Pathological, Morphometric and Correlation Analysis of the Modified Mankin Score, Tidemark Roughness and Calcified Cartilage Thickness in Rat Knee Osteoarthritis after Extracorporeal Shockwave Therapy

Abstract

The paper displayed the pathological changes and relationships of the modified Mankin score, tidemark roughness and calcified cartilage (CC) thickness by extracorporeal shockwave therapy (ESWT) (0.25 mJ/ mm² with 800 impulses) on different positions of the medial and lateral rat knee OA joint. After the experiments, the articular cartilage was assessed using histomorphometry, image analysis and statistical method. In the micro-CT analysis, ESWT on medial groups were better than lateral groups in the trabecular volume and trabecular number. The data showed a strong negative correlation between the modified Mankin score and tidemark roughness (r = -0.941; P < 0.001). In terms of the relationship of tidemark roughness with CC thickness, the medial and Sham groups showed a significant negative correlation (r = -0.788, P = 0.022). Additionally, the Euclidean distance derived from 3D scatter plot analysis was an indicator of chondropathic conditions, exhibiting a strong correlation with OA stage in the articular cartilage of the femur (r = 0.911, P < 0.001) and tibia (r = 0.890, P < 0.001) after ESWT. Principle component analysis (PCA) further demonstrated that ESWT applied to medial locations had a better outcome than treatment at lateral locations for knee OA by comparing with Sham and OA groups, and CC thickness was the most important factor affecting hyaline cartilage repair after ESWT.

Keywords: calcified cartilage thickness; extracorporeal shockwave therapy; osteoarthritis; statistical analysis.; tidemark roughness.

Figure 1

Micro-CT analysis. (A) Micro-CT images illustrated the positions of ESWT applied to the knee (white arrowheads) and the frontal-coronal view of the femur (F) and tibia (T) in each group by using CT-analyzer software (www.blue-scientific.com/bruker-ctan-micro-ct-software/). The anatomical side of the left knee OA joint was noted as medial (M). Osteophytes (white arrows), cysts (white star) and erosions (red arrowheads) formation were observed. The micro-CT data of the trabecular bone volume, and trabecular number in the subchondral bone of femur (B) (D) and tibia (C) (E) of the rat knee. ^*P < 0.05 was as compared with Sham group. ^#P < 0.05 was as compared with OA group. The lateral site is indicated by L. M-T as the medial tibia condyles. M-F as the medial femur condyle. M-FT as the medial femur and tibia condyle. ML-T as the medial and lateral tibia condyle. L-T as the lateral tibia condyle. L-F as the lateral femur condyle. L-FT as the lateral femur and tibia condyle. LM-F as the lateral and medial femur condyle.

Figure 2

Pathological analysis. (A) Knee sketch displays the 3 sub-regions, which are the anterior (a), central (c) and posterior (p) regions, on the femoral and tibial condyles in the sagittal plane. (B) A section of the hyaline cartilage in the sagittal plane is stained with safranin-O, where the layers of the uncalcified cartilage (UCC; dark red), calcified cartilage (CC; light red) and subchondral bone (SB; blue) are shown. The tidemark (white arrows) is located between the UCC and CC layers, and the cement line (black arrows) is located between the CC and SB layers. The magnification is 400×. (C) The tidemark length (black arrow) is the actual length of the tidemark, and the tidemark distance (white arrow) is the shortest length of the tidemark. The magnification is 400×. (D) The CC area (white box) is the region between the UCC and SB layers, and the distance of the CC area is the length of the CC area (white arrow). The magnification is 400×. n = 8 to 10 for each group.

Figure 3

Cartilage matrix conditions of the femur and tibia following ESWT application to various sites. The femoral (A) and tibial (B) condyle sections of the articular osteochondral tissues are stained with safranin-O and observed at 200× magnification and 400× magnification. n = 8 to10 for each group. M-F as the medial femur condyle. M-FT as the medial femur and tibia condyle. ML-T as the medial and lateral tibia condyle. L-T as the lateral tibia condyle. L-F as the lateral femur condyle. L-FT as the lateral femur and tibia condyle. LM-F as the lateral and medial femur condyle.

Figure 4

Changes in the articular cartilage evaluated by the modified Mankin score after ESWT at various sites of the femur (A) and tibia (B). * indicates the difference as compared with the Sham group, P < 0.05; # indicates the difference as compared with the OA group; P < 0.05. n = 8 to 10 for each group. M-F as the medial femur condyle. M-FT as the medial femur and tibia condyle. ML-T as the medial and lateral tibia condyle. L-T as the lateral tibia condyle. L-F as the lateral femur condyle. L-FT as the lateral femur and tibia condyle. LM-F as the lateral and medial femur condyle.

Figure 5

Tidemark roughness (R_tm) in the central regions of the femur (A) and tibia (B) condyles. * indicates the difference as compared with the Sham group, P < 0.05; # indicates the difference as compared with the OA group, P < 0.05. Undetermined Rtm is indicated as a score of 1. n = 8 to 10 for each group. The correlation of all groups between the modified Mankin score and R_tm are presented in the femur (C) and tibia (D) after ESWT. (E) The mean correlation between the modified Mankin score and R_tm was negative, with r = -0.941; P < 0.001. M-F as the medial femur condyle. M-FT as the medial femur and tibia condyle. ML-T as the medial and lateral tibia condyle. L-T as the lateral tibia condyle. L-F as the lateral femur condyle. L-FT as the lateral femur and tibia condyle. LM-F as the lateral and medial femur condyle.

Figure 6

Calcified cartilage thickness (CC_thick) in the central femur (A) and tibia (B) condyles. There were no significant differences when comparing the OA group with the other groups due to the high degree of fluctuation in standard deviation. * indicates the difference as compared with the Sham group, P < 0.05; # indicates the difference as compared with the OA group, P < 0.05. n = 8 to 10 for each group. The correlation of all groups between the modified Mankin score and CC_thick are presented in the femur (C) and tibia (D) after ESWT. (E) The mean correlation between the modified Mankin score and CC_thick was significant (r = 0.746; P = 0.034) in the Sham, M-FT, ML-T, M-T and M-F groups; however, there was no significant correlation (r = -0.372; P = 0.235) in the LM-F, OA, L-T, L-F and L-FT groups. (F) The mean correlation between the R_tm and CC_thick was significant (r = -0.788; P = 0.022) in the Sham, M-FT, ML-T, M-T and M-F groups; however, there was no significant correlation (r = 0.336; P = 0.501) in the LM-F, OA, L-T, L-F and L-FT groups. M-F as the medial femur condyle. M-FT as the medial femur and tibia condyle. ML-T as the medial and lateral tibia condyle. L-T as the lateral tibia condyle. L-F as the lateral femur condyle. L-FT as the lateral femur and tibia condyle. LM-F as the lateral and medial femur condyle.

Figure 7

Pathological and Morphometric correlations. (A) Correlations of three‐dimensional scatter plots with (B) Euclidean distance and percentage of cartilage involvement for the femur and tibia as assessed from the data of the modified Mankin score, CC_thick, and R_tm after ESWT at different positions of the rat OA knee. (C) Principle component analysis (PCA) of the femur and tibia with multiple inter-correlated variables including modified Mankin score, CC_thick, R_tm, Euclidean distance, and surface cartilage involvement by using Sigma blot version 14.0 (https://systatsoftware.com/products/sigmaplot/). n = 8 to 10 for each group. M-F as the medial femur condyle. M-FT as the medial femur and tibia condyle. ML-T as the medial and lateral tibia condyle. L-T as the lateral tibia condyle. L-F as the lateral femur condyle. L-FT as the lateral femur and tibia condyle. LM-F as the lateral and medial femur condyle.