Extracellular Matrix Produced by Osteoblasts Cultured Under Low-Magnitude, High-Frequency Stimulation is Favourable to Osteogenic Differentiation of Mesenchymal Stem Cells

Abstract

The effects of low-magnitude, high-frequency (LMHF) mechanical stimulation on osteoblastic cells are poorly understood. We have developed a system that generates very small (15-40 με), high-frequency (400 Hz, sine) deformations on osteoblast cultures (MC3T3-E1). We investigated the effects of these LMHF stimulations mainly on extracellular matrix (ECM) synthesis. The functional properties of this ECM after decellularization were evaluated on C3H10T1/2 mesenchymal stem cells (MSCs). LMHF stimulations were applied 20 min once daily for 1, 3, or 7 days in MC3T3-E1 culture (1, 3, or 7 dLMHF). Cell number and viability were not affected after 3 or 7 dLMHF. Osteoblast response to LMHF was assessed by an increase in nitric oxide secretion, alteration of the cytoskeleton, and focal contacts. mRNA expression for fibronectin, osteopontin, bone sialoprotein, and type I collagen in LMHF cultures were 1.8-, 1.6-, 1.5-, and 1.7-fold higher than controls, respectively (P < 0.05). In terms of protein, osteopontin levels were increased after 3 dLMHF and ECM organization was altered as shown by fibronectin topology after 7 dLMHF. After decellularization, 7 dLMHF-ECM or control ECM was reseeded with MSCs. Seven dLMHF-ECM improved early events such as cell attachment (2 h) and focal contact adhesion (6 h) and, later (16 h), modified MSC morphological parameters. After 5 days in multipotential medium, gene-expression changes indicated that 7 dLMHF-ECM promoted the expression of osteoblast markers at the expense of adipogenic marker. LMHF stimulations of osteoblasts are therefore efficient and sufficient to generate osteogenic matrix.

Vibrational stimulation induces osteoblast differentiation and the upregulation of osteogenic gene expression in vitro

“…In our model, silicone has several advantages compared with plastic cell culture plates. These advantages include: (1) a flexible silicone membrane to accurately apply a broad range of 1-100 l strain on the culture surface, while approximately 15-40 l strain was induced by resonant frequency on the plastic culture plates (Dumas et al 2010); (2) the phase of the dynamic straininduced sinusoidal wave was position-dependent, which suggests that the vibration wave was conducted through the entire membrane; (3) in our in vitro vibration system, the fluid shear stress induced by the vibration was not considerably large. We confirmed that there was no standing wave on the fluid surface within the range of accelerations and frequencies used in this study.…”

“…The manner by which vibrational strain was applied to cells on a plastic substrate is different. A resonant frequency was used to maximize vibrational displacement of the plastic culture plates (Dumas et al 2010). The amplitude of the vibrational strain is limited by the mechanical properties of the plastic substrate.…”

The effect of whole body vibration on fracture healing – a systematic review

“…2 )] and frequency ranged from 20 to 90 Hz (Bemben et al, 2010;Fuermaier et al, 2014;von Stengel et al, 2011), is also reported to have positive effects on osteogenic differentiation and osteogenesis (Dumas et al, 2010;Kim et al, 2012;Leung et al, 2014;Luu et al, 2009). Some previous studies using LMHFV for fracture healing in rats report its promising effects on callus formation, mineralisation and bone remodelling (Chow et al, 2011;Leung et al, 2009;Shi et al, 2010).…”

“…Some studies provide evidence that WBV has effects on the musculoskeletal system, including improving muscle function (Rees et al, 2008;Sitja-Rabert et al, 2015), increasing bone mineral density (BMD) (Lam et al, 2013;Verschueren et al, 2004), reducing risks of falls and improving muscle strength and balancing ability . Low-magnitude high-frequency vibration (LMHFV), a type of WBV with magnitude usually lower than 1 ×g [magnitude, in gravitational acceleration (m/s2 )] and frequency ranged from 20 to 90 Hz (Bemben et al, 2010;Fuermaier et al, 2014;von Stengel et al, 2011), is also reported to have positive effects on osteogenic differentiation and osteogenesis (Dumas et al, 2010;Kim et al, 2012;Leung et al, 2014;Luu et al, 2009). Some previous studies using LMHFV for fracture healing in rats report its promising effects on callus formation, mineralisation and bone remodelling (Chow et al, 2011;Leung et al, 2009;Shi et al, 2010).…”

Gene Expression Responses to Mechanical Stimulation of Mesenchymal Stem Cells Seeded on Calcium Phosphate Cement

“…[3][4][5] The majority of these effects have been attributed to the induction of osteoblast activity and function and increase in cell numbers. [6][7][8][9] Many transcription factors, growth factors, cell surface receptors, and signal transduction pathways have been implicated in the differentiation, proliferation, and functional activity of osteoblasts. 10,11 However, the molecular and cellular events that occur during signal transduction of mechanical load are not fully understood, but are likely to be key factors that may govern bone formation.…”