Conditional deletion of E11/podoplanin in bone protects against load-induced osteoarthritis

Abstract

Background: Subchondral bone (SCB) thickening is one of the earliest detectable changes in osteoarthritic joints and is considered a potential trigger for subsequent articular cartilage degeneration. In this manuscript, we examine whether disruption to the SCB osteocyte network contributes to the initiation and pathogenesis of osteoarthritis.

Methods: We examined expression patterns of the glycoprotein E11/podoplanin by immunohistochemical labelling in murine, human and canine osteoarthritis models. We also examined the effects of twice-weekly administration of Bortezomib, a proteasome inhibitor which stabilises osteocyte E11 levels, to C57/BL6 wild-type male mice (1 mg/kg/day) for 8 weeks after surgical destabilisation of the medial meniscus. By inducing osteoarthritis-like changes in the right knee joint of 12-week-old male E11 hypomorphic mice (and corresponding controls) using a post-traumatic joint loading model, we also investigated whether a bone-specific E11 deletion in mice increases joint vulnerability to osteoarthritis. Articular cartilage degradation and osteophyte formation were assessed by histology and in line with the OARSI grading system.

Results: Our studies reveal increased E11 expression in osteocytes of human and canine osteoarthritic SCB. We found that Bortezomib administration had no effect on surgically-induced osteoarthritis, potentially due to a lack of the expected stabilisation of E11 in the SCB. We also found, in concordance with our previous work, wild-type mice exhibited significant load-induced articular cartilage lesions on the lateral femoral condyle (p < 0.01) and osteophyte formation. In contrast, E11 hypomorphic mice did not develop osteophytes or any corresponding articular lesions.

Conclusions: Overall, these data suggest that an intact osteocyte network in the SCB contributes to the development of mechanically-driven osteoarthritis. Further, the data presented here indicate that the molecular pathways that preserve the osteocyte network, such as those driven by E11, may be targeted to limit osteoarthritis pathogenesis.

Keywords: E11/podoplanin; Osteoarthritis; Osteocytes; Subchondral bone.

Fig. 1

Immunohistochemical labelling for E11 in (a) DMM and non-operated C57/BL6 mice (b) human osteoarthritis samples from patients undergoing total knee replacement (c) canine osteoarthritis samples from the medial coronoid process of the elbow. Images are representative of n > 3. Arrows are representative of E11 positive osteocytes. Scale bar = 300 μm

Fig. 2

(a) Weights of mice treated with Bortezomib or vehicle for days post DMM surgery (b) Maximum OARSI score in the medial and lateral compartments of the left (contralateral control) knee joint of Bortezomib and vehicle treated mice (c) Maximum OARSI score in the medial and lateral compartments of the right (DMM) knee joint of Bortezomib and vehicle treated mice (d) Mean OARSI score in the medial and lateral compartments of the left non-operated (contralateral control) knee joint of Bortezomib and vehicle treated mice (e) Mean OARSI score in the medial and lateral compartments of the right (DMM) knee joint of Bortezomib and vehicle treated mice (f) Representative histology images of articular cartilage lesions (arrows) in the medial joint compartment. Data are presented as mean ± S.E.M (n = 8/group)

Fig. 3

MicroCT analysis of the epiphyseal region of the medial tibia in DMM-operated and non-operated controls (a) subchondral bone thickness (SCB Th.) (b) trabecular bone volume/tissue volume (Tb. BV/TV) (c) trabecular number (Tb. N.) (d) trabecular thickness (Tb. Th.) e trabecular separation (Tb. Sp.) f trabecular pattern factor (Tb. Pf.). MicroCT analysis of the epiphyseal region of the lateral tibia in DMM-operated and non-operated controls (g) subchondral bone thickness (SCB Th.) (h) trabecular bone volume/tissue volume (Tb. BV/TV) (I) trabecular number (Tb. N.) (J) trabecular thickness (Tb. Th.) (k) trabecular separation (Tb. Sp.) (l) trabecular pattern factor (Tb. Pf.). Data are presented as mean ± S.E.M (n = 8/group). P < 0.05*

Fig. 4

(a) E11 immunohistochemistry in the left non-operated (contralateral control) and right (DMM) knee joints of vehicle and Bortezomib treated mice (b) sclerostin immunohistochemistry in the left non-operated (contralateral control) and right (DMM) knee joints of vehicle and Bortezomib treated mice. Arrows are representative of E11 positive osteocytes and asterisks indicate positive E11 immunolabelling in the chondrocytes

Fig. 5

a Mean OARSI score in the lateral femur of control and loaded knee joints of WT mice. b Mean OARSI score in the lateral femur of control and loaded knee joints of cKO mice. c Maximum OARSI score in the lateral femur of control and loaded knee joints of WT mice. d Maximum OARSI score in the lateral femur of control and loaded knee joints of cKO mice. e Representative histology images of articular cartilage lesions (arrows) in the lateral femur. Data are presented as mean ± S.E.M (n > 3/group). P < 0.05*; P < 0.01**

Fig. 6

(a) Histology images of osteophyte formation (arrows) in loaded joints from WT mice (b) Articular cartilage thickness (LF – lateral femur, LT – lateral tibia, MF – medial femur, MT – medial tibia). MicroCT analysis of the epiphyseal region of the lateral femur (c) subchondral bone plate thickness (SCB Th.) (d) epiphyseal trabecular bone volume/tissue volume (Tb. BV/TV) (e) epiphyseal trabecular number (Tb. N.) (f) epiphyseal trabecular thickness (Tb. Th.) (g) epiphyseal trabecular separation (Tb. Sp.) (h) epiphyseal trabecular pattern factor (Tb. Pf.). i Number of osteoclasts / bone surface. Data are presented as mean ± S.E.M (n > 3/group). P < 0.05*; P < 0.001***