Dean Delphine, Han Lin, Grodzinsky Alan J, Ortiz Christine
Department of Electrical Engineering and Computer Science, 77 Massachusetts Avenue, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
J Biomech. 2006;39(14):2555-65. doi: 10.1016/j.jbiomech.2005.09.007. Epub 2005 Nov 9.
In this study, we have measured the nanoscale compressive interactions between opposing aggrecan macromolecules in near-physiological conditions, in order to elucidate the molecular origins of tissue-level cartilage biomechanical behavior. Aggrecan molecules from fetal bovine epiphyseal cartilage were chemically end-grafted to planar substrates, standard nanosized atomic force microscopy (AFM) probe tips (R(tip) approximately 50 nm), and larger colloidal probe tips (R(tip) approximately 2.5 microm). To assess normal nanomechanical interaction forces between opposing aggrecan layers, substrates with microcontact printed aggrecan were imaged using contact mode AFM, and aggrecan layer height (and hence deformation) was measured as a function of solution ionic strength (IS) and applied normal load. Then, using high-resolution force spectroscopy, nanoscale compressive forces between opposing aggrecan on the tip and substrate were measured versus tip-substrate separation distance in 0.001-1M NaCl. Nanosized tips enabled measurement of the molecular stiffness of 2-4 aggrecan while colloidal tips probed the nanomechanical properties of larger assemblies (approximately 10(4) molecules). The compressive stiffness of aggrecan was much higher when using a densely packed colloidal tip than the stiffness measured for using the nanosized tip with a few aggrecan, demonstrating the importance of lateral interactions to the normal nanomechanical properties. The measured stress at 0.1M NaCl (near-physiological ionic strength) increased sharply at aggrecan densities under the tip of approximately 40 mg/ml (physiological densities are approximately 20-80 mg/ml), corresponding to an average inter-GAG spacing of 4-5 Debye lengths (4-5 nm); this characteristic spacing is consistent with the onset of significant electrostatic interactions between GAG chains of opposing aggrecan molecules. Comparison of nanomechanical data to the predictions of Poisson-Boltzmann-based models further elucidated the regimes over which electrostatic and nonelectrostatic interactions affect aggrecan stiffness in compression. The most important aspects of this study include: the incorporation of experiments at two different length scales, the use of microcontact printing to enable quantification of aggrecan deformation and the corresponding nanoscale compressive stress vs. strain curve, the use of tips of differing functionality to provide insights into the molecular mechanisms of deformation, and the comparison of experimental data to the predictions of three increasingly refined Poisson-Boltzmann (P-B)-based theoretical models for the electrostatic double layer component of the interaction.
在本研究中，我们测量了近生理条件下相对的聚集蛋白聚糖大分子之间的纳米级压缩相互作用，以阐明组织水平软骨生物力学行为的分子起源。将来自胎牛骨骺软骨的聚集蛋白聚糖分子化学末端接枝到平面基底、标准纳米尺寸原子力显微镜（AFM）探针尖端（R（尖端）约50nm）和较大的胶体探针尖端（R（尖端）约2.5μm）上。为了评估相对的聚集蛋白聚糖层之间的正常纳米力学相互作用力，使用接触模式AFM对微接触印刷有聚集蛋白聚糖的基底进行成像，并测量聚集蛋白聚糖层高度（以及由此产生的变形）作为溶液离子强度（IS）和施加的法向载荷的函数。然后，使用高分辨率力谱，在0.001 - 1M NaCl中测量尖端和基底上相对的聚集蛋白聚糖之间的纳米级压缩力与尖端 - 基底分离距离的关系。纳米尺寸的尖端能够测量2 - 4个聚集蛋白聚糖的分子刚度，而胶体尖端则探测较大聚集体（约10⁴个分子）的纳米力学性质。当使用紧密堆积的胶体尖端时，聚集蛋白聚糖的压缩刚度比使用带有少数聚集蛋白聚糖的纳米尺寸尖端测量的刚度高得多，这表明横向相互作用对正常纳米力学性质的重要性。在0.1M NaCl（近生理离子强度）下测量的应力在尖端下方聚集蛋白聚糖密度约为40mg/ml时急剧增加（生理密度约为20 - 80mg/ml），对应于平均糖胺聚糖间距为4 - 5德拜长度（4 - 5nm）；这一特征间距与相对的聚集蛋白聚糖分子的糖胺聚糖链之间显著静电相互作用的开始一致。将纳米力学数据与基于泊松 - 玻尔兹曼模型的预测进行比较，进一步阐明了静电和非静电相互作用影响聚集蛋白聚糖压缩刚度的范围。本研究最重要的方面包括：在两个不同长度尺度上进行实验、使用微接触印刷来量化聚集蛋白聚糖变形以及相应的纳米级压缩应力与应变曲线、使用具有不同功能的尖端来深入了解变形的分子机制，以及将实验数据与三种日益精细的基于泊松 - 玻尔兹曼（P - B）的相互作用静电双层成分理论模型的预测进行比较。
