Effects of physiological mechanical strains on the release of growth factors and the expression of differentiation marker genes in human osteoblasts growing on Ti‐6Al‐4V

Abstract

Mechanical loading factors at the bone-implant interface are critical for the osseointegration and clinical success of the implant. The aim of the present investigation was to study the effects of mechanical strain on the orthopedic biomaterial Ti-6Al-4V/osteoblast interface, using an in vitro model. Homogeneous strain was applied to human bone marrow derived osteoblasts (HBMDOs) cultured on Ti-6Al-4V, at physiological levels (strain magnitudes 500 microstrain (microepsilon) and 1000 microepsilon, at frequencies of load application 0.5 Hz and 1 Hz), by a mechanostimulatory system, based on the principle of four-point bending. Semi-quantitative reverse transcription-polymerase chain reaction (sqRT-PCR) was used to determine the mRNA expression of Cbfa1 and osteocalcin at different loading conditions. The release of growth factors as a response to stretch was also investigated by transferring stretch-conditioned media to nonstretched cells and by measuring their effect on the regulation of DNA synthesis. Mechanical loading was found to contribute to the regulation of osteoblast differentiation by influencing the level of the osteoblast-specific transcription factor Cbfa1, both at the mRNA and protein level, and also the level of osteocalcin, which is regarded as the most osteoblast-specific gene. Both genes were differentially expressed shortly after the application of different mechanical stimuli, in terms of strain frequency, magnitude, and time interval. Media conditioned from mechanically stressed HBMDOs stimulate DNA synthesis more intensely compared to media conditioned from unstressed control cultures, indicating that mechanical strain induces the release of a mitogenic potential that regulates cell proliferation.

Mechanoresponses of human primary osteoblasts grown on carbon nanotubes

“…Mechanical loading of cells in vitro is a very informative approach in understanding the underlying mechanisms of load transduction at the tissue/implant interface and a variety of mechanisms have been used to produce strain. A few studies have applied homogenous strain to all cultured cells at levels which are measured experimentally on the external surface of bones and are regarded as physiological, on the basis of estimated deformation on hip stem prostheses …”

“…The cell-loading apparatus, used in this study, has been extensively described elsewhere. 22 Briefly, the cell-loading system applies four-point bending on a sheet of Ti-6Al-4V, resulting in a homogeneous strain of the sheet in the area between the inner span loading points. On its surface, cell culture wells have been created by attached tissue plastic rings.…”

“…Strain was applied at physiological levels [strain magnitudes 500 microstrain (μɛ), at frequency of load application 0.5 Hz]. Although frequency 0.5 Hz corresponds to very slow walking, it has been observed in a number of studies that these loading conditions (0.5 Hz and 500 μɛ) represent the most stimulating condition for Cbfa1 and osteocalcin mRNA expression in comparison to more intense stretching (1 Hz and 1000 μɛ) . We hypothesized that both the adhesion substrate (culture‐treated plastic and MWCNTs) as well as the application of mechanical stimulus affect the adhesion mechanisms employed by osteoblasts, perhaps by altering the expression of certain instrumental genes that mediate this adhesion.…”

“…Although frequency 0.5 Hz corresponds to very slow walking, it has been observed in a number of studies that these loading conditions (0.5 Hz and 500 lE) represent the most stimulating condition for Cbfa1 and osteocalcin mRNA expression in comparison to more intense stretching (1 Hz and 1000 lE). 22 We hypothesized that both the adhesion substrate (culture-treated plastic and MWCNTs) as well as the application of mechanical stimulus affect the adhesion mechanisms employed by osteoblasts, perhaps by altering the expression of certain instrumental genes that mediate this adhesion. The effect of mechanical strain and substrate was thus studied by measuring the mRNA expression of ALP, vinculin (VCL), collagen 1A (COL1A1), and integrins b1, b3, a4, av, 23 using real-time quantitative polymerase chain reaction (RT-qPCR).…”

The Comprehensive Biomechanics and Load-Sharing of Semirigid PEEK and Semirigid Posterior Dynamic Stabilization Systems

“…Semirigid instrumentations, such as polyetheretherketone (PEEK) rods and titanium rods with helical grooves, are designed to increase load-sharing in attempt to induce compression on the bone graft and promote bone remodeling as first credited by Wolff [4]. There is certainly evidence of osteoblastic response to mechanical activation, in various forms, as well as increases in bone formation rate following bouts of loading [5, 6]. Semirigid PEEK instrumentation attempts to allow loading through the anterior column, but most studies show the stiffness of these constructs to be relatively high [7].…”