex reagent to sections, using the VECTASTAIN Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) for BrdU staining and the VECTASTAIN Universal Quick Kit (Vector Laboratories) for other staining. Finally, the sections were incubated in substrate solution consisting of 0.05% diaminobenzidine tetrahydrochloride and 0.02% hydrogen peroxide in 0.05 mol/l Tris buffer solution (pH 7.6). Counterstaining was done with Mayer’s haematoxylin for 30 s.
Negative controls for immunohistochemistry were incubated with PBS instead of the primary antibodies. None of the negative controls showed a positive reaction.
Quantitative analysis
BrdU-labelled and non-labelled cells in the intermediate cell layers of both condyles in the fracture and sham control groups were counted with the aid of a squared eyepiece graticule (Nikon, Tokyo, Japan) at a magnification of 400×. For each animal, four coronal mid-condylar sections were prepared and at least 1000 cells per section were counted to obtain the average LI (labelled cells/total cells counted × 100%).
The number of Ihh-positive cells was also counted in a zone of flattened chondrocytes or a zone of hypertrophic chondrocytes by the same method as for the BrdU-labelled cells.
Statistical analysis
The Mann–Whitney U -test was used to analyse differences in body weight, LI values, and number of Ihh-positive cells between the fracture and sham control groups. All statistical analyses were performed using Statview version 5.0 for Macintosh (SAS Institute Inc., Cary, NC, USA) and a P -value of <0.05 was considered statistically significant.
Results
Gross findings
Throughout the postoperative course, neither specific malocclusion nor mandibular deformity was clinically detected in any of the animals. Mean body weight continued to increase postoperatively in both the fracture and sham groups of the 3- and 6-week-old rats ( Fig. 2 ). Body weight decreased in both the fracture and sham groups of 36-week-old rats from immediately postoperative to week 1, and then gradually increased in both groups. For rats of all ages, the fracture group weighed less than the sham control group, although only 3-week-old rats showed a significant difference between the fracture and sham control groups, at postoperative week 8.
Histological healing of the fracture
The healing process of the mandibular condyle fractures was almost the same in the right and left condyles in each fracture group.
In 3-week-old rats, at postoperative week 1, the condylar fragment was deviated and hypertrophy of the condylar cartilage was clearly apparent. Inflammatory fibrous connective tissues containing a variety of cells appeared around the diastasis of the fractured edge ( Fig. 3 A, a and f). At postoperative week 2, proliferation of cartilaginous and osseous tissue was observed at the fractured edge of each bone fragment. Each fragment was bridged by configured callus formation and the condylar fragment had returned to its normal position in the TMJ ( Fig. 3 A, b and g). At postoperative week 4, the segmental gap was bridged by bone trabeculae. The same trend was seen at postoperative week 8. On comparing the fracture group ( Fig. 3 A, c and h, d and i) to the sham control group ( Fig. 3 A, e and j), the condylar characteristics were normal and the position was centralized in the temporal fossa.
In 6-week-old rats, at postoperative week 1, the fracture fragments and condylar cartilage changes were the same as those observed in the 3-week-old rats ( Fig. 3 B, a and f). At postoperative week 2, osseous proliferation was observed at the fractured edge of each bone fragment, and callus formation was clearly evident at the condylar fragments. However, the fragments were still deviated and a bony gap was observed ( Fig. 3 B, b and g). At postoperative week 4, union by immature bone trabeculae was observed and the condylar process was centralized in the temporal fossa ( Fig. 3 B, c and h). Finally, at postoperative week 8, the normal characteristics of the condyle and condylar process were well preserved ( Fig. 3 B, d and i, compared to sham control e and j).
In 36-week-old rats, at postoperative week 1, the condylar fragment was deviated, and a variety of cells with inflammatory fibrous connective tissues had appeared, consistent with the findings in the 3- and 6-week-old rats. However, there was no significant hypertrophy of the condylar cartilage compared to the 3- and 6-week-old rats ( Fig. 3 C, a and f). At postoperative week 2, as with the 6-week-old rats, displacement of the condylar fragment, proliferation of osseous tissue, and diastasis of the bone edge were observed. However, part of the condylar fragment was absorbed ( Fig. 3 C, b) and deformity of the condylar cartilage, with cartilaginous proliferation, had occurred ( Fig. 3 C, b and g). At postoperative week 4, the condylar fragment was still deviated and a fracture gap was still clearly observed, without bony union. However, the condylar process had been formed in the direction of the median plane of the temporal fossa. Proliferation of osseous and cartilagenous tissue was observed around the diastasis of the fracture edge, but there was no callus formation ( Fig. 3 C, c and h). Finally, at postoperative week 8, repositioning of the condylar process was evident in the temporal fossa, although the subcondylar region showed discrete thickening compared with the sham control group ( Fig. 3 C, d and i, compared to sham control e and j).
BrdU- and Ihh-positive cells in the mandibular condyle
In rats of all ages, BrdU-positive cells were identified in the intermediate cell layer of the condylar cartilage by immunohistochemical staining (indicated in brown) ( Fig. 4 A). Ihh-positive cells were found in the zone of flattened chondrocytes and the zone of hypertrophic chondrocytes of the condylar cartilage ( Fig. 4 B).
With regard to changes in these positive cells, in all age groups, BrdU LI values were increased in the fracture groups during the period of hypertrophy and deformity of the condylar cartilage, before decreasing with fracture healing such that the BrdU LI values were lower in the fracture groups than in the sham control groups at postoperative week 8 ( Fig. 5 ). At postoperative week 8, recovery of the BrdU LI value (LI of fracture group/LI of sham control group) was evaluated because the fracture had already healed; it was found that LI values differed significantly between the fracture and sham control groups for all age cohorts. In the fracture groups, LI values had recovered by 45.8% in 3-week-old rats, 36.0% in 6-week-old rats, and 17.9% in 36-week-old rats ( Table 1 ).
| Rat age (weeks) | |||
|---|---|---|---|
| 3 | 6 | 36 | |
| LI of fracture group, mean ± SD, % | 1.84 ± 0.16 | 1.02 ± 0.16 | 0.15 ± 0.02 |
| LI of sham control group, mean ± SD, % | 4.02 ± 0.28 | 2.83 ± 0.18 | 0.84 ± 0.03 |
| Percentage of recovery of LI | 45.8% | 36.0% | 17.9% |
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