*4.3. Animals Experiments*

All animal experiments were performed following the approval of the Institutional Animal Care and Use Committee-IACUC from Kaohsiung Medical University (The project identification code and date: IACUC105127; 1 Aug 2017–31 July 2020). A total of 40 healthy male C57BL/6 mice (8-weeks-old) were obtained from the Jackson laboratory (National laboratory animal center, Taiwan), and were housed under the standard conditions in Animal Center of Kaohsiung Medical University (Kaohsiung, Taiwan). The animals were randomly assigned into four groups, including the sham group (sham operation treated by Normal saline, *n* = 10), ACLT group (OA induction treated by Normal saline, *n* = 10), ACLT plus 7 rh 6.9 nM (*n* = 10), and ACLT plus 7 rh 13.8 nM (*n* = 10). OA was surgically induced in 8-weeks-old C57BL/6 mice by ACLT in the right knee. According to study design, the mice either received IA injection with the same volume (10 μL) of Normal saline, 7 rh 6.8 nM, or 7 rh 13.8 nM three times per week from 7 to 9 weeks-old and then one dose per week until sacrifice at 13 weeks-old.

#### *4.4. Weight Bearing Distribution Test*

The weight-bearing ability after OA-induction was measured by Dual Channel Weight Averages (Singa Technology Corporation, Taiwan), which can analyze the weight-bearing of each hind paw. The changes in joint weight-bearing capacity represent the severity of the experimental joint symptoms. The weight-bearing test was performed at one week before ACLT and then every week until the mice were euthanized.

#### *4.5. Running Endurance*

Mice were subjected to run on a Columbus Instruments rodent treadmill (Columbus, OH, USA) once a week before and after OA-induction until the mice were euthanized. The running speed started from 8.5 m/min and lasted for 3 min. Thereafter, the treadmill speed increased by 2.5 m/min every 3 min with a treadmill angle of 5 degrees until the maximum running speed was 25 m/min. The limit of running endurance recording time was 15 min, and the experiment stopped after reaching the maximum duration. During the whole process, a mild electric shock caused an uncomfortable shock but not physical damage to the mice and was set according to the previous study [17].

#### *4.6. Histopathological Assessments*

The mice tibia samples were fixed in 10% buffered paraformaldehyde for 2 days and then decalcified with buffered ethylenediaminetetraacetic acid (0.5 M, pH 7.4). After dehydration and embedding in paraffin, the tissue blocks were cut in coronal with a thickness of 5 μm. Glycosaminoglycan (GAG) was stained by Safranin O-Fast Green (1% safranin O counterstained, 0.75% hematoxylin, and then 1% Fast Green; Sigma, St. Louis, MO, USA) and was used to evaluate the severity of OA. All of the histological results were assessed by two independent researchers blinded to any other information according to the histology grading system of Osteoarthritis Research Society International (OARSI) [18].
