A discoidin domain receptor 1 knock-out mouse as a novel model for osteoarthritis of the temporomandibular joint

Abstract

Discoidin domain receptor 1 (DDR-1)-deficient mice exhibited a high incidence of osteoarthritis (OA) in the temporomandibular joint (TMJ) as early as 9 weeks of age. They showed typical histological signs of OA, including surface fissures, loss of proteoglycans, chondrocyte cluster formation, collagen type I upregulation, and atypical collagen fibril arrangements. Chondrocytes isolated from the TMJs of DDR-1-deficient mice maintained their osteoarthritic characteristics when placed in culture. They expressed high levels of runx-2 and collagen type I, as well as low levels of sox-9 and aggrecan. The expression of DDR-2, a key factor in OA, was increased. DDR-1-deficient chondrocytes from the TMJ were positively influenced towards chondrogenesis by a three-dimensional matrix combined with a runx-2 knockdown or stimulation with extracellular matrix components, such as nidogen-2. Therefore, the DDR-1 knock-out mouse can serve as a novel model for temporomandibular disorders, such as OA of the TMJ, and will help to develop new treatment options, particularly those involving tissue regeneration.

Fig. 1

Structural and genomic aspects of OA of the TMJ in DDR-1 KO mice in vivo. a The micro-CT 3D reconstructions of the condylus mandibulae of the DDR-1 KO mice (KO, always on the left side) and the WT (WT, always on the right side). b, c Outline of the subchondral bone surface. The KO mice (b) exhibited a rough surface and an abnormal bone structure compared to the WT (c). d The measurements of the condyle bone mineral density revealed that the KO mice had a higher bone density. e–h Ultrastructural analysis: e, f the collagen fiber arrangement was altered in the superficial layer of DDR-1 KO mice compared to the parallel fiber alignment observed in the WT mice (g, h). i A short list of the fold changes of OA-relevant genes found in the microarray analysis. The complete lists can be found at GEO, GSE35297. j–m Toluidine blue histology of the condyles: j, k the DDR-1 KO condyle exhibits well-known signs of OA such as reduced staining of the superficial zone, cluster formation and surface fissures; l, m normal WT condyles. Three pairs of DDR-1 KO mice condyles and samples from two of their WT littermates were used for microarray analysis. (n = 6, including 3 KO mice and 3 WT mice, for electron microscopy; n = 5, including 3 KO mice and 2 WT mice for the microarray)

Fig. 2

The molecular changes in the ECM of the TMJ during OA in vivo of 9-week-old mice: Immunohistochemistry was performed for collagen type I (a–d), collagen type II (e–h), nidogen-1 (i–l) and nidogen-2 (m–p). a, c DDR-1 KO TMJ stained for collagen type I is shown. Note the fibrocartilaginous tissue as a sign of tissue regeneration in (c). b, d The WT TMJ exhibited the well-known collagen type I staining. e, g The KO TMJ showed less collagen type II staining than the WT (f, h). i Nidogen-1 is present in both the KO and the WT mice (j). However, there were no differences between the DDR-1 KO mice (k) and the corresponding WT mice (l). m Nidogen-2 staining was stronger in the TMJs of DDR-1-deficient mice than in the TMJs of WT mice (n). o Note the intense pericellular staining in the deeper zones of the DDR-1 KO TMJ cartilage. Less nidogen-2 was present in the pericellular matrix of the WT cartilage (p). The numbers of the animals evaluated at each time point are shown in Tables 1 and 2

Fig. 3

The histopathology and immunohistology of 24- and 28-week-old mice. a–d HE staining of 24-week-old mice. c Note the surface fissures in the KO compared with the intact joint surface of the WT mice. d Immunohistochemistry results for collagen type I of staining in 28-week-old mice. There was an increase of collagen type I in the KO (e) versus the WT mice (f). The joint surface of the KO is destroyed (g), while the WT joint was smooth and rounded shaped (h). i–l Toluidine blue histology of 24-weeks old mice: the DDR-1 KO condyle exhibited well-known signs of OA, e.g., reduced staining of the superficial zone (i), cluster formation and surface fissure (k); normal WT condyles were observed in (j, l). m–p Immunohistochemistry results of collagen type IV staining in 28-week-old KO mice: intense staining for collagen type IV was observed at the joint surface (m), especially in the pericellular matrix (o); nNormal WT staining was observed in (n, p). The numbers of the animals evaluated at each time point are shown in Tables 1 and 2

Fig. 4

Cell isolation and characterization. a The dissected mandible of a 9-week-old WT mouse is shown. Care was taken to sample only the translucent cartilage tissue, as shown in (b). c The cells growing out of a tissue sample after 10 days are shown. d–n These cells were identified as chondrocytes. However, DDR-1 KO cells exhibit reduced mRNA expression levels of sox-9 (d) and aggrecan (g) compared with WT cells. In contrast, the KO cells showed an increased expression of runx-2 (e) and collagen type I (f). A similar pattern was observed for the immunocytology; more pronounced staining for runx-2 was present in the DDR-1 KO (h) than in WT cells (k). Collagen type I (i, l), and aggrecan (j, m) were detected in both KO and WT cells. n Intracellular FACS analysis of cells in passage 6 identified aggrecan, collagen type II, COMP, and sox-9, as well as the osteoarthritic markers, runx-2 and collagen type I, in both cell types. A higher percentage of DDR-1 KO chondrocytes expressed runx-2 (second-to-last bars). *Significant differences (p ≤ 0.05); data are mean values with SD of three individual experiments (n = 10, including 4 KO mice, 4 WT mice, and 2 controls, for mRNA measurements; n = 6, including 3 KO mice and 3 WT mice, for immunocytochemistry and FACS-analysis)

Fig. 5

Differences in protein patterns in KO and WT cells. a The western blot for DDR-1 in the DDR-1 KO confirmed its absence; however, DDR-2 (b), MMP-13 (c) and collagen type I (d) were upregulated in the KO chondrocytes. Sox-9 (e), runx-2 (f) and COMP (g) were present in both the KO and WT cells. h β-actin staining confirmed the equal loading of the gels. i Coomassie blue staining was performed to evaluate the overall protein patterns. Protein isolation was performed using cells at passage 2 of the cells. Data are representatives of three individual experiments, or quantified as stated in “Results” (n = 6, including 3 KO mice and 3 WT mice, for western blots)

Fig. 6

The influence of three-dimensional alginate matrix, BMPs, laminin-1, nidogen-2 and the knockdown of runx-2 on TMJ chondrocytes: The expression patterns of runx-2 (a), collagen type I (b), sox-9 (c) and aggrecan (d) were similar to the results obtained in 2D-cultured cells (Fig. 3d–g). e Laminin-1 stimulation resulted in an upregulation of sox-9 in DDR-1 KO chondrocytes, while nidogen-2 down-regulates runx-2 (f). Therefore, the two basement membrane components promote the chondrogenesis of these cells. g BMP-6 reduced the relative mRNA levels of collagen type I in DDR-1 KO chondrocytes, as did laminin-1 (h), which is also a marker of chondrogenic differentiation. i Runx-2 protein expression was not detectable 24 h (lane 2) after the knockdown performed by the transient transfection with the siRNA vector. Lane 1 always represents the control. j Collagen type I was not detectable 24 h (lane 2) after runx-2 knockdown, as expected; k the amount of sox-9 was elevated compared with the control cells (lane 1). l Collagen type II was detectable (lane 2), after runx-2 knockdown, but not in the control cells, in which collagen type II was not detectable (lane 1). m Tubulin staining confirmed the equal loading of the gels. k–m are composite figures. *Significant differences (p ≤ 0.05); data are mean values with SD from three individual experiments. (n = 10, including 4 KO mice, 4 WT mice and 2 controls, for mRNA measurements)

Fig. 7

Possible players downstream of the DDRs in OA of the TMJ. a A PCR array identified the regulated signaling pathway molecules, with the IHH pathway prominently involved in SSH and BMP-2 interactions. VEGFA upregulation was observed. b Surface fissures (a) and proteoglycan degradation (b) of the TMJ of the DDR-1 KO mice revealed typical OA characteristics. The pathomechanism of OA in the TMJ of DDR-1 KO mice results in an upregulation of runx-2, collagen type I and DDR-2 (1). This leads to an increased activation of MMP-13 (2) to enhance matrix degradation, especially of collagens (3). *Also downregulated: Naip1, Brca1, Ccl2, Ccl20, Cd5, Cdh1, Csf2, Cxcl1, Cxcl9, Cyp19a1, En1, Fasl, Fgf4, Greb1, Hoxa1, Icam1, Il1a, Il2, Il2ra, Lef1, Lep, Lta, Mmp10, Mmp7, Nos2, Pparg, Rbp1, Tnf, Wnt1, Wnt2, MGDC, and Selp. Data are representative of three individual experiments. (n = 6, including 3 KO mice and 3 WT mice, for PCR array)