This is the first study which has investigated the effect of controlled fibrous architectures fabricated via melt electrospinning writing on stem cell behaviour and differentiation. After optimising the fabrication process and characterising scaffolds via SEM and mechanical testing, skeletal stem cells were seeded onto fibrous scaffolds with various micro-architectures. These architectures drove cell shape changes resulting in architecture dependent nuclear YAP localisation, suggesting altered mechanosensing at early time points. In agreement with these early markers, long term cell culture studies revealed for the first time that a 90° fibrous architecture is optimal for the osteogenic differentiation of skeletal stem cells.
Bone formation or regeneration requires the recruitment, proliferation, and osteogenic differentiation of stem/stromal progenitor cells. A potent stimulus driving this process is mechanical loading. Osteocytes are mechanosensitive cells that play fundamental roles in coordinating loading-induced bone formation via the secretion of paracrine factors. However, the exact mechanisms by which osteocytes relay mechanical signals to these progenitor cells are poorly understood. Therefore, this study aimed to demonstrate the potency of the mechanically stimulated osteocyte secretome in driving human bone marrow stem/stromal cell (hMSC) recruitment and differentiation, and characterize the secretome to identify potential factors regulating stem cell behavior and bone mechanobiology. We demonstrate that osteocytes subjected to fluid shear secrete a distinct collection of factors that significantly enhance hMSC recruitment and osteogenesis and demonstrate the key role of extracellular vesicles (EVs) in driving these effects. This demonstrates the pro-osteogenic potential of osteocyte-derived mechanically activated extracellular vesicles, which have great potential as a cell-free therapy to enhance bone regeneration and repair in diseases such as osteoporosis.
Limitations associated with current bone grafting materials has necessitated the development of synthetic scaffolds that mimic the native tissue for bone repair. Scaffold parameters such as pore size, pore interconnectivity, fibre diameter, and fibre stiffness are crucial parameters of fibrous bone tissue engineering (BTE) scaffolds required to replicate the native environment. Optimum values vary with material, fabrication method and cell type. Melt electrowriting (MEW) provides precise control over extracellular matrix (ECM)-like fibrous scaffold architecture. The goal of this study was to fabricate and characterise poly-ε-caprolactone (PCL) fibrous scaffolds with 100, 200, and 300 μm pore sizes using MEW and determine the influence of pore size on human bone marrow stem cell (hMSC) adhesion, morphology, proliferation, mechanosignalling and osteogenesis. Each scaffold was fabricated with a fibre diameter of 4.01 ± 0.06 μm. The findings from this study highlight the enhanced osteogenic effects of controlled micro-scale fibre deposition using MEW, where the benefits of 100 μm square pores in comparison with larger pore sizes are illustrated, a pore size traditionally reported as a lower limit for osteogenesis. This suggests a lower pore size is optimal when hMSCs are seeded in a 3D ECM-like fibrous structure, with the 100 μm pore size optimal as it demonstrates the highest global stiffness, local fibre stiffness, highest seeding efficiency, maintains a spread cellular morphology, and significantly enhances hMSC collagen and mineral deposition. Similarly, this platform represents an effective in vitro model for the study of hMSC behaviour to determine the significant osteogenic benefits of controlling ECM-like fibrous BTE scaffold pore size using MEW.
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