Hydrogel-Assisted 3D Model to Investigate the Osteoinductive Potential of MC3T3-Derived Extracellular Vesicles

Abstract

Effective and rapid regeneration of bone defects often pose substantial challenges in severe accidental injuries and disabilities occurring due to diseases and/or advanced age, especially in patients having reduced tissue regeneration competence. The success of mesenchymal stromal cell (MSC)-based research strategies in improving bone regeneration was hampered not only due to the limited knowledge of therapeutic actions of MSCs but also due to difficulties as well as expenses related to cell manufacturing and time taken for ethical approvals for clinical use of living cells and engineered tissues. The recent trend indicated that there is a shift from the direct usage of MSCs toward the application of paracrine factors and extracellular vesicles (EVs) isolated from their MSC secretome in bone tissue regeneration. This shift has directed research into the development of "cell-free" therapeutics, which could be a better alternative due to its several advantages over the use of their parental MSCs. Furthermore, accumulating evidence suggested that the 3D microenvironment influences the paracrine effects of MSCs. Although the osteogenic role of EVs has been explored recently, the current study showed, for the first time, that encapsulation of EVs along with MC3T3 cells in a 3D hydrogel-assisted culture with a distinct porous microenvironment having meso and macro (0.05−200 μm) pore size distribution resulted in an improved osteogenic response in vitro. The present work was primarily focused on investigating the influence of EVs isolated under distinct priming conditions to enhance the osteogenic potential. In addition, in the current work, the osteogenic ability of different types of EVs (microvesicles and exosomes) and total EVs isolated at different time points was also examined when encapsulated with MC3T3 cells in an alginate gel. Using various biochemical assays, such as alkaline phosphatase (ALP) production and calcium secretion, it was observed that both microvesicles and exosomes collected from MC3T3 cells independently had osteogenic potential; however, their collective activity was found to be superior. We further showed that EVs induce an early osteogenic response in MC3T3 cells as indicated by ALP and calcium secretion at a much earlier time point, compared to the controls. Our data suggested that this 3D hydrogel-assisted system provides close proximity of cells and EVs, and thus, mimics the in vivo scenario, making it clinically useful for bone tissue engineering.

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.…”

“…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 importance of the delivery vehicle on EV functionality was demonstrated by Davies et al, where osteoblast-derived EVs exhibited substantially enhanced mineralisation potency when delivered in mineralising medium compared to non-mineralising medium [ 65 ].…”

Preconditioning and Engineering Strategies for Improving the Efficacy of Mesenchymal Stem Cell-Derived Exosomes in Cell-Free Therapy

“…At present, 3D culture technology can be broadly divided into two types: material-free cultures (cell spheres formed by aggregation of cells) and material-supported cultures. Material-free 3D culture methods mainly include 3D spherical spatial boundary conditions [ 54 ], scaffold-free suspension cultures [ 55 ], systematic microcarrier screening, hydrogel-assisted 3D culture [ 56 , 57 ], and agitated culture conditions [ 58 ]. Material-supported culture methods are relatively diverse, including fibrous scaffolds [ 59 ], native extracellular matrix (ECM) bioscaffolds [ 60 ], hollow fiber bioreactors [ 61 ], quantum cell expansion systems [ 62 ], and computer-controlled bioreactors [ 63 ].…”

Mesenchymal Stem Cell-Derived Extracellular Vesicles for Bone Defect Repair

“…Hydrogels provide a 3D matrix for Exos that prevents the dispersion of Exos and maintains its local concentration, which enables controlled release of Exos for sustained efficacy. Hydrogel-released Exos at bone defect sites can reduce Exos consumption and ectopic effects to promote angiogenesis and osteogenesis [ 61 ]. Some scholars found gelatin nanoparticle hydrogels combined with hADSC-Exos can accurately transport the hADSC-Exos to the target site and effectively promote bone healing [ 62 ].…”

“…Yu et al [ 4 ] demonstrated that Exos derived from periodontal ligament stem cells (PDLSCs) encapsulated in a hydrogel 3D microenvironment exhibited enhanced osteoinductive ability and could significantly promote bone defect repair in rats. Another study demonstrated alginate hydrogels combined with EVs showed increased interactions with other cells, cell aggregation, and prolonged long-term viability, which in turn promoted osteogenesis [ 61 ]. Therefore, a variety of special properties of hydrogels can be combined with MSC-EVs to effectively treat bone defects.…”