*2.2. Experimental Design*

As shown in Figure 1A, ACLT + MMx (*n* = 48) or sham surgery (Sham-OP, *n* = 9) was performed at week 0. Body weights, widths of the knee joints, and weight-bearing symmetry were measured before the surgery as the baseline. After the surgery, ACLT + MMx rats were assigned as the nontreated control (OA-control, *n* = 12), the group treated with SNO (223.2 mg/kg, OA-SNO, *n* = 12, administered oral SNO daily after the surgery, the effective dose was derived from our SNO dose-dependent animal report [20]), the group treated with HA (50 μL per joint/week, OA-HA, *n* = 12, received IA injection of HA weekly at weeks 2–4 and 9–11) and a group with combined treatment, HA plus SNO (OA-SNOHA, IA injection of HA (50 μL per joint/week) at weeks 2–4 and 9–11 plus the daily oral SNO (223.2 mg/kg) beginning from the 2nd week). The SNO concentrate provided by Universal Integrated Corp. (Taipei, Taiwan) was administered by oral gavage with the aid of isoflurane anesthesia, and the HA (Seikagaku Corporation, Ibaraki, Japan) was injected into the OA knee joint with a 25 G-needle syringe. As an injection control, 50 μL of saline solution was injected to OA-control (*n* = 6) at week 2–4 (3 weekly injections), and it showed no statistical difference in both knee width and weight-bearing test from non-injected OA-control rats (*n* = 6) (Figure 1B). The 4th week and 12th week knee joint section confirmed the progressive deterioration of OA joint with a reactive chondrocytes hypertrophy, and increasing cartilage erosion accompanied by chondrocytes loss (Figure 1C).

#### *2.3. Knee Width and Weight-Bearing Test*

The width of the knee joint was measured using a steel caliper (resolution 0.01 mm, E-Base Measuring Tools Co., Taipei, Taiwan) biweekly after the surgery, and the width of the contralateral knee served as the naïve control. The data are expressed as the Δ knee width (mm); the value was derived from the OA rats (knee width difference of the operated knee and naïve knee) minus the mean value of the sham-OP rats (knee width difference of the operated knee and naïve knee) and was determined as the actual joint swelling induced by ACLT + MMx.

Hind paw static weight-bearing was measured using an incapacitance tester (Linton Instrumentation, Norfolk, UK) to detect OA-induced changes in postural equilibrium every two weeks. The rats were placed on their hind paws in a box containing an inclined plane (65◦ from horizontal) that was placed above the apparatus. After a brief accommodation period, the weight that the animals applied to each hind limb was measured independently by the apparatus. Five measurements were taken and averaged for each rat. The data are expressed as the difference between the weight applied to the naïve hind limb and the weight applied to the operated hind limb (Δ Force, g); the change in the weight distribution between the naïve and operated hind limb represents the OA pain of the rats [31,32].

**Figure 1.** (**A**) The graphic scheme of study. (**B**) The knee width/weight-bearing changes of OA (osteoarthritis) rats with 3 IA (intra-articular) injection (weekly) of 50 μL of saline, compared to non-injected control. (**C**) The representative section of histological change of ACLT + MMx (anterior cruciate ligament transection plus medial meniscectomy)-induced OA from 4 weeks to 12 weeks post-surgery as compared to sham-OP (sham operated) rats.

#### *2.4. Histopathological Examination of Knee Joint*

All rats were sacrificed via exsanguination under deep anesthesia on the 12th week post-surgery. The OA knee joints were removed and fixed in 10% formalin for 2 days, followed by a decalcifying solution based on EDTA disodium (12.5%, pH 7.0) for 4 weeks. After decalcification, the joints were embedded in paraffin blocks, and histological coronal sections (5 μm-thick serial section, slides interval: 200 μm) were obtained. Toluidine blue/fast green staining was used to examine morphological changes and the stained sections were digitalized using a Slide Scanner ZEISS Axio Scan Z1 image system (Jena, Germany) and ZEN lite 2.6 (blue edition). The severity of articular cartilage damage on medial tibial plateau was evaluated using the modified Osteoarthritis Research Society International (OARSI) scoring system [33]. The cartilage matrix loss width, tibia cartilage degeneration score, total and significant cartilage degeneration widths, and zonal depth ratio of the lesions and synovial reaction were evaluated.

#### *2.5. Metabolic Profile of Blood Biochemistry Assays*

The OA-SNO and OA-control rats fasted for 12 h before the blood sample withdrawal; blood samples were taken from the rat tail vein every 4 weeks post-ACLT + MMx surgery. The blood samples were centrifuged (8000× *g* for 5 min) to separate sera and stored in a −80 ◦C freezer prior to analysis. The serological levels of uric acid, glucose, total cholesterol (T-CHO), high-density lipoprotein (HDL), and triglyceride (TG) were measured using the FUJI DRI-CHEM 4000i analyzer (FUJIFILM Corporation, Tokyo, Japan) at the Taiwan Mouse Clinic (Academia Sinica, Taipei, Taiwan).
