Multi-peptide presentation and organization in 3D-printed scaffolds drives osteochondral tissue formation

Functional repair of osteochondral (OC) tissue remains challenging because the transition from bone to cartilage presents gradients in biochemical and physical properties necessary for joint function. Osteochondral regeneration requires strategies that restore the spatial composition and organization found in the native tissue. Several biomaterial approaches have been developed to guide chondrogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs). These strategies must be coupled with advanced manufacturing techniques to achieve precise spatial control of biochemical signaling. 3D printing has rapidly become a popular method for creating highly tunable multi-material constructs with complex architectures that can be customized and scaled for in vivo translation. Our lab has developed a platform that achieves surface functionalization of scaffolds in a single fabrication step by solvent-cast 3D printing peptide-functionalized polymers. This 3D printing strategy enables unprecedented control of surface peptide presentation and spatial organization within a continuous construct. Peptide-poly(caprolactone) (PCL) conjugates were synthesized bearing hyaluronic acid (HA)-binding (HAbind–PCL) or mineralizing (E3–PCL) peptides, which have been shown to promote hMSC chondrogenesis or osteogenesis, respectively. Scaffolds presenting both cartilage-promoting and bone-promoting peptides had a synergistic effect that enhanced hMSC chondrogenic and osteogenic differentiation in the absence of differentiation factors compared to scaffolds without peptides or only one peptide. Notably, multi-peptide organization significantly influenced hMSC response. Scaffolds presenting HAbind and E3 peptides in discrete opposing zones promoted hMSC osteogenic behavior. In contrast, presenting both peptides homogeneously throughout the scaffolds drove hMSC differentiation towards a mixed population of articular and hypertrophic chondrocytes. These significant results indicated that hMSC behavior was driven by dual-peptide presentation and organization. An ex vivo equine explant model was developed to evaluate scaffold-directed tissue formation in a native-like environment. The downstream potential of this platform is the ability to fabricate biomaterials with spatially controlled biochemical cues to guide functional tissue regeneration in situ without the need for differentiation factors.