Divergent chondro/osteogenic transduction laws of fibrocartilage stem cell drive temporomandibular joint osteoarthritis in growing mice

Abstract

The anterior disc displacement (ADD) leads to temporomandibular joint osteoarthritis (TMJOA) and mandibular growth retardation in adolescents. To investigate the potential functional role of fibrocartilage stem cells (FCSCs) during the process, a surgical ADD-TMJOA mouse model was established. From 1 week after model generation, ADD mice exhibited aggravated mandibular growth retardation with osteoarthritis (OA)-like joint cartilage degeneration, manifesting with impaired chondrogenic differentiation and loss of subchondral bone homeostasis. Lineage tracing using Gli1-CreER⁺; Tm^fl/-mice and Sox9-CreER⁺;Tm^fl/-mice showed that ADD interfered with the chondrogenic capacity of Gli1⁺ FCSCs as well as osteogenic differentiation of Sox9⁺ lineage, mainly in the middle zone of TMJ cartilage. Then, a surgically induced disc reposition (DR) mouse model was generated. The inhibited FCSCs capacity was significantly alleviated by DR treatment in ADD mice. And both the ADD mice and adolescent ADD patients had significantly relieved OA phenotype and improved condylar growth after DR treatment. In conclusion, ADD-TMJOA leads to impaired chondrogenic progenitor capacity and osteogenesis differentiation of FCSCs lineage, resulting in cartilage degeneration and loss of subchondral bone homeostasis, finally causing TMJ growth retardation. DR at an early stage could significantly alleviate cartilage degeneration and restore TMJ cartilage growth potential.

Fig. 1

TMJ ADD in growing mice led to mandibular growth retardation. a Schematic diagram of ADD-induced TMJOA mouse model generation and timing of animal sacrifice after ADD surgery. b Representative images of morphological changes of ADD condyle compared to the control condyle. c To analyze the condyle growth of ADD mice, mandibles were dissected and scanned by microCT and were reconstructed at 2/4/8 weeks post-surgery. d–f The ramus height (RH), condylar width (CW), and condylar length (CL) of ADD mice were analyzed. n = 5–7. ****P < 0.0001. The horizontal line and error bar indicates mean ± SD. g H&E staining of TMJ condylar cartilage 1/2/4/8 weeks after ADD surgery. FZ fibrocartilage zone, PZ proliferative zone, AC articular cartilage. h–j The semi-quantification of cell number in articular cartilage, the thickness of articular cartilage, and the ratio of (FZ + PZ) thickness/articular cartilage thickness. N = 6–9. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1

Fig. 2

TMJ ADD caused OA-like degeneration in cartilage in growing mice. a Safranin O staining of TMJ cartilage at 1/2/4/8 weeks after ADD surgery. Yellow arrows indicated the anterior displaced TMJ disc. b Semi-quantification of Safranin O⁺ area in ADD mice at different stages. N = 6–7. c Modified Mankin score of mice TMJ cartilage after ADD-induced TMJOA model generation at different stages. N = 7–8. d Immunofluorescent staining of Aggrecan and SOX9 expressions in the articular cartilage of ADD-TMJOA mouse at 1/2/4/8 weeks after ADD surgery. AC articular cartilage, SB subchondral bone. e Semi-quantitative of Aggrecan⁺ area and SOX9⁺ cell numbers in the articular cartilage. *P < 0.05, **P < 0.01, ***P < 0.001, ****P< 0.000 1

Fig. 3

TMJ ADD caused subchondral bone loss in growing mice. a Immunofluorescent staining of RUNX2 expressions in the subchondral bone area of TMJ in ADD-TMJOA mice at 2/4/8 weeks after ADD surgery. SB subchondral bone. b Semi-quantification of RUNX2⁺ cell ratio in TMJ SB area. c TRAP staining in the TMJ subchondral bone area. The black arrow indicated TRAP⁺ cells. d Semi-quantification of TRAP⁺ cell numbers in the SB area of ADD mice. N = 6–8. e Representative image of microCT analysis of ADD TMJ condyle. and selections for analysis. The white square indicates the region of interest (ROI). f The quantification analysis of ROI. BV/TV(%): bone volume fraction; Tb.Sp (mm): trabecular separation; Tb.N (1/mm): trabecular number; Tb.Th (mm): trabecular thickness; SMI structure model index. N = 6–7. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1

Fig. 4

FCSCs proliferation was stimulated by ADD-TMJOA. a The heatmap of mRNA expressions of inflammation-related genes, cartilage matrix-related genes, and proliferation-related genes in mice condylar cartilage at 1 week after ADD surgery. b Mating tactics for generating Gli1-CreER⁺; Tm^fl/- mice. c Representative images of RFP⁺ cell distributions in TMJ cartilage at 5/10/30 days after tamoxifen injection. d Semi-quantification of RFP⁺ cell numbers in articular cartilage at 5/10/30 days after tamoxifen injection. N = 6. ***P < 0.001, ****P < 0.000 1

Fig. 5

ADD-TMJOA led to activation of the chondrogenic capacity of Gli1⁺ FCSCs. a Expressions of Gli1-RFP, Aggrecan, and SOX9 were analyzed by immunofluorescent staining in TMJ cartilage in Gli1-CreER⁺; Tm^fl/- mice at 3 days/1 week/2 weeks/4 weeks after model generation. RFP⁺ cell% and RFP⁺/SOX9⁺ cell% were quantified in the b middle zone (MZ) and c posterior zone (PZ) of articular cartilage, respectively. N = 3–5. *P < 0.05, **P < 0.01, ****P < 0.000 1

Fig. 6

Sox9⁺ cell lineage exhibits distinct expression and functional patterns in ADD-TMJOA cartilage and subchondral bone. a Mating tactics for generating Sox9-CreER⁺; Tm^fl/- mice and strategies for generating the Sox9-CreER⁺; Tm^fl/− ADD-TMJOA mouse model. Tamoxifen injection was implemented right after ADD surgery, and mice were sacrificed at 1 week after ADD surgery. b Immunofluorescent staining of Aggrecan, SOX9, and RFP in the middle zone of TMJ cartilage in Sox9-CreER⁺; Tm^fl/− ADD-TMJOA mice. White solid arrows indicated RFP⁺/SOX9⁺ cells, and white hollow arrows indicated RFP⁺/SOX9⁻ cells. AC articular cartilage, SB subchondral bone. c Semi-quantification of RFP⁺ cell% and RFP⁺/SOX9⁺ cell% in the articular cartilage area. N = 6. d Immunofluorescent staining of RUNX2 and RFP in the middle zone of TMJ subchondral bone in Sox9-CreER⁺; Tm^fl/- ADD-TMJOA mice. White solid arrows indicated RFP⁺/RUNX2⁺ cells and white hollow arrows indicated RFP⁺/ RUNX2⁻ cells. AC articular cartilage, SB subchondral bone. e Semi-quantification of RFP⁺ cell% and RFP⁺/RUNX2⁺ cell% in the articular cartilage area. N = 6. *P < 0.05, **P < 0.01, ****P < 0.000 1

Fig. 7

Disc reposition in ADD-TMJOA mice alleviated cartilage degeneration during TMJ growth. a Schematic diagram of Disc reduction (DR) treatment in ADD-TMJOA mouse. Disc reposition surgery was performed 5 days after ADD model generation. Mice were sacrificed at 2/4/8 weeks. b Safranin O staining of temporomandibular joint condylar cartilage at 2/4/8 weeks after ADD surgery. c Semi-quantification of safranin O⁺ area in ADD and DR mice. N = 4–8. d Aggrecan and SOX9 immunostaining of TMJ cartilage of the Sham nice, ADD-TMJOA mice, and DR mice at 2/4/8 weeks after ADD surgery. e Quantitative analysis of Aggrecan⁺ area and SOX9⁺ cell rate in the three groups. N = 4–8. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1

Fig. 8

Disc reposition in ADD-TMJOA mice significantly stimulated the chondrogenic potential of Gli1⁺ FCSCs during TMJ growth. a Strategies for generating the DR mouse model using Gli1-CreER⁺; Tm^fl/− mice. b Expressions of Gli1-RFP, Aggrecan, and SOX9 were analyzed by immunofluorescent staining in TMJ cartilage in Gli1-CreER⁺; Tm^fl/− mice at 2 weeks after model generation. c RFP⁺ cell% and RFP⁺/SOX9⁺ cell% were semi-quantified in the middle zone (MZ) and posterior zone (PZ) of articular cartilage, respectively. N = 3–5. **P < 0.01, ***P < 0.001, ****P < 0.000 1

Fig. 9

Disc reposition alleviated TMJ subchondral bone destruction and promoted condylar growth in ADD-TMJOA mice. a Schematic diagram of Disc reduction (DR) in ADD-TMJOA mouse. b Immunofluorescent staining of RUNX2 expressions in TMJ subchondral bones in DR mice at 2/4/8 weeks after ADD surgery. c Semi-quantification of RUNX2⁺ cell% in subchondral bone area. N = 5–6. d TRAP staining in the subchondral bone area of DR mice at 2/4/8 weeks after ADD surgery. e Semi-quantification of TRAP⁺ cell% in TMJ subchondral bone area. N = 6–8. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1