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Figure 5. CLSM images (A) of MAEC and MC3T3-E1 cells in EO-MT; (B) and of aggrecan,

collagen I, and collagen II in C-MT. (C) H&E staining of EO-MT and C-MT.

To better recapitulate the native cartilage and subchondral bone microenvironment, we further

performed functionalized culture of the prepared EO-MTs and C-MTs. For EO-MTs, osteogenic

induction medium was formulated by supplementing complete medium with osteogenic growth

factors, including dexamethasone, ascorbic acid, and β-glycerophosphate sodium, followed by

long-term culture. ALP, a key and classical biomarker for osteoblast activity and bone formation,

directly reflects osteoblast maturation and functional status and plays a crucial role in bone matrix

mineralization; its expression is significantly upregulated when osteoblasts transition from the

proliferative to the mature differentiation phase. On day 7, quantitative analysis of ALP activity in

EO-MTs using an ALP assay kit (Figure 6A) revealed that the osteogenic induction group exhibited

significantly higher activity (0.141 μM) compared to the non-induced blank group (0.107 μM),

indicating that osteoblasts adhering to and proliferating on EO-MTs had entered an active bone-

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