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are representative. The cell adhesion rate was calculated using the following formula (Equation 1),

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as previously reported .

Cell adhesion rate (%) = Cell adhesion/Cell inoculation number ×100 (I)

MAEC and MC3T3-E1 cells were labeled with Qtracker®655 and Qtracker®525, respectively,

and the distribution of cells in EO-MT was observed by Leica TCS SP8 confocal laser scanning

microscopy (CLSM) (Leica Microsystems GmbH, Germany). Immunofluorescence staining was

used to observe the expression of aggrecan, collagen I, and collagen II in C-MT. CCK-8 method

was used to detect the proliferation of cells in the microtissues under static and dynamic culture.

The microtissues were fixed with paraformaldehyde and stained with DAPI for CLSM imaging.

The other microtissues were embedded in paraffin and sectioned for HE staining. After dehydration

with gradient ethanol solution, the fixed sealing plates were observed with a light microscope

(Nikon Eclipse Ts2, Japan). Assays were performed with triplicate cultures, and data are

representative of three experiments.

MAECs and MC3T3-E1 cells were co-cultured using the method described previously. After

5 days of co-culture, the cells were transferred to 24-well plates. Half of the wells were maintained

in regular complete medium (supplemented with 10% (v/v) FBS), while the other half were

switched to osteogenic induction medium, which contained 10% (v/v) FBS, 1 mM Dex, 10 mM

ascorbic acid, and 1 M β-glycerophosphate sodium. On day 7, alkaline phosphatase (ALP) activity

was detected using an ALP detection kit kit (Beyotime, Shanghai, China). On day 14, calcium

deposition on EO-MTs was stained with Alizarin Red-S (ARS). After thorough staining, the

staining solution was washed off, and the ARS-stained calcium deposits were dissolved in 10%

(w/v) cetylpyridinium chloride. The absorbance was then measured at a wavelength of 562 nm.

For C518 cells of the same passage, cells were harvested by trypsinization and seeded into 6-

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