Robust detection and high-resolution mapping of implanted alginate hydrogels in tissue with Raman spectroscopy

Alginate hydrogels are highly valuable for a variety of biomedical applications, such as scaffolds for tissue engineering and carriers for cell therapy. Alginate, a natural polysaccharide derived from brown algae, is unique for its ability to form a hydrogel in the presence of Ca²⁺ and is biocompatible and biodegradable. There is an unmet need for robust detection of hydrogels after implantation in cell-based therapies. In this study, we tested the feasibility of using Raman spectroscopy to detect alginate signatures and spatially mapping alginate in cryosectioned tissue samples following implantation of alginate scaffolds both with and without cells. We first characterized and validated Raman spectra of alginate. Then, we performed Raman spectroscopy on cell-laden alginate hydrogel microstrands that allow for high-density cell delivery. Furthermore, we used cyrosectioned mouse salivary gland tissue samples containing cell-laden alginate microstrands, which were prepared after in vivo implantation for 14 days, for Raman spectroscopy studies. We aimed to detect alginate and distinguish alginate in these tissue cryosections. We identified a region in the Raman spectra where alginate exhibited stronger signals than the tissue or freezing media. Specific Raman peaks at 816, 888, 959, 1300, and 1433 cm^-1 were associated with alginate. By analyzing Raman spectra from samples containing alginate, cells, tissue, and freezing media, we were able to distinguish alginate from the other components using either characteristic spectral peaks or Classical Least Squares (CLS) analysis. Additionally, we applied high-resolution mapping using line scanning Raman microscopy, demonstrating the capacity of spatial mapping of alginate in cryosectioned salivary gland tissue samples at the molecular level. The high sensitivity and specificity of Raman spectroscopy make it a promising tool for identifying engineered materials in tissue engineering applications.