Functional Properties Of Extracellular Matrix

软骨的受控水化或膨胀为确定软骨和其他细胞外基质的功能特性提供了一种手段。具体地说，我们使用受控的软骨水合作用来测量细胞外基质中独立的胶原网络和蛋白多糖(PG)的重要物理/化学性质。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：胶原网和困在其中的浓缩蛋白多糖溶液；施加各种已知水平的平衡渗透应力；使用物理化学原理和额外的实验来独立地确定PG和胶原蛋白两个相的“压力-体积”关系。在初步研究中，我们使用这种方法测定了天然和经胰酶处理的正常人软骨标本以及骨关节炎(OA)关节软骨标本中胶原网络和PG相的压力-体积曲线。在正常和胰酶处理的标本中，胶原网络硬度没有变化，而在骨性关节炎标本中，胶原网络硬度降低。我们的发现强调了胶原网络在限制正常软骨水化和确保基质中高PG浓度方面的作用，这两者都是软骨有效承载和润滑所必需的，但在OA中消失了。这些数据还表明，胶原网络僵硬的丧失，而不是PGs的丢失或修饰，可能是导致随后观察到的OA软骨解体的早期事件。 然而，这种方法的一个缺点是需要大量的组织来获得渗透滴定曲线。这导致研究单个软骨标本需要很长的平衡时间，这使得这种方法不适合常规的病理分析或组织工程应用。最近，我们开发了一种新的微渗透压计，以实用和快速的方式进行这些实验。该仪器可以测量微小组织样品(1微克)所吸收的微量水分，作为周围水蒸气的平衡活度(压力)的函数。石英晶体检测附着在其表面的样品的吸水率。其共振频率对吸附水量的微小变化高度敏感，这使我们能够精确地测量组织样品的质量摄取量。改变样品周围的平衡蒸汽压会导致组织层渗透压的受控变化。为了验证这一方法，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。 微渗透压计最终将使我们能够同时获得多个软骨标本的渗透可压缩性或硬度随深度从关节表面到骨界面处的分布。它还将使我们能够量化ECM的各个组成部分(如聚集素、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发育中的组织和组织工程软骨(或ECM)的渗透兼容性和机械完整性，以期改善植入后的整合。 最近，我们开发了一种原子力显微镜(AFM)技术来绘制组织的局部弹性和粘弹性属性。以前阻碍原子力显微镜用于高通量探测非均质样品，特别是生物组织的许多障碍已经得到解决。该技术利用商用AFM的精确扫描能力来生成大量的合规数据，并自动从数据中提取相关的弹性属性。结合微量渗透压和生化分析的结果，这项技术将使我们能够绘制软骨标本承载能力的空间变化图。

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.