Lubricin is Required for the Structural Integrity and Post-natal Maintenance of TMJ

Abstract

The Proteoglycan 4 (Prg4) product lubricin plays essential roles in boundary lubrication and movement in limb synovial joints, but its roles in temporomandibular joint (TMJ) are unclear. Thus, we characterized the TMJ phenotype in wild-type and Prg4(-/-) mouse littermates over age. As early as 2 weeks of age, mutant mice exhibited hyperplasia in the glenoid fossa articular cartilage, articular disc, and synovial membrane. By 1 month of age, there were fewer condylar superficial tenascin-C/Col1-positive cells and more numerous apoptotic condylar apical cells, while chondroprogenitors displayed higher mitotic activity, and Sox9-, Col2-, and ColX-expressing chondrocyte zones were significantly expanded. Mutant subchondral bone contained numerous Catepsin K-expressing osteoclasts at the chondro-osseous junction, increased invasive marrow cavities, and suboptimal subchondral bone. Mutant glenoid fossa, disc, synovial cells, and condyles displayed higher Hyaluronan synthase 2 expression. Mutant discs also lost their characteristic concave shape, exhibited ectopic chondrocyte differentiation, and occasionally adhered to condylar surfaces. A fibrinoid substance of unclear origin often covered the condylar surface. By 6 months of age, mutant condyles displayed osteoarthritic degradation with apical/mid-zone separation. In sum, lubricin exerts multiple essential direct and indirect roles to preserve TMJ structural and cellular integrity over post-natal life.

Keywords: Has2; Prg4; hyaluronic acid; osteoarthritis; superficial layer; temporomandibular joint.

Figure 1.

Glenoid fossa, articular disc, synovial membrane, and mandibular condyle are defective in Prg4^–/– TMJs. TMJs from 2-week (A-J), 3-month (K-N, P-S), and 6-month-old (O, T) control (A-E, K-O) and Prg4^–/– (F-J, P-T) mice were analyzed by histology. Hematoxylin and eosin (H&E) (A-T), Safranin O/Fast green (O, T, lower panel), and Masson’s trichrome (R, right panel) staining. Magnified sagittal views of glenoid fossa (B, G, L, Q), articular disc (C, H, M, R), and mandibular condyle (D, I, N, S, O, T) were obtained from each colored boxed area in the top panel of each line or corresponding sections. Note that H&E- or Masson’s trichrome-stained substance covers the surface of the glenoid fossa, disc, and condyle (G, H, Q, R, S arrowhead). There are no clear signs of inflammatory cell invasion into the synovial membrane (E, J). Scale bars: 250 µm in A for A, F, K, P, O, T; 65 µm in B for B, C, E, G, H, J, L, M, Q, R; and 150 µm in D for D, I, N, S. gf, glenoid fossa; di, disc; cd, condyle; sb, subchondral bone; pac, presumptive articular cartilage; ac, articular cartilage; in, intima; si, subintima; ab, anterior band; iz, intermediate zone; pb, posterior band.

Figure 2.

Prg4 mutants display defective superficial layer, abnormal subchondral bone, and increased chondrogenesis in the mandibular condyle. Serial parasagittal sections from 1-month (A-G, I-O) and 3-month-old (H, P) control (A-H) and Prg4–/– (I-P) TMJs. Sections were processed for Masson’s trichrome (A, B, I, J) and Safranin O/Fast green (H, P) staining and in situ hybridization (C-G, K-O) with isotope-labeled riboprobes for tenascin-C (Tn-C) (C, K), type I collagen (Col I) (C, K), Sox 9 (D, L), type II collagen (Col II) (E, M), type X collagen (Col X) (F, N), and Catepsin K (CtsK) (G, O). (Q) For quantification of the Safranin O-staining area and the thickness of chondrocyte zones, Sox9-expressing, Col II-expressing, and Col X-expressing zones along the longitudinal axis were measured from randomly selected 3-5 sections per sample (n = 3 for each mouse/each group) and presented as average ± SD. Control data were set as 100%. p values less than .05 were considered as statistically significant (*p < .01, **p < .005). Scale bars: 250 µm in A for A, D-H, I, L-P; 55 µm in B for B, C, H (right panel), J, K, and P (right panel). sf, superficial layer; pm, polymorphic layer; fc, flattened chondrocyte zone; hc, hypertrophic chondrocyte zone; sb, subchondral bone.

Figure 3.

Prg4 mutants display abnormal mitotic activity and apoptosis in TMJs. Serial parasagittal sections from wild-type (A-C) and Prg4^–/– (D-F) mice were processed for TUNEL (B, E, G), Edu (C, F), and staining. TUNEL-positive apoptotic cells in superficial and polymorphic layers of distinct TMJ sections from control and Prg4–/– mice at 2 wks, 1 mo, and 3 mos of age were counted (approximately 100-120 cells in the condyle). TUNEL fluorescence images were merged with the corresponding bright-field image, and the representative image was presented in (B, E). Data were collected from randomly selected 4-6 sections per sample (n = 3 for each mouse/each group) and presented as average ± SD; p values < .05 were considered statistically significant (*p < .01, **p < .005) (G). Edu-incorporated proliferating cells in distinct TMJ sections from control and mutant mice at 2 wks, 1 mo, and 3 mos of age were counted in articular disc (I), condylar superficial/polymorphic layers (J), glenoid fossa (K), and synovial membrane (L), corresponding to the illustrated image (H) (approximately 100-120 cells in the glenoid fossa, synovial membrane, and condyle and 40-70 cells in the disc). Images visualized with DAPI fluorescence for nuclei were merged with Edu-incorporated GFP-positive cells to visualize dividing cells, and the representative image was presented (C, F). Data were corrected and processed for statistical analysis as above. gf, glenoid fossa; ac, articular cartilage; cd, condyle; sy, synovial membrane; sf/pm, superficial/polymorphic layers; ant, anterior band; im, intermediate zone; post, posterior band.

Figure 4.

Prg4 mutants display excessive chondrogenesis in the glenoid fossa and articular disc and increased Has2 expression. Serial parasagittal sections from 3-month-old control (A-E, K) and Prg4^–/– (F-J) TMJs were processed for in situ hybridization with isotope-labeled riboprobes for Col II (A, B, F, G), Hyaluronic acid synthase 2 (Has2) (C-E, H-J), and Prg4 (K). Note the increased chondrogenesis in the Prg4–/–^- glenoid fossa (F) and thickened disc (G) and increased Has2 expression in the glenoid fossa (H), disk (J), synovial membrane (J), and mandibular condyle (I). (L) Summary of abnormalities identified in TMJ lacking Prg4. Scale bars: 180 µm in A for A-J; 350 µm in K for K. gf, glenoid fossa; di, articular disc; co, mandibular condyle; sm, synovial membrane; sf, superficial layer.