Department of Biomedical Engineering, University of Utah, Salt Lake City, Utah.
Scientific Computing and Imaging Institute, University of Utah, Salt Lake City, Utah.
J Orthop Res. 2019 Feb;37(2):431-438. doi: 10.1002/jor.24185. Epub 2019 Jan 3.
The purpose of this study was to develop a microplate assay for quantifying denatured collagen by measuring the fluorescence of carboxyfluorescein bound collagen hybridizing peptides (F-CHP). We have shown that F-CHP binds selectively with denatured collagen, and that mechanical overload of tendon fascicles causes collagen denaturation. Proteinase K was used to homogenize tissue samples after F-CHP staining, allowing fluorescence measurement using a microplate reader. We compared our new assay to our previous image analysis method and the trypsin-hydroxyproline assay, which is the only other available method to directly quantify denatured collagen. Relative quantification of denatured collagen was performed in rat tail tendon fascicles subjected to incremental tensile overload, and normal and ostoeoarthritic guinea pig cartilage. In addition, the absolute amount of denatured collagen was determined in rat tail tendon by correlating F-CHP fluorescence with percent denatured collagen as determined by the trypsin-hydroxyproline assay. Rat tail tendon fascicles stretched to low strains (<7.5%) exhibited minimal denatured collagen, but values rapidly increased at medium strains (7.5-10.5%) and plateaued at high strains (≥12%). Osteoarthritic cartilage had higher F-CHP fluorescence than healthy cartilage. Both of these outcomes are consistent with previous studies. With the calibration curve, the microplate assay was able to absolutely quantify denatured collagen in mechanically damaged rat tail tendon fascicles as reliably as the trypsin-hydroxyproline assay. Further, we achieved these results more efficiently than current methods in a rapid, high-throughput manner, with multiple types of collagenous tissue while maintaining accuracy. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:431-438, 2019.
本研究旨在开发一种通过测量与变性胶原杂交肽结合的羧基荧光素标记物（F-CHP）的荧光来定量变性胶原的微量板分析。我们已经表明，F-CHP 与变性胶原选择性结合，并且肌腱束的机械过载会导致胶原变性。在用 F-CHP 染色后，蛋白酶 K 用于组织样品的匀浆化，从而可以使用微孔板读取器进行荧光测量。我们将新的测定方法与以前的图像分析方法和胰蛋白酶-羟脯氨酸测定法进行了比较，后两者是唯一可直接定量变性胶原的方法。在递增拉伸过载的大鼠尾腱束以及正常和骨关节炎豚鼠软骨中，对变性胶原进行了相对定量。此外，通过将 F-CHP 荧光与胰蛋白酶-羟脯氨酸测定法确定的变性胶原百分比相关联，在大鼠尾腱中确定了变性胶原的绝对量。拉伸至低应变（<7.5％）的大鼠尾腱束表现出最小的变性胶原，但在中应变（7.5-10.5％）时迅速增加，并在高应变（≥12％）时达到平台。骨关节炎软骨的 F-CHP 荧光高于健康软骨。这些结果都与以前的研究一致。使用校准曲线，微量板测定法能够像胰蛋白酶-羟脯氨酸测定法一样可靠地绝对定量机械损伤的大鼠尾腱束中的变性胶原。此外，与当前方法相比，我们以更有效的方式，以快速、高通量的方式，使用多种类型的胶原组织，同时保持准确性，实现了这些结果。2018 年骨科研究协会。 Wiley Periodicals，Inc. 出版。J Orthop Res 37:431-438，2019 年。
