Targeting and differentiating stem cells at sites of injury and repair is an exciting and promising area for disease treatment and reparative medicine. We have investigated remote magnetic field activation of magnetic nanoparticle-tagged mechanosensitive receptors on the cell membrane of human bone marrow stromal cells (HBMSCs) for use in osteoprogenitor cell delivery systems and activation of differentiation in vitro and in vivo toward an osteochondral lineage. HBMSC-labeled with magnetic beads coated with antibodies or peptides to the transmembrane ion channel stretch activated potassium channel (TREK-1) or arginine–glycine–aspartic acid were cultured in monolayer or encapsulated into polysaccharide alginate/chitosan microcapsules. Upregulation in gene expression was measured in magnetic particle-labeled HBMSCs in response to TREK-1 activation over a short period (7 days) with an increase in mRNA levels of Sox9, core binding factor alpha1 (Cbfa1), and osteopontin. Magnetic particle-labeled HBMSCs encapsulated into alginate chitosan capsules were exposed to magnetic forces both in vitro and in vivo intermittently for 21 days. After 21 days the encapsulated, magnetic particle-labeled HBMSCs in vivo were viable as evidenced by extensive cell tracker green fluorescence. The mechanical stimulation of HBMSCs labeled with TREK-1 magnetic nanoparticle receptors enhanced expression of type-1 collagen in vitro with increases in proteoglycan matrix, core binding factor alpha1 (Cbfa1) and collagen synthesis, and extracellular matrix production and elevated the expression of type-1 and type-2 collagen in vivo. Additionally, the magnetically remote stimulation of HBMSCs labeled with magnetic nanoparticle arginine–glycine–aspartic acid considerably enhanced proteoglycan and collagen synthesis and extracellular matrix production and elevated the expression of type-1 and type-2 collagen in vivo and in vitro. Osteogenic mechanosensitive receptor manipulation by magnetic nanotechnology can induce the differentiation of osteoprogenitor cell populations toward an osteogenic lineage. These cell manipulation strategies offer tremendous therapeutic opportunities in soft and hard tissue repair.

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TREK-1 is a mechanosensitive member of the two-pore domain potassium channel family (2PK+) that is also sensitive to lipids, free fatty acids (including arachidonic acid), temperature, intracellular pH, and a range of clinically relevant compounds including volatile anaesthetics. TREK-1 is known to be expressed at high levels in excitable tissues, such as the nervous system, the heart and smooth muscle, where it is believed to play a prominent role in controlling resting cell membrane potential and electrical excitability. In this report, we use RT-PCR, Western blotting and immunohistochemistry to confirm that human derived osteoblasts and MG63 cells express TREK-1 mRNA and protein. In addition, we show gene expression of TREK2c and TRAAK channels. Furthermore, whole cell patch clamp electrophysiology demonstrates that these cells express a spontaneously active, outwardly rectifying potassium "background leak" current that shares many similarities to TREK-1. The outward current is largely insensitive to TEA and Ba2+, and is sensitive to application of lysophosphatidylcholine (LPC). In addition, blocking TREK-1 channel activity is shown to upregulate bone cell proliferation. It is concluded that human osteoblasts functionally express TREK-1 and that these channels contribute, at least in part, to the resting membrane potential of human osteoblast cells. We hypothesise a possible role for TREK-1 in mechanotransduction, leading to bone remodelling.

The regulatory pathways involved in the rapid response of the AP-1 transcription factor, c-fos, to mechanical load in human primary osteoblast-like (HOB) cells and the human MG-63 bone cell line were investigated using a four-point bending model. HOB and MG-63 cells showed upregulation of c-fos expression on fibronectin and collagen type I substrates; however, MG-63 cells did not respond on laminin YIGSR substrates. Addition of cytochalasin D and Arg-Gly-Asp peptides during loading did not inhibit the response, whereas addition of beta(1)-integrin antibodies inhibited the load response. The role of Ca(2+) signaling has been demonstrated by blocking upregulation with addition of 2 mM EGTA, which chelates extracellular Ca(2+), and gadolinium (10 microM), which inhibits stretch-activated channels. Addition of the Ca(2+) ionophore A-23187 induced upregulation without loading; however, addition of nifedipine (10 microM), the L-type channel blocker, failed to prevent the load response. Inhibitors of downstream pathways indicated the involvement of protein kinase C. Our results demonstrate a key involvement of Ca(2+) signaling pathways and integrin binding in the c-fos response to mechanical strain.