Functional Properties Of Extracellular Matrix

软骨的受控水合或溶胀提供了确定软骨和其它细胞外基质的功能性质的手段。具体而言，我们已经使用了软骨的控制水合作用来测量胶原蛋白网络和蛋白聚糖（PG）的重要物理/化学性质独立的细胞外基质。这种方法需要将软骨组织基质建模为由两个不同相组成的复合材料：胶原蛋白网络和被困在其中的浓缩蛋白聚糖溶液;应用各种已知水平的平衡渗透应力;并使用物理化学原理和额外的实验来独立地确定PG和胶原蛋白相的“压力-体积”关系。在初步研究中，我们使用这种方法来确定天然和胰蛋白酶处理的正常人软骨样本以及骨关节炎（OA）关节软骨样本中胶原网络和PG相的压力-体积曲线。在正常和胰蛋白酶处理的标本中，胶原网络刚度似乎没有变化，而在OA标本中，胶原网络刚度下降。我们的研究结果强调了胶原蛋白网络在限制正常软骨水合作用和确保基质中高PG浓度方面的作用，这两者对于软骨中的有效承重和润滑都是必不可少的，但在OA中丢失。这些数据还表明，胶原网络刚度的损失，而不是PG的损失或修饰可能是导致OA中观察到的软骨随后崩解的初始事件。 然而，这种方法的一个缺点是，它需要大量的组织来获得渗透滴定曲线。这导致长的平衡时间，需要人-天来研究单个软骨标本，使得这种方法不适合常规病理分析或用于组织工程应用。最近，我们开发了一种新的微渗压计，以实用和快速的方式进行这些实验。 该仪器可以测量小组织样本（< 1微克）吸收的微量水，作为周围水蒸气平衡活性（压力）的函数。石英晶体检测附着在其表面的样品的吸水量。其共振频率对吸附水量的微小变化的高灵敏度使我们能够精确地测量组织样本的质量吸收。改变样本周围的平衡蒸汽压会引起组织层渗透压的受控变化。 为了验证该方法，我们使用了具有已知渗透特性的合成聚合物凝胶。为了说明新装置的适用性，我们测量了组织工程软骨标本的膨胀压力。 微渗压计将最终允许我们同时获得多个软骨标本的渗透压缩性或刚度的曲线，作为从关节面到骨界面的深度的函数。 它还将使我们能够量化ECM的各个组分（如聚集蛋白聚糖、透明质酸和胶原蛋白）对总渗透压的贡献。此外，它应该使我们能够评估渗透相容性和机械完整性的发展中的组织和组织工程软骨（或ECM）的希望，提高整合后植入。 最近，我们已经开发了一种原子力显微镜（AFM）技术映射局部弹性和粘弹性的组织特性。 许多以前阻碍使用AFM在高通量探测不均匀的样品，特别是生物组织的障碍，已得到解决。 该技术利用商业AFM的精确扫描能力来生成大量的顺应性数据，并自动从数据中提取相关的弹性特性。 结合从微渗压和生化分析获得的结果，这种技术将使我们能够映射软骨标本的承载能力的空间变化。

Controlled hydration or swelling of cartilage provides a means of determining functional properties of cartilage and of other extracellular matrices. Specifically, we have used controlled hydration of cartilage to measure important physical/chemical properties of the collagen network and of the proteoglycans (PG) independently within the extracellular matrix. This approach entailed modeling the cartilage tissue matrix as a composite material consisting of two distinct phases: a collagen network and a concentrated proteoglycan solution trapped within it; applying various known levels of equilibrium osmotic stress; and using physical-chemical principles and additional experiments to determine a "pressure-volume" relationship for both the PG and collagen phases independently. In pilot studies, we used this approach to determine pressure-volume curves for the collagen network and PG phases in native and in trypsin-treated normal human cartilage specimen, as well as in cartilage specimen from osteoarthritic (OA) joints. In both normal and trypsin-treated specimen, collagen network stiffness appeared unchanged, whereas in the OA specimen, collagen network stiffness decreased. Our findings highlighted the role of the collagen network in limiting normal cartilage hydration, and in ensuring a high PG concentration in the matrix, both of which are essential for effective load bearing in cartilage and lubrication, but are lost in OA. These data also suggest that the loss of collagen network stiffness, and not the loss or modification of PGs may be the incipient event leading to the subsequent disintegration of cartilage observed in OA. One shortcoming of this approach, however, was that it required a significant amount of tissue to obtain the osmotic titration curves. This lead to long equilibration times requiring person-days to study a single cartilage specimen, making this approach unsuitable for routine pathological analysis or for use in tissue engineering applications. Recently, we developed a new micro-osmometer to perform these experiments in a practical and rapid manner. This instrument can measure minute amounts of water absorbed by small tissue samples (< 1 microgram) as a function of the equilibrium activity (pressure) of the surrounding water vapor. A quartz crystal detects the water uptake of a specimen attached to its surface. The high sensitivity of its resonance frequency to small changes in the amount of adsorbed water allows us to measure the mass uptake of the tissue specimen precisely. Varying the equilibrium vapor pressure surrounding the specimen induces controlled changes in the osmotic pressure of the tissue layer. To validate the methodology, we used synthetic polymer gels with known osmotic properties. To illustrate the applicability of the new apparatus, we measured the swelling pressure of tissue-engineered cartilage specimen. The micro-osmometer will eventually permit us to obtain a profile of the osmotic compressibility or stiffness of multiple cartilage specimens simultaneously as a function of depth from the articular surface to the bone interface. It will also allow us to quantify the contributions of individual components of ECM (such as aggrecan, hyaluronic acid and collagen) to the total osmotic pressure. Moreover, it should allow us to assess the osmotic compatibility and mechanical integrity of developing tissues and of tissue-engineered cartilage (or ECM) with the hope of improving integration following implantation. Recently, we have developed an atomic force microscopy (AFM) technique for mapping the local elastic and viscoelastic properties of tissues. Many of the impediments that have previously hindered the use of the AFM in high-throughput probing of inhomogeneous samples, particularly biological tissues, have been addressed. The technique utilizes the precise scanning capabilities of a commercial AFM to generate large volumes of compliance data and automatically extracts the relevant elastic properties from the data. In conjunction with results obtained from micro-osmometry and biochemical analysis, this technique will allow us to map spatial variations in the load-bearing capacity of cartilage specimens.