Effect of asymmetric force on the condylar cartilage, subchondral bone and collagens in the temporomandibular joints

archives of oral biology 60 (2015) 650–663

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Review

Effect of asymmetric force on the condylar cartilage, subchondral bone and collagens in the temporomandibular joints Caixia Zhang a, Yue Xu b, Yangxi Cheng c, Tuojiang Wu d,*, Huang Li a,* a

Institute and Hospital of Stomatology, Medical School of Nanjing University, Nanjing 210008, China Department of Orthodontics, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, China c Department of Orthodontics, West China College of Stomatology, Sichuan University, Chengdu, Sichuan, China d Department of Orthodontics, The Affiliated Hospital of Medical School, Nanjing Medical University, Nanjing, Jiangsu, China b

article info

abstract

Article history:

This study aimed to define the effects of asymmetric force on rat temporomandibular joints

Accepted 19 January 2015

(TMJs). A total of 232 10-week-old rats were used in the experiment. Their left TMJs were kept

Keywords:

the expression of type I, II and III collagens were observed. Our results showed that the curve

Asymmetric force

of the cartilage thickness changes in the anterior part of the treated side in the heavy force

Temporomandibular joints

group (HS) decreased first and increased later during the strength and the recovery periods,

forward and upward with 40 g or 120 g. The histological and osteogenic changes, as well as

Condylar cartilage

while the reverse changes were shown in the middle and posterior parts. The cartilage

Subchondral bone

thickness change on the other side in the heavy force group (HO) was the opposite.

Collagen

Additionally, the cartilage thickness change on the treated side and the other side of the light force group (LS and LO) were similar to but not as significantly changed as HS and HO. There were significant differences among the experimental groups. The subchondral bone trabecula also decreased after the pressure loading and removing, then recovered, without significant differences among these groups. Furthermore, more pathological changes such as fractures, bone cysts, the degradation of type II collagen and the increased expression of type III collagen were observed on the treated sides following the application of heavy force. In contrast, more osteogenesis and more active changes were found in the light force group. In conclusion, our study demonstrated that asymmetric force exerted different effects on the cartilage, subchondral bone and collagens of TMJs. Greater changes occurred in the heavy force group, and light force provided more benefits for TMJs remodelling. # 2015 Elsevier Ltd. All rights reserved.

* Corresponding authors. Tel.: +86 025 83620173. E-mail addresses: [email protected] (T. Wu), [email protected] (H. Li). http://dx.doi.org/10.1016/j.archoralbio.2015.01.008 0003–9969/# 2015 Elsevier Ltd. All rights reserved.

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archives of oral biology 60 (2015) 650–663

Contents 1. 2.

3.

4. 5.

1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Operative procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Sample preparation and histological measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Fluorescence vital staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Masson’s staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Sirius red F 3B(SR) in saturated carbazotic acid staining . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Clinical outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The effect of abnormal asymmetric force on the thickness of the condylar cartilage. . . . . 3.3. The effect of abnormal asymmetric force on the subchondral bone trabecula . . . . . . . . . . 3.4. The effect of abnormal asymmetric force on the expression of type I, II and III collagens. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction

The temporomandibular joint (TMJ) is a unique joint because it is the only linkage joint among all human joints that maintains the ability of lifelong reconstruction.1,2 Mechanical stresses on the TMJs, originating from daily functional activity or other sources, are thought to produce many different physical, electrical, and biochemical phenomena, which have significant impacts on the remodelling of the condyle.3–5 However, if the mechanical stress on the TMJs is asymmetric, the unbalanced force received by the TMJs can cause more complex remodelling changes. The effects of symmetric mechanical force on TMJs have been examined in many studies using different animal models. Chen et al.6 found that altered functional TMJ loading in 21-day-old mice led to obvious degradation of the condylar cartilage, a loss in the density of the mandibular subchondral bone, and a significant reduction in the expression of type II collagen. Ikeda7 found that mechanical loading from a jawopening device led to OA-like changes, including reduced thickness of the cartilage, irregularities in the chondrocytic layer, and decreased bone volume. Furthermore, Zhang,8 using in vitro organ culture, reported that the thickness of the proliferative layer in condylar cartilage significantly decreased under hydrostatic pressure of 100 kPa on the mandibular cartilage. We also confirmed that the thickness of the cartilage became thinner after 7 days of mechanical stress of 120 g in vivo.9,10 Different from the results mentioned above, Rabie11 noted that forward mandibular positioning by a bite-jumping device resulted in an anterior advancement of 4 mm, leading to condylar adaptation. However, there have been only a few studies on asymmetric mechanical force. Liu12 observed that reducing dietary loading decreased mouse TMJ degradation induced by a unilateral anterior crossbite prosthesis. Lu13 found that cartilage degradation and subchondral bone loss were caused by bonding a single metal tube or a pair of tubes to left incisor(s), but there were no differences

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in degrading changes between the left side and right side TMJs in experimental mice. Fuentes14 found that a lateral functional shift of the mandible resulted in increased proliferation of prechondroblastic cells on the protruding side, resulting in increased thickness of the condylar cartilage, while the thickness of the nonprotruded condyles remained the same. From the literature reviewed, we found that most animal models of asymmetric mechanical force on TMJs were created by unilateral occlusional changes and were only focused during the implementation phase. However, the different changes between the treated side and the other side, the different effects between heavy force and light force, the effect of different time courses, and the recovery phase changes were not clear until now. Here, to reveal the effects of abnormal asymmetric mechanical stress on TMJs, we developed an animal experimental model in which the intermaxillary asymmetric traction force was exerted in adult rats by applying energy storage springs on one side. We focused on the remodelling of the condylar cartilage, subchondral bone and collagen in the TMJs from heavy force and light force loading during a pressure period and a recovery period.

2.

Materials and methods

2.1.

Animals

A total of 232 male Sprague–Dawley (SD) rats (provided by Huaxi Medical Laboratory Animal Center of Sichuan University) aged 10 weeks (weight 300  25 g) were used in this study. All of the rats were acclimatized to the laboratory conditions with food and water available ad libitum 1 week before the experiment. The rats were randomly divided into three groups: a light force group (40 g, 0.39 N), a heavy force group (120 g, 1.18 N) and a control group (Table 1). Furthermore, in each group, rats were randomly selected for different experiments to exclude

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Table 1 – Schematic representation of the study protocol of 232 rats. Pressure period Days after receiving stress Light force group Heavy force group Control group

3 13 13 3

7 13 13 3

14 13 13 3

selection bias. The animals were housed in a light- and temperature-controlled room and given unrestricted access to food and water during the experimental period. For the experimental group, there was a pressure period of 28 days and a recovery period of 28 days (from 29 days to 56 days for this experiment). The use and care of the animals in this study were undertaken in compliance with the guidelines of and under the permission of the Animal Care Committee of Sichuan University.

2.2.

Recovering period

Operative procedure

The animals were anaesthetized intraperitoneally with 10% chloral hydrate monohydrate 3 ml kg 1, under sterile conditions. An incision parallel to the eyelid was made at the anterior inferior of the right orbit 4 mm in length. Another incision was made at the right mandibular angle, the subcutaneous tissues were dissected, and the periosteum was split to expose the zygomatic arch, maxillary zygomatic process and mandibular angle. Two 0.25 mm stainless steel wires were fixed after circling the zygomatic arch and mandibular angle separately with the desired strength (0.39 N and 1.18 N) of nickel–titanium (NiTi) alloy coil springs, and the left condyle was positioned anteriorly (protruded) relative to normal (Fig. 1a) For the control group, the same operation was performed, but force was not applied. The wound was closed in two layers with 4-0 Vicryl sutures. Cefazolin 10 mg kg 1 was administered intramuscularly to prevent infection. The implantation of the device was evaluated by X-ray (Fig. 1b). The midline of both the maxillary and mandibular teeth was deviated after the application of asymmetric traction (Fig. 1b). After 4 weeks, the force-applying

28 13 13 3

R7 13 13 3

R14 13 13 3

R28 13 13 3

coil springs were removed from the rats under anaesthetized conditions. The animals from all groups were killed at various postoperative stages as shown in Table 1: pressure period (the 3rd, 7th, 14th and 28th days) and recovery period (the R3rd, R7th, R14th and R28th day). The rats were killed using an excess of trichloroacetaldehyde monohydrate intraperitoneally, and the whole mandible and TMJs were resected en bloc with the surrounding soft tissues. The animals were exsanguinated to minimize bleeding into the wound tissue during the sacrifice. The harvested TMJs were evaluated using histological and immunohistochemical analysis.

2.3.

Sample preparation and histological measurements

The samples were perfused-fixed in 4% paraformaldehyde. Blocks containing the TMJ were fixed in 4% paraformaldehyde for 24 h. The specimens were decalcified in EDTA solution for 4 weeks and were embedded in paraffin after thorough rinsing. Blocks were cut into sagittal sections with a uniform thickness of 4 mm. The histological changes in the condylar cartilage were examined by H&E staining. The other specimens were stained using other methods listed as follows. For histological measurements, we drew a line from the section, starting from the attachment of the lateral pterygoid muscle to the attachment of bilaminar zone, and two other lines radially from the centre mark of the line to create three angles of 608 to divide the condyle into three parts: anterior, middle and posterior. The thicknesses of the condylar cartilage were measured along each of three axes; the measurement accuracy was 0.0001 mm, and the average of these values was recorded as the final depth of each layer on one section. The measurement of the square of the bone trabecula of the subchondral bone was the same as above: three different zones were taken from three parts, and the mean values were used for the final results.

2.4.

Fig. 1 – (a) Animal model: a nickel–titanium (Ni–Ti) coil spring was placed between the left mandibular angle and anterior left zygomatic arch of rats (above). (b) X-ray of the animal model (below). (c) The midline of maxillary and mandibular teeth deviated after the application of asymmetric force.

R3 13 13 3

Fluorescence vital staining

The operative procedures used on the rats for fluorescence vital staining were the same as reported elsewhere; the difference was that the rats were injected with tetracycline, calcein and xylenol orange 1 day before the experiment, on the 27th day and on the R27th day, respectively, during the experiment. Based on the different colours of the different colourants under different wavelengths of light (tetracycline, absorption spectrum 390–425 nm, emission monochromator 525–560 nm, yellow green; calcein, 494 nm, 517 nm, green; and xylenol orange, 546 nm, 580 nm, orange) deposited in the subchondral bone, the ability of osteogenesis of the different groups could be defined.

archives of oral biology 60 (2015) 650–663

2.5.

Masson’s staining

The specimens used in this experiment were those from the R28th day. After deparaffinization and hydration, sections were stained with haematoxylin for 5–10 min, differentiated with hydrochloric acid and ethyl alcohol in running tap water for 5 min and then rinsed in distilled water. Next, the specimens were placed in ponceau–acid fuchsin (ponceau 0.7 g + acid fuchsin 0.3 g + distilled water 99 ml + glacial acetic acid l ml) for 5–8 min and then washed in running tap water. One percent molybdophosphoric acid was used for 1–3 min on the specimens. They were then treated with aniline blue dye solution (aniline blue 2 g + distilled water 98 ml + glacial acetic acid 2 ml) for 5 min and washed in distilled water. They were quickly dehydrated with 95% ethyl alcohol and absolute ethyl alcohol and cleared in xylene. Stained samples were embedded on microscopic slides with neutral resins.

2.6. Sirius red F 3B(SR) in saturated carbazotic acid staining After the removal of the paraffin and the rehydration of slides as described above, the slides were incubated in iron alumcelestine blue solution (celestine blue B 1.25 g and ferric alum 1.25 g dissolved into 250 ml of ultrapure water, cooled and filtered after boiling, followed by adding 30 ml of glycerin and 0.5 ml of concentrated sulfuric acid) for 5–10 min and then washed with tap water three times. Next, the slides were placed in sirius red F3B dissolved in 0.05% (w/v) saturated picric acid for 15–30 min at room temperature in the dark. The slides were dehydrated with concentrated sulfuric acid and then mounted in neutral resins and observed under polarized light microscopy.

2.7.

Immunohistochemistry

After deparaffinization and hydration, endogenous peroxidase was quenched with 3% hydrogen peroxide for 10 min. Non-specific protein binding was blocked by incubation with normal sheep serum (Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. For immunostaining, primary rabbit polyclonal antibodies against type II collagen (Boster, Wuhan, China) were used. Sections were incubated with the primary antibody in phosphatebuffered saline (PBS) at a 1:100 dilution in a humidified chamber at 4 8C overnight. After three washes with PBS, the tissue sections were incubated with biotinylated secondary antibodies (Vector Labs) for 30 min in a humidified chamber, followed by staining with SP Reagent (Boster, Wuhan, China). Finally, the sections were developed with 3, 3¢-diaminobenzidine (DAB substrate kit, Vector Labs), counterstained with haematoxylin and examined under a light microscope. The condylar cartilage was the area of interest. The positive signal intensity (integrated optical density, IOD) of the condylar cartilage of the condyle was used as the parameter for semi-quantitative determination and was measured with the assistance of a computer-based Image-Pro Plus program (Media Cybernetics, Bethesda, MD, USA).

2.8.

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Statistical analysis

The measurement procedures for H&E staining and immunohistochemistry were performed by two observers (WTJ and ZCX) using Image-Pro Plus software, version 6.0 (Media Cybernetics, USA). The inter-observer reliability analysis was performed by calculating the intraclass correlation coefficient (ICC) of the measurements.15 The different measurements from the same sample were highly consistent. The inter-observer reliability was analysed by a two-way random model based on ‘‘absolute agreement’’ (ICC (2, 1) or ICC (agreement)). There was a high level of agreement between the two observers (Table 5, ICC > 0.95). ‘‘n’’ indicates the number of independent observations from different rats per group. All of the measurements were repeated three times. The average value of the three measurements from the same sample was used for further statistical analysis. The images from macroscopic and immunohistochemical analyses were analysed and quantified by Image-plus software. The data are expressed as the mean  SD, and comparisons were performed using t-tests or analysis of variance, depending on whether the data were normally distributed. All of the statistical analyses were performed using SPSS software, version 20.0 (Chicago, IL, USA), and p < 0.05 was considered to be statistically significant.

3.

Results

3.1.

Clinical outcomes

Most of the animals (226) tolerated the surgery and stress application. There were no postoperative infections. All of the devices were found to be intact and in place at the time of sacrifice. There were six deaths: two in the light force group and four in the heavy force group because of intolerability in the change of occlusion. All of the dead animals were replaced immediately. The animals were fed chipped food after surgery.

3.2. The effect of abnormal asymmetric force on the thickness of the condylar cartilage The thickness of the condylar cartilage of the experimental groups changed after the asymmetrical traction. For the thickness of the anterior part of condylar cartilage on the treated side in the heavy force group (HS), it decreased first and increased later during the strength and the recovery periods, and the change curve of the thickness presented as a ‘‘W’’ shape (Fig. 2a and c). After 3 days of a pressure of 120 g, the thickness (122.57  5.52) ( p < 0.05) was significantly reduced compared to the control group (153.78  6.39) and continued decreasing until the 7th day (66.43  5.53) ( p < 0.01). Then, the thickness of the cartilage recovered to some extent, but on the 28th day, it was still thinner than in the control group ( p < 0.05). However, after removing the asymmetric traction, there was a second decrease during the first 3 days of the recovery period ( p < 0.01). Later, the thickness increased again until the R28th day, and at the end of the experiment, the thickness was almost the same as that on the 3rd day, whereas

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Fig. 2 – (a) and (b) The thickness change of the anterior, middle, and posterior parts of the condylar cartilage in HS and HO after asymmetric mechanical stimulus on the 3rd, 7th, 14th, 28th days and the R3rd, R7th, R14th and R28th days. (c) The quantitative changes in the condylar cartilage thickness of HS and HO. The thickness of the anterior part in the HS decreased first and increased later, which presented as a ‘‘W’’ change curve; while the middle and posterior parts of the condylar cartilage in the HS showed reverse tendencies, which presented as ‘‘M’’ and ‘‘N’’ shapes, respectively. The cartilage thickness change on the other side of the heavy force group (HO) was the opposite, presenting an ‘‘M’’ shape in the anterior part and a ‘‘W’’ shape in the middle and posterior parts. HS: the treated side of the heavy force group (120 g); HO: the other side of the heavy force group (120 g) (compared with the control group, * indicates p < 0.05, # indicates p < 0.01).

archives of oral biology 60 (2015) 650–663

reverse changes of the thickness of the middle and posterior parts of condylar cartilage were found in the HS which presented as ‘‘M’’ and ‘‘N’’ curve shapes, respectively (Fig. 2a and c). The thickness of the middle part reached a peak on the 7th day ( p < 0.05) and then decreased. After removing the asymmetric traction, the thickness of the middle part first increased until the R7th day ( p < 0.05) and later decreased ( p < 0.05) The thickness of the posterior part increased until the 28th day ( p < 0.01) during the pressure period, and then there was a short and rapid decrease until the R3rd day ( p < 0.01), after which it increased until the end of the experiment, with the maximal thickness occurring on the R28th day ( p < 0.01). In contrast with the findings in the HS, the changes of the cartilage thickness of the anterior part of the other side in the heavy force group (HO) were almost opposite which presented as slightly ‘‘M’’ curve shape (Fig. 2b and c). After a short decrease during the first 3 days ( p < 0.01) of the pressure period, the thickness of the anterior part increased until the 14th day ( p < 0.05) and then slightly decreased until the 28th day. After removing the pressure, the thickness increased again until the R14th day ( p < 0.05) and remained unchanged during the later period. However, the curve of the thickness of the middle and posterior parts presented as a moderate ‘‘W’’ shape, decreasing continuously until the 14th day ( p < 0.01) and 7th day ( p < 0.05), respectively, and then remaining the same. When the pressures were removed, the cartilage thickness increased during the first 3 days ( p < 0.01) and fluctuated rapidly until R7th day ( p < 0.01), after which it remained unchanged. Compared with the thickness in the HS, the change curves of cartilage thickness on the treated side and the other side in the light force group (LS and LO) were similar but not as significantly changed as in the HS and HO (Fig. 3a and c). For the anterior part in the LS, the cartilage thickness fluctuated: it decreased until the 7th day ( p < 0.01) of the pressure period, increased until the R14th day ( p < 0.05) and then decreased ( p < 0.05), after removing the pressure, it increased until the R14th day ( p < 0.05) and later decreased ( p < 0.05), which was different from the changes in the HS. As for the middle and posterior parts, the thickness increased first and decreased later during the pressure and recovery periods, reaching the peak on the 14th day (both) ( p < 0.05 and p < 0.01, respectively) and on R7th day (the middle part) ( p < 0.01) and R14th day (the posterior part) ( p < 0.01), respectively. The tendencies of the thicknesses of the three parts in the LS were similar to those in the HS, while the changes in the LS were less than those in the HS. The changes in the thickness of the condylar cartilage on the other side in the light force group (LO) were different from those in other groups, with no significant changes observed during the entire experiment (Fig. 3b and c). The multivariate analysis of the thickness of the condylar cartilage revealed that there were significant differences among the different groups (HS, HO, LS, LO groups), sections (anterior, middle and posterior parts), groups and periods, and groups and sections ( p < 0.01), but no significant differences were observed among different periods, periods and sections, groups and periods or sections ( p > 0.05). There were significant differences among the HS, HO, LS, and LO groups except for between the HO and LS (Table 2).

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3.3. The effect of abnormal asymmetric force on the subchondral bone trabecula Next, we examined whether abnormal asymmetric force caused changes in the architecture of the mandibular condylar subchondral bone. The results of H&E staining revealed that an abnormal asymmetric mechanical stimulus caused obvious degenerative changes, including irregular changes to the order and morphology of the bone trabecula and even pathologic changes, such as fractures and cysts in the bone trabecula, compared to the control group (Fig. 4a and b). The percentage of the area of the bone trabecula of the four groups decreased after the pressure loading and removing, and recovered later, which also presented a ‘‘W’’ curve shape, for example, in the HS, it decreased until the 14th day ( p < 0.05), then increased until the R3rd day ( p < 0.05), and there was a second decrease till the R14th day ( p < 0.05), then slightly recovered; there was a loss during the early pressure period that recovered after the removal of the stimulus, with no significant differences among the HS, HO, LS and LO groups (Table 3). In the HS, there was an increase in density, disorders and fractures of the bone trabecula during the first 3 days of the pressure period. There was a loss in density on the 14th day, and disorders and bone cysts were observed on the R3rd day. No obvious change occurred on the R7th day (data not shown). However, on the R28th day, the end of the experiment, the disorder of the surface bone trabecula abated. In the HO, there were also fractures and disorders near the bone marrow cavity during the pressure period. However, after removing the pressure, there was recovery, and it returned to almost normal on the R28th day. The degeneration changes in the LS and LO were similar to those in the HS and HO (Fig. 4a and b). Next, to evaluate the osteogenesis after the stimulus, Masson’s staining and fluorescence vital staining were used. The results of Masson’s staining on the R28th day showed that the collagen in the condylar cartilage appeared blue, while that in the bone and calcified cartilage appeared red, and the more calcific the cartilage was, the redder it appeared. In the control group, there was no red staining above the hypertrophic layer, while in the HS and LS, there was a large amount of red staining near the hypertrophic layer; in the HO and LO, there was a large amount of blue staining, which indicated that there was more osteogenesis on the treated sides. Furthermore, in the subchondral bone, there was more red stained area in the HS and LS than in the HO and LO. There was the least osteogenesis in the HO and the most osteogenesis in the LS (Fig. 5a). The results of fluorescence vital staining also revealed that there was no yellow or green staining in any of the groups, but there was more green and orange staining, indicating more osteogenic activity in the experimental groups than the control groups. Furthermore, the treated sides had more staining than the other sides, and the most extensive staining was in the LS, particularly in the area near the condylar cartilage, which was in agreement with the Masson’s staining results (Fig. 5b).

3.4. The effect of abnormal asymmetric force on the expression of type I, II and III collagens Under polarized light microscopy, different types of collagens displayed different colours. In the control group, type I

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Fig. 3 – (a) and (b) The thickness changes in the anterior, middle, posterior parts of the condylar cartilage in LS and LO after asymmetric mechanical stimulus on the 3rd, 7th, 14th, and 28th days and the R3rd, R7th, R14th and R28th days. (c) The quantitative changes in the condylar cartilage thickness in LS and LO. The change tendency in the LS group was similar to but not as significant as in HS. The anterior part of LS increased first and decreased later, which presented as a slightly ‘‘M’’-shaped curve, while there was a moderate ‘‘W’’ shape of the middle and posterior parts. No significant changes were observed in the LO group. LS: the same side of the light force group (40 g); LO: the other side of the light force group (40 g) (compared with the control group, * indicates p < 0.05, # indicates p < 0.01).

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Table 2 – The sum of deviation square of different variables of the thickness of condylar cartilage.

Groups Periods Sections Groups and periods Groups and sections Periods and sections Groups and sections and sections

The sum of deviation square

Mean square

F

p

40,136.390 2912.951 282,054.518 13,676.374 95,306.378 6689.242 8884.262

13,378.797 2912.951 141,027.259 4558.791 15,884.396 3344.621 1480.710

12.818 2.790 135.115 4.368 15.218 3.204 1.419

0.000 0.096 0.000 0.005 0.000 0.042 0.208

The comparison of the variable of the thickness of condylar cartilage of different groups HN HN HP LN LP **

HP

LN

LP

–

**

**

0.413

**

–

**

**

**

**

0.413

**

– *

* –

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curve of the heavy force group was very gentle and almost on the same level. Type II collagen expression in the HS was significantly decreased during the first 14 days ( p < 0.01) after pressure compared to the control group, and it recovered until the R3rd day ( p < 0.05); it then decreased again from the R3rd day to the R7th day ( p < 0.05) and recovered later. Compared with the HS, the change of the HO showed opposite tendencies. The expression maintained very low levels in the heavy force group. In contrast, the expression of type II collagen in the LS and LO fluctuated more and changed rapidly. In the LO, it first increased and reached the peak on the 14th day ( p < 0.01) and then decreased until the R7th day ( p < 0.01), increasing again later, and the reverse changes were observed in the LS. The expression of type II collagen in the light force group was higher compared with the heavy force group. There were significant differences between the heavy force group and the light force group (Table 4). There were also significant differences ( p < 0.01) among the groups except for between the HO and HS and between the LO and LS (Table 4).

P < 0.01.

4. collagen presented a yellow, orange or red colour, type III collagen appeared green, and type II collagen showed a variable colour. Type I collagen was mainly located at the disc, the subchondral bone and the surface of the condylar cartilage, and it was ordered in a line, while type III collagen and type II collagen were mainly located at the transitional and hypertrophic layers of the condylar cartilage, and type II collagen was ordered as a grid. After abnormal asymmetric mechanical stimulus, type III collagen formed. In the HS, there was a large amount of type III collagen on the 28th day, which lasted until the R28th day, and at the same time, type II collagen became irregular. The results in the LS were similar to those in the HS and more obvious. In the HO, type III collagen mainly appeared during the recovery period (Fig. 6a). The results of immunohistochemical staining showed that type II collagen was mainly expressed at the junction between the transitional and hypertrophic layers. In the control group, the positive staining, which indicated the presence of type II collagen, was primarily located in the cytoplasm (figure not shown). As shown in Fig. 6b and c, the changes in the expression of type II collagen in the heavy force groups were more extensive than in the light force groups. The degradation of type II collagen was found in the early stage and recovered later in the HS and LS, and the change curves were also similar to the ‘‘W’’ shape. The reverse changes of type II collagen were observed in the HO and LO, which showed the ‘‘M’’ shape. The

Table 3 – The Multivariate analysis of the percentage of the square of bone trabecula of the subchondral bone.

Groups Periods Groups and periods

The sum of deviation square

Mean square

F

p

0.097 0.000 0.068

0.032 0.000 0.023

2.246 0.008 1.574

0.088 0.929 0.201

Discussion

The TMJs enable large relative movements, like many other synovial joints, during talking, chewing, clenching, and grinding. These complicated movements result in static and dynamic loading, which includes compression, tension, and shear. Moderate loadings are beneficial for the TMJs,16 but excessive loadings are likely to influence the onset and progression of OA.17 Moreover, because the TMJ is a linkage joint, these loadings on the TMJs need to maintain their balance under natural conditions. With one side of the joint moving forward, TMJs are subjected to unbalanced loading and different pressures, which causes different remodelling effects on the treated side and the other side. To reveal the effects of asymmetric traction force on SD rats, in this study, we developed an animal model that applied an intermaxillary mechanical force (a light force of 40 g and a heavy force of 120 g) on one side of the TMJs. This animal model allowed for suitable and feasible sample sizes, allowing us to use different types of and magnitudes of strength, and it was relatively inexpensive, permitted experiments for a short time period, and caused unbalanced intermaxillary asymmetric traction force on the TMJs. The TMJs include the condylar cartilage, subchondral bone and extracellular matrix (ECM). The condylar cartilage is responsible for outside force reaction because of its frontal position. The subchondral bone supports the condylar cartilage, which makes up the majority of the TMJs. Furthermore, ECM is a complex network of biomolecules that support and surround the chondrocytes and regulate their cellular activities.18 In ECM, collagens play important roles as structural proteins.19 Previously, many studies have focused on the effects of symmetric force on the cartilage, subchondral bone and collagens of the TMJs,12,13,20,21 and many studies have focused on asymmetric force.22–25 Some results have shown degradation or reduced thickness of cartilage, loss of subchondral bone, and reduction in the expression of type II collagen6,7; other results have demonstrated remodelling

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Fig. 4 – (a) Abnormal asymmetric force caused degenerative changes, which included irregularities in the order and morphology of the bone trabecula, particularly on the treated side, and even pathological changes. The star arrow indicates fractures, and the pentagram indicates a cyst. (b) The quantitative changes in the percentage of the area of the bone trabecula of the HS, HO, LS, and LO groups presented a ‘‘W’’ shape, and there was a loss during the early pressure period that recovered after the removal of the stimulus (compared with the control group, * indicates p < 0.05, # indicates p < 0.01).

effects on the TMJs.11,25 However, sufficient evidence has been lacking for the different effects on the treated side and the other side by asymmetric force. Moreover, the different effects between heavy force and light force and different changes during the time course and recovery phase were not clear until now. In the present study, we focused on asymmetric force loading on TMJs and obtained very interesting findings. The histological changes that we observed in the cartilage revealed an obvious difference on the treated side compared to

the other side. On the treated side, when the condylar cartilage was moved upward and forward, the anterior part received compressive stress, which decreased the thickness; it increased later as the organism adapted, and this result was consistent with those of previous studies.10 In the middle and posterior parts, the pressure decreased and opposed the anterior part. The posterior part even received distraction or tension force, so the thickness increased first because of the elasticity of the articular cartilage; it later decreased or

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Fig. 5 – The osteogenesis of subchondral bone in the HS, HO, LS and LO groups after asymmetric mechanical stimulus. (a) Masson’s Staining: In the HS and LS, there was much more red near the hypertrophic layer, and in the HO and LO, there was much more blue, indicating that there was more osteogenesis on the second sides. (b) Fluorescence vital staining: There was more green and orange, indicating more osteogenic activity, in the experimental groups than in the control groups and the staining on the treated sides were more than that on the other sides. XO: xylenol orange; Cal: calcein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

recovered because of the adaptability of the organism. The middle part received less stress than the posterior part, so there was a buffer; the thickness changed less than in the posterior part. The removal of pressure also caused a second change in pressure, which consequently had another effect on

the thickness of the cartilage. These pressure alternations could simply explain the ‘‘W’’ shape of the change curve in the anterior part versus the ‘‘M’’ or ‘‘N’’ shape in the middle and posterior parts in the HS and LS groups. Furthermore, because of the different values of the force, the thickness changes in

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Fig. 6 – (a) The synthesis of different types of collagens in the condylar cartilage of the HS, HO, LS and LO groups. After abnormal asymmetric mechanical stimulus, type III collagen came into existence and the expression increased in the HS and LS. The arrow indicates type III collagen. (b) The expression of type II collagen in the condylar cartilage of HS, HO, LS and LO by immunohistochemical staining. The changes in the expression of type II collagen in the heavy force groups were very large and were maintained at very low levels, while the light force group was more activated. (c) The quantitative changes in positive signal intensity (integrated optical density, IOD) of type II collagen. The expression of type II collagens decreased first and recovered later in the HS and LS, and the change curve was similar to the ‘‘W’’; while the opposite changes were observed in the HO and LO presenting as the ‘‘M’’ shape. It fluctuated more in the light force group (compared with the control group, * indicates p < 0.05, # indicates p < 0.01).

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Table 4 – The sum of deviation square of different variables of the expression of type II collagen.

Groups Periods Groups and periods

The sum of deviation square

Mean square

F

p

8157.441 358.646 558.641

2719.147 358.646 186.214

27.450 3.621 1.880

0.000 0.060 0.139

The comparison of the variable of the expression of type II collagen of different groups

HN HP LN LP **

HN

HP

– 0.668

0.668 –

**

**

**

**

**

**

**

**

– 0.214

0.214 –

LN

LP

P < 0.01.

the cartilage in the HS were obviously greater than those in the LS. On the other side, because of the limitation of the mandibular fossa, there was rotation when receiving the asymmetric traction on the treated side.26 The pressure on the other side was opposed by the treated side, and the anterior part received tension force, while the middle and posterior parts received compression force. Therefore, the thickness of the cartilage in the HO and LO groups changed differently from that in the HS and LS groups, which presented ‘‘M’’-shaped change patterns in the anterior part and a ‘‘W’’ shape in the middle and posterior parts. When indirectly receiving pressure, the changes on the other side were apparently weaker than on the treated side, and no significant changes were observed in the LO group because there was the least pressure on that side. In the subchondral bone, the order, density, morphology and even pathology of the bone trabecula changed significantly. The percentage change in the square of the bone trabecula also presented a slight ‘‘W’’ shape during the loading time and the recovery time. Unlike the large differences we

Table 5 – The inner and inter-observer reliability for measurements from stained images. Measurements

Condylar cartilage anterior third thickness Condylar cartilage middle third thickness Condylar cartilage posterior third thickness The percentage of the square of bone trabecula Collagen II-positive areas

Inter-observer reliability (ICC): mean (95% CI)

Numbers of total independent observations (n  3)

0.966 (0.951, 0.973)

18

0.967 (0.988, 0.972)

18

0.983 (0.963, 0.957)

18

0.989 (0.969, 0.964)

18

0.968 (0.955, 0.974)

18

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found in the cartilage among the HS, HO, LS, and LO groups, the changes in the subchondral bone trabecula were almost similar on both sides, and the value of the applied force did not affect it much. As support for the cartilage, the subchondral bone has a solid construction, and it has no elasticity like the cartilage. Under asymmetric force loading, the subchondral bone on both joint sides only barely responded to indirect mechanical stimulation,27 but the effects of different distributions of the pressure were not obvious. Although we did not find significant differences in the subchondral bone trabecula between heavy force and light force, more pathological changes, such as fractures and bone cysts were found on the treated side, which indicated that the subchondral bone was more fragile and could break earlier than the cartilage under excess mechanical stress stimuli.28 In contrast, more osteogenesis were found on the treated side on the R28th day, particularly in the light force group, which indicated that direct loading and light force could promote osteogenic processes. In normal rats, different distribution of type I, II, and III collagens meant different functional conditions.29 For type II collagen, previous studies have demonstrated that excessive static pressure could promote collagen expression over a short time period, but it eventually suppressed collagen expression after a longer duration.30 Although the change curves in the HS, LS, HO, and LO groups were similar, the synthesis of type II collagen when applying the heavy force of pressure was decreased with no apparent recovery compared to previous findings,31 indicating the degradation of type II collagen. The ability of chondrocytes to synthesize collagen was restrained under mechanical stress: the heavier the pressure was, the more significant the effect. On the other hand, increased levels of type III collagen were observed in the HS and LS groups. In TMJs pathological conditions such as bone sclerosis and cartilage degeneration, type III collagen comes into existence and its expressions increase, which may complement the loss of type II collagen.32,33 The results indicated more pathological changes on the treated side following the application of heavy mechanical force. In the light force group, the expression changes in type II collagen were very active and rapid, presenting as sharp ‘‘W’’ shapes and ‘‘N’’ shapes in the LS and LO groups. These changes were in agreement with the changes in the cartilage. The results revealed that light force causes more remodelling processes in the TMJs.

5.

Conclusion

In summary, we developed an asymmetric traction rat model and found that the thickness of the condylar cartilage, the morphology of the subchondral bone, and the collagen changes in the ECM changed and presented different curves, such as ‘‘W’’, ‘‘M’’ and ‘‘N’’ shapes, over the loading time and recovery time. Our results demonstrated that asymmetric force on the TMJs exerted different effects on the condylar cartilage, subchondral bone and collagens, which were closely related to the value, the distribution of the pressure and the character of tissue. Greater changes occurred in the heavy force group, and light force provided more benefits for TMJ remodelling.

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Funding This work was supported by grants from the National Nature Science Foundation of China (Nos. 81470712, 81200764).

Competing interests

13.

14.

The authors declare that there are no conflicts of interest. 15.

Ethical approval

16.

Not required. 17.

references

1. Blackwood HJ. Growth of the mandibular condyle of the rat studied with tritiated thymidine. Arch Oral Biol 1966;11(5):493–500. 2. Ravosa MJ, Kunwar R, Stock SR, Stack MS. Pushing the limit: masticatory stress and adaptive plasticity in mammalian craniomandibular joints. J Exp Biol 2007;210(Pt 4):628–41. 3. Jung JK, Sohn WJ, Lee Y, Bae YC, Choi JK, Kim JY. Morphological and cellular examinations of experimentally induced malocclusion in mice mandibular condyle. Cell Tissue Res 2014;355(2):355–63. 4. Wangsrimongkol T, Manosudprasit M, Pisek P, Chowchuen P, Chantaramungkorn M. Temporomandibular joint growth adaptation and articular disc positional changes in functional orthopedic treatment: magnetic resonance imaging investigation. J Med Assoc Thai 2012;95(Suppl. 11):S106–15. 5. Ma X, Fang B, Dai Q, Xia Y, Mao L, Jiang L. Temporomandibular joint changes after activator appliance therapy: a prospective magnetic resonance imaging study. J Craniofac Surg 2013;24(4):1184–9. 6. Chen J, Sorensen KP, Gupta T, Kilts T, Young M, Wadhwa S. Altered functional loading causes differential effects in the subchondral bone and condylar cartilage in the temporomandibular joint from young mice. Osteoarthritis Cartilage 2009;17(3):354–61. 7. Ikeda Y, Yonemitsu I, Takei M, Shibata S, Ono T. Mechanical loading leads to osteoarthritis-like changes in the hypofunctional temporomandibular joint in rats. Arch Oral Biol 2014;59(12):1368–76. 8. Zhang M, Chen FM, Chen YJ, Wu S, Lv X, Zhao RN. Effect of mechanical pressure on the thickness and collagen synthesis of mandibular cartilage and the contributions of G proteins. Mol Cell Biomech 2011;8(1):43–60. 9. Li H, Yang HS, Wu TJ, Zhang XY, Jiang WH, Ma QL, et al. Proteomic analysis of early-response to mechanical stress in neonatal rat mandibular condylar chondrocytes. J Cell Physiol 2010;223(3):610–22. 10. Li H, Zhang XY, Wu TJ, Cheng W, Liu X, Jiang TT, et al. Endoplasmic reticulum stress regulates rat mandibular cartilage thinning under compressive mechanical stress. J Biol Chem 2013;288(25):18172–83. 11. Rabie AB, Xiong H, Hagg U. Forward mandibular positioning enhances condylar adaptation in adult rats. Eur J Orthod 2004;26(4):353–8. 12. Liu YD, Liao LF, Zhang HY, Lu L, Jiao K, Zhang M, et al. Reducing dietary loading decreases mouse

18.

19. 20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

temporomandibular joint degradation induced by anterior crossbite prosthesis. Osteoarthritis Cartilage 2014;22(2): 302–12. Lu L, Huang J, Zhang X, Zhang J, Zhang M, Jing L, et al. Changes of temporomandibular joint and semaphorin 4D/Plexin-B1 expression in a mouse model of incisor malocclusion. J Oral Facial Pain Headache 2014;28(1):68–79. Fuentes MA, Opperman LA, Buschang P, Bellinger LL, Carlson DS, Hinton RJ. Lateral functional shift of the mandible: Part I. Effects on condylar cartilage thickness and proliferation. Am J Orthod Dentofacial Orthop 2003;123(2):153–9. Shrout PE, Fleiss JL. Intraclass correlations: uses in assessing rater reliability. Psychol Bull 1979;86(2):420–8. Newton PM, Mow VC, Gardner TR, Buckwalter JA, Albright JP. Winner of the 1996 Cabaud Award. The effect of lifelong exercise on canine articular cartilage. Am J Sports Med 1997;25(3):282–7. Sun HB. Mechanical loading, cartilage degradation, and arthritis. Ann N Y Acad Sci 2010;1211:37–50. Mlynarik V, Trattnig S. Physicochemical properties of normal articular cartilage and its MR appearance. Invest Radiol 2000;35(10):589–94. Eyre D. Collagen of articular cartilage. Arthritis Res 2002;4(1):30–5. Wang XD, Kou XX, He DQ, Zeng MM, Meng Z, Bi RY, et al. Progression of cartilage degradation, bone resorption and pain in rat temporomandibular joint osteoarthritis induced by injection of iodoacetate. PLoS ONE 2012;7(9):e45036. Zhang J, Jiao K, Zhang M, Zhou T, Liu XD, Yu SB, et al. Occlusal effects on longitudinal bone alterations of the temporomandibular joint. J Dent Res 2013;92(3):253–9. Yang T, Zhang J, Cao Y, Zhang M, Jing L, Jiao K, et al. Decreased bone marrow stromal cells activity involves in unilateral anterior crossbite-induced early subchondral bone loss of temporomandibular joints. Arch Oral Biol 2014;59(9):962–9. Jiao K, Zhang J, Zhang M, Wei Y, Wu Y, Qiu ZY, et al. The identification of CD163 expressing phagocytic chondrocytes in joint cartilage and its novel scavenger role in cartilage degradation. PLOS ONE 2013;8(1):e53312. Jiao K, Niu LN, Wang MQ, Dai J, Yu SB, Liu XD, et al. Subchondral bone loss following orthodontically induced cartilage degradation in the mandibular condyles of rats. Bone 2011;48(2):362–71. Wang GW, Wang MQ, Wang XJ, Yu SB, Liu XD, Jiao K. Changes in the expression of MMP-3, MMP-9, TIMP-1 and aggrecan in the condylar cartilage of rats induced by experimentally created disordered occlusion. Arch Oral Biol 2010;55(11):887–95. Harper RP, Bell WH, Hinton RJ, Browne R, Cherkashin AM, Samchukov ML. Reactive changes in the temporomandibular joint after mandibular midline osteodistraction. Br J Oral Maxillofac Surg 1997;35(1):20–5. Norrdin RW, Kawcak CE, Capwell BA, McIlwraith CW. Subchondral bone failure in an equine model of overload arthrosis. Bone 1998;22(2):133–9. Burr DB, Radin EL. Microfractures and microcracks in subchondral bone: are they relevant to osteoarthrosis? Rheum Dis Clin North Am 2003;29(4):675–85. Mizoguchi I, Takahashi I, Nakamura M, Sasano Y, Sato S, Kagayama M, et al. An immunohistochemical study of regional differences in the distribution of type I and type II collagens in rat mandibular condylar cartilage. Arch Oral Biol 1996;41(8–9):863–9. Smith RL, Lin J, Trindade MC, Shida J, Kajiyama G, Vu T, et al. Time-dependent effects of intermittent hydrostatic pressure on articular chondrocyte type II collagen and

archives of oral biology 60 (2015) 650–663

aggrecan mRNA expression. J Rehabil Res Dev 2000;37(2): 153–61. 31. Huang X, Liu H, Xiao P, Wang Y, Zhang H. Effect of psychological stress on the structure of the temporomandibular joint and the expression of MMP-3 and TIMP-3 in the cartilage in rats. Br J Oral Maxillofac Surg 2014;52(8):709–14.

663

32. Ali AM, Sharawy MM. An immunohistochemical study of collagen types III, VI and IX in rabbit craniomandibular joint tissues following surgical induction of anterior disk displacement. J Oral Pathol Med 1996;25(2):78–85. 33. Eyre DR, Weis MA, Wu JJ. Articular cartilage collagen: an irreplaceable framework. Eur Cell Mater 2006;12(1): 57–63.