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Figure 6. Functional characterization of microtissues during culture. (A) ALP activity of EO-MTs

at day 7 of osteogenic induction culture. (B) Quantitative analysis of ARS staining for calcium

deposition in EO-MTs at day 14 of osteogenic induction. (C) GAG content in the supernatants of

C518 cells cultured for 4 days under different conditions. (D) Temporal changes in GAG content

of C-MTs at different culture time points (n=3, *P<0.05, **P<0.01, ***P<0.001).

3.3. Characterization of MTs/GelMA Composite Bioink

GelMA was successfully synthesized via amidation of gelatin amino groups with methacrylic

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anhydride, as confirmed by H NMR spectrum, which showed characteristic peaks of methacrylate
vinyl protons (δ = 5.31, 5.55 ppm) and attenuation of the amide-associated signal (δ = 2.88 ppm),

indicating effective double-bond modification (Figure 7A). GelMA hydrogels exhibited an

interconnected porous structure after lyophilization (Figure 7B), favorable for nutrient diffusion

and cell infiltration. Subsequently, PMs/GelMA composite hydrogels were prepared, and SR and

MR tests were performed (Figure 7C, D). The results showed that the addition of PMs had no

significant effect on the bioink, possibly because the added PMs were already in an equilibrium

state, while the slight increase in the composite group might be due to the incorporation of MTs

increasing the internal space of the overall structure. Mechanical property test results (Figure 7E,

F) demonstrate that as the MTs concentration increases from 0 mg/mL to 20 mg/mL, the stress-

strain curves of the composite hydrogels exhibit a trend of increased stiffness, with a corresponding

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