Primary cilia expression in bone marrow in response to mechanical stimulation in explant bioreactor culture

Abstract

Bone marrow contains a multitude of mechanically sensitive cells that may participate in mechanotransduction. Primary cilia are sensory organelles expressed on mesenchymal stem cells (MSCs), osteoblasts, osteocytes, and other cell types that sense fluid flow in monolayer culture. In marrow, cilia could similarly facilitate the sensation of relative motion between adjacent cells or interstitial fluid. The goal of this study was to determine the response of cilia to mechanical stimulation of the marrow. Bioreactors were used to supply trabecular bone explants with low magnitude mechanical stimulation (LMMS) of 0.3 ×g at 30 Hz for 1 h/d, 5 d/week, inducing shear stresses in the marrow. Four groups were studied: unstimulated (UNSTIM), stimulated (LMMS), and with and without chloral hydrate (UNSTIM+CH and LMMS+CH, respectively), which was used to disrupt cilia. After 19 days of culture, immunohistochemistry for acetylated α-tubulin revealed that more cells expressed cilia in culture compared to in vivo controls. Stimulation decreased the number of cells expressing cilia in untreated explants, but not in CH-treated explants. MSCs represented a greater fraction of marrow cells in the untreated explants than CH-treated explants. MSCs harvested from the stimulated groups were more proliferative than in the unstimulated explants, but this effect was absent in CH treated explants. In contrast to the marrow, neither LMMS nor CH treatment affected bone formation as measured by mineralising surface. Computational models indicated that LMMS does not induce bone strain, and the reported effects were thus attributed to shear stress in the marrow. From a clinical perspective, genetic or pharmaceutical alterations of cilia expression may affect marrow health and function.

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“…The fixed explants were demineralized for 4 weeks in neutral buffered EDTA, processed, and sectioned at 5 µm. Immunohistochemistry was performed as previously described 72,73 with pan-cytokeratin (1:750) and p16 INK4a (1:100) antibodies. The fraction of the area exhibiting p16 INK4a staining was quantified in 20 regions (949 × 467 µm) of marrow in each explant using ImageJ (NIH).…”

Ex vivo Bone Models and Their Potential in Preclinical Evaluation

“…The compressive forces could be applied in a variety of different wave forms and frequencies to simulate walking or jumping for example, while also having the ability to measure mechanical properties, such as stiffness, in real time [58]. A different type of bioreactor system combined perfusion with low magnitude, high-frequency vibrations to evoke shear stresses within bone explants without associated deformations in the bone matrix [47,48]. The absence of…”

Effects of mechanobiological signaling in bone marrow on skeletal health

“…As discussed above, the mechanical behavior of bone marrow has not been definitively characterized, and various investigators have used either solid or fluid constitutive models . As such, the stress and motion of marrow modeled as a soft or viscoelastic solid within the trabecular pore space should also be considered.…”

“…Primary cilia exist in a relatively small fraction of marrow cells in vivo , but have been proposed to act as mechanosensory organelles in cells. Using the same bioreactor technique with low magnitude vibrations, mechanical stimulation affected the expression of the primary cilium on marrow cells . When cells were subjected to 50 Hz 0.3 g ( g is the Earth's gravitational field) vibration, the number of cells expressing primary cilia—defined as a cilium extending more than 1 µm from the nucleus—was about 25% lower than in unstimulated controls.…”

“…As such, the result does not provide definitive evidence that the altered bone formation was due to impaired mechanosensing in the marrow cells as opposed to impaired sensing of osteocyte secreted proteins. However, multiple studies have demonstrated that the expression of cilia in various cell types is sensitive to mechanical stimulation …”