Fig 1.
Schematic of the experimental design.
(A) A total of 72 rats were randomly divided into sham+PBS, DMM+PBS, and DMM+ZOL groups at 2, 4, 8, and 12 weeks after OA induction (n = 6 per time point). The bone resorption stage of OA was identified, and effects of ZOL on cartilage and subchondral bone were assessed. (B) Another 18 rats were randomly divided into three groups at 4 weeks after OA induction (n = 6 per group) to explore possible molecular mechanism of osteoclast activation in early OA.
Table 1.
Primer sequences for reverse transcription-quantitative polymerase chain reaction.
Fig 2.
DMM-induced abnormal subchondral bone remodeling is inhibited by ZOL.
(A) Reconstructed coronal μCT images of tibial subchondral bone at various time-points after OA induction. Scale bar, 2,000 μm. (B, C, D, E, and F) Quantitative μCT analyses of microarchitecture in tibial subchondral bone: (B) BV/TV (%), (C) Tb.Th, (D) Tb.Sp, (E) Tb.N and (F) Conn.D (n = 6 per group/time point). Quantitative analysis of (G) PINP and (H) CTX-I at various time-points (n = 6 per group/time point). *P < 0.05, **P < 0.01, and ***P < 0.001, compared with sham+PBS group; #P < 0.05, ##P < 0.01, and ###P < 0.001, compared with DMM+PBS group.
Fig 3.
ZOL inhibits the increase of calcified cartilage in DMM-induced OA rats.
Histological analysis of articular cartilage stained with (A, B and C) HE, in coronal sections of tibia at various time-points. Scale bar, 100 μm. Quantitative analysis of (D) CC/TAC at various time-points (n = 6 per group/time point). **P < 0.01 and ***P < 0.001, compared with sham+PBS group; ##P < 0.01 and ###P < 0.001, compared with DMM+PBS group.
Fig 4.
ZOL attenuates articular cartilage degeneration in DMM-induced OA rats.
Histological analysis of articular cartilage stained with (A, B and C) Safranin O-fast green in coronal sections of tibia at various time-points. Scale bar, 100 μm. Quantitative analysis of (D) OARSI scores at various time-points (n = 6 per group/time point). *P < 0.05, **P < 0.01, and ***P < 0.001, compared with sham+PBS group; #P < 0.05, compared with DMM+PBS group.
Fig 5.
ZOL inhibits osteoclast formation in DMM-induced OA rats.
Analysis of osteoclast formation in subchondral bone stained with (A, B and C) TRAP in coronal sections of tibia at various time-points. Scale bar, 100 μm. Quantitative analysis of (D) Oc.S/BS at various time-points (n = 6 per group/time point). **P < 0.01 and ***P < 0.001, compared with sham+PBS group; ##P < 0.01 and ###P < 0.001, compared with DMM+PBS group.
Fig 6.
ZOL attenuates BMM activity in vitro 4 weeks after DMM-induced OA.
(A) Ability of BMMs to differentiate into multinucleated osteoclasts and (D) quantitative analysis of TRAP staining. Scale bar, 200 μm. (B) Migrated and (C) adherent BMMs stained with crystal violet. Scale bar, 500 μm. Quantitation of BMM (E) migration and (F) adhesion (n = 6 per group). ***P < 0.001, compared with sham+PBS group; ###P < 0.001, compared with DMM+PBS group.
Fig 7.
ZOL attenuates the expression of Wnt5a and osteoclastogenesis genes 4 weeks after DMM-induced OA.
Expression of (A) Wnt5a, (B) CXCL12 and (C) NFATc1 determined by immunohistochemical staining and by (E) quantitative analysis. Scale bar, 200 μm. n = 6 per group. (D) Western blots and (F) quantitation of Wnt5a, RANKL, CXCL12 and NFATc1. Reverse transcription quantitative polymerase chain reaction of (G) Wnt5a, CXCL12, NFATc1, (H) RANKL, OPG, RANKL/OPGand (I) TRAP, Ctsk. n = 3 per group. *P < 0.05, **P < 0.01, and ***P < 0.001, compared with sham+PBS group; #P < 0.05, ##P < 0.01, and ###P < 0.001, compared with DMM+PBS group.