Label-free Raman monitoring of extracellular matrix formation in three-dimensional polymeric scaffolds

Abstract

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 noninvasive 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 21 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 proofof-principle that Raman microspectroscopy is a promising non-invasive tool to monitor ECM production and remodelling in three-dimensional porous cartilage tissue-engineered constructs.

Vibrational spectroscopy and imaging: applications for tissue engineering

“…The authors reported the use of Raman spectroscopy to quantify and image collagen, GAG and water distributions in the engineered constructs (Figure 7). Kunstar et al 139 reported on the use of Raman for monitoring extracellular matrix components in tissue engineered cartilage in addition to biochemical and histological analysis. Poly (ethylene oxide terephthalate)/poly (butylene terephthalate) scaffolds were seeded with primary bovine chondrocytes and evaluated at 7 and 21 days of culture.…”

Analysis of Synovial Fluid for Study of Diseases of Joints Using the Method of Raman Spectroscopy

“…At present, optical methods of research have become widely used for the study of bioobjects: backward scattering spectroscopy [4], microscopic analysis [5,6], and Raman spectroscopy [7,8]. Raman spectroscopy has certain advantages over other methods.…”

“…The authors of the articles [9,10], conducted studies of the structure of the synovial fluid and evaluated the content of their protein components.…”

1984 : on monitoring cell fate in three-dimensional polymeric scaffolds for tissue engineering applications

“…Careful selection of suitable laser wavelengths and laser intensity can eliminate cell damage allowing for the study of cells without inadvertently changing their phenotype or behavior caused by photo damage [191][192][193]. Raman spectral studies have been performed for structural analysis of ECM components such as collagen [194] and proteoglycans [189,195]. Raman peaks at distinct wavelengths exist, for example, for different types of carbon-carbon bonds, amide, carboxyl, sulfhydryl, and phenol groups [120].…”

“…Raman microspectroscopy is a label-free spectroscopic technique, which does not require special sample preparation and can be used for non-invasive characterization of cell and tissue biochemistry [189,190]. Careful selection of suitable laser wavelengths and laser intensity can eliminate cell damage allowing for the study of cells without inadvertently changing their phenotype or behavior caused by photo damage [191][192][193].…”