Inflammation-related collagen fibril destruction contributes to temporomandibular joint disc displacement via NF-κB activation

Abstract

Temporomandibular joint (TMJ) disc displacement is one of the most significant subtypes of temporomandibular joint disorders, but its etiology and mechanism are poorly understood. In this study, we elucidated the mechanisms by which destruction of inflamed collagen fibrils induces alterations in the mechanical properties and positioning of the TMJ disc. By constructing a rat model of TMJ arthritis, we observed anteriorly dislocated TMJ discs with aggravated deformity in vivo from five weeks to six months after a local injection of Freund's complete adjuvant. By mimicking inflammatory conditions with interleukin-1 beta in vitro, we observed enhanced expression of collagen-synthesis markers in primary TMJ disc cells cultured in a conventional two-dimensional environment. In contrast, three-dimensional (3D)-cultivated disc cell sheets demonstrated the disordered assembly of inflamed collagen fibrils, inappropriate arrangement, and decreased Young's modulus. Mechanistically, inflammation-related activation of the nuclear factor kappa-B (NF-κB) pathway occurs during the progression of TMJ arthritis. NF-κB inhibition reduced the collagen fibril destruction in the inflamed disc cell sheets in vitro, and early NF-κB blockade alleviated collagen degeneration and dislocation of the TMJ discs in vivo. Therefore, the NF-κB pathway participates in the collagen remodeling in inflamed TMJ discs, offering a potential therapeutic target for disc displacement.

Fig. 1

Chronic inflammation induces deformation and displacement of rat TMJ discs. a Anatomical diagram of the temporomandibular joint in rats subjected to the CFA intra-articular injection model. b Representative sagittal view of T₂ weighted-MRI images of the rat head at 5 weeks (w) and 6 months (m) after saline/1^st CFA injection: TMJ discs from the rats in the control group showed biconcave contours, whereas those from rats in the inflammation-induced group were elongated at 5 w and severely deformed at 6 m, both with a dislocated anterior section (n = 3). T: temporal bone; C: condyle; white arrows: the most anterior aspect of the condyle; yellow line with arrows: distance between the margin of the anterior band and the posterior band of the discs; yellow dotted line: disc contour. c Coronal view of T₂ weighted-MRI images of the rat head at 5 w and 6 m after saline/CFA 1^st injection. The control disc was located on the condyle with hypo intensity, and the upper cavity showed hyper intensity between disc and temporal bone. The lateral band in inflamed disc was thickened and folded (red arrow). yellow dotted line: disc contour. d Representative images of H&E staining of synovial tissue of normal and inflamed TMJ discs at 5 w and 6 m timepoint (n = 3 or 4). e Representative images of immunohistochemical staining of IL-1β of normal and inflamed TMJ discs and synovial tissue (n = 4). D: disc, Syno: synovial tissue, A: anterior band; In: intermediate zone, P: posterior band. Black arrows: positively stained cells

Fig. 2

IL-1β stimulation enhances cell proliferation and collagen synthesis abilities of disc cells. a The disc cells showed positive toluidine blue staining compared with FLS, but the staining was weaker than chondrocytes. FLS: fibroblast-like synoviocyte. b The expression of Col1a1 and Col1a2 mRNA in disc cells was comparable with FLS, and the expression of Col2a1 mRNA in disc cells significantly upregulated compared with FLS but significantly downregulated compared with chondrocytes. c The results of western blot showed that disc cells had higher expression of COL II and downregulation of vimentin compared with FLSs and no significant changes of COL I among the TMJ-derived cells. d Representative immunofluorescence images and semi-quantitative statistics of Ki-67 staining of disc cells stimulated with IL-1β for 24 h. e TMJ disc cell viability upon IL-1β stimulation over 24 h as assessed by the CCK8. f, g Expression of MMP3 and COL I assessed by western blot after IL-1β treatment at different concentrations (f) and timepoints (g). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

IL-1β stimulation impairs the content and nanostructure of cell-derived collagen fibrils. a Illustration depicting the fabrication of disc cell sheets. b Gross image of the disc cell sheets. c Expression of ECM related markers of disc cell sheets was assessed by qPCR after prolonged IL-1β treatment for 3 days (3 d) and 9 days (9 d). The results showed upregulated expression of Col1a1 and Col3a1, and downregulated expression of Mmp3 and Mmp13. d Representative immunofluorescence images and semi-quantitative statistics regarding COL I expression in cell sheets stimulated with IL-1β. e TEM images of ECM in cell sheets after prolonged treatment with IL-1β. Red arrows: thin microfibrils; yellow dotted line: periodic banding of collagen fibrils. f Representative spectra of cell sheets after baseline subtraction: the maximum peak of amide I shifted with a wide absorbance peak of amide II and weak peak of 1 338 cm ⁻¹ after the IL-1β treatment. g Representative AFM nanomechanical mapping features of disc cell sheets under fluid condition after stimulated with IL-1β. The IL-1β treated group showed a significant decrease in Young’s modulus compared with the control group. **P < 0.01, ***P < 0.001

Fig. 4

NF-κB is activated in TMJ disc cells under an inflammatory environment. a Representative immunofluorescence images and semi-quantitative statistics regarding NF-κB p65 staining in the posterior band of a TMJ disc at 5 week after saline/CFA 1st injection (n = 4). b The expression of p-p65 and p65 assessed via western blot. c Representative immunofluorescence images of p65 staining in disc cells stimulated with IL-1β for 0.5 h. d Expression of nuclear p65 assessed via western blot after IL-1β treatment. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Blocking NF-κB activation partly rescues IL-1β-induced abnormal collagen remodeling and nanostructure of collagen fibrils in vitro. a The expression of COL I and MMP3 of 2D-cultivated disc cells was assessed by western blot. b Representative immunofluorescence images and semi-quantitative statistics regarding COL I in cell sheets after a NF-κB blockade and stimulation with IL-1β. c SEM images showing recovery of the collagen ultrastructure in cell sheets after a NF-κB blockade during prolonged exposure to an inflammatory environment. Red arrow: collagen fibrils. d Representative spectra of cell sheets after baseline subtraction and quantitative statistics of 1 338 cm ⁻¹ /amide II area ratio. e Representative nanomechanical mapping features and non-parametric analysis of the Young’s modulus of the disc cell sheets under fluid condition. The results showed an increase in Young’s modulus after a NF-κB blockade during prolonged exposure to an inflammatory environment. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Blocking NF-κB activation alleviates the damage of collagen structure, disc deformation and displacement under inflammatory conditions in vivo. a Timeline of induction and PDTC treatment of chronic TMJ inflammation in rats. b Representative photographs and statistical analysis showing differences in head widths one day after the rats received saline/CFA injection. c Representative morphological features of the TMJ discs 5 weeks after PDTC administration. d H&E staining showing alterations in disc configuration; the statistical analysis showed differences in disc thickness among the three groups. The black lines indicate the locations where the thickness of the disc was measured (n = 4). A: anterior band, In: intermediate zone, P: posterior band. e Representative images of Sirius red staining under polarized light. f Representative sagittal view of T₂ weighted-MRI images of the rat head 6 m after saline/CFA 1^st injection (n = 3): the inflamed disc is deformed, thickened at the anterior band, and the disc is dislocated; compared with the CFA+Vehicle group, the disc in the CFA + PDTC group showed regular biconcave contours, and the posterior band was opposite to the top of the condyle. T: temporal bone, C: condyle, red dotted line: contour of the disc, yellow dotted line: contour of the bone. g Representative nanomechanical mapping features of the posterior band in TMJ discs. Non-parametric statistical analysis showed an increase in Young’s modulus after a NF-κB blockade. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

A schematic graph illustrating how sustained inflammation induces abnormal collagen remodeling and assembly via the NF-κB pathway, diminishes the mechanical properties of collagen fibrils, and aggravates degenerative changes in the collagen, ultimately resulting in disc displacement