Fig 1.
Inflammatory score and ankle perimeter.
Arthritic rats have a rapidly disease progression including ankle swelling, when compared with healthy control rats. Statistical differences were determined with non-parametric Mann Whitney test using GraphPad Prism (GraphPad, California, USA). Differences were considered statistically significant for p values ≤ 0.05. Healthy D11 N = 11, Healthy D22 N = 30, Arthritic D11 N = 16 and Arthritic D22 N = 31.
Fig 2.
Histological images of joints after 11 and 22 days of disease induction.
These patterns are merely illustrative of the type of histological features observed. Black arrow indicates the absence/presence of ankle swelling in rat hind paws. C–calcaneus, E–edema or erosion, S–synovia, Tb–tibia, Ts–tarso. Magnification of 50X. Bar: 100 μm.
Fig 3.
Semi-quantitative evaluation of histological sections of inflammation and tissue damage locally in the joints of AIA rats.
Notice that results demonstrate that arthritic rats after 11 and 22 days of disease induction increase cellular infiltration (A), number of lining layer cells (B), bone erosions (C) and cartilage surface damage (D). Global disease severity demonstrates this marked inflammation and progression between day 11 and 22 (E). Data are expressed as median with interquartile range. Differences were considered statistically significant for p-values<0.05, according to the Mann Whitney test. Healthy D11 N = 11, Healthy D22 N = 30, Arthritic D11 N = 16 and Arthritic D22 N = 31.
Fig 4.
Serum samples collected at day 11 and 22 post disease induction were analyzed by ELISA technique. IL6 was increased in arthritic rats at day 11 and 22 (p = 0.0003 and p<0.0001vs healthy controls, respectively). Differences were considered statistically significant for p-values<0.05, according to the Mann–Whitney tests Healthy D11 N = 11, Healthy D22 N = 21, Arthritic D11 N = 16 and Arthritic D22 N = 23.
Fig 5.
Bone turnover markers quantification.
Serum samples collected at day11 and 22 post disease induction were analyzed by ELISA technique. Bone resorption marker, CTX-I (A) and bone formation marker, P1NP (B) were increased in arthritic rats at day 22 (p<0.0001 and p = 0.0007, respectively). Results also demonstrate increased values of CTX-I in arthritic rats at day 11 when compared with healthy controls (p = 0.0218). Differences were considered statistically significant for p-values<0.05, according to the Mann–Whitney tests. Healthy D11 N = 11, Healthy D22 N = 18, Arthritic D11 N = 16 and Arthritic D22 N = 18.
Fig 6.
Micro-computed tomography (micro-CT)—Cortical analysis of tibiae rat sample.
The cortical bone area showed decreased values in the arthritic group at day 11 and 22(A), as well as the polar moment of inertia (D), the minimum (Imin) (mediolateral) (E) and the maximum (Imax) (anteroposterior) (F) moment of inertia. Arthritic group at day 22 presented a marked deterioration of bone tibia demonstrated by decreased average cortical thickness (B) and increased endocortical perimeter (C). Differences were considered statistically significant for p-values<0.05, according to the Mann–Whitney tests. Healthy D11 N = 11, Healthy D22 N = 30, Arthritic D11 N = 16 and Arthritic D22 N = 31.
Fig 7.
Micro-computed tomography (micro-CT)—Trabecular analysis of tibiae rat sample.
MicroCT images from healthy and arthritic tibiae rats (A). Images acquired with SkyScan 1272, Bruker microCT, Belgium. Results showed decreased values of the ratio bone volume/tissue volume (B), trabecular thickness (C) and number (D) in arthritic group at day 11 and 22 post disease induction. Trabecular bone also showed increased values of trabecular separation (E), porosity (F) and structural model index in both arthritic groups (G). Differences were considered statistically significant for p-values<0.05, according to the Mann–Whitney tests. Healthy D11 N = 11, Healthy D22 N = 30, Arthritic D11 N = 16 and Arthritic D22 N = 31.
Fig 8.
Bone mechanical properties assessed by three-point bending tests in rat femur.
Results showed that arthritic rats at day 22 have decreased properties at yield point, related to displacement (A), strength (B) and energy (elastic energy) (C). Arthritic bones at day 22 required a lower maximum load (D) to fracture, with a decreased elastic energy at maximum load (E) and toughness (F). Differences were considered statistically significant for p-values<0.05, according to the Mann–Whitney tests. Healthy D11 N = 5, Healthy D22 N = 14, Arthritic D11 N = 5and Arthritic D22 N = 10.
Fig 9.
Bone mechanical properties assessed by nanoindentation in rat femur at 11 and 22 days post disease induction and respective optical micrographs from the indentation tissue area.
Nano-mechanical tests revealed that arthritic rats have decreased cortical hardness at day 22 and of trabecular hardness at day 11 and 22 post disease induction (B). Results demonstrated that concentric lamellae (C) and ratio of area occupied by osteocyte lacunae in the total tissue (D) are increased when compared to healthy animals at day 22. Images are merely illustrative of the type of histological features observed. Concentric lamellas are identified in secondary osteons (SO), characteristic from arthritic animals at day 11 (G) and 22(H). On the contrary, parallel-lamellae (PL) are identified in healthy at day 11 (E) and 22 (F). Os—Osteocytes, SO—Secondary osteons, PL—Parallel-lamellae, CL—Concentric lamellas. Magnification 20X. Differences were considered statistically significant for p-values<0.05, according to the Mann–Whitney tests. Healthy D11 N = 11, Healthy D22 N = 28, Arthritic D11 N = 16 and Arthritic D22 N = 21.