Remotely controlled mechanotransduction via magnetic nanoparticles: applications for injectable cell therapies

摘要

Introduction: Mechanotransduction is a crucial stimulus in the formation of healthy bone, promoting the initial differentiation of mesenchymal stem cells and subsequent bone formation by osteoblasts. In this investigation we have used microinjections of human MSCs into the developing chick foetal femur to promote osteogenesis in the epiphysis. Mechanotransduction in these cells was stimulated by targeting the mechanosensitive domain of the TREK1 ion channel with magnetic nanoparticles, which were externally controlled by an oscillating magnetic field. A subsequent investigation was performed in which the MSCs were seeded into a collagen hydrogel to evaluate the nanoparticle approach as a method to provide mechaincal stimuli to cells in soft matrices. The objectives of this investigation were to demonstrate that targeted magnetic nanoparticles can stimulate mechanotransduction and enhance bone formation in a model of endochondral ossification and in tissue engineered hydrogels. Methods: Human MSCs tagged with TREK1-Ab-conjugated magnetic nanoparticles were injected into the cartilaginous epiphyses of an organotypically cultured ex vivo chick foetal femur using a glass capillary needle. 20nl of material was injected containing 10~3 cells per injection, and unlabelled (nanopartide-free) hMSCs were used as a control. The femurs were cultured organotypically for 14 days in the presence of an oscillating 25mT magnetic field for one hour per day, applying sufficient force to the conjugated nanoparticle to remotely activate TREK1. The cells were also seeded into a collagen hydrogel and cultured under the same magnetic field conditions for 28 days. Results: Control femurs were shown to mineralise in the diaphysis only, whilst secondary mineralisation sites were observed in the epiphyses of the femurs injected with TREKI-labelled hMSCs. Similarly, control hMSCs did not significantly mineralise the hydrogel constructs, whereas hMSCs labelled with nanoparticles showed significant increases in extracellular matrix production and mineralisation. Fig. 1: Tissue engineered collagen hydrogels containing hMSCs (top, a-c) and hMSCs labelled with magnetic nanoparticles (bottom, e-g). Remote activation of the mechanosensitive ion channel TREK1 by nanoparticles resulted in substantial formation of mineralised extracellular matrix, quantified by uCT (3D reconstruction of internal mineralisation, A & E), collagen staining (B & F) and calcium staining (C & G). The location of DiL-labelled hMSCs in the epiphyseal injection site within the chick foetal femur is shown in G. Following 14 days in vitro culture, the sites in which the TREK1-nanoparticle labelled cells were injected became mineralised (H, top; arrowed μCT image showing bone formation), whereas those injected with unlabelled hMSCs did not (H, bottom). Conclusion: In these experiments we targeted the TREK1 ion channel of hMSCs with magnetic nanoparticles to remotely activate mechanotransduction. When microinjected into an organotypically cultured chick foetal femur the MSCs differentiated and mineralised at the injection site - further analysis also suggests paracrine effects on surrounding chick cells. Subsequent research has shown that this method has the potential to act synergistically with other tissue engineering approaches, such as growth factor delivery, and can be used to drive osteogenic differentiation of hMSCs in hydrogel constructs.