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.
Highlights
Increases in life expectancy, or lifespan, seen throughout the developed world are a valuable indicator for the progress of modern medicine
Vibration of periodontal ligament stem cells at 50 Hz with a peak acceleration of 0.3 g showed increased markers of osteogenesis[17] whilst another study of adipose-derived stem cells stimulated using a feedback controlled vibration source at 50 and 100 Hz with a reported peak acceleration of 3 g, showed increased levels of alkaline phosphatase (ALP) activity and mineral deposition, not at the same level produced by osteogenic media[18]
The proposed bioreactor system consists of three main elements: a bioreactor vibration plate, a power supply to drive the piezo array underneath the vibration plate, and custom 6-well cultureware that can magnetically attach to the vibration plate
Summary
Lifespan, seen throughout the developed world are a valuable indicator for the progress of modern medicine. The controlled osteogenesis of MSCs through mechanical stimulation has been demonstrated through several methods including passive and active strategies Passive methods such as altered substrate topography and environmental stiffness provide one mechanism based on altering the adhesion profile[3,4,5,6,7], whilst active methods include exposure to variations of force from external sources[8,9,10,11,12,13]. Finite element analysis (FEA) was used to improve the bioreactor design as well as optimising its construction, minimising large amplitude variance arising from resonances or substrate deformation. These factors are important when using nanoscale amplitudes as consistent vibration ensures that all cells on the bioreactor experience the same levels of acceleration. Atomic force microscopy (AFM) measurements were carried out on collagen gel used in these experiments to determine that the vibrations were transmitting from the cultureware into the gel and that the stiffness of the gel didn’t significantly increase while being nanovibrated through non-Newtonian/strain hardening effects[24]
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