Extracorporeal Shockwave Therapy Modulates the Expressions of Proinflammatory Cytokines IL33 and IL17A, and Their Receptors ST2 and IL17RA, within the Articular Cartilage in Early Avascular Necrosis of the Femoral Head in a Rat Model

Abstract

Avascular necrosis (AVN) of the femoral head (AVNFH) is a disease caused by injury to the blood supply of the femoral head, resulting in a collapse with osteonecrosis and damage to the articular cartilage. Extracorporeal shockwave therapy (ESWT) has been demonstrated to improve AVNFH owing to its anti-inflammation activity, angiogenesis effect, and tissue regeneration in clinical treatment. However, there are still so many pieces of the jigsaw that need to be fit into place in order to ascertain the mechanism of ESWT for the treatment of AVNFH. The study demonstrated that ESWT significantly protected the trabecular bone volume fraction BV/TV (P < 0.01) and the trabecular thickness (P < 0.001), while in contrast, the trabecular number and trabecular separation were not significantly different after treatment as compared with AVNFH. ESWT protected the articular cartilage in animal model of AVNFH. The levels of IL1-β and IL33 were significantly induced in the AVNFH group (P < 0.001) as compared with Sham and ESWT groups and reduced in ESWT group (P < 0.001) as compared with AVNFH group. In addition, the expression of the receptor of IL33, ST2, was reduced in AVNFH and induced after ESWT (P < 0.001). The expression of IL17A was induced in the AVNFH group (P < 0.001) and reduced in the ESWT group (P < 0.001). Further, the expression of the receptor of IL17A, IL17RA, was reduced in the AVNFH group (P < 0.001) and improved to a normal level in the ESWT group as compared with Sham group (P < 0.001). Taken together, the results of the study indicated that ESWT modulated the expression of IL1-β, pro-inflammatory cytokines IL33 and IL17A, and their receptors ST2 and IL17RA, to protect against loss of the extracellular matrix in the articular cartilage of early AVNFH.

Figure 1

The study design and application of shockwave therapy. (a) The graph displayed the study design of the experiment, including ONFH surgery, shockwave application, and sacrificed animals. (b) The two focal points were approximately 0.5 cm apart and the corresponding locations on the skin in the groin area to make with a marker. Each of the two points was treated with 2000 impulses of shockwaves at 0.25 mJ/mm² energy flux density and total of 4000 impulses of shockwaves were applied to the affected femoral head. N = 8 for all groups.

Figure 2

Micro-CT scan of the left femur of subchondral bone in different groups. (a) The results showed photomicrographs of the femur head in sagittal and transverse views from micro-CT. The region of interesting was indicated by a red rectangle. (b) The data of subchondral bone of femur head displayed the graphic illustrations of the trabecular bone volume fraction (BV/TV), trabecular thickness, trabecular number, and trabecular separation.^★★P < 0.01 and ^★★★P < 0.001 as compared with the AVNFH group. The scale bar was 2 mm. N = 8 for all groups.

Figure 3

The microphotographs of the left femur head showed the changes of articular cartilage in the Sham, AVNFH, and ESWT groups. Some areas of disorganization, loss of extracellular matrix, and decreased number of chondrocytes are presented (arrow) and are protected in the ESWT group by (a) hematoxylin-eosin and (b) safranin-O stain. The scale bar was 200 μm. N = 8 for all groups.

Figure 4

Immunohistochemical analysis for type II colagen in the articular cartilage of the left femur head (a) and the level of expression was measured after treatment (b). The scale bar was 50 μm. N = 8 for all groups.

Figure 5

Immunohistochemical analysis for (a) IL1-β, (b) IL33, and (c) ST2 in the articular cartilage of the left femur head (right) and the level of expression was measured after treatment (left). ^∗∗∗P < 0.001 as compared with ESWT group and ^###P < 0.001 as compared with AVNFH group. The scale bar was 20 μm. N = 8 for all groups.

Figure 6

Immunohistochemical analysis for (a) IL17, (b) IL17RA in the articular cartilage of the left femur head (right), and the level of expression was measured after treatment (left). The expression of IL17A was major in superfacial zone and proliferation zone (arrowhead), while IL17RA was major expressed in the hypertrophic chondrocytes of the calcified cartilage zone (arrow). ^∗∗∗P < 0.001 as compared with the ESWT group and ^###P < 0.001 as compared with the AVNFH group. The scale bar was 50 μm. N = 8 for all groups.