*2.9. Cell cytotoxicity Test*

Cell cytotoxicity was carried out using the MTS method. In brief, chondrocytes were harvested from culture flask and seeded in a 24-well tissue culture plates at a density of 5 × 104 cells/well. HA-AGMCs were added to the cell culture in different concentrations (0, 0.05, 0.11, 0.21, 0.43, 0.85, 1.7, 3.4, 6.8, 13.6 mg mL−<sup>1</sup> ). After 1, 2, or 4 days incubation, culture medium was replaced with DMEM/F-12 containing 10% MTS and reacted for 2 h. Each medium was collected and centrifuged at 6000 rpm for 5 min to remove HA-AGMCs. The absorbance of supernatant was monitored by ELISA reader at wavelength 490 nm. The experiment was done in triplicate.

#### *2.10. 3D culture Methods*

The experiment method of agarose hydrogels followed the protocol of 3D Petri dish molds [27]. Briefly, agarose powder (1 g) was dissolved in PBS and then pipetted into micro-mold without creating any bubble in proper temperature. After solidification, the agarose hydrogels were placed in culture medium to equilibrate for at least 15 min, and then transferred to fresh medium for further use or storage. All steps were in sterile conditions. Cells at a final density of 2.56 × 105 cells/190 µL were seeded in each agarose hydrogel placing in 6 well plate and waited 10 min to settle cells. Finally, we added additional medium to the plate (2.5 mL/well).

### *2.11. Cell proliferation and Cell Compatibility Analysis*

MTS assay was performed to assess the proliferation of chondrocytes. In short, fresh media containing different concentrations of HA-AGMCs (42.5, 85, 170, 340, and 680 µg mL−<sup>1</sup> ) were mixed with chondrocytes before seeding in each agarose hydrogel placing in plate and waited 10 min to settle cells. Finally, additional medium was added to cover the hydrogel. After incubation for various time period, cell pellets were collected and counted before adding 10% MTS medium. Each medium was collected after 3 h reaction and the optical density was monitored by ELISA reader at wavelength 490 nm. The experiment was run in four times. Cell compatibility was also evaluated by Live/Dead assay. After days of incubation, cell pellets were collected and washed with PBS buffer. Staining reagent Calcein AM and Ethidium homodimer-1 in PBS covered the pellets at 37 ◦C for 30 min. The samples were observed under confocal microscopy. The experiment was done in triplicate. The fluorescence intensity in each pellet was measured by ImageJ and further analyzed by unpaired *t*-test.
