Page 24 - manuscript_ijb05580

forming phase. Notably, increased ALP activity does not necessarily indicate extensive mineralized

bone formation; thus, on day 14, ARS was performed to visualize calcium nodules on EO-MTs

(Figure S4), followed by quantitative analysis via dissolution with 10% (w/v) cetylpyridinium

chloride (Figure 6B). The results showed significantly higher calcium deposition in the osteogenic

induction group than in the blank group, confirming calcium salt deposition and indicating

mineralized bone formation, with ALP and ARS staining, markers of early and late osteogenesis,

respectively, mutually validating the successful osteogenic functionalization of EO-MTs. These

findings align with the regulatory role of RunX2, the master transcription factor of osteogenesis,

where ALP upregulation and ARS-positive mineralization represent the terminal events of RunX2-

mediated signaling cascade activation. For C-MTs, GAG, a critical component of chondrocyte

ECM, serves as a direct indicator of chondrogenic microtissue maturity and quality. On day 4 of

co-culture, GAG content in supernatants (Figure 6C) showed that 2D-cultured cells exhibited

higher GAG levels (106.8 μg/mL) than 3D co-cultured cells on PMs (49.5 μg/mL), attributed to

the fact that 3D PMs provide abundant attachment sites for secreted GAG, which gradually deposits

on the scaffold to form ECM, whereas 2D-cultured GAG diffuses freely into the medium.

Additionally, for 3D co-cultured C-MTs, deposited GAG content was detected at different time

points and normalized to DNA content (Figure 6D), revealing that chondrocytes gradually adapted

to the microenvironment, activated the synthetic phenotype, and initiated GAG and proteoglycan

synthesis over time, indicating microtissue formation and favorable cellular functionality. This

progressive GAG accumulation corresponds to the terminal output of SOX-9 signaling cascade, as

SOX-9, the central transcription factor for chondrogenesis, directly regulates the expression of

GAG-synthesizing enzymes and proteoglycan core proteins. In summary, functionalized culture of

microtissues enables improved recapitulation of the native cartilage and subchondral bone

microenvironment.

23