Label-free Raman monitoring of extracellular matrix formation in three-dimensional polymeric scaffolds
Label-free Raman monitoring of extracellular matrix formation in three-dimensional polymeric scaffolds
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DOI：
10.1098/rsif.2013.0464
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发表时间：
2013-09-06
影响因子：
3.9
通讯作者：
van Apeldoorn, Aart A.
中科院分区：
文献类型：
--
作者：
Kunstar, Aliz;Leferink, Anne M.;van Apeldoorn, Aart A.
监测细胞外基质（ECM）成分是用于确定再生医学和临床目的的三维支架中组织质量的关键方法之一。拉曼光谱可用于细胞和ECM生物化学的非侵入式感测。我们研究了使用传统的（共焦和双焦）拉曼显微光谱和光纤拉曼光谱在体外监测ECM形成的三维聚（环氧乙烷对苯二甲酸酯）-聚（对苯二甲酸丁二醇酯）（PEOT/PBT）支架。通过拉曼显微光谱、生化分析、组织学和扫描电子显微镜分析软骨细胞接种的PEOT/PBT支架的ECM形成。通过生物化学和组织学分析以及通过无标记非破坏性拉曼显微光谱法成功地检测了这些支架中的ECM沉积。在通过常规拉曼装置收集的光谱中，分别对应于胶原和硫酸化糖胺聚糖的937和1062 cm（-1）处的拉曼带可用作支架中ECM形成的拉曼标记。胶原蛋白的合成被发现是不同的单一软骨细胞接种的支架相比，微聚集体接种的样品。在21天的培养期间，单个细胞接种样品的胶原蛋白含量的归一化带面积比逐渐降低，而微聚集体接种样品的胶原蛋白含量在此期间显著增加。此外，光纤拉曼装置允许并行地从支架内部的多个孔收集拉曼光谱。这些光纤测量可以给出组织工程化构建体中存在的ECM拉曼信号的代表性平均值。本研究的结果提供了原理证明，拉曼显微光谱是一种很有前途的非侵入性工具，以监测ECM的生产和重建的三维多孔软骨组织工程建设。
Monitoring extracellular matrix (ECM) components is one of the key methods used to determine tissue quality in three-dimensional scaffolds for regenerative medicine and clinical purposes. Raman spectroscopy can be used for non-invasive sensing of cellular and ECM biochemistry. We have investigated the use of conventional (confocal and semiconfocal) Raman microspectroscopy and fibre-optic Raman spectroscopy for in vitro monitoring of ECM formation in three-dimensional poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) scaffolds. Chondrocyte-seeded PEOT/PBT scaffolds were analysed for ECM formation by Raman microspectroscopy, biochemical analysis, histology and scanning electron microscopy. ECM deposition in these scaffolds was successfully detected by biochemical and histological analysis and by label-free non-destructive Raman microspectroscopy. In the spectra collected by the conventional Raman set-ups, the Raman bands at 937 and at 1062 cm(-1) which, respectively, correspond to collagen and sulfated glycosaminoglycans could be used as Raman markers for ECM formation in scaffolds. Collagen synthesis was found to be different in single chondrocyte-seeded scaffolds when compared with microaggregate-seeded samples. Normalized band-area ratios for collagen content of single cell-seeded samples gradually decreased during a 21-day culture period, whereas collagen content of the microaggregate-seeded samples significantly increased during this period. Moreover, a fibre-optic Raman set-up allowed for the collection of Raman spectra from multiple pores inside scaffolds in parallel. These fibre-optic measurements could give a representative average of the ECM Raman signal present in tissue-engineered constructs. Results in this study provide proof-of-principle that Raman microspectroscopy is a promising non-invasive tool to monitor ECM production and remodelling in three-dimensional porous cartilage tissue-engineered constructs.