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Three-dimensional (3D) printing of biodegradable polymers has rapidly become a popular approach to create scaffolds for tissue engineering. This technique enables fabrication of complex architectures and layer-by-layer spatial control of multiple components with high resolution. The resulting scaffolds can also present distinct chemical groups or bioactive cues on the surface to guide cell behavior. However, surface functionalization often includes one or more post-fabrication processing steps, which typically produce biomaterials with homogeneously distributed chemistries that fail to mimic the biochemical organization found in native tissues. As an alternative, our laboratory developed a novel method that combines solvent-cast 3D printing with peptide-polymer conjugates to spatially present multiple biochemical cues in a single scaffold without requiring post-fabrication modification. Here, we describe a detailed, stepwise protocol to fabricate peptide-functionalized scaffolds and characterize their physical architecture and biochemical spatial organization. We used these 3D-printed scaffolds to direct human mesenchymal stem cell differentiation and osteochondral tissue formation by controlling the spatial presentation of cartilage-promoting and bone-promoting peptides. This protocol also describes how to seed scaffolds and evaluate matrix deposition driven by peptide organization. 

Three-dimensional (3D) printing of biodegradable polymers has rapidly become a popular approach to create scaffolds for tissue engineering. This technique enables fabrication of complex architectures and layer-by-layer spatial control of multiple components with high resolution. The resulting scaffolds can also present distinct chemical groups or bioactive cues on the surface to guide cell behavior. However, surface functionalization often includes one or more post-fabrication processing steps, which typically produce biomaterials with homogeneously distributed chemistries that fail to mimic the biochemical organization found in native tissues. As an alternative, our laboratory developed a novel method that combines solvent-cast 3D printing with peptide-polymer conjugates to spatially present multiple biochemical cues in a single scaffold without requiring post-fabrication modification. Here, we describe a detailed, stepwise protocol to fabricate peptide-functionalized scaffolds and characterize their physical architecture and biochemical spatial organization. We used these 3D-printed scaffolds to direct human mesenchymal stem cell differentiation and osteochondral tissue formation by controlling the spatial presentation of cartilage-promoting and bone-promoting peptides. This protocol also describes how to seed scaffolds and evaluate matrix deposition driven by peptide organization.