Surface patterns and topographies play a pivotal role in directing the stem cell fate and extracellular matrix (ECM) organization. Here, we present a cost-effective, 3D printing-assisted template strategy to generate macroscale surface patterns on hyaluronic acid methacrylate (HAMA) hydrogels. Polylactic acid (PLA) templates with honeycomb, rhombohedral, and triangular geometries were fabricated, and free-radically cross-linked HAMA hydrogels were cast and demolded to yield patterned constructs. Based on the rationale that such patterns can approximate aspects of cartilage zonal organization, we hypothesized that these customizable topographies would enhance the chondrogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells (hBM-MSCs). A comprehensive evaluation was performed using mechanical testing, swelling analysis, biochemical assays (sGAG, collagen content), metabolic activity (Alamar Blue), immunofluorescence staining, and RT-qPCR for chondrogenic (SOX9, COL2A1, ACAN) and hypertrophic (COL10A1) markers. Honeycomb hydrogels exhibited superior stiffness, swelling resistance, and laminin adsorption compared with rhombohedral, triangular, and nonpatterned controls. hBM-MSCs cultured on honeycomb hydrogels in a chondrogenic medium demonstrated enhanced viability, greater sGAG and collagen deposition normalized to DNA content, and robust upregulation of chondrogenic markers, while limiting hypertrophy and dedifferentiation. Immunostaining further confirmed cartilage-specific ECM organization, with spatial alignment and condensation of MSCs on honeycomb topographies, suggesting the activation of mechanotransduction pathways. This work establishes an innovative design principle linking hydrogel macrogeometry to chondrogenic outcomes, addressing a gap in the field where most studies have focused on micro- and nanoscale cues. Although these hydrogels do not fully replicate all four native cartilage zones, the honeycomb topography promoted alignment, condensation, and stratified ECM deposition, recapitulating key aspects of zonal organization. This 3D-printed template-assisted macropatterning strategy provides a cost-effective, reproducible, and scalable method for fabricating biomimetic scaffolds. These surface patterned hydrogels enable controlled differentiation and spatially organized ECM formation, underscoring their promise for cartilage tissue engineering and personalized repair strategies.
