Characterising Contraction in a Tissue-Engineered Buccal Mucosa Model with Raman Spectroscopy

Graft contracture of tissue engineered buccal mucosa models has been monitored and characterised with Raman Spectroscopy. Tissue engineered buccal mucosa grafts have been used clinically for the reconstruction of head and neck cancer and for extraoral applications such as replacement of scarred tissue in the urethra. However, both in the laboratory and clinically, these 3D models exhibit contraction compromising the proper functionality of the surrounding tissues. Graft contraction continues to be a poorly understood clinical problem and what is needed ideally is a better understanding of the underlying biochemical mechanism behind contraction. Since studying collagen crosslinking biochemically is technically difficult, assays are tedious and not very reliable, Raman spectroscopy was elected as the technique of choice to characterise and monitored the process of contraction. In this research project, the process of contraction was characterised as a function of time before and after incorporation of β-APN drug. The use of multivariate analysis (MVA) techniques highlighted the increase of -helix triple structure as a function of time and extent of crosslinking. A reduction of -helix formation was also noticed after incorporation of β-APN drug and inhibition of contraction. Together with this, formation of β-sheet structure was also highlighted by the statistical analysis after inhibiting crosslinking of collagen fibres. Together with this, the amide III region was also identified as a biomarker for the progression of contraction in all three layers of the oral mucosa graft. A turnover of the I1335/I1245 cm-1 doublet was noticed as crosslinking increased. No turnover was seen after incorporation of β-APN drug into the experiment. This region is representative of the peptidic bond of the polypeptide chains. Findings represent a stepping stone towards a better understanding behind the nature of this process. It opens up the possibility of considering the participation of these biomarkers, as a complementary route of collagen cross-linking progression, coupled with the important role that lysyl-oxidase (LOX) enzyme plays on it during the process of contraction.