Detection of Pentosidine Cross-Links in Cell-Secreted Decellularized Matrices Using Time Resolved Fluorescence Spectroscopy.
Detection of Pentosidine Cross-Links in Cell-Secreted Decellularized Matrices Using Time Resolved Fluorescence Spectroscopy.
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DOI：
10.1021/acsbiomaterials.6b00029
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发表时间：
2017-09-11
影响因子：
5.8
通讯作者：
Leach JK
中科院分区：
文献类型：
--
作者：
Mitra D;Fatakdawala H;Nguyen-Truong M;Creecy A;Nyman J;Marcu L;Leach JK
高血糖介导的非酶胶原交联（如戊糖苷（PENT））可能对细胞与细胞外基质（ECM）的相互作用产生有害影响。目前量化PENT的技术是有限的，因此需要改进方法来研究PENT对糖尿病临床挑战（如骨愈合受损）的积累和贡献。目前用于研究PENT的方法是破坏性的、费力的，并且经常采用缺乏天然ECM的复杂性的过度简化的胶原膜。本研究的主要目的是评估时间分辨荧光光谱法（TRFS）检测PENT在细胞分泌的ECM具有增强的组成复杂性的能力。为了证明这种方法的应用，我们评估了人间充质干细胞（MSC）对交联基质的反应，以探索检测到的PENT对成骨分化的作用。我们将MSC分泌的脱细胞基质（DM）暴露于0.66 M核糖2周，并使用TRFS检测PENT的积累。与对照DM相比，核糖处理导致峰值荧光发射蓝移30 nm，平均寿命显著降低（4.4 ± 0.3 ns vs 3.5 ± 0.09 ns）。用高效液相色谱法（HPLC）对样品进行评价，证实观察到的荧光变化是由于PENT蓄积所致。TRFS参数和PENT的HPLC测量之间存在很强的相关性，验证了TRFS作为PENT检测的替代方法的使用。在第7天和第14天，接种在核糖DM上的MSC中成骨细胞基因表达显著降低。然而，在对照和核糖DM之间没有检测到钙沉积的显著差异。这些数据证明了非破坏性荧光光谱法检查仿生培养平台内非酶胶原交联的形成的功效，并展示了一个例子，其中改进的仿生基质可用于探测胶原交联存在下的细胞-ECM相互作用。
Hyperglycemia-mediated, nonenzymatic collagen cross-links such as pentosidine (PENT) can have deleterious effects on cellular interactions with the extracellular matrix (ECM). Present techniques to quantify PENT are limited, motivating the need for improved methods to study the accumulation and contribution of PENT toward diabetic clinical challenges such as impaired bone healing. Current methods for studying PENT are destructive, laborious, and frequently employ oversimplified collagen films that lack the complexity of the native ECM. The primary goal of this study was to evaluate the capacity of time-resolved fluorescence spectroscopy (TRFS) to detect PENT in cell-secreted ECMs possessing enhanced compositional complexity. To demonstrate an application of this method, we assessed the response of human mesenchymal stem cells (MSCs) to cross-linked substrates to explore the role of detected PENT on osteogenic differentiation. We exposed MSC-secreted decellularized matrices (DMs) to 0.66 M ribose for 2 weeks and used TRFS to detect the accumulation of PENT. Ribose treatment resulted in a 30 nm blue shift in peak fluorescence emission and a significant decrease in average lifetime compared to that of control DMs (4.4 ± 0.3 ns vs 3.5 ± 0.09 ns). Evaluation of samples with high performance liquid chromatography (HPLC) confirmed that changes in observed fluorescence were due to PENT accumulation. A strong correlation was found between TRFS parameters and the HPLC measurement of PENT, validating the use of TRFS as an alternative method of PENT detection. Osteoblastic gene expression was significantly reduced in MSCs seeded on ribose DMs at days 7 and 14. However, no significant differences in calcium deposition were detected between control and ribose DMs. These data demonstrate the efficacy of nondestructive fluorescence spectroscopy to examine the formation of nonenzymatic collagen cross-links within biomimetic culture platforms and showcase one example where an improved biomimetic substrate can be used to probe cell-ECM interactions in the presence of collagen cross-links.
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影响因子：
5.1
作者：
Guariguata, L.;Whiting, D. R.;Shaw, J. E.
通讯作者：
Shaw, J. E.
影响因子：
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作者：
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DOI：
10.1016/j.biocel.2003.09.006
发表时间：
2004-05-01
影响因子：
4
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
Hedrick, MH
影响因子：
14
作者：
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通讯作者：
Leach JK