*2.7. Loading E*ffi*ciency of SPIOs*

SPIOs content was measured using UV-vis spectroscopy (UV-vis, Evolution 300, Thermo). Calibration curve was made by dissolving SPIOs (10 mg) and AG-g-HA (75 mg) in HCl (0.5 N). From UV-vis spectroscopy, the absorbance of the solution was measured at 363 nm. HA-AGMC was also diluted with HCl (0.5 N) to calculate the corresponding loaded SPIOs concentration.

### *2.8. Chondrocyte Isolation and Culture*

Chondrocytes were isolated from the articular cartilage of New Zealand White rabbits (0.4–0.8 kg). All procedures conformed to the guidelines of the Institute of Animal Care and Use Committee of I-Shou University in Taiwan. All the surgical instruments were sterilized before use. After rinsing thighbones with phosphate-buffered saline (PBS) two times, cartilage tissue from the joint was dissected and cut into pieces of approximately 1 <sup>×</sup> 1 mm<sup>2</sup> samples. These samples were digested with protease (20 mg in 10 mL DMEM/F-12) for 2 h and transferred to collagenase (20 mg in 10 mL DMEM/F-12) for another 3 h. The cell suspension was centrifuged at 2000 rpm for 5 min and resuspended in DMEM/F12 medium with 10% FBS in 75T culture flask.

Chondrocytes were seeded in monolayer and used within two passages. The cells were cultured in DMEM/F12 medium with 10% FBS in a 5% CO<sup>2</sup> incubator at 37 ◦C. The medium was renewed every 2 days and cells were passaged once it reached confluence.
