Fig 1.
Histopathological evaluation of cartilage of rat knee joints post-MMD at 4 and 6 weeks post surgeries.
(A) Representative images of histology sections from the medial side of knee joints isolated from, a control unoperated 17-week-old rat, a 15-week old rat at 4 weeks post-surgery (4-wks), and 17-week-old rat, at 6 weeks post-surgery (6-wks). The section from control unoperated knee shows smooth with no disruption of the articular cartilage surface integrity. Representative image of the medial condyle and tibia plateau of the MMD-knee, isolated at 4 weeks post-surgery, shows irregularities at the superficial layer of the articular cartilage (AC), fibrillation (black arrows) and loss of Safranin-O staining at the superficial zone (black stars). Image of the medial condyle and tibia plateau, isolated at 6 weeks post-surgery, shows severe cartilage degeneration, loss of Safranin-O staining (black stars) and depletion of chondrocytes (open arrows) with irregular trabecular bony between the junction of calcified cartilage (black arrowhead), and osteophyte formation (Of). Sections were stained with Safranin-O/Fast Green and observed using microscope objective lenses 1.25x and 4x., AC. for articular cartilage; Of. for osteophytes; T. for tibia plate and F. for femoral condyle. (B) Microscopic examination of the safranin-O stained sections. The changes in the articular cartilage were graded based on the changes in structure (0–6 points), cellularity (0–3 points), matrix staining (0–4 points), and tidemark integrity (0–1 points), and has a maximum of 14 points. The final score for each knee joint was based on the most severe histologic changes observed from multiple sections of each specimen. Data are presented as mean of 6 knees ± SE, n = 6. *P<0.05 vs control unoperated knees, #P<0.05 vs MMD-Knees at 4 wk-post-surgery.
Fig 2.
MMD caused recruitment of inflammatory cells to rat injured knees as early as one day post-MMD.
Hematoxylin-Eosin (H&E) staining of histology sections collected from sham-operated left knee joint (A), MMD-operated knee joint collected, at one day post-surgery (B) and 3 days post-surgery (C). Dashed black squares in the left panels indicate the regions magnified at the right panels. Inflammatory cells are obvious in the synovium of MMD-knee joint with abundant granulocytes (black arrows) and mononuclear cells (open arrowheads) at one day post-surgery. D. Cell density quantification at one and three days post-MMD in the synovial tissue of rat knee joints. Nuclei were counted in 2 fields per histology section, close to articular cartilage. 3 histology sections were evaluated from each knee. The sections were chosen in approximately the same knee joint area for comparison. We have counted the number of nuclei per mm2 synovial space in the MMD-operated right (Rk) knees and contralateral left knee (Lk) joints. Data are presented as mean ± SE, n = 6 right knees (Rk), *P<0.05 vs Lk sham-operated, #P<0.05 between Rks of 1d- and 3d- post surgery.
Fig 3.
Evaluation of the changes in mRNA expression levels of different genes in rodent knee joints at different time points post-MMD.
Two inflammatory cytokines, Il-6 and Tnfa (A) and two major matrix metalloproteinases, Mmp3 and Mmp13 in rat knees (B); The two chemokines of interest in this study in rat knees (C) and Ccl21 in mouse knees (D). Control unoperated knees and MMD-operated knees were collected from rats at three time points post-surgeries (A, B, C), and from mice at 4 time points (D). RNA was isolated from both MMD-operated and control unoperated knees, and mRNA expression level was evaluated by qPCR using pre-designed primers. Data are expressed as fold change in response to MMD as compared to control unoperated knees (Lk), and are presented as mean ± SE, n = 6 and *P<0.05 vs control un-operated knees.
Fig 4.
MMD caused the recruitment of CD4 and CD8 positive cells to rat injured knees at one day post-MMD.
Quantification of mRNA expression levels for the markers of three T cell subsets isolated from rat knee joint synovial tissue, at one day post-surgery. Both right MMD-operated knees and left sham-operated knees (Lk) were collected. Data are expressed as fold change vs control sham-operated knees and are presented as mean ± SE, n = 4–5, *P<0.05 vs control sham-operated knees (Lk).
Fig 5.
IL-6 synthesis is induced in response to MMD at three days post-surgery.
Immunohistochemistry staining was performed using antibodies against rat IL-6 for both rat MMD- (A) and the left sham-operated knees (B) collected at day 3 post-surgery. Representative images of histology sections collected from rat MMD- (A) and sham-operated knees (B), brown color represents IL-6 positive areas. Arrows show some of IL-6 positive stained areas. The squares in the left panels represent the areas magnified 20x on the right panels. T. tibia plateau, F. femur condyles, ST, synovial tissue. MCL, medial collateral ligament. A and B were taken using 1.25x microscope lenses.
Fig 6.
CCL21 synthesis was increased in response to MMD compared sham-operated knees.
Immunohistochemistry staining was performed using antibodies against rat CCL21 antibodies for both sham- (A) and MMD- operated knees (B and C), collected from rats at day 3 (A and B) and 4 weeks (C) post-surgeries. Representative images of histology sections collected from Sham- (A) and MMD-operated knees (B and C), brown color represents CCL21 positive areas. Arrowheads show some of CCL21 positive stained areas. The small squares on the left panels represent the areas magnified 20x on the right panels. T. tibia plateau, F. femur condyle, ST, synovial tissue. A, B and C images were taken using 1.25x microscope lenses.
Fig 7.
Treatment with CCL21 antibody ameliorates MMD-induced PTOA development in rat knees.
Representative images from histology sections isolated, at 4-wks post-surgery from MMD knees treated with PBS (A) showing irregularities, fibrillations at the articular cartilage (black arrows) and loss of safranin-o staining (black stars) at the medial side. The MMD-knees treated with10 μg CCL21-Ab (B), showing fewer irregularities and thinner fibrillations (black arrowheads) at the superficial zone of the articular cartilage and less loss of safranin-O staining (black stars). Bleu arrows show tide mark line. Sections were stained with Safranin-O/fast green and observed using microscope objectives 1.25x (A and B) and 4x (right panels). T. tibia plateau and F. femur condyle. AC for articular cartilage. Cartilage damage quantification (C). Histopathology Evaluation of MMD-operated knees at Four Week- Post-Surgery, for PBS and CCL21-Ab treated rats. Data are expressed as mean ± SE, n = 6–8, *P<0.05 vs controls, #P<0.05 vs MMD-PBS.
Fig 8.
The effect of local blockade of CCL21 function, on inflammation in rat knees, at three days post-surgery.
mRNA levels of the major pro-inflammatory cytokine Il-6 and the chemokines Ccl21 and Cxcl13 (A), Mmp3 and Mmp13 (B) and the markers of four inflammatory cell subsets (C). Right knees (Rk) were MMD-operated and left knees (Lk) were control un-operated from PBS treated rats. Data are expressed as fold change of the expression of each gene in the Rk treated with either 10 μg CCL21-Ab or PBS vs Lk of rats treated with PBS (Lk. PBS) and are represented as mean ± SE. n = 3–5, *P≤0.05 vs Lk. PBS and #P≤0.05 Rk-CCL21-Ab vs Rk-PBS.
Fig 9.
Effect of blocking the function of CCL21 in rat knees, on Mmp3 and Mmp13 and four markers of inflammatory cell subsets, at day five post-knee surgery.
Right knees (Rk) were MMD-operated and left knees (Lk) were un-operated. Data are expressed as fold change of the expression of each gene in the Rk treated with either CCL21-Ab or PBS vs Lk. PBS and are represented as mean ± SE. n = 4, *P≤0.05 vs Lk. PBS and #P≤0.05 Rk-CCL21-Ab vs Rk-PBS.
Fig 10.
MMD induced T cell infiltration to rat injured knees and treatment with CCL21-Ab reduced the number of CD4+ and CD8+ cells in MMD-knees at day 3 post surgery.
Images of areas from knee joints’ synovial tissues collected from; Sham-, control MMD-knees treated with PBS (MMD-PBS). and MMD-knees treated with CCL21-Ab (MMD-CCL21.Ab). Histology sections collected from rat knees at day 3 post-surgery were used. Immuno-histochemistry staining was performed using rat CD4 (A and C) and CD8 (B and D) antibodies, and counterstained with hematoxylin. Images in red squares represent areas from medial side of knee joints. Images in blue squares represent areas from collateral side of the knee joints. Images in green squares were taken at the frontal end of joint capsule, near the connection of the Patella ligament with femur. The exact position in knee joints is indicated in S1 Fig. The graph in the right bottom panel represents the number of positive stained cells per unit area at the joint capsules (E). Data are expressed as number of positive stained cells per unit area and are presented as mean ± SE. *P<0.05 vs Lk sham-operated knees. #P<0.05 vs MMD-saline, n = 5–6 knees.