Swift progress in biofabrication technologies has enabled unprecedented advances in the application of developmental biology design criteria in three-dimensional scaffolds for regenerative medicine. Considering that tissues and organs in the human body develop following specific physico-chemical gradients, in this study, we hypothesized that additive manufacturing (AM) technologies would significantly aid in the construction of 3D scaffolds encompassing such gradients. Specifically, we considered surface energy and stiffness gradients and analyzed their effect on adult bone marrow derived mesenchymal stem cell differentiation into skeletal lineages. Discrete step-wise macroscopic gradients were obtained by sequentially depositing different biodegradable biomaterials in the AM process, namely poly(lactic acid) (PLA), polycaprolactone (PCL), and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymers. At the bulk level, PEOT/PBT homogeneous scaffolds supported a higher alkaline phosphatase (ALP) activity compared to PCL, PLA, and gradient scaffolds, respectively. All homogeneous biomaterial scaffolds supported also a significantly higher amount of glycosaminoglycans (GAGs) production compared to discrete gradient scaffolds. Interestingly, the analysis of the different material compartments revealed a specific contribution of PCL, PLA, and PEOT/PBT to surface energy gradients. Whereas PEOT/PBT regions were associated to significantly higher ALP activity, PLA regions correlated with significantly higher GAG production. These results show that cell activity could be influenced by the specific spatial distribution of different biomaterial chemistries in a 3D scaffold and that engineering surface energy discrete gradients could be considered as an appealing criterion to design scaffolds for osteochondral regeneration.

Engineering the osteochondral tissue presents some challenges mainly relying in its function of transition from the subchondral bone to articular cartilage and the gradual variation in several biological, mechanical, and structural features. A possible solution for osteochondral regeneration might be the design and fabrication of scaffolds presenting a gradient able to mimic this transition. Covalent binding of biological factors proved to enhance cell adhesion and differentiation in two-dimensional culture substrates. Here, we used polymer brushes as selective linkers of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β3 (TGF-β3) on the surface of 3D scaffolds fabricated via additive manufacturing (AM) and subsequent controlled radical polymerization. These growth factors (GFs) are known to stimulate the differentiation of human mesenchymal stromal cells (hMSCs) toward the osteogenic and chondrogenic lineages, respectively. BMP-2 and TGF-β3 were covalently bound both homogeneously within a poly(ethylene glycol) (PEG)-based brush-functionalized scaffolds, and following a gradient composition by varying their concentration along the axial section of the 3D constructs. Following an approach previously developed by our group and proved to be successful to generate fibronectin gradients, opposite brush-supported gradients of BMP-2 and TGF-β3 were finally generated and subsequently tested to differentiate cells in a gradient fashion. The brush-supported GFs significantly influenced hMSCs osteochondral differentiation when the scaffolds were homogenously modified, yet no effect was observed in the gradient scaffolds. Therefore, this technique seems promising to maintain the biological activity of growth factors covalently linked to 3D scaffolds, but needs to be further optimized in case biological gradients are desired.