Therapeutic effects of kartogenin on temporomandibular joint injury by activating the TGF-β/SMAD pathway in rats

Abstract

Patients with temporomandibular dysfunction (TMD) usually suffer from pathology or malpositioning of the temporomandibular joint (TMJ) disk, leading to the degenerative lesion of condyles. Kartogenin can promote the repair of damaged cartilage. This study aimed to explore whether intra-articular injection of kartogenin could alleviate the TMJ injury induced by type II collagenase. We measured the head withdrawal threshold and found that kartogenin alleviated the pain around TMD induced by type II collagenase. We observed the morphology of the condylar surface and found that kartogenin protected the integration of the condylar surface. We analyzed the density of the subchondral bone and found that kartogenin minimized the damage of TMJ injury to the subchondral bone. We next explored the histological changes and found that kartogenin increased the thickness of the proliferative layer and more collagen formation in the superficial layer. Then, to further ensure whether kartogenin promotes cell proliferation in the condyle, we performed immunohistochemistry of proliferating cell nuclear antigen (PCNA). The ratio of PCNA-positive cells was significantly increased in the kartogenin group. Next, immunofluorescence of TGF-β1 and SMAD3 was performed to reveal that kartogenin activated the TGF-β/SMAD pathway in the proliferative layer. In conclusion, kartogenin may have a therapeutic effect on TMJ injury by promoting cell proliferation in cartilage and subchondral bone. Kartogenin may be promising as an intra-articular injection agent to treat TMD.

Keywords: Temporomandibular joint; cartilage; cell proliferation; collagenase; dysfunction; kartogenin.

Figure 1.

Pain in the TMJ region in rats treated with KGN. (A) Illustration of head withdrawal measurement by von Frey hairs. (B) The head withdrawal threshold of rats at four weeks (n = 5 in each group). ***P < 0.001 by comparing Sham group or Injury + KGN group to Injury group or Injury + DMSO group.

Figure 2.

Stereomicroscope and micro-CT analysis of TMJ treated with KGN. (A) Representative images of gross observation of TMJ specimens. Scale bar = 1 mm. (B) Representative images of sagittal plane images of micro-CT analysis for the changes of subchondral bones. Scale bar = 1 mm. (C) Bone parameter analysis of BV/TV, Tb. N, Tb. Th, and Tb. Sp (n = 5 in each group). ***p < 0.001 by comparing Sham group or Injury + KGN group to Injury group or Injury + DMSO group.

Figure 3.

Histological structure of TMJ treated with KGN. (A) Representative images of H&E, TBO, SO staining of TMJ treated with KGN. Scale bar = 400 μm for upper panel of H&E, TBO, and SO staining. Scale bar = 50 μm for lower panel of H&E staining. (B) Quantitative analysis of OARSI-modified Mankin score (n = 5 in each group). (C) Quantitative analysis of cartilage thickness (n = 5 in each group). ***P < 0.001 by comparing Sham group or Injury + KGN group to Injury group or Injury + DMSO group.

Figure 4.

Immunohistochemistry staining of PCNA in TMJ treated with KGN. (A) Representative images of immunohistochemistry staining of PCNA in the proliferative layer. Scale bar = 400 μm for upper panel and 50 μm for lower panel. (B) Quantitative analysis of the number of PCNA-positive cells in proliferative layer (n = 5 in each group). ***P < 0.001 by comparing Sham group or Injury + KGN group to Injury group or Injury + DMSO group.

Figure 5.

Immunofluorescent staining of TGF-β1 and SMAD3 in TMJ treated with KGN. (A) Representative images of immunofluorescent staining of TGF-β1 and SMAD3 in the proliferative layer. Scale bar = 50 μm for upper panel and 25 μm for lower panel. (B) Quantitative analysis of the number of TGF-β1-positive cells in proliferative layer (n = 5 in each group). ***P < 0.001 by comparing Sham group or Injury + KGN group to Injury group or Injury + DMSO group. (C) Quantitative analysis of the number of SMAD3-positive cells in proliferative layer (n = 5 in each group). **P < 0.01 by comparing Sham group to Injury group or Injury + DMSO group. *P < 0.05 by comparing Injury + KGN group to Injury group or Injury + DMSO group.