Using Dispersive Raman Spectroscopy to Assess Remodeling in Vascular Constructs used in Tissue Engineering
Theus, Andrea S.
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Tissue engineered vascular grafts (TEVGs) are a promising alternative to surgery for the treatment of thrombotic occlusion of small diameter (< 6 mm) arteries. It is important that TEVG composition prevent compliance mismatch. Both initial protein incorporation and cellular remodeling are important for blended TEVG compliance. Understanding the distribution of extracellular matrix (ECM) components is also important and may dictate the remodeling response. Raman spectroscopy has been used to assess components of engineered scaffolds non-destructively. However, Raman has not been applied to semi-quantitatively assess 3D engineered-tissues and cellular remodeling of blended materials with low-scattering natural and high-scattering synthetic components. This is important due to the increased use of synthetic biomaterials for TEVG applications. The overall goal of this study was to develop a technique to assess the cellular and matrix remodeling response. Electrospun meshes were prepared with 100% poly(ε-caprolactone) (PCL), 90% PCL/collagen, and 90% PCL/fibrinogen. XPS and Raman results showed differences of protein distribution when incorporated into a PCL scaffold and differences in the resulting effects with cell culturing and incubation. Raman spectra was acquired for all pure materials individually (e.g. PCL, collagen, and fibrinogen) to determine the unique spectral fingerprint of each material. Protein peaks of interest were identified (e.g. 740 and 1040 cm-¹ ) and were analyzed when protein was incorporated into the electrospun meshes. Raman spectra was acquired for the pure PCL control and blended scaffold conditions. The protein peaks remained relevant for proteins even when incorporated into the high scattering PCL polymer and showed remodeling due to incubation and cell culturing over 21 days. Spectra were also analyzed with multi-factor processing techniques (e.g. MFA) and showed that separation between appropriate sample groups can be achieved even with large amounts of spectral data sets. Biochemical analysis (e.g. Hyp, µ-BCA assay) was done to determine if specific remodeling responses, degradation and/or release occur with incubation and culturing of smooth muscle cells. These results showed a negligible loss of protein within electrospun meshes with incubation and culture. Overall, we have shown that Raman spectroscopy can be used to semi-quantitatively detect the incorporation of low levels of specific proteins (e.g. collagen and fibrinogen) within electrospun meshes as well as changes that occur with mesh remodeling.