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whereas 3D-printed scaffolds can form regular and interconnected macroporous channel networks

through parameter design (e.g., layer height, pore size, porosity), thereby providing superior mass

transfer capacity, including the transport of oxygen and nutrients as well as the removal of

metabolic waste. Furthermore, the mechanical microenvironment of 3D scaffolds is better suited

for cell proliferation. Mold-cast solid cylindrical hydrogels generally have high and uniformly

distributed overall mechanical strength, while the porous structure of 3D scaffolds allows

regulation of local stiffness through design, more closely mimicking the mechanical heterogeneity

of native tissues. The channel structure of the scaffold permits local elastic deformation, avoiding

“mechanical compression” of cells caused by excessive overall rigidity of solid cylinders, while

providing moderate mechanical stimulation. Meanwhile, the porous network of 3D scaffolds

facilitates cells to secrete their own ECM (e.g., collagen, fibronectin) and undergo matrix

remodeling, forming a “cell-matrix” positive feedback loop that further supports proliferation; in

contrast, the dense structure of solid cylinders limits the active-matrix remodeling capacity of cells.

Subsequently, to better recapitulate the osteochondral model, the scaffolds were continuously

subjected to functionalized culture. After one week of culture, the scaffold maintained good

structural integrity (Figure 8C i)). On day 14, ALP activity in the bone layer of the scaffold was

characterized using an ALP chromogenic kit (Figure 8C ii)), and on day 21, the mineralization

level of the bone layer was characterized via ARS staining (Figure 8C iii)). The results indicated

that the scaffold exhibited favorable osteogenic capacity.

EO-MTs and C-MTs were fluorescently labeled separately and then subjected to bioprinting.

After culturing for different time periods, the scaffolds were observed via CLSM. The primary

focus was on the interface region between the EO-MTs layer and C-MTs layer. On day 7 of culture

(Figure 8D i)), distinct upper and lower cell layers were observed. In addition, faint red

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