It is well established that the cells are able to sense and respond to mechanical loading via mechanotransduction, the process whereby mechanical stimuli are detected by cells and converted in chemical signals. Mechanotransduction plays a crucial role in the physiology of many tissues including bone. The human skeleton is constantly subjected to mechanical loading, such as vibration from the local environment or movement of the interstitial fluid to create shearing force and deformation of the bone matrix. In vitro loading systems, which aim at replicating the force values found within the bone microenvironment, allow us to better understand the processes of mechanotransduction. In the present project, an in vitro device will be developed and validated to replicate cell substrate deformation. This device will be able to apply cyclic uniaxial strains to cells cultured on silicone stretch chambers. The uniaxial stretch system will consist of three culture stretch chambers, two linear stepper motors, two linear slide rails, a plexiglass base and a PC. A software will be developed in LabVIEW to precisely controll the two motors, by regulating load parameters, i.e. the amplitude, the frequency and the waveform of the strain. The planar device structure will allow the cells to be visualized during the strain application. For the validation of the device we will use a digital image correlation (DIC) technique that will allow us to verify the local deformation imposed in the culture area of the three stretch chambers. This uniaxial strain device will be a versatile platform, which will yield to study the role of mechanical strain not only in bone cells but also in a variety of cells and tissues, like skeletal muscle, neural cells, fibroblasts and endothelial cells.
In this project I intend to develop a specific device able to provide uniaxial cyclic strain in bone cells cultured on a silicone stretch chambers. It is well established that bone cells respond to mechanical stimuli whereby physical forces are translated into chemical signals between cells, via mechanotransduction. Thus, several different types of systems are used both in vitro and in vivo in order to study the response of cells to mechanical strain. The central aim of a loading system is to recreate the conditions required to engender mechanotransduction in a controlled cell culture environment. A common criticism of in vitro studies is that the substrate and the surroundings of the cell are too different to the cell microenvironment for findings obtained to be relevant. However, the device that will be developed in this project will be versatile in order to be applied with cells cultured in monolayer, but also with 3D cell cultures on scaffold materials, that more closely resemble the cell microenvironment. Furthermore, the device will be used to study the response to mechanical strain both in bone healthy cells and bone cancer cells and the results will be compared in order to better understand the biomechanics and biophysics of the cancer cells. To date, only a few studies have investigated the response of cancer cells to cyclic stretching. For example, cyclic strain increased the proliferative ability of and decreased apoptosis in Lewis lung cancer cells (D. Ma et al., 2009). Furthermore, cyclic strain increased expression of alpha-smooth muscle actin in myofibroblasts and their ability to accelerate cancer cell migration (J.W. Huang et al., 2013). In this present context, the develop and the validation of the uniaxial strain device could result very useful to realize an increment of the knowledge about the application of the mechanical stimuli on bone cancer cells and the study of their response to loading. The effect of applied strains on bone is dictated by the amplitude and duration of the applied load. For instance, Rubin and Lanyon (Rubin CT and Lanyon LE, 1985; Rubin CT and Lanyon LE, 1984) demonstrated that an applied strain of 2050 µstrain applied for four cycles per day produced the same maintaining effect on bone mass in immobilized limbs as an applied strain of 1000 µstrain applied for 100 cycles per day. In this context, it is also interesting to study the response of bone cancer cells to mechanical loading by varying the parameters and the duration of the applied load. The strain device that will be developed in this project allows to change the displacement parameters in a very simple way with computer control and provides precise control of these parameters. Furthermore, the devices commercially available allow to obtain only fixed values of deformations (2, 4, 6, 8, 10, 12, 15, 20 %) and frequency (0.2, 0.25, 0.5, 1 Hz), only for two waveform types, i.e. sine wave and square wave (Yasuyuki Morita et al., 2013). The device developed in this project will be able to achieve values of deformation in the range 0.05-30% with the possibilities to select every value of strain and frequency and different types of waveform (sine wave, square wave, triangle wave ecc.). Since physiological bone matrix strains due to normal daily activity are typically on the order of 0.05% strain (D.B. Burr et al., 1996; S.P. Fritton et al., 2000) the device could be useful , for instance, to provide this value of substrate deformation during the cells growth inside the incubator, so as to recreate the physiological cells microenvironment. This aspect is very important since it will make the device useful for different type of applications. This will allow us to use the device with different types of cells and tissues that show different physiological levels of substrate deformation.