| The cell mechanics is an advancing realm of modern biomechanics that developes rapidly, it is an important part of tissue engineering, it studies mechanics of the cell membrane and the cytoskeleton, such as their deformation viscoelasticity and adhesion character, as well as various cell biochemical reactions, for instance, the change of mRNA expression, activation of transcription factors, alteration of cellular enzyme level and difference of membrane receptors. Because the diameter of human cell is between several microns and tens of microns and the thickness of cell membrane is between several nanometers and tens of nanometers, methods of the conventional experimental mechanics can not be applied directly to the research of cell mechanical characters, it is important to find suitable methods of cell loading and cell deformation measuring. Cell loading by fluid is an important method to study quantitatively the biomechanical response of cell morphology and function, and flowing fluid can simulate blood and interstitial fluid in vivo, the finding would become the theoretical basis for the clinical investigation of bone metabolism and vascular endothelial cells. In the realm of cell fluid mechanics, various kinds of experimental methods, measuring systems and apparatus have been introduced to research the mechanism of cells in response to fluid shearstress. Although the study of bone cells and vascular endothelial cells undergoing fluid shear stress with PPFC has been progressing and the PPFC has been improved, the report about the effect of shear stress on osteoclasts is scarce, and there are structures in classic PPFC that interfere experimentation, therefore the following four performances are to aim at tackling the above problems.(1) Designing parallel plate fluid system: On the basis of the well-established principles and techniques about flow chamber, a kind of parallel plate fluid system was designed and made to apply steady fluid shear stress to osteoclasts in vitro. The system consists of lower reservoir > peristaltic pump > upper reservoir and PPFC which are connected in turn with biocompatible silicon rubber tube. Biocompatible rubber gasket and metal clamping apparatus made of aluminum alloy are used to seal the flow chamber without changing its even height designed beforehand. The overflow structure and the overflow tube designed between the inlet and outlet in the upper reservoir guarantee the constant fluid surface height and fluid stabilization without vibrating in the upper reservoir. The influence of hydrostatic pressure on cells is minimized by adjusting the glass slide and the fluid surface in the upper reservoir to the same horizontal plane, comparability and repeatability elevate, so it is easy to analyze the experimental results. This system can be used to investigate the responses of osteoclasts etc. to fluid shear force in terms of morphology > physiology or biochemistry.(2) Culture and identification of rabbit osteoclasts: The smooth lower side of the silicone flexiPERM12 adhered to the dry slide when slightly pressed against it. In this way twelve chambers were formed which were isolated from each other. The slide with flexiPERM12 was moved into Petri dish before cell seeding. About one hour before cell suspension was removed into the chambers,the inner surface of each chamber bottom was coated with fibronectin. The bone marrow were induced with 1, 25(0H)2D3 to produce osteoclasts, osteoclasts were collected from New Zealand White rabbits, the femora, tibiae, humeri, and radii of two New Zealand white rabbits (1 day old) were dissected out. The shafts were cut longitudinally, and the interior surfaces were curetted to release the bone cells in a sterile petri dish containing a-MEM. Briefly, The bone fragments were removed by sedimentation under normal gravity, and cells were collected from the supernatant by centrifugation, the cells obtained from the bones of two rabbits were resuspended at a density of 1 xlO6 / ml in a-MEM ^6'1\ Osteoclasts were identified as cells containing three or more nuclei, culture of these cells stained intensely for tartrate-resistant acid phosphatase and formed resorption pits on bone slices in vitro. The osteoclasts obtained in this way are suitable for biomechanical study.(3) Study of the change of vacuolar area and osteoclast displacement in response to fluid shear stress: Osteoclasts in PPFC were observed with phase-contrast microscopy, osteoclasts were subjected to steady fluid shear stresses of 21.2 dyne/cm\ 9.7 dyne /cm2 and 1.1 dyne /cm2 for up to 120 min, respectively. The same osteoclast was photographed at 0, 5> \5> 30, 60 and 120 min, respectively. A software package Image-Pro Plus[8J was used to quantify vacuolar areas and displacement of osteoclasts selected randomly in each group. Osteoclasts hardly shedded while undergoing shear stress, they moved in the direction of fluid flow, the displacement extent was not the same among various kinds of cells, the osteoclasts were the greatest. The vacuolar area observed in osteoclasts increased with the passage of time and the raise of fluid shear stress. The results suggested that there is morphologic change ofosteoclasts in response to fluid shear stress, osteoclast is sensitive to fluid shear stress in terms of morphology.(4) Characteristics of free Ca2+ distribution in cultured osteoclasts: To study the spatial distribution of free Ca2+ in osteoclasts cultured on glass slide, the laser scanning confocal microscope and fluorescent probe to detect the free Ca2+ in osteoclast-like cells, the images were analyzed with image software^. At 37 °C the free Ca2+ in osteoclasts can be labelled effectively with lOumol/L Fluo-3/AM , the intensity of Ca2+ fluorescent signal in the central part is greater than that in the peripheral part and in the same section the signal was not distributed evenly. The intensity of Ca2+ fluorescent signal is different among various organellae in osteoclasts, which suggests the osteoclasts modulate its own function through the spatial difference of free Ca2+concentration.
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