TMJ degeneration in SAMP8 mice is accompanied by deranged Ihh signaling

Abstract

The temporomandibular joint (TMJ) functions as a load-bearing diarthrodial joint during mastication, and its continuous use and stress can lead to degeneration over age. Using senescence-accelerated (SAMP8) mice that develop early osteoarthritis-like changes in synovial joints at high frequency, we analyzed possible molecular mechanisms of TMJ degeneration and tested whether and how malocclusion may accelerate it. Condylar articular cartilage in young SAMP8 mice displayed early-onset osteoarthritic changes that included reductions in superficial/chondroprogenitor cell number, proteoglycan/collagen content, and Indian hedgehog (Ihh)-expressing chondrocytes. Following malocclusion induced by tooth milling, the SAMP8 condyles became morphologically defective, displayed even lower proteoglycan levels, and underwent abnormal chondrocyte maturation compared with malocclusion-treated condyles in wild-type mice. Malocclusion also induced faster progression of pathologic changes with increasing age in SAMP8 condyles as indicated by decreased PCNA-positive proliferating chondroprogenitors and increased TUNEL-positive apoptotic cells. These changes were accompanied by steeper reductions in Ihh signaling and by expression of matrix metalloproteinase 13 at the chondro-osseous junction in SAMP8 articular cartilage. In sum, we show for the first time that precocious TMJ degeneration in SAMP8 mice is accompanied by--and possibly attributable to--altered Ihh signaling and that occlusal dysfunction accelerates progression toward degenerative TMJ disease in this model.

Keywords: Indian hedgehog; degenerative joint disease; osteoarthritis; senescence-accelerated mouse; superficial cell; temporomandibular joint.

Figure 1.

Defective cellular organization and loss of proteoglycan and collagen content in SAMP8 condyles. Sections of mandibular condyles from 2-mo-old (A, D), 4-mo-old (B, E), 6-mo-old (C, F) wild-type (A-C) and SAMP8 (D-F) mice were stained with hematoxylin and eosin (H&E), Safranin O/Fast Green (Saf O/FG) and Masson’s trichrome (Mas’s). Magnified images are presented of boxed areas. SAMP8 condyles (D) at 2 mo do not display obvious histologic changes compared with wild type (A). Note the significant reduction of superficial and underlying chondroprogenitors in 4- and 6-mo-old SAMP8 articular cartilage (E, F, arrowhead) and the reduction in Safranin O–stained proteoglycans and Masson’s trichrome–stained collagens in territorial and interterritorial ECM in 6-mo-SAMP8 articular cartilage (F). sf, superficial layer; pm, polymorphic layer; fc/hc, flattened/hypertrophic chondrocyte zone; ac, articular cartilage. Scale bars: 125 µm in low-magnification pictures and 60 µm in high-magnification pictures.

Figure 2.

Deranged Ihh signaling in SAMP8 condyles. Mandibular condyles from 2-mo-old wild-type (A-H) and SAMP8 (I-P) mice were analyzed by in situ hybridization (A-P) with isotope-labeled RNA probes for type I collagen (Col I) (A, I), type II collagen (Col II) (B, J), type X collagen (Col X) (C, K), Indian hedgehog (Ihh) (D, L), Gli1 (E, M), Gli2 (F, N), Patched 1 (Ptch1) (G, O), and hedgehog interacting protein (Hip) (H, P). Note the significant reduction in Ihh-positive prehypertrophic chondrocytes and expression in Gli transcription factors and Hedgehog transcriptional targets Ptch1 and Hip in SAMP8 articular cartilage. pm, polymorphic layer; fc/hc, flattened/hypertrophic chondrocyte zone. Scale bars: 125 µm in P for A-P.

Figure 3.

Degenerative changes are accelerated by malocclusion in SAMP8 condyles. Mandibular condyles from 2-mo-old wild-type (A) and SAMP8 (H) mice exhibit no obvious histologic differences. Similar wild-type (B-G) and SAMP8 (I-N) mice were subjected to incisor tooth milling and sacrificed 2 wk later (2.5-mo overall age) (B-E, I-L) and 4 wk later (3.0 mo) (F, G, M, N). Condyles were analyzed by Safranin O/Fast Green (B, C, F, I, J, M) staining and in situ hybridization with isotope-labeled RNA probes for lubricin (lub) (D, K), type II collagen (Col II) (E, L), and Mmp13 (G, N). Boxed areas in B and I are presented with higher magnification in C and J, respectively. Note in 2.5-mo-old operated SAMP8 condyles a deranged cellular arrangement (J, arrowheads), fewer lubricin-expressing superficial cells (K), no major changes in Col II-expressing chondrocytes (L), and lower Safranin O–stained proteoglycans content in articular surface (M). Note the increased Mmp13 expression (N, arrowheads) in the anterior region of 3-mo-old SAMP8 condyles compared with wild-type condyles (G). Scale bars: 8 mm in A for A, H; 250 µm in B for B, I; 65 µm in C for C, J; and 125 µm in D for D, E, G, K, L, N.

Figure 4.

Degenerative changes are accelerated by malocclusion in SAMP8 condyles. Two-mo-old SAMP8 mice subdivided into control mock-milled (A-D, K, L, Q, R) and tooth-milled (F-I, M, N, S, T) groups and sacrificed at 2.5 mo (Q-T), 4 mo (C, H, K-N), and 6 mo of age (A, B, D, F, G, I). Condyles were analyzed by macroscopy (A, F), SEM (B, G), hematoxylin and eosin staining (H&E) (C, D, H, I; left panel), Safranin O/Fast Green staining (Saf O/FG) (C, H; right panel), aggrecan immunohistochemistry (D, I; right panel), and in situ hybridization with isotope-labeled probes for Ptch1 (K, M) and Mmp 13 (L, N). Note the flattening of the anterior end of the mandibular condyle in the tooth-milled group compared with controls (A, F, respectively). Note that the malocclusion-treated condyle is smaller along the anterior-posterior and mesolateral axes and that there is roughness of the articular surface compared with controls (B, G, respectively). Note also in malocclusion-subjected SAMP8 condyles that there is lower Safranin O staining (H), multiple tidemarks, and presence of resorption cavities (H, I, double arrowheads and arrowhead, respectively). Statistical analyses confirm changes of condylar size (^*p < .02, n = 4 for each group) and proteoglycan content (^**p < .01, n = 7 for each group) (E, J). Results are expressed as Safranin O–positive area/entire cartilage field obtained by 40× objectives (mean ± SD, n = 7 for each group). Note the reduced Ptch1 expression and increased Mmp13 expression. Serial parasagittal sections from 2.5-mo-old wild-type (O, P), mock- (Q, R), and malocclusion-treated (S, T) SAMP8 condyles were processed for PCNA (O, Q, S) and TUNEL staining (P, R, T). Data were collected from randomly selected 7 sections (approximately 120-150 cells/area) per sample and presented as averages ± SD; p values < .05 were considered statistically significant (^*p < .05, ^**p < .02). Note the significant decrease in proliferating chondroprogenitor cells in superficial/polymorphic layers and increased apoptosis in chondroprogenitors and chondrocytes in malocclusion-subjected SAMP8 condyles. Schematic depicting our current working model of early-onset degenerative TMJ disease in SAMP8 mice and its acceleration by malocclusion. Scale bars: 1.8 mm in A for A, F; 0.7 mm in B for B, G; 80 µm in C for C, D, H, I; 200 µm in K for K-N; and 40 µm in P for O-T.