Abstract
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.
Highlights
In the last decades regenerative medicine relied on the use of 3D structures, namely scaffolds, to support cell adhesion and tissue growth
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
In order to mimic the progressive variation of chemical characteristics of the osteochondral extra-cellular matrix (ECM), we propose here a scaffold fabrication based on AM of PCL, to create fully interconnected 3D supports, later on modified with POEGMA brushes applying SIATRP from initiator functions on the scaffold surface
Summary
In the last decades regenerative medicine relied on the use of 3D structures, namely scaffolds, to support cell adhesion and tissue growth. When the scaffolds were placed on a protein solution reservoir, an axial gradient was generated via diffusion due to capillary forces.[7] Growth factors such as bone morphogenetic protein (BMP) and transforming growth factor β (TGFβ) are known to trigger the differentiation of human mesenchymal stromal cells (hMSCs) toward the osteogenic or chondrogenic lineage.[8,9,10,11] A number of studies applied scaffolds in combination with growth factors (GFs)[12,13,14,15,16] to improve hMSCs differentiation These techniques are highly expensive, due to the great dosage of GFs used,[17] which can lead to adverse effects.[18,19] On the other hand, linking GFs to the surface[20,21,22] ensures a continuous mono-dose treatment, which may continuously trigger the underlying signaling pathways, instead of continued infusion of GFs during the culture time. The GF homogenously linked to the surface of the 3D scaffolds outperformed the simple addition of their soluble form to the cell culture media, determining the overexpression of osteogenic and chondrogenic markers in the shorter time span of 10 days, when normally the expression takes between 3 and 4 weeks with the soluble factors
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