定常流加载系统的设计制作及流体切应力对体外培养破骨细胞的影响
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摘要
细胞力学是现代生物力学发展迅速的一个科学前沿领域,是组织工程学的一个重要组成部分,它研究细胞在载荷作用下细胞膜和细胞骨架的变形、粘弹性和粘附力等力学性能,还有细胞的一系列生化反应,如mRNA表达的变化、转录因子的激活、细胞内酶水平的改变及膜受体变化等。由于人体细胞在十几至几十微米之间,细胞膜的厚度在几个纳米至几十纳米之间,常规实验力学方法无法直接应用于细胞力学特性研究,寻求合适的细胞加载方法和细胞变形的测量手段,是细胞力学面临的首要问题。应用流体对细胞加载是研究细胞生物力学反应的重要手段,模拟体内血液和组织液,从而定量研究切应力对细胞形态和功能影响,为骨代谢及血管内皮细胞的临床研究等提供理论基础。在细胞流体力学研究领域,人们提出各种实验方法和手段,开发出许多不同类型和功能的实验装置,通过对体外培养细胞施加不同方式的流体切应力,利用各种监测手段,研究细胞对切应力的反应机理。虽然运用流室研究流体切应力对骨组织来源细胞和血管内皮细胞形态及功能影响已取得诸多进展,流动装置也得以改进,但是关于流室中OC对切应力的生物力学反应报道很少,并且经典的流室系统中存在不利于实验顺利、快速进行的结构,本研究针对以上问题作了四方面工作:
(1)设计制作一套细胞流体切应力实验系统:基于传统的PPFC理论和方法,并做一些改进,设计制作一套细胞流体切应力实验系统,用于体
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 shear
stress. 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 of
osteoclasts 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.
(1)设计制作一套细胞流体切应力实验系统:基于传统的PPFC理论和方法,并做一些改进,设计制作一套细胞流体切应力实验系统,用于体
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 shear
stress. 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 of
osteoclasts 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.
引文
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