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TNF Accelerates Death of Mandibular Condyle Chondrocytes in Rats with Biomechanical Stimulation-Induced Temporomandibular Joint Disease.

Yang H, Zhang M, Wang X, Zhang H, Zhang J, Jing L, Liao L, Wang M - PLoS ONE (2015)

Bottom Line: TNF stimulated apoptosis of the isolated chondrocytes and up-regulated caspase-8 expression, but did not change caspase-9 expression levels.Local injection of TNF inhibitor down-regulated caspase-8 expression and reduced TUNEL-positive cell number, but did not reverse cartilage thickness reduction, caspase-9 up-regulation or cytochrome c release.However, anti-TNF therapy does not prevent cartilage loss in this model of temporomandibular joint.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Military Stomatology, Department of Oral Anatomy and Physiology and TMD, School of Stomatology, the Fourth Military Medical University, 145 Changle West Road, Xi'an, China.

ABSTRACT

Objective: To determine if temporomandibular joint chondrocyte apoptosis is induced in rats with dental biomechanical stimulation and what a role TNF takes.

Methods: Thirty-two rats were divided into 4 groups (n = 8/group) and exposed to incisor mal-occlusion induced by unilateral anterior crossbite biomechanical stimulation. Two groups were sampled at 2 or 4 weeks. The other two groups were treated with local injections of a TNF inhibitor or PBS into the temporomandibular joints area at 2 weeks and then sampled at 4 weeks. Twenty-four rats either served as unilateral anterior crossbite mock operation controls (n = 8/group) with sampling at 2 or 4 weeks or received a local injection of the TNF inhibitor at 2 weeks with sampling at 4 weeks. Chondrocytes were isolated from the temporomandibular joints of 6 additional rats and treated with TNF in vitro. Joint samples were assessed using Hematoxylin&eosin, Safranin O, TUNEL and immunohistochemistry staining, real-time PCR, fluorogenic activity assays and Western blot analyses. The isolated chondrocytes were also analyzed by flow cytometry.

Results: Unilateral anterior crossbite stimulation led to temporomandibular joint cartilage degradation, associated with an increase in TUNEL-positive chondrocytes number, caspase-9 expression levels, and the release of cytochrome c from mitochondria at 2 weeks without changes in TNF and caspase-8 levels until after 4 weeks. TNF stimulated apoptosis of the isolated chondrocytes and up-regulated caspase-8 expression, but did not change caspase-9 expression levels. Local injection of TNF inhibitor down-regulated caspase-8 expression and reduced TUNEL-positive cell number, but did not reverse cartilage thickness reduction, caspase-9 up-regulation or cytochrome c release.

Conclusions: Unilateral anterior crossbite stimulation induces mitochondrion-mediated apoptosis of articular chondrocytes. TNF accelerated the unilateral anterior crossbite induced chondrocytes apoptosis via death-receptor pathway. However, anti-TNF therapy does not prevent cartilage loss in this model of temporomandibular joint.

No MeSH data available.


Related in: MedlinePlus

Sagittal central section of the TMJ in the control group stained with hematoxylin & eosin.(a). Six squares denoting the areas on slides where immunohistochemical and TUNEL staining were performed. D, articular disc; C, mandibular condyle. (b). Sagittal central section of the TMJ condylar cartilage showing the four cellular layers.
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pone.0141774.g001: Sagittal central section of the TMJ in the control group stained with hematoxylin & eosin.(a). Six squares denoting the areas on slides where immunohistochemical and TUNEL staining were performed. D, articular disc; C, mandibular condyle. (b). Sagittal central section of the TMJ condylar cartilage showing the four cellular layers.

Mentions: Hematoxylin&eosin (H&E) and immunohistochemical (IHC) staining was performed, as previously reported [8]. Immunohistochemistry staining with an anti-TNF primary antibody (diluted 1:50, sc8436, Santa Cruz) was performed using an avidin-biotin complex (ABC) IHC staining protocol. Negative controls were stained with non-immune serum instead of the primary antibody. Sections were mounted with balsam after being dehydrated in serial alcohol solutions. H&E, Safranin O and IHC stained sections were analyzed under a light microscope (Leica DM 2500, Wetzlar, Germany) and images were acquired using a Leica DFC490 system, as reported previously [7]. Briefly, Photoshop CS3 software was used to divide the surface of the cartilage into three parts consisting of the anterior, center, and posterior thirds between the anterior and posterior attachment positions of the condyle to the disk. Three squares (300 pixels × 300 pixels) covering all hypertrophic layers were applied at the quartering points of the posterior and middle thirds of the condylar cartilage (Fig 1a). The lengths of three short lines crossing through the hypertrophic layer were measured in each portion and averaged to determine the thickness of the hypertrophic layer in each third. The percentage of stain-positive cells in the selected squares was detected with an image analyzing system (Leica Qwin.Plus, Leica Microsystem Imaging, Cambridge), and the average value of six squares was calculated for statistical analysis.


TNF Accelerates Death of Mandibular Condyle Chondrocytes in Rats with Biomechanical Stimulation-Induced Temporomandibular Joint Disease.

Yang H, Zhang M, Wang X, Zhang H, Zhang J, Jing L, Liao L, Wang M - PLoS ONE (2015)

Sagittal central section of the TMJ in the control group stained with hematoxylin & eosin.(a). Six squares denoting the areas on slides where immunohistochemical and TUNEL staining were performed. D, articular disc; C, mandibular condyle. (b). Sagittal central section of the TMJ condylar cartilage showing the four cellular layers.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4631347&req=5

pone.0141774.g001: Sagittal central section of the TMJ in the control group stained with hematoxylin & eosin.(a). Six squares denoting the areas on slides where immunohistochemical and TUNEL staining were performed. D, articular disc; C, mandibular condyle. (b). Sagittal central section of the TMJ condylar cartilage showing the four cellular layers.
Mentions: Hematoxylin&eosin (H&E) and immunohistochemical (IHC) staining was performed, as previously reported [8]. Immunohistochemistry staining with an anti-TNF primary antibody (diluted 1:50, sc8436, Santa Cruz) was performed using an avidin-biotin complex (ABC) IHC staining protocol. Negative controls were stained with non-immune serum instead of the primary antibody. Sections were mounted with balsam after being dehydrated in serial alcohol solutions. H&E, Safranin O and IHC stained sections were analyzed under a light microscope (Leica DM 2500, Wetzlar, Germany) and images were acquired using a Leica DFC490 system, as reported previously [7]. Briefly, Photoshop CS3 software was used to divide the surface of the cartilage into three parts consisting of the anterior, center, and posterior thirds between the anterior and posterior attachment positions of the condyle to the disk. Three squares (300 pixels × 300 pixels) covering all hypertrophic layers were applied at the quartering points of the posterior and middle thirds of the condylar cartilage (Fig 1a). The lengths of three short lines crossing through the hypertrophic layer were measured in each portion and averaged to determine the thickness of the hypertrophic layer in each third. The percentage of stain-positive cells in the selected squares was detected with an image analyzing system (Leica Qwin.Plus, Leica Microsystem Imaging, Cambridge), and the average value of six squares was calculated for statistical analysis.

Bottom Line: TNF stimulated apoptosis of the isolated chondrocytes and up-regulated caspase-8 expression, but did not change caspase-9 expression levels.Local injection of TNF inhibitor down-regulated caspase-8 expression and reduced TUNEL-positive cell number, but did not reverse cartilage thickness reduction, caspase-9 up-regulation or cytochrome c release.However, anti-TNF therapy does not prevent cartilage loss in this model of temporomandibular joint.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Military Stomatology, Department of Oral Anatomy and Physiology and TMD, School of Stomatology, the Fourth Military Medical University, 145 Changle West Road, Xi'an, China.

ABSTRACT

Objective: To determine if temporomandibular joint chondrocyte apoptosis is induced in rats with dental biomechanical stimulation and what a role TNF takes.

Methods: Thirty-two rats were divided into 4 groups (n = 8/group) and exposed to incisor mal-occlusion induced by unilateral anterior crossbite biomechanical stimulation. Two groups were sampled at 2 or 4 weeks. The other two groups were treated with local injections of a TNF inhibitor or PBS into the temporomandibular joints area at 2 weeks and then sampled at 4 weeks. Twenty-four rats either served as unilateral anterior crossbite mock operation controls (n = 8/group) with sampling at 2 or 4 weeks or received a local injection of the TNF inhibitor at 2 weeks with sampling at 4 weeks. Chondrocytes were isolated from the temporomandibular joints of 6 additional rats and treated with TNF in vitro. Joint samples were assessed using Hematoxylin&eosin, Safranin O, TUNEL and immunohistochemistry staining, real-time PCR, fluorogenic activity assays and Western blot analyses. The isolated chondrocytes were also analyzed by flow cytometry.

Results: Unilateral anterior crossbite stimulation led to temporomandibular joint cartilage degradation, associated with an increase in TUNEL-positive chondrocytes number, caspase-9 expression levels, and the release of cytochrome c from mitochondria at 2 weeks without changes in TNF and caspase-8 levels until after 4 weeks. TNF stimulated apoptosis of the isolated chondrocytes and up-regulated caspase-8 expression, but did not change caspase-9 expression levels. Local injection of TNF inhibitor down-regulated caspase-8 expression and reduced TUNEL-positive cell number, but did not reverse cartilage thickness reduction, caspase-9 up-regulation or cytochrome c release.

Conclusions: Unilateral anterior crossbite stimulation induces mitochondrion-mediated apoptosis of articular chondrocytes. TNF accelerated the unilateral anterior crossbite induced chondrocytes apoptosis via death-receptor pathway. However, anti-TNF therapy does not prevent cartilage loss in this model of temporomandibular joint.

No MeSH data available.


Related in: MedlinePlus