Non-damaging stretching combined with sodium pyruvate supplement accelerate migration of fibroblasts and myoblasts during gap closure.

BACKGROUND: Sustained, low- and mid-level (3-6%), radial stretching combined with varying concentrations of sodium pyruvate (NaPy) supplement increase the migration rate during microscale gap closure following an in vitro injury; NaPy is a physiological supplement often used in cell-culture media. Recently we showed that low-level tensile strains accelerate in vitro kinematics during en masse cell migration; topically applied mechanical deformations also accelerate in vivo healing in larger wounds. The constituents and nutrients at injury sites change. Thus, we combine a supplement with stretching conditions to effectively accelerate wound healing. METHODS: Monolayers of murine fibroblasts (NIH3T3) or myoblasts (C2C12) were cultured in 1 mM NaPy on stretchable, linear-elastic substrates. Monolayers were subjected to 0, 3, or 6% stretching using a custom three-dimensionally printed stretching apparatus, micro-damage was immediately induced, media was replaced with fresh media containing 0, 1, or 5 mM NaPy, and cell migration kinematics during gap-closure were quantitatively evaluated. FINDINGS: In myoblasts, the smallest evaluated strain (3%, minimal risk of damage) combined with preinjury (1 mM) and post-injury exogenous NaPy supplements accelerated gap closure in a statistically significant manner; response was NaPy concentration dependent. In both fibroblasts and myoblasts, when cells were pre-exposed to NaPy, yet no supplement was provided post-injury, mid-level stretches (6%) compensated for post-injury deficiency in exogenous NaPy and accelerated gap-closure in a statistically significant manner. INTERPRETATION: Small deformations combined with NaPy supplement prior-to and following cell-damage accelerate en masse cell migration and can be applied in wound healing, e.g. to preventatively accelerate closure of microscale gaps.

Low-level stretching accelerates cell migration into a gap.

We observed that radially stretching cell monolayers at a low level (3%) increases the rate at which they migrate to close a gap formed by in vitro injury. Wound healing has been shown to accelerate in vivo when deformations are topically applied, for example, by negative pressure wound therapy. However, the direct effect of deformations on cell migration during gap closure is still unknown. Thus, we have evaluated the effect of radially applied, sustained (static) tensile strain on the kinematics of en mass cell migration. Monolayers of murine fibroblasts were cultured on stretchable, linear-elastic substrates that were subjected to different tensile strains, using a custom-designed three-dimensionally printed stretching apparatus. Immediately following stretching, the monolayer was 'wounded' at its centre, and cell migration during gap closure was monitored and quantitatively evaluated. We observed a significant increase in normalised migration rates and a reduction of gap closure time with 3% stretching, relative to unstretched controls or 6% stretch. Interestingly, the initial gap area was linearly correlated with the maximum migration rate, especially when stretching was applied. Therefore, small deformations applied to cell monolayers during gap closure enhance en mass cell migration associated with wound healing and can be used to fine-tune treatment protocols.

Printable low-cost, sustained and dynamic cell stretching apparatus.

Deformations that are applied on body tissues during daily activities, as a result of morbid conditions, or during various medical treatments, affect cell viability and biological function. Such mechanobiological phenomena are often studied in vitro, in monolayer cultures. To facilitate such studies cost effectively, we have developed a novel, printable cell stretching apparatus. The apparatus is used to apply tensile strains on cells cultured on elastic, stretchable substrata, either by sustained or by dynamic-cyclic application. Most of the apparatus parts are three-dimensionally printed (excluding motors), and stretching is automatically performed by two direct current geared motors that are controlled by a programmable microcontroller platform. To demonstrate functionality of this novel printable device, which can be produced in multiple copies in research labs at a cost of under 100 US$ per unit, including motors and controller, we performed cell culture studies monitored by fluorescence microscopy. Specifically, we have applied sustained and cyclic, radial stretching at large strains to NIH3T3 mouse fibroblasts, and have demonstrated that cell viability, adhesion and morphology were maintained following stretching. Our apparatus is designed to be low-cost, rapidly manufactured at a university or small-company setting, and simple to use and control, where its flexible, versatile design allows users to experimentally induce different stretching regimes with varying amplitudes and frequencies.