Abstract
Decreased mechanical loading on bones, such as prolonged bed rest and microgravity during space flights, leads to the development of an osteoporotic-like phenotype. Although osteoblast hypo-functionality is reported to be involved in the progression of bone pathological conditions, the cellular mechanisms of this process remain largely unknown. The combined application of mass spectrometry “–omics” and histochemical and ultrastructural approaches have been employed to investigate the effects of the gravitational unloading on human bone-cell biology. Here we show, ex vivo, that simulated microgravity (Sμg) on human primary osteoblasts (hpOB) induces an alteration of pro-osteogenic determinants (i.e., cell morphology and deposit of hydroxyapatite crystals), accompanied by a downregulation of adhesive proteins and bone differentiation markers (e.g., integrin beta-1, protein folding Crystallin Alpha B (CRYα-B), runt-related transcription factor 2 (RUNX-2), bone morphogenic protein-2 (BMP-2), and receptor activator of nuclear factor kappa-B ligand (RANK-L)), indicating an impairment of osteogenesis. Further, we observed for the first time that Sμg can trigger a transition toward a mesenchymal-like phenotype, in which a mature osteoblast displays an hampered vitamin A metabolism, loses adhesive molecules, gains mesenchymal components (e.g., pre-osteoblast state marker CD44), morphological protrusions (filopodium-like), enhances GTPase activities, which in turn allows it to acquire migrating properties. Although this phenotypic conversion is not complete and can be reversible, Sμg environment proves a plasticity potential hidden on Earth. Overall, our results suggest that Sμg can be a powerful physical cue for triggering ex vivo a dedifferentiation impulse on hpOBs, opening a new scenario of possible innovative therapeutical biomechanical strategies for the treatment of osteo-degenerative diseases.
Highlights
Bone is a highly mechano-sensitive tissue, capable of undergoing rapid and robust rearrangement even in response to microscopic mechanical stimuli
To what reported for rat cells[32], our results show that for human primary osteoblasts (hpOB) the in vitro differentiation is impaired by Sμg
Cell inspection by inverted and up-right microscope revealed a morphological shift from flat-shape to spindleshape (Fig. 1), clearly showing that the majority of cellpopulation treated with Sμg displays a spindle-shaped cell morphology (Fig. 1b, d) rather than the cuboidal cell shape typical of the mature osteoblast phenotype (Fig. 1a, c)
Summary
Bone is a highly mechano-sensitive tissue, capable of undergoing rapid and robust rearrangement even in response to microscopic mechanical stimuli. Studies on stem cell differentiation have defined the principles of mechanobiology; cells sense extracellular stiffness through contraction of the actomyosin cytoskeleton, regulating the suitable response through focal adhesion, rho-GTPase signaling, cytoskeletal contractility, and nuclear rearrangement processes[5,6]. Empirical evidences report that mechanical unloading impairs osteoblasts differentiation of bone marrow mesenchymal stem cells, inhibiting osteogenesis[7,8]. Official journal of the Cell Death Differentiation Association. Humans have limited regenerative capacity, in principle, a mechanical induction of dedifferentiation may be a logical strategy to promote regeneration in tissues that lack osteogenic ability[17,18,19] Differentiation process is tightly intertwined to cellular motility, since they both involve (i) a peculiar actin organization forming specific cell-protrusions (such as lamellipodia, filopodia, or blebs)[9,10], (ii) a specific proteolytic set of enzymes[11,12,13], and (iii) adhesion proteins (e.g., cluster of differentiation proteins and integrins)[11,12,13,14,15,16].
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