RESUMEN
Blood vessel formation is an important initial step for bone formation during development as well as during remodelling and repair in the adult skeleton. This results in a heavily vascularized tissue where endothelial cells and skeletal cells are constantly in crosstalk to facilitate homeostasis, a process that is mediated by numerous environmental signals, including mechanical loading. Breakdown in this communication can lead to disease and/or poor fracture repair. Therefore, this study aimed to determine the role of mature bone cells in regulating angiogenesis, how this is influenced by a dynamic mechanical environment, and understand the mechanism by which this could occur. Herein, we demonstrate that both osteoblasts and osteocytes coordinate endothelial cell proliferation, migration, and blood vessel formation via a mechanically dependent paracrine mechanism. Moreover, we identified that this process is mediated via the secretion of extracellular vesicles (EVs), as isolated EVs from mechanically stimulated bone cells elicited the same response as seen with the full secretome, while the EV-depleted secretome did not elicit any effect. Despite mechanically activated bone cell-derived EVs (MA-EVs) driving a similar response to VEGF treatment, MA-EVs contain minimal quantities of this angiogenic factor. Lastly, a miRNA screen identified mechanoresponsive miRNAs packaged within MA-EVs which are linked with angiogenesis. Taken together, this study has highlighted an important mechanism in osteogenic-angiogenic coupling in bone and has identified the mechanically activated bone cell-derived EVs as a therapeutic to promote angiogenesis and potentially bone repair.
RESUMEN
INTRODUCTION: New approaches to treat osteoporosis have focused on promoting bone formation through the targeting of osteoblasts and their progenitors, mesenchymal stem cells (MSCs). The primary cilium is a singular cellular extension known to play an important role in biochemical and biophysical osteogenic induction of MSCs. Defects in ciliary structure have been associated with a plethora of diseases. Therefore targeting the cilium therapeutically (ciliotherapies) has emerged as a potential new treatment modality. Therefore, this study performed a comparison analysis on known ciliotherapies and their potential effects in mediating MSC osteogenic differentiation. METHODS: MSCs were treated with forskolin, lithium chloride (LiCl) or fenoldopam to investigate the effect on ciliogenesis and cilia-associated signalling. Moreover, both early and long term biochemical and biophysical (fluid shear) induced osteogenic differentiation was examined in terms of osteogenic gene expression and bone matrix deposition following each treatment. RESULTS: LiCl and fenoldopam were found to enhance MSC ciliogenesis to a similar degree. LiCl significantly altered hedgehog (HH) and Wnt signalling which was associated with inhibited osteogenic gene expression, while fenoldopam demonstrated enhanced early osteogenesis. Long term treatment with both ciliotherapies did not enhance osteogenesis, however LiCl had detrimental effects on cell viability. Intriguingly both ciliotherapies enhanced MSC mechanosensitivity as demonstrated by augmented osteogenic gene expression in response to fluid shear, which over longer durations resulted in enhanced matrix deposition per cell. CONCLUSIONS: Therefore, ciliotherapies can be utilised to enhance MSC ciliogenesis resulting in enhanced mechanosensitivity, however, only fenoldopam is a viable ciliotherapeutic option to enhance MSC osteogenesis.
RESUMEN
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.
