Functionalization of Electrospun Polycaprolactone Scaffolds with Matrix-Binding Osteocyte-Derived Extracellular Vesicles Promotes Osteoblastic Differentiation and Mineralization

Abstract

Synthetic polymeric materials have demonstrated great promise for bone tissue engineering based on their compatibility with a wide array of scaffold-manufacturing techniques, but are limited in terms of the bioactivity when compared to naturally occurring materials. To enhance the regenerative properties of these materials, they are commonly functionalised with bioactive factors to guide growth within the developing tissue. Extracellular matrix vesicles (EVs) play an important role in facilitating endochondral ossification during long bone development and have recently emerged as important mediators of cell-cell communication coordinating bone regeneration, and thus represent an ideal target to enhance the regenerative properties of synthetic scaffolds. Therefore, in this paper we developed tools and protocols to enable the attachment of MLO-Y4 osteocyte-derived EVs onto electrospun polycaprolactone (PCL) scaffolds for bone repair. Initially, we optimize a method for the functionalization of PCL materials with collagen type-1 and fibronectin, inspired by the behaviour of matrix vesicles during endochondral ossification, and demonstrate that this is an effective method for the adhesion of EVs to the material surface. We then used this functionalization process to attach osteogenic EVs, collected from mechanically stimulated MLO-Y4 osteocytes, to collagen-coated electrospun PCL scaffolds. The EV-functionalized scaffold promoted osteogenic differentiation (measured by increased ALP activity) and mineralization of the matrix. In particular, EV-functionalised scaffolds exhibited significant increases in matrix mineralization particularly at earlier time points compared to uncoated and collagen-coated controls. This approach to matrix-based adhesion of EVs provides a mechanism for incorporating vesicle signalling into polyester scaffolds and demonstrates the potential of osteocyte derived EVs to enhance the rate of bone tissue regeneration.

Controlled Release of Epigenetically-Enhanced Extracellular Vesicles from a GelMA/Nanoclay Composite Hydrogel to Promote Bone Repair

“…There is a growing body of evidence demonstrating the delivery of EV-functionalised biomaterials to promote bone regeneration [ 84 , 85 ], however, the vehicle employed often does not support EV-induced tissue regeneration. For example, Holkar et al reported the potential of an alginate hydrogel loaded with osteoblasts and their EVs for bone tissue engineering applications [ 85 ], however, alginate itself is osteogenically inert.…”

The Impact of the Extracellular Matrix Environment on Sost Expression by the MLO-Y4 Osteocyte Cell Line

“…The added dimension allows cellular polarisation and formation of apical and basal profiles with which to interact with matrix and adjacent cells. This has been realised in tissue engineering strategies investigating many different tissues such as those in bone, blood vessels, and the eye [ 11 , 13 , 19 , 36 , 37 , 47 , 52 , 53 , 54 , 55 , 56 , 57 ]. To create 3D scaffolds for this study, respective slurries were freeze-dried (VirTis Co., Gardiner, NY, USA) to a final freezing temperature of −10 °C [ 45 ].…”

Layer-by-Layer siRNA Particle Assemblies for Localized Delivery of siRNA to Epithelial Cells through Surface-Mediated Particle Uptake

“…Notably, the native extracellular matrix contains structures known as matrix-bound vesicles which contain proteins and nucleic acids. Matrix-bound vesicles are an often overlooked component of the functional extracellular matrix and may provide additional signaling cues to cells in a local and defined manner. − Our approach attempts to bridge the lack of functionality often present in synthetic biomaterial systems by replicating the role of matrix-bound vesicles as a component of the cellular niche. Combining particle delivery with film formats for surface modification has the benefit of leveraging the strengths of each approach to gene delivery.…”