Changes of articular cartilage and subchondral bone after extracorporeal shockwave therapy in osteoarthritis of the knee

Abstract

We assessed the pathological changes of articular cartilage and subchondral bone on different locations of the knee after extracorporeal shockwave therapy (ESWT) in early osteoarthritis (OA). Rat knees under OA model by anterior cruciate ligament transaction (ACLT) and medial meniscectomy (MM) to induce OA changes. Among ESWT groups, ESWT were applied to medial (M) femur (F) and tibia (T) condyles was better than medial tibia condyle, medial femur condyle as well as medial and lateral (L) tibia condyles in gross osteoarthritic areas (p<0.05), osteophyte formation and subchondral sclerotic bone (p<0.05). Using sectional cartilage area, modified Mankin scoring system as well as thickness of calcified and un-calcified cartilage analysis, the results showed that articular cartilage damage was ameliorated and T+F(M) group had the most protection as compared with other locations (p<0.05). Detectable cartilage surface damage and proteoglycan loss were measured and T+F(M) group showed the smallest lesion score among other groups (p<0.05). Micro-CT revealed significantly improved in subchondral bone repair in all ESWT groups compared to OA group (p<0.05). There were no significantly differences in bone remodeling after ESWT groups except F(M) group. In the immunohistochemical analysis, T+F(M) group significant reduced TUNEL activity, promoted cartilage proliferation by observation of PCNA marker and reduced vascular invasion through observation of CD31 marker for angiogenesis compared to OA group (P<0.001). Overall the data suggested that the order of the effective site of ESWT was T+F(M) ≧ T(M) > T(M+L) > F(M) in OA rat knees.

Keywords: cartilage histopathology; osteoarthritis; rats.; shockwave; subchondral bone.

Figure 1

The photographs showed macroscopic pathological osteoarthritic lesions of knee including the areas of osteophyte formation. (A) The knee photos demonstrated the gross pathological osteoarthritic lesions in distal femur and proximal tibia. The scale bar represented 5 mm. (B), (C) and (D) showed the gross appearance of OA lesion, osteophyte and lesion area as well as sclerotic bone volumes (n = 8 in each groups). The ESWT groups showed significantly lower lesion scores as compared to OA group and sham group. Amongst EWST groups, T+F(M) showed the lowest lesion score than other groups. ^**P < 0.001 compared to sham group. ^#P < 0.05, ^##P< 0.001 compared to OA group. ^※P < 0.05 compared to T(M).

Figure 2

The microphotographs of the knee showed articular cartilage degradation of the knee after ESWT. (A) Microphotographs of articular cartilage demonstrated cartilage damage in OA knee changes. The scale bar represented 200 μm. (B) and (C) showed graphic illustrations of cartilage area and modified Mankin score in histopathological examination. The ESWT groups showed significant increase in cartilage area and decrease in modified Mankin scoreas compared to OA group and sham group. Amongst ESWT groups, T+F(M) group showed the most dramatic changes than other groups. ^*P < 0.05, ^**P < 0.001 compared to sham group. ^#P < 0.05, ^##P < 0.001 compared to OA group. ^※P< 0.05 compared to T(M) group. All rats were n = 8.

Figure 3

The microphotographs showed un-calcified and calcified cartilages of the knee. (A) Microphotographs of the distal femur and proximal tibia showed un-calcified and calcified cartilage thickness in different groups. The magnification of the image was ×200. (B), (C) Graphic illustrations of un-calcified and calcified cartilage thickness in different groups were showed in this study. The ESWT groups showed significant increasing in un-calcified cartilage and decreasing in calcified cartilage. ^*P < 0.05, ^**P < 0.001 compared to sham group. ^#P < 0.05, ^##P < 0.001 compared to OA group. ^※P < 0.05 compared to T(M) group. All rats were n = 8.

Figure 4

The histopathology of cartilage assessment. The cartilage histopathology was measured from articular cartilage of the tibia by Safranin-O stain (A). The surface damage (B), the loss of cellularity (C), the loss of matrix stain (D) and the loss of tidemark integrity were measured. ^*P < 0.05, ^**P < 0.001 compared to sham group. ^#P < 0.05, ## P < 0.001 compared to OA group. ^※P< 0.05 compared to T(M) group. All rats were n = 8.

Figure 5

Photographs showed micro-CT scan of proximal tibia in different groups. (A) The result showed photomicrographs of the knee in saggital and transverse views from micro-CT. The subchondral bone medial compartment of each group was marked (red box). The scale bar represented 1 mm and rats n = 3. (B), (C), (D) and (E) showed the graphic illustrations of bone volume, bone porosity, trabecular bone thickness and trabecular number in different groups. ESWT groups showed significant increases in bone volume, and trabecular numbers, and decrease in bone porosity and trabecular thickness as compared to sham group and OA group. *P < 0.05, **P < 0.001 compared to sham group. ^#P < 0.05, ^##P < 0.001 compared to OA group. ^※P < 0.05 compared to T(M) group. All rats were n = 8.

Figure 6

Immunohistochemical analysis for molecular changes on different positions with ESWT. Microscopic features of immunohistochemical stains (left) and quantification (right) showed the effect of TUNEL assay (A) and the expression levels of PCNA (B) and CD31 (C) after ESWT on different positions. ^*P < 0.05, ^**P < 0.001 compared to sham group. ^#P < 0.05, ^##P < 0.001 compared to OA group. ^※P < 0.05, ^※※P < 0.001 compared to T(M) group. All rats were n = 8. The scale bar represented 100 μm.