Raman Spectroscopic Imaging for Quantification of Depth-dependent and Local Heterogeneities in Native and Engineered Cartilage

Articular cartilage possesses a remarkable, mechanically-robust extracellular matrix (ECM) that is organized and distributed throughout the tissue to resist physiologic strains and provide low friction during articulation. The ability to characterize the make-up and distribution of the cartilage ECM is critical to both understand the process by which articular cartilage undergoes disease-related degeneration and to develop novel tissue repair strategies to restore tissue functionality. However, the ability to quantitatively measure the spatial distribution of cartilage ECM constituents throughout the tissue has remained a major challenge. In this experimental investigation, we assessed the analytical ability of Raman micro-spectroscopic imaging to semi-quantitatively measure the distribution of the major ECM constituents in cartilage tissues. Raman spectroscopic images were acquired of two distinct cartilage tissue types that possess large spatial ECM gradients throughout their depth: native articular cartilage explants and large engineered cartilage tissue constructs. Spectral acquisitions were processed via multivariate curve resolution to decompose the "fingerprint" range spectra (800-1800 cm) to the component spectra of GAG, collagen, and water, giving rise to the depth dependent concentration profile of each constituent throughout the tissues. These Raman spectroscopic acquired-profiles exhibited strong agreement with profiles independently acquired via direct biochemical assaying of spatial tissue sections. Further, we harness this spectroscopic technique to evaluate local heterogeneities through the depth of cartilage. This work represents a powerful analytical validation of the accuracy of Raman spectroscopic imaging measurements of the spatial distribution of biochemical components in a biological tissue and shows that it can be used as a valuable tool for quantitatively measuring the distribution and organization of ECM constituents in native and engineered cartilage tissue specimens.