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

Aliz Kunstar, Anne M. Leferink, Paul I. Okagbare, Michael D. Morris,
Blake J. Roessler, Cees Otto, Marcel Karperien, Clemens
A. van Blitterswijk, Lorenzo Moroni and Aart A. van Apeldoorn

Journal of the Royal Society Interface 2013


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 scaffoldswere
analysed for ECMformation 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 Ramanmicrospectroscopy. In the spectra collected
by the conventional Raman set-ups, the Raman bands at 937 and at
1062 cm21 which, respectively, correspond to collagen and sulfated glycosaminoglycans
could be used as Raman markers for ECM formation in scaffolds.
Collagen synthesiswas 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.....