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Recent studies have highlighted the role of external biophysical cues on cell morphology and function. In particular, substrate geometry and rigidity in two dimensions has been shown to impact cell growth, death, differentiation, and motility. Knowledge of how these physical cues affect cell function in three dimensions is critical for successful development of novel regenerative therapies. In this work, the effect of discrete micromechanical cues in three-dimensional (3D) system on cell proliferation, gene expression, and extracellular matrix synthesis was investigated. Poly(ethylene glycol) dimethacrylate hydrogel microrods were fabricated using photolithography and suspended in gel to create a 3D culture with microscale cues of defined mechanical properties in the physiological range (2–50 kPa). These microrods significantly affected fibroblast proliferation, matrix production, and gene expression. Cultures with stiff …