Time Course of Expression of Bcl-2 and Bax in Rabbit Condylar Chondrocytes Following Forward Mandibular Positioning
Objective: To clarify the expression of Bcl-2 and Bax following forward mandibular positioning (FMP) in the condylar chondrocytes of rabbits.
Materials and Methods: Sixty rabbits at 8 weeks of age were randomly allocated to the experimental group (n = 36) and control group (n = 24). Rabbits in the experimental group were induced to FMP by a functional appliance. Six rabbits from the experimental group and four from the control group were sacrificed after 3 days and 1, 2, 4, 8, and 12 weeks, respectively. All the right temporomandibular joints (TMJs) were collected and the expression of Bcl-2 and Bax was evaluated by immunohistochemical staining.
Results: The results showed the expression pattern of Bcl-2 and Bax during 12 weeks after FMP. The expression of Bcl-2 reached the highest level at 1 week, whereas Bax reached its maximal expression after 4 weeks. Subsequently, the expression of Bcl-2 and Bax gradually decreased. The ratio of Bcl-2/Bax began to decrease 3 days after FMP and continued to decline until 12 weeks.
Conclusions: FMP with functional appliances could change the expression of Bcl-2 and Bax, which is related to apoptosis in condylar chondrocytes.Abstract
INTRODUCTION
Class II malocclusion that occurs in adolescents and young adults can be treated by fixed, rigid functional orthodontic appliances because of the remodeling of the mandible and muscles.1 The mandible is positioned forward during functional appliance therapy,2–4 and several arthroscopic studies have demonstrated that forward mandibular positioning (FMP) could result in adaptive changes in the condyle,56 which proceeds with the bimolecular pathway initiating with chondrogenesis and finalizing with osteogenesis.7 There is a certain association between malocclusion and the remodeling of the temporomandibular joint (TMJ), but controversial issues still remain regarding the compensatory mechanism of the TMJ after FMP.8
Indirect evidence has suggested that the selective process of physiologic cell deletion may contribute to the stabilization of the condylar cartilage.9 But the pathophysiologic mechanism of this specific phenomenon has not been entirely elucidated. Nowadays, the central point of caspases proteolytic cascade in the apoptosis has been confirmed, but its initiation is tightly regulated by a variety of other factors.10 Among these, Bcl-2 family proteins are known to play a key role in the induction of caspases activation and in the regulation of apoptosis.11 Bcl-2 and Bax are both important members in the Bcl-2 family, which can be activated by proinflammatory cytokines and environmental stresses.12 Overexpression of Bcl-2 has been shown to suppress apoptosis in response to different stimuli.13 Bax interaction with anti-apoptotic proteins from the Bcl-2 family (Bcl-2, Bcl-xl, Mcl-1, A1) was already described in 1995 by Sedlak et al.14
To identify whether the Bcl-2 related apoptosis plays a role in maintaining the cartilage cell stabilization during the condylar remodeling after FMP, we investigated the changes of Bcl-2 and Bax expression pattern in the condylar cartilage during a prolonged period after FMP.
MATERIALS AND METHODS
Experimental Animals
A total of 60 Japanese rabbits (8 weeks old, 30 male and 30 female, weighing 1.0–1.5 kg) were obtained from Zhejiang Experimental Animal Center (Grade II, Certificated SCXK2003-0001). The protocol of the experiment was in accordance with the Guideline for the Care and Use of Laboratory Animals of Zhejiang University. All rabbits were provided water and standard diet ad libitum for 1 week before the experiment. Among these rabbits, 24 rabbits served as a control group while another 36 were experimental animals. Food intake was monitored every day after FMP. Each rabbit was weighed daily. For sample collection, six experimental rabbits and four rabbits in the control group were sacrificed at 3 days and 1, 2, 4, 8, and 12 weeks after FMP.
Experimental Forward Mandibular Positioning in Condylar Cartilage
Functional appliances, made from polymethylmethacrylate with 20°–25° inclined planes, were cemented onto the maxillary central incisors of the experimental group with dental adhesive resin cement (3M, Monrovia, Calif). The appliances were worn 24 hours to produce a continuous forward and downward positioning of the mandible according to the method reported by Rabie et al.15 The control group was free from the appliances in order to retain natural growth.
Sample Preparation
The right TMJ samples were fixed in 4% paraformaldehyde, decalcified in 0.5 mol/L EDTA (ethylenediamine tetraacetic acid, pH 7.2), then dewaxed and soaked in 95% ethanol. After the TMJ samples were dissected into two halves, connective tissues were removed to expose the areas surrounding the mandibular condyle. Any excess tissues were removed and specimens were embedded in paraffin. Serial sections of 3–5 μm were cut through the TMJ at the parasagittal plane using a rotary microtome (Leica RM 2155, Leica, Vienna, Austria) and hematoxylin and eosin staining was applied. The middle part of the former inclined plane and posterior inclined plane of the condyle were selected.
Immunohistochemistry
Immunohistochemical analysis was performed using the Ultrasensitive streptavidin-peroxidase (SP) method with DAB-PO kit (Neumaker Co, Fremont, Calif). Briefly, specimens were treated with 0.3% hydrogen peroxide to inhibit the activity of endogenous peroxidase. Nonspecific protein staining was blocked by 1.5% goat serum (Sigma Chemical Co, St Louis, Mo). The tissues were incubated with rat anti-rabbit Bcl-2 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif) and goat anti-rabbit Bax polyclonal antibody (Santa Cruz Biotechnology) at 4°C overnight. The working titer of Bcl-2 and Bax was 1:100 for both. Samples were washed with PBS and incubated at room temperature with biotinated secondary antibody for 10 minutes, then washed extensively and incubated with the streptavidin-peroxidase complex for 10 minutes, and finally developed according to the manufacturer's directions (Neumaker). After washing in PBS, fresh DAB (Sigma) was added, and counterstained with hematoxylin. PBS was used as negative control.
Image Analysis and Statistical Analysis
Rabbits in our experiment were randomly assigned to each group. The immunostained sections were examined using an Olympus microscope (×400) coupled to a video camera, connected to a computer-aided color video image analysis (VIA) system. After being captured and digitized onto the video screen, microscopic images were quantitatively analyzed using an image analysis software program (HPIAS-1000, High Resolution Pathological Image & Word Analysis System, Beijing, China).
Twenty views in a high power field (×400), ie, 1 cm × 1 cm per view, were selected randomly and double-blindly. Then, the number of positive cells in the condylar cartilage was calculated. The gray values of the expression intensity were measured to indicate the expression of protein. The lower the gray value was, the stronger the intensity of the immunostaining. The data were processed with SPSS for Windows (Version 11.0, SPSS Inc, Chicago, Ill) using Student's t-test and analysis of variance (ANOVA). Values were expressed as the mean ± standard deviation. Differences at P < .05 were considered statistically significant.
RESULTS
Gross Examination of Animal Behavior
The experimental rabbits had some difficulties in mastication and took less food the first 2 days after wearing the functional appliance, but recovered 2 and 3 days after surgery. Observations during the experimental period found that a loss of weight did not occur.
X-ray of Forward Mandibular Positioning in Rabbit Mandible
The X-ray photos of the mandible from a normal animal and from a rabbit treated with a functional appliance are shown in Figure 1. It was obvious that the mandible of the rabbit in the experimental group showed forward and downward changes, as marked by arrows in Figure 1C.
Citation: The Angle Orthodontist 78, 3; 10.2319/012007-29.1
Expression of Bcl-2 and Bax Protein in Condylar Cartilage
Five tissue layers of condylar cartilage can be observed: the superficial layer, proliferative layer, transitive layer, hypertrophic layer, and mineralized layer.16 These are marked with S, P, T, H, and M, respectively, as shown in Figure 2. In the normal growing rabbits, positive signals of Bcl-2 protein can be observed in the P, T, H, and M layers, but not in the S layer; and the expression of Bcl-2 in the P and T layer was much more than in other layers (Figure 2A,C,E).
Citation: The Angle Orthodontist 78, 3; 10.2319/012007-29.1
Figure 3 showed the relative expression of Bcl-2 in the control and experimental groups during 12 weeks. In the control group the gray value of Bcl-2 reached 76 ± 1.93 one week after FMP and subsequently increased to the maximal level of 93 ± 2.23 at 12 weeks. Three days and 1 week after FMP, a significant decrease of Bcl-2 can be detected from 70 ± 1.18 to 58 ± 4.35 in the experimental group (Figure 2B,D). Then, Bcl-2 was continuously up-regulated, and 4 weeks after FMP the gray value arrived at the minimal level at 40 ± 1.41. During 4 weeks to 12 weeks, no change was evident. The statistical analysis shows that the expression of Bcl-2 in the experimental group at each time point was lower than that in the control group (P < .05).
Citation: The Angle Orthodontist 78, 3; 10.2319/012007-29.1
Bax expression was only observed in the T, H, and M layers, while the strongest signal was found in the M layer (Figure 4). The relative expression of Bax vs time in the experimental and control groups is shown in Figure 5. In the control group, the mean gray value varied from 99 to 119, indicating a decreasing expression of Bax. Meanwhile, in the experimental group, 1 week after FMP, the gray value of Bax reached the lowest level of 58 ± 4.19. As shown in Figure 4d, extensive expression of Bax appeared in the H and M layer. Then, the gray value of Bax gradually increased from 71 ± 4.22 at 2 weeks to 91 ± 1.82 at 12 weeks. Compared with the control group, Bax exhibited a significantly stronger expression in the experimental group at all monitored time points (P < .05).
Citation: The Angle Orthodontist 78, 3; 10.2319/012007-29.1
Citation: The Angle Orthodontist 78, 3; 10.2319/012007-29.1
The ratio of Bcl-2/Bax was also significantly affected by FMP. As shown in Figure 6, the ratio began to decrease from 1.07 ± 0.01 three days after surgery to 0.50 ± 0.037 at 4 weeks, and increased slowly to 0.62 ± 0.04 at 12 weeks. This alternation can be attributed to the intense decrease of Bcl-2 during the first 4 weeks after surgery and the increase of Bax during 2 to 12 weeks.
Citation: The Angle Orthodontist 78, 3; 10.2319/012007-29.1
DISCUSSION
Functional appliances are extensively applied in the treatment of Class II malocclusion by FMP.17 Earlier experiments have demonstrated that condylar growth after FMP mostly occurred within 3 months.18 Therefore, in the present study the appliances were kept for 12 weeks and samples were collected in this period. The rabbits were used at 8 weeks of age because their condyles are fast growing. After being fixed with the appliance, the mandible was maintained in a continuous advanced position and a different force transmitted to the condyle, so condylar adaptive responses are induced.19 Moreover, these biodynamic factors can also affect mandibular growth direction, size, and morphology.
The expression of Bcl-2 in the control group fluctuated slightly because of the ordinary growth of rabbits, and then consistently decreased within 4 weeks into the hypertrophic cartilage layer after FMP. Bax expression was extensively up-regulated at 3 days, then continual increased by hypertrophic chondrocytes throughout the same period.
In this period, alternation of Bcl-2 and Bax was associated with many more histologic changes and compensatory mechanisms. The process of endochondral ossification was excited, which then triggered the remodeling of the condylar cartilage.20 Bax was held by the formation of complexes with Bcl-2, which is an anti-apoptotic protein. When cells in cartilage receive death signals after abnormal loading, Bax moves to mitochondria and other membrane sites and triggers a transformation of mitochondrial function. This procession includes release of cytochrome, loss of transmembrane potential, and induction of mitochondrial permeability transition events that result in apoptotic cells.21 The remarkable decrease of Bcl-2 in late hypertrophic chondrocytes may be attributed to the lack of mechanical strain which caused failure of chondrogenesis.22 As reported by Chrysis et al,23 the downregulation of Bcl-2 expression was also found in the proliferative layer, possibly due to the presence of primary chondrocytes. In the cartilage growth plate, there is a decrease in the Bcl-2/Bax protein ratio from the proliferative to the hypertrophic zone.24
An abnormal expression of Bcl-2 in various rheumatic diseases and the increased expression of Bax in osteoarthritis has been observed.2526 However, in some critical conditions such as abnormal loading, ie, surgical trauma and stress, the synthesis of Bcl-2 is reduced while Bax increases, which ultimately contributes to the occurrence of arthritic disease and induces the degradation of the articular cartilage.27 Nevertheless, the ratio of Bcl-2/Bax holds the balance in the process. The ratio of Bcl-2/Bax has an important role on predicting therapeutic response and prognosis, and would be a valuable parameter for new therapeutic agents.28 Nowadays, the analysis of Bcl-2 and Bax by immunohistochemistry as a routine laboratory tool in the clinical management of malocclusion have some interesting studies.1229
Our earlier studies have shown that after FMP, apoptosis may be activated.30 The continuous joint position would result in a certain change of the condylar cartilage in response to a new mechanical stress. Biomechanical forces induced by FMP change the extracellular matrix and the form of the nondifferentiated mesenchymal cells.31 Subsequently, the increasing expression of Bcl-2 and decreasing expression of Bax make the chondrocytes achieve the balance between proliferation and apoptosis. The regulation of Bcl-2 and Bax might be the feedback of the survival of chondrocyte. This compensatory mechanism may play an important role through suppressing any increased enzyme activity and protecting the condylar cartilage during FMP.32 Thus, we can consider that fluctuation of Bcl-2 and Bax expression contributes to apoptosis of condylar chondrocytes after FMP, which is similar with the findings of Cheung et al.33
In this study, displacement of the articular disc was not found in the treated animals, and the future changes of the articular disc after FMP were unknown. But based on the present data, it is adequate to conclude that alteration of the condyle is enhanced by FMP. If the apoptosis could not compensate proliferation, it would lead to the incrassation of cartilage and the stagnation of ossification, and even become important pathogenesis of bone arthropathy.
CONCLUSIONS
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Biomechanical forces brought by FMP can induce changes in the Bcl-2 and Bax expression pattern of condylar chondrocytes.
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These changes would be involved in the apoptosis procedure, which may concern the remodeling of the mandibular condyle during the treatment of a Class II malocclusion.
X-ray photos of rabbit's mandible. (A) No treatment. (B) Functional appliances. (C) Overlap of A and B. The black arrow showed the mandibular central incisor having no treatment, while the white arrowhead showed the mandibular central incisor having functional appliances
Immunohistochemical micrographs of condyle cartilage in control and experimental group (DAB, ×200). Bcl-2 positive cells were identified as tissue with brown staining. A, C, E represent cartilage tissue of the control group in 3 days, 1 week and 4 weeks after surgery; B, D, F are tissues of the experimental animals
Mean gray value of Bcl-2 vs time in the cartilage for control (n = 4) and experimental group (n = 6). Bar represents standard deviations. * P < .05, comparing with the control group; ▵ P < .05 comparing with the former time point in the same group
Immunohistochemical micrographs of condyle cartilage in control and experimental group (DAB, ×200). Bax-2 positive cells were identified as tissue with brown staining
Mean gray value of Bax vs time in the cartilage for control (n = 4) and experimental group (n = 6). Bar represents standard deviations. * P < .05, comparing with the control group; ▵ P < .05, comparing with the former time point in the same group
The ratio of Bcl-2/Bax vs time in condyle cartilage for control (n = 4) and experimental group (n = 6). Bar represents standard deviations. * P < .05, comparing with the control group; ▵ P < .05, comparing with the former time point in the same group
Contributor Notes
Corresponding author: Zhi-Yuan Gu, PhD, Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Zhejiang University, Yan'an Road 395# Hangzhou 310006, China (gzy@zju.edu.cn)