Abstract
64 male Wistar rats were used: 24 for the removal of stem cells, 4 as a control group, and 36 for the experiment, in which either stem cells or bone graft was used. The rats were divided into groups according to the type of procedure and time span (15, 30 or 60 days). The joints were submitted to histological study in order to score the ankylosis. The mean differences between initial and final maximal mouth opening (MMO) were gradually increased from 15 to 60 days, for all times of evaluation for both groups, being statistically significant at 15 days ( p = 0.045) in the bone-graft group. When both groups were compared, the mean differences between initial and final MMO were statistically significant at 15 days ( p = 0.018) and 30 days ( p = 0.029). In relation to the histological scores, in the bone-graft group almost all animals had intra-articular fibrosis at all times of evaluation ( n = 17). In the stem-cell group, there was new bone at 15 days ( n = 4), 30 days ( n = 3) and 60 days ( n = 4). The study model permitted the development of fibrous ankylosis in the majority of animals for both groups and no bony bridge was observed.
Ankylosis of the temporomandibular joint (TMJ) is an intracapsular bonding of the disc–condyle complex to the temporal articular surface, including the fibrous adhesions or bony fusion between condyle, disc, glenoid fossa and eminence, which restricts mandibular movements . It is a serious and disabling condition that may cause problems with mastication, digestion, speech, appearance and hygiene. It can also cause disturbances of facial and mandibular growth, as well as acute compromise of the airway, invariably resulting in physical and psychological disability .
The treatment of TMJ ankylosis poses a significant challenge because of technical difficulties and a high incidence of recurrence. A variety of treatments have been described in the literature but no single method has produced uniformly successful results . More studies are needed to devise innovative treatments to reduce the recurrence rate.
Despite some studies in sheep and a few in rats , the development of an ankylosis model in rats would certainly be an advance in terms of cost and ease of conducting studies. Rats are small animals that are less expensive to maintain, so are convenient for experimental studies. In the literature reviewed, only one study model for the occurrence of fibrous ankylosis in rats was found, which used disc removal and articular damage . The purpose of the present study was to evaluate a more precise study model for inducing TMJ osseous ankylosis in rats.
Materials and methods
The trial protocol was approved by the University’s Ethics Committee (009686/2007-09). 64 male Wistar rats were used as an independent sample. 24 were used for the removal of stem cells and 4 as a control group. In the other 36 animals articular damage was induced and disc removal associated with placement of either stem cells or bone graft in the right joint to induce ankylosis. It was decided to remove the disc and induce articular damage either with stem cells, because they are able to generate differentiated cells possibly resulting in intra-articular bone, or with bone graft because this may offer osteogenic properties also promoting intra-articular bone.
The rats were divided into groups according to the type of procedure and time of death (15, 30 and 60 days) ( Fig. 1 ). They were killed with an intracardiac injection of 1 ml of potassium chloride (Baxter) following general anaesthesia.
The sample size was estimated using a computer program (PC-SIZE 1.1) based on similar variables of this study , such as the difference in the pre- and postoperative maximal mouth opening (MMO) in rats submitted to a process of ankylosis within 60 days of postoperative evaluation . Six animals per group were obtained with a correction factor of 20% and a significance rate of 5%.
The variables MMO, weight and mandibular deviation were recorded and evaluated at the time of surgery and death. Weight was measured using a digital balance (Kern & Sohn GmbH, Balingen-Frommern, Germany). Mandibular deviation was observed in MMO using dental floss in the middle line for reference after the general anaesthesia. The time of surgery was also recorded. MMO was measured from each incisal papilla because the teeth of this animal continue to grow throughout its lifetime.
36 animals underwent surgery under general anaesthesia, which was induced by muscular injection of ketamine (Francotar ® Virbac) and xylaxine (Virbaxyl ® Virbac) diluted 1:1 in a dose of 0.1 ml per 100 g of weight. For local anaesthesia 0.2 ml of lidocaine (1:200,000) (AstraZeneca) was infiltrated. A preauricular incision of approximately 10 mm was made on the right-hand side above the zygomatic arch. Blunt dissection was performed through the masseter muscle, just below the zygomatic arch, and the condylar process was exposed. Joint damage was induced with a small file and disc removal performed. In 18 animals, this procedure was followed by the placement of stem cells. The other 18 received a bone graft which was inserted into the right joint to induce ankylosis. Sutures were placed in layers, using 5-0 nylon.
Stem cell culture
The progenitor cells were collected from the medulla of the hind limbs (femur and tibia) of 24 rats, they were separated, both inside a laminar flow. The cells were cultured in a high glucose concentration solution (DMEM, Invitrogen) inside a hotbed at 37 °C and 5% carbon dioxide for 9 days, with changes of the solution every 3 days, which was the time necessary for a 70–80% confluence of cells in the culture vial. On the tenth day a solution with ascorbic acid (Invitrogen), β-glycerophosphate (Invitrogen) and dexametasone (Invitrogen) was added to the culture cells to transform them into osteoblast phenotypes . On the thirteenth day tripsine (Invitrogen) was added to the cells, to detach them from the vial and make it possible to use them. The number of cells was estimated using the Newbauer chamber. Approximately 1 million cells were placed in the TMJ of the experimental group.
Bone graft removal
The bone graft used was autogenous. In the same rat submitted to the TMJ procedure a sagittal incision of approximately 10 mm was made from the fronto-nasal region to the occipital protuberance. Blunt dissection was performed and the calvarium exposed. A defect of 5 mm on the right side of the median suture was created using a trephine, taking care to avoid injuries to the dura mater . The procedure was concluded with sutures in layers using nylon.
Histological study
After death, the joints underwent histological study to classify the ankylosis using scores ( Table 1 ) based on P orto et al. and in part on M atsuura et al. The area considered for this classification was the overall area in the middle of the joint. The purpose was to detect the presence or absence of the tissues ( Table 1 ) rather than quantify them.
| 0 | No evidence of fibrosis or other calcified tissues. |
| 1 | Presence of fibrosis. |
| 2 | Presence of dystrofic calcification or cartilage in formation. |
| 3 | Presence of bony tissue. |
The specimens were fixed in formaldehyde for 48 h and were decalcified in 5% nitric acid for approximately 10 days. Semi-serial sections 5 μm thick were cut in the sagittal plane and stained with haematoxylin and eosin.
Results
During the experimental period, the animals lost weight in the first 7 days, after which they started to grow and gain weight until the end of the 60 days for both groups. Mean values of body weight of the animals are shown in Table 2 . The mean differences between initial and final MMO gradually increased from 15 to 60 days, for all times of evaluation in both groups, being statistically significant at 15 days ( p = 0.045) in the bone-graft group. When the two groups were compared, the mean differences between initial and final MMO were statistically significant at 15 days ( p = 0.018) and 30 days ( p = 0.029). No mandibular deviation was observed at any of the times of evaluation for either group ( Table 3 ). The mean time duration of surgery for the bone-graft group was 20.22 ± 5.06 min and for the stem-cell group 7.31 ± 3.85 min.
| Time of evaluation | Mean weight (g) | |
|---|---|---|
| Stem cells | Bone graft | |
| 0 day | 369.94 | 426.5 |
| 7 days | 367.05 | 393.61 |
| 15 days | 384.83 | 405.28 |
| 30 days | 405.42 | 432.5 |
| 60 days | 432.17 | 476.66 |
| Time of evaluation | MMO | Groups | p value | |
|---|---|---|---|---|
| Bone graft | Stem cells | |||
| Mean ± SD | Mean ± SD | |||
| 15 days | Initial | 24.83 ± 2.56 | 24.50 ± 0.84 (A) | p (1) = 0.768 |
| Final | 21.67 ± 1.37 (A) | 24.83 ± 1.60 (a) | p (1) = 0.004 * | |
| Difference | −3.17 ± 2.93 | 0.33 ± 0.82 | p (1) = 0.018 * | |
| p value | p (3) = 0.045 * | p (3) = 0.363 | ||
| 30 days | Initial | 23.67 ± 1.37 | 25.50 ± 1.87 (AB) | p (1) = 0.081 |
| Final | 22.50 ± 1.22 (A) | 27.33 ± 0.82 (b) | p (1) < 0.001 * | |
| Difference | −1.17 ± 2.14 | 1.83 ± 1.94 | p (1) = 0.029 * | |
| p value | p (3) = 0.239 | p (3) = 0.069 | ||
| 60 days | Initial | 23.83 ± 2.04 | 23.17 ± 0.41 (B) | p (2) = 0.466 |
| Final | 28.33 ± 0.52 (B) | 26.50 ± 1.05 (ab) | p (1) = 0.003 * | |
| Difference | 4.50 ± 1.87 | 3.33 ± 1.03 | p (1) = 0.211 | |
| p value | p (3) = 0.002 * | p (3) = 0.001 * | ||
| p 1 value (4) | p (4) = 0.578 | p I (4) = 0.015 * | ||
| p 2 value (4) | p II (4) < 0.001 * | p II (4) = 0.008 * | ||
In relation to the histological scores, in the bone-graft group almost all animals had intra-articular fibrosis at all times of evaluation ( n = 17) ( Fig. 2 ). In the stem-cell group, there was new bone formation at 15 days ( n = 4) ( Fig. 3 A and B ) and 30 days ( n = 3) ( Fig. 2 B–D). At 60 days in the same group, there was new bone formation ( n = 4) ( Fig. 4 A and B ) and cartilage ( n = 2) ( Fig. 4 C and D) ( Table 4 ). A joint that is histologically normal is shown in Fig. 5 . Table 5 shows the means and medians of the histopathological scores according to group.
| Time of evaluation | Scores | Group | Total group | |||
|---|---|---|---|---|---|---|
| Bone graft | Stem cells | N | ||||
| n | % | n | % | |||
| 15 days | 1 | 5 | 2 | 7 | ||
| 3 | 1 | 4 | 5 | |||
| Total | 6 | 6 | 12 | |||
| 30 days | 1 | 6 | 3 | 9 | ||
| 3 | – | 3 | 3 | |||
| Total | 6 | 6 | 12 | |||
| 60 days | 1 | 6 | 1 | 7 | ||
| 2 | – | 2 | 2 | |||
| 3 | – | 3 | 3 | |||
| Total | 6 | 6 | 12 | |||
| Total group | 0 | 0 | – | 0 | – | |
| 1 | 17 | 94.44 | 6 | 33.33 | ||
| 2 | 0 | – | 2 | 11.11 | ||
| 3 | 1 | 5.55 | 10 | 55.55 | ||
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