【作者】 刘广源；

【导师】 邱贵兴；

【作者基本信息】 中国协和医科大学 ， 骨外科学， 2007， 博士

【摘要】 研究背景骨关节炎(Osteoarthritis，OA)是最常见关节疾病之一。其主要表现为关节软骨退变磨损和骨赘形成，导致关节肿胀、疼痛、畸形和功能障碍等。力学因素与骨关节炎之间有着密切的关系，在关节退行病变的开始和发展中起重要作用。软骨细胞力学是软骨细胞工程及软骨组织工程的基础和重要影响因素，种子细胞的三维培养和增殖与细胞的力学行为密切相关。了解软骨细胞在正常和病理条件下如何响应细胞外力信号及其调节情况，可进一步揭示软骨组织工程化细胞种植的最佳力学环境。而传统的单细胞力学研究多集中在单细胞整体平均力学特性的分析上，其常用的研究手段如微管吸吮法无法在细胞自然生活状态下进行力学检测，也不能提供细胞不同区域和部位更加精确的力学特性。为进一步分析细胞不同部位的力学特性和对应力刺激反应的差异，需要发展新的不损伤细胞的应力加载方法和生物学测量手段。这些方法所获得的基础信息将对实现体外大规模扩增培养软骨细胞以及软骨移植修复的发展和临床应用提供有益的帮助。研究目的运用原子力显微术(Atomic Force Microscopy，AFM)新近发展的磁驱动轻敲模式(Magnetic AC mode，MAC mode)能够对培养状态下的活细胞表面超微形貌结构进行更清晰的成像和对细胞不同部位进行力学测定的优点，来研究体外培养的正常人与骨关节炎患者活软骨细胞表面形貌结构及细胞核和细胞质不同部位力学特性的差异。研究方法体外培养正常人与骨关节炎患者的膝关节软骨细胞，运用MAC mode AFM在细胞培养的生活状态下对两组细胞的表面形貌进行高分辨成像，观察动态变化。并通过对细胞核与细胞质不同区域的力曲线(Force Curve)进行描述，分析软骨细胞核区与细胞质区的力学特性变化及细胞局部对力学刺激的反应情况。通过观察力曲线形态差异，分析正常与骨关节炎软骨细胞的力学特性差异。研究结果MAC mode AFM可实现生理条件下活软骨细胞的表面形貌超微结构的高分辨成像，同时可对液态培养环境中的附壁软骨细胞进行实时动态监测，观察细胞的生理活动现象。活软骨细胞细胞核区的可变形能力和弹性高于细胞质区(P＜0.001)，而表面粘滞性区别不显著(P＞0.05)。骨关节炎软骨细胞细胞核区与细胞质区的可变形能力和弹性均明显低于正常软骨细胞(P＜0.001)，表面粘滞性也低于正常软骨细胞(P＜0.001)。结论MAC Mode AFM不仅能进行活细胞的高分辨成像和实时动态观察，还可通过对单细胞局部施加微应力及描绘力曲线形态，来研究细胞不同区域的弹性和粘滞性等力学特性。活软骨细胞的细胞质区和细胞核区表现出不同的力学性质。骨关节炎软骨细胞的弹性和粘滞性等力学特性明显不同于正常软骨细胞。更多还原

【Abstract】 The Mechanical Properties of Living Osteoarthritis Chondrocytes by Using Magnetic AC mode Atomic Force MicroscopyBackground Osteoarthritis (OA) is one of the most common articular diseases, with major clinical symptoms of articular cartilage degeneration and abrasion, and brings on joint pain and dysfunction. Mechanical factor has a close relationship with OA and plays a crucial role in the development of joint degeneration. Mechanical properties of chondrocytes is the foundation and key influence in chondrocyte engineering and cartilage tissue-engineering. The three dimensional culture of seed cells is tightly related to the mechanical behavior of chondrocytes. Our understanding of how chondrocytes responds to force loading and adjusts correspondingly can further reveal the best mechanical circumstance for articular cartilage cell implantation. Traditional cytomechanics research focus upon average mechanical properties of a single cell, and the methods used such as micropipette aspiration are unable to undergo mechanical detection in terms of in vitro live adhered culture cells and can not provide mechanical properties in various parts of living cells. In order to make an in-depth investigation regarding the mechanical properties at various parts of cells and the difference between reaction of local force stimulation, it’s required to develop a new instrument of stress loading and biological measurement without damaging the cell. All these basic information obtained will greatly benefit the extensive spread of cultured chondrocytes in vitro and the development and clinical application of tissue-engineered cartilage repair and cells transplantation. Objective Studies on the topography and mechanical properties in different region of living normal and osteoarthritis chondrocytes in vitro by using the novel tapping mode atomic force microscopy (AFM) named magnetic AC mode AFM (MAC mode AFM).Methods The MAC mode AFM was carried out to observe the topographic details and dynamic state of the cultured normal and osteoarthritis human knee joint chondrocytes under the physiological state in vitro. Meantime, the force curve of MAC mode AFM was performed at certain regions to compare the mechanical properties of the nuclear area with its cytoplasm. Analyze the difference of mechanical properties and response of local force loading in different areas of normal and osteoarthritis chondrocytes.Results The high resolution imaging and time-lapse development of the living chondrocytes was successfully acquired by this experimental system. The force curve showed the much higher elasticity of nuclear region comparing with the body region in normal chondrocytes (P＜0.001) and there was no obvious difference of surface viscosity between them (P＞0.05). Either of nuclear and body region of osteoarthritis chondrocytes has a much lower elasticity (P＜0.001) and a lower surface viscosity (P＜0.001) than that of normal chondrocytes.Conclusions By using MAC Mode AFM, we can obtain high-resolution imaging, carry out real-time observation of living cells, and study such mechanical properties at various parts of the cell as elasticity and glutinosity through loading force on partial single cell and portraying change of force curve. Cytoplasmic and nuclear area of living chondrocytes represents different mechanical properties. The mechanical properties of osteoarthritis chondrocytes is obviously distinct with that of normal chondrocytes.更多还原