Design, construction and characterisation of a novel nanovibrational bioreactor and cultureware for osteogenesis

Abstract

In regenerative medicine, techniques which control stem cell lineage commitment are a rapidly expanding field of interest. Recently, nanoscale mechanical stimulation of mesenchymal stem cells (MSCs) has been shown to activate mechanotransduction pathways stimulating osteogenesis in 2D and 3D culture. This has the potential to revolutionise bone graft procedures by creating cellular graft material from autologous or allogeneic sources of MSCs without using chemical induction. With the increased interest in mechanical stimulation of cells and huge potential for clinical use, it is apparent that researchers and clinicians require a scalable bioreactor system that provides consistently reproducible results with a simple turnkey approach. A novel bioreactor system is presented that consists of: a bioreactor vibration plate, calibrated and optimised for nanometre vibrations at 1 kHz, a power supply unit, which supplies a 1 kHz sine wave signal necessary to generate approximately 30 nm of vibration amplitude, and custom 6-well cultureware with toroidal shaped magnets incorporated in the base of each well for conformal attachment to the bioreactor's magnetic vibration plate. The cultureware and vibration plate were designed using finite element analysis to determine the modal and harmonic responses, and validated by interferometric measurement. This helps ensure that the vibration plate and cultureware, and thus collagen and MSCs, all move as a rigid body, avoiding large deformations close to the resonant frequency of the vibration plate and vibration damping beyond the resonance. Assessment of osteogenic protein expression was performed to confirm differentiation of MSCs after initial biological experiments with the system, as well as atomic force microscopy of the 3D gel constructs during vibrational stimulation to verify that strain hardening of the gel did not occur. This shows that cell differentiation was the result of the nanovibrational stimulation provided by the bioreactor alone, and that other cell differentiating factors, such as stiffening of the collagen gel, did not contribute.

The use of nanovibration to discover specific and potent bioactive metabolites that stimulate osteogenic differentiation in mesenchymal stem cells

“…The vibration plate was secured on its underside to an array of low-profile, multilayer piezo actuators (NAC2022, Noliac A/S CTS, Denmark). To power the piezo array, a custom power supply unit was used, as detailed in a previous publication (56), consisting of a signal generator integrated circuit (AD9833, Analog Devices, MA, USA) to provide a 1000-Hz sine wave modulation. A parallel configuration of class AB audio amplifiers (TDA7293, STMicroelectronics, Geneva, Switzerland) was used to amplify the sine wave signal.…”

“…Vibrational amplitude was measured using a laser interferometry system previously used to accurately measure nanoscale displacements generated by the bioreactor platform used here (7,56). A USB interferometer (Model SP-S, SIOS Messtechnik GmbH, Ilmenau, Germany) was mounted on a frame, with the laser aimed downward at the measurement site.…”

The use of nanovibration to discover specific and potent bioactive metabolites that stimulate osteogenic differentiation in mesenchymal stem cells

“…The vibration plate was secured on its underside to an array of low-profile, multilayer piezo actuators (NAC2022, Noliac A/S CTS, Denmark). To power the piezo array, a custom power supply unit was used, as detailed in a previous publication 47 , consisting of a signal generator integrated circuit (AD9833, Analog Devices, Massachusetts, USA) to provide a 1000 Hz sine wave modulation. A parallel configuration of class AB audio amplifiers (TDA7293, STMicroelectronics, Geneva, Switzerland) was used to amplify the sine wave signal.…”

“…Interferometric measurement. Vibrational amplitude was measured using a laser interferometry system previously used to accurately measure nanoscale displacements generated by the bioreactor platform used here 7,47 . A USB interferometer (Model SP-S, SIOS Messtechnik GmbH, Ilmenau, Germany) was mounted on a frame, with the laser aimed downwards at the measurement site.…”

Magnetic particles for triggering insulin release in INS-1E cells subjected to a rotating magnetic field

“…Mechanical stimulation of live cells has been studied using various systems with a high degree of mechanical input including hydrostatic pressure, shear stress, substrate bending/distention, piezo-electric actuation, and magnetically induced actuation. [6][7][8][9][10] Several recent studies have dealt with their use in the field of cancer treatment to induce cancer cell death. [11][12][13][14] In this context, we propose a new approach for efficient stimulation of insulin granule secretion from β-cells based on mechanotransduction.…”